Non-small cell lung cancers harboring rearrangements involving the ALK gene are sensitive to treatment with the ALK inhibitor crizotinib. However, the emergence of drug resistance is universal and limits clinical applicability. The mechanisms of resistance include ALK gene amplification, acquired ALK missense mutations, bypass activation, and epithelial-mesenchymal transition (EMT). More than ten acquired ALK mutations have been identified from patients and preclinical cell lines resistant to crizotinib. Although the second generation of ALK inhibitors (ceritinib and alectinib) overcome some of the crizotinib resistant mutations, they are plagued by a different spectrum of ALK resistant mutations. For example, the ALK G1202R mutation confers resistance to crizotinib, ceritinib, and alectinib. None of the current ALK inhibitors approved or in clinical trials can overcome bypass and EMT resistance mechanisms. SRC kinase has been identified to contribute broadly to primary intrinsic resistance to ALK inhibitor treatment, the development of bypass resistance and EMT during ALK inhibitor treatment. Saracatinib, a selective SRC inhibitor, can re-sensitize ALK inhibitor-resistant cell lines, suggesting a therapeutic role. There are currently no available ALK/SRC dual inhibitors, and we designed a novel ALK and SRC inhibitor TPX-0005 to address the need to overcome resistance. TPX-0005 is a novel three-dimensional macrocycle with a much smaller size (MW <370) than current ALK inhibitors designed to efficiently target the center of the ATP binding site and circumvent the steric interference from mutations outside the ATP binding boundary. As expected, TPX-0005 potently inhibited WT ALK (IC50 1.01 nM) and mutant ALKs including ALK G1202R (1.26 nM) and ALK L1196M (1.08 nM). Moreover, TPX-0005 effectively inhibited tumor growth in vivo in ALK WT and ALK G1202R xenografts. TPX-0005 is also a potent SRC inhibitor (IC50 5.3 nM). The elevated SRC kinase activity in H2228 lung cancer cell line confers resistance to crizotinib (IC50 1200 nM) and ceritinib (IC50 1000 nM) in cell proliferation assays. TPX-0005 effectively overcame this primary resistance (IC50 100 nM in cell proliferation assay) with strong inhibition of the phosphorylation of EML4-ALK (IC50 13 nM) and the SRC substrate paxillin (IC50 107 nM), along with other downstream signaling targets. TPX-0005 inhibited H2228 cell migration in a wound healing assay with similar activity to saracatinib. Taken together, TPX-0005 was able to not only inhibit the wild-type and a broad spectrum of mutant ALKs, but also overcome primary resistance and suppress metastatic features by inhibiting SRC. Overall, TPX-0005 has a highly favorable profile with ability to overcome multiple ALK resistance mechanisms including secondary mutations, bypass signaling activation, EMT, and warrants clinical investigation.

Citation Format: Dayong Zhai, Wei Deng, Zhongdong Huang, Evan Rogers, J. Jean Cui. The novel, rationally-designed, ALK/SRC inhibitor TPX-0005 overcomes multiple acquired resistance mechanisms to current ALK inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2132.