Immune checkpoint inhibition (ICI) has revolutionized treatment in cancers that are naturally immunogenic by enabling infiltration of T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. Tumors possessing complex immunosuppressive TME’s such as breast and pancreatic cancers present unique therapeutic obstacles as response rates to ICI remain low. Such tumors often recruit myeloid-derived suppressor cells (MDSCs) whose functioning prohibits both T-cell activation and infiltration. We attempted to sensitize these tumors to ICI using epigenetic modulation to target MDSC trafficking and function to foster a less immunosuppressive TME. We showed that combining a histone deacetylase inhibitor, entinostat (ENT), with anti–PD-1, anti–CTLA-4, or both, significantly improved tumor-free survival in both the HER2/neu transgenic breast cancer and the Panc02 metastatic pancreatic cancer mouse models. Using flow cytometry, gene expression profiling, and ex vivo functional assays, we characterized populations of tumor-infiltrating lymphocytes (TILs) and MDSCs, as well as their functional capabilities. We showed that addition of ENT to checkpoint inhibition led to significantly decreased suppression by granulocytic-MDSCs in the TME of both tumor types. We also demonstrated an increase in activated granzyme-B–producing CD8+ T effector cells in mice treated with combination therapy. Gene expression profiling of both MDSCs and TILs identified significant changes in immune-related pathways. In summary, addition of ENT to ICI significantly altered infiltration and function of innate immune cells, allowing for a more robust adaptive immune response. These findings provide a rationale for combination therapy in patients with immune-resistant tumors, including breast and pancreatic cancers.

Therapeutic combinations to alter immunosuppressive, solid tumor microenvironments (TME), such as in breast cancer, are essential to improve responses to immune checkpoint inhibitors (ICI). Entinostat, an oral histone deacetylase inhibitor, has been shown to improve responses to ICIs in various tumor models with immunosuppressive TMEs. The precise and comprehensive alterations to the TME induced by entinostat remain unknown. Here, we employed single-cell RNA sequencing on HER2-overexpressing breast tumors from mice treated with entinostat and ICIs to fully characterize changes across multiple cell types within the TME. This analysis demonstrates that treatment with entinostat induced a shift from a protumor to an antitumor TME signature, characterized predominantly by changes in myeloid cells. We confirmed myeloid-derived suppressor cells (MDSC) within entinostat-treated tumors associated with a less suppressive granulocytic (G)-MDSC phenotype and exhibited altered suppressive signaling that involved the NFκB and STAT3 pathways. In addition to MDSCs, tumor-associated macrophages were epigenetically reprogrammed from a protumor M2-like phenotype toward an antitumor M1-like phenotype, which may be contributing to a more sensitized TME. Overall, our in-depth analysis suggests that entinostat-induced changes on multiple myeloid cell types reduce immunosuppression and increase antitumor responses, which, in turn, improve sensitivity to ICIs. Sensitization of the TME by entinostat could ultimately broaden the population of patients with breast cancer who could benefit from ICIs.

We have shown that KRAS-TP53 genomic co-alteration is associated with immune-excluded microenvironments, chemoresistance, and poor survival in pancreatic ductal adenocarcinoma (PDAC) patients. By treating KRAS-TP53 cooperativity as a model for high-risk biology, we now identify cell-autonomous Cxcl1 as a key mediator of spatial T-cell restriction via interactions with CXCR2+ neutrophilic myeloid-derived suppressor cells in human PDAC using imaging mass cytometry. Silencing of cell-intrinsic Cxcl1 in LSL-K-rasG12D/+;Trp53R172H/+;Pdx-1Cre/+(KPC) cells reprograms trafficking and functional dynamics of neutrophils to overcome T-cell exclusion, and controls tumor growth in a T-cell-dependent manner. Mechanistically, neutrophil-derived TNF is a central regulator of this immunologic rewiring, instigating feed-forward Cxcl1 overproduction from tumor-cells and cancer-associated fibroblasts (CAF), T-cell dysfunction, and inflammatory CAF polarization via transmembraneTNF-TNFR2 interactions. TNFR2 inhibition disrupts this circuitry and improves sensitivity to chemotherapy in vivo. Our results uncover cancer cell-neutrophil crosstalk in which context-dependent TNF signaling amplifies stromal inflammation and immune tolerance to promote therapeutic resistance in PDAC.

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.