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Mutations in epigenetic regulators are common in relapsed pediatric acute lymphoblastic leukemia (ALL). Here, we uncovered the mechanism underlying the relapse of ALL driven by an activating mutation of the NSD2 histone methyltransferase (p.E1099K). Using high-throughput drug screening, we found that NSD2-mutant cells were specifically resistant to glucocorticoids. Correction of this mutation restored glucocorticoid sensitivity. The transcriptional response to glucocorticoids was blocked in NSD2-mutant cells due to depressed glucocorticoid receptor (GR) levels and the failure of glucocorticoids to autoactivate GR expression. Although H3K27me3 was globally decreased by NSD2 p.E1099K, H3K27me3 accumulated at the NR3C1 (GR) promoter. Pretreatment of NSD2 p.E1099K cell lines and patient-derived xenograft samples with PRC2 inhibitors reversed glucocorticoid resistance in vitro and in vivo. PRC2 inhibitors restored NR3C1 autoactivation by glucocorticoids, increasing GR levels and allowing GR binding and activation of proapoptotic genes. These findings suggest a new therapeutic approach to relapsed ALL associated with NSD2 mutation. Significance: NSD2 histone methyltransferase mutations observed in relapsed pediatric ALL drove glucocorticoid resistance by repression of the GR and abrogation of GR gene autoactivation due to accumulation of K3K27me3 at its promoter. Pretreatment with PRC2 inhibitors reversed resistance, suggesting a new therapeutic approach to these patients with ALL. This article is highlighted in the In This Issue feature, p. 1

Primary headaches may be accompanied by increased intracranial blood flow induced by the release of the potent vasodilator calcitonin gene-related peptide (CGRP) from activated meningeal afferents. We aimed to record meningeal and medullary blood flow simultaneously and to localize the sites of CGRP release in rodent preparations in vivo and ex vivo. Blood flow in the exposed rat parietal dura mater and the medulla oblongata was recorded by laser Doppler flowmetry, while the dura was stimulated by topical application of 60mM potassium chloride (KCl). Samples of jugular venous plasma and cerebrospinal fluid (CSF) collected from the cisterna magna were analysed for CGRP concentrations using an enzyme immunoassay. In a hemisected rat skull preparation lined with dura mater the CGRP releasing effect of KCl superfusion was examined. Superfusion of the dura mater with KCl decreased meningeal blood flow unless alpha-adrenoceptors were blocked by phentolamine, whereas the medullary blood flow was increased. The same treatment caused increased CGRP concentrations in jugular plasma and CSF and induced significant CGRP release in the hemisected rat skull preparation. Anaesthesia of the trigeminal ganglion by injection of lidocaine reduced increases in medullary blood flow and CGRP concentration in the CSF upon meningeal KCl application. CGRP release evoked by depolarisation of meningeal afferents is accompanied by increased blood flow in the medulla oblongata but not the dura mater. This discrepancy can be explained by the smooth muscle depolarising effect of KCl and the activation of sympathetic vasoconstrictor mechanisms. The medullary blood flow response is most likely mediated by CGRP released from activated central terminals of trigeminal afferents. Increased blood supply of the medulla oblongata and CGRP release into the CSF may also occur in headaches accompanying vigorous activation of meningeal afferents.

Background: NSD2 (nuclear receptor binding SET domain protein 2) is a histone methyltransferase specific for dimethylation of histone H3 lysine 36 (H3K36me2), a modification associated with gene activation. In pediatric acute lymphoblastic leukemia (ALL), particularly at relapse, a gain of function mutation (E1099K) of NSD2 is found in 10-15% of cases. The NSD2 mutation is found in addition to fusion proteins such as E2A-PBX and ETV6-RUNX1. The mutation can be subclonal at diagnosis and dominant at relapse, suggesting a link to therapeutic resistance. The NSD2-E1099K mutation affects gene expression through an increase in H3K36me2 and a decrease in H3K27me3. Using CRISPR/Cas9-edited isogenic ALL cell lines, we found that NSD2-E1099K mutation drove oncogenic programming by altering chromatin architecture, gene expression and enhancing cell growth, migration and infiltration to the central neural system (CNS). NSD2 mutation caused resistance of ALL cells to glucocorticoids (GC) by blocking genome wide binding of the glucocorticoid receptor (GR, encoded by NR3C1 gene) preventing GC-mediated induction of pro-apoptotic genes. NR3C1 levels were depressed in NSD2-E1099K cells and GC failed to induce autoactivation of NR3C1. While H3K27me3 was globally decreased by NSD2-E1099K, increased H3K27me3 was noted at the promoter of NR3C1, suggesting a novel role of polycomb repressive complex 2 as a therapeutic target for relapsed ALL with NSD2 mutation. While NSD2 is highly expressed in B cells and NSD2 knockout causes defects in B cell development, how the NSD2 mutation affects B cell development and leukemia occurrence in vivo is uncertain. Aims: To determine the role of NSD2 mutation in the pathogenesis of lymphocytic malignancies and GC resistance in a mouse model. Methods: We generated a conditional NSD2-E1099K knock-in mouse model in which the NSD2-E1099K allele was placed in the Rosa26 locus and expressed in B cells under the control of Cd19-Cre (Cd19+/-NSD2E1099K/WT). The resulting phenotype was characterized through peripheral blood counts, cellular morphology and histology of blood smears, bone marrow (BM), spleen and liver, flow cytometric analysis, germinal center B cells (GCB) immunization, BM transplantation, and hematopoiesis analysis in a CD3-/- background. We further established mouse leukemia cell lines with NSD2 mutation for functional analysis. RNA-Seq, real time PCR, immunoblotting, and apoptosis analysis (Annexin V/PI staining) following GC treatment were performed to demonstrate the effects of NSD2 mutation on histone modifications, transcriptome and GC resistance. Results: The NSD2-E1099K mutation increased H3K36me2 and decreased H3K27me3 in isolated B cells from mouse BM and spleen. Mice were aged and did not develop signs of malignancy and RNA-sequencing showed few differences between B cells with or without the NSD2 mutation. However, after immunizing the mice with sheep red blood cells (SRBC), more GCBs were seen in the spleen of NSD2 mutant mice, suggesting mutant NSD2 stimulated germinal center hyperplasia. Transplantation of BM cells from mice expressing NSD2-E1099K into lethally irradiated recipients lead to an expansion of B cells while myeloid and T cells and life span of the recipients impaired. The NSD2 knock-in mouse model was crossed with Cd3-/- mice to create Cd19+/-Cd3-/-NSD2E1099K/WT mice, which within 2 months of birth developed a disease resembling an immature B lymphocytic leukemia (B220+CD19+IgM+IgD-CD5-) with infiltration of the spleen, liver and CNS and a median survival of 4.8 months. These tumors could be transplanted into immunodeficient mice but not immunocompetent mice. RNA seq analysis of these cells revealed 6,815 genes (3,295 upregulated and 3,520 downregulated) differentially expressed in NSD2 mutant B cells compared to normal B cells. The upregulated genes were related to abnormal immunoglobulin level , B cell activation, T-helper 1 physiology, and decreased B cell apoptosis. Importantly, the NSD2 mutant leukemic cells displayed depressed level of NR3C1 gene expression and GC resistance. Conclusions: The NSD2 mutation alters B cell development, particularly in an immunodeficient background and causes B cells to become resistant to glucocorticoids. The inability of the mutation to generate disease on its own except in an immunodeficient background suggests genes that collaborate with NSD2 in ALL may play a role in immune escape. Disclosures No relevant conflicts of interest to declare.

By examination of the cancer genomics database, we identified a new set of mutations in core histones that frequently recur in cancer patient samples and are predicted to disrupt nucleosome stability. In support of this idea, we characterized a glutamate to lysine mutation of histone H2B at amino acid 76 (H2B-E76K), found particularly in bladder and head and neck cancers, that disrupts the interaction between H2B and H4. Although H2B-E76K forms dimers with H2A, it does not form stable histone octamers with H3 and H4 in vitro, and when reconstituted with DNA forms unstable nucleosomes with increased sensitivity to nuclease. Expression of the equivalent H2B mutant in yeast restricted growth at high temperature and led to defective nucleosome-mediated gene repression. Significantly, H2B-E76K expression in the normal mammary epithelial cell line MCF10A increased cellular proliferation, cooperated with mutant PIK3CA to promote colony formation, and caused a significant drift in gene expression and fundamental changes in chromatin accessibility, particularly at gene regulatory elements. Taken together, these data demonstrate that mutations in the globular domains of core histones may give rise to an oncogenic program due to nucleosome dysfunction and deregulation of gene expression. Significance: Mutations in the core histones frequently occur in cancer and represent a new mechanism of epigenetic dysfunction that involves destabilization of the nucleosome, deregulation of chromatin accessibility, and alteration of gene expression to drive cellular transformation. See related commentary by Sarthy and Henikoff, p. 1346. This article is highlighted in the In This Issue feature, p. 1325

MMSET/WHSC1 is a histone methyltransferase (HMT) overexpressed in t(4;14)+ multiple myeloma (MM) patients, believed to be the driving factor in the pathogenesis of this MM subtype. MMSET overexpression in MM leads to an increase in histone 3 lysine 36 dimethylation (H3K36me2), and a decrease in histone 3 lysine 27 trimethylation (H3K27me3), as well as changes in proliferation, gene expression and chromatin accessibility. Prior work linked methylation of histones to the ability of cells to undergo DNA damage repair. In addition, t(4;14)+ patients frequently relapse after regimens that include DNA damage-inducing agents, suggesting that MMSET may play a role in DNA damage repair and response. In U2OS cells, we found that MMSET is required for efficient non-homologous end joining as well as homologous recombination. Loss of MMSET led to loss of expression of several DNA repair proteins, as well as decreased recruitment of DNA repair proteins to sites of DNA double-strand breaks (DSBs). By using genetically matched MM cell lines that had either high (pathological) or low (physiological) expression of MMSET, we found that MMSET-high cells had increased damage at baseline. Upon addition of a DNA-damaging agent, MMSET-high cells repaired DNA damage at an enhanced rate and continued to proliferate, whereas MMSET-low cells accumulated DNA damage and entered cell cycle arrest. In a murine xenograft model using t(4;14)+ KMS11 MM cells harboring an inducible MMSET shRNA, depletion of MMSET enhanced the efficacy of chemotherapy, inhibiting tumor growth and extending survival. These findings help explain the poorer prognosis of t(4;14) MM and further validate MMSET as a potential therapeutic target in MM and other cancers.

