Generation of conditional ALK F1174L mutant mouse models for the study of neuroblastoma pathogenesis

Abstract

Neuroblastoma, an embryonal tumor arising from the sympathetic ganglia and adrenal medulla, is among the most intractable pediatric cancers. Although a variety of genetic changes have been identified in neuroblastoma, how they contribute to its pathogenesis remains largely unclear. Recent studies have identified alterations of the anaplastic lymphoma kinase (ALK) gene in neuroblastoma; ALK F1174L (a phenylalanine-to-leucine substitution at codon 1174) represents one of the most frequent of these somatic mutations, and is associated with amplification of the MYCN gene, the most reliable marker for the poor survival. We engineered the mouse Alk locus so that ALK F1174L is expressed by its endogenous promoter and can be induced in a spatiotemporally controlled fashion using Cre-loxP system.Although expression of ALK F1174L resulted in enhanced proliferation of sympathetic ganglion progenitors and increased the size of the sympathetic ganglia, it was insufficient to cause neuroblastoma. However, lethal neuroblastoma frequently developed in mice co-expressing ALK F1174L and MYCN, even in a genetic background where MYCN alone does not cause overt tumors. These data reveal that physiological expression of ALK F1174L significantly potentiates the oncogenic ability of MYCN in vivo. Our conditional mutant mice provide a valuable platform for investigating the pathogenesis of neuroblastoma.

ALK ligand ALKAL2 potentiates MYCN‐driven neuroblastoma in the absence of ALK mutation

“…Further observation of heterozygous (n = 13 > 18 months) and homozygous (n = 19 > 18 months) Alk-F1178S animals up to 18 months of age did not reveal development of NB or any other type of cancer. Thus, while no gross tumour development is observed in Alk-F1178S mice, significant hyperplasia can be detected in Alk expressing neural crest-derived structures during development, such as the sympathetic ganglia, which is in agreement with other reports (Cazes et al, 2014;Ono et al, 2019).…”

“…Alk‐F1178S homozygous mice were obtained with expected Mendelian ratios, and a colony of Alk‐F1178S mice was established. Since previous reports of Alk gain‐of‐function mice have reported hyperplasia in the sympathetic ganglia (Cazes et al , 2014; Ono et al , 2019), we investigated ganglia from Alk‐F1178S mice and WT siblings at developmental stage P9. Alk‐F1178S heterozygous caeliac ganglia were significantly enlarged and displayed hyperplasia when compared with controls, which was enhanced in Alk‐F1178S homozygous animals (Fig 3A–C).…”

“…In the human genome, ALKAL2 is located on the distal portion of chromosome 2 (at 2p25), along with ALK and MYCN , in the “2p gain” region that has been associated with NB (Jeison et al , 2010; Javanmardi et al , 2019). We know from previous work that ALK activation drives transcription of MYCN and potentiates MYCN‐driven NB in mouse and zebrafish models (Weiss et al , 1997; Berry et al , 2012; Heukamp et al , 2012; Schonherr et al , 2012; Zhu et al , 2012; Cazes et al , 2014; Ono et al , 2019), and this is supported by analysis of NB tumours where coexistence of ALK activating mutations and MYCN amplification forms a high‐risk NB group with poor prognosis (De Brouwer et al , 2010). While our advances in genetic profiling of tumours have led to significant advances, this approach does not address signalling activity in cancer cells that may not be reflected by genetic mutation (Yaffe, 2019).…”

Defining Pathological Activities of ALK in Neuroblastoma, a Neural Crest-Derived Cancer

“…However, this was challenged in 2014, when Cazes et al showed that ALK F1174L and ALK R1275Q variants indeed lead to abnormal proliferation of sympathetic ganglia, but were not sufficient to initiate neuroblastoma formation. This suggests a requirement for additional factors, such as MYCN amplification, to trigger tumorigenesis [89,90]. Increased MYCN activity is likely to enhance neuroblast proliferation and survival [91].…”

“…Increased MYCN activity is likely to enhance neuroblast proliferation and survival [91]. Following this, ALK activation may potentiate the oncogenic capacity of MYCN [17,90,[92][93][94]. These two factors are the most common predisposition markers of neuroblastoma and, together, are associated with poor prognosis [77,95].…”