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Table S1 shows the cell viability inhibitory rate (%) in SCCOHT cell lines vs other cell lines in the epigenetic drug screen. Table S2 shows the effects of Quisinostat on the proteome of BIN67 cells after treatment for 24 or 72 hours. Table S3 shows the significantly altered proteins by Quisinostat treatment for 24 hours in BIN67 cells. Table S4 shows the significantly altered proteins by Quisinostat treatment for 72 hours in BIN67 cells. Table S5 shows the biological functions that were significantly affected by Quisinostat treatment for 72 hours in BIN67 cells. Table S6 shows the proteins that were significantly regulated by SMARCA4 re-expression in BIN67 cells. Table S7 shows the biological functions that were significantly affected by SMARCA4 re-expression in BIN67 cells. Table S8 shows the proteins that were significantly regulated by both Quisinostat treatment (72 hr) and SMARCA4 re-expression in BIN67 cells. Table S9 shows the biological functions that were significantly affected by both Quisinostat and SMARCA4 re-expression in BIN67 cells.

Fig.S1 shows that SCCOHT cells are sensitive to HDAC inhibition. Fig. S2 shows that SCCOHT cells are sensitive to HDAC inhibition or depletion. Fig.S3 shows that SMARCA4/A2 dual deficiency is not associated with hypersensitivity to HDAC inhibitors in lung and pancreatic cancer cell lines. Fig.S4 shows that pan-HDAC inhibitors induce cell morphology changes in SCCOHT cells. Fig.S5 shows that SMARCA2 activation is partially required for the hypersensitivity of SCCOHT cells to HDAC inhibition. Fig. S6 shows the effect of quisinostat on mouse body weights in BIN67 mouse xenograft models. Fig.S7 shows the synergistic effects between HDAC inhibitors and EZH2 inhibitors in SWI/SNF deficiency-driven tumor cells. Fig. S8 shows that depletion of HDAC2 increased sensitivity of BIN67 cells to EPZ-6438 treatment. Fig.S9 shows the synergistic effects between HDAC inhibitors and EZH2 inhibitors in SCCOHT cells. Fig.S10 shows the effect of combined treatment of EPZ-6438 and quisinostat on mouse body weight and survival.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but extremely lethal malignancy that mainly impacts young women. SCCOHT is characterized by a diploid genome with loss of SMARCA4 and lack of SMARCA2 expression, two mutually exclusive ATPases of the SWI/SNF chromatin-remodeling complex. We and others have identified the histone methyltransferase EZH2 as a promising therapeutic target for SCCOHT, suggesting that SCCOHT cells depend on the alternation of epigenetic pathways for survival. In this study, we found that SCCOHT cells were more sensitive to pan-HDAC inhibitors compared with other ovarian cancer lines or immortalized cell lines tested. Pan-HDAC inhibitors, such as quisinostat, reversed the expression of a group of proteins that were deregulated in SCCOHT cells due to SMARCA4 loss, leading to growth arrest, apoptosis, and differentiation in vitro and suppressed tumor growth of xenografted tumors of SCCOHT cells. Moreover, combined treatment of HDAC inhibitors and EZH2 inhibitors at sublethal doses synergistically induced histone H3K27 acetylation and target gene expression, leading to rapid induction of apoptosis and growth suppression of SCCOHT cells and xenografted tumors. Therefore, our preclinical study highlighted the therapeutic potential of combined treatment of HDAC inhibitors with EZH2 catalytic inhibitors to treat SCCOHT.

Supplementary Figure from The COX-2–PGE2 Pathway Promotes Tumor Evasion in Colorectal Adenomas

The mechanisms underlying the regulation of a checkpoint receptor, PD-1, in tumor-infiltrating immune cells during the development of colorectal cancer are not fully understood. Here we demonstrate that COX-2–derived PGE2, an inflammatory mediator and tumor promoter, induces PD-1 expression by enhancing NFκB's binding to the PD-1 promoter via an EP4–PI3K–Akt signaling pathway in both CD8+ T cells and macrophages. Moreover, PGE2 suppresses CD8+ T-cell proliferation and cytotoxicity against tumor cells and impairs macrophage phagocytosis of cancer cells via an EP4–PI3K–Akt–NFκB–PD-1 signaling pathway. In contrast, inhibiting the COX-2–PGE2–EP4 pathway increases intestinal CD8+ T-cell activation and proliferation and enhances intestinal macrophage phagocytosis of carcinoma cells accompanied by reduction of PD-1 expression in intestinal CD8+ T cells and macrophages in ApcMin/+ mice. PD-1 expression correlates well with COX-2 levels in human colorectal cancer specimens. Both elevated PD-1 and COX-2 are associated with poorer overall survival in patients with colorectal cancer. Our results uncover a novel role of PGE2 in tumor immune evasion. They may provide the rationale for developing new therapeutic approaches to subvert this process by targeting immune checkpoint pathways using EP4 antagonists. In addition, our findings reveal a novel mechanism explaining how NSAIDs reduce colorectal cancer risk by suppressing tumor immune evasion.Prevention Relevance:These findings provide a potential explanation underlying the chemopreventive effect of NSAIDs on reducing colorectal cancer incidence during premalignancy and provide a rationale for developing EP4 antagonists for colorectal cancer prevention and treatment. Simply targeting PGE2 signaling alone may be efficacious in colorectal cancer prevention and treatment, avoiding side effects associated with NSAIDs.

