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Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated. Gene expression analyses were performed on tumor samples from patients undergoing immunotherapy for metastatic melanoma. Finally, the effect of inhibiting the top targets on the efficacy of immunotherapy was investigated in multiple preclinical models. Aurora kinase was identified as a mediator of melanoma cell resistance to T-cell-mediated cytotoxicity in both complementary screens. Aurora kinase inhibitors were validated to synergize with T-cell-mediated cytotoxicity in vitro. The Aurora kinase inhibition-mediated sensitivity to T-cell cytotoxicity was shown to be partially driven by p21-mediated induction of cellular senescence. The expression levels of Aurora kinase and related proteins were inversely correlated with immune infiltration, response to immunotherapy and survival in melanoma patients. Aurora kinase inhibition showed variable responses in combination with immunotherapy in vivo, suggesting its activity is modified by other factors in the tumor microenvironment. These data suggest that Aurora kinase inhibition enhances T-cell cytotoxicity in vitro and can potentiate antitumor immunity in vivo in some but not all settings. Further studies are required to determine the mechanism of primary resistance to this therapeutic intervention. Electronic supplementary material The online version of this article (10.1007/s00262-020-02748-9) contains supplementary material, which is available to authorized users.

Purpose: Cancer immunotherapy has shown promising clinical outcomes in many patients. However, some patients still fail to respond, and new strategies are needed to overcome resistance. The purpose of this study was to identify novel genes and understand the mechanisms that confer resistance to cancer immunotherapy. Experimental Design: To identify genes mediating resistance to T-cell killing, we performed an open reading frame (ORF) screen of a kinome library to study whether overexpression of a gene in patient-derived melanoma cells could inhibit their susceptibility to killing by autologous tumor-infiltrating lymphocytes (TIL). Results: The RNA-binding protein MEX3B was identified as a top candidate that decreased the susceptibility of melanoma cells to killing by TILs. Further analyses of anti–PD-1–treated melanoma patient tumor samples suggested that higher MEX3B expression is associated with resistance to PD-1 blockade. In addition, significantly decreased levels of IFNγ were secreted from TILs incubated with MEX3B-overexpressing tumor cells. Interestingly, this phenotype was rescued upon overexpression of exogenous HLA-A2. Consistent with this, we observed decreased HLA-A expression in MEX3B-overexpressing tumor cells. Finally, luciferase reporter assays and RNA-binding protein immunoprecipitation assays suggest that this is due to MEX3B binding to the 3′ untranslated region (UTR) of HLA-A to destabilize the mRNA. Conclusions: MEX3B mediates resistance to cancer immunotherapy by binding to the 3′ UTR of HLA-A to destabilize the HLA-A mRNA and thus downregulate HLA-A expression on the surface of tumor cells, thereby making the tumor cells unable to be recognized and killed by T cells. Clin Cancer Res; 24(14); 3366–76. ©2018 AACR. See related commentary by Kalbasi and Ribas, p. 3239

Background Immunotherapy has increasingly become a staple in cancer treatment. However, substantial limitations in the durability of response highlight the need for more rational therapeutic combinations. The aim of this study is to investigate how to make tumor cells more sensitive to T-cell-based cancer immunotherapy. Methods Two pairs of melanoma patient-derived tumor cell lines and their autologous tumor-infiltrating lymphocytes were utilized in a high-throughput screen of 850 compounds to identify bioactive agents that could be used in combinatorial strategies to improve T-cell-mediated killing of tumor cells. RNAi, overexpression, and gene expression analyses were utilized to identify the mechanism underlying the effect of Topoisomerase I (Top1) inhibitors on T-cell-mediated killing. Using a syngeneic mouse model (n = 5 per group), the antitumor efficacy of the combination of a clinically relevant Top1 inhibitor, liposomal irinotecan (MM-398), with immune checkpoint inhibitors was also assessed. All statistical tests were two-sided. Results We found that Top1 inhibitors increased the sensitivity of patient-derived melanoma cell lines (n = 7) to T-cell-mediated cytotoxicity (P < .001, Dunnett’s test). This enhancement is mediated by TP53INP1, whose overexpression increased the susceptibility of melanoma cell lines to T-cell cytotoxicity (2549 cell line: P = .009, unpaired t test), whereas its knockdown impeded T-cell killing of Top1 inhibitor–treated melanoma cells (2549 cell line: P < .001, unpaired t test). In vivo, greater tumor control was achieved with MM-398 in combination with α-PD-L1 or α-PD1 (P < .001, Tukey’s test). Prolonged survival was also observed in tumor-bearing mice treated with MM-398 in combination with α-PD-L1 (P = .002, log-rank test) or α-PD1 (P = .008, log-rank test). Conclusions We demonstrated that Top1 inhibitors can improve the antitumor efficacy of cancer immunotherapy, thus providing the basis for developing novel strategies using Top1 inhibitors to augment the efficacy of immunotherapy.

