Activated ALK signals through the ERK–ETV5–RET pathway to drive neuroblastoma oncogenesis

Abstract

Activating mutations of the ALK receptor occur in a subset of neuroblastoma tumors. We previously demonstrated that Alk mutations cooperate with MYCN overexpression to induce neuroblastoma in mice and identified Ret as being strongly upregulated in MYCN/Alkmut tumors. By a genetic approach in vivo, we now document an oncogenic cooperation between activated Ret and MYCN overexpression in neuroblastoma formation. We show that MYCN/RetM919T tumors exhibit histological features and expression profiles close to MYCN/Alkmut tumors. We show that RET transcript levels decrease precedes RET protein levels decrease upon ALK inhibition in neuroblastoma cell lines. Etv5 was identified as a candidate transcription factor regulating Ret expression from murine MYCN/Alkmut tumor transcriptomic data. We demonstrate that ETV5 is regulated both at the protein and mRNA levels upon ALK activation or inhibition in neuroblastoma cell lines and that this regulation precedes RET modulation. We document that ALK activation induces ETV5 protein upregulation through stabilization in a MEK/ERK-dependent manner. We show that RNAi-mediated inhibition of ETV5 decreases RET expression. Reporter assays indicate that ETV5 is able to drive RET gene transcription. ChIP-seq analysis confirmed ETV5 binding on the RET promoter and identified an enhancer upstream of the promoter. Finally, we demonstrate that combining RET and ALK inhibitors reduces tumor growth more efficiently than each single agent in MYCN and AlkF1178L-driven murine neuroblastoma. Altogether, these results define the ERK–ETV5–RET pathway as a critical axis driving neuroblastoma oncogenesis downstream of activated ALK.

The ETS transcription factor ETV5 is a target of activated ALK in neuroblastoma contributing to increased tumour aggressiveness

“…1c). Our findings indicate that ETV5 is regulated downstream of activated ALK through MAPK-signalling, in line with previous studies reporting ETV5 as a downstream target of RAS/MAPK signalling in other cellular contexts 19,27,28 .…”

“…After stimulation, upregulated p-ALK levels are rapidly attenuated through dephosphorylation in keeping with previously established signalling kinetics 7 . Using artificial stimulation of the ALK receptor with a monoclonal antibody Lopez-Delisle et al also observed an increase in ETV5 mRNA levels after 6 hours 19 . Therefore, our data are in agreement with the activation of ALK leading to induction of expression of ETV5, as clearly shown here at the protein level.…”

“…Subsequently, we specifically sought ALK-upregulated genes implicated in normal neural crest and sympathetic nervous system development with putative oncogenic features and identified ETV5 as a strong candidate. Earlier reports established ETV5 as a downstream target of the RAS/MAPK pathway in neuroblastoma and other cell types 19,27,28 . We subsequently performed further experiments using pharmacological ALK inhibition and ALK ligand stimulation, and confirmed that ALK signalling regulates ETV5 expression levels regardless of whether wild-type or mutant ALK is expressed.…”

The paradox of cancer genes in non-malignant conditions: implications for precision medicine

“…Not listed [28] PIK3CA inhibitors such as alpelisib [26] Breast cancer PI3K-AKT-mTOR pathway activation ALK TPM3-ALK, TPM4-ALK Inflammatory myofibroblastic tumor ∼ 50% [29] ALK inhibitors [30] such as alectinib [31] Non-small cell lung cancer ALK pathway activation [32] NOTCH1 Loci not specified Aging esophagus 12-80% [33] No specific inhibitors approved Colon cancer Wnt-betacatenin pathway activation [34] KRAS G12V or G12D Arteriovenous malformations in brain ∼ 63% [35,36] MEK inhibitors such as trametinib [16] Colorectal and pancreatic cancer RAS-RAF-MEK-ERK pathway upregulation [15] G12C, G12V, G12A, G12D, G12R…”

ALK and neuroblastoma: from molecular genetics to clinics

“…На экспериментальных моделях установлено, что сочетание мутации ALK, в особенности F1174 [22], c амплификацией MYCN негативно влияет на прогноз, приводя к более быстрому формированию НБ с высокой агрессивностью и повышенной летальностью [23,24]. У детей с НБ, сочетающей амплификацию MYCN с мутацией ALK F1174, летальность достигала 90 % (9 из 10 детей).…”