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The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563–74. ©2016 AACR.

The poly (ADP-ribose) polymerase (PARP) family of enzymes are functionally implicated in DNA repair, transcriptional regulation, glucose metabolism, mitosis, and other cellular mechanisms. To date, the focus has been on defining the role of PARPs 1-3 in DNA damage repair. Currently, PARP1-3 inhibitors are in clinical trials for BRCA1/2 mutant ovarian and breast cancers. The roles of other PARP family members are starting to emerge. Using a cell-based assay measuring multi-polar spindle induction, PARP6 was identified as novel target with therapeutic potential. AZ482 and AZ108 were developed as potent PARP6 inhibitors. These compounds exhibited a unique and selective growth inhibition profile when screened in large panels of tumor cells and suitable pharmacokinetics in vivo. In sensitive hematological and solid tumor models, these compounds had low nanomolar potency in vitro and anti-tumor efficacy in vivo. Mechanistic studies suggest PARP6 inhibitors disrupt spindle pole clustering resulting in mitotic catastrophe and may show selectivity toward tumor cell lines with supernumary centrosomes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A221. Citation Format: Michele F. Mayo, Shaun Grosskurth, Xin Wang, Philip Petteruti, Prasad Nadella, Corinne Reimer, Keith Mikule. Novel PARP6 inhibitors demonstrate in vivo efficacy in xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A221.

Poly(ADP-ribose) polymerases (PARP) are a family of 17 enzymes that catalyze the transfer of the ADP-ribose from NADH to post-translationally modify (PTM) acceptor proteins. Currently, the PTM regulation of proteins by PARPs have been demonstrated to regulate numerous signaling cascades including but not limited to DNA damage response and tumor development for PARP1-3 as well as telomere maintenance, spindle assembly, vesicular movement, and regulation of beta-catenin destruction complex for PARP5a-5b. Although progress on the biological role and therapeutic potential for some PARPs has been made, the function of and possible therapeutic application for other PARPs are not fully understood. Using cell free enzyme and cell-base multi-polar spindle assays, semi-selective PARP6 inhibitors AZ482 and AZ108 were identified. From in vitro PARP6 knock-down and AZ108 pharmacological studies in breast cancer cell lines, PARP6 perturbation was demonstrated to disrupt spindle pole clustering. To identify PARP6 protein substrate that may contribute to the spindle pole clustering defect, an in vitro acceptor protein substrate screen was performed using high-density protein microarrays containing more than 8,000 unique proteins. From this screen, an enrichment for centromeric and microtubule organizing proteins was identified. Of these centromeric PARP6 protein substrate, CHEK1 and CENP were shown to be modulated by AZ108 treatment in breast cancer cell lines. These results implicate PARP6 in a role for stablization of the spindle assembly during mitosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A134. Citation Format: SHAUN GROSSKURTH, Philip Petteruti, Xin Wang, Keith Mikule. Functional perturbation of PARP6 affects centromeric proteins and is associated with increased multi-polar spindles in breast cancer cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A134.