Suppression Of Antitumor Immune Responses Research Articles

Autophagy and tumorigenesis.

Autophagy is a catabolic process that captures cellular waste and degrades them in the lysosome. The main functions of autophagy are quality control of cytosolic proteins and organelles, and intracellular recycling of nutrients in order to maintain cellular homeostasis. Autophagy is upregulated in many cancers to promote cell survival, proliferation, and metastasis. Both cell-autonomous autophagy (also known as tumor autophagy) and non-cell-autonomous autophagy (also known as host autophagy) support tumorigenesis through different mechanisms, including inhibition of p53 activation, sustaining redox homeostasis, maintenance of essential amino acids levels in order to support energy production and biosynthesis, and inhibition of antitumor immune responses. Therefore, autophagy may serve as a tumor-specific vulnerability and targeting autophagy could be a novel strategy in cancer treatment.

Abstract 1771: SIGLEC15 humanized knock-in mice are a powerful tool to evaluate new therapies for cancer and osteoporosis

Abstract SIGLEC15 (Sialic acid-binding immunoglobulin-like lectin 15) belongs to the Siglec family of sialic acid-binding, immunoglobulin-like proteins. SIGLEC15 is an effective immunosuppressive molecule which is highly expressed on M2 macrophages but can be upregulated in many human cancers. The SIGLEC15 signaling pathway is non-redundant to the PD-L1 pathway in cancer, and the surface expression of SIGLEC15 is generally non-overlapping with PD-L1 expression on tumors. SIGLEC15 engages a putative responder protein expressed on the surface of effector cells, such as CD8 T cells, inducing suppression of anti-tumor immune responses. Blocking of SIGLEC15 amplifies anti-tumor immunity in the tumor microenvironment and inhibits tumor growth. This opens a road for anti-SIGLEC15 antibody therapeutics which may provide a solution for patients with SIGLEC15-positive and PD-L1-negative tumors, a patient population less likely to respond to a PD-1/PD-L1 directed therapy. Biocytogen has generated a humanized SIGLEC15 mouse model to address the need for in vitro functional validation and in vivo efficacy evaluation of SIGLEC15-targeting strategies. In these mice, genomic DNA spanning exons 2~4 of mouse Siglec15 gene encoding the extracellular domain, was replaced by its human counterpart. Following the humanization procedure, human SIGLEC15 could be detected on bone marrow derived macrophages (BMDM). Basal leukocyte subpopulations, including T/B/NK cells, DC, granulocytes, and monocytes/macrophages, were similar between humanized and wild-type mice. In addition, Biocytogen has generated a humanized MC38 cell line, in which the mouse Siglec15 gene was replaced with the full-length human coding sequence. The successful humanization of M38 was confirmed by RT-PCR and flow cytometry. Using the hMC38 tumor model, we showed robust efficacy of the anti-SIGLEC15 antibodies in inhibiting tumor growth in vivo. Combined SIGLEC15 and PD-1/PD-L1 blockade showed greater tumor inhibition than monotherapies. In summary, SIGLEC15 humanized mice can be employed for in vivo efficacy evaluation of anti-SIGLEC15 antibodies in immune normalization approaches for cancer treatment. Besides the tumor models (B16F10 and other tumors underway), we are also actively exploring the feasibility of the SIGLEC15 humanized mice as a pre-clinical model for osteoporosis in a light of recent discovery of SIGLEC15's role in osteoclast differentiation. Citation Format: Huilin Li, Lei Zhao, Maopeng Tian, Veronika Chromikova, Zhaoxue (Luke) Yu. SIGLEC15 humanized knock-in mice are a powerful tool to evaluate new therapies for cancer and osteoporosis [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 1771.

Targeting Treg cells with GITR activation alleviates resistance to immunotherapy in murine glioblastomas

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma (GBM) trials. Here, we show that regulatory T (Treg) cells play a key role in GBM resistance to ICBs in experimental gliomas. Targeting glucocorticoid-induced TNFR-related receptor (GITR) in Treg cells using an agonistic antibody (αGITR) promotes CD4 Treg cell differentiation into CD4 effector T cells, alleviates Treg cell-mediated suppression of anti-tumor immune response, and induces potent anti-tumor effector cells in GBM. The reprogrammed GBM-infiltrating Treg cells express genes associated with a Th1 response signature, produce IFNγ, and acquire cytotoxic activity against GBM tumor cells while losing their suppressive function. αGITR and αPD1 antibodies increase survival benefit in three experimental GBM models, with a fraction of cohorts exhibiting complete tumor eradication and immune memory upon tumor re-challenge. Moreover, αGITR and αPD1 synergize with the standard of care treatment for newly-diagnosed GBM, enhancing the cure rates in these GBM models.

