Rina M. Mbofung

Loss of PTEN promotes resistance to T cell–mediated immunotherapy

UNLABELLED 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.

BRAF Inhibition Increases Tumor Infiltration by T cells and Enhances the Antitumor Activity of Adoptive Immunotherapy in Mice

Purpose: Treatment of melanoma patients with selective BRAF inhibitors results in objective clinical responses in the majority of patients with BRAF-mutant tumors. However, resistance to these inhibitors develops within a few months. In this study, we test the hypothesis that BRAF inhibition in combination with adoptive T-cell transfer (ACT) will be more effective at inducing long-term clinical regressions of BRAF-mutant tumors. Experimental Design: BRAF-mutated human melanoma tumor cell lines transduced to express gp100 and H-2Db to allow recognition by gp100-specific pmel-1 T cells were used as xenograft models to assess melanocyte differentiation antigen–independent enhancement of immune responses by BRAF inhibitor PLX4720. Luciferase-expressing pmel-1 T cells were generated to monitor T-cell migration in vivo. The expression of VEGF was determined by ELISA, protein array, and immunohistochemistry. Importantly, VEGF expression after BRAF inhibition was tested in a set of patient samples. Results: We found that administration of PLX4720 significantly increased tumor infiltration of adoptively transferred T cells in vivo and enhanced the antitumor activity of ACT. This increased T-cell infiltration was primarily mediated by the ability of PLX4720 to inhibit melanoma tumor cell production of VEGF by reducing the binding of c-myc to the VEGF promoter. Furthermore, analysis of human melanoma patient tumor biopsies before and during BRAF inhibitor treatment showed downregulation of VEGF consistent with the preclinical murine model. Conclusion: These findings provide a strong rationale to evaluate the potential clinical application of combining BRAF inhibition with T-cell–based immunotherapy for the treatment of patients with melanoma. Clin Cancer Res; 19(2); 393–403. ©2012 AACR.

The Effect of Topoisomerase I Inhibitors on the Efficacy of T-Cell-Based Cancer Immunotherapy

Abstract 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.

The RNA-binding Protein MEX3B Mediates Resistance to Cancer Immunotherapy by Downregulating HLA-A Expression

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

CpG-based immunotherapy impairs antitumor activity of BRAF inhibitors in a B-cell-dependent manner

Combining immunotherapy with targeted therapy has increasingly become an appealing therapeutic paradigm for cancer treatment due to its great potential for generating durable and synergistic antitumor response. In this study, however, we unexpectedly found that two types of CpG-based tumor peptide vaccine treatments consistently negated the antitumor activity of a selective BRAF inhibitor in tumors with BRAF mutation rather than showing a synergistic antitumor effect. Our further studies demonstrated that CpG alone was sufficient to dampen BRAF inhibitor-induced antitumor responses, suggesting that the impaired antitumor activity of the BRAF inhibitor observed in mice receiving CpG-based peptide vaccine is mainly dependent upon the use of CpG. Mechanistically, CpG increased the number of circulating B cells, which produced elevated amounts of tumor necrosis factor-α (TNFα) that contributed to the increased tumor resistance to BRAF inhibitors. More importantly, B-cell depletion or TNFα neutralization can restore the antitumor effect of BRAF inhibition in mice receiving CpG treatment, indicating that TNFα-secreting B cells play an indispensable role in BRAF inhibitor resistance induced by CpG. Taken together, our results strongly suggest that precautions must be implemented when designing combinatorial approaches for cancer treatment, because distinct regimens, despite their respective therapeutic benefit as monotherapy, may together provide antagonistic clinical outcomes.

Abstract B118: Using a high throughput T-cell cytotoxicity assay to develop combination strategies for immunotherapy

T-cells play a critical role in cancer immunosurveillance and control. As such, there has been a recent emergence of T cell based immunotherapies with durable clinical outcomes. These include the immune checkpoint inhibitors Ipilimumab (anti-CTLA-4) and Nivolumab (anti-PD-1), and adoptive cell therapy, which have conferred clinical response rates of up to 50%. Despite these encouraging outcomes, there is still a large cohort of patients for which these treatments are currently not applicable. They are either inherently resistant to or acquire resistance to immunotherapy. As such, understanding mechanisms of resistance to immune response and ascertaining efficacious therapy combinations is important in overcoming immune-resistance and developing novel treatment regimens. To address this need we performed preliminary screening of about 850 different bioactive compounds (Selleckem) to find candidate drugs that could modulate the sensitivity of tumor cells to T- cell mediated killing, using an established protocol for an in vitro T-cell mediated cytotoxicity screen. We hypothesized that either tumor cells treated with different compounds will increase or decrease their sensitivity to T cell mediated killing, and compounds that increase T cell killing can be used in combination with immunotherapy. Using patient-derived melanoma tumor cells and their autologous tumor infiltrating T cells (TILs), we assessed the increase or decrease of T cell killing of tumor cells following treatment with the test compounds via intracellular detection of active caspase-3 by flow cytometry in a 96-well format. This initial screen identified Heat shock protein 90 (Hsp90), Aurora Kinase and Topoisomerase I inhibitors as enhancers of T cell mediated killing, and studies to understand their mechanisms of action are currently underway. However, as this screening process is very time-intensive, we have increased the efficiency and feasibility of this screen by miniaturizing the workflow. This work highlights the development and initial findings of the screening method that is performed in 384-well plates. This format allows for a larger library of compounds, with a greater drug concentration range, and for more autologous tumor and T-cell pairs to be screened within a shorter time. The miniaturized assay also utilises robotics to streamline and increase output. Additionally, by understanding the signaling pathways and molecular factors that regulate tumor response to T cell mediated killing, we can translate these findings applicable to other cancer. This project aims to discover novel compounds that work synergistically with T-cell mediated tumor cell cytotoxicity, understand their mechanism of action and how the inhibited pathways contribute to resistance to immunotherapy and as a result better inform combination strategies with immunotherapy for clinical use. Citation Format: Leila Williams, Shruti Malu, Jodi McKenzie, Rina Mbofung, Jason Roszik, Patrick Hwu. Using a high throughput T-cell cytotoxicity assay to develop combination strategies for immunotherapy [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 B118.

