SF3B1 MUTATIONS IN MDS-RS ARISE IN MULTIPOTENT HEMOPOETIC STEM CELLS
(Abstract release date: 05/19/16)
EHA Library. Mortera-Blanco T. 06/11/16; 135201; S445
Dr. Teresa Mortera-Blanco
Contributions
Contributions
Abstract
Abstract: S445
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:15 - 12:30
Location: Hall C11
Background
Mutations in the RNA splicing gene SF3B1 are found in more than 80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS), and predict for a stable clinical course and a favourable survival (Malcovati L. et al, Blood 2015 126(2):233-41). Little is known about the cellular origin of these mutations or their hierarchy in relation to other somatic mutations.
Aims
To establish the origin of SF3B1 mutations within the bone marrow (BM) hematopoietic stem and progenitor cell compartment (HSPC) in MDS-RS.
Methods
We investigated BM mononuclear cells (MNC) from 13 MDS-RS patients (6 RARS and 7 RCMD-RS) and 4 healthy controls for mutational spectrum by targeted sequencing, and quantified HSPC subsets using flow cytometry. Functional analysis of the purified populations was performed in vivo, using xenotransplantation in NOD scid gamma (NSG) mice to establish repopulating and self-renewal ability, and ex vivo using clonogenic progenitor assays: colony forming-unit (CFU) and long-term culture initiating cell (LTC-IC). Hierarchy of molecularly and functionally distinct stem and progenitors cells was established using pyrosequencing.
Results
Screening for recurrently mutated genes in the MNC fractions revealed mutations in SF3B1 in 12/13 cases, combined with TET2 and DNMT3A in 4 and 2 patients, respectively. The frequencies of phenotypically defined MDS-RS HSPC in the BM did not differ from that of normal controls, whereas pro-B cells were significantly reduced (p<0.005) in MDS-RS. We tracked back all the mutations identified in the MNC fractions to each purified MDS-RS stem and progenitor population, and in all LTC-IC and CFU colonies generated in vitro. Importantly, we identified the SF3B1 mutation at the BM pro-B level in 5 investigated patients, and also in a smaller fraction, in purified peripheral blood B-cells from these patients. Analysis of peripheral blood B cells from the same patients 2-3 years after the first sampling showed an increase in SF3B1 allelic burden in all cases. Importantly, co-mutations including DNMT3A and B-COR were not detected in peripheral B cells. This demonstrates that the origin of SF3B1 mutation arises in the very primitive lymphoid-myeloid multipotent stem cell compartment.We transplanted purified hematopoietic stem cells (HSC) and lineage restricted cell populations: CMPs, GMPs and MEPs from 4 SF3B1 mutated MDS-RS patients into NSG mice; 2 of which also carried TET2 mutations. Engraftment, as defined by (hCD45+CD19+, hCD45+CD33+CD66b+CD15+ or GPA+CD71+) of all four HSC samples was detected, while all more mature cell populations failed to engraft. Importantly, morphological development of ring sideroblasts was observed in mice transplanted with HSCs from all 4 patients, proving that the self-renewal potential as well as commitment to the RS erythroid phenotype is restricted to MDS-RS HSC.
Conclusion
Our findings provide evidence of a multipotent stem cell origin of the SF3B1 mutation in MDS-RS patients and propose the existence of a mixed B-cell population formed by abnormal cells derived from the MDS-RS clone and a normal “pre-clonal” population. Stem cell potential and reproduction of the MDS-RS erythroid phenotype was restricted to MDS-RS stem cells only.
Session topic: Myelodysplastic syndromes - Biology
Keyword(s): MDS, Stem and progenitor cell
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:15 - 12:30
Location: Hall C11
Background
Mutations in the RNA splicing gene SF3B1 are found in more than 80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS), and predict for a stable clinical course and a favourable survival (Malcovati L. et al, Blood 2015 126(2):233-41). Little is known about the cellular origin of these mutations or their hierarchy in relation to other somatic mutations.
Aims
To establish the origin of SF3B1 mutations within the bone marrow (BM) hematopoietic stem and progenitor cell compartment (HSPC) in MDS-RS.
Methods
We investigated BM mononuclear cells (MNC) from 13 MDS-RS patients (6 RARS and 7 RCMD-RS) and 4 healthy controls for mutational spectrum by targeted sequencing, and quantified HSPC subsets using flow cytometry. Functional analysis of the purified populations was performed in vivo, using xenotransplantation in NOD scid gamma (NSG) mice to establish repopulating and self-renewal ability, and ex vivo using clonogenic progenitor assays: colony forming-unit (CFU) and long-term culture initiating cell (LTC-IC). Hierarchy of molecularly and functionally distinct stem and progenitors cells was established using pyrosequencing.
