Identification of Transcriptional Targets of NSD2 and the Underlying Regulatory Mechanism in t(4;14) Multiple Myeloma Cells Using the Slam-Seq and d-TAG Technologies
Authors: Wang Yubao , Liu Sanxiong , Ghamlouch Hussein , Davies Faith E , Morgan Gareth 
Published: December 2024

INTRODUCTION: Approximately 15% of multiple myeloma cases are featured with a t(4;14) translocation that places immunoglobulin enhancers in the vicinity of NSD2, resulting in gain of its expression which otherwise is silent. The translocation-driven expression of NSD2 is a common event in both precursor stage (i.e. MGUS) and multiple myeloma, suggesting that it is a primary oncogenic driver in t(4;14) multiple myeloma. Although NSD2 is characterized as a histone methyltransferase for H3K36me2, associated with active gene transcription, its specific transcriptional targets and regulatory mechanisms remain elusive. And few, if any, cellular functions of NSD2 have been unveiled yet. The major hindrance behind this is the lack of an experimental system in which its molecular and cellular function could be rigorously addressed. To that end, we have generated NSD2dTAG and NSD2KO cells, featuring acute and stable depletion of NSD2, respectively.
METHODS: To identify the primary target genes of NSD2 in transcriptional regulation, we have generated an inducible degron system of NSD2 in the t(4;14) KMS-11 cell line (i.e. NSD2dTAG) using CRISPR knock-in, enabling rapid NSD2 degradation upon addition of a small-molecule degrader, dTAGv1. To study the stable effects of NSD2 depletion, we have generated multiple clonal NSD2KO KMS-11 cells, thus eliminating the complication from inter-clonal heterogeneity.
To test to what extent NSD2 regulation of gene transcription is dependent on its SET domain function, we reconstituted NSD2dTAG and NSD2KO cells with doxycycline-inducible wild-type NSD2 (NSD2WT) or its SET-domain mutant (NSD2Y1179A) that is null for H3K36me2 methyl transferase activity.
We have used SLAM-seq to identify the transcriptional target genes of NSD2 and investigate the underlying mechanism. By leveraging metabolic labeling of newly synthesized mRNA, SLAM-seq profiles changes of mRNA at the level of both new mRNA and total mRNA.
RESULTS: Although NSD2 was acutely (i.e. &lt; 1 h) degraded in NSD2dTAG cells by the small-molecule degrader, dTAGv1, and the level of H3K36me2 was significantly reduced within 3 days, it needed 3 weeks for H3K36me2 to diminish to levels comparable to NSD2KO cells. However, NSD2 and H3K36me2 depletion only caused a slight reduction in cell growth. SLAM-seq analysis over this 3-week period revealed that NSD2 regulated the transcription of a selective set of a few hundred genes, the majority of which showed reduced transcription upon NSD2 depletion. We have further validated certain NSD2 target genes using Western blotting. These include R-RAS2, a RAS family member that mediates B cell-specific PI3Kδ signaling, and PHGDH, a rate-limiting metabolic enzyme in the serine synthesis pathway.
In NSD2dTAG cells, reconstitution with NSD2WT but not NSD2Y1179A reversed the effect of NSD2 depletion on gene transcription, highlighting a critical role of the SET domain function. Consistent with previous reports that deposition of H3K36me2 antagonizes that of H3K27me3, we observed a reciprocal increase of H3K27me3 in response to H3K36me2 depletion. We employed two PRC2 inhibitors, EPZ-6438 and MAK-683, targeting the EZH2 and EED subunits respectively, to determine the relative contributions of H3K36me2 and H3K27me3 to NSD2-mediated transcriptional regulation. Our data showed that transcriptional regulation by NSD2 is critically dependent on the level of H3K27me3, whereas H3K36me2 had little, if any, direct contribution. These findings suggest that NSD2 primarily functions by depositing H3K36me2, which in turn prevents H3K27me3 accumulation, thus indirectly promoting transcription.
CONCLUSION: Our study reveals that t(4;14) KMS-11 cells exhibit a slow H3K36me2 kinetics upon acute NSD2 depletion, facilitating the identification of the set of specific NSD2 transcriptional targets. We have further validated individual target genes at the protein level, including R-RAS2 and PHGDH that have potential implications in multiple myeloma. Mechanistically, we demonstrated that the transcriptional regulation by NSD2 is critically dependent on its SET domain function. Furthermore, this is directly dependent on the antagonistic effect of H3K36me2 on H3K27me3, rather than H3K36me2 itself, suggesting that H3K36me2 deposited by NSD2 creates a chromatin landscape conductive to transcription by preventing the deposition of H3K27me3.