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  • 1
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    Multiple Strategies Based on MEK, mTOR and BCL-2 Signal Transduction Inhibition in Acute Lymphoblastic Leukemia (ALL)
    American Society of Hematology ; 2008
    In:  Blood Vol. 112, No. 11 ( 2008-11-16), p. 5034-5034
    Details
    In: Blood, American Society of Hematology, Vol. 112, No. 11 ( 2008-11-16), p. 5034-5034
    Abstract: In spite of improvements in the last decade, relapses still occurs in the majority of ALL patients, with a long-term survival rate of only 30–40%. Recently, small-molecule inhibitors have been developed for targeting deregulated signal transduction pathways involved in proliferation and apoptosis. Current evidence identifies the Raf/MEK/ERK, the PI3K/AKT/mTOR, and the Bcl-2 pathways as potentially relevant targets for therapeutic intervention. We have previously demonstrated that constitutive ERK phosphorylation is an independent predictor of failure to achieve complete remission in adult ALL (Blood2007;109:5473) and thus we evaluated the in vitro activity of MEK-inhibitors. However, neither PD98059 nor PD0325901 affected cell growth, cell cycle distribution, and/or apoptosis in ALL cell lines (IC50 & gt;1 μM for PD0325901). These results were confirmed in primary ALL samples, in which PD98059 significantly (P=0.012) inhibited ERK phosphorylation in 8/12 samples, without inducing cell cycle changes or apoptosis. We next investigated the activity of the mTOR inhibitor Temsirolimus. Temsirolimus displayed a biphasic dose-response in a panel of different ALL cell lines, with a flat curve (35–55% of inhibition) at concentrations ranging between 1 and 5,000 nM and more pronounced growth inhibition at concentrations ≥ 10 μM. The CEM cell line was the most sensitive (IC50: 7 nM), Jurkat showed intermediate sensitivity (IC50: 200 nM), while MOLT-4 were resistant (IC50 & gt; 20,000 nM). Cell growth inhibition was associated with inhibition of cell cycle progression, while apoptosis induction was observed in less than 15% of the cells even at the highest concentration of Temsirolimus (20 μM). We then investigated the cell cycle and apoptotic effects of ABT-737 (kindly provided by Abbott Laboratories to A.T.), a Bcl-2/Bcl-xL (BH3 mimetic) inhibitor, in ALL cell lines and in 10 primary samples. ABT-737 showed a potent dose- and time-dependent growth-inhibitory activity in MOLT-4 (IC50: 198 nM), associated with loss of mitochondrial membrane potential, caspase activation, Bcl-2 cleavage, and ultimately apoptosis induction. Conversely, CEM cells proved resistant (IC50: 12,000 nM). We also found that ABT-737 was highly effective in primary adult and childhood ALL, independent of their chromosomal abnormalities, with a significant decrease in viability (p=0.008) and a remarkable induction of apoptosis (from a mean baseline value of 16.8±8.8% to 43.6±22.8%, p=0.04) at 10 nM ABT-737. A dose-dependent down-regulation of Bcl-2 and Bcl-xL expression was observed in sensitive samples, but not in the only resistant one. In summary, our study shows that ALL cells, do not undergo apoptosis in response to single-pathway inhibition, suggesting the presence of multiple, redundant pathways that preserve leukemic cell survival. The only notable exception was the Bcl-2/Bcl-xL inhibitor ABT-737. Studies are ongoing to identify mechanism-driven combinations of agents that would disrupt multiple signal transduction pathways, resulting in synergistic killing and, ultimately, in novel therapeutic strategies for ALL.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V112.11.5034.5034
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2008
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 2
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    Parp1 Localizes within the Dnmt1 Promoter and Protects Its Unmethylated State by Its Enzymatic Activity
    Public Library of Science (PLoS) ; 2009
    In:  PLoS ONE Vol. 4, No. 3 ( 2009-3-5), p. e4717-
    Details
    In: PLoS ONE, Public Library of Science (PLoS), Vol. 4, No. 3 ( 2009-3-5), p. e4717-
    Type of Medium: Online Resource
    ISSN: 1932-6203
    URL: Article
    URL: Article
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    DOI: 10.1371/journal.pone.0004717
    DOI: 10.1371/journal.pone.0004717.g001
    DOI: 10.1371/journal.pone.0004717.g002
    DOI: 10.1371/journal.pone.0004717.g003
    DOI: 10.1371/journal.pone.0004717.g004
    DOI: 10.1371/journal.pone.0004717.g005
    DOI: 10.1371/journal.pone.0004717.g006
    DOI: 10.1371/journal.pone.0004717.s001
    DOI: 10.1371/journal.pone.0004717.s002
    DOI: 10.1371/journal.pone.0004717.s003
    Language: English
    Publisher: Public Library of Science (PLoS)
    Publication Date: 2009
    detail.hit.zdb_id: 2267670-3
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  • 3
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    The BCL-2 Antagonist ABT-737 Is Highly Effective on Primary Acute Lymphoblastic Leukemia Cells.