Targeting the tumor microenvironment in chronic lymphocytic leukemia.

The tumor microenvironment (TME) plays an essential role in the development, growth, and survival of the malignant B-cell clone in chronic lymphocytic leukemia (CLL). Within the proliferation niches of lymph nodes, bone marrow, and secondary lymphoid organs, a variety of phenotypically and functionally altered cell types, including T cells, natural killer cells, monocytes/macrophages, endothelial and mesenchymal stroma cells, provide crucial survival signals, along with CLL-cellinduced suppression of antitumor immune responses. The B-cell receptor pathway plays a pivotal role in mediating the interaction between CLL cells and the TME. However, an increasing number of additional components of the multifactorial TME are being discovered. Although the majority of therapeutic strategies employed in CLL hitherto have focused on targeting the leukemic cells, emerging evidence implies that modulation of microenvironmental cells and CLL-TME interactions by novel therapeutic agents significantly affect their clinical efficacy. Thus, improving our understanding of CLL-TME interactions and how they are affected by current therapeutic agents may improve and guide treatment strategies. Identification of novel TME interactions may also pave the road for the development of novel therapeutic strategies targeting the TME. In this review, we summarize current evidence on the effects of therapeutic agents on cells and interactions within the TME. With a growing demand for improved and personalized treatment options in CLL, this review aims at inspiring future exploration of smart drug combination strategies, translational studies, and novel therapeutic targets in clinical trials.

Combined Effects of Myeloid Cells in the Neuroblastoma Tumor Microenvironment.

Simple SummaryNeuroblastoma is a type of childhood solid cancer often characterized by resistance to treatment and with poor prognosis. The tumors contain a mixture of cell types as well as cancer cells, and one of the most common is a type of blood cells called myeloid cells. In this review article we present a summary of what is known about the function of myeloid cells within neuroblastoma tumors.Despite multimodal treatment, survival chances for high-risk neuroblastoma patients remain poor. Immunotherapeutic approaches focusing on the activation and/or modification of host immunity for eliminating tumor cells, such as chimeric antigen receptor (CAR) T cells, are currently in development, however clinical trials have failed to reproduce the preclinical results. The tumor microenvironment is emerging as a major contributor to immune suppression and tumor evasion in solid cancers and thus has to be overcome for therapies relying on a functional immune response. Among the cellular components of the neuroblastoma tumor microenvironment, suppressive myeloid cells have been described as key players in inhibition of antitumor immune responses and have been shown to positively correlate with more aggressive disease, resistance to treatments, and overall poor prognosis. This review article summarizes how neuroblastoma-driven inflammation induces suppressive myeloid cells in the tumor microenvironment and how they in turn sustain the tumor niche through suppressor functions, such as nutrient depletion and generation of oxidative stress. Numerous preclinical studies have suggested a range of drug and cellular therapy approaches to overcome myeloid-derived suppression in neuroblastoma that warrant evaluation in future clinical studies.

Functional Analysis of Tumor-Infiltrating Myeloid Cells by Flow Cytometry and Adoptive Transfer.

The tumor-infiltrating myeloid cell compartment represents a heterogeneous population of broadly immunosuppressive cells that have been exploited by the tumor to support its growth. Their accumulation in tumor and secondary lymphoid tissue leads to the suppression of antitumor immune responses and is thus a target for therapeutic intervention. As it is known that the local cytokine milieu can dictate the functional programming of tumor-infiltrating myeloid cells, strategies have been devised to manipulate the tumor microenvironment (TME) to express a cytokine landscape more conducive to antitumor myeloid cell activity. To evaluate therapy-induced changes in tumor-infiltrating myeloid cells, this paper will outline the procedure to dissociate intradermal/subcutaneous tumor tissue from solid tumor-bearing mice in preparation for leukocyte recovery. Strategies for flow cytometric analysis will be provided to enable the identification of heterogeneous myeloid populations within isolated leukocytes and the characterization of unique myeloid phenotypes. Lastly, this paper will describe a means of purifying viable myeloid cells for functional assays and determining their therapeutic value in the context of adoptive transfer.

