The Combination Effect of Volasertib and Azacitidine, and the Resistant Mechanisms of Volasertib in Acute Myeloid Leukemia (AML)
Authors: Adachi Yoshiya , Ishikawa Yuichi , Kiyoi Hitoshi 
Published: October 2019

Abstract
Introduction:Polo-like kinase1 (PLK1) plays a crucial role in many steps of cell mitosis. Since PLK1 is overexpressed in various cancer cells, it is thought as an ideal target for cancer therapy and several PLK1 inhibitors are under development. Volasertib, a selective Plk1 inhibitor, was effective for elderly AML patients in clinical trials. However, its efficacy was limited in mono-therapy, and a higher incidence of severe adverse events was revealed in the combination therapy with low-dose cytarabine. Thus, it is necessary to optimize the combination therapies with other agents such as azacitizine (AZA) for its clinical use. Additionally, the resistant mechanism of volasertib is largely unknown. We evaluated the combination effect of volasertib and AZA on AML cells, and analyzed the resistant mechanism of volasertib using volasertib-resistant cell lines.
Methods:We evaluated the growth inhibitory effects of volasertib in mono- and combination-therapy with AZA on a series of cell lines and primary AML cells. Drug sensitivity was determined using MTS and an ATP quantification assays, and was compared with PLK1 protein and mRNA expression levels. Combination effect was determined by the combination index (CI) which was calculated by Compusyn software. Subsequently, to investigate the volasertib-resistant mechanism, we established volasertib-resistant leukemia cell lines by culturing with escalating concentrations of volasertib.
Results: Volasertib was highly potent against several kinds of cell lines and primary AML cells in mono-therapy, while its potency was not always associated with the expression and phosphorylation levels of PLK1 as well as cell proliferation rate. In the combination therapy, the efficacy of AZA was synergic in HEL and KG1, and additive in Marimo, K562 and HL-60, whereas it was antagonistic in MOLM14 and MV4;11. Consistently, the effect of combination therapy with AZA varied among primary AML cells, but it was prominent in the samples with relatively higher GI50values in volasertib mono-therapy.
To determine the volasertib-resistant mechanism, volasertib-resistant MOLM14, HL-60, MV4;11, HEL and K562 (R-MOLM14, R-HL-60, R-MV4;11, R-HEL and R-K562) were established. The GI50 values in resistant and parental cells were 149.8nM and 4.6nM, 164.0nM and 5.8nM, 42.8nM and 4.6nM, 277.7nM and 17.7nM and 1265.8nM and 14.1nM, respectively. Novel missense mutations in PLK1 were identified in R-MOLM14 (p.F183L), R-HL-60 (p.L59W) and R-MV4;11 (p.L59W). These amino acid residues were located in the ATP-binding site of PLK1, and mutant PLK1 transduced-U937 revealed resistance to volasertib: GI50 values in wild type, L59W and F183L transduced U937 cells were 37.1nM, 1150.9nM, and 363.6nM, respectively. Upon volasertib administration, the expression level of Wee1, a substrate of PLK1 degraded after its phosphorylation, was increased in parental cells, whereas it was not apparent in resistant cells. On the other hand, R-HEL and R-K562, which had no mutations in PLK1, showed the higher expression of MDR1 than parental cells. In these both cell lines, volasertib competitively inhibited MDR1-mediated transportation of a substrate in dose dependent manner, and the sensitivity to volasertib was restored by MDR1 inhibitor, zosquidar. We next evaluated the combination effect of volasertib and AZA in these volasertib-resistant cell lines. In the combination with AZA, the GI50values of volasertib were almost equal or increased compared to those of in mono-therapy. Thus, our results indicate that the combination effect of AZA and volesertib is not always consistent and can be antagonistic in volasertib-resistnat cells.
Conclusions: Volasertib inhibits the proliferation of most leukemia cells, but the combination effect with AZA is different among cell lines. Further investigation is required to identify the predictive factors for the efficacy of volasertib and its combination therapy with AZA. Our results indicated the mutations in the ATP-binding domain of PLK1 and the increased expression level of MDR1 were associated with resistance to volasertib in vitro. It is required to evaluate these alterations and the efficacy of combination therapy of AZA and volasertib in clinically volarsertib-resistant cells.

Disclosures
Kiyoi: Nippon Shinyaku Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Eisai Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; MSD K.K.: Research Funding; AlexionpharmaLLC.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Celgene Corporation: Consultancy; Zenyaku Kogyo Co., Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding.