A decade of checkpoint blockade immunotherapy in melanoma: understanding the molecular basis for immune sensitivity and resistance.

Ten years since the immune checkpoint inhibitor ipilimumab was approved for advanced melanoma, it is time to reflect on the lessons learned regarding modulation of the immune system to treat cancer and on novel approaches to further extend the efficacy of current and emerging immunotherapies. Here, we review the studies that led to our current understanding of the melanoma immune microenvironment in humans and the mechanistic work supporting these observations. We discuss how this information is guiding more precise analyses of the mechanisms of action of immune checkpoint blockade and novel immunotherapeutic approaches. Lastly, we review emerging evidence supporting the negative impact of melanoma metabolic adaptation on anti-tumor immunity and discuss how to counteract such mechanisms for more successful use of immunotherapy.

CD36-mediated metabolic adaptation supports regulatory T cell survival and function in tumors.

Depleting regulatory T cells (Treg cells) to counteract immunosuppressive features of the tumor microenvironment (TME) is an attractive strategy for cancer treatment; however, autoimmunity due to systemic impairment of their suppressive function limits its therapeutic potential. Elucidating approaches that specifically disrupt intratumoral Treg cells is direly needed for cancer immunotherapy. We found that CD36 was selectively upregulated in intrautumoral Treg cells as a central metabolic modulator. CD36 fine-tuned mitochondrial fitness via peroxisome proliferator-activated receptor-ß signaling, programming Treg cells to adapt to a lactic acid-enriched TME. Genetic ablation of Cd36 in Treg cells suppressed tumor growth accompanied by a decrease in intratumoral Treg cells and enhancement of antitumor activity in tumor-infiltrating lymphocytes without disrupting immune homeostasis. Furthermore, CD36 targeting elicited additive antitumor responses with anti-programmed cell death protein 1 therapy. Our findings uncover the unexplored metabolic adaptation that orchestrates the survival and functions of intratumoral Treg cells, and the therapeutic potential of targeting this pathway for reprogramming the TME.

Uptake of oxidized lipids by the scavenger receptor CD36 promotes lipid peroxidation and dysfunction in CD8+ T cells in tumors.

A common metabolic alteration in the tumor microenvironment (TME) is lipid accumulation, a feature associated with immune dysfunction. Here, we examined how CD8+ tumor infiltrating lymphocytes (TILs) respond to lipids within the TME. We found elevated concentrations of several classes of lipids in the TME and accumulation of these in CD8+ TILs. Lipid accumulation was associated with increased expression of CD36, a scavenger receptor for oxidized lipids, on CD8+ TILs, which also correlated with progressive T cell dysfunction. Cd36-/- T cells retained effector functions in the TME, as compared to WT counterparts. Mechanistically, CD36 promoted uptake of oxidized low-density lipoproteins (OxLDL) into T cells, and this induced lipid peroxidation and downstream activation of p38 kinase. Inhibition of p38 restored effector T cell functions in vitro, and resolution of lipid peroxidation by overexpression of glutathione peroxidase 4 restored functionalities in CD8+ TILs in vivo. Thus, an oxidized lipid-CD36 axis promotes intratumoral CD8+ T cell dysfunction and serves as a therapeutic avenue for immunotherapies.

Fundamental immune-oncogenicity trade-offs define driver mutation fitness.

