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Isocitrate dehydrogenase 1 mutations (mIDH1) are common in cholangiocarcinoma. (R)-2-hydroxyglutarate generated by the mIDH1 enzyme inhibits multiple α-ketoglutarate–dependent enzymes, altering epigenetics and metabolism. Here, by developing mIDH1-driven genetically engineered mouse models, we show that mIDH1 supports cholangiocarcinoma tumor maintenance through an immunoevasion program centered on dual (R)-2-hydroxyglutarate–mediated mechanisms: suppression of CD8+ T-cell activity and tumor cell–autonomous inactivation of TET2 DNA demethylase. Pharmacologic mIDH1 inhibition stimulates CD8+ T-cell recruitment and interferon γ (IFNγ) expression and promotes TET2-dependent induction of IFNγ response genes in tumor cells. CD8+ T-cell depletion or tumor cell–specific ablation of TET2 or IFNγ receptor 1 causes treatment resistance. Whereas immune-checkpoint activation limits mIDH1 inhibitor efficacy, CTLA4 blockade overcomes immunosuppression, providing therapeutic synergy. The findings in this mouse model of cholangiocarcinoma demonstrate that immune function and the IFNγ–TET2 axis are essential for response to mIDH1 inhibition and suggest a novel strategy for potentiating efficacy. Significance: Mutant IDH1 inhibition stimulates cytotoxic T-cell function and derepression of the DNA demethylating enzyme TET2, which is required for tumor cells to respond to IFNγ. The discovery of mechanisms of treatment efficacy and the identification of synergy by combined CTLA4 blockade provide the foundation for new therapeutic strategies. See related commentary by Zhu and Kwong, p. 604. This article is highlighted in the In This Issue feature, p. 587

Cancer immunotherapy with immune checkpoint blockade (ICB) has transformed the treatment of melanoma, although intrinsic or acquired resistance develops in nearly half of patients. Tumor-infiltrating CD8+ T lymphocytes (TILs) are key determinants of anti-tumor immunity in melanoma and other cancers, and single-cell RNA-sequencing has identified T cell states associated with improved clinical response to ICB, as well as adoptive T cell therapy (ACT). Despite these advances, strategies to identify and analyze tumor-reactive TILs in ICB-resistant patients remain limited. Here, we demonstrate that TILs from ICB-resistant melanoma patients can recognize and eliminate autologous tumor cells independent of class I HLA-TCR interactions. TILs eliminated matched melanoma cells in a time and dose-dependent fashion associated with secretion of effector cytokines. Strikingly, the deletion of B2M (resulting in loss of class I HLA surface expression) did not alter the activity of these TILs. Immunophenotyping studies confirmed that TILs are largely (>95%) effector memory (Tem) CD8 T cells (CD45RA-CD45RO+CCR7-) and give rise to terminal effector cells after co-culture with matched melanoma cells. Further, the elimination of melanoma cells by TILs required intact JAK1/2 signaling, although interferon-gamma (IFNγ) was neither necessary nor sufficient for tumor cell elimination. Together, these findings demonstrate that expanded TILs from ICB-resistant melanoma patients are capable of eliminating melanoma cells via a novel, class I MHC-independent mechanism. Citation Format: Hongyan Xie, Aiping Jiang, Anne Jenney, Yi Sun, Tatyana Sharova, Moshe Sade-Feldman, Or-yam Revach, Angelina Cicerchia, Martin Q. Rasmussen, Nir Hacohen, Robert T. Manguso, Russell W. Jenkins. Class I HLA-independent lysis of immunotherapy-resistant melanoma by CD8 T cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4072.