Abstract B105: HSP90 inhibitor, ganetespib, enhances responses to cancer immunotherapy through increased expression of interferon response genes

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 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.

Abstract B152: Increasing the antitumor efficacy of immunotherapy in melanoma by using topoisomerase I inhibitors

Melanoma is a highly aggressive form of skin cancer, whose rates of morbidity and mortality are continuously increasing. The development of immunotherapeutic agents like anti-PDL1 and anti-CTLA4 antibodies has resulted in fundamental advances in the treatment of melanoma. However, long lasting responses are only observed in a small subset of immunotherapy-treated melanoma patients. This shortfall highlights the need for a better understanding of the molecular mechanisms that govern tumor sensitivity or resistance to immunotherapy. To address this need, autologous patient-derived tumor cell lines and tumor infiltrating lymphocytes (TILs) were utilized in an in vitro activated caspase 3-based high-throughput screen, to identify compounds that increase the sensitivity of melanoma cells to T-cell mediated cytotoxicity. The screen consisted of a library of 850 bioactive compounds. One group of compounds that was most able to enhance T-cell killing of melanoma cells was topoisomerase I (Top1) inhibitors including: topotecan, and irinotecan. Topoisomerases are a family of DNA enzymes, which are involved in unwinding DNA and relieving torsional strain during replication and transcription. Our results indicate that treatment of melanoma tumor 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, and computed using CalcuSyn. 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 Programmed Death Ligand 1 (PDL1) in combination with a nanoparticle liposomal formulation of irinotecan, than in cohorts treated with either antibody or drug alone. These findings suggest synergism between Top1 inhibitors and immune-based therapies in the treatment of melanoma. Genomic and proteomic changes elicited by inhibition of Top1 are now being investigated to identify the molecular factors that mediate the effect of Top1 inhibitors on T cell-mediated killing of melanoma. Our goal is to identify molecular changes mediated by Top1 inhibition in melanoma tumor cells, and/or the tumor microenvironment, that relieves immunosuppression and potentiates the activity of cytotoxic T cell-based immunotherapy. 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, Patrick Hwu. Increasing the antitumor efficacy of immunotherapy in melanoma by using topoisomerase I inhibitors. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B152.

Abstract B110: Topoisomerase I inhibitors enhance efficacy of immunotherapy through a p53 regulatory pathway

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 mm 3 respectively, on day 21 after tumor inoculation, p 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.

Abstract IA19: Rational combinations of targeted and immunotherapies

Both targeted therapy and immunotherapy have shown great promise for the treatment of cancer patients, but neither approach results in durable success in the majority of patients. The combination of targeted therapies with immunotherapies has the potential to result in higher, more durable clinical responses and improved survival in patients with metastatic cancer. Besides inducing the release of antigens following tumor destruction, targeted therapies may also reverse the immunosuppressive microenvironment found at the tumor site. In previous studies, we found that BRAF inhibition enhances T-cell infiltration into tumors, partially by downregulating VEGF at the tumor site (CCR, Liu et al 2013). In addition, we have found other combinations in murine models that are effective, such as dasatinib plus anti-OX40 antibody in c-kit positive tumors (Blood, Yang et al 2012). Recently we reported that PTEN loss in tumors results in less infiltration by T-cells, and lower responses to anti-PD1 therapy in advanced melanoma patients (Cancer Discovery, Peng et al 2016). Importantly, this could be overcome in murine models using a combination of PI3K beta inhibitor and anti-PD1, a concept which will be tested in the clinic. We are now utilizing a high throughput screen of more than 1000 compounds to determine which can most effectively enhance the ability of tumor-specific T-cells to kill autologous tumor (poster #341). This screen evaluates tumor death using flow cytometry to detect intratumoral activated caspases 3 and 7, since T-cells kill using a caspase-dependent apoptotic pathway. Positive candidates are then confirmed and tested in murine tumor models. We are also evaluating the molecular basis of immune resistance using a similar in vitro screen as well as an in vivo screen with tumor cells transduced with shRNA libraries. Positive candidates will be tested for expression utilizing our banked samples from responding and non-responding patients who have received tumor-infiltrating lymphocytes (TIL). Using these screens we have thus far determined that the aurora kinase inhibitors, topoisomerase I inhibitors (poster #214), and HSP90 inhibitors (poster #276) enhance T-cell based immunotherapies. We are evaluating the mechanisms of action of these combination therapies. HSP90 inhibitors enhance anti-CTLA4 activity due to a mechanism involving the interferon response genes, for example. We have also found that altering metabolic pathways (eg, inhibiting glycolysis) in tumor cells enhances antitumor immune responses. In summary, we are currently performing high throughput screens to determine the best agents to combine with immunotherapy strategies, as well as molecular determinants of immune resistance. Citation Format: Weiyi Peng, Rina Mbofung, Leila Williams, Patrick Hwu. Rational combinations of targeted and immunotherapies [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 IA19.