ALK kinase inhibitors have achieved tremendous success in the treatment of lung cancer patients with abnormal ALK gene. However, the emergence of drug resistance limits their long-term clinical applications. The mechanisms of resistance often include gene amplification, acquired mutations, bypass signaling, and epithelial-mesenchymal transition (EMT). More than 10 different mutations have been identified in clinic, and recently the compound mutations represent new challenges after multiple ALK inhibitor treatments. The bypass and EMT-based resistance mechanisms constitute a large portion of the resistant patient population. None of the current ALK inhibitors can overcome bypass or EMT-based resistance when applied as a single-agent therapy. Therefore, a different strategy needs to be deployed for the design of new-generation ALK inhibitors to overcome multi-resistance mechanisms simultaneously. TPX-0005, a novel three-dimensional macrocycle with a much smaller size than current ALK inhibitors in the clinic, was designed to overcome clinical resistance mutations systematically. TPX-0005 potently inhibited both wild type and mutant ALK fusion proteins including gatekeeper, solvent front, and compound mutations as shown in the table. In addition to its primary targets, TPX-0005 is also a potent SRC/FAK inhibitor with IC50s of 70-80 nM in H2228 cells. H2228 lung cancer cell line, endogenously expressing EML4-ALKv3 protein, is refractory to crizotinib and ceritinib. The upregulation of multiple RTKs including EGFR and IGFR, as well as cancer stem cell marker CD44 and mesenchymal marker vimentin, is believed to induce the primary resistance to selective ALK inhibitors. Inhibition of SRC/FAK kinases will modulate bypass RTK expression, cancer stem-like properties, and EMT to restore the sensitivity to ALK inhibition. TPX-0005 dose-dependently downregulated EGFR, CD44, and vimentin expression levels via SRC/FAK inhibition. TPX-0005 effectively inhibited cancer cell migration in wound healing assays, and induced dramatic tumor regression in human PDX NSCLC tumor model. In addition, TPX-0005 significantly extended survival time in mouse H2228 orthotopic brain tumor model. Overall, TPX-0005 exerts unprecedented polypharmacology profile for combating multiple resistance mechanisms. A phase 1/2 clinical trial of TPX-0005 is actively being pursued (NCT03093116).

 EML4-ALK Ba/F3 Cell Proliferation Assay IC50 (nM) 
Inhibitor WT G1202R L1196M L1198F/C1156Y L1198F/G1202R L1198F/L1196M 
TPX-0005 17.8 20.5 50 1.1 0.2 34.8 
Crizotinib 74.8 359.4 713 22 43.7 350.7 
Ceritinib 2.1 388 1123 476.8 1794 
Alectinib 18.9 507 131 158 1369 1249 
Brigatinib 11.8 399 13 118 1187 341 
Lorlatinib 0.7 ND 18 89 131.6 1169 
 EML4-ALK Ba/F3 Cell Proliferation Assay IC50 (nM) 
Inhibitor WT G1202R L1196M L1198F/C1156Y L1198F/G1202R L1198F/L1196M 
TPX-0005 17.8 20.5 50 1.1 0.2 34.8 
Crizotinib 74.8 359.4 713 22 43.7 350.7 
Ceritinib 2.1 388 1123 476.8 1794 
Alectinib 18.9 507 131 158 1369 1249 
Brigatinib 11.8 399 13 118 1187 341 
Lorlatinib 0.7 ND 18 89 131.6 1169 

ND: not determined

Citation Format: Dayong Zhai, Wei Deng, Evan Rogers, Zhongdong Huang, Jeffrey Whitten, John Lim, Yishan Li, J. Jean Cui. TPX-0005, a polypharmacology inhibitor, overcomes ALK treatment resistance from acquired mutations, bypass signaling, and EMT [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B186.