NPM1 MUTATION AND FLT3/ITD IN A DOUBLE TRANSGENIC ZEBRAFISH RECAPITULATES FEATURES OF HUMAN ACUTE MYELOID LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Lu J. 06/09/16; 132443; E894
Dr. Jeng-Wei Lu
Contributions
Contributions
Abstract
Abstract: E894
Type: Eposter Presentation
Background
Acute myeloid leukemia (AML) is a clonal hematologic malignancy that shows great variability with regard to pathogenesis and treatment outcomes. One of the most common mutations associated with AML involves the class III receptor tyrosine kinase, FMS-like tyrosine kinase 3 (FLT3), which plays an important role in hematopoiesis. Constitutively activated FLT3 occurring as internal tandem duplication (ITD) within the juxtamembrane domain is observed in 20-25% of AML patients and refers to the poorest response to current standard treatment. FLT3-ITD mutant was reported to be a driver mutation in myeloid leukemogenesis. Zebrafish, sharing similar leukocyte compartment with human beings, recently emerged as a promising animal model for studying diseases and for drug discovery.
Aims
To establish a transgenic FLT3-ITD zebrafish line, and further explore their characteristics, on which future drug screening may be developed.
Methods
We established a transgenic zebrafish that is able to express human FLT3-ITD or NPM1-Mut under the control of a myeloid-specific promoter (5.3 kb spi-1). Specifically, we generated two constructs, referred to as spi1: FLT3-ITD-2A-EGFP/CG2 and spi1:NPM1-Mut-PA/CG2, respectively.
Results
Cytological analysis of kidney marrow (KM) and peripheral blood (PB) smears prepared from wildtype and FLT3-ITD zebrafish were examined at 4, 6 and 9 months. In comparison with wild type fish, the KM from some spi1:FLT3-ITD-2A-EGFP/CG2 6- and 9-month old transgenic zebrafish had a greater number of myeloid progenitors and an excess of blast cells with focal aggregation. Hematoxylin and eosin (H&E) staining of the 6 month-old FLT3-ITD transgenic fish kidney showed mild effacement and distortion of kidney structure as well as increased infiltration of myeloid cells. Further morphological analysis of cytospin marrow showed that AB-wild type fish possessed the normal complement of haematological cells, whereas 2 out of 6 spi1:FLT3-ITD-2A-EGFP/CG2 zebrafish had an abnormal number of myeloid progenitors and decreased synthesis of erythroid cells at 9 month-old. Finally, leukoerythroblastosis was observed in PB. It is well recognized that the NPM1 and FLT3-ITD mutations occur concurrently in AML patients, and the combined genotype that results from this (NPM1/FLT3-ITD) is the most powerful indicator of AML prognosis. To better delineate the two-hit model in zebrafish, we also generated FLT3-ITD/NPM1-Mut double transgenic mutants. In so doing, we found that FLT3-ITD and NPM1-Mut synergistically promoted myeloid blasts and the expansion of precursor cells in zebrafish as young as six months. Flow cytometric analysis and Liu's stains of the KM and PB smears from 6-month old FLT3-ITD/NPM1-Mut transgenic fish revealed myeloid hyperplasia with predominance of blast cells and depletion of erythroid series in KM, whereas immature blast cells were able to progressively infiltrate the KM and circulate into the PB. Taken above, we suggested that FLT3-ITD and NPM1-Mut synergistically promoted myeloid blasts and the expansion of precursor cells in zebrafish.
Conclusion
In conclusion, FLT3-ITD as a common FLT3 mutation in AML patients could results in expanded myelopoiesis with poor differentiation in adult zebrafish. These transgenic fish could potentially provide a valuable platform to investigate leukemogenesis and screen drugs in the near future.
Session topic: E-poster
Keyword(s): Flt3-ITD, Myeloid malignancies, Myelopoiesis, Zebra fish
Type: Eposter Presentation
Background
Acute myeloid leukemia (AML) is a clonal hematologic malignancy that shows great variability with regard to pathogenesis and treatment outcomes. One of the most common mutations associated with AML involves the class III receptor tyrosine kinase, FMS-like tyrosine kinase 3 (FLT3), which plays an important role in hematopoiesis. Constitutively activated FLT3 occurring as internal tandem duplication (ITD) within the juxtamembrane domain is observed in 20-25% of AML patients and refers to the poorest response to current standard treatment. FLT3-ITD mutant was reported to be a driver mutation in myeloid leukemogenesis. Zebrafish, sharing similar leukocyte compartment with human beings, recently emerged as a promising animal model for studying diseases and for drug discovery.
Aims
To establish a transgenic FLT3-ITD zebrafish line, and further explore their characteristics, on which future drug screening may be developed.
