Small-molecule Bromodomain Research Articles

Retinoblastoma protein as an intrinsic BRD4 inhibitor modulates small molecule BET inhibitor sensitivity in cancer

Bromodomain and extraterminal (BET) proteins including BRD4 play important roles in oncogenesis and immune inflammation. Here we demonstrate that cancer cells with loss of the retinoblastoma (RB) tumor suppressor became resistant to small molecule bromodomain inhibitors of BET proteins. We find that RB binds to bromodomain-1 (BD1) of BRD4, but binding is impeded by CDK4/6-mediated RB phosphorylation at serine-249/threonine-252 (S249/T252). ChIP-seq analysis shows RB knockdown increases BRD4 occupancy at genomic loci of genes enriched in cancer-related pathways including the GPCR-GNBIL-CREB axis. S249/T252-phosphorylated RB positively correlates with GNBIL protein level in prostate cancer patient samples. BET inhibitor resistance in RB-deficient cells is abolished by co-administration of CREB inhibitor. Our study identifies RB protein as a bona fide intrinsic inhibitor of BRD4 and demonstrates that RB inactivation confers resistance to small molecule BET inhibitors, thereby revealing a regulatory hub that converges RB upstream signaling onto BRD4 functions in diseases such as cancer.

Phase III MANIFEST-2: pelabresib+ ruxolitinib vs placebo+ ruxolitinib in JAK inhibitor treatment-naive myelofibrosis.

Myelofibrosis (MF) is a clonal myeloproliferative neoplasm, typically associated with disease-related symptoms, splenomegaly, cytopenias and bone marrow fibrosis. Patients experience a significant symptom burden and a reduced life expectancy. Patients with MF receive ruxolitinib as the current standard of care, but the depth and durability of responses and the percentage of patients achieving clinical outcome measures are limited; thus, a significant unmet medical need exists. Pelabresib is an investigational small-molecule bromodomain and extraterminal domain inhibitor currently in clinical development for MF. The aim of this article is to describe the design of the ongoing, global, phase III, double-blind, placebo-controlled MANIFEST-2 study evaluating the efficacy and safety of pelabresib and ruxolitinib versus placebo and ruxolitinib in patients with JAKi treatment-naiveMF. Clinical Trial Registration: NCT04603495 (ClinicalTrials.gov).

Chromatin Remodeling Induced by ARID1A Loss in Lung Cancer Promotes Glycolysis and Confers JQ1 Vulnerability.

This study links ARID1A loss with enhanced glycolysis in lung cancer and demonstrates the preclinical efficacy of BET inhibitor therapy as a strategy to combat tumor growth.

Abstract P033: CC-95775, a reversible, oral bromodomain and extra-terminal (BET) inhibitor in patients with advanced solid tumors (STs): Results of a phase 1 study

Abstract Background: BET proteins are epigenetic readers and activators of oncogenic pathways in cancer. CC-95775 is a novel oral small molecule bromodomain inhibitor. It is a non-specific inhibitor with potent activity against all 4 BET family members (BRD2, BRD3, BRD4, BRDT), and shows additional activity towards several non-BET bromodomain proteins. Methods: CC-95775-ST-001 is a phase 1 dose-escalation study of CC-95775 in patients with advanced STs. Primary objectives were to determine safety and recommended phase 2 dose (RP2D). Secondary and exploratory objectives were pharmacokinetics (PK), pharmacodynamics (PD) and antitumor activity. Four dose levels (DLs), from 400 to 1200 mg, administered on 4 consecutive days (Day 1 to Day 4) followed by 24 days off, every four weeks (Q4W), were evaluated. Results: As of 16 Apr 2021, 24 evaluable patients were enrolled and treated. The RP2D was 1200 mg (300 mg on days 1-4 and 24 days off, Q4W). One patient treated at 800 mg and two at 1200 mg had dose-limiting toxicities: QT prolongation, left ventricular ejection fraction (LVEF) decreased and abnormal T wave. The most common treatment-related adverse event (TRAE) was thrombocytopenia in 11 patients (45.8%), 2 of them grade 3 (8.3%) and 1 grade 4 (4.2%). Safety profile consisted mainly of gastrointestinal and general disorders. Five patients (20.8%) had transient serious TRAEs: nausea, QT prolongation, abnormal T wave, posterior reversible encephalopathy syndrome and acute kidney injury. Eleven patients 11 (45.8) had disease stabilization, 9 of them with a duration of ≥ 16 weeks and 4 of them ≥ 24 weeks: melanoma, chondrosarcoma, adenoid cystic carcinoma and chordoma. Plasma exposures increased in a dose-proportional manner across DLs. Across all dose groups, median Tmax was between ~ 2-4 h post-dose, indicating rapid absorption. The terminal half-life was approximately 30 h and repeated dosing leads to drug accumulation, as expected: ~2 - 3 fold for AUC and Cmax. CC-95775 induced ≥50% decrease of the PD biomarker CCR1 at the 4-hour timepoint in the 1200 mg cohort. Conclusions: CC-95775 was well tolerated and showed preliminary antitumor activity in heavily pretreated patients with advanced malignancies. The RP2D was 1200 mg Q4W. The favorable PD profile improved tolerability and enabled less frequent dosing. Further evaluation of CC-95775 alone or in combination in STs is warranted. Citation Format: Thierry Lesimple, María José de Miguel, Cristophe Le Tourneau, Mariano Ponz-Sarvise, Marie Paule Sablin, Diego Salas Benito, Bishoy Hanna, Henry Chang, Xin Wei, Marta Ocejo Garcia, Pilar Lardelli, Tania Sánchez, Josep Lluís Parra Palau, Zariana Nikolova, Emiliano Calvo. CC-95775, a reversible, oral bromodomain and extra-terminal (BET) inhibitor in patients with advanced solid tumors (STs): Results of a phase 1 study [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P033.

