- Email: [email protected]
Notch signaling dysregulation promotes multiple myeloma-associated bone disease
Abstracts significantly varies between BTZ adapted and CFZ adapted MM cells and can be increased by 2 inhibition.
PO-265 Notch signaling dysregulation promotes multiple myeloma-associated bone disease S. Garavelli,1 M. Colombo,1 K. Thümmler,2 L. Apicella,1 M. Lancellotti,1 E. Lazzari,1 K. Todoerti,3 R. Soutar,4 N. Platonova,1 M. Akbar,2 C.S. Goodyear,2 A. Neri,5 R. Chiaramonte1 1
Department of Health Sciences, Università degli Studi di Milano,
Milano; 2Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glas3
gow G12 8TA, UK; Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, Rionero in Vulture; 4Beatson West of Scotland Cancer Centre, Haemato5
oncology Service, Gartnavel Hospital, Glasgow G12 0YN, UK; Dept. Clinical Sciences and Community Health, Università degli Studi di
Finally, MM cell-derived Jag1-2 activate Notch signaling in BM stromal cells (BMSCs), which, in turn, enhance the osteoclastogenic potential of MM cells, by boosting their secretion of RANKL. Conclusion: This study provides the first evidence that Jag1 and 2 ligands aberrantly expressed by MM cells shape the BM niche and foster MM-induced OCLs differentiation and bone resorption activity. Our results suggest that inhibiting the engagement between the Jag1-2 ligands expressed by MM cells and Notch receptors may prevent MM-associated bone disease. This evidence strengthen the rational of a molecular therapy in MM myeloma directed to Notch ligands, Jag1 and 2.
PO-266 Notch pathway and Inteleukin-6 cooperate to support multiple myeloma cell proliferation S. Galletti,1 M. Colombo,2 S. Ravaioli,2 S. Garavelli,2 G. Bulfamante,2,3 M. Falleni,2,3 D. Tosi,2,3 A. Moschini,2 A. Paoli,2 K. Todoerti,4 A. Neri,1 R. Chiaramonte2
Milano. Hematology, Fondazione Cà Granda IRCCS Policlinico,
1
Milano, Italy
degli Studi di Milano; Hematology, Fondazione Cà Granda IRCCS Policlinico, Milano, Italy; 2Department of Health Sciences, Università
Introduction: The Notch pathway is dysregulated in multiple myeloma (MM), due to the hyperexpression of Notch receptors and Jag1/2 ligands. This alteration affects MM cell biology and improves the ability of MM cell to shape the bone marrow (BM) niche inducing a supportive behavior that promotes tumor progression. BM niche alterations include an increased osteolytic activity results in bone disease which affects patients quality of life and promotes tumor growth and survival, contributing to the fatal outcome of MM. Notch has been reported as a key regulator of bone tissue remodeling. The aim of this work was to study the role of Notch signaling in MM-associated bone disease and, specifically, to verify the effect of Notch signaling activated by the two dysregulated ligands, Jag1 and 2. Methods: The experiments took advantage of cell lines as well as primary cells including MM cells, osteoclasts (OCLs) and BM stromal cells (BMSCs). OCL differentiation was induced for 5-7 days cells by: i) adding 50ng/ml RANKL; ii) coculturing with MM cells or iii) addition of MM cell lines conditioned media (CM). OCLs were enumerated by TRAP staining. Notch signaling was inhibited by the gamma-secretase inhibitor, 25-50 microM DAPT, or by Jag1-2 silencing performed using the RNAiTM siRNA system (Invitrogen) Quantitative PCR reactions were carried with MaximaTM SYBR Green qPCR Master Mix. RANKL was quantified by ELISA and flow cytometry. Results: Our findings indicate that Notch activity induces the autonomous release of the osteoclastogenic factor RANKL by MM cells, inducing OCLs differentiation and osteolytic activity. In addition, MM-derived Jag1-2 directly activate the pro-osteoclastogenic Notch signaling in the neighboring OCL progenitors, thereby boosting their differentiation.
degli Studi di Milano, Milano, Italy; 3Unit of Pathology A.O. San Paolo,
Department of Clinical Sciences and Community Health, Università
via A. Di Rudinì 8, Milan, Italy; 4Laboratory of Pre-Clinical and 4Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, 85028 Rionero in Vulture (PZ), Italy
Introduction: The Notch pathway has a key role in multiple myeloma (MM) progression by positively regulating cell proliferation, drug resistance and bone marrow (BM) infiltration through the overexpression of both receptors (Notch1 and 2) and ligands (Jag1-2). MM cells are strictly dependent on the BM niche, that supports tumor growth and progression through adhesion molecules and soluble mediators, as interleukin-6 (IL6). MM cells initially depend on IL6 mainly produced by BM stromal cells (BMSCs) and later may acquire independence and/or the ability to autonomously produce IL6. Here, we investigated the cooperation between the Notch pathway and IL6 signaling in the promotion of MM cells proliferation. Material and Methods: Notch signaling modulation was induced in MM cell lines and primary MM cells as follows: upregulation by 5 g/mL soluble Jag1; down-regulation by 50 M DAPT or Jag1-2 RNA interference (RNAi). qPCR was performed using MaximaÔ SYBR Green Master Mix. Absolute cells count and evaluation of IL6 protein expression was achieved by flow cytometry. Immunohistochemistry (IHC) for HES6, IL6 and Ig light chain was performed on BM biopsies at different stages of MM. Results: By modulating Notch activity in MM cell lines, we demonstrated that dysregulated Notch proliferative signal can substitute IL6 stimulation. Indeed, upon Notch withdrawal, IL6indipendent cell lines became dependent on IL6 for their proliferation. On the opposite, Notch activation in IL6-dependent cell lines, may stimulate proliferation in the absence of IL6. Also, Notch
15th International Myeloma Workshop, September 23-26, 2015
- e233
PO-265 Notch signaling dysregulation promotes multiple myeloma-associated bone disease S. Garavelli,1 M. Colombo,1 K. Thümmler,2 L. Apicella,1 M. Lancellotti,1 E. Lazzari,1 K. Todoerti,3 R. Soutar,4 N. Platonova,1 M. Akbar,2 C.S. Goodyear,2 A. Neri,5 R. Chiaramonte1 1
Department of Health Sciences, Università degli Studi di Milano,
Milano; 2Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glas3
gow G12 8TA, UK; Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, Rionero in Vulture; 4Beatson West of Scotland Cancer Centre, Haemato5
oncology Service, Gartnavel Hospital, Glasgow G12 0YN, UK; Dept. Clinical Sciences and Community Health, Università degli Studi di
Finally, MM cell-derived Jag1-2 activate Notch signaling in BM stromal cells (BMSCs), which, in turn, enhance the osteoclastogenic potential of MM cells, by boosting their secretion of RANKL. Conclusion: This study provides the first evidence that Jag1 and 2 ligands aberrantly expressed by MM cells shape the BM niche and foster MM-induced OCLs differentiation and bone resorption activity. Our results suggest that inhibiting the engagement between the Jag1-2 ligands expressed by MM cells and Notch receptors may prevent MM-associated bone disease. This evidence strengthen the rational of a molecular therapy in MM myeloma directed to Notch ligands, Jag1 and 2.