Methods
We established a transgenic zebrafish that is able to express human FLT3-ITD or NPM1-Mut under the control of a myeloid-specific promoter (5.3 kb spi-1). Specifically, we generated two constructs, referred to as spi1: FLT3-ITD-2A-EGFP/CG2 and spi1:NPM1-Mut-PA/CG2, respectively.
Results
Cytological analysis of kidney marrow (KM) and peripheral blood (PB) smears prepared from wildtype and FLT3-ITD zebrafish were examined at 4, 6 and 9 months. In comparison with wild type fish, the KM from some spi1:FLT3-ITD-2A-EGFP/CG2 6- and 9-month old transgenic zebrafish had a greater number of myeloid progenitors and an excess of blast cells with focal aggregation. Hematoxylin and eosin (H&E) staining of the 6 month-old FLT3-ITD transgenic fish kidney showed mild effacement and distortion of kidney structure as well as increased infiltration of myeloid cells. Further morphological analysis of cytospin marrow showed that AB-wild type fish possessed the normal complement of haematological cells, whereas 2 out of 6 spi1:FLT3-ITD-2A-EGFP/CG2 zebrafish had an abnormal number of myeloid progenitors and decreased synthesis of erythroid cells at 9 month-old. Finally, leukoerythroblastosis was observed in PB. It is well recognized that the NPM1 and FLT3-ITD mutations occur concurrently in AML patients, and the combined genotype that results from this (NPM1/FLT3-ITD) is the most powerful indicator of AML prognosis. To better delineate the two-hit model in zebrafish, we also generated FLT3-ITD/NPM1-Mut double transgenic mutants. In so doing, we found that FLT3-ITD and NPM1-Mut synergistically promoted myeloid blasts and the expansion of precursor cells in zebrafish as young as six months. Flow cytometric analysis and Liu's stains of the KM and PB smears from 6-month old FLT3-ITD/NPM1-Mut transgenic fish revealed myeloid hyperplasia with predominance of blast cells and depletion of erythroid series in KM, whereas immature blast cells were able to progressively infiltrate the KM and circulate into the PB. Taken above, we suggested that FLT3-ITD and NPM1-Mut synergistically promoted myeloid blasts and the expansion of precursor cells in zebrafish.
Conclusion
In conclusion, FLT3-ITD as a common FLT3 mutation in AML patients could results in expanded myelopoiesis with poor differentiation in adult zebrafish. These transgenic fish could potentially provide a valuable platform to investigate leukemogenesis and screen drugs in the near future.
Session topic: E-poster
Keyword(s): Flt3-ITD, Myeloid malignancies, Myelopoiesis, Zebra fish
Abstract: E894
Type: Eposter Presentation
Background
Acute myeloid leukemia (AML) is a clonal hematologic malignancy that shows great variability with regard to pathogenesis and treatment outcomes. One of the most common mutations associated with AML involves the class III receptor tyrosine kinase, FMS-like tyrosine kinase 3 (FLT3), which plays an important role in hematopoiesis. Constitutively activated FLT3 occurring as internal tandem duplication (ITD) within the juxtamembrane domain is observed in 20-25% of AML patients and refers to the poorest response to current standard treatment. FLT3-ITD mutant was reported to be a driver mutation in myeloid leukemogenesis. Zebrafish, sharing similar leukocyte compartment with human beings, recently emerged as a promising animal model for studying diseases and for drug discovery.
Aims
To establish a transgenic FLT3-ITD zebrafish line, and further explore their characteristics, on which future drug screening may be developed.
Methods
We established a transgenic zebrafish that is able to express human FLT3-ITD or NPM1-Mut under the control of a myeloid-specific promoter (5.3 kb spi-1). Specifically, we generated two constructs, referred to as spi1: FLT3-ITD-2A-EGFP/CG2 and spi1:NPM1-Mut-PA/CG2, respectively.
Results
Cytological analysis of kidney marrow (KM) and peripheral blood (PB) smears prepared from wildtype and FLT3-ITD zebrafish were examined at 4, 6 and 9 months. In comparison with wild type fish, the KM from some spi1:FLT3-ITD-2A-EGFP/CG2 6- and 9-month old transgenic zebrafish had a greater number of myeloid progenitors and an excess of blast cells with focal aggregation. Hematoxylin and eosin (H&E) staining of the 6 month-old FLT3-ITD transgenic fish kidney showed mild effacement and distortion of kidney structure as well as increased infiltration of myeloid cells. Further morphological analysis of cytospin marrow showed that AB-wild type fish possessed the normal complement of haematological cells, whereas 2 out of 6 spi1:FLT3-ITD-2A-EGFP/CG2 zebrafish had an abnormal number of myeloid progenitors and decreased synthesis of erythroid cells at 9 month-old. Finally, leukoerythroblastosis was observed in PB. It is well recognized that the NPM1 and FLT3-ITD mutations occur concurrently in AML patients, and the combined genotype that results from this (NPM1/FLT3-ITD) is the most powerful indicator of AML prognosis. To better delineate the two-hit model in zebrafish, we also generated FLT3-ITD/NPM1-Mut double transgenic mutants. In so doing, we found that FLT3-ITD and NPM1-Mut synergistically promoted myeloid blasts and the expansion of precursor cells in zebrafish as young as six months. Flow cytometric analysis and Liu's stains of the KM and PB smears from 6-month old FLT3-ITD/NPM1-Mut transgenic fish revealed myeloid hyperplasia with predominance of blast cells and depletion of erythroid series in KM, whereas immature blast cells were able to progressively infiltrate the KM and circulate into the PB. Taken above, we suggested that FLT3-ITD and NPM1-Mut synergistically promoted myeloid blasts and the expansion of precursor cells in zebrafish.