Activity within the CNS can be quantified by a variety of methods. Here, we present an indirect method utilizing the neuro-vascular coupling via a continuous measurement of the vessel diameter. In anaesthetized rats, induced neuronal activity in the trigeminal system could be detected via arterial diameter measurements at the back of the medullary brainstem. Building upon the previously described diameter plugin, in images aligned to compensate for ventilation and heart-rate associated movement, automated processing allowed an evaluation immediately after acquisition. Electrical stimulation of facial trigeminal areas or the cranial dura mater caused vasodilatation of arteries in the dorsal medullary brainstem, which was abolished after application of the CGRP receptor antagonist olcegepant. No change in diameter was observed in corresponding veins. Intravenous infusion of sodium sulfide, which induces nitroxyl formation, also induced vasodilatation of brainstem-supplying arteries. Both experiments show a functional role of CGRP in the trigeminal nuclear brainstem complex. The presented method allows estimating central activity at the spinal level by vascular responses.

Unrestrained receptor tyrosine kinase (RTK) signaling and epigenetic deregulation are root causes of tumorigenesis. We establish linkage between these processes by demonstrating that aberrant RTK signaling unleashed by oncogenic HRas(G12V) or loss of negative feedback through Sprouty gene deletion remodels histone modifications associated with active typical and super-enhancers. However, although both lesions disrupt the Ras-Erk axis, the expression programs, enhancer signatures, and transcription factor networks modulated upon HRas(G12V) transformation or Sprouty deletion are largely distinct. Oncogenic HRas(G12V) elevates histone 3 lysine 27 acetylation (H3K27ac) levels at enhancers near the transcription factor Gata4 and the kinase Prkcb, as well as their expression levels. We show that Gata4 is necessary for the aberrant gene expression and H3K27ac marking at enhancers, and Prkcb is required for the oncogenic effects of HRas(G12V)-driven cells. Taken together, our findings demonstrate that dynamic reprogramming of the cellular enhancer landscape is a major effect of oncogenic RTK signaling.

Background and purpose Meningeal blood flow is controlled by CGRP released from trigeminal afferents and NO mainly produced in arterial endothelium. The vasodilator effect of NO may be due to the NO-derived compound, nitroxyl (HNO), generated through reaction with endogenous H2 S. We investigated the involvement of HNO in CGRP release and meningeal blood flow. Experimental approach Blood flow in exposed dura mater of rats was recorded by laser Doppler flowmetry. CGRP release from the dura mater in the hemisected rat head was quantified using an elisa. NO and H2 S were localized histochemically with specific sensors. Key results Topical administration of the NO donor diethylamine-NONOate increased meningeal blood flow by 30%. Pretreatment with oxamic acid, an inhibitor of H2 S synthesis, reduced this effect. Administration of Na2 S increased blood flow by 20%, an effect abolished by the CGRP receptor antagonist CGRP8-37 or the TRPA1 channel antagonist HC030031 and reduced when endogenous NO synthesis was blocked. Na2 S dose-dependently increased CGRP release two- to threefold. Co-administration of diethylamine-NONOate facilitated CGRP release, while inhibition of endogenous NO or H2 S synthesis lowered basal CGRP release. NO and H2 S were mainly localized in arterial vessels, HNO additionally in nerve fibre bundles. HNO staining was lost after treatment with L-NMMA and oxamic acid. Conclusions and implications NO and H2 S cooperatively increased meningeal blood flow by forming HNO, which activated TRPA1 cation channels in trigeminal fibres, inducing CGRP release. This HNO-TRPA1-CGRP signalling pathway may be relevant to the pathophysiology of headaches.

NSD2, a histone methyltransferase specific for methylation of histone 3 lysine 36 (H3K36), exhibits a glutamic acid to lysine mutation at residue 1099 (E1099K) in childhood acute lymphocytic leukemia (ALL), and cells harboring this mutation can become the predominant clone in relapsing disease. We studied the effects of this mutant enzyme in silico, in vitro, and in vivo using gene edited cell lines. The E1099K mutation altered enzyme/substrate binding and enhanced the rate of H3K36 methylation. As a result, cell lines harboring E1099K exhibit increased H3K36 dimethylation and reduced H3K27 trimethylation, particularly on nucleosomes containing histone H3.1. Mutant NSD2 cells exhibit reduced apoptosis and enhanced proliferation, clonogenicity, adhesion, and migration. In mouse xenografts, mutant NSD2 cells are more lethal and brain invasive than wildtype cells. Transcriptional profiling demonstrates that mutant NSD2 aberrantly activates factors commonly associated with neural and stromal lineages in addition to signaling and adhesion genes. Identification of these pathways provides new avenues for therapeutic interventions in NSD2 dysregulated malignancies.

Despite improvements in chemotherapy that have increased the 5-year survival rates of pediatric ALL to close to 90%, 15-20% of patients may relapse with a very poor prognosis. Pediatric ALL patients, particularly those in relapse can harbor a specific point mutation (E1099K) in NSD2 (nuclear receptor binding SET domain protein 2) gene, also known as MMSET or WHSC1, which encodes a histone methyl transferase specific for H3K36me2. To understand the biology of mutant NSD2, we used CRISPR-Cas9 gene editing to disrupt the NSD2E1099K mutant allele in B-ALL cell lines (RCH-ACV and SEM) and T-ALL cell line (RPMI-8402) or insert the E1099K mutation into the NSD2WT T-ALL cell line (CEM) and B-ALL cell line (697). Cell lines in which the NSD2E1099K mutant allele is present display increased global levels of H3K36me2 and decreased H3K27me3. NSD2E1099Kcells demonstrate enhanced cell growth, colony formation and migration. NSD2E1099K mutant cell lines assayed by RNA-Seq exhibit an aberrant gene signature, mostly representing gene activation, with activation of signaling pathways, genes implicated in the epithelial mesenchymal transition and prominent expression of neural genes not generally found in hematopoietic tissues. Accordingly, NSD2E1099K cell lines showed prominent tropism to the central neural system in xenografts. To understand why this NSD2 mutations are identified prominently in children who relapse early from therapy for ALL, we performed high-throughput screening in our isogenic cell lines with the National Center for Advancing Translation Science (NCATS) Pharmaceutical Collection and other annotated chemical libraries and found that NSD2E1099K cells are resistant to glucocorticoids (GC) but not to other chemotherapeutic agents used to treat ALL such as vincristine, doxorubicin, cyclophosphamide, methotrexate, and 6-mercaptopurine. Accordingly, patient-derived-xenograft ALL cells with NSD2E1099K mutation were resistant to GC treatment. Reversion of NSD2E1099K mutation to NSD2WT restored GC sensitivity to both B- and T-ALL cell lines, which was accompanied by cell cycle arrest in G1 and induced-apoptosis. Furthermore, knock-in of the NSD2E1099K mutation conferred GC resistance to ALL cell lines by triggering cell cycle progression, proliferation and anti-apoptotic processes. Mice with NSD2E1099K xenografts were completely resistant to GC treatment while treatment of mice injected with isogenic NSD2WT cells led to significant tumor reduction and survival benefit. To illustrate these biological phenotypes and understand the molecular mechanism of GC resistance driven by NSD2E1099Kmutation, we investigated the GC-induced transcriptome, GC receptor (GR) binding sites and related epigenetic changes in isogenic ALL cell lines in response to GC treatment. RNA-Seq showed that GC transcriptional response was almost completely blocked in NSD2E1099K cells, especially in T-ALL cell lines, correlating with their lack of biological response. GC treatment activated apoptotic pathways and downregulated cell cycle and DNA repair pathways only in NSD2WT cells. The critical pro-apoptotic regulators BIM and BMF failed to be activated by GC in NSD2E1099K cells but were prominently activated when the NSD2 mutation was removed. Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that, the NSD2E1099K mutation blocked the ability of GR and CTCF to bind most GC response elements (GREs) such as those within BIM and BMF. While GR binding in NSD2WT cells was accompanied by increased H3K27 acetylation and gene expression, this failed to occur in NSD2 mutant cells. Furthermore, we found that GR RNA and protein levels were repressed in ALL cells expressing NSD2E1099K and GC failed to induce GR expression in these cells. Paradoxically, while H3K27me3 levels were generally decreased in NSD2E1099K cells, we saw increased levels of H3K27me3 at the GRE within the GR gene body where GR itself and CTCF normally bind, suggesting a novel role for the polycomb repressive complex 2 and EZH2 inhibitors for this form of GC resistance. In conclusion, these studies demonstrate that NSD2E1099K mutation may play an important role in treatment failure of pediatric ALL relapse by interfering with the GR expression and its ability to bind and activate key target genes. Gene editing screens are being performed to understand how to overcome this resistance. Disclosures No relevant conflicts of interest to declare.

