NSD2 Mutation Research Articles

Epigenetic Regulation and Therapeutic Targeting in Myeloma

Genetic alterations of epigenetic regulators that alter the repressive trimethylation of lysine 27 on histone H3 (H3K27me3) are a recurrent feature in cancer. The histone demethylase UTX/KDM6A Is mutated in ~5% of multiple myeloma (MM) at diagnosis and is commonly absent in MM cell lines, derived from patients with advanced disease. KDM6A forms a complex containing H3K4 specific methyltransferases KMT2D and KMT2C, the histone acetyltransferase CBP/p300 and SWI/SNF chromatin-remodelers. Collectively the complex adds activation marks on histones and removes the gene repression associated H3K37me mark at enhancers. Removal of KDM6A from MM cells using CRISPR and re-expression of KDM6A in deficient cells showed that KDM6A has tumor suppressor function and regulates genes involved in cell adhesion. ChIP-studies showed that loss of KDM6A lead to decreased H3K27 acetylation and increased H3K27me at specific loci, but at other loci changes in H3K27Ac are the major effect of changes in KDM6A expression. KDM6A loss shifts the balance of gene expression to repression and KDM6A null cells had heightened sensitivity to EZH2 inhibitors. H3K27me/Ac chromatin modifications are also deregulated in MM associated with t(4;14) and overexpression of the NSD2/MMSET histone methyltransferase. NSD2 overexpression leads to a ~10-fold genome wide increase in histone 3 lysine 36 dimethylation (H3K36me2), a chromatin mark associated with gene activation and similar loss of H3K27me3. As a result, many genes are aberrantly activated and NSD2 aberrant stimulates cell growth in part through activation of c-myc. However, some loci are repressed in the presence of high NSD2 due to redistribution of EZH2. These genes may also be important for pathogenesis and growth since NSD2 high cells also show increased sensitivity to EZH2 inhibitors. Nevertheless, the global loss of H3K27me3 makes the chromatin structure of NSD2 overexpressing MM cells more “open” and these cells demonstrated increased DNA damage in response to genotoxic agents. At the same time NSD2 overexpression increased increases DNA repair processes making cells relatively resistant to chemotherapy, potentially explaining the poor prognosis of such patients. A point mutation in NSD2, E1099K is found in some MM patients, the commonly utilized MM.1 model cell line and in up to 15% of cases of relapsed childhood acute lymphocytic leukemia (ALL). This mutation increases the activity of the enzyme and also globally increases H3K36me2 and decreases H3K27me3. Removal of the mutant allele from ALL cells lines reverses of histone methylation patterns, deceased growth, altered cellular adhesion and increased susceptibility to therapy. RNA-seq analysis showed that similar to the UTX mutation, NSD2 mutation activated a program of cell adhesion, motility and signaling. Furthermore in in ALL, the activity of mutant NSD2 blocks glucocorticoid induced expression of pro-apoptotic genes, promoting tumor relapse. In summary deregulation of histone methylation patterns in MM plays an important role in disease pathogenesis and progression. Rebalancing histone modifications using appropriate inhibitors represent a potential therapeutic strategy. DisclosuresLicht:Celgene: Research Funding.

Long-term outcomes and mutation profiling of patients with mantle cell lymphoma (MCL) who discontinued ibrutinib.

