Metabolic imbalance leading to inflammatory hypoxia and stabilization of hypoxia-inducible transcription factors (HIFs) is a hallmark of inflammatory bowel diseases. We hypothesize that HIF could be stabilized in CD4+ T cells during intestinal inflammation and alter the functional responses of T cells via regulation of microRNAs. Our assays reveal markedly increased T cell-intrinsic hypoxia and stabilization of HIF protein during experimental colitis. microRNA screen in primary CD4+ T cells points us towards miR-29a and our subsequent studies identify a selective role for HIF-2α in CD4-cell-intrinsic induction of miR-29a during hypoxia. Mice with T cell-intrinsic HIF-2α deletion display elevated T-bet (target of miR-29a) levels and exacerbated intestinal inflammation. Mice with miR-29a deficiency in T cells show enhanced intestinal inflammation. T cell-intrinsic overexpression of HIF-2α or delivery of miR-29a mimetic dampen TH1-driven colitis. In this work, we show a previously unrecognized function for hypoxia-dependent induction of miR-29a in attenuating TH1-mediated inflammation.

Abstract The microenvironment that surrounds pancreatic ductal adenocarcinoma (PDAC) is profoundly desmoplastic and immunosuppressive. Understanding triggers of immunosuppression during the process of pancreatic tumorigenesis would aid in establishing targets for effective prevention and therapy. Here, we interrogated differential molecular mechanisms dependent on cell of origin and subtype that promote immunosuppression during PDAC initiation and in established tumors. Transcriptomic analysis of cell-of-origin–dependent epithelial gene signatures revealed that Nt5e/CD73, a cell-surface enzyme required for extracellular adenosine generation, is one of the top 10% of genes overexpressed in murine tumors arising from the ductal pancreatic epithelium as opposed to those rising from acinar cells. These findings were confirmed by IHC and high-performance liquid chromatography. Analysis in human PDAC subtypes indicated that high Nt5e in murine ductal PDAC models overlaps with high NT5E in human PDAC squamous and basal subtypes, considered to have the highest immunosuppression and worst prognosis. Multiplex immunofluorescent analysis showed that activated CD8+ T cells in the PDAC tumor microenvironment express high levels of CD73, indicating an opportunity for immunotherapeutic targeting. Delivery of CD73 small-molecule inhibitors through various delivery routes reduced tumor development and growth in genetically engineered and syngeneic mouse models. In addition, the adenosine receptor Adora2b was a determinant of adenosine-mediated immunosuppression in PDAC. These findings highlight a molecular trigger of the immunosuppressive PDAC microenvironment elevated in the ductal cell of origin, linking biology with subtype classification, critical components for PDAC immunoprevention and personalized approaches for immunotherapeutic intervention. Significance: Ductal-derived pancreatic tumors have elevated epithelial and CD8+GZM+ T-cell CD73 expression that confers sensitivity to small-molecule inhibition of CD73 or Adora2b to promote CD8+ T-cell–mediated tumor regression. See related commentary by DelGiorno, p. 977

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a profoundly immunosuppressive microenvironment. Innovative therapeutic strategies are urgently needed to stop the progression of precancerous lesions into aggressive PDAC, which remains a lethal malignancy. The goal of this research project is to test the immunopreventive strategies by targeting the ectonucleotidase CD73, one of the gatekeeper enzymes responsible for adenosine production and the formation of immunosuppressive tumor microenvironment in this malignancy. We hypothesize that inhibition of CD73 will prevent pancreatic intraepithelial neoplasia (PanIN) formation and progression to PDAC by reversing immunosuppression. Materials and methods: A syngeneic PDAC mouse model was employed by injecting 100k murine pancreatic cancer (KPC) cells in flanks of C57BL/6 female mice. Oral gavage of AB680 (small molecule CD73 inhibitor) was given three days/week at 10mg/kg starting the day after KPC injections and tumor sizes were measured weekly. Toxicity was analyzed by serum ALT analysis. At the time of death 6 weeks post-KPC inoculation, tumor volume and mass were recorded. In another experiment, AB680 was administered 3 days/week at 10mg/kg for 5 weeks. CyTOF immunoprofiling of digested tumors from control and AB680 treated mice and HPLC analysis on serum from the time of death were performed. Results: In experiment 1, by week three of the study, there was a significant difference in tumor volume between the control and AB680 treated groups (P=0.02). However, in weeks 4-5 AB680 reduced tumor growth compared to vehicle controls, but the grouped statistical analysis was not significant. When observing individual tumors, there was a reduction in tumor size in 30% of the AB680 treated mice between weeks 3-5, but the difference was not statistically different. ALT analysis determined AB680 does not induce liver toxicity. For experiment 2 (3x/week treatment with AB680), there was a significant reduction in tumor growth. Activated CD8-positive T cells, dendritic cells, and macrophages were significantly increased in the syngeneic tumors from AB680 treated mice. The intratumoral adenosine levels were significantly decreased in AB680 treated mice compared to vehicle treated mice. Conclusion: We conclude oral gavage delivery of CD73 inhibitor AB680 at 10mg/kg (6x/week) reduces tumor growth in KPC syngeneic tumor bearing mice. Treatment with AB680 at 10mg/kg 3x/week significantly increases tumor doubling time, significantly alters intratumoral immune cell populations, and results in a significant decrease in intratumoral adenosine levels. In addition, we observed a significant increase in infiltration of activated CD8-positive T cells indicating oral gavage delivery using AB680 reverses immunosuppression in vivo. [Supported by NCI 75N91019D00021/75N91020F00002] Citation Format: Kanchan Singh, Vidhi Chandra, Lincoln Ballew, Tingting Mills, Erika Y. Faraoni, Trent Clark, Lana A. Vornik, Michelle I. Savage, Holger K. Eltzschig, Altaf Mohammed, Shizuko Sei, Powel H. Brown, Florencia McAllister, Jennifer Bailey-Lundberg. Preclinical testing of CD73 inhibitors for pancreatic cancer immunoprevention [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 719.

