Jason J. Marineau

Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors.

Selective inhibition of protein methyltransferases is a promising new approach to drug discovery. An attractive strategy towards this goal is the development of compounds that selectively inhibit binding of the cofactor, S-adenosylmethionine, within specific protein methyltransferases. Here we report the three-dimensional structure of the protein methyltransferase DOT1L bound to EPZ004777, the first S-adenosylmethionine-competitive inhibitor of a protein methyltransferase with in vivo efficacy. This structure and those of four new analogues reveal remodelling of the catalytic site. EPZ004777 and a brominated analogue, SGC0946, inhibit DOT1L in vitro and selectively kill mixed lineage leukaemia cells, in which DOT1L is aberrantly localized via interaction with an oncogenic MLL fusion protein. These data provide important new insight into mechanisms of cell-active S-adenosylmethionine-competitive protein methyltransferase inhibitors, and establish a foundation for the further development of drug-like inhibitors of DOT1L for cancer therapy.

Phenothiazines induce PP2A-mediated apoptosis in T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that is frequently associated with activating mutations in NOTCH1 and dysregulation of MYC. Here, we performed 2 complementary screens to identify FDA-approved drugs and drug-like small molecules with activity against T-ALL. We developed a zebrafish system to screen small molecules for toxic activity toward MYC-overexpressing thymocytes and used a human T-ALL cell line to screen for small molecules that synergize with Notch inhibitors. We identified the antipsychotic drug perphenazine in both screens due to its ability to induce apoptosis in fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled with mass spectrometry, we identified protein phosphatase 2A (PP2A) as a perphenazine target. T-ALL cell lines treated with perphenazine exhibited rapid dephosphorylation of multiple PP2A substrates and subsequent apoptosis. Moreover, shRNA knockdown of specific PP2A subunits attenuated perphenazine activity, indicating that PP2A mediates the drugs antileukemic activity. Finally, human T-ALLs treated with perphenazine exhibited suppressed cell growth and dephosphorylation of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurring identification of phenothiazines as a class of drugs with anticancer effects. Furthermore, these data suggest that pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates has therapeutic potential.

Biased multicomponent reactions to develop novel bromodomain inhibitors.

BET bromodomain inhibition has contributed new insights into gene regulation and emerged as a promising therapeutic strategy in cancer. Structural analogy of early methyl-triazolo BET inhibitors has prompted a need for structurally dissimilar ligands as probes of bromodomain function. Using fluorous-tagged multicomponent reactions, we developed a focused chemical library of bromodomain inhibitors around a 3,5-dimethylisoxazole biasing element with micromolar biochemical IC50. Iterative synthesis and biochemical assessment allowed optimization of novel BET bromodomain inhibitors based on an imidazo[1,2-a]pyrazine scaffold. Lead compound 32 (UMB-32) binds BRD4 with a Kd of 550 nM and 724 nM cellular potency in BRD4-dependent lines. Additionally, compound 32 shows potency against TAF1, a bromodomain-containing transcription factor previously unapproached by discovery chemistry. Compound 32 was cocrystallized with BRD4, yielding a 1.56 Å resolution crystal structure. This research showcases new applications of fluorous and multicomponent chemical synthesis for the development of novel epigenetic inhibitors.

Structure-guided DOT1L probe optimization by label-free ligand displacement.

The DOT1L lysine methyltransferase has emerged as a validated therapeutic target in MLL-rearranged (MLLr) acute leukemias. Although S-adenosylmethionine competitive inhibitors have demonstrated pharmacological proof-of-principle in MLLr-leukemia, these compounds require further optimization to improve cellular potency and pharmacokinetic stability. Limiting DOT1L inhibitor discovery and ligand optimization have been complex biochemical methods often using radionucleotides and cellular methods requiring prolonged culture. We therefore developed a new suite of assay technologies that allows comparative assessment of chemical tools for DOT1L in a miniaturized format. Coupling these assays with structural information, we developed new insights into DOT1L ligand binding and identified several functionalized probes with increased cellular potency (IC50 values ∼10 nM) and excellent selectivity for DOT1L. Together these assay technologies define a platform capability for discovery and optimization of small-molecule DOT1L inhibitors.

Gene expression-based discovery of atovaquone as a STAT3 inhibitor and anti-cancer agent.

