![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Dipartimento di Scienze Anatomiche Umane (F.F., W.L.B., F.C., L.C., and A.M.M.) and Dipartimento di Ematologia e Scienze Oncologiche "L. e A. Seràgnoli" (G.M.), Università di Bologna, Italy; Centro Immunoematologia e Trasfusionale, Policlinico S. Orsola-Malpighi, Bologna, Italy (P.L.T.); Dipartimento di Scienze Motorie e della Salute, Università di Cassino, Italy (A.C.); Dipartimento di Biotecnologie ed Ematologia, Università degli Studi "La Sapienza," Roma, Italy (A.T.); and Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, North Carolina (J.A.M.)
Received for publication March 31, 2008.
Accepted for publication June 23, 2008.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). Lymphoblasts from more than 50% of patients with T-ALL contain an activating mutation in the transmembrane receptor, Notch-1 (Grabher et al., 2006; Weerkamp et al., 2006). Activating mutations as well as binding with its ligands of the Delta and Serrated/Jagged family result in the cleavage of Notch-1 and release of the intracellular portion of the receptor, which translocates to the nucleus, where it interacts with transcription factors to affect gene expression. Notch-1 signaling is critical for T-cell development, proliferation, and survival (Eagar et al., 2004). Recently, it was demonstrated that activated Notch-1 leads to constitutive activation of the PI3K/AKT/mTOR pathway by HES1-mediated transcriptional suppression of the MMAC1 gene, which encodes the dual-specificity, lipid and protein, PIP3 phosphatase known as PTEN (Palomero et al., 2007). In addition, PTEN is mutated in approximately 20% of patients with T-ALL, and virtually all T-ALL cell lines that are resistant to Notch-1 inhibition with 
-secretase inhibitors contain mutations leading to either no or low PTEN expression (Palomero et al., 2007). PTEN is a key modulator of the PI3K pathway and AKT activation through its regulation of the level of PIP3 generated from the available pool of phosphatidylinositol-4,5-phosphate by PI3K (Tokunaga et al., 2008). In the presence of PTEN, PIP3 is rapidly dephosphorylated to phosphatidylinositol-4,5-phosphate, blocking the recruitment of AKT to the membrane for activation. The loss of PTEN in T-ALL contributes to the hyperactivated state of AKT found in these cells, because activation of AKT by phosphorylation on Thr308 by PDK1 or on Ser473 by PDK2 (also referred to as the mTOR:Rictor complex) requires membrane recruitment via PIP3 (Martelli et al., 2006; Sale and Sale, 2008).
Activated AKT phosphorylates multiple targets involved in cell growth, inhibition of apoptosis and metabolism. In general, targets inhibited after phosphorylation by AKT are involved in cell cycle arrest, apoptosis induction, or homeostasis under low nutrient conditions. Targets inhibited by AKT phosphorylation include GSK-3
/β, FoxO transcription factors, Bad, p21Cip1, and p27Kip1 (Marone et al., 2008; Sale and Sale, 2008). Targets activated by AKT phosphorylation are involved in cell cycle progression, apoptosis inhibition, and metabolism in a high-energy environment and include murine double minute 2, X-linked inhibitor of apoptosis protein, mTOR (Marone et al., 2008; Sale and Sale, 2008). In tumors that contain low levels of, or no, PTEN, AKT activation is the lynchpin for growth and survival, as these tumors are extremely sensitive to AKT inhibition (Lopiccolo et al., 2008). Moreover, activation of the PI3K/AKT signaling pathway confers resistance to many types of cancer therapy and is a poor prognostic factor for many types of neoplastic disorders, making AKT an exciting target for innovative cancer treatment (Lindsley et al., 2008). In this study, we sought to analyze the efficacy of the novel AKT inhibitor A443654 (Luo et al., 2005) as a therapeutic agent in the treatment of T-ALL. We demonstrate that A443654 is highly cytotoxic against T-ALL cell lines (including a T-ALL drug-resistant cell line that overexpresses 170-kDa P-glycoprotein) and patient samples at doses well within the tolerated range in vivo. Moreover, it could synergize with standard therapeutic compounds to induce apoptotic cell death.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
MTT Assay. MTT assays were performed to assess the sensitivity of the T-ALL lines to either A443654 alone or in combination with another indicated compound using the MTT assay kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer's protocol.
Combined Drug Effects Analysis. To characterize the interactions between A443654 and etoposide, the combination effect and a potential synergy were evaluated from quantitative analysis of dose-effect relationships as described previously (Nyakern et al., 2006). For each A443654/etoposide drug combination experiment, a CI number was calculated using the Biosoft CalcuSyn computer program (Cambridge, UK) and the formula CI = Ca/Cxa + Cb/Cxb, where Cxa and Cxb are the concentrations of compound a and b alone, respectively, needed to achieve a given effect (x%) and Ca and Cb are the concentrations of A443654 and etoposide needed for the same effect (x%) when the drugs are combined. This method of analysis generally defines CI values of 0.9 to 1.1 as additive, 0.3 to 0.9 as synergistic, and <0.3 as strongly synergistic, whereas values >1.1 are considered antagonistic.
