Christine Will

The MMSET histone methyl transferase switches global histone methylation and alters gene expression in t(4;14) multiple myeloma cells

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.

Histone Methyltransferase MMSET/NSD2 Alters EZH2 Binding and Reprograms the Myeloma Epigenome through Global and Focal Changes in H3K36 and H3K27 Methylation

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.

The histone methyltransferase MMSET/WHSC1 activates TWIST1 to promote an epithelial–mesenchymal transition and invasive properties of prostate cancer

Epigenetic deregulation of gene expression has 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 with 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, whereas 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–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 and leads 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 has a role in PCa pathogenesis and progression through epigenetic regulation of metastasis-related genes.

MMSET stimulates myeloma cell growth through microRNA-mediated modulation of c-MYC

Multiple myeloma (MM) represents the malignant proliferation of terminally differentiated B cells, which, in many cases, is associated with the maintenance of high levels of the oncoprotein c-MYC. Overexpression of the histone methyltransferase MMSET (WHSC1/NSD2), due to t(4;14) chromosomal translocation, promotes the proliferation of MM cells along with global changes in chromatin; nevertheless, the precise mechanisms by which MMSET stimulates neoplasia remain incompletely understood. We found that MMSET enhances the proliferation of MM cells by stimulating the expression of c-MYC at the post-transcriptional level. A microRNA (miRNA) profiling experiment in t(4;14) MM cells identified miR-126* as an MMSET-regulated miRNA predicted to target c-MYC mRNA. We show that miR-126* specifically targets the 3′-untranslated region (3′-UTR) of c-MYC, inhibiting its translation and leading to decreased c-MYC protein levels. Moreover, the expression of this miRNA was sufficient to decrease the proliferation rate of t(4;14) MM cells. Chromatin immunoprecipitation analysis showed that MMSET binds to the miR-126* promoter along with the KAP1 corepressor and histone deacetylases, and is associated with heterochromatic modifications, characterized by increased trimethylation of H3K9 and decreased H3 acetylation, leading to miR-126* repression. Collectively, this study shows a novel mechanism that leads to increased c-MYC levels and enhanced proliferation of t(4;14) MM, and potentially other cancers with high MMSET expression.

MMSET/WHSC1 enhances DNA damage repair leading to an increase in resistance to chemotherapeutic agents.

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.

Abstract PL05-02: MMSET: A pathogenic factor and therapeutic target in multiple myeloma.

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.

An activating mutation of the NSD2 histone methyltransferase drives oncogenic reprogramming in acute lymphocytic leukemia

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.

Abstract 12: KMD6A/UTX loss enhances the malignant phenotype of multiple myeloma and sensitizes cells to EZH2 inhibition

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.

Abstract B32: Loss of the histone demethylase UTX alters the gene expression prolife and contributes to the malignant phenotype of multiple myeloma cells.

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.

Abstract 2209: Deregulation of the Ras-Erk signaling axis modulates the enhancer landscape

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