Multiple Myeloma: Combination Therapy of BET Proteolysis Targeting Chimeric Molecule with CDK9 Inhibitor

Abstract

22 Cyclin Dependent Kinase 9 (CDK9) associates with Bromodomain and Extra-Terminal Domain 23 (BET) proteins to promote transcriptional elongation by phosphorylation of serine 2 of RNAP II 24 C-terminal domain. We examined the therapeutic potential of selective CDK9 inhibitors (AZD 25 4573 and MC180295) against human multiple myeloma cells in vitro. Short-hairpin RNA silencing 26 of CDK9 in Multiple Myeloma (MM) cell lines reduced cell viability compared to control cells 27 showing the dependency of MM cells on CDK9. In order to explore synergy with the CDK9 28 inhibitor, proteolysis targeting chimeric molecule (PROTAC) ARV 825 was added. This latter 29 drug causes ubiquitination of BET proteins resulting in their rapid and efficient degradation.30 Combination treatment of MM cells with ARV 825 and AZD 4573 markedly reduced their protein 31 expression of BRD 2, BRD 4, MYC and phosphorylated RNA pol II as compared to each single 32 agent alone. Combination treatment synergistically inhibited multiple myeloma cells both in vitro 33 and in vivo with insignificant weight loss. The combination also resulted in marked increase of 34 apoptotic cells at low dose compared to single agent alone. Taken together, our studies show for 35 the first time that the combination of a BET PROTAC (ARV 825) plus AZD 4573 (CDK9 inhibitor) 36 is effective against MM cells.37 38 39 40 41 3 42 Introduction: 43 Multiple myeloma (MM) is a clonal plasma cell malignancy. It is the second most common 44 hematologic malignancy in United States 1 . Despite advances in treatment such as proteasome 45 inhibitors and immunomodulatory drugs, the disease remains incurable. Bromodomain and Extra-46 Terminal Domain (BET) family is composed of BRD-2, -3, -4 and -T. They facilitates 47 4 64 and producing relapse in these patients 10,11 . Therefore, CDK9 may represent a druggable target in 65 myeloma having dysregulated MYC expression 12,13 . 66 67 Inhibition of both CDK9 and BRD 4 has been reported synergistically to induce growth arrest and 68 apoptosis of cancer cells including MM 14,15 . Previously studied CDK inhibitors (eg. Flavopiridol 69 and SNS-032) are not selective to CDK9, inhibiting other CDKs and enzymes. Their lack of 70selectivity and decreased potency may contribute to many adverse effects in clinical trials 8 . 71Therefore, selective inhibitors of CDK9 are needed to prevent the undesirable off-target effects 72 and to enhance potency. 73 74 AZD 4573 is highly potent against CDK9 (<3 nM IC 50 ) and selective (>10 fold) against CDK9. 75The drug results in caspase activation and loss of viability across a diverse set of hematological 76 cancers including MM 16 . MC180295 is also a highly selective CDK9 inhibitor (> 22 fold, IC 50 = 77 5 nM) that has broad anti-cancer activity in vitro and in vivo 17 . In this study, we noted that AZD 78 4573 and MC180295 in vitro inhibited the viability of MM cells. We also showed that AZD 4573 79 is synergistic with ARV 825 in inducing apoptosis and inhibiting MM cell proliferation both in 80 vitro and in v...