Abstract 2113: Suppression of macrophage migration inhibitory factor (MIF) impairs tumor growth and overcomes immunotherapy resistance in KEAP1-deficient NSCLC tumors
Authors: Galan-Cobo Ana , Qian Yu , Peng Fuduan , McGrail Daniel James , Patel Sonia , Zhang Fahao , Zhang Xiang , Skoulidis Ferdinandos , Parra Edwin , Dang Minghao , Rodriguez Saxon , Reuben Alexandre , Wistuba Ignacio , Wang Linghua , Heymach John Victor 
Published: June 2022

Abstract
Background: KEAP1, which regulates the degradation of the antioxidant transcription factor NRF2, is the third most commonly mutated tumor suppressor in lung adenocarcinoma (LUAD). Recent reports have provided clinical evidence that mutations in STK11/LKB1 and KEAP1 are strongly associated with immune checkpoint blockade resistance in LUAD, particularly those harboring KRAS mutations. Nevertheless, the specific mechanisms by which loss of KEAP1 impacts anti-tumor immunity in KRAS mutant tumors remains to be determined.
Methods: KRAS-mutant (K) and LKB1 (KL), and/or KEAP1-deficient (KK and KLK) murine tumor models were profiled using single-cell RNA sequencing (scRNA-seq) and multiplex staining, and response to anti-PD1 treatment was assessed. Clinical samples from the MD Anderson ICON study (a cohort of 148 resected tumors from early-stage lung cancer patients) and TCGA lung cohorts were used to validate pre-clinical findings.
Results: While K tumors were sensitive to anti-PD1 treatment, KEAP1-deficient isogenic tumors (KK; KLK) were refractory. KEAP1-deficient tumors were found to exhibit low immune cell infiltration and an enrichment of cancer associated fibroblasts (CAFs) and endothelial cells. scRNA-seq analysis indicated that KEAP1-deficient tumors had reduced T cell infiltration, in particular, CD8 and NK T cells, decreased B cell populations, and a marked change in M2 macrophage polarization as compared to KEAP1-proficient tumors. Multiplex analysis of CD3 and F4/80 markers confirmed these findings. In the TCGA lung cancer cohort, CD8B expression was dramatically decreased while MIF (macrophage migration inhibitory factor) was upregulated in KK tumors as compared to K LUAD tumors, and expression of KEAP1 inversely correlated with CD163, ARG2 and IL10, which are mainly secreted by macrophages. KEAP1-deficient pre-clinical tumor models showed a significant upregulation of MIF expression and secretion. CRISPR-Cas9 deletion of MIF dramatically impaired in vivo tumor growth, and enhanced T cell cytotoxic effects, anti-tumor immune response and anti-PD1 treatment in KK and KLK tumor models.
Conclusions: These findings indicate that loss of KEAP1, alone or in combination with STK11/LKB1 alterations, contributes to an immunosuppressed tumor immune microenvironment. These changes appear to be mediated at least in part through MIF upregulation, providing a potential therapeutic strategy for overcoming KEAP1-dependent resistance to immunotherapy.
Citation Format: Ana Galan-Cobo, Yu Qian, Fuduan Peng, Daniel James McGrail, Sonia Patel, Fahao Zhang, Xiang Zhang, Ferdinandos Skoulidis, Edwin Parra, Minghao Dang, Saxon Rodriguez, Alexandre Reuben, Ignacio Wistuba, Linghua Wang, John Victor Heymach. Suppression of macrophage migration inhibitory factor (MIF) impairs tumor growth and overcomes immunotherapy resistance in KEAP1-deficient NSCLC tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2113.