NSD2 Interacts with SMARCA2 and Regulates Expression of Oncogenes CCND1 and PRL3 in T(4;14) Multiple Myeloma
Authors: Chooi Jing Yuan , Xie Zhigang , Gunaratne Jayantha , Keppei Dennis , Chng Wee Joo 
Published: October 2019

Abstract
Multiple Myeloma (MM), characterized by the uncontrolled proliferation of malignant plasma cells in the bone marrow, occurs mainly in the elderly population. Recurrent chromosomal translocations are central to the pathogenesis of MM, with t(4;14) being the second-most common and associated with poor prognosis. NSD2 is overexpressed as a result of the t(4;14) translocation and has been suggested to be the primary oncogenic factor in t(4;14) MM. However, the detailed molecular mechanism or signaling pathways describing how deregulation of NDS2 contributes to MM cell growth and survival is still uncertain. In this study, we performed Stable Isotope Labelling of Amino acid in Cell culture (SILAC)-based mass spectrometry analysis to determine NSD2 interacting proteins and identified 74 proteins in the immunoprecipitation (IP) of NSD2 protein complex. Gene Ontology (GO) analysis identified 3 enriched cluster, which are related to chromatin organization, RNA processing, and the translation. NSD2-interacting proteins from the most enriched functions were selected and verified by IP and Western-blot (WB) assays. The results indicated that there were interactions between NSD2 and SMARCA2, Topoisomerase IIa, ADAR1, DDX5 and RPS6. Micrococcal nuclease (MNase) accessibility assay indicated that NSD2 kncoked-down (KD) decreased level of mono- and di-nucleosome, suggesting a more compact chromatin structure. In silico analysis with STRING database showed that SMARCA2 interacts with NSD2. SMARCA2 is the ATPase subunit of the SWI/SNF chromatin remodeling complex, which harness the energy from ATP to remodel the position of nucleosomes, making the DNA accessible during transcription, replication and DNA repair. IP showed that NSD2 interacts with SMARCA2 but not other core subunits of SWI/SNF complex. In addition, comparison of SMARCA2 expression across MM cell lines with different translocation status, showed that SMARCA2 was highly expressed in t(4;14)+ MM cells but not in t(4;14)- MM cells. RNA-seq analysis showed that NSD2 KD decreased the expression of CCND1 and PRL3 in KMS11. Consistent with our RNA-seq analysis, qPCR confirmed that CCND1 and PRL3 levels were reduced, and WB assays showed their protein levels were decreased upon NSD2 KD. ChIP-PCR revealed that the reduction in CCND1 and PRL3 expression is due to the decrease of H3K36me2 level at the respective gene promoters, which subsequently decreases RNA Pol II promoter occupancy. Luciferase assay showed ectopic NSD2 significantly increased the promoter activity of CCND1 and PRL3 in HEK293T cells. Similar to NSD2 KD, there is a reduction in CCND1 and PRL3 RNA and protein level in SMARCA2 KD cells. CellTiter-Glo Luminescent Cell Viability Assay (CTG assay) indicated that SMARCA2 KD reduced t(4;14) MM cell growth and cell cycle S phase. This study reveals a protein-protein interaction between NSD2 and SMARCA2. NSD2 and SMARCA2 might cooperate to regulate the expression of CCND1 and PRL3. Our preliminary data suggested that SMARCA2 might be a novel therapeutic target for t(4;14) MM.

Disclosures
Chng: Merck: Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Aslan: Research Funding; Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.