EXPRESSION, REGULATION, AND FUNCTIONAL ROLE OF HERMES ADHESION RECEPTOR CD44 IN NEOPLASTIC MAST CELLS IN SYSTEMIC MASTOCYTOSIS
(Abstract release date: 05/19/16)
EHA Library. Mueller N. 06/11/16; 135266; S510
Niklas Mueller
Contributions
Contributions
Abstract
Abstract: S510
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 17:00 - 17:15
Location: Hall C13
Background
The Hermes receptor CD44 is a multifunctional adhesion molecule that plays an essential role in the homing and expansion of neoplastic stem- and progenitor cells in various myeloid malignancies. Although mast cells (MC) are known to express CD44, little is known about the regulation and function of this receptor on neoplastic stem cells and MC in patients with systemic mastocytosis (SM).
Aims
The aim of this study was to define the expression and function of CD44 on neoplastic MC.
Methods
CD44 expression on primary neoplastic MC and various human MC lines was analyzed by immunocytochemistry and flow cytometry.
Results
CD34+/CD38- stem cells, CD34+/CD38+ progenitor cells, and KIT+/CD34- MC invariably expressed CD44 in all patients with indolent SM (ISM, 12/12), SM with associated hematologic non-MC-disease (SM-AHNMD, 4/4), aggressive SM (ASM, 3/3), and MC leukemia (MCL, 6/6). In addition, all human MC lines examined, including HMC-1.1, HMC-1.2, ROSAKIT wt, and ROSAKIT D816V, were found to express cytoplasmic CD44 and cell surface CD44. We next examined the regulation of expression of CD44 in neoplastic MC. Incubation with the MEK inhibitor RDEA119 (0.1-5 µM) or the STAT5 blocker pimozide (2.5-10 µM) for 48 hours resulted in a dose-dependent and significant downregulation of surface expression of CD44 compared to control medium (100%) in all MC lines examined (RDEA119, 2.5 µM: 71±9% surface staining intensity in HMC-1.1; 82±3% in HMC-1.2, 33±13% in ROSAKIT wt, and 31±3% in ROSAKIT D816V cells; pimozide, 7.5 µM: 59±7% surface staining intensity in HMC-1.1; 68±3% in HMC-1.2, 62±16% in ROSAKIT wt, and 80±3% in ROSAKIT D816V cells, p<0.05). In contrast, incubation with the demethylating agents decitabine (0.1-5 µM) or azacitidine (0.1-5 µM) for 96 hours resulted in a dose-dependent and significant upregulation of CD44 expression compared to control medium (100%) in all MC lines examined (decitabine, 5 µM; 210±50% surface staining intensity in HMC-1.1; 282±78% in HMC-1.2, 236±56% in ROSAKIT wt, and 198±55% in ROSAKIT D816V cells; azacytidine, 5 µM: 379±103% surface staining intensity in HMC-1.1; 429±105% in HMC-1.2, 412±135% in ROSAKIT wt, and 292±136% in ROSAKIT D816V cells; p<0.05). We were also able to detect soluble CD44 in the sera of patients with ISM, SM-AHNMD, ASM, and MCL as well as in the supernatants of neoplastic MC lines. In order to define a functional role for CD44 on MC, an in vivo xenotransplantation model using severe combined immunodeficient (SCID) mice, HMC-1.2 cells, and shRNA against CD44, were employed. In this model, the shRNA-mediated knock-down of CD44 in HMC-1.2 cells was found to lead to reduced MC expansion, reduced tumor formation, delayed ulceration, and prolonged survival compared to cells transduced with control shRNA (median survival in the CD44 shRNA group: 110 days vs 97 days in the control group; p<0.05). In these experiments, the formation of lung metastasis, quantified by human Alu-sequence-specific qPCR, was found to be particularly decreased (15-fold) in the CD44 knock-down group compared to controls.
Conclusion
CD44 is a relevant MC homing molecule that is expressed in neoplastic MC as well as in neoplastic stem- and progenitor cells in advanced SM. Our data also suggest that CD44 may serve as an interesting new target of therapy in patients with advanced SM.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): CD44, Homing, Kit, Mastocytosis
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 17:00 - 17:15
Location: Hall C13
Background
The Hermes receptor CD44 is a multifunctional adhesion molecule that plays an essential role in the homing and expansion of neoplastic stem- and progenitor cells in various myeloid malignancies. Although mast cells (MC) are known to express CD44, little is known about the regulation and function of this receptor on neoplastic stem cells and MC in patients with systemic mastocytosis (SM).
Aims
The aim of this study was to define the expression and function of CD44 on neoplastic MC.
Methods
CD44 expression on primary neoplastic MC and various human MC lines was analyzed by immunocytochemistry and flow cytometry.
