ALBERT

Description: Abstract 1809 Glucocorticoids (GC) are common components in many chemotherapeutic protocols for lymphoid/myeloid malignancies, including acute lymphoblastic leukemia (ALL). However, patients often develop resistance to GC on relapse. Resistance to GC in ALL can be associated with defects in apoptosis machinery, but not in the GC receptor. Thus, targeting downstream molecules may lead to the development of new therapeutic strategies. GC-induced apoptosis is through the intrinsic mitochondria-dependent pathway. The BCL-2 family proteins are central regulatory proteins in this pathway. We hypothesized that targeting anti-apoptotic MCL-1 might be effective among the BCL-2 family proteins, since (1) we recognized that treatment with dexamethasone (Dex) in CCRF-CEM or Molt-4 T-ALL cells slightly induce MCL-1 and the expression level of MCL-1 is higher in Dex-resistant ALL cells compared with that in Dex-sensitive cells; (2) recent studies have demonstrated that increased expression of MCL-1 associates with GC resistance. In support of our hypothesis, down-regulation of MCL-1 by shRNA enhances Dex-induced cell death. We then pharmacologically inactivate MCL-1 function by GX15-070 (obatoclax), a BH3 mimetic small molecule that targets anti-apoptotic BCL-2 family proteins including BCL-2, BCL-XL, and MCL-1. Treatment with GX15-070 in both Dex-sensitive and -resistant ALL cells shows effective growth inhibition and cell death. GX15-070 induces caspase-3 cleavage and increases Annexin V-positive population, indicative of apoptosis. Before the onset of apoptosis, GX15-070 induces LC3 conversion as well as p62 degradation, both of which are autophagic cell death markers. A pro-apoptotic molecule BAK is released from BAK/MCL-1 complex following GX15-070 treatment. Consistently, down-regulation of BAK reduces caspase-3 cleavage and cell death, but does not alter LC3 conversion. In contrast, down-regulation of ATG5, an autophagy regulator, decreases LC3 conversion and cell death, but does not alter caspase-3 cleavage, suggesting that apoptosis and autophagy induced by GX15-070 are independently regulated. Down-regulation of Beclin-1, which is capable of crosstalk between apoptosis and autophagy, affects GX15-070-induced cell death through apoptosis but not autophagy. Taken together, GX15-070 treatment in ALL could be an alternative regimen to overcome glucocorticoid resistance by inducing BAK-dependent apoptosis and ATG5-dependent autophagy. Enhanced anti-apoptotic BCL-2 family protein expression has been observed in several types of tumors. Targeting these proteins is therefore an attractive strategy for restoring the apoptosis process in tumor cells. Among the small molecule BCL-2 inhibitors, ABT-737 and its analog ABT-263 are the leading compounds currently in clinical development. However, these molecules have an affinity only with BCL-2 and BCL-XL, but not with MCL-1. Thus, ABT-737 can not be effective as a single agent therapeutic for ALL when MCL-1 is overexpressed. In contrast, GX15-070 can overcome the resistance conferred by high level of MCL-1. Our results suggest that GX15-070 could be useful as a single agent therapeutic against ALL and that the activity/expression of anti-apoptotic proteins could be a biomarker to determine the treatment strategy to ALL patients. (Supported by NIH R01CA134473 and the William Lawrence and Blanche Hughes Foundation) Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3486 Glucocorticoids (GC) are common components in many chemotherapeutic protocols for lymphoid/myeloid malignancies, including acute lymphoblastic leukemia (ALL). However, patients often develop resistance to GC on relapse. Resistance to GC in ALL can be associated with defects in apoptosis machinery not in the GC receptor. Thus, targeting downstream molecules may lead to the development of new therapeutic strategies. GC-induced apoptosis is through the intrinsic mitochondria-dependent pathway. The BCL-2 family proteins are central regulatory proteins in this pathway. We and others have previously shown that BIM (BCL-2 Interacting Mediator of cell death), a pro-apoptotic BCL-2 family protein, is up-regulated by dexamethasone (Dex) treatment in ALL cells and plays an essential role in Dex-induced apoptosis (Lu et al., 2006). Furthermore, BIM is inactivated by extracellular signal-regulated kinase (ERK)-mediated phosphorylation by survival/growth factors (Harada et al., 2004). We therefore hypothesized that co-treatment with Dex and MEK/ERK inhibitors will promote apoptosis in ALL cells through BIM up-regulation and activation, resulting in cell death. Significantly, we have demonstrated that MEK inhibitors synergistically promote Dex lethality in a variety of ALL cell lines, in which BIM is a critical molecule in cell death (Rambal et al. 2009). Since BIM is capable to interact with all anti-apoptotic BCL-2 family proteins (i.e. BCL-2, BCL-XL, MCL-1), we hypothesized that down-regulation/inactivation of the anti-apoptotic BCL-2 family proteins is also effective to enhance the activity of BIM. We found that down-regulation of anti-apoptotic BCL-2 family proteins, especially MCL-1, enhanced GC-induced cell death. We then targeted MCL-1 in ALL cells by using GX15-070 (obatoclax), a small molecule inhibitor for anti-apoptotic BCL-2 family proteins. Treatment with GX15-070 in both Dex-sensitive and -resistant ALL cells showed effective growth inhibition and cell death (Heidari et al. 2010). GX15-070 treatment in ALL could be an alternative regimen to overcome GC-resistance. To further explore new treatments to overcome resistance to GC, it is important to elucidate entire signaling pathways for the activation of BIM in GC treatment. It has been indicated that Dex-induced BIM is regulated mainly by transcription, however the molecular mechanisms, including responsible transcription factors, of how Bim is induced are unclear. In this study, we examined the role of c-Jun in Dex-induced Bim transcriptional regulation in ALL cells. Dex treatment induced c-Jun mRNA and protein in parallel with BIM induction. BIM induction decreased in cells harboring dominant-negative c-Jun protein and increased in cells with overexpression of c-Jun. On the Bim promoter, c-Jun bound to and activated the AP-1 binding site at about −2.7kb from the transcription start site. Furthermore, pre-treatment with SB203580, a p38-MAPK inhibitor, decreased the amount of Dex-induced mRNAs and proteins of c-Jun and BIM, suggesting that p38-MAPK activation is upstream of c-Jun and BIM induction. Treatment with RU486, a GC receptor (GR) antagonist, blocked Dex-induced p38-MAPK phosphorylation, c-Jun and BIM induction, and apoptosis. In conclusion, we identified the critical signaling pathway for GC-induced apoptosis, i.e. GC–〉GR–〉p38-MAPK–〉c-Jun–〉BIM–〉Apoptosis. Targeting this pathway may be an alternative approach to overcome GC-resistance in leukemia treatment. Disclosures: No relevant conflicts of interest to declare.