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

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.

Figure 1

Chemical segregation of navitoclax activity in NHL cell lines; requirement for BCL-2 or BCL-X_L 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 BCL2^High or BCL2^Low 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 BCL2^High or BCL2^Low 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 BCL2^High 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

The MCL-1 inhibitor A-1210477 synergizes with navitoclax in BCL2^High NHL cell lines via BCL-2 and not BCL-X_L inhibition. NHL BCL2^High cell lines were co-treated with navitoclax (0–20 μM); (a), the BCL-2-selective inhibitor venetoclax (0–20 μM) or the BCL-X_L-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

The MCL-1 inhibitor A-1210477 synergizes with navitoclax in BCL2^Low NHL cell lines via BCL-X_L and not BCL-2 inhibition. NHL BCL2^Low cells were treated as in Figure 2 and the degree of synergy determined by Bliss analysis (a). BCL2^Low NHL cell lines were treated with navitoclax or A-1155463 (all 1 μM) for 8 h and the interaction of BIM with BCL-X_L 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

Synergy between A-1210477 and BCL-2 family inhibitors correlates with that observed between flavopiridol and BCL-2 family inhibitors. BCL2^High 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 BCL2^High and BCL2^Low 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

Flavopiridol-mediated downregulation of MCL-1 sensitizes BCL2^High NHL cell lines to navitoclax and venetoclax in a caspase-dependent manner. BCL2^High 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 BCL2^High 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.