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Supplementary table from Mutant IDH Inhibits IFNγ–TET2 Signaling to Promote Immunoevasion and Tumor Maintenance in Cholangiocarcinoma

Supplementary Figure from Mutant IDH Inhibits IFNγ–TET2 Signaling to Promote Immunoevasion and Tumor Maintenance in Cholangiocarcinoma

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

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

Syngeneic mouse models are tumors derived from murine cancer cells engrafted on genetically identical mouse strains. They are widely used tools for studying tumor immunity and immunotherapy response in the context of a fully functional murine immune system. Large volumes of syngeneic mouse tumor expression profiles under different immunotherapy treatments have been generated, although a lack of systematic collection and analysis makes data reuse challenging. We present Tumor Immune Syngeneic MOuse (TISMO), a database with an extensive collection of syngeneic mouse model profiles with interactive visualization features. TISMO contains 605 in vitro RNA-seq samples from 49 syngeneic cancer cell lines across 23 cancer types, of which 195 underwent cytokine treatment. TISMO also includes 1518 in vivo RNA-seq samples from 68 syngeneic mouse tumor models across 19 cancer types, of which 832 were from immune checkpoint blockade (ICB) studies. We manually annotated the sample metadata, such as cell line, mouse strain, transplantation site, treatment, and response status, and uniformly processed and quality-controlled the RNA-seq data. Besides data download, TISMO provides interactive web interfaces to investigate whether specific gene expression, pathway enrichment, or immune infiltration level is associated with differential immunotherapy response. TISMO is available at http://tismo.cistrome.org.

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.

Natural killer (NK) cells reject major histocompatibility complex class I (MHC-I)–deficient bone marrow through direct cytotoxicity but not solid organ transplants devoid of MHC-I. Here, we demonstrate an immediate switch in NK cell function upon exit from the circulation, characterized by a shift from direct cytotoxicity to chemokine/cytokine production. In the skin transplant paradigm, combining an NK cell–specific activating ligand, m157, with missing self MHC-I resulted in complete graft rejection, which was dependent on NK cells as potential helpers and T cells as effectors. Extracellular matrix proteins, collagen I, collagen III, and elastin, blocked NK cell cytotoxicity and promoted their chemokine/cytokine production. NK cell cytotoxicity against MHC-I–deficient melanoma in the skin was markedly increased by blocking tumor collagen deposition. MHC-I down-regulation occurred in solid human cancers but not leukemias, which could be directly targeted by circulating cytotoxic NK cells. Our findings uncover a fundamental mechanism that restricts direct NK cell cytotoxicity in peripheral tissues.

The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1

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.

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 tumour cells or host immune cells promotes anti-tumour immunity. However, inhibitors of PTPN2 with suitable pharmacokinetic properties for oral administration have not been described. Here, we present the characterization of ABBV-CLS-484 (A484), a potent active site inhibitor of PTPN2 and the closely related phosphatase PTPN1. A484 treatment in vitro amplifies the response to interferon gamma, and monotherapy A484 treatment generates robust anti-tumour immunity in several murine cancer models. Through in vivo studies and single cell transcriptional profiling of tumour-infiltrating lymphocytes (TIL) from A484-treated mice, we show that A484 inflames the tumour microenvironment and promotes CD8+ T cell function by enhancing cytokine signaling and decreasing T cell exhaustion and dysfunction. Our results demonstrate that oral administration of small molecule inhibitors of PTPN2/N1 can induce potent anti-tumour immunity in mouse models. PTPN2/N1 inhibitors offer a promising new strategy for cancer immunotherapy and are currently being evaluated clinically in patients with advanced solid tumours (NCT04777994). More broadly, our study shows that small molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to current antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge A484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics targeting this important class of enzymes. Citation Format: Hakimeh Ebrahimi-Nik, Arvin Iracheta-Vellve, Kira E. Olander, Thomas R.G. Davis, Sarah Y. Kim, Mitchell D. Yeary, James C. Patti, Tyler M. Balon, Omar Ismail Avila, Cun Lan Chuong, Meng-Ju Wu, Christina K. Baumgartner, Keith M. Hamel, Kathleen A. McGuire, Rebecca Mathew, Carey Backus, Ian C. Kohnle, Zhaoming Xiong, Elliot P. Farney, Jennifer M. Frost, Geoff T. Halvorsen, Matthew Rees, Andrew Boghossian, Melissa Ronan, Jennifer A. Roth, Todd R. Golub, Gabriel K. Griffin, Nabeel El-Bardeesy, Clay C. Beauregard, Philip R. Kym, Kathleen B. Yates, Robert T. Manguso. Small molecule inhibition of PTPN2/1 inflames the tumour microenvironment and unleashes potent CD8+ T cell immunity [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A41.

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, with concordant findings in matched patient-derived organotypic tumor spheroids (PDOTS) and matched patient-derived organoids (PDOs). Tumor 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, Robert T. Manguso, Russell W Jenkins. Targeting TBK1 to overcome resistance to cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B28.

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.