FAILURE OF MEGAKARYOCYTE APOPTOSIS IN MYELOPROLIFERATIVE NEOPLASMS
(Abstract release date: 05/19/16)
EHA Library. Malherbe J. 06/09/16; 132883; E1334
Disclosure(s): This work was supported by the Cancer Council Western Australia (Cancer Council WA Honours Scholarship 2013), Leukaemia Foundation Australia (Dominic Di Giacomo Honours Scholarship 2013), the University of Western Australia (Hackett Foundation Alumni Honours Scholarship 2013), the Royal College of Pathologists of Australasia (RCPA Scholarship in Pathology for Medical Schools 2015), Perpetual Foundation-Ann Helene Toakley Charitable Endowment and the Australian Leukaemia and Lymphoma Group and Novartis Pharmaceuticals Australia Pty. Ltd.
Mr. Jacques Malherbe
Contributions
Contributions
Abstract
Abstract: E1334
Type: Eposter Presentation
Background
Megakaryocytic hyperplasia and morphological atypia are characteristic features of the myeloproliferative neoplasms (MPN) and its entities: polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MF). Data from murine and ex vivo models suggest that multiple molecular abnormalities (e.g. JAK2V617F, CALR mutations) underpin megakaryocyte expansion in the MPN. Limited studies also show that megakaryocytes utilize apoptotic caspases to shed platelets and in the MPN, they may have apoptotic disturbances. Despite these claims, there is little data demonstrating the precise apoptotic defects and pathways affected in megakaryocytes of human MPN.
Aims
We investigated the apoptotic pathobiology of megakaryocytes in human MPN. We determined whether these disturbances targeted the intrinsic and/or extrinsic apoptotic pathways and examined their differential associations between MPN disease entities and the JAK2V617F and CALR driver mutations.
Methods
Bone marrow trephines of MPN (PV=20, ET=72, MF=53) and controls (n=15) were studied. These included JAK2V617F, CALR-mutated (CALRMut) and double negative (JAK2V617F-/CALRWT) cases. Sections were stained with antibodies to pro-apoptotic (BNIP-3, caspase-8, caspase-9, Diablo, p53) and anti-apoptotic (Bcl-XL, survivin) effectors involved in the intrinsic and extrinsic apoptotic pathways using an immuno-alkaline phosphatase and Fast Red chromogen detection system. Megakaryocyte positivity was assessed by light microscopy and the percent positive determined per case. Mean differences (MD) in megakaryocyte positivity between MPN entities and JAK2V617F/CALR mutation states were statistically evaluated. Megakaryocyte positivity was also correlated with platelet count.
Results
Megakaryocytes in MPN showed significantly greater positivity for anti-apoptotic Bcl-XL and survivin, and pro-apoptotic Diablo, caspase-8 and p53 than controls. However, MPN had significantly fewer pro-death BNIP-3 positive megakaryocytes (MD=42.8%, p<0.0001). Caspase-9 was only marginally increased in MPN (MD=4.3%, p=0.02). The apoptotic dysregulation was most marked in MF. Significantly fewer BNIP-3 positive megakaryocytes were seen in MF than ET (MD=14.7%, p=0.008). MF also had fewer caspase-9 positive megakaryocytes compared with both PV (MD=33.8%, p=0.003) and ET (MD=19.8%, p=0.02). However, p53 was increased ~2.7-fold in MF versus PV (p=0.007). CALRMut megakaryocytes typically had pyknotic chromatin and lower pro-apoptotic BNIP-3 levels relative to JAK2V617F (MD=24.2%, p=0.0008). Platelet counts correlated positively with caspase-9 megakaryocyte positivity in MPN (r=0.28, p=0.002), and strengthened among MF (r=0.34, p=0.03) and CALRMut cases (r=0.50, p=0.03).