Exposure-response analysis of adverse events associated with molibresib and its active metabolites in patients with solid tumors.

Molibresib (GSK525762) is an investigational orally bioavailable small‐molecule bromodomain and extraterminal (BET) protein inhibitor for the treatment of advanced solid tumors. In the first‐time‐in‐human BET115521 study of molibresib in patients with solid tumors, thrombocytopenia was the most frequent treatment‐related adverse event (AE), QT prolongation was an AE of special interest based on preclinical signals, and gastrointestinal (GI) AEs (nausea, vomiting, diarrhea, and dysgeusia) were often observed. The aims of this analysis were the following: (i) develop a population pharmacokinetic (PK)/pharmacodynamic (PD) model capable of predicting platelet time courses in individual patients after administration of molibresib and identify covariates of clinical interest; (ii) evaluate the effects of molibresib (and/or its two active metabolites [GSK3529246]) exposure on cardiac repolarization by applying a systematic modeling approach using high‐quality, intensive, PK time‐matched 12‐lead electrocardiogram measurements; (iii) evaluate the exposure–response (ER) relationship between molibresib and/or GSK3529246 exposures and the occurrence of Grade 2 or higher GI AEs. Overall, the PK/PD model (including a maximal drug effect model and molibresib concentration) adequately described platelet counts following molibresib treatment and was used to simulate the impact of molibresib dosing on thrombocytopenia at different doses and regimens. ER analyses showed no clinically meaningful QT interval prolongation with molibresib at up to 100 mg q.d., and no strong correlation between molibresib exposure and the occurrence of Grade 2 or higher GI AEs. The models described here can aid dosing/schedule and drug combination strategies and may support a thorough QT study waiver request for molibresib.

JQ-1 ameliorates schistosomiasis liver fibrosis by suppressing JAK2 and STAT3 activation

Schistosomiasis is a serious parasitic infection caused by Schistosoma. The parasite deposits eggs in the host liver, causing inflammation that activates hepatic stellate cells (HSCs), which leads to liver fibrosis. Currently, there is no effective therapy for liver fibrosis; thus, treatments are urgently needed. Therefore, in the present study, mice infected with Schistosoma japonicum were treated with JQ-1, a small-molecule bromodomain inhibitor with reliable anti-tumor and anti-inflammatory activities. The fibrotic area of the liver measured by computer-assisted morphometric analysis and the expression levels of the cytoskeletal protein alpha smooth muscle actin (α-SMA) and of collagen assessed by quantitative PCR, Western blot and immunohistochemistry were significantly decreased in the liver following JQ-1 treatment compared with vehicle-treated controls. Total RNA was extracted from the liver of JQ-1–treated Schistosoma-infected mice for RNA-sequencing analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that JQ-1 affected biological processes and the expression of cellular components known to play key roles in the transdifferentiation of HSCs to myofibroblasts. In vitro treatment with JQ-1 of JS-1 cells, a mouse HSC line, indicated that JQ-1 significantly inhibited JS-1 proliferation but had no effect on JS-1 activity, senescence, or apoptosis. Western blot results showed that JQ-1 inhibited the expression levels of phosphorylated JAK2 and phosphorylated STAT3 without altering expression levels of these non-phosphorylated proteins. Taken together, these findings suggested that JQ-1 treatment ameliorated S. japonicum egg–induced liver fibrosis, at least in part, by suppressing HSC activation and proliferation through the inhibition of JAK2/STAT3 signaling. These results lay a foundation for the development of novel approaches to treat and control liver fibrosis caused by S. japonicum.

