DEVELOPMENT OF RAS-INDUCED ZEBRAFISH LEUKEMIA MODELS
(Abstract release date: 05/19/16)
EHA Library. Lengerke C. 06/09/16; 134907; PB2007
Dr. Claudia Lengerke
Contributions
Contributions
Abstract
Abstract: PB2007
Type: Publication Only
Background
The zebrafish has emerged as a versatile novel experimental model for studies on developmental hematopoiesis and leukemogenesis. Several oncogenes involved in human leukemia have been successfully overexpressed in zebrafish embryos. However, despite first encouraging results, these models often fail to fully recapitulate human myeloid malignancy, perhaps due to early lethality caused by transgene expression or lack of secondary events necessary for full malignant transformation.
Aims
Here we present a RAS overexpression zebrafish model that we plan to use for investigations on molecular mechanisms involved in leukemia initiation and progression and for drug screening that can identify novel anti-leukemic compounds.
Methods
We take advantage of the Gal4/UAS binary system and of existing transgenic lines and overexpress human oncogenic HRAS in zebrafish hematopoietic cells under the control of specific promoters (fli.1, pu.1, runx.1, mpeg1). The generated HRAS-transgenic fish lines are followed microscopically until the time-point of death or sexual maturity and hematopoietic cell development is studied correspondingly at embryonic (fli.1, pu.1), larval (pu.1, runx1) and adult stages (runx1) also by in situ hybridization/real-time PCR analysis of hematopoietic gene expression, flow cytometry, immunohistochemistry and/or blood smear morphological assessment.
Results
Different phenotypes were observed depending on the promoter driving the oncogene expression. HRAS induction via the early hematopoietic promoter fli.1 affects primitive hematopoiesis inducing myelo-erythroid proliferation and delayed erythrocyte maturation resulting in an expansion of hematopoietic tissues (Alghisi et al. 2013). Unfortunately, studies at later stages are not possible in these fish due to their early lethality resulting from vascular defects and cardiac edema. Alternatively, HRAS expression driven by runx1, pu.1 and mpeg1 allows survival at these early stages permitting studies on larval and adult hematopoiesis. Interestingly, at 1 month, runx1-HRAS fish displayed a cellular expansion of hematopoietic stem/progenitor cells (HSPC) in the kidney marrow (KM), the zebrafish definitive hematopoietic compartment. Cytospin preparation and flow cytometric analyses revealed high numbers of undifferentiated stem/progenitor cells in both KM and peripheral blood of HRAS transgenic fish, suggesting that HRAS-overexpression in HSPCs expands this compartment by inducing its proliferation and perhaps additionally by impairing differentiation capacity. Furthermore, mpeg1-HRAS fish showed increased numbers of blood progenitors in the KM and abnormal gene expression of progenitor markers as demonstrated by qRT-PCR. Analysis of pu.1-HRAS juvenile fish is under way.
Conclusion
We are currently further investigating the effects of runx1-driven HRAS on the hematopoietic compartment and generate tools to explore potential cooperation of HRAS with other oncogenes during leukemogenesis. A screen analyzing compounds successfully suppressing the effects of HRAS on zebrafish hematopoiesis is underway and the results will be presented at the meeting.
Session topic: E-poster
Keyword(s): Leukemia, Ras, Zebra fish
Type: Publication Only
Background
The zebrafish has emerged as a versatile novel experimental model for studies on developmental hematopoiesis and leukemogenesis. Several oncogenes involved in human leukemia have been successfully overexpressed in zebrafish embryos. However, despite first encouraging results, these models often fail to fully recapitulate human myeloid malignancy, perhaps due to early lethality caused by transgene expression or lack of secondary events necessary for full malignant transformation.
Aims
Here we present a RAS overexpression zebrafish model that we plan to use for investigations on molecular mechanisms involved in leukemia initiation and progression and for drug screening that can identify novel anti-leukemic compounds.