Supplemental Table 5. SCCOHT-1 ABPP data; Supplemental Table 6. BIN67 ABPP data.

IC50 summary of validated drug hits in SCCOHT cell lines.

Purpose: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits SMARCA4 and SMARCA2. A great need exists for effective targeted therapies for SCCOHT.Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT.Results: The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%.Conclusions: Ponatinib is an effective agent for SMARCA4-mutant SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted. Clin Cancer Res; 24(8); 1932–43. ©2018 AACR.

Supplemental Figure 1. COV434-pIND20-Brg1 SMARCA4 induction optimization; Supplemental Figure 2. Kinase hits from siRNA screen mapped to kinome dendrogram; Supplemental Figure 3. ClueGo analysis of validated siRNA screen hits.

Supplemental Materials and Methods

Sustainable HIV remission after antiretroviral therapy (ART) withdrawal, or post-treatment control (PTC), remains a top priority for HIV treatment. We observed surprising PTC in an MHC-haplomatched cohort of MHC-M3+ SIVmac239+ Mauritian cynomolgus macaques (MCMs) initiated on ART at two weeks post-infection (wpi). For six months after ART withdrawal, we observed undetectable or transient viremia in seven of eight MCMs. In vivo depletion of CD8α+ cells induced rebound in all animals, indicating the PTC was mediated, at least in part, by CD8α+ cells. We found that MCMs had smaller acute viral reservoirs than a cohort of identically infected rhesus macaques, a population that rarely develops PTC. The mechanisms by which unusually small viral reservoirs and CD8α+ cell-mediated virus suppression enable PTC can be investigated using this MHC-haplomatched MCM model. Further, defining the immunologic mechanisms that engender PTC in this model may identify therapeutic targets for inducing durable HIV remission in humans.

The mechanisms underlying the regulation of a checkpoint receptor, PD-1, in tumor-infiltrating immune cells during the development of colorectal cancer are not fully understood. Here we demonstrate that COX-2–derived PGE2, an inflammatory mediator and tumor promoter, induces PD-1 expression by enhancing NFκB's binding to the PD-1 promoter via an EP4–PI3K–Akt signaling pathway in both CD8+ T cells and macrophages. Moreover, PGE2 suppresses CD8+ T-cell proliferation and cytotoxicity against tumor cells and impairs macrophage phagocytosis of cancer cells via an EP4–PI3K–Akt–NFκB–PD-1 signaling pathway. In contrast, inhibiting the COX-2–PGE2–EP4 pathway increases intestinal CD8+ T-cell activation and proliferation and enhances intestinal macrophage phagocytosis of carcinoma cells accompanied by reduction of PD-1 expression in intestinal CD8+ T cells and macrophages in ApcMin/+ mice. PD-1 expression correlates well with COX-2 levels in human colorectal cancer specimens. Both elevated PD-1 and COX-2 are associated with poorer overall survival in patients with colorectal cancer. Our results uncover a novel role of PGE2 in tumor immune evasion. They may provide the rationale for developing new therapeutic approaches to subvert this process by targeting immune checkpoint pathways using EP4 antagonists. In addition, our findings reveal a novel mechanism explaining how NSAIDs reduce colorectal cancer risk by suppressing tumor immune evasion. Prevention Relevance: These findings provide a potential explanation underlying the chemopreventive effect of NSAIDs on reducing colorectal cancer incidence during premalignancy and provide a rationale for developing EP4 antagonists for colorectal cancer prevention and treatment. Simply targeting PGE2 signaling alone may be efficacious in colorectal cancer prevention and treatment, avoiding side effects associated with NSAIDs.