Annexin V-FITC Assay. To assess the degree of apoptosis after treatment with A443654, either alone or in combination with an additional compound, the extent of Annexin V-FITC/PI staining was determined by flow cytometry as described previously using the Annexin V/PI staining kit from Bender MedSystems (Vienna, Austria) (Blalock et al., 2003). Samples were read on an Epics XL-MCL flow cytometer (Beckman Coulter, Fullerton, CA).
Cell Cycle Analysis. After their respective treatment, T-ALL cell lines and patient samples were prepared as described previously (Shelton et al., 2004). Samples were analyzed with a FC500 flow cytometer (Beckman Coulter) equipped with CXP software.
PDK1 siRNA Knockdown. Jurkat cells were washed two times in Opti-MEM (Invitrogen, Milan, Italy) and resuspended in Nucleofector Solution V (Amaxa Biosystems, Cologne, Germany) to a density of 5 x 106 cells/0.1 ml. Cells were electroporated with 1.5 µM SmartPool siRNA to PDK1 or control using a Nucleofector electro-porator (program X-05; Amaxa Biosystems, Gaithersburg, MD) according to the manufacturer's instructions. Cells were then cultured in growth media for 72 h before assaying.
Protein Extraction and Western Blotting. Protein lysates were prepared as described previously (Blalock et al., 2003). The protein concentration was determined by detergent-compatible protein assay (Bio-Rad Laboratories, Hercules, CA). Lysate (50 µg) was loaded onto an SDS-polyacrylamide gel, electrophoresed, and transferred onto nitrocellulose membranes, using a semidry transfer apparatus. The membranes were incubated overnight at 4°C in 5% nonfat dry milk in 1x PBST. The membranes were washed three times in 1x PBST and incubated overnight at 4°C in primary antibody diluted 1:1000 in 5% BSA in PBST. The following antibodies were from Cell Signaling Laboratories (Danvers, MA): p-Thr308 AKT, p-Ser473 AKT, AKT, p-Ser9 GSK-3β, GSK-3β, caspase-2, caspase-3, caspase-6, caspase-8, caspase-9, PKC-
, PDK1, β-actin. The membranes were washed three times in 1X PBST and incubated for 2 h in the appropriate peroxidase-conjugated secondary antibody (Cell Signaling) diluted 1:2000 in 5% milk in PBST. The blots were washed three times with PBST and visualized using ECL reagent (GE Healthcare). Band intensity was determined by densitometry using the public domain software Image J (a Java image processing program inspired by NIH Image for Macintosh; http://rsbweb.nih.gov/ij/) as described elsewhere (Nyakern et al., 2006).
Immunoprecipitation. This was performed as described previously (Neri et al., 2003).
Determination of the Levels of PTEN, p-Ser473 AKT, and Cleaved Caspase-3 in T-ALL Patient Samples. Viable lymphoblasts (5 x 105) from six patients with T-ALL or peripheral blood lymphocytes from healthy donors were fixed with reagent 1 of the Intraprep kit according to the manufacturer's instructions (Beckman Coulter) and permeabilized with saponin-based reagent 2. Cells were blocked in 5% BSA in PBS and incubated for 12 h at 4°C in primary antibody [p-Ser473 AKT (either Alexa Fluor 488 or Alexa Fluor 647 conjugate), PTEN (Alexa Fluor 647 conjugate), or cleaved caspase-3 (Alexa Fluor 488 conjugated), all from Cell Signaling, diluted 1:10 in 5% BSA in PBS]. Cells were then washed twice in 1x PBS and analyzed on a FC500 flow cytometer (Tazzari et al., 2007a,b; Papa et al., 2008).
Statistical Evaluation. The data are shown as mean values ± S.D. Data were statistically analyzed by Dunnett's test after one-way analysis of variance at a level of significance of p < 0.05 versus control samples.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Uddin et al., 2004). To ascertain the effectiveness of the novel AKT inhibitor A443654 as a therapeutic agent in T-ALL, we treated the T-ALL cell lines Jurkat, MOLT-4, CEM-S, and CEM-R with serially diluted concentrations of A443654 or the vehicle (DMSO) alone (control). After 24 h, the rates of proliferation and cell viabilities were measured using MTT assays. All three parental cell lines were sensitive to nanomolar doses of A443654 (IC50 = 80, 120, and 900 nM for MOLT-4, CEM-S, and Jurkat, respectively) well below 20 µM, the highest concentration reached in vivo in tumor (Fig. 1A) (Luo et al., 2005). In contrast, the drug-resistant CEM-R cell line, a cell line overexpressing the 170 kDa P-glycoprotein (Mantovani et al., 2006), showed increased resistance to A443654 (IC50 = 12 µM), but this IC50 was still below 20 µM (Fig. 1A).
