SUMMARY The ALKF1174L mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALKF1174L in the neural crest. Compared to ALKF1174L and MYCN alone, coexpression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance and enhanced lethality. ALKF1174L/MYCN tumors exhibited increased MYCN dosage due to ALKF1174L-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2, overcame the resistance of ALKF1174L/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALKF1174L in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.

Advanced prostate cancers are known to acquire not only invasive capabilities but also significant resistance to chemotherapy-induced apoptosis. To understand how microRNAs (miRNAs) may contribute to prostate cancer resistance to apoptosis, we compared microRNA expression profiles of a benign prostate cancer cell line WPE1-NA22 and a highly malignant WPE1-NB26 cell line (derived from a common lineage). We found that miR-205 and miR-31 are significantly downregulated in WPE1-NB26 cells, as well as in other cell lines representing advanced-stage prostate cancers. Antiapoptotic genes BCL2L2 (encoding Bcl-w) and E2F6 are identified as the targets of miR-205 and miR-31, respectively. By downregulating Bcl-w and E2F6, miR-205 and miR-31 promote chemotherapeutic agents-induced apoptosis in prostate cancer cells. The promoter region of the miR-205 gene was cloned and was found to be hypermethylated in cell lines derived from advanced prostate cancers, contributing to the downregulation of the gene. Treatment with DNA methylation inhibitor 5-aza-2′-deoxycytidine induced miR-205 expression, downregulated Bcl-w, and sensitized prostate cancer cells to chemotherapy-induced apoptosis. Thus, downregulation of miR-205 and miR-31 has an important role in apoptosis resistance in advanced prostate cancer.

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.