Development of BET Protein Bromodomain Inhibition for the Treatment of Hematologic Malignancies

Abstract Abstract 1331 BET (bromodomain and extra-terminal) proteins bind to acetylated lysine residues on histones and thereby directly and selectively regulate the expression of genes relevant to cancer. Small molecule inhibition of BET protein binding to chromatin suppresses the transcription of MYC, a subset of NF-κB-dependent genes, and BCL-2. Because of the clinical potential of this novel mechanism, we have discovered selective and potent small molecule inhibitors of BET bromodomains with physical and pharmacokinetic properties that are favorable for clinical development. CPI-267232 is representative of a series of small molecule BET inhibitors with these characteristics. In a biochemical binding assay it has an IC50 126 nM against BD1 of BRD4, and in the MV4-11 leukemia cell line it suppresses the transcription of MYC with an EC50 of 170 nM and inhibits its growth with a GI50of 120 nM. In a panel of more than 100 cancer cell lines CPI-267232 and other structurally dissimilar BET inhibitors demonstrate growth inhibitory activity most potently and consistently against cell lines of hematologic origin. Cells treated with CPI-267232 undergo a G1 arrest with the more sensitive lines undergoing apoptosis with longer periods of drug exposure (> 48 hrs). CPI-267232 has high oral bioavailability in mice (∼80%) but is cleared rapidly, with an elimination half-life of approximately 3 hrs (in dogs, oral bioavailability is 94% with an elimination half-life of 9 hrs). PK-PD studies conducted in mice bearing subcutaneous xenografts of Raji Burkitt's lymphoma demonstrated that maximal (80%) suppression of MYC expression was achieved 4 hours following a single 30 mg/kg oral dose; MYC expression returned to baseline levels by 12 hours, consistent with the rapid elimination of the compound. Efficacy studies in nude mice bearing xenografts were subsequently conducted, with a 30 mg/kg PO bid regimen yielding a %T/C value of 23% (Raji) or regression (MV4-11) after 21 days of treatment. Evaluation of the relationships between various measurements of drug exposure and efficacy revealed that efficacy is primarily driven by maintaining drug concentration above a minimum value rather by total AUC or Cmax. This observation is consistent with the importance of exposure duration in effecting growth arrest and cell killing in tissue culture. Although the transcription of MYC is frequently suppressed, the overall effects of BET inhibition on gene transcription vary across cell lines of different origin. Genome-wide expression profiling of 2 myeloma and 3 leukemia cells lines at 4 and 24 hours after exposure to a small molecule BET inhibitor resulted in the identification of approximately 200 genes that were commonly either up- or down-regulated by at least 2-fold with statistical significance. In addition, a small set of BET inhibitor-sensitive genes has been identified in peripheral blood mononuclear cells. The discovery of novel BET inhibitors with optimized potency, selectivity, and pharmacokinetic properties, coupled with these insights into the pharmacokinetic and pharmacodynamic determinants of anti-tumor activity, provides an opportunity for the rational development of BET inhibition in the treatment of patients with hematologic malignancies. Disclosures: Sims: Constellation Pharmaceuticals: Employment. Normant:Constellation Pharmaceuticals: Employment. Sandy:Constellation Pharmaceuticals: Employment. Mertz:Constellation Pharmaceuticals: Employment. Bryant:Constellation Pharmaceuticals: Employment. O'Meara:Constellation Pharmaceuticals: Employment. Green:Constellation Pharmaceuticals: Employment. Cooper:Constellation Pharmaceuticals: Employment. Audia:Constellation Pharmaceuticals: Employment.

