Combining the FLT3 inhibitor PKC412 and the triterpenoid CDDO-Me synergistically induces apoptosis in acute myeloid leukemia with the internal tandem duplication mutation

Abstract

Mutations of the FLT3 receptor tyrosine kinase consisting of internal tandem duplications (ITD) have been detected in blasts from 20% to 30% of patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. FLT3/ITD results in constitutive autophosphorylation of the receptor and factor-independent survival in leukemia cell lines. The C-28 methyl ester of the oleane triterpenoid (CDDO-Me) is a multifunctional molecule that induces apoptosis of human myeloid leukemia cells. Here, we report that CDDO-Me blocks targeting of NFkappaB to the nucleus by inhibiting IkappaB kinase beta-mediated phosphorylation of IkappaBalpha. Moreover, CDDO-Me blocked constitutive activation of the signal transducer and activator of transcription 3. We report the potent and selective antiproliferative effects of CDDO-Me on FLT3/ITD-positive myeloid leukemia cell lines and primary AML cells. The present studies show that CDDO-Me treatment results in caspase-3-mediated induction of apoptosis of FLT3/ITD-expressing cells and its antiproliferative effects are synergistic with PKC412, a FLT3-tyrosine kinase inhibitor currently in clinical trials. Taken together, our studies indicate that CDDO-Me greatly enhanced the efficacy of the FLT3 inhibitor PKC412, suggesting that combining two separate pathway inhibitors might be a viable therapeutic strategy for AML associated with a FLT3/ITD mutation.

Figure 1

A. BaF3-wt cells were treated with IL-3 for 24 hours. Nuclear lysates from BaF3/FLT3/ITD and BaF3-wt cells treated with IL-3 were analyzed by immunoblotting with anti-NFκB and anti-Lamin B antibodies. B. BaF3/FLT3/ITD cells were treated with 1 μM CDDO-Me for the indicated times. Nuclear lysates were prepared and analyzed by immunoblotting with anti-NFκB, anti-Lamin B and anti-IκBα antibodies. Cytosolic lysate (last lane) was also immunoblotted as a positive control for cytosol. C. BaF3/FLT3/ITD cells were treated with 1 μM CDDO-Me for the indicated times. Total cell lysates were analyzed by immunoblotting with anti-phospho-IKKβ and anti-IKKβ antibodies. D. BaF3/FLT3/ITD cells were treated with 1 μM CDDO-Me for the indicated times. Total cell lysates were then analyzed by immunoblotting with anti-phospho-STAT3 and anti-STAT3 antibodies.

Figure 2

A. BaF3/FLT3/ITD cells were passaged a day before and treated with different concentrations (1 μM to 5 μM) of CDDO-Me for the indicated times. The cells were assessed for viability using trypan blue exclusion method. B and C. Mv4-11 and MOLM-14 cells were treated with varying concentrations of CDDO-Me for the indicated times. The cells were assessed for viability using trypan blue exclusion. A, B and C: [Closed squares: untreated; open squares: 1 μM CDDO-Me; closed triangles: 2.5 μM CDDO; closed circles: 5.0 μM CDDO] D. BaF3/FLT3/ITD cells were treated with 1 μM CDDO-Me and 5 nM PKC412 for 24 hours, either alone or in combination (P + C) and the cells were seeded for colony formation. After 10 days, the number of colonies were counted and compared to that with untreated (control) cells. Isobologram analysis was performed to determine additive versus synergistic effects. Independent experiments were performed at least three times; error bars indicate the standard deviations.

Figure 3

A. BaF3/FLT3/ITD cells were treated with 1 μM CDDO-Me and 5 nM PKC412, alone or in combination (P + C), for 2 (closed bars) and 3 (open bars) days and analyzed for apoptosis by flow cytometry. Assessment of cells by annexin V/PI staining was used as a measure for apoptosis. B. MOLM-14 cells were treated with 1 μM CDDO-Me and 5 nM PKC412, alone or in combination (P + C), for 2 (closed bars) and 3 (open bars) days and analyzed for apoptosis by Flow cytometry. C. Mv4-11 cells were treated with 1 μM CDDO-Me and 5 nM PKC412, alone or in combination (P + C), for 3 days and analyzed for apoptosis by Flow cytometry. D. Cells from three different AML patients were treated with 1 μM CDDO-Me and 5 nM PKC412, either alone or in combination (P + C) for three days. The cells were assessed for viability using trypan blue exclusion and showed as percent survival of cells.

Figure 4

A. BaF3/FLT3/ITD cells were treated with CDDO-Me and PKC412 at various concentrations, alone or in combination, for the indicated times. Total cell lysates were analyzed by immunoblotting with anti-PARP or anti-Actin antibodies. B. BaF3/FLT3/ITD cells were treated with CDDO-Me and PKC412 and total cell lysates were analyzed by immunoblotting with anti-Caspase-3 or anti-Actin antibodies. C. MOLM-14 cells were treated with CDDO-Me and PKC412 at various concentrations, alone or in combination, for 48 hours. Total cell lysates were analyzed by immunoblotting with anti-PARP or anti-Actin antibodies.