- Email: [email protected]
Deficiency in DNA repair pathways of peripheral blood mononuclear cells correlates with better clinical outcome of myeloma patients
Abstracts
Hematology, Department of Internal Medicine, Juntendo University 4
School of Medicine, Tokyo, Japan; Department of Medicine and Clinical Science, Gunma University Graduate School of Medicine, 5
PO-284 Deficiency in DNA repair pathways of peripheral blood mononuclear cells correlates with better clinical outcome of myeloma patients M. Gkotzamanidou,1,2 E. Terpos,2 N.C. Munshi,1 M.A. Dimopoulos,2 V.L. Souliotis3 1
Department of Medical Oncology, Jerome Lipper Multiple Myeloma
Maebashi, Japan; Department of Hematology, Tokyo Women’s
Center, Dana-Farber Cancer Institute, Harvard Medical School,
Medical University, Tokyo, Japan; 6Department of Hematology, Jun-
Boston, USA; 2Department of Clinical Therapeutics, Alexandra Hos-
7
e242
ˇ
Division of Hematology, Department of Medicine, Nippon Medical
School, Tokyo, Japan; 2Department of Hematology, National Hospital Organization Nishigunma National Hospital, Shibui, Japan; 3Division of
ˇ
1
ˇ
A. Yamada,1 H. Tamura,1 M. Ishibashi,1 A. Isoda,2 M. Sasaki,3 H. Handa,4 Y. Imai,5 M. Koike,6 S. Ito,7 K. Moriya,1 Y. Hamada,1 T. Asayama,1 M. Matsumoto,2 N. Komatsu,3 J. Tanaka,5 Y. Ishida,7 S. Tanosaki,8 K. Inokuchi1
SLAMF3 high and SLAMF3 low cell fractions of each MM cell line were examined using FCM. 3) SLAMF3 and CD138 expression levels on clonal plasma cells from 144 newly diagnosed (18 asymptomatic and 126 symptomatic) MM patients, 25 refractory/ relapsed MM patients, and 44 patients with monoclonal gammopathy of undetermined significance (MGUS) were analyzed using FCM. Results: 1) SLAMF3 was expressed in most cell lines, even on the small CD138e subpopulation, which is immature and may represent the MM stem cell compartment. 2) SLAMF3 high cells transitioned from the G0/G1 to the G2/M phase of the cell cycle and had significantly increased levels of BrdU incorporation compared with SLAMF3 low cells. The percentage of melphalaninduced apoptosis in SLAMF3 high cells was lower than in SLAMF3 low cells. However, neither SAP nor EAT2 were detected in MM cell lines. 3) High SLAMF3 expression was detected in most patients with MGUS and MM, even in refractory/relapsed disease, although CD138 expression levels were significantly decreased in progressive disease such as advanced ISS stages and refractory/ relapsed status. Conclusions: SLAMF3 was highly expressed on MM cells and thus could become a new marker for identifying MM cells. Furthermore, high expression of SLAMF3 on MM cells was associated with a growth advantage and drug resistance. However, it remains unclear whether the SLAMF3eSLAMF3 interaction induces activation signaling in MM cells without SLAM-associated adaptor proteins. Further studies are underway to clarify the functions of SLAMF3 in MM pathogenesis and association of SLAMF3 with immune responses in the tumor microenvironment. ˇ
Expression and function of SLAM family molecule SLAMF3 (CD229) in myeloma
ˇ
PO-283
ˇ
[U266 (p53 mutant), OPM-2 (p53 mutant), KMS-18 (p53 WT/-), JJN3(p53 WT/-), MM.1S (p53 WT)]. Effects on downstream substrates were analysed by Western blotting. Elevated HUWE1 expression was confirmed in MM cell lines and primary CD138+ MM cells compared to healthy CD138+ cells at both the gene and protein level ( 2-fold). shRNA knockdown of HUWE1 significantly decreased (p0.01) the proliferation of all MM cell lines, regardless of p53 status; this effect was maintained when cells were cultured in the presence of bone marrow stromal cells. Conversely, we show that MM cell lines that carry mutant p53 were largely resistant to the MDM2 inhibitor Nutlin-3. Finally, we have demonstrated that HUWE1 depletion is associated with a significant increase of cells in G2/M, an accumulation of p53 and MIZ1 and a decrease in MYC protein levels (p0.05). The proteasome is an established target in MM and there are opportunities for more specific targeting of enzymes within the UPS. Here we demonstrate that the E3 ligase HUWE1 is important in the growth and survival of MM cells and offers a more valuable therapeutic target than MDM2. Future work will investigate the effect of small molecule inhibitors of HUWE1 in MM.