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Feasibility of Extrapolating Randomly Taken Plasma Samples to Trough Levels for Therapeutic Drug Monitoring Purposes of Small Molecule Kinase Inhibitors

Pharmaceuticals ◽

10.3390/ph14020119 ◽

2021 ◽

Vol 14 (2) ◽

pp. 119

Author(s):

Ruben A. G. van Eerden ◽

Esther Oomen-de Hoop ◽

Aad Noordam ◽

Ron H. J. Mathijssen ◽

Stijn L. W. Koolen

Keyword(s):

Therapeutic Drug Monitoring ◽

Small Molecule ◽

Trough Concentration ◽

Drug Monitoring ◽

Half Life ◽

Kinase Inhibitors ◽

Therapeutic Drug ◽

Blood Samples ◽

Elimination Half ◽

Trough Levels

Small molecule kinase inhibitors (SMKIs) are widely used in oncology. Therapeutic drug monitoring (TDM) for SMKIs could reduce underexposure or overexposure. However, logistical issues such as timing of blood withdrawals hamper its implementation into clinical practice. Extrapolating a random concentration to a trough concentration using the elimination half-life could be a simple and easy way to overcome this problem. In our study plasma concentrations observed during 24 h blood sampling were used for extrapolation to trough levels. The objective was to demonstrate that extrapolation of randomly taken blood samples will lead to equivalent estimated trough samples compared to measured Cmin values. In total 2241 blood samples were analyzed. The estimated Ctrough levels of afatinib and sunitinib fulfilled the equivalence criteria if the samples were drawn after Tmax. The calculated Ctrough levels of erlotinib, imatinib and sorafenib met the equivalence criteria if they were taken, respectively, 12 h, 3 h and 10 h after drug intake. For regorafenib extrapolation was not feasible. In conclusion, extrapolation of randomly taken drug concentrations to a trough concentration using the mean elimination half-life is feasible for multiple SMKIs. Therefore, this simple method could positively contribute to the implementation of TDM in oncology.

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The Dynamics of Circulating Heparin-Binding Protein: Implications for Its Use as a Biomarker

Journal of Innate Immunity ◽

10.1159/000521064 ◽

2021 ◽

pp. 1-14

Author(s):

Jane Fisher ◽

Fredrik Kahn ◽

Elena Wiebe ◽

Pontus Gustafsson ◽

Thomas Kander ◽

...

Keyword(s):

Half Life ◽

Binding Protein ◽

Hematologic Malignancies ◽

Heparin Binding ◽

Monocytic Cell ◽

Negative Results ◽

Heparin Binding Protein ◽

Elimination Half ◽

The Impact

Heparin-binding protein (HBP) is a promising biomarker for the development and severity of sepsis. To guide its use, it is important to understand the factors that could lead to false-positive or negative results, such as inappropriate release and inadequate clearance of HBP. HBP is presumably released only by neutrophils, and the organs responsible for its elimination are unknown. In this study, we aimed to determine whether non-neutrophil cells can be a source of circulating HBP and which organs are responsible for its removal. We found that in two cohorts of neutropenic patients, 12% and 19% of patients in each cohort, respectively, had detectable plasma HBP levels. In vitro, three leukemia-derived monocytic cell lines and healthy CD14+ monocytes constitutively released detectable levels of HBP. When HBP was injected intravenously in rats, we found that plasma levels of HBP decreased rapidly, with a distribution half-life below 10 min and an elimination half-life of 1–2 h. We measured HBP levels in the liver, spleen, kidneys, lungs, and urine using both ELISA and immunofluorescence quantitation, and found that the majority of HBP was present in the liver, and a small amount was present in the spleen. Immunofluorescence imaging indicated that HBP is associated mainly with hepatocytes in the liver and monocytes/macrophages in the spleen. The impact of hematologic malignancies and liver diseases on plasma HBP levels should be explored further in clinical studies.

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CDK9 Inhibition Reverses Resistance to ABT-199 (GDC-0199) By Down-Regulating MCL-1

Blood ◽

10.1182/blood.v124.21.2161.2161 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2161-2161 ◽

Cited By ~ 1

Author(s):

Jun Chen ◽

Sha Jin ◽

Paul Tapang ◽

Stephen K Tahir ◽

Morey Smith ◽

...

