Fenretinide inactivation of Mcl-1 enhanced activity of the Bcl-2 family inhibitor ABT-737 in acute lymphoblastic leukemia cell lines

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We previously reported that ABT-737, a BH3-mimetic, synergistically increased cytotoxcity in combination with 4-HPR, a cytotoxic retinoid in acute lymphobastic leukemia (ALL) cell lines (Proc AACR, abstract #1336, 2006). We further investigated the mechanism of synergy and the differential cytotoxicity between leukemia cells and normal cells of ABT-737 + 4-HPR. Cytotoxicity was determined using a fluorescence-based digital imaging assay (DIMSCAN). Changes in Bcl-2 family proteins were detected by immunoblotting. Reactive oxygen species (ROS) generation (DCFDA fluorescence) and phospho-Jun kinase (p-JNK) were measured by flow cytometry. ABT-737 increased Mcl-1 protein only in ABT-737-resistant ALL cell lines, while 4-HPR inactivated Mcl-1 via reactive oxygen species (ROS) generation and JNK phosphorylation. In CEM cells the antioxidant ascorbic acid (AA), completely abrogated ROS generation (43.5 ± 2.67-fold increase by 10 μM 4-HPR alone versus 0.75 ± 0.07-fold increase by AA + 4-HPR relative to untreated control), JNK phosphorylation and Mcl-1 inactivation, and attenuated the cytotoxicity of 4-HPR (p<0.001) and of ABT-737 + 4-HPR (p<0.001). Thus, 4-HPR enhanced ABT-737 cytotoxicity, at least in part through JNK activation via ROS generation that inactivated Mcl-1. Mcl-1 inactivation was not observed with non-cytotoxic retinoids (all-trans reninoic acid or 13-cis retinoic acid). Myriocin, a serinepalmitoyl transferase inhibitor at 10 μM blocked the increase in synthesis of ceramides by 4-HPR, but p-JNK expression was not affected (28.8 ± 0.5% for 4-HPR and 29.4 ± 1.4% for myriocin + 4-HPR), suggesting that ceramide is not an upstream signal of JNK phosphorylation. In normal resting lymphocytes, 4-HPR did not increase ROS or phosphorylate JNK. The cytotoxicity of 4-HPR correlated in ALL cell lines and normal resting lymphocytes with levels of ROS generated in response to 4-HPR (p< 0.002, r = - 0.92). These data identify in ALL cell lines a mechanism of resistance to ABT-737 that is overcome by 4-HPR and a novel drug combination that can inhibit the full spectrum of the Bcl-2 family of proteins.