Novel kinase fusion transcripts found in endometrial cancer

Abstract

Recent advances in RNA-sequencing technology have enabled the discovery of gene fusion transcripts in the transcriptome of cancer cells. However, it remains difficult to differentiate the therapeutically targetable fusions from passenger events. We have analyzed RNA-sequencing data and DNA copy number data from 25 endometrial cancer cell lines to identify potential therapeutically targetable fusion transcripts, and have identified 124 high-confidence fusion transcripts, of which 69% are associated with gene amplifications. As targetable fusion candidates, we focused on three in-frame kinase fusion transcripts that retain a kinase domain (CPQ-PRKDC, CAPZA2-MET, and VGLL4-PRKG1). We detected only CPQ-PRKDC fusion transcript in three of 122 primary endometrial cancer tissues. Cell proliferation of the fusion-positive cell line was inhibited by knocking down the expression of wild-type PRKDC but not by blocking the CPQ-PRKDC fusion transcript expression. Quantitative real-time RT-PCR demonstrated that the expression of the CPQ-PRKDC fusion transcript was significantly lower than that of wild-type PRKDC, corresponding to a low transcript allele fraction of this fusion, based on RNA-sequencing read counts. In endometrial cancers, the CPQ-PRKDC fusion transcript may be a passenger aberration related to gene amplification. Our findings suggest that transcript allele fraction is a useful predictor to find bona-fide therapeutic-targetable fusion transcripts.

Novel MXD4–NUTM1 fusion transcript identified in primary ovarian undifferentiated small round cell sarcoma

“…Given that the NUTM1 rearrangement was considered as an oncogenic driver event and an attractive therapeutic target in “NUT midline carcinoma (NMC),”28, 29 we focused on the MXD4–NUTM1 fusion transcript to perform subsequent analyses. The transcript allele fraction (TAF) score13 for the NUTM1 was 0.57, suggesting that both MXD4–NUTM1 and wild‐type NUTM1 were expressed in this tumor (Table 1). Next, we compared NUTM1 mRNA expression between this tumor and 10 epithelial ovarian cancer samples using quantitative real‐time PCR.…”

“…RT-PCR and Sanger sequencing were performed as previously reported. 13,14 In brief, total RNA (1 μg) was reverse-transcribed into cDNA (corresponding to 10 ng of total RNA) using Prime Script II Reverse Transcriptase (Takara Bio, Shiga, Japan Table 1. When most of cells were stained, the sample was considered positive.…”

“…Western blotting experiments for NUTM1 (C52B1, Cell signaling technology; dilution ratio 1:1000) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, clone 6C5 (MAB374, Merck Millipore Headquarters; dilution ratio 1:1000) were performed as previously reported. 13,14,26 Tissues were lysed in RIPA buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], and 1 mM EDTA) supplemented with protease inhibitors (Roche). The lysates were separated by SDS-polyacrylamide gel electrophoresis on 5%-20% gradient gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore).…”

“…We have used RNA sequencing data to identify numerous fusion genes in various types of tumors12, 13, 14 and developed the fusion gene data portal.…”

“…11 Recent advances in sequencing technology have enabled the comprehensive detection of fusion genes in the cancer genome and transcriptome. We have used RNA sequencing data to identify numerous fusion genes in various types of tumors [12][13][14] and developed the fusion gene data portal.…”