CCSG Pilot: Determining Mechanisms of Immunotherapy Resistance in KEAP1 and LKB1 Mutant Lung Cancers

ABSTRACT Immunotherapies, particularly those that inhibit the PD1/PD-L1 interaction and drive T cells to recognize and kill tumor cells, have shown striking responses in roughly 30% of non-small cell lung cancers. However, the other 70% of patients do not have a durable response. Genetic mutations in the tumor suppressor KEAP1 and LKB1, and high neutrophil to lymphocyte ratios in tumors have both been correlated to poor immunotherapy responses. What is less clear is what can be done to overcome immunotherapy resistance in tumors that have these biomarkers. One way to boost response is to include an epigenetic inhibitor, such as one targeting the Polycomb Repressive Complex 2 (PRC2), to influence the tumor cell and microenvironment heterogeneity. Our studies have shown that lung cancers that are squamous in lineage, even with LKB1 mutations, respond robustly to EZH2 inhibitor to become more responsive to immunotherapy. However, our in vitro models suggest that lung cancers that are adenocarcinoma in lineage may not respond similarly. Therefore, our Central Hypothesis is that regardless of LKB1 and KEAP1 genetic status, tumor histo-phenotype and microenvironment will determine response to immunotherapy combined with PRC2 inhibition. The overarching goal of the proposed study is to validate combining EZH2 inhibition with PD1/PD-L1 targeted immunotherapy in varied lung cancer subtypes, and learn the molecular mechanisms when the treatment is successful as well as when it is not successful. In Aim 1, we will use a newly developed mouse model of KRAS-driven tumors with deletion of Lkb1, Keap1 or both and generate lung tumors and tumoroid cultures. We will examine CXCL9/10/11, PD-L1 and MHC expression in tumoroids of similar genetics but differing lineages after EZH2 inhibitor treatment. We will also treat immune-competent tumor-bearing mice with the EZH2 inhibitor EPZ6438 (Tazemetostat), and the immunotherapy PD-1 antibody and follow tumor growth by magnetic resonance imaging. We will characterize tumor phenotypes in responders and non-responders and examine tumors that show resistance to this therapeutic drug combination. In Aim 2, we will focus on human samples. We will build several sets of isogeneic lines in which KEAP1 and LKB1 are mutated in the parental line and rescued by lentiviral transduction. We will examine markers of lineage fate, and use IFNγ with PRC2 inhibition in vitro to test ability of cells to up-regulate MHC and CXCL9/10/11 cytokines. In parallel, we will identify patient samples with NRF2, KRAS, LKB1 and KEAP1 mutations and determine cancer lineage, immune infiltration and PRC2 activity levels. Completion of these aims will solidify the efficacy of a promising therapeutic combination and uncover mechanisms by which tumor phenotypes and microenvironments are changed by EZH2 inhibitors. Given that a Phase 1/2 clinical trial combining EZH2 inhibition with anti-PDL1 will begin recruiting advanced-stage non-small cell lung cancer patients in May 2023, learning the phenotypes and mechanisms of responders and non-responders will be extremely timely for any Phase 2/3 trials that ensue. Brainson Fillmore, C 2