Abstract C033: KRAS-TP53 cooperativity regulates Cxcl1 to sustain tumor-permissive circuitry via granulocyte-derived CXCR2-TNF signaling in pancreatic cancer
Authors: Bianchi Anna , Silva Iago De Castro , Deshpande Nilesh U. , Mehra Siddharth , Garrido Vanessa T. , Singh Samara , Rafie Christine I. , Zhiqun Zhou , Ogobuiro Ifeanyichukwu C. , Dosch Austin R. , Nagathihalli Nagaraj , Merchant Nipun B. , Datta Jashodeep 
Published: November 2022

Abstract
Objective: We have recently shown that KRAS-TP53 genomic co-alteration is associated with innate immune-enriched and T-cell-excluded tumor microenvironments (TME), chemotherapy resistance, and poor survival in pancreatic ductal adenocarcinoma (PDAC) patients. We sought to define the multi-cellular crosstalk that underlies these effects by dissecting how cancer cell-autonomous transcriptional programs orchestrate tolerogenic circuitries to mediate chemoresistance in KRAS-TP53 cooperative PDAC. Methods: Spatial neighborhood analysis via Imaging Mass Cytometry (IMC) was performed in patient-derived PDAC sections. Immune profiling and bulk RNA-seq in whole tumors, as well as bulk-RNAseq in intratumoral F4/80-Ly6Ghi neutrophilic(PMN)-MDSCs in orthotopic KPC tumors with/without CRISPR/Cas9 editing of Cxcl1 was performed. Effect of TNFR2 inhibition via etanercept on ex vivo co-cultures of intratumoral PMN-MDSC with KPC tumor cells/CAFs and T-cells, as well as in orthotopic KPC models in vivo with/without gemcitabine+paclitaxel chemotherapy was performed. Results: Interrogation of cancer cell transcriptomes and IMC architecture in human tumors reveals disproportionate enrichment of Cxcl1 in KRAS-TP53 co-altered PDAC. IMC-enabled spatial neighborhood analysis in KRAS-TP53 co-altered human PDAC TMEs demonstrates strong spatial contiguity between PanCK+CXCL1+ tumor islands and cognate CD15+CXCR2+ PMN-MDSCs, with exclusion of CD8+ T-cells from tumor cell:PMN-MDSC communities. In murine orthotopic models that phenocopy T-cell excluded human PDAC, genetic silencing of tumor cell-intrinsic Cxcl1 overcomes CD8+ T-cell exclusion and controls tumor growth in a CD8+ T-cell dependent manner in vivo. Transcriptomes from KPC-Cxcl1KO tumors not only reveal enrichment in pathways encoding for T-cell effector activity but also attenuation in pathways related to innate immune function. These immune potentiating effects upon Cxcl1 silencing are driven in large part by reprogramming of trafficking dynamics and immunosuppressive potential in intratumoral CXCR2+ PMN-MDSCs. To identify neutrophil-intrinsic mechanisms that govern remodeling of the TME following Cxcl1 silencing, transcriptomes in intratumoral KPC-Cxcl1KO PMN-MDSCs reveal strong downregulation of MAPK and TNF pathways, with signaling studies implicating a novel Cxcr2-Ikk-Map3k8-Tnf axis. We uncover novel effects of neutrophil-derived TNF in promoting tumor cell-Cxcl1 production, inflammatory CAF polarization, and T-cell dysfunction in ex vivo co-cultures, predominantly via a membraneTNF-TNFR2 dependent mechanism. Systemic TNFR2 inhibition via etanercept not only augments T-cell activation, but also mitigates tumor-wide Cxcl1 production, stromal inflammation, and CAF:tumor cell IL6-STAT3 signaling to improve sensitivity to gemcitabine+paclitaxel chemotherapy in vivo. Conclusion: These data uncover novel tumor-permissive/chemoresistant circuitries in which cancer cell-intrinsic Cxcl1 sustains innate immunoregulatory and tolerogenic signaling via neutrophil-derived TNF in the PDAC TME.
Citation Format: Anna Bianchi, Iago De Castro Silva, Nilesh U. Deshpande, Siddharth Mehra, Vanessa T. Garrido, Samara Singh, Christine I. Rafie, Zhou Zhiqun, Ifeanyichukwu C. Ogobuiro, Austin R. Dosch, Nagaraj Nagathihalli, Nipun B. Merchant, Jashodeep Datta. KRAS-TP53 cooperativity regulates Cxcl1 to sustain tumor-permissive circuitry via granulocyte-derived CXCR2-TNF signaling in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C033.