FLT3-D835Y AND NPM1C ONCOGENES COOPERATE TO INDUCE AN AGGRESSIVE MPN IN MICE.
(Abstract release date: 05/19/16)
EHA Library. Rudorf A. 06/11/16; 135214; S458
Ms. Alina Rudorf
Contributions
Contributions
Abstract
Abstract: S458
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:45 - 12:00
Location: Hall C13
Background
Activating mutations in FLT3 and NPM1 are most common alterations in AML and frequently coincidental. The exon 12 mutation of Npm1 (NPM1c) leads to a mislocalization of the protein from the nucleus to the cytoplasm. Two types of mutations are present in FLT3: Tandem duplication of the juxtamembraneous domain (ITD) and point mutations of the tyrosine kinase domain (TKD). In murine models, both NPM1c and FLT3-ITD induce an MPN, while FLT3-TKD leads to a lymphoid disorder. Co-expression of NPM1c and FLT3-ITD rapidly induces an AML in mice.
Aims
In the present study, we investigated the impact of co-expression of NPM1c with the TKD mutation FLT3-D835Y in a mouse model.
Methods
For this purpose a heterozygous conditional Npm1c knockin mouse model was used where Npm1c can be induced via the Mx1Cre-system. Donor bone marrow was harvested from these mice and cells were retrovirally infected with the MiG-Flt3-D835Y vector. Infected bone marrow cells were injected into lethally irradiated C57Bl/6 wt recipient mice. The Npm1c expression was induced by p(I:C) injection post transplantation resulting in the activation of IFN-α and thereby Cre recombinase induction under the control of the Mx1 promotor. Control mice received no p(I:C) injection. Lymphatic/Leukemic organs of moribund mice were analyzed for lineage-specific cell surface markers by flow cytometry.
Results
Strikingly, Npm1c Flt3-D835Y mice rapidly developed a myeloproliferative neoplasia and succumbed after a median latency of 33 days. In contrast, Flt3-D835Y Npm wt control mice did not develop a leukemic disease. Detailed analysis Npm1c Flt3-D835Y mice revealed a high leukemic burden in the peripheral blood as well as in the bone marrow and a pronounced splenomegaly. Flow cytometry analysis revealed a myeloid phenotype in the peripheral blood, spleen and bone marrow of moribund mice, characterized by a high frequency of FLT3-D835Y+/CD11b+ and Gr1+ cells, whereas Flt3-D835Y control mice showed a normal immune phenotype. Interestingly, immunoblot analysis of spleen extracts from moribund mice indicated that Flt3-D835Y in combination with NPM1c induces STAT5 but not STAT3 signaling. These results suggest that the MPN induction in this model might be due to STAT5 activation.Previously, it has been shown that the myeloid leukemia of Npm1c Flt3-ITD mice is retransplantable. To discover whether the myeloid phenotype of NPM1c Flt3-D835Y mice is retransplantable as well, 1x105 and 1x106 spleen cells of an Npm1 Flt3-D835Y mouse were injected into sublethally irradiated C57Bl/6 wt recipient mice. Indeed, we were able to show retranplantability of Npm1c Flt3-TKD disease in the group transplanted with high cell numbers, whereas the retranplantability was not only partial inducible (15-30% of the mice) in the group transplanted with lower spleen cell numbers (1x105).
Conclusion
In summary, our data show that co-expression of NPM1c and FLT3-D835Y results in the development of a rapidly fatal MPN whereas FLT3-D835Y-expression alone is not sufficient to cause a myeloproliferative disease in C57Bl/6 mice. Moreover, the myeloid phenotype is retransplantable with 1x106 donor spleen cells in C57Bl/6 mice. Interestingly, myeloproliferative disease induction might be due to aberrant STAT5 activation by FLT3-D835Y in the background of NPM1c mutant.
Session topic: AML Biology Mutant FLT
Keyword(s): FLT3, Mouse model
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:45 - 12:00
Location: Hall C13
Background
Activating mutations in FLT3 and NPM1 are most common alterations in AML and frequently coincidental. The exon 12 mutation of Npm1 (NPM1c) leads to a mislocalization of the protein from the nucleus to the cytoplasm. Two types of mutations are present in FLT3: Tandem duplication of the juxtamembraneous domain (ITD) and point mutations of the tyrosine kinase domain (TKD). In murine models, both NPM1c and FLT3-ITD induce an MPN, while FLT3-TKD leads to a lymphoid disorder. Co-expression of NPM1c and FLT3-ITD rapidly induces an AML in mice.
Aims
In the present study, we investigated the impact of co-expression of NPM1c with the TKD mutation FLT3-D835Y in a mouse model.
