Neuroblastoma is one of the most common solid tumors in children and has a diverse clinical behavior that largely depends on the tumor biology. Neuroblastoma exhibits unique features, such as early age of onset, high frequency of metastatic disease at diagnosis in patients over 1 year of age and the tendency for spontaneous regression of tumors in infants. The high-risk tumors frequently have amplification of the MYCN oncogene as well as segmental chromosome alterations with poor survival. Recent advanced genomic sequencing technology has revealed that mutation of ALK, which is present in ~10% of primary tumors, often causes familial neuroblastoma with germline mutation. However, the frequency of gene mutations is relatively small and other aberrations, such as epigenetic abnormalities, have also been proposed. The risk-stratified therapy was introduced by the Japan Neuroblastoma Study Group (JNBSG), which is now moving to the Neuroblastoma Committee of Japan Children’s Cancer Group (JCCG). Several clinical studies have facilitated the reduction of therapy for children with low-risk neuroblastoma disease and the significant improvement of cure rates for patients with intermediate-risk as well as high-risk disease. Therapy for patients with high-risk disease includes intensive induction chemotherapy and myeloablative chemotherapy, followed by the treatment of minimal residual disease using differentiation therapy and immunotherapy. The JCCG aims for better cures and long-term quality of life for children with cancer by facilitating new approaches targeting novel driver proteins, genetic pathways and the tumor microenvironment.

Neuroblastoma is a pediatric solid tumor that originates from embryonic neural crest cells. The MYCN gene locus is frequently amplified in unfavorable neuroblastomas, and the gene product promotes the progression of neuroblastomas. However, the molecular mechanisms by which MYCN amplification contributes to stem cell‐like states of neuroblastoma remain elusive. In this study, we show that MYCN and its cis‐antisense gene, NCYM, form a positive feedback loop with OCT4, a core regulatory gene maintaining a multipotent state of neural stem cells. We previously reported that NCYM is co‐amplified with the MYCN gene in primary human neuroblastomas and that the gene product promotes aggressiveness of neuroblastoma by stabilization of MYCN. In 36 MYCN‐amplified primary human neuroblastomas, OCT4 mRNA expression was associated with unfavorable prognosis and was correlated with that of NCYM. The OCT4 protein induced both NCYM and MYCN in human neuroblastoma cells, whereas NCYM stabilized MYCN to induce OCT4 and stem cell‐related genes, including NANOG, SOX2, and LIN28. In sharp contrast to MYCN, enforced expression of c‐MYC did not enhance OCT4 expression in human neuroblastoma cells. All‐trans retinoic acid treatment reduced MYCN, NCYM, and OCT4 expression, accompanied by the decreased amount of OCT4 recruited onto the intron 1 region of MYCN. Knockdown of NCYM or OCT4 inhibited formation of spheres of neuroblastoma cells and promoted asymmetric cell division in MYCN‐amplified human neuroblastoma cells. These results suggest that the functional interplay between MYCN, NCYM, and OCT4 contributes to aggressiveness of MYCN‐amplified human neuroblastomas.

Genomic analysis–integrated whole-exome sequencing of neuroblastomas identifies genetic mutations in axon guidance pathway Genomic analysis–integrated whole-exome sequencing of neuroblastomas identifies genetic mutations in axon guidance pathway

KIF1Bβ, a member of the kinesin superfamily of motor proteins, is a haploinsufficient tumor suppressor mapped to chromosome 1p36.2, which is frequently deleted in neural crest–derived tumors, including neuroblastoma and pheochromocytoma. While KIF1Bβ acts downstream of the nerve growth factor (NGF) pathway to induce apoptosis, further molecular functions of this gene product have largely been unexplored. In this study, we report that KIF1Bβ destabilizes the morphological structure of mitochondria, which is critical for cell survival and apoptosis. We identified YME1L1, a mitochondrial metalloprotease responsible for the cleavage of the mitochondrial GTPase OPA1, as a physical interacting partner of KIF1Bβ. KIF1Bβ interacted with YME1L1 through its death‐inducing region, as initiated the protease activity of YME1L1 to cleave the long forms of OPA1, resulting in mitochondrial fragmentation. Overexpression of YME1L1 promoted apoptosis, while knockdown of YME1L1 promoted cell growth. High YME1L1 expression was significantly associated with a better prognosis in neuroblastoma. Furthermore, in NGF‐deprived PC12 cells, KIF1Bβ and YME1L1 were upregulated, accompanied by mitochondrial fragmentation and apoptotic cell death. Small interfering RNA–mediated knockdown of either protein alone, however, remarkably inhibited the NGF depletion–induced apoptosis. Our findings indicate that tumor suppressor KIF1Bβ plays an important role in intrinsic mitochondria–mediated apoptosis through the regulation of structural and functional dynamics of mitochondria in collaboration with YME1L1. Dysfunction of the KIF1Bβ/YME1L1/OPA1 mechanism may be involved in malignant biological features of neural crest–derived tumors as well as the initiation and progression of neurodegenerative diseases.

Neuroblastoma (NB) is the most common extracranial solid tumor that originates from multipotent neural crest cells. NB cell populations that express embryonic stem cell‐associated genes have been identified and shown to retain a multipotent phenotype. However, whether somatic reprogramming of NB cells can produce similar stem‐cell like populations is unknown. Here, we sought to reprogram NB cell lines using an integration‐free Sendai virus vector system. Of four NB cell lines examined, only SH‐IN cells formed induced pluripotent stem cell‐like colonies (SH‐IN 4F colonies) at approximately 6 weeks following transduction. These SH‐IN 4F colonies were alkaline phosphatase‐positive. Array comparative genomic hybridization analysis indicated identical genomic aberrations in the SH‐IN 4F cells as in the parental cells. SH‐IN 4F cells had the ability to differentiate into the three embryonic germ layers in vitro, but rather formed NBs in vivo. Furthermore, SH‐IN 4F cells exhibited resistance to cisplatin treatment and differentiated into endothelial‐like cells expressing CD31 in the presence of vascular endothelial growth factor. These results suggest that SH‐IN 4F cells are partially reprogrammed NB cells, and could be a suitable model for investigating the plasticity of aggressive tumors.

NCYM is a cis-antisense gene of MYCN and is amplified in human neuroblastomas. High NCYM expression is associated with poor prognoses, and the NCYM protein stabilizes MYCN to promote proliferation of neuroblastoma cells. However, the molecular mechanisms of NCYM in the regulation of cell survival have remained poorly characterized. Here we show that NCYM promotes cleavage of MYCN to produce the anti-apoptotic protein, Myc-nick, both in vitro and in vivo. NCYM and Myc-nick were induced at G2/M phase, and NCYM knockdown induced apoptotic cell death accompanied by Myc-nick downregulation. These results reveal a novel function of NCYM as a regulator of Myc-nick production in human neuroblastomas.