NPM1 AND FLT3-ITD MUTATIONS SINERGISTICALLY INDUCE GATA1 EPIGENETIC SILENCING IN A AML MOUSE MODEL
(Abstract release date: 05/19/16)
EHA Library. Sportoletti P. 06/11/16; 135213; S457
Dr. Paolo Sportoletti
Contributions
Contributions
Abstract
Abstract: S457
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:30 - 11:45
Location: Hall C13
Background
Mutations in NPM1 and FLT3-ITD are frequently associated in human AML and were also reported to induce leukemia in mice. However, the molecular consequences of this oncogenic cooperation in the development of AML still remain elusive.
Aims
We generated an NPM1/Flt3-ITD AML mouse model to address the following issues: i) to characterize the cellular origin of leukemia; ii) to identify the epigenetic/transcriptional signature in AML pathogenesis; iii) to analyze AML therapeutic response in vivo; and iv) to validate mouse data in human AML.
Methods
We crossed our NPM1 mutant and Flt3-ITD mice and used: 1) FACS analysis to study the HSC/progenitor cell compartments in bone marrow (BM); 2) real-time PCR and Western blot analysis to measure GATA1 mRNA and protein expression; 3) bisulfite conversion followed by DNA sequencing to study methylation; 4) 5-Aza-dC and the FLT3 inhibitor AC220 for treatment experiments.
Results
NPM1/Flt3-ITD double mutated mice progressed to AML recapitulating features of the human disease. The AML phenotype was preceded by a preleukemic stage of variable length that inversely correlated with the burden of the mutant alleles. Pre-leukemic mice displayed leukocytosis (WBC 20.8±18 x 109/L) and macrocytosis (MCV 58.74±5.6 fl). In NPM1/Flt3-ITD mice, FACS analysis revealed expansion of multipotent and granulocyte-macrophage progenitor compartments. Conversely, long-term hematopoietic stem cells and immature megakaryocytic/erythroid compartments were significantly reduced as compared to other genotypes combinations.Strikingly, we found that the key regulator of normal myelo-erythroid differentiation GATA1 was transcriptionally silenced in NPM1/Flt3-ITD mice. Similarly, AML patients with NPM1/FLT3-ITD mutations displayed a significantly lower mean GATA1 expression compared to patients with a different genotype (0.42±0.09 vs 5.3±1.3 respectively, p<0.05). Genomic bisulfite sequencing of the GATA1 promoter revealed more than 80%±0.12 methylated CpGs in BM cells from double mutated mice as compared to only 53%±0.22 methylation in other genotypes, thus clearly implicating epigenetic changes as the cause of GATA1 transcriptional silencing. In vivo treatment of leukemic mice with the DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-Aza-dC) reactivated GATA1 expression determining the normalization of leukocytosis and preventing a drop in platelets counts. Strikingly, real-time PCR analysis revealed a significant 3.45±1.01 upregulation of GATA1 mRNA levels in BM samples from NPM1/FLT3-ITD mutant AML patients that underwent 5-Aza-dC treatment (p<0.001).Finally, we assessed the sensitivity of NPM1/Flt3-ITD mice AML to targeted FLT3 inhibition. In vitro treatment with the FLT3 inhibitor AC220 significantly reduced the viability of BM cells isolated from NPM1/Flt3-ITD preleukemic or leukemic mice. In vivo, AC220 induced a reduction of WBC counts accompanied by a partial GATA1 re-expression in peripheral blood cells without improving mice survival. In a xenotransplant model of human NPM1/FLT3-ITD AML, the FLT3 inhibitor induced significant reduction in size of subcutaneous AML tumors with GATA1 upregulation.
Conclusion
We demonstrated that epigentic modification abrogated a GATA1-dependent transcriptional program and represented a prerequisite for NPM1/Flt3-ITD leukemogenesis. Mouse data are further supported by low GATA1 expression found in human AML samples and in vivo treatments rescues. Therefore, GATA1 represents a target for new tailored therapies in AML with NPM1 and FLT3-ITD mutations.
