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INTERLEUKIN-12 Enhances the antitumor effect of the tyrosine kinase inhibitor AG-490 in a murine myeloma model
Abstracts/Experimental Hematology 28 (2000) 31–131
clear survival advantage. We confirm that BCT is associated with a higher incidence of chronic GVHD an a better relapse control Longer follow-up will be presented with the extensive review focused on cGVHD which is currently under process. 232
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Chronic Myelogenous Leukemia
CHRONIC MYELOID LUKEMIA CELLS: TYROSINE KINASE INHIBITION USING AG 957 AND ARACHIDONIC ACID INDUCING APOPTOSIS V. Rizzoli, L. Mangoni, C. Caramatti*, E. Regazzi*, G. Sammarelli*, S. Colla* Cattedra di Ematologia Centro Trapianti Midollo Osseo Università di Parma - Italy Chronic myelogenous leukemia (CML) is characterized by Philadelphia chromosome that fuses genetic sequences of the BCR gene on chromosome 22. AG 957, a member of the tyrphostin compound produces a selective inhibition of P 210 BCR/ABL tyrosine phosphorylation. A group of ten patients with CML in chronic phase were treated after CD34 separation with 1 to 100 umol/L of AG 957. All patients were 100% Ph1 positive. Eight patients showed a b3a2 BCR/ABL junction and two cases showed a b2a2 junction when analyzed by RT-PCR. The effect of AG 957 on CML and normal CD34 cells in culture assay (CFU/MIX, BFUE and CFU-GM) is clearly demonstrated. The inhibition on CML untreated and AG957 treated colonies was from 2%–7% for CFUMIX, 38%–80% for BFU-E and 31%–18–% for CFU-GM. In the group of normal samples the inhibition ranged from 4%–8% for CFU-MIX, 24%–96% for BFU-E and 4%–64% for CFU-GM. The inhibitory effect of AG 957 on CML progenitors is statistically significant at the dose of 1 umol/L for CFU-MIX and BFU-E, 5 umol/L for CFU-GM. For colonies obtained from normal progenitors the inhibiting dose of AG 957 was more consistent: 5 umol/L for CFU-MIX and BFU-E, 10 umol/L for CFU-GM. These data demonstrated the possibility to select non clonal CML progenitors after incubation with the PTK inhibitor AG 957. This selection of “normal” progenitors from CML marrow could be used for autograft in patients without suitable allogeneic bone marrow donors. The research is in progression to explore the potential therapeutic effect of AG 957 in combination with other agents (IFN - ARA-C) and/or inductors of apoptosis as arachidonic acid in cell culture, to briefly translate the “in vitro” experimental approach to clinical therapeutic application. 233
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Cytokines, Growth Factors and Receptors
ONTOGENIC VARIATIONS OF CYTOKINES M. Michejda1,2, J. A. Bellanti1, G. J. Tchabo1,2, A. G. Wu1,2 Georgetown University School of Medicine1, Protocell Inc.2 We have previously shown that fetal stem cells derived from miscarriages exhibit high efficiency for transplantation. We reported that fetal bone marrow (FBM) has a higher number of CD34 cells, greater clonogenic capacity and lowered immunologic reactivity than adult bone marrow (ABM), cord blood (CB), and peripheral blood (PBSC). The present studies were designed to investigate whether similar ontogenic differences exist in the expression of cytokine in stromal cells from FBM, ABM, and CB using RI-PCR, and how the differences affect the repopulating ability of
105
the hematopoietic stem cells after long term bone marrow culture (LTBMC). Stromal cells were prepared from 8 samples each of FBM, ABM, CS. The expression for stem cell factor (SCF), GMCSF, G-CSF, M-CSF, IL-3, IL-6, IL-10, and IL-11 was detected by RT-PCR. Strong expression of SCF and IL-11 was observed in the stromal cells but was not detected in any of FBM specimens. IL-6 was positive in 6/8 ABM but not only 1/9 in FBM and 8 in CB. Although G-CSF was not observed in CB, it was expressed in all FBM and ABM specimens. In contrast, expression of GM-CSF, IL-3 and IL-10 was similar in all three sources. These results indicate that there are significant age-related changes in cytokine expression at different maturational stages. Further, LTBMC was performed to examine the ability of stromal cells to support hematopoietic stem cells from allogeneic bone marrows. The number of colonies derived from either FBM or ABM were greater when inoculated with fetal stromal cells. Our results suggest an important regulatory role of these cytokines in differentiation and proliferation of stem cells during ontogeny which may provide a basis for selection of stem cells for transplantation. 235
