Suppression Of Antitumor Immune Responses Research Articles

Abstract 5176: TIGIT expression increases with advancing clinical stages and does not differ across racial groups in resected pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a dismal 12% 5-year survival rate (SEER) due to a lack of early detection biomarkers and resistance to standard therapeutic options (surgery, chemotherapy, radiation). Black African Americans (BAA) have 20% increased incidence of PDAC compared to those with European Ancestry (EA). TIGIT, an immune checkpoint receptor, is a marker of T cell exhaustion and plays a key role in the inhibition of anti-tumor immune responses. Recent studies have demonstrated that immune checkpoint receptor expression (PD-1 and TIGIT) on specific T cell populations correlates to worse overall survival (OS). TIGIT inhibitors are being explored in clinical trials in pancreatic cancer due to implications of its ligand (CD155) promoting immune evasion. We hypothesize that targeted anti-TIGIT therapy, in conjunction with other therapies targeting the tumor microenvironment, could reverse the immune suppression that is characteristic of PDAC. We performed RNAscope in situ hybridization (ISH) with a probe specific for human TIGIT mRNA (combined with a nuclear counterstain) on 79 tissue samples. The cohort of tissue samples included 8 biopsies (endoscopic-guided fine needle biopsies at time of diagnosis), 66 primary (from surgical resection), and 5 metastatic (liver core biopsies from patients with a primary PDAC diagnosis) formalin-fixed paraffin-embedded (FFPE) tissue sections from patients with histologically confirmed PDAC. After cells were segmented based on nuclear recognition, the presence of TIGIT probe within the cytoplasm of each cell was determined and quantified. Percent positive cell values were then exported for statistical analysis in R. De-identified clinical metadata was obtained from REDCap, a cloud-based HIPAA-compliant database used to compile patient data for research purposes. We tested for associations between %TIGIT present and clinical covariates, using linear regression for continuous outcomes, and logistic regression for binary outcomes. ScRNAseq revealed that TIGIT mRNA is enriched in, but not exclusive to the T/NK cellular compartments in PDAC. Staining analysis showed that TIGIT expression did not differ significantly between racial groups (comparing BAA to non-BAA). The mean percentage of TIGIT positive cells was 64.0%. High expression of TIGIT was associated with clinical stage, where an increase in stage was associated with increasing %TIGIT (p < 0.05).The TIGIT biomarker assay can be conducted at time of diagnosis, time of surgical resection, and time of metastatic biopsy. If patients’ samples contain high levels of TIGIT expression, these patients may be candidates for anti-TIGIT drug therapy. Considering that TIGIT expression correlates with advancing clinical stage, patients with more advanced staging at diagnosis (stage IIB, III, IV) especially may benefit from anti-TIGIT therapy. Citation Format: Madison George, Julie Clark, Kendyll Gartrelle, Georges Nassif, Kailee Hartway, Daniel Long, Daniel Salas-Escabillas, Allison Wombwell, Thais Pichardo, Hui-Ju Wen, Simone Benitz, Samuel Zwernik, Rupen Shah, Hakmin Park, Philip, Gazala Khan, Howard Crawford, David Kwon, Brian Theisen, Nina Steele. TIGIT expression increases with advancing clinical stages and does not differ across racial groups in resected pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5176.

Abstract 5536: The role of p38 MAPK in the tumor-induced immune suppressive microenvironment in metastatic breast cancer

Abstract The tumor microenvironment (TME) in Metastatic Breast Cancer (MBC) is a major factor contributing to therapy resistance and suppression of antitumor immune response. Tumors promote expansion and recruitment of immune suppressive cells such as myeloid-derived suppressor cells (MDSCs) contributing to tumor invasion and suppressing anti-tumor T cells. Our prior work suggested a critical role of p38 MAPK in tumor-induced expansion and mobilization of myeloid cell populations thereby facilitating metastasis. The current study examined the role of p38 MAPK in tumor-induced changes in immune landscape and explored the mechanisms by which p38 mediates tumor-immune interactions. First, the contribution of T cells to anti-metastatic activity of p38 inhibitor (p38i) was addressed by depletion of CD8 T cells. Depletion of CD8+ T cells negated the effects of p38i on tumor growth and metastasis in the syngeneic 4T1 model. Next, we examined whether p38i exhibits a direct effect on T cells in vitro or in vivo. The in vitro assays showed that p38 blockade increased levels of CD44+ CD62L+ CD8+ T cells indicating enhancement of T cell differentiation. To determine the effects of p38 blockade on the immune landscape in vivo, single-cell RNA-seq and flow cytometry studies were performed in the 4T1 model with p38i or p38-deficient cells in which p38α/Mapk14 was inactivated by CRISPR/Cas9 (p38KO). The scRNA-seq data showed that p38 blockade increased the expression of T cell differentiation markers in the TME. Furthermore, p38 blockade significant decreased IL-6 signaling in myeloid cells, an important mediator of MDSC function. Assessment of myeloid cell populations showed a decreased expression of the immune suppressive signature. This observation was further validated in MDSCs isolated from the spleens of 4T1 tumor-bearing mice treated with p38i and in p38KO cohorts. Our prior work showed that p38i does not affect the generation of MDSCs in vitro. Therefore, we examined whether blockade of p38 affects the chemotactic capacity of conditioned media from MBC cells using mouse monocytes. The chemotaxis assays showed that p38i and p38KO largely reduced the ability of conditioned media to stimulate transwell migration of mouse monocytes. Notably, p38i and p38KO largely reduced expression of chemotactic cytokines in MBC cell models. Furthermore, p38i and p38KO reduced production and chemotactic ability of exosomes isolated from tumor cells. Together, these observations suggest that tumor p38 MAPK signaling promotes immune-suppressive TME and metastasis by facilitating expansion and mobilization of myeloid cell populations through the mechanisms involving production of exosomes and pro-myeloid chemokines/cytokines. This work highlights that p38 blockade can be utilized in combination with immune therapy or chemotherapy to enhance clinical benefits of immune therapy and reduce promyeloid effects of chemotherapy. Citation Format: Priyanka Rajan, Robert Zollo, Mackenzie Lieberman, Yanqi Guo, Mohammed Alruwaili, Mohammed Alqarni, Brian Morreale, Scott Olejniczak, Joseph Barbi, Scott Abrams, Andrei Bakin. The role of p38 MAPK in the tumor-induced immune suppressive microenvironment in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5536.

