- Email: [email protected]
P026 Different pattern of JNK activity in MDS-related versus de novo AML: implications for apoptosis-resistance and treatment failure in MDS-related AML
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Posters
of PKCz has been described as a conditioning step to signaling cascade. Methods: In the present study, we examined the cellular location of PKCz in immunomagnetically isolated and SDF1a-treated marrow CD34+ cells from 7 patients with MDS [3RA, 1RARS, 3RAEB(t)] by immunofluorescence. Human CB, mPB and BM CD34+ cells (n = 5) from healthy individuals were also studied as normal controls. Results: The majority of normal CD34+ cells showed translocation of PKCz from the cytoplasm to the membrane after incubation with SDF1a (200 ng/ml, 1 h). In contrast, the number of CD34+ cells from the MDS patients displaying membranal PKCz localization was either reduced (<40% positive cells in 3 of the 7 patients) or absent (4 of the 7 cases), indicating the lack of signaling upstream to PKCz translocation. Similar to our previous observation, when CD34+ cells from MDS patients were allowed to migrate toward a gradient of SDF1a (125 ng/ml), they displayed an impaired SDF1a chemotactic response compared to the normal CD34+ cells [MDS mean values of % input: 3% (0.5−6%), normal mean values of % input: 15% (4−35%)]. The membranal immunolocalization of PKCz was seen within migrated and non-migrated cell aliquots of normal CD34+ cells (>40% positive cells), whereas it was considerably reduced and only detected in 2 of the MDS samples examined (<15% positive cells). The addition of neutralizing monoclonal antibody anti-CXCR4 decreased the chemotactic response of normal and MDS CD34+ cells by 90% and the translocation of PKCz to the plasma membrane. Discussion: The lack of PKCz targeting to the plasma membrane may represent a defective signal transduction mechanism downstream to CXCR4 and therefore be responsible for the impaired SDF1a-mediated migration and development of marrow CD34+ cells from patients with MDS. P025 Dap-kinase hypermethylation and apoptosis in myelodysplastic syndromes M.T. Voso ° , D. Gumiero, F. D’Alo’, A. Scardocci, M. Greco, E. Fabiani, A. Di Ruscio, F. Guidi, S. Rutella, S. Bellesi, L. Fianchi, S. Hohaus, G. Leone. Istituto di Ematologia, Universita’ Cattolica S. Cuore, Roma *E-mail: [email protected] Introduction: Aberrant CpG island methylation leading to silencing of several genes has gained increasing recognition as important factor in development and progression in myelodysplastic syndromes (MDS). Among genes regulated by promoter hypermethylation, death-associated protein kinase (DAP-kinase) is a serine/threonine kinase that participates in several apoptotic pathways. We studied the effects of DAP-kinase promoter hypermethylation in MDS and the contribution of DAP-kinase re-activation due to demethylating agents in cell line models.
Methods: DNA was extracted from bone marrow mononuclear cells and CD34+ cells of 106 MDS patients at diagnosis and during follow-up. DAP-kinase promoter methylation and expression were analyzed by MS-PCR and real time RT-PCR. Apoptosis was evaluated by 7-AAD incorporation. Results: DAP-kinase was methylated in 37% of MDS patients. No association with patients’ characteristics was found. Sequential samples from 11 MDS patients were studied during follow-up, at a median of 12.4 months from initial diagnosis. Eight patients, 7 of whom with disease progression, maintained the same DAP-kinase unmethylated (n = 6) or methylated status. Freshly isolated CD34+ cells from MDS showed a DAP-kinase methylation status similar to the CD34− fractions. Moreover, hypermethylation was associated to reduced mRNA expression in CD34+ cells, when compared to unmethylated samples. Apoptosis was higher in BM samples from MDS patients compared to normal controls. In MDS patients, the apoptosis rate increased in BM mononuclear cells and CD34+ cells of patients “unmethylated” for DAP-kinase, when compared to “methylated” patients (p = 0.068, and p = 0.1). In the HL-60 cell line, restoration of the unmethylated state of DAP-kinase occurred after 72−96 hours exposure to 1 mM decitabine, and was associated to re-expression of DAP-kinase. Moreover, treatment was associated to cell growth inhibition and increase of apoptotic cells. In particular, following 4 days of treatment of HL60 cells with azacitidine, trichostatin A, or both drugs, we found a cell growth inhibition of 65%, 32% and 82%, respectively, compared to mock treated cells. Correspondingly, mean apoptosis increased in the presence of azacitidine (p = 0.02), with a synergistic effect when combined to trichostatin A (p = 0.009). Discussion: DAP-kinase promoter hypermethylation reduces apoptosis in MDS and its re-expression may have an important role in the response to epigenetic treatment. P026 Different pattern of JNK activity in MDS-related versus de novo AML: implications for apoptosis-resistance and treatment failure in MDS-related AML E.D. Lagadinou ° , P. Ziros, O. Tsopra, A. Klamargias, E. Thanopoulou, A. Kouraklis, A. Spyridonidis, M. Karakantza, N.C. Zoumbos. Department of Internal Medicine, Hematology Division, University Hospital of Patras, 26500 Patras, Greece *E-mail: [email protected] Introduction: AML arising from antecedent MDS (MDS/AML) is frequently refractory to treatment with standard chemotherapeutic agents. In a model of drugsensitive and resistant AML cell lines we have previously shown that the JNK signaling pathway mediates
2. Molecular mechanisms and pathophysiology I – Stromal cells, cytokines and apoptosis proapoptotic signals in response to anthracycline treatment. Moreover, in the resistant cell lines and primary AML blasts, we have observed that impairment of drug-related JNK activation was associated with a failure of anthracyclins to induce apoptosis. In the present study we investigated whether deregulation of JNK response is implicated in the comparative refractoriness of the MDS/AML to standard chemotherapy. Materials and Methods: Marrow blasts from10 MDS/AML and 14 de novo AML patients were exposed in vitro to anthracycline treatment (1 mM Daunorubicin, DNR) and the basal levels as well as the inducibility of JNK activity were detected at certain time points. Drug-induced apoptotic response of blast cells was determined by flow cytometry with Annexin staining. All patients were evaluated at diagnosis with the exception of one MDS/AML patient who was also evaluated at relapse. Results: Increased basal JNK activity levels were detected in 70% of the MDS/AML and in 42.6% of the de novo AML blasts. In 10 out of 14 (71.5%) of the de novo AML cases but only in 2 out of 10 (20%) of the MDS/AML samples the in vitro drug treatment resulted in an upregulation of basal JNK activity levels. DNR had no effect on basal JNK activity in 8 out of 10 (80%) of the MDS/AML samples and in 4 out of 14 (28.5%) of the de novo AML. In both anthracycline treated MDS/AML and de novo AML blasts the upregulation of basal JNK activity was followed by the induction of significant apoptosis, in relation to untreated controls (p = 0.0005 < 0.05). On the contrary, in the MDS/AML and de novo AML samples that JNK activity remained immutable in response to drug treatment, we observed a failure of DNR to induce significant apoptosis (p = 0.4697 > 0.05). One of the two MDS/AML patients that activated JNK and underwent DNR-induced apoptosis at diagnosis entered a CR and was also evaluated at relapse. Interestingly, neither JNK activation nor apoptosis induction was observed and patient failed to enter a second CR. Discussion: Our findings suggest that MDS/AML blasts exhibit a different pattern of basal and anthracycline induced JNK activity levels compared to the de novo AML blasts. Taking into account our previous observations defining JNK as a critical proapoptotic mediator of anthracycline treatment in AML cells, we suggest that the different pattern of the JNK response in MDS/AML samples may contribute to the worse prognosis of the MDS/AML patients.