Missense driver mutations in cancer are concentrated in a few hotspots1. Various mechanisms have been proposed to explain this skew, including biased mutational processes2, phenotypic differences3-6 and immunoediting of neoantigens7,8; however, to our knowledge, no existing model weighs the relative contribution of these features to tumour evolution. We propose a unified theoretical 'free fitness' framework that parsimoniously integrates multimodal genomic, epigenetic, transcriptomic and proteomic data into a biophysical model of the rate-limiting processes underlying the fitness advantage conferred on cancer cells by driver gene mutations. Focusing on TP53, the most mutated gene in cancer1, we present an inference of mutant p53 concentration and demonstrate that TP53 hotspot mutations optimally solve an evolutionary trade-off between oncogenic potential and neoantigen immunogenicity. Our model anticipates patient survival in The Cancer Genome Atlas and patients with lung cancer treated with immunotherapy as well as the age of tumour onset in germline carriers of TP53 variants. The predicted differential immunogenicity between hotspot mutations was validated experimentally in patients with cancer and in a unique large dataset of healthy individuals. Our data indicate that immune selective pressure on TP53 mutations has a smaller role in non-cancerous lesions than in tumours, suggesting that targeted immunotherapy may offer an early prophylactic opportunity for the former. Determining the relative contribution of immunogenicity and oncogenic function to the selective advantage of hotspot mutations thus has important implications for both precision immunotherapies and our understanding of tumour evolution.

CTLA-4 blockade drives loss of Treg stability in glycolysis-low tumours.

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 cells1. By contrast, the engagement of CTLA-4 has been shown to downregulate T cell glycolysis1. 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 (Treg) 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 Treg cells is dependent on Treg 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 Treg cell function in the presence of glucose.

Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer.

Pancreatic ductal adenocarcinoma is a lethal cancer with fewer than 7% of patients surviving past 5 years. T-cell immunity has been linked to the exceptional outcome of the few long-term survivors, yet the relevant antigens remain unknown. Here we use genetic, immunohistochemical and transcriptional immunoprofiling, computational biophysics, and functional assays to identify T-cell antigens in long-term survivors of pancreatic cancer. Using whole-exome sequencing and in silico neoantigen prediction, we found that tumours with both the highest neoantigen number and the most abundant CD8+ T-cell infiltrates, but neither alone, stratified patients with the longest survival. Investigating the specific neoantigen qualities promoting T-cell activation in long-term survivors, we discovered that these individuals were enriched in neoantigen qualities defined by a fitness model, and neoantigens in the tumour antigen MUC16 (also known as CA125). A neoantigen quality fitness model conferring greater immunogenicity to neoantigens with differential presentation and homology to infectious disease-derived peptides identified long-term survivors in two independent datasets, whereas a neoantigen quantity model ascribing greater immunogenicity to increasing neoantigen number alone did not. We detected intratumoural and lasting circulating T-cell reactivity to both high-quality and MUC16 neoantigens in long-term survivors of pancreatic cancer, including clones with specificity to both high-quality neoantigens and predicted cross-reactive microbial epitopes, consistent with neoantigen molecular mimicry. Notably, we observed selective loss of high-quality and MUC16 neoantigenic clones on metastatic progression, suggesting neoantigen immunoediting. Our results identify neoantigens with unique qualities as T-cell targets in pancreatic ductal adenocarcinoma. More broadly, we identify neoantigen quality as a biomarker for immunogenic tumours that may guide the application of immunotherapies.

Silibinin down-regulates PD-L1 expression in nasopharyngeal carcinoma by interfering with tumor cell glycolytic metabolism.

The upregulation of checkpoint inhibitor PD-L1 expression has recently been associated with nasopharyngeal carcinoma (NPC) resistance to therapy. The mechanism of induction of PD-L1 has also been linked to enhanced aerobic glycolysis promoted by HIF1-α dysregulation and LDH-A activity in cancer. Here, we investigated the effect of the anti-tumoral compound Silibinin on HIF-1α/LDH-A mediated cancer cell metabolism and PD-L1 expression in NPC. Our results demonstrate that exposure to Silibinin potently inhibits tumor growth and promotes a shift from aerobic glycolysis toward oxidative phosphorylation. The EBV + NPC cell line C666-1 and glycolytic human tumor explants treated with Silibinin displayed a reduction in LDH-A activity which consistently associated with a reduction in lactate levels. This effect was accompanied by an increase in intracellular citrate levels in C666-1 cells. Accordingly, expression of HIF-1α, a critical regulator of glycolysis, was down-regulated after treatment. This event associated with a down-regulation in PD-L1. Altogether, our results provide evidence that silibinin can alter PD-L1 expression by interfering with HIF-1α/LDH-A mediated cell metabolism in NPC. These results provide a new perspective for Silibinin use to overcome PD-L1 mediated NPC resistance to therapy.

