P3.02a-009 TPX-0005: A Multi-Faceted Approach to Overcoming Clinical Resistances from Current ALK or ROS1 Inhibitor Treatment in Lung Cancer: Topic: ALK Biomarkers

Abstract

ALK and ROS1 kinase inhibitors have achieved tremendous success in the treatment of lung cancer patients. 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). The bypass and EMT-based resistances constitute the majority of the resistant patient population, especially after multiple kinase inhibitor treatment. None of the current ALK or ROS1 inhibitors can overcome bypass or EMT-based resistance when applied as a single agent therapy. SRC kinase has been identified to contribute broadly to cancer treatment resistance via participation in signaling pathways required for DNA synthesis, control of receptor turnover, actin cytoskeleton rearrangement, migration, adhesion, invasion, motility, and survival. SRC/FAK signaling plays important roles in regulating antitumor immunity, cancer stem-like properties, and EMT. Here we deployed a polypharmacology approach to combatting multiple resistance mechanisms spontaneously. Recombined enzyme assays, engineered cell lines and H2228 cells were used to evaluate TPX-0005 in in vitro and in vivo models. TPX-0005 is a potent ALK/ROS1/TRK inhibitor with a rigid three-dimensional macrocyclic structure and a much smaller size (MW <370) than current ALK/ROS1/TRK inhibitors. The compact structure allows TPX-0005 efficiently target the center of ATP binding site and be able to circumvent the steric interference from clinical resistant mutations. Therefore, TPX-0005 potently inhibited both wild type and mutant ALK/ROS1/TRK fusion proteins including gatekeeper and solvent front mutations at low nanomolar concentration. In addition to its primary targets, TPX-0005 is also a potent SRC/FAK inhibitor. H2228 lung cancer cell line, endogenously expressing EML4-ALKv3 protein, is refractory to crizotinib and ceritinib in cell proliferation assay (IC50 ∼1 μM). The upregulation of multiple RTKs including EGFR and IGFR, as well as cancer stem cell marker CD44 in H2228 cells is believed to confer the primary resistance to selective ALK inhibitors. Inhibition of SRC/FAK kinases will modulate RTK expression and cancer stem-like properties to restore the sensitivity to ALK inhibitor. TPX-0005 inhibited the phosphorylation of EML4-ALK (IC50 13 nM), SRC and FAK (IC50s 70-80 nM), along with other downstream signaling targets in H2228 cells, leading to dose-dependent down-regulation of EGFR and CD44 expression levels. As a result, TPX-0005 overcame the primary resistance and effectively inhibited cell proliferation (IC50 ∼0.1 μM) and cell migration of H2228 cells. TPX-0005 exerts unprecedented polypharmacology profile for combatting multiple resistance mechanisms including acquired mutations, bypass signaling, cancer stemness, and metastasis, that warrants further clinical investigation.