- Email: [email protected]
Effective impairment of myeloma progenitors by hyperthermia: augmentation with bortezomib and Pim inhibition in combination
Abstracts Figure 1 The survival curves of 3 group mice recovered after surgery
in KMS11 cells. KMS11 cells with MMSET I shRNA treatment had markedly reduced ability to form colonies compared with control shLuc. MTS assay indicated that MMSET I knockdown enhanced KMS11 sensitivity to Bortezomib and oxidative stress induced by hydrogen peroxide. Moreover, ectopic expression of MMSET I increased colony formation and reduced sensitivity to Bortezomib and hydrogen peroxide in U266 cells. Conclusion: These preliminary results suggested that MMSET I might act as an oncogene in t(4;14) MM cells. The downstream target genes of MMSET I in t(4;14) MM cells are being pursued by our group.
PO-237 Effective impairment of myeloma progenitors by hyperthermia: augmentation with bortezomib and Pim inhibition in combination
PO-236 MMSET I acts as an oncogene in t(4;14) multiple myeloma cells
H. Miki,1 T. Harada,2 S. Nakamura,2 H. Tenshin,3 R. Amachi,3 K. Watanabe,3 D. Hanson,2 J. Teramachi,4 H. Fujino,2 K. Sogabe,2 M. Takahashi,2 T. Maruhashi,2 S. Fujii,2 K. Kagawa,2 M. Nakamura,5 T. Matsumoto,6 M. Abe2 1
Division of Transfusion Medicine and Cell Therapy, Tokushima Uni-
Z. Xie, W.J. Chng
versity Hospital, Tokushima, Japan; 2Department of Hematology,
Cancer Science Institute of Singapore, National University of
Endocrinology and Metabolism, Tokushima University, Tokushima, Japan; 3Department of Orthodontics and Dentofacial Orthopedics,
Singapore, Singapore 117599; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
The University of Tokushima Graduate School of Oral Science,
119228; Department of Haematology and Oncology, National Uni-
Tokushima, Japan; 4Department of Histology and Oral Histology, The
versity Cancer Institute of Singapore, National University Health
University of Tokushima Graduate School of Oral Science, Tokush-
System, Singapore 119228
ima, Japan; 5Department of Anatomy and Cell Biology Medical Informations, The University of Tokushima Graduate School,
Introduction/Background: Multiple myeloma (MM) is characterized by recurrent chromosomal translocations. Patients with t(4;14) have poor prognosis. The histone methyltransferase (HMTase) MMSET is overexpressed in MM as a result of the t(4;14) chromosomal translocation. MMSET is capable of producing 3 major isoforms, MMSET II, REIIBP and MMSET I. MMSET II contains the full-length protein of 1365 amino acids and possesses HMTase activity for H3K36 and H4K20. MMSET I, a short isoform with 647 amino acids, is identical to MMSET II Nterminus. REIIBP, a short isoform with 584 amino acids, has identical sequence of MMSET II C-terminus. Although the short isoform MMSET I is overexpressed in t(4;14) MM, currently we still understand little about its role in t(4;14) MM. Materials and Methods: Human MM cell lines KMS11 with t(4;14) and U266 without t(4;14) were used in this study. U266 cells with ectopic overexpression of MMSET I were established using puromycin selection after transfection with MMSET I gene. The functions of MMSET I in t(4;14) MM was determined by shRNA mediated knockdown combined with cell proliferation assay (MTS colorimetric assay), colony formation assay and cell cycle analysis. Results: MMSET I knockdown in KMS11 cells was confirmed by quantitative PCR and immunoblot analysis. Flow cytometric analysis indicated MMSET I knockdown could reduce cell cycle S phase
Tokushima, Japan; 6Fujii Memorial Institute for Medical Reseach, The University of Tokushima, Tokushima, Japan