Conclusion
Megakaryocytic hyperplasia in the MPN is assisted by disruptions of the intrinsic apoptotic pathway. Our data shows overexpression of anti-apoptotic Bcl-XL and survivin to be the key apoptotic inhibitors mediating megakaryocyte survival. In MF, apoptotic failure is exacerbated by reductions in pro-death BNIP-3 and caspase-9; this may relate to the marked formation of cohesive megakaryocyte sheets and abnormal nuclear morphology common in MF. CALR mutations also showed greater reductions in pro-apoptotic BNIP-3 compared with JAK2V617F. Finally, the upregulation of caspase-9 seen in megakaryocytes of CALR-mutated cases may account for the thrombocytosis associated with this molecular subgroup.
Session topic: E-poster
Keyword(s): Apoptosis, Megakaryocyte, Myeloproliferative disorder
Type: Eposter Presentation
Background
Megakaryocytic hyperplasia and morphological atypia are characteristic features of the myeloproliferative neoplasms (MPN) and its entities: polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MF). Data from murine and ex vivo models suggest that multiple molecular abnormalities (e.g. JAK2V617F, CALR mutations) underpin megakaryocyte expansion in the MPN. Limited studies also show that megakaryocytes utilize apoptotic caspases to shed platelets and in the MPN, they may have apoptotic disturbances. Despite these claims, there is little data demonstrating the precise apoptotic defects and pathways affected in megakaryocytes of human MPN.
Aims
We investigated the apoptotic pathobiology of megakaryocytes in human MPN. We determined whether these disturbances targeted the intrinsic and/or extrinsic apoptotic pathways and examined their differential associations between MPN disease entities and the JAK2V617F and CALR driver mutations.
Methods
Bone marrow trephines of MPN (PV=20, ET=72, MF=53) and controls (n=15) were studied. These included JAK2V617F, CALR-mutated (CALRMut) and double negative (JAK2V617F-/CALRWT) cases. Sections were stained with antibodies to pro-apoptotic (BNIP-3, caspase-8, caspase-9, Diablo, p53) and anti-apoptotic (Bcl-XL, survivin) effectors involved in the intrinsic and extrinsic apoptotic pathways using an immuno-alkaline phosphatase and Fast Red chromogen detection system. Megakaryocyte positivity was assessed by light microscopy and the percent positive determined per case. Mean differences (MD) in megakaryocyte positivity between MPN entities and JAK2V617F/CALR mutation states were statistically evaluated. Megakaryocyte positivity was also correlated with platelet count.
Results
Megakaryocytes in MPN showed significantly greater positivity for anti-apoptotic Bcl-XL and survivin, and pro-apoptotic Diablo, caspase-8 and p53 than controls. However, MPN had significantly fewer pro-death BNIP-3 positive megakaryocytes (MD=42.8%, p<0.0001). Caspase-9 was only marginally increased in MPN (MD=4.3%, p=0.02). The apoptotic dysregulation was most marked in MF. Significantly fewer BNIP-3 positive megakaryocytes were seen in MF than ET (MD=14.7%, p=0.008). MF also had fewer caspase-9 positive megakaryocytes compared with both PV (MD=33.8%, p=0.003) and ET (MD=19.8%, p=0.02). However, p53 was increased ~2.7-fold in MF versus PV (p=0.007). CALRMut megakaryocytes typically had pyknotic chromatin and lower pro-apoptotic BNIP-3 levels relative to JAK2V617F (MD=24.2%, p=0.0008). Platelet counts correlated positively with caspase-9 megakaryocyte positivity in MPN (r=0.28, p=0.002), and strengthened among MF (r=0.34, p=0.03) and CALRMut cases (r=0.50, p=0.03).
Conclusion
Megakaryocytic hyperplasia in the MPN is assisted by disruptions of the intrinsic apoptotic pathway. Our data shows overexpression of anti-apoptotic Bcl-XL and survivin to be the key apoptotic inhibitors mediating megakaryocyte survival. In MF, apoptotic failure is exacerbated by reductions in pro-death BNIP-3 and caspase-9; this may relate to the marked formation of cohesive megakaryocyte sheets and abnormal nuclear morphology common in MF. CALR mutations also showed greater reductions in pro-apoptotic BNIP-3 compared with JAK2V617F. Finally, the upregulation of caspase-9 seen in megakaryocytes of CALR-mutated cases may account for the thrombocytosis associated with this molecular subgroup.
