Discovery of Selective Dual Inhibitors of Bromodomain Protein BRD4 and JAK2 for Treatment of Hematologic Malignancies
Authors: Upadhayaya Ram S. , Kethiri Raghava Reddy , Vellanki Avanish , Lightfoot Jeff , Local Andrea , Rice William G. 
Published: October 2019

Abstract
The Bromodomain and Extra-Terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) are functional readers of acetylated lysine residues of histones, and have emerged as potential therapeutic targets in hematologic cancers and solid tumors characterized by dysregulated epigenetic processes. Targeted inhibition of BET proteins has proven to be an effective strategy for transcriptional downregulation of c-MYC, an oncogene that is frequently activated or overexpressed in leukemias, lymphomas, and multiple myeloma. Of the BET family members BRD4 is the most extensively studied for its role in cancer, furthermore C-MYC downregulation by BET inhibitors is attributed to inhibition of enhancer binding by BRD4 (Delmore et al., Cell. 2011. 146:904-17). BRD4 is a critical factor in AML disease maintenance (Zuber et al., Nature. 2011. 478:524-8), and its suppression is the dominant mechanism of BET inhibitor JQ1 activity in AML (Rathert et al., Nature. 2015. 525:543-7). Recent reports have shown that BRD4/BET inhibitors and kinase inhibitors act synergistically in a range of cancer types (Sun et al., Blood. 2015, 126:1565-74; Stratikopoulos et al., Cancer Cell. 2015, 27:837-51). Therefore, optimizing for this synergy by prospectively designing and developing multi-targeting BRD4-kinase inhibitors may prolong therapeutic efficacy and overcome tumor resistance of single-activity BET and oncogenic kinase inhibitors.
Dual inhibitors of BET proteins and Janus kinase 2 (JAK2), initially developed by Moffitt Cancer Center (Reuther et al., ASH 2015 Poster, Abstract #2826), demonstrated an opportunity for novel, potent dual inhibitors of BRD4 and JAK for treatment of myeloproliferative neoplasms (MPNs) and other disorders driven by the constitutively active somatic mutation, JAK2-V617F. Although JAK inhibitors such as ruxolitinib show clinical benefits in MPNs, these molecules demonstrate limited ability to induce remissions and are associated with significant toxicities such as myelosuppression. Therefore, selective JAK2 inhibitors that also target BRD4 hold promise as treatments of hematologic malignancies with improved activity and less off-target toxicity. While the dual inhibitor candidates from Moffitt exhibit strong potency, they possess a sub-optimal profile for inhibition of the thiamine transporter and other properties. Herein, we report that next generation, novel dual inhibitors of BRD4 and JAK2 have been discovered by Aptose, in a collaboration with Laxai Avanti Life Sciences (LALS), without observed thiamine transporter inhibition but with favourable drug-like properties.
In biochemical assays, the Aptose dual inhibitor compounds exhibit IC50 values ranging from 82-678 nM for BRD4 (BD1) and 0.7-35 nM for JAK2. Compounds exhibited selectivity of 2-7 fold for BRD4 compared to other BRD isoforms, and 3-152 fold selectivity for JAK2 over JAK1 or JAK3. Cellular IC50 values of the compounds in antiproliferative assays ranged from 6.6 - 118 nM in AML (MV4-11) and from 171-721 nM in myeloma (MM1.S) cell lines. Candidate structures show good solubility and metabolic stability in human, rat and mouse liver microsomes. Structure-activity relationship studies are ongoing to improve biochemical and antiproliferative activity and drug like properties. Collectively, we demonstrate that a rational design approach can be used to generate potent molecules with combined BRD4/JAK2 inhibitory activities for treatment of hematologic cancers.

Disclosures
No relevant conflicts of interest to declare.