Mucosal thickening of the maxillary sinus is frequently associated with diffuse glioma patients and correlates with poor survival prognosis of GBM patients: comparative analysis to meningioma patients

Glioma patients were frequently associated with mucosal thickening of the maxillary sinus (MTMS), which reflects mucosal inflammation. We suspected that MTMS is associated with impaired mucosal immune response and correlated with dysfunction in the anti-tumor immune response in diffuse glioma patients. Therefore, the aim of this study was to determine whether the occurrence of diffuse glioma is correlated with MTMS compared to meningioma and control groups. Furthermore, we investigated whether MTMS is associated with overall survival (OS) in glioblastoma (GBM) patients. This study included 343 patients with newly diagnosed diffuse gliomas and 218 patients with meningioma treated at our institution between 2015 and 2018. As control, 201 patients with headache who did not have an intracranial organic lesion were included. Using three-axis MR images, we evaluated the incidence of MTMS in all patients. Additionally, we investigated the relationship between MTMS and OS. The incidence of MTMS in patients with diffuse glioma was significantly higher than that in the meningioma (p < .0001) and control groups (p < .0001). In 128 patients with GBM, MTMS status correlated significantly with OS (p = .0064). We revealed that the incidence of MTMS is significantly associated with patients with diffuse glioma. This suggests that MTMS is indirectly involved in the occurrence of diffuse gliomas. Furthermore, the presence of MTMS correlated significantly with shorter OS in GBM patients, indicating that MTMS is involved in suppression of anti-tumor immune response. Preoperative recognition of MTMS might be useful for improving the clinical management of GBM patients.

Adenosinergic Pathway: A Hope in the Immunotherapy of Glioblastoma.

Simple SummaryGlioblastoma multiforme (GBM) is the most aggressive type of brain tumor with dismal survival and poor response to conventional therapies. Therefore, the development of immunotherapy for GBM treatment is necessary. However, the rigorous immunosuppression in the GBM-microenvironment (GME) is a crucial impediment for GBM immunotherapy. The adenosinergic pathway (AP) is a major player in suppressing antitumor immune responses in the GME. We reviewed the current GBM immunotherapies and elaborated on the role of AP in the immunopathogenesis, treatment, and even prognosis of GBM. Tumor cells metabolize pro-inflammatory ATP to anti-inflammatory adenosine using CD39 and CD73 enzymes. Adenosine suppresses immune responses through the signaling of adenosine receptors on immune cells. The preclinical results targeting AP in the GBM showed promising results in reinvigorating antitumor responses and overriding chemoresistance. We suggest that future clinical studies should consider this pathway in combination therapies along with other immunotherapeutic approaches.Brain tumors comprise different types of malignancies, most of which are originated from glial cells. Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor with a poor response to conventional therapies and dismal survival rates (15 months) despite multimodal therapies. The development of immunotherapeutic strategies seems to be necessary to enhance the overall survival of GBM patients. So far, the immunotherapies applied in GBM had promising results in the primary phases of clinical trials but failed to continue their beneficial effects in later phases. GBM-microenvironment (GME) is a heterogenic and rigorously immunosuppressive milieu wrapping by an impenetrable blood-brain barrier. Hence, in-depth knowledge about the dominant immunosuppressive mechanisms in the GME could foster GBM immunotherapy. Recently, the adenosinergic pathway (AP) is found to be a major player in the suppression of antitumor immune responses in the GME. Tumor cells evolve to metabolize pro-inflammatory ATP to anti-inflammatory adenosine. Adenosine can suppress immune responses through the signaling of adenosine receptors on immune cells. The preclinical results targeting AP in GBM showed promising results in reinvigorating antitumor responses, overriding chemoresistance, and increasing survival. We reviewed the current GBM immunotherapies and elaborated on the role of AP in the immunopathogenesis, treatment, and even prognosis of GBM. We suggest that future clinical studies should consider this pathway in their combination therapies along with other immunotherapeutic approaches.