Methods
We take advantage of the Gal4/UAS binary system and of existing transgenic lines and overexpress human oncogenic HRAS in zebrafish hematopoietic cells under the control of specific promoters (fli.1, pu.1, runx.1, mpeg1). The generated HRAS-transgenic fish lines are followed microscopically until the time-point of death or sexual maturity and hematopoietic cell development is studied correspondingly at embryonic (fli.1, pu.1), larval (pu.1, runx1) and adult stages (runx1) also by in situ hybridization/real-time PCR analysis of hematopoietic gene expression, flow cytometry, immunohistochemistry and/or blood smear morphological assessment.
Results
Different phenotypes were observed depending on the promoter driving the oncogene expression. HRAS induction via the early hematopoietic promoter fli.1 affects primitive hematopoiesis inducing myelo-erythroid proliferation and delayed erythrocyte maturation resulting in an expansion of hematopoietic tissues (Alghisi et al. 2013). Unfortunately, studies at later stages are not possible in these fish due to their early lethality resulting from vascular defects and cardiac edema. Alternatively, HRAS expression driven by runx1, pu.1 and mpeg1 allows survival at these early stages permitting studies on larval and adult hematopoiesis. Interestingly, at 1 month, runx1-HRAS fish displayed a cellular expansion of hematopoietic stem/progenitor cells (HSPC) in the kidney marrow (KM), the zebrafish definitive hematopoietic compartment. Cytospin preparation and flow cytometric analyses revealed high numbers of undifferentiated stem/progenitor cells in both KM and peripheral blood of HRAS transgenic fish, suggesting that HRAS-overexpression in HSPCs expands this compartment by inducing its proliferation and perhaps additionally by impairing differentiation capacity. Furthermore, mpeg1-HRAS fish showed increased numbers of blood progenitors in the KM and abnormal gene expression of progenitor markers as demonstrated by qRT-PCR. Analysis of pu.1-HRAS juvenile fish is under way.
Conclusion
We are currently further investigating the effects of runx1-driven HRAS on the hematopoietic compartment and generate tools to explore potential cooperation of HRAS with other oncogenes during leukemogenesis. A screen analyzing compounds successfully suppressing the effects of HRAS on zebrafish hematopoiesis is underway and the results will be presented at the meeting.
Session topic: E-poster
Keyword(s): Leukemia, Ras, Zebra fish
Abstract: PB2007
Type: Publication Only
Background
The zebrafish has emerged as a versatile novel experimental model for studies on developmental hematopoiesis and leukemogenesis. Several oncogenes involved in human leukemia have been successfully overexpressed in zebrafish embryos. However, despite first encouraging results, these models often fail to fully recapitulate human myeloid malignancy, perhaps due to early lethality caused by transgene expression or lack of secondary events necessary for full malignant transformation.
Aims
Here we present a RAS overexpression zebrafish model that we plan to use for investigations on molecular mechanisms involved in leukemia initiation and progression and for drug screening that can identify novel anti-leukemic compounds.
Methods
We take advantage of the Gal4/UAS binary system and of existing transgenic lines and overexpress human oncogenic HRAS in zebrafish hematopoietic cells under the control of specific promoters (fli.1, pu.1, runx.1, mpeg1). The generated HRAS-transgenic fish lines are followed microscopically until the time-point of death or sexual maturity and hematopoietic cell development is studied correspondingly at embryonic (fli.1, pu.1), larval (pu.1, runx1) and adult stages (runx1) also by in situ hybridization/real-time PCR analysis of hematopoietic gene expression, flow cytometry, immunohistochemistry and/or blood smear morphological assessment.