T cell–mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. We show that loss of PTEN in tumor cells in preclinical models of melanoma inhibits T cell–mediated tumor killing and decreases T-cell trafficking into tumors. In patients, PTEN loss correlates with decreased T-cell infiltration at tumor sites, reduced likelihood of successful T-cell expansion from resected tumors, and inferior outcomes with PD-1 inhibitor therapy. PTEN loss in tumor cells increased the expression of immunosuppressive cytokines, resulting in decreased T-cell infiltration in tumors, and inhibited autophagy, which decreased T cell–mediated cell death. Treatment with a selective PI3Kβ inhibitor improved the efficacy of both anti–PD-1 and anti–CTLA-4 antibodies in murine models. Together, these findings demonstrate that PTEN loss promotes immune resistance and support the rationale to explore combinations of immunotherapies and PI3K–AKT pathway inhibitors. Significance: This study adds to the growing evidence that oncogenic pathways in tumors can promote resistance to the antitumor immune response. As PTEN loss and PI3K–AKT pathway activation occur in multiple tumor types, the results support the rationale to further evaluate combinatorial strategies targeting the PI3K–AKT pathway to increase the efficacy of immunotherapy. Cancer Discov; 6(2); 202–16. ©2015 AACR. See related commentary by Rizvi and Chan, p. 128. This article is highlighted in the In This Issue feature, p. 109

The signaling mechanisms between prostate cancer cells and infiltrating immune cells may illuminate novel therapeutic approaches. Here, utilizing a prostate adenocarcinoma model driven by loss of Pten and Smad4, we identify polymorphonuclear myeloid-derived suppressor cells (MDSC) as the major infiltrating immune cell type, and depletion of MDSCs blocks progression. Employing a novel dual reporter prostate cancer model, epithelial and stromal transcriptomic profiling identified CXCL5 as a cancer-secreted chemokine to attract CXCR2-expressing MDSCs, and, correspondingly, pharmacologic inhibition of CXCR2 impeded tumor progression. Integrated analyses identified hyperactivated Hippo–YAP signaling in driving CXCL5 upregulation in cancer cells through the YAP–TEAD complex and promoting MDSC recruitment. Clinicopathologic studies reveal upregulation and activation of YAP1 in a subset of human prostate tumors, and the YAP1 signature is enriched in primary prostate tumor samples with stronger expression of MDSC-relevant genes. Together, YAP-driven MDSC recruitment via heterotypic CXCL5–CXCR2 signaling reveals an effective therapeutic strategy for advanced prostate cancer. Significance: We demonstrate a critical role of MDSCs in prostate tumor progression and discover a cancer cell nonautonomous function of the Hippo–YAP pathway in regulation of CXCL5, a ligand for CXCR2-expressing MDSCs. Pharmacologic elimination of MDSCs or blocking the heterotypic CXCL5–CXCR2 signaling circuit elicits robust antitumor responses and prolongs survival. Cancer Discov; 6(1); 80–95. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 1

Meeting abstracts Myeloid-derived suppressor cells (MDSCs) represent a phenotypically heterogeneous population of immature myeloid cells that play a tumor-promoting role by maintaining a state of immunological anergy and tolerance. Similar to other solid tumor types, Prostate cancer (PCa) is

The SWI/SNF multisubunit complex modulates chromatin structure through the activity of two mutually exclusive catalytic subunits, SMARCA2 and SMARCA4, which both contain a bromodomain and an ATPase domain. Using RNAi, cancer-specific vulnerabilities have been identified in SWI/SNF-mutant tumors, including SMARCA4-deficient lung cancer; however, the contribution of conserved, druggable protein domains to this anticancer phenotype is unknown. Here, we functionally deconstruct the SMARCA2/4 paralog dependence of cancer cells using bioinformatics, genetic, and pharmacologic tools. We evaluate a selective SMARCA2/4 bromodomain inhibitor (PFI-3) and characterize its activity in chromatin-binding and cell-functional assays focusing on cells with altered SWI/SNF complex (e.g., lung, synovial sarcoma, leukemia, and rhabdoid tumors). We demonstrate that PFI-3 is a potent, cell-permeable probe capable of displacing ectopically expressed, GFP-tagged SMARCA2-bromodomain from chromatin, yet contrary to target knockdown, the inhibitor fails to display an antiproliferative phenotype. Mechanistically, the lack of pharmacologic efficacy is reconciled by the failure of bromodomain inhibition to displace endogenous, full-length SMARCA2 from chromatin as determined by in situ cell