Abstract Adoptive cell transfer (ACT) has demonstrated potent anti-tumor efficacy in melanoma, yet therapeutic resistance frequently emerges within immunosuppressive tumor microenvironments. Indoleamine 2,3 dioxygenase 1 (IDO1) is a tryptophan catabolizing enzyme that generates kynurenine (Kyn), an immunomodulatory metabolite known to suppress effector T cell function. Here, we show that tumor overexpression of IDO1 undermines ACT efficacy through dual mechanisms of T cell exclusion and cytotoxic impairment. Using murine B16 melanoma cells engineered to overexpress IDO1 (B16IDO1), we observed elevated Kyn levels, reduced CXCL9/10 and CCL5 chemokine expression, and decreased intratumoral T cell infiltration. In vitro, IDO1 expressing tumor cells exhibited resistance to killing by activated CD8 PMEL T cells, a phenotype dependent on soluble factors in conditioned media and reversible with pharmacologic IDO1 inhibition. In vivo, adoptive transfer of either PMEL or TRP1 T cells, CD4 T cells specific for tyrosinase-related protein 1, failed to control B16IDO1 tumors, correlating with decreased infiltration, function, and survival. Pharmacologic blockade of IDO1 enhanced T cell infiltration and improved ACT mediated tumor control. Together, these findings identify IDO1 as a regulator of ACT resistance by suppressing T cell trafficking and effector activity. Complementary analyses of patient TIL, tumor, and serum samples are underway to assess correlations between IDO1/Kyn levels and immune cell burden, underscoring clinical relevance. Further studies extend this framework to human models, including IDO1+ melanoma xenografts and CAR T cells, to explore the translational potential of targeting the IDO1-Kyn-AHR axis to enhance cellular immunotherapy efficacy. Citation Format: Mamadou Alpha Bah, Rachana Maniyar, Jonathan F. Khan, Anais Assouvie, Sadna Budhu, Parwiz Abrahimi, Inna Serganova, Gabrielle A. Rizzuto, Taha Merghoub, Jedd D. Wolchok. Tumor IDO1 drives resistance to adoptive cell transfer by suppressing T cell recruitment and effector function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6537.

Effective combination immunotherapy regimens increase the success rate of treating cancer patients than either monotherapy alone. Ferroptosis is a novel form of cell death that has gained popularity in the last decade as a potential tool to be used against cancer progression but has not had much success clinically. Currently, several groups are studying the relevance of ferroptosis in the context of immunotherapy in an attempt to expand its clinical translatability as well as to overcome the lack of response to immunotherapy alone. The field though is divided regarding the nature of the effects of ferroptosis inducers on the immune system. While some reports suggest that ferroptosis inducers are immunosuppressive, others demonstrate the immune promoting effects elicited by ferroptosis, making it hard to effectively design a combination therapy with clinical potential. A possible explanation is the use of different tumor models or different types of ferroptosis inducers across these studies. In order to rigorously interrogate the use ferroptosis in an immunotherapy regimen, we have evaluated the impact of different classes of ferroptosis inducers with various different immune modulators in several pre-clinical cancer models both in vivo and in vitro. Interestingly, we observed that within the same preclinical model of metastatic melanoma, the choice of the immune modulator used can significantly alter the directionality of the effects of ferroptosis on tumor progression. We confirm that treatment with ferroptosis inducers increases the ability of tumor cells to activate T cells in vitro irrespective of the type of inducer. On the other hand, the quality of immune response in vivo, varies with the class of ferroptosis inducer. We subsequently, designed a regimen with a CD40 agonist that boosts the tumor immunogenic impact of ferroptosis and enhances the activation of the adaptive immune system. This regimen reduces the progression of the treated tumor, elicits an abscopal effect and improves the survival of mice as well. While FDA approved immune checkpoint blockade such as anti-PD1 therapy elicits variable responses based on the type of ferroptosis inducer used, our regimen is effective with either class of ferroptosis inducer in both melanoma and colon cancer pre-clinical models. We are also able to abrogate the effect of each class of ferroptosis inducers by using a ferroptosis inhibitor. Thus, our study evaluates for the first time the contribution of ferroptosis to immune based therapies without the confounding effects due to the chemistry of the small molecule used. In conclusion, our data resolves the conflicting reports about the immune effects of ferroptosis and paves the way for evaluating the clinical efficacy of ferroptosis in combination with appropriate immune modulators. Citation Format: Divya Venkatesh, Daniel Thach, Qian Wang, Rachana R Maniyar, Iken Takiyeddine, Mariam M George, Isabell Schulze, Hengrui Liu, Brent R Stockwell, Jedd D Wolchok, Taha Merghoub. Leveraging the tumor immunogenic effects of ferroptosis to rationally design effective immune based therapies [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B011.

