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Decreased sensitivity of 17p-deleted chronic lymphocytic leukemia cells to a small molecule BCL-2 antagonist ABT-737†
Article first published online: 14 JUL 2011
Copyright © 2011 American Cancer Society
Volume 118, Issue 4, pages 1023–1031, 15 February 2012
How to Cite
Kojima, K., Duvvuri, S., Ruvolo, V., Samaniego, F., Younes, A. and Andreeff, M. (2012), Decreased sensitivity of 17p-deleted chronic lymphocytic leukemia cells to a small molecule BCL-2 antagonist ABT-737. Cancer, 118: 1023–1031. doi: 10.1002/cncr.26360
We thank Teresa McQueen, Duncan Mak, and Twee Tsao for valuable technical help. We also acknowledge Dr. Jeffrey Medeiros at the Lymphoma Specialized Programs of Research Excellence (SPORE) sample core laboratory for providing samples.
- Issue published online: 3 FEB 2012
- Article first published online: 14 JUL 2011
- Manuscript Accepted: 26 MAY 2011
- Manuscript Revised: 25 MAY 2011
- Manuscript Received: 29 MAR 2011
- chronic lymphocytic leukemia;
- B-cell chronic lymphocytic leukemia/lymphoma 2;
- BCL-2-associated X protein;
- 17p deletion
Despite the high complete response rates achieved with fludarabine-based regimens, relapse is inevitable in chronic lymphocytic leukemia (CLL). Relapsed patients often acquire deletions of the short arm of chromosome 17 (del[17p]), which are closely associated with tumor protein 53 (TP53) mutations. Wild-type p53 up-regulates and activates B-cell CLL/lymphoma 2 (BCL-2)-associated X protein (BAX), and it down-regulates and inactivates BCL-2. The small-molecule BCL-2 inhibitor ABT-737 induces apoptosis in a BAX-dependent and BCL-2 homologous antagonist-killer (BAK)-dependent manner. The role of p53 in sensitivity of CLL cells to BCL-2 inhibition has not been extensively investigated.
The authors investigated the association of del(17p) with ABT-737 sensitivity in CLL cells from 50 patients. Stable p53 and BAX knockdown cells were used for mechanistic studies.
CLL cells with del(17p) were less sensitive to ABT-737-induced BAX activation and apoptosis than CLL cells without del(17p) (39% ± 7.3% vs 63.7% ± 2.9% [specific annexin V induction]; P < .01). A positive correlation between the degrees of apoptosis induced by ABT-737 and by the p53-activating binding protein homolog murine double minute (MDM2) antagonist nutlin-3a (correlation coefficient [r] = 0.75; P < .0001) was observed. CLL cells with del(17p) expressed lower levels of BAX than those without del(17p) (0.67 ± 0.12 vs 1.27 ± 0.10 in relative protein expression levels; P < .01). Knockdown of p53 or BAX in leukemia cells resulted in decreased apoptosis induced by ABT-737.
The current data indicated that p53 dysfunction may lead to decreased apoptosis induction by ABT-737. Cancer 2012;. © 2011 American Cancer Society.
Despite the high complete response rates achieved with fludarabine-based regimens, relapse is inevitable in chronic lymphocytic leukemia (CLL).1-3 Relapsed patients often acquire deletions in the short arm of chromosome 17 (del[17p]), which correspond to loss of the tumor suppressor TP53, commonly accompanied by TP53 mutation of the remaining allele.4 Relapsed CLL patients with del(17p) are resistant to most standard therapies, and patients with CLL who have del(17p) do not even benefit from first-line fludarabine-based regimens.2, 3
ABT-737, a small-molecule inhibitor of B-cell CLL/lymphoma 2 (BCL-2), BCL-like-1 long form (BCL-XL), and BCL-2 like (BCL-W), induces apoptosis in a BCL-2-associated X protein (BAX)-dependent and BCL-2 homologous antagonist killer (BAK)-dependent manner.5, 6 Primary CLL cells express high BCL-2 levels, and it is noteworthy that these cells depend on BCL-2 for survival.7, 8 Studies of sensitivity to ABT-737 have in common 2 results: 1) cells with BCL-2 primed with high levels of activators like Bcl-2-interacting mediator of cell death (BIM) tend to be sensitive to ABT-737, and 2) high expression of myeloid cell leukemia sequence 1 (BCL-2-related) (MCL-1) or the prosurvival Bcl-2 family member BFL-1/A1 can result in decreased sensitivity to ABT-737.8-13 It has been reported that BAX levels negatively correlate with sensitivity to ABT-737 in diffuse large B-cell lymphoma cell lines.9 However, it remains unknown whether cellular BAX levels in primary neoplastic cells affect susceptibility to BCL-2 inhibition.
