TARGETING EPITHELIAL TO MESENCHYMAL TRANSITION (EMT) THROUGH CXCR4 BLOCKADE IN MULTIPLE MYELOMA
(Abstract release date: 05/21/15)
EHA Library. Roccaro A. 06/13/15; 103406; S479
Dr. Aldo Roccaro
Contributions
Contributions
Abstract
Abstract: S479
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 12:15 to 13.06.2015 12:30
Location: Room A2+3
Background
Multiple osteolytic lesions are one of the main clinical features of patients with multiple myeloma (MM), thus suggesting the ability of clonal plasma cells to disseminate from bone to bone. The bone marrow (BM) homing process of MM cells is supported by the CXCR4/CXCL12 axis activation. Nevertheless, the role of CXCR4 in mediating MM cell bone metastasis, and the role of CXCR4-targeted therapy in inhibiting MM cell dissemination has not been previously reported.
Aims
1) To define the pro-metastatic role of CXCR4 in vivo; 2) to investigate the mechanisms of CXCR4-mediated EMT both in vitro and in vivo; 3) and to test the anti-CXCR4 monoclonal antibody (Ulocuplumab) in inhibiting CXCR4-mediated EMT phenotype in vivo.
Methods
Gene set enrichment analysis and Atlas of Cancer Signaling Networks were used to study publically available gene expression dataset of primary BM-derived MM plasma cells (GSE24080; n=577; FDR<0.25; P<0.05). The functional relevance of CXCR4 in mediating MM cell dissemination was studied by generating CXCR4-gain and -loss-of-function expressing cell lines, using an in vivo model of MM cell dissemination from bone to bone. Modulation of EMT-related genes and proteins was tested both in vitro and ex vivo, using qRT-PCR, western blot, confocal miscroscopy, IHC and flow cytometry. The anti-tumor effect of Ulocuplumab was tested both in vitro and in an in vivo model of MM cell bone metastasis, bioluminescence imaging (BLI) and intravital confocal miscroscopy.
Results
We categorized BM-MM-derived CD138+ cells (GSE24080; n=577) according to their CXCR4 mRNA expression; and identified an EMT-related pathway to be significantly activated in high-CXCR4- versus low-CXCR4-BM-MM CD138+ cells. We next performed CXCR4-gain-of-function studies and demonstrated that CXCR4+ cells presented with changes in actin cytoskeleton, with protrusion of cell pseudopodia, sustained by modulation of EMT-related markers (Slug/Snail/Twist up-regulation; E-cadherin down-regulation), compared to control cells. These findings were recapitulated using an in vivo model of MM cell bone-to-bone metastasis: CXCR4+ MM cells showed higher levels of bone-to-bone metastasescompared to control cells, as confirmed on harvested host femurs. Lower expression of human (h)-E-cadherin, and higher expression of h-Twist/h-Snail/h-Slug was confirmed within the BM of the host femurs. The monoclonal antibody, anti-CXCR4 (Ulocuplumab) exerted an anti-MM activity in situ, within the s.q. implanted bones; and also reduced MM cell dissemination from the implanted bone to the host bone. Importantly, Ulocuplumab modulated EMT-related genes in the MM cells that metastasized to the host bones, with enhanced mRNA expression of human (h)-E-cadherin, and reduced expression of h-Twist/h-Snail/h-Slug and h-CXCR4 in the BM cells harvested from the host bones of Ulocuplumab-treated mice. Similar findings were confirmed in an in vivo breast cancer model (MDA-MB-231 intra-cardiac injected). CXCR4-loss of function in MM cells led to inhibited BM homing and tumor growth in vivo; with prolonged survival compared to control mice injected with scramble-transfected control cells. Moreover, Ulocuplumab led to inhibition of MM tumor growth in vivo, using MM.1S- and RPMI.8226- xenograft s.q.models; with a synergistic effect when used in combination with lenalidomide or bortezomib.
