Marie-Andree Forget

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.

Uncovering a novel function of BTLA on tumor-infiltrating CD8+ T cells

Manipulation of T-cell co-inhibitory molecules, such as CTLA-4, PD-1, and BTLA has recently moved to the forefront of cancer immunotherapy. Although these molecules serve as inhibitors of T-cell activation, they are also biomarkers for activated T cells and may in fact have positive immune regulatory functions under certain circumstances. Recently, we demonstrated an unexpected positive association of CD8+ T cells expressing BTLA (B- and T- lymphocyte attentuator) with clinical response to adoptive T cell therapy in late-stage melanoma patients. We hypothesized that TIL may utilize the BTLA checkpoint differently and that key phenotypic and functional differences may exist between CD8+BTLA+ and CD8+BTLA- TIL subsets. In this study, we isolated and characterized BTLA+ and BTLA- CD8+ TIL from melanoma patients accrued in a Phase II clinical trial. We found that CD8+BTLA+ TIL had a superior proliferative response to IL-2 and a younger, more central memory T-cell behavior, such as secreting their own IL-2 after TCR stimulation. This younger and more robust memory phenotype was also associated with a longer persistence of T-cell clones in vivo in patients from the infused CD8+BTLA+ TIL subset. In contrast, CD8+BTLA- TIL were poorly proliferative, expressed killer-cell immunoglobulin-like receptors, and exhibited a gene expression signature of T cell deletion. As previously demonstrated, BTLA ligation with its cognate ligand herpes virus entry mediator (HVEM) resulted in decreased proliferation and inflammatory cytokine secretion. However, in a model of TIL activation induced cell death, we observed an enhanced survival of TIL co-cultured with HVEM+ target cells suggesting that BTLA ligation may also promote T-cell survival. Using a recombinant HVEM-Fc protein, we found that HVEM ligation of BTLA on CD8+ TIL activated the PI3K-Akt pathway resulting in the phosphorylation of Akt; this response was blocked using an anti-BTLA antibody. PI3K-Akt activation is most likely mediated by a unique GRB2-binding domain that exists in BTLA that can recruit PI3K via GRB2. As HVEM is expressed by many melanoma tumors and antigen-presenting cells these results suggest that HVEM ligation of BTLA on CD8+BTLA+ effector T-cells in the tumor microenvironment may play a dual role by reducing over-stimulation through the TCR and driving a PI3K-Akt-induced cell survival pathway. This survival signaling pathway may facilitate the longer persistence of the CD8+BTLA+ TIL subset in vivo in our patients. Our study has uncovered a novel role for BTLA both as a biomarker in TIL therapy and as a rheostat in perhaps fine tuning CD8+ T-cell responses rather than simply as a negative signaling pathway.

Molecular and immune heterogeneity in synchronous melanoma metastases.

Despite recent advances in the treatment of metastatic melanoma through targeted and immunotherapy, the majority of patients do not achieve a durable response. Research efforts to better understand responses are underway, and numerous molecular mechanisms of resistance to targeted therapy have been identified. There is a growing appreciation of genomic heterogeneity as a contributor to resistance to therapy, although immune heterogeneity has not been well characterized. The goal of the present study is to better understand genomic and immune heterogeneity in synchronous metastases within melanoma patients, with the potential to identify actionable strategies to overcome resistance. In this study, we prospectively evaluated 36 tumors from 16 melanoma patients (n=5 treatment-naive, n=6 targeted therapy, n=5 immunotherapy). Distinct synchronous metastases were evaluated by whole exome sequencing and NanoString analysis and showed up to 36% tumor-specific mutations as well as significant differences in expression of immune pathway effectors. Accordingly, we performed immune profiling by flow cytometry and immunohistochemistry demonstrating significant immune heterogeneity between synchronous melanoma tumors in all patients, most notably in the CD4+ and CD8+ T cell compartment. Deep TCR sequencing data revealed that T cell populations infiltrating synchronous metastases presented different specificities, with less than 10% of T cell clones shared between 2 tumors in the same patient. Additionally, the NetMHC 3.4 algorithm revealed that 10-30% of predicted neoantigens were unique to individual tumors and that over 10% of these presented high HLA-binding affinity. Together, these data suggest significant genomic and immune heterogeneity between synchronous metastases in melanoma patients – not only in the setting of therapy but also prior to its initiation. This has important clinical implications, and could help explain variable responses to therapy, however this hypothesis must be tested carefully in a larger data set. Nonetheless, these findings may have significant implications for the treatment of melanoma and other cancers.

