Functional disparities among BCL-2 members in tonsillar and leukemic B-cell subsets assessed by BH3-mimetic profiling

Abstract

For successful treatment of malignant B-cells it is crucial to understand intrinsic survival requirements in relation to their normal progenitors. Long-lived humoral immunity as well as most B-cell malignancies, originate in the germinal center (GC). Murine GC B-cells depend on pro-survival protein MCL-1, but not BCL-XL. In contrast, naive and memory B-cells depend on BCL-2, but not BCL-XL or MCL-1. For human B-cell subsets, the functional relationships among BCL-2 members are unclear, and also if and how they shift after malignant transformation. We here dissect these aspects in human tonsil and primary leukemia (CLL) cells by single and combined treatment with novel, highly specific BH3-mimetics. We found that MCL-1 expression in GC B-cells is regulated post-translationally and its importance is highlighted by preferential binding to pro-apoptotic BIM. In contrast, BCL-XL is transcriptionally induced and binds solely to weak sensitizer BIK, potentially explaining why BCL-XL is not required for GC B-cell survival. Using novel BH3-mimetics, we found that naive and memory B-cells depend on BCL-2, GC cells predominantly on MCL-1, whereas plasma cells need both BCL-XL and MCL-1 for survival. CLL cells switch from highly sensitive for BCL-2 inhibition to resistant after CD40-stimulation. However, combined inhibition of BCL-2, plus BCL-XL or MCL-1 effectively kills these cells, thus exposing a weakness that may be therapeutically useful. These general principles offer important clues for designing treatment strategies for B-cell malignancies.

Figure 1

Differential gene expression of BCL-2 family members in primary human B cells. (a) Gating strategy for isolation of naive B cells (Bn, IgD⁺CD38⁻), memory B cells (mem, IgD⁻CD38⁻), plasma cells (PC, IgD⁻CD38⁺⁺) and germinal center B cells (GC, IgD⁻CD38⁺) from the human tonsil after gating on CD19⁺CD3⁻ B cells. GC B cells were further subdivided in either centroblast (CB, CXCR4⁺FSC-A^+/−) or centrocyte (CC, CXCR4⁻FSC-A⁻) cells (right panel). (b) Expression profiling by RT-MLPA of human B cells purified directly ex vivo as shown in a. Gene induction of pro- and anti-apoptotic molecules is represented in a heat-map after log2 transformation of expression levels, relative to averaged values of naive B cells. Data is shown from four individual patients (three for memory B cells). (c) Averages of values shown in (b) relative to naive B cells. (d) Real time qPCR of indicated BCL-2 family members in purified cells as shown in a, corrected for expression of household gene Hprt and relative to naive B cells. Data are average of three (BCLX, BFL1) or five (BCL2, MCL1) experiments with s.e.m. Statistics were calculated in relation to Bn cells. *P⩽0.05, **P⩽0.01

Figure 2

Differential protein expression of BCL-2 family members in primary human B cells. (a) Western blot analysis for indicated BCL-2 family members in tonsil B cells, fluorescence-activated cell sorting (FACS)-purified as shown in Figure 1a. Data is representative of two (BFL-1, BIK and BIM) to four (BCL-2, MCL-1 and BCL-XL) independent experiments. (b) Quantified western blots shown in a corrected for expression of Actin and relative to naive B cells. BFL-1 expression could not be quantified due to the low expression level. Data are average values of three to four different experiments with S.E.M. (c) Fluorescence microscopy image of a tonsil section showing MCL-1 protein staining in a CD20⁺ GC in the tonsil. Scale bar, 100 μm in overview image (left panel) and 20 μm in selection (right panel). Images are representative of three individual experiments. Statistics were calculated in relation to Bn cells. *P⩽0.05, **P⩽0.01

