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Prior data have variably implicated the inactivation of the mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) complex with increased tumor sensitivity to immune checkpoint inhibitors (ICI). Herein, we examined the association between mSWI/SNF variants and clinical outcomes to ICIs. We correlated somatic loss-of-function (LOF) variants in a predefined set of mSWI/SNF genes (ARID1A, ARID1B, SMARCA4, SMARCB1, PBRM1, and ARID2) with clinical outcomes in patients with cancer treated with systemic ICIs. We identified 676 patients from Dana-Farber Cancer Institute (DFCI, Boston, MA) and 848 patients from a publicly available database from Memorial Sloan Kettering Cancer Center (MSKCC, New York, NY) who met the inclusion criteria. Multivariable analyses were conducted and adjusted for available baseline factors and tumor mutational burden. Median follow-up was 19.6 (17.6–22.0) months and 28.0 (25.0–29.0) months for the DFCI and MSKCC cohorts, respectively. Seven solid tumor subtypes were examined. In the DFCI cohort, LOF variants of mSWI/SNF did not predict improved overall survival (OS), time-to-treatment failure (TTF), or disease control rate. Only patients with renal cell carcinoma with mSWI/SNF LOF showed significantly improved OS and TTF with adjusted HRs (95% confidence interval) of 0.33 (0.16–0.7) and 0.49 (0.27–0.88), respectively, and this was mostly driven by PRBM1. In the MSKCC cohort, where only OS was captured, LOF mSWI/SNF did not correlate with improved outcomes across any tumor subtype. We did not find a consistent association between mSWI/SNF LOF variants and improved clinical outcomes to ICIs, suggesting that mSWI/SNF variants should not be considered as biomarkers of response to ICIs.

Clear cell renal cell carcinoma (ccRCC) is considered an immunotherapy-responsive disease; however, the reasons for this remain unclear. Studies have variably implicated PBRM1 mutations as a predictive biomarker of immune checkpoint blockade (ICB) response, and separate studies demonstrate that expression of human endogenous retroviruses (hERVs) might be an important class of tumor-associated antigens. We sought to understand if specific mutations were associated with hERV expression. Two large, annotated genomic datasets, TCGA KIRC and IMmotion150, were used to correlate mutations and hERV expression. PBRM1 mutations were consistently associated with increased hERV expression in primary tumors. In vitro silencing of PBRM1, HIF1A and HIF2A followed by RNA-seq was performed in UMRC2 cells, confirming that PBRM1 regulates hERVs in a HIF1α and HIF2α− dependent manner and that hERVs of the HERVERI superfamily are enriched in PBRM1-regulated hERVs. Our results uncover a role of PBRM1 in the negative regulation of hERVs in ccRCC. Moreover, the HIF-dependent nature of hERV expression explains the previously reported ccRCC-specific clinical associations of PBRM1 mutant ccRCC with both a good prognosis as well as improved clinical outcomes to ICB.

Programmed death 1 (PD-1) and PD ligand 1 (PD-L1) inhibitors have shown activity in metastatic clear cell renal cell carcinoma (ccRCC). Data on the activity of these agents in patients with non–clear cell RCC (nccRCC) or patients with sarcomatoid/rhabdoid differentiation are limited. In this multicenter analysis, we explored the efficacy of PD-1/PD-L1 inhibitors in patients with nccRCC or sarcomatoid/rhabdoid differentiation. Baseline and follow-up demographic, clinical, treatment, and radiographic data were collected. The primary endpoint was objective response rate. Secondary endpoints include time-to-treatment failure (TTF), overall survival (OS), and biomarker correlates. Forty-three patients were included: papillary (n = 14; 33%), chromophobe (n = 10; 23%), unclassified (n = 9; 21%), translocation (n = 3; 7%), and ccRCC with sarcomatoid differentiation (n = 7, 16%). Of those 43 patients, 11 patients (26%) had sarcomatoid and/or rhabdoid differentiation (n = 7 with ccRCC; n = 4 nccRCC). Overall, 8 patients (19%) objectively responded, including 4 patients (13%) who received PD-1/PD-L1 monotherapy. Responses were observed in patients with ccRCC with sarcomatoid and/or rhabdoid differentiation (n = 3/7, 43%), translocation RCC (n = 1/3, 33%), and papillary RCC (n = 4/14, 29%). The median TTF was 4.0 months [95% confidence interval (CI), 2.8–5.5] and median OS was 12.9 months (95% CI, 7.4–not reached). No specific genomic alteration was associated with clinical benefit. Modest antitumor activity for PD-1/PD-L1–blocking agents was observed in some patients with nccRCC. Further prospective studies are warranted to investigate the efficacy of PD-1/PD-L1 blockade in this heterogeneous patient population. Cancer Immunol Res; 6(7); 758–65. ©2018 AACR.

