Published online 8 October 2009
Haematologica, Vol 94, Issue 12, 1698-1707 doi:10.3324/haematol.2008.004028
Copyright © 2009 by Ferrata Storti Foundation

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Chronic Lymphocytic Leukemia

Akt inhibitors induce apoptosis in chronic lymphocytic leukemia cells

Mercè de Frias¹, Daniel Iglesias-Serret¹, Ana M. Cosialls¹, Llorenç Coll-Mulet¹, Antonio F. Santidrián¹, Diana M. González-Gironès¹, Esmeralda de la Banda², Gabriel Pons¹, Joan Gil¹

¹ Departament de Ciències Fisiològiques II, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, L’Hospitalet de Llobregat
² Servei d’Hematologia, IDIBELL–Hospital de Bellvitge, L’Hospitalet de Llobregat, Spain

Correspondence: Joan Gil, Ph.D., Departament de Ciències Fisiològiques II, IDIBELL-Universitat de Barcelona, Campus de Bellvitge, Pavelló de Govern, 4^a planta, E-08907 L'Hospitalet de Llobregat, Barcelona, Spain. E-mail: jgil{at}idibell.cat

TOP ABSTRACT Introduction Design and Methods Results Discussion References

 
Background: The phosphatidylinositol-3-kinase/Akt pathway has been described to be critical in the survival of chronic lymphocytic leukemia cells. In this study we analyzed the effect of two selective chemical inhibitors of Akt (Akti-1/2 and A-443654) on the survival of chronic lymphocytic leukemia cells.

Design and Methods: Using cytometry we studied the cytotoxic effects of Akt inhibitors on peripheral B and T lymphocytes from patients with chronic lymphocytic leukemia and from healthy donors. We studied the changes induced by Akti-1/2 and A-443654 at the mRNA level by performing reverse transcriptase multiplex ligation–dependent probe amplification. We also studied the changes induced by both Akt inhibitors in some BCL-2 protein family members on chronic lymphocytic leukemia cells by western blotting. Moreover, we analyzed the cytotoxic effect of Akt inhibitors in patients’ cells with deleted/mutated TP53.

Results: Both inhibitors induced apoptosis in chronic lymphocytic leukemia cells in a dose-dependent manner. Moreover, B cells from patients with chronic lymphocytic leukemia were more sensitive to Akt inhibitors than T cells from leukemic patients, and B or T cells from healthy donors. Survival factors for chronic lymphocytic leukemia cells, such as interleukin-4 and stromal cell-derived factor-1, were not able to block the apoptosis induced by either Akt inhibitor. Akti-1/2 did not induce any change in the mRNA expression profile of genes involved in apoptosis, while A-443654 induced some changes, including an increase in NOXA and PUMA mRNA levels, suggesting the existence of additional targets for A-443654. Both inhibitors induced an increase in PUMA and NOXA protein levels, and a decrease in MCL-1 protein level. Moreover, Akti-1/2 and A-443654 induced apoptosis irrespective of TP53 status.

Conclusions: These results demonstrate that Akt inhibitors induce apoptosis of chronic lymphocytic leukemia cells and might be a new therapeutic option for the treatment of chronic lymphocytic leukemia.

Key words: Akt, chronic lymphocytic leukemia, apoptosis.

TOP ABSTRACT Introduction Design and Methods Results Discussion References

 
The phosphatidylinositol-3-kinase (PI3K) pathway has been described to be critical in the survival of chronic lymphocytic leukemia (CLL) cells.^1–11 PI3K phosphorylates the D-3 position of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-diphosphate. The cellular levels of PI3K products are controlled by the balance between PI3K activity and the phosphatase activity of PTEN (phosphatase and tensin homolog deleted on chromosome ten).¹² Interestingly, PTEN protein is reduced or not detected in 48% of patients with CLL.¹³

One of the most important targets of PI3K products is the serine-threonine kinase Akt, also known as protein kinase B (PKB).¹⁴ Akt resides in the cytosol in a low-activity conformation, and it is activated through recruitment to cell membranes by PI3K lipid products and phosphorylation at Thr308 and Ser473. Once Akt is activated, it is able to promote cell survival through phosphorylation and inactivation of key components in the apoptotic cascade. Akt substrates include the members of the Forkhead family of transcription factors¹⁵ and glycogen synthase kinase-3 (GSK-3),¹⁶ which are inhibited by Akt. Furthermore, GSK-3 inhibition induces the up-regulation of the anti-apoptotic protein MCL-1.¹⁶ Activation of the PI3K/Akt pathway in CLL cells induces the phosphorylation of the Forkhead family member FoxO3a and GSK-3, and an increase in MCL-1 protein, while inhibition of PI3K induces a loss of cell viability, dephosphorylation of FoxO3a and GSK-3, and a decrease in the level of MCL-1 protein.^3,4,11 Importantly, a stronger activation of Akt pathway has been related to a higher capacity for cell cycle progression in CLL cells from patients with progressive disease.¹⁷ All together, these studies suggest that Akt plays a prominent role in the survival of CLL; however, the effect of selective chemical Akt inhibitors on the survival of CLL cells has not been reported yet.

