- Email: [email protected]
The chemokine CXCL9 (MIG) is an independent predictor of overall survival in newly diagnosed multiple myeloma
Abstracts PO-272
Hematology and Hematologic Malignancies, University of Utah,
Survivin suppressant YM155 induces cell death via proteasomal degradation of c-Myc in multiple myeloma cells
plantation, University Medical Center Hamburg-Eppendorf, Hamburg,
M. Asahi, S. Ito, Y. Ishida Hematology & Oncology, Department of Internal Medicine, Department of Medical Oncology, Iwate Medical University School of Medicine
Introduction: Survivin is a member of the inhibitor of apoptosis protein (IAP) family with its dual roles in mitosis and apoptosis, and emerges as an attractive target for cancer therapy. YM155, a novel molecular targeted agent, suppresses survivin, which is overexpressed in many tumor types. However, the effect of this agent on multiple myeloma (MM) cells remains unclear. Materials & Methods Results: Five human MM cell lines, RPMI8226, U266, KMS20, KMS28PE, and KMS34 were used. Cell proliferation and cell death were evaluated by MTT assay and by flow cytometric analysis with annexin V/PI staining. Protein expressions were analyzed with immunoblot. For proteasome inhibitory assay, cells were treated with YM155 and/or MG132 for 6 hours. Results: YM155 inhibited cell proliferation of these cells in a dose-dependent manner. Annexin V assay showed that YM155 induced apoptosis in these cells. To better understand these effects of YM155 on MM cells, we evaluated the intracellular signaling and apoptosis-associated protein status. Immunoblot analyses showed that YM155 reduced not only survivin but also Mcl-1 and XIAP expressions. We also observed the activation of caspase-3 and PARP in YM155-treated cells, indicating that YM155 induces caspasedependent apoptosis. In contrast, YM155 did not affect phosphorylation status of Erk1/2 and STAT3. Interestingly, YM155 suppressed c-Myc and IRF4 expressions, both of which are recognized as an important oncogene in the pathogenesis of MM. In addition, qPCR assay showed that YM155 treatment did not reduce c-Myc mRNA level. On the other hand, proteasome inhibitor prevented the suppression of c-Myc expression by YM155 treatment, indicating a proteasomal degradation of c-Myc by YM155. Conclusion: YM155 suppresses cell proliferation and survival in MM cells in part via not only inhibiting anti-apoptotic proteins such as survivin, Mcl-1 and XIAP but also suppressing c-Myc oncogene. Further study is needed to clarify the molecular mechanism of c-Myc degradation induced by YM155. Our results may provide a new concept in c-Myc-targeting therapy for MM.
Huntsman Cancer Institute, Salt Lake City, UT; Stem Cell TransGermany; Flow Cytometry Core Facility, University of Utah Health Sciences Center, Salt Lake City, UT; Oncology/Hematology/Bone Marrow Transplantation with the section Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Department of Medicine IV, University Hospital RWTH Aachen, Aachen, Germany; ARUP Laboratories, Hematopathology University of Utah, Salt Lake City, UT; Department of Pathology, University of Utah, Salt Lake City, UT
Multiple Myeloma (MM) is a plasma cell (PC) malignancy, which despite significant therapeutic advances, is still considered incurable. This is due to the persistence of chemotherapy-resistant minimal residual disease in the patients’ bone marrow (BM) after an effective induction therapy. Immunotherapies targeting surface molecules expressed on the bulk of tumor cells and the chemotherapy-resistant, myeloma-propagating cells could play a central role in this clinical setting. We recently described surface molecule CD229 as a potential therapeutic target for MM. In our current study we assessed the expression of CD229 on different PC subtypes and on cells with a myeloma-propagating phenotype in a total of 77 patients with PC dyscrasias independently at two different cancer centers. We found that CD229 was strongly and homogeneously overexpressed on the PC of patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma, MM, and PC leukemia. CD229 was particularly overexpressed on those PC showing an abnormal phenotype such as expression of CD56. Most importantly, CD229 was also highly expressed on those cells in the patients’ BM displaying the phenotype of chemotherapy-resistant and myeloma-propagating cells. In conclusion, our combined findings suggest that immunotherapies targeting CD229 will not only be effective for the bulk of tumor cells but will also help to eradicate chemotherapy-resistant cells remaining in the patients’ BM after induction treatment. Hopefully, the design of CD229-specific monoclonal antibodies or chimeric antigen receptor-transduced T cells will help to achieve prolonged remissions or even cures in MM patients.
