Telomere dysfunction drives aberrant hematopoietic differentiation and myelodysplastic syndrome

Abstract

Myelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres, but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including SRSF2. RNA-seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling, and histone modification, which are also enriched in mouse CMP haploinsufficient for SRSF2 and in CD34(+) CMML patient cells harboring SRSF2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing, and myeloid progenitor differentiation.

Figure 1. The hematopoietic compartment of telomere dysfunctional mice recapitulates hallmark features of human myelodysplastic syndrome

(A) Complete blood count evaluation of age-matched young (3 month old) and old (7 month old) mice of indicated genotypes (top panel) (error bars denote s.e.m). Relative abundance of neutrophils and lymphocytes in total white blood cells of young and old mice of indicated genotypes (bottom panel) (error bars denote s.e.m). (B) H&E stained sections of BM biopsies of representative G0 (left panel) and G5 (right panel) mice (scale bar, 50 μm). (C) Pattern of multilineage differentiation in a representative G0 BM cytospin (left panel); Dysplastic neutrophil (N), erythroblast (E) and megakaryocyte (M) in a representative G5 BM cytospin (central and right panel) (scale bar, 15 μm). (D) MPO (top, panel) and butyrate esterase (bottom panel) cytochemical staining of a representative G0 (on the left) or G5 (on the right) BM cytospin. Arrows indicate positive blasts (scale bar, 15 μm). (E) Pattern of multilineage differentiation in a representative G0 BM cytospin (left panel); increased number of BM blasts (> 20% of BM cellularity) in the BM cytospin of a representative G5 mouse in transformation (right panel) (scale bar, 15 μm). (F) H&E stained splenic section of a representative G0 mouse (top, left panel) or G5 mouse in transformation (top, right panel) (scale bar, 200 μm). Myeloid cells infiltrating the white-red pulp are positive for CD11b (bottom, left panel) and MPO (bottom, right panel) (scale bar, 15 μm). See also Figure S1.

Figure 2. Skewed myeloid-erythroid differentiation of CMP is reversed by telomerase reactivation

(A) KS⁻L frequency in the BM, as well as the CMP, GMP and MEP frequencies in the KS⁻L population of indicated genotypes and treatments (mean and s.e.m. of age-matched 3 month old mice from 6 independent experiments of telomerase reactivation in vivo; data are expressed as percentage of corresponding controls). (B) Anti-γH2AX immunofluorescence in CMP sorted from mice of indicated genotypes and treatments: numbers of γH2AX foci per cell (upper panel; error bars denote s.d); representative images (bottom panel): α-γH2AX: green; DAPI: blue; scale bar, 20 μm. (C) and (E) Frequency of the erythroid lineage in the BM (C, left panel) and spleen (E, upper panel) of indicated genotypes and treatments (mean and s.e.m of mice from 2 independent experiments of telomerase reactivation in vivo; data are expressed as percentage of corresponding controls); representative Ter119-stained sections of BM (C, right panel; scale bar, 100 μm) and spleen (E, bottom panel) from age-matched controls and experimental mice (scale bar, 200 μm). (D) H&E stained BM section of a representative G5 mouse without (left panel) or with OHT treatment (right panel; scale bar, 15 μm). See also Figure S2.

Figure 3. Defective CMP differentiation is due to cell intrinsic DNA damage signaling activation

(A) CMP, GMP and MEP frequencies in the KS⁻L population of G0 or G5 mice before and after HSC transplantation in wild type recipient mice (error bars denote s.e.m.; data are expressed as percentage of corresponding controls). (B) Representative BM cytospin of one recipient mouse transplanted with G5 HSCs (on the left). Arrows indicate blastic cells. Massive infiltration of CD11b positive myeloid precursors into the splenic white-red pulp architecture of a recipient mouse transplanted with G5 HSCs (on the right) (scale bar, 100 μm). (C) Clonogenic myeloid colony formation in methylcellulose from sorted CMP of indicated genotypes cultured in the presence of vehicle or OHT. Erythroid cells were scored by benzidine staining and expressed as frequency of the total number of colonies (mean and s.e.m of replicates from 6 experiments of telomerase reactivation in vitro; each experiment includes equal number of CMP sorted from independent mice of indicated genotypes; data are expressed as percentage of G0 control). (D) Clonogenic myeloid colony formation in methylcellulose from sorted CMP of indicated genotypes pre-treated with vehicle, ATR inhibitor (ATR-I, 1 μM) or ATM inhibitor (ATM-I, 2 μM) for 1 hr. Erythroid cells were scored by benzidine staining and expressed as frequency of the total number of colonies (mean and s.e.m of replicates from 3 independent experiments, each experiment includes equal number of CMP sorted from independent mice; data are expressed as percentage of G0 control). (E) Clonogenic myeloid colony formation in methylcellulose from sorted wild type CMP isolated from control or irradiated (IR, 3 Gy) mice (left panel), or pre-treated with vehicle or cisplatin (5 μM) for 4 hr (right panel). Erythroid cells were scored by benzidine staining and expressed as frequency of the total number of colonies (mean and s.e.m of replicates from 3 and 2 independent experiments, respectively; each experiment includes CMP sorted from a pool of three wild type mice; data are expressed as percentage of corresponding controls). (F) Clonogenic myeloid colony formation in methylcellulose from sorted CMP isolated from 9 month-old wild type mice 4 months after sub-lethal irradiation. Control mice indicate age-matched wild type mice without irradiation. Erythroid cells were scored by benzidine staining and expressed as frequency of the total number of colonies (mean and s.e.m of replicates from 4 or 5 mice for each condition; equal number of CMP was sorted from independent mice; data are expressed as percentage of control). See also Figure S3.

