ALK inhibitor resistance in ALKF1174L-driven neuroblastoma is associated with AXL activation and induction of EMT

Abstract

The crizotinib-resistant ALKF1174L mutation arises de novo in neuroblastoma (NB) and is acquired in ALK translocation-driven cancers, lending impetus to the development of novel anaplastic lymphoma kinase (ALK) inhibitors with different modes of action. The diaminopyrimidine TAE684 and its derivative ceritinib (LDK378), which are structurally distinct from crizotinib, are active against NB cells expressing ALKF1174L. Here we demonstrate acquired resistance to TAE684 and LDK378 in ALKF1174L-driven human NB cells that is linked to overexpression and activation of the AXL tyrosine kinase and epithelial-to-mesenchymal transition (EMT). AXL phosphorylation conferred TAE684 resistance to NB cells through upregulated extracellular signal-regulated kinase (ERK) signaling. Inhibition of AXL partly rescued TAE684 resistance, resensitizing these cells to this compound. AXL activation in resistant cells was mediated through increased expression of the active form of its ligand, GAS6, that also served to stabilize the AXL protein. Although ectopic expression of AXL and TWIST2 individually in TAE684-sensitive parental cells led to the elevated expression of mesenchymal markers and invasive capacity, only AXL overexpression induced resistance to TAE684 as well. TAE684-resistant cells showed greater sensitivity to HSP90 inhibition than did their parental counterparts, with downregulation of AXL and AXL-mediated ERK signaling. Our studies indicate that aberrant AXL signaling and development of an EMT phenotype underlie resistance of ALKF1174L-driven NB cells to TAE684 and its derivatives. We suggest that the combination of ALK and AXL or HSP90 inhibitors be considered to delay the emergence of such resistance.

Enhancer Remodeling and MicroRNA Alterations Are Associated with Acquired Resistance to ALK Inhibitors

“…We found that phosphorylation of AXL was remarkably increased in both H3122-LR and H2228-LR. Consistent with previous reports indicating that AXL was associated with the EMT (10,(20)(21)(22), both LR cells showed EMT-associated morphological changes; specifically, the shape of the cells changed from round to fibroblast-like following the development of treatment resistance (Fig. 1E).…”

AXL and GAS6 co-expression in lung adenocarcinoma as a prognostic classifier

“…AXL-related EMT resulting in drug resistance has been reported in patients with prior EGFR-TKI therapy, as well as in in vitro studies using NSCLC cell lines (15,16,30). Aberrant AXL signaling and the development of the EMT phenotype were also associated with ALK inhibitor resistance in ALK-driven neuroblastoma cells (31). Our clinical data support the concept that EMT under AXL or GAS6 high expression apparently exists in patients with prior surgical resection for lung AD, which consequently leads to de novo resistance to EGFR-TKI (15,30).…”

The second-generation ALK inhibitor alectinib effectively induces apoptosis in human neuroblastoma cells and inhibits tumor growth in a TH-MYCN transgenic neuroblastoma mouse model

“…The crizotinib-resistant ALK F1174L and ALK R1275Q mutations that arise de novo during crizotinib treatment in NB could result in acquired chemoresistance [10]. Thus alectinib may be very effective treatment option since it induces apoptosis in Kelly and SH-SY5Y, both of which harbor the ALK F1174L mutation.…”

“…In prior studies, alectinib has shown substantial inhibitory effects against tumors with ALK mutations, including ALK L1196M , ALK L1152R , ALK F1174L , and ALK R1275Q [28, 35, 40, 48, 55]. Furthermore, chemoresistant cells harboring crizotinib-mediated ALK mutations ALK F1174L [10, 47] are sensitive to alectinib. Alectinib demonstrated a favorable safety profile and clinically meaningful response in patients with ALK -positive metastatic NSCLC who progressed on crizotinib and, therefore, was granted accelerated approval by the United States Food and Drug Administration (FDA) on December 11, 2015 [25].…”