PO-274 The chemokine CXCL9 (MIG) is an independent predictor of overall survival in newly diagnosed multiple myeloma
PO-273
A. Bolomsky, M. Schreder, N. Zojer, H. Ludwig
CD229 is expressed on the surface of plasma cells carrying an aberrant phenotype and chemotherapy-resistant precursor cells in Multiple Myeloma
Wilhelminen Cancer Research Institute, Department of Medicine I,
S. Yousef, M. Kovacsovics-Bankowski, M.E. Salama, N. Bhardwaj, M. Steinbach, A. Langemo, T. Kovacsovics, J. Marvin, M. Binder, J. Panse, N. Kröger, T. Luetkens, D. Atanackovic
Wilhelminenhospital, Vienna, Austria
Background: The CXCR3 binding molecules CXCL9-11 have been associated with tumor progression, immune escape and angiogenesis in several malignancies. Information about the precise role of CXCR3 binding chemokines in MM is limited. We here aimed to evaluate the prognostic relevance of CXCL9 in MM.
15th International Myeloma Workshop, September 23-26, 2015
- e237
Abstracts Patients and Methods: CXCL9 serum levels were studied by ELISA in newly-diagnosed (ND) MM (n¼105) and MGUS patients (n¼17), and healthy donors (n¼37). CXCL9 expression was analyzed by qPCR in MM cell lines (HMCLs), MM-BMMNCs, MM-PBMCs and in a publically available GEP-dataset (GSE2658). Concurrent analysis of CXCL9-11 serum levels was performed by FACS-CBA array. Results: Median CXCL9 levels (161 pg/ml; range: 9-1966) were significantly elevated in MM patients compared to MGUS (93; 6-1303) and healthy donors (106; 51e391). CXCL9 levels significantly correlated with B2M, ISS stage, calcium, albumin, LDH, hemoglobin and age. High CXCL9 levels predicted adverse survival (17.0 months vs. not reached, P<0.001), which was upheld when age-adjusted cut-off levels were used. Similarly, in MM patients (n¼559) treated within the TT2 and 3 protocol (GSE2658) shorter OS was found in patients with a present compared to those with an absent detection call for CXCL9 (P¼0.004). Expression of CXCL9 was only detected in 1 of 7 HMCLs and 12% of previously published MM cell samples. Hence, increased serum levels of CXCL9 are likely derived from other cell types. We observed increased expression of CXCL9 in PBMCs compared to BMMNCs of MM patients, suggesting that elevated secretion of CXCL9 might result from aberrant cytokine production in cells of the PB. CXCL9 serum levels were also analyzed in conjunction with CXCL10 and 11 in 65 MM patients by FACS-CBA. Again, we observed a significant correlation with B2M for CXCL9-11. Survival was significantly worse in patients with high compared to low CXCL9 (median OS 25.3 months vs. not reached, P¼0.003) and CXCL10 (19.97 months vs. not reached, P¼0.0006). In multivariate analysis, CXCL9 (P¼0.03) and CXCL10 (P¼0.013) were revealed as independent predictors of survival. Conclusion: Our findings depict CXCL9 as novel prognostic marker for overall survival in ND-MM patients. High levels of CXCL9 in the PB probably increase TH1/TH2 ratios and may shield myeloma cells from immune attack by CD8+ cytotoxic T cells as previously shown for T cell lymphomas. Further experiments will aim to confirm these results and define the functional role of CXCL9 in MM.