Abstract 183: A novel bispecific antibody macrophage engager (BiME) designed for the treatment of solid tumors

Abstract Background: Anti-CD3-based bispecific T cell engagers (BiTE) showed limited clinical efficacy in solid tumors and caused significant cytokine storm. Similar to BiTE where T cells are activated by CD3 antibody, we constructed a novel bi-specific macrophage engager (BiME) platform where macrophage is activated by anti-SIRPa that is directed to a particular tumor via the targeting of the tumor-associated antigen (TAA), resulting in phagocytosis of the tumor cells. This BiME does not lead to cytokine storm and is particularly applicable for the treatment of solid tumors whose microenvironment contains plenty of macrophages. Tumor-associated macrophages are major component of immune cells in the tumor micro-environment (TME) that express an array of effector molecules leading to the inhibition of anti-tumor immune responses. Signal regulatory protein α (SIRPα) is a myeloid-lineage inhibitory receptor that restricts phagocytosis through engagement of its ligand CD47 expressed on tumors and normal tissues. Compared to anti-CD47 therapeutics, targeting myeloid-restricted SIRPα provides a differential pharmacokinetic, safety, and efficacy profile. Here, we report the construction of a SIRPα antagonist-based bispecific macrophage engager (BiME) platform, a potent tumor-killing bi-specific antibody platform. Methods: We have generated a panel of single domain antibody (sdAb), scFv or Fab antibodies targeting different TAAs and SIRPa. We constructed BiME antibodies using different orientations, ratios, and IgG isotypes of anti-TAA arm and anti-SIRPα arm. These bispecific antibodies were evaluated for homologue binding, and CD47-SIRPα blocking properties by ELISA and FACS. In vitro function activity was determined by phagocytosis assay using human monocyte derived macrophage and mouse bone marrow derived macrophage. In vivo anti-tumor efficacy was tested in a syngeneic tumor model with hSIRPα knock-in mice. The pharmacokinetic (PK) and safety profile were assessed in hSIRPα knock-in mice or cynomolgus monkeys. Results: Through Elpiscience proprietary BiME platform, we have generated a panel of TAA/SIRPα bispecific antibodies with TAA being Claudin18.2, EGFR, PDL1 and DLL-3. The BiME treatment demonstrated strong anti-tumor efficacy in multiple mouse syngeneic tumor models. Mechanistically, both myeloid and T cells were activated and contributed to antitumor activity of BiME bispecific antibody. the BiME bispecific antibodies were well tolerated in non-human primates (NHPs) and there is no hematotoxicity or cytokine release syndrome (CRS) observed after treatment of BiME antibodies. Conclusions: These findings provide novel mechanistic insights into how myeloid and T cells can be uniquely modulated by the bispecific macrophage engagers (BiMEs) and demonstrate the potential and advantage of BiME in future clinical development for solid tumor treatment as compared to BiTE approach. Citation Format: Dawei Sun, Haixia Jiang, Yanan Geng, Yue Wu, Rui Gao, Yefeng Lu, Quan Qiu, Yanfen Hu, Yingchao Liu, Xiaoli Guo, Xiaofeng Niu, Hongtao Lu. A novel bispecific antibody macrophage engager (BiME) designed for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 183.

The Role of Regulatory T cells (Tregs) in Tumorigenesis: A Comprehensive Literature Review

Introduction: Regulatory T cells (Tregs) are a subpopulation of CD4+ T lymphocytes that contribute to immune homeostasis by suppressing excessive immune activation. However, these immunosuppressive properties can lead to the suppression of anti-tumor immune responses. Depletion or blocking of Tregs through therapeutics has emerged as a possible method for enhancing anti-tumor immunity. However, the lack of selective targeting of Tregs in the tumor microenvironment is a significant limitation to the effectiveness of Treg therapies. Therefore, this investigation aims to review current literature on how Tregs suppress the antitumor immune response and how they can be targeted to promote anti-tumor immunity. Methods: This review examines recent literature on Tregs in the tumor microenvironment, focusing on both cell-contact dependent and independent mechanisms. Clinical trial studies were also included to assess therapeutic targeting of Tregs. The PubMed database was systematically searched for English articles from 2010 to present, supplemented by manual searches without date restrictions. Boolean expressions ensured comprehensive study retrieval. Results: The involvement of Tregs in the development of multiple cancer types is evident, and targeting these cells could potentially enhance the efficacy of antitumor immunity. In addition, we compiled a list of the novel approaches currently being used for Treg targeting in the context of cancer. Discussion: This review has identified the most promising targets for Treg-based therapies, opening avenues for accelerating the development of innovative cancer treatments. Conclusion: Our literature review offers insights into the complex interplay between the immune system and cancer. The understanding of this interaction is not just an endpoint but could potentially act as a steppingstone towards new scientific discoveries.

