Upregulation of MAPK Negative Feedback Regulators and RET in Mutant ALK Neuroblastoma: Implications for Targeted Treatment

Abstract

Purpose: Activating ALK mutations are present in almost 10% of primary neuroblastomas and mark patients for treatment with small-molecule ALK inhibitors in clinical trials. However, recent studies have shown that multiple mechanisms drive resistance to these molecular therapies. We anticipated that detailed mapping of the oncogenic ALK-driven signaling in neuroblastoma can aid to identify potential fragile nodes as additional targets for combination therapies.Experimental Design: To achieve this goal, transcriptome profiling was performed in neuroblastoma cell lines with the ALK F1174L or ALK R1275Q hotspot mutations, ALK amplification, or wild-type ALK following pharmacologic inhibition of ALK using four different compounds. Next, we performed cross-species genomic analyses to identify commonly transcriptionally perturbed genes in MYCN/ALK F1174L double transgenic versus MYCN transgenic mouse tumors as compared with the mutant ALKdriven transcriptome in human neuroblastomas.Results: A 77-gene ALK signature was established and successfully validated in primary neuroblastoma samples, in a neuroblastoma cell line with ALK F1174L and ALK R1275Q regulable overexpression constructs and in other ALKomas. In addition to the previously established PI3K/AKT/mTOR, MAPK/ERK, and MYC/ MYCN signaling branches, we identified that mutant ALK drives a strong upregulation of MAPK negative feedback regulators and upregulates RET and RET-driven sympathetic neuronal markers of the cholinergic lineage.Conclusions: We provide important novel insights into the transcriptional consequences and the complexity of mutant ALK signaling in this aggressive pediatric tumor. The negative feedback loop of MAPK pathway inhibitors may affect novel ALK inhibition therapies, whereas mutant ALK induced RET signaling can offer novel opportunities for testing ALK-RET oriented molecular combination therapies.

Proliferation and Survival of Embryonic Sympathetic Neuroblasts by MYCN and Activated ALK Signaling

“…As expected from the differentiated neuronal morphology of ALK F1174L cells, the pan-neuronal gene NEFM is strongly upregulated. The increase in RET and SLC18A3 (VACHT ), together with decreased TH expression suggests a shift toward cholinergic differentiation observed also in ALKinduced NB (Cazes et al, 2014;Lambertz et al, 2015;Fig. 6).…”

“…Upregulated RET has no proliferative function in ALK F1174L -induced sympathetic neurons, in contrast to MYCN/ALK F1174L neuroblasts and MYCN/ALK-induced mouse NB (Cazes et al, 2014;Lambertz et al, 2015). The longterm survival of ALK F1174L -induced neurons is surprising as sympathetic neuron death is tightly controlled by NGF/TRKA signaling with TRKA acting as an apoptosis-inducing dependence receptor (Nikoletopoulou et al, 2010).…”

Smoothened-independent activation of hedgehog signaling by rearranged during transfection promotes neuroblastoma cell proliferation and tumor growth

“…Though mutations in RET are rarely found in patients with NB, the occurrence of NB has been observed in RET transgenic mice [44]. Moreover, aberrant activation of RET signaling pathway frequently occurs in ALK-mutant NB and abnormalities of the RET signaling pathway rather than oncogenic RET activation by mutation are critical for primary NB tumorigenesis [11,12,45]. Though literatures pay more attention to the roles of RET upregulation and aberrant activation of RET signaling in NB differentiation, some previous studies still suggest that RET is actually involved in NB cell proliferation and that RET-mediated cell proliferation is critically important for the biological behaviors of NB [24,34,44,[46][47][48].…”

“…Loss of function mutations in RET cause Hirschsprung's disease, a developmental disorder characterized by absence of ganglia in the distal colon [7,8], whereas gain of function mutations in RET give rise to hereditary cancer syndrome known as multiple endocrine neoplasia type 2 (MEN2), including MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC) [9,10]. Regardless the importance of MYCN (N-myc) amplification and anaplastic lymphoma kinase (ALK) mutation in tumorigenesis of NB, because high expression of RET gene is frequently observed in NB, RET is considered to be another important player involved in the tumorigenesis and malignancy of NB [11,12].…”

“…Despite the fact that both GDNF/RET and HH/GLI pathways are crucial for the biological behaviors of NB and that activation of HH/GLI induces the expression of RET in NB cells [11,12,[16][17][18][19], whether GDNF/RET signal through HH pathway in biological behaviors of NB remains unknown. Here, we have shown that RET activates protein kinase B (AKT) to inhibit glycogen synthesis kinase-3β (GSK3β), resulting in activation of HH signaling and subsequent induction of NB cell proliferation and tumor growth.…”