Synergistic Cytotoxicity of the Multikinase Inhibitor Sorafenib with the DNA Alkylating Agent Busulfan, and Nucleoside Analogs in Human FLT3-ITD-Positive Acute Myeloid Leukemia Cell Lines

The nucleoside analogs clofarabine (Clo) and fludarabine (Flu), together with the DNA alkylating agent busulfan (Bu), are commonly used as part of the pre-transplant conditioning regimen in allogeneic hematopoietic stem cell transplantation (allo-HSCT). The multikinase inhibitor sorafenib (Sor) has clinical activity in FLT3-ITD-positive acute myeloid leukemia (AML). We previously reported that a combination of [Bu+Clo+Flu] has a synergistic cytotoxicity in AML cells. We now hypothesized that Sor, if combined with [Bu+Clo+Flu], will further enhance this cytotoxicity. We exposed the FLT3-ITD-mutated AML cell line MV4-11 to low concentrations of Bu (1 μg/ml), Clo (10 nM), Flu (2.5 μM) and Sor (1 nM) alone or in various combinations. Exposure of the cells to Bu, Clo, Flu or Sor alone, or to two-drug combinations [Bu+Clo], [Bu+Flu] and [Clo+Flu], did not significantly affect cell proliferation or apoptosis relative to control cells. The combination of [Bu+Clo+Flu] resulted in 20% inhibition of cell proliferation and demonstrated 10% increase in apoptosis. Addition of Sor to the [Bu+Clo+Flu] mixture further enhanced the inhibition of proliferation by 60%, and increased apoptosis to 50%, suggesting synergistic cytotoxicity. Biochemical analyses suggest that this cytotoxicity may be attributed to (1) activated DNA‑damage response (2) histone 3 modifications, (3) inhibition of different kinases, and (4) activation of the intrinsic apoptosis pathway. The phosphorylation of kinases, including FLT3, MEK and AKT, was significantly inhibited when cells were exposed to [Bu+Clo+Flu+Sor].  FLT3-ITD-activated STAT5 and its target gene Pim 2, a serine threonine kinase, were both down-regulated when cells were exposed to Sor alone, [Bu+Clo+Flu] and [Bu+Clo+Flu+Sor]. The level of the pro-apoptotic protein PUMA increased, while the pro-survival proteins MCL-1 and Bcl-xL were down-regulated when cells were exposed to the four-drug combination. The levels of phosphorylated and total P53 increased in the mitochondria when cells were exposed to [Bu+Clo+Flu] or [Bu+Clo+Flu+Sor]. The changes in the levels of these proteins involved in mitochondrial control of apoptosis may consequently cause mitochondrial outer membrane permeabilization (MOMP). The mitochondrial membrane potential (MMP), a marker of MOMP, decreased by 20% and 60% when cells were exposed to [Bu+Clo+Flu] and [Bu+Clo+Flu+Sor], respectively. This drug‑mediated decrease in MMP may have caused leakage of cytochrome c, SMAC/DIABLO and AIF from the mitochondria to the cytoplasm and/or nucleus, leading to caspase activation, nuclear fragmentation and cell death. These results provide a mechanistic basis for investigating the addition of sorafenib in future clinical trials of (double) nucleoside analog-busulfan combinations in pre-transplant conditioning therapy for patients undergoing allo-HSCT for FLT3-ITD positive AML.