Hypoxia as a potential inducer of immune tolerance, tumor plasticity and a driver of tumor mutational burden: Impact on cancer immunotherapy.

In cancer patients, immune cells are often functionally compromised due to the immunosuppressive features of the tumor microenvironment (TME) which contribute to the failures in cancer therapies. Clinical and experimental evidence indicates that developing tumors adapt to the immunological environment and create a local microenvironment that impairs immune function by inducing immune tolerance and invasion. In this context, microenvironmental hypoxia, which is an established hallmark of solid tumors, significantly contributes to tumor aggressiveness and therapy resistance through the induction of tumor plasticity/heterogeneity and, more importantly, through the differentiation and expansion of immune-suppressive stromal cells. We and others have provided evidence indicating that hypoxia also drives genomic instability in cancer cells and interferes with DNA damage response and repair suggesting that hypoxia could be a potential driver of tumor mutational burden. Here, we reviewed the current knowledge on how hypoxic stress in the TME impacts tumor angiogenesis, heterogeneity, plasticity, and immune resistance, with a special interest in tumor immunogenicity and hypoxia targeting. An integrated understanding of the complexity of the effect of hypoxia on the immune and microenvironmental components could lead to the identification of better adapted and more effective combinational strategies in cancer immunotherapy. Clearly, the discovery and validation of therapeutic targets derived from the hypoxic tumor microenvironment is of major importance and the identification of critical hypoxia-associated pathways could generate targets that are undeniably attractive for combined cancer immunotherapy approaches.

CMTM6 and CMTM7: New leads for PD-L1 regulation in breast cancer cells undergoing EMT.

Programmed death-ligand 1 (PD-L1) expression has long been used as a biomarker to stratify patients with cancer who will benefit from anti-PD-1/PD-L1 immunotherapy. However, the use of PD-L1 as a biomarker to guide treatment decisions has recently been called into question due to its dynamic and heterogeneous expression within each tumor and among different tumors as well as during tumor cell plasticity. Therefore, understanding the molecular basis of PD-L1 expression would enable delineating its value as a reliable biomarker in the clinic. Here, we provide our perspective on the involvement of CMTM6 and CMTM7 as new lead candidates for the regulation of PD-L1 in breast tumors undergoing an epithelial to mesenchymal transition.

Targeting autophagy inhibits melanoma growth by enhancing NK cells infiltration in a CCL5-dependent manner.

While blocking tumor growth by targeting autophagy is well established, its role on the infiltration of natural killer (NK) cells into tumors remains unknown. Here, we investigate the impact of targeting autophagy gene Beclin1 (BECN1) on the infiltration of NK cells into melanomas. We show that, in addition to inhibiting tumor growth, targeting BECN1 increased the infiltration of functional NK cells into melanoma tumors. We provide evidence that driving NK cells to the tumor bed relied on the ability of autophagy-defective tumors to transcriptionally overexpress the chemokine gene CCL5 Such infiltration and tumor regression were abrogated by silencing CCL5 in BECN1-defective tumors. Mechanistically, we show that the up-regulated expression of CCL5 occurred through the activation of its transcription factor c-Jun by a mechanism involving the impairment of phosphatase PP2A catalytic activity and the subsequent activation of JNK. Similar to BECN1, targeting other autophagy genes, such as ATG5, p62/SQSTM1, or inhibiting autophagy pharmacologically by chloroquine, also induced the expression of CCL5 in melanoma cells. Clinically, a positive correlation between CCL5 and NK cell marker NKp46 expression was found in melanoma patients, and a high expression level of CCL5 was correlated with a significant improvement of melanoma patients' survival. We believe that this study highlights the impact of targeting autophagy on the tumor infiltration by NK cells and its benefit as a novel therapeutic approach to improve NK-based immunotherapy.

The 'Holy Grail' in Immuno-Oncology: AC BioScience SA is Aiming to Potentiate Anti-PD-1 Therapy Efficacy through Tumor Cell Conditioning Strategy.