Despite the unprecedented success of immune checkpoint blockade (ICB) in melanoma and other cancers, therapeutic resistance remains a major challenge. CD38, an ecto-enzyme involved in NAD+ catabolism, is upregulated in dysfunctional/exhausted CD8+ T cells in human melanoma. As enrichment of dysfunctional CD8+ T cells is associated with lack of response to ICB, and NAD+ depletion via enhanced CD38 activity has been associated with diminished immune response, we hypothesized that CD38 might represent an attractive target to overcome resistance to ICB. While CD38 blockade has been shown to augment response to ICB in murine tumor models, the specific role of CD38 signaling in CD8 T cell exhaustion, and the therapeutic relevance of CD38 in human cancer have not been defined. Here, we confirm and extend previous observations that CD38 is enriched in exhausted CD8+ T cells during tumor progression and in response to ICB. Further, we demonstrate that disrupting CD38 signaling via neutralizing antibodies or pharmacological inhibition enhances response to ICB using established murine tumor models and patient-derived tumor explant models. Notably, we observed 54% response rate to combination PD-1/CD38 blockade using a cohort (n=35) of patient-derived organotypic tumor spheroids (PDOTS) from patients with ICB-refractory melanoma (11.4% response rate to single-agent PD-1 blockade). Supplementation of nicotinamide riboside (NR) to boost NAD+ levels mimicked the effects CD38 blockade/inhibition on PD-1 response, whereas FK866, an inhibitor of NAMPT a key step in NAD+ biosynthesis, blunted the response to combined PD-1/CD38 blockade, suggesting a major role for NAD+ in the efficacy of CD38 blockade/inhibition. Lastly, we demonstrate that in vitro CD38 inhibition/blockade in CD8+ T cells increases the levels of TCF7 (an effector/memory T cell transcription factor), decreases surface expression of co-inhibitory receptors (e.g., TIM-3, CD39, PD-1), and improves effector CD8 T cell function. Taken together, these data confirm a role for CD38 in CD8+ T cell exhaustion in melanoma and support further pre-clinical and clinical development of this novel therapeutic strategy to enhance anti-tumor immune responses. Citation Format: Or-Yam Revach, Angelina M. Cicerchia, Moshe Sade-Feldman, Seth Anderson, Robert T. Manguso, Nir Hacohen, Russell W. Jenkins. Targeting CD38 on exhausted T cells overcomes resistance to cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3250.

Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking. We identified the innate immune kinase TANK-binding kinase 1 (TBK1) as a candidate immune evasion gene in a pooled genetic screen. Using a suite of genetic and pharmacologic tools across multiple experimental model systems, we confirm a role for TBK1 as an immune evasion gene. Targeting TBK1 enhances response to PD-1 blockade by lowering the cytotoxicity threshold to effector cytokines (TNFα/IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumor models. Cancer cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNFα/IFNγ in a JAK/STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is a novel and effective strategy to overcome resistance to cancer immunotherapy. Citation Format: Yi Sun, Or-Yam Revach, Seth Anderson, Anne Jenney, Caitlin E. Mills, Payal Tiwari, Robert T. Manguso, Russell William Jenkins. Targeting TBK1 to overcome resistance to cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3285.

BackgroundImmune checkpoint blockade (ICB) response in recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) is limited to 15%–20% of patients and underpinnings of resistance remain undefined.MethodsStarting with an anti-PD1 sensitive murine HNSCC cell line, we generated an isogenic anti-PD1 resistant model. Mass cytometry was used to delineate tumor microenvironments of both sensitive parental murine oral carcinoma (MOC1) and resistant MOC1esc1 tumors. To examine heterogeneity and clonal dynamics of tumor infiltrating lymphocytes (TILs), we applied paired single-cell RNA and TCR sequencing in three HNSCC models.ResultsAnti-PD1 resistant MOC1esc1 line displayed a conserved cell intrinsic immune evasion signature. Immunoprofiling showed distinct baseline tumor microenvironments of MOC1 and MOC1esc1, as well as the remodeling of immune compartments on ICB in MOC1esc1 tumors. Single cell sequencing analysis identified several CD8 +TIL subsets including Tcf7 +Pd1− (naïve/memory-like), Tcf7 +Pd1+ (progenitor), and Tcf7-Pd1+ (differentiated effector). Mapping TCR shared fractions identified that successful anti-PD1 or anti-CTLA4 therapy-induced higher post-treatment T cell lineage transitions.ConclusionsThese data highlight critical aspects of CD8 +TIL heterogeneity and differentiation and suggest facilitation of CD8 +TIL differentiation as a strategy to improve HNSCC ICB response.