Abstract C012: TIGIT expression correlates to worse overall survival in primary and metastatic pancreatic ductal adenocarcinoma

Abstract Pancreatic Cancer has a 12% 5-year survival rate due to late-stage detection and lack of many chemotherapy or targeted therapy options. TIGIT is an immune checkpoint inhibitor being explored in clinical trials in pancreatic cancer due to the implications of its ligand (CD155) promoting immune evasion. TIGIT is a marker of T cell exhaustion and plays a key role in the inhibition of anti-tumor immune responses. We hypothesize that targeted anti-TIGIT therapy, in conjunction with other therapies targeting the tumor microenvironment, could reverse immune suppression that is characteristic of pancreatic ductal adenocarcinoma (PDAC). As such, here we aim to quantify TIGIT expression using automated RNAscope technology and to correlate it with overall patient survival. We performed RNAscope in situ hybridization and immunohistochemistry on 25 primary and 4 metastatic (liver) formalin-fixed paraffin-embedded (FFPE) tissue sections from patients with histologically confirmed PDAC. A nuclear counterstain combined with an RNAscope probe specific for the human TIGIT mRNA was utilized. Slides were scanned at 40X magnification with an automated slide scanner and quantified using the ISH-IHC module of the HALO-v3.5 software. After cells were segmented based on nuclear recognition, the presence of TIGIT probe within the cytoplasm of each cell was determined and quantified. Percent positive cell values were then exported for statistical analysis in SPSS. De-identified clinical metadata was obtained from REDCap, a cloud-based HIPAA-compliant database used to compile patient data for research purposes. Gender, survival months, ethnicity, race, tumor histology, stage, cancer history, treatment status, and comorbidities were evaluated. We analyzed a cohort of 29 histologically-confirmed surgically resected PDACs, comprised of 25 primary pancreatic tumor samples and 4 metastatic liver core biopsies. The mean percentage of TIGIT positive cells was 63%. From this, the TIGIT high versus low threshold was determined to be 70%. High TIGIT was associated with significantly lower overall survival (p=.013), suggesting that expression of TIGIT and T cell exhaustion may predict overall worse survival. The median overall survival months for the TIGIT low group was 321 months versus 106 months for the TIGIT high group. This data leads us to hypothesize that expression of TIGIT and T cell exhaustion may predict worse overall survival. Anti-TIGIT medications are currently in clinical trials for metastatic PDAC. Here we demonstrate that high TIGIT expression is associated with an overall worse prognosis and present a novel, automated TIGIT detection protocol that may be used to determine if patients are candidates for anti-TIGIT therapy. In a clinical setting, this novel, automated biomarker TIGIT detection protocol could be used to select candidates for anti-TIGIT therapy. This may lead to improved overall patient survival. Citation Format: Madison George, Julie Clark, Kendyll Gartrelle, Georges Nassif, Donald Rempinski, Daniel Long, Hui-Ju Wen, Simone Benitz, Samuel Zwernik, Rupen Shah, Hakmin Park, David Kwon, Philip Philip, Gazala Khan, Howard Crawford, Brian Theisen, Nina Steele. TIGIT expression correlates to worse overall survival in primary and metastatic pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C012.

High CTCF expression mediated by FGD5-AS1/miR-19a-3p axis is associated with immunosuppression and pancreatic cancer progression

The most common reason for cancer-related death globally is predicted to be pancreatic cancer (PC), one of the deadliest cancers. The CCCTC-binding factor (CTCF) regulates the three-dimensional structure of chromatin, was reported to be highly regulated in various malignancies. However, the underlying biological functions and possible pathways via which CTCF promotes PC progression remain unclear. Herein, we examined the CTCF function in PC and discovered that CTCF expression in PC tissues was significantly raised compared to neighboring healthy tissues. Additionally, Kaplan-Meier survival analysis demonstrated a strong connection between elevated CTCF expression and poor patient prognosis. A study of the ROC curve (receiver operating characteristic) revealed an AUC value for CTCF of 0.968. Subsequent correlation analysis exhibited a strong relationship between immunosuppression and CTCF expression in PC. CTCF knockdown significantly inhibited the malignant biological process of PC in vitro and in vivo, suggesting that CTCF may be a potential PC treatment target. We also identified the FGD5 antisense RNA 1 (FGD5-AS1)/miR-19a-3p axis as a possible upstream mechanism for CTCF overexpression. In conclusion, our data suggest that ceRNA-mediated CTCF overexpression contributes to the suppression of anti-tumor immune responses in PC and could be a predictive biomarker and potential PC treatment target.

Enhancing Cancer Chemo-Immunotherapy: Innovative Approaches for Overcoming Immunosuppression by Functional Nanomaterials.

Chemotherapy is a critical modality in cancer therapy to combat malignant cell proliferation by directly attacking cancer cells and inducing immunogenic cell death, serving as a vital component of multi-modal treatment strategies for enhanced therapeutic outcomes. However, chemotherapy may inadvertently contribute to the immunosuppression of the tumor microenvironment (TME), inducing the suppression of antitumor immune responses, which can ultimately affect therapeutic efficacy. Chemo-immunotherapy, combining chemotherapy and immunotherapy in cancer treatment, has emerged as a ground-breaking approach to target and eliminate malignant tumors and revolutionize the treatment landscape, offering promising, durable responses for various malignancies. Notably, functional nanomaterials have substantially contributed to chemo-immunotherapy by co-delivering chemo-immunotherapeutic agents and modulating TME. In this review, recent advancements in chemo-immunotherapy are thus summarized to enhance treatment effectiveness, achieved by reversing the immunosuppressive TME (ITME) through the exploitation of immunotherapeutic drugs, or immunoregulatory nanomaterials. The effects of two-way immunomodulation and the causes of immunoaugmentation and suppression during chemotherapy are illustrated. The current strategies of chemo-immunotherapy to surmount the ITME and the functional materials to target and regulate the ITME are discussed and compared. The perspective on tumor immunosuppression reversal strategy is finally proposed.

