Kimberly N. Smitheman

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27)

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 kcat/Km ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 kcat/Km ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 kcat/Km ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2.

Combinations of BRAF, MEK, and PI3K/mTOR inhibitors overcome acquired resistance to the BRAF inhibitor GSK2118436 dabrafenib, mediated by NRAS or MEK mutations

Recent results from clinical trials with the BRAF inhibitors GSK2118436 (dabrafenib) and PLX4032 (vemurafenib) have shown encouraging response rates; however, the duration of response has been limited. To identify determinants of acquired resistance to GSK2118436 and strategies to overcome the resistance, we isolated GSK2118436 drug-resistant clones from the A375 BRAFV600E and the YUSIT1 BRAFV600K melanoma cell lines. These clones also showed reduced sensitivity to the allosteric mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor GSK1120212 (trametinib). Genetic characterization of these clones identified an in-frame deletion in MEK1 (MEK1K59del) or NRAS mutation (NRASQ61K and/or NRASA146T) with and without MEK1P387S in the BRAFV600E background and NRASQ61K in the BRAFV600K background. Stable knockdown of NRAS with short hairpin RNA partially restored GSK2118436 sensitivity in mutant NRAS clones, whereas expression of NRASQ61K or NRASA146T in the A375 parental cells decreased sensitivity to GSK2118436. Similarly, expression of MEK1K59del, but not MEK1P387S, decreased sensitivity of A375 cells to GSK2118436. The combination of GSK2118436 and GSK1120212 effectively inhibited cell growth, decreased ERK phosphorylation, decreased cyclin D1 protein, and increased p27kip1 protein in the resistant clones. Moreover, the combination of GSK2118436 or GSK1120212 with the phosphoinositide 3-kinase/mTOR inhibitor GSK2126458 enhanced cell growth inhibition and decreased S6 ribosomal protein phosphorylation in these clones. Our results show that NRAS and/or MEK mutations contribute to BRAF inhibitor resistance in vitro, and the combination of GSK2118436 and GSK1120212 overcomes this resistance. In addition, these resistant clones respond to the combination of GSK2126458 with GSK2118436 or GSK1120212. Clinical trials are ongoing or planned to test these combinations. Mol Cancer Ther; 11(4); 909–20. ©2012 AACR.

BET Inhibition Silences Expression of MYCN and BCL2 and Induces Cytotoxicity in Neuroblastoma Tumor Models

BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.

Dabrafenib; Preclinical Characterization, Increased Efficacy when Combined with Trametinib, while BRAF/MEK Tool Combination Reduced Skin Lesions

Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer. BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436). Cellular inhibition of BRAFV600E kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G1 cell cycle arrest, followed by cell death. In a BRAFV600E-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition. However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors. In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models. Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor.

Discovery of Dabrafenib: A Selective Inhibitor of Raf Kinases with Antitumor Activity against B-Raf-Driven Tumors.

Hyperactive signaling of the MAP kinase pathway resulting from the constitutively active B-Raf(V600E) mutated enzyme has been observed in a number of human tumors, including melanomas. Herein we report the discovery and biological evaluation of GSK2118436, a selective inhibitor of Raf kinases with potent in vitro activity in oncogenic B-Raf-driven melanoma and colorectal carcinoma cells and robust in vivo antitumor and pharmacodynamic activity in mouse models of B-Raf(V600E) human melanoma. GSK2118436 was identified as a development candidate, and early clinical results have shown significant activity in patients with B-Raf mutant melanoma.

