Combined MEK and STAT3 Inhibition Uncovers Stromal Plasticity by Enriching for Cancer-Associated Fibroblasts With Mesenchymal Stem Cell-Like Features to Overcome Immunotherapy Resistance in Pancreatic Cancer

Abstract

BACKGROUND: We have shown that reciprocally activated RAS/MEK and JAK/STAT3 pathways mediate therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC), while combined MEK and STAT3 inhibition (MEKi+STAT3i) overcomes such resistance and alters stromal architecture. We now determine if MEKi+STAT3i reprograms the cancer-associated fibroblast (CAF) and immune microenvironment to overcome resistance to immune checkpoint inhibition in PDAC. METHODS: CAF and immune cell transcriptomes in MEKi (trametinib)+STAT3i(ruxolitinib)- vs. vehicle-treated Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) tumors were examined via single-cell RNA sequencing (scRNAseq). CRISPR/Cas9-silencing of CAF-restricted Map2k1/Mek1 and/or Stat3 enabled interrogation of CAF-dependent effects on immunologic remodeling in orthotopic models. Tumor growth, survival, and immune profiling via time-of-flight mass cytometry were examined in PKT mice treated with vehicle, anti-PD1 monotherapy, and MEKi+STAT3i combined with anti-PD1. RESULTS: MEKi+STAT3i attenuates Il6/Cxcl1-expressing pro-inflammatory and Lrrc15-expressing myofibroblastic CAF phenotypes while enriching for Ly6a/Cd34-expressing CAFs exhibiting mesenchymal stem cell-like features via scRNAseq in PKT mice. This CAF plasticity is associated with M2-to-M1 reprogramming of tumor-associated macrophages, and enhanced trafficking of CD8+ T-cells which exhibit distinct effector transcriptional programs. These MEKi+STAT3i-induced effects appear CAF-dependent, since CAF-restricted Mek1/Stat3 silencing mitigates inflammatory-CAF polarization and myeloid infiltration in-vivo. Addition of MEKi+STAT3i to PD-1 blockade not only dramatically improves anti-tumor responses and survival in PKT mice, but also augments recruitment of activated/memory T-cells while improving their degranulating and cytotoxic capacity, compared with anti-PD-1 monotherapy. Importantly, treatment of a patient with chemotherapy-refractory metastatic PDAC with MEKi (Trametinib), STAT3i (Ruxolitinib), and PD-1 inhibitor (Nivolumab) yielded clinical benefit. CONCLUSIONS: Combined MEKi+STAT3i mitigates stromal inflammation and enriches for CAF phenotypes with mesenchymal stem cell-like properties to overcome immunotherapy resistance in PDAC.