|
A443654 Results in Rapid Phosphorylation of Both Thr308 and Ser473 of AKT. A443654 was reported to lead to the dephosphorylation of the AKT substrate GSK-3β and most recently to induce phosphorylation of AKT on Ser473 (Luo et al., 2005; Han et al., 2007). We examined the effects of A443654 on AKT signaling in T-ALL cells by measuring the phosphorylation status of AKT and its downstream substrate GSK-3β (Fig. 2). Treatment of Jurkat, CEM-S, and CEM-R cells with A443654 resulted in a dose-dependent phosphorylation of AKT at both Thr308 and Ser473. Treatment of Jurkat cells with 0.1 µM A443654 for 24 h resulted in a dramatic increase in AKT phosphorylated at Thr308, whereas enhancement of AKT phosphorylated on Ser473 was less marked over control cells (Fig. 2A). In addition, 0.1 µM A443654 resulted in greatly diminished levels of GSK-3β phosphorylated at S9. Phosphorylation of AKT at Ser473 as well as loss of p-S9 GSK-3β occurred rapidly (within 30 min). Treatment of CEM-S (1 µM) or CEM-R (10 µM) cells with A443654 resulted in a robust increase in the amount of AKT phosphorylated at Ser473, respectively (Fig. 2A). In contrast to Ser473, where constitutive AKT phosphorylation was observed in CEM-S and, to a greater extent, in CEM-R cells, no constitutive phosphorylation of Thr308 was detected, using a phosphospecific antibody, but phosphorylation at this site was likewise rapidly induced. Moreover, GSK-3β was rapidly dephosphorylated, although dephosphorylation of GSK-3β occurred to a lesser extent in CEM-R cells, most likely as a result of the higher constitutive levels of p-Ser473 AKT in these cells (Fig. 2A).
|
). Likewise, we observed that part of the phosphorylation induced by treatment of Jurkat cells was PI3K-independent (Ser473) and PDK1-independent (Thr308), suggesting an additional kinase(s) responsible for the phosphorylation of AKT on Thr308 and Ser473 (Fig. 2B). Jurkat cells, which were pretreated with either DMSO (control), LY294002, wortmannin, or PI-103 (a dual PI3K/mTOR inhibitor; see Fan et al., 2006) for 1 h, were treated with 1 µM A443654 for 2 and 4 h, and the phosphorylation status of AKT was assayed. Pretreatment with wortmannin or PI-103 all but abolished the constitutive levels of p-Thr308 AKT in Jurkat cells, whereas LY294002 reduced Thr308 phosphorylation. It is noteworthy that, in the presence of these inhibitors, A443654 was able to induce phosphorylation of AKT on Thr308 to levels similar to or above that observed with A443654 alone, suggesting that membrane recruitment and PDK1 were not responsible for A443654-induced Thr308 phosphorylation (Fig. 2B). In addition, a similar lack of inhibition of A443654-induced Thr308 phosphorylation was observed when LY294002 and wortmannin were used at 50 µM and 300 nM, respectively (data not shown). The constitutive phosphorylation of AKT on Ser473 was not greatly affected by pretreatment with LY294002 (1.5 µM), whereas both wortmannin (100 nM) and PI-103 (50 nM) resulted in a marked decrease in AKT Ser473 phosphorylation. Treatment with A443654 was able to restore p-Ser473 AKT levels to that of the control (Fig. 2B). Higher concentrations of LY294002 (50 µM) and wortmannin (300 nM) abolished AKT Ser473 phosphorylation, but phosphorylation remained inducible by A443654, although not to levels of the control (data not shown). Each of the inhibitors, LY294002, wortmannin, and PI-103 (IC50 for PI3K, 1.4 µM, 4 nM, and 26 nM, respectively) have been shown to also inhibit mTOR [IC50, 5 µM, 300 nM, and 20 nM (mTOR:Raptor complex) or 86 nM (mTOR:Rictor complex)] (Bain et al., 2007). At doses at or above the IC50 for PI3K, the inhibitors had only a partial affect on AKT Ser473 phosphorylation but, at concentrations at or above the IC50 for mTOR, were capable of inhibiting AKT Ser473 phosphorylation; therefore, it is likely that mTOR:Rictor is the major kinase responsible for AKT Ser473 phosphorylation. This observation is in agreement with what was recently reported by others (Han et al., 2007). To rule out PDK1 as being responsible for Thr308 phosphorylation, siRNA directed to PDK1 was used to down-regulate PDK1 levels in Jurkat cells before treatment with 1 µM A443654. Although scrambled siRNA did not decrease PDK1 levels compared with untreated cells, as evaluated by Western blot, pretreatment with siRNA to PDK1 resulted in a marked decrease in PDK1 and in p-Thr308 AKT levels (Fig. 2C). After treatment with A443654, p-Thr308 AKT levels increased in samples pretreated with either scramble siRNA or PDK1 siRNA. By densitometric analysis of the blots, it was possible to determine that in control samples (scramble siRNA), the levels of p-Thr308 increased 3.2 times upon drug treatment, whereas in samples with down-regulated PDK1, the increase was 3.14-fold, despite a 10-fold reduction in the PDK1 levels.
