Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis and Inhibition of Proliferation of Ba/F3 FLT-3 ITD Mutant Cells.

FLT3 is an receptor tyrosine kinase of the subclass III family that plays a vital role in the regulation of the differentiation, proliferation and survival of normal hematopoietic cells. FLT3 mutations are often found in patients with Acute myelogenous leukemia (AML) and confer poor prognosis. Of these mutations, 15–35% are FLT3 ITD (internal tandem duplication) mutations and 5–7% are point mutations on the FLT3 kinase activation loop (e.g. D835V). Our laboratory is studying the signaling pathways associated with a newly identified multi-targeted tyrosine kinase receptor small molecule inhibitor (RTKI), ABT-869. Recently published work in our laboratory showed that using ABT-869 to treat MV4-11, a human AML FLT-3 ITD mutant cell line, resulted in the inhibition of phosphorylation of FLT-3 with a downstream inhibitory effect on the activation of STAT5, ERK, and Pim-1. Cell viability assays determined that MV-411 cells responded to ABT-869 in a concentration dependent manner (IC50 = 10nM). Apoptosis studies also showed an induction of apoptosis in ABT-869 treated cells. In vivo studies involving xenograft injections of MV-411 cells into SCID mice and subsequent treatment with ABT-869 demonstrated regression of tumor formation. In this study, a Ba/F3 mouse pro-B lymphocytic cell line harboring the FLT-3 ITD or FLT-3 D835V mutation is used as an isolated Flt-3 mutant model system. In vitro, ABT-869 is effective in inhibiting the proliferation of Ba/F3 Flt-3 ITD mutant cells when compared to Ba/F3 Flt-3 D835V mutant and Ba/F3 Flt-3 WT cells. Trypan Blue Exclusion and Alamar Blue assays were used to demonstrate that there is 50% inhibition of growth and proliferation (IC50) of Ba/F3 FLT3 ITD mutant cells at a concentration of 1nM after 48 hours of treatment. Ba/F3 FLT3 D835V mutant cells show an IC50 between 1μM and 10μM after 48 hours of treatment. In contrast, Ba/F3 FLT3 WT cells demonstrate an IC50 of 10μM only after 72 hours of treatment. Annexin V and propidium iodide staining of cells revealed that an increase in apoptosis (41.2%) occurred in Ba/F3 Flt-3 ITD mutant cells treated with 10nM ABT-869 after 24 hours when compared to untreated (6.5%) or vehicle control (6.1%) cells. Staining of Ba/F3 Flt-3 WT treated cell lines revealed no difference in apoptosis when compared to untreated Ba/F3 Flt-3 WT cell only and DMSO controls. PARP cleavage was observed in Ba/F3 FLT-3 ITD mutant cells following treatment with ABT-869 whereas no cleavage was observed with Ba/F3 WT cells treated with ABT-869. In vivo, the activity of ABT-869 treatment of SCID mice injected with Baf3 Flt-3 ITD, Baf3 Flt-3 D835V, or Baf3 Flt-3 WT cells is also being evaluated. Using bioluminescence imaging, it was determined that Ba/F3 FLT-3 ITD mutant and Ba/F3 Flt-3 D835Vmutant cell lines result in metastases and subsequent death in SCID mice after 2 weeks for ITD and 5 weeks for D835V, whereas mice injected with Ba/F3 WT survive longer than 5 weeks. Preliminary data demonstrated that ABT-869 prolonged survival in mice injected with the Ba/F3 FLT3-ITD cells compared to controls. Our preclinical data demonstrate that ABT-869 is effective specifically with FLT-3 ITD mutant cell lines in an isolated system. These studies provide rationale for the treatment of AML patients and the prevention of relapse.