ChemR23 activation reprograms macrophages toward a less inflammatory phenotype and dampens carcinoma progression.

Tumor Associated Macrophages (TAM) are a major component of the tumor environment and their accumulation often correlates with poor prognosis by contributing to local inflammation, inhibition of anti-tumor immune response and resistance to anticancer treatments. In this study, we thus investigated the anti-cancer therapeutic interest to target ChemR23, a receptor of the resolution of inflammation expressed by macrophages, using an agonist monoclonal antibody, αChemR23. Human GM-CSF, M-CSF and Tumor Associated Macrophage (TAM)-like macrophages were obtained by incubation of monocytes from healthy donors with GM-CSF, M-CSF or tumor cell supernatants (Breast cancer (BC) or malignant pleural mesothelioma (MPM) cells). The effects of αChemR23 on macrophages were studied at the transcriptomic, protein and functional level. Datasets from The Cancer Genome Atlas (TCGA) were used to study CMKLR1 expression, coding for ChemR23, in BC and MPM tumors. In vivo, αChemR23 was evaluated on overall survival, metastasis development and transcriptomic modification of the metastatic niche using a model of resected triple negative breast cancer. We show that ChemR23 is expressed at higher levels in M-CSF and tumor cell supernatant differentiated macrophages (TAM-like) than in GM-CSF-differentiated macrophages. ChemR23 activation triggered by αChemR23 deeply modulates M-CSF and TAM-like macrophages including profile of cell surface markers, cytokine secretion, gene mRNA expression and immune functions. The expression of ChemR23 coding gene (CMKLR1) strongly correlates to TAM markers in human BC tumors and MPM and its histological detection in these tumors mainly corresponds to TAM expression. In vivo, treatment with αChemR23 agonist increased mouse survival and decreased metastasis occurrence in a model of triple-negative BC in correlation with modulation of TAM phenotype in the metastatic niche. These results open an attractive opportunity to target TAM and the resolution of inflammation pathways through ChemR23 to circumvent TAM pro-tumoral effects.

Mitochondria targeted metabolic inhibitors promote lymphocyte infiltration into murine pancreatic tumors

Abstract Pancreatic ductal adenocarcinoma is a malignant carcinoma with a 5-year survival rate of 11% characterized with poor prognosis, rising incidence, and resistance to chemo- and immune-therapies. Cancer cell evasion and suppression of anti-tumor immune responses in pancreas cancer are regulated in part by metabolic reprogramming within cancer and tumor accessory cells. We sought to determine if targeted inhibition of mitochondrial oxidative phosphorylation by mito-metformin (MMe) and newly developed variant pCF3-MMe influence the tumor immune microenvironment. Our results showed that MMe strongly inhibited tumor growth in murine models of pancreas cancer. Flow cytometry revealed a significant increase in total CD45+ leukocytes, with elevated cytolytic granzyme B+ and CD44+ CD8+ T cells. Immunodepleting CD8+ T cells, reversed the anti-tumor effects of MMe. Immunofluorescence microscopy and immunoblot analysis indicated that MMe stimulated oligomerization of mitochondrial antiviral-signaling protein (MAVS) and phosphorylation of IRF3 and IRF7. Further, levels of the IRF3/7 target chemokines CXCL9, CXCL10, and CXCL11 were increased in MMe-treated tumor cells, consistent with a role in CD8+ T cell infiltration. These data indicate OXPHOS inhibition stimulated virus-independent MAVS oligomerization, activation of downstream proinflammatory signaling, and reignition of the suppressed pancreatic tumor immune microenvironment. Funding Provided by NIH (2211376.1-5 R01 CA226279-01.04) and Center for Immunology MCW

The crosstalk between autophagy and myeloid-derived suppressor cell responses in cancer.

The development of cancers is aided by the accumulation of myeloid-derived suppressor cells (MDSCs) within tumors, which are highly effective at suppressing anti-tumor immune responses. Direct cell-to-cell interaction and the production of immunosuppressive mediators have both been proposed as pathways for MDSC-mediated suppression of anti-tumor immune responses. The majority of current cancer treatments focus on altering the development and activity of MDSCs so that they have more of an immunogenic character. Autophagy is a catabolic system that contributes to the breakdown of damaged intracellular material and the recycling of metabolites. However, depending on the stage of tumor growth, autophagy can play both a prophylactic and a therapeutic function in carcinogenesis. However, several indirect lines of research have indicated that autophagy is a significant regulator of MDSC activity. The purpose of this work was to outline the interactions between MDSC and autophagy in cancer.

Abstract 3804: Linear PVT1 isoforms and circPVT1 regulate DNA damage response and immunological pathways in acute myeloid leukemia and have a potential role in the crosstalk between leukemic cells and the tumor microenvironment