|
It is noteworthy that, in Jurkat cells, but not in either CEM cell line, a degradation of AKT was observed after phosphorylation (Fig. 2, A and D and data not shown). As inhibition of AKT led to apoptosis, we analyzed the expression of AKT after A443654 treatment in the presence of caspase inhibitors. The loss of AKT after A443654 treatment was abolished by the inhibition of caspase-3, suggesting the loss of AKT in Jurkat cells was through a caspase-3 dependent mechanism (Fig. 2D). Moreover, a cleaved caspase-3/AKT complex was immunoprecipitated in Jurkat cells using an antibody to AKT, but not in either CEM cell line (Fig. 2E and data not shown). A similar mechanism to that of AKT may be responsible for the diminishing levels of GSK-3β observed in Jurkat cells after A443654 treatment (Fig. 2A).
GSK-3β Is Involved in A443654-Induced Cytotoxicity. Considering that GSK-3β regulates the expression of cyclin D1, Myc, and a number of other key regulatory proteins that are important for cell survival (Frame and Cohen, 2001), it was investigated whether GSK-3β played a role in A443654-dependent cytoxicity. Jurkat cells were pretreated with LiCl (a well established inhibitor of GSK-3β; see Bain et al., 2007), then treated with A443654, and cell survival was assessed by MTT assay. Whereas LiCl when employed alone at 10 µM slightly decreased cell survival, this was not statistically significant (p > 0.05). However, LiCl had a statistically significant (p < 0.01) inhibitory effect on A443654 induced cytotoxicity when employed in the range of 5 to 10 µM (i.e., concentrations at which most of GSK-3β activity is inhibited in vitro) (Bain et al., 2007) (Fig. 3). Overall, these findings indicated that apoptotic cell death elicited by A443654 is at least partially dependent on dephosphorylation and subsequent activation of GSK-3β.
|
|
Preincubation of cells with a pharmacological inhibitor selective for caspase-2 (Z-VDVAD-FMK) almost completely prevented cleavage of effector caspases 3 and 6 (Fig. 4C). The caspase-3 downstream target PKC- (DeVries-Seimon et al., 2007) was cleaved after treatment with A443654, and the cleavage was strongly reduced by preincubation with Z-VDVAD-FMK (Fig. 4D). Z-VDVAD-FMK also markedly reduced apoptotic cell death induced by A443654 in Jurkat cells, as demonstrated by Annexin V-FITC/PI staining (Fig. 4E). Taken together, these findings suggested an important role played by caspase-2 activation in response to A443654 treatment.
|
An additional complication of cancer therapy is drug resistance. Because we determined that coadministration of A443654 with etoposide resulted in synergistic killing of Jurkat cells, we examined whether sublethal doses of A443654 could enhance the effectiveness of etoposide against drug-resistant cells (Fig. 5B). CEM-S and CEM-R cells were cotreated for 48 h in the presence of 25 nM (CEM-S) or 3 µM A443654 (CEM-R) and serially diluted dosages of etoposide. Although enhanced cell death was observed in CEM-S cells when treated with both A443654 and etoposide, the enhancement was not significant. In contrast, a significant enhancement of etoposide induced cell killing was observed when CEM-R cells were cotreated with sublethal A443654. At etoposide concentrations between 25 to 100 µM, A443654 was able to enhance the extent of cell killing from 25% to 45% (Fig. 5B). By increasing the effectiveness of etoposide, a drug that is a substrate for 170-kDa P-glycoprotein, these results highlight the potential therapeutic applications A443654 has on drug-resistant cancers.