Keyword(s):

Clin Oncol ◽

Cell Lines ◽

Small Molecule ◽

Hodgkin’S Lymphoma ◽

Acquired Resistance ◽

Hematologic Malignancies ◽

Hodgkin's Lymphoma ◽

Employment Equity ◽

Non Hodgkin's Lymphoma ◽

Equity Ownership

Abstract All authors are employees of AbbVie and participated in the design, conduct, and interpretation of these studies. AbbVie and Genentech provided financial support for these studies and participated in the review and approval of this publication. The BCL-2-selective inhibitor ABT-199 has demonstrated efficacy in numerous preclinical models of hematologic malignancies without causing thrombocytopenia, a dose-limiting toxicity associated with the BCL-2/BCL-XL inhibitor navitoclax (Souers et al. 2013. Nat. Med. 19, 202-208). ABT-199 has also demonstrated clinical activity in chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) (Seymour et al. 2014. J. Clin. Oncol. 32, 448s; Davids et al. 2014. J. Clin. Oncol. 32, 544s). Despite these encouraging early clinical data, some subjects do not respond to ABT-199 or progress while on treatment. Pre-clinical models indicate that both intrinsic and acquired resistance may be a consequence of MCL-1 expression. Consequently, we have explored potent and selective small molecule inhibitors of CDK9, a kinase known to maintain the expression of MCL-1 through its role in p-TEFb-mediated transcription. Inhibition of CDK9 resulted in the rapid loss in RNA polymerase II phosphorylation (Serine 5) and MCL-1 expression that was closely followed by the induction of apoptosis in MCL-1-dependent cell lines, a cellular response that could be rescued by overexpression of BCL-2. Substantial synergy was observed between CDK9 inhibitors and ABT-199 in a number of hematologic cell lines with intrinsic or acquired resistance to ABT-199. Direct inhibition of MCL-1 with the small molecule BH3 mimetic A-1210477 was also highly synergistic with ABT-199, further validating the utility of co-inhibiting MCL-1 and BCL-2 function simultaneously in ABT-199 resistant tumors. Importantly, the CDK9 inhibitor-ABT-199 combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in xenograft models of non-Hodgkin’s lymphoma (NHL) and acute myelogenous leukemia (AML). These data indicate that CDK9 inhibitors may be highly efficacious when used in combination with ABT-199 for the treatment of hematologic malignancies. Disclosures Chen: Abbvie: Employment, Equity Ownership. Jin:Abbvie: Employment, Equity Ownership. Tapang:abbvie: Employment, Equity Ownership. Tahir:abbvie: Employment, Equity Ownership. Smith:abbvie: Employment, Equity Ownership. Xue:abbvie: Employment, Equity Ownership. Zhang:abbvie: Employment, Equity Ownership. Gao:abbvie: Employment, Equity Ownership. Tong:abbvie: Employment, Equity Ownership. Clark:abbvie: Employment, Equity Ownership. Ricker:abbvie: Employment, Equity Ownership. Penning:abbvie: Employment, Equity Ownership. Albert:abbvie: Employment, Equity Ownership. Phillips:abbvie: Employment, Equity Ownership. Souers:abbvie: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership.

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Computational Identification of Tractable Drug-like Inhibitors of eIF4A1

Blood ◽

10.1182/blood-2018-99-116829 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5802-5802

Author(s):

Derek Essegian ◽

Tyler A. Cunningham ◽

Jonathan H. Schatz ◽

Stephan Schurer

Keyword(s):