Abstract B88: A selective Raf kinase inhibitor induces cell death and tumor regression of human cancer cell lines encoding B‐RafV600E mutation

Activation of the Ras‐Raf‐MEK‐ERK pathway has been implicated in a large range of human cancers. Growth factor receptor stimulation by extracellular ligands activates Ras, which then sets in motion a signal transduction cascade through the Raf, MEK and ERK serine/threonine kinases. Mutation of the B‐Raf kinase constitutively activates MAPK signalling, thus bypassing the need for upstream stimuli. This has been genetically associated with several human cancers, especially occurrence of the B‐Raf V600E mutant and its high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. The ability to selectively and potently inhibit B‐Raf should provide a potential therapy for patients with mutant B‐Raf tumors, for which addictive dependency on this pathway is observed. We have identified a novel, potent, and selective Raf kinase inhibitor that is capable of inhibiting the kinase activity of wild‐type B‐Raf, B‐Raf V600E and c‐Raf with IC 50 values of 3.2, 0.8, and 5.0 nM, respectively. Kinase panel screening for over 270 kinases has indicated that this inhibitor is selective for Raf kinase, with ∼400 fold selectivity towards B‐Raf over 91% of the other kinases tested. Specific cellular inhibition of B‐Raf V600E kinase by this inhibitor leads to decreased ERK phosphorylation and inhibition of cell proliferation by an initial arrest in the G1 phase of the cell cycle, followed by cell death. This inhibition is selective for cancer cells that specifically encode the mutation for B‐Raf V600E . Oral compound administration inhibits the growth of B‐Raf V600E mutant melanoma (A375P) and colon cancer (Colo205) human tumor xenografts, growing subcutaneously in immuno‐compromised mice. This cell‐specific B‐Raf V600E inhibitor is currently being evaluated in a human Phase I clinical trial. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B88.

Development of potent B-RafV600E inhibitors containing an arylsulfonamide headgroup.

A potent series of inhibitors against the B-Raf(V600E) kinase have been developed that show excellent activity in cellular assays and good oral bioavailability in rats. The key structural features of the series are an arylsulfonamide headgroup, a thiazole core, and a fluorine ortho to the sulfonamide nitrogen.

LSD1 inhibition exerts its anti-leukemic effect by recommissioning PU.1- and C/EBPα-dependent enhancers in AML

Epigenetic regulators are recurrently mutated and aberrantly expressed in acute myeloid leukemia (AML). Targeted therapies designed to inhibit these chromatin-modifying enzymes, such as the histone demethylase lysine-specific demethylase 1 (LSD1) and the histone methyltransferase DOT1L, have been developed as novel treatment modalities for these often refractory diseases. A common feature of many of these targeted agents is their ability to induce myeloid differentiation, suggesting that multiple paths toward a myeloid gene expression program can be engaged to relieve the differentiation blockade that is uniformly seen in AML. We performed a comparative assessment of chromatin dynamics during the treatment of mixed lineage leukemia (MLL)-AF9-driven murine leukemias and MLL-rearranged patient-derived xenografts using 2 distinct but effective differentiation-inducing targeted epigenetic therapies, the LSD1 inhibitor GSK-LSD1 and the DOT1L inhibitor EPZ4777. Intriguingly, GSK-LSD1 treatment caused global gains in chromatin accessibility, whereas treatment with EPZ4777 caused global losses in accessibility. We captured PU.1 and C/EBPα motif signatures at LSD1 inhibitor-induced dynamic sites and chromatin immunoprecipitation coupled with high-throughput sequencing revealed co-occupancy of these myeloid transcription factors at these sites. Functionally, we confirmed that diminished expression of PU.1 or genetic deletion of C/EBPα in MLL-AF9 cells generates resistance of these leukemias to LSD1 inhibition. These findings reveal that pharmacologic inhibition of LSD1 represents a unique path to overcome the differentiation block in AML for therapeutic benefit.