Session topic: AML Biology Mutant FLT
Keyword(s): Epigenetic, Flt3-ITD, GATA-1, Mouse model
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:30 - 11:45
Location: Hall C13
Background
Mutations in NPM1 and FLT3-ITD are frequently associated in human AML and were also reported to induce leukemia in mice. However, the molecular consequences of this oncogenic cooperation in the development of AML still remain elusive.
Aims
We generated an NPM1/Flt3-ITD AML mouse model to address the following issues: i) to characterize the cellular origin of leukemia; ii) to identify the epigenetic/transcriptional signature in AML pathogenesis; iii) to analyze AML therapeutic response in vivo; and iv) to validate mouse data in human AML.
Methods
We crossed our NPM1 mutant and Flt3-ITD mice and used: 1) FACS analysis to study the HSC/progenitor cell compartments in bone marrow (BM); 2) real-time PCR and Western blot analysis to measure GATA1 mRNA and protein expression; 3) bisulfite conversion followed by DNA sequencing to study methylation; 4) 5-Aza-dC and the FLT3 inhibitor AC220 for treatment experiments.
Results
NPM1/Flt3-ITD double mutated mice progressed to AML recapitulating features of the human disease. The AML phenotype was preceded by a preleukemic stage of variable length that inversely correlated with the burden of the mutant alleles. Pre-leukemic mice displayed leukocytosis (WBC 20.8±18 x 109/L) and macrocytosis (MCV 58.74±5.6 fl). In NPM1/Flt3-ITD mice, FACS analysis revealed expansion of multipotent and granulocyte-macrophage progenitor compartments. Conversely, long-term hematopoietic stem cells and immature megakaryocytic/erythroid compartments were significantly reduced as compared to other genotypes combinations.Strikingly, we found that the key regulator of normal myelo-erythroid differentiation GATA1 was transcriptionally silenced in NPM1/Flt3-ITD mice. Similarly, AML patients with NPM1/FLT3-ITD mutations displayed a significantly lower mean GATA1 expression compared to patients with a different genotype (0.42±0.09 vs 5.3±1.3 respectively, p<0.05). Genomic bisulfite sequencing of the GATA1 promoter revealed more than 80%±0.12 methylated CpGs in BM cells from double mutated mice as compared to only 53%±0.22 methylation in other genotypes, thus clearly implicating epigenetic changes as the cause of GATA1 transcriptional silencing. In vivo treatment of leukemic mice with the DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-Aza-dC) reactivated GATA1 expression determining the normalization of leukocytosis and preventing a drop in platelets counts. Strikingly, real-time PCR analysis revealed a significant 3.45±1.01 upregulation of GATA1 mRNA levels in BM samples from NPM1/FLT3-ITD mutant AML patients that underwent 5-Aza-dC treatment (p<0.001).Finally, we assessed the sensitivity of NPM1/Flt3-ITD mice AML to targeted FLT3 inhibition. In vitro treatment with the FLT3 inhibitor AC220 significantly reduced the viability of BM cells isolated from NPM1/Flt3-ITD preleukemic or leukemic mice. In vivo, AC220 induced a reduction of WBC counts accompanied by a partial GATA1 re-expression in peripheral blood cells without improving mice survival. In a xenotransplant model of human NPM1/FLT3-ITD AML, the FLT3 inhibitor induced significant reduction in size of subcutaneous AML tumors with GATA1 upregulation.
Conclusion
We demonstrated that epigentic modification abrogated a GATA1-dependent transcriptional program and represented a prerequisite for NPM1/Flt3-ITD leukemogenesis. Mouse data are further supported by low GATA1 expression found in human AML samples and in vivo treatments rescues. Therefore, GATA1 represents a target for new tailored therapies in AML with NPM1 and FLT3-ITD mutations.
