Abstract C033: KRAS-TP53 cooperativity regulates Cxcl1 to sustain tumor-permissive circuitry via granulocyte-derived CXCR2-TNF signaling in pancreatic cancer

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.

Top advances of the year: Pancreatic cancer

“…Accumulation of tumor‐associated neutrophils was also found to be associated with loss of KDM6A protein (an important epigenetic regulator) in both human and murine pancreatic adenocarcinomas, and inhibition of CXCL1 (a neutrophil‐recruiting chemokine) effectively inhibited growth of KDM6A‐deficient tumors 48 . Furthermore, neutrophil‐derived tumor necrosis factor has been identified as a central regulator of immune tolerance and stromal inflammation in PDAC 49 . Inhibition of CCR2 (a receptor involved in chemokine‐mediated monocyte chemotaxis) affects the infiltration of multiple myeloid cell types, including myeloid‐derived suppressor cells (MDSCs), tumor‐associated macrophages, and neutrophils.…”

Targeting neutrophils: Mechanism and advances in cancer therapy

“…Research on resistance has never failed to attract scholars’ attention. The presence of NETs is partly responsible for the poor prognosis after treatment, as the mechanisms through which NETs coat tumour cells and promote resistance to chemotherapy, immunotherapy and radiotherapy have already been described 9,52–54 . Under certain chemical conditions, tumour cells secrete interleukin‐1β (IL‐1β), which subsequently induces the generation of NETs.…”

“…The presence of NETs is partly responsible for the poor prognosis after treatment, as the mechanisms through which NETs coat tumour cells and promote resistance to chemotherapy, immunotherapy and radiotherapy have already been described. 9,[52][53][54] Under certain chemical conditions, tumour cells secrete interleukin-1β (IL-1β), which subsequently induces the generation of NETs. The formation of a NET necessitates the presence of two essential proteins: integrin-αvβ1 and matrix metalloproteinase 9, which can trap and activate TGF-β.…”

“…58 However, the mechanism underlying the immunosuppressive effect was unclear until recently, when Bianchi et al elucidated the involvement of CXCL1 in eradicating T cells associated with pancreatic ductal adenocarcinoma. 9 Unlike normal neutrophils, MDSC plays only a protumour role and are harmful to the functioning of the immune system. Additionally, MDSCs are considered to be immature cells in the human peripheral blood circulation.…”

“…7 New clinical studies investigating the role of neutrophils have brought targeting neutrophil therapy into the spotlight. 8,9 The newest approaches for targeting neutrophils are based on the different subgroups of tumours that exert various functions. That is, they act by suppressing protumour subsets and promoting antitumour subsets.…”