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Constitutive activation of stat signaling in myeloid leukemia cells is associated with resistance to apoptosis
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236
Abstracts/Experimental Hematology 28 (2000) 31–131
Tuesday, July 11, 2000 (10:15–12:15) Session V-1: Apoptosis and Bone Marrow Failure
CONSTITUTIVE ACTIVATION OF STAT SIGNALING IN MYELOID LEUKEMIA CELLS IS ASSOCIATED WITH RESISTANCE TO APOPTOSIS M. Huang*, R. Catlett-Falcone*, L. Burdelya*, T. Landowski*, M. Oshiro*, L. Moscinski*, T. Loughran, H. Saba*, D. Sullivan*, H. Yu*, W. Dalton*, and R. Jove* H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL Growth factors that contribute to the pathogenesis of many types of cancers also induce activation of the JAK/STAT signaling pathway. Several STAT family members, most notably Stat3 and Stat5, have been found constitutively activated in various hematopoietic malignancies. Recent findings in multiple myeloma suggest that activation of Stat3 contributes to tumor cell proliferation and confers resistance to apoptosis. In the present study, blast cells from patients with various stages of myeloid leukemia and myelodysplastic syndrome (MDS) were analyzed for STAT activation. Our findings indicate that isoforms of STATs 1, 3, and 5 are found constitutively activated in the absence of exogenous growth factors. Similar patterns of STAT activation were also found in several factor-independent myeloid leukemia cell lines, including KG1a and HEL 92.1.7. Blocking tyrosine kinase signaling differentially affects STAT DNA-binding activity, cell proliferation, and apoptosis in the various cell lines, suggesting that divergent signaling pathways lead to constitutive STAT activation in myeloid malignancies. We conclude that constitutive activation of STAT proteins in some of the critical pathways involved in cellular transformation, and that interfering with STAT signaling results in apoptosis of tumor cells. These results provide evidence of a role for STAT signaling in the pathogenesis of myeloid leukemia, and thus identify STAT proteins as potential molecular targets for therapeutic intervention. 237
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Cytokines, Growth Factors and Receptors
PEG-MODIFIED ERYTHROPOIETIN WITH IMPROVED EFFICACY F. Malik, J. Brew*, S. A. Maidment*, C. Delgado* & G. E. Francis PolyMASC Pharmaceuticals plc, London NW3 2EZ, U.K. The objective of this study was to evaluate the efficacy of glycosylated erythropoietin (Epo) which had been modified using a biologically optimised PEGylation system (ProtoMASCTM) based on tresylated monomethoxypolyethylene glycol (TMPEG). TMPEG of chain length 12,000 daltons was used in this study. Six, ⫻1 weekly injections of PEG12000-Epo (1 g/kg) were administrated subcutaneously into normocythaemic Balb/c mice and the haematocrit levels monitored 1–2 times per week. After the initial dose of Epo, subsequent doses produced a well-sustained increase in haematocrit significantly above the controls. After cessation of treatment, the haematocrit declined but was above the controls in 12 of 13 samples taken over a period of 7.5 weeks after the last injection. Over this period, the test minus control difference fluctuated and there appeared to be a second wave of stimulation of haematocrit. This ‘second wave’ effect was not observed with untreated or MPEG-treated Epo (MPEG cannot covalently attach to the protein). We have shown that PEG-
modification of glycosylated erythropoietin by TMPEG-12K increased the t1/2 to 29 h in the linear elimination phase and found that circulating Epo was still detectable at 19 days. This ‘second wave’ effect may be due to the late circulating Epo, but further studies are needed to elucidate this. In conclusion, PEG-Epo showed improved efficacy over glycosylated Epo when dosed on a once weekly regime and had a surprisingly protracted effect on haematocrit. 238
Monday, July 10, 2000 (16:00–17:00) Poster Session II: Stem Cell Biology
