[3:15 PM] O33.05 | Anaplastic Lymphoma Kinase (ALK) dependent and co-dependent mechanisms of resistance to ALK kinase inhibitors in EML4-ALK non-small cell lung cancer
From abstracts in the World Lung Cancer Conference in July 2011
T. Sasaki1, J. Koivunen2, A. Ogino3, K. Okuda3, M. Yanagita3, M. Cappelletti3, K. Wilner4, J. Christensen4, M. Eck3, N. Gray3, P.A. Janne5
1Asahikawa Medical University Hospital/JAPAN, 2Oulu University Hospital/FINLAND, 3Dana-Farber Cancer Institute/UNITED STATES OF AMERICA, 4Pfizer Global Research and Development/UNITED STATES OF AMERICA, 5Dana Farber Cancer Institute/UNITED STATES OF AMERICA
Background:
Lung cancers harboring anaplastic lymphoma kinase (ALK) rearrangements represent a unique subpopulation of patients. Crizotinib, an ALK tyrosine kinase inhibitor (TKI) is clinically effective in this genomically defined patient subset. However, the clinical success of treatment with ALK-TKIs is often limited by the development of acquired drug resistance. In this study, we examined resistance mechanisms to ALK-TKIs. The understanding of resistance mechanism(s) is critical to the development of effective subsequent treatments.
Methods:
We examined tumor biopsies from patients treated with crizotinib that had developed acquired resistance to crizotinib. We also generated an ALK-TKI resistant H3122 cell (H3122TR3) by exposing the increasing dose of TAE684. Finally we generated a cell line (DFCI076) from a pleural effusion of a NSCLC patient that developed acquired resistance to crizotinib. We examined whether genomic changes (mutations or copy number changes) were present in the ALK TKI resistant tumors and cells. In addition, we evaluated for the presence of co-activated signaling pathways.
Results:
We identified a secondary mutation in ALK, F1174L (also detected in neuroblastoma), in a patient with an inflammatory myofibroblastic tumor (IMT) harboring a RANBP2-ALK translocation who progressed while on crizotinib therapy. When present in cis with an ALK translocation, the F1174L mutation causes an increase in ALK phosphorylation, cell growth and downstream signaling. This mutation likely promotes the active conformation of ALK which disfavors crizotinib binding as it binds the inactive conformation of ALK. Treatment with a structurally distinct ALK inhibitor, TAE684, or with 3-fold above the current efficacious concentrations of crizotinib was sufficient to inhibit the growth of Ba/F3 cells harboring the F1174L mutation. In contrast the DFCI076 cells (EML4-ALK), were resistant to both crizotinib and TAE684. These cells contained a novel L1152R ALK secondary mutation. Ba/F3 cells with EML4-ALK L1152R were also resistant to crizotinib and the L1152R mutation diminished crizotinib mediated inhibition of downstream phosphorylation. In addition, we determined that the DFCI076 cells had evidence of significant Epidermal Growth Factor Receptor (EGFR) phosphorylation. Infection of an ALK shRNA inhibited the growth of DFCI076 cells to 40% of control; however the combination of ALK knockdown and PF00299804, an irreversible pan-ERBB inhibitor, led to further growth inhibition (19% of control). The H3122TR3 cells were resistant in vitro to both TAE684 and crizotinib but did not contain a secondary ALK mutation. However, the H3122TR3 cells contained activation of EGFR signaling mediated by autocrine EGF production. The combination of crizotinib and PF00299804 effectively inhibited the growth of H3122TR3 cells.
Conclusion:
Multiple mechanisms of resistance, including secondary mutations in ALK and co-activation of parallel signaling pathways, can develop in response to ALK TKIs and can develop simultaneously. Our findings suggest the need to develop alternative strategies to inhibit ALK and/or to develop effective combination therapies that target both ALK and EGFR. A phase I clinical trial of crizotinib and PF00299804 is currently underway.