extraction, chromatin immunoprecipitation, and target gene expression studies. Furthermore, using inducible RNAi and cDNA complementation (bromodomain- and ATPase-dead constructs), we unequivocally identify the ATPase domain, and not the bromodomain of SMARCA2, as the relevant therapeutic target with the catalytic activity suppressing defined transcriptional programs. Taken together, our complementary genetic and pharmacologic studies exemplify a general strategy for multidomain protein drug-target validation and in case of SMARCA2/4 highlight the potential for drugging the more challenging helicase/ATPase domain to deliver on the promise of synthetic-lethality therapy. Cancer Res; 75(18); 3865–78. ©2015 AACR.

Recently, T cell based immunotherapies have moved to the forefront of cancer immunotherapy with the success of Adoptive T cell therapy (ACT) and Immune checkpoint blockade. ACT, where patients are treated with tumor infiltrating T cells (TILs), conferred a clinical response rate of ∼50%. Treatment with anti-CTLA4 therapy, Ipilimumab, conferred response rates of 10-20%, greatly improving the overall survival of patients with advanced melanoma. Despite the encouraging outcomes, there are relatively low response rates coupled with the delay of weeks to months before tumor shrinkage can be appreciated. Thus, understanding mechanisms of resistance to immune therapies, to improve response rates, shorten time to treatment effect and developing predictive biomarkers of response are vital to the care of melanoma patients. In order to identify possible resistance mechanisms to immunotherapy, a high-throughput in vitro screen with 850 different bio-active compounds (Selleckchem), was designed to search for agents that could either increase or decrease the resistance of melanoma tumor cells to T cell mediated killing. Paired patient derived human melanoma tumor samples and TILs were used to assess which compounds when used to treat the melanoma cell lines can enhance the cytotoxic activity of the TILs against the paired melanoma sample, using a flow cytometry based assay in which active caspase 3 was used as a read out of apoptosis. We identified heat shock protein 90 (HSP90) inhibitors amongst the top compounds that improved T cell mediated cytotoxicity of treated tumor cells. We show that treatment with the HSP90 inhibitor ganetespib (Synta) greatly improves T cell mediated cytotoxicity of human cancer cells lines in vitro. Furthermore, in vivo murine studies using the MC38/gp100 tumor model show that ganestespib in combination with anti-CTLA4, resulted in superior antitumor effect and survival compared to either treatment alone (Average tumor volume at day 21 of treatment: Vehicle 294.3mm3, α-CTLA4 193 mm3, Ganetespib 237.5 mm3 and Ganetespib + α-CTLA4 105.8 mm3, P < 0.0001). Microarray analysis of human cell lines treated with ganetespib in vitro revealed an increase in interferon response genes including IFIT1, IFIT2, IFIT3. We confirmed these findings with quantitative real time PCR and western blot analyses and found IFIT1, IFIT2 and IFIT3 to be consistently upregulated across multiple melanoma cell lines following treatment with ganetespib. We next sought to verify the importance of the IFIT genes in the synergy observed between ganetespib treatment and T cell killing. First, we overexpressed IFIT1, IFIT2 and IFIT3 in human melanoma cell lines to recapitulate the improved sensitivity of the human melanoma cell lines to T cell killing following treatment with ganetespib. We then co-cultured these cells with their autologous T cells and found that overexpressing IFIT1, IFIT2 and IFIT3 mimicked the effects of ganetespib by increasing the sensitivity to T cell killing over the GFP control. On the other hand, silencing IFIT1, IFIT2 and IFIT3 simultaneously, abrogated the synergy between ganetespib and T cell killing. We are further elucidating the role of these genes in lowering the apoptotic threshold of cancer cells and contributing to the synergy of ganetespib and immunotherapy. This will enable the emergence of a new combination therapy of HSP90 inhibitors and anti-CTLA4 for the treatment of melanoma patients that will increase the percentage of patients responding to immunotherapy and achieving long term responses. Citation Format: Rina M. Mbofung, Jodi A. McKenzie, Shruti Malu, Chengwen Liu, Weiyi Peng, Isere Kuiatse, Leila Williams, Seram Devi, Zhe Wang, Trang Tieu, Tim Heffernan, Richard E. Davis, Rodabe Amaria, Patrick Hwu. HSP90 inhibitor, ganetespib, enhances responses to cancer immunotherapy through increased expression of interferon response genes [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B105.