Background: Pharmacologic inhibition of PTPN2 and PTPN1 (PTPN2/N1) represents a novel therapeutic approach in immuno-oncology that augments innate and adaptive immune responses in addition to enhancing tumor cell sensitivity to immune-mediated killing. PTPN2/N1 emerged as top hits in an in vivo CRISPR screen to identify tumor-intrinsic targets that enhance sensitivity and overcome resistance to anti-PD-1 treatment. PTPN2/N1 are phosphatases that act as negative regulators in numerous pathways including immune activation. While phosphatases have long been of interest, they are challenging drug targets, and the active site had been considered undruggable. Results: Here we report the discovery of the highly selective, active site PTPN2/N1 small molecule inhibitor, ABBV-CLS-484. Highly optimized ligand-protein interactions have led to the design of sub-nanomolar PTPN2/N1 inhibitors, confirmed through x-ray crystallography. PTPN2/N1 inhibitors increase the activation and function of cytotoxic T cells as well as increase the pro-inflammatory properties of CD103+ dendritic cells and macrophages in vitro. However, they do not cause non-specific activation in the absence of stimulation; rather, they augment signaling in cells that are already activated. PTPN2/N1 inhibition also has effects directly on tumor cells, where it amplifies sensitivity to immune-mediated killing by enhancing the interferon response. ABBV-CLS-484 promotes anti-tumor immunity as monotherapy and in combination with anti-PD-1 leading to dramatic tumor regression, even in models resistant to anti-PD-1 treatment such as 4T1, or those with minimal inflammation such as EMT6. Single-cell RNAseq analyses of tumor-infiltrating immune cells confirmed activation of T cells and demonstrated switching of myeloid-derived suppressor cells towards a proinflammatory phenotype, thereby revealing a distinct mechanism of action of ABBV-CLS-484 compared with PD-1 blockade. Our results show that PTPN2/N1 inhibitors have complementary effects on the immune system and tumor microenvironment that act to promote effective tumor killing. Based on these robust preclinical data, phase I clinical trials of ABBV-CLS-484 alone and in combination with an anti-PD-1 agent have been initiated to establish the safety, tolerability, and efficacy in diverse solid tumor indications. Conclusions: We have discovered a first-in-class PTPN2/N1 inhibitor, which represents a promising novel immunotherapy that both enhances the immune response and increases tumor sensitivity to immune-mediated killing. ABBV-CLS-484 is currently being evaluated in phase I clinical trials in patients with advanced solid tumors, as a monotherapy or in combination with a PD-1 targeting agent (NCT04777994). Citation Format: Christina K. Baumgartner, Marcia N. Paddock, Jennifer M. Frost, Keith M. Hamel, Kathleen A. McGuire, Kyle Halliwill, Zhaoming Xiong, Liang Mu, Kelly Klinge, Prasanthi Geda, Jaqueline Aguado, Marinka Bulic, Elliot P. Farney, Kathleen B. Yates, Robert T. Manguso, Clay Beauregard, Philip R. Kym. ABBV-CLS-484: An active site PTPN2/N1 inhibitor that augments the immune response and sensitizes tumors to immune-mediated killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND06.

Immune checkpoint blockade is effective for a subset of patients across many cancers, but most patients are refractory to current immunotherapies and new approaches are needed to overcome resistance. The protein tyrosine phosphatase PTPN2 is a central regulator of inflammation, and genetic deletion of PTPN2 on either tumor cells or host immune cells promotes anti-tumor immunity. However, inhibitors of PTPN2 have not been described. Here, we present the validation of ABBV-CLS-484, a potent catalytic inhibitor of PTPN2 and the closely related phosphatase PTPN1. ABBV-CLS-484 treatment of tumor cells in vitro phenocopies the genetic deletion of PTPN2/N1, causing both amplified transcriptional responses to IFNg and reduced cell viability across human cancer cell lines. Monotherapy ABBV-CLS-484 treatment generates robust anti-tumor immunity in several murine cancer models with efficacy comparable to anti-PD-1 treatment. Through genetic studies, we show that while ABBV-CLS-484 can act on both tumor cells and the host immune system, IFN sensing and PTPN2/N1 expression on tumor cells are not always required for efficacy, suggesting that PTPN2/N1 inhibition on host immune cells may be sufficient for activity of the drug. Through scRNAseq profiling of TILs from both ABBV-CLS-484-treated and anti-PD-1-treated tumors, we show that ABBV-CLS-484 induces unique transcriptional changes to both myeloid and lymphoid populations in the tumor microenvironment which are dominated by enhanced IFN sensing and a shift from suppressive to pro-inflammatory phenotypes. ABBV-CLS-484 treatment enhances the activation and effector functions of CD8+ T cells while decreasing the expression of genes classically associated with T cell exhaustion and dysfunction such as Tox. The efficacy of ABBV-CLS-484 is critically dependent on CD8+ T cells and treatment with ABBV-CLS-484 results in greater levels of T cell infiltration into tumors and a more diverse repertoire of expanded T cell clones relative to anti-PD-1. Thus, the PTPN2/N1 inhibitor ABBV-CLS-484 is a highly effective immunotherapy with monotherapy efficacy across mouse tumor models. Small molecule inhibitors of PTPN2 offer a promising new strategy for cancer immunotherapy by targeting an IFN signaling checkpoint and are currently being evaluated clinically in patients with advanced solid tumors (NCT04777994). Citation Format: Arvin Iracheta-Vellve, Hakimeh Ebrahimi-Nik, Thomas R. Davis, Kira E. Olander, Sarah Y. Kim, Mitchell D. Yeary, James C. Patti, Ian C. Kohnle, Christina K. Baumgartner, Keith M. Hamel, Kathleen A. McGuire, Cun Lan Chuong, Zhaoming Xiong, Elliot P. Farney, Jennifer M. Frost, Matthew Rees, Andrew Boghossian, Melissa Ronan, Jennifer A. Roth, Todd R. Golub, Gabriel K. Griffin, Clay Beauregard, Philip R. Kym, Kathleen B. Yates, Robert T. Manguso. Targeting the immune checkpoint PTPN2 with ABBV-CLS-484 inflames the tumor microenvironment and unleashes potent CD8+ T cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 606.

