BCL-2 dependence and ABT-737 sensitivity in acute lymphoblastic leukemia

Abstract

Cancer cells acquire disruptions in normal signal transduction pathways and homeostatic mechanisms that would trigger apoptosis in normal cells. These abnormalities include genomic instability, oncogene activation, and growth factor independent proliferation. Therefore, cancer cells likely require a block in apoptosis in order to survive. Overexpression of the antiapoptotic protein BCL-2 provides a block in apoptosis that is frequently observed in cancer cells. We have developed methods for the detection and analysis of BCL-2 dependence and here apply them to acute lymphoblastic leukemia (ALL). BH3 profiling, a mitochondrial assay that classifies blocks in the intrinsic apoptotic pathway, indicated a dependence on BCL-2 of both ALL cell lines and primary samples. This dependence predicted that BCL-2 would be complexed with select pro-death BH3 family proteins, a prediction confirmed by the isolation of BCL-2 complexes with BIM. Furthermore, the BH3 profiling and protein analysis predicted that ALL cell lines and primary cells would be sensitive to ABT-737 as a single agent. Finally, BH3 profiling and protein studies accurately predicted a relative degree of sensitivity to BCL-2 antagonism in cell lines. The ALL cells studied exhibit BCL-2 dependence, supporting clinical trials of BCL-2 antagonists in ALL as single agents or combination therapies.

Figure 1

BH3 profiling determines BCL-2 dependence of ALL cell lines. (A) Summary of binding chart between BH3-only peptides and different antiapoptotic BCL-2 family members, adapted from Certo et al. (B-E) For BH3 profiling, at least 5 × 10⁸ cells were used to isolate mitochondria by differential centrifugation for each cell line. Mitochondria were then treated with 100 μM BH3 peptides for 35 minutes at room temperature and cytochrome c release assessed by ELISA. Profiles were put in order of most to least primed. (B) RS4;11 cells. (C) SEM-k2 cells. (D) 697 cells. (E) CEM-c1 cells. Values are representative of the mean of 3 independent experiments (+ SD).

Figure 2

Response of ALL cell lines demonstrates differential sensitivity to ABT-737 treatment. (A) 10⁶ cells were treated in triplicate with increasing amounts of ABT-737. Apoptosis was assessed by counting annexin-V–positive cells by FACS analysis. (B) EC50 values were plotted against the BAD BH3 peptide cytochrome c release values for each line.

Figure 3

Protein analysis in ALL cell lines reveals BCL-2, BIM, and BID are complexed. CHAPS lysates of the 4 ALL cell lines were used for protein experiments. (A) Western blot analysis of lysate (20 μg) reveals different expression of BCL-2, MCL-1, BIM, and BID among the cell lines. (B) Quantification of proteins bands by densitometry verifies that BCL-2 and BIM protein expression levels follow the rank order of whole-cell ABT-737 sensitivity and BAD-induced cytochrome c release: RS4;11 > SEM-k2 > 697 > CEMc-1. (C) Anti–BCL-2 immunoprecipitation of ALL cells lines (50 μg). Immunoprecipitation pellet (i.p.) and 10% of total lysate used for immunoprecipitation (input) were loaded. BIM and BID are complexed to BCL-2 in amounts proportional to whole-cell amounts.

Figure 4

ABT-737 induces apoptosis through the mitochondrial pathway in ALL cells. Cells were treated for 24 hours with 100 nM ABT-737 or DMSO, or pretreated for 1 hour with 200 μM ZVAD.fmk prior to addition of ABT-737. (A) PARP cleavage correlates with cell death, as analyzed by annexin-V staining and reported as percent alive. (B) Caspase-9 activation was measurable by a fluorometric assay. The amount of annexin-V–negative cells was recorded just prior to harvesting cells (percent alive). Note that in both panels A and B the amount of ABT-737 used was not sufficient to cause significant apoptosis in CEM-c1 cells, which is consistent with the results reported in Figure 2.

Figure 5

BH3 profiling of primary ALL cells also shows a BCL-2 pattern. 10⁹ cells from primary sample L1 (A) or 3.5 × 10⁸ cells from primary sample L8 (B) were subjected to mechanical disruption and differential centrifugation. Isolated mitochondria were brought to a final concentration of 0.5 mg/mL and incubated with 100 μM peptides for 35 minutes at room temperature. Mitochondria pellet and supernatant were separated and analyzed using a cytochrome c ELISA kit. (C) Primary ALL cells were plated with or without stromal cells (Mihara, 2003 #654) with increasing concentrations of ABT-737. Apoptosis was assessed at 24 and 48 hours by counting annexin-V–positive cells by FACS analysis.

Figure 6

Protein analysis of primary ALL cells is consistent with whole-cell ABT-737 response and BH3 profiles. (A) Western blot analysis of CHAPS lysates (20 μg) shows detectable expression of BCL-2, MCL-1, BIM, and BID among primary ALL cells. (B) Purified GST-BCL-XL and GST-BCL-2 (1 and 3 ng) were run next to 5 independent ALL lysates (10 μg) to quantitate BCL-XL (top panel) and BCL-2 (middle panel). Primary ALL lysates with better equal loading reveal clearer BIM levels among samples (bottom panel). (C) BCL-2, BIM, and BID are also complexed in primary ALL cells. Anti–BCL-2 immunoprecipitation of ALL primary cells (50 μg). Immunoprecipitation pellet (i.p.) and 10% of total lysate used for immunoprecipitation (input) were loaded. BIM and BID are complexed to BCL-2 in amounts proportional to whole-cell amounts.