Epigenetic deregulation of gene expression plays a role in the initiation and progression of prostate cancer (PCa). The histone methyltransferase MMSET/WHSC1 (Multiple Myeloma Set Domain) is overexpressed in a number of metastatic tumors, but its mechanism of action has not been defined. In this work, we found that PCa cell lines expressed significantly higher levels of MMSET compared to immortalized, non-transformed prostate cells. Knockdown experiments showed that, in metastatic PCa cell lines, dimethylation of lysine 36 and trimethylation of lysine 27 on histone H3 (H3K36me2 and H3K27me3, respectively) depended on MMSET expression, while depletion of MMSET in benign prostatic cells did not affect chromatin modifications. Knockdown of MMSET in DU145 and PC-3 tumor cells decreased cell proliferation, colony formation in soft agar, and strikingly diminished cell migration and invasion. Conversely, overexpression of MMSET in immortalized, non-transformed RWPE-1 cells promoted cell migration and invasion, accompanied by an epithelial to mesenchymal transition (EMT). Among a panel of EMT-promoting genes analyzed, TWIST1 expression was strongly activated in response to MMSET. Chromatin immunoprecipitation analysis demonstrated that MMSET binds to the TWIST1 locus, leading to an increase in H3K36me2, suggesting a direct role of MMSET in the regulation of this gene. Depletion of TWIST1 in MMSET-overexpressing RWPE-1 cells blocked cell invasion and EMT, indicating that TWIST1 was a critical target of MMSET, responsible for the acquisition of an invasive phenotype. Collectively, these data suggest that MMSET plays a role in PCa pathogenesis and progression through epigenetic regulation of metastasis-related genes.

The multiple myeloma SET domain (MMSET) protein is overexpressed in multiple myeloma (MM) patients with the translocation t(4;14). Although studies have shown the involvement of MMSET/Wolf-Hirschhorn syndrome candidate 1 in development, its mode of action in the pathogenesis of MM is largely unknown. We found that MMSET is a major regulator of chromatin structure and transcription in t(4;14) MM cells. High levels of MMSET correlate with an increase in lysine 36 methylation of histone H3 and a decrease in lysine 27 methylation across the genome, leading to a more open structural state of the chromatin. Loss of MMSET expression alters adhesion properties, suppresses growth, and induces apoptosis in MM cells. Consequently, genes affected by high levels of MMSET are implicated in the p53 pathway, cell cycle regulation, and integrin signaling. Regulation of many of these genes required functional histone methyl-transferase activity of MMSET. These results implicate MMSET as a major epigenetic regulator in t(4;14)+ MM.

Acute lymphoblastic leukemia (ALL) is the most common diagnosed pediatric cancer. Despite improvements in chemotherapy that have increased the 5-year survival rate to close to 90%, 15-20% of these patients may relapse with the majority of such children succumbing to this disease. Pediatric ALL patients, particularly those in relapse can harbor a specific point mutation (E1099K) in NSD2 (nuclear receptor binding SET domain protein 2) gene, also known as MMSET or WHSC1, which encodes a histone methyl transferase specific for H3K36me2. To understand the biological processes mediated by mutant NSD2, we used CRISPR-Cas9 gene editing to disrupt the NSD2E1099K mutant allele in two B-ALL cell lines (RCH-ACV and SEM) and one T-ALL cell line (RPMI-8402) and inserted the E1099K mutation into three ALL cell lines (697, CEM, MOLT4). Cell lines in which the NSD2E1099K mutant allele is present display increased global levels of H3K36me2 and decreased H3K27me3. NSD2E1099Kcells compared to cells in which the mutation is removed demonstrate enhanced cell growth, colony formation and migration. NSD2 mutant cell lines assayed by RNA-Seq exhibit an aberrant gene signature, mostly representing gene activation, with activation of signaling pathways, genes implicated in the epithelial mesenchymal transition and prominent expression of neural genes not generally found in hematopoietic tissues. Accordingly, NSD2E1099K cell lines showed prominent tropism to the central neural system (CNS) in xenografts. The NSD2 mutation is found prominently in children who relapse early from therapy for ALL, and NSD2E1099K cells are particularly resistant to glucocorticoids (GC). Reversion of NSD2E1099K mutation to wild type NSD2 conferred glucocorticoid sensitivity to both B and T cell lines. GC response upon disruption of mutant NSD2 was accompanied by cell cycle arrest and apoptosis. Mice xenografted with NSD2E1099K cells were completely resistant to GC treatment while treatment of mice injected with isogenic NSD2 wild-type cells led to significant tumor reduction and survival extension. RNA-Seq analysis showed that GC transcriptional response was almost completely blocked in NSD2E1099K cells, correlating with their lack of biological response. GC treatment activated apoptotic pathways and downregulated cell cycle and DNA repair pathways only in NSD2 wild-type cells. Furthermore, in NSD2 mutant cells, there was lower basal expression level of glucocorticoid receptor (GR) and GR levels were not significantly induced by GC. Accordingly, after treatment with GC, there was significantly less DNA-binding activity of the GR in NSD2E1099K cells than that of NSD2 wild-type cells. The key pro-apoptotic regulators Bim and BMF failed to be activated by GC in NSD2E1099K cells but were prominently activated when the NSD2 mutation was removed. In conclusion, these studies demonstrate that the NSD2E1099K mutation may play an important role in treatment failure of pediatric ALL relapse by causing GC resistance. Future studies will determine how NSD2 which generally activates genes paradoxically blocks the ability of GC and the GR to induce critical pro-death genes. Disclosures Licht: Celgene: Research Funding.

In multiple myeloma (MM), inactivating mutations and deletions encompassing the histone demethylase KDM6A locus are found in up to 10% of newly diagnosed patients and associated with poor prognosis. Future sequencing studies may show an increased incidence of mutation with disease relapse. KMD6A (also named UTX, Ubiquitously transcribed Tetratricopeptide repeat, X chromosome) belongs to a family of Jumonji-C (Jmj-C)-containing demethylases working as a scaffold for a multiprotein complex containing H3K4-specific methyltransferases KMT2D and/or KMT2C (MLL2/3), the histone acetyltransferase CBP/p300, and members of the SWI/SNF chromatin-remodeling complex. In a concerted manner this complex appears to remove the gene repression associated methylation of lysine 27 on histone H3 (H3K27me), a mark placed by EZH2. Hence loss-of-function mutations of KDM6A may affect the function of this complex, which is common in B-cell malignancies. We found that KDM6A act as a tumor suppressor in MM in vivo and in vitro, but how this exactly happens and which genes are affected is incompletely understood. We modeled the loss of KDM6A in MM in vitro, using a pair of cell lines, ARP-1 (KDM6A wild type) and ARD (homozygous KDM6A deletion), derived from the same MM patient. As well, we used CRISPR-Cas9 mediated genome editing to disrupt KDM6A gene in cell lines. We examined gene expression profiles in ARP-1 vs. ARD cells, and vs. ARD cells upon reexpression of KDM6A, in a doxycycline-inducible manner, by whole transcriptome (RNA sequencing). To understand how KDM6A demethylase activity contributes to tumor suppressive role of KDM6A, we abolished the demethylase activity using directed mutagenesis targeting the JmjC domain. Disruptive mutations in functional domain of KDM6A provide a growth advantage in MM cell in vitro. Many genes induced upon KDM6A add-back are found upregulated in KDM6A wild-type ARP-1 cells. Mass spectrometry analysis of these cell lines and the add-back system showed no difference in global H3K27me3 levels, suggesting that the tumor suppressive role of KDM6A does not involve alteration of H3K27 methylation or that the changes in this histone mark following KDM6A loss are loci-specific. Reexpression of JmjC-dead KDM6A and wt KDM6A had similar effect on growth and clonogenicity, suggesting that demethylase activity is dispensable. KDM6A demethylase activity is dispensable for the tumor-suppressive effect of KDM6A in MM. Loss of KDM6A alters the transcriptome of MM cells. We therefore hypothesized that the scaffolding properties of KDM6A may be essential for the tumor-suppressive role of KDM6A. We plan to use CRISPR-Cas9 mediated genome editing to create specific mutations and flag-insertion in KDM6A gene, in both cell lines and mouse model, to further investigate the molecular mechanism of KDM6A activity. Citation Format: Daphne Dupere-Richer, Teresa Ezponda, Christine Will, Eliza Caroline Small, Nobish Varghese, Patel Tej, Xiaoxiao Huang, Zheng Yupeng, Giovanni Tonon, Neil Kelleher, Jonathan Keats, Jonathan D. Licht. KMD6A/UTX loss enhances the malignant phenotype of multiple myeloma and sensitizes cells to EZH2 inhibition [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 12.