PURPOSE:Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) agonism in T cells may potentiate antitumor immune responses to immune checkpoint blockade therapy. This first-in-human, phase I/II dose escalation/expansion study assessed INCAGN01876, a humanized GITR-targeting agonistic mAb, for advanced solid tumors (NCT02697591). PATIENTS AND METHODS:Dose was escalated by 0.03 to 20 mg/kg every 2 weeks; flat doses of 400 mg every 4 weeks and 300 mg every 2 weeks were also evaluated. The primary objective was safety/tolerability; secondary objectives were pharmacokinetics and preliminary efficacy; and exploratory objectives were immunogenicity, GITR occupancy, and immune biomarker assessment. RESULTS:Among 100 patients enrolled [prior anti-PD-1/PD-L1 therapy, 47%; most common tumors: colorectal (19%) and melanoma (14%)], 2% had one dose-limiting toxicity (grade 4 hypoxia and grade 3 pleurisy). The MTD was not reached. Treatment-related adverse events (TRAE) occurred in 69% of patients, most frequently fatigue (17%) and pruritus (14%); 10% had grade ≥3 TRAEs, most commonly fatigue (3%); and 23% reported immune-related adverse events, most frequently generalized pruritus and generalized rash (7% each). Doses ≥5 mg/kg every 2 weeks resulted in full receptor occupancy at trough. INCAGN01876 elicited changes in immune parameters in some patients, including variable peripheral regulatory T-cell depletion and cytokine upregulation. Two patients achieved confirmed partial responses: one with appendiceal mucinous carcinoma and another with melanoma previously treated with pembrolizumab and glembatumumab; 36% of patients had disease control. CONCLUSIONS:INCAGN01876 was generally well tolerated; fatigue was the most frequent TRAE. INCAGN01876 elicited transient and variable regulatory T-cell depletion and limited antitumor activity. Future studies will explore combinatorial approaches.

Baseline demographics and characteristics.

Patient disposition. aTwo patients completed treatment [0.3 mg/kg every 2 weeks (Q2W) and 300 mg Q2W, respectively]. The death in the 300-mg Q2W cohort was coded as dyspnea secondary to disease progression. Reasons for patient ineligibility included meeting the following exclusion criteria: (i) laboratory and medical history parameters not within the protocol-defined range (39%; 17/44 patients); (ii) any condition that would, in the investigator’s judgment, interfere with full participation in the study (including administration of the study drug and attending required study visits), pose a significant risk to the subject, or interfere with the interpretation of study data (16%; 7/44 patients); (iii) known active central nervous system metastases and/or carcinomatous meningitis (14%; 6/44 patients); and (iv) evidence of hepatitis B virus (HBV) or hepatitis C virus (HCV) infection or risk of reactivation (positive testing for HBV DNA and/or HCV RNA; 11%; 5/44 patients) and not meeting the following inclusion criteria: willingness to provide a written informed consent form (7%; 3/44 patients) and having Eastern Cooperative Oncology Group performance status of 0 or 1 (5%; 2/44 patients). FAS, full analysis set.

A, Frequencies of Tregs. B, Changes in central memory (CM) and EM CD4+ T cells.

A, INCAGN01876 mean (±SE) concentration–time profiles after the first dose and at steady state. B, Relationship between serum concentrations of INCAGN01876 and GITR receptor occupancy. C, Percentage of INCAGN01876 receptor occupancy vs. time. The average receptor occupancy per cohort is depicted (± SD). PK analysis includes patients with ADA-negative status at the respective visit; first visit/steady state: 0.03 mg/kg, n = 4/n = 0; 0.1 mg/kg, n = 4/n = 0; 0.3 mg/kg, n = 3/n = 1; 1 mg/kg, n = 3/n = 1; 3 mg/kg, n = 15/n = 5; 5 mg/kg, n = 16/n = 5; 10 mg/kg, n = 14/n = 4; 20 mg/kg, n = 3/n = 0; 300 mg, n = 21/n = 7; and 400 mg, n = 9/n = 3. RO, receptor occupancy.

