Characterization of Rearrangements Involving the ALK Gene Reveals a Novel Truncated Form Associated with Tumor Aggressiveness in Neuroblastoma

Abstract

Activating mutations of the ALK gene have been identified in sporadic and familial cases of neuroblastoma (NB), a cancer of the peripheral nervous system, and are thought to be the primary mechanism of oncogenic activation of this receptor in this pediatric neoplasm. To address the possibility that ALK activation may occur through genomic rearrangements as detected in other cancers, we first took advantage of high-resolution arraycomparative genomic hybridization to search for ALK rearrangements in NB samples. Using complementary experiments by capture/paired-end sequencing and FISH experiments, various types of rearrangements were fully characterized, including partial gains or amplifications, in several NB cell lines and primary tumors. In the CLB-Bar cell line, we described a genomic rearrangement associated with an amplification of the ALK locus, leading to the expression of a 170 kDa protein lacking part of the extracellular domain encoded by exons 4 to 11, named ALK D4-11 . Analysis of genomic DNA from the tumor at diagnosis and relapse revealed that the ALK gene was amplified at diagnosis but that the rearranged ALK allele was observed at the relapse stage only, suggesting that it may be implicated in tumor aggressiveness. Consistently, oncogenic and tumorigenic properties of the ALK D4-11 variant were shown after stable expression in NIH3T3 cells. Moreover, we documented an increased constitutive kinase activity of this variant, as well as an impaired maturation and retention into intracellular compartments. These results indicate that genomic rearrangements constitute an alternative mechanism to ALK point mutations resulting in receptor activation. Cancer Res; 73(1); 195-204. Ó2012 AACR.

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].…”

Deep Sequencing Reveals Occurrence of Subclonal ALK Mutations in Neuroblastoma at Diagnosis

“…The most frequent substitutions, observed in approximately 85% of mutant ALK in neuroblastoma, are localized within the kinase domain of ALK at the F1174 (mutated to L, S, I, C, or V) and R1275 (mutated to Q or L) hotspots (12,21). In neuroblastoma, ALK can also be activated by genomic amplification (approximately 1%-2% of neuroblastomas) or more rarely following structural rearrangements (22).…”

Identification and Characterization of ALK Kinase Splicing Isoforms in Non–Small-Cell Lung Cancer

“…In this tumor type, characterized by mutations within the tyrosine kinase of ALK in a subset of samples, truncated ALK proteins with complete skipping of exon 2-3 ( ALK del2-3) and exons 4-11 ( ALK del4-11) of ALK have been described and characterized 24,25 . Different than the EML4-ALK del27-derived protein EML4-ALK T1312fs*0 described herein by us, these truncated extracellular domain proteins led to increased constitutive kinase activity of ALK and transformation potential (i.e., ALK del2-3 and ALK del4-11 were putative driver events).…”