Squamous cell carcinoma (SCC) is a subtype of non-small cell lung cancer (NSCLC) that often resists treatment, resulting in poor prognosis for the patient. Immunotherapies, including those targeting the PD1/PD-L1 immune checkpoint, have become popular treatment options, but they are effective for only 20% of squamous lung cancer patients. The likely cause of differential responses is tumor heterogeneity. This heterogeneity can present in the tumor cells, for example, if more or fewer tumor cells express PD-L1, and also in the microenvironment, for example, if the tumor is infiltrated by more immune-suppressive neutrophils or more immune-activating CD8+ T cells. In order to study SCC in immune-intact mice, our laboratory utilizes a mouse model whereby tumors are induced through biallelic deletion of Lkb1 (aka Stk11) and Pten in the mouse lung. These SCCs express PD-L1 at high levels on the surface of the tumor cells; however, treatment with anti-PD1 antibody caused only a delay in tumor growth, not a reduction. We believe the ineffectiveness of immunotherapy in this model is due to the presence of large populations of tumor-associated neutrophils (TANs). TANs have been shown to produce Arginase1, which has repressive effects on the T-cell populations by inhibiting proper signaling and activation. An inverse relationship between TAN and CD8+ T cell abundance has also been reported for squamous lung cancers. Finding a second therapy that can prime squamous lung cancers to respond to immunotherapy would be beneficial for a large number of patients. EZH2 is a histone methyl transferase that promotes differentiation of cells via lineage-specific gene repression. We hypothesize that using an anti-PD-1 immunotherapeutic in combination with an EZH2 inhibitor will increase the PD-L1 expression on the tumor cells, decrease the normal homing or T-cell suppression of neutrophil populations, and increase the efficacy of the immunotherapy. In order to test our hypothesis, we have treated lung cancer cells with EZH2 inhibitor both in vitro and in vivo. In vitro, treatment of lung cancer cell lines or patient-derived organoids of squamous lung cancers caused increased expression of PD-L1. In mice harboring Lkb1/Pten tumors, treatment with EZH2 inhibitor increased the proportion of tumor cells expressing PD-L1 but did not prevent the tumor from growing. In contrast, the mouse treated with EZH2 inhibitor and anti-PD-1 immunotherapy had tumor regression that was durable up to 8 weeks post-treatment initiation. We isolated TANs from untreated tumors and observed that they express very high levels of Arginase 1. Lung neutrophils isolated from EZH2 conditional knockout mice had significantly lower levels of Arginase 1 than WT lung neutrophils or Lkb1/Pten TANs. Together these data suggest that EZH2 inhibition will be an effective strategy to both increase the PD-L1+ tumor cell population, and to decrease TAN-mediate T cell suppression by decreasing TAN-derived Arginase 1 expression.

Citation Format: Tanner J. DuCote, Xiulong Song, Christine F. Brainson. Impact of EZH2 inhibition on the immune microenvironment of squamous lung cancers [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B42.