Abstract Cysteine-binding targeted drugs AMG-510 and MRTX-849 provide a new therapeutic approach for advanced KRASG12C mutant cancers. However, clinical response has been limited. Responders eventually develop adaptive resistance by gaining additional MAPK pathway mutations, resulting in limited therapeutic benefit. High-dose pulsatile treatment with MAPK pathway inhibitors has been proposed as an alternative strategy to alleviate adaptive resistance while maintaining tumor control. The sensitivity of tumor cells to pan-inhibitors of the MAPK pathway, crucial for proliferation, creates a therapeutic opportunity window for immune cells to restore their function during an intermittent regimen. Congruently, we have previously shown that intermittent pan-MAPK pathway inhibitor treatment delays drug resistance, supports T cell activation, and enhances tumor delay when combined with immunotherapy. In this study, we investigated the effects of continuous and pulsatile KRASG12C inhibition on tumor and immune cells in order to design combinatorial strategies with immune-modulating agents that overcome drug resistance. Our data suggest that a weekly single high-dose regimen of AMG-510 is less effective at achieving tumor control in preclinical mouse models of KRAS mutant lung cancer compared to daily treatments. However, supplementing this pulsatile regimen with lower maintenance doses markedly slows the growth of LLC KRASG12C lung cancer. Flow cytometry analysis revealed that high-dose pulse regimen enhances T cell activation, evidenced by increased expression of TCF-1, Ki67, PD-1, and GITR, along with lower expression of co-inhibitory molecules Lag-3 and Tim-3, 3 days after treatment. In vitro, AMG-510 also improves the antigen presentation of OVA-expressing LLC-KML lung cancer cells via MHC class I and, to a lesser extent, MHC class II. Furthermore, we found that while combinatorial therapy with PD-1 blockade alone does not significantly impact tumor control, the combination with CTLA-4 blockade shows efficacy across pulsatile and continuous AMG-510 dosing cohorts. Overall, our findings suggest that the utilization of intermittent dosing regimens for selective KRASG12C inhibitors is a promising approach for improving clinical responses in lung cancers with KRAS mutations. Future experiments will aim to delineate MAPK pathway behavior in vivo and in vitro under pulsatile and continuous treatment, assess immunogenicity, and further characterize immune changes in the different treatment regimens, with the goal of designing more efficient cancer therapies utilizing selective KRAS inhibitors. Citation Format: Valeria Estrada Navarro, Vincent Panneton, Lauren Dong, Hyejin Choi, Divya Venkatesh, Rachana Maniyar, Isabell Schulze, Jedd D. Wolchok, Taha Merghoub. Optimization of intermittent dosing strategies of KRAS G12C inhibitors in preclinical lung cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7274.

Abstract Immune checkpoint blockade (ICB) therapies, specifically anti PD-1 and anti CTLA-4, have resulted in tremendous success in the clinic. However, a substantial number of patients still relapse due to either inherent or acquired resistance to ICB therapies. In this scenario, activation of T cell co-stimulation pathways along with ICB emerges as a promising strategy to improve anti-tumor responses. Engagement of Glucocorticoid induced TNFR Related protein (GITR) costimulatory molecule, leads to enhanced activation of cytotoxic T cells while destabilizing suppressive T regulatory cells in the tumor microenvironment. Additionally, combining GITR agonism with PD-1 blockade improves the anti-tumor response in a murine model of advanced melanoma resistant to ICB, positioning GITR as an attractive immunotherapeutic target. In this study, we evaluated the therapeutic efficacy of combining blockade of the immune checkpoint LAG3 with GITR agonism for treating ICB-resistant advanced melanoma, using the B16 model. The combination of LAG3 blockade with GITR agonism was well tolerated, leading to better tumor control and improved survival. Our data revealed increased B cell infiltration and activation within tumors and draining lymph nodes (LNs) of treated mice. B cell depletion diminished the anti-tumor effect of the combination therapy, suggesting that B cells play an active role in controlling tumor growth. Spleens from treated mice exhibited increased lymphocytic hyperplasia, along with more and larger germinal centers. B cell receptor sequencing revealed an increased clonality and reduced entropy. Treating B cells with GITR agonism and LAG3 blockade in vitro led to increased activation and proliferation, suggesting a direct effect of these therapies on B cells. Furthermore, adding LAG3 blockade to the dual combination of GITR agonism and PD-1 blockade resulted in superior tumor control and increased B cell activity in the draining LNs. Collectively, our findings suggest that the humoral response significantly contributes to the anti-tumor responses elicited by ICB when combined with GITR agonism. This triple combination therapy (LAG3 blockade + PD1 blockade + GITR agonism) holds promise as a safe and potent therapeutic strategy to overcome ICB resistance. Further investigation into the mechanism of action by which GITR agonism leads to enhanced B cell responses when combined with ICB is warranted in order to design more effective therapies for cancer treatment. Citation Format: Rachana R. Maniyar, Yuval Elhanati, Levi Mangarin, Yacine Marouf, Ashley Ahmed, Benjamin Greenbaum, Jedd Wolchok, Taha Merghoub. Lag3 blockade enhances the anti-tumor effect of dual GITR agonism and PD-1 blockade in a preclinical melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4034.

