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

Proc Natl Acad Sci U S A. 2011 May 3;108(18):7535-40. doi: 10.1073/pnas.1019559108. Epub 2011 Apr 18.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents / pharmacology*
  • Antineoplastic Agents / therapeutic use
  • Apoptosis / drug effects
  • Carcinoma, Non-Small-Cell Lung / drug therapy*
  • Carcinoma, Non-Small-Cell Lung / genetics
  • Cell Line, Tumor
  • Crizotinib
  • DNA Primers / genetics
  • Dose-Response Relationship, Drug
  • Drug Resistance, Neoplasm / genetics*
  • Flow Cytometry
  • Humans
  • Immunoblotting
  • In Situ Hybridization, Fluorescence
  • Mice
  • Mice, Nude
  • Mutation / genetics
  • Oncogene Proteins, Fusion / antagonists & inhibitors
  • Oncogene Proteins, Fusion / genetics*
  • Organophosphorus Compounds / pharmacology
  • Phosphorylation / drug effects
  • Pyrazoles / pharmacology*
  • Pyrazoles / therapeutic use
  • Pyridines / pharmacology*
  • Pyridines / therapeutic use
  • Pyrimidines / pharmacology
  • RNA, Small Interfering / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction / drug effects
  • Survival Analysis
  • Transfection

Substances

  • Antineoplastic Agents
  • DNA Primers
  • EML4-ALK fusion protein, human
  • NVP-TAE684
  • Oncogene Proteins, Fusion
  • Organophosphorus Compounds
  • Pyrazoles
  • Pyridines
  • Pyrimidines
  • RNA, Small Interfering
  • Crizotinib
  • brigatinib