Abstract PO-055: Pan-cancer metabolic profiling of the tumor microenvironment

Abstract Tumors are heterogeneous cellular environments with entwined metabolic dependencies. However, it is challenging to differentially profile metabolism in cancer and infiltrating non-malignant cells inside patient tumors. Here, we used a tumor transcriptome deconvolution approach to profile the metabolic states of cancer and non-cancer (stromal) cells in bulk tumors of 20 solid tumor types. This analysis highlighted metabolic genes and processes recurrently upregulated in cancer cells across tumor types, including the mitochondrial dicarboxylate carrier SLC25A10. Intriguingly, the tryptophan-degrading dioxygenase, IDO1, was highly recurrently overexpressed in stroma compared to cancer cells and normal tissue, suggesting that tryptophan-mediated suppression of antitumor immune response is predominantly constrained by the stroma. Oxidative phosphorylation was unexpectedly the most upregulated metabolic process in cancer cells compared to both stromal cells and a large atlas of cancer cell lines, suggesting that the warburg effect may be less pronounced in cancer cells in vivo. Overall, our analysis highlights fundamental differences in metabolic states of cancer and stromal cells inside tumors and establishes a pan-cancer resource to interrogate tumor metabolism. Citation Format: Neha Rohatgi, Umesh Ghoshdastider, Probhonjon Baruah, Anders Jacobsen Skanderup. Pan-cancer metabolic profiling of the tumor microenvironment [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-055.

BIOM-30. DECREASED B CELL AND ALTERED CD4+ T CELL SUBSET RATIO PREDICT FAVORABLE RESPONSE TO RUXOLINITIB IN HIGH-GRADE GLIOMAS

Abstract Altered cellular growth and suppression of anti-tumor immune response collectively drive progression and therapeutic resistance of high-grade gliomas (HGGs), suggesting that successful treatments will likely target these key mechanisms. Janus-associated kinase (JAK)-STAT pathway comprises one such therapeutic node, as STAT3 signaling is upregulated in proliferating glioma cells and immunosuppressive cell types. We performed retrospective analysis on 13 HGG patients treated with the JAK1/2 inhibitor ruxolinitib in combination with radiation or radiation plus temozolomide in a Phase I setting (NCT03514069). Disease progression and treatment response was determined based on the Response Assessment in Neuro-Oncology (RANO) criteria. Peripheral blood mononuclear cells (PBMCs) cells were collected from patients prior to treatment, and 2- 4- and 6-week time points to characterize early immunological changes that are indicative of treatment response with flow cytometry. 6 patients responded favorably, define as progression-free survival &amp;gt;6 months. Investigation of myeloid and lymphoid cell populations revealed no differences in dendritic cell, myeloid-derived suppressor cell and CD8+ T cell subset frequencies in blood. Similarly, expression of checkpoint inhibitors PD-1, TIGIT, CTLA-4 and TIM3 were not altered with ruxolinitib treatment. However, there was a ~2-fold reduction in the frequency of peripheral B cells in favorable responders at 4-week time point, whereas the levels remained stable in non-responders (p&amp;lt;0.05). In addition, while 5 out of 7 non-responders had increased CD4+ T cells by 4-weeks, 5 out of 6 responders had stable or reduced CD4+ T cells. This effect was driven by reduction in immunosuppressive regulatory T cells (Tregs) as the relative ratio of immunostimulatory T helper 1 (Th1)-to-Treg ratio was 3-fold higher in responders compared to non-responders. These results support evaluation of B and CD4+ T cells in the peripheral blood as biomarkers to identify HGG patients likely to benefit from such approach.

Lactic Acid and an Acidic Tumor Microenvironment suppress Anticancer Immunity.

Immune evasion and altered metabolism, where glucose utilization is diverted to increased lactic acid production, are two fundamental hallmarks of cancer. Although lactic acid has long been considered a waste product of this alteration, it is now well accepted that increased lactic acid production and the resultant acidification of the tumor microenvironment (TME) promotes multiple critical oncogenic processes including angiogenesis, tissue invasion/metastasis, and drug resistance. We and others have hypothesized that excess lactic acid in the TME is responsible for suppressing anticancer immunity. Recent studies support this hypothesis and provide mechanistic evidence explaining how lactic acid and the acidic TME impede immune cell functions. In this review, we consider lactic acid’s role as a critical immunoregulatory molecule involved in suppressing immune effector cell proliferation and inducing immune cell de-differentiation. This results in the inhibition of antitumor immune responses and the activation of potent, negative regulators of innate and adaptive immune cells. We also consider the role of an acidic TME in suppressing anticancer immunity. Finally, we provide insights to help translate this new knowledge into impactful anticancer immune therapies.

