Abstract
Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALK(F1174L) mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALK(F1174L)/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALK(F1174L)/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.
Highlights
The discovery of activating mutations in the ALK gene in high-risk neuroblastoma (NB) has opened new opportunities for the development of novel therapeutic strategies against this often-fatal childhood cancer of the sympathetic nervous system [1]
Based on activation of the PI3K/mTOR pathway in a murine transgenic NB model expressing ALKF1174L and MYCN, we demonstrated that combining an ATP-competitive mTOR inhibitor with crizotinib induced tumor regression, the molecular basis for this result was unclear at the time [6]
The majority of the PI3K/mTOR pathway transcripts were not uniformly downregulated in the crizotinib-treated cells, suggesting inadequate suppression of the pathway by crizotinib even at high doses (Supplementary Table 1). To determine whether these findings extend to the protein level, we treated Kelly cells with doses of crizotinib similar to those used for the expression analysis www.impactjournals.com/oncotarget and analyzed the three main targets of both mTOR and PI3K signaling: pRPS6 and p4E-BP1, markers of mTORC1 activation, as well as phosphorylation of AKT at serine 473 and threonine 308, markers of mTORC2 and PI3K activation, respectively
Summary
The discovery of activating mutations in the ALK gene in high-risk neuroblastoma (NB) has opened new opportunities for the development of novel therapeutic strategies against this often-fatal childhood cancer of the sympathetic nervous system [1]. In the COG trial, 3 of the 4 patients with ALKF1174L–positive NB tumors developed progressive disease, compared with 2 of 5 whose tumors had a missense mutation at the R1275 locus [4]. This relative resistance of ALKF1174L to crizotinib has been attributed to the increased ATP-binding affinity of the mutant, with complete inhibition of constitutively active ALK attainable only at very high doses of the drug [5]. ALKF1174L is considered the most aggressive of all ALK mutations in NB, possessing higher transforming potential and segregating with MYCN oncogene amplification, itself a marker of aggressive disease in high-risk NB [7]. ALKF1174L arises secondarily as a mechanism of resistance after an initial response to crizotinib in patients with ALK-rearranged cancers [8]
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