The balance of antiapoptotic BCL-2 and proapoptotic BAX dictates cellular fate.14 Wild-type p53, but not mutant p53, up-regulates and activates BAX and down-regulates and inactivates BCL-2.15-17 The role of p53 in sensitivity of leukemic cells to BCL-2 inhibition is unknown. Here, we investigated the potential therapeutic utility of the BCL-2 antagonists in CLL, focusing on the possible association of defective p53 with sensitivity to ABT-737.
MATERIALS AND METHODS
ABT-737 was synthesized at the University of Texas M.D. Anderson Cancer Center based on the previously published structure.5 Nutlin-3a was kindly provided by Dr. Lyubomir Vassilev (Hoffmann-La Roche, Inc., New York, NY). The caspase inhibitor Z-VAD-FMK was purchased from Axxora (San Diego, Calif).
Clinical Data and Cell Culture
Heparinized peripheral blood samples were obtained from patients with CLL after we obtained informed consent through the Lymphoma Specialized Programs of Research Excellence (SPORE) grant sample core, according to institutional guidelines and the Declaration of Helsinki. Lymphocytes were purified and cultured in minimal essential medium-α medium supplemented with 10% fetal bovine serum at a density of 2.5 × 106 cell/mL. We treated cells with 10 nM ABT-737 based on a previous study in which it was determined that the effective concentration inducing 50% killing as measured by positive annexin V expression was 27 nM at 4 hours and 4.5 nM at 48 hours in CLL samples.8 Immunoglobulin heavy-chain variable gene (IgVH) somatic mutation status was determined as described previously.18 Somatic hypermutation in the IgVH heavy-chain variable locus was classified as absent when ≥98% homology to the germline sequence was measured. The mutation status was designated as unmutated if <2% mutations (>98% homology to germline sequences) or as mutated if ≥2% mutations (98% homology to germline sequences) were detected compared with the germline sequences in VBASE2 (Intergenomics, Bioinformatics Competence Center, Braunschweig, Germany; http://www.vbase2.org [accessed October 1, 2010]).19 Interphase fluorescence in situ hybridization was performed to detect 13q14, 11q22, 17p13, and trisomy 12. Patient samples were classified as positive for ζ-chain-associated protein kinase 70 (ZAP-70) when at least 20% of cells were positive and positive for cluster of differentiation 38 (CD38 [adenosine diphosphate ribose hydrolase]) when at least 30% of cells were positive. Human acute lymphoblastic leukemia REH cells (American Type Culture Collection, Rockville, Md) and human acute myeloid leukemia MOLM-13 cells (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were maintained in RPMI 1640 medium containing 10% fetal calf serum. REH and MOLM-13 cells were transduced with retroviruses encoding either p53-specific short-hairpin RNA (shRNA) (nucleotides 611-629; Genbank no. NM000546), BAX-specific shRNA (nucleotides 268-286; Genbank no. NM138761.3), or negative control shRNA, and stable shRNA-expressing cells were generated.20
Transfection of BAX Small-Interfering RNA
Five primary CLL samples were transfected with BAX small-interfering RNA (siRNA) oligonucleotides (SignalSilence Bax SiRNA; Cell Signaling Technology, Beverly, Mass) by the Amaxa electroporator Nucleofector II, using the Human B Cell Nucleofector Kit according to the manufacturer's instructions (Amaxa Biosystem, Cologne, Germany). Nonspecific, scrambled siRNA served as negative control. Twenty-four hours after transfection, BAX and β-actin expression levels were analyzed by Western blot analysis.
The following antibodies were used: mouse monoclonal anti-p53 (Santa Cruz Biotechnology, Santa Cruz, Calif), mouse monoclonal anti-BCL-2 (Dako Cytomation, Carpinteria, Calif), rabbit polyclonal anti-BAX (BD Biosciences, San Jose, Calif), mouse monoclonal anti-BAX (Trevigen, Gaithersburg, Md), mouse monoclonal anti-MCL-1 (BD Biosciences); rabbit polyclonal anti-p53-up-regulated modulator of apoptosis (anti-PUMA) (EMD Biosciences, San Diego, Calif), mouse monoclonal antiphorbol-12-myristate-13-acetate-induced protein 1 (anti-NOXA) (EMD Biosciences), and mouse monoclonal anti-β-actin (Sigma, St. Louis, Mo).
