Ectopic NSD2 Remodels H3K36me2 and DNA Methylation to Promote Oncogenic Gene Expression in Multiple Myeloma

Introduction: The t(4;14) translocation causes overexpression of NSD2 in ~12% of patients with multiple myeloma (MM) and is associated with poor clinical outcomes. NSD2 catalyzes dimethylation of histone H3 lysine 36 (H3K36me2), which is dramatically increased in t(4;14) MM. The molecular mechanisms by which altered H3K36me2 potentiates an oncogenic gene expression program are poorly understood. One function of H3K36me2 is the recruitment of DNA methyltransferase enzymes such as DNMT3B, leading to DNA methylation (DNAm). DNAm plays important roles in both plasma cell differentiation and oncogenesis, yet how DNAm is dysregulated in t(4;14) MM is largely unknown. Methods: We analyzed whole genome bisulfite sequencing (WGBS) DNAm data from 415 primary MM samples from the MMRF CoMMpass trial (NCT01454297) which had been integrated with genetic, transcriptional, and clinical information. Differential DNAm analysis between t(4;14) and non-t(4;14) MM samples was performed using the DSS Bioconductor package. KMS11 isogenic cell lines which express either high (NTKO) or low (TKO) levels of NSD2 were profiled for chromatin marks using cleavage under targets and tagmentation (CUT&Tag) and DNAm using WGBS. Three t(4;14) MM cell lines (KMS11, KMS18, H929) were treated with KTX-1031, a catalytic inhibitor of NSD2. Currently, KTX-1001 is a first-in-class catalytic NSD2 inhibitor in a phase 1 clinical trial (NCT05651932). Following 8 days of treatment, chromatin modifications, DNAm, and gene expression were assayed using CUT&Tag, WGBS, and RNAseq. DNMT3B protein levels were assayed by western blot. Results: Analysis of CoMMpass samples showed higher levels of global DNAm in the t(4;14) subtype compared to other subtypes (p = 2.9e-8) and >2M differentially methylated loci (DML) (FDR < 0.01), with 92% of DML having higher DNAm in t(4;14) MM samples. A correlation analysis between DNAm and gene expression determined that ~232K DML correlated with RNA levels of the nearest gene (FDR < 0.01). These RNA-correlated DML were enriched near genes identified by Zhan et al. (Blood, 2006) as defining the t(4;14) gene expression subtype (OR = 55.9, p < 1e-16), suggesting that changes in DNAm at these regions may facilitate the t(4;14) MM gene expression program. Epigenetic profiling of KMS11 NTKO (NSD2-high) and TKO (NSD2-low) cells showed higher global DNAm in NTKO cells (p < 1e-9). Furthermore, regions of elevated H3K36me2 in NTKO cells were enriched for increases in DNAm (OR = 1.85, p < 1e-15). Treating t(4;14) MM cell lines with the NSD2 inhibitor KTX-1031 reversed the chromatin changes observed in t(4;14) patients, resulting in reduced H3K36me2 and increased levels of the EZH2 modification H3K27me3. These epigenetic changes corresponded with downregulation of 539 genes and upregulation of 133 genes (FDR < 0.05) in KMS 11 t(4;14) cells. While the NSD2 inhibitor globally depleted H3K36me2 and elevated H3K27me3, these changes were most pronounced proximal to and in the gene bodies of downregulated genes. NSD2 inhibition for 8 days significantly reduced levels of DNMT3B protein, but only slightly reduced global DNAm (~2%). Importantly, the NSD2 inhibitor reversed much of the t(4;14) transcriptional program described by Zhan et al. (FDR = 5.5e-4). Furthermore, gene set enrichment analysis demonstrated that the NSD2 inhibitor resulted in reduced expression of genes involving NFKB (FDR < 0.001) and KRAS (FDR < 0.05) signaling. Conclusions: Through analyses of patient and cell line data, we provide evidence that NSD2-mediated increases in H3K36me2 directly dysregulate DNAm in t(4;14) MM, leading to DNAm hypermethylation as compared to non-t(4;14) MM. Many DNAm changes are correlated with transcriptional changes and occur primarily near disease-relevant genes. Treatment with catalytic NSD2 inhibitor for 8 days reverses much of the chromatin and gene expression changes associated with t(4;14) MM. NSD2 inhibition also reduces DNMT3B protein levels after 8 days and may disrupt the t(4;14) DNAm hypermethylation program after more prolonged treatment, resulting in further gene expression changes. We will present these results alongside data from extended NSD2 inhibitor treatments. We posit that elevated NSD2 in t(4;14) MM causes excessive H3K36me2 leading to relative DNA hypermethylation and transcriptional dysregulation which can be reversed through extended pharmacologic inhibition of NSD2.

