Abstract 744: Transactivation of HER3 via heterodimerization with epidermal growth factor receptor (EGFR) or insulin-like growth factor 1 receptor (IGF-1R) contributes to adaptive resistance to NVP-BKM120 in non-small cell lung cancer (NSCLC) and squamous cell carcinom

Purpose: To investigate adaptive resistant mechanisms to NVP-BKM120, a pan PI3K inhibitor, in NSCLC and SCCHN cells. Experimental Design: A panel of 16 NSCLC and 11 SCCHN cell lines, including PDX01 cell line developed from a biopsy sample of SCCHN patient showing resistance to NVP-BKM120, was used. The growth inhibitory effect of NVP-BKM120 was assessed using MTT assay and colony formation assays. Receptor tyrosine kinases (RTKs) array was used to identify candidate RTKs associated with adaptive resistance to NVP-BKM120. Signaling pathways were analyzed by immunoprecipitation, western blot, and siRNAs transfection. Results: Treatment of NVP-BKM120 showed the limited antitumor effects with IC50 of 0.5 to 1.5 μM in a panel of NSCLC and SCCHN cells. The sensitivity to BKM120 was not associated with genetic alterations of PI3K pathway (e.g. PIK3CA mutation). Although AKT phosphorylation decreased markedly upon NVP-BKM120 treatment, AKT activity was rapidly restored following 4 hrs of drug treatment. Moreover, using western blot analysis and RTK array, upregulation of HER3 expression and activity from 1 hr up to 72 hrs of NVP-BKM120 treatment was observed in the majority of NSCLC and SCCHN cells. The knockdown of HER3 with NVP-BKM120 resulted in a synergistic antitumor effect with marked suppression of NVP-BKM120-triggered AKT reactivation. Interestingly, the phosphorylation of kinase-inactive HER3 at 4 hrs of NVP-BKM120 treatment was completely suppressed by gefitinib, a selective EGFR TKI, or EGFR knockdown while gefitinib or EGFR knockdown failed to inhibit HER3 phosphorylation at 24 hrs of NVP-BKM120 treatment. In line with these results, immunoprecipitation assays demonstrated the NVP-BKM120-enhanced interaction of HER3 and EGFR only at 4 hrs with recruitment of p85 and c-SRC, leading to the transient phosphorylation of EGFR, specifically at Y845 and Y1173 sites. These results suggested that HER3 activation was driven by dimerization with other RTKs at later time points after drug treatment. In addition, OSI-906, a selective inhibitor of IGF-1R, and IGF-1R-specific siRNA also reduced NVP-BKM120-induced HER3 phosphorylation, resulting in a synergistic antitumor effect in a panel of NVP-BKM120 resistant cells. Conclusions: Transactivation of HER3 via EGFR or IGF-1R attenuates the effectiveness of NVP-BKM120 in multiple NSCLC and SCCHN cells. Our results suggest that either targeting HER3 directly or indirectly by inhibiting transactivation partners, such as