INVESTIGATING THE ROLE OF CONNEXIN43 IN MARROW Chris Jopling and Martin Rosendaal Department of Anatomy and Developmental Biology, University College London, England We have found connexin43 (Cx43) gap junctions in mouse haemopoietic tissue and proposed that they play a part in the division of primitive haemopoietic stem cells (PHSC, Rosendaal et al., 1997). Normal resting haemopoiesis, which produced the circulating blood cells, is carried out by progenitors, formed by the division of PHSC. They divide when their progeny are needed to repopulate a severely depleted blood-forming system, e.g. when the definitive haemopoietic system is founded in the embryo and neonate or after treating an adult with a cytotoxic drug which selectively deletes dividing haemopoietic cells (e.g. 5-fluorouracil). To divide, PHSC must receive signals and we believe that communication via Cx43 gap junctions is involved. We graft into toenotched reporter mice, all of whose stem cells have been deleted by busulphan, a competing mixture of two kinds of congenic stem cells with different glucose-phosphate isomerase markers, 1a and 1b. The competing populations of stem cells have different degrees of expression of Cx43, ⫹/⫹1a versus ⫹/⫹1b, or ⫹/⫹1a versus ⫹/⫺ 1b, or ⫹/⫹1a versus ⫺/⫺1b. At intervals thereafter we assay the proportions of Gpi-1a and 1b in five blood cells. This allows three levels of comparison, (1) of repopulation of donors by stem cells, (2) of repopulation of reporter mice by grafted stem cells and (3) of repopulation of reporter mice after subsequent challenge. (1) We treat donor 1a and 1b mice with 5-fluorouracil and then compete the same femoral fractions of their marrow in reporter mice. (2) We compete untreated 1a and 1b foetal liver stem cells from ⫹/⫹, ⫹/⫺ or ⫺/⫺ in reporter mice. (3) We stress with different cytotoxic drugs the blood-forming systems of reporter mice after they have been grafted with these combinations of stem cells. Rosendaal et al. (1997) Leukemia 11, 1281–9. 239
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Acute Leukemia: Clinical Research
SELECTION OF OPTIMAL REMISSION CONSOLIDATION THERAPY FOR INDIVIDUAL PATIENTS WITH ACUTE MYELOGENOUS LEUKEMIA H. D. Preisler, P. Venugopla, S. Sivaraman*, R. Larson*, G. Tricot, J. Goldberg*, K. Miller*, A. Galvez*, S. A. Gregory, S. Adler, S. Creech*, and A. Raza Rush Cancer Institute, Illinois Masonic Medical Cancer, Cooper Hospital, University of Chicago Prior to the initiation of remission induction therapy patients with AML a combination of 13-cis retinoic acid and a interferon
236
Abstracts/Experimental Hematology 28 (2000) 31–131
Tuesday, July 11, 2000 (10:15–12:15) Session V-1: Apoptosis and Bone Marrow Failure
CONSTITUTIVE ACTIVATION OF STAT SIGNALING IN MYELOID LEUKEMIA CELLS IS ASSOCIATED WITH RESISTANCE TO APOPTOSIS M. Huang*, R. Catlett-Falcone*, L. Burdelya*, T. Landowski*, M. Oshiro*, L. Moscinski*, T. Loughran, H. Saba*, D. Sullivan*, H. Yu*, W. Dalton*, and R. Jove* H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL Growth factors that contribute to the pathogenesis of many types of cancers also induce activation of the JAK/STAT signaling pathway. Several STAT family members, most notably Stat3 and Stat5, have been found constitutively activated in various hematopoietic malignancies. Recent findings in multiple myeloma suggest that activation of Stat3 contributes to tumor cell proliferation and confers resistance to apoptosis. In the present study, blast cells from patients with various stages of myeloid leukemia and myelodysplastic syndrome (MDS) were analyzed for STAT activation. Our findings indicate that isoforms of STATs 1, 3, and 5 are found constitutively activated in the absence of exogenous growth factors. Similar patterns of STAT activation were also found in several factor-independent myeloid leukemia cell lines, including KG1a and HEL 92.1.7. Blocking tyrosine kinase signaling differentially affects STAT DNA-binding activity, cell proliferation, and apoptosis in the various cell lines, suggesting that divergent signaling pathways lead to constitutive STAT activation in myeloid malignancies. We conclude that constitutive activation of STAT proteins in some of the critical pathways involved in cellular transformation, and that interfering with STAT signaling results in apoptosis of tumor cells. These results provide evidence of a role for STAT signaling in the pathogenesis of myeloid leukemia, and thus identify STAT proteins as potential molecular targets for therapeutic intervention. 237
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Cytokines, Growth Factors and Receptors