    American Society of Hematology ; 2007
    In:  Blood Vol. 110, No. 11 ( 2007-11-16), p. 155-155
    Details
    In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 155-155
    Abstract: The treatment of adult acute lymphoblastic leukemia (ALL) remains unsatisfactory. A potential hope is now given to Philadelphia-positive cases by targeted treatment modalities. Among other pathways involved in cell proliferation, we have recently demonstrated (Blood2007; 109:5473) the unfavorable role of ERK1/2 phosphorylation as an independent predictor of complete remission (CR) in adult ALL, suggesting the potential therapeutic value of other targeted therapies. The B-cell leukemia/lymphoma 2 (Bcl-2) family of proteins are important regulators of apoptosis and are frequently found aberrantly expressed, particularly in lymphoid malignancies. The role of Bcl-2 overexpression in tumorigenesis and chemoresistance prompted us to investigate whether the inhibition of the antiapoptotic function may result also in ALL in an attractive therapeutic strategy. In this study, we thus investigated the cell cycle and apoptotic effects of ABT-737 (kindly provided by Abbott Laboratories), a Bcl-2 (BH3) inhibitor, on both lymphoid leukemia cell lines and primary adult and childhood ALL cells. The lymphoid leukemia cell lines CEM and MOLT-4 were exposed to increasing concentrations of ABT-737 (from 0.1 to 1 μM) up to 72 hours. A dose- and time-dependent cell growth arrest and induction of apoptosis was found. In fact, measuring the subG0/1 peak at 48 hours, the levels of apoptosis increased in the CEM cell line from 14.1% (DMSO) to 34.4%, 64.5%, 86.5% and 98.6% at ABT-737 concentrations of 0.1, 0.25, 0.5 and 1 μM, respectively. Similarly, 48 hours of exposure to ABT-737 increased in MOLT-4 the Annexin V-positive cells from 7.2% to 64.2%. The effects of ABT-737 were then examined on primary blasts from 9 ALL patients (6 adults and 3 children). Bone marrow aspirates with a blast infiltration & gt;70% were obtained at diagnosis from patients broadly characterized for clinical and biological parameters, as well as therapeutic response. ALL cells were cultured in vitro with ABT-737 (at increasing concentrations from 0.01 to 1 μM) for 24 hours. A significant decrease in viability was observed at 0.01 μM (p=0.008) with a remarkable dose-dependent increase of apoptosis. In fact, Annexin V-positive cells increased from a mean baseline value of 16.8% ± 8.8 to 43.6% ± 22.8 (p=0.04), 66% ± 21.3 (p=0.0001), 70.3% ± 26.9 (p=0.04), 74.6% ± 18.9 (p=0.03) and 76.2% ± 11.8 (p & lt;0.0001) in the presence of ABT-737 at 0.01, 0.1, 0.25, 0.5 and 1 μM, respectively. A significant cell killing was demonstrated in all samples (9/9), including Ph-positive ALL. No significant cell cycle changes were instead detected even at higher concentration of ABT-737. In summary, our study shows for the first time a potent growth-inhibitory and pro-apoptotic activity of the Bcl-2 antagonist ABT-737, at nanomolar concentrations, on primary cells from adult and childhood ALL samples. These results prompt to further extend pre-clinical studies in the different biologically-defined subset of ALL and suggest a potential clinical development of a Bcl-2 family inhibitor in this disease.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V110.11.155.155
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 4
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    Functional Effects of the Bcl-2/Bcl-xL Inhibitor ABT-737 on Primary Cells from Smoldering Multiple Myeloma.
