P2.03-19 Co-Targeting PIM Kinase to Overcome MET Amplified Resistance to EGFR TKIs in NSCLC

Currently there are five EGFR tyrosine kinase inhibitors (TKIs) (erlotinib, gefitinib, afatinib, dacomitinib, and osimertinib) available for treatment of EGFR-mutated non-small cell lung cancer (NSCLC). However for virtually all patients, resistance is inevitable, and disease progression occurs within 1 to 2 years of starting a TKI. Efforts to overcome resistance define the landscape of TKI research resulting in the development of second-generation and now third-generation agents and combination regimens. Third-generation agents, such as osimertinib, show improved response rates and extended median overall survival (OS), with potential to overcome previously untreatable resistance mechanisms. However acquired resistance mutations and activation of bypass RTK signalling mechanisms such as MET can mediate primary and secondary resistance to all EGFR TKIs. MET amplification has been observed after prolonged exposure of HCC827 cell lines to third-generation EGFR-TKIs (osimertinib or CNX-2006). We have pinpointed a novel strategic downstream target that plays a key role in MET regulation, cancer progression, drug resistance and immune evasion namely PIM kinase (PIM). The PIM family of serine/threonine kinases constitute three major isoforms namely PIM-1, 2 and 3 and have been shown to synergise with c-Myc. We have shown that all three PIM isoforms are highly expressed in NSCLC cell lines and patient tumors and hypothesise that co-targeting PIM kinase and EGFR may provide a more durable response to treatment and overcome MET amplified EGFR TKI resistance. Quantification & localisation of MET, c-MYC, PIM kinases and downstream substrates were examined by Western blot analysis and high content analysis (HCA) in EGFR TKI sensitive (HCC827P) and resistant (HCC827ER) cell lines and selected resistant clones (HCC827ER clone 3, HCC827ER clone 10). Efficacy of pan-PIM inhibitor (AZD1208) & novel PI3K/mTOR/PIM inhibitor (IBL-302) alone and in combination with erlotinib were quantified using the CellTiter-Blue, cell viability assay, in all cell lines. Effect of PIM inhibitors on intracellular signalling was quantified by PathScan® Intracellular Signaling Arrays. Activation of PIM-1, PIM-3 and c-MYC expression was demonstrated in MET amplified EGFR TKI resistant cells (HCC827ER) and (HCC827ER clone 3) compared to EGFR TKI sensitive cells (HCC827P). Erlotinib resistant clone that had undergone EMT (HCC827ER clone 10) had reduced expression of PIM-1, PIM-3 and c-MYC compared to MET amplified cells (HCC827ER clone 3). HCC827P and HCC827ER cells were both sensitive to AZD1208 (IC50 47.1μM versus 48.2μM). HCC827ER cells and HCC827ER clone3 cells were more sensitive than HCC827P cells to IBL-302 in a dose-response cell viability assay (IC50 0.277μM vs 0.253μM vs 0.351μM). IBL-302 inhibited downstream intracellular signalling at significantly lower concentrations than AZD1208 (250nM versus 2μM respectively). We show here for the first time that PIM kinase is activated in MET amplified EGFR TKI resistant cells. Erlotinib resistant HCC827ER cells are sensitive to pan-PIM inhibitor AZD1208 and the novel triple targeted therapy IBL-302. These data demonstrate that PIM kinase is a pivotal mechanism involved in EGFR TKI resistance and is an ideal target for dual inhibition strategies.