Abstract The human Plasmacytoma Variant Translocation 1 (PVT1) gene encodes for 176 linear (PVT1, lncipedia.org) and 29 circular isoforms (circPVT1, www.circbase.org). The most common isoform of circPVT1 is 410bp and is a product of back-splicing containing the whole exon 2 of PVT1 in a closed loop-like structure (hsa_circ_0001821). PVT1 and circPVT1 have oncogenic roles in several tumor types and can contribute to the suppression of anti-tumor immune responses. Our study aims to investigate whether and how PVT1 isoforms and circPVT1 promote an aggressive phenotype in acute myeloid leukemia (AML) and are involved in the crosstalk between leukemic and immune cells. Six out of the 14 PVT1 isoforms expressed by white blood cells and lymphnodes (noncode.org) were detected in a panel of AML cell lines along with circPVT1. Downregulation (KD) of groups of linear isoforms (based on sequence homology) or of circPVT1 by antisense-oligonucleotides (ASOs) in the t(8;21) KASUMI-1 and the NPM1-mut OCI-AML3 cells induced an impairment of leukemia cell growth especially when all the 6 linear isoforms were silenced by ASO combination, in both cell lines, under normoxia and hypoxia mimicking the bone-marrow microenvironment. Interestingly circPVT1-KD induced apoptosis in OCI-AML3 cells. Moreover, MYC protein expression decreased by ASO combination or circPVT1_KD in OCI-AML3 under normoxia and in KASUMI-1 under hypoxia. To further investigate the biological consequences of PVT1/circPVT1-KD, we performed RNAseq. Analysis on AML cell lines revealed that downregulation of PVT1 and, especially circPVT1, altered the expression of genes involved in RNA transport and degradation, as expected for a long non-coding genes, but also in DNA damage response (DDR) and immunological pathways, as also confirmed by transcriptomic data of primary AML cases and proteomic analysis of both cell lines. Based on these novel findings, we tested the downregulation of PVT1/circPVT1 in combination with inhibition of ATM or ATR, key DDR transducers. Interestingly, circPVT1-KD sensitized OCI-AML3 to ATM (71% vs 95% live cells) or ATR (75% vs 90% live cells) inhibitors. PVT1 and circPVT1 expression was also detected at the level of cell-free RNA in primary AML samples, the latter showing higher levels. We then analyzed extracellular vesicles (EVs) from AML peripheral blood and healthy subjects. EVs from patients were larger, increased in number and expressed higher levels of myeloid lineage markers. Finally AML EVs had more circPVT1 cargo. In conclusion, we uncovered novel potential roles of PVT1 isoforms and circPVT1 in the DDR and in the tumor microenvironment that pave the way for novel therapeutic combinations. Citation Format: Martina Ghetti, Lorenzo Ledda, Ivan Vannini, Eugenio Fonzi, Maria Teresa Bochicchio, Tristan Cardon, Andrea Ghelli Luserna di Rorà, Matteo Paganelli, Chiara Servili, Francesco Fabbri, Sabina Marianini, Giovanni Marconi, Michela Rondoni, Roberta Chicchi, Rino Biguzzi, Francesco Lanza, Michel Salzet, Giovanni Martinelli, Giorgia Simonetti. Linear PVT1 isoforms and circPVT1 regulate DNA damage response and immunological pathways in acute myeloid leukemia and have a potential role in the crosstalk between leukemic cells and the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3804.

Abstract PD4-06: PD4-06 Obesity-associated changes in transcriptomic profile and immune landscape of primary breast cancer revealed by bulk and single-cell gene expression data

Abstract Background: Breast cancer (BC) is one of the cancer types recognized as an obesity-associated disease. Current understandings of molecular mechanisms underlying the BC-obesity connection however largely came from experimental models while systematic investigation of the impact of obesity on BC biology in large patient series is still lacking. The purpose of this study is to discover changes in the transcriptomic profile of primary BC according to patients’ body mass index (BMI). Data and Methods: Bulk and single-cell gene expression data from treatment-naïve primary breast tumors from non-underweight patients were retrieved from the MINDACT trial (NCT00433589; N = 1481) and the pre-treatment cohort of the BioKey trial (NCT03197389, N = 36), respectively. Three categories were considered for BMI: lean, overweight and obese. The main analyses focused on the invasive carcinoma of no special type (NST) estrogen receptor-positive/HER2-negative (ER+/HER2-, N_bulk = 735, N_single-cell(sc) = 10) and NST ER-/HER2- (N_bulk = 118, N_sc = 15) subgroups. The bulk expression data was subjected to differential gene expression analyses according to BMI which was adjusted for menopausal status and tumor grade, then followed by gene set enrichment analyses. Clustering and cluster annotation were performed on the single-cell profiling data before differentially expressed genes according to BMI were identified for each of the present cell types. Results: Obesity-associated differences in the transcriptomic profile of breast tumors, which were subtle but potentially indicative of a biological relationship, were revealed by the bulk data. In both investigated subgroups, tumors from obese patients were shown to be enriched in cell cycle hallmarks. In ER-/HER2- tumors, adiposity further increased MYC signaling. We also observed different obesity-associated changes according to the ER status. Among ER+/HER2- tumors, those from obese patients were enriched in hallmarks related to inflammatory response compared to those from lean patients. In contrast, these hallmarks appeared to be enriched in the ER-/HER2- tumors from lean patients. Our investigation of the single-cell data further revealed shifts in the cell composition of tumor tissue and cell type-specific transcriptomic differences according to BMI which were more pronounced than those detected from the bulk data. ER+/HER2- tumors from obese patients have a higher frequency of immunosuppressive and pro-tumoral cell subpopulations such as dendritic cells (DC) enriched in immunoregulatory molecules (p = .03), LYVE1+ macrophages (p = .02) and myofibroblasts (p = .03) than those from lean patients. Overexpression of Cyclin D1 and CD24 was found in cancer cells in ER+/HER2- tumors from obese patients. A reduction in anti-tumor immune responses was evident with downregulation of multiple interferons in CD8+ and CD4+ T cells as well as B cells. We observed in the ER-/HER2- subgroup increased infiltration of plasmacytoid DC (p = .01), CCL2+ macrophages (p = .01) in tumors from obese versus lean patients, while fibroblasts showed an opposite tendency. Additionally, significant obesity-associated downregulation of major histocompatibility complex (MHC) molecules class I in cancer cells and MHC class II molecules in B cells could be suggestive of deficient antigen presentation and activation of cytotoxic and helper T cells. Conclusion: We highlighted the impact of obesity on the remodeling of tumor and tumor microenvironment which might generally lead to a suppression of anti-tumor immune responses, albeit potentially via diverse axes according to the ER status. Although investigation on a larger cohort is warranted, our current results suggest that obesity-associated transcriptomic changes in BC could be highly cell type-specific, hence we recommend single-cell approaches in addition to spatial multi-omics analysis to further elucidate the interplay between obesity and BC. Citation Format: Ha-Linh Nguyen, Tatjana Geukens, Marion Maetens, Karen Van Baelen, Maxim De Schepper, Coralie Poncet, Mauro Delorenzi, Marjanka K. Schmid, Emiel Rutgers, Laura Van ’t Veer, Martine Piccart, Fatima Cardoso, Giuseppe Viale, Ayse Bassez, Hanne Vos, Patrick Neven, Ines Nevelsteen, Kevin Punie, Hans Wildiers, Giuseppe Floris, Diether Lambrechts, Ann Smeets, Elia Biganzoli, François Richard, Christine Desmedt. PD4-06 Obesity-associated changes in transcriptomic profile and immune landscape of primary breast cancer revealed by bulk and single-cell gene expression data [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD4-06.

