Efficacy Of The Polo-Like Kinase Inhibitor, Rigosertib Alone Or In Combination With ABL Tyrosine Kinase Inhibitor Against BCR-ABL-Positive Leukemia Cells
Authors: Okabe Seiichi , Tauchi Tetsuzo , Tanaka Yuko , Kitahara Toshihiko , Ohyashiki Kazuma 
Published: October 2019

Abstract
Chronic myeloid leukemia (CML) is characterized by the t(9:22) translocation known as the Philadelphia chromosome (Ph). ABL tyrosine kinase inhibitor (TKI), imatinib and second-generation ABL TKIs, nilotinib and dasatinib have demonstrated the potency against CML patients. However, resistance to ABL TKI can develop in CML patients due to BCR-ABL point mutations. Moreover, ABL TKIs do not eliminate the leukemia stem cells (LSCs). These leukemia cells are contained within a niche in the bone marrow and are often impervious to current treatments. Therefore, new approach against BCR-ABL mutant cells and LSCs may improve the outcome of Ph-positive leukemia patients. Polo-like kinases (PLK) are the family of serine threonine kinases and essential for mitosis. PLK is also critical regulator of cell cycle progression and DNA damage response. One of the PLK and phosphoinositide 3-kinase (PI3K) inhibitor, rigosertib (ON 01910.Na) is a novel synthetic benzyl styryl sulfone that is cytotoxic against a variety of human tumor cell lines. Rigosertib is currently being investigated in a pivotal phase 3 clinical trial against hematological malignancies such as myelodysplastic syndromes (MDS). We suggested that rigosertib mediated inhibition PLK and PI3K activity and in combination with ABL TKIs may abrogate the proliferation and survival of Ph-positive leukemia cells including T315I mutation and ABL TKI resistant. In this study, we investigated the combination therapy with a rigosertib and an ABL TKIs (imatinib, nilotinib and ponatinib) by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line with T315I mutant, nilotinib resistant K562 and ponatinib resistant Ba/F3 cells. 72 h treatment of rigosertib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. The treatment of imatinib, nilotinib and ponatinib exhibits cell growth inhibition partially against K562 cells in the presence of feeder cell (HS-5) conditioned media. We found that the treatment of rigosertib abrogated the protective effects of HS-5 conditioned media in K562 cells. We examined the intracellular signaling after treatment of rigosertib. Phosphorylation of BCR-ABL, Crk-L was not reduced. However, activity of caspase 3 was increased. We next investigated the efficacy between imatinib and rigosertib by using these cell line. Combined treatment of K562 cells with imatinib and rigosertib caused significantly more cytotoxicity than each drug alone. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved PARP and γH2A.X phosphorylation was increased after imatinib and rigosertib treatment. Anti-apoptotic protein, Mcl-1 was also decreased. We also found the phosphorylation of histone H3 was increased after rigosertib treatment suggested that the cells arrested in G2/M phase. We investigated the rigosertib activity against T315I positive cells. Rigosertib potently induced cell growth inhibition of Ba/F3 T315I cells. Combined treatment of Ba/F3 T315I cells with ponatinib and rigosertib caused significantly more cytotoxicity than each drug alone.We next investigated by using ponatinib resistant Ba/F3 cells and nilotinib resistant K562 cells. In the ponatinib resistant cell lines, IC50 of ponatinib was up to 200 nM. BCR-ABL triple point mutations (T315I, E255K and Y253H) were detected by direct sequence analysis. The treatment of rigosertib exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells and K562 nilotinib resistant cells. These results indicated that administration of the PLK and PI3K inhibitor, rigosertib may be a powerful strategy against ABL TKI resistant cells and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells.

Disclosures:
Ohyashiki: Novartis: Honoraria, Research Funding.