tendo University Shizuoka Hospital, Shizuoka, Japan; Division of
pital, National and Kapodistrian University of Athens, School of
Hematology and Oncology, Department of Internal Medicine, Iwate Medical University School of Medicine, Morioka, Japan; 8Department
Medicine, Athens, Greece; 3Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, Athens,
of Hematology, Fraternity Memorial Hospital, Tokyo, Japan
Greece
Introduction: The signaling lymphocyte-activation molecule family member 3 (SLAMF3), also known as CD229 or Ly9, is a member of the immunoglobulin superfamily expressed on T and B cells. SLAMF3 plays an important role in lymphocyte activation and cytotoxicity via SLAMF3eSLAMF3 binding. However, the function of SLAMF3 in multiple myeloma (MM) remains unclear. To clarify this, we investigated the expression and functions of SLAMF3 in MM. Methods: 1) The expression of SLAMF3 and its adaptor protein, SLAM-associated protein (SAP) and Ewing’s sarcoma-associated transcript 2 (EAT2) was analyzed using real-time PCR, flow cytometry (FCM), and Western blotting in 9 human MM cell lines. 2) The drug sensitivity and cell proliferation in
Introduction: DNA interstrand cross-linking (ICL) agents have an important impact in cancer treatment. However, the precise mechanisms contributing to differential response of patients to chemotherapy with these drugs are poorly understood. Patients and Methods: We studied peripheral blood mononuclear cells (PBMCs) from 25 healthy controls (HC) and 15 newly diagnosed multiple myeloma (MM) patients [8M/7F; median age 61 years; responders (PR, n¼9) and non-responders (
-
15th International Myeloma Workshop, September 23-26, 2015
Abstracts panel of 84 DNA damage response-related genes were measured. Results: The accumulation of melphalan-induced monoadducts was inversely correlated with the first-phase repair capacity of cells, being significantly higher in HC than in responders and lowest in non-responders (all P<0.001). In addition, the accumulation of ICLs was significantly higher in HC compared to responders (P<0.01), due to higher levels of monoadducts (precursors of ICLs) left unrepaired in these cells. Minimal amounts of ICLs were observed in non-responders. Moreover, DSBs burden was significantly higher in HC than in responders, due to higher accumulation of ICLs (precursors of DSBs) and lower rates of DSB repair in these cells (P<0.01). Minimal amounts of ICLs were observed in non-responders. More interestingly, apoptotic rates were inversely correlated with the DSBs repair efficiency being significantly higher in HC compared to responders and lowest in non-responders (all P<0.05). PBMCs without exposure to melphalan, showed a progressive and significant increase in the looseness of the local chromatin structure, from HC to MM-responders and finally to non-responders (all P<0,05). To note, microarray analysis of untreated PBMCs consistently points to an altered expression of several DNA damage response-related genes in MM patients. Responders showed significant upregulation of ATR, CHEK2, XPA, XRCC1 and CHEK2 genes and downregulation of ATM, MPG, UNG, CDKN1A and CDC25C compared to non-responders. Conclusion: MM patients with deficient DNA repair pathways exhibit simultaneous accumulation of the extremely cytotoxic ICLs and DSBs lesions, which in turn triggers the induction of the apoptotic pathway, a priority for successful clinical outcome.
cells in cocultures with bone marrow stromal cells. Although Pim-2 expression in MM cells was marginally affected by doxorubicin and lenalidomide, bendamustine potently reduced Pim-2 expression along with inducing cell death in a portion of MM cell lines, including INA-6 and TSPC-1, even under cocultures with bone marrow stromal cells. Interestingly, bendamustine more potently down-regulated Pim-2 protein levels in MM cells and induced their death in acidic conditions; the Pim inhibitor SMI-16a further enhanced bendamustine’s antiMM effects in acidic conditions. Although RPMI8226 and U266 cells were resistant to bendamustine at pH7.4, bendamustine was able to exert cytotoxic effects on these cells along with decreasing Pim-2 expression in acidic conditions. In contrast, bortezomib dose-dependently increased Pim-2 protein levels in MM cells without enhancing Pim-2 mRNA expression, while inducing ER stress. However, ER stress inducer tunicamycin only weakly increased Pim-2 protein levels in MM cells, suggesting suppression of proteasomal degradation of Pim-2 by bortezomib. Interestingly, the Pim inhibitor SMI-16a suppressed the levels of ubiquitinylated proteins and ATF4 increased by bortezomib. Thus, bortezomib appears to increase Pim-2 protein levels in MM cells while Pim inhibition may mitigate bortezomib-induced ER stress. Although susceptibility to bendamustine considerably varies among MM cells, the present study demonstrates that anti-MM effects of bendamustine are augmented in acidic conditions at least in part through down-regulation of Pim-2, and that Pim inhibition further enhances the bendamustine’s anti-MM activity especially in acidic conditions. Combinatory treatment with bortezomib and Pim inhibitors seems interesting; however, efficacy of sequential treatment with Pim inhibitors after bortezomib warrants further study.
PO-286 PO-285 Alteration of Pim-2 expression by clinically available anti-myeloma agents: combinatory anti-myeloma effects with Pim inhibition S. Nakamura,1 H. Miki,2 H. Tenshin,3 R. Amachi,3 D. Hanson,1 K. Watanabe,3 J. Teramachi,4 A. Oda,1 K. Sogabe,1 H. Fujino,1 T. Maruhashi,1 S. Fujii,1 K. Kagawa,1 M. Abe1
Relation between lymphocytes and dendritic cells - implications for the effectiveness of an immune response to neoplastic antigens in patients suffering from multiple myeloma E. Grywalska, M. Pasiarski, B. Pasiarska, S. Gozdz, J. Rolinski Department of Clinical Immunology and Immunotherapy, Medical University of Lublin; Department of Hematology, Holycross Cancer Centre in Kielce
1
Department of Hematology, Endocrinology and Metabolism,
Tokushima University, Tokushima, Japan; 2Division of of transfusion medicine and cell therapy, Tokushima University hospital, Tokushima, Japan; 3Department of Orthodontics and Dentofacial Orthopedics, Tokushima University, Tokushima, Japan; 4Department of Histology and Oral Histology, Tokushima University, Tokushima, Japan
The serine/threonine kinase Pim-2 is over-expressed as a pro-surviving mediator in myeloma (MM) cells, and has drawn considerable attention as a novel therapeutic target in MM. Pim-2 kinase is constitutively active without post-translational modification, and its activity is dependent on its protein levels. In the present study, we aimed to explore the effects of clinically available anti-MM agents on Pim-2 expression in MM cells and their anti-MM activity in combination with Pim inhibitors. Pim-2 was markedly up-regulated in MM
Interactions between T lymphocytes, NK, NKT-like cells, and dendritic cells (DCs), constitute crucial elements of the anti-cancer response. In the present study, we determined the frequencies of activated T CD4+, T CD8+, NK, and NKT-like cells and their correlation with myeloid and lymphoid populations of DCs in patients suffering from multiple myeloma (MM). The study involved 50 patients diagnosed with MM at the Department of Haematology in the Holycross Cancer Centre in Kielce. The research samples were collected after the MM diagnosis before the initiation of anticancer therapy. Peripheral blood mononuclear cells were separated by density gradient centrifugation and stained with monoclonal antibodies conjugated with relevant fluorochromes for flow cytometric assessment.