Monday, July 10, 2000 (12:45–14:15) Session IV-4: Lymphocyte Biology and Immunomodulation
INTERLEUKIN-12 ENHANCES THE ANTITUMOR EFFECT OF THE TYROSINE KINASE INHIBITOR AG-490 IN A MURINE MYELOMA MODEL Lyudmila Burdelya*, Robyn Catlett-Falcone*, Alexander Levitzki*1, Richard Jove* and Hua Yu* (intro by Karen Fields) H. Lee Moffitt Cancer Center & Research Institute, USF, Tampa, FL, 1 Institute of Life Sciences, The Hebrew University, Jerusalem, Israel Constitutive activation of Jak kinases (JAKs) and signal transducer and activator of transcription 3 (STAT3) has been found in various hematopoietic malignancies and solid tumors. In this study, we investigated the effect of blocking JAK/STAT signaling with AG-490 on the survival of murine myeloma cells and on interleukin-12 (IL-12)-mediated immune response in a syngenic murine model. We found that in vivo treatment with AG-490 selectively induced apoptosis of the murine myeloma cell lines that harbored constitutively activated STAT3. Because the murine myeloma tumors are fast growing and poorly immunogenic, AG-490induced tumor regression was only transient. In contrast to AG490, cytokine-based immunotherapy usually generates long-term antitumor immunity but fails to induce regression of established tumors. However, many cytokines, including IL-12, are known to signal through JAK/STAT pathways. Our results demonstrate that in vivo administration of AG-490 does not reduce IL-12-mediated macrophage and splenocyte activation. Furthermore, combinational therapy with AG-490 and IL-12 resulted in prolonged tumor regression. These results suggest that combining AG-490 and IL-12 may posses clinical potential as an effective approach for treatment of human cancers harboring constitutively activated JAK/ STAT signaling.
clear survival advantage. We confirm that BCT is associated with a higher incidence of chronic GVHD an a better relapse control Longer follow-up will be presented with the extensive review focused on cGVHD which is currently under process. 232
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Chronic Myelogenous Leukemia
CHRONIC MYELOID LUKEMIA CELLS: TYROSINE KINASE INHIBITION USING AG 957 AND ARACHIDONIC ACID INDUCING APOPTOSIS V. Rizzoli, L. Mangoni, C. Caramatti*, E. Regazzi*, G. Sammarelli*, S. Colla* Cattedra di Ematologia Centro Trapianti Midollo Osseo Università di Parma - Italy Chronic myelogenous leukemia (CML) is characterized by Philadelphia chromosome that fuses genetic sequences of the BCR gene on chromosome 22. AG 957, a member of the tyrphostin compound produces a selective inhibition of P 210 BCR/ABL tyrosine phosphorylation. A group of ten patients with CML in chronic phase were treated after CD34 separation with 1 to 100 umol/L of AG 957. All patients were 100% Ph1 positive. Eight patients showed a b3a2 BCR/ABL junction and two cases showed a b2a2 junction when analyzed by RT-PCR. The effect of AG 957 on CML and normal CD34 cells in culture assay (CFU/MIX, BFUE and CFU-GM) is clearly demonstrated. The inhibition on CML untreated and AG957 treated colonies was from 2%–7% for CFUMIX, 38%–80% for BFU-E and 31%–18–% for CFU-GM. In the group of normal samples the inhibition ranged from 4%–8% for CFU-MIX, 24%–96% for BFU-E and 4%–64% for CFU-GM. The inhibitory effect of AG 957 on CML progenitors is statistically significant at the dose of 1 umol/L for CFU-MIX and BFU-E, 5 umol/L for CFU-GM. For colonies obtained from normal progenitors the inhibiting dose of AG 957 was more consistent: 5 umol/L for CFU-MIX and BFU-E, 10 umol/L for CFU-GM. These data demonstrated the possibility to select non clonal CML progenitors after incubation with the PTK inhibitor AG 957. This selection of “normal” progenitors from CML marrow could be used for autograft in patients without suitable allogeneic bone marrow donors. The research is in progression to explore the potential therapeutic effect of AG 957 in combination with other agents (IFN - ARA-C) and/or inductors of apoptosis as arachidonic acid in cell culture, to briefly translate the “in vitro” experimental approach to clinical therapeutic application. 233
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Cytokines, Growth Factors and Receptors
ONTOGENIC VARIATIONS OF CYTOKINES M. Michejda1,2, J. A. Bellanti1, G. J. Tchabo1,2, A. G. Wu1,2 Georgetown University School of Medicine1, Protocell Inc.2 We have previously shown that fetal stem cells derived from miscarriages exhibit high efficiency for transplantation. We reported that fetal bone marrow (FBM) has a higher number of CD34 cells, greater clonogenic capacity and lowered immunologic reactivity than adult bone marrow (ABM), cord blood (CB), and peripheral blood (PBSC). The present studies were designed to investigate whether similar ontogenic differences exist in the expression of cytokine in stromal cells from FBM, ABM, and CB using RI-PCR, and how the differences affect the repopulating ability of
105
the hematopoietic stem cells after long term bone marrow culture (LTBMC). Stromal cells were prepared from 8 samples each of FBM, ABM, CS. The expression for stem cell factor (SCF), GMCSF, G-CSF, M-CSF, IL-3, IL-6, IL-10, and IL-11 was detected by RT-PCR. Strong expression of SCF and IL-11 was observed in the stromal cells but was not detected in any of FBM specimens. IL-6 was positive in 6/8 ABM but not only 1/9 in FBM and 8 in CB. Although G-CSF was not observed in CB, it was expressed in all FBM and ABM specimens. In contrast, expression of GM-CSF, IL-3 and IL-10 was similar in all three sources. These results indicate that there are significant age-related changes in cytokine expression at different maturational stages. Further, LTBMC was performed to examine the ability of stromal cells to support hematopoietic stem cells from allogeneic bone marrows. The number of colonies derived from either FBM or ABM were greater when inoculated with fetal stromal cells. Our results suggest an important regulatory role of these cytokines in differentiation and proliferation of stem cells during ontogeny which may provide a basis for selection of stem cells for transplantation. 235
Monday, July 10, 2000 (12:45–14:15) Session IV-4: Lymphocyte Biology and Immunomodulation
INTERLEUKIN-12 ENHANCES THE ANTITUMOR EFFECT OF THE TYROSINE KINASE INHIBITOR AG-490 IN A MURINE MYELOMA MODEL Lyudmila Burdelya*, Robyn Catlett-Falcone*, Alexander Levitzki*1, Richard Jove* and Hua Yu* (intro by Karen Fields) H. Lee Moffitt Cancer Center & Research Institute, USF, Tampa, FL, 1 Institute of Life Sciences, The Hebrew University, Jerusalem, Israel Constitutive activation of Jak kinases (JAKs) and signal transducer and activator of transcription 3 (STAT3) has been found in various hematopoietic malignancies and solid tumors. In this study, we investigated the effect of blocking JAK/STAT signaling with AG-490 on the survival of murine myeloma cells and on interleukin-12 (IL-12)-mediated immune response in a syngenic murine model. We found that in vivo treatment with AG-490 selectively induced apoptosis of the murine myeloma cell lines that harbored constitutively activated STAT3. Because the murine myeloma tumors are fast growing and poorly immunogenic, AG-490induced tumor regression was only transient. In contrast to AG490, cytokine-based immunotherapy usually generates long-term antitumor immunity but fails to induce regression of established tumors. However, many cytokines, including IL-12, are known to signal through JAK/STAT pathways. Our results demonstrate that in vivo administration of AG-490 does not reduce IL-12-mediated macrophage and splenocyte activation. Furthermore, combinational therapy with AG-490 and IL-12 resulted in prolonged tumor regression. These results suggest that combining AG-490 and IL-12 may posses clinical potential as an effective approach for treatment of human cancers harboring constitutively activated JAK/ STAT signaling.