Results
Screening for recurrently mutated genes in the MNC fractions revealed mutations in SF3B1 in 12/13 cases, combined with TET2 and DNMT3A in 4 and 2 patients, respectively. The frequencies of phenotypically defined MDS-RS HSPC in the BM did not differ from that of normal controls, whereas pro-B cells were significantly reduced (p<0.005) in MDS-RS. We tracked back all the mutations identified in the MNC fractions to each purified MDS-RS stem and progenitor population, and in all LTC-IC and CFU colonies generated in vitro. Importantly, we identified the SF3B1 mutation at the BM pro-B level in 5 investigated patients, and also in a smaller fraction, in purified peripheral blood B-cells from these patients. Analysis of peripheral blood B cells from the same patients 2-3 years after the first sampling showed an increase in SF3B1 allelic burden in all cases. Importantly, co-mutations including DNMT3A and B-COR were not detected in peripheral B cells. This demonstrates that the origin of SF3B1 mutation arises in the very primitive lymphoid-myeloid multipotent stem cell compartment.We transplanted purified hematopoietic stem cells (HSC) and lineage restricted cell populations: CMPs, GMPs and MEPs from 4 SF3B1 mutated MDS-RS patients into NSG mice; 2 of which also carried TET2 mutations. Engraftment, as defined by (hCD45+CD19+, hCD45+CD33+CD66b+CD15+ or GPA+CD71+) of all four HSC samples was detected, while all more mature cell populations failed to engraft. Importantly, morphological development of ring sideroblasts was observed in mice transplanted with HSCs from all 4 patients, proving that the self-renewal potential as well as commitment to the RS erythroid phenotype is restricted to MDS-RS HSC.
Conclusion
Our findings provide evidence of a multipotent stem cell origin of the SF3B1 mutation in MDS-RS patients and propose the existence of a mixed B-cell population formed by abnormal cells derived from the MDS-RS clone and a normal “pre-clonal” population. Stem cell potential and reproduction of the MDS-RS erythroid phenotype was restricted to MDS-RS stem cells only.
Session topic: Myelodysplastic syndromes - Biology
Keyword(s): MDS, Stem and progenitor cell
Abstract: S445
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:15 - 12:30
Location: Hall C11
Background
Mutations in the RNA splicing gene SF3B1 are found in more than 80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS), and predict for a stable clinical course and a favourable survival (Malcovati L. et al, Blood 2015 126(2):233-41). Little is known about the cellular origin of these mutations or their hierarchy in relation to other somatic mutations.
Aims
To establish the origin of SF3B1 mutations within the bone marrow (BM) hematopoietic stem and progenitor cell compartment (HSPC) in MDS-RS.
Methods
We investigated BM mononuclear cells (MNC) from 13 MDS-RS patients (6 RARS and 7 RCMD-RS) and 4 healthy controls for mutational spectrum by targeted sequencing, and quantified HSPC subsets using flow cytometry. Functional analysis of the purified populations was performed in vivo, using xenotransplantation in NOD scid gamma (NSG) mice to establish repopulating and self-renewal ability, and ex vivo using clonogenic progenitor assays: colony forming-unit (CFU) and long-term culture initiating cell (LTC-IC). Hierarchy of molecularly and functionally distinct stem and progenitors cells was established using pyrosequencing.
Results
Screening for recurrently mutated genes in the MNC fractions revealed mutations in SF3B1 in 12/13 cases, combined with TET2 and DNMT3A in 4 and 2 patients, respectively. The frequencies of phenotypically defined MDS-RS HSPC in the BM did not differ from that of normal controls, whereas pro-B cells were significantly reduced (p<0.005) in MDS-RS. We tracked back all the mutations identified in the MNC fractions to each purified MDS-RS stem and progenitor population, and in all LTC-IC and CFU colonies generated in vitro. Importantly, we identified the SF3B1 mutation at the BM pro-B level in 5 investigated patients, and also in a smaller fraction, in purified peripheral blood B-cells from these patients. Analysis of peripheral blood B cells from the same patients 2-3 years after the first sampling showed an increase in SF3B1 allelic burden in all cases. Importantly, co-mutations including DNMT3A and B-COR were not detected in peripheral B cells. This demonstrates that the origin of SF3B1 mutation arises in the very primitive lymphoid-myeloid multipotent stem cell compartment.We transplanted purified hematopoietic stem cells (HSC) and lineage restricted cell populations: CMPs, GMPs and MEPs from 4 SF3B1 mutated MDS-RS patients into NSG mice; 2 of which also carried TET2 mutations. Engraftment, as defined by (hCD45+CD19+, hCD45+CD33+CD66b+CD15+ or GPA+CD71+) of all four HSC samples was detected, while all more mature cell populations failed to engraft. Importantly, morphological development of ring sideroblasts was observed in mice transplanted with HSCs from all 4 patients, proving that the self-renewal potential as well as commitment to the RS erythroid phenotype is restricted to MDS-RS HSC.