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

Prospective Analysis of Adoptive TIL Therapy in Patients with Metastatic Melanoma: Response, Impact of Anti-CTLA4, and Biomarkers to Predict Clinical Outcome

Purpose: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has consistently demonstrated clinical efficacy in metastatic melanoma. Recent widespread use of checkpoint blockade has shifted the treatment landscape, raising questions regarding impact of these therapies on response to TIL and appropriate immunotherapy sequence. Patients and Methods: Seventy-four metastatic melanoma patients were treated with autologous TIL and evaluated for clinical response according to irRC, overall survival, and progression-free survival. Immunologic factors associated with response were also evaluated. Results: Best overall response for the entire cohort was 42%; 47% in 43 checkpoint-naïve patients, 38% when patients were exposed to anti-CTLA4 alone (21 patients) and 33% if also exposed to anti-PD1 (9 patients) prior to TIL ACT. Median overall survival was 17.3 months; 24.6 months in CTLA4-naïve patients and 8.6 months in patients with prior CTLA4 blockade. The latter patients were infused with fewer TIL and experienced a shorter duration of response. Infusion of higher numbers of TIL with CD8 predominance and expression of BTLA correlated with improved response in anti-CTLA4 naïve patients, but not in anti-CTLA4 refractory patients. Baseline serum levels of IL9 predicted response to TIL ACT, while TIL persistence, tumor recognition, and mutation burden did not correlate with outcome. Conclusions: This study demonstrates the deleterious effects of prior exposure to anti-CTLA4 on TIL ACT response and shows that baseline IL9 levels can potentially serve as a predictive tool to select the appropriate sequence of immunotherapies. Clin Cancer Res; 24(18); 4416–28. ©2018 AACR.

Combined analysis of antigen presentation and T cell recognition reveals restricted immune responses in melanoma

The quest for tumor-associated antigens (TAA) and neoantigens is a major focus of cancer immunotherapy. Here, we combine a neoantigen prediction pipeline and human leukocyte antigen (HLA) peptidomics to identify TAAs and neoantigens in 16 tumors derived from seven patients with melanoma and characterize their interactions with their tumor-infiltrating lymphocytes (TIL). Our investigation of the antigenic and T-cell landscapes encompassing the TAA and neoantigen signatures, their immune reactivity, and their corresponding T-cell identities provides the first comprehensive analysis of cancer cell T-cell cosignatures, allowing us to discover remarkable antigenic and TIL similarities between metastases from the same patient. Furthermore, we reveal that two neoantigen-specific clonotypes killed 90% of autologous melanoma cells, both in vitro and in vivo, showing that a limited set of neoantigen-specific T cells may play a central role in melanoma tumor rejection. Our findings indicate that combining HLA peptidomics with neoantigen predictions allows robust identification of targetable neoantigens, which could successfully guide personalized cancer immunotherapies.Significance: As neoantigen targeting is becoming more established as a powerful therapeutic approach, investigating these molecules has taken center stage. Here, we show that a limited set of neoantigen-specific T cells mediates tumor rejection, suggesting that identifying just a few antigens and their corresponding T-cell clones could guide personalized immunotherapy. Cancer Discov; 8(11); 1366-75. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333.

The Role of the Immune System and Immunoregulatory Mechanisms Relevant to Melanoma

A hallmark of melanoma is its inherent immunogenicity through both innate and adaptive immune mechanisms. However, a number of factors inhibit these immune responses through intrinsic mechanisms in tumor cells or adaptive resistance triggered by the immune response via negative feedback. Understanding how these processes are balanced in context of pathways regulating T-cell activation, migration, and differentiation, and T-cell dysfunction in tumors has become a critical area of research. This chapter describes the immunoregulatory mechanisms in the melanoma tumor microenvironment and how positive and negative signaling elements can be harnessed to facilitate enhanced anti-tumor immune responses through immunotherapy.

Abstract 4363: Loss of PTEN promotes resistance to T cell-mediated 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. 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 Lizee, 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.

You can run but you can't hide: tracking T cells in metastatic melanoma patients treated with tumor-infiltrating lymphocytes

Meeting abstracts Adoptive T cell therapy using tumor-infiltrating lymphocytes (TIL) for patients with metastatic melanoma has been shown to have a 45-50% response rate as demonstrated by multiple institutions worldwide. However, understanding how long infused T cells persist in patients remains