Abstract 5353: PIK3CA and PTEN mutations as drivers of osimertinib resistance in patients with NSCLC
Authors: Grazini Ursula , O'Neill Daniel J. , Martin Matthew , Xu Chintia , Floch Nicolas , Smith Paul D. , Cuomo Emanuela 
Published: June 2022

Abstract
Osimertinib is a third-generation EGFR-TKI approved for the first-line treatment of EGFR-driven lung adenocarcinomas. High response rates to osimertinib are observed in patients with activating EGFR mutations. However, there is variability in duration of response and patients eventually develop acquired resistance.Preclinical validation of potential acquired resistance mutations identified in relapsed patients treated with osimertinib is a key challenge to develop new strategies to improve outcomes for patients with EGFR mutation positive NSCLC. Tissue and ctDNA NGS analysis from relapsed patients treated first-line with osimertinib in the FLAURA and ORCHARD trials identified PIK3CA gain of function (GOF 11%) and PTEN loss of function (LOF 5%) mutations.To test whether PIK3CA activating mutations can drive resistance to osimertinib we introduced PIK3CA activating variants (PIK3CA-H1047R and PIK3CA-E453K) or knocked-out PTEN in NSCLC lung cancer cell lines harbouring EGFR activating mutations and WT PIK3CA/PTEN using CRISPR/Cas9 technology. Our results show that PIK3CA GOF and PTEN LOF mutations conferred resistance to osimertinib in vitro. Resistance was associated with increased EC50 for osimertinib and diminished apoptotic response in NSCLC PIK3CA GOF and PTEN LOF mutant CRISPR cell lines when compared to parental cells. Protein analysis by western blot also showed an increase in the basal and osimertinib treated levels of pAKT and pS6 in PIK3CA/PTEN CRISPR engineered cell lines, indicating activation of downstream PI3K/AKT and MAPK signalling pathways in the osimertinib-resistant cells. Importantly, our in vitro experiments from multiple cell line models indicated that PIK3CA-mediated resistance to osimertinib could be partially reversed by co-treatment with AKT (capivasertib, AstraZeneca) and PI3K alpha (alpelisib, Novartis) inhibitors; PTEN-mediated resistance could be rescued by co-treatment with capivasertib and PI3K beta (AZD8186, AstraZeneca) inhibitors. We next examined the in vivo efficacy of osimertinib + AKT/PI3K inhibitors therapies in NSCLC xenograft models. Our in vivo analysis show that PIK3CA GOF CRISPR engineered cells displayed diminished response to osimertinib when implanted in mice; importantly, combination with AKT (capivasertib) or PI3K alpha (alpelisib) inhibitors enhanced response on treatment and delayed outgrowth after withdrawal treatment. In addition, we observed that combination treatment with capivasertib induced tumour stasis in a PIK3CA-E454K and two PTENloss PDX models.Altogether our in vitro and in vivo data provide evidence of PIK3CA mutants and PTEN loss-driven mechanisms of resistance to osimertinib and offer possible therapeutic combination strategies for those patients that develop resistance or experience a sub-optimal response to osimertinib through PIK3CA/PTEN mutations
Citation Format: Ursula Grazini, Daniel J. O'Neill, Matthew Martin, Chintia Xu, Nicolas Floch, Paul D. Smith, Emanuela Cuomo. PIK3CA and PTEN mutations as drivers of osimertinib resistance in patients with NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5353.