Targeting therapeutic vulnerabilities with PARP inhibition and radiation in IDH-mutant gliomas and cholangiocarcinomas – Science Advances

Abstract

Mutations in isocitrate dehydrogenase (IDH) genes occur in multiple cancer types, lead to global changes in the epigenome, and drive tumorigenesis. Yet, effective strategies targeting solid tumors harboring IDH mutations remain elusive. Here, we demonstrate that IDH-mutant gliomas and cholangiocarcinomas display elevated DNA damage. Using multiple in vitro and preclinical animal models of glioma and cholangiocarcinoma, we developed treatment strategies that use a synthetic lethality approach targeting the reduced DNA damage repair conferred by mutant IDH using poly(adenosine 5-diphosphate) ribose polymerase inhibitors (PARPis). The therapeutic effects are markedly enhanced by cotreatment with concurrent, localized radiation therapy. PARPi-buttressed multimodality therapies may represent a readily applicable approach that is selective for IDH-mutant tumor cells and has potential to improve outcomes in multiple cancers.

Neomorphic mutations in the genes encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) have been identified in multiple cancer types, including lower grade glioma (LGG) (1), secondary glioblastoma (2), intrahepatic cholangiocarcinoma (ICC) (3, 4), acute myeloid leukemia (AML) (5), chondrosarcoma (CS) (6), and others. The mutant IDH enzyme (IDHmut) converts the Krebs cycle intermediate -ketoglutarate (KG) into 2-hydroxyglutarate (2-HG), which functions as an oncometabolite. 2-HG can induce global DNA hypermethylation, inhibition of histone lysine demethylases, and block of cell differentiation (710). One strategy to treat IDHmut tumors is to inhibit the mutant IDH protein and 2-HG production. This is being tested in IDH-mutated cancers. Recently, inhibitors of IDH2 (enasidenib) and IDH1 (ivosidenib) have been shown to induce differentiation of cancer cells in patients with recurrent or refractory AML (11, 12). However, this approach has been much less effective for solid tumors in both clinical and experimental contexts. Paradoxically, exogenous 2-HG can cause toxicity and slow down cell proliferation by inhibiting mammalian target of rapamycin signaling and mRNA m6A modification (13, 14). Furthermore, 2-HG directly inhibits homologous recombination (HR), thus weakening DNA damage repair (DDR) and potentially improving the outcome from DNA damaging agents in patients receiving standard-of-care cytotoxic therapies (15, 16). IDH mutations are associated with better outcomes from radiation therapy (RT) and chemotherapy in patients with glioma. It has been hypothesized that therapeutic modalities that inhibit 2-HG production in gliomas may abolish such protection and promote unfavorable evolution of the disease. Our previous work demonstrated that IDHmut causes genetic instability linked to accelerated copy number alterations throughout the genome (10). Biochemical studies showed that 2-HG inhibits HR-dependent repair and confers poly(adenosine 5-diphosphate) ribose polymerase inhibitor (PARPi) sensitivity (1619). However, the therapeutic potential for this phenomenon remains ill-defined. Moreover, it remains to be seen whether this approach is sufficient by itself or needs to be combined with other therapeutic modalities.

PARP1 (and other PARPs) play critical roles in the repair of DNA single-strand breaks (SSBs) through base excision repair, nucleotide excision repair, and other DNA damage response pathways (20). PARP inhibition leads to persistence of unrepaired SSBs and cytotoxic PARP-DNA complexes, which, when encountered at the replication fork, leads to the formation of potentially lethal DNA double-strand breaks (DSBs) (21). Cells with deficient HR, the main compensatory mechanism to manage the increased DSB stress imposed by PARPi, are unable to efficiently repair these DSB and enter mitotic catastrophe and apoptosis (22). We hypothesized that the HR deficiency induced by 2-HG across different solid tumors would fit this model of synthetic lethality, leading to marked sensitivity to PARPi in IDHmut tumors. Moreover, ionizing radiation (IR), alone or in combination with surgical resection, is routinely used in the clinic as a part of standard of care in treating cancers including LGG and ICC. By rapidly introducing high numbers of exogenous DNA SSB and DSB, IR exacerbates the effects of deficiency of DDR in HR-deficient tumors. Early trials combining IR with PARPi have shown promise in the BRCA-mutant context (23). However, PARPis as radiosensitizers for other types of HR-deficient tumors have not been thoroughly tested in preclinical or clinical settings. In this study, we showed that IDHmut tumor samples from patients with LGG and ICC harbor markedly elevated levels of DNA damage. We demonstrate in multiple in vitro contexts that expression of mutant IDH1 sensitizes the cell to radiation and PARPi. Last, we used two orthotopic LGG and one heterotopic ICC xenograft animal model to show that PARPi sensitizes the tumors to IR and that this sensitization is specifically associated with IDH mutation status. Overall, our study demonstrates that IR markedly augments the therapeutic effects of PARPi and provides evidence supporting the combinatorial use of PARPi with IR to treat IDH-mutant tumors.

Previous studies have suggested that repair of DNA damage by HR is impaired by mutant IDH1 expression in a human colon cancer cell line through the oncometabolite 2-HG (16). To ascertain whether this effect is generalizable, we first used an immortalized human astrocyte (IHA) isogenic cell line system, which includes one line that expresses mutant IDH1 R132H (IHA-IDH1mut), and a matching isogenic control, which does not express mutant IDH1 (IHA-EV) (10). Expression of mutant IDH1 induces changes in the DNA methylation and histone landscape, which recapitulates those in IDH1-mutant tumors and blocks differentiation (7, 10). We first subjected IHA-EV and IHA-IDH1mut to staining of -H2AX histones. As shown, IHA-EV demonstrates low levels of -H2AXpositive foci, whereas IHA-IDH1mut exhibits elevated levels of -H2AX staining (Fig. 1A and fig. S1A). These cells were fixed at the exponentially proliferative stage without being exposed to exogenous DNA damaging agents. The DSBs marked by -H2AX positivity in IDH1mut cells indicates a higher level of unrepaired DNA damage. To further support this finding, we performed Western blots to examine the level of phosphoKRAB-associated protein 1 (KAP1), an enzyme downstream of ATM (ataxia telangiectasia mutated). IHA-IDH1mut displayed notably higher levels of KAP1 phosphorylation compared to IHA-EV, suggesting increased engagement of the replication stress pathway (Fig. 1, B and C). However, consistent with previous reports (10, 14, 16), these unrepaired DNA damage sites did not induce significant change in cell death, likely due to concurrent inactivation of p53 as a part of immortalization. IDH-dependent defects in DDR may function as a driving force to produce additional mutations in the founder population during malignant progression. Next, we hypothesized that this rescue by p53 inactivation can be overcome by excessive DNA damage that accumulates in IDH1mut cells beyond a critical threshold. As observed in BRCA-mutant malignancies, the defective DSB repair in HR-defective tumors is often compensated for by other DDR pathways such as nonhomologous end joining, which, in turn, may themselves contribute to disease progression by inaccurate repair. We reasoned that the IDH1mut-induced DDR deficiency can be targeted by PARP inhibition similar to the scenario in BRCA-mutant breast and ovarian cancer and that this synthetic lethality could be augmented by inflicting further DNA damage through radiotherapy.

(A) Quantification of -H2AX positivity based on the number of foci. Fifty nuclei were quantified under each condition. (B) Western blots of phospho-KAP1 (p-KAP1) and the loading control (-actin). Samples were loaded in duplicates. (C) Relative intensity of each condition in (B) was quantified and plotted. (D) Immunostaining of -H2AX in vehicle (veh), olaparib (ola), IR-, or IR + olaparibtreated IHA cells expressing either EV or IDH1mut. DAPI, 4,6-diamidino-2-phenylindole. Scale bar,10 m. (E) Quantification of (D), performed by measuring the percentage of nuclei with more than 10 foci (left) (the numbers at bottom of bar graphs are correspondent to numbers in the panel) or average foci number of 50 nuclei (right). (F) Neutral Comet assays determining DNA breaks in IHA-EV and IHA-IDH1mut. Scale bars, 200 m. (G) The length of comet tails was measured and represented on the plot. (H) Apoptotic activities of IHA after radiation and/or olaparib treatment were measured for annexin V and propidium iodide (PI) positivity. (I) The PI+ and annexin V+ double positive populations were plotted on the bar graph. (J) IHA, expressing EV or IDH1mut, was subjected to soft agar colony formation assay, treated with four conditions: vehicle, veliparib (20 M), IR [1 to 4 grays (Gy)], or IR + veliparib in combination. Plot showed radiosensitization by veliparib in IDH1mut IHA versus EV. Where applicable, error bars represent the SEM. P values were determined by Students t test and represented using **P < 0.01, ***P < 0.001, and ****P < 0.0001. n.s., not significant.

To test our hypothesis, we assessed whether a combination of PARPi (olaparib), with or without IR, induces significant increases in levels of DNA damage. Elevated -H2AX positivity was observed in IHA-IDH1mut compared to IHA-EV at baseline (Fig. 1, D and E). The differential DDR abilities in IHA-EV and IHA-IDH1mut were more marked when treated with olaparib, radiation, or the combination of both, leading to differences in the amount of unrepaired DSB (Fig. 1, D and E). IDH mutation was associated with a markedly reduced ability to repair DNA damage from IR and PARPi, as measured by the neutral Comet assay (Fig. 1, F and G). The combination of IR and PARPi displayed a cooperative effect. Moreover, the deficiency in DDR found in IHA-IDH1mut cells leads to a greater extent of cell death when treated with the combination as shown by annexin V flow cytometry (Fig. 1, H and I).

Mechanistic studies show that PARPis can be classified on the basis of their ability to trap PARP proteins to DNA, thus preventing the recycling of PARP (24). Olaparib has potent PARP trapping activity and consequential cytotoxicity (21). However, treatment with strong PARP-trapping agents, such as olaparib and talazoparib, tends to confer resistance through genetic mutation (25, 26). Veliparib has demonstrated weaker trapping activity but strong inhibition of PARylation, effecting an alternative type of mechanistic target manipulation (27). In addition, veliparib shows superior penetration through the blood-brain barrier (BBB) (28)a key feature that is important for brain tumor therapeutics. Therefore, we also tested the efficacy of veliparib. Similar to olaparib, IR + veliparib generated a significantly higher level of -H2AX foci in IHA-EV treated with 2-HG than IHA-EV without 2-HG receiving the same IR + veliparib treatment (fig. S1, B and C). We next compared the clonogenic ability of IHA in response to increasing doses of radiation [0, 1, 2, and 4 gray (Gy)] with or without 20 M veliparib. Under all IR conditions, IHA-EV yielded modest reduction of colonies when simultaneously treated with veliparib, while this reduction was markedly enhanced in IHA-IDH1mut (Fig. 1J and fig. S1D). Similar to our results with veliparib, IHA-IDH1mut showed enhanced sensitivity toward IR when treated with olaparib (fig. S2A). Moreover, we tested the clonogenic ability of two glioma stem cell (GSC) lines, TS543 [IDH wild-type (IDHwt)] and TS603 (IDH1mut), which provide a more clinically relevant model. TS603 GSC also showed notably amplified synthetic lethality when treated with IR and olaparib (fig. S2B). These results indicate that IR + PARPi preferentially inhibits the clonogenic growth of IDH-mutant cells.

We tested whether the synthetic lethality conferred by PARPi in the setting of mutant IDH is observed in other tumor types that commonly harbor IDH mutations. ICC is a highly lethal malignancy with a 5-year overall survival (OS) rate of less than 20% (29). The current standard of care for most patients with unresectable disease at presentation is still limited to standard chemotherapy and radiation with median overall survival of only 7 to 12 months and no currently available targeted therapy (3032). Genomic studies have observed that a substantial portion of ICCs harbor mutations in IDH genes (3, 33). We tested whether expression of mutant IDH1 sensitizes ICC cancer cells to PARPis. First, we expressed IDH1-R132H in a human cholangiocarcinoma cell line (HUCCT1) that is wild type for IDH. The expression of mutant IDH1 was confirmed by Western blot (Fig. 2A). Similar to what we observed in the IHA isogenic cell lines, IDH1mut expression significantly increased -H2AX positivity. This increase was amplified by olaparib, IR, and the combination treatment (Fig. 2, B and C). The unrepaired DSBs, in turn, led to increased fragmentation of genomic DNA shown by Comet assays (Fig. 2, D and E). Clonogenic capacity of HUCCT1 cells was severely decreased by IDH1mut expression, demonstrated by a 100-fold difference in clonogenicity when IDH1mut HUCCT1 cells were exposed to 6-Gy radiation and 4 M olaparib (Fig. 2, F and G). In addition, using patient-derived ICC cell lines of IDHwt (HUCCT1) and IDH1-R132C (SNU-1079), we showed different levels of -H2AX staining (Fig. 2H) and clonogenicity (Fig. 2, I and J) in response to IR and olaparib, consistent with the other in vitro models tested above. Together, we showed in two different cancers, using both engineered isogenic cells and native IDH-mutant tumor cell lines, that mutant IDH1 expression leads to hypersensitivity to PARPi, and this hypersensitivity is markedly amplified by radiation.

(A) Confirmation of IDH1mut expression in HUCCT1 cholangiocarcinoma cell line. Lysates from HUCCT1-EV or HUCCT1-IDH1mut were subjected to Western blots determining expression of IDH1 R132H. Loading control is performed with anti-vinculin. (B) Immunostaining of -H2AX in HUCCT1-EV and HUCCT1-IDH1mut after IR (4 Gy) or olaparib (4 M), or both, showing synergy specifically in HUCCT1-IDH1mut. Scale bar, 10 m. (C) The average number of -H2AX foci in (B) were quantified and shown as means SEM. (D) Neutral Comet assays showed different levels of DNA damage between the indicated treatments. Scale bars, 200 m. (E) The Comet tail moment lengths were individually quantified and compared. (F) Representative results of colony formation assay with HUCCT1-EV or HUCCT1-IDH1mut treated with increasing doses of IR (2, 4, and 6 Gy), with or without olaparib (4 M). (G) The colonies of all conditions were quantified and represented on a survival plot showing synergestic effect of olaparib and IR specifically in HUCCT1-IDH1mut cells. Photo credit: Yuxiang Wang, Memorial Sloan Kettering Cancer Center (MSKCC). (H) Immunostaining of -H2AX in IDHwt (HUCCT1) and IDH1mut (SNU-1079) cell lines, treated with IR (4 Gy) + olaparib (4 M). wt, wild-type. (I) Results from clonogenic assays with IDHwt (HUCCT1) and IDH1mut (SNU-1079) cholangiocarcinoma cell lines. Panel shows representative results when cells were treated with IR (4 Gy) + olaparib 4 M. Photo credit: Yuxiang Wang, MSKCC. (J) The colonies in IR (4 Gy) + olaparib (4 M) were quantified and divided by the IR-alone control. P values were determined by Students t test and represented using **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Having confirmed that expression of mutant IDH1 is associated with increased levels of DNA damage in vitro, we sought to ascertain whether this is true in patient tumors. We took primary LGG and ICC specimens from patients who underwent surgical resection at Memorial Sloan Kettering Cancer Center (MSKCC) without any previous treatment. With assistance from expert clinical pathologists at MSKCC, we determined the IDH mutation status of the tumors and ensured that the IDH-mutant and wild-type tumors were matched for similar disease stage, grade, and pathologic features. We then subjected the paired tissue samples to -H2AX staining, a marker for DNA damage. IDH1mut World Health Organization (WHO) grade III glioma sections showed elevated -H2AX signals compared to their IDHwt controls, regardless of their histopathologic classification as oligodendroglioma or astrocytoma (Fig. 3, A and B). Similarly, ICC tumor pairs collected at similar disease stage (T1, no lymph node or distant metastases, no neoadjuvant therapy, and no intrahepatic therapy before resection) demonstrated that IDH mutations lead to significantly augmented -H2AX staining (Fig. 3, C and D).

(A) Frozen glioma specimens were collected during routine surgeries at MSKCC (see also the Human pathology section under Materials and Methods). Four grade III oligodendroglioma (top) and six grade III astrocytoma (bottom) samples were stained for -H2AX positivity, and representative images are shown in the panels. (B) H-scores of five 20 fields of each sample were calculated and reported on the bar graphs as means SEM. Top: Comparison of H-scores of the oligodendroglioma sample pair. Bottom: Comparison of H-scores of the astrocytoma sample pair. ****P < 0.0001, determined by Students t test. (C) Sections from six cholangiocarcinoma specimens (three IDHwt and three IDHmut) were stained for -H2AX positivity, and representative images are shown in the panels. (D) Top: For top panels of (C), H-scores of five 20 fields of each sample were calculated and represented on the bar graphs as means SEM. Bottom: Comparison of H-scores of the bottom panels in (C). ****P < 0.0001, determined by Students t test.

