Simultaneous Targeting of Bcl-2/Bcl-Xl and MEK/ERK Results in Highly Synergistic Induction of Apoptosis in Human AML, Both in Vitro and In Vivo.

Abstract 2757

Poster Board II-733

Previous experience from our group suggests that simultaneous inhibition of MEK-to-ERK signaling and interference with the anti-apoptotic activity of Bcl-2/Bcl-xL, using a variety of different agents, may result in synergistic anti-leukemic activity (Milella M, Blood 2002; Ricciardi MR, ASH 2004; Konopleva M, Cancer Cell 2006; Milella M, ASH 2008). Here, we screened AML cell lines (OCI-AML3, HL-60, MOLM-13, and U937) for the growth inhibitory/pro-apoptotic effects of the combination of the BH3 mimetic ABT-737 and the selective MEK inhibitor PD0325901. With the notable exception of U937 cells all the cell lines tested displayed highly synergistic growth inhibition/induction of apoptosis in response to the combination (CIs ranging from 0.01 to 0.43); the 25:1 and 1:2 ABT-737/PD0325901 ratios appeared to have optimal synergistic effects in OCI-AML3 and MOLM-13 cells, respectively. Cell growth inhibition was primarily due to the highly synergistic induction of apoptosis in sensitive cell line models. From a mechanistic standpoint, ABT-737 induced ERK activation and Mcl-1 protein expression, two putative resistance mechanisms, both of which were efficiently abrogated by co-treatment with PD0325901. In addition to the modulation of Mcl-1, both single agent PD0325901 and combined ABT-737/PD0325901 treatment rapidly (1-6 hrs) induced BimEL dephosphorylation and Bak expression, thereby contributing to Mcl-1 inactivation. To further assess the role of pro-apoptotic Bcl-2 family members in the observed proapoptotic synergism betweeen ABT-737 and PD0325901, we analyzed the effects of the combination in Bim-, Bak-, and Bax-KO MEFs, as well as in double (Bak/Bax) KO; while these experiments demonstrated that all three proapoptotic proteins play a role in apoptosis induction by combined ABT-737/PD0325901, siRNA-mediated silencing of either Bim or Bak clearly indicated Bim as the most important player in the AML cell line OCI-AML3. In addition to cell line models of leukemia, striking apoptosis induction (20-75% net apoptosis induction) was also observed with the combination of Bcl-2 and MEK inhibitors in ex vivo-cultured primary AML samples (n=8); most interestingly, the ABT-737/PD0325901 combination appeared to selectively kill leukemic stem cells, with < 20% of CD34+/CD38- cells surviving after exposure to relatively low doses of the combination (50 nM for each agent). Finally, we tested the combination of ABT-737 and the MEK inhibitor CI-1040 in nude mice injected with GFP/luciferase bearing MOLM-13 human leukemia cells. Two weeks after leukemia transplantation, mice were randomized and treated with liposomal ABT-737 (i.v. 20 mg/kg, qod for three weeks), CI1040 (i.p. 50 mg/kg qod for three weeks), ABT-737 in combination with CI1040 (ABT-737 + CI1040), or with empty liposomes (i.v.; control). Engraftment of MOLM-13 cells was shown by immunohistochemical detection of GFP-positive cells in the spleen of control mice five weeks after transplantation. Notably, while control and CI1040 treated mice demonstrated progressive increases in leukemia-derived bioluminescence, ABT-737 treated mice, and to a greater extent ABT-737 + CI1040 treated mice, appeared to resist tumor burden progression. In addition, quantitation of leukemia-derived bioluminescence demonstrated that ABT-737 + CI1040 treated mice had significantly (p<0.00001) lower leukemia burden than control mice or ABT-737 treated mice at all time points (7, 14 and 21 days of treatment). Overall our data demonstrate that an anti-apoptotic crosstalk between the Bcl-2 and the MEK/ERK pathway is operative in AML cells and could be exploited therapeutically by targeting both pathways simultaneously. The combination of BH3 mimetics (such as ABT-737) and MEK inhibitors warrants clinical testing as a novel therapeutic strategy for patients with AML.