引言：t(4;14)易位导致约12%的多发性骨髓瘤（MM）患者中NSD2过度表达，并与不良临床结果相关。NSD2催化组蛋白H3赖氨酸36（H3K36me2）的二甲基化，在t(4;14) MM中该二甲基化显著增加。改变的H3K36me2增强致癌基因表达程序的分子机制尚不清楚。H3K36me2的一个功能是招募如DNMT3B等DNA甲基转移酶，导致DNA甲基化（DNAm）。DNAm在浆细胞分化和肿瘤发生中都起重要作用，但在t(4;14) MM中DNAm如何失调在很大程度上是未知的。 方法：我们分析了来自MMRF CoMMpass试验（NCT01454297）的415例原发性MM样本的全基因组亚硫酸氢盐测序（WGBS）DNAm数据，这些数据已与遗传、转录和临床信息整合。使用DSS生物信息学软件包对t(4;14)和非t(4;14) MM样本进行差异DNAm分析。对表达高水平（NTKO）或低水平（TKO）NSD2的KMS11同基因细胞系，使用靶向切割和转座酶标记（CUT&Tag）分析染色质标记，使用WGBS分析DNAm。用NSD2的催化抑制剂KTX - 1031处理3种t(4;14) MM细胞系（KMS11、KMS18、H929）。目前，KTX - 1001是一种处于1期临床试验（NCT05651932）的首创NSD2催化抑制剂。处理8天后，使用CUT&Tag、WGBS和RNA测序分析染色质修饰、DNAm和基因表达。通过蛋白质印迹法检测DNMT3B蛋白水平。 结果：对CoMMpass样本的分析显示，与其他亚型相比，t(4;14)亚型的全局DNAm水平更高（p = 2.9e - 8），且有>200万个差异甲基化位点（DML）（错误发现率<0.01），其中92%的DML在t(4;14) MM样本中具有更高的DNAm。DNAm和基因表达之间的相关性分析确定，约23.2万个DML与最近基因的RNA水平相关（错误发现率<0.01）。这些与RNA相关的DML在Zhan等人（《血液》，2006年）确定的定义t(4;14)基因表达亚型的基因附近富集（优势比 = 55.9，p < 1e - 16），表明这些区域的DNAm变化可能促进t(4;14) MM基因表达程序。KMS11 NTKO（NSD2高表达）和TKO（NSD2低表达）细胞的表观遗传学分析显示，NTKO细胞中的全局DNAm更高（p < 1e - 9）。此外，NTKO细胞中H3K36me2升高的区域DNAm增加更为富集（优势比 = 1.85，p < 1e - 15）。用NSD2抑制剂KTX - 1031处理t(4;14) MM细胞系逆转了在t(4;14)患者中观察到的染色质变化，导致H3K36me2降低和EZH2修饰的H3K27me3水平升高。这些表观遗传学变化与KMS11 t(4;14)细胞中539个基因的下调和133个基因的上调相对应（错误发现率<0.05）。虽然NSD2抑制剂全局消耗H3K36me2并升高H3K27me3，但这些变化在下调基因的近端和基因体中最为显著。NSD2抑制8天显著降低了DNMT3B蛋白水平，但仅略微降低了全局DNAm（约2%）。重要的是，NSD2抑制剂逆转了Zhan等人描述的大部分t(4;14)转录程序（错误发现率 = 5.5e - 4）。此外，基因集富集分析表明，NSD2抑制剂导致涉及NFKB（错误发现率<0.001）和KRAS（错误发现率<0.05）信号通路的基因表达降低。 结论：通过对患者和细胞系数据的分析，我们提供证据表明，在t(4;14) MM中，NSD2介导的H3K36me2增加直接失调DNAm，导致与非t(4;14) MM相比DNAm过度甲基化。许多DNAm变化与转录变化相关，并且主要发生在疾病相关基因附近。用NSD2催化抑制剂处理8天逆转了与t(4;14) MM相关的大部分染色质和基因表达变化。NSD2抑制8天后也降低了DNMT3B蛋白水平，并且在更长时间的治疗后可能破坏t(4;14) DNAm过度甲基化程序，导致进一步的基因表达变化。我们将结合延长NSD2抑制剂治疗的数据呈现这些结果。我们认为，在t(4;14) MM中升高的NSD2导致过度的H3K36me2，从而导致相对的DNA过度甲基化和转录失调，这可以通过对NSD2的长期药物抑制来逆转。