Multiple myeloma (MM) remains incurable despite recent advances in the treatment of MM. MM-initiating cells or MM progenitors are considered to contribute to disease relapse through their drug-resistant nature. We recently developed superparamagnetic nanoparticles which selectively accumulate in MM tumors to kill them by heat generated with magnetic resonance (Theranostics, 2013). In the present study, we aimed to clarify the therapeutic efficacy of hyperthermia on MM cells, focusing on its efficacy on drug-resistant MM progenitor cells. When MM cell lines, RPMI8226, KMS11 and OPM2, were incubated at 41, 43, and 45 for 30 or 60 minutes in a water bath, the cells underwent death in a time- and temperature-dependent manner. Heat treatment at 43 leaded to the phospholylation of eIF2 , and the induction of ATF4 and CHOP along with heat shock protein (HSP) 60, HSP70 and HSP90, indicating induction of ER stress. The treatment also substantially reduced the protein levels of IRF4, Pim-2, c-Myc and Mcl-1 to contribute to MM cell death. Bortezomib, an ER stress inducer, further potentiated ER stress to enhance MM cell death in combination with the heat treatment. RPMI8226 and KMS11 cells exhibited distinct fractions of “side population (SP)” cells, which
15th International Myeloma Workshop, September 23-26, 2015
- e217
Abstracts were substantially minimized after the heat treatment. The heat treatment also suppressed the clonogenic or self-renewal capacity of these MM cells as determined by in vitro colony formation and in vivo tumor formation in SCID mice, suggesting targeting MM progenitors. Further, the Pim inhibitor SMI16a also reduced the SP sizes and the ability of colony formation in RPMI8226 and KMS11 cells. Interestingly, the Pim inhibition in combination with heat treatment enhanced the induction of CHOP, a suicide mediator, while further reducing the protein levels of IRF4 and c-Myc to facilitate MM cell death. These results collectively demonstrated that hyperthermia is able to impair clonogenic drug-resistant fractions of MM cells, which may be augmented in combination with ER stress inducers, such as bortezomib, as well as Pim inhibition. We are now developing superparamagnetic mesoporous nanoparticles, which are able to deliver tumor-selective hyperthermia and drug release. A new strategy with tumor-selective hyperthermia and drug release warrants further study especially in the setting of drugresistant extramedullary plasmacytomas.
PO-238 Preclinical Antimyeloma Activity of EDO-S101 A.A. Lopez-Iglesia,1 L. San-Segundo,1 A.B. Herrero,1 L. González,1 S. Hernández,1 D. Primo,2 T. Paíno,1 M. Martín,1 M. Algarín,1 M. Garayoa,1 T. Mehrling,3 J.F. San Miguel,4 M.V. Mateos,1 E.M. Ocio1 1
University Hospital of Salamanca-IBSAL, IBMCC (USAL-CSIC),
University Hospital & Cancer Research Center. Salamanca. Spain; 2
Vivia Biotech, Madrid, Spain; 3Mundipharma; 4Clínica Universidad de
Navarra, Centro de Investigaciones Médicas Aplicadas (CIMA), IDISNA, Pamplona, Spain
resulted in the accumulation of double strand breaks (DSBs) and increased of p-chk1, p-chk2 and p53. Interestingly, we found that EDO-S101 was able to inhibit DSB repair by the homologous recombination (HR) pathway, as revealed by functional assays performed in two different MM cell lines (JJN3 and U266). An increase in the acetylation of histones H3, H4 and alpha-tubuline were also observed. In vivo EDO-S101 induced more DSB (pH2AX), PARP cleavage and H3 acetylation than bendamustine or vorinostat treatments. Ki67 index was lower in tumors obtained from mice receiving treatment with EDO-S101. We also studied combinations of EDO-S101 with different standard of care in MM (lenalidomide, pomalidomide, oprozomib, carfilzomib and bortezomib). The triple combination with bortezomib and dexamethasone was the one with higher efficacy not only in vitro experiments but also in vivo, in terms of growth inhibition and prolongation of survival. Conclusions: The in vitro, ex vivo and in vivo efficacy of EDO-S101 and its combination with bortezomib and dexamethasone, provides the rationale for the investigations of this compound in clinical trials in MM.