Meningeal blood flow is controlled by CGRP released from trigeminal afferents and NO mainly produced in arterial endothelium. The vasodilator effect of NO may be due to the NO-derived compound, nitroxyl (HNO), generated through reaction with endogenous H2 S. We investigated the involvement of HNO in CGRP release and meningeal blood flow.Blood flow in exposed dura mater of rats was recorded by laser Doppler flowmetry. CGRP release from the dura mater in the hemisected rat head was quantified using an elisa. NO and H2 S were localized histochemically with specific sensors.Topical administration of the NO donor diethylamine-NONOate increased meningeal blood flow by 30%. Pretreatment with oxamic acid, an inhibitor of H2 S synthesis, reduced this effect. Administration of Na2 S increased blood flow by 20%, an effect abolished by the CGRP receptor antagonist CGRP8-37 or the TRPA1 channel antagonist HC030031 and reduced when endogenous NO synthesis was blocked. Na2 S dose-dependently increased CGRP release two- to threefold. Co-administration of diethylamine-NONOate facilitated CGRP release, while inhibition of endogenous NO or H2 S synthesis lowered basal CGRP release. NO and H2 S were mainly localized in arterial vessels, HNO additionally in nerve fibre bundles. HNO staining was lost after treatment with L-NMMA and oxamic acid.NO and H2 S cooperatively increased meningeal blood flow by forming HNO, which activated TRPA1 cation channels in trigeminal fibres, inducing CGRP release. This HNO-TRPA1-CGRP signalling pathway may be relevant to the pathophysiology of headaches.

Overexpression of the histone methyltransferase MMSET in t(4;14)+ multiple myeloma patients is believed to be the driving factor in the pathogenesis of this subtype of myeloma. MMSET catalyzes dimethylation of lysine 36 on histone H3 (H3K36me2), and its overexpression causes a global increase in H3K36me2, redistributing this mark in a broad, elevated level across the genome. Here, we demonstrate that an increased level of MMSET also induces a global reduction of lysine 27 trimethylation on histone H3 (H3K27me3). Despite the net decrease in H3K27 methylation, specific genomic loci exhibit enhanced recruitment of the EZH2 histone methyltransferase and become hypermethylated on this residue. These effects likely contribute to the myeloma phenotype since MMSET-overexpressing cells displayed increased sensitivity to EZH2 inhibition. Furthermore, we demonstrate that such MMSET-mediated epigenetic changes require a number of functional domains within the protein, including PHD domains that mediate MMSET recruitment to chromatin. In vivo, targeting of MMSET by an inducible shRNA reversed histone methylation changes and led to regression of established tumors in athymic mice. Together, our work elucidates previously unrecognized interplay between MMSET and EZH2 in myeloma oncogenesis and identifies domains to be considered when designing inhibitors of MMSET function., Author Summary Precise spatial and temporal gene expression is required for normal development, and aberrant regulation of gene expression is a common factor in many diseases, including cancer. Histone modifications contribute to the control of gene expression by altering chromatin structure and affecting the recruitment of transcriptional regulators. In this study, we demonstrate interplay between two oncogenic proteins, MMSET and EZH2, known to methylate histone H3 on lysine 36 (H3K36) and lysine 27 (H3K27), respectively. Overexpression of MMSET in myeloma cells increases global levels of H3K36 methylation, alters its normal distribution throughout the genome and decreases global levels of H3K27 methylation. We found that while the majority of the genome loses H3K27 methylation in the presence of MMSET, certain loci have augmented recruitment of EZH2 and enhanced H3K27 methylation, leading to transcriptional repression. Repression of these genes likely plays an important role in the disease because MMSET-overexpressing cells show higher sensitivity to small molecule inhibitors targeting EZH2-mediated methylation. Thus, our study suggests that the specific local changes may outweigh the gross global changes we frequently observe in cancer and implicates EZH2 as a novel therapeutic target in myeloma cells.

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences–Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis., National Cancer Institute (U.S.) (Grant U54CA143874)

Abstract 469 In multiple myeloma recurrent chromosomal translocations lead to the overexpression of oncogenes such as MAF, MAFB and MMSET. An underlying paradox of MM is the continued proliferation and self-renewal of a highly differentiated cell. Plasma cell development requires the BLIMP1 transcription factor, which stimulates terminal B cell differentiation and represses expression of c-MYC, a master regulator of cell growth. By contrast, malignant plasma cells express c-MYC and proliferate while maintaining the specialized machinery required for immunoglobulin production. MMSET gene is overexpressed in about 15% of cases of MM due to chromosomal translocation (4;14). In most cases upregulation of MMSET is accompanied by the overexpresson of FGFR3. Overexpression of FGFR3 would be expected to increase MAP kinase signaling and c-MYC expression. However, in 30% of cases of t(4;14) myeloma, only MMSET is overexpressed. Hence we hypothesized that MMSET might also stimulate c-MYC expression. We showed that MMSET is a histone methyltransferase that has promiscuous activity in vitro. In vivo, MMSET overexpression was correlated with elevated dimethylation of H3K36 and depressed trimethylation of H3K27 levels, altered chromatin structure, gene expression profiles, and cell growth. However, the exact genetic targets underlying the MMSET activity are not well understood. We found that MMSET overexpression was associated with increased c-MYC protein but not mRNA expression. Depletion of MMSET from myeloma cells was associated with decreased levels of c-MYC protein but not mRNA and no change in the half-life of the MYC proteins. This suggested that MMSET controlled MYC at the post-transcriptional level by preventing its translation, an effect reminiscent of the action of microRNAs. miRNA profiling of t(4;14) myeloma cells before and after shRNA-mediated depletion of MMSET showed that MMSET repressed the expression of many miRNAs including miR-126*, which was predicted to target c-MYC. We therefore hypothesized that by repressing miR126* expression MMSET could stimulate c-MYC protein expression. Accordingly, miR126* but not a point mutant of mir126* inhibited translation of a construct fusing the 3' untranslated region of c-MYC to luciferase. Furthermore a point mutation within the 3'UTR of c-MYC abrogated the effect of miR126* overexpression on the reporter. Overexpression of miR126* in t(4;14) myeloma cells suppressed c-MYC expression and cell growth. Furthermore, growth suppression and inhibition of c-MYC expression in myeloma cells after MMSET knock-down was partially blocked by an antagomir directed against mir126*. Importantly, growth suppression mediated by MMSET knockdown was reversed by expression of exogenous miRNA-resistant form of c-MYC. MMSET was recruited to the miR126* promoter along with KAP1 a partner protein identified by affinity chromatography and mass spectroscopic analysis. KAP1 mediated repression has been associated with increased H3K27 and H3K9 methylation at promoter sites and accordingly on the miR126/126* promoter, MMSET expression was associated with MMSET and KAP1 recruitment to the promoter, increased H3K9 and H3K27 methylation and decreased histone acetylation. KAP1 depletion in myeloma cells was associated with increased expression of mir126*, decreased c-MYC levels and decreased myeloma growth. Lastly to model the effects of an MMSET inhibitor in combination with HDAC inhibitors cells were subjected to trichostatin A, MMSET depletion or both. HDAC inhibitor treatment on its own elevated mir126* levels and decreased c-MYC expression but the combination of a sub-optimal dose of the HDAC inhibitor combined with MMSET depletion led to complete silencing of c-MYC expression and increased cell death. Collectively, these data suggest a new mechanism of oncogenesis by MMSET in MM, and emphasize the key role of c-MYC overexpression in this disease. Disclosures: No relevant conflicts of interest to declare.

Chromatin regulators including methyltransferases are frequently disrupted by inactivating mutations in a wide spectrum of cancers. In contrast gain of function mutations that increase enzyme activity are much more rare and found in only two enzymes within the family of approximately 60 SET domain methyltransferases, MMSET and EZH2. Specific recurrent mutations in either MMSET or EZH2 are detected in hematological malignancies in conjunction with increased methylation of their histone substrates, H3K36 and H3K27, respectively. We and others characterized the MMSET E1099K mutant, which generates a clear molecular hallmark of increased dimethylation of H3K36 (H3K36me2). This gain of function also induces a corresponding decrease in trimethylation at H3K27 (H3K27me3). The altered balance between H3K36me2 and H3K27me3 mimics global chromatin effects that we previously reported in multiple myeloma lines that overexpress wild type MMSET due to the t(4:14) translocation. Research led by the Pediatric Cancer Genome Project has shown that MMSET E1099K is detected in ~10% of B-cell acute lymphoblastic leukemia (ALL) and particularly enriched in samples following disease relapse. Therefore, we hypothesize that this mutation is a driver of ALL progression and mediates resistance to therapy. To address this question, we have rescued or disrupted the endogenous E1099K mutation in cell lines from relapsed ALL and characterized the resulting molecular and phenotypic effects. The sequence context of the E1099K mutation allows allele-specific CRISPR targeting, which we have exploited to create isogenic lines that differ by MMSET E1099K status. We isolated subclones from three parental lines and sequencing analysis revealed distinct outcomes: indel mutations that disrupt the E1099K allele or rescue to WT by interchromosomal gene conversion. Comparing these subclones to non-targeted controls showed that loss of E1099K causes a 3-fold reduction in H3K36me2 levels and 5-fold increase in H3K27me3. Mass spectrometry-based measurement of methylation kinetics revealed that E1099K accelerates the rate constant of conversion from unmodified H3K36 to monomethylation. This corresponds to an increased ability of the mutant enzyme to turnover substrate in vitro. Because H3K36 and H3K27 methylation both contribute to transcriptional regulation, we compared expression profiles and identified a common set of genes overexpressed in cells lines harboring the mutant allele. These overexpressed genes included components of the SLIT/ROBO, WNT/Beta-Catenin, and cell adhesion pathways. Correlating with these expression changes, several phenotypic changes resulted from loss of E1099K, such as reduction in proliferation, colony-forming ability, and adhesive properties. Loss of E1099K also increased sensitivity to chemotherapeutic agents used to treat ALL, such as doxorubicin and dexamethasone. One gene consistently upregulated in the presence of E1099K was the transcription factor ETV1. In support of ETV1 being a key mediator of E1099K-driven phenotypes we found that parental lines treated with an ETV1 inhibitor displayed reduced viability and adhesion. To further our phenotypic analysis, subclones and parental lines have been tagged with luciferase or fluorescent markers to assess invasion in models of metastasis and allow in vivo monitoring of tumors. Collectively, we have developed gene-targeting reagents specific for the MMSET E1099K mutation and used these tools to show its impact on global chromatin environment and cell phenotypes. Disclosures No relevant conflicts of interest to declare.