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P027 Analysis of cell cycle regulatory genes expression in the bone marrow of adult de novo myelodysplastic syndromes (MDS) C. Economopoulou1 ° , V. Pappa1 , F. Kontsioti1 , V. Kapsimali2 , P. Tsirigotis1 , S. Papageorgiou1 , V. Giannopoulou1 , K. Girkas1 , E. Papageorgiou, J. Dervenoulas1 , T. Economopoulos1 . 1 Second Department of Internal Medicine Propaedeutic, University of Athens, Attikon University General Hospital, Athens, Greece; 2 Immunology Department, Evangelismos Hospital, Athens, Greece *E-mail: [email protected] Introduction: Alterations of cell cycle regulatory genes, such as cyclin-dependent kinases (CDKs) and CDK inhibitor proteins (CDKIs) have been suggested to play a role in oncogenesis. The aim of the present study was to examine CDK1−4, p16, p21, p27 and PISSLRE expression and apoptosis in the bone marrow (BM) of adult de novo MDS and to correlate our findings with clinical parameters and prognosis. Methods: We studied 46 cases of MDS including 14 RA, 21 RAEB, 6 CMML and 5 RARS according to FAB criteria. Apoptosis was determined by Annexin method on BM mononuclear cells. The expression of CDK1−4, p16, p21, p27 and PISSLRE was determined using an RNase Protection System. A pool of RNA from normal BM mononuclear cells was used as a normal control. The expression of each gene was compared to that of two housekeeping genes (GAPDH and L32) using Image Master Software. The level of each gene expression was compared to that obtained from normal RNA pool. Results: Cases expressing higher levels of CDK1, 2 and 3 had significantly lower levels of apoptosis (p = 0.01, 0.04 and 0.02 respectively). A positive correlation was observed between all CDKs’ levels of expression (p < 0.001). A significant negative correlation was observed between p27 and both CDK2 and CDK4 levels (p = 0.03 and 0.05 respectively). Moreover PISSLRE expression did correlate with CDK3,4 and p16 expression (p = 0.02, 0.008 and <0.001 respectively). The level of expression as well as the percentage of positive cases for all genes was not significantly different between FAB and IPSS categories. CDK2 and p27 levels were higher in patients with abnormal and normal karyotype respectively (p = 0.04 and 0.02); p27 expression was also higher in patients with no BM blasts (p = 0.05). Higher PISSLRE levels correlated positively with normal WBC values (p = 0.03). Discussion: The expression levels of all genes did not correlate with FAB and IPSS groups in patients with MDS. Cell cycle inhibitory gene p27 expression was significantly higher in cases with no BM blasts and normal karyotype, while cell cycle progression gene CDK2 was significantly higher in cases with abnormal karyotype. Cases expressing
Posters
of PKCz has been described as a conditioning step to signaling cascade. Methods: In the present study, we examined the cellular location of PKCz in immunomagnetically isolated and SDF1a-treated marrow CD34+ cells from 7 patients with MDS [3RA, 1RARS, 3RAEB(t)] by immunofluorescence. Human CB, mPB and BM CD34+ cells (n = 5) from healthy individuals were also studied as normal controls. Results: The majority of normal CD34+ cells showed translocation of PKCz from the cytoplasm to the membrane after incubation with SDF1a (200 ng/ml, 1 h). In contrast, the number of CD34+ cells from the MDS patients displaying membranal PKCz localization was either reduced (<40% positive cells in 3 of the 7 patients) or absent (4 of the 7 cases), indicating the lack of signaling upstream to PKCz translocation. Similar to our previous observation, when CD34+ cells from MDS patients were allowed to migrate toward a gradient of SDF1a (125 ng/ml), they displayed an impaired SDF1a chemotactic response compared to the normal CD34+ cells [MDS mean values of % input: 3% (0.5−6%), normal mean values of % input: 15% (4−35%)]. The membranal immunolocalization of PKCz was seen within migrated and non-migrated cell aliquots of normal CD34+ cells (>40% positive cells), whereas it was considerably reduced and only detected in 2 of the MDS samples examined (<15% positive cells). The addition of neutralizing monoclonal antibody anti-CXCR4 decreased the chemotactic response of normal and MDS CD34+ cells by 90% and the translocation of PKCz to the plasma membrane. Discussion: The lack of PKCz targeting to the plasma membrane may represent a defective signal transduction mechanism downstream to CXCR4 and therefore be responsible for the impaired SDF1a-mediated migration and development of marrow CD34+ cells from patients with MDS. P025 Dap-kinase hypermethylation and apoptosis in myelodysplastic syndromes M.T. Voso ° , D. Gumiero, F. D’Alo’, A. Scardocci, M. Greco, E. Fabiani, A. Di Ruscio, F. Guidi, S. Rutella, S. Bellesi, L. Fianchi, S. Hohaus, G. Leone. Istituto di Ematologia, Universita’ Cattolica S. Cuore, Roma *E-mail: [email protected] Introduction: Aberrant CpG island methylation leading to silencing of several genes has gained increasing recognition as important factor in development and progression in myelodysplastic syndromes (MDS). Among genes regulated by promoter hypermethylation, death-associated protein kinase (DAP-kinase) is a serine/threonine kinase that participates in several apoptotic pathways. We studied the effects of DAP-kinase promoter hypermethylation in MDS and the contribution of DAP-kinase re-activation due to demethylating agents in cell line models.