Apoptosis was measured by using the annexin V binding assay. The extent of apoptosis was quantified as percentage of annexin V-positive cells, and the extent of drug-specific apoptosis was calculated by the following formula: percentage of specific apoptosis = (test − control) × 100/(100 − control). In the formula, the numerator is the actual amount of killing that occurred, and the denominator is the potential amount of killing that could occur.
Quantification of Intracellular Proteins by Flow Cytometry
For intracellular p53 detection, cells were fixed with 2% paraformaldehyde, permeabilized with 100% ice-cold methanol, and incubated for 1 hour at 4°C with antibody against p53 or its isotypic control (BD Biosciences). Involvement of BAX conformational change was analyzed by means of an antibody directed against the NH2-terminal region of BAX (YTH-6A7; Trevigen).21
Western Blot Analysis
Equal amounts of protein lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% gel) for 2 hours at 80 V. Proteins were transferred onto nitrocellulose membrane, immunoblotted with primary antibodies followed by infrared secondary antibodies (LI-COR Biosciences, Lincoln, Neb), and detected by an Odyssey infrared imaging system (LI-COR Biosciences). An anti-β-actin blot was used in parallel as a loading control. Visualized blots were analyzed with the ImageJ 1.44 software package (National Institutes of Health, Bethesda, Md).
Real-Time Quantitative Polymerase Chain Reaction
RNA was prepared from cells using an RNeasy Mini Kit (Qiagen, Valencia, Calif), and first-strand combinational DNA was generated using random hexamers (SuperScript III First-Strand Synthesis SuperMix; Invitrogen, Carlsbad, Calif) from 1 μg total RNA. Messenger RNA (mRNA) expression levels of MCL-1, BAX, PUMA, NOXA, p21, FAS (tumor necrosis receptor superfamily, member 6 (FAS), apoptotic protease activating factor 1 (APAF1), tumor necrosis factor-related apoptosis-inducing ligand death receptor 5 (DR5), murine double minute (MDM2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were quantified using Taq human gene expression assays (MCL-1, Hs03043899_m1; BAX, Hs00180269_m1; PUMA, Hs00248075_m1; NOXA, Hs00382168_m1; p21, Hs00171132_m1; FAS, Hs00163653_m1; APAF1, Hs00185508_m1; DR5, Hs00366272_m1; MDM2, Hs00242813_m1; GAPDH, Hs99999905_m1; Applied Biosystems, Foster City, Calif) on a 7900HT Fast Real-Time polymerase chain reaction (PCR) System. According to the manufacturer's guidelines, the level of expression was calculated based on the PCR cycle number (Ct) at which the exponential growth in fluorescence from the probe passes a certain threshold value. For each sample, relative gene expression level was determined by subtracting the Ct value of the housekeeping gene GAPDH to the Ct value of the target gene (ΔCt = Cttarget gene − Ct18S ribosomal RNA). Relative quantification (fold change) between different samples (eg, treated vs control) was then determined according to the 2−ΔΔCt method (ΔΔCt = ΔCttreated sample − Ctcontrol sample).
TP53 mutation analysis
PCR for p53 gene expression followed by direct sequencing was performed as described previously.21
Statistical analysis was performed using the 2-tailed Student t test, the Mann-Whitney U test, and Pearson correlation. Results were considered statistically significant at P values < .01. Unless otherwise indicated, average values are expressed as the mean ± standard error of the mean.
