MAP Kinase inhibition reshapes tumor microenvironment of mouse pancreatic cancer by depleting anti-inflammatory macrophages

Abstract

2The RAF/MEK/ERK (MAP Kinase) pathway is the index oncogenic signaling towards which many 3 compounds have been developed and tested for the treatment of KRAS-driven cancers, including 4 pancreatic ductal adenocarcinoma (PDA). Here, we explored the immunological changes induced 5 by targeted MEK1/2 inhibition (MEKi) using trametinib in preclinical mouse models of PDA. We 6 evaluated the dynamic changes in the immune contexture of mouse PDA upon MEKi using a 7 multidimensional approach (mRNA analyses, flow cytometry, and immunophenotyping). Effect of 8 MEKi on the viability and metabolism of macrophages was investigated in vitro. We showed that 9 transcriptional signatures of MAP Kinase activation are enriched in aggressive human PDA 10 subtype (squamous/basal-like/quasimesenchymal), while short term MEKi treatment in mouse 11 PDA induced subtype switching. Integrative mRNA expression and immunophenotypic analyses 12 showed that MEKi reshapes the immune landscape of PDA by depleting rather than 13 reprogramming macrophages, while augmenting infiltration by neutrophils. Depletion of 14 macrophages is observed early in the course of in vivo treatment and is at least partially due to 15 their higher sensitivity to MEKi. Tumor-associated macrophages were consistently reported to 16 interfere with gemcitabine uptake by PDA cells. Here, our in vivo studies show a superior 17 antitumor activity upon combination of MEKi and gemcitabine using a sequential rather than 18 simultaneous dosing protocol. Our results show that MEK inhibition induces a dramatic 19 remodeling of the tumor microenvironment of mouse PDA through depletion of macrophages, 20 which substantially improves the antitumor activity of gemcitabine. 22Pancreatic ductal adenocarcinoma (PDA) has the lowest survival rate of all cancers (1). 25Current therapeutic strategies are mostly based on combination of chemotherapeutic agents, 26 which provides intermittent response and modest survival benefit in the advanced disease setting 27 (2,3). Results from recent clinical trials showed that immune-checkpoint based therapy is rarely 28 active in PDA (4). The complex and heterogenous PDA microenvironment is reported to play a 29 major role in mediating resistance to both chemotherapy and immune therapy (5). In human PDA 30 stromal non-malignant cells often outnumber neoplastic cells, with fibroblasts and macrophages 31 being the dominants cellular components (6). 32Activating mutations of the GTPase KRAS are a nearly universal feature of PDA (7). Activated 33 KRAS engages a multitude of pathways, including the RAF/MEK/ERK (MAP Kinase) pathway, 34 which regulates cellular processes such as proliferation and cell survival (8). Preclinical mouse 35 models show that the oncogenic KRAS-induced cytokine milieu likely contributes to the highly 36 immunosuppressive microenvironment observed already at the preneoplastic PanIN stage (9). In 37 addition to non-cell autonomous effects, KRAS activation and increased activity through the MAP 38Kinase pathway induce cell-intrinsic u...