BH3 Profiling Predicts On-Target Cell Death Due To Selective Inhibition Of BCL-2 By ABT-199 In Acute Myelogenous Leukemia
Authors: Hogdal Leah , DeAngelo Daniel J. , Stone Richard M. , Gaidzik Verena I. , Bucci Donna , Döhner Konstanze , Marcucci Guido , Ryan Jeremy , Letai Anthony 
Published: October 2019

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. The B-cell leukemia /lymphoma 2 (BCL-2) protein acts to prevent commitment to programmed cell death initiated in the mitochondrion. Inhibiting the function of this protein is an attractive approach to cancer therapy, but it remains a challenge to identify those tumors that will best be treated with such a strategy. Here, we test the sensitivity of AML cell lines and primary patient samples to the selective BCL-2 inhibitor, ABT-199, and test for correlation between ABT-199 sensitivity with BCL-2 dependence measured by BH3 profiling. We hypothesized that cells that are more dependent on BCL-2 will be more sensitive to inhibition by ABT-199.
To this end, we tested the sensitivity of seven AML cell lines and 34 primary patient samples to ABT-199. We found that both cell lines and primary patient samples were sensitive to ABT-199 (Figure 1). In the case of primary patient cells, the median EC-50 was approximately 20 nM, and cell death could be seen within 2 hours. Importantly, we found that sensitivity to ABT-199 was independent of cytogenetics and NPM1 and FLT3 mutations of the primary patient cells suggesting that treatment with ABT-199 could be useful for a variety of AML patients, even those with an intrinsically poor prognosis.
We also measured dependence on BCL-2-mediated apoptosis in cell lines and primary patient samples by 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. Therefore, we tested whether ABT-199 sensitivity correlates with functional dependence on BCL-2 in both cell lines and primary patient samples. We found that cells that released cytochrome c following incubation with the BAD peptide were more sensitive to ABT-199 treatment (Figure 2). This demonstrates that ABT-199 functions on target at the mitochondria since the ABT-199 mitochondrial activity correlates well with ABT-199 cytotoxicity data.
Since ABT-199 does not inhibit MCL-1, increased expression of MCL-1 could be a potential source of upfront resistance to BCL-2 inhibition. Therefore, we asked if there was a correlation between MCL-1 dependence and ABT-199 sensitivity. We observed a weak anti-correlation between mitochondrial sensitivity to ABT-199 and sensitivity to the MCL-1 selective peptide NOXA BH3. This suggests that there is a minor tendency for MCL-1 dependent mitochondria to be less sensitive to ABT-199.
The ex-vivo sensitivity of AML cells to ABT-199, which appears to be BCL-2 specific, is similar to that observed in CLL, a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, the BH3 profiling studies demonstrate ABT-199 activity at the mitochondrion that correlates very well with cytotoxicity, supporting a mitochondrial mechanism of action. Our BH3 profiling studies will be undertaken to determine if the results will serve as a predictive biomarker in an upcoming phase II clinical trial of ABT-199 in AML.

Disclosures:
Letai: AbbVie: Consultancy; Dana-Farber Cancer Institute: Patents & Royalties.