Intrahepatic cholangiocarcinoma (ICC) is a cancer of the liver bile ducts, which has been rising in incidence and carries a 5-year survival rate of less than 10%. Mutations in isocitrate dehydrogenase 1 and 2 (mIDH1/2) are among the most common genetic alterations in ICC, present in 20% patients. The hot-spot point mutations of IDH result in aberrant enzymatic function, reducing alpha-ketoglutarate (alphaKG) to the oncometabolite, 2-hydroxyglutarate (2-HG), which accumulates to millimolar concentrations within the cell. 2-HG competitively inhibits alphaKG-dependent histone and DNA demethylating enzymes, thereby altering chromatin regulation. The first-in-class small molecule selective IDH1m inhibitor, ivosidenib, has shown benefit in phase I and III trials for patients with IDH1m ICC. In order to gain insight into the mechanisms underlying the response to IDH1m inhibition, we developed the first genetically engineered mouse model (GEMM) of IDH1m ICC. We found that ivosidenib reduced the overall tumor burden and increased overall survival of syngeneic allografts of ICCs derived from the GEMM. The positive response to ivosidenib was associated with an induction of mature hepatocyte lineage markers and a reduction of tumor produced 2-HG >85%, supporting observations from the ivosidenib phase I data in ICC patients that mIDH1 inhibition may promote ICC tumor cell differentiation. In addition, there was pronounced induction of immune stimulatory interferon signaling, increased recruitment of CD8+ T cells to the tumors and strong PD-L1 upregulation. Thus, ivosidenib treatment leads to a sustained inhibition of 2-HG production, which correlated with an increased immune stimulation compared to vehicle treatment. We will present these data as well as ongoing studies interrogating the immune and transcriptomic changes with further resolution via fluorescence-activated cell sorting and single-cell RNA-sequencing analysis, direct functional testing of contributions of specific immune populations to the therapeutic efficacy of ivosidenib, and testing ivosidenib in combination with checkpoint inhibition. These studies highlight the impact of ivosidenib on hepatocyte differentiation and immune stimulation in IDH1m ICC and support the key role of IDHm in ICC tumor maintenance. Citation Format: Meng-Ju Wu, Lei Shi, Juan Dubrot, Christine Hudson, Joshua Merritt, Ramzi Adil, Qibiao Wu, Sebastien Ronseaux, Rohini Narayanaswamy, Robert Manguso, Brandon Nicolay, Nabeel Bardeesy. The mutant IDH1 inhibitor ivosidenib promotes tumor cell differentiation and anti-tumor immunity in intrahepatic cholangiocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1810.