Immunotherapies such as anti-PD-1 antibodies can induce durable responses in a subset of cancer patients, but intrinsic or acquired resistance occurs in most cases. Understanding the molecular mechanisms that drive resistance and response to immunotherapies could therefore lead to alternative treatment strategies exhibiting greater efficacy and precision. To identify new tumor cell-intrinsic mechanisms of resistance to PD-1 blockade, we evaluated metastatic melanoma patients who exhibited complete regression of all but one metastatic site following anti-PD-1 therapy. In one patient, we observed a loss-of-function mutation in the tumor suppressor gene F-Box and WD repeat domain containing 7 (FBXW7)—specifically in the resistant tumor. Using an immunocompetent, anti-PD-1 sensitive melanoma mouse model, we demonstrated that Fbxw7 loss of function in tumor cells causes resistance to PD-1 blockade. We showed that the genetic deletion of Fbxw7 in tumor cells alters the tumor immune microenvironment—including decreased effector cells and increased suppressive cells—in addition to diminished responses to viral sensing and interferon signaling pathways in vivo. Mechanistically, we found that Fbxw7 is necessary for the expression of the dsRNA sensors Mda5 and Rig-I. As a consequence, Fbxw7 is required for dsRNA-induced type I interferon production and interferon signaling in tumor cells. Conversely, restoration of the dsRNA signaling pathway in Fbxw7-deficient cells is sufficient to increase MHC-I expression and to suppress growth of Fbxw7-deficient tumors, thereby promoting antitumor immunity. Our findings establish a hitherto unrecognized role of the tumor suppressor gene FBXW7 in tumor immunity and sensitivity to immunotherapy. Collectively, these findings establish a novel tumor-intrinsic pathway of resistance and suggest that therapeutic reactivation of viral sensing pathways could improve clinical responses to checkpoint inhibitors in genomically defined populations. Citation Format: Cécile Gstalder, David Liu, Diana Miao, Alexander Devine, Bart Lutterbach, Priya Pancholi, Megha Shettigar, Elizabeth Buchbinder, Scott Carter, Michael Manos, Vanesa Rojas-Rudilla, Ryan Brennick, Evisa Gjini, Ana Lako, Scott Rodig, Charles Yoon, Gordon Freeman, F. Stephen Hodi, Eliezer M. Van Allen, Rizwan Haq. Inactivation of Fbxw7 impairs dsRNA sensing and confers resistance to PD-1 blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A48.

Immune checkpoint inhibitors (ICI) produce durable responses in some melanoma patients, but many patients derive no clinical benefit. The molecular underpinnings of ICI resistance involve intricate cell-cell interactions that are yet elusive. To systematically map the interactions between malignant and immune cells in the tumor ecosystem, we applied single-cell RNA sequencing to 31 human melanoma tumors, profiling thousands of malignant, immune, and stromal cells. We identified a transcriptional program in malignanT-cells that is strongly associated with T-cell exclusion and immunotherapy resistance. Using highly multiplexed in situ imaging we first demonstrated that this program characterizes malignanT-cells in “cold” niches. Next, we showed that the program predicts clinical responses to ICI according to multiple independent validation cohorts, including a new cohort that we obtained from 112 melanoma patients treated with anti-PD-1 therapy. We then identified CDK4/6 as master regulators of this resistance program, and found that CDK4/6 inhibitors repress the program and shift melanoma cells into a senescence-associated secretory phenotype. Lastly, we showed that CDK4/6-inhibition leads to a substantial reduction in melanoma tumor outgrowth in a B16 mouse model when given in combination with immunotherapy. Taken together, our study provides a high-resolution landscape of ICI-resistant cell states, identifies clinically predictive signatures, and forms a basis for the development of novel therapeutic strategies that could overcome immunotherapy resistance. Citation Format: Livnat Jerby, Parin Shah, Michael S. Cuoco, Christopher Rodman, Mei-Ju Su, Johannes M. Melms, Rachel Leeson, Abhay Kanodia, Shaolin Mei, Jia-Ren Lin, Shu Wang, Bokang Rabasha, David Liu, Gao Zhang, Claire Margolais, Orr Ashenberg, Patrick A. Ott, Elizabeth I. Buchbinder, Riz Haq, Stephen Hodi, Genevieve M. Boland, Ryan J. Sullivan, Dennie Frederick, Benchun Miao, Tabea Moll, Keith Flaherty, Meenhard Herlyn, Russell S. Jenkins, Rohit Thummalapalli, Monika S. Kowalczyk, Israel Canadas, Bastian Schilling, Adam N.R Cartwright, Adrienne M. Luoma, Shruti Malu, Patrick Hwu, Chantale Bernatchez, Marie-Andree Forget, David A. Barbie, Alex K. Shalek, Itay Tirosh, Peter K. Sorger, Kai W. Wucherpfennig, Eliezer M. Van Allen, Dirk Schadendorf, Bruce E. Johnson, Asaf Rotem, Orit Rosenblatt-Rozen, Levi A. Garraway, Charles H. Yoon, Benjamin Izar, Aviv Regev. Single-cell RNA-sequencing of metastatic melanoma identifies a cancer cell-intrinsic program associated with immune checkpoint inhibitor resistance [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A082.