PO-239 Bone Marrow Angiogenic Potential and Molecular Expression of Cell-Cell Adhesion Molecules and Matrix-Metallo Proteinases in Multiple Myeloma M.C. Rapanotti,2 L. Franceschini,2 T.M. Suarez Viguria,1 C. Ialongo,1 S. Vaccarini,2 M. Rizzo,2 L. Cicconi,2 I. Onnis,2 F. Meconi,2 R. Cerretti,3 G. De Angelis,3 B. Mariotti,3 L. Pupo,2 M. Cantonetti,2 M. Postorino,2 F. Lo-Coco1 1
Department of Laboratory Medicine; 2Department of Biomedicine
and Prevention; 3Stem Cell Transplant Unit for Rome Transplant
Background: Alkylators such as melphalan are part of the backbone treatment of MM patients. EDO-S101 is a novel drug resulting from the fusion of a molecule of bendamustine, an alkylator, with one of the deacetylase inhibitor vorinostat. Aims: To study the efficacy and mechanism of action of EDO-S101 in Multiple Myeloma. Methods: Sensitivity to EDO-S101 was assessed by MTT in HMCLs and by flow cytometry in freshly isolated cells from MM patients. The efficacy in vivo was analyzed in a xenograft plasmocitoma model of MM1S in CB-17 SCID mice. Immunohistochemical analyses were performed in selected tumors obtained from mice receiving the vehicle control and different drugs. Results: EDO-S101 efficacy was observed in a panel of 6 HMCLs MM, it was independent of p53 status and it was able to overcome resistance to melphalan. Furthermore, EDOS101 was also active in cells isolated from MM patients with different stage and cytogenetic. Regarding the in vivo efficacy, three weekly doses of EDO-S101 were able to significantly decrease tumor growth and also to prolong survival (p<0.05) compared with bendamustine or vorinostat treatment. Regarding the mechanism of action in vitro, treatment with EDO-S101 induced apoptosis, modified cell cycle profile and
e218
-
15th International Myeloma Workshop, September 23-26, 2015
Network, - University
Background: Bone Marrow (BM) angiogenesis is involved in the pathogenesis and progression of multiple myeloma (MM). Increase of angiogenesis pathway, modulation of specific cell-cell adhesion molecules and secretion of matrix-metalloproteinase-degrading enzymes (MMPs) play an important role to change BM composition from benign conditions such as M-GUS to early stage multiple myeloma (smouldering myeloma, SM) and to advanced multiple myeloma (MM). Aims: We compared the “angiogenic potential”, the cell-cell adhesion and MMPs gene expression in BM and peripheral blood plasma cells (PC) from M-GUS, SM and active MM patients. Patients and Methods: Among the pro-angiogenic factors, the cell-cell adhesion molecules and MMP enzymes, we analysed by qualitative and semi-quantitative RT-PCR, the expression of VEGF, Ang-2, MCAM/MUC18, bFGF, and MMP-2 and MMP-9 eCADH (epithelial cadherin) and the VEeCADH (vascular endothelial cadherin) genes. Epithelial and myeloma cell lines were used as positive controls. Bone marrow healthy donors PC were separated from BM and PB specimen with antibodies against CD138 as described by the manufacturer (Voden-Stem Cell Technology Inc.,
in KMS11 cells. KMS11 cells with MMSET I shRNA treatment had markedly reduced ability to form colonies compared with control shLuc. MTS assay indicated that MMSET I knockdown enhanced KMS11 sensitivity to Bortezomib and oxidative stress induced by hydrogen peroxide. Moreover, ectopic expression of MMSET I increased colony formation and reduced sensitivity to Bortezomib and hydrogen peroxide in U266 cells. Conclusion: These preliminary results suggested that MMSET I might act as an oncogene in t(4;14) MM cells. The downstream target genes of MMSET I in t(4;14) MM cells are being pursued by our group.
PO-237 Effective impairment of myeloma progenitors by hyperthermia: augmentation with bortezomib and Pim inhibition in combination
PO-236 MMSET I acts as an oncogene in t(4;14) multiple myeloma cells
H. Miki,1 T. Harada,2 S. Nakamura,2 H. Tenshin,3 R. Amachi,3 K. Watanabe,3 D. Hanson,2 J. Teramachi,4 H. Fujino,2 K. Sogabe,2 M. Takahashi,2 T. Maruhashi,2 S. Fujii,2 K. Kagawa,2 M. Nakamura,5 T. Matsumoto,6 M. Abe2 1
Division of Transfusion Medicine and Cell Therapy, Tokushima Uni-
Z. Xie, W.J. Chng
versity Hospital, Tokushima, Japan; 2Department of Hematology,
Cancer Science Institute of Singapore, National University of
Endocrinology and Metabolism, Tokushima University, Tokushima, Japan; 3Department of Orthodontics and Dentofacial Orthopedics,
Singapore, Singapore 117599; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