Background: Immune checkpoint blockade therapeutics have revolutionized cancer therapy. However, most patients are either unresponsive or develop acquired resistance. Understanding patient-specific temporal T cell repertoires in the context of immune interventions will contribute to a better understanding of potential improvement strategies for better T cell anti-tumor immunity. Current methods to understand the T cell repertoire changes over time are limited due to the limited amount of material and prohibitive cost. In this study, we designed and performed longitudinal TCR sequencing in mice treated with multiple therapies. Methods: C57BL/6 mice were used in each treatment group, with blood drawn overtime before, during and after treatment. Mice were treated with a combination of immunotherapeutic agents and OVA peptide immunization, 100µl blood was drawn at multiple pre-, on- and post-treatment time points followed subsequently by TCR sequencing. Cyclophosphamide (CTX) is known to cause lymphodepletion followed by homeostatic proliferation. Thus, we also treated B16-F10 tumor bearing mice with CTX, and performed TCR sequencing on blood draws pre-, on-, and post-treatment to track TCR clone diversity and clonality over time. Results: We find that our method is sensitive enough to detect effects of immunotherapies on TCR diversity in small sample quantities. Using our method, we discovered that certain immunotherapeutic agents, most evidently CD40, have a significant but transient effect on the diversity of the T cell repertoire. We observed an initial increase in repertoire clonality, seven days post CD40 treatment followed by contraction. We show how we can identify antigen specific T cells and follow reacting T cell clones. In addition, we find that CTX treatment is associated with increased clonality in tumor bearing mice particularly in the week following treatment reflecting changes in the TCR repertoire. Conclusion: We have developed a method by which we can follow the TCR repertoire in mice treated with immunotherapy or chemotherapy. This enabled us to isolate and study the effects of different immune modulatory drugs of the endogenous T cell repertoire overtime. Citation Format: Yuval Elhanati, Mariam M. George, Hyejin Choi, Rachana Maniyar, Linda Hamadane, Neeman Mohibullah, Benjamin D. Greenbaum, Taha Merghoub. Immune modulation of the T cell repertoire in mice following immunotherapy and chemotherapy combination treatments [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 2282.

GITR is a TNF receptor, and its activation promotes immune responses and drives antitumor activity. The receptor is activated by the GITR ligand (GITRL), which is believed to cluster receptors into a high-order array. Immunotherapeutic agonist antibodies also activate the receptor, but their mechanisms are not well characterized. We solved the structure of full-length mouse GITR bound to Fabs from the antibody DTA-1. The receptor is a dimer, and each subunit binds one Fab in an orientation suggesting that the antibody clusters receptors. Binding experiments with purified proteins show that DTA-1 IgG and GITRL both drive extensive clustering of GITR. Functional data reveal that DTA-1 and the anti-human GITR antibody TRX518 activate GITR in their IgG forms but not as Fabs. Thus, the divalent character of the IgG agonists confers an ability to mimic GITRL and cluster and activate GITR. These findings will inform the clinical development of this class of antibodies for immuno-oncology.