Results
CD34+/CD38- stem cells, CD34+/CD38+ progenitor cells, and KIT+/CD34- MC invariably expressed CD44 in all patients with indolent SM (ISM, 12/12), SM with associated hematologic non-MC-disease (SM-AHNMD, 4/4), aggressive SM (ASM, 3/3), and MC leukemia (MCL, 6/6). In addition, all human MC lines examined, including HMC-1.1, HMC-1.2, ROSAKIT wt, and ROSAKIT D816V, were found to express cytoplasmic CD44 and cell surface CD44. We next examined the regulation of expression of CD44 in neoplastic MC. Incubation with the MEK inhibitor RDEA119 (0.1-5 µM) or the STAT5 blocker pimozide (2.5-10 µM) for 48 hours resulted in a dose-dependent and significant downregulation of surface expression of CD44 compared to control medium (100%) in all MC lines examined (RDEA119, 2.5 µM: 71±9% surface staining intensity in HMC-1.1; 82±3% in HMC-1.2, 33±13% in ROSAKIT wt, and 31±3% in ROSAKIT D816V cells; pimozide, 7.5 µM: 59±7% surface staining intensity in HMC-1.1; 68±3% in HMC-1.2, 62±16% in ROSAKIT wt, and 80±3% in ROSAKIT D816V cells, p<0.05). In contrast, incubation with the demethylating agents decitabine (0.1-5 µM) or azacitidine (0.1-5 µM) for 96 hours resulted in a dose-dependent and significant upregulation of CD44 expression compared to control medium (100%) in all MC lines examined (decitabine, 5 µM; 210±50% surface staining intensity in HMC-1.1; 282±78% in HMC-1.2, 236±56% in ROSAKIT wt, and 198±55% in ROSAKIT D816V cells; azacytidine, 5 µM: 379±103% surface staining intensity in HMC-1.1; 429±105% in HMC-1.2, 412±135% in ROSAKIT wt, and 292±136% in ROSAKIT D816V cells; p<0.05). We were also able to detect soluble CD44 in the sera of patients with ISM, SM-AHNMD, ASM, and MCL as well as in the supernatants of neoplastic MC lines. In order to define a functional role for CD44 on MC, an in vivo xenotransplantation model using severe combined immunodeficient (SCID) mice, HMC-1.2 cells, and shRNA against CD44, were employed. In this model, the shRNA-mediated knock-down of CD44 in HMC-1.2 cells was found to lead to reduced MC expansion, reduced tumor formation, delayed ulceration, and prolonged survival compared to cells transduced with control shRNA (median survival in the CD44 shRNA group: 110 days vs 97 days in the control group; p<0.05). In these experiments, the formation of lung metastasis, quantified by human Alu-sequence-specific qPCR, was found to be particularly decreased (15-fold) in the CD44 knock-down group compared to controls.
Conclusion
CD44 is a relevant MC homing molecule that is expressed in neoplastic MC as well as in neoplastic stem- and progenitor cells in advanced SM. Our data also suggest that CD44 may serve as an interesting new target of therapy in patients with advanced SM.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): CD44, Homing, Kit, Mastocytosis
Abstract: S510
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 17:00 - 17:15
Location: Hall C13
Background
The Hermes receptor CD44 is a multifunctional adhesion molecule that plays an essential role in the homing and expansion of neoplastic stem- and progenitor cells in various myeloid malignancies. Although mast cells (MC) are known to express CD44, little is known about the regulation and function of this receptor on neoplastic stem cells and MC in patients with systemic mastocytosis (SM).
Aims
The aim of this study was to define the expression and function of CD44 on neoplastic MC.
Methods
CD44 expression on primary neoplastic MC and various human MC lines was analyzed by immunocytochemistry and flow cytometry.
Results
CD34+/CD38- stem cells, CD34+/CD38+ progenitor cells, and KIT+/CD34- MC invariably expressed CD44 in all patients with indolent SM (ISM, 12/12), SM with associated hematologic non-MC-disease (SM-AHNMD, 4/4), aggressive SM (ASM, 3/3), and MC leukemia (MCL, 6/6). In addition, all human MC lines examined, including HMC-1.1, HMC-1.2, ROSAKIT wt, and ROSAKIT D816V, were found to express cytoplasmic CD44 and cell surface CD44. We next examined the regulation of expression of CD44 in neoplastic MC. Incubation with the MEK inhibitor RDEA119 (0.1-5 µM) or the STAT5 blocker pimozide (2.5-10 µM) for 48 hours resulted in a dose-dependent and significant downregulation of surface expression of CD44 compared to control medium (100%) in all MC lines examined (RDEA119, 2.5 µM: 71±9% surface staining intensity in HMC-1.1; 82±3% in HMC-1.2, 33±13% in ROSAKIT wt, and 31±3% in ROSAKIT D816V cells; pimozide, 7.5 µM: 59±7% surface staining intensity in HMC-1.1; 68±3% in HMC-1.2, 62±16% in ROSAKIT wt, and 80±3% in ROSAKIT D816V cells, p<0.05). In contrast, incubation with the demethylating agents decitabine (0.1-5 µM) or azacitidine (0.1-5 µM) for 96 hours resulted in a dose-dependent and significant upregulation of CD44 expression compared to control medium (100%) in all MC lines examined (decitabine, 5 µM; 210±50% surface staining intensity in HMC-1.1; 282±78% in HMC-1.2, 236±56% in ROSAKIT wt, and 198±55% in ROSAKIT D816V cells; azacytidine, 5 µM: 379±103% surface staining intensity in HMC-1.1; 429±105% in HMC-1.2, 412±135% in ROSAKIT wt, and 292±136% in ROSAKIT D816V cells; p<0.05). We were also able to detect soluble CD44 in the sera of patients with ISM, SM-AHNMD, ASM, and MCL as well as in the supernatants of neoplastic MC lines. In order to define a functional role for CD44 on MC, an in vivo xenotransplantation model using severe combined immunodeficient (SCID) mice, HMC-1.2 cells, and shRNA against CD44, were employed. In this model, the shRNA-mediated knock-down of CD44 in HMC-1.2 cells was found to lead to reduced MC expansion, reduced tumor formation, delayed ulceration, and prolonged survival compared to cells transduced with control shRNA (median survival in the CD44 shRNA group: 110 days vs 97 days in the control group; p<0.05). In these experiments, the formation of lung metastasis, quantified by human Alu-sequence-specific qPCR, was found to be particularly decreased (15-fold) in the CD44 knock-down group compared to controls.