Targeting the Bromodomain of BRG-1/BRM Subunit of the SWI/SNF Complex Increases the Anticancer Activity of Temozolomide in Glioblastoma.

Glioblastoma (GBM) is a deadly and incurable brain cancer with limited therapeutic options. PFI-3 is a small-molecule bromodomain (BRD) inhibitor of the BRM/BRG1 subunits of the SWI/SNF chromatin remodeling complex. The objective of this study is to determine the efficacy of PFI-3 as a potential GBM therapy. We report that PFI-3 binds to these BRDs when expressed in GBM cells. PFI-3 markedly enhanced the antiproliferative and cell death-inducing effects of temozolomide (TMZ) in TMZ-sensitive GBM cells as well as overcame the chemoresistance of highly TMZ-resistant GBM cells. PFI-3 also altered gene expression in GBM and enhanced the basal and interferon-induced expression of a subset of interferon-responsive genes. Besides the effects of PFI-3 on GBM cells in vitro, we found that PFI-3 markedly potentiated the anticancer effect of TMZ in an intracranial GBM animal model, resulting in a marked increase in survival of animals bearing GBM tumors. Taken together, we identified the BRG1 and BRM subunits of SWI/SNF as novel targets in GBM and revealed the therapeutic potential of applying small molecule inhibitors of SWI/SNF to improve the clinical outcome in GBM using standard-of-care chemotherapy.

Optimization of Naphthyridones into Selective TATA-Binding Protein Associated Factor 1 (TAF1) Bromodomain Inhibitors.

Bromodomain containing proteins and the acetyl-lysine binding bromodomains contained therein are increasingly attractive targets for the development of novel epigenetic therapeutics. To help validate this target class and unravel the complex associated biology, there has been a concerted effort to develop selective small molecule bromodomain inhibitors. Herein we describe the structure-based efforts and multiple challenges encountered in optimizing a naphthyridone template into selective TAF1(2) bromodomain inhibitors which, while unsuitable as chemical probes themselves, show promise for the future development of small molecules to interrogate TAF1(2) biology. Key to this work was the introduction and modulation of the basicity of a pendant amine which had a substantial impact on not only bromodomain selectivity but also cellular target engagement.

Synthesis, evaluation and in silico studies of novel BRD4 bromodomain inhibitors bearing a benzo[d]isoxazol scaffold

The BRD4 protein is associated with various diseases, which has been an attractive target for the treatment of cancer and inflammation. This paper is a follow-up to our previous studies, in which we report the structure-based design, synthesis, and evaluation of a new class of small-molecule BRD4 bromodomain inhibitors bearing a benzo[d]isoxazol scaffold. The SARs focused on exploration of the 2′ or 3′ position to afford novel inhibitors that may avoid potential metabolically unstable site. The most potent inhibitor 13f exhibited high binding affinity to BRD4(1) with a ΔTm value of 7.8 °C as evaluated in thermal shift assay (TSA). The potent activity was also demonstrated by a peptide competition assay with an IC50 value of 0.21 μM. The docking studies revealed the binding mode of the compounds with the active site of BRD4(1). In addition, in silico predictions indicated that these compounds possessed good drug-likeness and pharmacokinetic profile. This paper is a follow-up to our previous studies, in which we report the structure-based design, synthesis, and evaluation of a new class of small-molecule BRD4 bromodomain inhibitors bearing a benzo[d]isoxazol scaffold.

The long noncoding RNA lnc-HLX-2-7 is oncogenic in Group 3 medulloblastomas.

Medulloblastoma (MB) is an aggressive brain tumor that predominantly affects children. Recent high-throughput sequencing studies suggest that the noncoding RNA genome, in particular long noncoding RNAs (lncRNAs), contributes to MB subgrouping. Here we report the identification of a novel lncRNA, lnc-HLX-2-7, as a potential molecular marker and therapeutic target in Group 3 MBs. Publicly available RNA sequencing (RNA-seq) data from 175 MB patients were interrogated to identify lncRNAs that differentiate between MB subgroups. After characterizing a subset of differentially expressed lncRNAs in vitro and in vivo, lnc-HLX-2-7 was deleted by CRISPR/Cas9 in the MB cell line. Intracranial injected tumors were further characterized by bulk and single-cell RNA-seq. Lnc-HLX-2-7 is highly upregulated in Group 3 MB cell lines, patient-derived xenografts, and primary MBs compared with other MB subgroups as assessed by quantitative real-time, RNA-seq, and RNA fluorescence in situ hybridization. Depletion of lnc-HLX-2-7 significantly reduced cell proliferation and 3D colony formation and induced apoptosis. Lnc-HLX-2-7-deleted cells injected into mouse cerebellums produced smaller tumors than those derived from parental cells. Pathway analysis revealed that lnc-HLX-2-7 modulated oxidative phosphorylation, mitochondrial dysfunction, and sirtuin signaling pathways. The MYC oncogene regulated lnc-HLX-2-7, and the small-molecule bromodomain and extraterminal domain family‒bromodomain 4 inhibitor Jun Qi 1 (JQ1) reduced lnc-HLX-2-7 expression. Lnc-HLX-2-7 is oncogenic in MB and represents a promising novel molecular marker and a potential therapeutic target in Group 3 MBs.

