Anaplastic Lymphoma Kinase (ALK) regulates initiation of transcription of MYCN in neuroblastoma cells

Abstract

Neuroblastoma is a neural crest-derived embryonal tumour of the postganglionic sympathetic nervous system and a disease with several different chromosomal gains and losses, which include MYCN-amplified neuroblastoma on chromosome 2, deletions of parts of the chromosomes 1p and 11q, gain of parts of 17q and triploidy. Recently, activating mutations of the ALK (Anaplastic Lymphoma Kinase) RTK (Receptor Tyrosine Kinase) gene have been described in neuroblastoma. A meta-analysis of neuroblastoma cases revealed that ALK mutations (49 of 709 cases) in relation to genomic subtype were most frequently observed in MYCN amplified tumours (8.9%), correlating with a poor clinical outcome. MYCN proteins target proliferation and apoptotic pathways, and have an important role in the progression of neuroblastoma. Here, we show that both wild-type and gain-of-function mutants in ALK are able to stimulate transcription at the MYCN promoter and initiate mRNA transcription of the MYCN gene in both neuronal and neuroblastoma cell lines. Further, this stimulation of MYCN gene transcription and de novo MYCN protein expression is abrogated by specific ALK inhibitors, such as crizotinib (PF-2341066), NVP-TAE684, and by small interfering RNA to ALK resulting in a decrease in proliferation rate. Finally, co-transfection of ALK gain-of-function mutations together with MYCN leads to an increase in transformation potential. Taken together, our results indicate that ALK signalling regulates initiation of transcription of the MYCN gene providing a possible explanation for the poor clinical outcome observed when MYCN is amplified together with activated ALK.Oncogene (2012) 31, 5193 --5200; doi:10.1038/onc.2012.12; published online 30 January 2012Keywords: neuroblastoma; anaplastic lymphoma kinase; ALK; MYCN; transcription factor INTRODUCTION Neuroblastoma is a neural crest-derived embryonal tumour of the postganglionic sympathetic nervous system and accounts for B15% of all deaths due to paediatric tumours. 1,2 Genetically, many cases of neuroblastoma show amplification of the MYCN gene (B24% of all cases), deletions of parts of the chromosomes 1p and 11q, gain of parts of 17q and triploidy. 1,3 --6 Anaplastic Lymphoma Kinase (ALK) Receptor Tyrosine Kinase (RTK) is mutated in both familial and somatic neuroblastoma. 7 --11 Moreover, these reports also indicated that downregulation or inhibition of ALK led to a marked decrease of cell proliferation, suggesting ALK as a critical factor in the initiation and progression of neuroblastoma. The ALK RTK was first described in the mid-nineties and aberrant ALK protein activity is now implicated in a range of nonhematopoietic, hematopoietic as well as neuroendocrine tumours (for a review see Palmer et al. 12 ). A recent meta-analysis of neuroblastoma has reported ALK gain-of-function mutations to be present at a frequency of 6.9% of investigated neuroblastoma tumours. Further, a comparison of the ALK mutation frequency in relation to genomic subtype revealed that ALK mutations were most frequently obs...

Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK positive neuroblastoma cells, Drosophila and mice

“…In order to investigate the therapeutic efficacy of brigatinib in a neuroblastoma setting we employed several neuroblastoma cell lines, including CLB-BAR (MYCN amplification, ALK (Δ4-11) and amplified, ALK addicted), CLB-GE (MYCN amplification, ALK (F1174V) amplification, ALK addicted), IMR32 (MYCN amplification, WT ALK) and CLB-PE (MYCN amplified, WT ALK) [21,[31][32][33]. We have earlier shown that both CLB-BAR and CLB-GE cell lines are ALK addicted, while IMR32 and CLB-PE are not [34,35]. ALK addicted cell lines were treated with either brigatinib or crizotinib, as a positive control for ALK inhibition, and their effect on ALK signaling analyzed by immunoblotting ( Figure 1A, 1B).…”

Advances in ALK Targeted Therapy for Neuroblastoma

“…12 different ALK active mutations have been reported in sporadic neuroblastoma, including two most common ALK variants, ALKF1174L and ALKR1275Q [26]. Most of these pathogenic variants are found within the tyrosine kinase domain of ALK and cause constitutive autophosphorylation and activation of the ALK protein and downstream cellular pathways, including the MAPK and RASrelated protein 1 signal pathways [9,27]. The PI3K (phosphatidylinositol 3-kinase)/Akt [10] and the JAK/STAT (Janus activated kinase/signal transducer and activator of transcription) pathways [28].…”

“…It has been shown that ALK and MYCN drive tumor malignancy cooperatively. Activation of ALK increases the expression of MYCN by enhancing the activity of the MYCN promoter and stabilizing MYCN protein likely via activation of AKT and ERK pathways [9][10][11]. In vivo, compared to ALKF1174L and MYCN alone, co expression of these two oncogenes leads to the development of neuroblastoma tumors with earlier onset, higher penetrance and enhanced lethality [10,12,13].…”

Anaplastic lymphoma kinase: Role in cancer and therapy perspective

“…[14][15][16][17] MYCN is identified in neuroblastoma as a transcriptional target of activated full-length ALK. 18 There are reports of ALK signaling involving microRNAs, with miR-135b, miR-29a and miR-16 being downstream of NPM-ALK, and miR-96-mediated regulation of ALK expression. [19][20][21][22] Two proteins, midkine and pleiotrophin, have been reported to be activating ligands for mammalian ALK.…”