Session topic: E-poster
Keyword(s): Apoptosis, Megakaryocyte, Myeloproliferative disorder
Abstract: E1334
Type: Eposter Presentation
Background
Megakaryocytic hyperplasia and morphological atypia are characteristic features of the myeloproliferative neoplasms (MPN) and its entities: polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MF). Data from murine and ex vivo models suggest that multiple molecular abnormalities (e.g. JAK2V617F, CALR mutations) underpin megakaryocyte expansion in the MPN. Limited studies also show that megakaryocytes utilize apoptotic caspases to shed platelets and in the MPN, they may have apoptotic disturbances. Despite these claims, there is little data demonstrating the precise apoptotic defects and pathways affected in megakaryocytes of human MPN.
Aims
We investigated the apoptotic pathobiology of megakaryocytes in human MPN. We determined whether these disturbances targeted the intrinsic and/or extrinsic apoptotic pathways and examined their differential associations between MPN disease entities and the JAK2V617F and CALR driver mutations.
Methods
Bone marrow trephines of MPN (PV=20, ET=72, MF=53) and controls (n=15) were studied. These included JAK2V617F, CALR-mutated (CALRMut) and double negative (JAK2V617F-/CALRWT) cases. Sections were stained with antibodies to pro-apoptotic (BNIP-3, caspase-8, caspase-9, Diablo, p53) and anti-apoptotic (Bcl-XL, survivin) effectors involved in the intrinsic and extrinsic apoptotic pathways using an immuno-alkaline phosphatase and Fast Red chromogen detection system. Megakaryocyte positivity was assessed by light microscopy and the percent positive determined per case. Mean differences (MD) in megakaryocyte positivity between MPN entities and JAK2V617F/CALR mutation states were statistically evaluated. Megakaryocyte positivity was also correlated with platelet count.
Results
Megakaryocytes in MPN showed significantly greater positivity for anti-apoptotic Bcl-XL and survivin, and pro-apoptotic Diablo, caspase-8 and p53 than controls. However, MPN had significantly fewer pro-death BNIP-3 positive megakaryocytes (MD=42.8%, p<0.0001). Caspase-9 was only marginally increased in MPN (MD=4.3%, p=0.02). The apoptotic dysregulation was most marked in MF. Significantly fewer BNIP-3 positive megakaryocytes were seen in MF than ET (MD=14.7%, p=0.008). MF also had fewer caspase-9 positive megakaryocytes compared with both PV (MD=33.8%, p=0.003) and ET (MD=19.8%, p=0.02). However, p53 was increased ~2.7-fold in MF versus PV (p=0.007). CALRMut megakaryocytes typically had pyknotic chromatin and lower pro-apoptotic BNIP-3 levels relative to JAK2V617F (MD=24.2%, p=0.0008). Platelet counts correlated positively with caspase-9 megakaryocyte positivity in MPN (r=0.28, p=0.002), and strengthened among MF (r=0.34, p=0.03) and CALRMut cases (r=0.50, p=0.03).
Conclusion
Megakaryocytic hyperplasia in the MPN is assisted by disruptions of the intrinsic apoptotic pathway. Our data shows overexpression of anti-apoptotic Bcl-XL and survivin to be the key apoptotic inhibitors mediating megakaryocyte survival. In MF, apoptotic failure is exacerbated by reductions in pro-death BNIP-3 and caspase-9; this may relate to the marked formation of cohesive megakaryocyte sheets and abnormal nuclear morphology common in MF. CALR mutations also showed greater reductions in pro-apoptotic BNIP-3 compared with JAK2V617F. Finally, the upregulation of caspase-9 seen in megakaryocytes of CALR-mutated cases may account for the thrombocytosis associated with this molecular subgroup.