CLL Cells With Del(17p) Are Resistant to Nutlin-3a and Less Sensitive to ABT-737
We examined the proapoptotic effects of the MDM2 inhibitor nutlin-3a and the BCL-2 antagonist ABT-737 on primary cells from 50 patients with CLL. Table 1 summarizes the clinical characteristics of the patients. Cells were exposed to 0.1% dimethyl sulfoxide, 10 μM nutlin-3a, or 10 nM ABT-737 for 48 hours. The status of p53 is the major determinant of response to nutlin-3a in CLL21, 22; and as expected, CLL cells with del(17p) (n = 8) were resistant to nutlin-3a versus CLL cells without del(17p) (n = 42; 5.5% ± 1.0% vs 59% ± 3% specific apoptosis; P < .01). It is noteworthy that CLL cells with del(17p) also were less sensitive to ABT-737 (39% ± 7.3% vs 63.7% ± 2.9% specific apoptosis; P < .01). We also observed that the extent of apoptosis induced by ABT-737 correlated with that induced by nutlin-3a (r = 0.75; P < .0001) (Fig. 1a). Sensitivity to ABT-737 was independent of other prognostic parameters, including IgVH status, 11q deletion, previous treatments, and CD38 or ZAP-70 positivity (Table 2). We also investigated whether these prognostic parameters were associated with sensitivity to nutlin-3a. The presence of del(17p) was the only factor that affected nutlin-3a sensitivity (Table 3). The involvement of BAX conformational change was analyzed in 21 primary samples (7 samples with del[17p] and 14 samples without del[17p] as controls). We could not obtain the eighth del(17p) sample, because the patient did not return to the hospital during the study period. Z-VAD-FMK (100 μM) was used to inhibit caspase-promoted conformational change in BAX. Increases in the percentage of conformationally active, BAX-positive cells were significantly smaller in 17p-deleted samples than in samples without del(17p) (39% ± 6.2% vs 64.6% ± 3.7% specific apoptosis; P < .01) (Fig. 1b). There was a significant, positive correlation between the percentage of specific annexin V induction and that of specific BAX activation (Fig. 1c) (r = 0.72; P < .01), suggesting a direct role of BAX activation in apoptosis induction by ABT-737.
|Characteristic||No. of Patients|
|Median age (range), y||67 (45-88)|
|Positive: ≥20% (positive)||19|
|Median β2 microglobulin (range), mg/mLa||3.7 (1.9-15.0)|
|Sex||Men vs women||.089|
|Age, y||0-65 vs >65||.749|
|Rai stage||0-2 vs 3-4||.197|
|Prior treatment||Yes vs no||.484|
|Deletion 17p||Yes vs no||.006|
|Deletion 11q||Yes vs no||.857|
|Deletion 13q||Yes vs no||.868|
|CD38||Positive vs negative||.060|
|IgVH status||Mutated vs not mutated||.197|
|ZAP70||Positive vs negative||.768|
|β2-microglobulin||0-4 mg/mL vs >4 mg/mL||.115|
|Sex||Men vs women||.759|
|Age, y||0-65 vs > 65||.515|
|Rai stage||0-2 vs 3-4||.374|
|Prior treatment||Yes vs no||.627|
|Deletion 17p||Yes vs no||<.0001|
|Deletion 11q||Yes vs no||.839|
|Deletion 13q||Yes vs no||.293|
|CD38||Positive vs negative||235|
|IgVH status||Mutated vs not mutated||.939|
|ZAP70||Positive vs negative||.768|
|β2 microglobulin||0-4 mg/mL vs >4 mg/mL||.181|
CLL Cells With Del(17p) Have Lower BAX Levels Than Cells Without Del(17p)
To investigate the contribution of p53-influenced BCL-2 family members to ABT-737-induced apoptosis, we analyzed expression levels of BCL-2, MCL-1, BAX, PUMA, and NOXA. BAX levels were lower in 17p-deleted samples than in samples without del(17p) (Fig. 2a). Levels of BCL-2, MCL-1, PUMA, and NOXA were independent of del(17p). Because MCL-1 protein levels, which reportedly determine cell sensitivity to ABT-737, were low in CLL cells (Fig. 2a),8 real-time quantitative PCR was used to measure MCL-1 transcription. MCL-1 mRNA levels were not affected significantly by the presence or absence of del(17p) (Fig. 2b).
ABT-737 Does Not Induce p53 Responses in CLL Cells
To exclude the possibility that ABT-737 induces p53 responses in CLL cells, 3 patient samples with known wild-type p53 were treated with 10 nM ABT-737 for 6 hours, and cellular p53 levels and mRNA levels of p53 targets were determined. Figure 3a,b indicated that ABT-737 treatment did not increase p53 levels or induce p53-related genes BAX, PUMA, cyclin-dependent kinase inhibitor 1A (CDKN1A [p21]), FAS, or MDM2.
