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. 2015 Nov 13;5(11):e368.
doi: 10.1038/bcj.2015.88.

Loss in MCL-1 function sensitizes non-Hodgkin's lymphoma cell lines to the BCL-2-selective inhibitor venetoclax (ABT-199)

D C Phillips  1 , Y Xiao  1 , L T Lam  1 , E Litvinovich  2 , L Roberts-Rapp  1 , A J Souers  3 , J D Leverson  3
Affiliations

Loss in MCL-1 function sensitizes non-Hodgkin's lymphoma cell lines to the BCL-2-selective inhibitor venetoclax (ABT-199)

D C Phillips et al. Blood Cancer J. .

Erratum in

Abstract

As a population, non-Hodgkin's lymphoma (NHL) cell lines positive for the t(14;18) translocation and/or possessing elevated BCL2 copy number (CN; BCL2(High)) are exquisitely sensitive to navitoclax or the B-cell lymphoma protein-2 (BCL-2)-selective inhibitor venetoclax. Despite this, some BCL2(High) cell lines remain resistant to either agent. Here we show that the MCL-1-specific inhibitor A-1210477 sensitizes these cell lines to navitoclax. Chemical segregation of this synergy with the BCL-2-selective inhibitor venetoclax or BCL-XL-selective inhibitor A-1155463 indicated that MCL-1 and BCL-2 are the two key anti-apoptotic targets for sensitization. Similarly, the CDK inhibitor flavopiridol downregulated MCL-1 expression and synergized with venetoclax in BCL2(High) NHL cell lines to a similar extent as A-1210477. A-1210477 also synergized with navitoclax in the majority of BCL2(Low) NHL cell lines. However, chemical segregation with venetoclax or A-1155463 revealed that synergy was driven by BCL-XL inhibition in this population. Collectively these data emphasize that BCL2 status is predictive of venetoclax potency in NHL not only as a single agent, but also in the adjuvant setting with anti-tumorigenic agents that inhibit MCL-1 function. These studies also potentially identify a patient population (BCL2(Low)) that could benefit from BCL-XL (navitoclax)-driven combination therapy.

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Conflict of interest statement

DCP, YX, LTL, LR-R, AJS and JDL are AbbVie employees and are stock holders. The design, study conduct and financial support were provided by AbbVie. AbbVie participated in the data generation, interpretation of data, review and approval of this publication. EL is now an employee of Abbott Molecular Inc.

Figures

Figure 1
Figure 1
Chemical segregation of navitoclax activity in NHL cell lines; requirement for BCL-2 or BCL-XL for survival. The efficacy of navitoclax, venetoclax or A-1155463 was determined in NHL cell lines as described in the Materials and Methods section. EC50s were calculated from the resulting sigmoidal dose/response curves (see Supplementary Table 1) and segregated according to their BCL2High or BCL2Low status. Data are presented as the mean of at least three independent experiments. Cell lines with navitoclax EC50s >2 μM were deemed resistant. Data are presented as the mean of at least three independent experiments (a). Expression of anti-apoptotic BCL-2 family proteins was determined by Luminex as described and segregated according to the BCL2High or BCL2Low status. The median is shown in red and the Mann–Whitney U-test was used to determine statistical significance. NS, not significant (b). Navitoclax-resistant BCL2High cells were treated with navitoclax or venetoclax (both 1 μM) for 8 h and the interaction of BIM with BCL-2 or MCL-1 assessed using an Electrochemiluminescent ELISA (MSD) as described in the Materials and Methods section. Data are presented as the mean±s.e.m. of three independent experiments (c).
Figure 2
Figure 2
The MCL-1 inhibitor A-1210477 synergizes with navitoclax in BCL2High NHL cell lines via BCL-2 and not BCL-XL inhibition. NHL BCL2High cell lines were co-treated with navitoclax (0–20 μM); (a), the BCL-2-selective inhibitor venetoclax (0–20 μM) or the BCL-XL-selective inhibitor A-1155463 (0–20 μM); (b), in combination with the MCL-1-specific inhibitor A-1210477 (0, 5, 10 and 15 μM) for 48 h and the effect on viability determined. Synergy was quantified using the Bliss algorithm (c). Data are presented as the mean±s.e.m. of three independent experiments.
Figure 3
Figure 3
The MCL-1 inhibitor A-1210477 synergizes with navitoclax in BCL2Low NHL cell lines via BCL-XL and not BCL-2 inhibition. NHL BCL2Low cells were treated as in Figure 2 and the degree of synergy determined by Bliss analysis (a). BCL2Low NHL cell lines were treated with navitoclax or A-1155463 (all 1 μM) for 8 h and the interaction of BIM with BCL-XL or MCL-1 assessed using an electrochemiluminescent ELISA (MSD) as described in the Materials and Methods section (b). Data are presented as the mean±s.e.m. of three independent experiments.
Figure 4
Figure 4
Synergy between A-1210477 and BCL-2 family inhibitors correlates with that observed between flavopiridol and BCL-2 family inhibitors. BCL2High NHL cell lines were co-treated with navitoclax (0–20 μM), venetoclax (0–20 μM) or A-1155463 (0.20 μM) in combination with flavopiridol (0, 16.67, 50 and 150 nM) for 48 h and the effect on viability determined (a). Synergy was quantified by Bliss analysis (b). Data are presented as the mean±s.e.m. of three independent experiments. Bliss sums obtained in both BCL2High and BCL2Low NHL cell lines treated with A-1210477 in combination with navitoclax, venetoclax or A-1155463 were then correlated with those obtained with flavopiridol and navitoclax, venetoclax or A-1155463 (c). Data points represent the mean of three independent experiments. Spearman rank correlation co-efficient and associated statistical significance was determined using GraphPad Prism.
Figure 5
Figure 5
Flavopiridol-mediated downregulation of MCL-1 sensitizes BCL2High NHL cell lines to navitoclax and venetoclax in a caspase-dependent manner. BCL2High NHL cell lines were co-treated with navitoclax (1 μM) or venetoclax (1 μM) in combination with flavopiridol at 0, 16.67, 50 or 150 nM (a) or 50 nM (b) for 24 h before assessing effects on MCL-1, BCL-2, caspase-3, PARP and β-actin by western blot. Alternatively BCL2High cell lines were pre-treated with z-VAD-fmk (50 μM) for 1 h and then co-treated with navitoclax (1 μM) or venetoclax (1 μM) in combination with A-1210477 (5 μM) or flavopiridol (50 nM) for a further 24 h and the effect on apoptosis determined by flow cytometric analysis of Annexin-V/7-AAD staining. Representative flow cytometry histograms in SU-DHL-4 cells from three independent experiments are shown in c and quantified in d.
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