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A. Al-Zebeeby et al.
formed to compare sensitive and resistant cells. For samples from CLL patients, one-way repeated measures ANOVA with the Fisher least significant difference test (P≤0.01) was used and statistics analyzed using GraphPad Prism 6 software (La Jolla, CA, USA).
Results
Hematologic malignancies rapidly acquire resistance to BH3 mimetics
The potential of BH3 mimetic therapy in cancer was first demonstrated in the treatment of BCL-2-dependent CLL using navitoclax/ABT-263. In a phase I/II clinical trial of navitoclax, CLL patients were treated for an initial lead-in period of 7 days with a low dose of navitoclax (100 mg daily) followed by five to seven cycles of treat- ment, with each cycle lasting 21 days during which the patients received 250 mg navitoclax daily. Analysis of blood samples collected from these patients, either prior to the first in vivo dose of navitoclax or 4 h after dosing, during the different cycles of therapy revealed marked
changes in the ability of navitoclax to induce apoptosis in the CLL cells (Figure 1A). The first in vivo dose of navito- clax on day 1 of the lead-in period (L1D1) resulted in a time-dependent induction of apoptosis, as assessed by phosphatidylserine externalization and ultrastructural changes, in comparison to that of CLL cells from the same patients prior to treatment (Figure 1A and Online Supplementary Figure S1). A progressive increase in resist- ance to navitoclax was observed in CLL cells in vivo during the different cycles of treatment (Figure 1A). Since these studies were carried out in whole blood, we wanted to ascertain whether the decrease in ABT-263-induced apoptosis in CLL cells could be attributed to chemoresis- tance. To test this, CLL cells were isolated from these patients at the beginning of each treatment cycle and exposed to increasing concentrations of ABT-263. A sig- nificant decrease [3-fold difference in the half maximal inhibitory concentration (IC50) values between the lead-in period and cycle 5] was observed in their ability to under- go ABT-263-induced apoptosis (Figure 1B), demonstrating that continued dosing of patients with ABT-263 resulted in a modest, yet significant increase in chemoresistance.
ABC
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Figure 1. Hematologic malignancies rapidly acquire resistance to BH3 mimetics. (A) Blood samples collected from patients with chronic lymphocytic leukemia (CLL) (n=5), either prior to the first in vivo dose of navitoclax or 4 h after dosing during different stages of treatment - day 1 of the initial lead-in-period (L1D1), day 1 of cycle 1 (C1D1), day 1 of cycle 3 (C3D1) or day 1 of cycle 5 (C5D1) - were incubated ex vivo and the extent of apoptosis in the CD19+ CLL cells was assessed at the indicated time points by measuring phosphatidylserine (PS) externalization. (B) CLL cells isolated from these patients at the beginning of each treatment cycle, as indicated in the figure, were exposed in vitro to increasing concentrations of ABT-263 and the extent of apoptosis was assessed: half maximal inhibitory concentration (IC50) values are shown. (C) Scheme for establishing resistance to specific BH3 mimetics in relevant hematologic cell lines, as explained in the Methods section. Sensitive [A] and resistant [E] cells of MAVER-1, K562 and H929 cell lines were exposed for 4 h to ABT-199 (10 nM), A-1331852 (10 nM) and A-1210477 (5 mM), respectively, and apoptosis was assessed. (D-F) Combinations with some but not all BH3 mimetics restored apoptotic sensitivity of resistant [E] MAVER-1, K562 and H929 cells exposed for 4 h to ABT-199 (10 nM), A-1331852 (10 nM) or A-1210477 (5 mM), respectively. ***P⩽0.001, **P⩽0.01. Error bars = mean ± standard error of mean (n=3).
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haematologica | 2019; 104(5)