Summary
Our study supports the pro-metastatic role of CXCR4, due to its effect in mediating EMT in both MM and breast cancer in vivo models, thus suggesting the potential role for using CXCR4-neutralizing agents in order to delay or prevent tumor cell metastasis to bones.
Session topic: Multiple myeloma - Biology
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 12:15 to 13.06.2015 12:30
Location: Room A2+3
Background
Multiple osteolytic lesions are one of the main clinical features of patients with multiple myeloma (MM), thus suggesting the ability of clonal plasma cells to disseminate from bone to bone. The bone marrow (BM) homing process of MM cells is supported by the CXCR4/CXCL12 axis activation. Nevertheless, the role of CXCR4 in mediating MM cell bone metastasis, and the role of CXCR4-targeted therapy in inhibiting MM cell dissemination has not been previously reported.
Aims
1) To define the pro-metastatic role of CXCR4 in vivo; 2) to investigate the mechanisms of CXCR4-mediated EMT both in vitro and in vivo; 3) and to test the anti-CXCR4 monoclonal antibody (Ulocuplumab) in inhibiting CXCR4-mediated EMT phenotype in vivo.
Methods
Gene set enrichment analysis and Atlas of Cancer Signaling Networks were used to study publically available gene expression dataset of primary BM-derived MM plasma cells (GSE24080; n=577; FDR<0.25; P<0.05). The functional relevance of CXCR4 in mediating MM cell dissemination was studied by generating CXCR4-gain and -loss-of-function expressing cell lines, using an in vivo model of MM cell dissemination from bone to bone. Modulation of EMT-related genes and proteins was tested both in vitro and ex vivo, using qRT-PCR, western blot, confocal miscroscopy, IHC and flow cytometry. The anti-tumor effect of Ulocuplumab was tested both in vitro and in an in vivo model of MM cell bone metastasis, bioluminescence imaging (BLI) and intravital confocal miscroscopy.
Results
We categorized BM-MM-derived CD138+ cells (GSE24080; n=577) according to their CXCR4 mRNA expression; and identified an EMT-related pathway to be significantly activated in high-CXCR4- versus low-CXCR4-BM-MM CD138+ cells. We next performed CXCR4-gain-of-function studies and demonstrated that CXCR4+ cells presented with changes in actin cytoskeleton, with protrusion of cell pseudopodia, sustained by modulation of EMT-related markers (Slug/Snail/Twist up-regulation; E-cadherin down-regulation), compared to control cells. These findings were recapitulated using an in vivo model of MM cell bone-to-bone metastasis: CXCR4+ MM cells showed higher levels of bone-to-bone metastasescompared to control cells, as confirmed on harvested host femurs. Lower expression of human (h)-E-cadherin, and higher expression of h-Twist/h-Snail/h-Slug was confirmed within the BM of the host femurs. The monoclonal antibody, anti-CXCR4 (Ulocuplumab) exerted an anti-MM activity in situ, within the s.q. implanted bones; and also reduced MM cell dissemination from the implanted bone to the host bone. Importantly, Ulocuplumab modulated EMT-related genes in the MM cells that metastasized to the host bones, with enhanced mRNA expression of human (h)-E-cadherin, and reduced expression of h-Twist/h-Snail/h-Slug and h-CXCR4 in the BM cells harvested from the host bones of Ulocuplumab-treated mice. Similar findings were confirmed in an in vivo breast cancer model (MDA-MB-231 intra-cardiac injected). CXCR4-loss of function in MM cells led to inhibited BM homing and tumor growth in vivo; with prolonged survival compared to control mice injected with scramble-transfected control cells. Moreover, Ulocuplumab led to inhibition of MM tumor growth in vivo, using MM.1S- and RPMI.8226- xenograft s.q.models; with a synergistic effect when used in combination with lenalidomide or bortezomib.
Summary
Our study supports the pro-metastatic role of CXCR4, due to its effect in mediating EMT in both MM and breast cancer in vivo models, thus suggesting the potential role for using CXCR4-neutralizing agents in order to delay or prevent tumor cell metastasis to bones.