Methods: DNA was extracted from bone marrow mononuclear cells and CD34+ cells of 106 MDS patients at diagnosis and during follow-up. DAP-kinase promoter methylation and expression were analyzed by MS-PCR and real time RT-PCR. Apoptosis was evaluated by 7-AAD incorporation. Results: DAP-kinase was methylated in 37% of MDS patients. No association with patients’ characteristics was found. Sequential samples from 11 MDS patients were studied during follow-up, at a median of 12.4 months from initial diagnosis. Eight patients, 7 of whom with disease progression, maintained the same DAP-kinase unmethylated (n = 6) or methylated status. Freshly isolated CD34+ cells from MDS showed a DAP-kinase methylation status similar to the CD34− fractions. Moreover, hypermethylation was associated to reduced mRNA expression in CD34+ cells, when compared to unmethylated samples. Apoptosis was higher in BM samples from MDS patients compared to normal controls. In MDS patients, the apoptosis rate increased in BM mononuclear cells and CD34+ cells of patients “unmethylated” for DAP-kinase, when compared to “methylated” patients (p = 0.068, and p = 0.1). In the HL-60 cell line, restoration of the unmethylated state of DAP-kinase occurred after 72−96 hours exposure to 1 mM decitabine, and was associated to re-expression of DAP-kinase. Moreover, treatment was associated to cell growth inhibition and increase of apoptotic cells. In particular, following 4 days of treatment of HL60 cells with azacitidine, trichostatin A, or both drugs, we found a cell growth inhibition of 65%, 32% and 82%, respectively, compared to mock treated cells. Correspondingly, mean apoptosis increased in the presence of azacitidine (p = 0.02), with a synergistic effect when combined to trichostatin A (p = 0.009). Discussion: DAP-kinase promoter hypermethylation reduces apoptosis in MDS and its re-expression may have an important role in the response to epigenetic treatment. P026 Different pattern of JNK activity in MDS-related versus de novo AML: implications for apoptosis-resistance and treatment failure in MDS-related AML E.D. Lagadinou ° , P. Ziros, O. Tsopra, A. Klamargias, E. Thanopoulou, A. Kouraklis, A. Spyridonidis, M. Karakantza, N.C. Zoumbos. Department of Internal Medicine, Hematology Division, University Hospital of Patras, 26500 Patras, Greece *E-mail: [email protected] Introduction: AML arising from antecedent MDS (MDS/AML) is frequently refractory to treatment with standard chemotherapeutic agents. In a model of drugsensitive and resistant AML cell lines we have previously shown that the JNK signaling pathway mediates
2. Molecular mechanisms and pathophysiology I – Stromal cells, cytokines and apoptosis proapoptotic signals in response to anthracycline treatment. Moreover, in the resistant cell lines and primary AML blasts, we have observed that impairment of drug-related JNK activation was associated with a failure of anthracyclins to induce apoptosis. In the present study we investigated whether deregulation of JNK response is implicated in the comparative refractoriness of the MDS/AML to standard chemotherapy. Materials and Methods: Marrow blasts from10 MDS/AML and 14 de novo AML patients were exposed in vitro to anthracycline treatment (1 mM Daunorubicin, DNR) and the basal levels as well as the inducibility of JNK activity were detected at certain time points. Drug-induced apoptotic response of blast cells was determined by flow cytometry with Annexin staining. All patients were evaluated at diagnosis with the exception of one MDS/AML patient who was also evaluated at relapse. Results: Increased basal JNK activity levels were detected in 70% of the MDS/AML and in 42.6% of the de novo AML blasts. In 10 out of 14 (71.5%) of the de novo AML cases but only in 2 out of 10 (20%) of the MDS/AML samples the in vitro drug treatment resulted in an upregulation of basal JNK activity levels. DNR had no effect on basal JNK activity in 8 out of 10 (80%) of the MDS/AML samples and in 4 out of 14 (28.5%) of the de novo AML. In both anthracycline treated MDS/AML and de novo AML blasts the upregulation of basal JNK activity was followed by the induction of significant apoptosis, in relation to untreated controls (p = 0.0005 < 0.05). On the contrary, in the MDS/AML and de novo AML samples that JNK activity remained immutable in response to drug treatment, we observed a failure of DNR to induce significant apoptosis (p = 0.4697 > 0.05). One of the two MDS/AML patients that activated JNK and underwent DNR-induced apoptosis at diagnosis entered a CR and was also evaluated at relapse. Interestingly, neither JNK activation nor apoptosis induction was observed and patient failed to enter a second CR. Discussion: Our findings suggest that MDS/AML blasts exhibit a different pattern of basal and anthracycline induced JNK activity levels compared to the de novo AML blasts. Taking into account our previous observations defining JNK as a critical proapoptotic mediator of anthracycline treatment in AML cells, we suggest that the different pattern of the JNK response in MDS/AML samples may contribute to the worse prognosis of the MDS/AML patients.
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P027 Analysis of cell cycle regulatory genes expression in the bone marrow of adult de novo myelodysplastic syndromes (MDS) C. Economopoulou1 ° , V. Pappa1 , F. Kontsioti1 , V. Kapsimali2 , P. Tsirigotis1 , S. Papageorgiou1 , V. Giannopoulou1 , K. Girkas1 , E. Papageorgiou, J. Dervenoulas1 , T. Economopoulos1 . 1 Second Department of Internal Medicine Propaedeutic, University of Athens, Attikon University General Hospital, Athens, Greece; 2 Immunology Department, Evangelismos Hospital, Athens, Greece *E-mail: [email protected] Introduction: Alterations of cell cycle regulatory genes, such as cyclin-dependent kinases (CDKs) and CDK inhibitor proteins (CDKIs) have been suggested to play a role in oncogenesis. The aim of the present study was to examine CDK1−4, p16, p21, p27 and PISSLRE expression and apoptosis in the bone marrow (BM) of adult de novo MDS and to correlate our findings with clinical parameters and prognosis. Methods: We studied 46 cases of MDS including 14 RA, 21 RAEB, 6 CMML and 5 RARS according to FAB criteria. Apoptosis was determined by Annexin method on BM mononuclear cells. The expression of CDK1−4, p16, p21, p27 and PISSLRE was determined using an RNase Protection System. A pool of RNA from normal BM mononuclear cells was used as a normal control. The expression of each gene was compared to that of two housekeeping genes (GAPDH and L32) using Image Master Software. The level of each gene expression was compared to that obtained from normal RNA pool. Results: Cases expressing higher levels of CDK1, 2 and 3 had significantly lower levels of apoptosis (p = 0.01, 0.04 and 0.02 respectively). A positive correlation was observed between all CDKs’ levels of expression (p < 0.001). A significant negative correlation was observed between p27 and both CDK2 and CDK4 levels (p = 0.03 and 0.05 respectively). Moreover PISSLRE expression did correlate with CDK3,4 and p16 expression (p = 0.02, 0.008 and <0.001 respectively). The level of expression as well as the percentage of positive cases for all genes was not significantly different between FAB and IPSS categories. CDK2 and p27 levels were higher in patients with abnormal and normal karyotype respectively (p = 0.04 and 0.02); p27 expression was also higher in patients with no BM blasts (p = 0.05). Higher PISSLRE levels correlated positively with normal WBC values (p = 0.03). Discussion: The expression levels of all genes did not correlate with FAB and IPSS groups in patients with MDS. Cell cycle inhibitory gene p27 expression was significantly higher in cases with no BM blasts and normal karyotype, while cell cycle progression gene CDK2 was significantly higher in cases with abnormal karyotype. Cases expressing