Crystal Structure ◽

Crystal Structures ◽

Cell Lines ◽

Small Molecule ◽

Nucleotide Binding ◽

Hematologic Malignancies ◽

Dead Box ◽

Binding Cleft ◽

Homology Models

Abstract Targeted signaling inhibitors for hematologic malignancies often lead to limited clinical efficacy due to the outgrowth of subpopulations with alternative pathways independent of the drug target. The eIF4F complex responsible for translation initiation is a convergence point for many cancer-promoting signaling pathways and its inhibition leads to decreased expression of key oncoproteins and apoptosis. Lymphomas and leukemias show particular dependence on constitutive eIF4F activation. Indeed, natural compounds targeting the eIF4F enzymatic component, eIF4A1, demonstrate activities both in vitro and in vivo against lymphoma and leukemia model systems, among other tumor types. eIF4A1 is a noteworthy target for hematologic malignancies based on the finding that BCR stimulation leads to increased mRNA translation primary CLL patient samples. Additionally, eIF4F components eIF4A1 and eIF4G1 had increased expression upon IgM-induced BCR activation. The natural compound silvestrol is a potent inhibitor of eIF4A1, results in cancer cell cytotoxicity, and has an established therapeutic window in vivo. Silvestrol shows potent antitumor activity against 924 pan-cancer tumor cell lines with 830/924 (90%) sensitive at IC50 <100nM with lymphoma and leukemia cell lines being particularly sensitive. Silvestrol and other natural compounds, however, lack core drug-like properties and synthetic tractability. To discover new, specific and tractable inhibitors of eIF4A1 that are more drug-like, we have constructed several molecular models that we used to virtually screen more than 20 million compounds. eIF4A1 is the founding member of the DEAD-box RNA helicases, which include its paralogs eIF4A2 (91% amino-acid identity with eIF4A1) and eIF4A3 (60% identity). All DEAD-box helicases contain two RecA-like domains separated by a flexible linker. The cleft between these domains is lined with helicase motifs that mediate nucleotide binding and hydrolysis. In an absence of RNA or nucleotide, eIF4A proteins adopt diffuse open conformations; binding of RNA and ATP triggers transition to a more stable closed state. Modeling small-molecule interactions in the nucleotide cleft of eIF4A1 therefore assesses ability of molecules to lock eIF4A1 in a conformation unable to cycle through ATPase and helicase activities. Although no experimentally derived structure of human eIF4A1 co-crystalized with ATP exists, crystal structures of other DEAD-box family members with similar motifs permit detailed studies of nucleotide and ligand-binding and the development of homology models. We have used four available high-resolution crystal structures to build models predicting interactions of small molecules in the interdomain nucleotide-binding cleft. We identified nucleotide binding-site residues and accurately reproduced ATP interactions for all four models (derived from PDB: 2J0S, 1FUU, 2VSO, 2DB3). We then performed all-atom explicit-water molecular dynamics (MD) simulations for 500-700 ns to study conformational dynamics and atomic interactions of ATP-bound and ATP-unbound states. Yeast eif4A crystal structure (PDB:1FUU) in the open state, for example, illustrated closure of the two RecA domains upon ATP binding. As expected, ATP makes strong interactions with the N-terminal, while phosphate groups extend to the C-terminal interacting with arginines, bringing the two RecA domains together. In contrast, we did not observe domain closure in the same simulation with 1FUU without ATP bound. We also assessed 2J0S, a crystal structure of human closed eIF4A3 bound to ANP. ATP docked to this active site followed by 500 ns MD held the protein in the closed state with several interdomain interactions. Upon nucleotide removal, marked RecA separation occurred. We observed similar domain closure and opening for PDB: 2VSO and 2DB3. Pooling these results, we constructed two homology models of human eIF4A1 with both open (2VSO, 1FUU) and closed conformations (2J0S) as structural templates. We therefore have developed accurate and novel in silico models of eIF4A1 highly useful in assessing interactions of small-molecule ATPase inhibitors, with focus on the ATP-binding cleft. Disclosures No relevant conflicts of interest to declare.

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The bromodomain and extra-terminal domain degrader MZ1 exhibits preclinical anti-tumoral activity in diffuse large B-cell lymphoma of the activated B cell-like type

Exploration of Targeted Anti-tumor Therapy ◽

10.37349/etat.2021.00065 ◽

2021 ◽

Vol 2 (6) ◽

pp. 586-601

Author(s):

Chiara Tarantelli ◽

Eleonora Cannas ◽

Hillarie Ekeh ◽

Carmelo Moscatello ◽

Eugenio Gaudio ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Bet Inhibitor ◽