Methods
For this purpose a heterozygous conditional Npm1c knockin mouse model was used where Npm1c can be induced via the Mx1Cre-system. Donor bone marrow was harvested from these mice and cells were retrovirally infected with the MiG-Flt3-D835Y vector. Infected bone marrow cells were injected into lethally irradiated C57Bl/6 wt recipient mice. The Npm1c expression was induced by p(I:C) injection post transplantation resulting in the activation of IFN-α and thereby Cre recombinase induction under the control of the Mx1 promotor. Control mice received no p(I:C) injection. Lymphatic/Leukemic organs of moribund mice were analyzed for lineage-specific cell surface markers by flow cytometry.
Results
Strikingly, Npm1c Flt3-D835Y mice rapidly developed a myeloproliferative neoplasia and succumbed after a median latency of 33 days. In contrast, Flt3-D835Y Npm wt control mice did not develop a leukemic disease. Detailed analysis Npm1c Flt3-D835Y mice revealed a high leukemic burden in the peripheral blood as well as in the bone marrow and a pronounced splenomegaly. Flow cytometry analysis revealed a myeloid phenotype in the peripheral blood, spleen and bone marrow of moribund mice, characterized by a high frequency of FLT3-D835Y+/CD11b+ and Gr1+ cells, whereas Flt3-D835Y control mice showed a normal immune phenotype. Interestingly, immunoblot analysis of spleen extracts from moribund mice indicated that Flt3-D835Y in combination with NPM1c induces STAT5 but not STAT3 signaling. These results suggest that the MPN induction in this model might be due to STAT5 activation.Previously, it has been shown that the myeloid leukemia of Npm1c Flt3-ITD mice is retransplantable. To discover whether the myeloid phenotype of NPM1c Flt3-D835Y mice is retransplantable as well, 1x105 and 1x106 spleen cells of an Npm1 Flt3-D835Y mouse were injected into sublethally irradiated C57Bl/6 wt recipient mice. Indeed, we were able to show retranplantability of Npm1c Flt3-TKD disease in the group transplanted with high cell numbers, whereas the retranplantability was not only partial inducible (15-30% of the mice) in the group transplanted with lower spleen cell numbers (1x105).
Conclusion
In summary, our data show that co-expression of NPM1c and FLT3-D835Y results in the development of a rapidly fatal MPN whereas FLT3-D835Y-expression alone is not sufficient to cause a myeloproliferative disease in C57Bl/6 mice. Moreover, the myeloid phenotype is retransplantable with 1x106 donor spleen cells in C57Bl/6 mice. Interestingly, myeloproliferative disease induction might be due to aberrant STAT5 activation by FLT3-D835Y in the background of NPM1c mutant.
Session topic: AML Biology Mutant FLT
Keyword(s): FLT3, Mouse model
Abstract: S458
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:45 - 12:00
Location: Hall C13
Background
Activating mutations in FLT3 and NPM1 are most common alterations in AML and frequently coincidental. The exon 12 mutation of Npm1 (NPM1c) leads to a mislocalization of the protein from the nucleus to the cytoplasm. Two types of mutations are present in FLT3: Tandem duplication of the juxtamembraneous domain (ITD) and point mutations of the tyrosine kinase domain (TKD). In murine models, both NPM1c and FLT3-ITD induce an MPN, while FLT3-TKD leads to a lymphoid disorder. Co-expression of NPM1c and FLT3-ITD rapidly induces an AML in mice.
Aims
In the present study, we investigated the impact of co-expression of NPM1c with the TKD mutation FLT3-D835Y in a mouse model.
Methods
For this purpose a heterozygous conditional Npm1c knockin mouse model was used where Npm1c can be induced via the Mx1Cre-system. Donor bone marrow was harvested from these mice and cells were retrovirally infected with the MiG-Flt3-D835Y vector. Infected bone marrow cells were injected into lethally irradiated C57Bl/6 wt recipient mice. The Npm1c expression was induced by p(I:C) injection post transplantation resulting in the activation of IFN-α and thereby Cre recombinase induction under the control of the Mx1 promotor. Control mice received no p(I:C) injection. Lymphatic/Leukemic organs of moribund mice were analyzed for lineage-specific cell surface markers by flow cytometry.
Results
Strikingly, Npm1c Flt3-D835Y mice rapidly developed a myeloproliferative neoplasia and succumbed after a median latency of 33 days. In contrast, Flt3-D835Y Npm wt control mice did not develop a leukemic disease. Detailed analysis Npm1c Flt3-D835Y mice revealed a high leukemic burden in the peripheral blood as well as in the bone marrow and a pronounced splenomegaly. Flow cytometry analysis revealed a myeloid phenotype in the peripheral blood, spleen and bone marrow of moribund mice, characterized by a high frequency of FLT3-D835Y+/CD11b+ and Gr1+ cells, whereas Flt3-D835Y control mice showed a normal immune phenotype. Interestingly, immunoblot analysis of spleen extracts from moribund mice indicated that Flt3-D835Y in combination with NPM1c induces STAT5 but not STAT3 signaling. These results suggest that the MPN induction in this model might be due to STAT5 activation.Previously, it has been shown that the myeloid leukemia of Npm1c Flt3-ITD mice is retransplantable. To discover whether the myeloid phenotype of NPM1c Flt3-D835Y mice is retransplantable as well, 1x105 and 1x106 spleen cells of an Npm1 Flt3-D835Y mouse were injected into sublethally irradiated C57Bl/6 wt recipient mice. Indeed, we were able to show retranplantability of Npm1c Flt3-TKD disease in the group transplanted with high cell numbers, whereas the retranplantability was not only partial inducible (15-30% of the mice) in the group transplanted with lower spleen cell numbers (1x105).