T-All Blasts Display Elevated p-AKT and Are Sensitive to A443654. To determine the effectiveness of A443654 as a therapeutic agent in T-ALL, we examined T-ALL patient samples (age, 19-59 years; sex, 5 male, 1 female) isolated from bone marrow or peripheral blood, for the presence of PTEN and p-Ser473 AKT and their sensitivity to A443654, using flow cytometry. All patient samples (6/6) had levels of p-Ser473 AKT higher than those observed in peripheral blood lymphocytes from healthy donors (Fig. 6, A and B). In addition, although we did not determine whether these patient samples harbored activating mutations in Notch-1, as it was beyond the scope of this study, we did determine that all six samples had decreased levels of PTEN compared with peripheral blood lymphocytes (Fig. 6, A and B). Jurkat cells served as an additional control, in that they have elevated p-Ser473 AKT and they do not express PTEN. To determine the susceptibility of these cells to inhibition of AKT, the patient samples were treated with 1 µM A443654 for 72 h, and the percentage of sub-G1/G0 cells was analyzed by flow cytometry. Each of the patient samples displayed at least 50% of the cells in sub-G1/G0 after treatment (Fig. 6C). In addition, increased levels of cleaved caspase-3 were detected in patient samples treated with A443654 (Fig. 6D). Flow cytometric analysis of samples double-stained for p-Ser473 AKT and cleaved caspase-3 demonstrated that cells with increased levels of Akt phosphorylation as a result of drug treatment actually underwent apoptotic cell death (Fig. 6E).
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Grabher et al., 2006; Chan et al., 2007; Palomero et al., 2007). Because AKT seems to play a central role in T-ALL pathology, we sought to determine whether the novel AKT inhibitor, A443654, might be a useful therapy in treating T-ALL. We found that A443654 was able to inhibit cellular proliferation in the T-ALL cell lines tested at nanomolar concentrations. Preclinical studies performed in vivo in a mouse model have shown that A443654 could be well tolerated up to a maximum concentration of approximately 20 µM in tumor (Luo et al., 2005). It is remarkable that in the drug-resistant T-ALL cell line CEM-R, A443654 had an IC50 of approximately 12 µM; this is still well within the maximum tolerated dose, suggesting that this particular AKT inhibitor may be extremely useful in treating a majority of T-ALLs as well as drug-resistant T-ALL. It is worth emphasizing here that 170 kDa P-glycoprotein is detected in approximately 24% of patients with T-ALL and negatively correlates with the achievement of complete remission (Tafuri et al., 2002; Vitale et al., 2006a). A443654 also induced a significant amount of apoptosis in all six T-ALL patient samples.
A443654 induced cell cycle arrest in the G2/M phase followed by subsequent apoptosis in T-ALL cell lines. It is interesting that cells were arrested in G2/M, in that AKT is often thought of as a kinase responsible for entering into S-phase, but AKT has been demonstrated to have additional targets that control entry into G2 and subsequent entry into the M-phase of the cell cycle (Okumura et al., 2002; Katayama et al., 2005). The observed G2/M arrest is intriguing in that the CI of A443654 and etoposide in Jurkat cells showed synergy when the two drugs were either added simultaneously or etoposide was added before A443654. Addition of A443654 before etoposide had an additive/antagonistic effect. The fact that A443654 arrests pretreated cells in G2/M may preclude any response to etoposide, a drug that requires DNA to be replicating to have its cytotoxic effects (Markovits et al., 1987; D'Arpa et al., 1990). Moreover, because caspase-2 was the first caspase observed to be cleaved after treatment with A443654, the G2/M arrest may be indicative of mitotic catastrophe, an event that results in the formation of the PIDD-osome and activation of caspase-2 followed by caspase-8 and caspase-9 activation (Tinel and Tschopp, 2004). The use of a selective capase-2 inhibitor allowed us to establish that activation of this apical caspase was very important for the activation of effector caspases 3 and 6, and for PKC- cleavage. It is noteworthy that, besides caspase-3, caspase-2 has been reported to possibly directly cleave PKC- (Zhivotovsky and Orrenius, 2005). In contrast, our unpublished data showed that inhibition of apical caspase-8 and -9 only slightly diminished caspase-3 and -6 cleavage (data not shown).