Figure 3

MCL-1 protein is stabilized in GC B cells. (a) Ratios of protein (average of quantified western blot experiments shown in Figure 2b, with n=2–4) over mRNA (average of real time qPCR experiments shown in Figure 1d, with n=3–5) in B-cell subsets for MCL-1, BCL-2 and BCL-XL expressed in arbitrary units (a.u.) (b) Western blot analysis on FACS-purified naive or GC B cells for MCL-1 and Actin after culture for 0, 2 or 4 h (h) with cycloheximide (CHX), an inhibitor of protein synthesis. Data are representative of two individual experiments. (c) Real time qPCR of known MCL-1-specific ubiquitin ligases Mule (HUWE1), FBW7 (FBXW7) and βTrCP1 (BTRC) in purified cells as shown in Figure 1a, corrected for expression of household gene Hprt and relative to naive B cells. Data are average of five experiments with S.E.M. Statistics were calculated in relation to Bn cells. (d) Western blot analysis on MACS-enriched GC B cells for MCL-1 and Actin after culture for 0, 2 or 4 h (h) with cycloheximide (CHX), with or without PP2A-inhibitor okadaic acid (OA). Cells in (b) and (d) were cultured in standard tissue culture medium (Iscove's modified Dulbecco's media) supplemented with 10% (v/v) heat-inactivated fetal calf serum and antibiotics, but without additional cytokines. *P⩽0.05, **P⩽0.01

Figure 4

MCL-1 associates with BIM and BCL-XL associates with BIK in tonsil B cells. (a) Western blots after IP experiments in total B cells purified from the tonsil. Shown are the indicated proteins in the lysate after IP (cleared lysate) or the indicated proteins that were pulled down (IP). Background bands in the IP fraction were visualized by incubation with only the labeled secondary antibody and are indicated by (*). The experiment shown is representative of four individual experiments. (b) IP as in (a), but either with or without treatment of 30 μM A-1210477 for 4 h at 37 ºC. Experiment is representative of two individual experiments. (c) IP as in (a), but either with or without treatment of 10 μM WEHI-539 for 4 h at 37 °C. Experiment is representative of two individual experiments

Figure 5

Differential sensitivity of healthy and leukemic B cells upon inhibition of MCL-1, BCL-XL or BCL-2. (a) Total B cells purified from the tonsil were incubated with MCL-1-specific inhibitor A-1210477, BCL-XL-specific inhibitor WEHI-539 or BCL-2-specific inhibitor ABT-199 for 24 h at 37 °C with indicated drug concentrations (1 nM–10 μM for WEHI-539 and ABT-199, 50 nM–50 μM for A-1210477). B-cell populations (naive B cells; Bn, GC B cells; GC, PC or memory B cells; mem) were discriminated using antibodies recognizing IgD and CD38 as shown in Figure 1a. Specific apoptosis was calculated by measuring the altered percentage of TOPRO3⁻ (live) cells within indicated B-cell populations, compared with untreated cells. Data are average values of four different patient samples with S.E.M. and is representative of two independent experiments. Statistics were calculated in relation to Bn cells. (b) Experiment as in a, using primary CLL cells co-cultured with 3T3 cells without (unstimulated) or with (stimulated) expression of CD40L (3T40) in the presence of indicated drug concentrations (1 nM–10 μM). Data are average values of three different patient samples with S.E.M. and is representative of two independent experiments. Statistics were calculated in relation to unstimulated CLL cells. (c) Evaluation of combination effects of A-1210477 (5000 nM), WEHI-539 (5000 nM) or ABT-199 (1000 nM) on stimulated CLL cells. Experiment performed as in (b) with CLL cells co-cultured with 3T40 cells, shown are average values of nine different patient samples with S.E.M. Statistics were calculated in relation to single treatment with indicated drug. *P⩽0.05, **P⩽0.01

Figure 6

Model of expression and dependence on BCL-2 family proteins. Schematic representation of the dependence of naive B cells, GC B cells, PCs or memory B cells on pro-survival proteins BCL-2, BCL-XL or MCL-1 (black bold). Pro-apoptotic proteins that associate with indicated pro-survival proteins are show in gray. Interaction between BCL-XL and BIK in GC B cells (between brackets) seems not involved in cell survival. Arrows indicate possible routes of differentiation after activation of naive B cells