HSPH1 inhibition downregulates Bcl-6 and c-Myc and hampers the growth of human aggressive B-cell non-Hodgkin lymphoma.

We have shown that human B-cell non-Hodgkin lymphomas (B-NHLs) express heat shock protein (HSP)H1/105 in function of their aggressiveness. Here, we now clarify its role as a functional B-NHL target by testing the hypothesis that it promotes the stabilization of key lymphoma oncoproteins. HSPH1 silencing in 4 models of aggressive B-NHLs was paralleled by Bcl-6 and c-Myc downregulation. In vitro and in vivo analysis of HSPH1-silenced Namalwa cells showed that this effect was associated with a significant growth delay and the loss of tumorigenicity when 10(4) cells were injected into mice. Interestingly, we found that HSPH1 physically interacts with c-Myc and Bcl-6 in both Namalwa cells and primary aggressive B-NHLs. Accordingly, expression of HSPH1 and either c-Myc or Bcl-6 positively correlated in these diseases. Our study indicates that HSPH1 concurrently favors the expression of 2 key lymphoma oncoproteins, thus confirming its candidacy as a valuable therapeutic target of aggressive B-NHLs.

Pleiotropic antitumor effects of the pan-HDAC inhibitor ITF2357 against c-Myc-overexpressing human B-cell non-Hodgkin lymphomas.

Histone deacetylases (HDAC) extensively contribute to the c-Myc oncogenic program, pointing to their inhibition as an effective strategy against c-Myc-overexpressing cancers. We, thus, studied the therapeutic activity of the new-generation pan-HDAC inhibitor ITF2357 (Givinostat®) against c-Myc-overexpressing human B-cell non-Hodgkin lymphomas (B-NHLs). ITF2357 anti-proliferative and pro-apoptotic effects were analyzed in B-NHL cell lines with c-Myc translocations (Namalwa, Raji and DOHH-2), stabilizing mutations (Raji) or post-transcriptional alterations (SU-DHL-4) in relationship to c-Myc modulation. ITF2357 significantly delayed the in vitro growth of all B-NHL cell lines by inducing G1 cell-cycle arrest, eventually followed by cell death. These events correlated with the extent of c-Myc protein, but not mRNA, downregulation, indicating the involvement of post-transcriptional mechanisms. Accordingly, c-Myc-targeting microRNAs let-7a and miR-26a were induced in all treated lymphomas and the cap-dependent translation machinery components 4E-BP1, eIF4E and eIF4G, as well as their upstream regulators, Akt and PIM kinases, were inhibited in function of the cell sensitivity to ITF2357, and, in turn, c-Myc downregulation. In vivo, ITF2357 significantly hampered the growth of Namalwa and Raji xenografts in immunodeficient mice. Noteworthy, its combination with suboptimal cyclophosphamide, achieved complete remissions in most animals and equaled or even exceeded the activity of optimal cyclophosphamide. Collectively, our findings provide the rationale for testing the clinical advantages of adding ITF2357 to current therapies for the still very ominous c-Myc-overexpressing lymphomas. They equally provide the proof-of-concept for its clinical evaluation in rational combination with the promising inhibitors of B-cell receptor and PI3K/Akt/mTOR axis currently in the process of development.

APR-246 increases tumor antigenicity independent of p53.

