The BCL-2 Inhibitor ABT-199 Enhances Imatinib-Induced Cell Death In Chronic Phase CML Progenitors
Authors: Ko Tun Kiat , Chuah Charles , Huang John , Ng King-Pan , Ong S. Tiong 
Published: October 2019

Abstract
The advent of tyrosine kinase inhibitors (TKI) that specifically target BCR-ABL has significantly prolonged the life of patients with CML. We and others have previously shown that the BH3 mimetic ABT-737 or its clinical equivalent, ABT-263, can significantly enhance TKI-induced cell death in both CML cell lines and primary cells (Ng et al. Nature Medicine, 18: 521-528, 2012). ABT-737 and ABT-263 are BH3 mimetics that have broad specificity against anti-apoptosis regulators such as BCL-2 and its related proteins (BCL-XL & BCL-W). However, in clinical trials employing ABT-263, a dose-limiting toxicity is thrombocytopenia. This is due to the inhibitory effect of ABT-263 on BCL-XL that is pivotal for platelet survival. As a result, a new BH3 mimetic, ABT-199, has been developed that binds with high affinity to BCL-2 but not BCL-XL, and thus does not harm platelets.
Here, we evaluated the effectiveness of ABT-199, as a single agent or in combination with imatinib, in reducing the viability of CD34+ cells from the different phases of CML (chronic, accelerated and blast). We also assessed the cytotoxic effect of ABT-199 on normal cord blood CD34+ cells. The number of samples used in this study was: (1) chronic phase CML (CP, n=4), (2) accelerated phase CML (AP, n=2), (3) blast phase CML (BP, n=1), and (4) normal cord blood (NCB, n=3). We evaluated the viability of progenitor cells by colony formation assay. A broad concentration range of ABT-199 (in nM: 0, 1, 2, 5, 10, 20, 50, 100, 200, 500, 2000) was used in this study. The concentration of imatinib used was 2uM, which is in line with the plasma concentrations achievable in most patients with CML.
Below are three tables that summarize the results obtained from this study.Table 1Treatment with 2uM imatinib only.CP (n=4)AP & BC (n=3)NCB (n=3)Average CFU reduction65%23%20%(Relative to DMSO control)Table 2Treatment with ABT-199 only: concentration required to achieve the following CFU reduction.Average CFU reductionCP (n=4)AP & BC (n=3)NCB (n=3)50%500nM2000nM10nM90%2000nMNot achieved within the concentration range used500nMTable 3Combined treatment with ABT-199 and 2uM imatinib (IM): concentration required to achieve the following CFU reduction.Average CFU reductionCP (n=4)AP & BC (n=3)NCB (n=3)50%Not achieved within the concentration range used2nM ABT-199 + 2uM IM10nM ABT-199 + 2uM IM90%10nM ABT-199 + 2uM IM200nM ABT-199 + 2uM IM100nM ABT-199 + 2uM IM
In summary, we found that: (1) Compared to CML, NCB progenitors were very sensitive to ABT-199, with a reduction of average cell viability to 50% at just 10nM (with or without imatinib); (2) When the dose-limiting cytotoxic effect of ABT-199 on NCB was taken into consideration, we were able to discover an ABT-199 concentration (10nM) that significantly enhanced imatinib-induced cell death of CP CML progenitors without causing NCB viability to fall below 50%; and (3) The inferior efficacy of the combination on AP/BC vs CP progenitors suggests that BCL2-independent mechanisms contribute to the survival of advanced phase CML progenitors.
Finally, our in vitro data may also explain some of the adverse hematologic effects observed in clinical trials that employ ABT-199, including neutropenia and thrombocytopenia. Nevertheless, our data suggest that ABT-199, when used at a concentration that is not cytotoxic to normal progenitors and in combination with imatinib, can be beneficial in treating patients with chronic phase CML.

Disclosures:
Chuah: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.