P2.03-26 Elucidating Mechanisms of Resistance to Targeted Therapies in Mutant EGFR or KRAS Driven Lung Adenocarcinoma Harboring Dual Loss of p53 and RB1

Abstract

Inactivation of the two canonical tumor suppressors, p53 and RB1, is a genetic hallmark of small-cell lung cancer (SCLC). In contrast, lung adenocarcinomas (LUADs) preferentially harbor alterations in the p16 pathway over RB1. Nonetheless, despite being rare, concurrent loss of p53 and RB1 occurs in a subset of LUADs and this is hypothesized to be necessary for the histological transformation of LUAD to SCLC, observed during treatment with tyrosine kinase inhibitors (TKIs). However, whether the dual loss of p53 and RB1 is sufficient for this histological transformation remains unknown. Furthermore, loss of RB1 in LUADs with EGFR mutations is associated with poor response to TKIs in the absence of SCLC transformation. Here, we aimed to explore how loss of p53/RB1 affects the biology of p16 pathway-altered LUAD, particularly in the context of acquired resistance mechanisms to targeted therapies. Four TP53-mutated LUAD cell lines were used: two EGFR mutation-positive (PC9 and H1975) and two KRAS mutation-positive (H1792 and H358). All these cell lines possess p16 pathway alterations: p16 (CDKN2A) mutations in PC9 and H1975, CDK4 amplification in H1792, and silenced p16 in H358. Inactivation of RB1 was carried out using CRISPR-Cas9 and RB1 knockout monoclonal cells were established. Cell proliferative and clonogenic abilities were assessed. In addition, osimertinib-resistant (PC9 and H1975) and trametinib-resistant (H1792 and H358) cells were generated (initial high dose and/or stepwise dose escalation methods). Acquired resistance mechanisms were evaluated by MSK-IMPACT profiling. Two RB1 knockout clones were established for each cell line. No advantageous effects were observed for proliferative and clonogenic abilities after RB1 knockout. Although loss of p53 and RB1 has been reported to result in lineage shift in prostate cancer through the upregulation of SOX2, deregulation of SOX2 expression was not observed upon RB1 knockout in the TP53-mutant LUAD cells. In addition, although loss of RB1 caused a modest reduction in osimertinib and trametinib sensitivities of H1975 and H358 cells, respectively, no effect was observed in PC9 and H1792 cells. After becoming resistant to osimertinib or trametinib, transformation to SCLC was not observed. Individual resistance mechanisms are currently being assessed by MSK-IMPACT. Dual loss of p53 and RB1 caused no advantageous effects in TP53-mutated and p16 pathway-altered LUAD cells and did not initiate transformation to SCLC as a resistance mechanism to targeted therapies. TP53/RB1 deficiency-related acquired resistance mechanisms to osimertinib or trametinib will be further explored and presented.