Inhibition of OCT4 binding at the MYCN locus induces neuroblastoma cell death accompanied by downregulation of transcripts with high-open reading frame dominance.

Amplification of MYCN is observed in high-risk neuroblastomas (NBs) and is associated with a poor prognosis. MYCN expression is directly regulated by multiple transcription factors, including OCT4, MYCN, CTCF, and p53 in NB. Our previous study showed that inhibition of p53 binding at the MYCN locus induces NB cell death. However, it remains unclear whether inhibition of alternative transcription factor induces NB cell death. In this study, we revealed that the inhibition of OCT4 binding at the MYCN locus, a critical site for the human-specific OCT4-MYCN positive feedback loop, induces caspase-2-mediated cell death in MYCN-amplified NB. We used the CRISPR/deactivated Cas9 (dCas9) technology to specifically inhibit transcription factors from binding to the MYCN locus in the MYCN-amplified NB cell lines CHP134 and IMR32. In both cell lines, the inhibition of OCT4 binding at the MYCN locus reduced MYCN expression, thereby suppressing MYCN-target genes. After inhibition of OCT4 binding, differentially downregulated transcripts were associated with high-open reading frame (ORF) dominance score, which is associated with the translation efficiency of transcripts. These transcripts were enriched in splicing factors, including MYCN-target genes such as HNRNPA1 and PTBP1. Furthermore, transcripts with a high-ORF dominance score were significantly associated with genes whose high expression is associated with a poor prognosis in NB. Because the ORF dominance score correlates with the translation efficiency of transcripts, our findings suggest that MYCN maintains the expression of transcripts with high translation efficiency, contributing to a poor prognosis in NB. In conclusion, the inhibition of OCT4 binding at the MYCN locus resulted in reduced MYCN activity, which in turn led to the downregulation of high-ORF dominance transcripts and subsequently induced caspase-2-mediated cell death in MYCN-amplified NB cells. Therefore, disruption of the OCT4 binding at the MYCN locus may serve as an effective therapeutic strategy for MYCN-amplified NB.

Secondary Structure of Human De Novo Evolved Gene Product NCYM Analyzed by Vacuum-Ultraviolet Circular Dichroism.

NCYM, a cis-antisense gene of MYCN, encodes a Homininae-specific protein that promotes the aggressiveness of human tumors. Newly evolved genes from non-genic regions are known as de novo genes, and NCYM was the first de novo gene whose oncogenic functions were validated in vivo. Targeting NCYM using drugs is a potential strategy for cancer therapy; however, the NCYM structure must be determined before drug design. In this study, we employed vacuum-ultraviolet circular dichroism to evaluate the secondary structure of NCYM. The SUMO-tagged NCYM and the isolated SUMO tag in both hydrogenated and perdeuterated forms were synthesized and purified in a cell-free in vitro system, and vacuum-ultraviolet circular dichroism spectra were measured. Significant differences between the tagged NCYM and the isolated tag were evident in the wavelength range of 190-240 nm. The circular dichroism spectral data combined with a neural network system enabled to predict the secondary structure of NCYM at the amino acid level. The 129-residue tag consists of α-helices (approximately 14%) and ß-strands (approximately 29%), which corresponded to the values calculated from the atomic structure of the tag. The 238-residue tagged NCYM contained approximately 17% α-helices and 27% ß-strands. The location of the secondary structure predicted using the neural network revealed that these secondary structures were enriched in the Homininae-specific region of NCYM. Deuteration of NCYM altered the secondary structure at D90 from an α-helix to another structure other than α-helix and ß-strand although this change was within the experimental error range. All four nonsynonymous single-nucleotide polymorphisms (SNPs) in human populations were in this region, and the amino acid alteration in SNP N52S enhanced Myc-nick production. The D90N mutation in NCYM promoted NCYM-mediated MYCN stabilization. Our results reveal the secondary structure of NCYM and demonstrated that the Homininae-specific domain of NCYM is responsible for MYCN stabilization.

Utility of a scoring system for differentiating cancer-associated stroke from cryptogenic stroke in patients with cancer.

