Abstract 2487: Novel ZNF-fusions in NUT carcinoma

INTRODUCTION. NUT Carcinoma (NC) is a rare, aggressive squamous carcinoma characterized by chromosome translocations involving rearrangement of the NUT gene (also known as NUTM1) on chromosome 15. While the BRD4-NUT fusion oncoprotein drives the majority of NCs, approximately 25% of NCs harbor non-BRD4 fusion partner genes, including BRD3-, NSD3-, and ZNF532-NUT. All of these partner genes encode proteins that are key components of the BRD4-NUT oncogenic complex and have led to greater understanding of the oncogenic mechanism of NC. Despite these advances, several NCs harbor NUT-fusions with genes of unknown identity, here termed NUT-variant, offering further opportunity to discover additional NUT-fusion partners that may play a key role in BRD4-NUT complex function. METHODS. In this study, we identified 7 NCs with NUT-variant fusions. Archer® FusionPlex®, a next generation sequencing-based derivation of rapid amplification of cDNA ends (RACE), was used to identify the NUT-fusions using RNA extracted from formalin-fixed paraffin-embedded (FFPE) archival tumor tissue. Confirmatory studies included RT-PCR, Sanger sequencing, and fluorescence in situ hybridization (FISH). RESULTS. Of the 7 NCs with unknown NUT- fusion partners, Archer® FusionPlex® identified two tumors with classic BRD4-NUT fusions, two BRD3-NUT, one non-fusion, one technical failure, and one novel ZNF592-NUT fusion. The tumor harboring the ZNF592-NUT fusion was biopsied from a pelvic tumor in an 18 year old female patient. She was initially diagnosed with NC based on positive NUT immunohistochemical staining of her pelvic tumor, and died 13 months later. RT-PCR and Sanger sequencing confirmed the ZNF592-NUT fusion detected in Archer® FusionPlex® analysis. The ZNF592-NUT fusion was further confirmed by DNA-FISH performed on the same tissue. ZNF592 is known to form a “Z4” coregulator complex with ZNF532 (a NUT-fusion partner), ZNF687 and ZMYND8. Interestingly, we have previously reported that all Z4 members co-purify with the BRD4-NUT chromatin complex. Here we show that wild type ZNF532 and wild type ZNF592 co-localize with BRD4-NUT by immunofluorescence in the BRD4-NUT NC cell line TC-797. Furthermore, knockdown of ZNF532 and ZNF592 reduced cell viability/growth of TC-797 cells, indicating these are critical factors for NC growth and/or viability. CONCLUSION. Here we report, through the identification of a novel NUT-fusion partner, ZNF592, evidence that supports a critical role of Z4 factors in BRD4-NUT complex oncogenic function, providing the opportunity for future mechanistic insights and targeted therapeutic inhibition. Secondly, the findings suggest that Archer® FusionPlex® may in certain circumstances have a greater sensitivity for the detection of NUT-fusions than conventional FISH, and should be considered as an alternative approach for fusion-partner detection and discovery in NC. Citation Format: Hitoshi Shiota, Janine E. Elya, Kristina Danga, Kelly Becht, Pauline Chou, Artyom A. Alekseyenko, Valentina Nardi, Christopher A. French. Novel ZNF-fusions in NUT carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2487.