Setd2 deficiency impairs hematopoietic stem cell self-renewal and causes malignant transformation

Abstract

The histone H3 lysine 36 methyltransferase SETD2 is frequently mutated in various cancers, including leukemia. However, there has not been any functional model to show the contribution of SETD2 in hematopoiesis or the causal role of SETD2 mutation in tumorigenesis. In this study, using a conditional Setd2 knockout mouse model, we show that Setd2 deficiency skews hematopoietic differentiation and reduces the number of multipotent progenitors; although the number of phenotypic hematopoietic stem cells (HSCs) in Setd2-deleted mice is unchanged, functional assays, including serial BM transplantation, reveal that the self-renewal and competitiveness of HSCs are impaired. Intriguingly, Setd2-deleted HSCs, through a latency period, can acquire abilities to overcome the growth disadvantage and eventually give rise to hematopoietic malignancy characteristic of myelodysplastic syndrome. Gene expression profile of Setd2-deleted hematopoietic stem/progenitor cells (HSPCs) partially resembles that of Dnmt3a/Tet2 double knockout HSPCs, showing activation of the erythroid transcription factor Klf1-related pathway, which plays an important role in hematopoietic malignant transformation. Setd2 deficiency also induces DNA replication stress in HSCs, as reflected by an activated E2F gene regulatory network and repressed expression of the ribonucleotide reductase subunit Rrm2b, which results in proliferation and cell cycle abnormalities and genomic instability, allowing accumulation of secondary mutation(s) that synergistically contributes to tumorigenesis. Thus, our results demonstrate that Setd2 is required for HSC self-renewal, and provide evidence supporting the causal role of Setd2 deficiency in tumorigenesis. The underlying mechanism shall advance our understanding of epigenetic regulation of cancer and provide potential new therapeutic targets.

Fig. 1

Setd2 deficiency impairs multiple-hematopoietic compartment development. a qPCR detection of Setd2 expression level in different BM cell subsets, including HSCs (LT-HSC, CD150⁺CD48^-Lin⁻c-Kit⁺Sca1⁺), MPPs (CD48⁺Lin⁻c-Kit⁺Sca1⁺), B cells (B220⁺), T cells (CD3ε⁺), Ery cells (Ter119⁺), GM cells (Gr1⁺Mac1⁺). b, c The absolute number of PB leukocyte and lymphocyte subsets. WT, n = 18; KO, n = 19. d Statistical comparison of the proportions of different lineages between WT and KO BM. n = 4. e Colony-forming unit (CFU) assay of total BM cells. n = 3. f, g Representative FACS analysis (f) and absolute cell count (g) of BM HSPCs from bilateral femurs and tibias. WT, n = 8; KO, n = 9. h CFU assay of BM FLT3⁻-LSKs. n = 3. i In vitro growth curve of BM FLT3⁻-LSK cells. n = 3. All the samples are prepared using mice at 4−5 weeks after pI–pC induction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 2

Setd2 deficiency abolishes HSC self-renewal capacity. a Long-term follow-up of donor-derived cell (CD45.2) proportion in PB of competitive BM transplanted (cBMT) mice. n = 16. b Proportion analysis of donor-derived cells in different BM subsets of cBMT mice. n = 7. c Long-term follow-up of donor-derived cell proportion in PB of primary (WT, n = 19; KO, n = 18) and secondary (n = 14) noncompetitive BM transplanted (ncBMT) mice. d Proportion analysis of donor-derived cells in different BM subsets of primary ncBMT mice. WT, n = 5, KO, n = 4. e Absolute cell count of donor-derived HSPC subsets in BM of primary ncBMT mice. WT, n = 5; KO, n = 4. f Proportion analysis of donor-derived cells in different BM subsets of secondary ncBMT mice. n = 4. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

