A SOMATIC MUTATION OF GFI1B IDENTIFIED IN LEUKEMIA ALTERS CELL FATE VIA A SPI1 (PU.1) CENTERED GENETIC REGULATORY NETWORK
(Abstract release date: 05/19/16)
EHA Library. Anguita E. 06/10/16; 133165; P177
Dr. Eduardo Anguita
Contributions
Contributions
Abstract
Abstract: P177
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
Establishing a link between leukemia mutations and the malignant process requires functional assessment of their biological impact in the context of appropriate normal and malignant primary cells. Against this backdrop, we have identified a mutation (D262N) in the erythroid/megakaryocytic affliated transcriptional repressor GFI1B in acute myeloid leukemia (AML) and explored its biological properties.
Aims
Establishing a link between a GFI1B mutation and leukemia in the context of normal and malignant primary cells.
Methods
Human Material: Human material and clinical information was obtained with informed consent and approval of institutional ethics committee from 69 patients with AMLPoint mutation analysis: GFI1B coding regions were analyzed by DHPLC. Next generation sequencing performed with Hiseq2000 (Illumina) allowed the identification of a novel mutation.Cloning: GFI1B cDNA was amplified, cloned in pGEM-T easy vector and sequenced. Point mutation was introduced by PCR. Wild-type or mutated coding cDNA was cloned into pHRSINCSGW lentivirus vector, expressing green fluorescent protein (GFP).The SPI1 hairpin was cloned into the SLX vector, with or without wild-type or mutant GFI1B.Enrichment of hematopoietic stem and progenitors: Enrichment of human CD34+ cells from mobilized peripheral blood was by magnetic selection (Miltenyi Biotec). Transduced GFP/CD34 positive cells were sorted.Methylcellulose Colony-Forming Cell (CFC) assays: CFC assays were performed with MethoCult H4436 (StemCell Technologies), 15 days.Liquid differentiation assays: CD34+ cells expressing mutant or wild type GFI1B were maintained under liquid culture conditions that support multi-lineage differentiation: Myelocult® H5100 with human cytokines, SCF, FLT3L (50 ng/ml); IL-3, GM-CSF, M-CSF (10 ng/ml); G-CSF (0.1 MUI); EPO (100 ng/ml) for 5-6 days.MDS cell culture: CD34+ cells were cultured in MyeloCult® H5100 with 10% (v/v) HS-5 conditioned medium, human SCF and IL-3 (both 50 ng/ml).AML patients gene expression profiling and data analysis: This has been published (www.ncbi.nlm.nih.gov/geo, GSE1159).
Results
We identified a GFI1B new mutation (D262N) in an AML patient with antecedent myelodysplastic syndrome (MDS). The GFI1B-D262N mutant functionally antagonizes the transcriptional activity of wild-type GFI1B. GFI1B-D262N promoted myelomonocytic versus erythroid output from primary human hematopoietic precursors and enhanced cell survival of both normal and MDS derived human precursors. Re-analysis of 285 AMLs transcriptome data revealed a significant subset of patients in which expression of wild-type GFI1B was inversely correlated with that of SPI1 (PU.1). In delineating this GFI1B-SPI1 relationship we show (I) SPI1 is a direct target of GFI1B, (II) expression of GFI1B-D262N produces elevated expression of SPI1, (III) SPI1-knockdown restores balanced lineage output from GFI1B-D262N-expressing precursors, (IV) we also observed that GFI1B produced an increase in MLLT3, while expression of mutant GFI1B reduced MLLT3 expression.
Conclusion
In conclusion, our data links GFI1B to both the myeloid and erythroid transcriptional networks by repressing SPI1 and increasing MLLT3 expression, helping to understand its role in lineage specification and its potential in promoting blood malignancy. In fact, our clinical findings and experimental data show that the GFI1B D262N mutant plays a role in AML in humans and does so primarily through the agency of master transcriptional regulator SPI1, reflecting GFI1B physiological function in SPI1 regulation.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Acute myeloid leukemia, Bone marrow-derived stem cell, Gene regulation, PU.1
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
Establishing a link between leukemia mutations and the malignant process requires functional assessment of their biological impact in the context of appropriate normal and malignant primary cells. Against this backdrop, we have identified a mutation (D262N) in the erythroid/megakaryocytic affliated transcriptional repressor GFI1B in acute myeloid leukemia (AML) and explored its biological properties.
Aims
Establishing a link between a GFI1B mutation and leukemia in the context of normal and malignant primary cells.