A-443654 is a potent, ATP competitive and reversible inhibitor of Akt catalyzed phosphorylation activity. It is a pan-Akt inhibitor and has equal potency against Akt1, Akt2 or Akt3.^18,19 Together with the decrease in phosphorylation of Akt targets, a concomitant increase in the phosphorylation of Ser473 and Thr308 Akt residues has been observed. This increase is PI3K-dependent, as demonstrated by the fact that incubating the cells with LY294002 blocks it.²⁰ Recently, it was reported that this inhibitor also inhibits other protein kinases, albeit with slightly lower potency, such as PKA, PRK2, MSK1 and DYRK1A.²¹ It was also reported that Akti-1/2, an ATP non-competitive Akt inhibitor, was a highly selective Akt inhibitor, blocking Akt1 and Akt2 but not Akt3 activity; the pleckstrin homology domain is required for the activity of this inhibitor.^22,23

In this study we examined the effect of these two Akt inhibitors on CLL cells.

TOP ABSTRACT Introduction Design and Methods Results Discussion References

 
Patients with chronic lymphocytic leukemia, healthy donors and cell isolation
Samples from patients with CLL (Table 1) or healthy donors were studied. CLL was diagnosed according to standard clinical and laboratory criteria. Blood samples were obtained from the Hospital de Bellvitge, L’Hospitalet de Llobregat, Spain. Written informed consent was obtained from all patients in accordance with the Hospital de Bellvitge Ethical Committee’s requirements.

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Table 1. Characteristics of the chronic lymphocytic leukemia patients studied.

Mononuclear cells from peripheral blood samples were isolated by centrifugation on a Ficoll-Hypaque (Seromed, Berlin, Germany) gradient and cryopreserved in liquid nitrogen in the presence of 10% dimethyl sulfoxide (Sigma-Aldrich, Steinheim, Germany), and are referred to as CLL cells throughout this article. B-cells (CD19⁺ cells) were purified from samples from three patients (n. 49, 50 and 51) by using the RosetteSep Human B Cell Enrichment Cocktail (StemCell Technologies, Vancouver, Canada). Blood samples or isolated mononuclear cells were incubated with RosetteSep Human B Cell Enrichment Cocktail at 50 µL/mL for 20 min prior to centrifugation on a Ficoll-Hypaque, according to the manufacturer’s protocol.

Immunological and genetic analyses
CD38 and ZAP-70 were determined by flow cytometry in fixed cells with conjugated antibodies (PE clone HB7, Becton Dickinson, Franklin Lakes, NJ, USA and Alexa-Fluor 488, Caltag Laboratories, Burlingame, CA, USA, respectively). CD19 was determined by flow cytometry with a conjugated antibody [phycoerythrin (PE)–conjugated anti-CD19; Becton Dickinson].

Genomic alterations were detected by fluorescence in situ hybridization (FISH). Fluorescent-labeled DNA probes were used in interphase cytogenetic analyses. Locus-specific probes (LSI P53/Spectrum-Orange, LSI ATM/Spectrum-Green, LSI 13S319/Spectrum-Orange, LSI 13q34/SpectrumAqua) were used to determine loss of these genetic regions within interphase nuclei. Trisomy 12 was detected in interphase nuclei using a chromosomal centromere enumeration probe (CEP) labeled with Spectrum-Green. These five probes are packaged together in a commercially available kit (Vysis Chronic Lymphocytic Leukemia Multicolor Kit) and were used in accordance with the manufacturer’s specifications. Cells were fixed with fresh fixative before placement onto slides. The probe mixture was applied directly to slides. These slides were denaturated at 74ºC for 2 min and incubated overnight at 37ºC. Slides were then washed with 0.4 x saline sodium citrate-0.3% nonidet P-40 (NP-40) at 73±1ºC for 2 min and 2 x saline sodium citrate-0.1% nonidet P-40 at room temperature for 1 min. 4’6-diamidino-2-phenylindole (DAPI) II counterstain was applied to the target area. Slides were stored at 20ºC in the dark. Two hundred nuclei were analyzed for each probe, using a NIKON fluorescent microscope. Cut-off levels used were 5% for CEP 12 and 7% for locus-specific probes. The karyotype of all samples was determined.

Reagents
Akti-1/2 (previously known as Akt-I-1/2) was purchased from Calbiochem-Novabiochem (San Diego, CA, USA), A-443654 was kindly provided by Abbott (North Chicago, IL, USA), recombinant human interleukin-4 (IL-4) and stromal cell-derived factor-1 (SDF-1) were purchased from Immunotools (Friesoythe, Germany), annexin V–fluorescein isothiocyanate and propidium iodide were from Bender MedSystems (Vienna, Austria). Nutlin-3a was provided by Hoffmann-La Roche. Z-VAD.fmk was purchased from Bachem (Bubendorf, Switzerland). Ethanol and RNase A were from Sigma-Aldrich.

Cell culture
Lymphocytes were cultured immediately after thawing or isolation in RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U penicillin and 100 ng/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. To avoid differences in cell viability due to the cell concentration, flow cytometry experiments were performed at a concentration of 1x10⁶ cells/mL, whereas the concentration used for reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) and the experiments to obtain cell extracts to perform western blotting was 2.5 to 3x10⁶ cells/mL.

Analysis of apoptosis by flow cytometry
Apoptosis was assessed by exposure of phosphatidylserine and membrane integrity. This was determined by annexin V-fluorescein isothiocyanate and propidium iodide double staining. Flow cytometric analysis was performed using FACSCalibur and CellQuest software (Becton Dickinson), as described previously.²⁴ Cell viability was measured as the percentage of annexin V and propidium iodide double-negative cells. The results of flow cytometric analysis of three representative samples are shown in Online Supplementary Figure S1A.

