Cyrus Calosing

Downregulation of the Complement Cascade In Vitro, in Mice and in Patients with Cardiovascular Disease by the BET Protein Inhibitor Apabetalone (RVX-208)

Apabetalone (RVX-208) is an epigenetic regulator developed to treat cardiovascular disease (CVD) that targets BET proteins. Through transcriptional regulation RVX-208 modulates pathways that underlie CVD including reverse cholesterol transport, vascular inflammation, coagulation, and complement. Using transcriptomics and proteomics we show that complement is one of the top pathways downregulated by RVX-208 in primary human hepatocytes (PHH) and in plasma from CVD patients. RVX-208 reduces basal and cytokine-driven expression of complement factors in PHH and in chimeric mice with humanized livers. Plasma proteomics of CVD patients shows that RVX-208 decreases complement proteins and regulators, including complement activators SAP and CRP. Circulating activated fragments C5a, C3b, and C5b-C6 are reduced by 51, 32, and 10%, respectively, indicating decreased activity of complement in patients. As complement components are linked to CVD and metabolic syndrome, including major acute cardiac events, modulating their levels and activity by RVX-208 may alleviate risks associated with these diseases.

Abstract LB-207: Preclinical characterization of ZEN-3694, a novel BET bromodomain inhibitor entering phase I studies for metastatic castration-resistant prostate cancer (mCRPC)

Metastatic castration resistant prostate cancer (mCRPC) is a major unmet medical need due to its widespread occurrence and incurable status. Current standard of care for advanced prostate cancer is androgen-deprivation therapy (ADT), and upon failure, patients are administered secondary ADT with androgen receptor (AR) antagonists such as enzalutamide and abiraterone. While most patients display an initial response to these agents, eventually all become resistant via various mechanisms that often result in constitutive AR signaling including mutations of the AR, and the generation of AR splice variants that bypass the ligand binding domain. Other mechanisms of resistance to AR antagonists include up-regulation of the glucocorticoid receptor (GR), and partial to complete loss of AR signaling through neuroendocrine differentiation. Recent evidence suggests that BET bromodomain inhibitors (BETi) could be efficacious in AR-signaling positive or negative mCRPC that are resistant to current therapies. ZEN-3694 is an orally bioavailable, potent BETi that selectively binds to both bromodomains of the BET proteins. In vitro, ZEN-3694 has demonstrated strong activity against several prostate cancer cell lines with submicromolar potency, including AR positive and AR negative, neuroendocrine, and enzalutamide resistant cell lines. In VCaP AR-positive prostate cancer cells, ZEN-3694 inhibited proliferation synergistically with enzalutamide, resulting in potent up-regulation of the CDKN1C/KIP2 tumor suppressor gene. In 22Rv1 cells displaying constitutive AR signaling through the AR-V7 splice variant, ZEN-3694 inhibited AR signaling, and in an in vitro LNCaP model of acquired resistance to enzalutamide characterized by GR up-regulation, ZEN-3694 decreased levels of GR in a dose-dependent manner. Furthermore, in the PC3 AR-null cell line, the expression of a subset of NF-KB-dependent genes reported to be involved in mCRPC bone metastasis was found to be inhibited by ZEN-3694. In vivo, using multiple prostate cancer cell line xenografts such as 22Rv1, and VCaP, ZEN-3694 showed efficacy in inhibiting tumor progression at well-tolerated doses, and modulating target gene expression. ZEN-3694 also inhibited progression of a patient-derived xenograft (PDX) LuCaP 35CR that is resistant to enzalutamide. In summary, our results indicate that ZEN-3694 demonstrates potent activity in advanced metastatic prostate cancer targeting multiple mechanisms of enzalutamide resistance in CRPC, including AR-V7 signaling and GR up-regulation in different preclinical models. This together supports the clinical development of ZEN-3694 as a single agent, and in combination with enzalutamide in mCRPC patients that have failed first line ADT. We are implementing a robust translational medicine program in the phase 1 study to measure target engagement and explore mechanisms of enzalutamide resistance and sensitivity to ZEN-3694 in patients. Citation Format: Sarah Attwell, Ravi Jahagirdar, Karen Norek, Cyrus Calosing, Laura Tsujikawa, Olesya A. Kharenko, Reena G. Patel, Emily M. Gesner, Eva Corey, Holly M. Nguyen, Sanjay Lakhotia, Henrik C. Hansen, Eric Campeau. Preclinical characterization of ZEN-3694, a novel BET bromodomain inhibitor entering phase I studies for metastatic castration-resistant prostate cancer (mCRPC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-207.

Abstract LB-038: Preclinical development and clinical validation of a whole blood pharmacodynamic marker assay for the BET bromodomain inhibitor ZEN-3694 in metastatic castration-resistant prostate cancer (mCRPC) patients

Detection of drug activity in patients is essential to confirm its mechanism of action, as well as to ensure proper target engagement at the selected dose to elicit optimal clinical activity. Pharmacodynamic (PD) markers are often developed to detect pharmacological responses and optimize drug dosing. Whole blood is an easily attainable and minimally invasive source of biological material to measure clinical activity of drugs. We designed, developed, and validated a whole blood PD marker assay to detect the activity of ZEN-3694, an orally available inhibitor of the bromodomain and extra-terminal (BET) domain family of proteins currently in phase I clinical trials in mCRPC (NCT02705469 and NCT02711956). Potential BET-specific PD markers were first identified via comparative microarray analysis using a PI3K inhibitor, a BET inhibitor, and a dual PI3K/BET inhibitor in an MV4-11 acute myeloid leukemia (AML) cell line. Further microarray analysis of subsequent in house data and published data of BET inhibitors from different chemical scaffolds in hematologic cell lines allowed us to develop a short list of ~20 candidate genes. Further testing was done by measuring the modulation of these PD markers by various Zenith BET inhibitors from different chemical scaffolds in a number of human cell lines derived from hematological cancers and solid tumors, as well as cryopreserved human peripheral blood mononuclear cells (PBMCs). In vivo validation was also done in whole blood obtained from xenograft mice, and cynomolgus monkeys that were dosed orally with ZEN-3694, as well as ex-vivo treated human blood derived from normal donors or patients diagnosed with either AML or diffuse large B cell lymphoma. There was also robust target engagement in tumors of mouse AML xenografts, making them suitable tumor PD markers. A quantitative real-time PCR assay was developed for human whole blood matrix with parameters defined based on the multiplex efficiency (85-115%), coefficient of correlation of the standard curve (R2>0.98), and dynamic copy number range (10-106). Assay validation testing demonstrated an inter-assay variability (operator/day/machine) of Citation Format: Laura Tsujikawa, Karen Norek, Cyrus Calosing, Sarah Attwell, Dean Gilham, Nimisha Sharma, Jennifer Tobin, Michelle Haager, Ravi Jahagirdar, Sanjay Lakhotia, Henrik C. Hansen, Eric Campeau. Preclinical development and clinical validation of a whole blood pharmacodynamic marker assay for the BET bromodomain inhibitor ZEN-3694 in metastatic castration-resistant prostate cancer (mCRPC) patients [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 LB-038. doi:10.1158/1538-7445.AM2017-LB-038

Abstract C86: The clinical candidate ZEN-3694, a novel BET bromodomain inhibitor, is efficacious in the treatment of a variety of solid tumor and hematological malignancies, alone or in combination with several standard of care and targeted therapies

ZEN-3694 is an orally bioavailable small molecule discovered and developed from a BET bromodomain inhibitor discovery platform. In vitro, ZEN-3694 selectively binds to both bromodomains of the BET proteins, inhibiting the interaction of acetylated histone peptide with IC50 values in low nM range. ZEN-3694 inhibits proliferation of MV4-11 AML cells with an IC50 of 0.2 uM, and inhibits MYC mRNA expression with an IC50 of 0.16 uM. ZEN-3694 has also demonstrated strong activity against many solid tumor and hematological cell lines with sub-uM IC50 values. In vitro synergy with Standard of Care (SOC) agents has been shown in a wide variety of malignancies including Breast, Prostate, Lung, Melanoma, AML, and DLBCL. Xenograft studies conducted with ZEN-3694 in AML, prostate and breast cancer models have demonstrated that it is efficacious at well-tolerated doses, modulating target gene expression and halting tumor growth in a dose-dependent manner. In the AR positive VCAP prostate cancer cell line, ZEN-3694 inhibits proliferation synergistically with the AR antagonists enzalutamide and ARN-509. In an in vitro enzalutamide resistance model characterized by the up-regulation of the glucocorticoid receptor (GR), GR expression was inhibited by ZEN-3694 in a dose-dependent manner. Sensitivity to ZEN-3694 was unaltered, suggesting that it could be a valid therapeutic approach in patients developing resistance to AR antagonists through GR induction. Robust PD modulation has been observed across multiple matrices for ZEN-3694 and will be explored further in the clinic. Promising target validation data, excellent pharmacological properties, and robust activity of ZEN-3694 across a variety of hematological malignancy and solid tumor settings support the clinical development of ZEN-3694 in various oncologic indications. Citation Format: Sarah Attwell, Eric Campeau, Ravi Jahagirdar, Olesya Kharenko, Karen Norek, Laura Tsujikawa, Cyrus Calosing, Reena Patel, Emily Gesner, Sanjay Lakhotia, Henrik Hansen. The clinical candidate ZEN-3694, a novel BET bromodomain inhibitor, is efficacious in the treatment of a variety of solid tumor and hematological malignancies, alone or in combination with several standard of care and targeted therapies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C86.

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.

APABETALONE (RVX-208) LOWERS CARDIOVASCULAR DISEASE (CVD) IN DIABETES MELLITUS BY A MECHANISM INVOLVING MICROBIOME MEDIATED ACTIVITY ON THE COMPLEMENT PATHWAY

Background: In our phase 2b trials, patients (n=499) given 200 mg/d apabetalone (RVX-208) for 6 months were observed to have a 55% relative risk reduction of major adverse cardiovascular events (MACE) that was further reduced in diabetes mellitus (DM) patients. These findings underpin our interest