Characterization of novel potent and selective anaplastic lymphoma kinase (ALK) inhibitors.

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e13600 Background: Aberrant forms of the anaplastic lymphoma kinase (ALK) have been implicated in the pathogenesis of multiple human cancers and represent rational therapeutic targets. Crizotinib (PF-02341066), a dual ALK/MET tyrosine kinase inhibitor (TKI), has demonstrated significant activity in ALK-fusion positive lung cancer patients. Unfortunately, as with other TKIs in clinical use, patients who initially respond to crizotinib eventually display disease progression. New agents and combinations of targeted therapies are needed to improve the care of these patients. Methods: We report identification and characterization of X-376 and X-396, two potent and highly specific ALK small molecule tyrosine kinase inhibitors (TKIs). These agents were tested against crizotinib in cancer cell lines harboring ALK fusions as well as activating ALK point mutations. Xenograft studies were completed in mice harboring EML4-ALK E13;A20 fusion positive tumors. In addition, X-376 and X-396 were tested in combination with the mTOR inhibitor, rapamycin. Results: X-376 and X-396 were 3-fold and 10-fold more potent, respectively, than PF-1066 in H3122 lung adenocarcinoma cells harboring an EML4-ALK E13;A20 fusion (IC50: PF-1066 180nM, X-376 77nM, X-396 15nM). Similar results were obtained with H2228 lung cancer cells, SUDHL-1 lymphoma cells, and SY5Y neuroblastoma cells, which harbor an EML4-ALK E6a/b;A20 fusion, an NPM-ALK fusion, and an activating point mutation within the ALK kinase domain (F1174L), respectively. In H3122 xenograft studies, both X-376 and X-396 demonstrated potent anti-tumor activity in vivo with favorable pharmacokinetic and toxicity profiles. The gatekeeper mutant (L1196M) engineered into the EML4-ALK E13;A20 fusion variant was more potently inhibited by X-396 compared to PF-1066. Finally, X-396 displayed synergistic anti-tumor activity in vitro when combined with the mTOR inhibitor, rapamycin. Conclusions: These novel ALK inhibitors and combinations of ALK inhibitors plus mTOR inhibitors have the potential to improve the therapeutic outcomes of patients with mutant ALK-driven malignancies. X-396 is expected to enter clinical trials later this year.