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EphA3 is a therapy target for leukaemia and other cancers
S52
Poster Presentations/ Experimental Hematology 41 (2013) S23–S75
P1113 - EFFECTIVELY TARGETING TREATMENT-NA€ıVE CML STEM/ PROGENITOR CELLS FROM IMATINIB-NONRESPONDERS WITH COMBINATION TREATMENTS OF NEW JAK2 AND ABL INHIBITORS IN VITRO AND IN VIVO Hanyang Lin1,2, Min Chen1, Katharina Rothe1,3, Matthew Lorenzi4, Adrian Woolfson4, and Xiaoyan Jiang1,2,3 1 Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada; 2 Medicine, University of British Columbia, Vancouver, British Columbia, Canada; 3 Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada; 4Discovery Medicine Oncology, Bristol-Myers Squibb, Princeton, New Jersey, USA Introduction of Imatinib Mesylate (IM) and other tyrosine kinase inhibitor (TKI) therapies has had a major impact on treatment of chronic phase CML, but early relapses and persistence of leukemic stem cells remain problematic. We recently identified a new AHI-1-BCR-ABL-JAK2 protein complex that contributes to the transforming activity of BCR-ABL and IM-resistance of CML stem/progenitor cells. We have therefore hypothesized that combined suppression of BCR-ABL and JAK2 activities might be more effective in eliminating primitive CML cells in vitro and in vivo. We have now examined the biological effects of an orally bioavailable, selective JAK2 inhibitor (BMS-911543) in combination with TKIs, including IM, dasatinib (DA) and nilotinib, on treatment-na€ıve CML IM-nonresponder cells. Intracellular staining analyses showed that combined exposure of CD34+ CML cells to BMS-911543 and a TKI produced a deeper, more prolonged suppression of pSTAT5 activity than single agents (2.5 fold, p!0.05). Interestingly, combination treatment resulted in greater inhibition in colony growth compared to single agents (74-86% vs. 40-50%, p!0.05). Long-term cultureinitiating cell assays showed that the more primitive cells were also more significantly eliminated by combination treatments (2-3 fold, p!0.05). Importantly, BMS-911543 is less toxic to normal bone marrow CD34+ cells (n54) than CML samples (n57, 2-3 fold, p!0.05). Finally, oral admission of BMS-911543 and TKIs significantly eliminated primitive CML cells and enhanced survival of leukemic mice compared to mice treated with single agents (median survival of IM + BMS-911543 vs IM: 70 days vs 53 days, p! 0.05; DA + BMS-911543 vs DA: 85 days vs 78 days). This study indicates that simultaneously targeting BCR-ABL and JAK2 activities in CML stem/progenitor cells may improve outcomes in patients, especially those destined to develop TKI resistance. P1114 - THE ROLE OF BIN1 TUMOR SUPPRESSOR ISOFORMS IN REGULATION OF PROLIFERATION AND APOPTOSIS OF HUMAN CUTANOUS T-CELL LYMPHOMA CELLS Sharmin Esmailzadeh1,2, Youwen Zhou3, and Xiaoyan Jiang1,2 1 Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada; 2 Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada; 3Dermatology and Skin Science, University of British Columbia, Vancouver, British Columbia, Canada The two main types of cutaneous T-cell lymphomas (CTCLs) are mycosis fungoides (MF) and its leukemic variant Sezary Syndrome (SS). We recently demonstrated the importance of AHI-1 oncogene in tumorigenic activity of Sezary cells (Hut78) using in vitro and in vivo assays. BIN1 tumor suppressor was identified through microarray analysis as one of the genes that may be involved in AHI-1-mediated leukemic transformation in CTCL cells. BIN1 is a nucleocytosolic adaptor protein with more than ten isoforms; some isoforms, including the BIN1 isoform (+10, +13), act as tumor suppressors, but the BIN1 (+12A) isoform behaved as a cancer-related isoform in solid tumor models. However, the role of BIN1 in normal hematopoiesis and lymphomagenesis remains unknown. We have recently demonstrated that transcript levels of BIN1 isoforms are significantly lower in patients with MF or SS compared to controls. Four isoforms of BIN1 have been identified in Hut78 and primary CD4+CD7- Sezary cells. To investigate the role of BIN1 in CTCL, the BIN1 isoforms (+10, +13) and BIN1 (+12A) lentiviral constructs were transduced into Hut78 and HH cells. Overexpression of BIN1 isoforms led to a significant reduction in cell proliferation, as assessed by colony forming cell and 3H-Thymidine uptake assays (2-3 ford, p!0.05). Furthermore, a significant increase in spontaneous and specific apoptosis was observed in BIN1-transduced cells, with and without exogenous FAS-ligand (2-3 ford, p!0.05). Interestingly, a significant reduction in protein expression of c-FLIP (inhibitor of the FAS-mediated apoptosis pathway) and upregulation of downstream cleaved caspase-8 and caspase-3 was demonstrated in BIN1-transduced cells, suggesting that BIN1 isoforms induce apoptosis by downregulating the expression of c-FLIP, which leads to activation of the FAS-mediated apoptosis pathway. In addition, subcellular fractionation and confocal microscopy further shown that BIN1 (+10, +13) is localized in both the cytoplasm and nucleus, whereas BIN1 (+12A) is mostly located in the nucleus. These findings indicate antiproliferative and pro-apoptotic roles for BIN1 isoforms in human CTCL cells.
P1115 - EPHA3 IS A THERAPY TARGET FOR LEUKAEMIA AND OTHER CANCERS Sara Charmsaz1, Bryan Day1, Fares El-Ajeh1, Varghese Palath3, Geoff Yarranton3, Chris Bebbington3, Andrew Scott4, Martin Lackmann2, and Andrew Boyd1 1 Queensland Institute of Medical Research and University of Queensland, Brisbane, Queensland, Australia; 2Monash University, Melbourne, Victoria, Australia; 3 KaloBios Pharmaceuticals Inc, San Francisco, California, USA; 4Ludwig Institute for Cancer Research, Melbourne, Victoria, Australia Eph receptor tyrosine kinases (RTKs) interact with cell-surface ephrin ligands to direct cell migration and orchestrate developmental patterning during embryogenesis by modulating cell shape and adhesion. High expression in a number of cancers has been linked to progression through facilitation of invasiveness and metastatic spread. EphA3 protein, which was originally described in leukaemia, is also reported to be expressed in sarcomas, lung cancer, melanoma and glioblastoma. The EphA3specific monoclonal antibody, IIIA4, binds and activates the human and mouse EphA3 with similar affinities. Binding is followed by internalization of receptor-antibody complexes. In a mouse xenograft model of leukaemia, LK63, administration of the IIIA4 antibody leads to inhibition of tumour growth and spread from bone marrow to the spleen and other organs and increases the latency of the disease. A more impressive anti-tumour effect is delivered by targeting of a bound radio-active isotope, illustrated by analpha particle emitting bismuth isotope-linked to IIIA4 in leukaemia or a beta particle-emmitting lutetium isotope linked to IIIA4 in glioma xenograft models. Importantly, little toxicity to normal tissues was observed as EphA3 is expressed at very low/no levels on normal tissues. A high-affinity recombinant human antibody, derived by Antibody Humaneering from the a-EphA3 mouse monoclonal antibody IIIA4 has been developed and shows enhanced Antibodydependent cellular cytotoxicity (ADCC) against leukaemic targets. This antibody is now in a phase I trial in subjects with haematological tumours.
