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Tumor reliance on glycolysis is a hallmark of cancer. Immunotherapy is more effective in controlling glycolysis-low tumors lacking lactate dehydrogenase (LDH) due to reduced tumor lactate efflux and enhanced glucose availability within the tumor microenvironment (TME). LDH inhibitors (LDHi) reduce glucose uptake and tumor growth in preclinical models, but their impact on tumor-infiltrating T cells is not fully elucidated. Tumor cells have higher basal LDH expression and glycolysis levels compared with infiltrating T cells, creating a therapeutic opportunity for tumor-specific targeting of glycolysis. We demonstrate that LDHi treatment (a) decreases tumor cell glucose uptake, expression of the glucose transporter GLUT1, and tumor cell proliferation while (b) increasing glucose uptake, GLUT1 expression, and proliferation of tumor-infiltrating T cells. Accordingly, increasing glucose availability in the microenvironment via LDH inhibition leads to improved tumor-killing T cell function and impaired Treg immunosuppressive activity in vitro. Moreover, combining LDH inhibition with immune checkpoint blockade therapy effectively controls murine melanoma and colon cancer progression by promoting effector T cell infiltration and activation while destabilizing Tregs. Our results establish LDH inhibition as an effective strategy for rebalancing glucose availability for T cells within the TME, which can enhance T cell function and antitumor immunity.

BackgroundTumor reliance on glycolytic metabolism represents a hallmark of cancer and a mechanism of resistance to immune based therapies.1 2 Competition for glucose between T cells and tumor cells as well as lactate-mediated immune suppression contribute to this resistance. Thus, immune checkpoint blockade (ICB) more effectively controls glycolysis-low tumors lacking lactate dehydrogenase (LDH), primarily due to enhanced glucose availability within the tumor microenvironment.3 The development of LDH inhibitors (LDHi) have been reported to reduce glucose uptake and growth of preclinical models of cancer,4 but their impact on infiltrating T cells within the tumor microenvironment remains unexplored.MethodsB16F10 and MC38 tumor-bearing C57BL/6 mice were treated with LDHi GNE-140 and/or anti-CTLA-4 and tumor volume was measured twice per week. Tumors were also processed for flow cytometric analysis of tumor-infiltrating lymphocytes to assess infiltration, activation, and function. Glucose uptake, LDH, and Glut1 expression was assessed in tumor and T cells by flow cytometry. Seahorse analysis was performed on mouse and human tumor and T cells.ResultsTumor cells exhibit higher basal levels of LDH and glucose consumption compared to tumor-infiltrating T cells, exposing a therapeutic window for tumor-specific targeting of glycolysis. We show that effective LDH inhibition relies on tumoral overexpression of LDH and the adaptive immune system to impede murine tumor progression. Mechanistically, we demonstrate that LDHi treatment 1) decreases tumor cell glucose uptake and expression of the glucose transporter GLUT1 while 2) increasing glucose uptake and GLUT1 expression in tumor-infiltrating CD8+ T cells. Compared to activated T cells, tumor cells display significantly higher metabolic sensitivity to LDH inhibition via reduction of glucose uptake and extracellular acidification rates while also downregulating LDH and GLUT1 expression. Strikingly, LDH inhibition combined with anti-CTLA-4 effectively controls murine melanoma and colon cancer progression. This dual therapy also promotes effector T cell infiltration and activation while functionally destabilizing regulatory T cells (Tregs). Accordingly, increasing glucose availability leads to improved T cell killing of tumor cells, and impaired Treg-mediated suppression of T cells.ConclusionsIncreasing intratumoral glucose levels contributes to anti-tumor efficacy of CTLA-4 blockade by supporting effector T cell function while impairing Tregs. Our results establish LDH inhibition as an effective tumor-specific strategy to reduce tumor glucose uptake and increase glucose availability within the tumor microenvironment, thereby boosting T cell glucose uptake. This study provides a comprehensive rationale for combining immune checkpoint blockade with inhibitors of glycolysis for patients with highly glycolytic cancers.ReferencesHanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–674.Zappasodi R, Merghoub T, Wolchok JD. Emerging Concepts for Immune Checkpoint Blockade-Based Combination Therapies. Cancer Cell. 2018;34:690.Zappasodi R, et al. CTLA-4 blockade drives loss of Treg stability in glycolysis-low tumours. Nature. 2021;591:652–658.Boudreau A, et al. Metabolic plasticity underpins innate and acquired resistance to LDHA inhibition. Nat Chem Biol. 2016;12:779–786.