A Phase 1 Study of Tasquinimod in Patients with Relapsed or Refractory Multiple Myeloma

Background Multiple myeloma (MM) is a hematologic malignancy resulting from clonal proliferation of plasma cells in the bone marrow (BM). The BM tumor microenvironment confers therapeutic resistance through survival and growth signals to myeloma cells and suppression of anti-tumor immune responses. A population of myeloid-derived suppressor cells (MDSCs), abundantly producing the pro-inflammatory S100A9 protein, promotes myeloma progression. Tasquinimod (TASQ) is an investigational drug that targets MDSCs via the S100A9 protein. In pre-clinical models of MM, TASQ has significant anti-tumor effects as a single agent and in combination with bortezomib, a proteasome inhibitor (PI) and lenalidomide, an immunomodulator (IMiD) (Lin C, et al. 25th Annual Meeting of European Hematology Association June 11-21 2020: EP896). TASQ has previously been studied as a novel, once-daily oral anti-cancer agent, including in a phase-3 randomized trial in patients (pts) with metastatic prostate cancer that showed improvement in radiographic progression-free survival (Sternberg C, et al. JCO 2016; 34(22): 2636-43). The side effect profile of TASQ is well-characterized based on this previous experience. We therefore designed a phase 1 trial (NCT04405167) to establish the maximum tolerated dose (MTD) and optimal schedule for TASQ in MM and then investigate the MTD of TASQ in combination with a standard oral myeloma regimen of ixazomib, lenalidomide, and dexamethasone (IRd). For both single agent TASQ and the combination of TASQ with IRd, exploratory expansion cohorts will be enrolled to preliminarily characterize the antimyeloma activity of each regimen. Study Design Enrolled pts must have MM relapsed or refractory after at least one prior line of anti-MM therapy. Other key inclusion criteria include measurable disease and Eastern Cooperative Oncology Group performance status ≤2. Key exclusion criteria include failure to have fully recovered from clinically significant adverse effects of prior chemotherapy; active graft versus host disease; treatment with cytotoxic chemotherapy within 3 weeks prior to initiation of study treatment; treatment with PIs, IMiDs, or monoclonal antibodies within 2 wks, experimental therapy or plasmapheresis within 4 wks, or systemic corticosteroids or radiotherapy within 7 days; known central nervous system involvement by myeloma; or a diagnosis of smoldering MM or POEMS syndrome, active plasma cell leukemia, symptomatic primary amyloidosis, or myelodysplastic or myeloproliferative syndrome. Single agent TASQ and the combination of TASQ with IRd will be investigated in two parts. Part A will start with dose-escalation of single agent TASQ using a standard 3+3 design, with 3 to 6 pts evaluable for dose-limiting toxicity at each dose-level (see table). In the subsequent expansion cohort, additional pts will enroll at the MTD/optimal schedule, so that 12 pts total evaluable for response will have received the MTD/optimal schedule of single agent TASQ. Part B combines TASQ with standard-dose IRd: 28-day cycles of ixazomib 4 mg on days 1, 8, and 15, lenalidomide 25 mg on days 1-21, and dexamethasone 40 mg on days 1, 8, 15, and 22. The part B dose-escalation starts at the lower of either dose-level 1 or one dose-level below the single agent MTD. Additional pts with disease refractory to their most recent PI/IMiD combination will enroll into a part B expansion cohort, so that 12 pts total who are both evaluable for response and PI/IMiD refractory will have received the MTD/optimal schedule of TASQ in combination with IRd. Treatment will continue until unacceptable toxicity or myeloma progression. The primary study endpoint is the MTD of TASQ as single agent and in combination with IRd. Key secondary endpoints include toxicity (treatment-emergent grade 3/4 adverse events using the National Cancer Institute Common Toxicity Criteria for Adverse Events, version 5), preliminary antimyeloma activity (using the response criteria of the International Myeloma Working Group), changes in the BM microenvironment, and systemic TASQ exposure during therapy with single-agent TASQ and during therapy with TASQ in combination with IRd. These results will determine whether TASQ, alone or combined with standard anti-MM therapy, represents a promising novel treatment strategy in MM. Table 1 Disclosures Vogl: Active Biotech: Consultancy, Research Funding; Janssen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Oncopeptides: Consultancy; MorphoSys: Consultancy. Nefedova:Active Biotech: Consultancy, Research Funding. Bondesson:Active Biotech: Current Employment. Eriksson:Active Biotech: Current Employment. Tuvesson:Active Biotech: Current Employment. OffLabel Disclosure: Tasquinimod for multiple myeloma

Deep immunophenotyping at the single-cell level identifies a combination of anti-IL-17 and checkpoint blockade as an effective treatment in a preclinical model of data-guided personalized immunotherapy

BackgroundAlthough immune checkpoint blockade is effective for several malignancies, a substantial number of patients remain refractory to treatment. The future of immunotherapy will be a personalized approach adapted to each...