Conclusion
In conclusion, FLT3-ITD as a common FLT3 mutation in AML patients could results in expanded myelopoiesis with poor differentiation in adult zebrafish. These transgenic fish could potentially provide a valuable platform to investigate leukemogenesis and screen drugs in the near future.
Session topic: E-poster
Keyword(s): Flt3-ITD, Myeloid malignancies, Myelopoiesis, Zebra fish
Type: Eposter Presentation
Background
Acute myeloid leukemia (AML) is a clonal hematologic malignancy that shows great variability with regard to pathogenesis and treatment outcomes. One of the most common mutations associated with AML involves the class III receptor tyrosine kinase, FMS-like tyrosine kinase 3 (FLT3), which plays an important role in hematopoiesis. Constitutively activated FLT3 occurring as internal tandem duplication (ITD) within the juxtamembrane domain is observed in 20-25% of AML patients and refers to the poorest response to current standard treatment. FLT3-ITD mutant was reported to be a driver mutation in myeloid leukemogenesis. Zebrafish, sharing similar leukocyte compartment with human beings, recently emerged as a promising animal model for studying diseases and for drug discovery.
Aims
To establish a transgenic FLT3-ITD zebrafish line, and further explore their characteristics, on which future drug screening may be developed.
Methods
We established a transgenic zebrafish that is able to express human FLT3-ITD or NPM1-Mut under the control of a myeloid-specific promoter (5.3 kb spi-1). Specifically, we generated two constructs, referred to as spi1: FLT3-ITD-2A-EGFP/CG2 and spi1:NPM1-Mut-PA/CG2, respectively.
Results
Cytological analysis of kidney marrow (KM) and peripheral blood (PB) smears prepared from wildtype and FLT3-ITD zebrafish were examined at 4, 6 and 9 months. In comparison with wild type fish, the KM from some spi1:FLT3-ITD-2A-EGFP/CG2 6- and 9-month old transgenic zebrafish had a greater number of myeloid progenitors and an excess of blast cells with focal aggregation. Hematoxylin and eosin (H&E) staining of the 6 month-old FLT3-ITD transgenic fish kidney showed mild effacement and distortion of kidney structure as well as increased infiltration of myeloid cells. Further morphological analysis of cytospin marrow showed that AB-wild type fish possessed the normal complement of haematological cells, whereas 2 out of 6 spi1:FLT3-ITD-2A-EGFP/CG2 zebrafish had an abnormal number of myeloid progenitors and decreased synthesis of erythroid cells at 9 month-old. Finally, leukoerythroblastosis was observed in PB. It is well recognized that the NPM1 and FLT3-ITD mutations occur concurrently in AML patients, and the combined genotype that results from this (NPM1/FLT3-ITD) is the most powerful indicator of AML prognosis. To better delineate the two-hit model in zebrafish, we also generated FLT3-ITD/NPM1-Mut double transgenic mutants. In so doing, we found that FLT3-ITD and NPM1-Mut synergistically promoted myeloid blasts and the expansion of precursor cells in zebrafish as young as six months. Flow cytometric analysis and Liu's stains of the KM and PB smears from 6-month old FLT3-ITD/NPM1-Mut transgenic fish revealed myeloid hyperplasia with predominance of blast cells and depletion of erythroid series in KM, whereas immature blast cells were able to progressively infiltrate the KM and circulate into the PB. Taken above, we suggested that FLT3-ITD and NPM1-Mut synergistically promoted myeloid blasts and the expansion of precursor cells in zebrafish.
Conclusion
In conclusion, FLT3-ITD as a common FLT3 mutation in AML patients could results in expanded myelopoiesis with poor differentiation in adult zebrafish. These transgenic fish could potentially provide a valuable platform to investigate leukemogenesis and screen drugs in the near future.
Session topic: E-poster
Keyword(s): Flt3-ITD, Myeloid malignancies, Myelopoiesis, Zebra fish
{{ help_message }}
{{filter}}