    American Society of Hematology ; 2007
    In:  Blood Vol. 110, No. 11 ( 2007-11-16), p. 4782-4782
    Details
    In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 4782-4782
    Abstract: Smoldering multiple myeloma (MM) is an asymptomatic plasma-cell disorder with a high risk of progression to symptomatic MM. The time to progression and the underlying mechanisms are still unclear. The bcl-2 family proteins are key regulators of cell survival and are frequently found aberrantly expressed in MM, conferring resistance to chemotherapy. The activity of bcl-2 family inhibitors is currently under investigation in MM. In contrast, the activity in smoldering MM cells is still unexplored. We investigated the cell cycle and apoptotic effects of the bcl-2/bcl-xL inhibitor, ABT-737 (kindly provided by Abbott Laboratories) on primary CD138+ malignant plasma cells purified from smoldering MM samples. In addition, we extended this study to MM cell lines and to symptomatic MM. The MM cell lines (KMS18, ARH-77) exposed for 24, 48 and 72 hours to scalar concentrations of ABT-737 (from 0.1 to 1 μM) showed a dose- and time-dependent cell growth inhibition (IC50s 0.286 and 0.346 μM, respectively, at 72 hours) due to a significant increase of apoptotic cells. In addition, cell cycle analysis on KMS18 demonstrated at 72 hours a significant G1-phase depletion: from 54.6% ± 4.5 (DMSO) to 26.9% ± 1.4 (1 μM ABT-737) (p=0.021). The effects of ABT-737 were then examined on primary cells from 7 smoldering MM. Bone marrow cells, following CD138 enrichment ( 〉 80% of purity), were cultured in vitro with ABT-737 (at scalar concentrations from 0.1 to 1 μM) up to 72 hours. A statistically significant reduction in cell growth and increase in the percentage of apoptotic cells was observed in response to 0.1μM ABT-737. In particular, a statistically significant pro-apoptotic activity of ABT-737 was demonstrated by the increment of the subG0/1 peak (Acridine-Orange) at 24 hours from 24.0% ± 8.9 in DMSO to 51.7% ± 17.7 (p=0.02) and 77.6% ± 15.6 (p=0.001) in the presence of ABT-737 at 0.1 and 1 μM, respectively. The effects of ABT-737 on smoldering MM were then compared to those obtained following exposure of CD138+ cells from symptomatic MM (14 cases) to the bcl-2/bcl-xL inhibitor. The results obtained demonstrated that ABT-737 acts in smoldering MM as well as in symptomatic MM in a similar manner. In fact, a comparable increment of the subG0/1 peak was also recorded in symptomatic MM: from 31.2% ± 17.8 in DMSO to 56.4% ± 18.7 (p=0.017) and 69.9% ± 16.4 (p=0.0002) in the presence of ABT-737 at 0.1 and 1 μM, respectively, at 24 hours. These data were confirmed by measuring Annexin V+ cells. In conclusion, ABT-737 shows potent in vitro growth-inhibitory and pro-apoptotic activity at nanomolar concentrations in smoldering MM. This effects was similarly achieved in symptomatic MM. These data warrant a further pre-clinical/clinical development of the bcl-2 family inhibitor ABT-737 in patients with MM regardless of the disease status, aiming also at delaying disease progression.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V110.11.4782.4782
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
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  • 5
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    Protein Expression and Methylation Status of the CKI p21, p15 and p16 in Adult Acute Lymphoblastic Leukemia (ALL) Patients: Prognostic Implications.