Long-term Setd2 deficiency is sufficient to induce MDS-like disease. a–c Routine blood test follow-up. n = 10. d Representative displays of Giemsa staining of blood smears. 1: Howell–Jolly body; 2: Microspherocyte; 3: large spherocyte. e Representative displays of hematoxylin–eosin (HE) staining of spleen tissue sections. f CFU assay of total spleen cells at 8 months after pI–pC induction. n = 3. g Proportion analysis of spleen and BM erythrocytes in different development stages at 8 months after pI–pC induction. n = 3. h Proportion analysis of BM erythrocytes in different development stages at 4 weeks after pI–pC induction. n = 4. i FACS and absolute cell count analyses of BM myeloid progenitor compartments at 4 weeks after pI–pC induction. GMP: CD34⁺CD16/32⁺LK, CMP: CD34⁺CD16/32^lowLK, MEP: CD34⁻CD16/32⁻LK. n = 3. j Ki67 staining for the detection of MEP cell cycle at 4 weeks after pI–pC induction. n = 9. k Absolute cell count analyses of spleen MEP cells at 4 weeks after pI–pC induction. n = 6. l CFU assay of BM MEPs at 4 weeks after pI–pC induction. n = 3. m Representative display of BM smears. 1: the myeloid cell with megaloblastic change; 2: bilobulated cell; 3: the erythroid cell with megaloblastic change. n CFU assay of total BM cells at 8 months after pI–pC induction. n = 3. o, p Representative displays of HE (o) and silver staining (p) of BM tissue sections. q Absolute cell count of HSPCs in spleen and BM. n = 3. r Proportion analysis of EGFP-positive cells in spleen erythroid cells of WT and KO mice at different ages. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

MDS-like disease of Setd2 deficiency is reproducible in primary ncBMT mice. a Routine blood test follow-up. Setd2 KO ncBMT mice are divided to two groups: mice in group1 (KO1) did not develop MDS-like disease (n = 6), whereas mice in group2 (KO2) did (n = 4). WT = 10. b Proportion follow-up of donor-derived myeloid cells. WT, n = 10; KO2, n = 4. c Spleen size and morphology analysis. Representative morphology is displayed. n = 3. d Proportion analysis of different mature lineages in total or donor-derived spleen cells. n = 3. e Proportion analysis of EGFP-positive cells in spleen erythroid cells. n = 3. f Absolute cell count of LSK and LK cells in total or donor-derived spleen cells. n = 3. g Proportion analysis of different erythroid subsets in total and donor-derived BM cells. n = 3. h Proportion analysis of donor-derived cells in different spleen cell subsets. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

Setd2 KO HSPCs share some common transcriptional signatures with Dnmt3a/Tet2DKO HSPCs. a GSEA analysis of the transcriptional signature of HSCs (left) and mature cells (right) in KO BM LSKs relative to WT group. NES normalized enrichment score. b Plot representing the comparison of upregulated and downregulated genes between Setd2 KO LSKs and other epigenetic regulator KO HSPCs cells. *Nom P < 0.05, **Nom P < 0.01, ***Nom P < 0.001. Nom P, Nominal P-Value. c Number of hypo-methylated and hyper-methylated DMRs in Setd2 KO LSKs. d The comparison of average methylation level of all DMRs between WT and KO LSKs. e qPCR validation of deregulated genes associated with erythroid cells. n = 3. f GSEA analysis of Klf1 target genes (top) and heme metabolism-related genes (bottom) in Setd2 KO BM LSKs relative to WT control. g qPCR detection of Klf1, Hbb-b1, and Hba-a1 expression in Setd2 KO Lin⁻ BM cells transfected with Klf1-shRNA. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Setd2 deficiency leads to HSPC proliferation defects. a, b GSEA analysis of E2F3 target genes (a) and deoxyribonucleotide (dNTP) metabolic process (b) in Setd2 KO BM LSKs relative to WT control. c qPCR detection of dNTP metabolism-related genes in KO and WT BM LSKs at 4 weeks after pI–pC induction. d CFU assay of Setd2 KO Lin⁻ BM cells transfected with empty vector or Rrm2b. e Cell cycle analysis of BM FLT3⁻-LSKs (left) and FLT3⁺-LSKs (right) from WT and KO mice at 4 weeks after pI–pC induction by using Ki67 staining. n = 4. f Statistical comparison of absolute cell count of BM HSPCs between WT or KO group at day 20 after single-time 5-FU treatment. n = 5. g Proliferation potential analysis of BM FLT3⁻-LSKs (left) and FLT3⁺-LSKs (right) by in vivo BrdU administration at day 20 after single-time 5-FU treatment. n = 4. h Genome distribution of mutations (SNVs and indels) in KO BM cells at 8 months after pI–pC induction. i Percentages of distinct mutation type of SNVs in KO BM cells. j Measurement of the ROS level in WT and KO LSKs at 4 weeks after pI–pC induction by using CellROX Deep Red Reagent staining. k Genome distribution of CNVs in KO BM cells at 8 months after pI–pC induction. l, m GSEA analysis of NUP98-HOXA9 targets (l) and interferon gamma pathway (m) in 8 months-old KO BM LSK cells relative to WT group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001