To analyze apoptosis in T cells and B cells from the samples, 5x10⁵ cells were incubated for 24 h with the indicated factors. Cells were then washed in annexin-binding buffer, and incubated in 50 µL annexin-binding buffer with allophycocyanin-conjugated anti-CD3 and phycoerythrin-conjugated anti-CD19 from Becton Dickinson, for 10 min in the dark. Cells were then diluted with annexin-binding buffer to a volume of 150 µL and incubated with 1 µL annexin V-fluorescein isothiocyanate for 15 min in the dark. Cells were analyzed using FACSCalibur and CellQuest software. Results of flow cytometric analysis of one CLL sample and one sample from a healthy donor are shown in Online Supplementary Figure S1B.

Apoptosis in T cells and B cells was also assessed by subdiploid DNA analysis. Briefly, 1x10⁶ cells were harvested, washed twice in phosphate-buffered saline containing 1% fetal bovine serum (PBS/1% FBS) and fixed in 70% ethanol. The cells were centrifuged, washed in PBS/1% FBS, and resuspended in 0.5 mL PBS/1% FBS, containing allophycocyanin–conjugated anti-CD3 or allophycocyanin–conjugated anti-CD19. Tubes were incubated for 20 min at room temperature in the dark and then propidium iodide (50 µg/mL) and RNase A (100 µg/mL) were added and incubated for 30 min at room temperature in the dark before flow cytometry analysis to identify the sub-G₀ peak corresponding to apoptosis in CD3⁺ or CD19⁺ cells. Cells were analyzed using FACSCalibur and CellQuest software.

Western blot analysis
Cells were lysed with Laemmli sample buffer, and western blotting was performed as described previously.² We used antibodies against p53 (Ab-5, Neomarkers, Fremont, CA, USA), MCL-1 and Akt (Santa Cruz Biotechnology, Santa Cruz, CA, USA), BCL-2 (Dako, A/S, Glostrup, Denmark), PUMA and NOXA (Abcam, Cambridge, UK), P-Ser473-Akt, P-GSK-3/β and P-FoxO1/FoxO3a (Cell Signaling Technologies, Beverly, MA, USA), and β-actin (Sigma-Aldrich). Antibody binding was detected using a secondary antibody conjugated to horseradish peroxidase and an enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK).

Reverse transcriptase multiplex ligation-dependent probe amplification
RNA was analyzed by RT-MLPA using a SALSA MLPA KIT R011 Apoptosis mRNA from MRC-Holland (Amsterdam, The Netherlands) for the simultaneous detection of 38 mRNA molecules.²⁵ In brief, RNA samples (200 ng total RNA) were first reverse transcribed using a gene-specific probe mix. The resulting cDNA was annealed overnight at 60°C to the MLPA probe mix. Annealed oligonucleotides were ligated by adding Ligase-65 (MRC-Holland, Amsterdam, The Netherlands) and incubated at 54°C for 15 min. Ligation products were amplified by polymerase chain reaction (PCR; 35 cycles, 30 s at 95°C; 30 s at 60°C, and 1 min at 72°C) with one unlabeled and one FAM labeled primer. The final PCR fragments amplified were separated by capillary electrophoresis on a 48-capillary ABI-Prism 3730 Genetic Analyzer (Applied Biosystems/Hitachi, Foster City, CA, USA). Peak area and height were measured using GeneScan analysis software (Applied Biosystems). The sum of all peak data was set at 100% to normalize for fluctuations in total signal between samples, and individual peaks were calculated relative to the 100% value.

Statistical analysis
Results are shown as the mean ± standard error of the mean (SEM) of values obtained in independent experiments. The paired Student’s t test was used to compare differences between paired samples. Data were analyzed using the SPSS 14.0 software package (Chicago, IL, USA).

TOP ABSTRACT Introduction Design and Methods Results Discussion References

 
Akti-1/2 and A-443654 inhibit Akt in chronic lymphocytic leukemia cells
To assess the effect of Akti-1/2 and A-443654 on Akt activity in CLL cells, we examined the phosphorylation status of Akt or Akt substrates which are used to assess the activation status of Akt. Akti-1/2 inhibited Ser473 phosphorylation in a dose-dependent manner (Figure 1A). As previously described for other cell types,^18,20 A-443654 induced an increase in Ser473 phosphorylation. In order to confirm that A-443654 was inhibiting Akt, we analyzed the status of the Akt substrates GSK3/β and FoxO1/FoxO3a. Both inhibitors reduced the phosphorylation of GSK3/β and FoxO1/FoxO3a (Figure 1B), demonstrating that they inhibited Akt activity.

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Figure 1. Akti-1/2 and A-443654 effects on the phosphorylation of Akt and Akt substrates. CLL cells were incubated with or without a range of doses of Akti-1/2 for 2 h. (A) Cells were lysed and whole extracts were analyzed by western blot as described in the Design and Methods section. Results from three patients are shown (n=3). (B) CLL cells were incubated with or without 5 µM Akti-1/2 and 0.5 µM (patients 2, 4 and 5) or 1 µM (patient 3) A-443654 for 2 h. Cells were lysed and whole extracts were analyzed by western blot as described in the Design and Methods section. Results from four representative patients are shown (n=9).

