Abstract 4454: The AXL tyrosine kinase receptor contributes to ALK-inhibitor resistance in neuroblastoma.
Authors: Bhatnagar Namrata , Luther William , Sharma Bandana , Liu Qingsong , Gray Nathanael , George Rani E. 
Published: August 2014

The crizotinib-resistant ALKF1174L mutation occurs de novo in neuroblastoma (NB) and as an acquired mechanism of resistance in ALK translocation-driven cancers, lending impetus to the development of novel ALK inhibitors with different modes of action. One such molecule is TAE684, a structurally unrelated ATP-competitive diaminopyrimidine derivative that has shown activity against ALKF1174L in both in vitro and in vivo models of ALK-driven cancers, such as lung cancer and NB.

To identify mechanisms of acquired resistance to TAE684, we established in vitro models of resistance by serially exposing TAE684-sensitive SH-SY5Y NB cells (expressing ALKF1174L) to increasing concentrations of the compound over a prolonged period (&gt; 1 year). The resulting TAE684-resistant SY5Y-TR cells exhibited &gt;10-fold reduced sensitivity to TAE684 compared with parental cells. Three TAE684-resistant clonal lines (TR1-TR3) were analyzed, all of which showed downregulation of pALK. In addition, TR1 cells showed downregulation of pAKT but increased expression of pERK1/2 compared to parental cells, suggesting activation of compensatory signaling pathways. To identify candidate receptor tyrosine kinases (RTKs) that may account for this observation, we analyzed TR1 cells using phospho-RTK arrays. Upregulation of the AXL RTK was seen in the resistant cells as compared to the parental cells, and this was confirmed by immunoblotting in all the resistant clones. Concurrent with the role of AXL in tumor cell invasion, TR1 cells exhibited increased invasiveness in matrigel assays. Depletion of AXL expression by shRNA knockdown in TR1 cells led to their growth inhibition. TR1 cells were more sensitive to SKI-606, a Src/Abl inhibitor with activity against AXL, than parental cells. However, exposure to an Hsp90 inhibitor led to significant cytotoxicity in all the clones (IC50s&lt;30nM), as compared to parental cells (IC50=304nM). Inhibiting Hsp90 in the TR1 cells led to a decrease in pAXL and pERK1/2 levels with a concomitant decrease in the binding of AXL to Hsp90. Analysis of AXL in TR1 cells ruled out mutation, genomic amplification or promoter demethylation as a basis for its increased expression; however, the AXL ligand, GAS6, was increased in the resistant clones.

These studies demonstrate that AXL activation contributes to TAE684 resistance in ALKF1174L-expressing cells, and suggests that the same would be true for derivatives of TAE684 in clinical development. The finding that AXL is also involved in EGFR-targeted therapy in lung cancer suggests that AXL activation could be a general mechanism of resistance to tyrosine kinase inhibitor therapy.

Citation Format: Namrata Bhatnagar, William Luther, Bandana Sharma, Qingsong Liu, Nathanael Gray, Rani E. George. The AXL tyrosine kinase receptor contributes to ALK-inhibitor resistance in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4454. doi:10.1158/1538-7445.AM2013-4454