Abstract B10: Studying BCL-2 dependence using BH3 profiling in a phase 2 clinical trial of ABT-199 in acute myeloid leukemia.
Authors: Hogdal Leah , Chyla Brenda , McKeegan Evelyn , Leverson Joel , Potluri Jalaja , Humerickhouse Rod , Mack Mabry , DeAngelo Daniel , Galinsky Ilene , Stone Richard , Letai Anthony 
Published: August 2015

Abstract
Most patients with acute myeloid leukemia (AML) become resistant to chemotherapy at some point in their course and succumb to their disease. It is necessary to prevent chemo-resistance or enhance chemosensitivity in a selective fashion to lead to a higher cure rate and a lower toxic burden. ABT-199 is a potent and selective inhibitor of the anti-apoptotic, protein B-cell leukemia /lymphoma 2 (BCL-2). We have previously shown that AML cell lines, primary patient samples and primary xenografts are very sensitive to ABT-199 treatment (Pan et al. 2014. Cancer Discovery). We have also measured dependence on BCL-2-mediated apoptosis in cell lines and primary patient samples using BH3 profiling. BH3 profiling is a method to determine the mitochondrial priming level of a cell by exposing cellular mitochondria to standardized amounts of peptides derived from the BH3 domains of BH3-only proteins and determining the rate of cytochrome c release. BH3 profiling can also identify which anti-apoptotic species are critical in mediating cell death in a given cell type. For instance, the BAD BH3-only protein binds with high affinity to BCL-2, BCL-XL and BCL-W. Thus, release of cytochrome c following BAD peptide incubation suggests an anti-apoptotic dependency on BCL-2, BCL-XL or BCL-W. Mitochondrial studies using BH3 profiling demonstrate ABT-199 activity at the mitochondrion that correlates well with cytotoxicity in our in vitro assays suggesting that BH3 profiling may prove useful as a predictive biomarker in the treatment of AML patients with ABT-199.
ABT-199 is in clinical development in relapsed/refractory AML. Since our pre-clinical results suggest that BCL-2 dependence measured by BH3 profiling may be predictive of sensitivity to ABT-199, we BH3 profiled pre-treatment bone marrow aspirate samples. We found that AML primary cells release varying amounts of cytochrome c following BAD peptide incubation, suggesting that different AML patient samples possess different dependencies on BCL-2.
ABT-199 promotes apoptosis by displacing BIM from BCL-2 to other anti-apoptotics and the pro-apoptotic effector proteins BAX and BAK. To test if the proposed mechanism of action was active in vivo, we BH3 profiled bone marrow aspirates collected while the patients were on treatment and analyzed the response to the NOXA, HRK and BIM peptides. We found that blasts isolated after ABT-199 exposure have an increased response to the HRK (measures BCL-XL dependence) and NOXA (measures MCL-1 dependence) peptides compared to the pre-treatment samples. This finding suggests that ABT-199 is working on-target as it displaces BIM from BCL-2 to BCL-XL and MCL-1. Furthermore, we find that there is an increase in overall priming as measured by the BIM peptide, also suggesting an on-target mechanism of action as ABT-199 displaces BIM from BCL-2 increasing the overall pro-apoptotic reserve.
Briefly, our BH3 profiling data show that ABT-199 is acting on-target at the mitochondria in vivo. Furthermore, we find that the overall priming level of the residual AML blasts that remain following ABT-199 treatment have increased overall priming. This suggests that a combination of ABT-199 with other agents may be beneficial in AML, since ABT-199 increases the pro-apoptotic reserve which might push AML cells towards cell death in clinical settings.

Citation Format: Leah Hogdal, Brenda Chyla, Evelyn McKeegan, Joel Leverson, Jalaja Potluri, Rod Humerickhouse, Mabry Mack, Daniel DeAngelo, Ilene Galinsky, Richard Stone, Anthony Letai. Studying BCL-2 dependence using BH3 profiling in a phase 2 clinical trial of ABT-199 in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B10.