Synergistic Cytotoxicity of Sorafenib with Busulfan and Nucleoside Analogs in Human FMS-like Tyrosine Kinase 3 Internal Tandem Duplications–Positive Acute Myeloid Leukemia Cells

Abstract

Clofarabine (Clo), fludarabine (Flu), and busulfan (Bu) are used in pre-transplant conditioning therapy for patients with myeloid leukemia. To further improve their efficacy in FLT3-ITD-positive AML, we investigated their synergism with sorafenib (Sor). Exposure of FLT3-ITD-positive MV-4-11 and MOLM 13 cells to [Bu+Clo+Flu+Sor] resulted in synergistic cytotoxicity; no such synergism was observed in the FLT3-wild type THP-1 and KBM3/Bu2506 cell lines. The drug synergism in MV-4-11 cells could be attributed to activation of DNA-damage response, histone 3 modifications, inhibition of pro-survival kinases, and activation of apoptosis. Further, the phosphorylation of kinases, including FLT3, MEK and AKT, was inhibited. The FLT3-ITD-substrate STAT5 and its target-gene PIM 2 product decreased 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 level of pro-survival protein MCL-1 decreased when cells were exposed to [Bu+Clo+Flu+Sor]. The interactions of PUMA with MCL-1 and/or BCL-2 were enhanced when cells were exposed to [Bu+Clo+Flu] or [Bu+Clo+Flu+Sor]. The changes in the level of these proteins, which are involved in mitochondrial control of apoptosis, correlate with changes in mitochondrial membrane potential (MMP). [Bu+Clo+Flu+Sor] decreased MMP by 60% and caused leakage of cytochrome c, SMAC/DIABLO and AIF from the mitochondria to the cytoplasm, caspase activation and cell death suggesting the activation of apoptosis. Analogous, synergistic cytotoxicity in response to Bu, Clo, Flu and Sor was observed in mononuclear cells isolated from FLT3-ITD-positive AML patients. While our previous studies were aimed at standardizing the conditioning regimen, the new findings suggest that patients with abnormal expression of FLT3 might further benefit from individualizing treatment through the addition of Sorafenib to [Bu+Clo+Flu], thereby providing personalized pre-transplant therapy.

Association between increased mutation rates in DNMT3Α and FLT3‑ITD and poor prognosis of patients with acute myeloid leukemia

“…There were no significant differences in the platelet counts and hemoglobin levels in the peripheral blood of patients in the four groups (P>0.05), suggesting that simultaneous mutation of DNMT3A and FLT3-ITD contributes to increased WBC and myeloid progenitor cells. It was previously demonstrated that FLT3-ITD mutation can activate a downstream kinase through the continuous activation of protein tyrosine kinase, thereby triggering signal transduction and further inducing the spontaneous and non-receptor-dependent proliferation of leukocytes (27). However, the role of DNMT3A in cell proliferation remains unclear.…”

Expression Levels of Human Equilibrative Nucleoside Transporter 1 and Deoxycytidine Kinase Enzyme as Prognostic Factors in Patients with Acute Myeloid Leukemia Treated with Cytarabine

“…Therefore, new target drugs were developed and evaluated in AML patients either alone or in combination with chemotherapy [28]. As an example, the addition of cladribine to regimens containing Ara-C and anthracyclines (idarubicine) allows not only a higher complete remission rate, including patients with unfavorable karyotype, but also has a more significant advantage in OS [29].…”

“…Advances in cancer genomics have revealed a spectrum of somatic mutations that give rise to human AML, drawing our attention to its molecular evolution and clonal architecture [26,27,28]. Therefore, new target drugs were developed and evaluated in AML patients either alone or in combination with chemotherapy [28].…”

“…Clofarabine primarily requires hENT2 , and only a minor proportion of hENT1 , to be transported into the cell, and it also requires phosphorylation by dCK and deoxyguanosine kinase. The sensitivity of Ara-C-resistant leukemia to clofarabine may be related to the affinity of this drug for other transporters and activation by other kinases [26,27,28]. …”

Romidepsin enhances the cytotoxicity of fludarabine, clofarabine and busulfan combination in malignant T-cells

“…Since [Flu+Clo+Bu] activates the DDR in AML cells [20], we examined if [Flu+Clo+Bu+Rom] would have similar effects in neoplastic T-cells. Since the activation of DDR is an early event, we exposed PEER cells to drugs for 24 hrs and analyzed them by Western blotting.…”