The canonical WNT pathway plays an
important role in cancer pathogenesis.
Inhibition of poly(ADP-ribose) polymerase catalytic activity of the
tankyrases (TNKS/TNKS2) has been reported to reduce the Wnt/β-catenin
signal by preventing poly ADP-ribosylation-dependent degradation of
AXIN, a negative regulator of Wnt/β-catenin signaling. With
the goal of investigating the effects of tankyrase and Wnt pathway
inhibition on tumor growth, we set out to find small-molecule inhibitors
of TNKS/TNKS2 with suitable drug-like properties. Starting from 1a, a high-throughput screening hit, the spiroindoline derivative 40c (RK-287107) was discovered as a potent TNKS/TNKS2 inhibitor
with >7000-fold selectivity against the PARP1 enzyme, which inhibits
WNT-responsive TCF reporter activity and proliferation of human colorectal
cancer cell line COLO-320DM. RK-287107 also demonstrated dose-dependent
tumor growth inhibition in a mouse xenograft model. These observations
suggest that RK-287107 is a promising lead compound for the development
of novel tankyrase inhibitors as anticancer agents.
Epstein–Barr virus (EBV)‐associated gastric cancer (EBVaGC), whose prognosis remains controversial, is diagnosed by in situ hybridization of EBV‐derived EBER1/2 small RNAs. In The Cancer Genome Atlas (TCGA) Stomach Adenocarcinoma (STAD) project, the EBV molecular subtype was determined through a combination of multiple next‐generation sequencing methods, but not by the gold standard in situ hybridization method. This leaves unanswered questions regarding the discordance of EBV positivity detected by different approaches and the threshold of sequencing reads. Therefore, we reanalyzed the TCGA‐STAD RNA sequencing (RNA‐seq) dataset including 375 tumor and 32 normal samples, using our analysis pipeline. We defined a reliable threshold for EBV‐derived next‐generation sequencing reads by mapping them to the EBV genome with three different random arbitrary alignments. We analyzed the prognostic impact of EBV status on the histopathological subtypes of gastric cancer. EBV‐positive cases identified by reanalysis comprised nearly half of the cases (49.6%) independent from infiltrating lymphocyte signatures, and showed significantly longer overall survival for adenocarcinomas of the ‘not‐otherwise‐specified’ type [P = 0.016 (log‐rank test); hazard ratios (HR): 0.476; 95% CI: 0.260–0.870, P = 0.016 (Cox univariate analysis)], but shorter overall survival for the tubular adenocarcinoma type [P = 0.005 (log‐rank test); HR: 3.329; 95% CI: 1.406–7.885, P = 0.006 (Cox univariate analysis)]. These results demonstrate that the EBV positivity rates were higher when determined by RNA‐seq than when determined by EBER1/2 in situ hybridization. The RNA‐seq‐based EBV positivity demonstrated distinct results for gastric cancer prognosis depending on the histopathological subtype, suggesting its potential to be used in clinical prognoses.
Gene abnormalities, including mutations and fusions, are important determinants in the molecular diagnosis of myeloid neoplasms. The use of bone marrow (BM) smears as a source of DNA and RNA for next-generation sequencing (NGS) enables molecular diagnosis to be done with small amounts of bone marrow and is especially useful for patients without stocked cells, DNA or RNA. The present study aimed to analyze the quality of DNA and RNA derived from smear samples and the utility of NGS for diagnosing myeloid neoplasms. Targeted DNA sequencing using paired BM cells and smears yielded sequencing data of adequate quality for variant calling. The detected variants were analyzed using the bioinformatics approach to detect mutations reliably and increase sensitivity. Noise deriving from variants with extremely low variant allele frequency (VAF) was detected in smear sample data and removed by filtering. Consequently, various driver gene mutations were detected across a wide range of allele frequencies in patients with myeloid neoplasms. Moreover, targeted RNA sequencing successfully detected fusion genes using smear-derived, very low-quality RNA, even in a patient with a normal karyotype. These findings demonstrated that smear samples can be used for clinical molecular diagnosis with adequate noise-reduction methods even if the DNA and RNA quality is inferior.
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