Abstract 3513: Inhibition of LSD1 for the treatment of cancer

Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression and has been shown to play a role in hematopoietic differentiation. LSD1 is also overexpressed in multiple tumor types. These studies implicate LSD1 as a key regulator of the epigenome that modulates gene expression through post-translational modification of histones and its presence in transcriptional complexes. The current study describes the anti-tumor effects of a novel, irreversible, GSK LSD1 inhibitor (GSK2879552) in acute myeloid leukemia (AML) and small cell lung cancer (SCLC). GSK2879552 is a potent, selective, mechanism-based inhibitor of LSD1. Screening of over 150 cancer cell lines revealed that AML and SCLC cells have a unique requirement for LSD1. While GSK2879552 treatment did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of genes whose expression increased with LSD1 inhibition. Treatment of AML cell lines with GSK2879552 increased cell surface expression of CD11b and CD86, markers associated with a differentiated immunophenotype. Six days of GSK2879552 treatment resulted in potent anti-proliferative growth effects in 19 of 25 AML cell lines representing a range of AML subtypes. Treating for longer time periods revealed sensitivity in all AML cell lines. AML blast colony forming ability was also inhibited in 4 of 5 bone marrow samples derived from primary AML patient samples. The effects of LSD1 inhibition were further characterized in vivo using a mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into a cohort of secondary recipient mice and were treated upon engraftment. After 17 days of treatment, control mice had 80% GFP+ cells in the bone marrow whereas treated mice had only 2.8% GFP positive cells (p Growth inhibition was also observed in a subset of SCLC cell lines. GSK2879552 treatment of mice engrafted with SCLC lines resulted in greater than 80% tumor growth inhibition. Studies using patient derived primary SCLC showed similar efficacy demonstrating the growth inhibition of SCLC with an LSD1 inhibitor extended beyond cell lines. Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML and SCLC. A Phase I clinical trial using GSK2879552 was initiated in March, 2014. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Citation Format: Kimberly Smitheman, Monica Cusan, Yan Liu, Michael Butticello, Melissa Pappalardi, James Foley, Kelly Federowicz, Glenn Van Aller, Jiri Kasparec, Xinrong Tian, Dominic Suarez, Jess Schneck, Jeff Carson, Patrick McDevitt, Thau Ho, Charles McHugh, William Miller, Scott Armstrong, Christine Hann, Neil Johnson, Ryan G. Kruger, Helai P. Mohammad, Shekhar Kamat. Inhibition of LSD1 for the treatment of cancer. [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 3513. doi:10.1158/1538-7445.AM2015-3513

Abstract 1057: Mutation of EZH2 A677 in human B-cell lymphoma promotes hyper-trimethylation of H3K27

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive post-translational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the Polycomb Repressive Complex 2 (PRC2) and its expression and catalytic activity are dysregulated in cancers. While EZH2 may be over-expressed as a result of multiple mechanisms in tumors, only somatic mutation of the EZH2 Y641 residue has thus far been reported to alter its substrate preference and enhance its catalytic efficiency to generate H3K27me3. Herein, we report mutation of the A677 residue of EZH2 to a glycine (A677G) in a lymphoma cell line with aberrantly elevated H3K27me3 levels. Additional EZH2 sequence analysis in 41 primary lymphoma specimens identified another occurrence of this mutation. Biochemical evaluation of recombinant EZH2 complexes revealed that A677G EZH2 possesses catalytic activity with substrate specificity that is novel and distinct from those of wild-type and Y641 mutants. Whereas wild-type EZH2 displayed a preference for substrates with less methylation (i.e. H3K27me0>me1>me2), the Y641 mutants exhibited greatly decreased activity with H3K27me0 and increased activity with H3K27me2. The A677G EZH2, on the other hand, exhibited nearly equal efficiency for all three substrates. A677G EZH2, but not wild-type EZH2, was shown to be capable of significantly increasing global H3K27me3 when transiently expressed in an EZH2 wild-type cancer cell line. Finally, structural modeling suggests that the mutation results in a larger lysine tunnel capable of accommodating the H3K27me2 substrate while retaining the ability to properly orient H3K27me0 and H3K27me1 with the Y641 residue. In addition, functional and biochemical analyses are performed with reversible SAM-competitive EZH2 inhibitors. Therefore, this mutation appears to contribute to the aberrant epigenetic profile observed in certain lymphomas. 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 1057. doi:1538-7445.AM2012-1057