Abstract 3079: FT-6876, a potent and selective inhibitor of CBP/p300 with antitumor activity in AR-positive breast cancer

Abstract Triple negative breast cancer (TNBC) remains a high unmet medical need, commonly treated with chemotherapies. Biomarker-driven treatment strategies have led to the recent approvals of both PARP inhibitors and an immune checkpoint inhibitor for a subset of women with TNBC. Androgen receptor (AR) is expressed in a 20-30% of patients with TNBC. Preclinical studies indicate that AR in this context can substitute for estrogen receptor (ER) to drive transcription of ER target genes, supporting TNBC tumor proliferation and survival. AR-driven transcription is dependent upon multiple essential cofactors, among which are the structurally related, bromodomain (BRD) and lysine acetyl transferase containing proteins, CBP and p300. Here we report the discovery of FT-6876, a potent and selective small molecule bromodomain inhibitor of CBP/p300. Our hypothesis is that modulation of CBP/p300 with FT-6876 will alter chromatin compaction and, when in the vicinity of AR responsive genes, selectively reduce AR transcriptional activity at these loci. This should translate to growth inhibition in vitro and in vivo in AR+ breast cancer models. Consistent with this hypothesis, a rapid reduction in H3K27Ac was observed in an AR+ breast cancer cell line following treatment with FT-6876. Removal of the compound led to the restoration of this mark to baseline levels reflecting the dynamic state of histone modification in cells. High content analysis revealed that the reduction in H3K27Ac occurred in the majority of the cells. In order to have a greater understanding of the dynamic and selective impact of FT-6876 on AR/ER transcriptional activity, we used precision run-on (PRO) seq to quantify nascent RNA and infer transcription factor modulation genome-wide. GSEA analysis of nascent RNA showed a high enrichment for AR and ER pathway genes upon exposure with FT-6876. Using ChIPseq, we confirmed that the reduction of H3K27Ac at specific promoter sites was concurrent with a decrease in CBP/p300 on the chromatin and reduction in nascent RNA and eRNA. FT-6876 reduced proliferation in a time-dependent manner with optimal growth inhibition observed after 10 days drug exposure. This was maintained even after removal of the drug for several days. Across a panel of breast cancer cell lines, there was a good correlation between growth inhibition and AR expression. In vivo, FT-6876 induced tumor stasis at well tolerated doses in AR+ breast cancer models. This was associated with a reduction in H3K27Ac, AR and ER target gene modulation, and reduction in Ki67. FT-6876 is a promising new CBP/p300 bromodomain inhibitor demonstrating efficacy in preclinical models of AR+ breast cancer. Citation Format: Maureen Caligiuri, Grace L. Williams, Jennifer Castro, Linda Battalagine, Kristina Muskiewicz, Erik Wilker, Lili Yao, Shawn Schiller, Angela Toms, Ping Li, Eneida Pardo, Bradford Graves, Kenneth W. Wood, Sylvie M. Guichard. FT-6876, a potent and selective inhibitor of CBP/p300 with antitumor activity in AR-positive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3079.

BET inhibitor molibresib for the treatment of advanced solid tumors: Final results from an open-label phase I/II study.