Methods
Human Material: Human material and clinical information was obtained with informed consent and approval of institutional ethics committee from 69 patients with AMLPoint mutation analysis: GFI1B coding regions were analyzed by DHPLC. Next generation sequencing performed with Hiseq2000 (Illumina) allowed the identification of a novel mutation.Cloning: GFI1B cDNA was amplified, cloned in pGEM-T easy vector and sequenced. Point mutation was introduced by PCR. Wild-type or mutated coding cDNA was cloned into pHRSINCSGW lentivirus vector, expressing green fluorescent protein (GFP).The SPI1 hairpin was cloned into the SLX vector, with or without wild-type or mutant GFI1B.Enrichment of hematopoietic stem and progenitors: Enrichment of human CD34+ cells from mobilized peripheral blood was by magnetic selection (Miltenyi Biotec). Transduced GFP/CD34 positive cells were sorted.Methylcellulose Colony-Forming Cell (CFC) assays: CFC assays were performed with MethoCult H4436 (StemCell Technologies), 15 days.Liquid differentiation assays: CD34+ cells expressing mutant or wild type GFI1B were maintained under liquid culture conditions that support multi-lineage differentiation: Myelocult® H5100 with human cytokines, SCF, FLT3L (50 ng/ml); IL-3, GM-CSF, M-CSF (10 ng/ml); G-CSF (0.1 MUI); EPO (100 ng/ml) for 5-6 days.MDS cell culture: CD34+ cells were cultured in MyeloCult® H5100 with 10% (v/v) HS-5 conditioned medium, human SCF and IL-3 (both 50 ng/ml).AML patients gene expression profiling and data analysis: This has been published (www.ncbi.nlm.nih.gov/geo, GSE1159).
Results
We identified a GFI1B new mutation (D262N) in an AML patient with antecedent myelodysplastic syndrome (MDS). The GFI1B-D262N mutant functionally antagonizes the transcriptional activity of wild-type GFI1B. GFI1B-D262N promoted myelomonocytic versus erythroid output from primary human hematopoietic precursors and enhanced cell survival of both normal and MDS derived human precursors. Re-analysis of 285 AMLs transcriptome data revealed a significant subset of patients in which expression of wild-type GFI1B was inversely correlated with that of SPI1 (PU.1). In delineating this GFI1B-SPI1 relationship we show (I) SPI1 is a direct target of GFI1B, (II) expression of GFI1B-D262N produces elevated expression of SPI1, (III) SPI1-knockdown restores balanced lineage output from GFI1B-D262N-expressing precursors, (IV) we also observed that GFI1B produced an increase in MLLT3, while expression of mutant GFI1B reduced MLLT3 expression.
Conclusion
In conclusion, our data links GFI1B to both the myeloid and erythroid transcriptional networks by repressing SPI1 and increasing MLLT3 expression, helping to understand its role in lineage specification and its potential in promoting blood malignancy. In fact, our clinical findings and experimental data show that the GFI1B D262N mutant plays a role in AML in humans and does so primarily through the agency of master transcriptional regulator SPI1, reflecting GFI1B physiological function in SPI1 regulation.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Acute myeloid leukemia, Bone marrow-derived stem cell, Gene regulation, PU.1
Abstract: P177
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
Establishing a link between leukemia mutations and the malignant process requires functional assessment of their biological impact in the context of appropriate normal and malignant primary cells. Against this backdrop, we have identified a mutation (D262N) in the erythroid/megakaryocytic affliated transcriptional repressor GFI1B in acute myeloid leukemia (AML) and explored its biological properties.
Aims
Establishing a link between a GFI1B mutation and leukemia in the context of normal and malignant primary cells.
Methods
Human Material: Human material and clinical information was obtained with informed consent and approval of institutional ethics committee from 69 patients with AMLPoint mutation analysis: GFI1B coding regions were analyzed by DHPLC. Next generation sequencing performed with Hiseq2000 (Illumina) allowed the identification of a novel mutation.Cloning: GFI1B cDNA was amplified, cloned in pGEM-T easy vector and sequenced. Point mutation was introduced by PCR. Wild-type or mutated coding cDNA was cloned into pHRSINCSGW lentivirus vector, expressing green fluorescent protein (GFP).The SPI1 hairpin was cloned into the SLX vector, with or without wild-type or mutant GFI1B.Enrichment of hematopoietic stem and progenitors: Enrichment of human CD34+ cells from mobilized peripheral blood was by magnetic selection (Miltenyi Biotec). Transduced GFP/CD34 positive cells were sorted.Methylcellulose Colony-Forming Cell (CFC) assays: CFC assays were performed with MethoCult H4436 (StemCell Technologies), 15 days.Liquid differentiation assays: CD34+ cells expressing mutant or wild type GFI1B were maintained under liquid culture conditions that support multi-lineage differentiation: Myelocult® H5100 with human cytokines, SCF, FLT3L (50 ng/ml); IL-3, GM-CSF, M-CSF (10 ng/ml); G-CSF (0.1 MUI); EPO (100 ng/ml) for 5-6 days.MDS cell culture: CD34+ cells were cultured in MyeloCult® H5100 with 10% (v/v) HS-5 conditioned medium, human SCF and IL-3 (both 50 ng/ml).AML patients gene expression profiling and data analysis: This has been published (www.ncbi.nlm.nih.gov/geo, GSE1159).