Next, we used several animal models to experimentally test our therapeutic approach for IDH-mutant tumors in vivo. First, we intracranially implanted glioma tumorsphere lines (TS543 and TS603) and monitored the tumor growth with bioluminescent imaging (BLI). Mice with tumors were randomized into four-armed trials: control, veliparib (25 mg/kg; 5 days per week until moribund), fractionated RT (2 Gy 5 fractions, days 1 to 5) or the combination of RT and veliparib (Fig. 4A). Veliparib was used here because of its ability to cross the BBB. Intracranial tumor growth was followed using weekly BLI, and mice were also monitored for OS. Mice with TS543 (IDHwt) tumors showed similar OS in veliparib and control groups (median OS, 11 days versus 10 days) (Fig. 4B), and while RT successfully prolonged OS (median, 19 days) compared to control, the addition of veliparib did not prolong OS (median, 17 days) (Fig. 4B). However, the IDH1mut tumors (TS603) showed significant improvement of OS in veliparib treated group (11 days) compared to control (8.5 days), as well as in RT + veliparib (21 days) versus RT alone (14 days) (Fig. 4C). At day 7 (2 days after the last RT dose), 11 of 16 mice bearing TS603 implantation that received RT and veliparib treatment had reduction in BLI signal, among which 4 of 16 mice showed marked reduction of >90% (Fig. 4, D to F), whereas only 3 of 13 mice receiving RT alone showed reduction and no mice showed >90% reduction (Fig. 4, D to F). In addition, 3 of 13 mice in the RT arm showed BLI signal increase of >800%, while none in the RT + veliparib arm showed this level of tumor growth (Fig. 4, E and F). These data suggest that combination therapy with veliparib and RT has greater efficacy against glioma than RT alone in the IDH1-mutant setting. Pathologic studies performed with tumor tissues collected at day 6 (18 hours after the last RT dose) supported the idea that synergy between RT and veliparib was specific to the IDH1-mutant context (figs. S3, A to D, and S4) as shown by quantification of mitotic index (fig. S3, A and B), apoptosis (cleaved caspase 3; fig. S3, C and D), and DNA damage (-H2AX; fig. S4).

(A) Work flow of treatments. Mice received GSC implantation. All mice received weekly BLI scans, and the results were recorded. All mice with established tumors (over the defined threshold) were equally distributed to vehicle, veliparib (veli), RT, or RT + veliparib arms. TX, treatment; PATH, pathologic analysis. (B) Kaplan-Meier analysis of mice bearing TS543 GSC (IDHwt) cells, starting from the day they entered trials. P values were determined by log-rank (Mantel-Cox) test. (C) Kaplan-Meier analysis of mice bearing TS603 GSC (IDH1mut), starting from the day they entered trials. (D) Representative BLI scans of paired mice receiving RT or RT + veliparib. Top: BLI scans at day 0. Bottom: Scans at day 7. (E) Responses based on BLI reads for RT + veliparibtreated (left) (n = 16) and RT-treated (right) (n = 13) mice. Dashed line indicates a 90% reduction in tumor BLI signal. (F) Pie graphs showing the percentage of any reduction (top), >90% reduction (middle), or increase of >800% (bottom) in BLI. Statistics were performed with chi-square test, and the P values are presented. D, day.

To rule out the possibility that the observed sensitivity could be due to different genetic backgrounds (i.e., TS543 and TS603), we performed similar trials in a genetically engineered mouse model of glioma with RCAS-TVA (replication competent avian sarcoma-leukosis virus long terminal repeat with a splice acceptor)mediated gene transfer of mutant IDH in an isogenic setting (34, 35). This is a previously established model where mutant IDH is expressed in endogenously generated gliomas. In these animal models, tumors that express the wild-type or mutant IDH1 were generated through intracranial injection of DF1 cells that carry the corresponding expression cassette (Fig. 5A). The brain tumors were allowed to grow for 5 weeks before magnetic resonance imaging (MRI) scans (Fig. 5A). After the initial MRI scan, the mice were randomized to one of four treatment arms testing tumor sensitivity to veliparib and radiation with MRI scans every week for follow-up (Fig. 5A). The data showed that IDHwt gliomas are sensitive to radiation but relatively insensitive to PARPi, either as monotherapy or in combination with RT (Fig. 5B). On the contrary, IDH1mut gliomas are somewhat sensitive to both RT or veliparib as monotherapy (median OS, 22 days versus 22 days versus 14 days for vehicle control) and the combination of RT and veliparib substantially extended OS (median, 66 days, >4-fold longer OS than vehicle control and 3-fold longer than RT or veliparib alone) (Fig. 5C). Representative MRI images of IDH1mut gliomas show similar initial sizes (Fig. 5D, circled areas) and demonstrate that veliparib limited the tumor growth compared to vehicle-treated tumors (Fig. 5D). Combination treatment with RT and veliparib was able to achieve marked tumor regression over time with some tumors undergoing reduction in tumor size so that they became undetectable at 3 weeks from the start of treatment (Fig. 5D).

(A) Mice receiving intracranial injection of RCAS virus-producing cells carrying platelet-derived growth factor A (PDGFA), shTP53, and either IDHwt or IDHmut expression cassettes were maintained for 5 weeks before their initial MRI scans. After MRI, mice were equally distributed into four-arm treatment groups based on tumor volume. (B) Kaplan-Meier analysis of mice bearing IDHwt gliomas, starting from the day they entered trials. P values were determined by log-rank (Mantel-Cox) test. (C) Kaplan-Meier analysis of mice bearing IDH1mut gliomas. (D) Representative images of MRI scans from (C) at days 0, 7, and 21, showing overall effect of treatments. (E) Kaplan-Meier analysis of mice bearing IDH1mut gliomas, receiving BGB PARPi + RT treatments. (F) Representative images of MRI scans from (E) at days 0, 7, and 21, showing overall effect of treatments.

BGB-290 (Pamiparib) is a potent PARPi with good oral bioavailability and excellent BBB penetration (36). In contrast to veliparib, BGB-290 displayed potent PARP-trapping activity at nanomolar level (36). Thus, we also tested BGB-290 in our RCAS-IDH glioma model. BGB-290 prolonged the OS of mice with IDH1mut glioma, both as monotherapy (median OS, 28 days) or in combination with RT (median OS, 44 days), with 4 of 13 mice living more than 90 days (Fig. 5E). MRI also showed decelerated tumor growth in the BGB-290treated group and marked reduction of tumor volume in the RT + BGB-290 group (Fig. 5F). Histopathologic studies showed reduced mitotic activity (fig. S5, A and B), as well as increased apoptotic activity (fig. S5, C and D) and accumulation of DSB (fig. S6) specifically in veliparib- and BGB-290treated tumors. Again, these findings suggest cooperativity between PARPi and radiation in the setting of IDHmut tumors.

Last, we tested our synthetic lethal approach in the context of IDH-mutant ICC in vivo using an animal model. As shown above (Fig. 2, H to J), the IDH1-mutant SNU-1079 ICC line showed increased sensitivity to olaparib. Unfortunately, none of the mice (n = 40) that received subcutaneous injection of SNU-1079 cells developed detectable tumor after 3 months, making it impossible to test the effect of PARPi on this ICC cell line in vivo. However, HUCCT1 cells competently form subcutaneous tumors in athymic nude mice regardless of the mutational status of IDH1. Therefore, we subcutaneously implanted isogenic IDHwt and IDH1mut tumors in the hind flank region and treated mice with vehicle, olaparib, RT, or RT + olaparib (Fig. 6A). The IDH1mut tumors grew slightly slower than the wild-type tumors (median survival, 31 days versus 21 days; Fig. 6, B and D), consistent with previously reported 2-HG toxicity. Measurements of tumor volume over time showed that the IDH1wt tumors are insensitive to olaparib treatment. Moreover, although RT slowed down tumor growth, addition of olaparib failed to further delay tumor growth (Fig. 6, B and C). However, the HUCCT1-IDH1mut tumors grew slower when treated with olaparib alone (median time to reach four times initial tumor volume, 41 days versus 31 days). Moreover, RT and olaparib treatment (median OS, 60 days) significantly delayed growth compared to RT alone (median OS, 47 days) (Fig. 6, D and E). Histopathologic analyses with tumor tissues collected at day 6 showed, specifically in IDH1mut tumors, greater reduction in mitotic index (Fig. 6, F and G), increase in apoptosis (fig. S7A), reduction in Ki-67 positivity (fig. S7B), and increase in DSB (fig. S7C). Together, these in vivo findings support, in ICC cells, that IDH mutations confer vulnerability to PARPi, which can be further exploited by introducing DNA damaging agents, such as radiation.

(A) Mice received subcutaneous injection of HUCCT1 cells expressing EV or IDH1mut. Three weeks after injection, the hind flank tumors were measured and equally distributed to four-arm treatment groups when tumors exceeded the defined threshold of 100 mm3. The tumor sizes were measured twice a week. (B) Tumor growth of HUCCT1-EV xenografts with the indicated treatments. P values were calculated using two-way ANOVA. (C) Kaplan-Meier analysis of HUCCT1-EV xenografts with the indicated treatments. P values were determined by log-rank (Mantel-Cox) test. (D) Tumor growth of HUCCT1-IDH1mut xenografts with the indicated treatments. (E) Kaplan-Meier analysis of HUCCT1-IDH1mut xenografts. (F) Mice were sacrificed at day 6, and tumor tissues were subjected to pathological analyses. Hematoxylin and eosin (H&E) staining was performed. (G) The mitotic cell numbers per 400 field were counted, and means SEM was shown on the bar graphs. For each condition, 10 400 fields were quantified. *P < 0.05 and **P < 0.01. P values were determined by Students t test.

Despite current standard-of-care multimodal treatment approaches for glioma, including surgery, radiation, and chemotherapy, the outcomes for these patients remain poor (37). The case for ICC is similar, and researchers have struggled to find effective targeted therapies with none standardly in clinical use for these diseases at the present time. The low patient survival is multifactorial but likely stems from the similar clinical challenges presented by LGG and ICC. Both diseases occur in critical organs, often cannot be completely resected, tend to recur locally, and commonly cause death through local progression. Furthermore, both diseases have complicated tumor microenvironments and heterogenicity predisposing to therapeutic resistance. Although directly targeting the mutant IDH enzyme with small-molecule inhibitors has been shown to have benefits in patients with AML, concerns exist regarding their application to solid tumors, such as systemic availability of the drug, ability to penetrate into tumor, and lack of efficacy. In vitro studies of IDH1 inhibitor AGI-5027 have failed to show increased sensitivity in IDH1mut versus IDH1wt ICC cells (38). Moreover, in solid tumors, mutation of IDH typically portends a better prognosis compared to tumors with wild-type IDH. This has been shown to be the case for both glioma (2) and ICC (4), which begs the question of whether targeting the IDH phenotype for reversal makes sense. We examine an approach to treating IDHmut tumors that takes advantage of their unique metabolic, genomic, and epigenetic state through exploitation of impaired HR associated with mutations in IDH1. While a randomized phase 1/2 study of temozolomide (TMZ) veliparib showed no benefit in recurrent TMZ-refractory glioblastoma, IDH mutation status was not considered in the enrollment criteria nor reported in the manuscript (39). To our knowledge, combined treatment with PARPi + RT has not been examined in the specific setting of IDH-mutant tumors at either the laboratory or clinical level.

We developed both in vitro and in vivo models that mimic IDH-mutant tumors found in the patients. Using these models, we were able to determine that IDH mutation confers sensitivity to DNA damaging agents and PARP inhibitors, and we established preclinical strategies to target these therapeutic vulnerabilities. Most LGG recurrences (up to 92%) occur within the RT field (40). Our results nominate PARPi-based systemic therapy as a way to increase control of IDHmut gliomas. We hypothesize that combination therapy with PARPi + RT, as evaluated in our study, could substantially lengthen OS and progression-free survival for patients with IDHmut LGG. In the setting of ICC, unresectable and recurrent intrahepatic disease poses an imminent threat to patient lives, given their proclivity to cause liver failure through biliary, portal vein, or hepatic vein obstruction. Nonoperative therapies produce median OS of only 7 to 12 months (31, 41). Conventional RT doses, even with concurrent chemotherapy, achieve only modest improvement in outcomes and few long-term survivors (32) with most patients experiencing local progression as site of first failure (42). Similar to LGGs, given the propensity of ICCs to cause death through local progression, the ability to further increase tumor cell killing within the RT field for unresectable and recurrent intrahepatic disease through combined PARPi + RT in the IDHmut setting can be explored.

Ongoing clinical trials have been set up to test this concept (e.g., NCT03212274, NCT03561870, NCT03749187, etc.), yet the strategies and designs of these trials still lack critical rationale or insights from preclinical studies. For example, in our animal studies, PARPi alone, whether it is veliparib, BGB-290, or olaparib, provided modest benefit for OS. Yet, the combination with radiation markedly amplified this benefit. Our data suggest that concurrent RT needs to be considered to yield maximal benefit of PARPi therapy for IDHmut tumors. While the mechanisms underlying this are still being worked out, Sulkowski et al. (16) show that 2-HGdependent inhibition of KDM4A and KDM4B may contribute to the observed synthetic lethality. These two proteins are key KG-dependent histone demethylases that are important for proper DNA damage response. Our study suggests that these biological phenomena may potentially be clinically actionable and should be tested.

The objective of this study was to determine the therapeutic efficacy of PARPis, as monoagent or in combination with IR, for IDH-mutant tumors. This was a controlled, laboratory-based, experimental study using cell line models in culture, tumor specimens, tumor cell xenograft, and genetically engineered mouse models. Isogenic cell lines and genetically engineered tumors were produced by introducing mutant IDH1, along with appropriate controls. IDHwt and IDHmut patient-derived glioma and cholangiocarcinoma cell lines were compared and provided additional, disease-related evidence. Randomization of animals varied depending on individual assays and is described separately below. Pharmaceutical agents against PARP and/or IR were applied. Sample sizes were determined independently for each experiment without formal power calculation. No data were excluded from analyses. Unless otherwise specified, experiments used three replicates per sample. End points varied by experiment and are described below, in figure legends, or in Results. Histopathological and immunohistochemical review of xenografts was conducted by pathologist (C.S., D.S.K., and J.T.H.) in a nonblinded fashion. Quantification of mitotic index, -H2AX, Ki67 and cleaved caspase 3 immunostaining, length of Comet tails, colony numbers, and BLI signals was blinded.

The source of antibodies, chemicals, plasmids, cell lines, and mouse strains used in this study are listed in table S1.

All cell lines used in this study were regularly tested for mycoplasma contamination at the Antibody and Bioresource Core of MSKCC. Parental IHAs (a gift from R. O. Peiper, University of California, San Francisco) were infected with a viral vector carrying expression cassette for IDH1-R132H or the empty vector control (10). TS543 and TS603 are patient-derived GSCs (4345) maintained in NeuroCult NS-A Proliferation media (no. 05751, STEMCELL Technologies). For intracranial injection, TS543 or TS603 was infected with pHIV-Luc-ZsGreen (a gift from B. Welm; no. 39196, Addgene) and fluorescence-activated cell sortingsorted for top 10% ZsGreen expression. Two well-characterized patient-derived human ICC cell lines were obtained as follows: (i) SNU-1079 (endogenous R132C mutation in IDH1) was purchased from the Korean Cell Line Bank (Cancer Research Institute, Seoul National University, Seoul, Korea; http://cellbank.snu.ac.kr), and (ii) HUCCT1 was purchased from the RIKEN BioResource Research Center Cell Bank (Tsukuba, Japan; https://en.brc.riken.jp/). To generate HUCCT1 isogenic cells, the parental HUCCT1 cells were infected with pLNCX2 retroviruses expressing IDH1-R132H or the empty vector control (7). Olaparib (no. S1060) and veliparib (no. S1004) were purchased from Selleckchem. BGB-290 (no. C-1286) was purchased from Chemgood. For in vitro use, olaparib, veliparib, and BGB-290 were diluted with dimethyl sulfoxide (DMSO). For in vivo use, olaparib was diluted with DMSO as a stock and further diluted with 10% (w/v) 2-hydroxy-propyl--cyclodextrin (no. H107, Sigma-Aldrich). Veliparib and BGB-290 were diluted with DMSO and further diluted with phosphate-buffered saline (PBS). The final solutions were prepared fresh before each injection. For 2-HG treatment, Octyl-d-2-HG (no. 16366, Cayman Chemical, MI) was initially diluted into DMSO and further diluted with culture medium to achieve a final concentration of 1 mM.

All mouse experiments were approved by Institutional Animal Care and Use Committee at MSKCC strictly following its guidelines. Female nude mice (age 4 to 6 weeks) were purchased from Taconic Biosciences and maintained in the xenograft suite. Nestin-TVA mice were obtained from E. Holland (Fred Hutchinson).

All tumors were obtained following surgical resection at the MSKCC as part of routine clinical care in accordance with the Institutional Review Board policies at the MSKCC. Informed consent was obtained from all patients. Ten glioma (five wild-type and six mutant) and six cholangiocarcinoma (three wild-type and three mutant) samples were included in this study. The clinical determination, classification, and grouping were performed by pathologists at MSKCC and MD Anderson. For glioma patient samples, 10-m sections of frozen tissues was directly fixed with 4% paraformaldehyde in PBS for 30 min, followed by staining procedures as described below in the Immunofluorescent imaging section. For cholangiocarcinoma patient samples, formalin-fixed paraffin-embedded (FFPE) sections were stained following antigen retrieval with boiling citrate buffer (10 mM) (pH 6), following procedures in the Immunofluorescent imaging section. After staining, the sections were scanned with Pannoramic 250 (3DHISTECH, Budapest, Hungary) using Zeiss 20/0.8 numerical aperture objective. The scans were viewed and exported to .tif images using CaseViewer software (3DHISTECH, Budapest, Hungary). -H2AX positivity was quantified as H-score (1 A + 2 B + 3 C), where A is the percentage of cells with no staining, B is the percentage of cells with weak to moderate staining, and C is the percentage of cells with strong staining. The quantification was performed by the Molecular Cytology Core, and the score determination was double checked by Y.W.