3618 Background: Molibresib is an orally available, small molecule bromodomain and extra-terminal domain (BET) protein inhibitor under investigation for treatment of advanced solid tumors. Methods: This was an open-label, single- and repeat-dose, 2-part, Phase 1/2 study including patients (aged ≥16 years) with advanced solid tumors. Part 1: patients received different oral doses of molibresib (2–100mg QD; amorphous free-base formulation) to determine recommended Phase 2 dose. Part 2 (expansion cohort): patients with various tumor types received the bioequivalent besylate formulation (75mg) to explore clinical activity at recommended dose. Safety and efficacy (response rate [RR] based on RECIST 1.1 criteria, progression-free survival [PFS], and overall survival [OS]) were evaluated for the total cohort (patients from Part 1 and 2). Safety, pharmacokinetic, pharmacodynamic, and efficacy per tumor type were evaluated in Part 2. Results: Part 1 only data have previously been reported. Overall, 196 patients were included in the total cohort (1 patient in Part 1 was counted twice). In the all treated population, 195 patients (median age 58 years; 46% male) received ≥1 dose of molibresib (Part 1: n = 93; Part 2: n = 102). Adverse events (AEs) were experienced by 193/196 (98%) patients; 180/196 (92%) had a treatment-related AE (TRAE). AEs led to permanent treatment discontinuation in 38/196 (19%) patients. Of different tumor types in Part 2, NUT carcinoma (NC) had the lowest frequency of TRAEs (10/12 [83%]) and AEs leading to permanent treatment discontinuation (1/12 [8%]). In total cohort, 3/31 NC patients and 1/35 with castration-resistant prostate cancer (CRPC) achieved a confirmed partial response. A further 67/196 (34%) achieved stable disease (SD). In Part 2, RR in 12 NC patients was 8% (CI: 0.2–38.5); 50% had SD and median PFS was 4.8 months with median OS of 5.0 months. In CRPC patients, RR was 4% (CI: 0.1–21.9); 22% had SD; median PFS was 8.0 months with median OS of 9.1 months. Plasma concentrations for molibresib and active metabolites were similar between different tumor types. Gene expression analysis from pre- and post-dose biopsy samples collected from 10 mCRPC patients showed transcriptional downregulation of Myc target genes upon treatment with molibresib. Conclusions: Molibresib demonstrated a manageable safety and tolerability profile with single agent activity observed in selected patients with NC and CRPC. Clinical trial information: NCT01587703 .

P5509Apabetalone (RVX-208) inhibits key drivers of vascular inflammation, calcification, and plaque vulnerability through a BET-dependent epigenetic mechanism

Abstract Apabetalone (RVX-208) is an orally available small molecule bromodomain & extraterminal (BET) protein inhibitor that targets the second bromodomain (BD2) of BET proteins. Apabetalone returns dysregulated BET-dependent transcription toward normal physiological levels. In phase 2 trials, apabetalone treatment reduced the incidence of major adverse cardiac events by 44% in CVD patients and by 57% in diabetic CVD patients. Previous studies have highlighted apabetalone's positive impact on vascular calcification (VC) and inflammation (VI) marker expression in vitro, as well as its ability to lower serum alkaline phosphatase (ALP) levels, and improve atherosclerotic plaque stability parameters in treated patients. In CVD, elevated inflammatory mediators and cell surface adhesion molecules drive VI, resulting in leukocyte adhesion, infiltration, uptake of oxLDL, and ultimately plaque formation. Here we show in vitro that THP-1 monocyte adhesion to human aortic endothelial cells (HAECs) increases with TNFα stimulation and is attenuated by apabetalone treatment, with fewer monocytes attaching to HAECs under flow conditions. This functional outcome is attributed to apabetalone's reduction of key endothelial adhesion genes, VCAM-1 (50%, p=0.0001) and SELE (37%, p=9x10–5). Apabetalone also prevents TNFα induction of endothelial recruitment genes (MCP-1; 75%, p=0.0002) and genes involved in plaque rupture (IL8; 24%, p=2x10–5). Basal HAEC ALP expression, a potential contributor to endothelial dysfunction and VC, also decreases with apabetalone treatment (70%, p=0.005). Induction of VI genes by TNFα is BET-dependent as degradation of BET proteins by MZ-1 prevents an increase in transcripts in response to TNFα treatment. Ingenuity® Pathway Analysis (IPA®), GSEA, and GO analysis of HAEC gene expression data predicts apabetalone inhibition of pro-atherogenic pathways, gene sets, and upstream regulators induced by TNFα. These include cytokine and chemokine, Toll-Like Receptor (TLR), NFkβ, Interferon and TNFα signaling. In addition, IPA® disease and biological function analysis predicts inhibition of immune cell activation and recruitment by apabetalone. Plasma proteomics (SOMAscan®) and IPA® analysis from apabetalone-treated CVD patients in ASSERT and ASSURE phase 2 trials indicate that apabetalone inhibits pro-atherogenic upstream regulators (IL-6 and IFNy), canonical pathways, and diseases and functions. Serum ALP also decreases dose dependently with apabetalone treatment (ASSERT). Epigenetic inhibition of VI and VC driven atherogenesis likely contributes to the reduction in MACE observed in phase 2 apabetalone treated patients. The ongoing phase 3 post-acute coronary syndrome (ACS) clinical trial in T2DM patients, BETonMACE, is currently testing this hypothesis.