Akt inhibitors induce apoptosis in chronic lymphocytic leukemia cells
To examine the ability of Akt inhibitors to induce apoptosis in CLL cells, we incubated CLL cells with a range of concentrations of Akti-1/2 (0.5–20 µM) and A-443654 (0.1–1 µM) for 24 h and measured cell viability. Both inhibitors induced apoptosis in a dose-dependent manner (Figure 2A and 2B) and in a time-dependent manner (Online Supplementary Figure S2). The half-maximal effective concentration (EC₅₀) was 9.85±0.67 µM (range, 5 to 15 µM) for Akti-1/2, and 0.63±0.03 µM (range, 0.40 to 0.85 µM) for A-443654. Similar results were obtained in purified CD19⁺ CLL cells (Online Supplementary Figure S3). Akti-1/2 and A-443654 induced apoptosis in all the analyzed samples, independently of sex, ZAP70 status, CD38 status or genomic alterations (data not shown).

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Figure 2. Cytotoxic effect of Akt inhibitors on CLL cells and on peripheral blood lymphocytes (PBL) from healthy donors. Cells from CLL patients were incubated for 24 h with or without various doses of Akti-1/2 (A, n=15) and A-443654 (B, n=15). Viability was measured by analysis of phosphatidylserine exposure and PI uptake as described in the Design and Methods section. Cells from CLL patients and healthy donors were incubated for 24 h with or without various doses of Akti-1/2 (C, n=9 and n=3, respectively) and A-443654 (D, n=13 and n = 8, respectively). Viability was measured as non-apoptotic CD3+/CD19– T cells from PBL () and CLL () or CD3–/CD19+ B cells from PBL () and CLL () as described in the Design and Methods section. Viability is expressed as the percentage of the viability of untreated cells. Data are shown as the mean value ± SEM. *p<0.005, treated versus untreated cells (A, B), or B cells versus T cells from patients with CLL (C, D).

Differential effect of Akt inhibitors on B and T cells from patients with chronic lymphocytic leukemia and healthy donors
Next, we analyzed the sensitivity of normal B and T cells to Akt inhibitors, in terms of induction of apoptosis. The number of apoptotic T cells (CD3⁺ cells) was measured in CLL samples and blood samples from healthy donors exposed to several doses of Akti-1/2 (up to 10 µM) and A-443654 (up to 1 µM) for 24 h. Incubation with 5 µM Akti-1/2 reduced the percentage of viable CLL cells to 64.64±6.12%. In contrast, the percentage of viable T cells from CLL samples was 102.4±3.46%. Interestingly, B and T cells derived from healthy donors were resistant to Akti-1/2-induced apoptosis. Thus, after incubation with 5 µM Akti-1/2 the percentage of viable B cells and T cells were 91.6 ± 2.1% and 100.7±7.2%, respectively (Figure 2C). Similar results were obtained in the treatment of CLL cells and cells from healthy donors with A-443654, but the difference between CLL cells and B or T cells from healthy donors was less pronounced than in the case of Akti-1/2. Incubation with 0.5 µM A-443654 reduced the percentage of viable CLL cells to 64.2±4.9%. In contrast, the percentage of viable T cells was 91.3±3.7%. Furthermore, B and T cells derived from healthy donors were less sensitive than cells from CLL samples to A-443654-induced apoptosis. Thus, after incubation with 0.5 µM A-443654 the percentages of viable B and T cells were 83.4±5.8% and 87.1±5.9%, respectively (Figure 2D). Similar results were obtained when CLL cells and cells from healthy donors were treated for 48 h with both Akt inhibitors (Online Supplementary Figure S4). We confirmed this differential induction of apoptosis between CLL cells and normal lymphocytes by performing an analysis of the DNA content. We observed that CLL cells were more sensitive than T cells and normal B cells to the Akt inhibitor-induced increase in sub-G₀ peak (Online Supplementary Figure S5). These results indicate that B cells from CLL samples are more sensitive than normal B and T cells to Akt inhibitor-induced apoptosis.

Effect of survival factors in combination with Akt inhibitors
We studied the effect of two well-known survival factors in CLL cells, IL-4²⁶ and SDF-1,²⁷ in combination with Akt inhibitors. We used selected CLL samples in which these factors induced a survival effect. Thus, we treated CLL cells with 10 ng/mL IL-4 (Figure 3A) or 50 ng/mL SDF-1 (Figure 3B) and with or without 10 µM Akti-1/2 or 0.5 µM A-443654 for 48 h, and then measured cell viability. Neither IL-4 nor SDF-1 was able to inhibit the apoptosis induced by both Akt inhibitors. These results show that Akt inhibitors can induce apoptosis in CLL cells even in the presence of survival signals.

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Figure 3. Effect of survival signals on the apoptotic activity of Akt inhibitors. CLL cells were untreated or treated with 10 ng/mL IL-4 (A, n = 10) or 50 ng/mL SDF-1 (B, n = 7) without (white filled bars) or with 10 µM Akti-1/2 (gray filled bars) or 0.5 µM A-443654 (black filled bars) for 48 h. Viability was measured by analysis of phosphatidylserine exposure and propidium iodide uptake as described in the Design and Methods section. Data are shown as the mean value±SEM. **p<0.001 control versus IL-4 and Akt inhibitors treated cells versus control, IL-4 or SDF-1; *p<0.01 control versus SDF-1-treated cells.