Terminal Domain ◽

Large B Cell Lymphoma ◽

Bet Inhibitors ◽

Large B Cell

Aim: Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that play a fundamental role in transcription regulation. Preclinical and early clinical evidence sustain BET targeting as an anti-cancer approach. BET degraders are chimeric compounds comprising of a BET inhibitor, which allows the binding to BET bromodomains, linked to a small molecule, binder for an E3 ubiquitin ligase complex, triggering BET proteins degradation via the proteasome. These degraders, called proteolysis-targeting chimeras (PROTACs), can exhibit greater target specificity compared to BET inhibitors and overcome some of their limitations, such as the upregulation of the BET proteins themselves. Here are presented data on the anti-tumor activity and the mechanism of action of the BET degrader MZ1 in diffuse large B cell lymphoma (DLBCL) of the activated B-cell like (ABC, ABC DLBCL), using a BET inhibitor as a comparison. Methods: Established lymphoma cell lines were exposed for 72 h to increasing doses of the compounds. Cell proliferation was evaluated by using an 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) assay. Fluorescent-Activated Cell Sorter (FACS) analysis was performed to measure apoptotic activation and RNA sequencing (RNA-Seq) to study the transcriptional changes induced by the compounds. Results: MZ1, and not its negative control epimer cisMZ1, was very active with a median half maximal inhibitory concentration (IC50) of 49 nmol/L. MZ1 was more in vitro active than the BET inhibitor birabresib (OTX015). Importantly, MZ1 induced cell death in all the ABC DLBCL cell lines, while the BET inhibitor was cytotoxic only in a fraction of them. BET degrader and inhibitor shared partially similar changes at transcriptome level but the MZ1 effect was stronger and overlapped with that caused cyclin-dependent kinase 9 (CDK9) inhibition. Conclusions: The BET degrader MZ1 had strong cytotoxic activity in all the ABC DLBCL cell lines that were tested, and, at least in vitro, it elicited more profound effects than BET inhibitors, and encourages further investigations.

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Pharmacological Blockade of NF-kB with AS602868, a Potent Small Molecule Inhibitor of IKK2 Leads to Apoptosis of Sensitive and Resistant Chronic Myeloid Leukemia Cells.

Blood ◽

10.1182/blood.v106.11.2882.2882 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2882-2882

Author(s):

Veronique Imbert ◽

Nadege Gonthier ◽

Catherine Frelin ◽

Nicolas Sirvent ◽

Michael Hummelsberger ◽

...

Keyword(s):

Cell Lines ◽

Small Molecule ◽

Hematologic Malignancies ◽

Cytogenetic Response ◽

Small Molecule Inhibitor ◽

Cellular Targets ◽

Promising Target ◽

Molecule Inhibitor ◽

First Choice ◽

Dependent Growth

Abstract The Bcr-Abl inhibitor imatinib mesylate is now the first choice treatment for all newly diagnosed CML patients. Nevertheless not all patients reach a complete cytogenetic response (CCR) and many develop resistance for the drug upon disease progression. Additional cellular targets should be thus identified to develop alternative therapeutic strategies. The transcription factor NF-kB appears as a promising target for the treatment of cancer since it is a pro-survival factor, found abnormaly active in numerous hematologic malignancies and solid tumors. NF-kB activation relies on ubiquitin-mediated degradation of its inhibitor IkB after phosphorylation by 2 specific kinases, IKK1 and IKK2. We disrupted NF-kB signaling pathway by the use of AS602868, a small molecule inhibitor of IKK2 on two CML cell lines, LAMA84 and BaF3/Bcr-Abl and on primary CML cells. First we showed that NF-kB is constitutively activated in both CML cell lines and that this constitutive activation is under the control of the Bcr-Abl kinase. We next demonstrated that IKK2 targeting by AS602868 led to apoptosis of CML cells. AS602868 induced a dose dependent growth inhibition (IC50 3–4μM) of CML cell lines. This inhibition was associated with activation of cellular caspases 3, 8 and 9 and induction of DNA fragmentation. Interestingly AS602868 could induce death of imatinib resistant cellular clones derived from LAMA84 and BaF3-Bcr-Abl cells (IC50 1–2μM). Finally AS602868 selectively inhibited the proliferation and the hematopoietic colony formation of primary CML cells derived from imatinib sensitive or resistant patients. Although the exact mechanism of NF-kB activation by Bcr-Abl remains to be elucidated, our data strongly suggest that targeting NF-kB with the IKK2 inhibitor AS602868 may represent a new promising therapeutic tool for the treatment of CML.

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Stromal induction of BRD4 phosphorylation Results in Chromatin Remodeling and BET inhibitor Resistance in Colorectal Cancer

Nature Communications ◽

10.1038/s41467-021-24687-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Wenyu Wang ◽

Yen-An Tang ◽

Qian Xiao ◽

Wee Chyan Lee ◽

Bing Cheng ◽

...