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. CTLA-4 promotes glucose uptake by tumour-infiltrating regulatory T cells, making them unstable.

Altered cellular metabolism is both a hallmark of cancer and a mechanism of resistance to immunotherapy. Cancer cells preferentially engage in aerobic glycolysis, which can cause immunosuppression due to lactate-mediated acidification and decreased glucose availability within the tumor microenvironment (TME). Therefore, immunotherapy is more effective in controlling glycolysis-low tumors: our group has previously reported that the efficacy of immune checkpoint blockade (ICB) therapy is increased when key glycolytic enzyme lactate dehydrogenase (LDH) is knocked down within the tumor. However, it remains to be determined whether pharmacologic inhibition of glycolysis in combination with immunotherapy is an efficacious strategy to blunt cancer progression.Inhibitors of glycolysis, such as LDH inhibitors (LDHi), reduce tumor lactate efflux, glucose uptake, and growth in preclinical models, but their impact on tumor-infiltrating immune cells is not fully elucidated. There remains a critical need to understand how pharmacologic LDH inhibition affects anti-tumor immune cells, such as T cells. We show that LDH is a rational target for immunotherapy combinations. Tumor cells have higher basal LDH expression and glycolysis levels compared to activated T cells, creating a therapeutic opportunity for tumor-specific targeting of glycolysis. In vitro, tumor cells are significantly more glycolytically sensitive to LDH inhibition than T cells. We demonstrate that LDHi treatment in vivo 1) decreases tumor cell glucose uptake, expression of the glucose transporter GLUT1, and tumor cell proliferation, while 2) increasing glucose uptake, GLUT1 expression, and proliferation of tumor-infiltrating T cells. Accordingly, increasing glucose availability in the microenvironment via LDH inhibition leads to improved CD8+ T cell-mediated killing ability and impaired regulatory T cells (Treg) immunosuppressive activity in vitro. Moreover, combining LDH inhibition with immune checkpoint blockade therapy effectively controls murine melanoma and colon cancer progression by promoting effector T cell infiltration and activation while destabilizing Tregs. Our results establish LDH inhibition as an effective strategy to rebalance glucose availability for T cells within the TME, which can enhance anti-tumor immunity.

BackgroundDurable clinical responses to immune checkpoint blockade (ICB) occur in a limited fraction of patients. We thus hypothesized that the characteristic tumor metabolic switch towards aerobic glycolysis could contribute to ICB resistance. High glucose consumption and lactate production by tumor cells can indeed restrict nutrient availability for tumor-infiltrating T cells, which also rely on glycolysis to proliferate and function. Therefore, we investigated whether targeting tumor glucose metabolism potentiates ICB anti-tumor activity.MethodsWe modeled tumor-selective glycolysis inhibition by knocking down the critical glycolytic enzyme lactate dehydrogenase A (LDHA-KD) in the murine metastatic breast carcinoma 4T1 and melanoma B16, which are known immune-refractory tumor models. Anti-CTLA-4 and anti-PD-1 were tested in immunocompetent mice orthotopically implanted with control vs. LDHA-KD tumor cells. Changes in glucose metabolism were assessed by Seahorse and fluorescent-glucose flow-cytometry staining. Changes in immune cells were measured by multiparameter flow cytometry. Glucose-dependent effects of anti-CTLA-4 in regulatory T cells (Tregs) were tested in standard suppression assays with increasing glucose concentration (0.5–10 mM). Pearson correlations between glycolysis and intra-tumor immune-cell infiltration by CIBERSORT immune-deconvolution method were analyzed in bulk RNA-sequencing data sets from human and murine tumors treated with ICB.ResultsComparison of ICB activity in LDHA-KD vs. control tumor-bearing mice revealed improved anti-tumor effects and overall survival in the setting of glycolysis-defective tumors specifically upon CTLA-4 blockade. Anti-tumor CD8+ T-cell responses correlated with Treg phenotypic and functional destabilization in anti-CTLA-4-treated LDHA-KD tumors. CTLA-4 blockade led to CTLA-4 and CD25 downregulation associated with increased IFN-gamma and TNF-alpha production in Tregs from glycolysis-defective vs. control tumors. We next mimicked high- vs. low-glycolysis tumor microenvironment (TME) in vitro using control vs. LDHA-KD tumor co-cultures with Tregs, control vs. LDHA-KD tumor-conditioned media or directly modulating glucose concentrations. In these assays, we observed that CTLA-4 blockade promotes IFN-gamma±TNF-alpha production and glucose uptake by Tregs and more efficiently counteracts Treg suppression and enhances CD28 co-stimulation at higher glucose concentrations. Lastly, by interrogating transcriptomic data from human melanoma and murine 4T1 tumors, we found that CTLA-4 blockade promotes immune-cell infiltration and metabolic fitness especially in glycolysis-defective tumors.ConclusionsOur findings indicate that increasing glucose availability in the TME may improve anti-CTLA-4 therapeutic activity and reveal a new mechanism through which CTLA-4 blockade interferes with Treg immunosuppression in a glucose-dependent manner. These results suggest that CTLA-4 blockade can be more effective in tumors with low glycolysis and/or can be best exploited in combination with inhibitors of tumor glycolysis.

