Abstract 7206: Inhibition of CAD, an enzyme of de novo pyrimidine synthesis, to overcome EGFR-TKI resistance in NSCLC

Abstract

Abstract Given the fast-growing cancer cells, there is an increased demand for large amounts of energy and nucleotides for DNA and RNA synthesis. Thus, targeting nucleotide metabolism, specifically de novo nucleotide biosynthesis, is a promising strategy for cancer treatment. Hyperactivation of Carbamoyl-phosphate synthetase, aspartate transcarbamylase (CAD), a key enzyme catalyzing the first three steps of de novo pyrimidine synthesis, is a common event in tumors and has been associated with chemoresistance in tumors. Our analysis, using TCGA data, revealed that increased expression of CAD is associated with poor prognosis in non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have demonstrated significant benefits to patients with NSCLC harboring EGFR-activating mutations; however, acquired resistance limits their long-term efficacy. Therefore, it remains urgent to discover the underlying mechanisms for discovering novel therapeutic strategies for overcoming TKI resistance. Our previous findings demonstrated that blockade of S6K1 overcomes acquired resistance to EGFR-TKIs in NSCLC. It was reported that S6K1 is involved in de novo pyrimidine biosynthesis. In this study, we aimed to investigate whether CAD mediates S6K1-induced EGFR-TKI resistance in NSCLC. Immunohistochemistry analysis on a tissue microarray of lung adenocarcinoma tissues indicated that increased co-expression of p-CAD and S6K1 proteins is associated with clinicopathological parameters and poor survival outcomes. Our in vitro results showed an increased expression level of CAD and p-CAD proteins in TKI-resistant cells compared with parental cells upon TKI treatment. Knockdown of CAD by siRNA led to sensitizing TKI-resistant cells to osimertinib, as indicated by BrdU cell proliferation assay. Inhibition of S6K1 by a siRNA or specific inhibitors PF-470867 and LY2584702 effectively reduced the phosphorylation levels of CAD protein in TKI-resistant cells, suggesting that S6K1 may serve as an upstream regulator of CAD for TKI resistance. The String database predicted a protein-protein interaction between S6K1 and CAD. Indeed, our co-immunoprecipitation (co-IP) results revealed a direct association between S6K1 and CAD proteins. Importantly, TKI treatment enhanced the binding between S6K1 and CAD. Taken together, these findings provide evidence that CAD may mediate S6K1-mediated EGFR-TKI resistance in NSCLC. Our ongoing and future work will assess whether inhibition of CAD sensitizes resistant cells to TKI in vivo and elucidate the associated mechanism. Citation Format: Leili Saeednejad Zanjani, Lei Zhao, Shaomin Wang, Margot Federoff, Xin Sun, Jun He. Inhibition of CAD, an enzyme of de novo pyrimidine synthesis, to overcome EGFR-TKI resistance in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7206.