PHF19 Inhibits Multiple Myeloma Cell Response to Immunotherapy Via Promoting Immunosuppressive Microenvironment

Introduction: The multiple myeloma (MM) bone marrow (BM) microenvironment promotes tumor cell growth, survival, and drug resistance, while also conferring immunosuppression. This involves crosstalk between the myeloma cells and the components of BM microenvironment which can be facilitated or hindered by epigenetic modifiers altering the gene expression of receptors. Among those, regulatory T (Tregs) cells are essential to control peripheral tolerance and response to foreign and tumor antigens. Tregs within the BM niche play a significant role in the suppression of antitumor immune responses and limiting the beneficial response of immunotherapy in MM. Here, we investigated the impact of the Polycomb-like protein PHF19/PCL3, an epigenetic gene whose high level predicts poor prognosis in patients with MM, on the immunosuppressive BM microenvironment. Methods & Results: We first investigated how PHF19 overexpression would affect the microenvironment by using an ex vivo co-culture of MM cells with PBMCs or purified CD3+ T cells. MM cells that overexpressed PHF19 by lenti-virus infection (PHF19-OE cells) induced 2-4-fold higher conversion of conventional T cells (Tcon) into induced regulatory T cells (iTregs) (n=5, day 4). In addition, PHF19-OE MM cells increased the frequency of iTreg at later time points (day 7) and upregulated cell proliferation (CCND1, CCND2), anti-apoptosis (BCL2, BCL2L1), and Treg-related genes (TGFβ1, IL-10, Foxp3) (P < .0001). Moreover, enhanced checkpoint marker (PD1, TIM3, LAG3) expression was observed on Tcon co-cultured with PHF19-OE vs control MM cells. There were elevated IL-10 and TGFβ1 levels in the culture supernatants of PHF19-OE MM cells, alone and in co-cultures, and antibodies to these cytokines partially neutralized the Treg inhibition of Tcon proliferation triggered by PHF19-OE MM cells. Thus, PHF19 seems to be important in facilitating the crosstalk between MM cells and immune cells in the BM. CD38 expression on the MM cell surface is commonly targeted in immunotherapy. However, CD38 expression was downregulated on PHF19-OE vs -KD MM cells, and PHF19 mRNA expression was negatively correlated to CD38 expression in patient MM cells (Spearman's ρ = -0.38 and -0.2 for GSE5900 and GSE31161, P < .004). As expected, PHF19-OE MM cells were less susceptible than PHF19-KD MM cells to in vitro NK-mediated lysis induced by daratumumab (dara) or isatuximab, which target CD38, as evidenced by fewer CD107a+ and IFN γ+ NK effector cells. Moreover, dara-induced cytotoxicity was diminished in xenografted NSG mice with PHF19-OE vs control cells and reconstituted with human NK cells. These resistant PHF19-OE AMO1 tumors had lower expression of CD38, STAT1, and IFN-related genes, which produced higher levels of human κ light chain, soluble BCMA, and TGFβ1 in matched serum samples. Accordingly, PHF19-OE resulted in significantly decreased mouse survival. To find the mediators of the immunosuppression induced by PHF19, we used ATAC-seq and CHIP-seq analysis in MM cell lines. We found that a number of genes essential for T cell-mediated tumor immunity was downregulated in the PHF19 OE group vs control, including key genes in the interferon-γ (IFN-γ) signaling pathway, which is a pathway that leads to cytokine-mediated cell death. Among the genes of the IFN-γ pathway, 56 genes had less chromatin accessibility in PHF19 OE vs control cells. RNA-seq analysis confirmed that PHF19 OE repressed the expression of CD38 and the IFN-γ and IFN-α signaling pathway. Furthermore, PHF19 OE altered the expression of genes in NF-κB signaling triggered by TNF-α signaling pathway, which are associated with resistance to immunotherapy. Finally, activation of the PI3K/AKT pathway was also identified in PHF19 OE cells, which directly promotes MM cell proliferation and survival, as well as enhances resistance to immunotherapy via upregulation of PD-L1 on the MM cell surface. Conclusions: Taken together, these data indicate that PHF19 promotes an immunosuppressive BM milieu by promoting iTreg frequency and the expression of immunosuppressive cytokines, as well as decreasing CD38 expression on MM cells and diminishing their sensitivity to IFN-γ-mediated killing. These results suggest the potential clinical utility of targeting PHF19 to overcome immunosuppression and thereby enhance or restore sensitivity to CD38-targeted therapy in MM.

USP7 Inhibitors in Cancer Immunotherapy: Current Status and Perspective.

