CARD8/BCL-2 Cascade in Early Adaptive Drug Resistant Tumor Escape Against Targeted Inhibitors in NSCLC.

e22032 Background: Lung cancer targeted therapy is largely limited by inevitable recurrent resistant disease after initial response to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), typically accompanied with divergent late acquired resistance mechanisms. We now focused on studying the emergence of early adaptive resistance to uncover attractive therapeutic targets to overcome drug resistance. Methods: HCC827 cells were treated with EGFR-TKI (0-9 days) with apoptosis pathway-specific QPCR array and TLVM analysis performed. MTS and crystal violet assays were performed. Western blot analysis was performed to examine prosurvival signaling developed against erlotinib, alone or in combination with MET inhibitor SU11274. IHC was performed on lung cancer tumor microarray (TMA) using BCL-2 and caspase-recruitment domain-containing protein 8 (CARD8) antibodies and graded (4 tier scoring system). NSCLC cell lines and murine xenograft models (HCC827, H1975) were developed for resistance biomarkers expression analysis in pre-/post-TKI treatment using anti-human CARD8, p-STAT3 and BCL-2 antibodies. Results: We characterized the emergence of early resistant lung cancer cells in escape against targeted TKIs with 100-fold higher IC50 in adaptive drug resistance. The resistant cells that evaded EGFR-TKI based targeted inhibition exhibited MET-independent induction of CARD8 and STAT3/BCL-2 mitochondrial prosurvival signaling in cellular quiescence, and inhibited cytoskeletal functions. Expression analysis studies demonstrated common tumor-associated expression of CARD8 but relatively low BCL-2 level in NSCLC. In vitro cell line studies suggest that CARD8 induction was preceded by a resurgence of STAT3 activation. In vivo xenograft model (HCC827/erlotinib; H1975/erlotinib+ SU11274) also verified upregulated CARD8/BCL-2 activation within early resistant cells. Conclusions: Resistant tumor cells that evaded EGFR inhibitors, alone or in combination with MET inhibitors, exhibited increased expression of CARD8-STAT3/BCL-2 prosurvival signaling cascade. Further studies to define the mechanism of CARD8 in promoting adaptive tumor drug resistance would be warranted.