For soft agar colony formation assays, 50,000 cells were seeded in six-well plates containing 1% bottom layer and 0.5% top layer soft agar. Cells were then cultured in growth media with or without olaparib (1 M) or veliparib (20 M). Radiation dosing of 0, 1, 2, or 4 Gy was immediately applied after plating. The 1.5 ml of growth media covering the agar cultures was replenished every week. At day 21, colonies were fixed with 4% paraformaldehyde for 30 min and stained with 0.005% crystal violet in PBS overnight. Stained colonies were then washed extensively in PBS and water and quantified on a GelCount colony counter (Oxford Optronix).

Clonogenic assays were performed by plating cells in exponential growth phase at 125 to 1000 cells per 10-cm dish depending on the radiation dose level. Olaparib (4 M) was added 24 hours after plating with IR (0 to 6 Gy) delivered 24 hours later. Colonies (>50 cells) were counted with GelCount colony counter (Oxford Optronix) 10 to 14 days after IR by fixing and staining with a solution of 0.1% crystal violet in 4% paraformaldehyde in PBS. Surviving fraction was calculated by dividing colonies by cells plated with adjustment for plating efficiency.

Comet assays were performed using OxiSelect Comet Assay Kit (STA-350, Cell Biolabs) according to the manufacturers instruction. Briefly, cells were mixed with agarose, dropped onto the glass slides provided by the kit, and lysed with prechilled lysis buffer for 60 min at 4C in the dark. The electrophoresis was performed with prechilled tris-borate EDTA buffer, followed by five times washes with ddH2O. The slides were then incubated in cold 70% ethanol for 5 min and air-dried. Representative pictures were taken with a wide-field microscope with fluorescein isothiocyanate channel (Nikon) and analyzed with OpenComet plug-in in ImageJ (46).

Cells were grown in chamber slides (Nunc Lab-Tek II, cat no. 154526, Thermo Fisher Scientific) before fixation (4% paraformaldehyde in PBS for 10 min) and permeabilization (0.5% Tween 20 and 0.2% Triton X-100 in PBS for 10 min). Cells were blocked with goat serum (Sigma-Aldrich) for 4 hours at room temperature and incubated with -H2AX antibody (1:500; no. 05-636, Millipore) overnight at 4C and secondary antibody (1:2000; goat anti-mouse Alexa Fluor 488 or Alexa Fluor 568) for 2 hours. The slides were mounted with coverslips using ProLong Gold antifade reagent and 4,6-diamidino-2-phenylindole counterstain (Invitrogen).

BLI was performed weekly following intraperitoneal injection of d-luciferin (PerkinElmer) and measured using Xenogen IVIS Spectrum in vivo imaging system (PerkinElmer). Living Image software (PerkinElmer) was used to acquire and analyze the BLI data.

Brains of injected mice were scanned on a 200-MHz Bruker 4.7 T Biospec MRI scanner (Bruker Biospin Corp., Ettlingen, Germany) and equipped with a 300-mT/m ID 20-cm gradient (Resonance Research Inc., Billerica, MA). Mice were anaesthetized by 2% isoflurane in oxygen. Sedated animals were physiologically monitored during scan period (SA Instruments Inc., Stony Brook, NY). For mouse brain imaging, brain axial T2-weighted images using fast spin-echo RARE (Rapid Acquisition with Relaxation Enhancement) sequence were acquired by sequential scanning with a slice thickness of 1 mm.

TS543 or TS603 cells expressing pHIV-Luc-ZsGreen (described above) were implanted into the brain of nude mice (5 105 cells per brain), with a fixed stereotactic device (Stoelting, Illinois). Injections were made to the right frontal cortex, 3-mm lateral and 3-mm caudal, and at a depth of 3 mm with respect to bregma. Two weeks after the implantation, the tumor growth is monitored by BLI and MRI once every week, respectively. Tumors over the BLI threshold were correlated with MRI signal for confirmation of location and actual volume. The mice with confirmed tumors enter a randomized trial consists of the following: (i) vehicle; (ii) intraperitoneal injection of veliparib (25 mg/kg, 5 days per week); (iii) RT (2 Gy 5 fractions), delivered to the whole head using an X-RAD 320 Irradiation Platform (Precision X-Ray Inc., North Branford, CT; http://www.pxinc.com) in combination with a QUAD Fixture and Shield Set specifically designed with lead shielding of the body to allow for cranial irradiation (Precision X-Ray Inc., Connecticut); and (iv) RT + veliparib (concurrent treatment for the initial 5 days and then veliparib injection 5 days per week). The BLI signals were continuingly followed up weekly until defined end of trial (death). For pathological analyses, three mice of each group were sacrificed at day 6 after the initial trial start, and the brains were collected and subjected to standard FFPE processing.

Nestin-TVA mice were a gift from E. Holland (Fred Hutchinson, Seattle, WA) (47). RCAS vectors carrying expression cassette for platelet-derived growth factor A (PDGFA), IDH1wt-shTP53, and IDH1R132H-shTP53 were gifts from E. Holland (35). RCAS viral vectors were introduced into DF1 cells separately, and the expression of PDGFA, IDH1wt, and IDH1-R132H was verified by Western blots. Cells expressing PDGFA were mixed with cells expressing IDH1wt-shTP53 or IDH1R132H-shTP53 at a ratio of 1:1 (3 105 total) and intracranially injected as described above. Mice received MRI at week 5 after the initial injection, and the tumors were randomized on the basis of size so that tumors of different sizes are equally distributed across groups: (i) vehicle; (ii) intraperitoneal injection of veliparib (25 mg/kg, 5 days per week) or BGB-290 (6 mg/kg, 5 days per week); (iii) RT (2 Gy 5 days), delivered to the whole head using X-RAD 320 Irradiation Platform; and (iv) RT + veliparib or BGB-290 (concurrent treatment with RT delivered 1 hour after veliparib/BGB-290 injection for the initial 5 days and then veliparib/BGB-290 injection 5 days per week). The MRI signals were followed weekly until defined end of trial (death). For pathological analyses, three mice from each group were sacrificed at day 6 after the initial trial start, and the brains were collected and subjected to standard FFPE processing.

HUCCT1 cells expressing IDH1R132H or the empty vector control were harvested at exponentially proliferative stage and mixed 1:1 (v/v) with Matrigel (no. 356231, Corning). A total of 5 106 cells were injected into nude mice flanks in a 100-l volume. The size of tumors was measured with caliber and calculated using the formula (l w2)/2, where w is width and l is length in millimeters. Tumors that reached threshold (100 mm3) were randomized to the following: (i) vehicle; (ii) intraperitoneal injection of olaparib (50 mg/kg, 5 days per week); (iii) RT (2 Gy 5 days), delivered to the posterior through the X-RAD 320 Irradiation Platform; and (iv) RT + olaparib (concurrent treatment for the initial 5 days and then olaparib injection 5 days per week). The tumor volume was continuingly measured twice a week until the defined end of trial (400 mm3). For pathological analyses, three mice of each group were sacrificed at day 6 after the initial trial start, and the xenograft tumors were collected and subjected to standard FFPE processing.

Statistical analysis was performed using GraphPad Prism 7. Where applicable, P value was determined by unpaired, two-tailed t tests, if not otherwise specified. Difference of tumor growth curves was determined by two-way analysis of variance (ANOVA). Log-rank (Mantel-Cox) test were used to determine the significance of differences in Kaplan-Meier analysis of GSC xenograft, RCAS-induced gliomas, and ICC hind flank xenograft experiments. Unless otherwise stated, all results, representing at least three independent experiments, were plotted as means SEM. P values are represented either directly on figures or using *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Acknowledgments: We thank the Chan lab for discussions. We thank C. Thompson for advice. Funding: This work was supported in part by the U.S. NIH (R01 CA177828) (to T.A.C.) and NIH Core Grant P30 CA008748. Author contributions: The study was designed by T.A.C., Y.W., and A.T.W. Experiments were performed by Y.W., A.T.W., S.T., W.H.W., X.M., and Y.G. Results were interpreted by T.A.C., Y.W., A.T.W., and J.T.H. Pathologic samples were provided and characterized by D.S.K. and C.S. T.A.C., Y.W., and A.T.W. wrote the paper with input from all authors. Competing interests: T.A.C. is an inventor on provisional patent application (62/569,053) submitted by MSKCC that covers use of tumor mutational burden as a predictive biomarker for cancer immunotherapy. T.A.C. is an inventor on patent application (PCT/US2015/062208) filed by MSKCC, relating to the use of TMB in lung cancer immunotherapy that has been licensed to Personal Genome Diagnostics, and MSKCC and T.A.C. receive royalties. T.A.C. is a cofounder of Gritstone Oncology and holds equity in An2H. T.A.C. has served as an advisor for Bristol-Myers Squibb, Illumina, Eisai, and An2H. The other authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Targeting therapeutic vulnerabilities with PARP inhibition and radiation in IDH-mutant gliomas and cholangiocarcinomas - Science Advances

Scientists in Singapore to Use Immunotherapy Cells in Targeting COVID-19 as It Works with SARS – Science Times

(Photo : fernandozhiminaicela from Pixabay)

Scientists from Duke-NUS Medical School in Singapore are exploring the body's immunotherapy cells as a way to destroy the Coronavirus Disease 2019 (COVID-19).

According to the Daily Mail, the team will use chimeric antigen receptors (CAR) and T cell receptors (TCR T) to control the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and protect patients from its symptomatic effects.

In the study Challenges of CAR- and TCR-T cell-based therapy for chronic infections published in the Journal of Experimental Medicine (JEM), the aforementioned cells are engineered to lyse the targeted cells directly. Their purpose is to lessen or completely eliminate a tumor.

According to Dr. Anthony Tanoto Tan, a senior research fellow at Duke's Emerging Infectious Diseases (EID) program, "This therapy is classically used in cancer treatment, where the lymphocytes of the patients are redirected to find and kill the cancer cells."

However, these have to be used with caution. The study mentioned, "In an infectious disease setting where organs essential for life are infected and where the T cells mediate protection but also organ pathology, the use of CAR/TCR-T cells has to be evaluated with caution."

Read now: CDC Director Warns A Second Wave of Coronavirus May Happen This Winter As It Coincides With the Flu Season

The laboratory generates CARs, which are artificial T-cell receptors that allow the immune system to recognize cells that have been infected by the virus. Antiviral bodies coupled with CAR/TCR T cells are the most cost-effective way of eliminating them.

The study said, "It is logical that research efforts targeting these pathogens are diverted mainly toward vaccine development or therapeutic small molecules that target their replication. However, in some infectious diseases, CAR/TCR-T cells might offer a rational and practical approach despite the inherent drawbacks."

According to the study, some of these drawbacks were viral infections and relapses. These were seen in immunosuppressed patients that had "hematopoietic stem cell or organ transplantation with human cytomegalovirus or EBV ( Epstein-Barr Virus) reactivation.

The researchers said, "there's no timeline for how long a patient would need to take the treatment so it could be indefinitely." Fortunately for government and health agencies, it will be easier because the therapy is not that expensive.

Read now: Contaminated Coronavirus Testing Kits from China Force UW School of Medicine to Pull Out Use Despite Limited Supply

The process was published in the Journal of Experimental Medicine. The therapy will extract immune cells called T lymphocytes from the patient's bloodstream. It will train the cells to recognize the virus once it enters the body.

With over 800,000 cases and almost 43,000 deaths in the US because of COVID-19, going anywhere is a risk. Using this therapy, the patient will not go to a hospital anymore, lowering the risk of spreading and acquiring the disease.

The therapy, while it's in the works, has not yet been tested against other infectious diseases and viruses. Dr. Tan said, "We argue that some infections, such as HIV and [Hepatitis B virus], can be a perfect target for this therapy, especially if lymphocytes are engineered using an approach that keeps them active for a limited amount of time to minimize potential side effects."

However, what researchers are sure of is the therapy works against SARS, the cousin of COVID-19. The study's senior author Dr. Antonio Bertoletti, also from the EID said, "We demonstrated that T cells can be redirected to target the coronavirus responsible for SARS.

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Scientists in Singapore to Use Immunotherapy Cells in Targeting COVID-19 as It Works with SARS - Science Times

Gilead, Kite and oNKO-innate Announce Pact to Focus on NK Cells for Immunotherapy – BioSpace

Gilead Sciences, Kite Pharma, and oNKo-innate announced on Wednesday that they were entering a three-year cancer immunotherapy research collaboration. The goal is to support the discovery and development of next-generation drug and engineered cell therapies, specifically with a focus on natural killer (NK) cells.

Gilead is pleased to partner with oNKo-innate as a leader in this new and highly promising area of cancer immunotherapy, said William A. Lee, PhD, Executive Vice President, Research, Gilead Sciences. We have a strategic focus of growing both our expertise and pipeline in immuno-oncology and we believe this exciting collaboration will support each of these objectives as we work to discover and develop novel cancer therapies for patients.

Most existing cancer immunotherapy approaches focus on T cell mediated anti-tumor immunity. NK cells are a class of white blood cells that have an effector role in the immune system. Together, NK and T cells can potentially attack cancer cells, but they ultimately have different approaches to killing tumor cells. For this reason, activated and targeted NK cells may represent a different approach to attacking cancer at its source.

Kite is committed to building upon our leadership in cell therapy as we seek to meet the needs of patients with cancer, said Peter Emtage, PhD, Senior Vice President of Research at Kite. Early clinical data utilizing adoptively transferred NK cells has been encouraging and we are excited by the opportunity to scientifically expand our capabilities in this area and to identify novel NK cell therapies to advance toward clinical development.

As a result of the collaboration, oNKo-innate will use genome-wide screening techniques to reveal immune cell targets that enhance NK cell anti-tumor immunity. It will also execute screens for Gilead to identify and validate targets for internal immune-oncology discovery programs. For Kite, oNKo-innate intends to create and evaluate NK constructs to develop next-generation cell therapies.

With more than 20 years of collective academic expertise in NK cell biology, we have long believed in the potential for NK cells to play a role in cancer immunotherapy, said Jai Rautela, PhD, Co-founder and Chief Executive Officer of oNKo-innate. We look forward to bringing this NK cell expertise and our unique screening techniques into a collaboration with Gilead and Kite to serve a common goal of discovering new treatments for patients.

A study published in March of this year revealed that NK cells can be more effective the earlier they are in development. Senior Author Christopher M. Sturgeon, PhD, an assistant professor of medicine, stated that there is evidence that suggests future immunotherapy will not utilize cells from patients or a matched donor. Instead, it may potentially be developed from existing supplies of human pluripotent stem cells.

There is early evidence that they are more consistent in their effectiveness, and we would not need to process cells from a donor or the patient, said Sturgeon. They could be manufactured from existing cell supplies following the strict federal guidelines for good manufacturing practices. The characteristics of these cells let us envision a supply of them ready to pull off the shelf whenever a patient needs them.

Earlier this month, a study published in Targeted Oncology showed results that suggest that the number of NK cells and their high activity could potentially be a biomarker to predict the effectiveness of immunotherapy in non-small cell lung cancer patients. Choi Chang-min of Asan Medical Center, who led the research, told the Korea Biomedical Review that an NK cell activity test could eventually become a biomarker to predict immune checkpoint inhibitors and serve as criteria to provide various cancer treatment options.

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Gilead, Kite and oNKO-innate Announce Pact to Focus on NK Cells for Immunotherapy - BioSpace

Mogrify and Sangamo in license agreement for ‘off-the-shelf’ CAR-Treg – BioPharma-Reporter.com

The two partners have entered into a collaboration and exclusive license agreement to use Mogrifys proprietary induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) to develop allogeneic cell therapies.

Sangamo Therapeutics plans to utilize the stem cells with its zinc finger protein (ZFP) technology to create gene-engineered chimeric antigen receptor regulatory T cell (CAR-Treg) technology.

In return for the license to use Mogrifys technology, Sangamo will pay an upfront fee and will be eligible to receive further additional milestone payments. A spokesperson was unable to reveal any further details on the financials.

Mogrifys technology allows iPSCs and ESCs to be transformed using transcriptions factors or small molecules into any human cell type.

In the example of this agreement, Mogrify will be responsible for the discovery and optimization of iPSCs or ESCs to regulatory T cells, which can be transformed into novel off-the-shell allogeneic CAR-Treg cell therapy candidates.

Mogrify states that this method of production potentially makes therapies more cost-effective.

A spokesperson told BioPharma-Reporter why this is: The use of optimized transcription factor combinations helps to drive the change of the cells transcriptome and can, therefore, increase the efficiency (yield) and speed of conversion.

This means more of the target cells can be made quicker and from a smaller amount of starting material. This includes both the source cell population and other necessary materials, such as culture media, which are costly.

Jason Fontenot, head of cell therapy at Sangamo, stated that access to Mogrifys technology diversifies the companys options for developing CAR-Treg cell therapies.

In 2018, Sangamo acquired TxCell to gain the latters own CAR-Treg technology.

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Mogrify and Sangamo in license agreement for 'off-the-shelf' CAR-Treg - BioPharma-Reporter.com

Lozier praises promising, and ethical, blindness study – OneNewsNow

New research results show promise in treating people who are blind.

The National Eye Institute funded the study, which is research considered to be ethical.

Dr. David Prentice of the Charlotte Lozier Institute says there have been discussions over using adult stem cells to restore sight, which he calls a different tack for advancing science and medicine.