Ubiquitin-specific protease 7 (USP7) regulates the stability of a plethora of intracellular proteins involved in the suppression of anti-tumor immune responses and its overexpression is associated with poor survival in many cancers. USP7 impairs the balance of the p53/MDM2 axis resulting in the proteasomal degradation of the p53 tumor suppressor, a process that can be reversed by small-molecule inhibitors of USP7. USP7 was shown to regulate the anti-tumor immune responses in several cases. Its inhibition impedes the function of regulatory T cells, promotes polarization of tumor-associated macrophages, and reduces programmed death-ligand 1 (PD-L1) expression in tumor cells. The efficacy of small-molecule USP7 inhibitors was demonstrated in vivo. The synergistic effect of combining USP7 inhibition with cancer immunotherapy is a promising therapeutic approach, though its clinical efficacy is yet to be proven. In this review, we focus on the recent developments in understanding the intrinsic role of USP7, its interplay with other molecular pathways, and the therapeutic potential of targeting USP7 functions.

Abstract 3572: Oncolytic virotherapy with the HSV-1 (VC2) vaccine strain induces anti-tumor T-cell responses in the murine 4T1 breast cancer model

Abstract The tumor microenvironment (TME) plays important roles in suppression of anti-tumor immune responses. Immune suppression is achieved by multiple mechanisms including interfering with antigen presentation and inhibition of effector T-cell tissue infiltration. Altering specific TME properties can reduce immune-suppression and improve anti-tumor immunity with abscopal effects. Oncolytic Virotherapy (OV) is a promising approach for cancer treatment. We evaluated the efficacy of the HSV-1 (VC2) vaccine strain for the treatment of breast cancer using the murine stage-4 (4T1) syngeneic and immunocompetent breast cancer mouse model. Following Intratumoral (IT) injection of VC2 in mice bearing implanted 4T1 tumors, treatment efficacy was evaluated by monitoring tumor weight, immune infiltration, and metastasis in the lung. Animals treated with VC2 via the IT route exhibited significantly reduced metastasis and improved intra-tumoral T-cell infiltration. Improved T-cell infiltration was not due to viral replication since improved parameters were observed during late times post VC2 treatment when no viral replication was detectable. The total area of metastasis in the lung was significantly reduced compared to control groups. VC2 treated animals via the IT route had significantly higher cytotoxic splenocytes, evident by the in-vitro killing of 4T1 cells, compared to the control group. This improved cytotoxic activity, intratumoral T-cell infiltration, and reduced metastasis in the lung suggest that IT administration of VC2 altered TME in a way that induced better antigen recognition, processing, and presentation resulting in improved anti-tumor immune responses with an abscopal effect in the lung. Further understanding of the mechanisms involved in the observed improved anti-tumor immune responses will help improve treatment efficacy and provide new approaches for the treatment of solid tumors. Citation Format: Rafiq Nabi, Farhana Musarrat, Vladimir Chouljenko, Konstantin Kousoulas. Oncolytic virotherapy with the HSV-1 (VC2) vaccine strain induces anti-tumor T-cell responses in the murine 4T1 breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3572.

Abstract 1995: β-Hydroxy-β-methylbutyrate promotes immunotherapy response and pro-inflammatory macrophage polarization in a mouse model of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) continues to represent a critical unmet therapeutic need in the US, and is exacerbated by obesity. Current chemotherapeutic regimens poorly control PDAC progression, and immunotherapy has yet to be widely successful against PDAC. Obesity promotes a potently protumor PDAC microenvironment including suppression of antitumor immune response. The purpose of our studies was to test whether dietary supplementation with β-hydroxy-β-methylbutyrate (HMB), shown to be safe and well tolerated in cancer patients, could offset obesity-mediated exacerbation of PDAC and promote antitumor immunosurveillance. Methods: Lean and obese C57BL/6 mice were treated with HMB alone or in combination with anti-PD1 immunotherapy following subcutaneous injection of Panc02 PDAC cells. Bone marrow-derived macrophages (BMDM) were treated with HMB in vitro with or without lipopolysaccharide (LPS). Tumor/macrophage transcriptomic analysis using Affymetrix arrays followed by gene set enrichment was performed, and tumor microenvironment immune cell composition was determined by mass cytometry. Results: a) HMB cooperates with obesity and immunotherapy to reduce PDAC progression, as indicated by reduced tumor growth with the combination regimen of HMB + anti-PD1 relative to obese or anti-PD1 immunotherapy treated mice without HMB supplementation; b) HMB mitigates PDAC immunosuppression and promotes tumor immune surveillance, as demonstrated by induction of an antitumor immune-related gene expression profile in response to HMB supplementation; c) HMB promotes pro-inflammatory polarization of tumor-associated macrophages and BMDM, as shown by increased CD38 positivity and reduced Arg1 positivity in tumor-associated macrophages from HMB supplemented mice relative to control. Further, BMDM transcriptional response to HMB alone or HMB in combination with LPS paralleled that of BMDM treated with LPS relative to untreated LPS. Conclusion: Overall, our findings suggest HMB is an adjuvant for antitumor immune responses in PDAC. Moreover, HMB promotes immune surveillance in PDAC, synergizing with immunotherapy. Hence, HMB-induced suppression of PDAC tumor growth and promotion of immune surveillance may offer significant synergy with chemotherapies or immunotherapies in PDAC. Funding: This work was supported by R35CA197627 to SDH. Citation Format: Michael F. Coleman, Kristyn A. Liu, Alexander J. Pfeil, Suhas K. Etigunta, Xiaohu Tang, Laura M. Lashinger, Zhengrong Cui, Stephen D. Hursting. β-Hydroxy-β-methylbutyrate promotes immunotherapy response and pro-inflammatory macrophage polarization in a mouse model of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1995.