Cancer immunotherapy has transformed the treatment landscape for a number of cancer patients, with some achieving durable and long lasting clinical benefit. Cancer immunotherapy engages and intensifies the host immune response to attack and kill tumor cells. However, as evidenced by the heterogeneous response to immunotherapy, tumor cells have evolved a host of known and unknown mechanisms to evade, inhibit or supersede the immune response. Consequently, scientists and clinicians are unable to accurately predict which patients will respond, or how well they will respond to cancer immunotherapy.To address this shortfall, we have asked the question of how we can modulate tumor cells in order to make them more amenable to immunotherapy, thereby increasing its efficacy. We approached this question by conducting a high throughput drug screen of 850 compounds, to identify bioactive drugs that can increase T cell mediated killing of tumor cells. The goal here is to develop rational combination treatment strategies involving T cell based cancer immunotherapy that will increase the breadth and depth of the clinical response to cancer immunotherapy. One of three top hits from the screen was Topoisomerase I (Top1) inhibitors including irinotecan, topotecan, and camptothecin. We then utilized multiple patient-derived cell lines in an in vitro cytotoxicity assay to validate that treatment of melanoma tumor cells with a Top1 inhibitor, before incubation with their autologous tumor infiltrating lymphocytes (TILs) results in a synergistic increase in T cell mediated killing of tumor cells.These findings were further corroborated in a pre-clinical mouse model, where we found that tumor-bearing mice treated with a combination of a clinically relevant Top1 inhibitor nal-IRI (nano-liposomal irinotecan) and an anti-PD-L1 antibody, showed enhanced tumor regression compared to mice treated with either single agent (mean tumor volume: combo vs nal-IRI vs α-PDL1 = 40.04 ± 5.66 vs 136.30 ± 28.96 vs 373.04 ± 23.96 mm3 respectively, on day 21 after tumor inoculation, p < 0.0001). Significantly longer survival was also achieved in tumor-bearing mice treated with the combination in comparison to cohorts treated with either single agent. To investigate the molecular changes being mediated by Top1 inhibitors in the tumor cells, we conducted gene expression analysis on Top1 inhibitor-treated tumor cells. One striking gene expression change in Top1 inhibitor-treated tumor cells was an upregulation of a number of genes known to be functionally important for p53 signaling including TP53INP1 (Teap). We then focused on the functional relevance of Teap to the increased T cell mediated killing of Top1 inhibitor-treated melanoma cells. Overexpression of Teap in melanoma cells resulted in increased T cell mediated killing, recapitulating the phenotype observed in Top1 inhibitor-treated melanoma cells. Complementary to this, silencing of Teap via shRNA in melanoma cells, inhibited T cell mediated killing of Top1 inhibitor-treated cells, indicating that the enhancement of T cell mediated killing observed in Top1 inhibitor-treated cells is dependent on the p53 regulatory gene Teap. These results support our goal of developing combinations involving T cell based cancer immunotherapy to improve therapeutic efficacy in cancer patients. We have demonstrated that Top1 inhibitors can be effectively combined with T cell based cancer immunotherapy. The results are also indicative of a role for p53 signaling in regulating response to T cell based immunotherapy. By understanding the molecular mechanisms in the tumor that can dictate response or resistance to immunotherapy, we can develop a more comprehensive picture of the cancer immunity response cycle and develop more effective strategies to combat not only melanoma, but also other tumor types where immunotherapy is not yet applicable. Citation Format: Jodi A. McKenzie, Rina M. Mbofung, Shruti Malu, Rodabe N. Amaria, Emily L. Ashkin, Seram N. Devi, Weiyi Peng, Leila J. Williams, Richard E. Davis, Jason Roszik, Trang N. Tieu, Timothy Heffernan, Patrick Hwu. Topoisomerase I inhibitors enhance efficacy of immunotherapy through a p53 regulatory pathway [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B110.

