ALK inhibitor resistance in ALK(F1174L)-driven neuroblastoma is associated with AXL activation and induction of EMT.

D N Debruyne,W Luther,B Sharma,N S Gray,R E George,N-K Cheung,N F Moore,N Bhatnagar

doi:10.1038/onc.2015.434

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.

Highlights

The predictable emergence of resistance to tyrosine kinase inhibitors (TKIs), leading to disease progression or relapse, has hindered their long-term therapeutic impact.[1]
TAE684 resistance is associated with the loss of anaplastic lymphoma kinase (ALK) activity but maintenance of downstream signaling NB cells that express the ALKF1174L mutation are relatively resistant to crizotinib but are sensitive to TAE684.5,9 To elucidate the
SK-N-SH cells rendered resistant to TAE684 (SK-N-SH-TAE-R) showed upregulation of AXL as well as extracellular signal-regulated kinase (ERK) signaling despite downregulation of phosphorylated ALK (Supplementary Figures S2a and b). These results indicate that TAE684-resistant NB cells express higher levels of alternative receptor tyrosine kinase (RTK) such as AXL to compensate for the loss of ALK phosphorylation

Summary

Introduction

The predictable emergence of resistance to tyrosine kinase inhibitors (TKIs), leading to disease progression or relapse, has hindered their long-term therapeutic impact.[1]. This aggressive childhood tumor is characterized by mutations in the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK) in 10% of cases.[4,5,6,7] Many of these point mutations are considered ‘drivers’ of the malignant process: do they induce constitutive, ligand-independent activation of ALK signaling, and their inhibition leads to tumor cell death and tumor regression.[5,6] The most common somatic mutation in NB, ALKF1174L, is highly tumorigenic, both by itself and when coexpressed with the MYCN oncogene, a combination that increases the penetrance of the disease and accelerates tumor formation.[8,9] This mutation confers primary resistance to the ALK inhibitor crizotinib in NB9 and serves as a mechanism of acquired resistance to crizotinib in patients with ALK-translocated cancers.[10]

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Conclusion