We previously reported that activation of p53 by APR-246 reprograms tumor-associated macrophages to overcome immune checkpoint blockade resistance. Here, we demonstrate that APR-246 and its active moiety, methylene quinuclidinone (MQ) can enhance the immunogenicity of tumor cells directly. MQ treatment of murine B16F10 melanoma cells promoted activation of melanoma-specific CD8+ T cells and increased the efficacy of a tumor cell vaccine using MQ-treated cells even when the B16F10 cells lacked p53. We then designed a novel combination of APR-246 with the TLR-4 agonist, monophosphoryl lipid A, and a CD40 agonist to further enhance these immunogenic effects and demonstrated a significant antitumor response. We propose that the immunogenic effect of MQ can be linked to its thiol-reactive alkylating ability as we observed similar immunogenic effects with the broad-spectrum cysteine-reactive compound, iodoacetamide. Our results thus indicate that combination of APR-246 with immunomodulatory agents may elicit effective antitumor immune response irrespective of the tumor's p53 mutation status.

Pharmacologic LDH inhibition redirects intratumoral glucose uptake and improves antitumor immunity in solid tumor models.

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.

Loss of CREBBP and KMT2D cooperate to accelerate lymphomagenesis and shape the lymphoma immune microenvironment.

Despite regulating overlapping gene enhancers and pathways, CREBBP and KMT2D mutations recurrently co-occur in germinal center (GC) B cell-derived lymphomas, suggesting potential oncogenic cooperation. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d induces a more severe mouse lymphoma phenotype (vs either allele alone) and unexpectedly confers an immune evasive microenvironment manifesting as CD8+ T-cell exhaustion and reduced infiltration. This is linked to profound repression of immune synapse genes that mediate crosstalk with T-cells, resulting in aberrant GC B cell fate decisions. From the epigenetic perspective, we observe interaction and mutually dependent binding and function of CREBBP and KMT2D on chromatin. Their combined deficiency preferentially impairs activation of immune synapse-responsive super-enhancers, pointing to a particular dependency for both co-activators at these specialized regulatory elements. Together, our data provide an example where chromatin modifier mutations cooperatively shape and induce an immune-evasive microenvironment to facilitate lymphomagenesis.

Germinal Center Dark Zone harbors ATR-dependent determinants of T-cell exclusion that are also identified in aggressive lymphoma.

The germinal center (GC) dark zone (DZ) and light zone (LZ) regions spatially separate expansion and diversification from selection of antigen-specific B-cells to ensure antibody affinity maturation and B cell memory. The DZ and LZ differ significantly in their immune composition despite the lack of a physical barrier, yet the determinants of this polarization are poorly understood. This study provides novel insights into signals controlling asymmetric T-cell distribution between DZ and LZ regions. We identify spatially-resolved DNA damage response and chromatin compaction molecular features that underlie DZ T-cell exclusion. The DZ spatial transcriptional signature linked to T-cell immune evasion clustered aggressive Diffuse Large B-cell Lymphomas (DLBCL) for differential T cell infiltration. We reveal the dependence of the DZ transcriptional core signature on the ATR kinase and dissect its role in restraining inflammatory responses contributing to establishing an immune-repulsive imprint in DLBCL. These insights may guide ATR-focused treatment strategies bolstering immunotherapy in tumors marked by DZ transcriptional and chromatin-associated features.

Serological identification of HSP105 as a novel non-Hodgkin lymphoma therapeutic target.

We reported that the clinical efficacy of dendritic cell-based vaccination is strongly associated with immunologic responses in relapsed B-cell non-Hodgkin lymphoma (B-NHL) patients. We have now investigated whether postvaccination antibodies from responders recognize novel shared NHL-restricted antigens. Immunohistochemistry and flow cytometry showed that they cross-react with allogeneic B-NHLs at significantly higher levels than their matched prevaccination samples or nonresponders' antibodies. Western blot analysis of DOHH-2 lymphoma proteome revealed a sharp band migrating at approximately 100 to 110 kDa only with postvaccine repertoires from responders. Mass spectrometry identified heat shock protein-105 (HSP105) in that molecular weight interval. Flow cytometry and immunohistochemistry disclosed HSP105 on the cell membrane and in the cytoplasm of B-NHL cell lines and 97 diagnostic specimens. A direct correlation between HSP105 expression and lymphoma aggressiveness was also apparent. Treatment of aggressive human B-NHL cell lines with an anti-HSP105 antibody had no direct effects on cell cycle or apoptosis but significantly reduced the tumor burden in xenotransplanted immunodeficient mice. In vivo antilymphoma activity of HSP105 engagement was associated with a significant local increase of Granzyme B(+) killer cells that very likely contributed to the tumor-restricted necrosis. Our study adds HSP105 to the list of nononcogenes that can be exploited as antilymphoma targets.