PEG-MODIFIED ERYTHROPOIETIN WITH IMPROVED EFFICACY F. Malik, J. Brew*, S. A. Maidment*, C. Delgado* & G. E. Francis PolyMASC Pharmaceuticals plc, London NW3 2EZ, U.K. The objective of this study was to evaluate the efficacy of glycosylated erythropoietin (Epo) which had been modified using a biologically optimised PEGylation system (ProtoMASCTM) based on tresylated monomethoxypolyethylene glycol (TMPEG). TMPEG of chain length 12,000 daltons was used in this study. Six, ⫻1 weekly injections of PEG12000-Epo (1 g/kg) were administrated subcutaneously into normocythaemic Balb/c mice and the haematocrit levels monitored 1–2 times per week. After the initial dose of Epo, subsequent doses produced a well-sustained increase in haematocrit significantly above the controls. After cessation of treatment, the haematocrit declined but was above the controls in 12 of 13 samples taken over a period of 7.5 weeks after the last injection. Over this period, the test minus control difference fluctuated and there appeared to be a second wave of stimulation of haematocrit. This ‘second wave’ effect was not observed with untreated or MPEG-treated Epo (MPEG cannot covalently attach to the protein). We have shown that PEG-
modification of glycosylated erythropoietin by TMPEG-12K increased the t1/2 to 29 h in the linear elimination phase and found that circulating Epo was still detectable at 19 days. This ‘second wave’ effect may be due to the late circulating Epo, but further studies are needed to elucidate this. In conclusion, PEG-Epo showed improved efficacy over glycosylated Epo when dosed on a once weekly regime and had a surprisingly protracted effect on haematocrit. 238
Monday, July 10, 2000 (16:00–17:00) Poster Session II: Stem Cell Biology
INVESTIGATING THE ROLE OF CONNEXIN43 IN MARROW Chris Jopling and Martin Rosendaal Department of Anatomy and Developmental Biology, University College London, England We have found connexin43 (Cx43) gap junctions in mouse haemopoietic tissue and proposed that they play a part in the division of primitive haemopoietic stem cells (PHSC, Rosendaal et al., 1997). Normal resting haemopoiesis, which produced the circulating blood cells, is carried out by progenitors, formed by the division of PHSC. They divide when their progeny are needed to repopulate a severely depleted blood-forming system, e.g. when the definitive haemopoietic system is founded in the embryo and neonate or after treating an adult with a cytotoxic drug which selectively deletes dividing haemopoietic cells (e.g. 5-fluorouracil). To divide, PHSC must receive signals and we believe that communication via Cx43 gap junctions is involved. We graft into toenotched reporter mice, all of whose stem cells have been deleted by busulphan, a competing mixture of two kinds of congenic stem cells with different glucose-phosphate isomerase markers, 1a and 1b. The competing populations of stem cells have different degrees of expression of Cx43, ⫹/⫹1a versus ⫹/⫹1b, or ⫹/⫹1a versus ⫹/⫺ 1b, or ⫹/⫹1a versus ⫺/⫺1b. At intervals thereafter we assay the proportions of Gpi-1a and 1b in five blood cells. This allows three levels of comparison, (1) of repopulation of donors by stem cells, (2) of repopulation of reporter mice by grafted stem cells and (3) of repopulation of reporter mice after subsequent challenge. (1) We treat donor 1a and 1b mice with 5-fluorouracil and then compete the same femoral fractions of their marrow in reporter mice. (2) We compete untreated 1a and 1b foetal liver stem cells from ⫹/⫹, ⫹/⫺ or ⫺/⫺ in reporter mice. (3) We stress with different cytotoxic drugs the blood-forming systems of reporter mice after they have been grafted with these combinations of stem cells. Rosendaal et al. (1997) Leukemia 11, 1281–9. 239
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Acute Leukemia: Clinical Research
SELECTION OF OPTIMAL REMISSION CONSOLIDATION THERAPY FOR INDIVIDUAL PATIENTS WITH ACUTE MYELOGENOUS LEUKEMIA H. D. Preisler, P. Venugopla, S. Sivaraman*, R. Larson*, G. Tricot, J. Goldberg*, K. Miller*, A. Galvez*, S. A. Gregory, S. Adler, S. Creech*, and A. Raza Rush Cancer Institute, Illinois Masonic Medical Cancer, Cooper Hospital, University of Chicago Prior to the initiation of remission induction therapy patients with AML a combination of 13-cis retinoic acid and a interferon