IN VITRO EVALUATION OF BORTEZOMIB MOLECULAR EFFECTS IN LARGE GRANULAR LYMPHOCYTE LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Calabretto G. 06/09/16; 132581; E1032
Mrs. Giulia Calabretto
Contributions
Contributions
Abstract
Abstract: E1032
Type: Eposter Presentation
Background
Large Granular Lymphocyte Leukemia (LGLL) is a chronic lymphoproliferative disorder characterized by clonal expansion of Large Granular Lymphocytes (LGLs) with cytotoxic activity. LGLs proliferation is maintained through the activation of several survival signaling pathways. Among these, one of the most important deregulated pathway is the JAK/STAT axis. STAT3 activation, mediated by IL-6 signaling or activating STAT3 mutations, promotes LGLs survival by inducing several anti-apoptotic genes transcription and represents a hallmark of this disease.Nowadays, LGLL therapy is based on immunosuppressive drugs, such as Methotrexate (MTX), Cyclophosphamide (CTX) and Cyclosporine A (CyA), showing however only a partial efficacy with an Overall Response Rate (ORR) of about 50-60%. Bortezomib (Bz), the first FDA-approved proteasome inhibitor used in Multiple Myeloma (MM) therapy, is not an approved treatment opportunity for LGLL, but the rare co-association of LGLL with MM offered the opportunity to retrospectively investigate the effect of this drug on leukemic LGLs. The literature reports that patients diagnosed with both LGLL and MM showed a decrease in the LGLs count after Bz treatment, suggesting that this drug may have an effect on inhibiting the LGL clone.
Aims
The aim of this study is to investigate the in vitro effects of Bz on PBMCs from LGLL patients, in order to understand its molecular mechanism of action in reducing the LGL clone.
Methods
Peripheral blood mononuclear cells (PBMCs) of 20 LGLL patients (8 out of 20 characterized by STAT3 mutations), isolated through Ficoll density centrifugation, were cultured and treated with Bz (5.2 nM) for 24 or 48 hours. Real Time-PCR, western blot (WB) assays and immunofluorescence assay were performed to analyze mRNA and protein expression. Cell apoptosis was evaluated by Flow cytometry, using Annexin V staining.
Results
We analyzed IL-6 mRNA expression levels in patients-derived PBMCs by Real Time-PCR. Our results demonstrated that IL-6 gene expression significantly decreases in Bz-treated cells, as compared to a not treated condition (p<0.01). The transcriptional data were supported by immunofluorescence assay, showing that this cytokine is mainly produced by the monocyte/macrophage lineage and that its expression is reduced after Bz treatment.To investigate the Bz effect on IL-6-induced JAK/STAT pathway, we performed WB analysis to evaluate STAT3 activation by measuring pSTAT3 Tyr705. Our results showed that pSTAT3 Tyr705 levels strongly decrease in Bz-treated condition, irrespective to the presence of STAT3 mutations. This pattern strictly differs from untreated PBMCs, in which STAT3 remains over-activated. Then, by Real Time-PCR and WB assays, we analyzed the expression of STAT3 downstream targets, focusing on the anti-apoptotic MCL-1 and BCL-2 gene expression. Our results demonstrated a significant reduction of the expression levels of both these genes following Bz treatment (p<0.01).Finally, we evaluated Bz effect on cell survival by Annexin V staining. Our results showed a significant time-dependent increase in LGL apoptosis after Bz treatment, as compared to a not treated condition. Moreover, we observed that this drug is highly specific in inducing leukemic LGLs apoptosis, not affecting the other PBMCs.
Conclusion
Our results provide evidence that Bortezomib might represent a new intriguing therapeutic option to be added to the immunosuppressive drugs already used in LGLL therapy.
Session topic: E-poster
Keyword(s): Bortezomib, IL-6, Large granular lymphocytic leukaemia, STAT3
Type: Eposter Presentation
Background
Large Granular Lymphocyte Leukemia (LGLL) is a chronic lymphoproliferative disorder characterized by clonal expansion of Large Granular Lymphocytes (LGLs) with cytotoxic activity. LGLs proliferation is maintained through the activation of several survival signaling pathways. Among these, one of the most important deregulated pathway is the JAK/STAT axis. STAT3 activation, mediated by IL-6 signaling or activating STAT3 mutations, promotes LGLs survival by inducing several anti-apoptotic genes transcription and represents a hallmark of this disease.Nowadays, LGLL therapy is based on immunosuppressive drugs, such as Methotrexate (MTX), Cyclophosphamide (CTX) and Cyclosporine A (CyA), showing however only a partial efficacy with an Overall Response Rate (ORR) of about 50-60%. Bortezomib (Bz), the first FDA-approved proteasome inhibitor used in Multiple Myeloma (MM) therapy, is not an approved treatment opportunity for LGLL, but the rare co-association of LGLL with MM offered the opportunity to retrospectively investigate the effect of this drug on leukemic LGLs. The literature reports that patients diagnosed with both LGLL and MM showed a decrease in the LGLs count after Bz treatment, suggesting that this drug may have an effect on inhibiting the LGL clone.
