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. 2008 May 1;68(9):3413-20.
doi: 10.1158/0008-5472.CAN-07-1919.

Mechanisms of antileukemic activity of the novel Bcl-2 homology domain-3 mimetic GX15-070 (obatoclax)

Marina Konopleva  1 , Julie Watt, Rooha Contractor, Twee Tsao, David Harris, Zeev Estrov, William Bornmann, Hagop Kantarjian, Jean Viallet, Ismael Samudio, Michael Andreeff
Affiliations

Mechanisms of antileukemic activity of the novel Bcl-2 homology domain-3 mimetic GX15-070 (obatoclax)

Marina Konopleva et al. Cancer Res. .

Abstract

In this study, we investigated the mechanism of apoptosis induction of obatoclax (GX15-070), a novel Bcl-2 homology domain-3 (BH3) mimetic, in acute myeloid leukemia (AML) cell lines and primary AML samples. Obatoclax inhibited cell growth of HL-60, U937, OCI-AML3, and KG-1 cell lines. Apoptosis induction contributed to the observed antiproliferative effects at concentrations of this agent that mirror its affinity for antiapoptotic Bcl-2 proteins. We show that obatoclax can promote the release of cytochrome c from isolated leukemia cell mitochondria and that apoptosis induced by this agent is preceded by the release of Bak from Mcl-1, liberation of Bim from both Bcl-2 and Mcl-1, and the formation of an active Bak/Bax complex. Notably, apoptosis was diminished, but not fully prevented, in the absence of Bak/Bax or Bim, suggesting that obatoclax has additional targets that contribute to its cytotoxicity. At growth inhibitory doses that did not induce apoptosis or decrease viability, obatoclax induced an S-G(2) cell-cycle block. Obatoclax induced apoptosis in AML CD34+ progenitor cells with an average IC(50) of 3.59 +/- 1.23 micromol/L although clonogenicity was inhibited at concentrations of 75 to 100 nmol/L. Obatoclax synergized with the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells and synergistically induced apoptosis in combination with AraC in leukemic cell lines and in primary AML samples. In conclusion, we show that obatoclax potently induces apoptosis and decreases leukemia cell proliferation and may be used in a novel therapeutic strategy for AML alone and in combination with other targeted agents and chemotherapeutics.

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Figures

Figure 1
Figure 1
Obatoclax induces apoptosis in AML. A - OCI-AML3 cells were incubated with various concentrations of obatoclax (0, 1, 2.5, 5, and 10 μM) and the number of viable cells was determined as described in the Materials and Methods. B - OCI-AML3 cells were treated with increasing concentrations of obatoclax for various times and phosphatydil serine (PS) externalization was monitored by flow cytometry by staining with Annexin V-APC. C – OCI-AML3 cells were treated with 0.5 and 1.0 μM of obatoclax for 48 h and BrdU incorporation was quantitated by flow cytometry as described in the Materials and Methods. Cell volume was determined from the average diameter measured by the ViCell XR analyzer. D – Cells were treated with obatoclax for 1 h and washed twice in serum containing media. Cells were then cultured under standard conditions for 48 h, and cell viability and apoptosis were quantitated as described in the Materials and Methods.
Figure 2
Figure 2
Obatoclax activates the intrinsic apoptotic pathway. A - Succinate/rotenone-energized HL60 mitochondria were exposed to obatoclax (10 μM) for 15 minutes and the levels of cytochrome C in the pellet and corresponding supernatant were determined as described in the Materials and Methods. B - Mcl-1 was immunoprecipitated from OCI-AML3 cells treated with obatoclax for 6 h, and the presence of Bak was determined by Western blot. C – Active Bak was immunoprecipitated from OCI-AML3 cells treated with obatoclax for 6 h, and the presence of Bax was determined by Western blot. D – Wild-type (WT)- or Bak-deficient MEFs were treated with obatoclax for 48 h and Annexin V-positivity was monitored by flow cytometry as described in the Materials and Methods.
Figure 3
Figure 3
Obatoclax induces release of Bim from anti-apoptotic Bcl-2 and Mcl-1 proteins. A – Bcl-2 was immunoprecipitated from obatoclax treated OCI-AML3 cells and the presence of Bim was investigated by Western blot. B – Mcl-1 was immunoprecipitated from obatoclax treated OCI-AML3 cells and the presence of Bim was investigated as above. C - Bim-deficient MEFs were treated with obatoclax for 48 h and Annexin V-positivity was monitored by flow cytometry as described in the Materials and Methods. D – Bak, Bim, Bak and Bim siRNA or control (NS) siRNA was transfected into HL-60 cells using Amaxa nucleofection, and the levels of Bim/Bak expression were analyzed by Western blotting. Cells were treated with 5μM obatoclax for 48 h, and induction of apoptosis was assessed by annexin V flow cytometry.
Figure 4
Figure 4
Obatoclax synergizes with ABT-737 and AraC to induce cell death in OCI-AML3 cells. A - OCI-AML3 cells were treated simultaneously with ABT-737 and obatoclax using a fixed ratio (1:1), and Annexin V-positivity was monitored by flow cytometry after 48 h and CI values were determined by isobologram analysis. B - ABT-737-resistant OCI-AML3 cells were treated simultaneously with AraC and obatoclax and CI values were determined as above.
Figure 5
Figure 5
Obatoclax induces apoptosis in primary AML samples. A - Primary AML samples were treated with increasing concentrations of obatoclax and annexin V was measured in the CD34-positive compartment by flow cytometry after 24 h. Specific apoptosis was determined as described in the Materials and Methods. B – Primary AML samples were cultured with obatoclax as indicated and clonogenicity was determined as described in the Materials and Methods. C – The clonogenic potential of normal bone marrow samples treated with obatoclax was determined as above. D – Bcl-2 was immunoprecipitated from a primary AML sample treated with obatoclax and the presence of Bim was assessed by Western blot. Results are representative of three independent samples examined.
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