Session topic: Multiple myeloma - Biology
Abstract: S479
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 12:15 to 13.06.2015 12:30
Location: Room A2+3
Background
Multiple osteolytic lesions are one of the main clinical features of patients with multiple myeloma (MM), thus suggesting the ability of clonal plasma cells to disseminate from bone to bone. The bone marrow (BM) homing process of MM cells is supported by the CXCR4/CXCL12 axis activation. Nevertheless, the role of CXCR4 in mediating MM cell bone metastasis, and the role of CXCR4-targeted therapy in inhibiting MM cell dissemination has not been previously reported.
Aims
1) To define the pro-metastatic role of CXCR4 in vivo; 2) to investigate the mechanisms of CXCR4-mediated EMT both in vitro and in vivo; 3) and to test the anti-CXCR4 monoclonal antibody (Ulocuplumab) in inhibiting CXCR4-mediated EMT phenotype in vivo.
Methods
Gene set enrichment analysis and Atlas of Cancer Signaling Networks were used to study publically available gene expression dataset of primary BM-derived MM plasma cells (GSE24080; n=577; FDR<0.25; P<0.05). The functional relevance of CXCR4 in mediating MM cell dissemination was studied by generating CXCR4-gain and -loss-of-function expressing cell lines, using an in vivo model of MM cell dissemination from bone to bone. Modulation of EMT-related genes and proteins was tested both in vitro and ex vivo, using qRT-PCR, western blot, confocal miscroscopy, IHC and flow cytometry. The anti-tumor effect of Ulocuplumab was tested both in vitro and in an in vivo model of MM cell bone metastasis, bioluminescence imaging (BLI) and intravital confocal miscroscopy.
Results
We categorized BM-MM-derived CD138+ cells (GSE24080; n=577) according to their CXCR4 mRNA expression; and identified an EMT-related pathway to be significantly activated in high-CXCR4- versus low-CXCR4-BM-MM CD138+ cells. We next performed CXCR4-gain-of-function studies and demonstrated that CXCR4+ cells presented with changes in actin cytoskeleton, with protrusion of cell pseudopodia, sustained by modulation of EMT-related markers (Slug/Snail/Twist up-regulation; E-cadherin down-regulation), compared to control cells. These findings were recapitulated using an in vivo model of MM cell bone-to-bone metastasis: CXCR4+ MM cells showed higher levels of bone-to-bone metastasescompared to control cells, as confirmed on harvested host femurs. Lower expression of human (h)-E-cadherin, and higher expression of h-Twist/h-Snail/h-Slug was confirmed within the BM of the host femurs. The monoclonal antibody, anti-CXCR4 (Ulocuplumab) exerted an anti-MM activity in situ, within the s.q. implanted bones; and also reduced MM cell dissemination from the implanted bone to the host bone. Importantly, Ulocuplumab modulated EMT-related genes in the MM cells that metastasized to the host bones, with enhanced mRNA expression of human (h)-E-cadherin, and reduced expression of h-Twist/h-Snail/h-Slug and h-CXCR4 in the BM cells harvested from the host bones of Ulocuplumab-treated mice. Similar findings were confirmed in an in vivo breast cancer model (MDA-MB-231 intra-cardiac injected). CXCR4-loss of function in MM cells led to inhibited BM homing and tumor growth in vivo; with prolonged survival compared to control mice injected with scramble-transfected control cells. Moreover, Ulocuplumab led to inhibition of MM tumor growth in vivo, using MM.1S- and RPMI.8226- xenograft s.q.models; with a synergistic effect when used in combination with lenalidomide or bortezomib.
Summary
Our study supports the pro-metastatic role of CXCR4, due to its effect in mediating EMT in both MM and breast cancer in vivo models, thus suggesting the potential role for using CXCR4-neutralizing agents in order to delay or prevent tumor cell metastasis to bones.