BACKGROUND: The commonest type of ischemic cerebral stroke in patients with cancer is cryptogenic stroke (CRS), the majority of which are considered cancer-associated strokes (CAS) caused by multiple microemboli associated with hypercoagulation, known as Trousseau syndrome. However, detection of microemboli and diagnosing CAS is difficult. We have therefore developed a scoring system for diagnosing CAS. METHODS: We retrospectively examined data of patients with cancer and stroke between 2006 and 2017. We identified risk factors for CRS, assigned them one or two points, and calculated total scores (Trousseau score) for each patient. We used overall survival after stroke (OSs) to validate the utility of the system. RESULTS: In 181 consecutive strokes, CRS was the commonest type (43.6%) and had a short OSs (median 56 days). We identified the following five risks for CRS: high D-dimer concentration (≥ 10.0 µg/mL) and lesions in multiple territories (two points each); and active cancer, low platelet count (150,000/µL>) and female sex (one point each). Trousseau score ≥ 3 indicated CAS (50.3%), which had a median OSs of 50 days. Only CAS (hazard ratio 3.44 [2.34-5.10], P < 0.0001) and poor performance status (3 or 4) (2.27 [1.50-3.39], P = 0.0002) were risk factors for OSs; CRS was not. OSs of patients with non-CAS/CRS was significantly longer than that of those with CAS/CRS (404.5 days vs. 47 days, P = 0.0114), whereas OSs of CAS/non-CRS was much shorter than that of non-CAS/non-CRS (53 days vs. 547 days, P < 0.0001). CONCLUSION: Trousseau scores simply and clearly identify CAS.

Clinical significance of the 2016 WHO classification in Japanese patients with gliomas.

In this study, we retrospectively compared the prognostic value of the 2016 WHO classification with the former classification in 387 patients with glioma treated at our institution. According to the new classification, diagnoses included oligodendroglioma with isocitrate dehydrogenase (IDH) mutation and 1p/19q co-deletion (5.4%), anaplastic oligodendroglioma with IDH mutation and 1p/19q co-deletion (3.4%), diffuse astrocytoma IDH-mutated (3.9%), anaplastic astrocytoma IDH-mutated (2.8%), glioblastoma IDH-mutated (7.8%), glioblastoma IDH-wildtype (58.4%), diffuse midline glioma H3 K27M mutation (2.6%), oligodendroglioma NOS (1.3%), anaplastic oligodendroglioma NOS (0.8%), diffuse astrocytoma IDH-wildtype (2.8%), and anaplastic astrocytoma IDH-wildtype (10.9%). The prognoses of IDH-mutated astrocytomas clearly varied according to tumor grade. However, we identified no survival difference between IDH-wildtype anaplastic astrocytomas and glioblastomas; additionally, these tumors showed similar gene expression profiles. After exclusion of those without 1p/19q co-deletion, patients with oligodendroglial tumors showed excellent survival regardless of tumor grade. Our evaluation of chromosomal aberrations suggests that the MAPK/PI3K pathway plays a role in acquired malignancy of astrocytic tumors, whereas TP53 participates in tumorigenesis. We suspect the RB pathway also plays a role in tumorigenesis of IDH-mutated gliomas. The new WHO classification more clearly reflects the tumorigenesis of gliomas and improves the prognostic power of classification.

Life-Threatening Intracranial Hypotension after Skull Base Surgery with Lumbar Drainage.

Although lumbar drainage (LD) is widely used in skull base surgery (SBS), no cases with intracranial hypotension (IH) following LD-assisted SBS have been reported, and skull base surgeons lack awareness of this potentially life-threatening condition. We report two cases of IH after LD-assisted SBS, a spheno-orbital meningioma and an osteosarcoma in the orbit. Despite a minimal amount of cerebrospinal fluid (CSF) drainage and early LD removal, severe postural headache and even a deteriorating consciousness level were observed in the early postoperative course. Neuroimages demonstrated epidural fluid collections, severe midline shift, and tonsillar sag compatible with IH. Epidural blood patch (EBP) immediately and completely reversed the clinical and radiologic findings in both patients. IH should be included in the differential diagnosis of postural headache after LD-assisted SBS that can be managed successfully with EBP. Persistent leakage of CSF at the LD-inserted site leads to IH. Broad dural dissection and wide removal of bony structure may be involved in the midline shift. EBP should be performed soon after conservative management fails. Further reports will determine the risk factors for IH development following LD-assisted SBS.