T cell-mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. Here, we interrogated the role of loss of expression of the tumor suppressor, PTEN, in immune resistance. In preclinical studies, we found that silencing PTEN in tumor cells inhibited T cell-mediated tumor killing and decreased T cell trafficking into tumors. In clinical studies, we observed that tumors with loss of PTEN had significantly less CD8+ T cell infiltration than PTEN-present tumors. In addition, 26% of melanomas that did not yield successful TIL growth demonstrated PTEN loss, which was more frequent than was observed in tumors that yielded successful TIL growth (11%). We further validated the association between reduced number and impaired function of TIL with PTEN loss using another independent cohort, TCGA dataset for SKCM. More importantly, we analyzed clinical outcomes of metastatic melanoma patients treated with the FDA-approved anti-PD-1 antibodies. Our analysis demonstrates that a greater reduction in tumor burden was achieved by PD-1 blockade in PTEN present patients, when compared with PTEN absent patients. To decipher the factors mediating the immunosuppressive effects of PTEN loss, we determined the expression profiles of tumor cells with or without PTEN expression. Our results indicated that PTEN loss increased the production of immunosuppressive factors, including CCL2 and VEGF. Anti-VEGF blocking antibody improved anti-tumor activity of transferred tumor-reactive T cells and enhanced tumor infiltration of transferred T cells in PTEN-silenced tumors. These results suggest that loss of PTEN can facilitate the resistance of T cell-mediated immune responses by increasing the expression of immunosuppressive factors. Given that PTEN loss results in activation of the PI3K pathway, we evaluated the efficacy of immunotherapy in combination with a selective PI3Kβinhibitor to treat spontaneously developed BRAF mutant, PTEN null melanomas in genetically engineered mouse models. Our result showed that the combination of PI3Kβ inhibitor and anti-PD-1 significantly delayed tumor growth in tumor-bearing mice. Mice treated with this combination had a median survival time of 28 days, which is longer than the survival time of mice treated with either therapy. Increased numbers of T cells at tumor sites were found in mice receiving the combination therapy compared with mice receiving either agent alone. Taken together, our results demonstrate that PTEN loss contributes to the generation of immunosuppressive tumor microenvironment. Notably, this study provides the first direct clinical evidence to support the association between PTEN loss and poor clinical outcome in immunotherapy treated patients. In addition, our study indicates that inhibition of the PI3K-AKT pathway can improve the efficacy of immunotherapy in cancer. Citation Format: Weiyi Peng, Jie Qing Chen, Chengwen Liu, Shruti Malu, Caitlin Creasy, Michael Tetzlaff, Chunyu Xu, Jodi McKenzie, Chunlei Zhang, Xiaoxuan Liang, Leila Williams, Wanleng Deng, Guo Chen, Rina Mbofung, Alexander Lazar, Carlos Torres-Cabala, Zachary Cooper, Pei-Ling Chen, Trang Tieu, Stefani Spranger, Xiaoxing Yu, Chantale Bernatchez, Marie-Andree Forget, Cara Haymaker, Rodabe Amaria, Jennifer McQuade, Isabella Glitza, Tina Cascone, Haiyan Li, Lawrence Kwong, Timothy Heffernan, Jianhua Hu, Roland Bassett, Marcus Bosenberg, Scott Woodman, Willem Overwijk, Gregory Lizée, Jason Roszik, Thomas Gajewski, Jennifer Wargo, Jeffrey Gershenwald, Laszlo Radvanyi, Michael Davies, Patrick Hwu. Loss of PTEN promotes resistance to T cell-mediated immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4363.

