Abstract
ALK is a receptor tyrosine kinase with an oncogenic role in various types of human malignancies. Despite constitutive activation of the kinase through gene alterations, such as chromosomal translocation, gene amplification or mutation, treatments with kinase inhibitors invariably lead to the development of resistance. Aiming to develop new tools for ALK targeting, we took advantage of our previous demonstration identifying ALK as a dependence receptor, implying that in the absence of ligand the kinase-inactive ALK triggers or enhances apoptosis. Here, we synthesized peptides mimicking the proapoptotic domain of ALK and investigated their biological effects on tumor cells. We found that an ALK-derived peptide of 36 amino acids (P36) was cytotoxic for ALK-positive anaplastic large-cell lymphoma and neuroblastoma cell lines. In contrast, ALK-negative tumor cells and normal peripheral blood mononuclear cells were insensitive to P36. The cytotoxic effect was due to caspase-dependent apoptosis and required N-myristoylation of the peptide. Two P36-derived shorter peptides as well as a cyclic peptide also induced apoptosis. Surface plasmon resonance and mass spectrometry analysis of P36-interacting proteins from two responsive cell lines, Cost lymphoma and SH-SY5Y neuroblastoma, uncovered partners that could involve p53-dependent signaling and pre-mRNA splicing. Furthermore, siRNA-mediated knockdown of p53 rescued these cells from P36-induced apoptosis. Finally, we observed that a treatment combining P36 with the ALK-specific inhibitor crizotinib resulted in additive cytotoxicity. Therefore, ALK-derived peptides could represent a novel targeted therapy for ALK-positive tumors.
Highlights
anaplastic lymphoma kinase (ALK) was first identified in 1994 as a rearranged gene fusion (NPM–ALK) resulting from the t(2;5)(p23;q35) translocation occurring in 75% human anaplastic large-cell lymphomas (ALCLs).[5,6] Other translocations or gene inversions involving ALK were later described in solid tumors including 50–60% inflammatory myofibroblastic tumors, and a small proportion of diffuse large B-cell lymphomas, breast and renal carcinomas.[7,8] Recently, 4–8% non-small-cell lung cancer (NSCLC) were found to harbor an echinoderm microtubuleassociated protein-like 4 (EML4)–ALK fusion.[7,9] Resulting fusion proteins associate the N-terminal portion of a protein partner to the entire intracellular portion of ALK, including its tyrosine kinase domain
We identified a total of 13 proteins from Cost ALCL (Supplementary Table S1) and 179 proteins from SH-SY5Y neuroblastoma (Supplementary Table S2) that bound to P36, respectively
To determine whether crizotinib (PF) and P36 could synergize in ALK-positive tumor cells, we examined the effect of single drug and combination treatments on the viability of Cost ALCL and SH-SY5Y neuroblastoma cells after 48 h exposure
Summary
ALK was first identified in 1994 as a rearranged gene fusion (NPM–ALK) resulting from the t(2;5)(p23;q35) translocation occurring in 75% human anaplastic large-cell lymphomas (ALCLs).[5,6] Other translocations or gene inversions involving ALK were later described in solid tumors including 50–60% inflammatory myofibroblastic tumors, and a small proportion of diffuse large B-cell lymphomas, breast and renal carcinomas.[7,8] Recently, 4–8% non-small-cell lung cancer (NSCLC) were found to harbor an echinoderm microtubuleassociated protein-like 4 (EML4)–ALK fusion.[7,9] Resulting fusion proteins associate the N-terminal portion of a protein partner (containing in most cases a dimerization domain) to the entire intracellular portion of ALK, including its tyrosine kinase domain Subsequent dimerization of this fusion protein leads to constitutive activation of ALK kinase, resulting in enhanced signaling for cell proliferation, survival and oncogenicity.[10]. One promising strategy would be to impair distinct functions of the oncogenic tyrosine kinase through targeting different sites of the ALK protein
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call