AC BioScience is a Swiss biotech company based at the EPFL Innovation Park and Biopôle, dedicated to developing groundbreaking therapies to fight a range of cancers and infectious diseases. We are about to start clinical trials with two of four leading-edge cancer drugs mainly focusing on immune-oncology and tumor vascular normalization with multi-billion $ sales potential. Here, we present our strategy and one of our pioneering drug candidates that has already shown exceptional results with tumor cell conditioning to improve the efficacy of immune checkpoint inhibitors.

Thioridazine inhibits autophagy and sensitizes glioblastoma cells to temozolomide.

Glioblastoma multiforme (GBM) has a poor prognosis with an overall survival of 14-15 months after surgery, radiation and chemotherapy using temozolomide (TMZ). A major problem is that the tumors acquire resistance to therapy. In an effort to improve the therapeutic efficacy of TMZ, we performed a genome-wide RNA interference (RNAi) synthetic lethality screen to establish a functional gene signature for TMZ sensitivity in human GBM cells. We then queried the Connectivity Map database to search for drugs that would induce corresponding changes in gene expression. By this approach we identified several potential pharmacological sensitizers to TMZ, where the most potent drug was the established antipsychotic agent Thioridazine, which significantly improved TMZ sensitivity while not demonstrating any significant toxicity alone. Mechanistically, we show that the specific chemosensitizing effect of Thioridazine is mediated by impairing autophagy, thereby preventing adaptive metabolic alterations associated with TMZ resistance. Moreover, we demonstrate that Thioridazine inhibits late-stage autophagy by impairing fusion between autophagosomes and lysosomes. Finally, Thioridazine in combination with TMZ significantly inhibits brain tumor growth in vivo, demonstrating the potential clinical benefits of compounds targeting the autophagy-lysosome pathway. Our study emphasizes the feasibility of exploiting drug repurposing for the design of novel therapeutic strategies for GBM.

Cutting Edge: NANOG Activates Autophagy under Hypoxic Stress by Binding to BNIP3L Promoter.

Hypoxia upregulates the core pluripotency factors NANOG, SOX2, and OCT4, associated with tumor aggressiveness and resistance to conventional anticancer treatments. We have previously reported that hypoxia-induced NANOG contributed in vitro to tumor cell resistance to autologous-specific CTL and in vivo to the in situ recruitment of immune-suppressive cells. In this study, we investigated the mechanisms underlying NANOG-mediated tumor cell resistance to specific lysis under hypoxia. We demonstrated the tumor-promoting effect of hypoxia on tumor initiation into immunodeficient mice using human non-small lung carcinoma cells. We next showed a link between NANOG and autophagy activation under hypoxia because inhibition of NANOG decreased autophagy in tumor cells. Chromatin immunoprecipitation and luciferase reporter assays revealed a direct binding of NANOG to a transcriptionally active site in a BNIP3L enhancer sequence. These data establish a new link between the pluripotency factor NANOG and autophagy involved in resistance to CTL under hypoxia.

Inhibition of HIF1α-Dependent Upregulation of Phospho-l-Plastin Resensitizes Multiple Myeloma Cells to Frontline Therapy.

The introduction of novel frontline agents in multiple myeloma (MM), like immunomodulatory drugs and proteasome inhibitors, has improved the overall survival of patients. Yet, MM is still not curable, and drug resistance (DR) remains the main challenge. To improve the understanding of DR in MM, we established a resistant cell line (MOLP8/R). The exploration of DR mechanisms yielded an overexpression of HIF1α, due to impaired proteasome activity of MOLP8/R. We show that MOLP8/R, like other tumor cells, overexpressing HIF1α, have an increased resistance to the immune system. By exploring the main target genes regulated by HIF1α, we could not show an overexpression of these targets in MOLP8/R. We, however, show that MOLP8/R cells display a very high overexpression of LCP1 gene (l-Plastin) controlled by HIF1α, and that this overexpression also exists in MM patient samples. The l-Plastin activity is controlled by its phosphorylation in Ser5. We further show that the inhibition of l-Plastin phosphorylation restores the sensitivity of MOLP8/R to immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs). Our results reveal a new target gene of DR, controlled by HIF1α.

