Abstract 4650: High intratumoral levels of Notch3 increase tumorigenesis and promote an immunosuppressive TME in EGFR-mutant NSCLC
Authors: Suman Shankar , Kaufman Jacob , Arasada Rajeswara , Varikuti Sanjay , Navari Nastaran , Guan Shuxiao , Amann Joseph , Dikov Mikhail , Carbone David , Memmott Regan 
Published: April 2023

Abstract
Activating mutations in the Epidermal Growth Factor Receptor (EGFR) are one of the most common driver mutations in non-small cell lung cancer (NSCLC). They are present in the tumors of ~15% of Caucasian patients and in up to 50% of Asian patients. Notch3 is a member of a family of transmembrane receptors that has been shown to increase cell proliferation and inhibit cell death and differentiation. Notch3 also promotes the formation of cancer cells with stem-like properties. Studies have shown that a high level of Notch3 in tumors is associated with poor prognosis of patients with NSCLC. However, its role in EGFR-mutant NSCLC tumorigenesis has not been specifically evaluated. We analyzed the TCGA, Moffit, and ORIEN datasets, which include tumor samples from patients with EGFR-mutant lung cancer. We found that high levels of Notch3 were associated with decreased overall survival (HR=2.0, p=0.04). Interestingly, analysis of two additional datasets derived from the phase 3 clinical trials OAK and POPLAR showed that patients whose EGFR-mutant tumors had the highest levels of Notch3 had worse clinical outcomes when treated with immunotherapy (HR 18.6, p=4e-04) but not chemotherapy. This could suggest that Notch3 promotes EGFR-mutant lung tumorigenesis by creating an immunosuppressive tumor microenvironment (TME). To test this, we generated a novel mouse model, c/EGFRL858RNotch3, that expresses tetracycline-inducible Notch3 and mutant EGFR under the control of the Clara cell-secretory protein (CCSP) promoter. CT-based imaging showed that mice whose tumors harbored high levels of Notch3 had significantly increased lung tumor burden at eight weeks. These mice also had worse overall survival (HR 2.3, p=0.04). To determine if increased Notch3 expression in tumors promotes a more immunosuppressive TME, we did immunophenotyping analysis that revealed a significant increase in regulatory T cells and a decrease in cytotoxic CD8+T cells. Furthermore, T cells in the TME expressed higher levels of markers of exhaustion, specifically, PD-1 and Tim-3. We also found that mice whose tumors had high levels of Notch3 had a greater density of immunosuppressive macrophages in the TME, which was associated with elevated levels of TNF-α. Overall, our data suggest that patients with EGFR-mutant NSCLC whose tumors harbor high levels of Notch3 may have worse clinical outcomes and response to immune checkpoint inhibitors due to Notch3 promoting an immunosuppressive TME.
Citation Format: Shankar Suman, Jacob Kaufman, Rajeswara Arasada, Sanjay Varikuti, Nastaran Navari, Shuxiao Guan, Joseph Amann, Mikhail Dikov, David Carbone, Regan Memmott. High intratumoral levels of Notch3 increase tumorigenesis and promote an immunosuppressive TME in EGFR-mutant NSCLC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4650.