PO-266 Notch pathway and Inteleukin-6 cooperate to support multiple myeloma cell proliferation S. Galletti,1 M. Colombo,2 S. Ravaioli,2 S. Garavelli,2 G. Bulfamante,2,3 M. Falleni,2,3 D. Tosi,2,3 A. Moschini,2 A. Paoli,2 K. Todoerti,4 A. Neri,1 R. Chiaramonte2
Milano. Hematology, Fondazione Cà Granda IRCCS Policlinico,
1
Milano, Italy
degli Studi di Milano; Hematology, Fondazione Cà Granda IRCCS Policlinico, Milano, Italy; 2Department of Health Sciences, Università
Introduction: The Notch pathway is dysregulated in multiple myeloma (MM), due to the hyperexpression of Notch receptors and Jag1/2 ligands. This alteration affects MM cell biology and improves the ability of MM cell to shape the bone marrow (BM) niche inducing a supportive behavior that promotes tumor progression. BM niche alterations include an increased osteolytic activity results in bone disease which affects patients quality of life and promotes tumor growth and survival, contributing to the fatal outcome of MM. Notch has been reported as a key regulator of bone tissue remodeling. The aim of this work was to study the role of Notch signaling in MM-associated bone disease and, specifically, to verify the effect of Notch signaling activated by the two dysregulated ligands, Jag1 and 2. Methods: The experiments took advantage of cell lines as well as primary cells including MM cells, osteoclasts (OCLs) and BM stromal cells (BMSCs). OCL differentiation was induced for 5-7 days cells by: i) adding 50ng/ml RANKL; ii) coculturing with MM cells or iii) addition of MM cell lines conditioned media (CM). OCLs were enumerated by TRAP staining. Notch signaling was inhibited by the gamma-secretase inhibitor, 25-50 microM DAPT, or by Jag1-2 silencing performed using the RNAiTM siRNA system (Invitrogen) Quantitative PCR reactions were carried with MaximaTM SYBR Green qPCR Master Mix. RANKL was quantified by ELISA and flow cytometry. Results: Our findings indicate that Notch activity induces the autonomous release of the osteoclastogenic factor RANKL by MM cells, inducing OCLs differentiation and osteolytic activity. In addition, MM-derived Jag1-2 directly activate the pro-osteoclastogenic Notch signaling in the neighboring OCL progenitors, thereby boosting their differentiation.
degli Studi di Milano, Milano, Italy; 3Unit of Pathology A.O. San Paolo,
Department of Clinical Sciences and Community Health, Università
via A. Di Rudinì 8, Milan, Italy; 4Laboratory of Pre-Clinical and 4Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, 85028 Rionero in Vulture (PZ), Italy
Introduction: The Notch pathway has a key role in multiple myeloma (MM) progression by positively regulating cell proliferation, drug resistance and bone marrow (BM) infiltration through the overexpression of both receptors (Notch1 and 2) and ligands (Jag1-2). MM cells are strictly dependent on the BM niche, that supports tumor growth and progression through adhesion molecules and soluble mediators, as interleukin-6 (IL6). MM cells initially depend on IL6 mainly produced by BM stromal cells (BMSCs) and later may acquire independence and/or the ability to autonomously produce IL6. Here, we investigated the cooperation between the Notch pathway and IL6 signaling in the promotion of MM cells proliferation. Material and Methods: Notch signaling modulation was induced in MM cell lines and primary MM cells as follows: upregulation by 5 g/mL soluble Jag1; down-regulation by 50 M DAPT or Jag1-2 RNA interference (RNAi). qPCR was performed using MaximaÔ SYBR Green Master Mix. Absolute cells count and evaluation of IL6 protein expression was achieved by flow cytometry. Immunohistochemistry (IHC) for HES6, IL6 and Ig light chain was performed on BM biopsies at different stages of MM. Results: By modulating Notch activity in MM cell lines, we demonstrated that dysregulated Notch proliferative signal can substitute IL6 stimulation. Indeed, upon Notch withdrawal, IL6indipendent cell lines became dependent on IL6 for their proliferation. On the opposite, Notch activation in IL6-dependent cell lines, may stimulate proliferation in the absence of IL6. Also, Notch
15th International Myeloma Workshop, September 23-26, 2015
- e233