Characterization of Akt inhibitor-induced apoptosis in chronic lymphocytic leukemia
We investigated the effect of Akt inhibitors on the overall apoptosis mRNA expression profile by performing RT-MLPA.²⁵ Treatment of CLL cells with 5 µM Akti-1/2 for 24 h did not induce any significant change in the RT-MLPA profile (Figure 4 and Online Supplementary Figure S6A). The results indicated that Akt inhibition has minimal effects on the apoptosis mRNA expression profile. Surprisingly, incubation of CLL cells with 0.5 µM A-443654 for 24 h induced an increase in the mRNA levels of NOXA and PUMA and a decrease in the mRNA levels of BMF, BID, one BAX probe, BCLw, NAIP, AIF, APAF and APAF_L (Figure 4 and Online Supplementary Figure S6B). Similar results were obtained in purified CD19⁺ CLL cells (Online Supplementary Figure S7). Thus, A-443654-induced mRNA changes are likely independent of its activity of inhibiting Akt.

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Figure 4. Apoptosis-related gene expression profile induced by Akti-1/2 and A-443654. Cells from CLL patients were untreated (open bars) or treated with 5 µM Akti-1/2 or 0.5 µM A-443654 (black filled bars) for 24 h. Cells were lysed, and NOXA (A) and PUMA (B) mRNA expression was analyzed by RT-MLPA as described in the Design and Methods section. The results are shown as the mean value ± SEM of four and eight different experiments for Akti-1/2 and A-443654, respectively. *p<0.005 treated versus untreated cells.

We then investigated the effect of Akt inhibitors on the expression of BCL-2 family proteins. Western blot analysis revealed that Akti-1/2 and A-443654 increased NOXA protein levels and decreased MCL-1 protein levels in all the samples analyzed. PUMA protein levels were also increased in 50% of the samples after treatment with Akt inhibitors (Figure 5A). Furthermore, p53 protein was increased by A-443654 but not by Akti-1/2 in all the samples analyzed.

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Figure 5. Apoptosis profile induced by Akti-1/2 and A-443654. (A) Apoptosis-related protein expression profile induced by Akti-1/2 and A-443654. Cells were untreated (ct) or treated with 5 µM Akti-1/2 (n = 10) and 0.5 µM A-443654 (n = 13) for 24 h, and MCL-1, NOXA, PUMA, p53 and BCL-2 expression was determined by western blot. Cell viability is expressed at the top of the figure. β-actin was used to standardize protein levels. Results from three representative patients are shown, two corresponding to Ficoll isolated cells (patients 7 and 37) and one corresponding to purified CD19+ CLL cells (patient 50). (B) Effect of Z-VAD.fmk on Akti-1/2 and A-443654-induced MCL-1 decrease. Cells were pretreated with or without 200 µM Z-VAD.fmk for 30 min and then treated with 5 µM Akti-1/2 and 0.5 µM A-443654 for 24 h (results from two representative patients are shown, n = 3), and MCL-1 levels were determined by western blot. BCL-2 and β-actin were used to standardize protein levels. Cell viability is expressed at the top of the figure.

We further analyzed the apoptosis-related protein expression profile induced by Akti-1/2 and A-443654 at different times. We did not observe any change in MCL-1, NOXA and PUMA protein levels after 3 h of treatment with Akt inhibitors (data not shown). NOXA protein was increased by Akt inhibitors at 6 h while PUMA protein levels were increased at 12 h. Moreover, MCL-1 protein was decreased after incubation with A-443654 for 6 h (Online Supplementary Figure S8). Finally, pretreatment with 200 µM Z-VAD.fmk for 30 min blocked the decrease in MCL-1 induced by Akti-1/2 but not that induced by A-443654 (Figure 5B). These results demonstrate that the decrease in MCL-1 induced by Akti-1/2 is caspase-dependent whereas that induced by A-443654 is caspase-independent and precedes the activation of caspases.

Akti-1/2 and A-443654 induce apoptosis irrespective of TP53 status in chronic lymphocytic leukemia cells
To study the role of p53 in Akti-1/2 and A-443654-induced apoptosis we analyzed the effect of these compounds on CLL samples with deleted/mutated TP53. Patients’ samples with deleted/mutated TP53 or altered expression have been described previously.^28–30 Patient 8 had a 17p deletion in one allele in 43% of peripheral blood lymphocytes, patient 41 had a 17p deletion in one allele in 94% of peripheral blood lymphocytes, and patient 40 had a frame-shift mutation in one allele (nucleotide deletion in codon 272) and a 17p deletion in the other allele in 86% of peripheral blood lymphocytes. First, we incubated these CLL cells with 5 µM Akti-1/2 or 0.5 µM A-443654 for 24 h. Interestingly, we observed a decrease in viability in two of the three samples with Akti-1/2 treatment and in all three samples with A-443654 treatment (Figure 6A).

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Figure 6. Apoptosis profile induced by Akti-1/2 and A-443654 in TP53 deleted/mutated samples. (A) Protein expression profile and cytotoxic effect of Akti-1/2, A-443654 and Nutlin-3a. Cells were untreated (ct) or treated with 5 µM Akti-1/2, 0.5 µM A-443654 or 5 µM Nutlin-3a for 24 h, and MCL-1, NOXA and PUMA expression was determined by western blot. BCL-2 and β-actin were used to standardize protein levels. Viability was measured by analysis of phosphatidylserine exposure and propidium iodide uptake as described in the Design and Methods section. (B) Apoptosis-related gene expression profile induced by Akti-1/2 and A-443654 in TP53 deleted/mutated CLL samples. Cells were untreated or treated with 5 µM Akti-1/2 (white filled bars) or 0.5 µM A-443654 (gray filled bars) and 5 µM Nutlin-3a (black filled bars) for 24 h. Cells were lysed, and the expression of apoptosis-related genes was analyzed by RT-MLPA as described in the Design and Methods section. The results are shown as fold induction relative to untreated cells.