Aims
The aim of this study is to investigate the in vitro effects of Bz on PBMCs from LGLL patients, in order to understand its molecular mechanism of action in reducing the LGL clone.
Methods
Peripheral blood mononuclear cells (PBMCs) of 20 LGLL patients (8 out of 20 characterized by STAT3 mutations), isolated through Ficoll density centrifugation, were cultured and treated with Bz (5.2 nM) for 24 or 48 hours. Real Time-PCR, western blot (WB) assays and immunofluorescence assay were performed to analyze mRNA and protein expression. Cell apoptosis was evaluated by Flow cytometry, using Annexin V staining.
Results
We analyzed IL-6 mRNA expression levels in patients-derived PBMCs by Real Time-PCR. Our results demonstrated that IL-6 gene expression significantly decreases in Bz-treated cells, as compared to a not treated condition (p<0.01). The transcriptional data were supported by immunofluorescence assay, showing that this cytokine is mainly produced by the monocyte/macrophage lineage and that its expression is reduced after Bz treatment.To investigate the Bz effect on IL-6-induced JAK/STAT pathway, we performed WB analysis to evaluate STAT3 activation by measuring pSTAT3 Tyr705. Our results showed that pSTAT3 Tyr705 levels strongly decrease in Bz-treated condition, irrespective to the presence of STAT3 mutations. This pattern strictly differs from untreated PBMCs, in which STAT3 remains over-activated. Then, by Real Time-PCR and WB assays, we analyzed the expression of STAT3 downstream targets, focusing on the anti-apoptotic MCL-1 and BCL-2 gene expression. Our results demonstrated a significant reduction of the expression levels of both these genes following Bz treatment (p<0.01).Finally, we evaluated Bz effect on cell survival by Annexin V staining. Our results showed a significant time-dependent increase in LGL apoptosis after Bz treatment, as compared to a not treated condition. Moreover, we observed that this drug is highly specific in inducing leukemic LGLs apoptosis, not affecting the other PBMCs.
Conclusion
Our results provide evidence that Bortezomib might represent a new intriguing therapeutic option to be added to the immunosuppressive drugs already used in LGLL therapy.
Session topic: E-poster
Keyword(s): Bortezomib, IL-6, Large granular lymphocytic leukaemia, STAT3
Abstract: E1032
Type: Eposter Presentation
Background
Large Granular Lymphocyte Leukemia (LGLL) is a chronic lymphoproliferative disorder characterized by clonal expansion of Large Granular Lymphocytes (LGLs) with cytotoxic activity. LGLs proliferation is maintained through the activation of several survival signaling pathways. Among these, one of the most important deregulated pathway is the JAK/STAT axis. STAT3 activation, mediated by IL-6 signaling or activating STAT3 mutations, promotes LGLs survival by inducing several anti-apoptotic genes transcription and represents a hallmark of this disease.Nowadays, LGLL therapy is based on immunosuppressive drugs, such as Methotrexate (MTX), Cyclophosphamide (CTX) and Cyclosporine A (CyA), showing however only a partial efficacy with an Overall Response Rate (ORR) of about 50-60%. Bortezomib (Bz), the first FDA-approved proteasome inhibitor used in Multiple Myeloma (MM) therapy, is not an approved treatment opportunity for LGLL, but the rare co-association of LGLL with MM offered the opportunity to retrospectively investigate the effect of this drug on leukemic LGLs. The literature reports that patients diagnosed with both LGLL and MM showed a decrease in the LGLs count after Bz treatment, suggesting that this drug may have an effect on inhibiting the LGL clone.
Aims
The aim of this study is to investigate the in vitro effects of Bz on PBMCs from LGLL patients, in order to understand its molecular mechanism of action in reducing the LGL clone.
Methods
Peripheral blood mononuclear cells (PBMCs) of 20 LGLL patients (8 out of 20 characterized by STAT3 mutations), isolated through Ficoll density centrifugation, were cultured and treated with Bz (5.2 nM) for 24 or 48 hours. Real Time-PCR, western blot (WB) assays and immunofluorescence assay were performed to analyze mRNA and protein expression. Cell apoptosis was evaluated by Flow cytometry, using Annexin V staining.