The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is frequently activated inappropriately in a wide range of hematological and solid cancers, but clinically available therapies targeting STAT3 are lacking. Using a computational strategy to identify compounds opposing the gene expression signature of STAT3, we discovered atovaquone (Mepron), an antimicrobial approved by the US Food and Drug Administration, to be a potent STAT3 inhibitor. We show that, at drug concentrations routinely achieved clinically in human plasma, atovaquone inhibits STAT3 phosphorylation, the expression of STAT3 target genes, and the viability of STAT3-dependent hematological cancer cells. These effects were also observed with atovaquone treatment of primary blasts isolated from patients with acute myelogenous leukemia or acute lymphocytic leukemia. Atovaquone is not a kinase inhibitor but instead rapidly and specifically downregulates cell-surface expression of glycoprotein 130, which is required for STAT3 activation in multiple contexts. The administration of oral atovaquone to mice inhibited tumor growth and prolonged survival in a murine model of multiple myeloma. Finally, in patients with acute myelogenous leukemia treated with hematopoietic stem cell transplantation, extended use of atovaquone for Pneumocystis prophylaxis was associated with improved relapse-free survival. These findings establish atovaquone as a novel, clinically accessible STAT3 inhibitor with evidence of anticancer efficacy in both animal models and humans.

7,9-Diaryl-1,6,8-trioxaspiro[4.5]dec-3-en-2-ones: Readily accessible and highly potent anticancer compounds

7,9-Diaryl-1,6,8-trioxaspiro[4.5]dec-3-en-2-ones are a recently described group of spirocyclic butenolides that can be generated rapidly and as a single diastereomer through a cascade process between γ-hydroxybutenolides and aromatic aldehydes. The following outlines our findings that these spirocycles are potently cytotoxic and have a dramatic structure-function profile that provides excellent insight into the structural features required for this potency.

Abstract 1143: Targeting the transcriptional kinases CDK12 and CDK13 in breast and ovarian cancer

CDK12 and CDK13 regulate expression of large transcripts requiring substantial processing to produce mature mRNA. This transcriptional regulation includes coordinated phosphorylation of specific repeats within the C-terminal domain of RNA polymerase II and association with RNA processing factors (Chila, 2016). RNAi knockdown of CDK12 in cell culture decreases expression of DNA damage response genes, including BRCA1 and ATR, while enhancing sensitivity to DNA damaging agents (Blazek, 2011; Liang, 2015). Recently THZ531, a selective covalent inhibitor of CDK12 and CDK13, was shown to decrease expression of DNA damage response genes in cell culture (Zhang, 2016). Here we present further studies with THZ531 to guide our discovery program toward molecules suitable for clinical development and to explore mechanistic rationales for combining a CDK12/13 inhibitor with PARP inhibitors or DNA damaging agents for difficult-to-treat cancers such as high-grade serous ovarian cancer and triple-negative breast cancer. Using THZ531 as a benchmark, we developed assays capable of discriminating sub-nM inhibitors, including quantifying time-dependent covalent inhibition and cell-based CDK occupancy. Since CDK7, like CDK12 and CDK13, contains a cysteine residue proximal to the kinase active site, these approaches are critical to understand covalent inhibitor selectivity. Furthermore, we performed kinome paneling studies to better understand selectivity of this scaffold in support of our ongoing efforts to optimize CDK12/13 potency and selectivity. To pharmacologically investigate the previously reported effects of CDK12 RNAi, growth inhibition of a panel of ovarian and breast cancer cell lines was assessed following treatment with THZ531 (OVA EC 50 = 50-200 nM (n=6); BRCA EC 50 400) to reveal relationships between inhibitor sensitivity, mutation status, gene expression, and potential oncology indications that may be addressed by these different mechanisms. Finally THZ531 was synergistic with both PARP inhibitors and DNA damaging agents in ovarian and breast cancer cell lines. These data highlight cancer indications and combinations that may be particularly amenable to treatment with CDK12/13 inhibitors. While the pharmacokinetic properties of THZ531 preclude adequate target engagement in tumor tissue at tolerated doses in mouse model systems, our ongoing medicinal chemistry program is progressing to identify and optimize CDK12/13 inhibitors suitable for clinical evaluation. Citation Format: Michael Bradley, Jason Marineau, Yoon Choi, Kristin Hamman, Goran Malojcic, David Orlando, Yixuan Ren, Nan Ke, Shanhu Hu, Eric Olson, Christian Fritz, Christopher Roberts. Targeting the transcriptional kinases CDK12 and CDK13 in breast and ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1143. doi:10.1158/1538-7445.AM2017-1143