THERAPEUTIC TARGETING OF HYPOXIA-INDUCIBLE FACTOR SIGNALING PATHWAYS- A PROMISING APPROACH IN CANCER TREATMENT

Oxygen and nutrients are delivered to the cells with the help of the vascular networking system, which makes availability of oxygen as primary regulator for many processes. Low oxygen availability condition activates the Hypoxia Inducible Factors (HIF), which are transcription regulators helping in the expression of genes related to cell cycle regulation and angiogenesis. HIF is hence regarded as the master regulator of angiogenesis. The oxygen deprival is due to the increased consumption of oxygen in the tumor microenvironment and in turn leads to hypoxia. A thorough understanding of how hypoxia influences angiogenesis mediated by several pathways has become essential for identifying novel strategies targeting HIF thereby blocking angiogenesis. In this review we would discuss about the HIF signaling pathways and altered functions of immune cells due to hypoxia by considering that reducing or targeting hypoxia may in turn prevent the suppression of anti-tumor immune response.

Abstract 1942: AGX73 is an effective treatment for hepatocellular carcinoma (HCC) by selective inhibition of immunokinases that play crucial roles in the crosstalk between cancer and immune cells in the tumor microenvironment

Abstract HCC is the second leading cause of cancer-related death and the fifth-most-common cancer worldwide. Unlike almost all other tumor types, the vast majority (90%) of HCCs develop due to underlying chronic inflammation, the induction of fibrosis and/or subsequent cirrhosis. In established disease, persistent upregulation of inflammatory signals in the tumor microenvironment leads to suppression of antitumor immune response by various mechanisms such as reprogramming of tumor-associated macrophages (TAMs) and loss of critical immune cell subsets (e.g. tumor-infiltrating lymphocytes). We identified 6 key immunokinases (AXL, DDR1, DDR2, FMS, KIT and MER) that are frequently amplified in HCC and are significant predictive markers of poor outcome (TCGA). We design small molecule compounds that potently and selectively inhibit these kinases. AGX73, optimized for drug-like properties, is an orally bioavailable compound currently undergoing IND evaluation as potential clinical candidate for first-in-human trial. In in vitro assay, carried out at high ATP concentration (100 μM), AGX73 inhibited the activity of recombinant human AXL, DDR1, DDR2, FMS, KIT and MER with IC50 values of 30, 12, 0.63, 96, 54, and 40 nM, respectively, but was highly selective against a panel of 374 kinases. In an in vitro primary macrophage assays, AGX73 promoted M1 macrophage polarization and increased the secretion of IL-6 and TNFα. In contrast, AGX73 suppressed the activation of M2 macrophages and decreased the secretion of IL-10. Importantly, AGX73 prevented the reprogramming of human LPS/IFN-γ polarized M1 macrophages back to M2 macrophages. In contrast, AGX73 had no effect on the reprogramming of IL-4/IL-13 polarized M2 macrophages back to M1. In vivo, in the MC38 colon carcinoma model, AGX73 treatment reduced the number of TAMs, from 46 to 22% of the total CD45+ cells in the tumors. Moreover, the M1/M2 ratio was favorably increased, from 1.3 to 1.8. AGX73 demonstrated robust antitumor activity in multiple (11) patient-derived xenograft (PDX) models of HCC, with tumor growth inhibition (TGI) ranging 62-105% (Median = 84%) and was effective at multiple dose levels, from 30-150 mg/kg. In combination with anti-PD1 (10 mg/kg) in the MC38 model, AGX73 produced synergistic activity at multiple dose levels. In nonclinical ADME studies, AGX73 demonstrated favorable drug-like properties: including good bioavailability (F&amp;gt;50% in mouse, rat and monkey) and systemic exposure following oral administration in multiple species, acceptable metabolic stability, lack of significant drug-drug interaction liabilities (transporters, CYP inhibition, CYP induction) and excellent cardiac safety profile. IND toxicologic studies are currently in progress. Citation Format: Francis Y.F. Lee, Wenlian Wu, Zhiqiang Yang, John Tan. AGX73 is an effective treatment for hepatocellular carcinoma (HCC) by selective inhibition of immunokinases that play crucial roles in the crosstalk between cancer and immune cells in the tumor microenvironment [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 1942.

