Michael R. McKeown

Selective inhibition of BET bromodomains

Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic ‘writers’ and ‘erasers’. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein–protein interactions of epigenetic ‘readers’, and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.

BET Bromodomain Inhibition as a Therapeutic Strategy to Target c-Myc

MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.

Discovery and Characterization of Super-Enhancer Associated Dependencies in Diffuse Large B-Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) is a biologically heterogeneous and clinically aggressive disease. Here, we explore the role of bromodomain and extra-terminal domain (BET) proteins in DLBCL, using integrative chemical genetics and functional epigenomics. We observe highly asymmetric loading of bromodomain 4 (BRD4) at enhancers, with approximately 33% of all BRD4 localizing to enhancers at 1.6% of occupied genes. These super-enhancers prove particularly sensitive to bromodomain inhibition, explaining the selective effect of BET inhibitors on oncogenic and lineage-specific transcriptional circuits. Functional study of genes marked by super-enhancers identifies DLBCLs dependent on OCA-B and suggests a strategy for discovering unrecognized cancer dependencies. Translational studies performed on a comprehensive panel of DLBCLs establish a therapeutic rationale for evaluating BET inhibitors in this disease.

Small-Molecule Inhibition of BRDT for Male Contraception

Summary A pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception. PaperClip

Genetic resistance to JAK2 enzymatic inhibitors is overcome by HSP90 inhibition

Hsp90 inhibition in B cell acute lymphoblastic leukemia overcomes resistance to JAK2 inhibitors.

Therapeutic Strategies to Inhibit MYC

MYC is a master regulator of stem cell state, embryogenesis, tissue homeostasis, and aging. As in health, in disease MYC figures prominently. Decades of biological research have identified a central role for MYC in the pathophysiology of cancer, inflammation, and heart disease. The centrality of MYC to such a vast breadth of disease biology has attracted significant attention to the historic challenge of developing inhibitors of MYC. This review will discuss therapeutic strategies toward the development of inhibitors of MYC-dependent transcriptional signaling, efforts to modulate MYC stability, and the elusive goal of developing potent, direct-acting inhibitors of MYC.

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.

Antitumor synergy with SY-1425, a selective RARα agonist, and hypomethylating agents in retinoic acid receptor pathway activated models of acute myeloid leukemia

Acute myeloid leukemia (AML) and biologically related myelodysplastic syndrome (MDS) are hematologic malignancies with poor outcomes. While recent approvals of new targeted therapies have increased options for some patients, those unfit for intensive treatment have few options, with single agent hypomethylating agents (HMAs) remaining as standard of care. The retinoic acid receptor alpha (RARα) transcription factor, encoded by the RARA gene, plays a critical role in myeloid cells and shows dysregulation in a subset of AML and MDS tumors. We recently demonstrated that the selective RARα agonist SY-1425 (tamibarotene) had biologic and clinical activity in 43% of evaluable relapsed or refractory AML and higher-risk MDS patients with activation of the RARA pathway. In this study, we sought to determine whether HMAs and SY-1425 exerted synergistic antiproliferative effects in AML models of RARA pathway activation in vitro and in vivo. Addition of HMAs and SY-1425 to RARA-high or IRF8-high, but not RARAlow, AML cell lines resulted in synergistic antiproliferative effects supported by evidence of DNA damage and apoptosis to a far greater extent than either agent alone. Studies in a patient-derived xenograft mouse model also demonstrated deeper and more durable responses with the combination than either agent alone. Furthermore, preclinical testing of various regimens determined that treating with azacitidine for one week followed by treatment with SY-1425 for three weeks maximized tumor suppression and tolerability. These findings directly support the ongoing clinical study of SY-1425 in combination with azacitidine. Both AML and MDS arise, in part, due to genetic alterations in transcription factors (i.e., RUNX1, NPM1) and epigenetic modifying genes (i.e., MLL, DNMT3A) leading to inactivation of tumor suppressor genes, thus enabling proliferation of immature cells. Alterations in DNA methyltransferases (DNMTs) specifically result in DNA hypermethylation which contributes to gene silencing through promoter inactivation, and can be targeted by HMAs that mimic native nucleoside residues and incorporate into DNA. Once incorporated, HMAs are recognized by DNMT1 as a cytosine, however this interaction creates an irreversible DNA-DNMT1 adduct that requires DNA damage repair to resolve. This then results in loss of DNMT1, as the DNA-protein adduct is degraded by the DNA damage response pathway. After loss of

Abstract 1511: AML patient clustering by super-enhancers reveals an RARA associated transcription factor signaling partner