P1116 - MN1 REGULATES SELF-RENEWAL AND DIFFERENTIATION THROUGH DISTINCT DOMAINS Courteney Lai1, Gyeongsin Park1, Florian Kuchenbauer1, Bob Argiropoulos1, Eric Yung1, Philip Beer1, Adrian Schwarzer2, Yeonsook Moon3, Malina Leung1, Grace Lin1, Sarah Vollett1, Stephen Fung1, Andrew Weng1, R. Keith Humphries1, and Michael Heuser1 1 Terry Fox Lab., BCCRC, Van., British Columbia, Canada; 2Inst. of Expt. Hem., Hannover Med. School, Hannover, Germany; 3Dept. of Lab. Med., Med. School of Inha Univ., Incheon, Republic of Korea Meningioma 1 (MN1) is an independent prognostic marker for normal karyotype acute myeloid leukemia, with high expression linked to all-trans retinoic acid (ATRA) resistance and poor overall and relapse-free survival. MN1 is also a potent and sufficient oncogene in murine leukemia, able to block myeloid differentiation, promote leukemic stem cell self-renewal and transform cells to the common myeloid progenitor level. Yet despite its established leukemic role, little is known about its mechanism of action. To elucidate the role(s) of each portion of MN1, we divided the gene into 7 regions of 200 amino acids. We cloned 16 mutants as distinct region deletions or cumulative deletions from the N- or C-termini. Mouse bone marrow was retrovirally transduced and assayed for (1) proliferation/self-renewal by CFU colony formation and in vivo engraftment; (2) erythro-megakaryocyte differentiation by red blood cell (RBC) engraftment and CFU-Mk colony formation; (3) myeloid/lymphoid differentiation by immunophenotypic analysis of transplanted mice; and (4) in vitro ATRA resistance. We show that the MN1 N-terminus is required for proliferation and self-renewal, as the N-terminus deletion (MN1D1) showed decreasing in vivo engraftment and did not induce leukemia after 181 days. MN1D1 cells contained a 1.9-fold higher fraction of GFP+ cells in RBCs over white blood cells, compared to equivalent ratios in control and MN1 cells (n55, P!.05), and preferentially formed CFU-Mk colonies in vitro, suggesting the N-terminus also blocks erythromegakaryocyte differentiation. MN1 C-terminal regions are crucial to the myeloid differentiation block, as C-terminal deletions showed increased Gr1 and CD11b expression in GFP+ peripheral blood, delayed disease onset, and reversal of ATRA resistance to control levels (n56, P!.05). Gene expression profiling showed Cterminally deleted MN1 cells clustering with Gr1+CD11b+ cells, underscoring the importance of the C-terminus in blocking myeloid differentiation. This study
Poster Presentations/ Experimental Hematology 41 (2013) S23–S75
P1113 - EFFECTIVELY TARGETING TREATMENT-NA€ıVE CML STEM/ PROGENITOR CELLS FROM IMATINIB-NONRESPONDERS WITH COMBINATION TREATMENTS OF NEW JAK2 AND ABL INHIBITORS IN VITRO AND IN VIVO Hanyang Lin1,2, Min Chen1, Katharina Rothe1,3, Matthew Lorenzi4, Adrian Woolfson4, and Xiaoyan Jiang1,2,3 1 Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada; 2 Medicine, University of British Columbia, Vancouver, British Columbia, Canada; 3 Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada; 4Discovery Medicine Oncology, Bristol-Myers Squibb, Princeton, New Jersey, USA Introduction of Imatinib Mesylate (IM) and other tyrosine kinase inhibitor (TKI) therapies has had a major impact on treatment of chronic phase CML, but early relapses and persistence of leukemic stem cells remain problematic. We recently identified a new AHI-1-BCR-ABL-JAK2 protein complex that contributes to the transforming activity of BCR-ABL and IM-resistance of CML stem/progenitor cells. We have therefore hypothesized that combined suppression of BCR-ABL and JAK2 activities might be more effective in eliminating primitive CML cells in vitro and in vivo. We have now examined the biological effects of an orally bioavailable, selective JAK2 inhibitor (BMS-911543) in combination with TKIs, including IM, dasatinib (DA) and nilotinib, on treatment-na€ıve CML IM-nonresponder cells. Intracellular staining analyses showed that combined exposure of CD34+ CML cells to BMS-911543 and a TKI produced a deeper, more prolonged suppression of pSTAT5 activity than single agents (2.5 fold, p!0.05). Interestingly, combination treatment resulted in greater inhibition in colony growth compared to single agents (74-86% vs. 40-50%, p!0.05). Long-term cultureinitiating cell assays showed that the more primitive cells were also more significantly eliminated by combination treatments (2-3 fold, p!0.05). Importantly, BMS-911543 is less toxic to normal bone marrow CD34+ cells (n54) than CML samples (n57, 2-3 fold, p!0.05). Finally, oral admission of BMS-911543 and TKIs significantly eliminated primitive CML cells and enhanced survival of leukemic mice compared to mice treated with single agents (median survival of IM + BMS-911543 vs IM: 70 days vs 53 days, p! 0.05; DA + BMS-911543 vs DA: 85 days vs 78 days). This study indicates that simultaneously targeting BCR-ABL and JAK2 activities in CML stem/progenitor cells may improve outcomes in patients, especially those destined to develop TKI resistance. P1114 - THE ROLE OF BIN1 TUMOR SUPPRESSOR ISOFORMS IN REGULATION OF PROLIFERATION AND APOPTOSIS OF HUMAN CUTANOUS T-CELL LYMPHOMA CELLS Sharmin Esmailzadeh1,2, Youwen Zhou3, and Xiaoyan Jiang1,2 1 Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada; 2 Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada; 3Dermatology and Skin Science, University of British Columbia, Vancouver, British Columbia, Canada The two main types of cutaneous T-cell lymphomas (CTCLs) are mycosis fungoides (MF) and its leukemic variant Sezary Syndrome (SS). We recently demonstrated the importance of AHI-1 oncogene in tumorigenic activity of Sezary cells (Hut78) using in vitro and in vivo assays. BIN1 tumor suppressor was identified through microarray analysis as one of the genes that may be involved in AHI-1-mediated leukemic transformation in CTCL cells. BIN1 is a nucleocytosolic adaptor protein with more than ten isoforms; some isoforms, including the BIN1 isoform (+10, +13), act as tumor suppressors, but the BIN1 (+12A) isoform behaved as a cancer-related isoform in solid tumor models. However, the role of BIN1 in normal hematopoiesis and lymphomagenesis remains unknown. We have recently demonstrated that transcript levels of BIN1 isoforms are significantly lower in patients with MF or SS compared to controls. Four isoforms of BIN1 have been identified in Hut78 and primary CD4+CD7- Sezary cells. To investigate the role of BIN1 in CTCL, the BIN1 isoforms (+10, +13) and BIN1 (+12A) lentiviral constructs were transduced into Hut78 and HH cells. Overexpression of BIN1 isoforms led to a significant reduction in cell proliferation, as assessed by colony forming cell and 3H-Thymidine uptake assays (2-3 ford, p!0.05). Furthermore, a significant increase in spontaneous and specific apoptosis was observed in BIN1-transduced cells, with and without exogenous FAS-ligand (2-3 ford, p!0.05). Interestingly, a significant reduction in protein expression of c-FLIP (inhibitor of the FAS-mediated apoptosis pathway) and upregulation of downstream cleaved caspase-8 and caspase-3 was demonstrated in BIN1-transduced cells, suggesting that BIN1 isoforms induce apoptosis by downregulating the expression of c-FLIP, which leads to activation of the FAS-mediated apoptosis pathway. In addition, subcellular fractionation and confocal microscopy further shown that BIN1 (+10, +13) is localized in both the cytoplasm and nucleus, whereas BIN1 (+12A) is mostly located in the nucleus. These findings indicate antiproliferative and pro-apoptotic roles for BIN1 isoforms in human CTCL cells.
