Overcoming Resistance To Osimertinib By Tusc2 Gene Therapy In Egfr Mutant Nsclc.

Abstract Osimertinib, the only third-generation EGFR-TKI approved for the treatment of T790M-mutant non-small cell lung cancer, shows robust clinical activity, yet patients inevitably develop secondary resistance. TUSC2, an immunogene therapy, has multifunctional activity, which a) directly blocks downstream signaling through inhibiting MAPK and mTOR, b) arrests growth and proliferation of cancer cells, c) induces direct tumor cell death, and d) activates both innate and adaptive immunity. An osimertinib resistant H1975-OsiR isogenic cell line was developed through continuous exposure to osimertinib, and an osimertinib resistant clone was selected which showed 100 fold higher resistance to osimertinib compared with its parental counterpart (H1975-parental). Xenograft tumors from both H1975-parental and H1975-OsiR cells were developed in NSG mice and were treated with osimertinib. H1975-OsiR tumors were significantly less sensitive than their parental counterparts. To maintain the resistance in H1975-OsiR, the cells were cultured in the presence of osimertinib, and the osimertinib pressure was maintained in vivo throughout the experiments. Synergistic antitumor activity of TUSC2+osimertinib was found in H1975-OsiR tumors where both TUSC2+osimertinib (5mg/kg) and TUSC2+osimertinib (10mg/kg) combinations showed a robust antitumor effect compared with single agent treatment groups. No synergistic effect was observed for H1975-parental tumors. RPPA analysis of residual tumors showed a distinct set of proteins including CD44, VEGFR-2, SHP2, Akt2, and YAP-pS127 overexpressed in H1975-OsiR vs H1975-parental. Among H1975-OsiR tumors, RPPA data showed that PDK1 protein was significantly altered in osimertinib treated groups as compared with controls. PDK1 was also found to be significantly upregulated in the TUSC2+osimertinib group when compared with either control, osimertinib alone, or TUSC2 alone treated H1975-OsiR tumors indicating that PDK1 may be associated with osimertinib resistance. PDK1 was not altered by TUSC2 alone treatment. PDK1 was not altered in any treatment groups in H1975-parental tumors. In order to validate the role of PDK1 in H1975-OsiR, we performed XTT assays of osimertinib and TUSC2+osimertinib combinations in the presence or absence of a PDK1 inhibitor (BX-795). Only the osimertinib and TUSC2+osimertinib groups showed significantly increased sensitivity to osimertinib in the presence of BX-795 as compared with the same treatment without the inhibitor. No PDK1 inhibitor effect was found in the TUSC2 treated group validating the specific role of PDK1 in osimertinib resistance. In conclusion, TUSC2 therapy in combination with osimertinib showed synergistic antitumor efficacy in EGFR mutant osimertinib resistant NSCLC tumors, and upregulation of PDK1 was associated with osimertinib resistance. Citation Format: Ismail M. Meraz, Mourad Majidi, RuPing Shao, Lihui Gao, Meng Feng, Huiqin Chen, Min Jin Ha, Jack A. Roth. Overcoming resistance to osimertinib by TUSC2 gene therapy in EGFR mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1105.