Long term outcomes and mutations in patients with mantle cell lymphoma (MCL) who discontinued ibrutinib have not been described. Using deep targeted next generation sequencing, we performed somatic mutation profiling from 15 MCL patients (including 5 patients with paired samples; before and after progression on ibrutinib). We identified 80 patients with MCL who discontinued ibrutinib therapy for various reasons. Median follow-up after ibrutinib discontinuation was 38months. The median duration on ibrutinib was 7·6months. Forty-one (51%) patients discontinued ibrutinib due to disease progression/transformation, 20 (25%) for intolerance, 7 (9%) due to patient choice, 5 (6%) for stem cell transplant, 4 (5%) due to second cancers and 3 (4%) other causes. The median survival after ibrutinib was 10 and 6months for disease progression and transformation, respectively, and 25months for patients with ibrutinib intolerance. Overall, BTK mutations were observed in 17% patients after progression on ibrutinib. Notably, TP53 alterations were observed after progression in 75% patients. Among the 4 patients with blastoid transformation, 3 (75%) had NSD2 mutations (co-existing with TP53). Ibrutinib-refractory MCL patients had a short survival. Demonstration of TP53 and NSD2 mutations in patients who developed blastoid transformation and ATM and TP53 mutations in patients who progressed, opens the door for future investigations in ibrutinib-refractory MCL.

Abstract IA27: Deregulation and oncogenic functions of the NSD2/MMSET histone methyl transferase in hematologic malignancies

Abstract Epigenetic regulators of gene expression are among the commonest class of proteins mutated in human cancer. Most of these mutations lead to compete or total loss of function of the proteins leading to deregulated gene expression. Gain-of-function mutations in regulators are rare. The NSD2/MMSET histone methyltranferase is overexpressed in multiple myeloma associated with t(4;14) and leads to a global increase in histone 3 lysine 36 dimethylation, a chromatin mark associated with gene activation and a decrease of the inhibitory H3K27 methyl mark. As a result, gene expression is reprogrammed and many genes are aberrantly activated. Furthermore, the chromatin structure of cells with NSD2 overexpression is more “open.” In MM cells NSD2 overexpression increases MM cell growth and strikingly increases DNA repair processes making cells relatively resistant to chemotherapy, potentially explaining the poor prognosis of such patients. Proteomic studies show that NSD2 is associated with a variety of proteins involved in RNA metabolism, suggesting roles in both transcriptional and post-transcriptional processes. NSD2 is recurrently mutated in up to 15% of cases of relapsed childhood acute lymphocytic leukemia, in mantle cell lymphoma, and rarely in multiple myeloma and other tumors. This mutation increases the turnover rate of the enzyme in vitro and in vivo accelerates the conversion of unmodified histone to H3K36me1.The result is a global increase of H3K36me2 levels in cells with a concomitant decrease in H3k27me3 levels. Using CRISPR-mediated gene editing we eliminated this mutant allele, yielding reversal of histone methylation patterns, deceased growth, altered cellular adhesion, and increased susceptibility to chemotherapy. RNA-seq analysis showed that NSD2 mutation activated an aberrant program of cell adhesion resembling the epithelial-mesenchymal transition, increased motility, and augmented signaling and glucocorticoid and chemotherapy resistance likely associated with tumor progression. Mice harboring ALL cells with the E1099K mutation succumbed to disseminated leukemia faster than mice injected with cells in which the mutation was corrected. Furthermore, there was enhanced accumulation of cells with E1099K mutation in the brain, a common site of relapse in childhood ALL. Animal experiments suggest that knockdown of NSD2 could augment chemotherapy sensitivity motivating the search for NSD2 inhibitors. While modestly effective inhibitors have been identified to work in vitro, there are no inhibitors that are active in cells. A recently published crystal structure of an NSD2 structure is motivating our new in silico searches for candidate inhibitors. Citation Format: Jonathan D. Licht. Deregulation and oncogenic functions of the NSD2/MMSET histone methyl transferase in hematologic malignancies [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA27.

NMR backbone resonance assignment and solution secondary structure determination of human NSD1 and NSD2.

Proteins of the NSD family are histone-methyl transferases with critical functions in the regulation of chromatin structure and function. NSD1 and NSD2 are homologous proteins that function as epigenetic regulators of transcription through their abilities to catalyse histone methylation. Misregulation of NSD1 and NSD2 expression or mutations in their genes are linked to a number of human diseases such as Sotos syndrome, and cancers including acute myeloid leukemia, multiple myeloma, and lung cancer. The catalytic domain of both proteins contains a conserved SET domain which is involved in histone methylation. Here we report the backbone resonance assignments and secondary structure information of the catalytic domains of human NSD1 and NSD2.