Prior studies have shown that the RARA gene is associated with a super-enhancer (SE) and has upregulated mRNA expression in a subset of AML patients. Furthermore, this has been found to confer increased sensitivity to SY-1425, a potent and selective RARα agonist. We sought to better characterize the cell state and transcription factor circuitry in these RARA-high AML cells. Clustering of 62 primary AML patient samples based on their genome wide SE maps identified six discrete clusters. RARA-high patients partitioned principally into cluster 2, and to a lesser extent 1, suggesting that RARA upregulation is associated with a specific transcription factor (TF) network and cell state. To start unraveling the TF circuitry in the RARA-high cluster, we investigated which other TFs were SE associated with clusters 1 and 2. In particular, interferon regulatory factor 8 (IRF8) was found to be strongly associated with clusters 1 and 2 by SE and mRNA expression, similar to RARA. Moreover, the expression of both genes is correlated in primary patient samples. IRF8 is involved in interferon signaling and previous studies have shown crosstalk between interferon and retinoic acid signaling. Furthermore, aberrant IRF8 pathway signaling is implicated in AML and CML pathogenesis. The tight clustering of RARA and IRF8 in patient subgroups defined by genome wide enhancer maps suggests RARα and IRF8 may form an integrated transcriptional circuit. Indeed, treatment with SY-1425 was found to strongly induce interferon-like gene expression changes in AML cells with high RARA or IRF8 levels, including the tumor suppressive IFN responsive gene IRF1. While RARA-high AML cell line models have been previously shown to respond to SY-1425, we found that models with high IRF8 expression and low levels of RARA were also found to respond to SY-1425. Such IRF8-high, RARA-low AML cell lines showed activation of similar transcriptional pathways as RARA-high cell lines in response to SY-1425 based on GSEA. IRF8-high AML also had comparable low nM EC50 anti-proliferative effects following SY-1425 treatment. In addition, SY-1425 was found to elicit differentiation in both RARA-high and IRF8-high AML cell lines based on flow cytometry. While RARA and IRF8 expression appear correlated, this data suggests that IRF8 levels may predict for sensitivity to SY-1425 in addition to RARA levels, particularly in cases of AML with high IRF8 expression but low RARA levels. Insights derived from enhancer analysis, transcriptional profiling and differentiation response in preclinical models support the recently initiated Phase 2 trial of SY-1425 (NCT02807558) in which we are evaluating the SE based patient selection strategies and gene circuitry derived pharmacodynamics clinical measurements, including differentiation markers, in patients with AML and MDS. Citation Format: Michael R. McKeown, Matthew L. Eaton, Chris Fiore, Emily Lee, Katie Austgen, Darren Smith, M. Ryan Corces, Ravindra Majeti, Christian C. Fritz. AML patient clustering by super-enhancers reveals an RARA associated transcription factor signaling partner [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 1511. doi:10.1158/1538-7445.AM2017-1511

Abstract 3085: SY-1425 (tamibarotene), a selective RARα agonist, shows synergistic anti-tumor activity with hypomethylating agents in a biomarker selected subset of AML

In patients with acute myeloid leukemia (AML) (≥ 60 years) and myelodysplastic syndrome (MDS), the use of hypomethylating agents (HMAs) may extend survival, but cure rates are very low and new treatment approaches are needed. HMAs, such as azacitidine, work by inhibiting DNMT1, leading to depletion of DNA methylation in the tumor cells. Hypomethylation, in turn, leads to the re-expression of genes associated with differentiation and growth arrest. We have recently explored the potent and selective RARα agonist SY-1425 in a genomically defined subset of AML. SY-1425 binds to RARα and causes a transition from repression to strong activation of target genes, thus reprogramming the tumor cells toward terminal maturation in RARA-high AML models, supporting our recently initiated Phase 2 trial in a biomarker-selected subset of AML and MDS (NCT02807558). Based on potential mechanistic synergy, we evaluated SY-1425 in combination with HMAs and identified a synergistic anti-proliferative effect. In RARA-high AML cell lines, but not RARA-low, the combination of SY-1425 with either azacitidine or decitabine showed synergistic anti-proliferative effects on the cells, with combination indices less than 0.5 over a range of concentrations from 0.01 to 100nM of SY-1425 and 0.1 to 1µM of HMAs. SY-1425 and azacitidine were also co-administered to a disseminated patient-derived xenograft (PDX) mouse model of RARA-high AML. The combination demonstrated superior reduction of tumor burden vs either therapy alone, leading to deeper and more durable responses with less than 1% detectable tumor burden. A follow-up study in the RARA-high PDX model investigated different treatment schedules of SY-1425 and azacitidine over a period of 56 days, supporting a regimen that maximizes anti-tumor activity and tolerability. Mechanistic studies using RNA-seq and ChIP-seq in AML cell line models have revealed that while azacitidine had only moderate suppressive or activating effects over a broad set of genes, the addition of SY-1425 in RARA-high models resulted in strong and specific induction of genes bound by RARα. It is hypothesized that azacitidine acts to prime the tumor cells for reprogramming by SY-1425. The loss of methyl-cytosine residues following azacitidine treatment lowers the barrier to SY-1425 mediated gene induction. It was observed that the two agents work cooperatively to promote terminal differentiation and decrease proliferation of the AML tumor cells, with the potential for increased clinical benefit in a subset of AML defined by a RARA super-enhancer. Based on the largely non-overlapping clinical toxicity profiles of azacitidine and SY-1425, supported by the observed tolerability of the combination in preclinical models, these findings provide a strong rationale for a planned study of this combination in biomarker selected, newly diagnosed AML patients. Citation Format: Michael R. McKeown, Emily Lee, Chris Fiore, Matthew L. Eaton, Christian C. Fritz, Eric Olson. SY-1425 (tamibarotene), a selective RARα agonist, shows synergistic anti-tumor activity with hypomethylating agents in a biomarker selected subset of AML [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 3085. doi:10.1158/1538-7445.AM2017-3085