The ability to make rapid genetic alterations to tumor cells with CRISPR for screens and single-gene studies has been transformative for studying cancer biology. Recently, we and others have used CRISPR in murine tumor models in vivo to identify targets and resistance mechanisms for immunotherapy. However, the use of these techniques in immunocompetent mice is limited by endogenous immune recognition of CRISPR components such as Cas9. Here we report the design of a screening-compatible lentiviral vector system which allows selective CRISPR antigen removal (SCAR) from tumor cells before they are implanted in vivo. The SCAR system enables highly efficient genome engineering and reverses CD8+ T cell-mediated rejection of CRISPR modified tumor cells in vivo. Using this technology, we conducted a pooled, in vivo screen designed to probe tumor-immune interactions in the highly immunogenic CT26 colon carcinoma model and discovered that these tumors require intact interferon signaling to evade immune destruction. The SCAR system enables the study of tumor-immune interactions in any cancer model and can be more broadly applied to generate non-immunogenic lentivirally-engineered cells. Citation Format: Juan Dubrot, Sarah Kate Lane-Reticker, Emily Kessler, Clara Wolfe, Animesh Mahapatra, Peter Du, Robert T. Manguso. SCAR (Selective CRISPR Antigen Removal) vector system expands cancer immunology discovery with in vivo functional genomics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5866.

Despite the unprecedented success of immune checkpoint blockade (ICB) in melanoma and other cancers, tackling innate (primary) resistance remains a major challenge and robust biomarkers to guide treatment are lacking. Clinical trials are already underway evaluating novel immune modulatory agents in combination with anti-PD-1/PD-L1 therapies in an effort to overcome innate resistance. Despite increasing reports of ‘rational’ combination strategies, these therapies remain “one size fits all”, due the lack of robust biomarkers to guide clinical decision-making. There is an unmet need for novel approaches, tools, techniques, and methods for pre-clinical and clinical use to better understand mechanisms of response and resistance to immune checkpoint inhibitors and next-generation anti-tumor immune modulatory drugs. We have adapted a 3D microfluidic device to the short-term culture of murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS)for functional ex vivo profiling of PD-1 blockade in a model tumor immune microenvironment (TIME) to facilitate identification of mediators of response and resistance to ICB and to guide development of next-generation immunotherapy combinations. The MDOTS/PDOTS platform provides a window into the complex and dynamic events of the TIME during response (and resistance) to ICB and is an ideal model system for pre-clinical evaluation of novel cancer immunotherapy targets. Focused evaluation of novel treatment combinations using MDOTS identified TANK-binding kinase 1 (TBK1) as a novel cancer immunotherapy target, mirroring in vivoefficacy of this treatment approach. TBK1 is a Ser/Thr kinase involved in innate immune signaling and is an emerging target for anti-cancer therapy. Importantly, independent orthogonal data from two different laboratories has also identified TBK1 as a cancer immunotherapy target.We have confirmed that Tbk1 deletion and/or TBK1 pharmacologic inhibition enhances response to in vivo anti-PD-1 therapy, and have characterized the impact of deletion of Tbk1 (CRISPR) or pharmacologic inhibition of TBK1 (Cmpd1) on the tumor-immune microenvironment.Lastly, we confirmed that resistance to PD-1 blockade can be overcome with pharmacologic TBK1 inhibition using murine- and patient-derived organotypic tumor spheroids in 3D microfluidic culture. These findings confirm TBK1 as a target to overcome resistance to PD-1 blockade, further supporting the pre-clinical and clinical development of this novel combination strategy. REFERENCES 1. Jenkins RW, Barbie DA, Flaherty KT. Mechanisms of resistance to immune checkpoint inhibitors. Br J Cancer. 2018;118(1):9-16. doi: 10.1038/bjc.2017.434. PubMed PMID: 29319049; PMCID: PMC5765236. 2. Manguso RT, Pope HW, Zimmer MD, Brown FD, Yates KB, Miller BC, Collins NB, Bi K, LaFleur MW, Juneja VR, Weiss SA, Lo J, Fisher DE, Miao D, Van Allen E, Root DE, Sharpe AH, Doench JG, Haining WN. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature. 2017. doi: 10.1038/nature23270. PubMed PMID: 28723893. 3. Pan D, Kobayashi A, Jiang P, Ferrari de Andrade L, Tay RE, Luoma AM, Tsoucas D, Qiu X, Lim K, Rao P, Long HW, Yuan GC, Doench J, Brown M, Liu XS, Wucherpfennig KW. A major chromatin regulator determines resistance of tumor cells to T cell-mediated killing. Science. 2018;359(6377):770-5. doi: 10.1126/science.aao1710. PubMed PMID: 29301958. Citation Format: Ajaykumar Vishwakarma, Yi Sun, Amina Fu, Emily Robitschek, Arvin Iracheta-Vellve, Susanna Stinson, Aliasger Salem, Robert Manguso, Russell Jenkins. TANK-Binding Kinase 1 (TBK1) as a novel cancer immunotherapy target [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B065. doi:10.1158/1535-7163.TARG-19-B065