    American Society of Hematology ; 2006
    In:  Blood Vol. 108, No. 11 ( 2006-11-16), p. 1843-1843
    Details
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 1843-1843
    Abstract: Cyclin-dependent kinase inhibitors (CKI) regulate cell division resulting aberrantly expressed in many types of cancer. Alterations of CKI have been reported in acute leukemia, as the result of gene promoter methylation. Despite the common frequency of these alterations, little has been reported on the role of CKI aberrant protein expression and results are less clear, especially in acute lymphoblastic leukemia (ALL). The aim of this study was to analyze p21, p15 and p16 protein expression and their gene methylation status in primary cells from adult ALL cases enrolled in the LAL2000 GIMEMA protocol. Normal peripheral blood lymphocytes (PBL) and 91 primary samples from untreated ALL patients were evaluated in this study. The p21, p15 and p16 protein expression was analyzed by Western blot using the specifically MoAbs. The CKI gene methylation status was investigated using a widely accepted method based on bisulfite modification of DNA, followed by the use of the methylation-specific PCR assay (MSP). This assay was further validated in vitro by SSI methylase. Normal PBL from 10 healthy donors, as described, did not expressed all CKIs and resulted unmethylated. The p21 expression was found in 28/91 cases (30.8%); in contrast, samples were found constantly unmethylated. The p15 expression was found in 44/85 cases (51.8%) and its gene methylated in 41.7%; a significant correlation was found between absence of protein expression and gene methylation (P=0.040). The p16 resulted never expressed in adult ALL, while its promoter was found methylated in 8/42 cases (19.1%). A significant association (P=0.037) was observed between p21 expression and immunophenotype; in fact, 3/24 (12.5%) T-ALL and 24/65 (36.9%) B-lineage ALL expressed this protein. The p16 methylation was associated with T-ALL (P=0.082). Achievement of CR was not influenced by single protein expression, nor by gene methylation status. However, the co-expression of p15 and p21 was associated with failure to induction treatment; in fact, only 6/67 (9%) of patients co-expressing p15 and p21 achieved CR (P=0.021). In summary, in adult ALL p21 is not methylated and p16 is never found expressed, and CR achievement is adversely affected by the co-expression of p21 and p15. In conclusion, we report that in addition to CKI methylation, aberrant expression of CKI, namely p21 and p15, is associated with poor outcome in adult ALL, suggesting that chemotherapy resistance may be promoted in these cases by cell cycle arrest and/or abnormal survival.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V108.11.1843.1843
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
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  • 6
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    Breast Cancer Resistance Protein (BCRP) in Adult Acute Lymphoblastic Leukemia Patients.
    American Society of Hematology ; 2004
    In:  Blood Vol. 104, No. 11 ( 2004-11-16), p. 1897-1897
    Details
    In: Blood, American Society of Hematology, Vol. 104, No. 11 ( 2004-11-16), p. 1897-1897
    Abstract: The transporter breast cancer resistance protein (BCRP) is a member of the ATP-binding cassette which has been recently described as a protein involved in the multidrug resistance (MDR) phenotype. There are currently no reports concerning the role of this protein in adult acute lymphoblastic leukemia (ALL). The aim of this study was to evaluate the frequency of BCRP expression, its correlation with other MDR-related proteins and their prognostic role in 93 untreated adult ALL patients enrolled in the GIMEMA protocols LAL 0496 and LAL 2000. BCRP protein expression was detected by flow cytometry using the monoclonal antibody BXP-34 (Kamiya, Seattle, WA) and the analysis was performed by the Kolmogorov-Smirnov (KS) statistic test (D-value). Detection of BCRP in the cell lines MCF7 pcDNA3 and MDA231 pcDNA3 showed a D-value of 0.12 ± 0.11 and 0.09 ± 0.06, respectively (negative controls). In contrast, the cell lines MCF7 pcDNA3 clone 8 and MDA231 pcDNA3 clone 23 overexpressed BCRP with a D-value of 0.44 ± 0.21 and 0.33 ± 0.11, respectively (positive controls). Analysis of primary ALL samples showed a BCRP expression (D-value 〉 0.20) in 70/93 (75.3%) cases, with a mean value of 0.33 ± 0.19 (range 0.00-0.87, median 0.33) in the overall population analyzed. BCRP