Abstract 3272: Differential sensitivities to heat shock protein 90(HSP90) inhibitors in anaplastic lymphoma kinase(ALK)-positive non-small cell ling cancer(NSCLC) cells.
Authors: Lee SunHwa , Kim Soyeon , Kim Tae Min , Kim Dong-Wan , Heo Dae Seog 
Published: August 2014

Objectives: HSP90 is an abundant cellular chaperone protein and its inhibition results in the instability and degradation of client proteins such as HER2, mutant KIT, mutant EGFR, and ALK. Recently, HSP90 inhibitors showed clinical activity against echinoderm microtubule-associated protein-like 4 (EML4)-ALK NSCLC. However, it is unknown whether HSP90 inhibitors remain sensitive against ALK-positive NSCLC cells that show acquired resistance to ALK inhibitors, such as crizotinib. Here, we evaluated the activity of two clinical stage HSP90 inhibitors in ALK-positive NSCLC cells that were sensitive and resistant to crizotinib.

Methods: ALK-positive NSCLC cells that were crizotinib-sensitive (SNU-2292 and NCI-H3122) and crizotinib-resistant (SNU-2535 and H3122CR1) were used. In vitro cytotoxicities of HSP90 inhibitors alone (ganetespib and NVP-AUY922), or in combination with crizotinib, were evaluated against ALK-positive NSCLC cells using a modified MTT assay. Cell cycle and Annexin-V binding assays were evaluated by flow cytometry. Expression of ALK and its downstream effectors were detected by Western blot assay after exposure to HSP90 inhibitors ± crizotinib.

Results: HSP90 inhibitors were highly potent against crizotinib sensitive SNU-2292 and NCI-H3122 cell lines and displayed enhanced activity when combined with 0.1 μM crizotinib. Consistent with previous reports, crizotinib resistant H3122 cells were ∼5x fold less sensitive to HSP90 inhibitors compared to parental H3122 cells, with IC50 still in the low nanomolar range. In a concentration dependent manner, the HSP90 inhibitors did not suppress ALK downstream signals in H3122CR1 and SNU-2535 cell lines compared to effects on NCI-H3122 and SNU-2292 cell lines. In addition, phospho-ALK signals were significantly diminished in Ba/F3 cells expressing EML4-ALK compared to cell lines expressing L1196M or G1269A after exposure to HSP90 inhibitors. Cell cycle analysis demonstrated HSP90 inhibitors and/or crizotinib significantly increased the sub-G1 populations of NCI-H3122 and SNU-2292 cells compared with those of H3122CR1 and SNU-2535. Similarly, apoptotic cells significantly increased in crizotinib-sensitive cells than in crizotinib-resistant cells treated with HSP90 inhibitors and/or crizotinib.

Conclusions: HSP90 inhibitors showed differential sensitivities in ALK-positive NSCLC cells. Our results suggest that HSP90 inhibitors alone or in combination treatment with crizotinib are effective against crizotinib-sensitive, ALK-positive NSCLC cells.

Citation Format: SunHwa Lee, Soyeon Kim, Tae Min Kim, Dong-Wan Kim, Dae Seog Heo. Differential sensitivities to heat shock protein 90(HSP90) inhibitors in anaplastic lymphoma kinase(ALK)-positive non-small cell ling cancer(NSCLC) cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3272. doi:10.1158/1538-7445.AM2013-3272