Histone Profiling in Leukemia Using LC-MS/MS

It has become increasingly evident that epigenetic changes, including aberrant histone modifications, play a crucial role in malignant transformation and may contribute to relapsed and refractory disease. There are over 160 known histone modifications, most of which remain poorly explored. Post-translational histone modifications (PTMs) have been shown to be altered in hematologic malignancies, and the protein network that regulates these PTMs offers potential therapeutic opportunities through pharmacologic inhibition. Prominent examples include aberrant histone acetylation in multiple malignancies, mutations in the histone acetyl transferase CEBBP in relapsed acute lymphoblastic leukemia (ALL), and activation mutations of the H3K27 lysine methyltransferase (KMT) EZH2 in lymphoma. Several histone deacetylase (HDAC) and KMT inhibitors are currently in clinical trials. Most mutations in the histone modifying enzymes have been identified by whole genome sequencing efforts. More recently, mass spectrometry has been proposed as a technique to identify patterns of histone modifications in cancer. Jaffe et al (Nature Genetics, 2013) developed a mass spectrometry approach to global chromatin profiling using leukemia cell lines. Their initial analysis focused on lysine methylation and acetylation, including some of the best characterized residues (H3K4, H3K9, H3K27, H3K36, H3K79). This proof of principle study showed that histone profiles matched what would be predicted based on our knowledge of genetic mutations. More importantly, profiles could serve as leads to discover previously unknown mutations in chromatin modifying enzymes.We adapted the methods established by Jaffe et al to histone modification profiling using small amounts of primary material from leukemia patients. Our work flow involves enrichment of blasts by ficoll centrifugation or fluorescent activated cell sorting, isolation highly purified histones from patient samples using a resin column followed by gel electrophoresis, digestion with ArgC and Trypsin, and nanoscale liquid chromatography followed by tandem mass spectrometry (nano LC-MS/MS). We established a leukemia cell line panel labeled with heavy (13C6) arginine and heavy (13C6) lysine for SILAC based internal standardization. Initially focusing our analysis on H3K27 and H3K36 methylation and using published retention times and m/z ratios, we were able to identify almost all major combinatorial acetylation and methylation states for the two residues (Table 1). We also identified methylation/acetylation states for H3K9 (ac), H3K14 (ac), H3K18 (ac, me), H3K23 (ac), and H3K79(me, me2) (data not shown). Preliminary experiments using a cell line with an activating NSD2 mutation correctly identified increased H3K36 dimethylation and decreased H2K27 trimethylation compared to the SILAC standard. We will use this method to correlate histone profiles of patient leukemia samples with clinical and molecular information as a means of interrogating the potential role for a number of known histone modifying enzymes in a single sample. In the future, we hope to extend our analysis to less well characterized histone PTMs. Table 1Lysine modifications found on histone H3.1 in Jurkat cell line. Percentages in BOLD = Arg-C digestion; Regular = Trypsin digestion.H3.1 ResidueCombinationsUnmodifiedAcetylMethylDimethylTrimethylK27 total K27unmod/K36 K27me1/K36 K27me2/K36 K27me3/K36 K27ac/K3614%/8% 71%66%33%100%/100%36%41%100% 22%60%31%/25% 29%33%44%40%14%/31%K36 total63%/44%6%/14%31%/20% DisclosuresNo relevant conflicts of interest to declare.

