PO-505 Targeting PIM kinase to overcome resistance to PI3K-mTOR inhibition in NSCLC

Abstract

IntroductionNon-small cell lung cancer (NSCLC) is the leading cause of cancer mortality globally, having a 5 year survival rate of less than 15%. PI3K-mTOR signalling has been implicated in various hallmarks of cancer and is frequently dysregulated in NSCLC. Efforts to therapeutically target the PI3K-mTOR pathway have been hampered by the inevitable emergence of drug resistance inhibiting a durable response to treatment. Our group developed NSCLC cell line models of acquired resistance to PI3K-mTOR inhibitor GDC-0980. Resistant cells (H1975GR) were also less sensitive to PI3K-mTOR dual targeting inhibitor, BEZ235 compared to matched parent cells (H1975P) making them an ideal model to identify and interrogate drug resistance mechanisims.Material and methodsThe sensitivity of GDC-0980 resistant cells (H1975GR) versus age-matched parent cells (H1975P) to BEZ235 following a 72 hour treatment was compared using a Cell Titre Blue cell viability assay (n=3). Alterations to the mRNA expression profile of H1975GR versus H1975P were examined using an IL-6/STAT3 signalling-specific RT2 gene profiler array (n=1). Selected genes from the array were validated by SYBR-based qPCR, immunofluorescence (IF) and western blot analysis (n=3–4). 11 miRNAs (regulated by or regulators of c-Myc/PIM) plus housekeeping control miRNAs were quantified in the H1975P/GR model by QPCR. IC50 values of pan-PIM kinase inhibitors AZD1208, IBL101 and novel PI3K/mTOR/PIM inhibitor IBL301 inhibiting growth in H1975P versus H1975GR were compared using the BrdU assay. The effect of these drugs on RTK and c-Myc expression were examined in H1975GR by IF and western blot analysis.Results and discussionsIn-depth characterisation of the H1975GR cells identified activation of several receptor tyrosine kinases (RTKs) including EGFR, IGF1R, EPHA2 and members of the IL-6/STAT3 pathway including c-Myc and PIM1, PIM2 and PIM3 kinases. IL-6/STAT3 overexpression in cancer stimulates angiogenesis, migration, invasiveness, cytokine signalling and notably drug resistance. A miRNA signature of PI3K/mTOR inhibitor resistance was validated which can be used to monitor patients on treatment. IC50 values of IBL301 were significantly less for H1975GR cells (0.02 uM) compared to H1975P cells (0.75 uM) highlighting activated PIM kinase as a mechanism of resistance to PI3K-mTOR inhibition. Targeting PIM kinase in H1975GR cells inhibits activated c-Myc and RTKs.ConclusionDual targeting of both PIM and PI3K-mTOR may provide a more durable response to treatment for patients with NSCLC.