Introduction: The t(4;14) multiple myeloma (MM) subgroup, characterised by expression of the histone methyltransferase MMSET, has a poor prognosis. We have identified that an epithelial-to-mesenchymal transition (EMT)-like process plays a critical role in t(4;14)-positive MM disease pathogenesis. This EMT-like phenotype is, in part, characterised by increased expression of the cell adhesion molecule N-cadherin (CDH2). In this study, we investigated N-cadherin as a therapeutic target in t(4;14)positive MM. Materials and Methods: The effect of shRNAmediated Cdh2 knock-down was assessed in vitro in the MM plasma cell line 5TGM1. In addition, the effect of targeting N-cadherin on MM plasma cell bone marrow homing, tumour establishment and tumour growth was assessed in vivo using the C57BL/KaLwRijHsd mouse model of MM. Results: When compared with 5TGM1-scramble-shRNA cells, 5TGM1-Cdh2shRNA cells had significantly reduced adhesion to bone marrow endothelial cells. However, N-cadherin knock-down did not affect 5TGM1 cell proliferation, migration or adhesion to bone marrow stromal cells in vitro. Mice intravenously inoculated with 5TGM1Cdh2-shRNA cells showed significantly decreased tumour burden after four weeks, as assessed by bioluminescence imaging, compared with animals bearing 5TGM1-scramble-shRNA cells. Notably, daily administration of the N-cadherin antagonist ADH-1 (100mg/ kg/day), commencing at the time of tumour inoculation, significantly decreased tumour burden after 4 weeks compared with PBStreated mice. In contrast, ADH-1 had no effect on tumour burden in the established disease setting. Conclusion: Our findings demonstrate a potential role for N-cadherin in MM plasma cell extravasation and bone marrow homing. Furthermore, these studies suggest that N-cadherin represents a novel therapeutic target to prevent the dissemination of MM plasma cells and delay disease progression in MM patients with high N-cadherin expression.
PO-276 Myeloma-derived CCL27 induces stroma-dependent resistance against bortezomib via upregulation of IL-10 S. Thangavadivel,1 A. Olivier,1 B. Postert,1 C. Zelle-Rieser,1 J. Kern,2 G. Untergasser,1,2 A. Brunner,3 W. Willenbacher,4 R. Biedermann,5 R. Greil,1,6 K. Jöhrer1
PO-275 N-cadherin is a therapeutic target in t(4;14)-positive multiple myeloma K.M. Mrozik,1,2 C.M. Cheong,1,2 D. Hewett,1,2 C.H. Kok,2,3 A.W.S. Chow,1 O. Blaschuk,4 J.D. Licht,5 A.C.W. Zannettino,1,2,3,6 K. Vandyke1,2,3,6
for Experimental Oncology, Internal Medicine V, Medical University
1
bruck, Austria; 4Department of Internal Medicine V, Hematology and
Myeloma Research Laboratory, Discipline of Physiology, School of
Medical Sciences, Faculty of Health Science, University of Adelaide, 2
Adelaide, South Australia; Cancer Theme, South Australian Health 3
1
Tyrolean Cancer Research Institute, Innsbruck, Austria; 2Laboratory
Innsbruck, Austria; 3Institute for Pathology, Medical University InnsOncology, Medical University Innsbruck, Austria; 5Department of Orthopedic Surgery, Medical University Innsbruck, Austria; 6Labora-
and Medical Research Institute, Adelaide, South Australia; School of Medicine, University of Adelaide, Adelaide, South Australia; 4Division
tory for Immunological and Molecular Cancer Research, Salzburg Cancer Research Institute, 3rd Medical Department, Paracelsus
of Urology, Department of Surgery, McGill University, Montreal,
Medical University, Salzburg, Austria
Canada; 5Division of Hematology/Oncology, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Feinberg School of Medicine, Chicago, USA; 6Centre for Cancer Biology and Hanson Institute, SA Pathology, Adelaide, South Australia
e238
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15th International Myeloma Workshop, September 23-26, 2015
Introduction: Multiple Myeloma is still incurable and drugresistance is the major cause for relapse and early death of the patients. The bone marrow microenvironment plays a fundamental role in shaping tumor progression. Chemokines are soluble
Hematology and Hematologic Malignancies, University of Utah,
Survivin suppressant YM155 induces cell death via proteasomal degradation of c-Myc in multiple myeloma cells
plantation, University Medical Center Hamburg-Eppendorf, Hamburg,
M. Asahi, S. Ito, Y. Ishida Hematology & Oncology, Department of Internal Medicine, Department of Medical Oncology, Iwate Medical University School of Medicine
