Neil W. Johnson

Identification of Potent, Selective, Cell-Active Inhibitors of the Histone Lysine Methyltransferase EZH2.

The histone H3-lysine 27 (H3K27) methyltransferase EZH2 plays a critical role in regulating gene expression, and its aberrant activity is linked to the onset and progression of cancer. As part of a drug discovery program targeting EZH2, we have identified highly potent, selective, SAM-competitive, and cell-active EZH2 inhibitors, including GSK926 (3) and GSK343 (6). These compounds are small molecule chemical tools that would be useful to further explore the biology of EZH2.

Transition state for the NSD2-catalyzed methylation of histone H3 lysine 36

Significance Epigenetic control by methylation of histones is essential in development. Loss of regulation of methylation pathways is involved in developmental disorders and oncogenesis. Despite interest in NSD2, there have been no selective inhibitors reported. Analogs designed to mimic the NSD2 transition state structure are potential enzyme inhibitors. A combination of experimental kinetic isotope effects and quantum chemistry was used to define the subangstrom details of reaction chemistry at the transition state of NSD2. Electrostatic potential maps of reactants and transition states provide a high-resolution map of reaction chemistry and a blueprint for design of transition state analogs for this mechanism of epigenetic regulation. Nuclear receptor SET domain containing protein 2 (NSD2) catalyzes the methylation of histone H3 lysine 36 (H3K36). It is a determinant in Wolf–Hirschhorn syndrome and is overexpressed in human multiple myeloma. Despite the relevance of NSD2 to cancer, there are no potent, selective inhibitors of this enzyme reported. Here, a combination of kinetic isotope effect measurements and quantum chemical modeling was used to provide subangstrom details of the transition state structure for NSD2 enzymatic activity. Kinetic isotope effects were measured for the methylation of isolated HeLa cell nucleosomes by NSD2. NSD2 preferentially catalyzes the dimethylation of H3K36 along with a reduced preference for H3K36 monomethylation. Primary Me-14C and 36S and secondary Me-3H3, Me-2H3, 5′-14C, and 5′-3H2 kinetic isotope effects were measured for the methylation of H3K36 using specifically labeled S-adenosyl-l-methionine. The intrinsic kinetic isotope effects were used as boundary constraints for quantum mechanical calculations for the NSD2 transition state. The experimental and calculated kinetic isotope effects are consistent with an SN2 chemical mechanism with methyl transfer as the first irreversible chemical step in the reaction mechanism. The transition state is a late, asymmetric nucleophilic displacement with bond separation from the leaving group at (2.53 Å) and bond making to the attacking nucleophile (2.10 Å) advanced at the transition state. The transition state structure can be represented in a molecular electrostatic potential map to guide the design of inhibitors that mimic the transition state geometry and charge.

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

Activation of the p53-MDM4 regulatory axis defines the anti-tumour response to PRMT5 inhibition through its role in regulating cellular splicing

Evasion of the potent tumour suppressor activity of p53 is one of the hurdles that must be overcome for cancer cells to escape normal regulation of cellular proliferation and survival. In addition to frequent loss of function mutations, p53 wild-type activity can also be suppressed post-translationally through several mechanisms, including the activity of PRMT5. Here we describe broad anti-proliferative activity of potent, selective, reversible inhibitors of protein arginine methyltransferase 5 (PRMT5) including GSK3326595 in human cancer cell lines representing both hematologic and solid malignancies. Interestingly, PRMT5 inhibition activates the p53 pathway via the induction of alternative splicing of MDM4. The MDM4 isoform switch and subsequent p53 activation are critical determinants of the response to PRMT5 inhibition suggesting that the integrity of the p53-MDM4 regulatory axis defines a subset of patients that could benefit from treatment with GSK3326595.

Abstract 5379: A potent EZH2 inhibitor exhibits long residence time and anti-tumor activity

The EZH2 histone methyltransferase is frequently mutated in diffuse large B-cell lymphoma leading to increased trimethylation of histone H3 lysine 27 (H3K27me3). Drug discovery efforts have previously identified a pyridone-based chemical series of EZH2 inhibitors that potently and selectively inhibit EZH2 catalytic activity. These compounds are capable of globally decreasing H3K27me3 levels, de-repressing EZH2 target genes, and inducing growth inhibition of many lymphoma cell lines both in cell culture and in vivo. Through medicinal chemistry optimization, we have developed EZH2 inhibitors with significantly improved potency in both biochemical and cellular assays. These compounds exhibit a prolonged enzyme residence time that can be further extended in vitro through the addition of an H3K27me3 peptide. Herein, we report the biochemical and cellular activity of these new EZH2 inhibitors. Citation Format: Heidi Ott, Glenn van Aller, Jessica Ward, BaoChau Le, Cynthia Rominger, James Foley, Susan Korenchuk, Charles McHugh, Michael Butticello, Charles Blackledge, James Brackley, Joelle Burgess, Celine Duquenne, Neil Johnson, Jiri Kasparec, Louis LaFrance, Mei Li, Kenneth McNulty, Kenneth Newlander, Stuart Romeril, Stanley Schmidt, Mark Schulz, Dai-Shi Su, Dominic Suarez, Xinrong Tian, Christopher Carpenter, Juan Luengo, Ryan Kruger, Steven Knight, Michael T. McCabe. A potent EZH2 inhibitor exhibits long residence time and anti-tumor activity. [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 5379. doi:10.1158/1538-7445.AM2015-5379

Abstract 2939: Discovery and synthesis of highly potent and selective small molecule inhibitors of the histone methyltransferase EZH2

The histone methyltransferases are a group of enzymes which catalyze the transfer of a methyl group from the co-factor S-Adenosylmethionine (SAM) to the lysine and arginine residues of histone tails. This post-translational modification is a key event in maintaining gene expression patterns. In recent years, the relationships between aberrant histone methylation patterns and cancer progression have been recognized. These developments, along with an improved understanding of the underlying structural biology, have made histone methyltransferases highly attractive targets for therapeutic intervention. The histone lysine methyltransferase EZH2 (Enhancer of Zeste Homolog 2) is frequently over-expressed in a wide variety of cancerous tissues. There is a strong correlation between overexpression of EZH2 and aberrant transcriptional signaling in cells, ultimately resulting in poor clinical prognosis. Inhibition of EZH2 is expected to alter transcriptional expression and ultimately lead to an improved clinical outcome. This presentation will describe medicinal chemistry efforts in the development of highly potent and selective small molecule inhibitors of EZH2. The synthesis, SAR, and identification of a clinical candidate will be discussed. 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 2939. doi:1538-7445.AM2012-2939