It's still an ethical way to go about this, he observes. There's no embryonic stem cells, no fetal tissue, none of this unethical type of research direction.

What the scientists did was turn a skin cell directly into a photoreceptor for vision then transplanted it.

Prenticeadvises the testing is very preliminary after the experiment on mice.

But what they find was when they transplanted this newly formed type of vision cell into the eyes of these blind mice, he says, they restored their vision.

The researchers applied chemicals that transformed one cell type into another needed for vision, and there is now potential to help people with all forms of vision blindness or vision correction, which would include macular degeneration and other retinal disorders.

Editor's note: Original posting attribute comments to wrong person.

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Lozier praises promising, and ethical, blindness study - OneNewsNow

A potent CBP/p300-Snail interaction inhibitor suppresses tumor growth and metastasis in wild-type p53-expressing cancer – Science Advances

INTRODUCTION

Metastasis is the major cause of cancer motility and accounts for about 90% of cancer-associated death (1). Cancer metastasis is a multistep and inefficient process in which tumor cells disseminate from the primary tumors, survive in the circulation, and settle and grow at the distant vital organs (24). One key event of metastasis is the epithelial-mesenchymal transition (EMT), a highly conserved developmental program that enables cancer cells to acquire malignancy-associated traits and the properties of tumor-initiating cells (TICs) [also known as cancer stem cells (CSCs)] during tumor initiation and progression (59). A hallmark of EMT is the loss of expression of the key epithelial cell-cell adhesion protein E-cadherin, and the expression levels of mesenchymal markers vimentin, fibronectin, and N-cadherin are also up-regulated in cancer cells undergoing EMT (10). While EMT therapeutics that efficiently reverse EMT and impair EMT-associated therapeutic resistance and tumor-initiating ability (i.e., stemness) are recently proven to be an effective therapeutic strategy for cancer treatment, the therapeutic target of these agents remains unclear (11, 12).

Snail is recognized as a major transcriptional factor that induces EMT by repressing E-cadherin protein (13, 14). Emerging evidence suggests that Snail has a substantially broader impact on tumor progression and metastasis. Following its overexpression in mammary epithelial cells, Snail promotes an EMT program and acquisition of tumor-initiating properties while enhancing tumor invasion, metastasis, tumorigenicity, and therapeutic resistance (9, 10, 15, 16). In addition, Snail accelerates tumor metastasis by suppressing host immune surveillance and inducing tumor microenvironment modulation (17, 18). Snail is also known to promote cancer cell survival by enhancing resistance to apoptosis under the genotoxic stress condition (19). We recently found that Snail deletion stabilizes wild-type, but not mutant, p53 and identified Snail as a molecular bypass that suppresses the antiproliferative and proapoptotic effect executed by wild-type p53 in breast cancer (BrCa) (20). However, it remains largely elusive whether p53 signaling pathway actively participates in Snail-mediated EMT, stemness, migration, and metastasis in cancer cells.

Snail is aberrantly activated in many human cancers and strongly associated with poor prognosis (2023). Many oncogenic signaling pathways, such as hypoxia/hypoxia-inducible factor1, transforming growth factor (TGF), epidermal growth factor (EGF), fibroblast growth factor2, and Notch, are implicated in the regulation of Snail gene expression (8, 24). In many cases, the posttranslational modification actively participates in the regulation of Snail protein. For instance, glycogen synthase kinase 3 (GSK3) and protein kinase D1 (PKD1) can phosphorylate Snail and promote its polyubiquitination and degradation by forming a complex with E3 ligases beta-transducin repeats-containing proteins (-TrCP) and F-Box protein 11 (FBXO11), respectively (2529). Another E3 ligase F-box and leucine-rich repeat protein 14 (Fbxl14), the human homolog of the partner of paired gene product in Xenopus, is also known to degrade Snail in a phosphorylation-independent manner (30, 31). On the other hand, histone acetyltransferases (HATs) such as adenosine 3,5-monophosphate response elementbinding proteinbinding protein (CBP) and p300 interact with Snail and acetylate Snail at lysine-146 (K146) and K187, which consequently reduces Snail ubiquitination and thus enhances its protein stability (18). Given the important role of Snail in driving cancer progression, targeting Snail may exert potent therapeutic benefit in patients with cancer. In the present study, we have successfully identified a small-molecule compound CYD19 as a potent CBP/p300-Snail protein-protein interaction inhibitor. We further demonstrate that CYD19 restores Snail-dependent repression of wild-type p53 and thus impairs tumor cell growth and survival in vitro and in vivo. In addition, CYD19 reverses Snail-mediated EMT in aggressive cancer cells and thus diminishes tumor invasion and metastasis. Our findings demonstrate that Snail protein is a druggable target and that pharmacologically targeting Snail by compound CYD19 may exert potent therapeutic effects in patients with locally advanced and metastatic cancer.

To identify small-molecule compounds having high-affinity binding with Snail, we performed a virtual ligand screening assay based on compound docking into the potential binding pocket of Snail (32). Using the FTMap, an online computational solvent mapping software for predicting the binding hotspots of a protein (http://ftmap.bu.edu/login.php), we identified the evolutionarily conserved arginine-174 (R174) pocket (in red) as a key hotspot in the binding site of Snail protein. Meanwhile, the leucine-178 (L178) side pocket (in yellow) and the serine-257 (S257) hydrophobic pocket (in blue) are also important for the potential binding (Fig. 1, A and B, and fig. S1A). We then performed an established fragment-based virtual screening of the DrugBank database to seek the novel scaffolds (fig. S1B). We retrieved the fragment-like small molecules from the in-house chemical library and docked them in the Snail crystal structure [Protein Data Bank (PDB) ID: 3W5K] (32) using Glide docking algorithms. Small molecules that were able to form binding interactions (e.g., hydrogenic, hydrophobic, or noncovalent interactions) with R174 pocket were scored and ranked according to their Glide results. The docking poses of the top 200 ranked molecules were visually inspected. Fifty molecules representing 23 structural clusters with prior Glide scores were found to bind with R174 pocket (fig. S1C). Notably, we found that pyrrole-pyrimidine fragment (drugbank_431) may also occupy L178 side pocket and its amide group forms a hydrogenic binding interaction with the flexible R174 residue (Fig. 1B). However, the fragment is small and only occupies two binding pockets. As each pocket can describe the binding interaction between the pocket and its preferred moieties, we introduced a hydrophobic moiety to the pyrrole-pyrimidine fragment. Using a small library featured with hydrophobic fragments, we identified N-phenylsubstituted benzamide fragment as a suitable moiety that was predicted to occupy S257 hydrophobic pocket and maintain the compounds ability to form a hydrogenic binding interaction with R174 pocket (Fig. 1B). Using pyrrole-pyrimidine and N-phenylsubstituted benzamide fragments as the core scaffold, we designed and synthesized 17 compounds (fig. S1D). These compounds were docked into R174 pocket of Snail for the second round of filtration, and four compounds (i.e., CYD16 to CYD19) were found to form interaction with R174. As shown, the most potent compound CYD19 was predicted to anchor into Snail cavity by forming binding interactions with hotspot R174 pocket, L178 side pocket, and S257 hydrophobic pocket (Fig. 1, B and C). Next, we performed the biolayer interferometry (BLI) and microscale thermophoresis (MST) assays to measure the dissociation kinetics of CYD19. BLI analysis revealed that CYD19 had a submicromolar potency (Kd = 0.18 M), while the inactive analog CYD18 was approximately 80-fold less potent toward Snail (Kd = 14.1 M) (Fig. 1D). Similarly, MST assay showed that CYD19 was 55-fold more potent toward Snail than CYD18 (0.2 M versus 11.1 M in Kd) (fig. S1E). To further test whether R174 of Snail is important for its interaction with CYD19, we generated the Snail R174A174 mutant (Snail-R174A mutant) and performed the BLI assay. We observed that the R174A mutation caused steric conformation alteration due to dissimilarity of the side chain of residue, and thus, the compound CYD19 could not fit well with Snail-R174A mutant (Fig. 1C, compare right panel and left and middle panels). As expected, CYD19 showed a 16-fold lower binding affinity to Snail-R174A mutant (Kd = 3.0 M) than wild-type Snail (Snail-WT), as assessed by a BLI assay (fig. S1F). Together, the results from the in silico and BLI assays suggest that R174 is essential for the high-affinity binding of Snail with CYD19. Because the C2H2-type zinc fingers (ZFs) are highly conserved across Snail family members, we used BLI assay to examine the dissociation kinetics of CYD19 toward Slug (alternatively termed Snail2), another member of the Snail family (6, 21, 33). As shown, CYD19 had a submicromolar potency (Kd = 0.6 M), while the inactive analog CYD18 was approximately 145-fold less potent toward Slug protein (fig. S1G), suggesting that CYD19 also forms a binding interaction with Slug protein.

(A) Diagram showing that R174 is evolutionarily conserved across species. Hu, human; Ms, mouse; Rt, rat; Ch, chimpanzee; Zf, zebra fish; Cf, clawed frog; Rd., rock dove. (B) Close-up view of three predicted binding pockets of Snail protein (left) and presumed interaction surface of pyrrole-pyrimidine fragment (middle) and hit compound (right) with Snail. (C) Molecular docking analysis. (D) BLI analysis to measure dissociation kinetics of compounds toward Snail recombinant proteins. (E and F) Immunoblot analysis of Snail expression in cancer cells treated with vehicle or CYD19 for 48 hours (E) or in MMTV-PyMT cells treated with vehicle or 50 nM CYD19 and then with cycloheximide (CHX; 100 g/ml) for a total of 48 hours (F). MDA231, MDA-MB-231. (G) Densitometry of Snail protein in cells as described in (F). (H and I) Comparison of exogenous Snail-WT and Snail-R174A expressions in human embryonic kidney (HEK) 293T cells treated with vehicle or CYD19 for 48 hours (H) or in cells treated with vehicle or 50 nM CYD19 for different times (I). (J) Comparison of ubiquitinated Snail-WT and Snail-R174A proteins in HEK293T cells treated with vehicle or 50 nM CYD19 for 48 hours. MG132 (10 M) was added 4 hours before harvesting. IgG, immunoglobulin G; IP, immunoprecipitation; HA-ubi, hemagglutinin-ubiquitin. (K) Comparison of acetylated and phosphorylated Snail-WT versus Snail-R174A proteins in HEK293T cells as described in (J). (L and M) Binding interaction of exogenous (L) or endogenous (M) Snail with endogenous CBP/p300 was monitored in cells that were treated with vehicle or 50 nM CYD19 for 48 hours. (N) His pulldown assay to assess CYD19s impact on association of CBP-HAT with Snail-WT or Snail-R174A. Arrows and asterisks mark specific and nonspecific bands, respectively. (O) Immunoblot analysis of exogenous Snail expression in HEK293T cells treated with vehicle or 50 nM CYD19 and then with CHX (100 g/ml) for a total of 48 hours. (P) Densitometry of exogenous Snail protein in cells described in (O). All representative blots as shown are from three independent experiments.

Next, we asked whether compound CYD19 could affect Snail expression in carcinoma cell cultures. Immunoblot analysis revealed that CYD19 dose-dependently decreased Snail protein levels in freshly isolated human BrCa primary cells, mouse and human BrCa cell lines, and colorectal cancer cell lines (Fig. 1E and fig. S2A). In addition, we observed that CYD19 reduced Snail protein levels in a time-dependent manner (fig. S2B). As expected, CYD18 did not affect Snail protein levels in the tested cell lines (fig. S2C). No significant changes in Snail mRNA levels were detected in CYD19-treated cells relative to control cells, suggesting that CYD19 regulated Snail expression at posttranslational level (fig. S2D). To directly test whether CYD19 could affect Snail protein stability, we cultured vehicle- or CYD19-treated mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) cells in the presence of cycloheximide (CHX; 100 g/ml) to block newly protein synthesis and examined Snail degradation. After treatment with CHX, Snail became unstable and degraded rapidly in CYD19-treated cells, while the protein was relatively stable in vehicle-treated cells, suggesting that CYD19 indeed reduces Snail protein stability (Fig. 1, F and G). Because CYD19 showed a significantly lower affinity with Snail-R174A mutant than Snail-WT, we compared the protein stability of Snail-R174A mutant versus Snail-WT following CYD19 treatment. Treatment of transfected human embryonic kidney (HEK) 293T cells with CYD19 diminished FLAG-tagged Snail-WT protein levels in a dose- and time-dependent manner (Fig. 1, H and I, top). However, treatment with CYD19 at up to 150 nM or for up to 48 hours failed to decrease Snail-R174A mutant protein levels (Fig. 1, H and I, bottom), confirming that R174 is a key amino acid for Snails binding with CYD19. To test whether this CYD19 effect is mediated through a ubiquitination of Snail, we cotransfected HEK293T cells with FLAG-tagged Snail-WT (or Snail-R174A mutant) and hemagglutinin (HA)ubiquitin and treated them with vehicle or CYD19 for 48 hours. MG132 (10 M) was added to the cells 4 hours before cell harvesting, and the cell lysates were subjected to immunoprecipitation (IP) assay using an anti-FLAG antibody. Notably, we observed that CYD19 remarkably increased the ubiquitination levels of Snail-WT but failed to affect the ubiquitination of Snail-R174A mutant (Fig. 1J). The acetylation of Snail has been reported to stabilize Snail protein (18). We therefore asked whether CYD19 could affect Snail acetylation. We found that CYD19 remarkably decreased acetylation of Snail-WT but not Snail-R174A mutant proteins (Fig. 1M). GSK3 and PKD1 can phosphorylate Snail and promotes its ubiquitination and degradation (2529). Snail acetylation can reduce its phosphorylation, which consequently results in increased protein stability (18). Here, we showed that treatment with CYD19 markedly increased phosphorylation levels of Snail-WT protein but had negligible effects on phosphorylation levels of Snail-R174A mutant protein (Fig. 1K). CBP/p300 has been reported to function as the primary HATs that may acetylate Snail at K146 and K187 (18). We therefore hypothesized that CYD19 binds to Snail protein, which consequently interrupts the interaction of Snail with CBP/p300 and results in impairment of Snail acetylation. To test this, we treated exogenous Snail-transfected HEK293T and HCT116 cells with vehicle or CYD19 and subjected the cell lysates to IP assays using anti-FLAG or anti-Snail antibodies, followed by immunoblot analysis using anti-CBP and anti-p300 antibodies (Fig. 1, L and M). We observed that the treatment of HEK293T and HCT116 cells with CYD19 did not affect total CBP/p300 expressions but markedly reduced Snail-bound CBP/p300 levels (Fig. 1, L and M). In notable contrast, CYD19 did not affect the binding of Snail-R174A mutant with CBP/p300 (Fig. 1L, right). To directly evaluate the ability of CYD19 to interfere the interaction between Snail and CBP, we expressed and purified glutathione S-transferase (GST)CBP-HAT (containing HAT domain of CBP protein) and His-tagged Snail-WT and Snail-R174A (His-Snail-WT and His-Snail-R174A, respectively) mutant recombinant proteins in Escherichia coli bacteria and performed in vitro His pulldown experiments. We observed that CYD19 dose-dependently diminished the interaction of CBP-HAT with His-Snail-WT but not His-Snail-R174A mutant recombinant proteins, suggesting that CYD19 directly interferes the binding between CBP and Snail in a dose-dependent manner (Fig. 1N). To examine whether CBP/p300-mediated acetylation of Snail is actively involved in the regulation of Snail protein stability by CYD19, we generated the Snail-K146R/K187R (Snail-2KR) mutant and performed the CHX chase assay. We observed that the half-life of Snail-2KR mutant protein and Snail-WT protein was comparable in vehicle-treated cells (Fig. 1, O and P). However, Snail-2KR mutant protein degraded more rapidly than Snail-WT protein in CYD19-treated cells, suggesting that CBP/p300-mediated acetylation stabilizes Snail protein in the presence of CYD19 (Fig. 1, O and P). Because CYD19 can also form a binding interaction with Slug, we asked whether CYD19 has an impact on Slug protein expression. Unexpectedly, CYD19 did not affect Slug protein expression in a variety of cancer cell lines (fig. S2E). We demonstrated that Slug, unlike Snail, did not form a binding interaction with CBP/p300 (fig. S2F), suggesting that there should exist other potential regulator proteins (not CBP/p300) responsible for modulating Slug protein expression. These findings suggest that compound CYD19 does not interrupt Slugs interaction with its potential regulator proteins and thus loses the ability to affect Slug protein expression.