15th International Myeloma Workshop, September 23-26, 2015
- e243
Hematology, Department of Internal Medicine, Juntendo University 4
School of Medicine, Tokyo, Japan; Department of Medicine and Clinical Science, Gunma University Graduate School of Medicine, 5
PO-284 Deficiency in DNA repair pathways of peripheral blood mononuclear cells correlates with better clinical outcome of myeloma patients M. Gkotzamanidou,1,2 E. Terpos,2 N.C. Munshi,1 M.A. Dimopoulos,2 V.L. Souliotis3 1
Department of Medical Oncology, Jerome Lipper Multiple Myeloma
Maebashi, Japan; Department of Hematology, Tokyo Women’s
Center, Dana-Farber Cancer Institute, Harvard Medical School,
Medical University, Tokyo, Japan; 6Department of Hematology, Jun-
Boston, USA; 2Department of Clinical Therapeutics, Alexandra Hos-
7
e242
ˇ
Division of Hematology, Department of Medicine, Nippon Medical
School, Tokyo, Japan; 2Department of Hematology, National Hospital Organization Nishigunma National Hospital, Shibui, Japan; 3Division of
ˇ
1
ˇ
A. Yamada,1 H. Tamura,1 M. Ishibashi,1 A. Isoda,2 M. Sasaki,3 H. Handa,4 Y. Imai,5 M. Koike,6 S. Ito,7 K. Moriya,1 Y. Hamada,1 T. Asayama,1 M. Matsumoto,2 N. Komatsu,3 J. Tanaka,5 Y. Ishida,7 S. Tanosaki,8 K. Inokuchi1
SLAMF3 high and SLAMF3 low cell fractions of each MM cell line were examined using FCM. 3) SLAMF3 and CD138 expression levels on clonal plasma cells from 144 newly diagnosed (18 asymptomatic and 126 symptomatic) MM patients, 25 refractory/ relapsed MM patients, and 44 patients with monoclonal gammopathy of undetermined significance (MGUS) were analyzed using FCM. Results: 1) SLAMF3 was expressed in most cell lines, even on the small CD138e subpopulation, which is immature and may represent the MM stem cell compartment. 2) SLAMF3 high cells transitioned from the G0/G1 to the G2/M phase of the cell cycle and had significantly increased levels of BrdU incorporation compared with SLAMF3 low cells. The percentage of melphalaninduced apoptosis in SLAMF3 high cells was lower than in SLAMF3 low cells. However, neither SAP nor EAT2 were detected in MM cell lines. 3) High SLAMF3 expression was detected in most patients with MGUS and MM, even in refractory/relapsed disease, although CD138 expression levels were significantly decreased in progressive disease such as advanced ISS stages and refractory/ relapsed status. Conclusions: SLAMF3 was highly expressed on MM cells and thus could become a new marker for identifying MM cells. Furthermore, high expression of SLAMF3 on MM cells was associated with a growth advantage and drug resistance. However, it remains unclear whether the SLAMF3eSLAMF3 interaction induces activation signaling in MM cells without SLAM-associated adaptor proteins. Further studies are underway to clarify the functions of SLAMF3 in MM pathogenesis and association of SLAMF3 with immune responses in the tumor microenvironment. ˇ
Expression and function of SLAM family molecule SLAMF3 (CD229) in myeloma
ˇ
PO-283
ˇ
[U266 (p53 mutant), OPM-2 (p53 mutant), KMS-18 (p53 WT/-), JJN3(p53 WT/-), MM.1S (p53 WT)]. Effects on downstream substrates were analysed by Western blotting. Elevated HUWE1 expression was confirmed in MM cell lines and primary CD138+ MM cells compared to healthy CD138+ cells at both the gene and protein level ( 2-fold). shRNA knockdown of HUWE1 significantly decreased (p0.01) the proliferation of all MM cell lines, regardless of p53 status; this effect was maintained when cells were cultured in the presence of bone marrow stromal cells. Conversely, we show that MM cell lines that carry mutant p53 were largely resistant to the MDM2 inhibitor Nutlin-3. Finally, we have demonstrated that HUWE1 depletion is associated with a significant increase of cells in G2/M, an accumulation of p53 and MIZ1 and a decrease in MYC protein levels (p0.05). The proteasome is an established target in MM and there are opportunities for more specific targeting of enzymes within the UPS. Here we demonstrate that the E3 ligase HUWE1 is important in the growth and survival of MM cells and offers a more valuable therapeutic target than MDM2. Future work will investigate the effect of small molecule inhibitors of HUWE1 in MM.