Regulation of hypoxia-induced autophagy in glioblastoma involves ATG9A.

BACKGROUND: Hypoxia is negatively associated with glioblastoma (GBM) patient survival and contributes to tumour resistance. Anti-angiogenic therapy in GBM further increases hypoxia and activates survival pathways. The aim of this study was to determine the role of hypoxia-induced autophagy in GBM. METHODS: Pharmacological inhibition of autophagy was applied in combination with bevacizumab in GBM patient-derived xenografts (PDXs). Sensitivity towards inhibitors was further tested in vitro under normoxia and hypoxia, followed by transcriptomic analysis. Genetic interference was done using ATG9A-depleted cells. RESULTS: We find that GBM cells activate autophagy as a survival mechanism to hypoxia, although basic autophagy appears active under normoxic conditions. Although single agent chloroquine treatment in vivo significantly increased survival of PDXs, the combination with bevacizumab resulted in a synergistic effect at low non-effective chloroquine dose. ATG9A was consistently induced by hypoxia, and silencing of ATG9A led to decreased proliferation in vitro and delayed tumour growth in vivo. Hypoxia-induced activation of autophagy was compromised upon ATG9A depletion. CONCLUSIONS: This work shows that inhibition of autophagy is a promising strategy against GBM and identifies ATG9 as a novel target in hypoxia-induced autophagy. Combination with hypoxia-inducing agents may provide benefit by allowing to decrease the effective dose of autophagy inhibitors.

Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts.

Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin-positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts.

The Selective Degradation of Synaptic Connexin 43 Protein by Hypoxia-induced Autophagy Impairs Natural Killer Cell-mediated Tumor Cell Killing.

Although natural killer (NK) cells play an important role in the control of melanoma, hypoxic stress in the tumor microenvironment may impair NK-mediated tumor cell killing by mechanisms that are not fully understood. In this study, we investigated the effect of hypoxia on the expression and channel activity of connexin 43 (Cx43) in melanoma cells and its impact on their susceptibility to NK cell-mediated lysis. Our results demonstrated that hypoxic stress increases Cx43 expression in melanoma cells via hypoxia-inducible factor-1α (HIF-1α) transcriptional activity. Hypoxic cells displaying increased Cx43 expression were less susceptible to NK cell-mediated lysis compared with normoxic cells expressing a moderate level of Cx43. Conversely, when overexpressed in normoxic tumor cells, Cx43 improves their susceptibility to N cell-mediated killing. We show that the NK cell immune synapse formed with normoxic melanoma cells is more stable and contains a high level of gap-junctional Cx43 whereas that formed with hypoxic cells is less stable and contains a significant lower level of gap-junctional Cx43. We provide evidence that the activation of autophagy in hypoxic melanoma cells selectively degrades gap-junctional Cx43, leading to the destabilization of the immune synapse and the impairment of NK cell-mediated killing. Inhibition of autophagy by genetic or pharmacological approaches as well as expression of the non-degradable form of Cx43 significantly restore its accumulation at the immune synapse and improves N cell-mediated lysis of hypoxic melanoma cells. This study provides the first evidence that the hypoxic microenvironment negatively affects the immune surveillance of tumors by NK cells through the modulation of Cx43-mediated intercellular communications.

Granzyme B degradation by autophagy decreases tumor cell susceptibility to natural killer-mediated lysis under hypoxia.

Recent studies demonstrated that autophagy is an important regulator of innate immune response. However, the mechanism by which autophagy regulates natural killer (NK) cell-mediated antitumor immune responses remains elusive. Here, we demonstrate that hypoxia impairs breast cancer cell susceptibility to NK-mediated lysis in vitro via the activation of autophagy. This impairment was not related to a defect in target cell recognition by NK cells but to the degradation of NK-derived granzyme B in autophagosomes of hypoxic cells. Inhibition of autophagy by targeting beclin1 (BECN1) restored granzyme B levels in hypoxic cells in vitro and induced tumor regression in vivo by facilitating NK-mediated tumor cell killing. Together, our data highlight autophagy as a mechanism underlying the resistance of hypoxic tumor cells to NK-mediated lysis. The work presented here provides a cutting-edge advance in our understanding of the mechanism by which hypoxia-induced autophagy impairs NK-mediated lysis in vitro and paves the way for the formulation of more effective NK cell-based antitumor therapies.