Tag and Capture Flow Hydrogen Exchange Mass Spectrometry with a Fluorous-Immobilized Probe

Analysis of complex mixtures of proteins by hydrogen exchange (HX) mass spectrometry (MS) is limited by ones ability to resolve the protein(s) of interest from the proteins that are not of interest. One strategy for overcoming this problem is to tag the target protein(s) to allow for rapid removal from the mixture for subsequent analysis. Here we illustrate a new solution involving fluorous conjugation of a retrievable probe. The appended fluorous tag allows for facile immobilization on a fluorous surface. When a target protein is passed over the immobilized probe molecule, it can be efficiently captured and then exposed to a flowing stream of deuterated buffer for hydrogen exchange. The utility of this method is illustrated for a model system of the Elongin BC protein complex bound to a peptide from HIV Vif. Efficient capture is demonstrated, and deuteration when immobilized was identical to deuteration in conventional solution-phase hydrogen exchange MS. Protein captured from a crude bacterial cell lysate could also be deuterated without the need for separate purification steps before HX MS. The advantages and disadvantages of the method are discussed in light of miniaturization and automation.

Abstract IA8: A new class of drugs active in T-ALL is revealed in a zebrafish screen

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer frequently associated with activating NOTCH1 mutations and dysregulation of MYC. We performed two complementary screens to identify novel agents with activity against T-ALL: i) a zebrafish screen for small molecules toxic to MYC-overexpressing thymocytes, and ii) a human T-ALL cell line screen for small molecules that synergize with Notch inhibitors. “Hits” common to both screens included perphenazine, a phenothiazine antipsychotic that induced apoptosis of fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled to mass spectrometry, we identified protein phosphatase 2A (PP2A) as the critical perphenazine target. In line with this finding, T-ALL cell lines treated with perphenazine underwent apoptosis associated with rapid dephosphorylation of multiple PP2A substrates, indicating that perphenazine binds and activates the PP2A tumor supressor. Moreover, shRNA knockdown of the scaffolding or catalytic subunits of PP2A attenuated the activity of perphenazine, indicating that PP2A is required for its antileukemic activity. Finally, treatment of primary human T-ALLs with pherphenazine suppressed cell growth and caused dephosphorylated of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurrent “rediscovery” of phenothiazines as a class of drugs with anti-cancer effects and highlight the therapeutic potential of pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates. Citation Format: Alejandro Gutierrez, Li Pan, Richard Groen, Frederic Baleydier, Alex Kentsis, Jason Marineau, Ruta Grebliunaite, Elena Kozakewich, Casie Reed, Francoise Pflumio, Sandrine Poglio, Benjamin Uzan, Paul Clemons, Lynn Verplank, Frank An, Jason Burbank, Stephanie Norton, Nicola Tolliday, Hanno Steen, Andrew P. Weng, Huipin Yuan, James E. Bradner, Constantine Mitsiades, A. Thomas Look, Jon C. Aster. A new class of drugs active in T-ALL is revealed in a zebrafish screen. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA8.

Abstract 3230: Genome-wide localization of anti-cancer drugs

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA A vast number of small-molecule ligands, including therapeutic drugs under development and in clinical use, elicit their effects by binding specific proteins associated with the genome. An ability to map the direct interactions of a chemical entity with chromatin genome-wide could provide new and important insights into the mechanisms by which such small molecules interfere with tumor cell functions. We have developed a method that couples affinity capture of chemical entities and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small molecules throughout the human genome. Using Chem-seq, we have uncovered the full repertoire of the genomic sites bound by a BET bromodomain inhibitor, a cyclin-dependent kinase (CDK) inhibitor and a DNA intercalating drug. Moreover, by combining Chem-seq with ChIP-seq, we have characterized the interactions of drugs with their targets throughout the genome of tumor cells. These methods provide a powerful approach to enhance understanding of therapeutic action and characterize the specificity of drugs that interact with DNA or genome-associated proteins. Citation Format: Lars Anders, Matthew G. Guenther, Jun Qi, Zi Peng Fan, Jason J. Marineau, Peter B. Rahl, Jakob Loven, Alla A. Sigova, William B. Smith, Tong Ihn Lee, James E. Bradner, Richard A. Young. Genome-wide localization of anti-cancer drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3230. doi:10.1158/1538-7445.AM2014-3230