Introduction: Survivin is a member of the inhibitor of apoptosis protein (IAP) family with its dual roles in mitosis and apoptosis, and emerges as an attractive target for cancer therapy. YM155, a novel molecular targeted agent, suppresses survivin, which is overexpressed in many tumor types. However, the effect of this agent on multiple myeloma (MM) cells remains unclear. Materials & Methods Results: Five human MM cell lines, RPMI8226, U266, KMS20, KMS28PE, and KMS34 were used. Cell proliferation and cell death were evaluated by MTT assay and by flow cytometric analysis with annexin V/PI staining. Protein expressions were analyzed with immunoblot. For proteasome inhibitory assay, cells were treated with YM155 and/or MG132 for 6 hours. Results: YM155 inhibited cell proliferation of these cells in a dose-dependent manner. Annexin V assay showed that YM155 induced apoptosis in these cells. To better understand these effects of YM155 on MM cells, we evaluated the intracellular signaling and apoptosis-associated protein status. Immunoblot analyses showed that YM155 reduced not only survivin but also Mcl-1 and XIAP expressions. We also observed the activation of caspase-3 and PARP in YM155-treated cells, indicating that YM155 induces caspasedependent apoptosis. In contrast, YM155 did not affect phosphorylation status of Erk1/2 and STAT3. Interestingly, YM155 suppressed c-Myc and IRF4 expressions, both of which are recognized as an important oncogene in the pathogenesis of MM. In addition, qPCR assay showed that YM155 treatment did not reduce c-Myc mRNA level. On the other hand, proteasome inhibitor prevented the suppression of c-Myc expression by YM155 treatment, indicating a proteasomal degradation of c-Myc by YM155. Conclusion: YM155 suppresses cell proliferation and survival in MM cells in part via not only inhibiting anti-apoptotic proteins such as survivin, Mcl-1 and XIAP but also suppressing c-Myc oncogene. Further study is needed to clarify the molecular mechanism of c-Myc degradation induced by YM155. Our results may provide a new concept in c-Myc-targeting therapy for MM.
Huntsman Cancer Institute, Salt Lake City, UT; Stem Cell TransGermany; Flow Cytometry Core Facility, University of Utah Health Sciences Center, Salt Lake City, UT; Oncology/Hematology/Bone Marrow Transplantation with the section Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Department of Medicine IV, University Hospital RWTH Aachen, Aachen, Germany; ARUP Laboratories, Hematopathology University of Utah, Salt Lake City, UT; Department of Pathology, University of Utah, Salt Lake City, UT
Multiple Myeloma (MM) is a plasma cell (PC) malignancy, which despite significant therapeutic advances, is still considered incurable. This is due to the persistence of chemotherapy-resistant minimal residual disease in the patients’ bone marrow (BM) after an effective induction therapy. Immunotherapies targeting surface molecules expressed on the bulk of tumor cells and the chemotherapy-resistant, myeloma-propagating cells could play a central role in this clinical setting. We recently described surface molecule CD229 as a potential therapeutic target for MM. In our current study we assessed the expression of CD229 on different PC subtypes and on cells with a myeloma-propagating phenotype in a total of 77 patients with PC dyscrasias independently at two different cancer centers. We found that CD229 was strongly and homogeneously overexpressed on the PC of patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma, MM, and PC leukemia. CD229 was particularly overexpressed on those PC showing an abnormal phenotype such as expression of CD56. Most importantly, CD229 was also highly expressed on those cells in the patients’ BM displaying the phenotype of chemotherapy-resistant and myeloma-propagating cells. In conclusion, our combined findings suggest that immunotherapies targeting CD229 will not only be effective for the bulk of tumor cells but will also help to eradicate chemotherapy-resistant cells remaining in the patients’ BM after induction treatment. Hopefully, the design of CD229-specific monoclonal antibodies or chimeric antigen receptor-transduced T cells will help to achieve prolonged remissions or even cures in MM patients.