It was previously reported that A443654 induces rapid phosphorylation of AKT on Ser473 and induces a rapid dephosphorylation of the AKT substrate, GSK-3β (Luo et al., 2005; Han et al., 2007). The phosphorylation of Ser473 was reportedly elicited through the rapamycin insensitive mTOR: Rictor complex. We found that, in addition to dephosphorylation of GSK-3β and phosphorylation of AKT at position Ser473, Thr308 phosphorylation was rapidly induced by A443654. A443654 induced phosphorylation of Thr308 was not significantly inhibited by the PI3K/mTOR inhibitors, wortmannin, LY294002, and PI-103, or down-regulation of PDK1 through siRNA, indicating that membrane recruitment by PIP3 and subsequent phosphorylation by PDK1 were not a requirement for A443654-mediated Thr308 phosphorylation. In contrast, phosphorylation of Ser473 was partially inhibited by LY294002, wortmannin, or PI-103 when used at or above the IC50 for PI3K but was greatly inhibited when these compounds were used at or above the IC50 for mTOR, suggesting that, in Jurkat cells, some Ser473 phosphorylation may not be completely dependent on mTOR: Rictor, but a majority is. The phosphorylation of AKT after addition of A443654 may imply that AKT functions as a stress/ATP indicator. A443654 functions as an AKT ATP-binding site analog (Luo et al., 2005; Han et al., 2007), and the inability of AKT to phosphorylate downstream substrates is similar to a low ATP state in the cell. Such a state could occur during poor growth conditions (low O2 or a sugar source) or after mitochondrial damage resulting in AKT phosphorylation. Such stress-induced AKT phosphorylation has been reported recently to be dependent on eukaryotic initiation factor 2 phosphorylation after activation of protein kinase R or protein kinase R-like endoplasmic reticulum kinase (Kazemi et al., 2007). Further analysis is needed to identify the kinase(s) responsible for stress-induced AKT phosphorylation.
A443654-induced GSK-3β dephosphorylation, despite concomitant hyperphosphorylation of AKT, could occur because A443654-bound AKT is locked into a conformation that is not amenable to dephosphorylation, and thus the phosphorylated forms of AKT rapidly accumulate. If indeed AKT relocalization is necessary for its subsequent dephosphorylation, A443654 might lead to an accumulation of inactive but highly phosphorylated AKT by simply preventing its release from sites of activation (Han et al., 2007). In this connection, unpublished results of F. Falà have revealed that immunoprecipitated AKT from A443654 cells actually increased phosphorylation (and hence inhibition) of recombinant GSK-3β in an in vitro assay. Thus, this finding could support the hypothesis of Han et al. (2007), in that immunoprecipitated AKT would be presumably freed from interactions with A443654. The occurrence of in vivo inhibition of GSK-3β by A443654 was demonstrated by the effects of concomitant incubation with LiCl, which resulted in a mitigation of the drug-induced cytotoxicity.
It should be stated that, in vitro, assays of A443654 inhibitory activity identified 16 kinases that were significantly affected (Bain et al., 2007). Although these results have not been repeated in vivo, it is possible that some effects of A443654 are not due directly to AKT inhibition. In our experience, however, we did not detect modifications in the activation status of the ERK kinases or JNK in T-ALL cell lines immediately after treatment with A443654 (data not shown). In conclusion, our preclinical studies support the concept that inhibition of Akt signaling may have clinical application for treatment of T-ALLs. Based on these data, it is conceivable that A443654, or other Akt inhibitors, may serve as efficient therapeutic agents to combat T-ALLs that require elevated levels of AKT for their survival and growth. Moreover, A443654 may be an effective adjuvant to combine with current chemotherapy regimens to enhance their therapeutic efficiency, especially in drug-resistant T-ALLs.
|
Footnotes |
|---|
F.F. and W.L.B. contributed equally to this work.
ABBREVIATIONS: T-ALL, T-cell acute lymphoblastic leukemia; PI3K, phosphatidylinositol-3 kinase; mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homolog deleted on chromosome 10; PIP3, phosphatidylinositol-3,4,5-phosphate; GSK-3/β, glycogen synthetase kinase-3/β; A443654, (2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan2-amine; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; Z-VDVAD-FMK, N-benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethyl ketone; PI-103, 3-(4-(4-morpholinyl)pyrido[3',2',4,5]furo[3,2-d]pyrimidin-2-yl)phenol; CI, combination index; FITC, fluorescein isothiocyanate; PI, propidium iodide; PAGE, polyacrylamide gel electrophoresis; PBST, PBS containing 0.05% Tween 20; BSA, bovine serum albumin; PKC, protein kinase C; PDK1, phosphatidylinositol-dependent kinase 1; PBS, phosphate-buffered saline; CEM-S, drug-sensitive CEM cells; CEM-R, drug-resistant CEM cells; siRNA, short interfering RNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.
Address correspondence to: Prof. Alberto M. Martelli, Department of Human Anatomical Sciences, Cell Signaling Laboratory, University of Bologna, via Irnerio 48, 40126 Bologna, Italy. E-mail: alberto.martelli{at}gmail.com
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Blalock WL, Navolanic PM, Steelman LS, Shelton JG, Moye PW, Lee JT, Franklin RA, Mirza A, McMahon M, White MK, et al. (2003) Requirement for the PI3K/Akt pathway in MEK1-mediated growth and prevention of apoptosis: identification of an Achilles heel in leukemia. Leukemia 17: 1058-1067.[CrossRef][Medline]
Chan SM, Weng AP, Tibshirani R, Aster JC, and Utz PJ (2007) Notch signals positively regulate activity of the mTOR pathway in T-cell acute lymphoblastic leukemia. Blood 110: 278-286.