Results
We analyzed IL-6 mRNA expression levels in patients-derived PBMCs by Real Time-PCR. Our results demonstrated that IL-6 gene expression significantly decreases in Bz-treated cells, as compared to a not treated condition (p<0.01). The transcriptional data were supported by immunofluorescence assay, showing that this cytokine is mainly produced by the monocyte/macrophage lineage and that its expression is reduced after Bz treatment.To investigate the Bz effect on IL-6-induced JAK/STAT pathway, we performed WB analysis to evaluate STAT3 activation by measuring pSTAT3 Tyr705. Our results showed that pSTAT3 Tyr705 levels strongly decrease in Bz-treated condition, irrespective to the presence of STAT3 mutations. This pattern strictly differs from untreated PBMCs, in which STAT3 remains over-activated. Then, by Real Time-PCR and WB assays, we analyzed the expression of STAT3 downstream targets, focusing on the anti-apoptotic MCL-1 and BCL-2 gene expression. Our results demonstrated a significant reduction of the expression levels of both these genes following Bz treatment (p<0.01).Finally, we evaluated Bz effect on cell survival by Annexin V staining. Our results showed a significant time-dependent increase in LGL apoptosis after Bz treatment, as compared to a not treated condition. Moreover, we observed that this drug is highly specific in inducing leukemic LGLs apoptosis, not affecting the other PBMCs.
Conclusion
Our results provide evidence that Bortezomib might represent a new intriguing therapeutic option to be added to the immunosuppressive drugs already used in LGLL therapy.
Session topic: E-poster
Keyword(s): Bortezomib, IL-6, Large granular lymphocytic leukaemia, STAT3
Type: Eposter Presentation
Background
Large Granular Lymphocyte Leukemia (LGLL) is a chronic lymphoproliferative disorder characterized by clonal expansion of Large Granular Lymphocytes (LGLs) with cytotoxic activity. LGLs proliferation is maintained through the activation of several survival signaling pathways. Among these, one of the most important deregulated pathway is the JAK/STAT axis. STAT3 activation, mediated by IL-6 signaling or activating STAT3 mutations, promotes LGLs survival by inducing several anti-apoptotic genes transcription and represents a hallmark of this disease.Nowadays, LGLL therapy is based on immunosuppressive drugs, such as Methotrexate (MTX), Cyclophosphamide (CTX) and Cyclosporine A (CyA), showing however only a partial efficacy with an Overall Response Rate (ORR) of about 50-60%. Bortezomib (Bz), the first FDA-approved proteasome inhibitor used in Multiple Myeloma (MM) therapy, is not an approved treatment opportunity for LGLL, but the rare co-association of LGLL with MM offered the opportunity to retrospectively investigate the effect of this drug on leukemic LGLs. The literature reports that patients diagnosed with both LGLL and MM showed a decrease in the LGLs count after Bz treatment, suggesting that this drug may have an effect on inhibiting the LGL clone.
Aims
The aim of this study is to investigate the in vitro effects of Bz on PBMCs from LGLL patients, in order to understand its molecular mechanism of action in reducing the LGL clone.
Methods
Peripheral blood mononuclear cells (PBMCs) of 20 LGLL patients (8 out of 20 characterized by STAT3 mutations), isolated through Ficoll density centrifugation, were cultured and treated with Bz (5.2 nM) for 24 or 48 hours. Real Time-PCR, western blot (WB) assays and immunofluorescence assay were performed to analyze mRNA and protein expression. Cell apoptosis was evaluated by Flow cytometry, using Annexin V staining.
Results
We analyzed IL-6 mRNA expression levels in patients-derived PBMCs by Real Time-PCR. Our results demonstrated that IL-6 gene expression significantly decreases in Bz-treated cells, as compared to a not treated condition (p<0.01). The transcriptional data were supported by immunofluorescence assay, showing that this cytokine is mainly produced by the monocyte/macrophage lineage and that its expression is reduced after Bz treatment.To investigate the Bz effect on IL-6-induced JAK/STAT pathway, we performed WB analysis to evaluate STAT3 activation by measuring pSTAT3 Tyr705. Our results showed that pSTAT3 Tyr705 levels strongly decrease in Bz-treated condition, irrespective to the presence of STAT3 mutations. This pattern strictly differs from untreated PBMCs, in which STAT3 remains over-activated. Then, by Real Time-PCR and WB assays, we analyzed the expression of STAT3 downstream targets, focusing on the anti-apoptotic MCL-1 and BCL-2 gene expression. Our results demonstrated a significant reduction of the expression levels of both these genes following Bz treatment (p<0.01).Finally, we evaluated Bz effect on cell survival by Annexin V staining. Our results showed a significant time-dependent increase in LGL apoptosis after Bz treatment, as compared to a not treated condition. Moreover, we observed that this drug is highly specific in inducing leukemic LGLs apoptosis, not affecting the other PBMCs.
Conclusion
Our results provide evidence that Bortezomib might represent a new intriguing therapeutic option to be added to the immunosuppressive drugs already used in LGLL therapy.
Session topic: E-poster
Keyword(s): Bortezomib, IL-6, Large granular lymphocytic leukaemia, STAT3
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