Deconvoluting the contributions of the IFNy pathway components to lung TME and tumor immunity

Project Summary The approval of anti-PD1/PD-L1 and anti-CTLA immune checkpoint blockade (ICB) therapies for those with non- small cell lung cancer (NSCLC) has demonstrated that immunotherapies can generate robust responses for a subset of patients with advanced disease. However, while nearly 45% of patients demonstrate a major

pathological response to ICB, the median progression-free survival for NSCLC patients remains dismal at around 7 months. It is now well documented that persistent interferon gamma (IFNy) cytokine pathway signaling plays a paradoxical role within the tumor microenvironment and has been associated with both resistance and

response to ICB leading us to believe that there is greater nuance to dissect within this pathway. Accordingly, this cytokine pathway is quite complex as IFNy stimulates the expression of ~200 hallmark interferon-stimulated genes (ISGs); thus, it is still unclear how the majority of ISGs influence tumor immunity as it has been difficult to

study this pathway in a relevant biologic context. Using a novel functional genomics platform developed in our lab (Perturb-map) my sponsor lab was able to knock-out (KO) 35 cytokine genes in parallel in the KP mouse model of NSCLC and investigate spatial and temporal phenotypes at a single-cell resolution. It was found that

KO of Socs1, a negative regulator of the IFNy pathway, caused an overt growth advantage and a paradoxical increase in both CD8+ and CD4+ T-cell infiltration into tumors. Further, these tumors were found to be more sensitive to PD-L1 blockade than wildtype tumors. We hypothesize that Socs1 KO causes chronic upregulation

of tumor cell IFNy signaling leading to cytokine-mediated infiltration of the TME which is followed by an increase in immune-inhibitory signal expression on tumor cells such as PD-L1. In Aim 1, we will investigate the influence of Socs1 KO on tumor immune-composition of the TME and tumor phenotype using Perturb-map and

transcriptomics. We will then corroborate these data with CITE-seq of human lung cancer specimens to further explore the role of SOCS1 in human malignancy. In Aim 2, we will use Perturb-map to KO all 162 ISGs and determine how each impacts tumor immunity in the context of immune checkpoint blockade therapy and TME

biology at depth. These experiments will include assessment of immune & stromal recruitment, tumor organization & differentiation, and unbiased analysis by spatial transcriptomics to identify mechanistic programs controlled by each ISG.