expression resulted higher (mean 0.34 ± 0.03) in samples from patients with WBC counts ≥100 x 109/L compared to the values (mean 0.26 ± 0.13) found in those with lower WBC counts (P=0.06). No significant difference was found between BCRP expression and the clinical characteristics of the patients. The analysis was then extended to the Multidrug Resistance Associated protein (MRP1) and to the MDR1/P-glycoprotein-170 (MDR1). A D-value ≥0.20 and ≥0.05 was found in 53.4% (39/73) and 30.2% (26/86) of cases, respectively. Samples analyzed for both BCRP and MRP1 expression (73/93) showed a significant correlation (R2 = 0.25; P=0.0001): 12.3% of samples were negative for both proteins, while 43.8% expressed both BCRP and MRP1 proteins. In addition, MRP1 negative samples showed lower BCRP levels (mean 0.30, range 0–0.60) compared to MRP1 positive cases (mean 0.38, range 0–0.87) (P=0.017). BCRP expression did not correlate with MDR1 expression. None of these MDR markers correlated separately with achievement of complete remission (CR). In contrast, BCRP/MRP1 co-expression correlated significantly (P=0.034) with failure to respond to induction treatment: 47.8% (11/23) of BCRP+/MRP1+ patients failed to achieve CR, while 78.4% (29/37) of cases negative for only one protein (BCRP-/MRP1+ or BCRP+/MRP1−) or for both (BCRP-/MRP1-) responded to induction treatment. Multivariate analysis (Backward method) confirmed the unfavorable prognostic role on CR of these two proteins concomitantly expressed (P=0.029; OR 0.27, 95% CI, 0.081–0.87). In conclusion, our study shows that BCRP is expressed in a significant proportion of adult ALL. The co-expression of BCRP and MRP1 plays an unfavorable prognostic role on achievement of CR in ALL. These data suggest that the resistance phenotype may be the result of the combined effects of several transporter proteins involved in the MDR process and therefore detection of all these proteins may better predict clinical response to induction treatment.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V104.11.1897.1897
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2004
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  • 7
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    Molecular and Functional Effects of the Novel MEK Inhibitor PD0325901 in Preclinical Models of Human Leukemias.
    American Society of Hematology ; 2006
    In:  Blood Vol. 108, No. 11 ( 2006-11-16), p. 254-254
    Details
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 254-254
    Abstract: The Raf/MEK/ERK signaling module plays a pivotal role in the regulation of cell proliferation, survival, and differentiation. Our group, among others, has recently demonstrated that this pathway is frequently dysregulated in hematological malignancies and may constitute an attractive therapeutic target, particularly in AML. Here we investigated the effects of PD0325901, a novel MEK inhibitor, on phospho-protein expression, gene expression profiles, cell proliferation, and apoptosis in cell line models of AML, ALL, multiple myeloma (MM), ex vivo-cultured primary AML blasts, and oncogene-transformed hematopoietic cells. AML cell lines (OCI-AML2, OCI-AML3, HL-60) were strikingly sensitive to PD0325901 (IC50: 5–19 nM), NB4 (APL) and U266 (MM) showed intermediate sensitivity (IC50: 822 and 724 nM), while all the lymphoid cell lines tested and the myeloid cell lines U937 and KG1 were resistant (IC50 〉 1000 nM). Cell growth inhibition was due to inhibition of cell cycle progression and induction of apoptosis. A statistically significant reduction in the proportion of S-phase cells (p=0.01) and increase in the percentage of apoptotic cells (p=0.019) was also observed in 18 primary AML samples in response to 100 nM PD0325901. Analysis of the correlation between sensitivity/resistance to PD0325901 and Ras/Raf mutation status is currently ongoing. PD0325901 effects were also examined in a panel of IL-3-dependent murine myeloid FDC-P1 cell lines transformed to grow in response to 11 different oncogenes in the absence of IL-3. Fms-, Ras-, Raf-1-, B-Raf-, MEK1-, IGF-1R-, and STAT5a-transformed FDC-P1 cells were very sensitive to PD0325901 (IC50: ~ 1 nM), while A-Raf-, BCR-ABL-, EGFR- or Src-transformed cells were 10 to 100 fold less sensitive (IC50: 10 to 100 nM); the parental, IL-3 dependent FDC-P1 cell line had an IC50 〉 1000 nM. Analysis of the phosphorylation levels of 18 different target proteins after treatment with 10 nM PD0325901 showed a 5- to 8-fold reduction in ERK-1/2, observed