Session topic: Multiple myeloma - Biology
Type: Oral Presentation
Presentation during EHA20: From 13.06.2015 12:15 to 13.06.2015 12:30
Location: Room A2+3
Background
Multiple osteolytic lesions are one of the main clinical features of patients with multiple myeloma (MM), thus suggesting the ability of clonal plasma cells to disseminate from bone to bone. The bone marrow (BM) homing process of MM cells is supported by the CXCR4/CXCL12 axis activation. Nevertheless, the role of CXCR4 in mediating MM cell bone metastasis, and the role of CXCR4-targeted therapy in inhibiting MM cell dissemination has not been previously reported.
Aims
1) To define the pro-metastatic role of CXCR4 in vivo; 2) to investigate the mechanisms of CXCR4-mediated EMT both in vitro and in vivo; 3) and to test the anti-CXCR4 monoclonal antibody (Ulocuplumab) in inhibiting CXCR4-mediated EMT phenotype in vivo.
Methods
Gene set enrichment analysis and Atlas of Cancer Signaling Networks were used to study publically available gene expression dataset of primary BM-derived MM plasma cells (GSE24080; n=577; FDR<0.25; P<0.05). The functional relevance of CXCR4 in mediating MM cell dissemination was studied by generating CXCR4-gain and -loss-of-function expressing cell lines, using an in vivo model of MM cell dissemination from bone to bone. Modulation of EMT-related genes and proteins was tested both in vitro and ex vivo, using qRT-PCR, western blot, confocal miscroscopy, IHC and flow cytometry. The anti-tumor effect of Ulocuplumab was tested both in vitro and in an in vivo model of MM cell bone metastasis, bioluminescence imaging (BLI) and intravital confocal miscroscopy.
Results
We categorized BM-MM-derived CD138+ cells (GSE24080; n=577) according to their CXCR4 mRNA expression; and identified an EMT-related pathway to be significantly activated in high-CXCR4- versus low-CXCR4-BM-MM CD138+ cells. We next performed CXCR4-gain-of-function studies and demonstrated that CXCR4+ cells presented with changes in actin cytoskeleton, with protrusion of cell pseudopodia, sustained by modulation of EMT-related markers (Slug/Snail/Twist up-regulation; E-cadherin down-regulation), compared to control cells. These findings were recapitulated using an in vivo model of MM cell bone-to-bone metastasis: CXCR4+ MM cells showed higher levels of bone-to-bone metastasescompared to control cells, as confirmed on harvested host femurs. Lower expression of human (h)-E-cadherin, and higher expression of h-Twist/h-Snail/h-Slug was confirmed within the BM of the host femurs. The monoclonal antibody, anti-CXCR4 (Ulocuplumab) exerted an anti-MM activity in situ, within the s.q. implanted bones; and also reduced MM cell dissemination from the implanted bone to the host bone. Importantly, Ulocuplumab modulated EMT-related genes in the MM cells that metastasized to the host bones, with enhanced mRNA expression of human (h)-E-cadherin, and reduced expression of h-Twist/h-Snail/h-Slug and h-CXCR4 in the BM cells harvested from the host bones of Ulocuplumab-treated mice. Similar findings were confirmed in an in vivo breast cancer model (MDA-MB-231 intra-cardiac injected). CXCR4-loss of function in MM cells led to inhibited BM homing and tumor growth in vivo; with prolonged survival compared to control mice injected with scramble-transfected control cells. Moreover, Ulocuplumab led to inhibition of MM tumor growth in vivo, using MM.1S- and RPMI.8226- xenograft s.q.models; with a synergistic effect when used in combination with lenalidomide or bortezomib.
Summary
Our study supports the pro-metastatic role of CXCR4, due to its effect in mediating EMT in both MM and breast cancer in vivo models, thus suggesting the potential role for using CXCR4-neutralizing agents in order to delay or prevent tumor cell metastasis to bones.
Session topic: Multiple myeloma - Biology
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