Results
Different phenotypes were observed depending on the promoter driving the oncogene expression. HRAS induction via the early hematopoietic promoter fli.1 affects primitive hematopoiesis inducing myelo-erythroid proliferation and delayed erythrocyte maturation resulting in an expansion of hematopoietic tissues (Alghisi et al. 2013). Unfortunately, studies at later stages are not possible in these fish due to their early lethality resulting from vascular defects and cardiac edema. Alternatively, HRAS expression driven by runx1, pu.1 and mpeg1 allows survival at these early stages permitting studies on larval and adult hematopoiesis. Interestingly, at 1 month, runx1-HRAS fish displayed a cellular expansion of hematopoietic stem/progenitor cells (HSPC) in the kidney marrow (KM), the zebrafish definitive hematopoietic compartment. Cytospin preparation and flow cytometric analyses revealed high numbers of undifferentiated stem/progenitor cells in both KM and peripheral blood of HRAS transgenic fish, suggesting that HRAS-overexpression in HSPCs expands this compartment by inducing its proliferation and perhaps additionally by impairing differentiation capacity. Furthermore, mpeg1-HRAS fish showed increased numbers of blood progenitors in the KM and abnormal gene expression of progenitor markers as demonstrated by qRT-PCR. Analysis of pu.1-HRAS juvenile fish is under way.
Conclusion
We are currently further investigating the effects of runx1-driven HRAS on the hematopoietic compartment and generate tools to explore potential cooperation of HRAS with other oncogenes during leukemogenesis. A screen analyzing compounds successfully suppressing the effects of HRAS on zebrafish hematopoiesis is underway and the results will be presented at the meeting.
Session topic: E-poster
Keyword(s): Leukemia, Ras, Zebra fish
Type: Publication Only
Background
The zebrafish has emerged as a versatile novel experimental model for studies on developmental hematopoiesis and leukemogenesis. Several oncogenes involved in human leukemia have been successfully overexpressed in zebrafish embryos. However, despite first encouraging results, these models often fail to fully recapitulate human myeloid malignancy, perhaps due to early lethality caused by transgene expression or lack of secondary events necessary for full malignant transformation.
Aims
Here we present a RAS overexpression zebrafish model that we plan to use for investigations on molecular mechanisms involved in leukemia initiation and progression and for drug screening that can identify novel anti-leukemic compounds.
Methods
We take advantage of the Gal4/UAS binary system and of existing transgenic lines and overexpress human oncogenic HRAS in zebrafish hematopoietic cells under the control of specific promoters (fli.1, pu.1, runx.1, mpeg1). The generated HRAS-transgenic fish lines are followed microscopically until the time-point of death or sexual maturity and hematopoietic cell development is studied correspondingly at embryonic (fli.1, pu.1), larval (pu.1, runx1) and adult stages (runx1) also by in situ hybridization/real-time PCR analysis of hematopoietic gene expression, flow cytometry, immunohistochemistry and/or blood smear morphological assessment.
Results
Different phenotypes were observed depending on the promoter driving the oncogene expression. HRAS induction via the early hematopoietic promoter fli.1 affects primitive hematopoiesis inducing myelo-erythroid proliferation and delayed erythrocyte maturation resulting in an expansion of hematopoietic tissues (Alghisi et al. 2013). Unfortunately, studies at later stages are not possible in these fish due to their early lethality resulting from vascular defects and cardiac edema. Alternatively, HRAS expression driven by runx1, pu.1 and mpeg1 allows survival at these early stages permitting studies on larval and adult hematopoiesis. Interestingly, at 1 month, runx1-HRAS fish displayed a cellular expansion of hematopoietic stem/progenitor cells (HSPC) in the kidney marrow (KM), the zebrafish definitive hematopoietic compartment. Cytospin preparation and flow cytometric analyses revealed high numbers of undifferentiated stem/progenitor cells in both KM and peripheral blood of HRAS transgenic fish, suggesting that HRAS-overexpression in HSPCs expands this compartment by inducing its proliferation and perhaps additionally by impairing differentiation capacity. Furthermore, mpeg1-HRAS fish showed increased numbers of blood progenitors in the KM and abnormal gene expression of progenitor markers as demonstrated by qRT-PCR. Analysis of pu.1-HRAS juvenile fish is under way.
Conclusion
We are currently further investigating the effects of runx1-driven HRAS on the hematopoietic compartment and generate tools to explore potential cooperation of HRAS with other oncogenes during leukemogenesis. A screen analyzing compounds successfully suppressing the effects of HRAS on zebrafish hematopoiesis is underway and the results will be presented at the meeting.
Session topic: E-poster
Keyword(s): Leukemia, Ras, Zebra fish
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