Results
We identified a GFI1B new mutation (D262N) in an AML patient with antecedent myelodysplastic syndrome (MDS). The GFI1B-D262N mutant functionally antagonizes the transcriptional activity of wild-type GFI1B. GFI1B-D262N promoted myelomonocytic versus erythroid output from primary human hematopoietic precursors and enhanced cell survival of both normal and MDS derived human precursors. Re-analysis of 285 AMLs transcriptome data revealed a significant subset of patients in which expression of wild-type GFI1B was inversely correlated with that of SPI1 (PU.1). In delineating this GFI1B-SPI1 relationship we show (I) SPI1 is a direct target of GFI1B, (II) expression of GFI1B-D262N produces elevated expression of SPI1, (III) SPI1-knockdown restores balanced lineage output from GFI1B-D262N-expressing precursors, (IV) we also observed that GFI1B produced an increase in MLLT3, while expression of mutant GFI1B reduced MLLT3 expression.
Conclusion
In conclusion, our data links GFI1B to both the myeloid and erythroid transcriptional networks by repressing SPI1 and increasing MLLT3 expression, helping to understand its role in lineage specification and its potential in promoting blood malignancy. In fact, our clinical findings and experimental data show that the GFI1B D262N mutant plays a role in AML in humans and does so primarily through the agency of master transcriptional regulator SPI1, reflecting GFI1B physiological function in SPI1 regulation.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Acute myeloid leukemia, Bone marrow-derived stem cell, Gene regulation, PU.1
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
Establishing a link between leukemia mutations and the malignant process requires functional assessment of their biological impact in the context of appropriate normal and malignant primary cells. Against this backdrop, we have identified a mutation (D262N) in the erythroid/megakaryocytic affliated transcriptional repressor GFI1B in acute myeloid leukemia (AML) and explored its biological properties.
Aims
Establishing a link between a GFI1B mutation and leukemia in the context of normal and malignant primary cells.
Methods
Human Material: Human material and clinical information was obtained with informed consent and approval of institutional ethics committee from 69 patients with AMLPoint mutation analysis: GFI1B coding regions were analyzed by DHPLC. Next generation sequencing performed with Hiseq2000 (Illumina) allowed the identification of a novel mutation.Cloning: GFI1B cDNA was amplified, cloned in pGEM-T easy vector and sequenced. Point mutation was introduced by PCR. Wild-type or mutated coding cDNA was cloned into pHRSINCSGW lentivirus vector, expressing green fluorescent protein (GFP).The SPI1 hairpin was cloned into the SLX vector, with or without wild-type or mutant GFI1B.Enrichment of hematopoietic stem and progenitors: Enrichment of human CD34+ cells from mobilized peripheral blood was by magnetic selection (Miltenyi Biotec). Transduced GFP/CD34 positive cells were sorted.Methylcellulose Colony-Forming Cell (CFC) assays: CFC assays were performed with MethoCult H4436 (StemCell Technologies), 15 days.Liquid differentiation assays: CD34+ cells expressing mutant or wild type GFI1B were maintained under liquid culture conditions that support multi-lineage differentiation: Myelocult® H5100 with human cytokines, SCF, FLT3L (50 ng/ml); IL-3, GM-CSF, M-CSF (10 ng/ml); G-CSF (0.1 MUI); EPO (100 ng/ml) for 5-6 days.MDS cell culture: CD34+ cells were cultured in MyeloCult® H5100 with 10% (v/v) HS-5 conditioned medium, human SCF and IL-3 (both 50 ng/ml).AML patients gene expression profiling and data analysis: This has been published (www.ncbi.nlm.nih.gov/geo, GSE1159).
Results
We identified a GFI1B new mutation (D262N) in an AML patient with antecedent myelodysplastic syndrome (MDS). The GFI1B-D262N mutant functionally antagonizes the transcriptional activity of wild-type GFI1B. GFI1B-D262N promoted myelomonocytic versus erythroid output from primary human hematopoietic precursors and enhanced cell survival of both normal and MDS derived human precursors. Re-analysis of 285 AMLs transcriptome data revealed a significant subset of patients in which expression of wild-type GFI1B was inversely correlated with that of SPI1 (PU.1). In delineating this GFI1B-SPI1 relationship we show (I) SPI1 is a direct target of GFI1B, (II) expression of GFI1B-D262N produces elevated expression of SPI1, (III) SPI1-knockdown restores balanced lineage output from GFI1B-D262N-expressing precursors, (IV) we also observed that GFI1B produced an increase in MLLT3, while expression of mutant GFI1B reduced MLLT3 expression.
Conclusion
In conclusion, our data links GFI1B to both the myeloid and erythroid transcriptional networks by repressing SPI1 and increasing MLLT3 expression, helping to understand its role in lineage specification and its potential in promoting blood malignancy. In fact, our clinical findings and experimental data show that the GFI1B D262N mutant plays a role in AML in humans and does so primarily through the agency of master transcriptional regulator SPI1, reflecting GFI1B physiological function in SPI1 regulation.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Acute myeloid leukemia, Bone marrow-derived stem cell, Gene regulation, PU.1
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