The University of Tokushima Graduate School of Oral Science,
119228; Department of Haematology and Oncology, National Uni-
Tokushima, Japan; 4Department of Histology and Oral Histology, The
versity Cancer Institute of Singapore, National University Health
University of Tokushima Graduate School of Oral Science, Tokush-
System, Singapore 119228
ima, Japan; 5Department of Anatomy and Cell Biology Medical Informations, The University of Tokushima Graduate School,
Introduction/Background: Multiple myeloma (MM) is characterized by recurrent chromosomal translocations. Patients with t(4;14) have poor prognosis. The histone methyltransferase (HMTase) MMSET is overexpressed in MM as a result of the t(4;14) chromosomal translocation. MMSET is capable of producing 3 major isoforms, MMSET II, REIIBP and MMSET I. MMSET II contains the full-length protein of 1365 amino acids and possesses HMTase activity for H3K36 and H4K20. MMSET I, a short isoform with 647 amino acids, is identical to MMSET II Nterminus. REIIBP, a short isoform with 584 amino acids, has identical sequence of MMSET II C-terminus. Although the short isoform MMSET I is overexpressed in t(4;14) MM, currently we still understand little about its role in t(4;14) MM. Materials and Methods: Human MM cell lines KMS11 with t(4;14) and U266 without t(4;14) were used in this study. U266 cells with ectopic overexpression of MMSET I were established using puromycin selection after transfection with MMSET I gene. The functions of MMSET I in t(4;14) MM was determined by shRNA mediated knockdown combined with cell proliferation assay (MTS colorimetric assay), colony formation assay and cell cycle analysis. Results: MMSET I knockdown in KMS11 cells was confirmed by quantitative PCR and immunoblot analysis. Flow cytometric analysis indicated MMSET I knockdown could reduce cell cycle S phase
Tokushima, Japan; 6Fujii Memorial Institute for Medical Reseach, The University of Tokushima, Tokushima, Japan
Multiple myeloma (MM) remains incurable despite recent advances in the treatment of MM. MM-initiating cells or MM progenitors are considered to contribute to disease relapse through their drug-resistant nature. We recently developed superparamagnetic nanoparticles which selectively accumulate in MM tumors to kill them by heat generated with magnetic resonance (Theranostics, 2013). In the present study, we aimed to clarify the therapeutic efficacy of hyperthermia on MM cells, focusing on its efficacy on drug-resistant MM progenitor cells. When MM cell lines, RPMI8226, KMS11 and OPM2, were incubated at 41, 43, and 45 for 30 or 60 minutes in a water bath, the cells underwent death in a time- and temperature-dependent manner. Heat treatment at 43 leaded to the phospholylation of eIF2 , and the induction of ATF4 and CHOP along with heat shock protein (HSP) 60, HSP70 and HSP90, indicating induction of ER stress. The treatment also substantially reduced the protein levels of IRF4, Pim-2, c-Myc and Mcl-1 to contribute to MM cell death. Bortezomib, an ER stress inducer, further potentiated ER stress to enhance MM cell death in combination with the heat treatment. RPMI8226 and KMS11 cells exhibited distinct fractions of “side population (SP)” cells, which
15th International Myeloma Workshop, September 23-26, 2015
- e217
Abstracts were substantially minimized after the heat treatment. The heat treatment also suppressed the clonogenic or self-renewal capacity of these MM cells as determined by in vitro colony formation and in vivo tumor formation in SCID mice, suggesting targeting MM progenitors. Further, the Pim inhibitor SMI16a also reduced the SP sizes and the ability of colony formation in RPMI8226 and KMS11 cells. Interestingly, the Pim inhibition in combination with heat treatment enhanced the induction of CHOP, a suicide mediator, while further reducing the protein levels of IRF4 and c-Myc to facilitate MM cell death. These results collectively demonstrated that hyperthermia is able to impair clonogenic drug-resistant fractions of MM cells, which may be augmented in combination with ER stress inducers, such as bortezomib, as well as Pim inhibition. We are now developing superparamagnetic mesoporous nanoparticles, which are able to deliver tumor-selective hyperthermia and drug release. A new strategy with tumor-selective hyperthermia and drug release warrants further study especially in the setting of drugresistant extramedullary plasmacytomas.
