Preclinical Evaluation of Sorafenib in Combination with Cytarabine and Clofarabine in Acute Myeloid Leukemia (AML).
Authors: Hu Shuiying , Niu Hongmei , Orwick Shelley , Rubnitz Jeffrey , Schuetz John , Baker Sharyn D. 
Published: October 2019

Abstract
Deregulation of receptor tyrosine kinases and downstream signal transduction pathways may promote leukemogenesis by conferring cell proliferation and survival advantages in AML. The multitargeted tyrosine kinase inhibitor sorafenib (with activity against FLT3, c-kit, PDGFR, VEGFR, RAF) has been shown to have potent antitumor effects in AML cell lines with c-kit and FLT3 mutations, possibly by inducing G0/G1 cell cycle block and apoptosis and inhibiting STAT and/or MAPK signaling (S. Hu Proc AACR 2007). Sorafenib has shown single-agent activity in AML, but clinical responses have not been durable, suggesting that sorafenib is likely to be more clinically effective if combined with other antileukemic agents. In this study, we evaluated the antitumor activity of sorafenib in combination with the nucleoside analogues clofarabine and cytarabine in three AML cell lines (MV4-11, THP-1 and U937), which represent different FAB types and variable alterations in tyrosine kinases. Three sequences of administration including simultaneous, pre-chemotherapy, and pre-sorafenib were evaluated with a MTT cellular proliferation assay using 24- to 72-hr exposures (duplicate experiments performed with 8 replicates each). Combined drug effects were analyzed using the computer software CalcuSyn. The combination of clofarabine with sorafenib at a fixed ratio of 5:1 was synergistic to additive or antagonistic in MV4-11 cells (with FLT-3 ITD) depending on sequence; simultaneous > pre-sorafenib > pre-clofarabine with combination index (CI) values at ≥ ED50 of 0.53–0.80, 0.54–1.12, and 0.93–1.20, respectively. The combination of sorafenib and clofarabine was antagonistic in THP-1 and U937 cells with all three sequences evaluated (CI values at ≥ ED50 of 1.25–1.96). The combination of cytarabine with sorafenib at a fixed ratio of 250:1 was synergistic in MV4-11 cells regardless of the sequence of administration with CI values at ≥ ED50 of 0.28–0.77 in all 3 cell lines, representing an approximate 3–4- fold dose reduction index for both drugs. Although studies evaluating cytarabine and sorafenib in THP-1 and U937 cells are pending, results in MV4-11 cells indicate that cytarabine with sorafenib may be more effective than the combination of clofarabine with sorafenib. Combination studies of clofarabine or cytarabine with sorafenib in blasts from children with AML are ongoing. To understand potential differences between the nucleoside analogues when combined with sorafenib, studies were performed to determine the effect of sorafenib 5 μM (pre-treatment for 15 min) on the cellular accumulation (1-hr exposure) of clofarabine and cytarabine in MV4-11 cells (2 to 3 independent experiments performed in duplicate). Sorafenib decreased the intracellular accumulation of clofarabine by about 2-fold, but increased the intracellular accumulation of cytarabine by approximately 2-fold; transporter studies are ongoing to identify the mechanism(s) of the observed differential drug interaction. In conclusion, sorafenib in combination with cytarabine may represent a promising treatment strategy for AML, where simultaneous or sequential administration appears to be equally effective.