Aberrant epigenetic gene regulation is now considered a hallmark of many diseases, including cancer. Mutations and disruptions of epigenetic modifier enzymes are among the commonest mutation in human cancer. MMSET/NSD2/WHSC1 is a histone methyltransferase overexpressed in many cancers, including a subset of multiple myeloma (MM) harboring the t(4;14) translocation. This increased expression of MMSET is linked to a poorer prognosis and response to therapy than other MM subtypes. More recently activating mutations of MMSET were identified in relapsed pediatric ALL. The major substrate for MMSET in vivo is lysine 36 on histone H3 (H3K36). The ability of MMSET to methylate H3 depends on the enzymatic activity of its SET domain, found in most histone methyltransferases. Overexpression or activating mutations of MMSET lead to a global increase in dimethylation of H3K36 (H3K36me2) and a concomitant global loss of trimethylated H3K27 (H3K27me3), which leads to a more open chromatin confirmation. We, and others, have shown that MMSET has a role in a number of pathways including cell cycle, apoptosis, cell adhesion, and DNA repair. The loss of MMSET in myeloma cells decreases cell proliferation and leads to a reduced clonogenic capacity. These data highlight MMSET as a promising therapeutic target for t(4;14)+ MM. A small molecule inhibitor could also prove advantageous in our efforts to understand the various mechanisms of MMSET's enzymatic activity. Here we performed two high throughput screens on two different small molecule libraries. The screens were conducted by using the recombinant SET methyltransferase domain of MMSET (aa980-1214), generated in vitro, in combination with H4 (aa36-50) as the peptide substrate and S-adenosylmethionine (SAM) as the methyl donor. These screens were designed to avoid SAM competitors by screening under high SAM concentrations (50μM). The first screen used SAMDI technology, which combines mass spectrometry with high-density arrays. This method allows us to identify compounds within the libraries that inhibit MMSET in a rapid, label-free format. The second screen used alphalisa technology, a luminescent proximity assay that provides a homogeneous, sensitive method to screen for molecules that reduce MMSET activity. A total of 88 compounds from both libraries were identified as potential MMSET inhibitors (>3-standard deviation) and were counter-screened against the histone demethylase LSD1 to confirm their specificity. Numerous candidate compounds promiscuously inhibited the enzymatic activity of LSD1, yielding eleven putative MMSET inhibitors, with IC50 values ranging from 6-42μM. These eleven compounds were then screened against full-length MMSET enzyme using HeLa-derived mono/di-nucleosomes as the substrate. Eight of the compounds demonstrated a 50% or greater inhibition of MMSET at 10 μM and three compounds exhibited a dose-dependent inhibition upon dilution. Future in vitro, cellular, and animal based assays will determine the potency and selectivity of these compounds against MMSET and gauge their effectiveness as targeted therapies in cancers characterized by MMSET overexpression and activating mutations. Disclosures No relevant conflicts of interest to declare.

Genetic alterations of epigenetic regulators have become a recurrent theme in hematological malignancies. In particular, aberrations that lead to altered levels or distribution of methylation of lysine 27 on histone H3 (H3K27me) have emerged as a common feature of a wide variety of cancers, including multiple myeloma (MM). The histone demethylase UTX/KDM6A activates gene expression by removing the H3K27me3 repressive histone mark, counteracting the activity of EZH2, the enzyme that places this modification. UTX somatic inactivating mutations and deletions are found in up to 10% of multiple myeloma (MM) cases; nevertheless, the epigenetic impact of UTX loss in the MM cell and the mechanisms by which it contributes to this disease remain to be elucidated. To ascertain the biological impact of UTX loss, we used paired isogenic cell lines isolated from a MM patient before (ARP-1, UTX wild-type) and after (ARD, UTX null) relapse. The UTX-mutant ARD cells were engineered to express UTX in a doxycycline-inducible manner. UTX add-back slowed the proliferation rate of ARD cells, without affecting their viability. Soft agar assays demonstrated that UTX mutant ARD cells have increased clonogenic abilities compared to ARP-1 cells. Re-expression of UTX partially reversed this effect, decreasing the number and size of colonies formed. ARD cells also showed increased adhesion to bone marrow stromal cells Hs-5 and to fibronectin than ARP-1 cells, an ability associated with cell survival and drug resistance. UTX add-back decreased the adhesive properties of ARD mutant cells demonstrating this effect is dependent on UTX loss. Mass spectrometry analysis of the add-back system and a panel of UTX wild-type and mutant MM cell lines showed that global levels of H3K27me are not altered after UTX loss or upon its add-back, indicating that UTX depletion may lead to alteration of H3K27me only at specific loci, and thus only control the expression of a limited number of genes. To identify the genes and pathways altered upon UTX loss we performed RNA-seq on the paired MM cell lines and the add-back system. This analysis revealed about 5,000 genes differentially expressed between ARP-1 and ARD cells. Re-expression of UTX was able to reverse the expression of about 1,400 genes, most of them being upregulated upon reintroduction of this factor. Gene ontology analysis of the genes responsive to UTX manipulation demonstrated that many of these genes were involved in the regulation of pathways such as the JAK-STAT, the cadherin, the integrin or the Wnt pathway. Many of the pathways were related to cell adhesion properties, correlating with the effects observed in vitro. Single locus chromatin immunoprecipitation (ChIP) demonstrated changes in the levels of H3K27me3 at specific target genes, with the levels of this modification decreasing or increasing in genes activated or repressed upon UTX loss, respectively. Overall, our data shows that loss of UTX leads to altered H3K27me3 levels at specific loci, resulting in deregulation of gene expression, ultimately promoting the proliferation, clonogenicity and adhesion of MM cells. The characterization of UTX target genes in this disease may help in the identification of targeted therapies for the treatment of this type of MM. Citation Format: Teresa Ezponda, Relja Popovic, Yupeng Zheng, Behnam Nabet, Christine Will, Eliza Small, Manuela Occhionorelli, Giovanni Tonon, Jonathan Keats, Neil Kelleher, Jonathan D. Licht. Loss of the histone demethylase UTX alters the gene expression prolife and contributes to the malignant phenotype of multiple myeloma cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B32.

Unrestrained activation of receptor tyrosine kinase (RTK) pathways drives tumorigenesis through the persistent activation of critical signaling pathways, including the Ras-ERK axis. This aberrant signaling is unleashed by mutations in signaling effectors such as Ras, as well as through disruption of feedback control by regulatory proteins such as the Sprouty family. Mutations in epigenetic regulators also commonly occur during cancer progression, modulating chromatin architecture and gene expression. While unrestrained signaling and epigenetic deregulation are root causes of tumorigenesis, the relationship between aberrant RTK signaling and chromatin modifications at cis-regulatory elements remains to be fully elucidated. We establish linkage between these processes by examining the effects of oncogenic HRasG12V or loss of feedback regulation by Sprouty on global changes in gene expression and enhancer-associated chromatin modifications. Using RNA-sequencing and ChIP-sequencing, we demonstrate that aberrant RTK signaling unleashed by oncogenic HRasG12V or Sprouty gene deletion disrupts gene expression programs and remodels histone modifications associated with active enhancers, including histone 3 lysine 27 acetylation (H3K27ac). Abolishing persistent Ras-Erk signaling through chemical inhibition of MEK activity reverses the aberrant transcriptional program and H3K27ac remodeling at deregulated enhancers. While both lesions disrupt the Ras-Erk axis, the specific target genes and enhancers modulated upon HRasG12V-transformation or Sprouty deletion are largely distinct. Specifically, oncogenic HRasG12V significantly elevates the expression and H3K27ac levels near key target genes encoding the transcription factor Gata4 and the kinase Prkcb. Gata4 is necessary for the HRasG12V expression program and coordinates H3K27ac marking at enhancers of deregulated target genes. We further show that HRasG12V-driven cells are sensitive to chemical inhibition of Prkcb, which reduces the viability and clonogenicity of HRasG12V-transformed cells. These oncogenic effects upon HRasG12V-transformation are also reduced by chemical inhibition of the chromatin regulators BET bromodomain proteins and p300/CBP, which recognize and deposit H3K27ac, respectively. Taken together, our data support a model in which dynamic reprogramming of the cellular enhancer landscape is a key effect of oncogenic RTK signaling. Furthermore, our study shows that identification of enhancers regulated by oncogenic Ras yields insight into targeting key epigenetic regulators and downstream factors that promote Ras-driven malignancies. Citation Format: Behnam Nabet, Pilib O Broin, Jaime Reyes, Kevin Shieh, Charles Y. Lin, Christine M. Will, Relja Popovic, Teresa Ezponda, James E. Bradner, Aaron A. Golden, Jonathan D. Licht. Deregulation of the Ras-Erk signaling axis modulates the enhancer landscape. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2209. doi:10.1158/1538-7445.AM2015-2209

Genetic alterations of epigenetic regulators have become a recurrent theme in hematological malignancies. In particular, aberrations that alter the levels or distribution of methylation of lysine 27 on histone H3 (H3K27me) have emerged as a common feature of a wide variety of cancers, including multiple myeloma (MM). The histone demethylase UTX/KDM6A activates gene expression by removing the H3K27me3 repressive histone mark, counteracting the activity of EZH2, the enzyme that places this modification. UTX somatic inactivating mutations and deletions are found in up to 10% of MM cases; nevertheless, the epigenetic impact of UTX loss in MM and the mechanisms by which it contributes to this disease remain to be elucidated. To ascertain the biological impact of UTX loss, we used a recently identified isogenic cell line pair: ARP-1 (UTX wild-type) and ARD (UTX null). UTX-null ARD cells were engineered to express UTX in a doxycycline-inducible manner. UTX add-back slowed the proliferation rate of ARD cells, without affecting their viability. Soft agar assays demonstrated that UTX-null ARD cells have increased clonogenicity compared to UTX-wild-type ARP-1 cells. Re-expression of UTX partially reversed this effect, decreasing the number and size of colonies formed. ARD cells also showed increased adhesion to Hs-5 bone marrow stromal cells and to fibronectin than ARP-1 cells, an ability associated with cell survival and drug resistance. UTX add-back decreased the adhesive properties of ARD cells demonstrating this effect is dependent on UTX loss. Mass spectrometry analysis of the add-back system and a panel of UTX wild-type and mutant MM cell lines showed that global levels of H3K27me are not altered after UTX loss or upon its add-back. Therefore, UTX depletion may alter H3K27me at specific loci, and control the expression of a limited number of genes. To identify the genes and pathways that are altered upon UTX loss, we performed RNA-sequencing (RNA-seq) on the paired MM cell lines and the add-back system. This analysis revealed approximately 5,000 genes differentially expressed between ARP-1 and ARD cells. Re-expression of UTX in the UTX-null ARD cells reversed the expression of approximately 1,400 genes, most of them being upregulated upon reintroduction of UTX. Gene ontology analysis of genes responsive to UTX manipulation identified pathways such as JAK-STAT, cadherin, integrin and Wnt pathways. Many of these pathways are related to cell adhesion properties, correlating with the effects observed in vitro. Some examples of the genes which expression was restored upon UTX add-back are E-cadherin, whose loss has been associated with MM progression; and PTPN6, a negative regulator of the JAK-STAT pathway. Chromatin immunoprecipitation (ChIP) experiments at UTX target genes revealed a decrease in H3K27me3 and a concomitant increase in H3K4me3 upon UTX add-back, correlating with the observed changes in gene expression. As loss of UTX leads to a failure in the removal of H3K27me3, we hypothesized that UTX-null cells may be more dependent on EZH2 to maintain high H3K27me3 levels at specific loci. Treatment of the paired cell lines with the EZH2 inhibitor GSK343 for 7 days significantly decreased the viability of UTX-null ARD cells, but had no effect on the UTX wild-type ARP-1 cells. This effect was not exclusive to these cell lines, as treatment of a panel of UTX wild-type and mutant MM cells corroborated the increased sensitivity in UTX-mutant cells. RNA-seq of ARD cells treated with GSK343 for 7 days identified approximately 2,000 genes with altered expression in response to this drug, most of them being upregulated upon EZH2 inhibition. These genes partially overlapped with the genes that were responsive to UTX add-back, including E-cadherin, suggesting that treatment with EZH2 inhibitors is somewhat similar to UTX add-back. Collectively, this work demonstrates that loss of UTX alters the epigenetic landscape of MM cells, leading to altered expression of a specific set of genes, ultimately benefiting cells through increased proliferation, clonogenicity and adhesion. Moreover, inhibition of EZH2 partially reverses aberrations promoted by UTX loss and may represent a rationale therapy for the treatment of this type of MM. Disclosures No relevant conflicts of interest to declare.

MMSET/WHSC1 is a histone methyltransferase (HMT) overexpressed in t(4;14)+ multiple myeloma (MM) patients, and is believed to be the driving factor in the pathogenesis of this subtype of MM. Overexpression of MMSET also occurs in solid cancers, including neuroblastoma, colon and prostate. MMSET overexpression in MM and prostate cells leads to an increase in histone 3 lysine 36 dimethylation (H3K36me2), and a decrease in histone 3 lysine 27 trimethylation (H3K27me3). This altered epigenetic landscape is accompanied by changes in proliferation, gene expression, and chromatin accessibility. Prior work linked methylation of histones, including H3K36, to the ability of cells to undergo DNA damage repair. In addition, t(4;14)+ patients frequently relapse after regimens that include DNA damage-inducing agents, suggesting that MMSET might play a role in DNA damage repair and response. To investigate the role of MMSET in DNA damage repair, we transfected U2OS cells with a linearized vector expressing a neomycin-resistant gene. In the presence of G418, only cells that are able to integrate this plasmid through non-homologous end joining (NHEJ) can survive. siRNA knockdown of MMSET led to a decrease in cell survival, suggesting that MMSET is necessary for efficient DNA repair. We also used U2OS cells engineered to express the AsiSI enzyme fused to an estrogen receptor hormone-binding domain. Upon tamoxifen treatment, double strand breaks (DSBs) are induced at multiple AsiSI recognition sites, accompanied by an increase in γH2AX foci. The extent of repair after AsiSI-induced damage was ascertained by the ability of a DNA fragment that spans a specific cut site to be PCR amplified. With MMSET knockdown, there was a >10 fold increase in unrepaired DNA. ChIP analysis showed that with the depletion of MMSET, γH2AX persisted at the cut site. ChIP for specific effectors of DNA damage showed a marked decrease of recruitment of CtIP and RAD51 to the DSB. However, immunoblot analysis showed that CtIP and RAD51 levels were drastically decreased with MMSET depletion, thus explaining the loss of their recruitment to DSBs. In contrast, XRCC4 levels were maintained with MMSET siRNA, but its recruitment to the DSB decreased. CtIP is important for both NHEJ and homologous recombination (HR), RAD51 is critical for HR, and XRCC4 is necessary for NHEJ, suggesting that MMSET is important in multiple pathways of DNA repair. To study the effect of MMSET in MM, we used the t(4;14)+ KMS11 cell line, NTKO, and genetically matched TKO cells in which the overexpressed MMSET allele was knocked out. NTKO cells have elevated levels of DNA damage at baseline, as measured by a comet assay and by the presence of elevated numbers of 53BP1-positive foci. Upon addition of the DNA damaging agent melphalan, NTKO cells showed increased damage as measured by an increase in the tail moment by the comet assay. Paradoxically, upon treatment of these cells with the DNA damaging agents, NTKO cells survived better than TKO cells. NTKO repaired DNA damage at an enhanced rate and continued to proliferate after a significant DNA damage insult, whereas TKO cells accumulated DNA damage and entered cell cycle arrest. We repleted TKO cells with constructs expressing either wild-type MMSET or an HMT-dead (Y1118A) isoform. Upon treatment, cells expressing the wild-type MMSET have showed enhanced DNA repair and continued proliferation after DNA damage, whereas cells expressing the HMT-dead protein repaired DNA damage more slowly and entered cell cycle arrest. The HMT activity of MMSET was critical for the induction of expression of genes required for multiple DNA repair pathways including CHEK2, DDB2, DDIT3, RAD51, and MRE11, again suggesting that MMSET modulates DNA repair by affecting expression of critical components of the repair machinery. The clinical relevance of these finds becomes more apparent in vivo. Luciferase-tagged KMS11 cells harboring doxycycline-inducible MMSET shRNA were injected into nude mice. After one week, mice were treated with doxycycline and injected with melphalan or saline. Knockdown of MMSET or melphalan treatment alone decreased tumor growth but eventually all mice had progressive disease. Only when MMSET was knocked down and chemotherapy given were the mice rendered tumor free. These findings indicate a new mechanism for the ability of MMSET to enhance DNA repair and identify the protein as a potential therapeutic target in MM and other cancers. Disclosures: No relevant conflicts of interest to declare.

Epigenetic regulation of gene expression involves covalent modifications of histones and DNA in a way that does not alter the underlying DNA sequence. Recently, misregulation of epigenetic mechanisms has been linked to a number of diseases, including cancer. These data signify the importance of understanding normal and aberrant epigenetic regulation and for development of tools that allow us to modulate various aspects of epigenetic machinery. One approach to do this is by targeting epigenetic enzymes with small molecule inhibitors. MMSET (Multiple Myeloma SET domain), is a histone methyltransferase overexpressed in many cancers, including a subset of multiple myelomas (MM) harboring the t(4;14) translocation. Overexpression of MMSET leads to a global increase in dimethylation of lysine 36 on histone H3 (H3K36me2). The ability of MMSET to methylate H3 depends on a functional SET (Suppressor of variegation, Enhancer of zeste and Trithorax) domain, found in most histone methyltransferases. Methylation of H3K36 by MMSET affects overall chromatin structure thereby affecting the expression of many genes, including genes that play a role in cellular proliferation and adhesion. Conversely, the loss of MMSET in t(4;14)+ cells suppresses cell growth and induces apoptosis thus supporting the idea that inhibition of MMSET activity is a viable approach to treatment of this particular type of myeloma. Currently there are not any known inhibitors of MMSET. Using the previously identified structure of the SET domain of NSD1, an MMSET homologue, we performed an in silico screen against a library of compounds to identify those that may fit in the substrate binding pocket of MMSET. We tested the in silico hits in a high throughput screen (HTS) that combines Self-Assembled Monolayers and matrix-assisted laser Desorption Ionization time-of-flight (SAMDI). This method allows us to test the ability of the compounds to inhibit MMSET's methylation activity in a label-free format. From this screen two potential hits have been identified. These compounds have IC50 values in the micromolar range and Differential Scanning Fluorimetry (DSF) confirmed the binding of the small molecule to MMSET. Future in vivo work will determine the potency and selectivity of these compounds towards MMSET. Identifying an effective inhibitor of MMSET could make way for new potential therapeutics in the treatment of t(4;14)+ multiple myeloma, as well as other cancers that overexpress this protein, and could also prove to be highly useful in our efforts to understand the various mechanisms of epigenetic regulation. Citation Format: Christine Will, Michael Scholle, Roodolph St. Pierre, Ji Hyun Shim, Zhong Jun Cheng, Relja Popovic, Dinshaw J. Patel, James E. Bradner, Alex D. MacKerell, Milan Mrksich, Jonathan D. Licht. MMSET: Can we flip the switch. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1127. doi:10.1158/1538-7445.AM2013-1127