Conclusion
CD44 is a relevant MC homing molecule that is expressed in neoplastic MC as well as in neoplastic stem- and progenitor cells in advanced SM. Our data also suggest that CD44 may serve as an interesting new target of therapy in patients with advanced SM.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): CD44, Homing, Kit, Mastocytosis
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 17:00 - 17:15
Location: Hall C13
Background
The Hermes receptor CD44 is a multifunctional adhesion molecule that plays an essential role in the homing and expansion of neoplastic stem- and progenitor cells in various myeloid malignancies. Although mast cells (MC) are known to express CD44, little is known about the regulation and function of this receptor on neoplastic stem cells and MC in patients with systemic mastocytosis (SM).
Aims
The aim of this study was to define the expression and function of CD44 on neoplastic MC.
Methods
CD44 expression on primary neoplastic MC and various human MC lines was analyzed by immunocytochemistry and flow cytometry.
Results
CD34+/CD38- stem cells, CD34+/CD38+ progenitor cells, and KIT+/CD34- MC invariably expressed CD44 in all patients with indolent SM (ISM, 12/12), SM with associated hematologic non-MC-disease (SM-AHNMD, 4/4), aggressive SM (ASM, 3/3), and MC leukemia (MCL, 6/6). In addition, all human MC lines examined, including HMC-1.1, HMC-1.2, ROSAKIT wt, and ROSAKIT D816V, were found to express cytoplasmic CD44 and cell surface CD44. We next examined the regulation of expression of CD44 in neoplastic MC. Incubation with the MEK inhibitor RDEA119 (0.1-5 µM) or the STAT5 blocker pimozide (2.5-10 µM) for 48 hours resulted in a dose-dependent and significant downregulation of surface expression of CD44 compared to control medium (100%) in all MC lines examined (RDEA119, 2.5 µM: 71±9% surface staining intensity in HMC-1.1; 82±3% in HMC-1.2, 33±13% in ROSAKIT wt, and 31±3% in ROSAKIT D816V cells; pimozide, 7.5 µM: 59±7% surface staining intensity in HMC-1.1; 68±3% in HMC-1.2, 62±16% in ROSAKIT wt, and 80±3% in ROSAKIT D816V cells, p<0.05). In contrast, incubation with the demethylating agents decitabine (0.1-5 µM) or azacitidine (0.1-5 µM) for 96 hours resulted in a dose-dependent and significant upregulation of CD44 expression compared to control medium (100%) in all MC lines examined (decitabine, 5 µM; 210±50% surface staining intensity in HMC-1.1; 282±78% in HMC-1.2, 236±56% in ROSAKIT wt, and 198±55% in ROSAKIT D816V cells; azacytidine, 5 µM: 379±103% surface staining intensity in HMC-1.1; 429±105% in HMC-1.2, 412±135% in ROSAKIT wt, and 292±136% in ROSAKIT D816V cells; p<0.05). We were also able to detect soluble CD44 in the sera of patients with ISM, SM-AHNMD, ASM, and MCL as well as in the supernatants of neoplastic MC lines. In order to define a functional role for CD44 on MC, an in vivo xenotransplantation model using severe combined immunodeficient (SCID) mice, HMC-1.2 cells, and shRNA against CD44, were employed. In this model, the shRNA-mediated knock-down of CD44 in HMC-1.2 cells was found to lead to reduced MC expansion, reduced tumor formation, delayed ulceration, and prolonged survival compared to cells transduced with control shRNA (median survival in the CD44 shRNA group: 110 days vs 97 days in the control group; p<0.05). In these experiments, the formation of lung metastasis, quantified by human Alu-sequence-specific qPCR, was found to be particularly decreased (15-fold) in the CD44 knock-down group compared to controls.
Conclusion
CD44 is a relevant MC homing molecule that is expressed in neoplastic MC as well as in neoplastic stem- and progenitor cells in advanced SM. Our data also suggest that CD44 may serve as an interesting new target of therapy in patients with advanced SM.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): CD44, Homing, Kit, Mastocytosis
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