tendo University Shizuoka Hospital, Shizuoka, Japan; Division of
pital, National and Kapodistrian University of Athens, School of
Hematology and Oncology, Department of Internal Medicine, Iwate Medical University School of Medicine, Morioka, Japan; 8Department
Medicine, Athens, Greece; 3Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, Athens,
of Hematology, Fraternity Memorial Hospital, Tokyo, Japan
Greece
Introduction: The signaling lymphocyte-activation molecule family member 3 (SLAMF3), also known as CD229 or Ly9, is a member of the immunoglobulin superfamily expressed on T and B cells. SLAMF3 plays an important role in lymphocyte activation and cytotoxicity via SLAMF3eSLAMF3 binding. However, the function of SLAMF3 in multiple myeloma (MM) remains unclear. To clarify this, we investigated the expression and functions of SLAMF3 in MM. Methods: 1) The expression of SLAMF3 and its adaptor protein, SLAM-associated protein (SAP) and Ewing’s sarcoma-associated transcript 2 (EAT2) was analyzed using real-time PCR, flow cytometry (FCM), and Western blotting in 9 human MM cell lines. 2) The drug sensitivity and cell proliferation in
Introduction: DNA interstrand cross-linking (ICL) agents have an important impact in cancer treatment. However, the precise mechanisms contributing to differential response of patients to chemotherapy with these drugs are poorly understood. Patients and Methods: We studied peripheral blood mononuclear cells (PBMCs) from 25 healthy controls (HC) and 15 newly diagnosed multiple myeloma (MM) patients [8M/7F; median age 61 years; responders (PR, n¼9) and non-responders (
-
15th International Myeloma Workshop, September 23-26, 2015
Abstracts panel of 84 DNA damage response-related genes were measured. Results: The accumulation of melphalan-induced monoadducts was inversely correlated with the first-phase repair capacity of cells, being significantly higher in HC than in responders and lowest in non-responders (all P<0.001). In addition, the accumulation of ICLs was significantly higher in HC compared to responders (P<0.01), due to higher levels of monoadducts (precursors of ICLs) left unrepaired in these cells. Minimal amounts of ICLs were observed in non-responders. Moreover, DSBs burden was significantly higher in HC than in responders, due to higher accumulation of ICLs (precursors of DSBs) and lower rates of DSB repair in these cells (P<0.01). Minimal amounts of ICLs were observed in non-responders. More interestingly, apoptotic rates were inversely correlated with the DSBs repair efficiency being significantly higher in HC compared to responders and lowest in non-responders (all P<0.05). PBMCs without exposure to melphalan, showed a progressive and significant increase in the looseness of the local chromatin structure, from HC to MM-responders and finally to non-responders (all P<0,05). To note, microarray analysis of untreated PBMCs consistently points to an altered expression of several DNA damage response-related genes in MM patients. Responders showed significant upregulation of ATR, CHEK2, XPA, XRCC1 and CHEK2 genes and downregulation of ATM, MPG, UNG, CDKN1A and CDC25C compared to non-responders. Conclusion: MM patients with deficient DNA repair pathways exhibit simultaneous accumulation of the extremely cytotoxic ICLs and DSBs lesions, which in turn triggers the induction of the apoptotic pathway, a priority for successful clinical outcome.
