Abstract 5354: 3-phosphoinositide-dependent kinase-1 (PDK1, PDPK1) is a driver of osimertinib acquired resistance in EGFR mutant NSCLC
Authors: Meraz Ismail M. , Majidi Mourad , Fang Bingliang , Meng Feng , Gao Lihui , Shao RuPing , Song Renduo , Li Feng , Ha Min Jin , Wang Qi , Wang Jing , Shpall Elizabeth , Jung Sung Yun , Haderk Franziska , Gui Philippe , Riess Jonathan W. , Olivas Victor , Bivona Trever G. , Roth Jack A. 
Published: June 2022

Abstract
Osimertinib, the only third-generation EGFR-TKI, showed incomplete responses to T790M-mutant NSCLC due to acquired resistance caused by activation of bypass pathways. We developed osimertinib-acquired resistant H1975-OSIR (T790M/L858R mutant) isogenic cells and TC386-OSIR isogenic PDXs. Neither H1975-OSIR nor TC386-OSIR PDXs developed additional mutations in EGFR. The H1975-OSIR clone showed 100 fold higher resistance to osimertinib compared with H1975 cells. TC386-OSIR PDX was developed through continuous in-vivo treatment for 8 months and the residual PDXs were passaged for several generations under continuous osimertinib treatment. TC386-OSIR fourth resistant generation (RG4) showed significantly higher resistance than initial generations (RG1). H1975-OSIR xenografts were developed in non-humanized and humanized NSG mice under osimertinib pressure. H1975-OsiR tumors were significantly less sensitive to osimertinib than their parental counterparts in both mouse models. Dose dependent antitumor activity of osimertinib (5mg/kg and 10mg/kg) was observed in H1975-parental tumors, whereas no treatment effect was observed for H1975-OsiR tumors with increasing doses. The tumor microenvironment was enriched with higher infiltration of tumor associated macrophages (TAM) and lower numbers of tumor infiltrating lymphocytes (TIL) in H1975-OSIR vs H1975 tumors. RPPA analysis of residual tumor tissues showed a distinct set of proteins upregulated in H1975-OsiR vs H1975-parental, among which PDK1 was the most upregulated. PDK1 was also significantly upregulated in H1975-OsiR tumors treated with osimertinib vs controls. PDK1 was not altered in any treatment groups in H1975-parental tumors. PDK1 and pPDK1 expression was many-fold higher in both H1975-OSIR cells and TC386-OSIR PDXs as compared to their parental counterparts by western blot and mass spec proteomics. Selective inhibition by the PDK inhibitor, BX 795, and CRISPR knock-out (KO) restored osimertinib sensitivity in resistant cells. Colony forming assays showed that the PDK1 KO clone was as sensitive as H1975-parental cells whereas a PDK overexpressing clone (OE) restored resistance. In-vivo inhibition of PDK1 by treating mice with BX-795 in both H1975-OSIR xenografts and TC386-OSIR PDXs significantly enhanced the antitumor activity of osimertinib. PDK1 KO dysregulated PI3K/Akt/mTOR signaling by downregulating Akt and mTOR phosphorylation and promoted cell cycle arrest at the G1 phase. NCI-H1975-OSIR and PDK1 OE cells showed a high level of nuclear localization of the activated Yes-associated protein pYAP(Y357). PDK1 KO cells significantly reduced nuclear localization of pYAP(Y357). The level of YAP and pYAP was upregulated in osimertinib resistant xenograft tumors and residual tumor biopsies. Taken together, we identified PDK1 as a drug able target to treat osimertinib acquired resistance.
Citation Format: Ismail M. Meraz, Mourad Majidi, Bingliang Fang, Feng Meng, Lihui Gao, RuPing Shao, Renduo Song, Feng Li, Min Jin Ha, Qi Wang, Jing Wang, Elizabeth Shpall, Sung Yun Jung, Franziska Haderk, Philippe Gui, Jonathan W. Riess, Victor Olivas, Trever G. Bivona, Jack A. Roth. 3-phosphoinositide-dependent kinase-1 (PDK1, PDPK1) is a driver of osimertinib acquired resistance in EGFR mutant 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 5354.