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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

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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

Aerobic exercise is associated with decreased cancer incidence and cancer-associated mortality. However, little is known about the effects of exercise on pancreatic ductal adenocarcinoma (PDA), a disease for which current therapeutic options are limited. Herein, we show that aerobic exercise reduces PDA tumor growth, by modulating systemic and intra-tumoral immunity. Mechanistically, exercise promotes immune mobilization and accumulation of tumor-infiltrating IL15Rα

The immune microenvironment plays a critical role in cancer development, progression and resistance to therapy. Specific tumor genotypes can profoundly influence the composition of the immune microenvironment. After oncogenic KRAS, TP53 is the most frequently mutated gene in pancreatic ductal adenocarcinoma (PDAC). KRASG12D mutations in tumor cells are known to modify the immune landscape of PDAC, however the immune effects of neomorphic TP53 mutations have not been defined. Focusing on the most common neomorphic TP53 mutation in human PDAC, we sought to evaluate the non-cell-autonomous role of gain-of-function mutant Trp53R172H in modulating the immune microenvironment of pancreatic tumors by using KrasG12D-mutated mouse pancreatic ductal epithelial cells (PDEC) genetically engineered to express the Trp53R172H gain-of-function. Orthotopically implanted tumors derived from CRISPR-generated KrasG12D/+;Trp53R172H/+ PDEC had a distinct immune profile in comparison to KrasG12D/+;Trp53+/+ tumors, characterized by an influx of intratumoral CD11b+Ly6G+ neutrophils and concomitant decreases in CD3+ T cells, CD8+ T cells, and CD4+ T helper 1 (Th1) cells. Analysis of publicly available human PDAC cohorts revealed enrichment of genes in neutrophil-related pathways in TP53-mutated tumors. Knockdown of CXCL2, a neutrophil chemoattractant, in the tumor epithelial compartment of CRISPR KrasG12D/+;Trp53R172H/+ PDEC tumors reversed the neutrophil phenotype. Depleting neutrophils in mice bearing CRISPR KrasG12D/+;Trp53R172H/+ PDEC tumors augmented sensitivity to combined CD40 immunotherapy and chemotherapy. Collectively, these data link gain-of-function mutant Trp53R172H to the presence of intratumoral neutrophils in pancreatic cancer and suggests that tumor genotypes could inform patient selection for immunotherapy. Citation Format: Despina Siolas, Emily Vucic, Emma Kurz, Cristina Hajdu, Dafna Bar-Sagi. Gain-of-function p53R172H mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment promoting resistance to immunotherapy [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 6142.

Tumor genotype can influence the immune microenvironment, which plays a critical role in cancer development and therapy resistance. However, the immune effects of gain-of-function Trp53 mutations have not been defined in pancreatic cancer. We compare the immune profiles generated by Kras(G12D)-mutated mouse pancreatic ductal epithelial cells (PDECs) engineered genetically to express the Trp53(R172H) mutation with their p53 wild-type control. Kras(G12D/+);Trp53(R172H/+) tumors have a distinct immune profile characterized by an influx of CD11b(+)Ly6G(+) neutrophils and concomitant decreases in CD3(+) T cells, CD8(+) T cells, and CD4(+) T helper 1 cells. Knockdown of CXCL2, a neutrophil chemokine, in the tumor epithelial compartment of CRISPR Kras(G12D/+;)Trp53(R172H/+) PDEC tumors reverses the neutrophil phenotype. Neutrophil depletion of mice bearing CRISPR Kras(G12D/+);Trp53(R172H/+) tumors augments sensitivity to combined CD40 immunotherapy and chemotherapy. These data link Trp53(R172H) to the presence of intratumoral neutrophils in pancreatic cancer and suggest that tumor genotypes could inform selection of affected individuals for immunotherapy.

Background: The application of a multidimensional integrated analysis of recurring genomic and expression alterations can identify new insights into the molecular mechanisms involved in the pathogenesis of NSCLC. Distinguishing the key mechanisms and causal events driving tumorigenesis will lead not only to a better understanding of lung cancer phenotypes and biology, but also new molecular markers and therapeutic targets. Using this approach, we identified the chromosomal region at 12q13-15, and more specifically Glioma amplified sequence 41 (GAS41) to be frequently amplified and overexpressed in NSCLC. A putative transcription factor, amplification of GAS41 has been reported in dedifferentiated liposarcomas and in the earliest stages of glioma and astrocytoma. While the oncogene MDM2 has long been believed to be the driver of this amplicon, we hypothesize GAS41 is an oncogene capable of driving selection of the 12q15 amplicon, and not merely a passenger event. Methods: An integrative genomics approach, examining 261 NSCLC tumors (169 adenocarcinomas (AC) and 92 squamous cell carcinomas (SqCC)) profiled for copy number and gene expression alterations was used to identify novel candidate oncogenes in NSCLC. Recurrent DNA amplifications were identified using the GISTIC algorithm and integrated with gene expression data to identify genes frequently amplified and overexpressed. Genes were classified as overexpressed if the fold change between tumor and matched non-malignant tissues was greater than 2 fold. The functional significance of GAS41 was assessed by lentiviral knockdown and ectopic overexpression in lung cancer cell lines with and without GAS41 amplification, and human bronchial epithelial cells respectively. In vitro assays measuring proliferation, anchorage independent growth, senescence and apoptosis were used to assess the phenotypic effect of gene dosage manipulation. Survival analysis was performed using the Cox regression model for multiple independent cohorts. Results: GAS41 is gained or amplified in over 20% of NSCLC tumors, with similar frequencies of amplification in both AC (26%) and SqCC (24%). Although frequently co-amplified with MDM2, amplification of GAS41 was observed to occur in the absence of MDM2 amplification. Overexpression of GAS41 in human bronchial epithelial cells abrogated senescence, whereas knockdown reduced cell proliferation, impaired colony formation and induced cellular senescence only in lung cancer cell lines with amplification. Western blotting revealed increased p21, cleaved PARP and reduced levels of phospho-p53 in knockdown lines as compared to empty vector controls, suggesting GAS41 is implicated in the regulation of the p21-p53 pathway. Consistent with in vitro results, patients expressing high levels of GAS41 displayed poorer outcomes compared to those with lower levels of GAS41. Conclusions: Our findings reveal GAS41 as a candidate oncogene frequently amplified and overexpressed in NSCLC, both in the presence and absence of MDM2 amplification. Gene dosage manipulation resulted in distinct phenotypic changes characteristic of oncogenes, and thus implicate amplification of GAS41 as a novel mechanism of NSCLC tumorigenesis.