PO-274 The chemokine CXCL9 (MIG) is an independent predictor of overall survival in newly diagnosed multiple myeloma
PO-273
A. Bolomsky, M. Schreder, N. Zojer, H. Ludwig
CD229 is expressed on the surface of plasma cells carrying an aberrant phenotype and chemotherapy-resistant precursor cells in Multiple Myeloma
Wilhelminen Cancer Research Institute, Department of Medicine I,
S. Yousef, M. Kovacsovics-Bankowski, M.E. Salama, N. Bhardwaj, M. Steinbach, A. Langemo, T. Kovacsovics, J. Marvin, M. Binder, J. Panse, N. Kröger, T. Luetkens, D. Atanackovic
Wilhelminenhospital, Vienna, Austria
Background: The CXCR3 binding molecules CXCL9-11 have been associated with tumor progression, immune escape and angiogenesis in several malignancies. Information about the precise role of CXCR3 binding chemokines in MM is limited. We here aimed to evaluate the prognostic relevance of CXCL9 in MM.
15th International Myeloma Workshop, September 23-26, 2015
- e237
Abstracts Patients and Methods: CXCL9 serum levels were studied by ELISA in newly-diagnosed (ND) MM (n¼105) and MGUS patients (n¼17), and healthy donors (n¼37). CXCL9 expression was analyzed by qPCR in MM cell lines (HMCLs), MM-BMMNCs, MM-PBMCs and in a publically available GEP-dataset (GSE2658). Concurrent analysis of CXCL9-11 serum levels was performed by FACS-CBA array. Results: Median CXCL9 levels (161 pg/ml; range: 9-1966) were significantly elevated in MM patients compared to MGUS (93; 6-1303) and healthy donors (106; 51e391). CXCL9 levels significantly correlated with B2M, ISS stage, calcium, albumin, LDH, hemoglobin and age. High CXCL9 levels predicted adverse survival (17.0 months vs. not reached, P<0.001), which was upheld when age-adjusted cut-off levels were used. Similarly, in MM patients (n¼559) treated within the TT2 and 3 protocol (GSE2658) shorter OS was found in patients with a present compared to those with an absent detection call for CXCL9 (P¼0.004). Expression of CXCL9 was only detected in 1 of 7 HMCLs and 12% of previously published MM cell samples. Hence, increased serum levels of CXCL9 are likely derived from other cell types. We observed increased expression of CXCL9 in PBMCs compared to BMMNCs of MM patients, suggesting that elevated secretion of CXCL9 might result from aberrant cytokine production in cells of the PB. CXCL9 serum levels were also analyzed in conjunction with CXCL10 and 11 in 65 MM patients by FACS-CBA. Again, we observed a significant correlation with B2M for CXCL9-11. Survival was significantly worse in patients with high compared to low CXCL9 (median OS 25.3 months vs. not reached, P¼0.003) and CXCL10 (19.97 months vs. not reached, P¼0.0006). In multivariate analysis, CXCL9 (P¼0.03) and CXCL10 (P¼0.013) were revealed as independent predictors of survival. Conclusion: Our findings depict CXCL9 as novel prognostic marker for overall survival in ND-MM patients. High levels of CXCL9 in the PB probably increase TH1/TH2 ratios and may shield myeloma cells from immune attack by CD8+ cytotoxic T cells as previously shown for T cell lymphomas. Further experiments will aim to confirm these results and define the functional role of CXCL9 in MM.
