Differential Protein Stability and ALK Inhibitor Sensitivity of EML4-ALK Fusion Variants

Abstract

Purpose: ALK rearrangement-positive lung cancers can be effectively treated with ALK inhibitors. However, the magnitude and duration of response is heterogeneous. In addition, acquired resistance limits the efficacy of ALK inhibitors, with most upfront resistance mechanisms being unknown.Experimental Design: By making use of the Ba/F3 cell line model, we analyzed the cytotoxic efficacy of ALK kinase inhibitors as a function of different EML4-ALK fusion variants v1, v2, v3a, and v3b as well as of three artificially designed EML4-ALK deletion constructs and the ALK fusion genes KIF5b-ALK and NPM1-ALK. In addition, the intracellular localization, the sensitivity to HSP90 inhibition and the protein stability of ALK fusion proteins were studied.Results: Different ALK fusion genes and EML4-ALK variants exhibited differential sensitivity to the structurally diverse ALK kinase inhibitors crizotinib and TAE684. In addition, differential sensitivity correlated with differences in protein stability in EML4-ALK-expressing cells. Furthermore, the sensitivity to HSP90 inhibition also varied depending on the ALK fusion partner but differed from ALK inhibitor sensitivity patterns. Finally, combining inhibitors of ALK and HSP90 resulted in synergistic cytotoxicity.Conclusions: Our results might explain some of the heterogeneous responses of ALK-positive tumors to ALK kinase inhibition observed in the clinic. Thus, targeted therapy of ALK-positive lung cancer should take into account the precise ALK genotype. Furthermore, combining ALK and HSP90 inhibitors might enhance tumor shrinkage in EML4-ALK-driven tumors.

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

“…The current standard diagnostic test for the presence of ALK rearrangements identifies fragmentation of ALK by a fluorescent in situ hybridization assay (27). Previous work (12) and our data suggest that knowledge of the specific fusion variant that is present in patient tumors may allow more effective stratification of Hsp90 therapy in EML4-ALK NSCLC. We plan to correlate variant-specific genotype with response to ganetespib treatment in an ongoing phase II clinical trial involving patients with ALK-positive NSCLC (NCT01562015).…”

“…Indeed, EML4-ALK variant 1 has been shown to be highly unstable, and it may be more sensitive to Hsp90 inhibition than any other client protein yet observed (8,9,25). The sensitivity of variants 1 and 2 are due to protein mis-folding resulting in exposure of hydrophobic residues that recruit Hsp90, which is supported by the observation that this sensitivity is not found in variants 3 and 5 (12). These differences are also found in patient-derived NSCLC cells that express variant 1 or 3 endogenously (Fig.…”

“…Furthermore, clinical efficacy of an Hsp90 inhibitor in EML4-ALK NSCLC has been confirmed (11), and clinical trials are ongoing. However, because neither ALK nor EML4 are native Hsp90 clients, it was proposed that Hsp90 sensitivity of EML4-ALK fusions was due to their protein-folding properties, which might expose hydrophobic residues that lead to Hsp90 recruitment (12). EML4-ALK variants incorporating various portions of EML4 differ in sensitivity to Hsp90 inhibitors, suggesting that they have different protein-folding properties (12).…”

Tackling ALK in non-small cell lung cancer: the role of novel inhibitors

“…Although more uncommon, other ALK fusion partners have been identified as TFG (TRK-fused gene), KIF5B, PTPN3 and KLC1 (21)(22)(23). The different EML4 variants and the further partner genes do not seem to significantly impact on biology and sensitivity of ALK-rearranged malignant cells and tumors to specific inhibitors (24-26) although a putative role in conditioning response to treatments has been reported in vitro (27).…”

Tyrosine kinase inhibitors for EGFR- and ALK-mutated non-small cell lung cancer

“…At least 8 different EML4-ALK fusion variants have been identified. All variants harbor a breakpoint at intron 19 of ALK and variable intron breakpoints in EML4 [109,110]. The EML4-ALK oncogene generates the EML4-ALK fusion protein and all variants of this protein contain an EML4 transmembrane domain and an intracellular ALK tyrosine kinase domain.…”