The Novel Human Double Minute (HDM)-2 Inhibitor DS-5272 Is Active Both Alone, and In Combination With Other Novel Agents, Against Multiple Myeloma
Authors: Gu Dongmin , Wang Hua , Wang Zhiqiang , Davis Richard E. , Cai Shengli , Seki Takahiko , Tse Archie , Orlowski Robert Z. 
Published: October 2019

Abstract

Background
The ubiquitin-proteasome pathway is now a validated target for myeloma therapy given the regulatory approvals of proteasome inhibitors such as bortezomib and carfilzomib. Another logical set of targets are the E3 ligases, whose role is to facilitate the protein poly-ubiquitination typically needed for recognition by the constitutive or immunoproteasome prior to proteolysis. One of the more attractive such targets is HDM-2, the E3 ligase best known for its role in turnover of the p53 tumor suppressor, in part because p53 deletion or mutation is less common in myeloma than in solid tumors, and because it has been possible to develop agents targeting the HDM-2 p53 binding pocket.

Methods
Activity of the specific HDM-2 inhibitor DS-5272 (Daiichi-Sankyo) was evaluated using a panel of p53 wild-type (wt) and mutant (mut) myeloma cell lines and also against primary patient samples. Studies were performed with DS-5272 both alone, and also in combination regimens with novel agents. Tetrazolium dye-based assays were employed to determine cell viability, Annexin V staining was used to examine apoptosis, and quantitative PCR and Western blotting were used to study selected transcripts and gene products, respectively. This study was supported in part by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma.

Results
DS-5272 induced potent cytotoxicity in wt p53 MM1.S, H929, and MOLP-8 myeloma cell lines, with a median inhibitory concentration (IC50) in the single micromolar range, which was reproduced in studies of primary CD138+ plasma cells but not CD138- cells from myeloma patients. This cytotoxicity was both time- and concentration-dependent, and DS-5272 was more potent than the prototypical HDM-2 inhibitors, Nutlin-3a and MI-219. A dependence of DS-5272 activity on wt p53 was demonstrated by the much lower IC50 in the wt p53 than mut p53 cell lines, and the finding that suppression of p53 with a shRNA, as well as mutation of p53 with a sequence-specific zinc finger nuclease, significantly increased the IC50. Notably, DS-5272 remained active in the presence of conditioned media from stromal cells, or key myeloma cytokines such as IL-6. The reduced viability after exposure to DS-5272 was due at least in part to activation of type I cell death, as determined by increased staining for Annexin V, activation of caspases 9 and 3, and cleavage of PARP. Other downstream effects included induction of transcription of p21, Bax, HDM-2, NOXA, and PUMA. These proteins, as well as p27 and p53 were induced, while the abundance of Survivin, CHK1, Aurora A and B, PLK1, and KIF11 was reduced. Consistent with these latter effects, DS-5272 induced some accumulation of cells at the G2/M phase, and the appearance of cells with a disordered spindle. Suppression of KIF11 (kinesin spindle protein (KSP; Eg5)), which is responsible for centromere separation and bipolar spindle formation, seemed to occur through the binding of p21 to the cell cycle genes homology region (CHR) within the KIF11 promoter. Finally, combinations of DS-5272 with the specific KIF11 inhibitor Ispinesib produced enhanced G2/M arrest, as well as a synergistic reduction in cell viability with increased levels of apoptosis.

Conclusion
DS-5272 is a potent and novel agent with activity against multiple myeloma, and is active both alone and in rationally designed combination regimens with other drugs. These findings provide a rationale for the clinical translation of HDM-2 inhibitors as monotherapy, and possibly with other agents such as the KSP inhibitor ARRY-520, which is also active against myeloma, for patients with wt p53 relapsed and/or refractory myeloma.

Disclosures:
Cai: Daiichi-Sankyo Pharma Development: Employment, Equity Ownership. Seki:Daiichi Sankyo Co., Ltd.: Employment, Equity Ownership. Tse:Daiichi-Sankyo Pharma Development: Employment, Equity Ownership.