Introduction: The t(4;14) multiple myeloma (MM) subgroup, characterised by expression of the histone methyltransferase MMSET, has a poor prognosis. We have identified that an epithelial-to-mesenchymal transition (EMT)-like process plays a critical role in t(4;14)-positive MM disease pathogenesis. This EMT-like phenotype is, in part, characterised by increased expression of the cell adhesion molecule N-cadherin (CDH2). In this study, we investigated N-cadherin as a therapeutic target in t(4;14)positive MM. Materials and Methods: The effect of shRNAmediated Cdh2 knock-down was assessed in vitro in the MM plasma cell line 5TGM1. In addition, the effect of targeting N-cadherin on MM plasma cell bone marrow homing, tumour establishment and tumour growth was assessed in vivo using the C57BL/KaLwRijHsd mouse model of MM. Results: When compared with 5TGM1-scramble-shRNA cells, 5TGM1-Cdh2shRNA cells had significantly reduced adhesion to bone marrow endothelial cells. However, N-cadherin knock-down did not affect 5TGM1 cell proliferation, migration or adhesion to bone marrow stromal cells in vitro. Mice intravenously inoculated with 5TGM1Cdh2-shRNA cells showed significantly decreased tumour burden after four weeks, as assessed by bioluminescence imaging, compared with animals bearing 5TGM1-scramble-shRNA cells. Notably, daily administration of the N-cadherin antagonist ADH-1 (100mg/ kg/day), commencing at the time of tumour inoculation, significantly decreased tumour burden after 4 weeks compared with PBStreated mice. In contrast, ADH-1 had no effect on tumour burden in the established disease setting. Conclusion: Our findings demonstrate a potential role for N-cadherin in MM plasma cell extravasation and bone marrow homing. Furthermore, these studies suggest that N-cadherin represents a novel therapeutic target to prevent the dissemination of MM plasma cells and delay disease progression in MM patients with high N-cadherin expression.
PO-276 Myeloma-derived CCL27 induces stroma-dependent resistance against bortezomib via upregulation of IL-10 S. Thangavadivel,1 A. Olivier,1 B. Postert,1 C. Zelle-Rieser,1 J. Kern,2 G. Untergasser,1,2 A. Brunner,3 W. Willenbacher,4 R. Biedermann,5 R. Greil,1,6 K. Jöhrer1
PO-275 N-cadherin is a therapeutic target in t(4;14)-positive multiple myeloma K.M. Mrozik,1,2 C.M. Cheong,1,2 D. Hewett,1,2 C.H. Kok,2,3 A.W.S. Chow,1 O. Blaschuk,4 J.D. Licht,5 A.C.W. Zannettino,1,2,3,6 K. Vandyke1,2,3,6
for Experimental Oncology, Internal Medicine V, Medical University
1
bruck, Austria; 4Department of Internal Medicine V, Hematology and
Myeloma Research Laboratory, Discipline of Physiology, School of
Medical Sciences, Faculty of Health Science, University of Adelaide, 2
Adelaide, South Australia; Cancer Theme, South Australian Health 3
1
Tyrolean Cancer Research Institute, Innsbruck, Austria; 2Laboratory
Innsbruck, Austria; 3Institute for Pathology, Medical University InnsOncology, Medical University Innsbruck, Austria; 5Department of Orthopedic Surgery, Medical University Innsbruck, Austria; 6Labora-
and Medical Research Institute, Adelaide, South Australia; School of Medicine, University of Adelaide, Adelaide, South Australia; 4Division
tory for Immunological and Molecular Cancer Research, Salzburg Cancer Research Institute, 3rd Medical Department, Paracelsus
of Urology, Department of Surgery, McGill University, Montreal,
Medical University, Salzburg, Austria
Canada; 5Division of Hematology/Oncology, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Feinberg School of Medicine, Chicago, USA; 6Centre for Cancer Biology and Hanson Institute, SA Pathology, Adelaide, South Australia
e238
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15th International Myeloma Workshop, September 23-26, 2015
Introduction: Multiple Myeloma is still incurable and drugresistance is the major cause for relapse and early death of the patients. The bone marrow microenvironment plays a fundamental role in shaping tumor progression. Chemokines are soluble