Antibodies targeting coinhibitory receptors on T cells (“checkpoint blockade”) have emerged as some of the most promising therapies for a broad range of malignancies, including melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin’s lymphoma, and bladder cancer. These coinhibitory molecules include CTLA-4, PD-1, LAG-3, TIM-3, and others. The anti-CTLA-4 antibody ipilimumab was approved in 2011 and the anti-PD-1 antibodies pembrolizumab and nivolumab were approved in 2014 for patients with advanced melanoma. Single agent checkpoint blockade is associated with 20–40 % objective response rates in advanced melanoma with improved overall survival. The combination of anti-CTLA-4 and anti-PD-1 antibodies leads to an increased durable response rate compared to either antibody alone, supporting the concept that combination therapy may result in increased clinical benefit. An important goal in the field is to combine checkpoint blockade with other immunotherapies and other types of therapy (e.g., radiation, targeted therapy, chemotherapy, surgery) to increase the fraction of patients that have objective and durable responses. Here, we discuss the current understanding of the mechanisms underlying checkpoint blockade and the rationale for combination therapy. We then discuss potential immunotherapeutic and non-immunotherapeutic combination therapies. Finally, we discuss critical issues that need to be addressed in order to develop combination strategies to induce long-term clinical responses in patients with cancer.

T-cell exhaustion is a heterogeneous state, with recent work in chronic viral infections revealing at least two subtypes with different functional properties: "stem-like" and "terminal" exhausted cells. Whether these two populations exist in tumors and have different roles to control tumor growth remains unknown. We performed single-cell RNA-seq on exhausted CD8+ T cells from chronic virally infected mice, generating unique transcriptional signatures of both exhausted populations. By transcriptional enrichment and flow cytometry, we discovered both the stem-like and terminal exhausted populations in the B16 mouse melanoma model and confirmed their presence in human melanoma samples with multiplex immunofluorescence. ATAC-seq revealed that these two subpopulations are defined by state-specific changes in chromatin accessibility. These states are shared between exhausted CD8+ T cells from both viral and tumor models, demonstrating a common epigenetic program of T-cell dysfunction. The two populations have different functional properties, with the stem-like cells having a more polyfunctional cytokine response whereas terminal cells are more cytotoxic. When transferred into new tumor-bearing mice, stem-like cells were better able to control tumor growth, suggesting superior long-term functionality. Checkpoint blockade with anti-PD-1 is known to increase CD8+ T cell numbers in the tumor microenvironment, but it is not known which subpopulation responds to therapy. Treatment with anti-PD-1 antibody resulted in proliferation of the stem-like population, which differentiate into the more cytotoxic terminal exhausted cells. We have shown that two subpopulations of exhausted CD8+ T cells exist in mouse and human tumors, and anti-PD-1 therapy activates the stem-like CD8+ T cells to promote tumor control. Citation Format: Brian C. Miller, Debattama R. Sen, Rose Al-Abosy, Kevin Bi, Kathleen B. Yates, Evisa Gjini, Kristen Felt, Robert T. Manguso, Scott J. Rodig, Arlene H. Sharpe, Nicholas Haining. Distinct subsets of dysfunctional CD8+ T cells underlie response to checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4683.