Importins (e.g., importin ) are reported to transport Snail protein into the nucleus by tightly interacting with several key amino acid residues within Snails ZF domains, including K161, K170, K187, R191, W193 (tryptophan-193), Q196 (glutamine-196), R220, R224, and Q228 (32, 34, 35). Single mutation, double mutations, or multiple mutations in these residues efficiently (or completely) reduce the binding of Snail with importin , thus severely impairing importin mediated nuclear import of Snail protein (32, 34). To assess whether R174 is required for Snail binding to importin and whether CYD19 that specifically binds to R174 could affect Snailimportin binding interaction, we performed serial His pulldown assays, followed by immunoblots using antiimportin and anti-Snail antibodies (34). To this end, His-Snail-WT or His-Snail-R174A mutant recombinant proteins were purified, immobilized on Ninitrilotriacetic acid (NTA) agarose, and incubated, either in the absence or presence of various concentrations of CYD19, with a complete HEK293T cell lysates used as a source of importin . As shown, both Snail-WT and Snail-R174A mutant proteins physically bound with importin indistinguishably (fig. S2G), suggesting that R174 is not required for Snail binding to importin . Furthermore, compound CYD19 at various concentrations failed to affect binding of Snail-WT with importin (fig. S2H). In addition, we performed in-cell experiments to test whether mutation in R174 could affect Snail subcellular localization. To completely exclude the possibility that small molecules (smaller than 50 kDa) such as Snail protein can diffuse into the nucleus through nuclear pore complexes, we increased the sizes of green fluorescent protein (GFP)Snail-WT and GFP-Snail-R174A proteins by fusing them to GST and transfected them into MCF7 BrCa cells (32, 34). Although GFP-GST was detected in the nucleus and cytoplasm, both GFP-Snail-WT and GFP-Snail-R174A mutant proteins were exclusively localized in the nucleus (fig. S2I), suggesting that R174 is not required for Snail binding to importin and plays no role in importin mediated Snail nuclear import. Intracellular localization of Snail protein was also examined by cell fractionation. As shown, FLAG-tagged Snail-WT and Snail-R174A mutant proteins were both exclusively localized in the nucleus of vehicle- and CYD19-treated cells (fig. S2J). These findings suggest that compound CYD19 that forms binding interaction with R174 pocket of Snail protein does not affect Snailimportin interaction and subsequent Snail subcellular localization. Together, our data support the mode of action by on-target effect of compound CYD19; that is, CYD19 specifically binding to hotspot R174 pocket of Snail protein disrupts the interaction of Snail with CBP/p300 and eventually triggers Snail protein degradation without affecting Snailimportin interaction and subsequent Snail subcellular localization.

Snail has been shown to induce EMT and promote migration and metastasis in various cancer types (5, 8). TGF signaling is known to activate EMT in epithelial-like cancer cells through transcriptionally inducing Snail (8). We therefore tested whether CYD19 could block TGF1/Snail-driven EMT phenotypes in cancer cells. To do this, we pretreated cells with vehicle or TGF1 (2 ng/ml) for 24 hours and further treated them with vehicle or various concentrations of CYD19 in combination with TGF1 (2 ng/ml) for another 48 hours. Notably, we found that CYD19 efficiently blocked TGF1/Snail-driven EMT phenotypes in freshly isolated human BrCa primary cells and various cancer cell lines, as evidenced by increased expression of epithelial marker (E-cadherin) and decreased expressions of mesenchymal markers such as vimentin, N-cadherin, and fibronectin (Fig. 2, A and B, and fig. S3A). Snail is also known to transcriptionally activate inflammatory cytokine genes such as tumor necrosis factor (TNF), extension repair cross-complementation group 1 (ERCC1), C-C motif chemokine ligand 2 (CCL2), CCL5, and interleukin-8 (IL8) (18, 36, 37). We next examined the impact of CYD19 on TGF1/Snail-modulated cytokinome in cancer cells. We observed that CYD19 treatment completely abolished TGF1/Snail-mediated activation of the indicated inflammatory cytokine genes in human BrCa primary cells and various cancer cell lines (fig. S3B), indicating the impact of CYD19 on tumor microenvironment remodeling during cancer progression. TNF has been demonstrated to stabilize Snail protein by modulating nuclear factor B signaling pathway (27). Thus, we evaluated the impact of CYD19 on TNF-stimulated Snail expression. To do this, we treated cells with vehicle or CYD19 for 48 hours and added TNF (10 ng/ml) to stimulate the cells 8 hours before cell harvesting. We found that CYD19 efficiently blocked TNF-stimulated Snail protein expression (Fig. 2C). Together, these findings suggest the important role of CYD19 in suppressing the external stimulusinduced Snail expression. Given that Snail-induced EMT is closely related to migration and invasion of cancer cells, we examined the impact of CYD19 on cancer cell migration. To do this, equal numbers of vehicle- or CYD19-pretreated cells were cultured in serum-free medium supplemented with vehicle or CYD19 in the upper chambers of transwell inserts, while the lower chambers were filled with medium containing 10% serum. We found that CYD19 dose-dependently reduced migration of a variety of cancer cell lines (fig. S3C). To test whether CYD19 inhibited cell migration by specifically targeting Snail protein, we infected Snailfl/fl MMTV-PyMT cancer cells, a cell line that was previously established in our laboratory (20), with adeno-galactosidase (Gal) or adeno-Cre to generate control or Snail-deleted cells, treated them with CYD19 (or vehicle), and subjected them to cell migration assay (Fig. 2, D to F). As expected, migration of Snail-deleted cells was markedly reduced compared to control cells, and CYD19 remarkably suppressed migration of control cells but largely failed to inhibit migration of Snail-deleted cells (Fig. 2, E and F). Moreover, we silenced Snail expression in HCT116 and SUM159 cells and then subjected the cells to migration analysis. As shown, cell migration was slightly reduced in HCT116 cells where Snail was moderately silenced but significantly reduced in cells where Snail was almost completely depleted; CYD19 efficiently reduced migration of control and Snailmoderately silenced HCT116 cells but did not affect migration of Snailcompletely silenced cells (Fig. 2, G and H). A similar phenotype was also observed in SUM159 cells (Fig. 2, I and J). These results suggest that CYD19 inhibits cell migration by specifically targeting Snail protein. Recently, Snail has been reported to play a critical role in regulating aldehyde dehydrogenasepositive (ALDH+) CSC expansion in established MMTV-PyMT breast tumors (20, 38). Here, we observed substantially reduced numbers of ALDH+ CSCs in CYD19-treated cells compared to vehicle-treated cells, suggesting that CYD19 blocked Snail-driven CSC expansion in MMTV-PyMT cells (Fig. 2, K and L).

(A) Immunoblot analysis of Snail, E-cadherin, and vimentin expressions in primary cancer cells and cancer cell lines that were treated with vehicle (Veh.) or TGF1 (2 ng/ml) for 24 hours and then with vehicle or CYD19 in the presence of TGF1 for another 48 hours. (B) Immunofluorescence staining of E-cadherin and vimentin in MMTV-PyMT (left) and 4T1 (right) cells as described in (A). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (blue). (C) Immunoblotting of Snail expression in MMTV-PyMT and HCT116 cells. Cells were treated with vehicle or CYD19 for 48 hours, and TNF (10 ng/ml) was added 8 hours before cell harvesting. (D) Immunoblotting of Snail expression in Snailfl/fl MMTV-PyMT cells that were infected with adeno-Gal or adeno-Cre vectors. (E and F) Equal numbers (2 105 cells per well) of control and Snail-deleted MMTV-PyMT cells pretreated with vehicle or CYD19 for 48 hours were subjected to cell migration assays, and invaded cells were quantified (F). (G and I) Immunoblot analysis of Snail expression in HCT116 (G) and SUM159 (I) cells that were infected with lentiviral vectors expressing controlshort hairpinmediated RNA (shRNA) or two independent Snail-shRNAs. (H and J) Equal numbers (2 105 cells per well) of HCT116 (H) and SUM159 (J) cells were subjected to cell migration assays, and invaded cells were quantified. (K and L) Representative histogram (K) and quantification (L) of ALDH+ subpopulation in control and Snail-deleted MMTV-PyMT cells. All representative blots, images, and histograms as shown are from three independent experiments. All data are presented as means SD (n = 3 independent experiments). *P < 0.05 and **P < 0.01. N.S., not significant. Differences are tested using one-way analysis of variance (ANOVA) with Tukeys post hoc test (H and J) and unpaired two-tailed Students t test (L).

We previously showed that Snail interacts directly with wild-type, but not mutant, p53, thereby triggering its proteasome degradation in BrCa cells (20). Therefore, we asked whether CYD19 has an impact on expression of wild-type and mutant p53. Immunoblot analysis revealed that CYD19 dose-dependently increased wild-type p53 protein levels in various cell lines (Fig. 3A, left). In notable contrast, CYD19 did not affect mutant p53 protein expression in MDA-MB-231, SW620, and DLD1 cells (Fig. 3A, right). Immunofluorescence analysis revealed markedly decreased Snail expression in tandem with increased p53 expression in CYD19-treated MMTV-PyMT and HCT116 cells relative to control cells (Fig. 3B). Although CYD19 did not affect TP53 expression, the compound did increase the mRNA and protein levels of p53 targets p21 and MDM2 in MMTV-PyMT and HCT116 cells in a dose- and time-dependent manner (Fig. 3, C and D, and fig. S4, A and B). To test whether CYD19 could affect wild-type p53 protein stability, vehicle- or CYD19-treated MMTV-PyMT cells were cultured in the presence of CHX (100 g/ml) to block newly protein synthesis, and p53 degradation was examined. After treatment with CHX, p53 protein in vehicle-treated cells was unstable and degraded rapidly starting from 1/2 hours after CHX treatment, while p53 protein in CYD19-treated cells was more stable and started to degrade 2 hours after CHX treatment (Fig. 3, E and F), suggesting that CYD19 increases wild-type p53 protein stability. Consistently, we observed that CYD19 robustly decreased the ubiquitination of endogenous p53 in MMTV-PyMT cells (Fig. 3G). Notably, increase in p53 protein levels and activity are associated with increased levels of p53 acetylation (20, 39), and following Snail deletion, p53 acetylation levels increase (20). We found that CYD19 treatment of MMTV-PyMT cells exhibited increased levels of acetylated p53 (Fig. 3H), suggesting that CYD19 promotes p53 acetylation and thus stabilizes p53 protein by inhibiting Snail protein expression. We previously demonstrated that Snail binds to wild-type p53 and triggers p53 deacetylation by recruiting histone deacetylases (HDACs) to the complex (20). Here, we observed that CYD19 robustly diminished Snail-mediated binding interaction of wild-type p53 with HDAC1 (Fig. 3I), indicating that CYD19 disrupts the HDAC1 recruitment to wild-type p53 and thus increases p53 acetylation and protein levels. To directly test whether Snail is required for CYD19-mediated up-regulation on wild-type p53 expression, we compared expressions of p53 and its target protein p21 in control and Snail-deleted MMTV-PyMT cells in the presence of increasing concentrations of CYD19. Notably, we found that CYD19 robustly increased p53 and p21 expressions in control cells but largely failed to increase their expressions in Snail-deleted cells (Fig. 3J), suggesting that CYD19-mediated up-regulation on p53 pathway heavily depends on Snail expression. Snail silencing robustly increased expression of wild-type p53 protein in HCT116 cells but did not affect mutant p53 expression in DLD1 and SUM159 cells (fig. S4, C to E), confirming our previous observations (20).

(A) Immunoblot analysis of p53 expression in wild-type (left) and mutant (right) p53-expressing cells that were treated with vehicle or CYD19 for 48 hours. (B) Immunofluorescence staining of Snail and p53 in MMTV-PyMT (left) and HCT116 (right) cells treated with vehicle or 50 nM CYD19 for 48 hours. (C) Reverse transcription quantitative polymerase chain reaction (qPCR) analysis of p53, p21, and MDM2 expressions in MMTV-PyMT (top) and HCT116 (bottom) cells as described in (B). (D) Immunoblot analysis of p53, p21, and MDM2 expressions in MMTV-PyMT and HCT116 cells treated with vehicle or CYD19 for 48 hours. (E) Immunoblot analysis of p53 expression in MMTV-PyMT cells treated with vehicle or 50 nM CYD19 and then with CHX (100 g/ml) for a total of 48 hours. (F) Densitometry of p53 protein in cells as described in (E). (G) Comparison of ubiquitinated p53 protein in vehicle- and CYD19-treated MMTV-PyMT cells. MG132 (10 M) was added 4 hours before harvesting. Lysates from vehicle- and CYD19-treated cells loaded at ratios of 2:1 and 1:1 were subjected to IP assay using an anti-p53 antibody. (H) Comparison of acetylated p53 protein in vehicle- and CYD19-treated MMTV-PyMT cells as described in (G). (I) Comparison of binding interaction of p53 with HDAC1 in vehicle- and CYD19-treated MMTV-PyMT cells as described in (G). (J) Comparison of Snail, p53, and p21 expressions in control (left) and Snail-deleted (right) MMTV-PyMT cells that were treated with vehicle or CYD19 for 48 hours. All representative blots and images as shown are from three independent experiments. All data are presented as means SD (n = 3 independent experiments). **P < 0.01. Differences are tested using unpaired two-tailed Students t test (C).

We previously identified Snail as a molecular bypass that suppresses the antiproliferative and proapoptotic effects exerted by wild-type p53 in BrCa (20). Because compound CYD19 increases protein expression of wild-type, but not mutant, p53, we asked whether the compound could affect proliferation and survival of cancer cells harboring wild-type or mutant p53. Notably, we observed that cells harboring wild-type p53 were significantly more sensitive to CYD19 treatment than cells expressing mutant p53, as assessed by the CCK-8 (cell counting kit-8) proliferation assay (Fig. 4A). Furthermore, CYD19 induced apoptosis in a dose-dependent manner in cells expressing wild-type p53 but essentially failed to induce apoptosis in cells with mutant p53 (Fig. 4B and fig. S5A). Consistently, treatment of wild-type p53-expressing MMTV-PyMT and HCT116 cells with compound CYD19 dose-dependently increased expressions of p53-inducible proapoptotic proteins Puma and Bax and triggered the release of cytochrome c (Cyt-c) from mitochondria, thus inducing the activation (cleavage) of caspase 9 and caspase 3, a dominant executor of cell apoptosis (Fig. 4C). CYD19 also increased Bax expression and induced caspase 3 activation in a time-dependent manner (fig. S5B). To determine whether Snail is required for CYD19-mediated up-regulation on proapoptotic protein expressions, we compared their expressions in control and Snail-deleted MMTV-PyMT cells in the presence of increasing concentrations of CYD19. As shown, we observed that CYD19 dose-dependently increased Bax and activated caspase 3 expressions in control MMTV-PyMT cells, while the compound essentially failed to increase proapoptotic protein expressions in Snail-deleted cells (Fig. 4D). The CCK-8 cell proliferation assay further revealed that Snail-deleted MMTV-PyMT cells were substantially less sensitive to CYD19 treatment than control cells (Fig. 4E). To directly test whether Snail is required for CYD19-mediated inhibition on cell proliferation and survival, Snail expression was silenced in HCT116 cells, and cell proliferation and survival were assessed in control and Snail-silenced cells in the presence of vehicle or increasing concentrations of CYD19. As shown, we found that CYD19 dose-dependently induced apoptosis in control HCT116 cells but essentially failed to induce apoptosis in Snail-silenced cells (Fig. 4F and fig. S5C). Consistently, the CCK-8 cell proliferation assay revealed that Snail-silenced HCT116 cells were significantly less sensitive to CYD19 treatment than control cells (Fig. 4G). To further test whether p53 is required for CYD19-mediated inhibition on cell survival and proliferation, p53 expression were silenced in HCT116, and cell survival and proliferation were assessed in control and p53-silenced cells in the presence of vehicle or increasing concentrations of CYD19. As compared with control cells, p53-silenced HCT116 cells had significantly diminished responsiveness to CYD19 to inhibit cell survival and proliferation (Fig. 4, H and I, and fig. S5D). Notably, Snail silencing efficiently reduced proliferation of wild-type p53-expressing tumor cells but did not affect growth of mutant p53-expressing cells (fig. S5E), which confirms and extends our previous observations (20). Given that Snail-driven EMT confers tumor resistance toward many chemotherapeutics (10, 15, 16), the impact of CYD19 on EMT-driven chemoresistance was therefore examined. We found that low-dose taxol (doses ranging from 0.5 to 4.0 nM in MMTV-PyMT cells and from 1.0 to 8.0 nM in HCT116 cells) or CYD19 (20 nM in both cell lines) had no impact on cell proliferation, while low-dose taxol in combination with CYD19 (25 nM) yielded a strong and superior antiproliferation activity in both cell lines (Fig. 4J), suggesting that CYD19 reverses EMT-driven chemoresistance and thus sensitizes cancer cells to low-dose chemotherapy. Together, our findings suggest that CYD19 reduces proliferation and survival of tumor cells in a TP53 wild typedependent fashion.

(A) CCK-8 cell proliferation assay for wild-type and mutant p53-expressing cells treated with vehicle or CYD19 for 48 hours. (B) Quantification of apoptotic subpopulation in various cell lines treated with vehicle or CYD19 for 48 hours. (C) Immunoblot analysis of the indicated protein expressions in MMTV-PyMT (left) and HCT116 (right) cells as described in (B). C-casp9, cleaved caspase 9. (D) Immunoblot analysis of the indicated protein expressions in control and Snail-deleted MMTV-PyMT cells treated with vehicle or CYD19 for 48 hours. (E) CCK-8 analysis for control and Snail-deleted MMTV-PyMT cells treated with vehicle or CYD19 for 48 hours. (F) Quantification of apoptotic subpopulation in control and Snail-silenced HCT116 cells treated with vehicle or CYD19 for 48 hours. (G) CCK-8 analysis for control and Snail-silenced HCT116 cells treated with vehicle or CYD19 for 48 hours. (H) Quantification of apoptotic subpopulation in control and p53-silenced HCT116 cells treated with vehicle or CYD19 for 48 hours. (I) CCK-8 analysis for control and p53-silenced HCT116 cells treated with vehicle or CYD19 for 48 hours. (J) CCK-8 analysis for MMTV-PyMT and HCT116 cells that were treated with vehicle or taxol in combination with vehicle or 25 nM CYD19 for 48 hours. All representative blots as shown are from three independent experiments. All data are presented as means SD (n = 3 independent experiments). **P < 0.01. Differences are tested using one-way ANOVA with Tukeys post hoc test (B, F, and H).