only in sensitive cell lines, and a 2-fold reduction in JNK and STAT3 phosphorylation. PD0325901 (10 nM) treatment also profoundly altered the gene expression profile of the sensitive cell line OCI-AML3: 96 genes were modulated after 24 h (37 up- and 59 down-regulated), most of which involved in cell cycle regulation. Changes in cyclin D1 and D3, cyclin E, and cdc 25A were also validated at the protein level. Overall, PD0325901 shows potent growth-inhibitory and pro-apoptotic activity, indicating that MEK may be an appropriate therapeutic target in an array of different hematological malignancies. Further preclinical/clinical development of this compound is warranted, particularly in myeloid leukemias.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V108.11.254.254
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 8
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    ERK1/2 phosphorylation is an independent predictor of complete remission in newly diagnosed adult acute lymphoblastic leukemia
    American Society of Hematology ; 2007
    In:  Blood Vol. 109, No. 12 ( 2007-06-15), p. 5473-5476
    Details
    In: Blood, American Society of Hematology, Vol. 109, No. 12 ( 2007-06-15), p. 5473-5476
    Abstract: Extracellular signal-regulated kinase-1/2 (ERK1/2) is frequently found constitutively activated (p-ERK1/2) in hematopoietic diseases, suggesting a role in leukemogenesis. The aim of this study was to assess the expression and clinical role of p-ERK1/2 in adult acute lymphoblastic leukemia (ALL). In 131 primary samples from adult de novo ALL patients enrolled in the Gruppo Italiano per le Malattie Ematologiche dell'Adulto (GIMEMA) Leucemia Acute Linfoide (LAL) 2000 protocol and evaluated by flow cytometry, constitutive ERK1/2 activation was found in 34.5% of cases; these results were significantly associated with higher white blood cell (WBC) values (P = .013). In a multivariate analysis, p-ERK1/2 expression was an independent predictor of complete remission achievement (P = .027). Effective approaches toward MEK inhibition need to be explored in order to evaluate whether this may represent a new therapeutic strategy for adult ALL patients.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2006-05-021071
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
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  • 9
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    Role of Extracellular Signal-Regulated Kinase-1/2 (ERK) on Complete Remission Achievement of Primary Adult Acute Lymphoblastic Leukemia Patients Enrolled in the GIMEMA Protocol LAL 2000: In Vitro Activity of the MEK Inhibithor PD98059.
    American Society of Hematology ; 2005
    In:  Blood Vol. 106, No. 11 ( 2005-11-16), p. 853-853
    Details
    In: Blood, American Society of Hematology, Vol. 106, No. 11 ( 2005-11-16), p. 853-853
    Abstract: The mitogen-activated protein kinase (MAPK) pathway contributes to the proliferative and differentiative control of hematopoietic cells. Extracellular signal-regulated kinase-1/2 (ERK) is one of the members of the MAPK pathway; its constitutive activation (p-ERK) has been found in a broad spectrum of hematological malignancies, particularly in acute myeloid leukemia (AML). A molecular therapeutic approach based on MAPK inhibitors has been therefore attempted. The aim of this study was to assess the expression and the clinical role of p-ERK in adult acute lymphoblastic leukemia (ALL) evaluating, in addition, the in vitro effects of the MEK inhibitor PD98059, used alone or in combination with chemotherapy, on the proliferation and apoptosis of ALL blast cells. Primary samples from 131 uniformly treated de novo patients enrolled in the GIMEMA LAL 2000 protocol were evaluated, using a p-ERK1/2-specific MoAb (clone E10), by Western blot analysis and flow cytometric assay which allowed single cell phospho-protein determination by the Kolmogorov-Smirnov statistic test (D-value). Lymphoid cell lines (Jurkat, CEM, RAJI and RPMI8866), as well as normal PMA-activated PBL expressed p-ERK, whereas resting PBL exhibited minimal levels (D-value & lt;0.10). In clinical ALL samples p-ERK ranged between 0 and 0.83, constitutive activation (D-value ≥ 0.10) was found in 45/131 samples (34.5%) and resulted significantly associated with higher WBC values ( & gt;50x109/L; P=0.0128) and lower RNA-index of G1 phase (P=0.0296). Age, leukemia phenotype and molecular characteristics did not associate with p-ERK expression, while phospho-protein levels, measured as