PO-238 Preclinical Antimyeloma Activity of EDO-S101 A.A. Lopez-Iglesia,1 L. San-Segundo,1 A.B. Herrero,1 L. González,1 S. Hernández,1 D. Primo,2 T. Paíno,1 M. Martín,1 M. Algarín,1 M. Garayoa,1 T. Mehrling,3 J.F. San Miguel,4 M.V. Mateos,1 E.M. Ocio1 1
University Hospital of Salamanca-IBSAL, IBMCC (USAL-CSIC),
University Hospital & Cancer Research Center. Salamanca. Spain; 2
Vivia Biotech, Madrid, Spain; 3Mundipharma; 4Clínica Universidad de
Navarra, Centro de Investigaciones Médicas Aplicadas (CIMA), IDISNA, Pamplona, Spain
resulted in the accumulation of double strand breaks (DSBs) and increased of p-chk1, p-chk2 and p53. Interestingly, we found that EDO-S101 was able to inhibit DSB repair by the homologous recombination (HR) pathway, as revealed by functional assays performed in two different MM cell lines (JJN3 and U266). An increase in the acetylation of histones H3, H4 and alpha-tubuline were also observed. In vivo EDO-S101 induced more DSB (pH2AX), PARP cleavage and H3 acetylation than bendamustine or vorinostat treatments. Ki67 index was lower in tumors obtained from mice receiving treatment with EDO-S101. We also studied combinations of EDO-S101 with different standard of care in MM (lenalidomide, pomalidomide, oprozomib, carfilzomib and bortezomib). The triple combination with bortezomib and dexamethasone was the one with higher efficacy not only in vitro experiments but also in vivo, in terms of growth inhibition and prolongation of survival. Conclusions: The in vitro, ex vivo and in vivo efficacy of EDO-S101 and its combination with bortezomib and dexamethasone, provides the rationale for the investigations of this compound in clinical trials in MM.
PO-239 Bone Marrow Angiogenic Potential and Molecular Expression of Cell-Cell Adhesion Molecules and Matrix-Metallo Proteinases in Multiple Myeloma M.C. Rapanotti,2 L. Franceschini,2 T.M. Suarez Viguria,1 C. Ialongo,1 S. Vaccarini,2 M. Rizzo,2 L. Cicconi,2 I. Onnis,2 F. Meconi,2 R. Cerretti,3 G. De Angelis,3 B. Mariotti,3 L. Pupo,2 M. Cantonetti,2 M. Postorino,2 F. Lo-Coco1 1
Department of Laboratory Medicine; 2Department of Biomedicine
and Prevention; 3Stem Cell Transplant Unit for Rome Transplant
Background: Alkylators such as melphalan are part of the backbone treatment of MM patients. EDO-S101 is a novel drug resulting from the fusion of a molecule of bendamustine, an alkylator, with one of the deacetylase inhibitor vorinostat. Aims: To study the efficacy and mechanism of action of EDO-S101 in Multiple Myeloma. Methods: Sensitivity to EDO-S101 was assessed by MTT in HMCLs and by flow cytometry in freshly isolated cells from MM patients. The efficacy in vivo was analyzed in a xenograft plasmocitoma model of MM1S in CB-17 SCID mice. Immunohistochemical analyses were performed in selected tumors obtained from mice receiving the vehicle control and different drugs. Results: EDO-S101 efficacy was observed in a panel of 6 HMCLs MM, it was independent of p53 status and it was able to overcome resistance to melphalan. Furthermore, EDOS101 was also active in cells isolated from MM patients with different stage and cytogenetic. Regarding the in vivo efficacy, three weekly doses of EDO-S101 were able to significantly decrease tumor growth and also to prolong survival (p<0.05) compared with bendamustine or vorinostat treatment. Regarding the mechanism of action in vitro, treatment with EDO-S101 induced apoptosis, modified cell cycle profile and
e218
-
15th International Myeloma Workshop, September 23-26, 2015
Network, - University
Background: Bone Marrow (BM) angiogenesis is involved in the pathogenesis and progression of multiple myeloma (MM). Increase of angiogenesis pathway, modulation of specific cell-cell adhesion molecules and secretion of matrix-metalloproteinase-degrading enzymes (MMPs) play an important role to change BM composition from benign conditions such as M-GUS to early stage multiple myeloma (smouldering myeloma, SM) and to advanced multiple myeloma (MM). Aims: We compared the “angiogenic potential”, the cell-cell adhesion and MMPs gene expression in BM and peripheral blood plasma cells (PC) from M-GUS, SM and active MM patients. Patients and Methods: Among the pro-angiogenic factors, the cell-cell adhesion molecules and MMP enzymes, we analysed by qualitative and semi-quantitative RT-PCR, the expression of VEGF, Ang-2, MCAM/MUC18, bFGF, and MMP-2 and MMP-9 eCADH (epithelial cadherin) and the VEeCADH (vascular endothelial cadherin) genes. Epithelial and myeloma cell lines were used as positive controls. Bone marrow healthy donors PC were separated from BM and PB specimen with antibodies against CD138 as described by the manufacturer (Voden-Stem Cell Technology Inc.,