Conclusion
In summary, our data show that co-expression of NPM1c and FLT3-D835Y results in the development of a rapidly fatal MPN whereas FLT3-D835Y-expression alone is not sufficient to cause a myeloproliferative disease in C57Bl/6 mice. Moreover, the myeloid phenotype is retransplantable with 1x106 donor spleen cells in C57Bl/6 mice. Interestingly, myeloproliferative disease induction might be due to aberrant STAT5 activation by FLT3-D835Y in the background of NPM1c mutant.
Session topic: AML Biology Mutant FLT
Keyword(s): FLT3, Mouse model
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:45 - 12:00
Location: Hall C13
Background
Activating mutations in FLT3 and NPM1 are most common alterations in AML and frequently coincidental. The exon 12 mutation of Npm1 (NPM1c) leads to a mislocalization of the protein from the nucleus to the cytoplasm. Two types of mutations are present in FLT3: Tandem duplication of the juxtamembraneous domain (ITD) and point mutations of the tyrosine kinase domain (TKD). In murine models, both NPM1c and FLT3-ITD induce an MPN, while FLT3-TKD leads to a lymphoid disorder. Co-expression of NPM1c and FLT3-ITD rapidly induces an AML in mice.
Aims
In the present study, we investigated the impact of co-expression of NPM1c with the TKD mutation FLT3-D835Y in a mouse model.
Methods
For this purpose a heterozygous conditional Npm1c knockin mouse model was used where Npm1c can be induced via the Mx1Cre-system. Donor bone marrow was harvested from these mice and cells were retrovirally infected with the MiG-Flt3-D835Y vector. Infected bone marrow cells were injected into lethally irradiated C57Bl/6 wt recipient mice. The Npm1c expression was induced by p(I:C) injection post transplantation resulting in the activation of IFN-α and thereby Cre recombinase induction under the control of the Mx1 promotor. Control mice received no p(I:C) injection. Lymphatic/Leukemic organs of moribund mice were analyzed for lineage-specific cell surface markers by flow cytometry.
Results
Strikingly, Npm1c Flt3-D835Y mice rapidly developed a myeloproliferative neoplasia and succumbed after a median latency of 33 days. In contrast, Flt3-D835Y Npm wt control mice did not develop a leukemic disease. Detailed analysis Npm1c Flt3-D835Y mice revealed a high leukemic burden in the peripheral blood as well as in the bone marrow and a pronounced splenomegaly. Flow cytometry analysis revealed a myeloid phenotype in the peripheral blood, spleen and bone marrow of moribund mice, characterized by a high frequency of FLT3-D835Y+/CD11b+ and Gr1+ cells, whereas Flt3-D835Y control mice showed a normal immune phenotype. Interestingly, immunoblot analysis of spleen extracts from moribund mice indicated that Flt3-D835Y in combination with NPM1c induces STAT5 but not STAT3 signaling. These results suggest that the MPN induction in this model might be due to STAT5 activation.Previously, it has been shown that the myeloid leukemia of Npm1c Flt3-ITD mice is retransplantable. To discover whether the myeloid phenotype of NPM1c Flt3-D835Y mice is retransplantable as well, 1x105 and 1x106 spleen cells of an Npm1 Flt3-D835Y mouse were injected into sublethally irradiated C57Bl/6 wt recipient mice. Indeed, we were able to show retranplantability of Npm1c Flt3-TKD disease in the group transplanted with high cell numbers, whereas the retranplantability was not only partial inducible (15-30% of the mice) in the group transplanted with lower spleen cell numbers (1x105).
Conclusion
In summary, our data show that co-expression of NPM1c and FLT3-D835Y results in the development of a rapidly fatal MPN whereas FLT3-D835Y-expression alone is not sufficient to cause a myeloproliferative disease in C57Bl/6 mice. Moreover, the myeloid phenotype is retransplantable with 1x106 donor spleen cells in C57Bl/6 mice. Interestingly, myeloproliferative disease induction might be due to aberrant STAT5 activation by FLT3-D835Y in the background of NPM1c mutant.
Session topic: AML Biology Mutant FLT
Keyword(s): FLT3, Mouse model
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