Abstract 2930: Global chromatin profiling reveals NSD2 mutation in pediatric ALL

Abstract Epigenetic dysregulation is an emerging hallmark of cancers. We developed a high-information-content mass spectrometry approach to profile global histone modifications in human cancers. When applied to 115 lines of the Cancer Cell Line Encyclopedia1, this approach identified distinct molecular chromatin signatures. One signature was characterized by increased H3K36 dimethylation, exhibited by several lines harboring NSD2 translocations. A novel NSD2 p.E1099K variant was identified in non-translocated acute lymphoblastic leukemia (ALL) lines sharing this signature. Ectopic expression of the variant induced a chromatin signature characteristic of NSD2 hyperactivation and promoted transformation. NSD2 knockdown selectively inhibited the proliferation of NSD2-mutant lines and impaired the in vivo growth of an NSD2-mutant ALL xenograft. Sequencing analysis of >1000 pediatric cancer genomes identified the NSD2 p.E1099K mutation in 14% of t(12;21)[ETV6-RUNX1]-containing ALLs. These findings identify NSD2 as a potential therapeutic target for pediatric ALL and provide a general framework for the functional annotation of cancer epigenomes. 1. Barretina,J. et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603-607 (2012). Citation Format: Ho Man Chan, Jacob D. Jaffe, Yan Wang, Jinghui Zhang, Robert Huether, Gregory V. Kryukov, Hyo-eun C. Bhang, Jordan E. Taylor, Min Hu, Nathan P. Englund, Feng Yan, Zhaofu Wang, E Robert McDonald III, Lei Wei, Jing Ma, John Easton, Zhengtian Yu, Rosalie deBeaumount, Veronica Gibaja, Kavitha Venkatesan, Robert Schlegel, William R. Sellers, Nicholas Keen, Jun Liu, Giordano Caponigro, Jordi Barretina, Vesselina G. Cooke, Charles Mullighan, Steven A. Carr, James R. Downing, Levi A. Garraway, Frank Stegmeier. Global chromatin profiling reveals NSD2 mutation in pediatric ALL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2930. doi:10.1158/1538-7445.AM2014-2930

Abstract PR01: Global chromatin profiling identifies NSD2 mutations in pediatric acute lymphoblastic leukemia

Abstract Epigenetic dysregulation is an emerging hallmark of cancers, and the identification of recurrent somatic mutations in chromatin modifying enzymes implies a causal role for altered chromatin states in tumorigenesis. While the genomic characterization of cancers is well advanced, our current understanding of cancer epigenomes is limited. Here, we developed a high-information-content mass spectrometry approach to profile global histone modifications in human cancer cells. When applied to 115 cell lines of the Cancer Cell Line Encyclopedia, this approach identified distinct molecular chromatin signatures (MCS) that provided novel insights into their epigenetic states. One MCS cluster, characterized by high H3K27 trimethylation, strongly correlated with EZH2 gain-of-function mutations, whereas another MCS cluster provided functional epigenetic correlates of EZH2 loss-of-function mutations. A third MCS cluster was characterized by increased H3K36 dimethylation and contained several cell lines harboring NSD2 translocations. Analysis of genomic correlates of the remaining cell lines within this third cluster identified a novel NSD2 E1099K variant in acute lymphoblastic leukemia (ALL) lines. Ectopic expression of the NSD2 E1099K variant induced a MCS profile characteristic of NSD2 hyperactivation and promoted transformation. Moreover, NSD2 knockdown selectively inhibited the proliferation of cell lines harboring NSD2 mutations. Massively parallel sequencing analysis of over 1000 pediatric cancer genomes identified the same NSD2 E1099K mutation in 14% of t(12;21)[ETV6-RUNX1]-containing ALLs. Together, these findings identify NSD2 as a potential therapeutic target for pediatric ALL and provide a general framework for the functional annotation of cancer epigenomes. Citation Format: Jacob Jaffe, Yan Wang, HoMan Chan, Jinghui Zhang, Roberts Huether, Gregory Kryukov, Jordan Taylor, Min Hu, Nathan Englund, Feng Yan, Zhaofu Wang, Lei Wei, Lei Wei, Jing Ma, John Easton, William R. Sellers, Nicholas Keen, Jun Liu, Jun Liu, Charles Mullighan, Steven Carr, James Downing, Levi Garraway, Frank Stegmeier. Global chromatin profiling identifies NSD2 mutations in pediatric acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr PR01.