Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis and Suppresses Proliferation of Ba/F3 FLT-3 ITD Mutant Cells in Vitro and in Vivo through Inhibition of FLT3 and AKT.

FLT3 is a receptor tyrosine kinase of the subclass III family that plays a vital role in the regulation of differentiation, proliferation and survival of normal hematopoietic cells. FLT3 mutations are often found in patients with acute myelogenous leukemia (AML) and confer a poor prognosis. Of these mutations, 15–35% are FLT3 ITD (internal tandem duplication) mutations and 5–7% are point mutations in the FLT3 kinase activation loop, e.g. D835V. We are studying the signaling pathways associated with a small molecule multi-targeted receptor tyrosine kinase inhibitor (RTKI), ABT-869. To determine the effects of ABT-869 in vitro and in vivo, a Ba/F3 mouse pro-B lymphocytic cell line harboring the FLT-3 ITD or FLT-3 D835V mutation was 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 (IC50 value of 1 nM) when compared to Ba/F3 Flt-3 D835V mutant (IC50 value between 1 and 10 μM) and Ba/F3 Flt-3 wildtype (WT) cells (IC50 value of 10 μM). Annexin V and propidium iodide staining of cells revealed that an increase in apoptosis occurred in Ba/F3 Flt-3 ITD mutant cells treated with 1μM ABT-869 for 24 hours (42.8%) when compared to untreated (4.7%) or vehicle control (4.0%) cells. Ba/F3 Flt-3 D835V mutant cell lines demonstrated a 12.5% rate of apoptosis at 1μM, compared to untreated (1.99%) and vehicle control (2.1%) cell lines. Propidium iodide staining of treated Ba/F3 Flt-3 WT cell lines revealed no difference in apoptosis when compared to untreated Ba/F3 Flt-3 WT cells or DMSO controls. PARP cleavage was observed in Ba/F3 FLT-3 ITD mutant cells, following 6 hours of treatment with 1 to 100 nM ABT-869, whereas no cleavage was observed in Ba/F3 WT cells treated with ABT-869. To study the effects of ABT-869 in vivo, we treated SCID mice injected with Ba/F3 Flt-3 ITD, Ba/F3 Flt-3 D835V, or Ba/F3 Flt-3 WT cells and monitored disease progression using bioluminescence imaging. The mice injected with the Ba/F3 FLT-3 ITD mutant cells and treated with vehicle control developed metastases and had a median survival time of 2 weeks. In contrast, the ABT-869 treated group had slower disease progression with median survival of 6.2 weeks (P<0.008). Both control and treated mice injected with Ba/F3 FLT-3 D835V mutant cell lines developed metastases and had similar survival (median 1.7 and 1.9 weeks, respectively). Survival times of control and treated mice injected with Ba/F3 FLT-3 WT cells were also similar (median 8.4 and 8.1 weeks, respectively). Previous work identified that ABT-869 induced apoptosis of acute myeloid leukemia cells through inhibition of FLT-3 reception phosphorylation, which is observed as early as 3 hours after treatment. In Ba/F3 cells expressing FLT-3 ITD, ABT-869 also inhibited phosphorylation of AKT, which is upstream of the pro-apoptotic protein Bad. Therefore, our preclinical data suggest that ABT-869 induces apoptosis of FLT-3 ITD mutant cells both in vitro and in vivo. These studies provide rationale for the treatment of acute leukemia patients harboring the FLT3-ITD mutation with ABT-869 and the potential benefit of combining small molecule inhibitors that target both RTKs and AKT.