Session topic: AML Biology Mutant FLT
Keyword(s): Epigenetic, Flt3-ITD, GATA-1, Mouse model
Abstract: S457
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:30 - 11:45
Location: Hall C13
Background
Mutations in NPM1 and FLT3-ITD are frequently associated in human AML and were also reported to induce leukemia in mice. However, the molecular consequences of this oncogenic cooperation in the development of AML still remain elusive.
Aims
We generated an NPM1/Flt3-ITD AML mouse model to address the following issues: i) to characterize the cellular origin of leukemia; ii) to identify the epigenetic/transcriptional signature in AML pathogenesis; iii) to analyze AML therapeutic response in vivo; and iv) to validate mouse data in human AML.
Methods
We crossed our NPM1 mutant and Flt3-ITD mice and used: 1) FACS analysis to study the HSC/progenitor cell compartments in bone marrow (BM); 2) real-time PCR and Western blot analysis to measure GATA1 mRNA and protein expression; 3) bisulfite conversion followed by DNA sequencing to study methylation; 4) 5-Aza-dC and the FLT3 inhibitor AC220 for treatment experiments.
Results
NPM1/Flt3-ITD double mutated mice progressed to AML recapitulating features of the human disease. The AML phenotype was preceded by a preleukemic stage of variable length that inversely correlated with the burden of the mutant alleles. Pre-leukemic mice displayed leukocytosis (WBC 20.8±18 x 109/L) and macrocytosis (MCV 58.74±5.6 fl). In NPM1/Flt3-ITD mice, FACS analysis revealed expansion of multipotent and granulocyte-macrophage progenitor compartments. Conversely, long-term hematopoietic stem cells and immature megakaryocytic/erythroid compartments were significantly reduced as compared to other genotypes combinations.Strikingly, we found that the key regulator of normal myelo-erythroid differentiation GATA1 was transcriptionally silenced in NPM1/Flt3-ITD mice. Similarly, AML patients with NPM1/FLT3-ITD mutations displayed a significantly lower mean GATA1 expression compared to patients with a different genotype (0.42±0.09 vs 5.3±1.3 respectively, p<0.05). Genomic bisulfite sequencing of the GATA1 promoter revealed more than 80%±0.12 methylated CpGs in BM cells from double mutated mice as compared to only 53%±0.22 methylation in other genotypes, thus clearly implicating epigenetic changes as the cause of GATA1 transcriptional silencing. In vivo treatment of leukemic mice with the DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-Aza-dC) reactivated GATA1 expression determining the normalization of leukocytosis and preventing a drop in platelets counts. Strikingly, real-time PCR analysis revealed a significant 3.45±1.01 upregulation of GATA1 mRNA levels in BM samples from NPM1/FLT3-ITD mutant AML patients that underwent 5-Aza-dC treatment (p<0.001).Finally, we assessed the sensitivity of NPM1/Flt3-ITD mice AML to targeted FLT3 inhibition. In vitro treatment with the FLT3 inhibitor AC220 significantly reduced the viability of BM cells isolated from NPM1/Flt3-ITD preleukemic or leukemic mice. In vivo, AC220 induced a reduction of WBC counts accompanied by a partial GATA1 re-expression in peripheral blood cells without improving mice survival. In a xenotransplant model of human NPM1/FLT3-ITD AML, the FLT3 inhibitor induced significant reduction in size of subcutaneous AML tumors with GATA1 upregulation.
Conclusion
We demonstrated that epigentic modification abrogated a GATA1-dependent transcriptional program and represented a prerequisite for NPM1/Flt3-ITD leukemogenesis. Mouse data are further supported by low GATA1 expression found in human AML samples and in vivo treatments rescues. Therefore, GATA1 represents a target for new tailored therapies in AML with NPM1 and FLT3-ITD mutations.