Abstract 523 Multiple myeloma (MM) is associated with recurrent chromosomal translocations that lead to overexpression of known and putative oncogenes. The MMSET (Multiple Myeloma SET domain) protein is overexpressed in MM patients harboring the translocation t(4;14), and is believed to be the driving factor in the pathogenesis of this subtype of MM. Additionally, through unknown mechanisms, MMSET expression is enhanced in many different tumors including neuroblastoma, colorectal carcinomas and prostate cancer. Our initial studies showed that overexpression of MMSET in myeloma cells induces a global increase in H3K36 dimethylation, a mark associated with actively transcribed genes, and a concomitant genome-wide loss of H3K27 trimethylation. This effect is due to an enhanced rate of H3K36 methylation as well as an increased rate of H3K27 demethylation, and causes a physical loosening of the chromatin structure, leading to altered gene expression. Using genomic-wide chromatin precipitation/next generation sequencing (ChIP-seq), and genetically matched cells in which the overexpressed MMSET allele was knocked out (KMS11-TKO), we found that increased H3K36 methylation due to MMSET disrupts the normal genomic H3K36me2 architecture, from a mostly promoter-enriched mark to a modification spread throughout gene bodies. Additionally, many loci activated in response to MMSET showed the loss of H3K27 methylation near the transcriptional start site. Surprisingly, while MMSET-overexpressing cells lose the H3K27me3 mark globally, specific loci display enrichment of this modification and are transcriptionally repressed. This is associated with a shift in the genomic localization of the H3K27 methyltransferase, EZH2, in the presence of MMSET. Thus, in addition to gene activation through increased H3K36 methylation, overexpression of MMSET can also induce gene repression by imposing EZH2 and H3K27me3 accumulation at specific genomic loci. Using KMS11-TKO cells, we performed a structure-function study to identify important domains of MMSET. Re-introduction of the wild-type MMSET into the TKO cells induces the H3K36/H3K27 epigenetic switch, enhances proliferation and colony formation of these cells, and leads to differential gene expression. These biological activities require all PHD finger domains of the protein (putative chromatin binding regions), the second PWWP domain (a non-specific DNA binding domain) and the functional SET/histone methyltransferase domain. When MMSET constructs containing mutations or deletions in one of these domains were overexpressed in TKO cells, the resulting protein failed to methylate histones, alter growth or change gene expression. Furthermore, point mutations in the PHD domains 2 or 3, analogous to those found in the MMSET homologue NSD1 in Sotos syndrome, prevented MMSET from binding to chromatin and altering histone methylation. By contrast, an MMSET construct lacking the C-terminal PHD4 domain bound chromatin, induced methylation of H3K36, but was unable to mediate a complete loss of the H3K27me3 mark or fully stimulate growth. Together this suggests that pathogenic activity of MMSET could be interrupted by blocking its binding to chromatin, its intrinsic H3K36 methylation activity or by blocking demethylases of H3K27 that might interact with MMSET. To validate MMSET as a therapeutic target, we used KMS11 t(4;14)+ cells that express MMSET specific shRNA in the presence of doxycycline. In vitro, doxycycline-mediated knockdown of MMSET leads to loss of H3K36 methylation, gain of H3K27 methylation and decreased proliferation. KMS11 cells rapidly formed tumors when injected into immunocompromised mice. Doxycycline treatment caused a dramatic decrease in the volume of established tumors and extended survival in mice. Interestingly, removal of the doxycycline treatment restarted tumor growth in some, but not all animals. This suggests that, at least in some cases, targeting MMSET alone may be sufficient to completely eliminate the tumor burden. Together, our work elucidates some of the mechanisms used by MMSET to induce an oncogenic phenotype and identifies domains to be considered in designing novel inhibitors of MMSET function. Disclosures: No relevant conflicts of interest to declare.

Prostate cancer (PCa) remains the most common neoplasia diagnosed among men in the USA, and the second leading cause of cancer deaths. Recent evidence has indicated that epigenetic alterations contribute to the initiation and progression of PCa; however, the mechanisms by which chromatin modifiers influence this disease are still poorly understood. The histone methyl transferase MMSET (Multiple Myeloma Set Domain) is overexpressed in a number of different tumor types, including PCa. While the precise mechanism of MMSET action is still not well defined, it is believed to involve alteration of chromatin structure through dimethylation of lysine 36 on histone H3 (H3K36me2). Analysis of microarray databases indicates that MMSET expression rises in the progression from benign prostatic epithelium to localized and metastatic PCa. We found that PCa cell lines express significantly higher levels of MMSET compared to immortalized but non-transformed prostate cells. In t(4;14)+ myelomas, MMSET overexpression changes global levels of histone modifications, promoting H3K36me2, and repressing trimethylation of lysine 27 (H3K27me3). Similarly, H3K36me2 and H3K27me3 levels in PCa cell lines depended on the expression of MMSET, while depletion of MMSET in benign prostatic epithelial cells did not affect chromatin modifications. Knockdown of MMSET in DU145 and PC-3 tumor cells decreased soft agar colony formation and cell proliferation. Furthermore, inhibition of MMSET expression strikingly decreased cell migration and invasion, while overexpression of MMSET in immortalized non-transformed RWPE-1 cells promoted migratory and invasive properties. These findings suggest that high levels of MMSET play a pathogenic role in PCa progression. Overexpression of MMSET in RWPE-1 cells was accompanied by an epithelial to mesenchymal transition (EMT), characterized by a change in cell morphology and increased expression of mesenchymal markers vimentin and N-cadherin. Among a panel of EMT promoting genes analyzed, Twist1 expression was strongly regulated by MMSET both in the knockdown and the overexpression models. Chromatin immunoprecipitation analysis demonstrated that MMSET binds to the Twist1 locus, leading to an increase in H3K36me2 and a decrease in the repressive mark H3K27me3, suggesting a direct role of MMSET in regulation of Twist1. siRNA depletion of Twist1 in MMSET-overexpressing RWPE-1 cells blocked invasion, indicating that Twist1 was a critical target of MMSET responsible for the acquisition of EMT properties. Intriguingly, Twist1 expression was also elevated in a panel of myeloma cell lines overexpressing MMSET due to chromosomal translocation t(4;14). Collectively, these data suggest that MMSET has a critical role in cancer pathogenesis and progression through epigenetic regulation of metastasis-related genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1046. doi:1538-7445.AM2012-1046

The SPROUTY (SPRY) proteins, key negative regulators of receptor tyrosine kinase (RTK) signaling, control developmental processes such as organ branching and maintain cellular homeostasis. The SPRY proteins inhibit critical signaling pathways that are downstream of RTKs, including the Ras-MAPK, PI3K-AKT, and the PLCα-PKC pathway. These pathways are highly deregulated in various cancers including HER2 overexpressing and PIK3CA mutant breast cancers. Importantly, the SPRY genes are significantly down-regulated in many cancers including 90% of human breast cancer cases, suggesting that they may function as putative tumor suppressors. To understand the role of Spry in normal mammary development, we examined the mammary glands of our Spry1 mutant mice at various developmental stages. In virgin Spry1-/- mice we observed a trend towards more branching of developing breast ducts. Upon pregnancy there was striking overgrowth of the mammary epithelium in Spry1+/− mice (Spry1-/- mice are sterile), suggesting that Spry regulates mammary development. To investigate the role of Spry in breast cancer we explored the consequence of Spry loss in vitro and in vivo. We found that Spry deficient cells possess accelerated cell growth, enhanced cell survival, and increased tumorigenic potential. In addition these cells were hypersensitive to a number of growth factors including EGF, showing enhanced and prolonged activation of the MAPK and PI3K-AKT pathways. To examine the role of Spry loss in promoting mammary tumorigenesis in vivo, we crossed Spry1 mutant mice with mice in which we can induce HER2/neu or PIK3CA expression. We found that Spry1 loss cooperates with oncogenic activation of HER2/neu or PIK3CA to increase cell proliferation and accelerate mammary hyperplasia. Breast tissues from the Spry1-/- mice with oncogenic activation of HER2/neu exhibited a trend towards increased levels of activated Mapk and Akt compared to their Spry1+/+ counterparts. These Spry1-/- breast tissues also accumulated significantly more DNA damage. Finally, microarray gene expression analysis of these tissues identified that genes differentially expressed in Spry1-/- breasts are involved in cell cycle regulation and the DNA damage response. Collectively, this data suggests that Spry functions as a tumor suppressor in breast cancer opposing HER2/neu and PIK3CA by suppressing MAPK and AKT activation to prevent DNA damage. Our work demonstrates that the misregulation of critical signaling pathways, through loss of Spry, leads to increased cell growth, enhanced cell survival, and accelerated tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2165. doi:1538-7445.AM2012-2165