cells in cocultures with bone marrow stromal cells. Although Pim-2 expression in MM cells was marginally affected by doxorubicin and lenalidomide, bendamustine potently reduced Pim-2 expression along with inducing cell death in a portion of MM cell lines, including INA-6 and TSPC-1, even under cocultures with bone marrow stromal cells. Interestingly, bendamustine more potently down-regulated Pim-2 protein levels in MM cells and induced their death in acidic conditions; the Pim inhibitor SMI-16a further enhanced bendamustine’s antiMM effects in acidic conditions. Although RPMI8226 and U266 cells were resistant to bendamustine at pH7.4, bendamustine was able to exert cytotoxic effects on these cells along with decreasing Pim-2 expression in acidic conditions. In contrast, bortezomib dose-dependently increased Pim-2 protein levels in MM cells without enhancing Pim-2 mRNA expression, while inducing ER stress. However, ER stress inducer tunicamycin only weakly increased Pim-2 protein levels in MM cells, suggesting suppression of proteasomal degradation of Pim-2 by bortezomib. Interestingly, the Pim inhibitor SMI-16a suppressed the levels of ubiquitinylated proteins and ATF4 increased by bortezomib. Thus, bortezomib appears to increase Pim-2 protein levels in MM cells while Pim inhibition may mitigate bortezomib-induced ER stress. Although susceptibility to bendamustine considerably varies among MM cells, the present study demonstrates that anti-MM effects of bendamustine are augmented in acidic conditions at least in part through down-regulation of Pim-2, and that Pim inhibition further enhances the bendamustine’s anti-MM activity especially in acidic conditions. Combinatory treatment with bortezomib and Pim inhibitors seems interesting; however, efficacy of sequential treatment with Pim inhibitors after bortezomib warrants further study.
PO-286 PO-285 Alteration of Pim-2 expression by clinically available anti-myeloma agents: combinatory anti-myeloma effects with Pim inhibition S. Nakamura,1 H. Miki,2 H. Tenshin,3 R. Amachi,3 D. Hanson,1 K. Watanabe,3 J. Teramachi,4 A. Oda,1 K. Sogabe,1 H. Fujino,1 T. Maruhashi,1 S. Fujii,1 K. Kagawa,1 M. Abe1
Relation between lymphocytes and dendritic cells - implications for the effectiveness of an immune response to neoplastic antigens in patients suffering from multiple myeloma E. Grywalska, M. Pasiarski, B. Pasiarska, S. Gozdz, J. Rolinski Department of Clinical Immunology and Immunotherapy, Medical University of Lublin; Department of Hematology, Holycross Cancer Centre in Kielce
1
Department of Hematology, Endocrinology and Metabolism,
Tokushima University, Tokushima, Japan; 2Division of of transfusion medicine and cell therapy, Tokushima University hospital, Tokushima, Japan; 3Department of Orthodontics and Dentofacial Orthopedics, Tokushima University, Tokushima, Japan; 4Department of Histology and Oral Histology, Tokushima University, Tokushima, Japan
The serine/threonine kinase Pim-2 is over-expressed as a pro-surviving mediator in myeloma (MM) cells, and has drawn considerable attention as a novel therapeutic target in MM. Pim-2 kinase is constitutively active without post-translational modification, and its activity is dependent on its protein levels. In the present study, we aimed to explore the effects of clinically available anti-MM agents on Pim-2 expression in MM cells and their anti-MM activity in combination with Pim inhibitors. Pim-2 was markedly up-regulated in MM
Interactions between T lymphocytes, NK, NKT-like cells, and dendritic cells (DCs), constitute crucial elements of the anti-cancer response. In the present study, we determined the frequencies of activated T CD4+, T CD8+, NK, and NKT-like cells and their correlation with myeloid and lymphoid populations of DCs in patients suffering from multiple myeloma (MM). The study involved 50 patients diagnosed with MM at the Department of Haematology in the Holycross Cancer Centre in Kielce. The research samples were collected after the MM diagnosis before the initiation of anticancer therapy. Peripheral blood mononuclear cells were separated by density gradient centrifugation and stained with monoclonal antibodies conjugated with relevant fluorochromes for flow cytometric assessment.
15th International Myeloma Workshop, September 23-26, 2015
- e243