Introduction: The all stages combined five-year survival rate for pancreatic adenocarcinoma (PDA) is 11%; however, the five-year survival rate for localized PDA is 42%. These statistics highlight the importance of early prevention strategies to prevent disease progression and metastatic dissemination. Through the NCI PREVENT program, this research program explores immunoprevention strategies for PDA by targeting CD73, a gatekeeper ectoenzyme responsible for production of extracellular adenosine. We have recently shown aggressive subtypes of pancreatic intraepithelial neoplasia (PanIN) and PDA arising in ductal pancreatic epithelium have elevated CD73 and intrapancreatic adenosine indicating adenosine generation may be an early trigger of immunosuppression. We hypothesize inhibition of CD73 and adenosine generation will promote a more robust anti-tumor immune response and prevent PanIN and PDA progression. Methods: We tested three small molecule CD73 inhibitors (APCP, OP-5244, and AB680) in a syngeneic PDA mouse model by injecting 100-200k murine PDA cells derived from KrasG12D;Trp53R172H/+;Pdx:Cre (KPC) mice in the flanks of C57BL/6 female mice. Tumor sizes were measured weekly and tumor volume and mass were recorded at time of death. Dosage: APCP oral gavage (3x/week at 20mg/kg) and intraperitoneal (IP) (3x/week at 20 mg/kg). OP-5244 oral (3x/week at 25mg/kg and 10mg/kg). AB680 oral gavage (3x/week at 10mg/kg). HPLC analysis was performed for each inhibitor to quantify adenosine levels. Results: IP delivery of APCP significantly reduced tumor growth and intratumoral adenosine levels; however oral gavage delivery did not reduce tumor growth. Similarly, oral gavage delivery of OP-5244 did not reduce tumor growth. AB680 significantly reduced tumor volume and intratumoral adenosine levels and CyTOF immunoprofiling showed activated CD8+ T cells, dendritic cells, and macrophages were significantly increased in the tumors from AB680 treated mice. Conclusion: APCP IP delivery is more effective than oral gavage delivery and OP-5244 oral gavage delivery does not significantly decrease tumor growth. AB680 oral gavage delivery significantly decreases tumor growth and tumor adenosine concentrations. We observed a significant increase in infiltration of activated CD8+ T cells. AB680 shows high translational potential for preclinical testing in spontaneous GEM models. Citation Format: Lincoln Ballew, Kanchan Singh, Vidhi Chandra, Tingting Mills, Erika Y. Faraoni, Victoria Mota, Trent Clark, Lana Vornik, Michelle I. Savage, Shizuko Sei, Altaf Mohammed, Holger K. Eltzschig, Powel H. Brown, Florencia McAllister, Jennifer M. Bailey-Lundberg. Preclinical testing of CD73 inhibitors for pancreatic cancer immunoprevention [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA013.

ABSTRACTThe microenvironment that surrounds pancreatic ductal adenocarcinoma (PDAC) is profoundly desmoplastic and immunosuppressive. Understanding initial triggers of immunosuppression during the process of pancreatic tumorigenesis would aid in establishing novel targets for effective prevention and therapy. Here, we interrogate the differential molecular mechanisms dependent on cell of origin and pathology subtype that determine immunosuppression during PDAC initiation and in established tumors. Transcriptomic analysis of cell of origin dependent-epithelial gene signatures revealed that Nt5e/CD73, a cell surface enzyme that is the pacemaker for extracellular adenosine generation, is one of the top 10% of genes over-expressed in murine tumors arising from ductal pancreatic epithelium as opposed to those rising from acinar cells. These findings were confirmed by Imaging Mass Cytometry and High-Performance Liquid Chromatography. Our data indicate that ductal activation of oncogenic mutant Kras results in loss of PTEN and elevated AKT signaling which ultimately releases CD73 suppression. Delivery of CD73 small molecule inhibitors through various delivery routes reduced tumor development and growth in genetically engineered and syngeneic mouse models. Analysis in human PDAC subtypes indicates that high Nt5e in murine ductal PDAC models overlaps with high NT5E in human PDAC Squamous and Basal Subtypes, considered to have the highest immunosuppression and worst prognosis. These findings highlight a molecular trigger of the immunosuppressive PDAC microenvironment which is dependent on ductal cell of origin, linking biology with pathological subtype classification, critical components to personalized approaches for PDAC prevention and immunotherapeutic intervention.Abstract Figure