Snail has been known to play an essential role in controlling tumor progression and metastasis as well as the expansion of TICs in MMTV-PyMT transgenic mice (20), a mouse model of BrCa that mirrors the multistep progression of human BrCa (40). Here, we asked whether CYD19 could affect Snail-driven progression and metastasis of spontaneous breast tumors in MMTV-PyMT transgenic mice. To do this, we treated 2-month-old female littermates that developed palpable breast tumors in a total volume of ~0.4 cm3 with vehicle or CYD19 (30 mg/kg) for consecutive 25 days and examined the formation of primary and metastasized tumors. As shown, tumor volumes and weights were robustly reduced in CYD19-treated mice compared to vehicle-treated mice (Fig. 5, A and B). Notably, CYD19 did not affect body weights of tumor-bearing mice or induce detectable histological alterations in their vital organs such as the heart, liver, spleen, or kidneys, supporting the absence of toxicity in CYD19-treated mice (fig. S6, A and B). Furthermore, we observed that CYD19 substantially decreased the percentages of proliferative (Ki67-positive) and mitotic (phospho-histone H3positive) cells but increased the percentages of apoptotic (cleaved caspase 3positive) cells (Fig. 5, C and D, and fig. S6, C and D). As expected, tumors of CYD19-treated mice exhibited remarkably reduced Snail expression in tandem with increased wild-type p53 protein levels, as assessed by immunoblot and immunofluorescence analyses (Fig. 5E and fig. S6, E and F). Histological analysis revealed that vehicle-treated tumors progressed to poorly differentiated adenocarcinomas at the end of the treatment, while CYD19-treated tumors exhibited a more differentiated phenotype (Fig. 5F). Consistently, tumors of CYD19-treated mice showed an increase in E-cadherin expression in tandem with reduced vimentin expression, suggesting that CYD19 suppresses Snail-driven EMT in the in vivo setting (Fig. 5, G and H). We observed that CYD19 remarkably impaired ALDH+ CSC expansion in primary tumors (Fig. 5, I and J), which is consistent with the in vitro observations (Fig. 2, M and N). Snail is known to promote recruitment of tumor-associated macrophages (TAMs), thus facilitating tumor progression (18). We observed that CYD19 reduced intratumoral infiltration of F4/80+ TAMs and CD31+ endothelial cells (Fig. 5, K and L). CYD19 also reduced metastatic potential of primary tumors, as evidenced by remarkably fewer and smaller metastatic nodules in the lungs of CYD19-treated mice relative to vehicle-treated mice (Fig. 5, M to O). Collectively, the findings suggest that CYD19 suppressed Snail-driven tumor progression, pulmonary metastasis, and CSC expansion in MMTV-PyMT transgenic mice that express wild-type p53.

(A and B) Primary tumor volumes (A) and weights (B) were measured in MMTV-PyMT mice that were intraperitoneally treated with vehicle or CYD19 (30 mg/kg) for 25 consecutive days (n = 6 mice, each). (C) Immunohistochemical staining of Ki67 (top) and cleaved caspase 3 (bottom) in primary tumors of vehicle- and CYD19-treated mice (n = 6 mice, each). (D) Quantification of Ki67-positive (Ki67+; top) and cleaved caspase 3positive (C-casp3+; bottom) cells in tumors as described in (C). (E) Immunoblot analysis of Snail and p53 expressions in tumor lysates of vehicle- and CYD19-treated mice (n = 3 pools from six mice, each). (F) Hematoxylin and eosin (H&E) staining for primary tumors as described in (C) (n = 6 mice, each). Magnified areas of boxed sections are shown in the bottom panels. (G) Immunofluorescence staining of E-cadherin and vimentin in primary tumors as described in (C) (n = 6 mice, each). (H) Quantification of staining intensity in primary tumors as described in (G). (I and J) Representative histogram (I) and quantification (J) of ALDH+ subpopulation in primary tumors as described in (C) (n = 6 mice, each). DEAB, diethylaminobenzaldehyde. (K) Immunofluorescence staining of F4/80 and CD31 in primary tumors as described in (C) (n = 6 mice, each). (L) Quantification of staining intensity in primary tumors as described in (K). (M) H&E staining for vehicle- and CYD19-treated lungs (n = 6 mice, each). (N) Magnified areas of boxed sections in (M) are shown. (O) Quantification of nodules in vehicle- and CYD19-treated lungs as described in (M). All data are presented as means SD (n = 6 independent experiments). *P < 0.05 and **P < 0.01. Differences are tested using Mann-Whitney U test.

Next, we asked whether CYD19 had a similar impact on colon cancer growth and hepatic metastasis using a HCT116 xenograft model in which 1 106 HCT116 cells in 50 l of diluted Matrigel were injected subcutaneously into the dorsal flank of athymic BALB/c nude mice. We observed that CYD19 dose-dependently reduced the growth of HCT116 xenograft tumors (Fig. 6, A and B), without eliciting body weight loss or histological alterations in the vital organs such as the heart, liver, spleen, lung, and kidney (fig. S7, A and B). Furthermore, we found that CYD19 reduced the percentages of proliferative and mitotic cells while increasing the percentages of apoptotic cells in xenograft tumors (Fig. 6, C and D, and fig. S7, C and D). Notably, CYD19 suppressed Snail expression while increasing p53 expression in xenograft tumors, as assessed by immunoblot and immunohistochemical analyses (Fig. 6E and fig. S7, E to H). In addition, impaired EMT was detected in CYD19-treated xenograft tumors, as illustrated by increased E-cadherin expression in tandem with a reduction in vimentin expression (Fig. 6, F and G). We next examined the impact of CYD19 on ALDH+ CSC expansion in HCT116 xenograft tumors. To do this, we sorted ALDH+ and ALDH cells from HCT116 xenograft tumors and performed in vitro tumorsphere assay. The results demonstrated that ALDH+ but not ALDH cells had the potential to form tumorspheres, confirming that ALDH can be used for identification of CSCs in HCT116 xenograft tumors (fig. S7I). Notably, we observed that CYD19 severely impaired ALDH+ CSC expansion in HCT116 xenograft tumors (Fig. 6, H and I). To further examine whether the in vivo anticancer effect of CYD19 is Snail-dependent, we subcutaneously implanted 1 106 control or 2 106 Snail-silenced HCT116 cells into nude mice, treated mice with vehicle or CYD19 (30 mg/kg) for two consecutive weeks starting at 7 days after implantation, and monitored tumor growth. The volumes of xenograft tumors formed by 1 106 control or 2 106 Snail-silenced cells were comparable (Fig. 6J). Notably, CYD19 suppressed tumor growth of control cells but largely failed to affect tumor growth of Snail-silenced cells (Fig. 6J), suggesting that CYD19 suppresses tumor growth by specifically targeting Snail protein. Furthermore, immunoblot analysis of xenograft tumor lysates revealed that Snail expression was efficiently silenced in Snailshort hairpinmediated RNA 2 (shRNA2)expressing cells where p53 protein was robustly increased (Fig. 6K, compare lane 3 versus lane 1). As expected, CYD19 decreased Snail expression while increasing p53 protein in control cells (Fig. 6K, compare lane 2 versus lane 1), and the compound lost its ability to increase p53 expression in Snail-silenced cells (Fig. 6K, compare lane 4 versus lane 3). In addition, equal numbers (1 106) of control or Snail-silenced HCT116 cells were implanted into nude mice; the mice were treated with vehicle or CYD19, and tumor growth was monitored. As shown in fig. S7J, CYD19 suppressed tumor growth of control cells by 60.3% at the end point of treatment (compare curve 2 versus curve 1), and Snail silencing itself reduced tumor growth by 64.8% (compare curve 3 versus curve 1). While CYD19 remarkably reduced control tumor growth by 60.3%, the compound inhibited tumor growth of Snail-silenced cells by 4% (compare curve 4 versus curve 3), further confirming that CYD19 suppresses tumor growth by specifically targeting Snail protein. Next, we assessed the impact of CYD19 on tumor metastasis using a hepatic metastasis model in which 1 106 GFP-labeled HCT116 cells were intrasplenically injected to nude mice. The results demonstrated that CYD19 treatment for three consecutive weeks robustly reduced tumor metastasis and nodule formation in the livers (Fig. 6, L and M). Together, these findings suggest that CYD19 reduces Snail-driven tumor growth, hepatic metastasis, and CSC expansion in colon cancer xenografts expressing wild-type p53.

(A and B) HCT116 xenograft tumor volumes (A) and weights (B) were measured in athymic nude mice that were intraperitoneally treated with vehicle or CYD19 for two consecutive weeks (n = 6 mice, each). (C) Immunohistochemical staining of Ki67 (top) and cleaved caspase 3 (bottom) in xenograft tumors of vehicle- and CYD19-treated mice (n = 6 mice, each). (D) Quantification of Ki67+ (top) and C-casp3+ (bottom) cells in tumors as described in (C). (E) Immunoblot analysis of Snail and p53 expressions in tumor lysates of vehicle- and CYD19-treated mice (n = 3 pools from six mice, each). (F) Immunofluorescence staining of E-cadherin and vimentin in xenograft tumors of vehicle- and CYD19-treated mice (n = 6 mice, each). (G) Quantification of staining intensity in xenograft tumors as described in (F). (H and I) Representative histogram (H) and quantification (I) of ALDH+ subpopulation in xenograft tumors as described in (C) (n = 6 mice, each). (J) Growth of HCT116 xenograft tumors derived from 1 106 control cells or 2 106 Snail-silenced cells was monitored in nude mice treated with vehicle or CYD19 for two consecutive weeks (n = 6 mice, each). (K) Immunoblot analysis of Snail and p53 expressions in lysates of xenograft tumors as described in (J). (L) Representative phase contrast (top), GFP fluorescence (middle), and H&E (bottom) images of vehicle- and CYD19-treated livers (n = 6 mice, each). Mice were treated with vehicle or CYD19 for three consecutive weeks starting from the third day after surgery. (M) Quantification of fluorescence intensity in livers as described in (L). All data are presented as means SD (n = 6 independent experiments). **P < 0.01. Differences are tested using Mann-Whitney U test.

The ZF transcription factor Snail is aberrantly activated in a variety of malignant tumor types (2023) and plays an essential role in EMT, metastasis, stem celllike properties, cancer metabolism, microenvironment modulation, immune evasion, cancer recurrence, and therapeutic resistance (9, 10, 1318, 41, 42). Snail is also known to promote cancer cell survival by enhancing resistance to apoptosis under the genotoxic stress condition (19). We recently identified Snail as a molecular bypass that suppresses the antiproliferative and proapoptotic effect in BrCa (20). Given the important role of Snail in driving cancer progression, we propose that targeting Snail would be an attractive anticancer therapeutic approach. However, to our knowledge, the development of small molecules to inhibit Snails functions is unsuccessful, as there is no clear ligand-binding domain for targeting Snail (43). In the current study, we have identified the evolutionarily conserved R174 pocket as a key hotspot in the binding site of Snail. Using fragment-based virtual screening analysis in combination with Glide docking algorithms, we have screened 50 small molecules that represent 23 structural clusters. Using the pyrrole-pyrimidine fragment and N-phenylsubstituted benzamide fragment as the core scaffold, we then designed 17 small-molecule compounds. Using BLI and MST analyses, the compound CYD19 that is predicted to form both hydrogenic and hydrophobic binding interactions with R174 pocket has been eventually identified as a lead compound showing the highest binding affinity with recombinant Snail protein among these compounds. BLI analysis reveals that Snail-R174A mutant protein is 16-fold less potent toward CYD19 than Snail-WT protein. Serial biochemical analyses further show that Snail-WT protein can be efficiently captured by CYD19 and is consequently degraded through the ubiquitin-proteasome pathway, while Snail-R174A mutant protein is essentially resistant to degradation following CYD19 treatment because of its inefficient interaction with CYD19. On the basis of these observations, we conclude that the evolutionarily conserved R174 pocket instead of the ligand-binding domain within Snail protein is critical for its interaction with the compound CYD19.

CBP/p300 HATs have been shown to bind to acetylate and stabilize Snail by repressing its polyubiquitination and subsequent proteasome degradation (18). Note that CYD19 binding to Snail has no impact on the interaction of Snail with importin 1, thus failing to affect importin 1mediated nuclear import of Snail protein. On the basis of Snailimportin 1 cocrystal structure (32), we propose that CYD19 binds to the outer surface of Snailimportin 1 complex and thus impairs the surface contactmediated Snail-CBP/p300 interaction. Following treatment of cancer cells with CYD19, Snail acetylation level is reduced while its levels of phosphorylation and ubiquitination are increased, thereby promoting proteasome degradation of Snail. Two phosphorylation-dependent E3 ligases -TRCP and FBXO11 and one phosphorylation-independent E3 ligase FBXL14 have been identified that mediate Snail degradation (2531). Although we observed that CYD19-treated cells increased the phosphorylation levels of Snail, we could not exclude the possibility that FBXL14 is also responsible for Snail degradation. Snail is abundantly expressed in specific cell lineages during embryonic development, becomes essentially undetectable in normal adult tissues, and is reactivated in cancerous tissues, revealing the spatial and temporal expression pattern of Snail in normal and neoplastic states (7, 2023, 44, 45). Notably, we have observed that CYD19 potently suppresses Snail-driven cancer growth and metastasis without eliciting obvious side toxicity in tumor-bearing mice. This can be attributed to the high selectivity of the compound for targeting Snail protein and the spatial expression pattern of Snail in cancerous tissues versus normal tissues (7, 2023, 44, 45). Since CYD19 specifically interrupts the binding interaction of CBP/p300 with Snail without affecting its enzymic activity, we expect that CYD19 may have a significantly lower toxicity than the enzyme inhibitors of CBP/p300 or deubiquitinases 3, two enzymes that may affect expression of many downstream proteins including Snail protein (18, 43). Notably, Slug, unlike Snail, cannot form a binding interaction with CBP/p300, and there should exist other potential regulator proteins responsible for modulating Slug protein expression. We therefore propose that compound CYD19 does not interrupt Slugs interaction with its regulator proteins and thus loses the ability to affect Slug protein expression. Future work is needed to identify the regulator proteins that are responsible for modulating Slug protein expression.

The tumor suppressor p53 protein is stabilized and activated in response to cellular stress, thereby triggering growth arrest and apoptosis in cancer cells. TP53 is a frequent mutational target in human cancers (~50%), and mutant p53 loses the function of wild-type p53 but functions as an oncoprotein instead (46). The EMT-associated transcription factors, including Slug, Zinc Finger E-Box Binding Homeobox 1 (ZEB1), and Twist, have been reported to indirectly or directly affect p53 function, but the outcome of these interactions has varied (19, 47, 48). Using a MMTV-PyMT BrCa mouse model, we recently find that Snail deletion stabilizes wild-type, but not mutant, p53 and identify Snail as a molecular bypass that suppresses the antiproliferative and proapoptotic effects executed by wild-type p53 (20). Here, we further present in vitro data demonstrating that silencing Snail robustly reduces growth of wild-type p53expressing tumor cells but does not affect growth of tumor cells expressing mutant p53. Snail deficiency in embryonic endothelial cells epigenetically enhances Delta Like Canonical Notch Ligand 4 (DLL4)/Notch signaling but does not affect wild-type p53 protein expression, which consequently represses embryonic vascular remodeling without affecting proliferation or survival of endothelial cells (44). On the basis of these observations, we propose that Snail functions as a key regulator in tumor progression and embryonic vascular development through two distinct mechanisms.

In the present study, we found that compound CYD19 specifically binds to hotspot R174 pocket of Snail protein and thus disrupts the binding interaction of Snail with CBP/p300, which eventually triggers Snail protein degradation through the ubiquitin-proteasome pathway. CYD19 restores Snail-dependent repression of wild-type p53 and thus reduces tumor cell growth and survival. CYD19 also reverses Snail-driven EMT and impairs EMT-associated tumor invasion and metastasis. Given that aberrantly activated Snail is associated with poor prognosis and that more than 50% of patients with cancer express wild-type p53, pharmacologically targeting Snail by CYD19 may exert good therapeutic benefits in patients with cancer especially harboring wild-type p53. Moreover, pharmacologically targeting Snail by CYD19 may also diminish EMT-associated therapeutic resistance and thus sensitizes tumors to low-dose chemotherapy, supporting the rationale for the combination of CYD19 with nontoxic low-dose chemotherapeutics for cancer treatment in the clinic.

Mice were housed under standard specific pathogenfree conditions, and all animal experiments were performed in accordance with protocols approved by the Animal Ethics Committee of China Pharmaceutical University. MMTV-PyMT transgenic mice on FVB background were purchased from the Jackson laboratory (#002374), and the colony was maintained in our laboratory. Male athymic BALB/c nu/nu nude mice were obtained from Qinglongshan Animal Facility (Nanjing, China). The maximal tumor sizes permitted under the approved protocols are 3 cm (length) by 3 cm (width). The clinical study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University, and written informed consents were obtained from each participant before procedure.

