Abstract 3919: SHP2-DDX3X complexes contribute to translational control of CD274 mRNA in KRAS-active non-small cell lung cancer

Abstract

Abstract Background: Immune checkpoint inhibitors (ICIs) have shifted the landscape in management for non-small cell lung cancer (NSCLC) and improve treatment outcomes. However, the 5-year overall survival rate of 23.2% for treatment-naïve patients with late-stage NSCLC on Pembrolizumab therapy can likely be improved. Clinical benefit is correlated with tumoral expression of PD-L1 and mutation burden. Many groups seek to identify other therapeutic targets that could improve response and overall survival. Ongoing work from our group demonstrated that SHP2, a tyrosine phosphatase, can negatively regulate PD-L1 expression in KRAS-active NSCLC, and we seek to understand the mechanism by which this occurs. SHP2 was ectopically expressed in H460 cells, immunoprecipitated and identification of interacting proteins was achieved by mass spectrometry. DDX3X, an RNA helicase, and eukaryotic translation initiation factor 1a (eIF-1a) were identified as high-ranking, potential partners of SHP2. We hypothesized that SHP2 complexes with DDX3X and eIF-1a on newly transcribed CD274 mRNA to negatively regulate PD-L1 expression. Materials and Methods: To confirm SHP2 interactions with DDX3X and eIF-1a, two pull-down assays were performed: 1. bacterially-expressed, his-tagged and immobilized proteins was used as bait for proteins contained in A549 and H460 extracts, and 2. immunoprecipitation of each endogenous protein in A549 and H460 cell extracts. Western blot analysis was performed to evaluate interacting proteins. To determine whether phosphatase activity of SHP2 is required for these interactions, immunoprecipitation was also carried out in the presence of allosteric inhibitors of SHP2 (SHP-099 and RMC-0455). SHP2 ablation was carried out with CRISPR and siRNA to examine PD-L1 expression, also determined by western blot assay. Results: His-tagged DDX3X from bacterial expression systems was able to capture SHP2 protein from both A549 cells and H460 cells, and vice versa with his-tagged SHP2. Immunoprecipitation of endogenous SHP2 was able to co-precipitate DDX3X and vice versa. Phosphatase activity of SHP2 was not required for complex formation nor does it change PD-L1 expression in A549 or H460 cells. Ablation of SHP2 leads to increased PD-L1 expression. Conclusion: A complex containing SHP2 and DDX3X may regulate the translation of CD274 in concert with eukaryotic initiation factors. Other groups have previously reported the control of translation of CD274 mRNA carried out at the 3’ UTR, and we believe this is novel evidence of a translation initiation complex monitoring the expression of CD274. Ribosome footprint and RNA immunoprecipitations are underway to address these questions. Citation Format: Palapoom Wongsaengpiboon. SHP2-DDX3X complexes contribute to translational control of CD274 mRNA in KRAS-active non-small cell lung cancer. [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 3919.