Hypoxia: a key player in antitumor immune response. A Review in the Theme: Cellular Responses to Hypoxia.

The tumor microenvironment is a complex system, playing an important role in tumor development and progression. Besides cellular stromal components, extracellular matrix fibers, cytokines, and other metabolic mediators are also involved. In this review we outline the potential role of hypoxia, a major feature of most solid tumors, within the tumor microenvironment and how it contributes to immune resistance and immune suppression/tolerance and can be detrimental to antitumor effector cell functions. We also outline how hypoxic stress influences immunosuppressive pathways involving macrophages, myeloid-derived suppressor cells, T regulatory cells, and immune checkpoints and how it may confer tumor resistance. Finally, we discuss how microenvironmental hypoxia poses both obstacles and opportunities for new therapeutic immune interventions.

Tumor plasticity interferes with anti-tumor immunity.

Since tumor cell plasticity was first shown to be crucial in tumor promotion and immune surveillance evasion, it has become an issue of intense investigation. Several mechanisms are associated with the acquisition of tumor cell plasticity and immune evasion, including loss of epithelial phenotype through epithelial-to-mesenchymal transition (EMT). We discuss recent evidence revealing that tumor cell plasticity may lead to the emergence of immunoresistant variants and how the tumor microenvironment evolves to shape this plasticity. We argue that targeting carcinoma cell plasticity represents a novel strategy to better control the emergence of resistant variants and to ensure more effective cancer therapies. In this context, the design of innovative integrative immunotherapy approaches is warranted.

Improving STING agonist-based cancer therapy by inhibiting the autophagy-related protein VPS34.

We have recently demonstrated that inhibiting VPS34 enhances T-cell-recruiting chemokines through the activation of the cGAS/STING pathway using the STING agonist ADU-S100. Combining VPS34 inhibitors with ADU-S100 increased cytokine release and improved tumor control in mouse models, suggesting a potential synergy between VPS34 inhibition and therapies based on STING agonists.

Combining VPS34 inhibitors with STING agonists enhances type I interferon signaling and anti-tumor efficacy.

An immunosuppressive tumor microenvironment promotes tumor growth and is one of the main factors limiting the response to cancer immunotherapy. We have previously reported that inhibition of vacuolar protein sorting 34 (VPS34), a crucial lipid kinase in the autophagy/endosomal trafficking pathway, decreases tumor growth in several cancer models, increases infiltration of immune cells and sensitizes tumors to anti-programmed cell death protein 1/programmed cell death 1 ligand 1 therapy by upregulation of C-C motif chemokine 5 (CCL5) and C-X-C motif chemokine 10 (CXCL10) chemokines. The purpose of this study was to investigate the signaling mechanism leading to the VPS34-dependent chemokine increase. NanoString gene expression analysis was applied to tumors from mice treated with the VPS34 inhibitor SB02024 to identify key pathways involved in the anti-tumor response. We showed that VPS34 inhibitors increased the secretion of T-cell-recruitment chemokines in a cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes protein (STING)-dependent manner in cancer cells. Both pharmacological and small interfering RNA (siRNA)-mediated VPS34 inhibition increased cGAS/STING-mediated expression and secretion of CCL5 and CXCL10. The combination of VPS34 inhibitor and STING agonist further induced cytokine release in both human and murine cancer cells as well as monocytic or dendritic innate immune cells. Finally, the VPS34 inhibitor SB02024 sensitized B16-F10 tumor-bearing mice to STING agonist treatment and significantly improved mice survival. These results show that VPS34 inhibition augments the cGAS/STING pathway, leading to greater tumor control through immune-mediated mechanisms. We propose that pharmacological VPS34 inhibition may synergize with emerging therapies targeting the cGAS/STING pathway.

Lighting Up the Fire in the Microenvironment of Cold Tumors: A Major Challenge to Improve Cancer Immunotherapy.

