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. 2017 Aug 17;130(7):881-890.
doi: 10.1182/blood-2017-03-776070. Epub 2017 Jun 20.

SF3B1-initiating Mutations in MDS-RSs Target Lymphomyeloid Hematopoietic Stem Cells

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Free PMC article

SF3B1-initiating Mutations in MDS-RSs Target Lymphomyeloid Hematopoietic Stem Cells

Teresa Mortera-Blanco et al. Blood. .
Free PMC article

Abstract

Mutations in the RNA splicing gene SF3B1 are found in >80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS). We investigated the origin of SF3B1 mutations within the bone marrow hematopoietic stem and progenitor cell compartments in patients with MDS-RS. Screening for recurrently mutated genes in the mononuclear cell fraction revealed mutations in SF3B1 in 39 of 40 cases (97.5%), combined with TET2 and DNMT3A in 11 (28%) and 6 (15%) patients, respectively. All recurrent mutations identified in mononuclear cells could be tracked back to the phenotypically defined hematopoietic stem cell (HSC) compartment in all investigated patients and were also present in downstream myeloid and erythroid progenitor cells. While in agreement with previous studies, little or no evidence for clonal (SF3B1 mutation) involvement could be found in mature B cells, consistent involvement at the pro-B-cell progenitor stage was established, providing definitive evidence for SF3B1 mutations targeting lymphomyeloid HSCs and compatible with mutated SF3B1 negatively affecting lymphoid development. Assessment of stem cell function in vitro as well as in vivo established that only HSCs and not investigated progenitor populations could propagate the SF3B1 mutated clone. Upon transplantation into immune-deficient mice, SF3B1 mutated MDS-RS HSCs differentiated into characteristic ring sideroblasts, the hallmark of MDS-RS. Our findings provide evidence of a multipotent lymphomyeloid HSC origin of SF3B1 mutations in MDS-RS patients and provide a novel in vivo platform for mechanistically and therapeutically exploring SF3B1 mutated MDS-RS.

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
Analysis of hematopoietic stem and progenitor cells in MDS-RS. (A) Representative FACS analysis of SF3B1-mutated MDS-RS BM (patient 4). Shown are FACS analyses of viable, lineage-negative, CD34-enriched cells further gated as CMPs (7AADLinCD34+CD38+CD90CD123+CD45RA), MEPs (7AADLinCD34+CD38+CD90CD123CD45RA), GMPs (7AADLinCD34+CD38+CD90CD123+CD45RA+), and HSCs (7AADLinCD34+CD38CD90+CD45RA). (B) Mean (standard error of the mean [SEM]) frequencies of HSCs and progenitors in SF3B1-mutated MDS-RS patients (n = 9) and healthy NBM (n = 4). (C) Mean (SEM) erythroid and myeloid colony formation from purified CMPs, GMPs, and MEPs from 8 MDS-RS patients and 4 NBMs. (D) Representative images of an erythroid colony (BFU-E) from an MDS-RS patient and a healthy NBM control. Original magnification for light microscopy ×10. (E) LTC-CFC activity in HSCs, CMPs, GMPs, and MEPs from MDS-RS patients (n = 8) and healthy NBM controls (n = 5). Results are mean (SEM) values for all patients and healthy controls, in each case derived from the mean of 3 technical replicates for each cell population and patient. **P < .005.
Figure 2.
Figure 2.
Evidence that SF3B1 mutations are initiating mutations targeting rare HSCs in MDS-RS. (A) Computational prediction of fraction of cells with identified genomic lesions within total BM MNCs from SF3B1-mutated MDS-RS patients based on VAF. Error bars indicate 95% confidence intervals. Shown are cases where SF3B1 is the only identified recurrent mutation, SF3B1 is predicted to be the first of multiple recurrent mutations, or SF3B1 is predicted to be secondary to other mutations. Inconclusive results (overlapping 95% confidence intervals) are included in supplemental Figure 2. (B) Tracking of SF3B1 mutations in individually picked HSC-derived LTC-CFCs or MEP- and GMP-derived CFCs. 7 SF3B1 mutated and 1 SF3B1 wild-type (WT) patients were analyzed using pyrosequencing to screen for identified SF3B1 mutations in each case and scored as positive (gray), negative (white), or inconclusive (I) (see supplemental Methods for definitions and cutoffs). (C) Sequenom analysis from 4 MDS-RS samples of FACS-purified stem and progenitor cell populations to assess SF3B1 VAF. PAT, patient.
Figure 3.
Figure 3.
SF3B1 mutations in lymphoid and myeloid progenitors in MDS-RS. (A) Representative FACS analysis of BM MNCs from healthy NBM and SF3B1-mutated MDS-RS patient 6, showing gating strategy for viable LinCD34+CD19+ pro–B cells. (B) Mean (SEM) frequencies of pro–B cells in SF3B1-mutated MDS-RS patients (n = 9) and healthy NBM (n = 4). (C) VAF of SF3B1 mutations in MDS-RS pro–B cells (MDS-RS PROB, n = 5) and mature PB B cells from MDS-RS (MDS-RS B cells, n = 3) compared with MDS-RS bulk BM MNCs (n = 5). Healthy NBM pro–B cells (NBM PROB, n = 4) were included as a negative control for pyrosequencing. PAT, patient. (D) Representative gating strategy for FACS purification of PB CD3+CD8+ T cells, CD19+ B cells, and CD33/66B/15+ myeloid cells from MDS-RS patient 4. (E) VAF of identified mutations in FACS-purified PB myeloid, B, and T cells from MDS-RS patient 4 at diagnosis and 16 months later using droplet digital PCR. Healthy normal DNA was negative, as indicated by the dotted line (<0.1%).
Figure 4.
Figure 4.
In vivo reconstitution of RSs from MDS-RS HSCs. (A) Representative FACS gating strategy used for analysis of human myeloid (CD33/CD66b/CD15+) and lymphoid (CD19+) engraftment in xenotransplanted nonobsese diabetic/LtSz-scid IL2Rγc−/− (NSG) mice transplanted with healthy or SF3B1-mutated MDS-RS human stem and progenitor cells. The number of purified HSCs, CMPs, GMPs, and MEPs transplanted into NSG mice was according to their relative ratios in the patient BM. Patient 4: 1400, 5000, 21 000, and 1400 cells; patient 5: 25 000, 55 000, 49 300, and 25 200 cells, respectively. Top panels show gating in a nontransplanted NSG mice (negative control); bottom panels show analysis in an NSG mouse transplanted with MDS-RS HSCs from patient 4. (B) In vivo human B-lymphoid and myeloid engraftment in BM of NSG mice 20 to 22 weeks posttransplantation of FACS-purified HSCs or indicated progenitors (mean values from 2 or 3 mice per cell population per patient) from 2 SF3B1-mutated MDS-RS. (C) Mean VAF in myeloid cells derived from patient 4 HSCs transplanted in NSG mice. (D) Prussian blue stains from sections of paraffin-embedded BM tissue from NSG mice with human reconstitution from healthy (top left) or SF3B1-mutated MDS-RS HSCs (top right; patient 5). The bottom left panel shows positive control from BM of MDS-RS patient; scale bar, 50 μm. The bottom right panel shows characteristic Prussian blue–positive cells in BM of NSG mice transplanted with MDS-RS HSCs from patient 5; scale bar, 10 μm. (E) Cytospins of erythroid cells purified from BM of patient 5 (supplemental Figure 6), stained with Prussian blue; scale bar, 20 μm. (F) Percentage of RSs of total nucleated BM cells in BM of NSG mice transplanted with purified HSCs. PAT, patient.

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