Multiple Myeloma (MM), a neoplasm of plasma cells accounts for up to 10% of hematological malignancies. The MMSET (Multiple Myeloma SET domain) protein is a histone methyl transferase (HMT) overexpressed as a result of the translocation t(4;14), present in about 15% of multiple myeloma patients. MMSET is a nuclear protein with multiple domains critical for gene regulation including the SET domain, which encodes histone methyl transferase activity, and protein interaction domain including PHD, and PWWP domains. Although studies have shown the involvement of MMSET in heart development and tumor progression, its mode of action in the pathogenesis of MM is largely unknown. We found that overexpression of MMSET induces global increase in H3K36 methylation with concomitant loss of global H3K27 methylation. This is also accompanied by significant changes in over DNA methylation in these cells. These changes cause physical loosening of the chromatin structure, increased micrococcal nuclease accessibility, leading to altered gene expression. Pathways affected by MMSET overexpression include cell cycle, apoptosis and response to DNA damage. The HMT activity of MMSET is important for growth stimulation by MMSET since reexpression of MMSET in a t(4;14) myeloma cell line in which the rearranged MMSET allele was disrupted by homologous recombination (KMS11-KO) rescued growth only when the HMT activity of the protein was intact. Conversely shRNA-mediated deletion of MSMET in t(4;14)+ cell lines leads to growth arrest and cell death, together suggesting that MMSET is a therapeutic target in multiple myeloma. We investigated the mechanism by which MMSET induces global chromatin changes. KMS-11-KO cells were stably repleted with wild-type MMSET or deletion constructs. The complete H3K36/H3K27 switch mediated by MMSET requires all 4 PHD finger domains of the protein, the second PWWP domain and the functional SET domain. Furthermore, these domains are also required for the increased proliferation as well as the aberrant gene expression caused by overexpression of the wild type MMSET. We also found that the PHD and PWWP domains of MMSET were required for its ability to bind to specific chromatin marks and likely play a role in firmly binding MMSET to chromatin. Furthermore deletion of PHD domain 4 yielded a form of MMSET able to increase H3K36 methylation but unable to reduce H3K37 methylation. Wild-type MMSET but not the PHD4 deletion mutant could bind to the H3K27 histone demethyalse JMJD3. This further implicates a histone demethylase as a possible target in this form of myeloma. Recently, it has been shown that double stranded DNA breaks lead to unwinding of chromatin in a manner regulated by the DNA damage response (DDR). Considering this and the global changes induced by MMSET in chromatin structure, we hypothesized that MMSET could affect the DDR by inducing global changes in chromatin structure. We observed that cells overexpressing MMSET displayed more DNA damage at baseline as measured by alkaline electrophoresis comet assay and had higher levels of phosphorylated H2AX, a common DNA damage marker. To try to explain the observed resistance of t(4;14)+ myelomas to chemotherapy, we incubated cells in the presence of melphalan, an alkylating agent currently used in myeloma treatment. Paradoxically, despite the higher baseline level of phosphorylated H2AX and higher levels of single- and double-strand breaks of DNA upon melphalan treatment, MMSET overexpressing cells show better survival in response to the drug. The cells with high MMSET levels were more resistant to the action of melphalan and displayed less apoptosis after the treatment. After repleting MMSET expression in the KMS11-KO cells, we observed that the SET domain of the protein is important for these effects as KO cells expressing the HMT inactive mutant grew more slowly and formed fewer colonies in the presence of melphalan than KO cells expressing wild type MMSET. We also profiled gene expression in cells with high and low MMSET levels using Illumina HumanWG-6 v3.0 expression arrays and performed functional annotations using Ingenuity Pathway analysis. There was an enrichment of genes implicated in cell cycle regulation (CCNE2, E2F2, TP53INP1, CDC25A) and DNA damage-repair (DNA-PK, GADD45G). We then studied the cell cycle profile of KMS11-KO treated with high dose melphalan for two hours. We performed cell cycle profile with BrdU staining two days after the release from the drug. We observed that cells with high MMSET levels could cycle normally and overcame the cell cycle arrest but cells with low MMSET were arrested in G0-G1 and G2-M phases. Collectively this information suggests that the normal role of MMSET in DNA repair is dysregulated by its overexpression in myeloma. An underlying paradox of MM is the continued proliferation and self-renewal of a highly differentiated cell. Plasma cell development requires the BLIMP1 transcription factor, which stimulates terminal B cell differentiation and represses c-MYC, a master regulator of cell growth. By contrast, malignant plasma cells express c-MYC and proliferate while maintaining the specialized machinery required for immunoglobulin production. We found that MMSET overexpression was associated with increased c-MYC protein but not mRNA expression and no change in the half-life of the MYC proteins. miRNA profiling of t(4;14) myeloma cells before and after depletion of MMSET showed that MMSET repressed expression of miR-126*, which was predicted to target c-MYC. miR126* but not a point mutant of mir126* inhibited translation of a c-MYC-UTR-luciferase reporter. Overexpression of miR126* in t(4;14) myeloma cells suppressed c-MYC expression and cell growth, while growth suppression and inhibition of c-MYC expression in myeloma cells after MMSET knock-down was partially blocked by an antagomir directed against mir126*. Growth suppression mediated by MMSET knockdown was reversed by expression of exogenous miRNA-resistant form of c-MYC. MMSET was recruited to the miR126* promoter along with a co-repressor complex and in this case repressed miR126* expression. To model the effects of an MMSET inhibitor in combination with histone deactylase (HDAC) inhibitors, cells were subjected to trichostatin A, MMSET depletion or both. HDAC inhibitor treatment on its own elevated mir126* levels and decreased c-MYC expression while an HDAC inhibitor combined with MMSET depletion led to complete silencing of c-MYC expression and increased cell death. These studies motivate the development of MMSET inhibitors. We assayed MMSET in vitro using bacterially purified enzyme and recombinant histones. In vitro, MMSET is a promiscuous HMT altering H3K36 and H3K27 residues and methylating itself on C-terminal lysine residues. Previously identified HMT inhibitors including chaetocin and BIX-01294 inhibited MMSET activity in vitro. We developed a screen for additional MMSET inhibitors using a high throughput mass spectroscopic assay in which the HMT activity of MMSET was detected by the presence of a 14Da shift in molecular weight of a small peptide representing the H3K36 methylation site of histone H3. Of 10,000 compounds screened, 7 showed >50% inhibition, none of these structurally similar to known HMT inhibitors. Secondary assays confirmed activity of at least three of these compounds that are undergoing further testing in vitro and in cell culture. In conclusion MMSET mediates extensive changes in chromatin structure and function that drive malignancy. MMSET inhibition, alone in combination with chemotherapy or other agents that affect chromatin such as HDAC inhibitors may represent future therapies for t(4;14)-associated multiple myeloma Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL05-02.

Abstract 675 The multiple myeloma SET domain (MMSET) gene is fused to the immunoglobulin locus in t(4;14)-associated multiple myeloma, and MMSET levels are elevated in these patients relative to other myeloma cases and normal cells. MMSET contains several domains commonly found in chromatin regulators including the PHD domain, PWWP domain and SET domain; responsible for histone methyl transferase (HMT) activity. What histone residues are methylated by MMSET in vivo has been uncertain. A well-folded, highly active form of the MMSET SET domain made in bacteria was promiscuous, methylating the H3K36, H3K27 and H4K20 residues of native histone as well as itself. To determine how MMSET affects chromatin in vivo and to identify genes regulated by MMSET, we engineered t(4;14)+ KMS11 cells with a tetracycline-inducible shRNA, leading to a >90% decrease in MMSET expression. Upon loss of MMSET expression, there was a striking decrease of trimethylated histone 3, lysine 36 (H3K36me3), a mark of transcriptional elongation and repression of intragenic transcription. At the same time, loss of MMSET expression was associated with a strong increase in H3K27me3, a chromatin mark associated with gene repression. For gain-of-function studies, the overexpressed MMSET allele in KMS11 cells was disrupted by homologous recombination (KMS11-TKO). KMS11-TKO cells, stably infected with a retrovirus carrying MMSET, displayed high levels of H3K36me3 and loss of H3K27me3. A specific mutation (Y1118A) in the SET domain of MMSET, predicted from crystal structure models to be required for histone binding, abrogated HMT activity of MMSET in vitro. Accordingly expression of MMSET Y1118A in KMS11-TKO cells failed to increase H3K36me3 levels. Another mutant, F1177A actually increased H3K36me3 levels above those stimulated by wild-type MMSET itself, most likely due to an expanded “pocket” within the SET domain that removed steric obstacles to the conversion of H3K36 from the mono-methyl to tri-methyl state. Collectively, these data indicate that H3K36 is a major methylation target of MMSET in vivo. To determine the genes regulated by MMSET and the importance of histone methylation in MMSET action, we profiled gene expression in both gain and loss-of-function systems using Illumina HumanWG-6 v3.0 expression arrays. We compared these gene lists with the top 2000 genes bound by MMSET as determined in a ChIP-on-chip assay using NimbleGen 2.7kb promoter arrays. MMSET knockdown affected expression of 1845 genes (FC>1.5, p Disclosures: No relevant conflicts of interest to declare.