Background Immune checkpoint blockade (ICB) therapies anti-PD1 and anti CTLA-4 have had tremendous successes in clinic. However, many patients are either inherently resistant or acquire resistance to these therapies. Appropriately activating co-stimulation pathways of T cells together with blocking immune checkpoints can provide substantial anti-tumor responses. Glucocorticoid induced TNFR Related protein (GITR), is a costimulatory molecule whose engagement with agonist antibodies leads to proliferation, cytokine production and survival of cytotoxic T cells, and destabilization and depletion of suppressive T regulatory cells in the tumor microenvironment, making it an attractive target for cancer immunotherapy. GITR agonism as a monotherapy in murine models of advanced melanoma leads to increased effector T cell dysfunction with a marked upregulation in expression of exhaustion markers PD-1 and Lag3, making them rational targets to combine with GITR agonism. Methods C57BL/6 mice were implanted with B16-F10 melanoma and were treated with a single dose of GITR Agonism with or without anti Lag3 on Day 7 post tumor implant, followed by anti Lag3 every 3 days. Spectral flow cytometry and immunohistochemistry was used to study immune cell repertoires. Splenic B cell repertoire was studied using BCR IgH sequencing. Results We show that combining GITR agonism with Lag3 ICB therapy, leads to better tumor control, and improved survival in mice with advanced ICB resistant B16 melanoma. Additionally, mice treated with GITR agonism monotherapy show a marked increase in activated B cells infiltrating the tumor microenvironment. This infiltration is further increased when GITR agonism is combined with Lag3 blockade therapy. These tumor-infiltrating B cells are highly activated with an increased expression of activation markers CD86, MHC-II, and CD38. The spleens from mice treated with GITR agonism in combination with Lag3 blockade demonstrate increased hyperplasia, and an increase in size and number of germinal centers. B cell receptor sequencing from the spleens of these mice revealed an increased clonality and reduced entropy in mice treated with GITR agonism + Lag3 blockade therapy. Conclusions Increased B cell activity observed in these mice warrants further investigation into the role and mechanism of action of a GITR/Lag3 combination therapy. Our results suggest that combining GITR agonism with Lag3 blockade is a safe and potent therapeutic strategy to overcome ICB resistance in mice with advanced melanoma. Ethics Approval This study was approved by the Institutional Animal Care and Use Committee (IACUC) and Memorial Sloan Kettering Cancer Center

Melanoma is the most aggressive form of skin cancer with an estimated 106,110 newly diagnosed cases in the United States of America in 2021 leading to an approximated 7180 melanoma-induced deaths. Cancer typically arises from an accumulation of somatic mutations and can be associated with mutagenic or carcinogenic exposure. A key characteristic of melanoma is the extensive somatic mutation rate of 16.8 mutations/Mb, which is largely attributed to UV exposure. Bearing the highest mutational load, many of them occur in key driver pathways, most commonly the BRAFV600E in the mitogen-activated protein kinase (MAPK) pathway. This driver mutation is targeted clinically with FDA-approved therapies using small molecule inhibitors of oncogenic BRAFV600E and MEK, which has greatly expanded therapeutic intervention following a melanoma diagnosis. Up until 2011, therapeutic options for metastatic melanoma were limited, and treatment typically fell under the spectrum of surgery, radiotherapy, and chemotherapy.Attributed to the extensive mutation rate, as well as having the highest number of neoepitopes, melanoma is deemed to be extremely immunogenic. However, despite this highly immunogenic nature, melanoma is notorious for inducing an immunosuppressive microenvironment which can be relieved by checkpoint inhibitor therapy. The two molecules currently approved clinically are ipilimumab and nivolumab, which target the molecules CTLA-4 and PD-1, respectively.A plethora of immunomodulatory molecules exist, many with redundant functions. Additionally, these molecules are expressed not only by immune cells but also by tumor cells within the tumor microenvironment. Tumor profiling of these cell surface checkpoint molecules is necessary to optimize a clinical response. The presence of immunomodulatory molecules in melanoma, using data from The Cancer Genome Atlas and validation of expression in two model systems, human melanoma tissues and patient-derived melanoma cells, revealed that the expression levels of B and T lymphocyte attenuator (BTLA), TIM1, and CD226, concurrently with the BRAFV600E mutation status, significantly dictated overall survival in melanoma patients. These molecules, along with herpesvirus entry mediator (HVEM) and CD160, two molecules that are a part of the HVEM/BTLA/CD160 axis, had a higher expression in human melanoma tissues when compared to normal skin melanocytes and have unique roles to play in T cell activation. New links are being uncovered between the expression of immunomodulatory molecules and the BRAFV600E genetic lesion in melanoma. Small molecule inhibitors of the MAPK pathway regulate the surface expression of this multifaceted molecule, making BTLA a promising target for immuno-oncology to be targeted in combination with small molecule inhibitors, potentially alleviating T regulatory cell activation and improving patient prognosis.

