Abstract PR10: Inhibition of MEK1/2 overcomes resistance to aPD-1 blockade in pancreatic ductal adenocarcinoma through modulation of NETosis in tumor-associated neutrophils

Abstract

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC), defined by an immunosuppressive desmoplastic tumor microenvironment (TME) orchestrated by oncogenic KRAS signaling, is notoriously resistant to both conventional therapies and immune-checkpoint blockade. We overcame this resistance through inhibition of the downstream KRAS effector MEK1/2 in combination with aPD-1 blockade. Methods: Tumor cell lines derived from LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mice were orthotopically transplanted into C57BL/6J mice for efficacy, survival, and downstream analytical procedures. All mice were computationally randomized into treatment groups, tumor volume was tracked ultrasonically. Mice were treated with aPD-1 (BMS, 5 mg/kg) or IgG isotype control (BMS 5 mg/kg), the MEK1/2 inhibitor (GSK1120212, 0.5 mg/kg) or vehicle alone (DMSO). A CXCR2 blocking antibody (BMS 12.5 mg/kg) was used to assess effects of direct CXCR2 blockade. Immunophenotyping and functional study of tumor-infiltrating leukocytes (TILs) were performed on both orthotopic and hemi-spleen TILs. Pooled bulk-tumor RNA from orthotopic tumors was used for RT-qPCR and RNA-seq analysis. Immunofluorescent staining of neutrophil extracellular traps in murine tumors was performed on formalin-fixed paraffin-embedded (FFPE) orthotopic tumors. Sections were stained for Ly-6G (CD66b in human samples), myeloperoxidase (MPO), histone H2-B (H2B), and DNA. NET(+) areas were identified as extracellular co-localization of DNA/H2B and MPO or Ly-6G(+) cells with > 75% nuclear MPO co-localization with HALO software. Results: aPD-1+MEKi therapy routinely eradicated tumors in >80% of mice, with tumor-free survival and protection from tumor re-challenge extending as far as one-year post-treatment. Initial gene expression data showed a reduction in IL-8 homologs in aPD-1+MEKi tumors. Subsequent immunophenotyping data showed increased CXCR2(+) neutrophils in aPD-1+MEKi tumors compared to aPD-1+DMSO. Given the functional role of CXCR2 in IL-8-mediated NETosis, we compared tumors from aPD-1+MEKi sensitive (KPC_S) and resistant (KPC_R) KPC cell lines for NETosis signatures via multiplex IF. Within KPC_S groups, mean NET scores w/SEM fell from 2.29 +/- 0.12 to 0.51 +/- 0.10 between aPD-1+DMSO and aPD-1+MEKi groups (p <= 0.0001). No difference was found within KPC_R treatment groups (2.10 +/- 0.11 vs 2.07 +/- 0.12, p = n.s.). Furthermore, 23.41 +/- 3.04% of ex vivo stimulated CD8(+) TILs from aPD-1+MEKi KPC_S tumors were IFNy(+) vs 9.09 +/- 2.02% in aPD-1+DMSO KPC_S and 6.55 +/- 1.38% in aPD-1+MEKi KPC_R cells (p = 0.016 and 0.011 respectively). Conclusions: We have demonstrated that resistance to aPD-1 blockade in PDAC can be annulled with combined MEK1/2 inhibition. The elimination of immunosuppressive NETs from sensitive KPC tumors but not resistant tumors suggests that MEKi works by modulating the TME rather than neutrophils directly. Work is ongoing to evaluate aCXCR2 and NETosis inhibition in these resistant tumors. Correlative IHC/IF analysis of our J1568 clinical trial cohort is also near completion. Citation Format: Brian Herbst, Elizabeth Jaffee, Lei Zheng. Inhibition of MEK1/2 overcomes resistance to aPD-1 blockade in pancreatic ductal adenocarcinoma through modulation of NETosis in tumor-associated neutrophils [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr PR10.