APABETALONE, AN EPIGENETIC BET INHIBITOR IN A PHASE 3 TRIAL, INHIBITS VASCULAR INFLAMMATION AND CELLULAR ADHESION LEADING TO BENEFICIAL OUTCOMES IN CVD PATIENTS

Apabetalone (RVX-208) is a small molecule bromodomain & extraterminal (BET) protein inhibitor, targeting the second bromodomain (BD2) of BET proteins. In cardiovascular disease (CVD) patients enrolled in phase 2 trials apabetalone treatment reduced the relative risk of a CV event by 44% (Nicholls

Disulfide bridge formation influences ligand recognition by the ATAD2 bromodomain.

The ATPase family, AAA domain-containing protein 2 (ATAD2) has a C-terminal bromodomain, which functions as a chromatin reader domain recognizing acetylated lysine on the histone tails within the nucleosome. ATAD2 is overexpressed in many cancers and its expression is correlated with poor patient outcomes, making it an attractive therapeutic target and potential biomarker. We solved the crystal structure of the ATAD2 bromodomain and found that it contains a disulfide bridge near the base of the acetyllysine binding pocket (Cys1057-Cys1079). Site-directed mutagenesis revealed that removal of a free C-terminal cysteine (C1101) residue greatly improved the solubility of the ATAD2 bromodomain in vitro. Isothermal titration calorimetry experiments in combination with the Ellman's assay demonstrated that formation of an intramolecular disulfide bridge negatively impacts the ligand binding affinities and alters the thermodynamic parameters of the ATAD2 bromodomain interaction with a histone H4K5ac peptide as well as a small molecule bromodomain ligand. Molecular dynamics simulations indicate that the formation of the disulfide bridge in the ATAD2 bromodomain does not alter the structure of the folded state or flexibility of the acetyllysine binding pocket. However, consideration of this unique structural feature should be taken into account when examining ligand-binding affinity, or in the design of new bromodomain inhibitor compounds that interact with this acetyllysine reader module.

Design and Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors Targeting a Methionine.

BET proteins are key epigenetic regulators that regulate transcription through binding to acetylated lysine (AcLys) residues of histones and transcription factors through bromodomains (BDs). The disruption of this interaction with small molecule bromodomain inhibitors is a promising approach to treat various diseases including cancer, autoimmune and cardiovascular diseases. Covalent inhibitors can potentially offer a more durable target inhibition leading to improved in vivo pharmacology. Here we describe the design of covalent inhibitors of BRD4(BD1) that target a methionine in the binding pocket by attaching an epoxide warhead to a suitably oriented noncovalent inhibitor. Using thermal denaturation, MALDI-TOF mass spectrometry, and an X-ray crystal structure, we demonstrate that these inhibitors selectively form a covalent bond with Met149 in BRD4(BD1) but not other bromodomains and provide durable transcriptional and antiproliferative activity in cell based assays. Covalent targeting of methionine offers a novel approach to drug discovery for BET proteins and other targets.

Abstract P3-06-07: The BET bromodomain inhibitors ZEN-3694 and ZEN-3309 targets several mechanisms of resistance to endocrine therapies in preclinical models of ER+ breast cancers