Targeting GITR in cancer immunotherapy - there is no perfect knowledge.

Glucocorticoid-induced TNFR-related protein (GITR) belongs to the TNFR superfamily (TNFRSF) and stimulates both the acquired and innate immunity. GITR is broadly expressed on immune cells, particularly regulatory T cells (Tregs) and natural killer (NK) cells. Given its potential to promote T effector function and impede Treg immune suppression, GITR is an attractive target for cancer immunotherapy. Preclinically, GITR agonists have demonstrated potent anti-tumor efficacy singly and in combination with a variety of agents, including PD-1 blockade. Multiple GITR agonists have been advanced into the clinic, although the experience with these agents has been disappointing. Recent mechanistic insights into the roles of antibody structure, valency, and Fc functionality in mediating anti-tumor efficacy may explain some of the apparent inconsistency or discordance between preclinical data and observed clinical efficacy.

Modulating Treg stability to improve cancer immunotherapy.

Immunosuppressive regulatory T cells (Tregs) provide a main mechanism of tumor immune evasion. Targeting Tregs, especially in the tumor microenvironment (TME), continues to be investigated to improve cancer immunotherapy. Recent studies have unveiled intratumoral Treg heterogeneity and plasticity, furthering the complexity of the role of Tregs in tumor immunity and immunotherapy response. The phenotypic and functional diversity of intratumoral Tregs can impact their response to therapy and may offer new targets to modulate specific Treg subsets. In this review we provide a unifying framework of critical factors contributing to Treg heterogeneity and plasticity in the TME, and we discuss how this information can guide the development of more specific Treg-targeting therapies for cancer immunotherapy.

Society for Immunotherapy of Cancer (SITC) consensus definitions for resistance to combinations of immune checkpoint inhibitors.

Immunotherapy is the standard of care for several cancers and the field continues to advance at a rapid pace, with novel combinations leading to indications in an increasing number of disease settings. Durable responses and long-term survival with immunotherapy have been demonstrated in some patients, though lack of initial benefit and recurrence after extended disease control remain major hurdles for the field. Many new combination regimens are in development for patients whose disease progressed on initial immunotherapy. To guide clinical trial design and support analyses of emerging molecular and cellular data surrounding mechanisms of resistance, the Society for Immunotherapy of Cancer (SITC) previously generated consensus clinical definitions for resistance to single-agent anti-PD-1 immune checkpoint inhibitors (ICIs) in three distinct scenarios: primary resistance, secondary resistance, and progression after treatment discontinuation. An unmet need still exists, however, for definitions of resistance to ICI-based combinations, which represent an expanding frontier in the immunotherapy treatment landscape. In 2021, SITC convened a workshop including stakeholders from academia, industry, and government to develop consensus definitions for resistance to ICI-based combination regimens for improved outcome assessment, trial design and drug development. This manuscript reports the minimum drug exposure requirements and time frame for progression that define resistance in both the metastatic setting and the perioperative setting, as well as key caveats and areas for future research with ICI/ICI combinations. Definitions for resistance to ICIs in combination with chemotherapy and targeted therapy will be published in companion volumes to this paper.

Updates on radiotherapy-immunotherapy combinations: Proceedings of 6th annual ImmunoRad conference.

Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference.