Potential mechanisms of resistance to venetoclax and strategies to circumvent it

Abstract

Background: Venetoclax (ABT-199), a first-in-class orally bioavailable BCL-2-selective inhibitor, was recently approved by the FDA for use in patients with 17p-deleted chronic lymphocytic leukemia who have received prior therapy. It is also being evaluated in numerous clinical trials for treating patients with various hematologic malignancies. As with any targeted cancer therapy, it is critically important to identify potential mechanisms of resistance, both for patient stratification and developing strategies to overcome resistance, either before it develops or as it emerges.

Methods: In order to gain a more comprehensive insight into the nature of venetoclax resistance mechanisms, we evaluated the changes in the BCL-2 family members at the genetic and expression levels in seven different venetoclax-resistant derived leukemia and lymphoma cell lines.

Results: Gene and protein expression analyses identified a number of different alterations in the expression of pro- and anti-apoptotic BCL-2 family members. In the resistant derived cells, an increase in either or both the anti-apoptotic proteins BCL-X_L or MCL-1, which are not targeted by venetoclax was observed, and either concomitant or exclusive with a decrease in one or more pro-apoptotic proteins. In addition, mutational analysis also revealed a mutation in the BH3 binding groove (F104L) that could potentially interfere with venetoclax-binding. Not all changes may be causally related to venetoclax resistance and may only be an epiphenomenon. For resistant cell lines showing elevations in BCL-X_L or MCL-1, strong synergistic cell killing was observed when venetoclax was combined with either BCL-X_L- or MCL-1-selective inhibitors, respectively. This highlights the importance of BCL-X_L- and MCL-1 as causally contributing to venetoclax resistance.

Conclusions: Overall our study identified numerous changes in multiple resistant lines; the changes were neither mutually exclusive nor universal across the cell lines tested, thus exemplifying the complexity and heterogeneity of potential resistance mechanisms. Identifying and evaluating their contribution has important implications for both patient selection and the rational development of strategies to overcome resistance.

Keywords: Apoptosis; BCL-2; BCL-XL; MCL-1.

Fig. 1

Generation of venetoclax-resistant cancer cell lines. Hematologic cancer cells lines were incubated in gradually increasing concentrations of venetoclax to isolate populations resistant to μM concentrations. Cell killing EC₅₀ values are plotted for parental cell lines and the venetoclax-resistant sublines. Data are presented as the mean +/- S.E.M. of three independent experiments. Asterisks denote p < 0.05

Fig. 2.

BCL-2 family protein expression in parental cell lines and venetoclax-resistant populations. Equivalent amounts of protein from whole cell lysates generated from parental cell lines and their venetoclax-resistant sublines growing in (a) human serum and (b) fetal bovine serum were assessed by immunoblotting for BCL-2 family proteins as indicated

Fig. 3.

Sub-population of parental RS4;11 cells lack BAX expression. BAX protein levels were quantified in parental RS4;11 cells by quantitative flow cytometry. a Dot plot of naïve parental RS4;11 cells stained with APC-isotype control. b Dot plot of naïve parental RS4;11 cells stained for BAX. Data are representative of three separate experiments

Fig. 4.

Mutation in the BH3-binding domain of BCL-2 in SC-1 cells confer resistance to venetoclax. a Table showing region/domain mutations reside in the cell lines identified by sequencing. b X-ray crystal structure of BCL-2 with F104L mutation, which is predicted to lower affinity for venetoclax. c General domain structure of BCL2 showing the amino acids mutationally altered in DLBCL. A, Ala; F, Phe; I, Ile; L, Lys; T, Thr; V, Val

Fig. 5.

Combination of MCL-1- or BCL-X_L-selective inhibitors resensitizes venetoclax-resistant cells to venetoclax. Venetoclax-resistant (199R) cells were incubated in varying concentrations of venetoclax with or without increasing concentrations of the MCL-1-selective inhibitor A-1208746 (a-c) or the BCL-X_L-selective inhibitor A-1155463 (d) for 24 hours before assessing cell viability. Data are presented as the percent viability compared to untreated cells. Bliss scores were calculated as described in Materials and Methods. Data are representative of three separate experiments. Asterisk designates significant difference (p<0.05) as compared to venetoclax-treatment alone.