a continuous variable, correlated with failure to achieve CR (P=0.069). According to cell availability, we evaluated the effects of the MEK inhibitor PD98059 at 25μM on 30 fresh ALL samples, cultured in vitro for 24 hours. Eight of them were characterized by constitutive ERK activation, which was efficiently abrogated in 6; however, this effect did not induce cell cycle changes or apoptosis either alone or in combination with chemotherapeutic agents (Ara-C and/or Dexametasone). In summary, our study demonstrated that p-ERK is expressed in one third of adult ALL samples at presentation. Patients with p-ERK expression are characterized by higher leukemic mass, with a trend to induction treatment failure. MEK inhibition by PD98059 is capable of modulating p-ERK in primary ALL cells, though it does not result in cell cycle/apoptotic changes. In conclusion, ERK signaling appears only partially involved in the control of proliferative signals in adult ALL and modulation of additional pathways may result in more effective therapeutic strategies.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V106.11.853.853
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2005
    detail.hit.zdb_id: 1468538-3
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  • 10
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    Protein Expression of p15 and p21 Plays an Unfavorable Prognostic Role in Adult Acute Lymphoblastic Leukemia (ALL) Patients Independently of Their Gene Promoter Methylation Status.
    American Society of Hematology ; 2007
    In:  Blood Vol. 110, No. 11 ( 2007-11-16), p. 2802-2802
    Details
    In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 2802-2802
    Abstract: Epigenetic silencing of tumor suppressor (TS) genes is a hallmark in human leukemias, particularly through DNA methylation. Cyclin-dependent kinase inhibitors (CKI) are, among other genes, frequently found methylated in their promoter region. This epigenetic modification has been described also in acute lymphoblastic leukemia (ALL). However, the relationship between aberrant DNA methylation and protein expression of TS genes has not yet been extensively evaluated in adult ALL series. The aim of this study was to analyze in primary cells from newly diagnosed adult ALL patients, uniformly treated according to the LAL2000 GIMEMA protocol, the promoter methylation status of p73, p21, p15 and p16, evaluating in addition the p21, p15 and p16 protein expression. The DNA methylation status of promoter regions was investigated, according to cell availability, using a widely accepted method based on bisulfite modification of DNA, followed by methylation-specific PCR assay (MSP). Protein expression was evaluated by Western blot. Normal peripheral blood lymphocytes, as already described, resulted unmethylated for p73, p21, p15 and p16, and did not express the p21, p15 and p16 proteins. In ALL patients, in contrast, only the p21 promoter region was found constantly unmethylated. The p15, p16 and p73 promoter genes were found methylated in 15/37 (40.5%), 8/43 (18.6%) and 9/36 (25%) patients, respectively. Only 2/23 cases (8.6%) resulted simultaneously methylated for p15, p16 and p73. The p21 and p15 protein expression was found in 28/85 (32.9%) and 44/85 cases (51.8%), respectively. The p16 protein, in contrast, was never expressed. The p16 methylation was associated with the T-ALL (P=0.005) phenotype and with higher white blood cell (WBC) counts (P=0.027). Resistance to spontaneous induction of apoptosis was significantly associated with p21 protein expression (P=0.019) and its co-expression with p15 (P=0.049). Achievement of CR was not influenced by gene methylation status, nor by single protein expression. Interestingly, the co-expression of p15 and p21 was associated with failure to induction treatment: only 6/63 (9.5%) patients co-expressing p15 and p21 obtained a CR (P=0.027). Multivariate analysis confirmed the unfavorable role of this protein co-expression (P=0.059) on CR achievement. In contrast, once patients achieved remission, p21 protein expression was associated with a prolonged DFS, as confirmed by multivariate analysis for DFS (P=0.039). In conclusion, p15 and p21 protein expression plays an unfavorable prognostic role in adult ALL patients independently of the p73, p21, p15 and p16 gene promoter methylation status.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V110.11.2802.2802
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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