The crystal structure of Snail has been reported (32), thus offering an opportunity for structure-based drug design. FTMap, an online computational solvent mapping software (http://ftmap.bu.edu/login.php), was applied to predict the binding hotspots of a protein by using a set of 16 small organic molecules (that is, probes) that vary in size, shape, and polarity. The probes were applied to find favorable positions using an empirical energy function and the CHARMM potential with a continuum electrostatics term. The regions that bind several small organic probe clusters are defined as the predicted hotspots. The residues with the highest number of interactions are defined as the main hotspots. The druggable binding cleft of Snail (PDB ID: 3W5K) (32) mainly consists of three main subpockets: R174 pocket, L178 side pocket, and S257 hydrophobic pocket. For each pocket, a set of chemically related fragments were identified. On the basis of the DrugBank database for virtual screening, an in-house chemical library containing fragment-like molecules was prepared to explore the potential small molecules that form a high-affinity binding interaction with Snail protein. The DrugBank database (http://www.drugbank.ca), which consists of 7736 drug items (including 1584 Food and Drug Administrationapproved small-molecule drugs), was applied for drug screening. For virtual screening, the simulations were applied through the software Schrdinger 2016. Preparation of the crystal structures of Snail (32) was carried out using the Protein Preparation Wizard module. Proper preparation of the ligands was accomplished by the LigPrep module. All other parameters were set to the default values. The cavity that surrounds within 15 of the R174 pocket was defined as the binding site. Top-ranking 200 molecules were picked up for visual observation based on docking scores of Glide_SP module. These molecules were then filtered on the basis of the predefined interaction to the Snail crystal structure. The pyrrole-pyrimidine (DrugBank_431) fragment could form close atomic contacts with residues in both R174 binding pocket and L178 binding pocket. The molecules were further optimized to improve the compounds shape complementarity to the third S257 hydrophobic binding pocket. A small-molecule library featured by hydrophobic fragments was applied to screen the appropriate hit compounds. Both pyrrole-pyrimidine and N-phenylsubstituted benzamide fragments were predicted to match Snail protein: (i) engaging in H bondacceptor interactions with the backbone residue of R174 (hinge binding region), (ii) occupying S257 hydrophobic pocket, and (iii) positioning an aromatic group to make edge-to-face interaction with L178 side pocket. Last, 17 candidate compounds were selected and synthesized for further docking and experimental validation.

Details of the organic synthesis and chemical characterization of the compounds are available upon reasonable request. Compounds used in assays were dissolved in 100% dimethyl sulfoxide and kept as 50 mM stock solutions for in vitro studies.

All cell lines used in the study were purchased from the American Type Culture Collection. Cells were tested for mycoplasma contamination every 1 month, and only mycoplasma-negative cells were used. Wild-type and Snailfl/fl MMTV-PyMT cancer cells were generated and maintained in our laboratory as described previously (20). MMTV-PyMT cancer cells were cultured in Dulbeccos modified Eagles medium (DMEM)/F12 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, #10099-147), EGF (10 ng/ml; PeproTech, #315-09), hydrocortisone (500 ng/ml; Sigma-Aldrich, #H0888), insulin (5 mg/ml; #I9278), cholera toxin (20 ng/ml; #C8052), and 1% penicillin-streptomycin (Thermo Fisher Scientific, #15140122). HEK293T, HCT116, RKO, 4T1, DLD1, SW620, SUM159, and MDA-MB-231 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. For isolation of human BrCa primary cells, freshly isolated breast tumors were rinsed extensively three times in cold phosphate-buffered saline (PBS) supplemented with 1% penicillin-streptomycin and chopped into small fragments (~1 mm3). Tissue fragments were digested into single-cell suspension by incubation in DMEM containing 10% FBS, 1% penicillin-streptomycin, collagenase type 1 (1 mg/ml; Sigma-Aldrich, #C0130), and hyaluronidase (125 U/ml; STEMCELL Technologies, #07919) for 12 to 18 hours at 37C with slow agitation. After incubating for 5 min at room temperature without agitation, the stromal cellenriched supernatant was discarded, and the epithelial cellrich pellets were filtered with a 40-m nylon mesh to remove cell clumps. Tumor epithelial cells were washed three times, resuspended, and cultured in DMEM/F12 medium containing 5% heat-inactivated FBS, EGF (10 ng/ml), hydrocortisone (500 ng/ml), insulin (5 mg/ml), cholera toxin (20 ng/ml), and 1% penicillin-streptomycin.

The binding of various concentrations of CYD19 to Snail-R174A mutant proteins was determined using BLI assays with an Octet RED96 instrument (ForteBio). Briefly, recombinant Snail-R174A mutant proteins were dissolved in PBS. For biotin labeling, EZ-Link NHS-Biotin was incubated for 60 min with proteins at room temperature (1:3 molar ratio of protein to biotin). Desalination was used to remove the excess of biotin. The biotinylated protein was immobilized onto Super Streptavidin (SSA) biosensors for further measurement. A duplicate set of SSA sensors incubated in the buffer without protein were used as negative binding control. The assay was determined in black 96-well plates at different concentrations of CYD19 and PBS as a nonspecific interaction control. The binding event was recorded according to the shift in the interference pattern of the light. Data were then analyzed in ForteBio Data Analysis to calculate the association and dissociation rates using 1:1 binding model, and Kd was represented by the ratio Koff/Kon.

A Monolith NT.115 purchased from NanoTemper Technologies was used for MST assays. The concentration of GFP-tagged Snail recombinant protein was diluted according to the manufacturers instructions. The selected compounds at different concentrations were incubated with GFP-tagged Snail protein for 5 min at room temperature in assay buffer containing 0.05% Tween 20. Thermophoresis was then determined at 25C with 20 to 50% excitation power and 40 to 60% MST power.

Recombinant His-tagged Snail protein was purified from E. coli (BL21) by Ni-NTA affinity chromatography. Cells were lysed in lysis buffer [containing 10 mM MgCl2, 150 mM NaCl, 20 mM tris-HCl (pH 8.0), and 10 mM imidazole] and eluted stepwise using 50, 300, and 500 mM imidazole in wash buffer. The eluted protein was further purified by size exclusion chromatography using a Superdex 75 (Millipore) equilibrated with 20 mM Hepes (pH 7.0), 50 mM NaCl, and 2 mM tris(2-carboxyethyl)phosphine (TCEP). Recombinant GST-tagged CBP-HAT protein was purified from E. coli (BL21) by affinity glutathione-agarose chromatography. Cells were lysed in STE buffer [containing 10 mM tris-HCl buffer, 100 mM NaCl, 1 mM EDTA, 0.01% Triton X-100, and 1 mM dithiothreitol (pH 7.5)] and eluted stepwise using elute buffer [200 mM tris-HCl and 30 mM l-glutathione reduced (pH 8.0)].

For apoptosis analysis, cancer cells were treated with vehicle or various concentrations of CYD19 for 48 hours, and the percentage of apoptotic cells was determined by the fluorescein isothiocyanate annexin V apoptosis detection kit I (BD Biosciences, #556547) according to the manufacturers instructions. The cell apoptosis was analyzed with FlowJo software. For ALDH activity analysis, tumors were chopped into small fragments (around 1 mm3), digested into single-cell suspension by incubation in digestion buffer [0.1% collagenase type 2 (Sigma-Aldrich, #C6885) and deoxyribonuclease I (3 U/ml; Sigma-Aldrich, #D5025)] for 30 min at 37C, and then filtered with a 40-m nylon mesh to remove cell clumps. The single-cell suspensions or cancer cell lines were subjected to serial incubations with an antibody cocktail containing CD31, CD45, and Ter119 (STEMCELL Technologies, #19757C.1); a secondary biotin-labeled antibody cocktail (STEMCELL Technologies, #19153); and magnetic beads (15 min each) on ice (STEMCELL Technologies, #19150). The unbound cells were collected, and the bound cells were discarded. Cells were washed extensively and subjected to ALDH activity assay using a kit from STEMCELL Technologies according to the manufacturers instructions. For each sample, half of the cells were treated with diethylaminobenzaldehyde (DEAB), and the other half were incubated with an activated ALDEFLUOR reagent. Gating was established using fixable viability dye exclusion for viability, and DEAB-treated cells were used to define negative gates. Flow cytometry data were collected with a MACSQuant flow cytometer (BD Biosciences), and analysis was conducted using FlowJo software.

Cells were treated with vehicle or various concentrations of CYD19 for 48 hours, and equal numbers (2 105 cells per well) of the cells were seeded in FBS-free DMEM culture medium in the presence of vehicle or various concentrations of CYD19 in the upper chambers of transwell inserts with an 8-m pore size (BD Biosciences, #354480). The lower chambers were filled with 1 ml of complete medium supplemented with 10% FBS. Cells were allowed to invade the bottom chamber for 12 or 18 hours. Noninvading cells in the upper surface were removed, and invaded cells on the lower surface were fixed with 90% methanol and stained with 0.1% crystal violet for 5 min. The stained cells were photographed and quantified.

Cell proliferation was measured by a CCK-8 kit (Yeasen, #40203ES60) according to the manufacturers instructions. Briefly, cells were seeded in 96-well plates at 4 103 cells per well in culture medium supplemented with 10% FBS. Cells were allowed to adhere for 12 hours and then treated with vehicle or various concentrations of CYD19 for another 48 hours. Cell proliferation was measured, and absorbance intensity was determined with a Molecular Devices microplate reader at 450 nm.

Single-cell suspensions of 1 106 HCT116 cells in 50 l of diluted Matrigel (1:1; BD Biosciences, #356234) were injected subcutaneously into the dorsal flank of male nude mice at 6 to 8 weeks of age. Mice were randomized into three groups until their tumors reached a size of approximately 100 mm3. Mice were then treated with vehicle [formulated in ethanol/cremophor/water at 10:10:80 (v/v/v)], CYD19 (30 mg/kg), or CYD19 (50 mg/kg) via intraperitoneal injection for two consecutive weeks. Tumor volumes were measured every 1 day using the formula length width2/6. At the end point of treatment, mice were euthanized, and tumors and key organs were dissected, photographed, and weighed. Tissues were either fixed in 4% paraformaldehyde (PFA) for immunohistochemical and histological analyses or snap-frozen in liquid N2 and stored at 80C for immunoblot analysis. In some experiments, 1 106 control-shRNAexpressing cells and 2 106 (or 1 106) Snail-shRNA2expressing cells were used to form tumor xenografts in comparable sizes. For liver metastasis assay, a left subcostal surgical incision was created, and 1 106 GFP-labeled HCT116 cells were intrasplenically injected into the spleen of male nude mice (6 to 8 weeks of age). Mice were then treated intraperitoneally with vehicle or CYD19 (30 mg/kg) for three consecutive weeks starting from the third day after surgery, and livers were then harvested for analysis.

MMTV-PyMT female mice bearing primary tumors with an average volume of 400 mm3 were divided into two groups and intraperitoneally injected with vehicle or CYD19 (30 mg/kg) for 25 consecutive days. Tumors were measured every 1 day using a caliper, and the volumes were calculated using the formula length width2/6. At the end of treatment point, mice were euthanized, and tumors, lungs, and key organs were dissected for further use.

p3XFLAG-Snail-WT, p3XFLAG-Slug-WT, and pLKO.1-ms.p53-shRNA vectors were generated and used as described previously (20, 21). pET23a(+)-His-Snail-WT, His-Snail-R174A, p3XFLAG-Snail-R174A, FLAG-Snail-K147R/K186R, pLKO.1-hu.p53-shRNA (targeting mRNA sequence from ATG, 176 to 196), pLKO.1-Snail-shRNA1 (468 to 486), pLKO.1-Snail-shRNA2 (1515 to 1533), pCDN3.1-GST-Snail-WT-GFP, and pCDN3.1-GST-Snail-R174A-GFP vectors were generated by GenScript Biotech Inc. (Nanjing, China). HA-ubiquitin (#18712), GST-CBP-HAT (#21093), pLKO.1-TRC (#10879), psPAX2 (#12260), and pMD2.G (#12259) were purchased from Addgene. To produce pLKO.1 lentiviral particles, HEK293T cells were cotransfected with pLKO.1-shRNA, psPAX2, and pMD2.G at a ratio of 4:3:1 using Lipofectamine 2000 Reagent (Invitrogen, #11668027). Cells were fed with fresh medium 24 hours after transfection, and conditioned medium containing viral particles was harvested 48 and 72 hours after transfection. Viral particles were stored at 80C for further use or immediately used. For lentiviral infection, target cells were incubated with a mixture of conditioned medium (containing viral particles) and culture medium at a ratio of 1:1 for 24 hours in the presence of polybrene (8 g/ml; Sigma-Aldrich, #H9268). Cells were reinfected with viral particles for another 24 hours and harvested for further use. For adenoviral infection, cells were infected with complete medium supplemented with adeno-Gal or adeno-Cre viral particles for 24 hours, refed with fresh medium containing viral particles, and further cultured for another 24 hours. Cells were collected for further use.

For immunoblot analysis, cells were lysed in RIPA lysis buffer (Thermo Fisher Scientific, #89901) supplemented with protease inhibitor cocktail (#87786), and total cell lysates were collected for further uses. In some experiments, nuclear and (or) cytoplasmic proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, #78833) according to the manufacturers instructions. The cell lysates were subjected to immunoblot assay using primary antibodies against Snail (#3895; 1:1000), Slug (#9585; 1:1000), Cyt-c (#4280; 1:1000), caspase 3 (#9665; 1:1000), caspase 9 (#9508; 1:1000), cleaved caspase 3 (#9661; 1:500), cleaved caspase 9 (#52873; 1:500), p53 (#2524; 1:1,000), Bax (#2772; 1:1000), Puma (#24633; 1:1000), pan-acetyl-Lys (pan-AcK; #9441; 1:500), CBP (#7389; 1:1000), p300 (#70088; 1:1000), ubiquitin (#3936; 1:1000), HDAC1 (#5356; 1:1000), vimentin (#5741; 1:1000), histone H3 (#4499; 1:2000), HA-tag (#3724S; 1:2000), -tubulin (#2128; 1:2000) (all from Cell Signaling Technology), E-cadherin (BD Biosciences, #610181; 1:5000), p21 (#ab7903; 1: 200), MDM2 (#ab16895; 1:500), phospho-Ser/Thr (#ab17464; 1:1000) (all from Abcam), FLAG (#F3165; 1:1000), importin (Thermo Fisher Scientific, #MA3-070), and -actin (#A5316; 1:10,000) (both from Sigma-Aldrich), followed by incubation with appropriate horseradish peroxidase (HRP)conjugated secondary antibodies. Blots were detected by enhanced chemiluminescence (Thermo Fisher Scientific, #32106). For IP assay, cells were lysed in IP lysis buffer [50 mM tris-HCl, 150 mM NaCl, 1 mM EDTA, and 1% NP-40 (pH 7.4)] containing protease inhibitor cocktail for 20 min on ice. The cell lysates were sonicated, clarified, and incubated with antibodies against control immunoglobulin G, FLAG (1:100), Snail (1:100), HDAC1 (1:100), or p53 (1:100), followed by incubation with precleared Protein A/G agarose beads (Santa Cruz Biotechnology, #sc-2003). The immunocomplexes were subjected to immunoblot analysis using antibodies against ubiquitin, HA, pan-AcK, phospho-Ser/Thr, CBP, p300, FLAG, or p53. For His pulldown assay, GST-CBP-HAT, His-Snail-WT, and His-Snail-R174A mutant recombinant proteins were expressed and purified from E. coli (BL21). The bead-bound His-tagged proteins were preincubated with various concentrations of CYD19 for 15 min at 4C on a rotator, and eluted GST-CBP-HAT protein was added to the reaction mixtures and incubated for another 2 hours. The beads were collected, extensively washed, eluted, electrophoresed, and subjected to Coomassie staining. In some experiments, His-Snail-WT and His-Snail-R174A mutant recombinant proteins were immobilized to Ni-NTA agarose and incubated with whole lysates of HEK293T cells for 3 hours (34). After extensive washes, the bound proteins were eluted with SDS sample buffer, resolved by SDSpolyacrylamide gel electrophoresis, and analyzed by immunoblotting.