Western blot analysis revealed an increase in NOXA levels in the three samples when treated with both inhibitors, while PUMA levels increased in only one sample (patient 8) with 5 µM Akti-1/2 treatment. MCL-1 levels decreased with Akti-1/2 in patients 8 and 41, where Akti-1/2 induced apoptosis. In patient 40, MCL-1 levels increased with Akti-1/2 treatment, and the inhibitor did not induce apoptosis. A-443654 treatment induced a decrease in MCL-1 levels in the three samples (Figure 6A). As a control of TP53 status we used 5 µM Nutlin-3a, which has been described to induce apoptosis and p53 accumulation in cells with wild-type TP53 but not in those with deleted/mutated TP53.²⁸

Next, we examined the apoptosis mRNA expression profile by performing RT-MLPA. Incubation with 5µM Akti-1/2 or 0.5 µM A-4436545 induced almost the same mRNA expression profile as that in CLL cells with the wild-type TP53, except that PUMA mRNA levels did not increase after treatment with A-443654 (Figure 6B).

TOP ABSTRACT Introduction Design and Methods Results Discussion References

 
In this study we investigated the effect of two Akt inhibitors on the viability of CLL cells. Both Akt inhibitors induced apoptosis in primary CLL cells. These results are in agreement with a recent report describing that introduction of constitutively active myr.Akt increases the viability of CLL cells.³¹

The mechanism of action of the two Akt inhibitors is somewhat different (Online Supplementary Figure S9). Surprisingly, Akti-1/2 inhibits Akt but does not induce changes in the RT-MLPA profile. Akt modulates the transcriptional activity of at least one transcription factor, FoxO3a,¹⁵ which would induce changes in the mRNA levels of its transcriptional targets BIM³² and PUMA.³³ However, RT-MLPA experiments suggest that dephosphorylation of FoxO3a induced by Akti-1/2 is not sufficient to induce the transcription of BIM and PUMA in CLL cells. As A-443654 is a less specific Akt-inhibitor,²¹ the simplest model would be to consider that inhibition of Akt (common to both inhibitors) does not induce changes in the RT-MLPA profile, and that A-443654 has additional targets to explain its effects on the expression of genes. Thus, A-443654 induces an increase in the levels of p53 protein and the induction of PUMA mRNA, a transcriptional target of TP53 in CLL.^28,34 In agreement with these data, the induction of PUMA mRNA by A-443654 was decreased in CLL cells with deleted/mutated TP53. Interestingly, Akti-1/2 induced PUMA and NOXA proteins without affecting PUMA and NOXA mRNA. The mechanism for this effect is unknown and could be explained by increased translation or decreased proteolysis of these proteins.

A common effect of both Akt inhibitors is the modulation of NOXA/MCL-1 balance. It has been reported that in primary CLL cells, the majority of NOXA protein is associated with MCL-1.³⁵

Thus, Akti-1/2 and A-443654 treatment induced an increase in NOXA protein levels and a down-regulation of the levels of MCL-1, a critical survival protein in CLL cells. Furthermore, inhibition of caspases prevents the down-regulation of MCL-1 induced by Akti-1/2, suggesting that MCL-1 cleavage participates in an amplification loop that increases cytochrome c release and apoptosis in CLL cells, as described for PKC inhibitors.³⁶ In agreement with our results, the introduction of constitutively active myr.Akt increases MCL-1 protein, and inhibition of MCL-1 by treatment with siRNA induces apoptosis in CLL.³¹ The mechanism of regulation of MCL-1 protein by Akt in CLL cells is unknown. The Akt substrate GSK-3 has been reported to induce destabilization of MCL-1 protein.¹⁶ However, inhibition of GSK-3 does not inhibit the apoptotic effect of PI3K inhibitors.⁷ As GSK-3 is inhibited by PKC,³⁷ perhaps the over-expression of active PKC-βII³⁸ blocks this pathway in CLL cells.

We found that B cells from CLL samples were more sensitive to Akt inhibitors than T cells from CLL samples, and B or T cells from healthy donors. Chemotherapeutic drugs, including fludarabine, chlorambucil, and doxorubicin induce apoptosis equally in both B and T cells, leading to immunosuppression.^39,40 Thus, the differential effect of Akti-1/2 and A-443654 in B and T lymphocytes is of interest. In conclusion, the results presented here suggest that clinically suitable small-molecule inhibitors of Akt alone or in combination with chemotherapeutic drugs might be a new therapeutic option for the treatment of CLL.

 
the authors thank Alba Pérez Perarnau, Camila Rubio and Dr. Esther Castaño for helpful discussions and suggestions; Michael Maudsley for language assistance; and Abbott for kindly providing A-443654. We also thank the Unitat de Biologia and the Unitat de Genòmica from the Serveis Cientificotècnics at the Universitat de Barcelona for their technical support.

 
Funding: this study was supported by grants from the Ministerio de Ciencia e Innovación and FEDER (SAF2007-60964), the Ministerio de Sanidad y Consumo (ISCIII-RTICC RD06/0020), and the AGAUR-Generalitat de Catalunya (2005SGR-00549). MdeF is a recipient of a fellowship from the AGAUR-Generalitat de Catalunya, AMC and DMGG are recipients of research fellowships from the Ministerio de Educación y Ciencia. DIS and LCM are recipients of fellowships from the José Carreras International Leukemia Foundation (FIJC-07/ESP-FCAJAMADRID).