Keyword(s):

Colorectal Cancer ◽

Chromatin Remodeling ◽

Cancer Drug ◽

Bet Inhibitor ◽

Cancer Associated Fibroblast ◽

Bet Inhibitors ◽

Inhibitor Resistance ◽

Bet Protein

AbstractBRD4, a Bromodomain and Extraterminal (BET) protein family member, is a promising anti-cancer drug target. However, resistance to BET inhibitors targeting BRD4 is common in solid tumors. Here, we show that cancer-associated fibroblast (CAF)-activated stromal signaling, interleukin-6/8-JAK2, induces BRD4 phosphorylation at tyrosine 97/98 in colorectal cancer, resulting in BRD4 stabilization due to interaction with the deubiquitinase UCHL3. BRD4 phosphorylation at tyrosine 97/98 also displays increased binding to chromatin but reduced binding to BET inhibitors, resulting in resistance to BET inhibitors. We further show that BRD4 phosphorylation promotes interaction with STAT3 to induce chromatin remodeling through concurrent binding to enhancers and super-enhancers, supporting a tumor-promoting transcriptional program. Inhibition of IL6/IL8-JAK2 signaling abolishes BRD4 phosphorylation and sensitizes BET inhibitors in vitro and in vivo. Our study reveals a stromal mechanism for BRD4 activation and BET inhibitor resistance, which provides a rationale for developing strategies to treat CRC more effectively.

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Pharmacokinetics of Tinidazole in Chinese subjects: Comparison of Mongolian, Korean, Hui, Uighur and Han nationalities

Journal of Pharmacy & Pharmaceutical Sciences ◽

10.18433/j3kk50 ◽

2009 ◽

Vol 12 (2) ◽

pp. 175 ◽

Cited By ~ 5

Author(s):

Xin-Yu Chang ◽

Tao Guo ◽

Dong-Ya Xia

Keyword(s):

Oral Dose ◽

Single Oral Dose ◽

Half Life ◽

Peak Plasma ◽

Peak Plasma Concentration ◽

Elimination Rate ◽

Area Under The Curve ◽

Elimination Half ◽

Pharmacokinetic Properties ◽

Chinese Subjects

ABSTRACT - Purpose. This study investigated the pharmacokinetics of tinidazole in subjects of five different Chinese nationalities (Han, Mongolian, Korean, Hui, and Uighur). Methods. Fifty healthy subjects (five male and five female of each nationality) were recruited for the study, and each received 1 g tinidazole. A total of 14 blood samples were collected over a 72-hour period after administration. Results. Pharmacokinetic profiles, including area under the curve from time zero to infinity (AUC0-inf), peak plasma concentration (Cmax), time to reach Cmax (tmax), oral clearance (CL/F), elimination rate constant (Ke), and elimination half-life (t1/2), were determined following a single oral dose of tinidazole. The respective pharmacokinetic properties of Han, Mongolian, Korean, Hui, and Uighur nationalities were: half-life (h): 16.94±2.40, 16.40±1.79, 16.63±1.82, 16.81±1.56, 14.34±1.92; Cmax (μg/mL): 19.04±2.42, 19.22±4.93, 20.83±3.33, 20.25±4.05, 18.81±3.10; AUC0-inf (h•μg/mL): 483.13±65.65, 479.70±99.74, 511.07±53.47, 514.25±130.78, 388.58±37.37. The t1/2 and AUC0-inf of Uighur subjects were significantly lower (p =0.023, 0.011) and the CL/F and Ke were significantly higher (p = 0.003, 0.013) than those of other nationalities. After normalization by weight, the differences in AUC0-inf and CL/F between Uigur subjects and those of other races were still significant. Conclusions. The results indicate that ethnicity had significant impact on the pharmacokinetics of tinidazole after a single oral dose in healthy volunteers of different nationalities in China.

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FT-1101: A Structurally Distinct Pan-BET Bromodomain Inhibitor with Activity in Preclinical Models of Hematologic Malignancies

Blood ◽

10.1182/blood.v126.23.1367.1367 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1367-1367 ◽

Cited By ~ 6

Author(s):

David S Millan ◽

Monica A Alvarez Morales ◽

Kenneth J Barr ◽

Daniel Cardillo ◽

Alan Collis ◽

...