Conclusion
Our findings provide evidence of a multipotent stem cell origin of the SF3B1 mutation in MDS-RS patients and propose the existence of a mixed B-cell population formed by abnormal cells derived from the MDS-RS clone and a normal “pre-clonal” population. Stem cell potential and reproduction of the MDS-RS erythroid phenotype was restricted to MDS-RS stem cells only.
Session topic: Myelodysplastic syndromes - Biology
Keyword(s): MDS, Stem and progenitor cell
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:15 - 12:30
Location: Hall C11
Background
Mutations in the RNA splicing gene SF3B1 are found in more than 80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS), and predict for a stable clinical course and a favourable survival (Malcovati L. et al, Blood 2015 126(2):233-41). Little is known about the cellular origin of these mutations or their hierarchy in relation to other somatic mutations.
Aims
To establish the origin of SF3B1 mutations within the bone marrow (BM) hematopoietic stem and progenitor cell compartment (HSPC) in MDS-RS.
Methods
We investigated BM mononuclear cells (MNC) from 13 MDS-RS patients (6 RARS and 7 RCMD-RS) and 4 healthy controls for mutational spectrum by targeted sequencing, and quantified HSPC subsets using flow cytometry. Functional analysis of the purified populations was performed in vivo, using xenotransplantation in NOD scid gamma (NSG) mice to establish repopulating and self-renewal ability, and ex vivo using clonogenic progenitor assays: colony forming-unit (CFU) and long-term culture initiating cell (LTC-IC). Hierarchy of molecularly and functionally distinct stem and progenitors cells was established using pyrosequencing.
Results
Screening for recurrently mutated genes in the MNC fractions revealed mutations in SF3B1 in 12/13 cases, combined with TET2 and DNMT3A in 4 and 2 patients, respectively. The frequencies of phenotypically defined MDS-RS HSPC in the BM did not differ from that of normal controls, whereas pro-B cells were significantly reduced (p<0.005) in MDS-RS. We tracked back all the mutations identified in the MNC fractions to each purified MDS-RS stem and progenitor population, and in all LTC-IC and CFU colonies generated in vitro. Importantly, we identified the SF3B1 mutation at the BM pro-B level in 5 investigated patients, and also in a smaller fraction, in purified peripheral blood B-cells from these patients. Analysis of peripheral blood B cells from the same patients 2-3 years after the first sampling showed an increase in SF3B1 allelic burden in all cases. Importantly, co-mutations including DNMT3A and B-COR were not detected in peripheral B cells. This demonstrates that the origin of SF3B1 mutation arises in the very primitive lymphoid-myeloid multipotent stem cell compartment.We transplanted purified hematopoietic stem cells (HSC) and lineage restricted cell populations: CMPs, GMPs and MEPs from 4 SF3B1 mutated MDS-RS patients into NSG mice; 2 of which also carried TET2 mutations. Engraftment, as defined by (hCD45+CD19+, hCD45+CD33+CD66b+CD15+ or GPA+CD71+) of all four HSC samples was detected, while all more mature cell populations failed to engraft. Importantly, morphological development of ring sideroblasts was observed in mice transplanted with HSCs from all 4 patients, proving that the self-renewal potential as well as commitment to the RS erythroid phenotype is restricted to MDS-RS HSC.
Conclusion
Our findings provide evidence of a multipotent stem cell origin of the SF3B1 mutation in MDS-RS patients and propose the existence of a mixed B-cell population formed by abnormal cells derived from the MDS-RS clone and a normal “pre-clonal” population. Stem cell potential and reproduction of the MDS-RS erythroid phenotype was restricted to MDS-RS stem cells only.
Session topic: Myelodysplastic syndromes - Biology
Keyword(s): MDS, Stem and progenitor cell
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