p53 Knockdown in Leukemia Cells Leads to Reduced BAX Levels and Decreased ABT-737 Sensitivity
Because there are no CLL cell lines available, we used the REH and MOLM-13 cell lines for knockdown experiments. REH cells with p53 knockdown displayed reduced levels of BAX and were less susceptible to ABT-737-induced apoptosis than parental cells or cells that expressed negative control shRNA (Fig. 4a), suggesting that functional p53 expression may be required for full induction of apoptosis by ABT-737. Desensitization of leukemia cells to ABT-737 also was observed in p53-knockdown MOLM-13 cells, in which steady-state Bax mRNA levels were reduced by 30.2% compared with negative control shRNA-expressing cells (Fig. 4b,c). To determine whether BAX levels affect leukemia cell sensitivity to ABT-737, BAX levels were reduced in REH and MOLM-13 cells. Figure 5 indicates that BAX knockdown cells were less sensitive to ABT-737-induced apoptosis than cells that expressed scrambled shRNA in both cell lines (P < .01), suggesting that BAX levels affect cell susceptibility to ABT-737 in leukemia cells. Gene silencing by RNA interference could not be accomplished in primary CLL cells because of poor transfection efficiency and high cellular toxicity.
We, here report that CLL cells with del(17p), an adverse prognostic factor closely associated with TP53 mutations,23 are less sensitive to ABT-737 than those without del(17p). We also observed a positive correlation between apoptosis induced by ABT-737 and nutlin-3a. Because sensitivity to nutlin-3a largely relies on p53 status in CLL, our data suggest that BCL-2 inhibition may require functional p53 signaling to fully induce apoptosis. Other prognostic markers or clinical parameters did not affect ABT-737 sensitivity, in agreement with previous studies.8, 13 An association of p53 status with ABT-737 sensitivity has not been investigated extensively in CLL. Mason et al, in a series of 30 patients with CLL, reported that 3 patients who had del(17p) displayed sensitivity to ABT-737 similar to that displayed by patients without del(17p).24 Conversely, Balakrishnan et al, in a series of 32 patients, reported a single patient with del(17p) who had limited apoptosis in response to ABT-737.25 Because del(17p) and p53 mutations are closely associated with fludarabine resistance and disease progression,26 the hypothesis that p53 status affects clinical response to ABT-737 could be validated in patients with refractory CLL undergoing clinical trials with ABT-737.
Although it has been reported that the expression of BAX is up-regulated at the transcriptional level by p53,27 CLL cells with del(17p) expressed BAX to some extent, possibly because nondeleted or nonmutated alleles would maintain low levels of BAX. In addition, it has been reported that BAX can be post-translationally regulated by BCL-2,28 which is overexpressed in CLL.7, 8 Furthermore, TA-p73, a p53 family protein, reportedly transcriptionally up-regulated BAX in the absence of p53.29 Future studies are required to clarify whether p53-independent regulators of BAX contribute to ABT-737 resistance. Bax expression may not be the only determinant of reduced sensitivity to ABT-737 in CLL cells with del(17p). Because steady-state mRNA expression levels of “apoptotic” p53 target genes were modestly but widely repressed in association with stable p53 knockdown (Fig. 4c), cumulative changes in aberrantly expressed apoptosis-regulating proteins may directly or indirectly reduce ABT-737 sensitivity in patients with defective p53.
In conclusion, our data indicate that p53 dysfunction may lead to decreased apoptosis induction by ABT-737 in CLL cells and that BAX levels may dictate sensitivity to BCL-2 inhibition. Recently, Letai et al proposed 3 classes of apoptotic blocks used to maintain cancer survival against BCL-2 inhibition.30 Our case may represent the class B block, in which cancer cells eliminate the effector arm of the mitochondrial apoptotic pathway by reducing or eliminating BAX and BAK. Because the conventional agents used in CLL treatment, including alkylating agents, fludarabine, and rituximab, kill CLL cells through the mitochondrial apoptotic pathway, decreased BAX in del(17p) cells may contribute to resistance to these agents.3 Patients with defective p53 may be considered for alternative treatment approaches, including early stem cell transplantation,31 if BCL-2 inhibitors and other promising agents in clinical trials do not provide sufficient clinical responses in this specific subgroup.
This work was supported in part by grants from the National Institutes of Health Lymphoma Specialized Programs of Research Excellence (SPORE) (CA136411), P01 “The Therapy of AML” (CA55164), Leukemia SPORE (CA100632), and the Paul and Mary Haas Chair in Genetics (M. Andreeff).
CONFLICT OF INTEREST DISCLOSURES
The authors made no disclosures.
- 26Detailed analysis of p53 pathway defects in fludarabine-refractory chronic lymphocytic leukemia (CLL): dissecting the contribution of 17p deletion, TP53 mutation, p53-p21 dysfunction, and miR34a in a prospective clinical trial. Blood. 2009; 114: 2589-2597., , , et al.