Authorship and Disclosures

MdeF performed the research and contributed to data analysis and manuscript writing. DIS, AMC, LCM, AFS and DMGG performed the research and contributed with analytical tools. EdelaB contributed with patients’ samples and data. GP designed the research and contributed to data analysis. JG designed the research and contributed to data analysis and manuscript writing. The authors reported no potential conflicts of interest.

The online version of this article contains a supplementary appendix.

Received for publication November 25, 2008. Revision received June 4, 2009. Accepted for publication June 10, 2009.

TOP ABSTRACT Introduction Design and Methods Results Discussion References

 

1. Barragan M, Campas C, Bellosillo B, Gil J. Protein kinases in the regulation of apoptosis in B-cell chronic lymphocytic leukemia. Leuk Lymphoma 2003;44:1865–70.[CrossRef][Web of Science][Medline]
2. Barragan M, Bellosillo B, Campas C, Colomer D, Pons G, Gil J. Involvement of protein kinase C and phosphatidylinositol 3-kinase pathways in the survival of B-cell chronic lymphocytic leukemia cells. Blood 2002;99:2969–76.[Abstract/Free Full Text]
3. Bernal A, Pastore RD, Asgary Z, Keller SA, Cesarman E, Liou HC, et al. Survival of leukemic B cells promoted by engagement of the antigen receptor. Blood 2001;98:3050–7.[Abstract/Free Full Text]
4. Ringshausen I, Schneller F, Bogner C, Hipp S, Duyster J, Peschel C, et al. Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the defect of apoptosis in B-CLL: association with protein kinase C. Blood 2002;100:3741–8.[Abstract/Free Full Text]
5. Jones DT, Ganeshaguru K, Anderson RJ, Jackson TR, Bruckdorfer KR, Low SY, et al. Albumin activates the AKT signaling pathway and protects B-chronic lymphocytic leukemia cells from chlorambucil- and radiation-induced apoptosis. Blood 2003;101:3174–80.[Abstract/Free Full Text]
6. Cuni S, Perez-Aciego P, Perez-Chacon G, Vargas JA, Sanchez A, Martin-Saavedra FM, et al. A sustained activation of PI3K/NF-kappaB pathway is critical for the survival of chronic lymphocytic leukemia B cells. Leukemia 2004;18:1391–400.[CrossRef][Web of Science][Medline]
7. Plate JM. PI3-kinase regulates survival of chronic lymphocytic leukemia B-cells by preventing caspase 8 activation. Leuk Lymphoma 2004;45:1519–29.[CrossRef][Web of Science][Medline]
8. Nedellec S, Renaudineau Y, Bordron A, Berthou C, Porakishvili N, Lydyard PM, et al. B cell response to surface IgM cross-linking identifies different prognostic groups of B-chronic lymphocytic leukemia patients. J Immunol 2005;174:3749–56.[Abstract/Free Full Text]
9. Hu X, Haney N, Kropp D, Kabore AF, Johnston JB, Gibson SB. Lysophosphatidic acid (LPA) protects primary chronic lymphocytic leukemia cells from apoptosis through LPA receptor activation of the anti-apoptotic protein AKT/PKB. J Biol Chem 2005;280:9498–508.[Abstract/Free Full Text]
10. Barragan M, de Frias M, Iglesias-Serret D, Campas C, Castano E, Santidrian AF, et al. Regulation of Akt/PKB by phosphatidylinositol 3-kinase-dependent and -independent pathways in B-cell chronic lymphocytic leukemia cells: role of protein kinase Cβ. J Leukoc Biol 2006;80:1473–9.[Abstract/Free Full Text]
11. Petlickovski A, Laurenti L, Li X, Marietti S, Chiusolo P, Sica S, et al. Sustained signaling through the B-cell receptor induces Mcl-1 and promotes survival of chronic lymphocytic leukemia B cells. Blood 2005;105:4820–7.[Abstract/Free Full Text]
12. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet 2006;7:606–19.[CrossRef][Web of Science][Medline]
13. Leupin N, Cenni B, Novak U, Hugli B, Graber HU, Tobler A, et al. Disparate expression of the PTEN gene: a novel finding in B-cell chronic lymphocytic leukaemia (B-CLL). Br J Haematol 2003;121:97–100.[CrossRef][Web of Science][Medline]
14. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell 2007;129:1261–74.[CrossRef][Web of Science][Medline]
15. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 1999;96:857–68.[CrossRef][Web of Science][Medline]
16. Maurer U, Charvet C, Wagman AS, Dejardin E, Green DR. Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1. Mol Cell 2006;21:749–60.[CrossRef][Web of Science][Medline]
17. Longo PG, Laurenti L, Gobessi S, Petlickovski A, Pelosi M, Chiusolo P, et al. The Akt signaling pathway determines the different proliferative capacity of chronic lymphocytic leukemia B-cells from patients with progressive and stable disease. Leukemia 2007;21:110–20.[CrossRef][Web of Science][Medline]
18. Luo Y, Shoemaker AR, Liu X, Woods KW, Thomas SA, de Jong R, et al. Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo. Mol Cancer Ther 2005;4:977–86.[Abstract/Free Full Text]