Abstract Melanoma, the most aggressive form of skin cancer, is still a challenge despite the advent of recent immunotherapies using checkpoint inhibitors. The presence of checkpoint molecules on immune cells serve to regulate optimal immune responses, however, when present on tumor cells, they serve as immune evasion molecules. As such, this presents challenges to the specific inhibitors being used in relation to dose, side effects, and specificity. We hypothesize that tumor cell profiling of co-activators and co-inhibitors will serve as an important step in the identification and use of specific checkpoint inhibitors in personalized medicine. To this end, we used five patient-derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM, and GLM-2 as a model and screened them for the expression of a comprehensive list of twenty-nine co-stimulatory and co-inhibitory molecules. We compared the differentially expressed molecules on tumor cells with the expression of the co-stimulatory and co-inhibitors on normal non-transformed adult melanocytes. We see a differential mRNA expression of many of these immune-regulatory molecules, including BTLA, HVEM, CD160, CD226 and TIM1. Western blots and immunofluorescence confirmed the presence of these molecules at the protein level. A flow cytometry analysis demonstrated that BTLA, HVEM, CD160, TIM1 and CD226 are present on the membrane of these patient derived melanoma cells; implying that they are capable of engaging their respective ligands and exerting a functional role in immunomodulation. The expression of these molecules by immunohistochemistry in patient melanoma issues provided additional validation. Analysis of protein expression in various cancers from the Human protein atlas was conducted so as to evaluate the expression of these immune regulators across cancers. HVEM expression in melanoma was high. Interestingly, treatment of MEL-2, MEL-V, KFM and GLM-2, our BRAFV600E containing patient-derived cells, with BRAFV600E inhibitor PLX4032 led to the upregulation of these molecules. Since BRAFV600E is the most common mutation, and some response to small molecule inhibitors has been clinically verified, we undertook an analysis of the expression of immune regulators with overall survival using the cancer genome atlas. We also evaluated the interferon regulatory factor binding site so as to molecularly validate that expression of these molecules is dependent on secreted cytokine in the tumor microenvironment. We conclude that the BTLA-HVEM axis is a significant novel target in melanoma that can be used in combinatorial therapy with small molecule inhibitors of cell survival. Our results advocate the need for profiling of immune modulators on tumor cells prior to immunotherapy. Citation Format: Tara Jarboe, Rachana R. Maniyar, Sanjukta Chakraborty, John J. Degliuomini, Anitha Srinivasan, Marc Wallack, Jan Geliebter, Raj K. Tiwari. Novel immune targets in melanoma by profiling of co-activators and co-receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1624.

The incidence of thyroid cancer in the United States is on the rise with an appreciably high disease recurrence rate of 20-30%. Anaplastic thyroid cancer (ATC), although rare in occurrence, is an aggressive form of cancer with limited treatment options and bleak cure rates. This chapter uses discussions of in vitro models that are representative of papillary, anaplastic, and follicular thyroid cancer to evaluate the crosstalk between specific cells of the tumor microenvironment (TME), which serves as a highly heterogeneous realm of signaling cascades and metabolism that are associated with tumorigenesis. The cellular constituents of the TME carry out varying characteristic immunomodulatory functions that are discussed throughout this chapter. The aforementioned cell types include cancer-associated fibroblasts (CAFs), endothelial cells (ECs), and cancer stem cells (CSCs), as well as specific immune cells, including natural killer (NK) cells, dendritic cells (DCs), mast cells, T regulatory (Treg) cells, CD8+ T cells, and tumor-associated macrophages (TAMs). TAM-mediated inflammation is associated with a poor prognosis of thyroid cancer, and the molecular basis of the cellular crosstalk between macrophages and thyroid cancer cells with respect to inducing a metastatic phenotype is not yet known. The dynamic nature of the physiological transition to pathological metastatic phenotypes when establishing the TME encompasses a wide range of characteristics that are further explored within this chapter, including the roles of somatic mutations and epigenetic alterations that drive the genetic heterogeneity of cancer cells, allowing for selective advantages that aid in their proliferation. Induction of these proliferating cells is typically accomplished through inflammatory induction, whereby chronic inflammation sets up a constant physiological state of inflammatory cell recruitment. The secretions of these inflammatory cells can alter the genetic makeup of proliferating cells, which can in turn, promote tumor growth.This chapter also presents an in-depth analysis of molecular interactions within the TME, including secretory cytokines and exosomes. Since the exosomal cargo of a cell is a reflection and fingerprint of the originating parental cells, the profiling of exosomal miRNA derived from thyroid cancer cells and macrophages in the TME may serve as an important step in biomarker discovery. Identification of a distinct set of tumor suppressive miRNAs downregulated in ATC-secreted exosomes indicates their role in the regulation of tumor suppressive genes that may increase the metastatic propensity of ATC. Additionally, the high expression of pro-inflammatory cytokines in studies looking at thyroid cancer and activated macrophage conditioned media suggests the existence of an inflammatory TME in thyroid cancer. New findings are suggestive of the presence of a metastatic niche in ATC tissues that is influenced by thyroid tumor microenvironment secretome-induced epithelial to mesenchymal transition (EMT), mediated by a reciprocal interaction between the pro-inflammatory M1 macrophages and the thyroid cancer cells. Thus, targeting the metastatic thyroid carcinoma microenvironment could offer potential therapeutic benefits and should be explored further in preclinical and translational models of human metastatic thyroid cancer.