Immunotherapy includes immune checkpoint inhibitors (ICI) such as antibodies targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or the programmed cell death protein/programmed death ligand 1 (PD-1/PD-L1) axis. Experimental and clinical evidence show that immunotherapy based on immune checkpoint inhibitors (ICI) provides long-term survival benefits to cancer patients in whom other conventional therapies have failed. However, only a minority of patients show high clinical benefits via the use of ICI alone. One of the major factors limiting the clinical benefits to ICI can be attributed to the lack of immune cell infiltration within the tumor microenvironment. Such tumors are classified as "cold/warm" or an immune "desert"; those displaying significant infiltration are considered "hot" or inflamed. This review will provide a brief summary of different tumor properties contributing to the establishment of cold tumors and describe major strategies that could reprogram non-inflamed cold tumors into inflamed hot tumors. More particularly, we will describe how targeting hypoxia can induce metabolic reprogramming that results in improving and extending the benefit of ICI.

Microenvironmental hypoxia orchestrating the cell stroma cross talk, tumor progression and antitumor response.

Hypoxia, a common feature of solid tumors and one of the hallmarks of tumor microenvironment, favors tumor survival and progression. Although hypoxia has been reported to play a major role in the acquisition of tumor resistance to cell death, the molecular mechanisms that control the survival of hypoxic cancer cells and the role of hypoxic stress in shaping the cross talk between immune cells and stroma components are not fully elucidated. Recently, several lines of investigation are pointing to yet another ominous outcome of hypoxia in the tumor microenvironment involving suppression of antitumor immune effector cells and enhancement of tumor escape from immune surveillance. Although the identification of tumor-associated antigens provided a new arsenal of approaches to enhance antigen-specific response, the immunotherapy approaches that are currently used in the clinic have only limited success. In fact, tumor stroma components including hypoxia are engaged in an active molecular cross talk that has serious implications for immunological recognition of tumor in shaping the microenvironment. In this review, we will focus on the impact of hypoxia on the regulation of the antitumor response and the subsequent tumor progression. We will also in particular discuss data that indicate that manipulation of hypoxic stress may represent an innovative strategy for a better immunotherapy of cancer.

The Promise of Targeting Hypoxia to Improve Cancer Immunotherapy: Mirage or Reality?

Almost all solid tumors display hypoxic areas in the tumor microenvironment associated with therapeutic failure. It is now well established that the abnormal growth of malignant solid tumors exacerbates their susceptibility to hypoxia. Therefore, targeting hypoxia remains an attractive strategy to sensitize tumors to various therapies. Tumor cell adaptions to hypoxia are primarily mediated by hypoxia-inducible factor-1 alpha (HIF-1α). Sensing hypoxia by HIF-1α impairs the apoptotic potential of tumor cells, thus increasing their proliferative capacity and contributing to the development of a chaotic vasculature in the tumor microenvironment. Therefore, in addition to the negative impact of hypoxia on tumor response to chemo- and radio-therapies, hypoxia has also been described as a major hijacker of the tumor response by impairing the tumor cell susceptibility to immune cell killing. This review is not intended to provide a comprehensive overview of the work published by several groups on the multiple mechanisms by which hypoxia impairs the anti-tumor immunity and establishes the immunosuppressive tumor microenvironment. There are several excellent reviews highlighting the value of targeting hypoxia to improve the benefit of immunotherapy. Here, we first provide a brief overview of the mechanisms involved in the establishment of hypoxic stress in the tumor microenvironment. We then discuss our recently published data on how targeting hypoxia, by deleting a critical domain in HIF-1α, contributes to the improvement of the anti-tumor immune response. Our aim is to support the current dogma about the relevance of targeting hypoxia in cancer immunotherapy.

A role for EMT in CD73 regulation in breast cancer.

CD73 is an emerging target in cancer due to its role in generating adenosine, a potent immunosuppressor. We found that SNAI1, a driver of epithelial-to-mesenchymal transition (EMT), upregulates CD73 in triple negative breast cancer cells. Here, we discuss the relevance of improving CD73-based therapy by combining with inhibitors of EMT.