Session topic: AML Biology Mutant FLT
Keyword(s): Epigenetic, Flt3-ITD, GATA-1, Mouse model
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:30 - 11:45
Location: Hall C13
Background
Mutations in NPM1 and FLT3-ITD are frequently associated in human AML and were also reported to induce leukemia in mice. However, the molecular consequences of this oncogenic cooperation in the development of AML still remain elusive.
Aims
We generated an NPM1/Flt3-ITD AML mouse model to address the following issues: i) to characterize the cellular origin of leukemia; ii) to identify the epigenetic/transcriptional signature in AML pathogenesis; iii) to analyze AML therapeutic response in vivo; and iv) to validate mouse data in human AML.
Methods
We crossed our NPM1 mutant and Flt3-ITD mice and used: 1) FACS analysis to study the HSC/progenitor cell compartments in bone marrow (BM); 2) real-time PCR and Western blot analysis to measure GATA1 mRNA and protein expression; 3) bisulfite conversion followed by DNA sequencing to study methylation; 4) 5-Aza-dC and the FLT3 inhibitor AC220 for treatment experiments.
Results
NPM1/Flt3-ITD double mutated mice progressed to AML recapitulating features of the human disease. The AML phenotype was preceded by a preleukemic stage of variable length that inversely correlated with the burden of the mutant alleles. Pre-leukemic mice displayed leukocytosis (WBC 20.8±18 x 109/L) and macrocytosis (MCV 58.74±5.6 fl). In NPM1/Flt3-ITD mice, FACS analysis revealed expansion of multipotent and granulocyte-macrophage progenitor compartments. Conversely, long-term hematopoietic stem cells and immature megakaryocytic/erythroid compartments were significantly reduced as compared to other genotypes combinations.Strikingly, we found that the key regulator of normal myelo-erythroid differentiation GATA1 was transcriptionally silenced in NPM1/Flt3-ITD mice. Similarly, AML patients with NPM1/FLT3-ITD mutations displayed a significantly lower mean GATA1 expression compared to patients with a different genotype (0.42±0.09 vs 5.3±1.3 respectively, p<0.05). Genomic bisulfite sequencing of the GATA1 promoter revealed more than 80%±0.12 methylated CpGs in BM cells from double mutated mice as compared to only 53%±0.22 methylation in other genotypes, thus clearly implicating epigenetic changes as the cause of GATA1 transcriptional silencing. In vivo treatment of leukemic mice with the DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-Aza-dC) reactivated GATA1 expression determining the normalization of leukocytosis and preventing a drop in platelets counts. Strikingly, real-time PCR analysis revealed a significant 3.45±1.01 upregulation of GATA1 mRNA levels in BM samples from NPM1/FLT3-ITD mutant AML patients that underwent 5-Aza-dC treatment (p<0.001).Finally, we assessed the sensitivity of NPM1/Flt3-ITD mice AML to targeted FLT3 inhibition. In vitro treatment with the FLT3 inhibitor AC220 significantly reduced the viability of BM cells isolated from NPM1/Flt3-ITD preleukemic or leukemic mice. In vivo, AC220 induced a reduction of WBC counts accompanied by a partial GATA1 re-expression in peripheral blood cells without improving mice survival. In a xenotransplant model of human NPM1/FLT3-ITD AML, the FLT3 inhibitor induced significant reduction in size of subcutaneous AML tumors with GATA1 upregulation.
Conclusion
We demonstrated that epigentic modification abrogated a GATA1-dependent transcriptional program and represented a prerequisite for NPM1/Flt3-ITD leukemogenesis. Mouse data are further supported by low GATA1 expression found in human AML samples and in vivo treatments rescues. Therefore, GATA1 represents a target for new tailored therapies in AML with NPM1 and FLT3-ITD mutations.
Session topic: AML Biology Mutant FLT
Keyword(s): Epigenetic, Flt3-ITD, GATA-1, Mouse model
{{ help_message }}
{{filter}}