Only a subset of cancer patients responds to checkpoint blockade inhibition in the clinic. Strategies to overcome resistance are promising areas of investigation. Targeting glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) has shown efficacy in preclinical models, but GITR engagement is ineffective in controlling advanced, poorly immunogenic tumors, such as B16 melanoma, and has not yielded benefit in clinical trials. The alkylating agent cyclophosphamide (CTX) depletes regulatory T cells (Tregs), expands tumor-specific effector T cells (Teffs) via homeostatic proliferation, and induces immunogenic cell death. GITR agonism has an inhibitory effect on Tregs and activates Teffs. We therefore hypothesized that CTX and GITR agonism would promote effective antitumor immunity. Here we show that the combination of CTX and GITR agonism controlled tumor growth in clinically relevant mouse models. Mechanistically, we show that the combination therapy caused tumor cell death, clonal expansion of highly active CD8+ T cells, and depletion of Tregs by activation-induced cell death. Control of tumor growth was associated with the presence of an expanded population of highly activated, tumor-infiltrating, oligoclonal CD8+ T cells that led to a diminished TCR repertoire. Our studies show that the combination of CTX and GITR agonism is a rational chemoimmunotherapeutic approach that warrants further clinical investigation.

BackgroundImmune checkpoint blockade (ICB) has revolutionized the treatment of many cancer types; however, many patients do not benefit from these therapies due to inherent or acquired resistance. Preferential engagement in glycolysis is a hallmark of cancer cells and contributes to the progression and metastasis of many tumor types, including melanoma and triple-negative breast cancer (TNBC). Tumor reliance on glycolysis is emerging as a mechanism of resistance to immunotherapy, due in part to lactate-mediated acidification and competition for glucose in the tumor microenvironment. We recently found that knocking down the glycolytic enzyme lactate dehydrogenase A (LDHA) in 4T1 TNBC results in improved and long-lasting anti-tumor responses to CTLA-4 blockade in mice. These LDHA-defective tumors consume less glucose than wild-type tumors, leaving more glucose available in the TME for effector T cells to exert their tumoricidal function directly and via lineage destabilization of regulatory T cells. However, it remains to be determined whether systemic pharmacologic targeting of LDH can improve the efficacy of immunotherapy.MethodsLactate production and glucose consumption was quantified by YSI enzymatic analysis of metabolites. LDHA was detected by immunoblot in tumor and T cells. A clonogenic killing assay was used to assess T-cell killing. B16-F10-bearing mice were treated daily with LDH inhibitor GNE-140 and/or biweekly with anti-CTLA-4.ResultsSince activated T cells rely on glycolysis, we first determined that glycolytic cancers overexpress LDH compared to immune cells by analyzing single-cell transcripts from patient melanoma biopsies. LDHA gene expression is significantly higher in malignant cells than infiltrating CD8+ T cells, and we replicated these findings at the protein level in whole cell lysate from B16-F10 melanoma and 4T1 TNBC tumor cells vs. activated tumor-antigen specific T cells in vitro. We showed that the LDH inhibitor GNE-140 reduces tumor lactate production and glucose consumption without inhibiting anti-tumor T-cell killing in vitro. Daily treatment with GNE-140 results in reduced growth in immunocompetent but not immune deficient mice. Additionally, our preliminary findings indicate that targeting LDHA in combination with CTLA-4 blockade is more effective in slowing B16-F10 growth compared with CTLA-4 blockade alone.ConclusionsThese results suggest that targeting LDH with GNE-140 is a safe, efficacious method for relieving tumor glycolysis-mediated immunosuppression within the TME, without adversely affecting immune cell function within the tumor. Our long-term goal is to determine optimal combinations of metabolic inhibitors with ICB to alleviate the metabolic competition between tumor and infiltrating immune cells and better potentiate anti-tumor immune responses.