Session topic: E-poster
Keyword(s): Apoptosis, Megakaryocyte, Myeloproliferative disorder
Type: Eposter Presentation
Background
Megakaryocytic hyperplasia and morphological atypia are characteristic features of the myeloproliferative neoplasms (MPN) and its entities: polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MF). Data from murine and ex vivo models suggest that multiple molecular abnormalities (e.g. JAK2V617F, CALR mutations) underpin megakaryocyte expansion in the MPN. Limited studies also show that megakaryocytes utilize apoptotic caspases to shed platelets and in the MPN, they may have apoptotic disturbances. Despite these claims, there is little data demonstrating the precise apoptotic defects and pathways affected in megakaryocytes of human MPN.
Aims
We investigated the apoptotic pathobiology of megakaryocytes in human MPN. We determined whether these disturbances targeted the intrinsic and/or extrinsic apoptotic pathways and examined their differential associations between MPN disease entities and the JAK2V617F and CALR driver mutations.
Methods
Bone marrow trephines of MPN (PV=20, ET=72, MF=53) and controls (n=15) were studied. These included JAK2V617F, CALR-mutated (CALRMut) and double negative (JAK2V617F-/CALRWT) cases. Sections were stained with antibodies to pro-apoptotic (BNIP-3, caspase-8, caspase-9, Diablo, p53) and anti-apoptotic (Bcl-XL, survivin) effectors involved in the intrinsic and extrinsic apoptotic pathways using an immuno-alkaline phosphatase and Fast Red chromogen detection system. Megakaryocyte positivity was assessed by light microscopy and the percent positive determined per case. Mean differences (MD) in megakaryocyte positivity between MPN entities and JAK2V617F/CALR mutation states were statistically evaluated. Megakaryocyte positivity was also correlated with platelet count.
Results
Megakaryocytes in MPN showed significantly greater positivity for anti-apoptotic Bcl-XL and survivin, and pro-apoptotic Diablo, caspase-8 and p53 than controls. However, MPN had significantly fewer pro-death BNIP-3 positive megakaryocytes (MD=42.8%, p<0.0001). Caspase-9 was only marginally increased in MPN (MD=4.3%, p=0.02). The apoptotic dysregulation was most marked in MF. Significantly fewer BNIP-3 positive megakaryocytes were seen in MF than ET (MD=14.7%, p=0.008). MF also had fewer caspase-9 positive megakaryocytes compared with both PV (MD=33.8%, p=0.003) and ET (MD=19.8%, p=0.02). However, p53 was increased ~2.7-fold in MF versus PV (p=0.007). CALRMut megakaryocytes typically had pyknotic chromatin and lower pro-apoptotic BNIP-3 levels relative to JAK2V617F (MD=24.2%, p=0.0008). Platelet counts correlated positively with caspase-9 megakaryocyte positivity in MPN (r=0.28, p=0.002), and strengthened among MF (r=0.34, p=0.03) and CALRMut cases (r=0.50, p=0.03).
Conclusion
Megakaryocytic hyperplasia in the MPN is assisted by disruptions of the intrinsic apoptotic pathway. Our data shows overexpression of anti-apoptotic Bcl-XL and survivin to be the key apoptotic inhibitors mediating megakaryocyte survival. In MF, apoptotic failure is exacerbated by reductions in pro-death BNIP-3 and caspase-9; this may relate to the marked formation of cohesive megakaryocyte sheets and abnormal nuclear morphology common in MF. CALR mutations also showed greater reductions in pro-apoptotic BNIP-3 compared with JAK2V617F. Finally, the upregulation of caspase-9 seen in megakaryocytes of CALR-mutated cases may account for the thrombocytosis associated with this molecular subgroup.
Session topic: E-poster
Keyword(s): Apoptosis, Megakaryocyte, Myeloproliferative disorder
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