Exploiting GLAAD molecules to drive an antitumor immune response in a colorectal cancer mouse model.

2565 Background: Only a minority of colorectal cancer (CRC) patients benefit from immune checkpoint inhibitors (ICI), with less than half of eligible patients responding to the treatment. Abnormal glycosylation of tumor cells is a signature of cancer development. Here, we described novel immunomodulatory molecules, named GLAAD (glycopeptides with adjuvant and tumor-associated antigen delivery). We hypothesized that GLAAD, by sharing similarities with aberrant glycans expressed in many cancers, may be a safe and effective treatment strategy to promote response and overcome resistance to ICI, thus addressing an urgent clinical need. Using a CRC mouse model, we tested whether two GLAAD candidates reduce tumor burden when orally administered alone or in combination with ICI. Methods: C57/BL6J mice were supplemented with 3% GNU-201 or 3% GNU-101 in the drinking water starting 14 days prior to tumor cell injection (d-14), while control mice received normal drinking water. On day 0, all mice were injected subcutaneously with MC38 CRC cells in each flank. On days 6, 9, 12, and 15, mice in each group received anti-PD1 antibody (aPD1) or an isotype control. Tumor size was measured every 3rd day. On day 18, the mice were sacrificed. Results: The aPD1 therapy alone showed a limited anti-tumor efficacy overall. Groups treated with GNU-201, GNU-101, GNU-201+aPD1 or GNU-101+aPD1 exhibited reduced growth, resulting in a significantly reduced tumor volume vs control on day 18. In contrast, GLAAD candidates alone significantly reduced tumor volume compared with control and led to a numerically larger decrease than aPD1 treatment alone, suggesting a higher efficacy. When analyzing tumor infiltrating myeloid cells on day 18, no differences were seen for neutrophils or dendritic cells but the proportion of macrophages was decreased in mice receiving GNU-201 or GNU-101. GNU-101 resulted in reduced serum levels of IL-10, a molecule participating in the suppression of antitumor immune responses. The tested GLAAD candidates stimulated robust anti-tumor T cell responses. GNU-201 and GNU-101 supplementation alone or in combination with aPD1 increased the proportion of T cells and among those CD8+ T cells, IFNγ producing CD4+ T cells, IFNγ+ and perforin+ CD8+ T cells. GNU-101 and GNU-201 elicited memory T cell responses after priming of the mice, measured as the ex vivo recall response (CD69 expression) of CD8+ T cells isolated from the tumor. Conclusions: GNU-201 and GNU-101 as standalone treatment induce a strong anti-tumor response that is as good as or even better than aPD1 alone. The therapeutic efficacy is associated with increased tumor infiltration and activation of T cells, in particular CD8+ T cells, highlighting the role of these GLAAD candidates as potent adjuvants in supporting specific tumor-associated antigen recognition by the immune system.

Cross-Talk Between Tumor Cells Undergoing Epithelial to Mesenchymal Transition and Natural Killer Cells in Tumor Microenvironment in Colorectal Cancer

Tumor cells undergoing epithelial to mesenchymal transition (EMT) and immune cells in tumor microenvironment (TME) reciprocally influence each other. Immune cells, by supplying TME with bioactive molecules including cytokines, chemokines, enzymes, metabolites, and by physical interactions with tumor cells via their receptors, represent an important factor that affects EMT. Chronical inflammation in TME favorizes tumor growth and invasiveness and stimulates synthesis of EMT promoting transcription factors. Natural killer (NK) cells, owing to their unique ability to exert cytotoxic function independent of major histocompatibility (MHC)-mediated antigen presentation, play a significant role in the control of metastasis in colorectal cancer (CRC). Although, the cross-talk between immune cells and tumor cells in general favors the induction of EMT and inhibition of antitumor immune responses, there are some changes in the immunogenicity of tumor cells during EMT of CRC cells that increase their susceptibility to NK cell cytotoxic lysis. However, suppressive TME downmodulates the expression of activating NK cell receptors, decreases the expression of activating and increases the expression of inhibitory NK cell ligands on tumor cells, and impairs NK cell metabolism that altogether negatively affects the overall NK cell function. Furthermore, process of EMT is often associated with increased expression of programmed cell death ligand (PD-L) and expression of immune checkpoint molecules PD-1, TIGIT, and TIM3 on functionally exhausted NK cells in TME in CRC. In this review we discuss modalities of cross-talk between tumor cells and NK cells, with regard of EMT-driven changes.

Regulatory T-Cells and Multiple Myeloma: Implications in Tumor Immune Biology and Treatment.

Multiple myeloma (MM) is associated with both cellular and humoral immune deficiencies and, despite significant advances in treatment, remains an incurable disease. Regulatory T-cells (Tregs) represent a critical subset of CD4 T-cells, characterized by CD4 + CD25+ Forkhead box P3+ (FoxP3+) phenotype, able to control peripheral tolerance and responses to foreign and tumor antigens. Tregs are elevated in various types of cancer, including hematological malignancies; in MM, data regarding Tregs function and numbers and their correlation with survival parameters are controversial. Advances in cancer biology have shown that the tumor microenvironment plays an important role in tumor progression. In MM, the highly immunosuppressive nature of the bone marrow microenvironment has been significantly elucidated in the past decade and it is now well acknowledged that targeting only the tumor clone may not be able to cure MM. Tregs within the tumor microenvironment might play a significant role in the suppression of antitumor immune responses against cancer cells and are considered to predict poor outcome in cancer patients; nonetheless the exact prognostic significance of this cell subpopulation in malignancies is still a matter of debate. In this review, we discuss the role of Tregs as an essential cell population of the MM immune microenvironment.

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.