Figure 4. Telomere dysfunction induces aberrant RNA splicing by repressing splicing gene expression in CMP

(A) Significantly downregulated and upregulated pathways identified by GSEA in G4/G5 compared to G0 CMP (FDR =0.05). (B) Fluidigm-based gene expression analysis of single cells (rows) for representative genes in the mRNA processing/ spliceosome pathways (columns) from GSEA, which are significantly altered in sorted CMP from the G0 and G5 mice with or without OHT treatment (n=3). Genes analyzed were (from left to right): ACTB, β2M, GAPDH (housekeeping genes; internal controls), U2AF2, SF3B2, SF3A3, SRSF2, SFPQ, SFRS10, SFRS2IP, CDC51, DDX46, WBP11, SMC1A, PAPOLA, SRRM1, FUS, RBM5 and NUP54. A full gene list is shown in Table S2. Color scale on the right shows correspondence between color code and Ct values. (C) Interaction network of splicing genes significantly downregulated in G4/G5 CMP (blue color). Size of the nodes is proportional to the number of interactions of a given protein with other splicing components. * indicates splicing factors mutated in MDS. (D) Fluidigm-based gene expression analysis of single cells (rows) for representative genes in the mRNA processing / spliceosome pathways (columns), which are significantly altered in sorted CMP from the G0 and G5 mice with or without ATR inhibitor treatment (n=2 or 3 mice for each condition). Genes analyzed were (from left to right): ACTB, β2M, GAPDH (housekeeping genes; internal controls), U2AF2, SF3B2, SF3A3, SRSF2, SFPQ, SFRS10, SFRS2IP, CDC51, DDX46, PAPOLA, SRRM1 and RBM5. Color scale on the right shows correspondence between color code and Ct values. (E) Significantly enriched pathways relative to the 1,940 aberrantly spliced genes (p <0.05). See also Figure S4, Table S1, S2, S3, S4 and S5.

Figure 5. SRSF2 haploinsufficiency induces skewed myeloid differentiation of CMP

(A) Dysplastic monolobated and hyperlobated (upper panel, on the left) or multinucleated (upper panel, on the right) megakaryocytes (M), dysplastic erythroblast (E, bottom panel on the left) and abnormal (bottom panel, in the middle) and mitotic (bottom panel, on the right) blasts (B) in a representative Vav-cre/ SRSF2^L/+ BM cytospin (scale bar, 15 μm). (B) Butyrate esterase cytochemical staining of a representative Vav-cre (upper panel, on the left) or Vav-cre/ SRSF2^L/+ BM cytospin (upper panel, on the right). CD11b stained sections of BM biopsies of representative Vav-cre (bottom panel on the left) and Vav-cre/ SRSF2^L/+ (bottom panel on the right) mice (scale bar, 15 μm). (C) KS⁻L frequency in the BM, as well as the CMP, GMP and MEP frequencies in the KS⁻L compartment of 2 month old Vav-cre (n=5) or Vav-cre/ SRSF2^L/+ (n=5) mice (error bars denote s.e.m.; data are expressed as percentage of the Vav-cre control). (D) Clonogenic myeloid colony formation in methylcellulose from CMP sorted from Vav-cre or Vav-cre/ SRSF2^L/+ mice. Erythroid cells were scored by benzidine staining and expressed as frequency of the total number of colonies (mean and s.e.m of replicates from 5 independent mice; data are expressed as percentage of Vav-cre control). See also Figure S5.

Figure 6. Aberrant RNA splicing due to altered SRSF2 function induces telomere dysfunction

(A) Significantly enriched pathways relative to the 1,357 aberrantly spliced genes (p <0.05) in the Vav-cre/ SRSF2^L/+ mice. (B) Telomere-FISH and anti-γH2AX immunofluorescence in CMP sorted from 5 month-old mice of indicated genotypes (telomere: red; anti-γH2AX: green; co-localization: yellow; n=5 Vav-cre and n=7 Vav-cre/ SRSF2^L/+); numbers of telomere dysfunction-induced foci per cell (left panel) (error bars denote s.e.m.); representative images (right panel) (scale bar, 10 μm). (C) Mean value of telomere length in primary BM cells of 5 month old mice of indicated genotypes, as determined by flow-FISH analysis (error bars denote s.e.m.; data are expressed as percentage of the Vav-cre control). (D) Significantly enriched pathways relative to the 1,355 aberrantly spliced genes (p <0.01) in CMML patients with SRSF2 (P95) mutation. See also Figure S6, Table S6 and S7