D'Arpa P, Beardmore C, and Liu LF (1990) Involvement of nucleic acid synthesis in cell killing mechanisms of topoisomerase poisons. Cancer Res 50: 6919-6924.
DeVries-Seimon TA, Ohm AM, Humphries MJ, and Reyland ME (2007) Induction of apoptosis is driven by nuclear retention of protein kinase C . J Biol Chem 282: 22307-22314.
Eagar TN, Tang Q, Wolfe M, He Y, Pear WS, and Bluestone JA (2004) Notch 1 signaling regulates peripheral T cell activation. Immunity 20: 407-415.[CrossRef][Medline]
Fan QW, Knight ZA, Goldenberg DD, Yu W, Mostov KE, Stokoe D, Shokat KM, and Weiss WA (2006) A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma. Cancer Cell 9: 341-349.[CrossRef][Medline]
Frame S and Cohen P (2001) GSK3 takes centre stage more than 20 years after its discovery. Biochem J 359: 1-16.[CrossRef][Medline]
Grabher C, von Boehmer H, and Look AT (2006) Notch 1 activation in the molecular pathogenesis of T-cell acute lymphoblastic leukaemia. Nat Rev Cancer 6: 347-359.[CrossRef][Medline]
Han EK, Leverson JD, McGonigal T, Shah OJ, Woods KW, Hunter T, Giranda VL, and Luo Y (2007) Akt inhibitor A-443654 induces rapid Akt Ser-473 phosphorylation independent of mTORC1 inhibition. Oncogene 26: 5655-5661.[CrossRef][Medline]
Katayama K, Fujita N, and Tsuruo T (2005) Akt/protein kinase B-dependent phosphorylation and inactivation of WEE1Hu promote cell cycle progression at G2/M transition. Mol Cell Biol 25: 5725-5737.
Kazemi S, Mounir Z, Baltzis D, Raven JF, Wang S, Krishnamoorthy JL, Pluquet O, Pelletier J, and Koromilas AE (2007) A novel function of eIF2 kinases as inducers of the phosphoinositide-3 kinase signaling pathway. Mol Biol Cell 18: 3635-3644.
Lindsley CW, Barnett SF, Layton ME, and Bilodeau MT (2008) The PI3K/Akt pathway: recent progress in the development of ATP-competitive and allosteric Akt kinase inhibitors. Curr Cancer Drug Targets 8: 7-18.[CrossRef][Medline]
Lopiccolo J, Blumenthal GM, Bernstein WB, and Dennis PA (2008) Targeting the PI3K/Akt/mTOR pathway: Effective combinations and clinical considerations. Drug Resist Updat 11: 32-50.[CrossRef][Medline]
Luo Y, Shoemaker AR, Liu X, Woods KW, Thomas SA, de Jong R, Han EK, Li T, Stoll VS, Powlas JA, et al. (2005) Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo. Mol Cancer Ther 4: 977-986.
Mantovani I, Cappellini A, Tazzari PL, Papa V, Cocco L, and Martelli AM (2006) Caspase-dependent cleavage of 170-kDa P-glycoprotein during apoptosis of human T-lymphoblastoid CEM cells. J Cell Physiol 207: 836-844.[CrossRef][Medline]
Markovits J, Pommier Y, Kerrigan D, Covey JM, Tilchen EJ, and Kohn KW (1987) Topoisomerase II-mediated DNA breaks and cytotoxicity in relation to cell proliferation and the cell cycle in NIH 3T3 fibroblasts and L1210 leukemia cells. Cancer Res 47: 2050-2055.
Marone R, Cmiljanovic V, Giese B, and Wymann MP (2008) Targeting phosphoinositide 3-kinase-Moving towards therapy. Biochim Biophys Acta 1784: 159-185.[Medline]
Martelli AM, Nyakern M, Tabellini G, Bortul R, Tazzari PL, Evangelisti C, and Cocco L (2006) Phosphoinositide 3-kinase/Akt signaling pathway and its therapeutical implications for human acute myeloid leukemia. Leukemia 20: 911-928.[CrossRef][Medline]
Neri LM, Borgatti P, Tazzari PL, Bortul R, Cappellini A, Tabellini G, Bellacosa A, Capitani S, and Martelli AM (2003) The phosphoinositide 3-kinase/AKT1 pathway involvement in drug and all-trans-retinoic acid resistance of leukemia cells. Mol Cancer Res 1: 234-246.
Nyakern M, Cappellini A, Mantovani I, and Martelli AM (2006) Synergistic induction of apoptosis in human leukemia T cells by the Akt inhibitor perifosine and etoposide through activation of intrinsic and Fas-mediated extrinsic cell death pathways. Mol Cancer Ther 5: 1559-1570.