Melanoma is a highly aggressive form of skin cancer, whose rates of morbidity and mortality are increasing. The development of immunotherapies like anti-PDL1 and anti-CTLA4 antibodies has resulted in fundamental advances in the treatment of some cancers. However, long lasting responses are only observed in a subset of immunotherapy-treated patients. This shortfall highlights the need for a better understanding of the molecular mechanisms that govern tumor response to immunotherapy. To address this need, autologous patient-derived tumor cell lines and tumor infiltrating lymphocytes (TILs) were utilized in an in vitro high throughput screen, to identify compounds that increase the sensitivity of melanoma cells to T cell mediated cytotoxicity. The screen consisted of an 850 compound library. One group of compounds that was most able to enhance T cell killing of melanoma cells was topoisomerase I (Top1) inhibitors such as topotecan and irinotecan. Our results indicate that treatment of melanoma cells with a Top1 inhibitor prior to exposure to autologous T cells produced a synergistic increase in tumor cell death, as measured by intracellular staining of activated caspase 3. We have also recapitulated this finding in an in vivo model, where a better anti-tumor effect was observed in tumor bearing mice treated with an antibody against the co-inhibitory molecule PDL1 in combination with MM398 (nanoliposomal irinotecan), than in cohorts treated with either α-PDL1 or Top1 inhibitor alone. These findings suggest synergism between Top1 inhibitors and immune-based therapies in the treatment of melanoma. Molecular changes elicited by inhibition of Top1 are now being investigated to identify the factors that mediate the effect of Top1 inhibitors on T cell-mediated killing of melanoma. We have identified a p53-driven gene signature in Top1 inhibitor-treated melanoma cell lines and are investigating the functional relevance of Tumor Protein p53 Inducible Nuclear Protein 1 (TP53INP1) in mediating increased T cell killing of Top1 inhibitor-treated melanoma cells. Our results indicate that TP53INP1 is a critical component of this apoptotic response, as overexpression of TP53INP1 in melanoma cells increased their susceptibility to T cell mediated cytotoxicity. Complementary to this observation, we have also found that knockdown of TP53INP1 by shRNA, impedes the sensitivity of Top1 inhibitor-treated melanoma cells to T cell mediated killing. Understanding how Top1 inhibitors enhance melanoma killing by immunotherapy will allow for the development of predictive biomarkers, and also augment immune-based therapeutic strategies to ensure durable responses in a larger population of melanoma patients. By using melanoma as a model disease system, we can gain valuable insights into the dynamics of cancer immune response that may be applied to other cancers where effective treatment strategies are also lacking. Citation Format: Jodi A. McKenzie, Rina M. Mbofung, Shruti Malu, Rodabe N. Amaria, Richard E. Davis, Li Zhang, Trang N. Tieu, Tim P. Heffernan, Patrick Hwu. Enhancing the antitumor efficacy of immunotherapy by using the topoisomerase I inhibitor MM398. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4002.

Recently, T cell based immunotherapies have moved to the forefront of cancer immunotherapy with the success of Adoptive T cell therapy (ACT) and Immune checkpoint blockade. ACT, where patients are treated with tumor infiltrating T cells (TILs), conferred a clinical response rate of ∼50%. Treatment with anti-CTLA4 therapy, Ipilimumab, conferred response rates of 10-20%, greatly improving the overall survival of patients with advanced melanoma. Despite the encouraging outcomes, there are relatively low response rates coupled with the delay of weeks to months before tumor shrinkage can be appreciated. Thus, understanding mechanisms of resistance to immune therapies, to improve response rates, shorten time to treatment effect and developing predictive biomarkers of response are vital to the care of melanoma patients. In order to identify possible resistance mechanisms to immunotherapy, a high-throughput in vitro screen with 850 different bio-active compounds (Selleckchem), was designed to search for agents that could either increase or decrease the resistance of melanoma tumor cells to T cell mediated killing. Paired tumor samples and TILs from melanoma patients were used to assess which compounds when used to treat the melanoma cell lines can enhance the cytotoxic activity of the