The New Era of Cancer Immunotherapy: Targeting Myeloid-Derived Suppressor Cells to Overcome Immune Evasion.

Suppression of antitumor immune responses is one of the main mechanisms by which tumor cells escape from destruction by the immune system. Myeloid-derived suppressor cells (MDSCs) represent the main immunosuppressive cells present in the tumor microenvironment (TME) that sustain cancer progression. MDSCs are a heterogeneous group of immature myeloid cells with a potent activity against T-cell. Studies in mice have demonstrated that MDSCs accumulate in several types of cancer where they promote invasion, angiogenesis, and metastasis formation and inhibit antitumor immunity. In addition, different clinical studies have shown that MDSCs levels in the peripheral blood of cancer patients correlates with tumor burden, stage and with poor prognosis in multiple malignancies. Thus, MDSCs are the major obstacle to many cancer immunotherapies and their targeting may be a beneficial strategy for improvement the efficiency of immunotherapeutic interventions. However, the great heterogeneity of these cells makes their identification in human cancer very challenging. Since both the phenotype and mechanisms of action of MDSCs appear to be tumor-dependent, it is important to accurately characterized the precise MDSC subsets that have clinical relevance in each tumor environment to more efficiently target them. In this review we summarize the phenotype and the suppressive mechanisms of MDSCs populations expanded within different tumor contexts. Further, we discuss about their clinical relevance for cancer diagnosis and therapy.

Signaling of Tumor-Derived sEV Impacts Melanoma Progression.

Small extracellular vesicles (sEV or exosomes) are nanovesicles (30–150 nm) released both in vivo and in vitro by most cell types. Tumor cells produce sEV called TEX and disperse them throughout all body fluids. TEX contain a cargo of proteins, lipids, and RNA that is similar but not identical to that of the “parent” producer cell (i.e., the cargo of exosomes released by melanoma cells is similar but not identical to exosomes released by melanocytes), possibly due to selective endosomal packaging. TEX and their role in cancer biology have been intensively investigated largely due to the possibility that TEX might serve as key component of a “liquid tumor biopsy.” TEX are also involved in the crosstalk between cancer and immune cells and play a key role in the suppression of anti-tumor immune responses, thus contributing to the tumor progression. Most of the available information about the TEX molecular composition and functions has been gained using sEV isolated from supernatants of cancer cell lines. However, newer data linking plasma levels of TEX with cancer progression have focused attention on TEX in the patients’ peripheral circulation as potential biomarkers of cancer diagnosis, development, activity, and response to therapy. Here, we consider the molecular cargo and functions of TEX as potential biomarkers of one of the most fatal malignancies—melanoma. Studies of TEX in plasma of patients with melanoma offer the possibility of an in-depth understanding of the melanoma biology and response to immune therapies. This review features melanoma cell-derived exosomes (MTEX) with special emphasis on exosome-mediated signaling between melanoma cells and the host immune system.

Preventing ATP Degradation by ASO-Mediated Knockdown of CD39 and CD73 Results in A2aR-Independent Rescue of T Cell Proliferation

The adenosine axis contributes to the suppression of antitumor immune responses. The ectonucleotidase CD39 degrades extracellular adenosine triphosphate (ATP) to adenosine monophosphate (AMP), which is degraded to adenosine by CD73. Adenosine binds to, e.g., the A2a receptor (A2aR), which reportedly suppresses effector immune cells. We investigated effects of ATP, AMP, and adenosine analogs on T cell proliferation, apoptosis, and proinflammatory cytokine secretion. CD39 and CD73 expression were suppressed using antisense oligonucleotides (ASOs), and A2aR was blocked using small molecules. Addition of ATP to T cells reduced proliferation and induced apoptosis. Intriguingly, those effects were reverted by suppression of CD39 and/or CD73 expression but not A2aR inhibition. Adenosine analogs did not suppress proliferation but inhibited secretion of proinflammatory cytokines. Here, we suggest that suppression of T cell proliferation is not directly mediated by A2aR but by intracellular downstream metabolites of adenosine, as blockade of the equilibrative nucleoside transporter (ENT) or adenosine kinase rescued proliferation and prevented induction of apoptosis. In conclusion, adenosine might primarily affect cytokine secretion directly via adenosine receptors, whereas adenosine metabolites might impair T cell proliferation and induce apoptosis. Therefore, inhibition of CD39 and/or CD73 has evident advantages over A2aR blockade to fully revert suppression of antitumor immune responses by the adenosine axis.

