Obatoclax (GX15-070) Overcomes Glucocorticoid-Resistance In Acute Lymphoblastic Leukemia through Induction of Apoptosis and Autophagy

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-X_L, 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-X_L, 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)