Okumura E, Fukuhara T, Yoshida H, Hanada Si S, Kozutsumi R, Mori M, Tachibana K, and Kishimoto T (2002) Akt inhibits Myt1 in the signalling pathway that leads to meiotic G2/M-phase transition. Nat Cell Biol 4: 111-116.[CrossRef][Medline]
Palomero T, Sulis ML, Cortina M, Real PJ, Barnes K, Ciofani M, Caparros E, Buteau J, Brown K, Perkins SL, et al. (2007) Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia. Nat Med 13: 1203-1210.[CrossRef][Medline]
Papa V, Tazzari PL, Chiarini F, Cappellini A, Ricci F, Billi AM, Evangelisti C, Ottaviani E, Martinelli G, Testoni N, et al. (2008) Proapoptotic activity and chemosensitizing effect of the novel Akt inhibitor perifosine in acute myelogenous leukemia cells. Leukemia 22: 147-160.[CrossRef][Medline]
Sale EM and Sale GJ (2008) Protein kinase B: signalling roles and therapeutic targeting. Cell Mol Life Sci 65: 113-127.[CrossRef][Medline]
Seminario MC, Precht P, Wersto RP, Gorospe M, and Wange RL (2003) PTEN expression in PTEN-null leukaemic T cell lines leads to reduced proliferation via slowed cell cycle progression. Oncogene 22: 8195-8204.[CrossRef][Medline]
Shelton JG, Blalock WL, White ER, Steelman LS, and McCubrey JA (2004) Ability of the activated PI3K/Akt oncoproteins to synergize with MEK1 and induce cell cycle progression and abrogate the cytokine-dependence of hematopoietic cells. Cell Cycle 3: 503-512.[Medline]
Tafuri A, Gregorj C, Petrucci MT, Ricciardi MR, Mancini M, Cimino G, Mecucci C, Tedeschi A, Fioritoni G, Ferrara F, et al. (2002) MDR1 protein expression is an independent predictor of complete remission in newly diagnosed adult acute lymphoblastic leukemia. Blood 100: 974-981.
Tazzari PL, Cappellini A, Ricci F, Evangelisti C, Papa V, Grafone T, Martinelli G, Conte R, Cocco L, McCubrey JA, et al. (2007a) Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts. Leukemia 21: 427-438.[CrossRef][Medline]
Tazzari PL, Tabellini G, Bortul R, Papa V, Evangelisti C, Grafone T, Martinelli G, McCubrey JA, and Martelli AM (2007b) The insulin-like growth factor-I receptor kinase inhibitor NVP-AEW541 induces apoptosis in acute myeloid leukemia cells exhibiting autocrine insulin-like growth factor-I secretion. Leukemia 21: 886-896.[Medline]
Tinel A and Tschopp J (2004) The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress. Science 304: 843-846.
Tokunaga E, Oki E, Egashira A, Sadanaga N, Morita M, Kakeji Y, and Maehara Y (2008) Deregulation of the Akt pathway in human cancer. Curr Cancer Drug Targets 8: 27-36.[CrossRef][Medline]
Uddin S, Hussain A, Al-Hussein K, Platanias LC, and Bhatia KG (2004) Inhibition of phosphatidylinositol 3'-kinase induces preferentially killing of PTEN-null T leukemias through AKT pathway. Biochem Biophys Res Commun 320: 932-938.[CrossRef][Medline]
Vitale A, Guarini A, Ariola C, Mancini M, Mecucci C, Cuneo A, Pane F, Saglio G, Cimino G, Tafuri A, et al. (2006a) Adult T-cell acute lymphoblastic leukemia: biologic profile at presentation and correlation with response to induction treatment in patients enrolled in the GIMEMA LAL 0496 protocol. Blood 107: 473-479.
Vitale A, Guarini A, Chiaretti S, and Foa R (2006b) The changing scene of adult acute lymphoblastic leukemia. Curr Opin Oncol 18: 652-659.[Medline]
Weerkamp F, van Dongen JJ, and Staal FJ (2006) Notch and Wnt signaling in T-lymphocyte development and acute lymphoblastic leukemia. Leukemia 20: 1197-1205.[CrossRef][Medline]
Zhivotovsky B and Orrenius S (2005) Caspase-2 function in response to DNA damage. Biochem Biophys Res Commun 331: 859-867.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  F. Chiarini, F. Fala, P. L. Tazzari, F. Ricci, A. Astolfi, A. Pession, P. Pagliaro, J. A. McCubrey, and A. M. Martelli Dual Inhibition of Class IA Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin as a New Therapeutic Option for T-Cell Acute Lymphoblastic Leukemia Cancer Res., April 15, 2009; 69(8): 3520 - 3528. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