TILs against the paired melanoma sample, using a flow cytometry based assay in which active caspase 3 was used as a read out of apoptosis. We identified heat shock protein 90 (HSP90) inhibitors amongst compounds that improved T cell mediated cytotoxicity. We show that treatment with the HSP90 inhibitor ganetespib (Synta) greatly improves T cell mediated cytotoxicity of both human and murine cancer cells lines in vitro. Furthermore, in vivo murine studies using the MC38/gp100 tumor model show that ganestespib in combination with anti-CTLA4, resulted in superior antitumor effect and survival compared to either treatment alone (Average tumor volume at day 21 of treatment: Vehicle 294.3mm3, α-CTLA4 193 mm3, Ganetespib 237.5 mm3 and Ganetespib + α-CTLA4 105.8 mm3, P < 0.0001). Microarray analysis of human cell lines treated with ganetespib in vitro revealed an increase in interferon alpha (IFN-α) response genes including IFIT1, IFIT2, IFIT3 and IFIH1. Silencing IFIT2 abrogated the synergy observed with ganetespib treatment and T cell mediated killing, suggesting that the IFN-α response pathway plays an important role in this combination therapy. We are further elucidating the role of these genes in the synergy observed. This will enable the emergence of a new combination therapy of HSP90 inhibitors and anti-CTLA4 for the treatment of melanoma patients that will increase the percentage of patients responding to immunotherapy and achieving long term responses. Citation Format: Rina M. Mbofung, Jodi A. McKenzie, Shruti Malu, Chengwen Liu, Leila Williams, Weiyi Peng, Zhe Wang, Satyendra Tripathi, Trang Tieu, Shuping Zhao, Seram Devi, Isere Kuiatse, Emily Ashkin, Leah Bailey, Jason Roszik, Samir Hanash, Timothy Heffernan, Richard E. Davis, Rodabe N. Amaria, Patrick Hwu. Inhibition of HSP90 enhances T cell-mediated antitumor immune responses through expression of interferon-alpha response Genes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4360.

The SWI/SNF multi-subunit complex modulates chromatin structure through the activity of two mutually exclusive catalytic subunits, SMARCA2 and SMARCA4, which both contain a bromodomain and an ATPase domain. Using RNAi, cancer-specific vulnerabilities have been identified in SWI/SNF mutant tumors, including SMARCA4-deficient lung cancer, however, the contribution of conserved, druggable protein domains to this anticancer phenotype is unknown. Here, we functionally deconstructed the SMARCA2/4 paralog dependence of cancer cells using bioinformatics, genetic and pharmacological tools. We evaluated a potent and selective SMARCA2/4 bromodomain inhibitor (PFI-3) and characterized its activity in chromatin-binding and cell-functional assays focusing on cells with altered SWI/SNF status (e.g. Lung, Synovial Sarcoma, Leukemia, and Rhabdoid tumors). We demonstrated that PFI-3 is a cell-permeable probe capable of displacing ectopically expressed, GFP-tagged SMARCA2-bromodomain from chromatin, yet contrary to target knockdown, the inhibitor failed to display an antiproliferative phenotype. Mechanistically, the lack of pharmacological efficacy was reconciled by the failure of bromodomain inhibition to displace endogenous, full-length SMARCA2 from chromatin as determined by in situ cell extraction, chromatin immunoprecipitation (ChIP) and target gene expression and promoter occupancy studies. Using RNAi and cDNA complementation (bromodomain and ATPase-dead constructs), we identified the catalytic ATPase domain, and not the bromodomain of SMARCA2, as a relevant therapeutic target. Taken together, our complementary genetic and pharmacological studies exemplify a general strategy for bromodomain drug-target validation and in case of SMARCA2/4 highlight the requirement for drugging the more challenging helicase/ATPase domain affording potential synthetic-lethal treatment options to cancer patients with genetically defined alterations in SWI/SNF. Citation Format: Bhavatarini Vangamudi, Thomas Paul, Parantu K. Shah, Maria K. Alimova, Lisa Nottebaum, Xi Shi, Yanai Zhan, Elisabetta Leo, Harshad S. Mahadeshwar, Alexei Protopopov, Andrew Futreal, Trang N. Tieu, Mike Peoples, Alessia Petrocchi, Joseph R. Marszalek, Carlo Toniatti, Timothy P. Heffernan, Dominique Verhelle, Giulio Draetta, Dafydd Owen, Philip Jones, Wylie Palmer, Shikhar Sharma, Jannik N. Andersen. The SMARCA2/4 catalytic activity, but not the bromodomain, is a drug target in SWI/SNF mutant cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3528. doi:10.1158/1538-7445.AM2015-3528