Abstract More than 200,000 women are diagnosed with breast cancer every year in the United States. About 80% of these cases are estrogen receptor positive (ER+), which is characterized by the up-regulation of ER signaling and downstream activation of cyclin-dependent kinases CDK4/6 and cyclin D1 (CCND1). Current lines of therapies include either endocrine therapies or CDK4/6 inhibitors, which have resulted in great improvement in the treatment of ER+ breast cancer. As expected, resistance to these therapies occurs over time and the development of additional therapeutic strategies is needed. Recently, the bromodomain and extra terminal (BET) proteins BRD3 and BRD4 were shown to be involved in the transcription of ER, and BET inhibitor (BETi) treatment can suppress ER-mediated signaling, offering a potential strategy to overcome endocrine resistance by further shutting down ER signaling regardless of ESR1 mutation status. However, it is still unclear if BRD3/4 are involved in the mechanisms of resistance to endocrine therapies, and whether BETi can potently inhibit the proliferation of ER+ breast cancer cells that are resistant to CDK4/6 inhibitors. ZEN-3694 is an orally bioavailable small molecule bromodomain BET inhibitor currently undergoing clinical development in metastatic castration-resistant prostate cancer (mCRPC). ZEN-3694 showed efficacy in a panel of ER+ breast cancer cell lines and synergized with both tamoxifen and fulvestrant to inhibit proliferation and induce apoptosis. ZEN-3694 also showed activity in several models of resistance to ER+ breast cancer therapies, including tamoxifen, fulvestrant, estrogen-deprivation, as well as with CDK4/6 inhibitors. Analysis of the major pathways that were up-regulated upon acquisition of resistance to endocrine therapies and down-regulated by ZEN-3694 revealed several key players previously involved in developing resistance to endocrine therapies in breast cancer patients, including: inflammatory cytokines (IL-6, IL-1A, IL-1B), inducers and regulators of epithelial-mesenchymal transition (EMT) (Slug and ZEB-1), cancer stem cell marker (CD44), and angiogenesis regulators (VEGFA, VEGFC). Therefore, several of the transcriptional programs linked to the acquisition of resistance to endocrine therapies are regulated by BET proteins and inhibited by ZEN-3694. Together, these results suggest that BET inhibitors have the potential to be a novel therapeutic strategy to treat ER+ breast cancer patients developing resistance to endocrine therapies as well as CDK4/6 inhibitors. Citation Format: Kharenko OA, Patel RG, Jahagirdar R, Attwell S, Calosing C, Tsujikawa L, Campeau E, Lakhotia S, Hansen HC. The BET bromodomain inhibitors ZEN-3694 and ZEN-3309 targets several mechanisms of resistance to endocrine therapies in preclinical models of ER+ breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-06-07.

Bisphosphonate-Functionalized Hydroxyapatite Nanoparticles for the Delivery of the Bromodomain Inhibitor JQ1 in the Treatment of Osteosarcoma.

Osteosarcoma (OS) is one of the most common neoplasia among children, and its survival statistics have been stagnating since the combinatorial anticancer therapy triad was first introduced. Here, we report on the assessment of the effect of hydroxyapatite (HAp) nanoparticles loaded with medronate, the simplest bisphosphonate, as a bone-targeting agent and JQ1, a small-molecule bromodomain inhibitor, as a chemotherapeutic in different 2D and 3D K7M2 OS in vitro models. Both additives decreased the crystallinity of HAp, but the effect was more intense for medronate because of its higher affinity for HAp. As the result of PO43--NH+ binding, JQ1 shielded the surface phosphates of HAp and pushed its surface charge to more positive values, exhibiting the opposite effect from calcium-blocking medronate. In contrast to the faster and more exponential release of JQ1 from monetite, its release from HAp nanoparticles followed a zero-order kinetics, but 98% of the payload was released after 48 h. The apoptotic effect of HAp nanoparticles loaded with JQ1, with medronate and with both JQ1 and medronate, was selective in 2D culture: pronounced against the OS cells and nonexistent against the healthy fibroblasts. While OS cell invasion was significantly inhibited by all of the JQ1-containing HAp formulations, that is, with and without medronate, all of the combinations of the targeting compound, medronate, and the chemotherapeutic, JQ1, delivered using HAp, but not HAp alone, inhibited OS cell migration from the tumor spheroids. JQ1 delivered using HAp had an effect on tumor migration, invasion, and apoptosis even at extremely low, subnanomolar concentrations, at which no effect of JQ1 per se was observed, meaning that this form of delivery could help achieve a multifold increase of this drug's efficacy. More than 80% of OS cells internalized JQ1-loaded HAp nanoparticles after 24 h of coincubation, suggesting that this augmentation of the activity of JQ1 may be due to the intracellular delivery and sustained release of the drug enabled by HAp. In addition to the reduction of the OS cell viability, the reduction of the migration and invasion radii was observed in OS tumor spheroids challenged with even JQ1-free medronate-functionalized HAp nanoparticles, demonstrating a definite anticancer activity of medronate alone when combined with HAp. The effect of medronate-functionalized JQ1-loaded HAp nanoparticles was most noticeable against OS cells differentiated into an osteoblastic lineage, in which case they surpassed in effect pure JQ1 and medronate-free compositions. The activity of JQ1 was mediated through increased Ezrin expression and decreased RUNX2 expression and was MYC and FOSL1 independent, but these patterns of gene expression changed in cells challenged with the nanoparticulate form of delivery, having been accompanied by the upregulation of RUNX2 and downregulation of Ezrin in OS cells treated with medronate-functionalized JQ1-loaded HAp nanoparticles.