Keyword(s):

Gene Expression ◽

Proliferative Activity ◽

Xenograft Model ◽

Hematologic Malignancies ◽

In Vivo Efficacy ◽

Bet Inhibitor ◽

Super Enhancer ◽

Bet Inhibitors ◽

Myc Gene

Abstract Members of the Bromodomain and Extra-Terminal (BET) family of bromodomain-containing proteins (BRD2, BRD3, BRD4, and BRDT) bind to acetylated lysine residues on histone tails and act as epigenetic readers to regulate chromatin structure and gene expression. In particular, BET family member BRD4 has been shown to positively regulate the expression of MYC and other critical cancer-associated genes through the localization of BRD4 to super-enhancer regulatory elements. Results from preclinical studies conducted using the small molecule tool BET inhibitor (+)-JQ1, and emerging data from clinical trials of BET inhibitors in leukemia and lymphoma patients, have provided support for the therapeutic potential of BET inhibitors in hematologic malignancies. A protein structure-guided drug design approach was employed to explore small molecule acetyl lysine mimetics and led to the identification of the BET inhibitor FT-1101, which is structurally unrelated to reported clinical-stage BET inhibitors in the (+)-JQ1 class. In biochemical binding assays, FT-1101 displayed equipotent inhibition of binding of both bromodomains in all four BET family members to a known bromodomain ligand (Kd ≤ 20 nM). In vitro, FT-1101 displayed potent anti-proliferative activity across a broad panel of human leukemia, lymphoma, and multiple myeloma cell lines, with 66 out of 123 cell lines having IC50 values < 500 nM. For the MV-4-11 acute myeloid leukemia cell line, the anti-proliferative activity of FT-1101 (IC50 = 40 nM) correlated with down-regulation of MYC gene and protein expression, suggesting that its anti-proliferative activity was at least in part due to suppression of MYC. Additionally, genome-wide mapping of BRD4 super-enhancer binding sites in MV-4-11 cells by ChIP-sequencing identified several potential pharmacodynamic biomarkers outside of the MYC signaling network. In vivo, in the MV-4-11 xenograft model, oral treatment with FT-1101 at its maximum tolerated dose resulted in significant tumor growth inhibition, including tumor regressions, on schedules ranging from once daily to once weekly, and similar activity was also observed in the THP-1 AML xenograft model. Superior in vivo efficacy in the MV-4-11 model was observed for FT-1101 relative to BET inhibitors of the (+)-JQ1 class. In vivo efficacy was associated with prolonged drug exposure and a >75% decrease in MYC gene expression in tumors that was sustained for at least 12 hours. FT-1101 also crossed the blood-brain barrier in mice, achieving a pharmacologically relevant free drug concentration in the brain. The promising preclinical profile of FT-1101 warranted its rapid progression into human clinical trials, and a Phase 1 study in patients with relapsed refractory acute leukemia or high-risk myelodysplastic syndrome is currently underway. Disclosures No relevant conflicts of interest to declare.

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Viral E Protein Neutralizes BET Protein-Mediated Post-Entry Antagonism of SARS-CoV-2

10.1101/2021.11.14.468537 ◽

2021 ◽

Author(s):

Irene Chen ◽

James Edward Longbotham ◽

Sarah McMahon ◽

Rahul Suryawanshi ◽

Jared Carlson-Stevermer ◽

...

Keyword(s):

Angiotensin Converting Enzyme 2 ◽

Entry Level ◽

Bet Inhibitor ◽

Protein Inhibition ◽

E Protein ◽

Bet Inhibitors ◽

Post Entry ◽

Bet Protein ◽

Interferon Responses ◽

Inhibitor Treatment

Inhibitors of Bromodomain and Extra-terminal domain (BET) proteins are possible anti-SARS-CoV-2 prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here, we show that BET proteins should not be inactivated therapeutically as they are critical antiviral factors at the post-entry level. Knockouts of BRD3 or BRD4 in cells overexpressing ACE2 exacerbate SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection, and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

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