Local and Systemic Immune Dysregulation Alters Glioma Growth in Hyperglycemic Mice.

Unlike most cancers, no clear epidemiological correlation between diabetes (Db) and malignant glioma progression exists. Because hyperglycemia activates proinflammatory pathways through the receptor for advanced glycation endproducts (RAGE), we hypothesized that Db can also promote malignant glioma progression. We compared the growth of two phenotypically diverse syngeneic glioma models in control and diabetic mice. Tumor growth and antitumor immune responses were evaluated in orthotopic and heterotopic models and correlated to RAGE and RAGE ligand expression. Irrespective of tumor implantation site, growth of a "classical" glioma model, GL261, increased in hyperglycemic mice and was mediated by upregulation of RAGE and its ligand, HMGB1. However, growth of a "mesenchymal" glioma subtype, K-Luc, depended on tumor implantation site. Whereas heterotopic K-Luc tumors progressed rapidly in Db mice, intracranial K-Luc tumors grew slower. We further showed that hyperglycemia inhibited the innate antitumor inflammatory responses in both models. Although this contributed to the accelerated growth of heterotopic tumors, suppression of tumor inflammatory responses dampened the growth of orthotopic K-Luc gliomas. Hyperglycemia may enhance glioma growth through promotion of RAGE expression and suppression of antitumor immune responses. However, abrogation of the proinflammatory milieu in tumors may also dampen the growth of inflammatory glioma subtypes in the brains of diabetic mice. This dichotomy in glioma growth response to hyperglycemia may partly explain why conflicting epidemiological studies show both an increased risk and a protective effect of Db in patients with malignant gliomas.

Abstract A72: IFNAR1 signaling regulates PMN-MDSCs immune suppressive activity in cancer

Abstract Polymorphnuclear myeloid-derived suppressor cells (PMN-MDSCs) are a subset of MDSCs that are expanded and accumulated in several cancers. The main role of PMN-MDSCs in cancer is the suppression of antitumor immune responses, which leads to tumor progression, angiogenesis and metastasis. PMN-MDSCs within the tumor microenvironment (TME) have potent immune-suppressive effects compared to PMN-MDSCs in peripheral organs. However, the mechanism regulating the immune-suppressive activity of PMN-MDSCs within the TME remain poorly understood. Recently, it was shown that PMN-MDSCs from spleen of tumor-bearing mice treated with type 1 interferon (IFN1) have a decrease in their immune-suppressive activity. It has been established that tumor-associated PMN-MDSCs are more suppressive than those residing in the spleen; however, their response to IFN1 remains unclear. Therefore, our goal is to understand the role of IFN1 in the regulation of PMN-MDSCs’ function within the TME. We found that IFN1 receptor, IFNAR1, is downregulated in tumor-associated PMN-MDSCs compared to their counterparts in the spleen. In cancer patients IFNAR1 on PMN-MDSC was substantially lower than on neutrophils from healthy donors. IFNAR1 downregulation in PMN-MDSCs correlated with their immune-suppressive activity. To understand the importance of IFNAR1 signaling in PMN-MDSC biology, we utilized a knock-in mouse expressing a mutant form of IFNAR1 (Ifnar1SA) that is resistant to downregulation upon stimuli and stress. PMN-MDSCs from Ifnar1SA mice were defective in their immune-suppressive activity whereas PMN-MDSCs from Ifnar1-/- mice are as immunosuppressive as PMN-MDSCs from WT mice. In this study, we described the possible role of IFN1 in negative regulation of PMN-MDSCs function in cancer. Citation Format: Serge Fuchs, Kevin M. Alicea-Torres, Dmitry I. Gabrilovich. IFNAR1 signaling regulates PMN-MDSCs immune suppressive activity in cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A72.