Genomic targeting of epigenetic probes using a chemically tailored Cas9 system

Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells. The versatility of this technology is demonstrated by delivering dCas9 fusions that include either the small-molecule bromodomain and extra-terminal family bromodomain inhibitor JQ1 or a peptide-based PRC1 chromodomain ligand, which are capable of recruiting endogenous copies of their cognate binding partners to targeted genomic binding sites. We expect that this technology will allow for the genomic localization of a wide array of small molecules and modified proteinaceous materials.

Targeting BET Bromodomains in Pre-Clinical Models of Burkitt Lymphoma

Background: Burkitt lymphoma (BL) is the most common NHL type in children. Although treatment for pediatric BL has improved significantly, there is an urgent need for novel therapies that reduce the toxicity of modern treatment regimens and improve on the dismal survival observed in the relapsed/refractory setting where only about 20-30% of patients survive their disease. Recent reports have implicated co-activation of c-Myc and PI3K in Burkitt lymphomagenesis. Genomic analysis of recurrent oncogenic mutations in BL have identified tonic B-cell receptor signaling and the over-expression of Myc induced microRNAs from the MIR17-92 family, e.g. mir17 and mir19, as possible mechanisms of PI3K activation in BL. Mir17 and mir19, have been implicated in Burkitt lymphomagenesis and their overexpression may be associated with a higher risk of relapse. The protein phosphatase and tensin homolog (PTEN) is a major regulator of PI3K pathway activation. MIR17-92 cluster members have been shown to target PTEN leading to increased PI3K activation. We have previously identified increased expression of mir17 and mir19 along with increased activation of AKT in cell line models of chemotherapy resistant BL suggesting a potential mechanism for increased resistance. BET bromodomains interact with chromatin and enhance transcriptional activation of numerous genes including c-Myc. Thus, BET bromodomains represent a promising target in BL. To investigate the effects of inhibition of c-Myc driven activation of the PI3K/AKT/mTOR pathway, we characterized the activity of the highly potent small molecule bromodomain inhibitor JQ1 in chemotherapy sensitive and resistant BL cell lines. Additionally, we analyzed the ability to enhance anti-lymphoma activity of PI3K/Akt/mTOR pathway inhibition in BL by the combination of BET bromodomain inhibition and targeted inhibition of the PI3K/AKT/mTOR pathway.Methods: The in vitro activity of JQ1 was investigated in the BL cell lines Raji, Raji 4RH (chemotherapy-rituximab resistant), Raji 8RH (rituximab resistant), Ramos, and Daudi. Cell Viability following exposure to JQ1 alone and in combination with the PI3K/mTOR inhibitor omipalisib (GSK458)) was analyzed using Cell-Titer Glo or Alamar Blue assays following 24, 48, and 72 hour exposure over a range of inhibitor concentrations. Induction of apoptosis was analyzed using western blotting for cleaved PARP. C-Myc expression following JQ1 exposure was determined by western blot following 48 hour JQ1 exposure. The effect of JQ1 exposure on the expression of c-Myc induced microRNA expression was determined by qRT-PCR in cells exposed to JQ1 for 48hours. Synergy of combination exposures was determined using CalcuSyn to generate combination index (CI) values.Results: In vitro exposure of BL cell lines to JQ1 for 24, 48, and 72 hours resulted in a significant dose and time dependent decrease in viable cells (72 hour IC50 values: Raji 0.12µM, Raji 4RH 1.7µM, Raji 8RH 0.7µM, Ramos 0.22µM and Daudi 4µM). There was an increase in cleaved PARP after 72 hour exposure indicating induction of apoptosis. While single agent effect was seen in the resistant Raji 4RH cell line, activity was noted to primarily occur at the higher end of the dosing. Western blot analysis demonstrated a reduction in c-Myc expression following exposure to JQ1 1µM for 24 hours (relative band intensity normalized to control: Raji=0.12, Raji 4RH=0.18, Raji 8RH=0.11). qPCR showed a reduction in Mir17 relative transcription levels after 48 hours of exposure to JQ1 2.5mM (relative expression compared to control: Raji=0.72, Raji 4RH=0.98, Raji 8RH=0.83, Ramos=0.57, Daudi=0.46). When combined with omipalisib, an increased effect on cell viability was noted. The combination effect was noted to be synergistic (CI<0.9) at multiple dose combinations while other combinations exhibited primarily additive effects.Conclusion: In vitro inhibition of BET bromodomains with JQ1 results in a decrease in c-Myc expression and a decrease in c-Myc dependent miR expression with impaired proliferation and induction of apoptosis in chemotherapy-sensitive and -resistant BL cell lines. Augmented, and in some cases synergistic, activity is also noted with dual inhibition of BET bromodomains and the PI3K/Akt/mTOR pathway in BL cell lines. DisclosuresNo relevant conflicts of interest to declare.