Abstract Introduction: With the improvement of treatment outcome in pediatric acute lymphoblastic leukemia (ALL), about 80% of the patients can be cured. However, around 10% of patients have poor outcome due to drug resistance which calls for novel drugs such as BH3-mimetics. We have previously demonstrated that GX15-070 (GX), a BH3-mimetic, could effectively induce cell death. In this study, we investigate the mechanisms of GX induced cell death in ALL cells. Method: Seven ALL cell lines were used in this study. Cell viability was determined by MTS assay (Promega). Western blot was used to detect protein expression level. Cholesterol (CHO) level was measured by Amplex® Red Cholesterol Assay Kit (Promega). RT-PCR was done by Roche Light Cycler using SYBR Green. Results: There was a dose-dependent cell death induced in all 7 ALL cell lines after treatment with GX. Interestingly, genes that are involved in the CHO synthesis pathway such as LDLR, HMGCR, HMGSC1 and SQLE were upregulated after GX treatment. Furthermore, GX mediated reduction in cellular CHO levels was observed. To verify that CHO depletion could induce cell death in ALL cells, Lovastatin was applied. Our results showed that Lovastatin not only induced a dose-dependent cell death in all 7 ALL cell lines but also upregulated the genes mentioned above. Co-treatment of 0.1µM GX and 25µM Lovastatin could enhance the cell death by further decreasing CHO level. In addition, GX-induced cell death could also be enhanced by 10mM 2-Deoxy-D-glucose (2-DG) co-treatment. 2-DG is an inhibitor of glycolysis which provides metabolites for CHO metabolism. Next, to mimic a culture condition whereby CHO is depleted, we incubated the cells in HBSS without Fetal bovine serum (FBS) for 24h. This CHO depleted condition decreased the cellular CHO level to 46% compared to the cells that were cultured in 10% FBS and RPMI medium. After incubation in HBSS for 24h, 0.1µM GX could eliminate more cells (80%) compared to the same dose of GX in serum supplemented RPMI medium (20%). Both HBSS incubation and GX treatment caused a decrease in anti-apoptotic protein Mcl-1 expression levels, while pro-apoptotic protein BIM and cleaved-PARP protein levels were upregulated. The two treatments did not alter the protein levels of other Bcl-2 family members. These results indicate that Mcl-1 expression is tightly controlled by cellular metabolism and CHO depletion can result in cell death. The mechanism of GX induced cell death may entail the suppression of Mcl-1 expression, hence sensitizing ALL cells to apoptosis. Conclusion: GX can regulate CHO metabolism to induce cell death in ALL cells which are normally dependent on aerobic glycolysis. This highlights the possibility of manipulating CHO metabolism to provide a specific way to treat leukemic cells. The administration of GX in the clinic, alone or in combination, may improve the treatment outcome of leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3227. doi:1538-7445.AM2012-3227