BackgroundThe majority of JAK2V617F-negative myeloproliferative neoplasms (MPN) have disease-initiating frameshift mutations in calreticulin (CALR) resulting in a common novel C-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in CALRMUT MPN patients, but the underlying reasons for this phenomenon are unknown.MethodsIn this study, we examine class-I major histocompatibility complex (MHC-I) allele frequency in CALRMUT MPN patients from two independent cohorts and observed that MHC-I alleles that present CALRMUT neoepitopes with high affinity are under-represented in CALRMUT MPN patients. We speculate that this is due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifests. As a consequence of this MHC-I allele restriction, we reasoned that CALRMUT MPN patients would not efficiently respond to cancer vaccines composed of the CALRMUT fragment, but could do so when immunized with a properly modified CALRMUT heteroclitic peptide vaccine approach.ResultsWe found that heteroclitic CALRMUT peptides specifically designed for CALRMUT MPN patient MHC-I alleles efficiently elicited a cross-reactive CD8+ T cell response in human PBMC samples otherwise unable to respond to the matched weakly immunogenic CALRMUT native peptides. We also modeled this effect in mice and observed that C57BL/6J mice, which are unable to mount an immune response to the human CALRMUT fragment, can mount a cross-reactive CD8+ T cell response against a CALRMUT-derived peptide upon heteroclitic peptide immunization and this was further amplified by combining the heteroclitic peptide vaccine with blockade of the immune checkpoint molecule PD-1.ConclusionsTogether, our data underscore the therapeutic potential of heteroclitic peptide-based cancer vaccines in CALRMUT MPN patients.Ethics ApprovalApproval was obtained for the use of patient-derived specimens and access to clinical data extracted from patient charts by the Institutional Review Boards at Memorial Sloan Kettering Cancer Center, the Dana-Farber Cancer Institute and the Massachusetts General Hospital, as well as by the Danish Regional Science Ethics Committee. Mouse experiments were performed in accordance with institutional guidelines under a protocol approved by the Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use Committee.

The dynamics of CAR-T cells remain incompletely understood. Novel methods are urgently needed to longitudinally monitor transferred cells non-invasively for biodistribution, functionality, proliferation, and persistence and for improving their cytotoxic potency in case of treatment failure. Here we engineered CD19 CAR-T cells (\"Thor\"-cells) to express a membrane-bound scFv, huC825, that binds DOTA-haptens with picomolar affinity suitable for labeling with imaging or therapeutic radionuclides. We assess its versatile utility for serial tracking studies with PET and delivery of α-radionuclides to enhance anti-tumor killing efficacy in sub-optimal adoptive cell transfer using Thor-cells in lymphoma models. We show that this reporter gene/probe platform enables repeated, sensitive, and specific assessment of the infused Thor-cells in the whole-body using PET/CT imaging with exceptionally high contrast. The uptake on PET correlates with the Thor-cells on a cellular and functional level. Furthermore, we report the ability of Thor-cells to accumulate cytotoxic alpha-emitting radionuclides preferentially at tumor sites, thus increasing therapeutic potency. Thor-cells are a new theranostic agent that may provide crucial information for better and safer clinical protocols of adoptive T cell therapies, as well as accelerated development strategies.

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 . By contrast, the engagement of CTLA-4 has been shown to downregulate T cell glycolysis . 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 ) 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 cells is dependent on T 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 cell function in the presence of glucose.