Anti-PD-1 therapy is an important new treatment option for many different types of malignancies, but overall response rates are less than 40%. Limited understanding of how anti-PD-1 treatment changes the tumor immune microenvironment is a barrier to identifying rational combination therapies and understanding mechanisms of immunotherapy resistance. To overcome this barrier, we set out to understand the mechanisms by which anti-PD-1 therapy augments the anti-tumor immune response using single-cell genomics. We have developed a massively parallel single-cell RNA-sequencing platform (“Seq-Well”) that uses a fabricated chip with nearly 100,000 nanowells into which barcoded beads and individual cells are distributed prior to lysis and RNA capture. We used this platform to comprehensively define the global expression profile of all major immune lineages in the tumor microenvironment in a mouse tumor model of immunotherapy. Mice were implanted with the B16F10 melanoma transplantable model of cancer and treated with anti-PD-1 or control antibodies. Tumors were harvested and CD45+ tumor-infiltrating leukocytes isolated by FACS. In a single experiment we were able to sequence the transcriptomes of over 600 individual cells, allowing us to clearly distinguish different immune lineages within the tumor microenvironment. We detect two transcriptionally distinct populations of CD8+ T cells, one that is highly proliferative (as marked by Ki-67), and one that has higher expression of cytotoxic markers (i.e. perforin). The Ki-67hi population is enriched for a gene expression signature from terminally exhausted CD8+ T cells in chronic viral infection, suggesting that this is a more exhausted subset. Treatment with anti-PD-1 globally alters the tumor microenvironment, including enriching for CD8+ T cells in the Prfhi subpopulation compared with the Ki-67hi more terminally exhausted population. Studies to understand changes in the immune infiltrate of immunotherapy resistant tumors are currently ongoing. In conclusion, massively parallel single-cell RNA-sequencing allows us to dissect the mechanisms by which checkpoint blockade controls tumor growth, revealing shifts in the differentiation state of exhausted CD8+ T cells induced by checkpoint blockade. Citation Format: Brian C. Miller, Marc H. Wadsworth, Kevin Bi, Travis K. Hughes, Robert Manguso, Arlene H. Sharpe, Alex K. Shalek, Nicholas Haining. Dissecting mechanisms of anti-PD-1 therapy with massively parallel single-cell RNA-sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3027. doi:10.1158/1538-7445.AM2017-3027

OVERVIEW: Despite the impact of cancer immunotherapy, expanding its clinical utility requires a rational method for identifying combination therapies and resistance mechanisms. This is pertinent to glioblastoma (GBM), where initial trials show uncertain response rates. Functional genomic screens used to identify new therapies and escape mechanisms were generally conducted in vitro, where interaction with the immune system is absent. Here we present the development of a high-throughput, in vivo, loss-of-function screening platform for GBM immune-escape mechanisms. METHODS: CT2A- and GL261-astrocytoma cells bearing Cas9-endonuclease were engineered to express a library of barcoded single guide RNAs (sgRNAs). These cells form tumors when implanted intracranially in immunocompetent mice. Tumor-bearing mice were treated with vaccination or PD-1 checkpoint blockade. Dropout of sgRNAs targeting putative immune evasion molecules or enrichment of sgRNAs mimicking resistance mechanisms were detected using next-generation sequencing at the time of tumor implantation and harvest post-immunotherapy. RESULTS: We first developed an in vivo, pooled, loss-of-function genetic screen using Cas9/CRISPR genome editing in mouse transplantable tumors subjected to titratable, selective immune-pressure. Screening 2,400 genes expressed by melanoma cells for those that synergize with or cause resistance to checkpoint blockade recovered known immune-evasion molecules PD-L1 and CD47. Novel immunotherapy targets validated individually, identifying essential pathways of immune-evasion. We then sought to recapitulate this approach in the CNS, and showed that 500-1000 genes can be functionally screened under graded immune-pressure. In optimized immune-competent CT2A and GL261 GBM models, tumors derived from cancer stem cell CD133hi-rich neurospheres were sensitive to immunotherapy and more aggressive and infiltrative than tumors derived from adherent tumor cells. A neurosphere based immune-competent model can be scaled up to a whole-genome screen due to a shorter experimental time requirement and improved engraftment allowing for functional screening of >1000 genes. We curated a GBM-specific library based on differential in vitro and in vivo gene expression profiles of CT2A and GL261 cells exposed to graded immune pressure. We are now screening this GBM-specific library in our optimized in vivo pooled loss-of-function genetic screen using immune-pressure titration to identify novel immunotherapy targets in GBM. CONCLUSIONS: This assay provides the first high-throughput method for systematically identifying resistance mechanisms and new candidate targets for immunotherapy in CNS tumors. Our optimized model could be scaled up to whole-genome loss of function screens, serving as an important tool for identification of next-generation and combination immunotherapies. Citation Format: Martha R. Neagu, Robert T. Manguso, Hans Pope, Maria C. Speranza, Gordon J. Freeman, John Doench, Arlene H. Sharpe, William Nicholas Haining. Defining molecular mechanisms of resistance to glioblastoma immunity using a novel CRISPR/Cas9 in vivo loss-of-function screening platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 417. doi:10.1158/1538-7445.AM2017-417