Limiting metabolic competition in the tumour microenvironment may increase the effectiveness of immunotherapy. Owing to its crucial role in the glucose metabolism of activated T cells, CD28 signalling has been proposed as a metabolic biosensor of T cells 1 . By contrast, the engagement of CTLA-4 has been shown to downregulate T cell glycolysis 1 . Here we investigate the effect of CTLA-4 blockade on the metabolic fitness of intra-tumour T cells in relation to the glycolytic capacity of tumour cells. We found that CTLA-4 blockade promotes metabolic fitness and the infiltration of immune cells, especially in glycolysis-low tumours. Accordingly, treatment with anti-CTLA-4 antibodies improved the therapeutic outcomes of mice bearing glycolysis-defective tumours. Notably, tumour-specific CD8 + T cell responses correlated with phenotypic and functional destabilization of tumour-infiltrating regulatory T (T reg ) cells towards IFNγ- and TNF-producing cells in glycolysis-defective tumours. By mimicking the highly and poorly glycolytic tumour microenvironments in vitro, we show that the effect of CTLA-4 blockade on the destabilization of T reg cells is dependent on T reg cell glycolysis and CD28 signalling. These findings indicate that decreasing tumour competition for glucose may facilitate the therapeutic activity of CTLA-4 blockade, thus supporting its combination with inhibitors of tumour glycolysis. Moreover, these results reveal a mechanism by which anti-CTLA-4 treatment interferes with T reg cell function in the presence of glucose.

Only a limited fraction of patients derives durable clinical benefit upon immune checkpoint blockade. Deepening our understanding of mechanisms of response and resistance to these therapies is thus needed to optimize their employment in rational combinations. Cellular energy metabolism reprogramming is a critical hallmark of cancer. High glucose consumption and lactate production by tumor cells restrict nutrient availability in the tumor microenvironment (TME) for effector T cells, which also rely on glycolysis to replicate and function. In addition, immune checkpoints and co-stimulatory molecules are emerging as important regulators of T cell metabolism. Exploiting the capacity of immune checkpoint blockade to perturb T cell metabolism in combination with inhibition of tumor glycolysis may thus be a rational and more effective anti-cancer approach. We investigated the link between tumor glycolysis and immune cell function using RNA sequencing data sets from patients treated with checkpoint blockade and in tumor:T cell co-culture systems. We then studied the effects of checkpoint blockade in syngeneic glycolysis-defective murine tumor models and explored mechanisms underlying anti-tumor activity. We found that expression of glycolysis-related genes is inversely correlated with infiltration of most immune cell types in melanomas from patients before CTLA-4 blockade. However, after treatment, immune-related and glycolysis-related genes were more often co-expressed, suggesting that anti-CTLA-4 may partially restore immune cell fitness in the glycolytic TME. To directly assess the effect of tumor metabolism on T cell function, we co-cultured activated T cells with the highly glycolytic murine mammary carcinoma 4T1 and observed that tumor cells, or similar non-toxic concentrations of exogenous lactate, significantly limited T cell activation and viability. We thus investigated whether inhibition of tumor glycolysis could potentiate the activity of CTLA-4 blockade. We found that neoadjuvant treatment with anti-CTLA-4 significantly prolonged survival in mice bearing glycolysis defective 4T1 tumors, where lactate dehydrogenase A (LDH-A) - the critical enzyme controlling lactate production in aerobic glycolysis - was knocked down (4T1-KD). Intriguingly, tumor protection was associated with intratumoral regulatory T cell (Treg) functional phenotypic destabilization towards IFN-gamma and TNF-alpha producing Tregs. By mimicking the LDH-A-KD and control 4T1 TME in vitro using higher and lower concentrations of glucose, respectively, we showed that CTLA-4 blockade promotes glucose uptake by Tregs and more efficiently counteracts Treg suppression and enhances CD28 co-stimulation at higher glucose concentrations. These findings indicate that rebalancing glucose utilization in favor of immune cells in the TME may facilitate the activity of CTLA-4 blockade and reveal an additional mechanism through which anti-CTLA-4 interferes with Treg function in the presence of glucose. Citation Format: Roberta Zappasodi, Inna Serganova, Ivan Cohen, Masatomo Maeda, Masahiro Shindo, Yasin Senbabaoglu, Avigdor Leftin, Rachana Maniyar, Svena Verma, Matthew Lubin, Myat Kyaw Ko, Mayuresh M. Mane, Arnab Ghosh, Ellen Ackerstaff, Jason A. Koutcher, Ping-Chih Ho, Greg M. Delgoffe, Ronald Blasberg, Jedd D. Wolchok, Taha Merghoub. CTLA-4 blockade drives loss of regulatory T cell functional stability in glycolysis defective tumors [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 3257.