CXCR2 blockade overcomes the NETosis-mediated resistance to MEK inhibition in pancreatic cancer models
Brian Herbst, Alex Blair, Yiming Li, Elizabeth M. Jaffee, Lei Zheng
Brian Herbst, Alex Blair, Yiming Li, Elizabeth M. Jaffee, Lei Zheng
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Research Article Immunology Oncology

CXCR2 blockade overcomes the NETosis-mediated resistance to MEK inhibition in pancreatic cancer models

Abstract

Single-agent anti-PD-1 antibodies are ineffective for pancreatic ductal adenocarcinoma (PDAC) due to the immunosuppressive tumor-microenvironment (TME). KRAS mutations contribute to the inflammatory TME and therapeutic resistance by upregulating IL-8 via MAPK pathways. Thus, this study attempted to overcome the resistance to anti-PD-1 antibodies by targeting downstream KRAS-effectors. The study found that the resistance to anti-PD-1 antibodies can be overcome through MEK1/2-inhibition. The combination of anti-PD-1 antibodies and MEK inhibitors displayed antitumor activity in Kras mutated (Krasmut) KPC mouse tumors, but not WT (KrasWT) Panc02 tumors. The combination of anti-PD-1 antibodies and MEK inhibitors induced recruitment of tumor-associated neutrophils (TANs) via CXCR2, an IL-8 receptor, and increased memory CD8+ T cells and IFN-γ production in treatment-sensitive tumors. However, larger tumors still resisted the combination of anti-PD-1 antibody and MEK inhibitor, likely due to hypoxia/necrosis-induced NETosis and associated paucity of CD8+ T cells. The subsequent addition of anti-CXCR2 antibody overcame this resistance by blocking TAN-infiltration to hypoxic/necrotic areas. Consistently, a risk-score based on the NETosis-MAPK signaling interaction is significantly associated with poorer survival in human PDAC. This study thus provides a new venue for overcoming resistance to strategies targeting KRAS signaling.

Authors

Brian Herbst, Alex Blair, Yiming Li, Elizabeth M. Jaffee, Lei Zheng

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Figure 6

Characterization of NETosis in the PDACs treated by aPD-1 with or without MEKi.

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Characterization of NETosis in the PDACs treated by aPD-1 with or withou...
(A) Singscore for the NETosis gene sets through a pooled bulk-tumor RNA-seq analysis following different treatments, as indicated (n = 5 pooled tumors/group). KPC_S: KPC001BH; KPC_R: KPC3403F, as indicated here and below. MPAS, MAPK Pathway Activity Score; Neutrophil_mcp, Neutrophil Microenvironment Cell Populations counter; TAN versus NN, Fold changes of genes upregulated in TANs versus Tissue Naive (circulating) Neutrophil. Necroptosis score was calculated with a panel of genes previously described. (B) Multiplexed immunofluorescence staining of NETs and CD8+ T cells in the same orthotopic tumors as in A. Blue, DAPI; White, MPO; Green, CD8. Note that areas of necrosis are adjacent to the areas enriched with NETosis, but less infiltration of CD8+ T cells. Scale bars: 1 mm (note that the sizes of tumors vary due to different treatment effects and, therefore, tumor images were amplified at different scales to similar sizes, resulting in different sizes of 1-mm scale bars). (C) Densities of TANs, NETs and CD8+ T cells counted in random 20 × HPFs in tumors from different treatment groups, as indicated. Statistics were conducted by Kruskal-Wallis tests without multiple comparisons. *P < 0.05. (D) Correlation between CD8+ T cell density and NET density. Pearson correlation was conducted with R and P value indicated. B–D, n = 3 or 4 mice/group. Experiment was repeated twice.