Synergistic antileukemic effects between ABT-869 and chemotherapy involve downregulation of cell cycle-regulated genes and c-Mos-mediated MAPK pathway

Abstract

Internal tandem duplications (ITDs) of fms-like tyrosine kinase 3 (FLT3) receptor play an important role in the pathogenesis of acute myeloid leukemia (AML) and represent an attractive therapeutic target. ABT-869 has demonstrated potent effects in AML cells with FLT3-ITDs. Here, we provide further evidence that ABT-869 treatment significantly downregulates cyclins D and E but increases the expression of p21 and p27. ABT-869 induces apoptosis through downregulation of Bcl-xL and upregulation of BAK, BID and BAD. We also evaluate the combinations of ABT-869 and chemotherapy. ABT-869 demonstrates significant sequence-dependent synergism with cytarabine and doxorubicin in cell lines and primary leukemia samples. The optimal combination was validated in MV4-11 xenografts. Low-density array analysis revealed the synergistic interaction involved in downregulation of cell cycle and mitogen-activated protein kinase pathway genes. CCND1 and c-Mos were the most significantly inhibited targets on both transcriptional and translational levels. Treatment with short hairpin RNAs targeting either CCND1 or c-Mos further sensitized MV4-11 cells to ABT-869. These findings suggest that specific pathway genes were further targeted by adding chemotherapy and support the rationale of combination therapy. Thus, a clinical trial using sequence-dependent combination therapy with ABT-869 in AML is warranted.

Aberrant RNA splicing and mutations in spliceosome complex in acute myeloid leukemia

“…FLT3 mutations are identified in about one-third of adult (AML) (69,70). FLT3 mutations induce constitutive activation of phosphoinositide 3-kinase (PI3K)-AKT, RAS-MEK-mitogen-activated protein kinase (MAPK), and signal transducers and activators of transcription (STAT) five pathways, leading uncontrolled cell proliferation, blockage of differentiation and cell survival (52).…”

Enhanced activation of STAT pathways and overexpression of survivin confer resistance to FLT3 inhibitors and could be therapeutic targets in AML

“…For in vitro and in vivo experiments, the preparation for ABT-869 was previously published. 15 Indirubin derivative (IDR) E804, Tyrene CR4, AG490, AG1296, JAK3 inhibitor II, NU6140, and FLT3 inhibitor III were purchased from Calbiochem (Gibbstown, NJ) and dissolved in dimethyl sulfoxide (DMSO) before use. SU5416 and 5-aza-deoxycytidine (5-aza) were purchased from Sigma-Aldrich (St Louis, MO).…”

“…Colorimetric CellTiter 96 AQ ueous One Solution Cell Proliferation assay (MTS assay; Promega, Madison, WI) was used to determine the cytotoxicity as described previously. 15 IC 50 values were determined by MTS assay and calculated with CalcuSyn software (Biosoft, Cambridge, United Kingdom). Each experiment was in triplicate.…”

“…15 Briefly, RNA was extracted from cells using the Purescript RNA isolation kit (Genetra Systems, Minneapolis, MN). First strand cDNA was synthesized with SuperScript III First-Strand Synthesis SuperMix (Invitrogen).…”

“…ABT-869 was administrated at 15 mg/kg per day by oral gavage daily. 15,20 IDR E804 was prepared and given the same as ABT-869, but at dose of 10 mg/kg per day. In the combination group, mice were treated with both compounds at the same dose as monotherapy.…”