Abstract 3716: Overexpression of the adaptor protein CRKL as a mechanism of acquired resistance to Crizotinib in ALK-positive NSCLC

Abstract

Abstract Background: The fusion of the anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) is a known oncogenic driver in a subset of non-small cell lung cancer (NSCLC). An ALK/ROS1/MET kinase inhibitor, Crizotinib, was approved in 2011 for the treatment of patients with metastatic ALK+ NSCLC. Despite the dramatic clinical effects of Crizotinib in EML4-ALK positive NSCLC, vast majority of patients subsequently acquire resistance and new therapeutic strategies are needed. Methods and Results: We performed a kinase (n=597) open reading frame screen to identify kinases and phosphoproteins capable of circumventing ALK inhibition in H3122 cell line sensitive to the ALK kinase inhibitor TAE684. The top hit of the screen imparting resistance to TAE684 was the adaptor phospho-protein CRK-like (CRKL), a known substrate of BCR-ABL. To validate our screen, we constructed a tagged form of CRKL and stably expressed it in H3122. H3122CRKL acquired a migratory phenotype, however, its growth rate was comparable to that of the parental cell line. The viability of H3122CRKL was assessed in response to drug dose escalation, confirming that overexpression of CRKL decreased sensitivity of H3122 to Crizotinib 47-fold compared to the control. Analysis of downstream signaling in response to Crizotinib treatment further revealed sustained activation of PI3K/AKT and MAPK/ERK1/2 pathways in CRKL overexpressing cells. Closer examination of signaling in H3122CRKL revealed additional signaling alterations. Most significantly, we found that overexpressing CRKL in H3122 resulted in activation of EGFR. We and others have previously demonstrated that EGFR signaling can mediate Crizotinib resistance. We further examined primary tumor cell lines (n=6), generated from ALK rearranged NSCLC patients who developed resistance to Crizotinib, and 4 demonstrated increased levels of both CRKL and phosphorylated EGFR. To determine if EGFR inhibition could be a therapeutic approach to overcome Crizotinib resistance in CRKL overexpressing cells, cell viability was assessed in response to dual treatment with Crizotinib and Gefitinib. The drug combination displayed robust synergistic effects on growth inhibition of ALK+ CRKL overexpressing cell lines but not of CRKLlow Crizotinib sensitive H3122, highlighting their co-dependence on EGFR. Finally, activation of FAK and SRC family kinases correlated with EGFR activation and CRKL overexpression in H3122CRKL, an observation further corroborated by a 9-fold increase in migratory capacity of H3122CRKL vs. control. Conclusion: Our findings indicate that CRKL overexpression can lead to acquired resistance to ALK inhibition in ALK rearranged NSCLC. CRKL overexpression results in EGFR activation suggesting that therapy directed at both EGFR and EML4-ALK may be effective in treating and/or preventing acquired resistance to Crizotinib. Citation Format: Magda Bahcall, Takaaki Sasaki, Atsuko Ogino, Marzia Capelletti, Mohit Butaney, Pasi A. Jänne. Overexpression of the adaptor protein CRKL as a mechanism of acquired resistance to Crizotinib in ALK-positive NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3716. doi:10.1158/1538-7445.AM2014-3716