Blockade of the PD1/PD-L1 T cell immune checkpoint yields responses and survival benefits exceeding mTOR blockade alone in metastatic renal cell carcinoma (mRCC). However, predictors of response to anti-PD1/PD-L1 therapy are mostly unknown, and pathways of acquired resistance to immune checkpoint therapy are poorly understood. In this study, we undertook whole exome sequencing and neoantigen analysis of matched “trios” (primary tumor resection, immune-checkpoint-refractory metastasis, and germline tissue) from 3 patients treated with anti-PD1 or anti-PD-L1 therapy for mRCC. Patients RCC-001 and RC-002 received anti-PD1 therapy after VEGF-targeted therapy and patient RCC-003 received anti-PD-L1 first-line for mRCC. All 3 patients experienced tumor regression on immune checkpoint therapy, with treatment durations of 10 months, 2 years, and 3 months, respectively. Progressive disease samples were sequenced for analysis of genomic mechanisms of treatment resistance. Whole transcriptome sequencing was obtained on primary and metastatic tumors from RCC-003. Nonsynonymous mutation load was moderate in all samples (range: 33-46 primary, 36-67 resistant). Alterations in VHL were seen in RCC-001 and RCC-002 before and after treatment. RCC-003 had a frameshift deletion in the tumor suppressor NF2 and a nonsense mutation in the MHCI antigen-processing protein TAP1 in both primary and refractory tumors. Resistance-associated mutations in RCC-002 included a frameshift deletion in MR1, involved in MHC I antigen presentation, and missense mutations in TJP1, implicated in JAK/STAT signaling, and PIAS2, implicated in STAT2 and PTEN regulation. JAK2 amplifications have been found to upregulate PD-L1 expression (Green et al. 2010, Blood), and PTEN loss mediates resistance to T cell-mediated immmunotherapy in vitro (Peng et al. 2016, Cancer Discovery). None of the primary tumors harbored alterations in microsatellite-instability-associated genes, and loss of β2 microglobulin was not observed in any sample. Despite moderate mutational loads, each sample harbored a sizeable number of neoantigens (range: 24-76 primary, 50-104 resistant). Across the 6 samples, 28 to 69% of nonsynonymous mutations were predicted to yield at least one neoantigen, with one alteration yielding a maximum of 12 unique neoantigens. Up to 60% of neoantigens observed in the primary tumor were not seen in the resistant setting (range: 20.8-59.2%). Whole transcriptome sequencing in RCC-003 showed 18/24 predicted neoantigens were expressed in the primary tumor and 24/50 in the refractory sample. 13 neoantigens were shared between the two samples, while 5 were unique to the pre-treatment tumor. Integrated whole exome and whole transcriptome sequencing with matched germline profiling identified 5 neoantigens in RCC-003 that were found exclusively in the primary tumor (lost in the treatment-resistant tumor). This could represent immune evasion via deletion of immunogenic mutations, cytotoxic killing of immunogenic tumor clones, or intrinsic resistance to immune checkpoint therapy in heterogenous tumors. Ongoing in vitro analysis of reactivity of predicted neoantigens from all 3 samples with patient-derived T cells will further clarify the biological significance these putative tumor antigens. Citation Format: Diana Miao, Guillermo De Velasco, Dennis Adeegbe, Craig Norton, Dylan Martini, Stephanie Mullane, Raphael Moreira, Sabina Signoretti, Kwok-Kin Wong, Toni Choueiri, Eliezer Van Allen. Genomic and neoantigen evolution and resistance to immune checkpoint blockade in metastatic renal cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A18.

The rise of multiple immunotherapies, such as immune checkpoint blockade, has significantly improved outcomes for multiple patient populations, including melanoma, lung adenocarcinoma, and urothelial cancers, among others. In parallel, the expansion of precision cancer medicine through robust interrogation of cancer genomes at the point of care has enhanced patient stratification for multiple treatment selection strategies and cancer types. The intersection of clinical cancer genomics and immuno-oncology has created an opportunity to learn how the tumor and germline genomes, along with transcriptomic measurements of the immune microenvironment, impact clinical immune checkpoint blockade response. Here, we describe the current landscape of tumor and microenvironmental genomic correlates of immune checkpoint blockade across cancer subtypes, and examine how emerging genomic studies of immunotherapy responders and non-responders may inform patient selection strategies for clinical trial design in these diverse clinical settings. We explore how these approaches, paired with emerging computational biology methodology, may also enable discovery of new mechanisms of selective treatment response. Finally, we examine emerging genomic data on clinical resistance to immunotherapies, from both signaling pathway and neoantigen evolution perspectives, and discuss the implications for combination treatment strategies. Overall, the convergence of comprehensive clinical genomics and immuno-oncology treatment strategies may inform precision medicine for immunotherapies across cancer types. Citation Format: Eliezer M. Van Allen. Genomic correlates of response and resistance to immune checkpoint blockade. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr IA26.