P1115 - EPHA3 IS A THERAPY TARGET FOR LEUKAEMIA AND OTHER CANCERS Sara Charmsaz1, Bryan Day1, Fares El-Ajeh1, Varghese Palath3, Geoff Yarranton3, Chris Bebbington3, Andrew Scott4, Martin Lackmann2, and Andrew Boyd1 1 Queensland Institute of Medical Research and University of Queensland, Brisbane, Queensland, Australia; 2Monash University, Melbourne, Victoria, Australia; 3 KaloBios Pharmaceuticals Inc, San Francisco, California, USA; 4Ludwig Institute for Cancer Research, Melbourne, Victoria, Australia Eph receptor tyrosine kinases (RTKs) interact with cell-surface ephrin ligands to direct cell migration and orchestrate developmental patterning during embryogenesis by modulating cell shape and adhesion. High expression in a number of cancers has been linked to progression through facilitation of invasiveness and metastatic spread. EphA3 protein, which was originally described in leukaemia, is also reported to be expressed in sarcomas, lung cancer, melanoma and glioblastoma. The EphA3specific monoclonal antibody, IIIA4, binds and activates the human and mouse EphA3 with similar affinities. Binding is followed by internalization of receptor-antibody complexes. In a mouse xenograft model of leukaemia, LK63, administration of the IIIA4 antibody leads to inhibition of tumour growth and spread from bone marrow to the spleen and other organs and increases the latency of the disease. A more impressive anti-tumour effect is delivered by targeting of a bound radio-active isotope, illustrated by analpha particle emitting bismuth isotope-linked to IIIA4 in leukaemia or a beta particle-emmitting lutetium isotope linked to IIIA4 in glioma xenograft models. Importantly, little toxicity to normal tissues was observed as EphA3 is expressed at very low/no levels on normal tissues. A high-affinity recombinant human antibody, derived by Antibody Humaneering from the a-EphA3 mouse monoclonal antibody IIIA4 has been developed and shows enhanced Antibodydependent cellular cytotoxicity (ADCC) against leukaemic targets. This antibody is now in a phase I trial in subjects with haematological tumours.
P1116 - MN1 REGULATES SELF-RENEWAL AND DIFFERENTIATION THROUGH DISTINCT DOMAINS Courteney Lai1, Gyeongsin Park1, Florian Kuchenbauer1, Bob Argiropoulos1, Eric Yung1, Philip Beer1, Adrian Schwarzer2, Yeonsook Moon3, Malina Leung1, Grace Lin1, Sarah Vollett1, Stephen Fung1, Andrew Weng1, R. Keith Humphries1, and Michael Heuser1 1 Terry Fox Lab., BCCRC, Van., British Columbia, Canada; 2Inst. of Expt. Hem., Hannover Med. School, Hannover, Germany; 3Dept. of Lab. Med., Med. School of Inha Univ., Incheon, Republic of Korea Meningioma 1 (MN1) is an independent prognostic marker for normal karyotype acute myeloid leukemia, with high expression linked to all-trans retinoic acid (ATRA) resistance and poor overall and relapse-free survival. MN1 is also a potent and sufficient oncogene in murine leukemia, able to block myeloid differentiation, promote leukemic stem cell self-renewal and transform cells to the common myeloid progenitor level. Yet despite its established leukemic role, little is known about its mechanism of action. To elucidate the role(s) of each portion of MN1, we divided the gene into 7 regions of 200 amino acids. We cloned 16 mutants as distinct region deletions or cumulative deletions from the N- or C-termini. Mouse bone marrow was retrovirally transduced and assayed for (1) proliferation/self-renewal by CFU colony formation and in vivo engraftment; (2) erythro-megakaryocyte differentiation by red blood cell (RBC) engraftment and CFU-Mk colony formation; (3) myeloid/lymphoid differentiation by immunophenotypic analysis of transplanted mice; and (4) in vitro ATRA resistance. We show that the MN1 N-terminus is required for proliferation and self-renewal, as the N-terminus deletion (MN1D1) showed decreasing in vivo engraftment and did not induce leukemia after 181 days. MN1D1 cells contained a 1.9-fold higher fraction of GFP+ cells in RBCs over white blood cells, compared to equivalent ratios in control and MN1 cells (n55, P!.05), and preferentially formed CFU-Mk colonies in vitro, suggesting the N-terminus also blocks erythromegakaryocyte differentiation. MN1 C-terminal regions are crucial to the myeloid differentiation block, as C-terminal deletions showed increased Gr1 and CD11b expression in GFP+ peripheral blood, delayed disease onset, and reversal of ATRA resistance to control levels (n56, P!.05). Gene expression profiling showed Cterminally deleted MN1 cells clustering with Gr1+CD11b+ cells, underscoring the importance of the C-terminus in blocking myeloid differentiation. This study