Total RNAs were extracted and reversely transcribed using TRIzol reagent (Invitrogen, #15596018) and the PrimeScript RT reagent kit (Takara, #RR037A), respectively, according to the manufacturers instructions. Quantitative polymerase chain reaction (qPCR) was performed on an Applied Biosystems QuantStudio 3 qPCR (Thermo Fisher Scientific) using the SYBR Green PCR Master Mix (Takara, #RR820A), and relative mRNA expressions were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). qPCR primers for amplifying the indicated genes are used as follows: GAPDH, 5-CACCGTCAAGGCTGAGAACGG-3/5-GACTCCACGACGTACTCAGCC-3; Gapdh, 5-CCCTGGCCAAGGTCATCCATG-3/5-TGATGTTCTGGGCAGCCCCAC-3; SNAI1, 5-TCGGAAGCCTAACTACAGCGA-3/5-AGATGAGCATTGGCAG CGAG-3; Snai1, 5-AAGATGCACATCCGAAGC-3/5-ATCTCTTCACATCCGAGTGG-3; TP53, 5-GTTCCGAGAGCTGAATGAGG-3/5-TCTGAGTCAGGCCCTTCTGT-3; Trp53, 5-AGCTCCCTCTGAGCCAGGAGA-3/5-TCCTCAACATCCTGGGGCAGC-3; CDKN1A, 5-TCTTGTACCCTTGTGCCTCG-3/5-GTTCCTGTGGGCGGATTAGG-3; Cdkn1a, 5-TGCCGTTGTCTCTTCGGTCCC-3/5-TAGACCTTGGGCAGCCCTAGG-3; MDM2, 5-GTGAATCTACAGGGACGCCATC-3/5-CTGATCCAACCAATCACCTGA A-3; Mdm2, 5-CGCTGAGTGAGAGCAGACGTC-3/5-GCTCCCCAGGTAGCTCATCTG-3; CDH1, 5-GTCAGTTCAGACTCCAGCCCG-3/5-CGTGTAGCTCTCGGCGTCAA-3; Cdh1, 5-GAAGTCCATGGGGCACCACCA-3/5-CTGAGACCTGGGTACACGCTG-3; CDH2, 5-CGACCCAAACAGCAACGACGC-3/5-CGGGTGCTGAATTCCCTTGGC-3; Cdh2, 5-TGTGCACGAAGGACAGCCCCT-3/5-CCTGCTCTGCAGTGAGAGGGA-3; VIM, 5-GCCCTAGACGAACTGGGTC-3/5-GGCTGCAACTGCCTAATGAG-3; Vim, 5-AGCGTGGCTGCCAAGAACCTC-3/5-GCAGGGCATCGTGTTCCGGT-3; FN1, 5-CATCCCTGACCTGCTTCCTGG-3/5-CTGTACCCTGTGATGGGAGCC-3; Fn1, 5-GGGTGACACTTATGAGCGCCC-3/5-GACTGACCCCCTTCATGGCAG-3; ERCC1, 5-GCATCATTGTGAGCCCTCGGC-3/5-GTGCAGGTTGTGGTAGCGGAG-3; Ercc1, 5-CCACAACCTCCATCCAGACTA-3/5-GCTTCTGCT CATACGCCTTGTA-3; CCL2, 5-AGTCTCTGCCGCCCTTCTGTG-3/5-CGCGAGCCTCTGCACTGAGAT-3; Ccl2, 5-CTGTCATGCTTCTGGGCCTGC-3/5-CAGC AGGTGAGTGGGGCGTTA-3; CCL5, 5-CAGCCCTCGCTGTCATCCTCA-3/5-GTGGGCGGGCAATGTAGGCAA-3; Ccl5, 5-AGCAATGACAGGGAAGCTATAC-3/5-AGGACTCTGAGACAGCACAT-3; TNFA, 5-GATTCTGAGCAAAATAGCCAGCA-3/5-GGCTTCCTTCTTGTTGTGTGT-3; Tnfa, 5-CCCTCACACTCAGATCATCTTCT-3/5-GCTACGACGACGTGGGCTACA-3; IL8, 5-ACTGAGAGTGATTGAGAGTGGAC-3/5-AACCCTCTGCACCCAGTTTTC-3; and Il8, 5-TGTGAGGCTGCAGTTCTGGCAAG-3/5-GGGTGGAAAGGTGTGGAATGCGT-3. The specificity of the PCR amplification was validated by the presence of a single peak in the melting curve analyses.

For histological assays, tumor and normal tissues were fixed in 4% PFA and embedded in paraffin. The embedded tissues were sectioned at 5 m, deparaffinized, and subjected to hematoxylin and eosin (H&E) staining according to the manufacturers instructions. For immunocytochemical analysis, cells were grown on chamber slides, fixed with 4% PFA, and incubated with primary antibodies against E-cadherin (1:1000), vimentin (1:200), Snail (1:200), or p53 (1:200), followed by incubation with goat anti-mouse and anti-rabbit Alexa secondary antibodies (all from Thermo Fisher Scientific, 1:300). Cells were then counter stained with 4it6-diamidino-2-phenylindole (DAPI), and images were acquired on a Zeiss LSM 800 microscope. For immunohistochemical analysis, deparaffinized sections were rehydrated and subjected to antigen heat retrieval with citric acidbased Antigen Unmasking Solution (pH 6.0; Vector Laboratories, #H-3300). The sections were incubated in 0.3% H2O2 (in PBS) and then in blocking buffer (5% goat serum in PBS). The sections were then incubated in blocking buffer containing primary antibodies against Ki67 (Abcam, #ab15580; 1:1000), cleaved caspase 3 (1:100), phosphohistone H3 (Cell Signaling Technology, #9849; 1:200), and Snail (1:100), followed by incubation with biotinylated goat anti-mouse (Vector Laboratories, #BA-9200; 1:200) and goat anti-rabbit (Vector Laboratories, #BA-1000; 1:200) secondary antibodies. Standard avidin-biotin complex (ABC) kit (Vector Laboratories, #PK-6101) and 3,3-diaminobenzidine (DAB) HRP Substrate Kit (Vector Laboratories, #SK-4105) were used for the detection of HRP activity. Slides were counterstained with hematoxylin, dehydrated, and mounted. For immunofluorescence analysis, rehydrated tissues were incubated in blocking buffer containing primary antibodies against E-cadherin (1:400), vimentin (1:200), F4/80 (Thermo Fisher Scientific, #14-4801-81; 1:100), CD31 (Dianova, #DIA310; 1:100), or p53 (1:800), followed by incubation with goat anti-mouse, anti-rabbit, and anti-rat Alexa Fluor secondary antibodies (all from Thermo Fisher Scientific; 1:300). The sections were then counter stained with DAPI, and images were acquired on a Zeiss LSM 800 microscope.

Data were presented as means SD. Statistical analysis was carried out as described in each corresponding figure legend, and sample size were shown in each figure legend.

Differences were evaluated by Mann-Whitney U test, unpaired two-sided Students t test, or one-way analysis of variance (ANOVA) with Tukeys post hoc test. P < 0.05 was considered statistically significant.

Acknowledgments: Funding: This research was supported by grants from the National Natural Science Foundation of China (81973363, 81973188, 81803033, 81572745, and 81603134), the Jiangsu Province Natural Science Funds for Distinguished Young Scholar (BK20170029), the Jiangsu Province Natural Science Funds for Young Scholar (BK20180573 and BK20160758), the Jiangsu Province Innovative Research Program, the State Key Laboratory of Natural Medicines of China Pharmaceutical University (SKLNMZZCX201808), and the Double First-Class University project (CPU2018GF02). Author contributions: Z.-Q.W. and T.L. conceived the project, designed experiments, interpreted data, and wrote the manuscript. H.-M.L., Y.-R.B., Y.L., and R.F. performed experiments and interpreted data with the help from W.-C.L., N.J., Y.X., and B.-X.R. Y.-D.C. designed and synthesized the compounds. S.W. and H.X. provided fresh human breast tumor samples. Competing interests: T.L., Y.-D.C., Z.-Q.W., and H.-M.L. are inventors on three pending patents (no. PCT/CN2019/102696, 27 August 2019; no. 201811623605.0, 28 December 2018; and no. 201811212157.5, 23 October 2018) related to this work. Z.-Q.W., T.L., Y.-D.C., R.F., H.-M.L., and Y.L. are inventors on a pending patent (no. 202010050205.6, 16 January 2020) related to this work. The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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A potent CBP/p300-Snail interaction inhibitor suppresses tumor growth and metastasis in wild-type p53-expressing cancer - Science Advances

HealthDay Reports: More Good News on Remdesivir’s Power to Treat COVID-19 – HealthDay Coronavirus Liveblog

The U.S. Food and Drug Administration (FDA) authorized the first at-home sample collection test for COVID-19 today.

The FDA re-issued the emergency use authorization (EUA) for LabCorp's COVID-19 RT-PCR Test, which will now be permitted to test the at-home samples people take using LabCorp's Pixel by LabCorp COVID-19 Test home collection kit.

The at-home sample collection kit includes designated nasal swabs and saline. After someone takes a sample from their nose with the special nasal swab, they mail the sample inside of an insulated package to a LabCorp lab for testing.

According to a tweet from LabCorp, kits will initially be made available to frontline healthcare workers and first responders. Kits will be available to consumers in most states, with a doctor's order, in the coming weeks, according to the FDA release.

This authorization is only for the LabCorp COVID-19 RT-PCR Test for at-home collection of nasal swab specimens using the Pixel by LabCorp COVID-19 home collection kit. This is not a general authorization for at-home COVID-19 sample collection tests.

Read the full press release.

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HealthDay Reports: More Good News on Remdesivir's Power to Treat COVID-19 - HealthDay Coronavirus Liveblog

Baby Hair or Breakage: How to Tell the Difference – Allure

Christine Shaver, a dermatologist at Bernstein Medical Center for Hair Loss in New York City reminds us that "it's important to differentiate genetic 'baby hair' from broken hair and miniaturized hair as they all can look similar. If hair is broken, then styling practices need to be reassessed as heat, chemical, and over-styling can cause more brittleness and cracking," she says. "Miniaturization is the shrinking of hair in genetic hair loss and can occur in both men and women." Thankfully, this can be reversed to some extent with medications liketopical 5 percent minoxidil ( a.k.a Rogaine). In contrast to baby hairs, which are stable, the amount of miniaturized hair on a persons head will tend to increase over time.

Additionally, Shaver adds that platelet-rich plasma (PRP) scalp injections can help women reverse miniaturizing hairs. "Both these therapies need to be maintained for their benefit to continue as the hair is always growing and cycling," she shares. "While Rogaine can be applied at home, PRP injections require periodic in-office treatments with your dermatologist."

Also watch for changes in texture, Hill notes. "The changes in the texture around the hairline are individual and impacted by your genetic makeup, hair type, texture, and hairstyling habits. Extensive tension, overuse of heat and styling tools, as well styling products can impact the texture as well as cause those finer hairs to grow in coarser and thicker."

Pregnancy can create baby (-looking) hairs temporarily. "Following pregnancy, there is often a large shed period of hair as the plentiful pregnancy hormones decrease in the body," explains Shaver. "Following this shed, hair initially grows back more wispy and fine and then over time strengthens, darkens, and returns to normal."

Ah, the golden question: Do you actually have baby hair or just breakage? Emmanuel weighs in: "If it is breakage, the hair along the edge of your hairline will feel dry, it will also be uneven in length and brittle. You will also notice split, frayed hairs," she says.

"If it is hair loss, you may notice a smooth, shiny surface with little or no hair this may be due to traction, pulling your hair too tight, or overusing hot tools really close to the scalp. The scalp may look red and inflamed as well," Emmanuel clarifies.

So what to do if your baby hairs are really breakage hairs? First, cut back on heat styling. "These hair-care practices weaken the strength of bonding among hairs and can create brittle nodes which lead to premature cracking and breakage," Shaver explains. But if you absolutely cannot help yourself, "You should always try to use the lowest temperature possible when styling hair to avoid additional trauma."

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Baby Hair or Breakage: How to Tell the Difference - Allure

Global Stem Cells Market with Focus on Clinical Therapies, 2020-2030 – Presents a Detailed Clinical Trial Analysis on More Than 540 Completed, Ongoing…

Dublin, April 22, 2020 (GLOBE NEWSWIRE) -- The "Global Stem Cells Market: Focus on Clinical Therapies, 2020 - 2030" report has been added to ResearchAndMarkets.com's offering.

Stem cell therapies are viable alternatives to conventional treatments with substantial therapeutic potential; market opportunities are huge, as multiple product candidates are expected to be approved over the coming decade

This report features an extensive study of the current market landscape, offering an informed opinion on the likely adoption of these therapeutics over the next decade. The report features an in-depth analysis, highlighting the capabilities of various stakeholders engaged in this domain.

One of the key objectives of the report was to estimate the existing market size and identify the future opportunity for stem cell therapies over the next decade. The research, analyses and insights presented in this report are based on revenue generation trends based on the sales of approved stem cell therapies.

The report also features the likely distribution of the current and forecasted opportunity within stem cell therapies market across:

According to the WHO, in 2020, nearly 75% of fatalities that are estimated to be reported across the globe, are likely to be caused due to diseases, such as chronic stroke disease, diabetes, cancer, heart disease, and certain mental health conditions. In addition, as per a report published by the Center for Managing Chronic Disease, University of Michigan, more than 50% of the global population was estimated to be living with some form of chronic illness.

The rising prevalence of these clinical conditions has resulted in dire need for the identification of effective therapeutic options. Despite advances in healthcare, there is an evident lack of permanent treatment solutions for many aforementioned diseases. Majority of the currently available treatment options focus on palliative care and are incapable of addressing the root cause of disease, therefore, are unable to improve quality of life of patients.

Since the first bone marrow transplant in 1950s, these regenerative cellular therapies have garnered significant attention within the biopharmaceutical industry. Over the years, advances in the field of cell biology and regenerative medicine have led to the development of a number of stem cell therapies, which are believed to possess the potential to address several unmet needs related to the treatment of a wide range of disease conditions. Stem cell-based treatments are known for their ability to replace damaged cells and tissues, thereby, curing affiliated disease symptoms.

In fact, such interventions have also been shown to enable cell regeneration, restoring normal functioning capabilities in affected organs. Till date, nearly 25 stem cell-based therapies have been approved; popular examples include EYE-01M (2019), Alofisel (2018), MACI (2016), Stempeucel (2016) and Strimvelis (2016). Further, several such therapies are presently being evaluated across 540 active clinical trials worldwide. This emerging field of research has received significant capital investments from several big pharma companies and venture capital funds / investors.

Despite the associated optimism, the growth of this market is stunted by a number of development and manufacturing related challenges, primarily revolving around the limited availability of the required expertise and infrastructure to produce such products. However, the availability of innovative technology platforms, large target patient population, encouraging clinical trial results, and extensive government support, the stem cell therapies market is poised to grow in the long-term.

In addition to other elements, the study includes:

To account for the uncertainties associated with the manufacturing of stem cell therapies and to add robustness to our model, we have provided three forecast scenarios, portraying the conservative, base and optimistic tracks of the market's evolution.

The opinions and insights presented in the report were influenced by discussions held with senior stakeholders in the industry.

The report features detailed transcripts of interviews held with the following industry stakeholders:

Key Topics Covered

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Global Stem Cells Market with Focus on Clinical Therapies, 2020-2030 - Presents a Detailed Clinical Trial Analysis on More Than 540 Completed, Ongoing...

Antibodies of COVID-19 survivors could hold the key to potential treatment – The Hudson Reporter

A digital scientific rendering of antibodies attacking COVID-19.

A digital scientific rendering of antibodies attacking COVID-19.

As the COVID-19 pandemic rages across the nation, researchers are searching for cures, vaccines and other treatments to prevent or kill the virus. In Hudson County, some local researchers are searching for anything to help stop the disease.

Researchers and clinical experts at Hackensack Meridian Health (HMH) are examining the blood of COVID-19 survivors for a potential treatment for current COVID-19 patients.HMH operates Palisades Medical Center in North Bergen, where staff has been working to save patients lives.

Recently, HMH was approved to begin recruiting recovered and recovering COVID-19 patients to assess their blood and test it for antibodies in response to the virus. These antibodies may help other patients who are infected with COVID-19.

Patients with promising antibodies will be asked to come back to donate an additional blood sample, according to a statement from Palisades Medical Center.

The research will scrutinize the antibodies within the serum of the surviving patients in an attempt to discover more about the disease. The findings could perhaps develop new ways to fight the virus.

If you were a COVID-19 patient, treated at Palisades Medical Center or not, and were officially diagnosed with the virus and have recovered, the hospital is looking for your help.

A race against time

Dr. Michele Donato, chief of stem cell transplantation and cellular therapy at Hackensack University Medical Center John Theurer Cancer Center, is leading the potential treatment part of the work.

It really is a race against time, Donato said. People are getting sick right now, and we are working night and day to save as many lives as possible.

Convalescent plasma treatments have previously been used to fight other viral outbreaks, including those of severe acute respiratory syndrome (SARS), according to HMH.

The researchers will first seek a small blood sample from those recovered or recovering patients who volunteer for the study, with the goal of finding those who developed the highest levels of targeted antibodies in response to the virus.

Those patients with the highest level of antibodies will be asked to return to provide a larger plasma donation, which may be utilized to infuse into very sick COVID-19 patients.

Hoping to save lives

Taking part in this work will be doctors from Hackensack Meridian John Theurer Cancer Center including Donato, who are experts in stem cell transplantation and cellular therapy.

They will be joined by scientists from the Hackensack Meridian Health Center for Discovery and Innovation (CDI), who have developed a test to assess the presence and levels of the antibodies in the blood samples. The CDI also previously developed a diagnostic test for detecting the virus which has been used to diagnose more than a thousand patients so far in the HMH network, according to a press release.

This is applied science in real-time, as this pandemic continues to spread, said David Perlin, chief scientific officer of the CDI. Our scientists at the CDI are responding to needs, and were hoping to save lives.

Robert Garrett, CEO of Hackensack Meridian Health, said he was so proud of his staffs robust and innovative response to this unprecedented global challenge.

Our scientists have been at the forefront of the latest innovations, including developing our own test and taking part in clinical trials of antiviral drugs, Garrett said. Now theyre taking a leadership role in this advanced antibody work, which could prove to be a breakthrough.

The patients sought for the studies will be between the ages of 18 and 60, and have a prior laboratory diagnosis of COVID-19. They must also be at least 14 days without symptoms, according to the guidelines.

Potential donors can fill out an online form available online for the initial screening.

For updates on this and other stories, check http://www.hudsonreporter.com and follow us on Twitter @hudson_reporter. Daniel Israel can be reached at disrael@hudsonreporter.com.

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Antibodies of COVID-19 survivors could hold the key to potential treatment - The Hudson Reporter