19. Shi Y, Liu X, Han EK, Guan R, Shoemaker AR, Oleksijew A, et al. Optimal classes of chemotherapeutic agents sensitized by specific small-molecule inhibitors of akt in vitro and in vivo. Neoplasia 2005;7:992–1000.[CrossRef][Web of Science][Medline]
20. Han EK, Leverson JD, McGonigal T, Shah OJ, Woods KW, Hunter T, et al. Akt inhibitor A-443654 induces rapid Akt Ser-473 phosphorylation independent of mTORC1 inhibition. Oncogene 2007;26:5655–61.[CrossRef][Web of Science][Medline]
21. Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, et al. The selectivity of protein kinase inhibitors: a further update. Biochem J 2007;408:297–315.[Medline]
22. Barnett SF, Defeo-Jones D, Fu S, Hancock PJ, Haskell KM, Jones RE, et al. Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors. Biochem J 2005;385:399–408.[CrossRef][Web of Science][Medline]
23. Logie L, Ruiz-Alcaraz AJ, Keane M, Woods YL, Bain J, Marquez R, et al. Characterization of a protein kinase B inhibitor in vitro and in insulin-treated liver cells. Diabetes 2007;56:2218–27.[CrossRef][Web of Science][Medline]
24. Bellosillo B, Pique M, Barragan M, Castano E, Villamor N, Colomer D, et al. Aspirin and salicylate induce apoptosis and activation of caspases in B-cell chronic lymphocytic leukemia cells. Blood 1998;92:1406–14.[Abstract/Free Full Text]
25. Eldering E, Spek CA, Aberson HL, Grummels A, Derks IA, de Vos AF, et al. Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways. Nucleic Acids Res 2003;31:e153.[Abstract/Free Full Text]
26. Dancescu M, Rubio-Trujillo M, Biron G, Bron D, Delespesse G, Sarfati M. Interleukin 4 protects chronic lymphocytic leukemic B cells from death by apoptosis and upregulates Bcl-2 expression. J Exp Med 1992;176:1319–26.[Abstract/Free Full Text]
27. Burger JA, Tsukada N, Burger M, Zvaifler NJ, Dell’Aquila M, Kipps TJ. Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1. Blood 2000;96:2655–63.[Abstract/Free Full Text]
28. Coll-Mulet L, Iglesias-Serret D, Santidrian AF, Cosialls AM, de Frias M, Castano E, et al. MDM2 antagonists activate p53 and synergize with genotoxic drugs in B-cell chronic lymphocytic leukemia cells. Blood 2006;107:4109–14.[Abstract/Free Full Text]
29. Coll-Mulet L, Santidrian AF, Cosialls AM, Iglesias-Serret D, de Frias M, Grau J, et al. Multiplex ligation-dependent probe amplification for detection of genomic alterations in chronic lymphocytic leukaemia. Br J Haematol 2008;142:793–801.[CrossRef][Web of Science][Medline]
30. Santidrian AF, Cosialls AM, Coll-Mulet L, Iglesias-Serret D, de Frias M, Gonzalez-Girones DM, et al. The potential anticancer agent PK11195 induces apoptosis irrespective of p53 and ATM status in chronic lymphocytic leukemia cells. Haematologica 2007;92:1631–8.[Abstract/Free Full Text]
31. Longo PG, Laurenti L, Gobessi S, Sica S, Leone G, Efremov DG. The Akt/Mcl-1 pathway plays a prominent role in mediating antiapoptotic signals downstream of the B-cell receptor in chronic lymphocytic leukemia B cells. Blood 2008;111:846–55.[Abstract/Free Full Text]
32. Dijkers PF, Medema RH, Lammers JW, Koenderman L, Coffer PJ. Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr Biol 2000;10:1201–4.[CrossRef][Web of Science][Medline]
33. You H, Pellegrini M, Tsuchihara K, Yamamoto K, Hacker G, Erlacher M, et al. FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal. J Exp Med 2006;203:1657–63.[Abstract/Free Full Text]
34. Mackus WJ, Kater AP, Grummels A, Evers LM, Hooijbrink B, Kramer MH, et al. Chronic lymphocytic leukemia cells display p53-dependent drug-induced Puma upregulation. Leukemia 2005;19:427–34.[CrossRef][Web of Science][Medline]
35. Hallaert DY, Spijker R, Jak M, Derks IA, Alves NL, Wensveen FM, et al. Crosstalk among Bcl-2 family members in B-CLL: seliciclib acts via the Mcl-1/Noxa axis and gradual exhaustion of Bcl-2 protection. Cell Death Differ 2007;14:1958–67.[CrossRef][Web of Science][Medline]
36. Snowden RT, Sun XM, Dyer MJ, Cohen GM. Bisindolylmaleimide IX is a potent inducer of apoptosis in chronic lymphocytic leukaemic cells and activates cleavage of Mcl-1. Leukemia 2003;17:1981–9.[CrossRef][Web of Science][Medline]
37. Christian SL, Sims PV, Gold MR. The B cell antigen receptor regulates the transcriptional activator β-catenin via protein kinase C-mediated inhibition of glycogen synthase kinase-3. J Immunol 2002;169:758–69.[Abstract/Free Full Text]
38. Abrams ST, Lakum T, Lin K, Jones GM, Treweeke AT, Farahani M, et al. B-cell receptor signaling in chronic lymphocytic leukemia cells is regulated by overexpressed active protein kinase CβII. Blood 2007;109:1193–201.[Abstract/Free Full Text]
39. Keating MJ. Immunosuppression with purine analogues – the flip side of the gold coin. Ann Oncol 1993;4:347–8.[Free Full Text]
40. Hamblin AD, Hamblin TJ. The immunodeficiency of chronic lymphocytic leukaemia. Br Med Bull 2008;87:49–62.[Abstract/Free Full Text]

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