Abstract A43: Identifying a role for Ntrk1 in regulating immune functionality and checkpoint blockade resistance in Kras/p53 mutant lung cancer
Authors: Konen Jessica , Rodriguez Bertha L. , Gibson Laura , Minelli Rosalba , Kovacs Jeffrey , Carugo Alessandro , Bristow Christopher , Heffernan Timothy , Gibbons Don L. 
Published: June 2021

Lung cancer is the leading cause of cancer-related deaths in the United States, with the 5-year survival rate of all lung cancer patients combined at only 18%. The implementation of cancer immunotherapeutics for solid tumors such as lung cancers has shown great promise for improving clinical outcome. Immune checkpoint blocking antibodies targeting the PD-1/PD-L1 axis have been FDA approved for the treatment of late-stage metastatic disease, and clinical trial results indicate durable response in a small percentage of patients (~15-20%) compared to standard-of-care chemotherapy. However, the majority of patients show little to no response, or they acquire resistance during treatment with checkpoint inhibitors delivered as a monotherapy. Therefore, identifying resistance mechanisms and potential combination therapy strategies is imperative to improve response rates to immune checkpoint inhibitors and patient prognosis. To address this, we developed a clinically relevant in vivo shRNA screen that focuses on genes encoding for FDA-approved drug targets (FDAome). We implanted Kras/p53 (KP) mutant murine lung cancer cells expressing the FDAome shRNA library into syngeneic mice treated with isotype control or anti-PD-1 antibody. Sequencing for the unique barcoded shRNAs revealed that neurotrophic receptor tyrosine kinase 1 (Ntrk1) was significantly depleted from tumors challenged with PD-1 blockade, suggesting that it provides a survival advantage to tumor cells when under immune system pressure. To confirm Ntrk1 as a hit from the screen, we analyzed tumors treated with anti-PD-1 antibody and found that transcript levels of Ntrk1 are significantly upregulated with treatment, and cell lines derived from tumors with acquired resistance to anti-PD-1 treatment also have higher levels of Ntrk1 expression. Additionally, in vitro co-culture assays revealed that Ntrk1 overexpressing cells upregulate PD-L1 and other immunosuppressive markers when cultured with splenocytes, and this upregulation was inhibited through pharmacologic targeting of either Trk or Jak signaling. Stable knockdown of Ntrk1 in murine KP mutant lung cancer cells reduced Jak signaling and downregulation of PD-L1. In vivo, Ntrk1 knockdown tumors demonstrated significantly reduced tumor growth, and analysis of tumor-infiltrating T cell populations via flow cytometry revealed significantly fewer CD25+/FoxP3+ T regulatory cells in Ntrk1 knockdown tumors, as well as reduced exhaustion of CD8+ cytotoxic T cells. In contrast, Ntrk1 overexpressing tumors had a significant increase in tumor growth, as well as a hyperexhaustive phenotype in the intratumoral CD8+ T cells. Together, these data suggest that Ntrk1 activity can modulate Jak/Stat signaling to regulate PD-L1 expression as well as the expression of other immunosuppressive molecules, thereby promoting an exhausted and suppressed tumor microenvironment.

Citation Format: Jessica Konen, Bertha L. Rodriguez, Laura Gibson, Rosalba Minelli, Jeffrey Kovacs, Alessandro Carugo, Christopher Bristow, Timothy Heffernan, Don L. Gibbons. Identifying a role for Ntrk1 in regulating immune functionality and checkpoint blockade resistance in Kras/p53 mutant lung cancer [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 A43.