Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK

Abstract

The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion oncogene represents a molecular target in a small subset of non-small cell lung cancers (NSCLCs). This fusion leads to constitutive ALK activation with potent transforming activity. In a pivotal phase 1 clinical trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib (PF-02341066) demonstrated impressive antitumor activity in the majority of patients with NSCLC harboring ALK fusions. However, despite these remarkable initial responses, cancers eventually develop resistance to crizotinib, usually within 1 y, thereby limiting the potential clinical benefit. To determine how cancers acquire resistance to ALK inhibitors, we established a model of acquired resistance to crizotinib by exposing a highly sensitive EML4-ALK–positive NSCLC cell line to increasing doses of crizotinib until resistance emerged. We found that cells resistant to intermediate doses of crizotinib developed amplification of the EML4-ALK gene. Cells resistant to higher doses (1 μM) also developed a gatekeeper mutation, L1196M, within the kinase domain, rendering EML4-ALK insensitive to crizotinib. This gatekeeper mutation was readily detected using a unique and highly sensitive allele-specific PCR assay. Although crizotinib was ineffectual against EML4-ALK harboring the gatekeeper mutation, we observed that two structurally different ALK inhibitors, NVP-TAE684 and AP26113, were highly active against the resistant cancer cells in vitro and in vivo. Furthermore, these resistant cells remained highly sensitive to the Hsp90 inhibitor 17-AAG. Thus, we have developed a model of acquired resistance to ALK inhibitors and have shown that second-generation ALK TKIs or Hsp90 inhibitors are effective in treating crizotinib-resistant tumors harboring secondary gatekeeper mutations.

Treatment Efficacy and Resistance Mechanisms Using the Second-Generation ALK Inhibitor AP26113 in Human NPM-ALK–Positive Anaplastic Large Cell Lymphoma

“…The gatekeeper L1196M was one of the first causes of Crizotinib resistance found in patients. A moderate AP26113 resistance was predicted in vitro (29,34,35); however, the IC 50 value is low enough to consider this mutation sensitive to AP26113. Because in SUP-M2AR500B cells both L1122V and L1196M were found, we hypothesize that the first is the mutation driving resistance, while the latter was selected at low doses and never counterselected, remaining as a passenger in the high-dose-resistant clone.…”

Molecular pathology of lung cancer: key to personalized medicine

“…However, despite these remarkable initial clinical responses, these cancers eventually developed resistance to crizotinib, usually within 1 year, thereby limiting the potential clinical benefit of this drug. Katayama et al 165 found that cells resistant to intermediate doses of crizotinib develop either amplification of the EML4-ALK gene or a gatekeeper mutation, L1196M, within the kinase domain. Sasaki et al 164 proposed two mechanisms of ALK TKI resistance based on evidence from a crizotinib-treated ALK-positive lung cancer patient and in a cell line generated from the resistant tumor.…”

Crystal structure of EML1 reveals the basis for Hsp90 dependence of oncogenic EML4-ALK by disruption of an atypical β-propeller domain

“…Hsp90 inhibitors induce the degradation of EML4-ALK variant 1 and regression in some EML4-ALK-positive tumor models (7,9,10). Furthermore, clinical efficacy of an Hsp90 inhibitor in EML4-ALK NSCLC has been confirmed (11), and clinical trials are ongoing.…”

“…These fusions display potent transforming activity due to constitutive activation of the tyrosine kinase but confer addiction to the oncogene, and inhibition of the tyrosine kinase induces apoptosis in transformed cells (4,5). The ALK inhibitor crizotinib is a highly effective first-line therapy for NSCLC patients with EML4-ALK fusions, but because the acquirement of resistance is inevitable, often involving secondary mutations in the kinase portion of the fusion protein, additional therapeutic strategies must be identified (6,7).…”