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Phf6-null hematopoietic stem cells have enhanced self-renewal capacity and oncogenic potentials

Yueh-Chwen Hsu, Tsung-Chih Chen, Chien-Chin Lin, Chang-Tsu Yuan, Chia-Lang Hsu, Hsin-An Hou, Chein-Jun Kao, Po-Han Chuang, Yu-Ren Chen, Wen-Chien Chou and Hwei-Fang Tien

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Cell counts in the peripheral blood of mice 8 to 12 weeks of age. (A) Western hybridization of BMCs harvested from 3 wild-type mice (left 3 lanes) and 3 Phf6 knockout (KO) mice (right 3 lanes). Phf6 (∼41 kDa) was markedly depleted in the bone marrow of Phf6 KO mice. (B) Female Phf6 KO mice had higher WBC, lymphocyte (LYMPH), and monocyte (MONO) counts than wild-type littermates. (C) Phf6 KO mice (n = 19), male and female, had higher B220+ B-cell counts (left panel), lower CD4+ T-cell counts (middle panel), and lower CD8+ T-cell counts (right panel) in their peripheral blood than wild-type mice (n = 17). *P < .05; **P < .01; ***P < .001; ****P < .0001. BASO, basophil; CBC, complete cell count; EOS, eosinophil; NEUT, neutrophil.

  • Figure 2.

    Lymphocyte composition in the spleen and thymus of mice 8 to 12 weeks of age. (A) The percentages of T cells (P = .0372), especially CD8+ T cells (P = .001), in the spleen were lower in Phf6 knockout (KO) mice than in Phf6 wild-type mice (wild-type, n = 12; Phf6 KO, n = 12), whereas the B-cell and CD4+ T-cell percentages did not differ. (B) Within CD4+ splenocytes, Phf6 KO mice had higher percentages of naive regulatory T cells (Treg) (left panel; P = .0076) but lower percentages of effector T cells (right panel; P = .0043; wild-type, n = 12; Phf6 KO, n = 12). (C) Within thymocytes, Phf6 KO mice had lower percentages of CD4 and CD8 double-negative (DN) cells compared with wild-type mice (P = .0316; both, n = 5). *P < .05; **P < .01; ***P < .001. DP, CD4 and CD8 double-positive cells.

  • Figure 3.

    The composition of HSPCs under steady state. (A) Phf6 knockout (KO) mice had higher percentages of pro-B cells in their bone marrow (P = .0429; wild-type, n = 6; Phf6 KO, n = 6). (B) Phf6 KO mice had less GMP (P = .0184; wild-type, n = 11; Phf6 KO, n = 11). (C) Phf6 KO mice had higher percentages of LSK BMCs compared with wild-type mice (P = .0187; wild-type, n = 11; Phf6 KO, n = 11). (D) The percentages of MPP2 were higher in Phf6 KO mice (P = .0035; wild-type, n = 11; Phf6 KO, n = 11). (E) Cell cycle analysis of the subpopulations of Phf6 KO HSPCs exhibited a lower proportion of G0 phase cells compared with the wild-type counterparts. Representative FACS plots showed more active proliferation in Phf6 KO MPP3 compared with the wild-type cells.*P < .05; **P < .01.

  • Figure 4.

    Analysis of aged wild-type and Phf6 knockout (KO) mice. (A) At 18 months of age, Phf6 KO mice had lower lymphocyte (LYMPH) counts (P = .0237; wild-type, n = 20; Phf6 KO, n = 21). (B) Aged wild-type mice had higher platelet (PLT) counts compared with aged Phf6 KO mice (P = .0002; wild-type, n = 20; Phf6 KO, n = 21). (C) Aged Phf6 KO mice had lower B220+ B-cell counts in their peripheral blood compared with wild-type mice (P = .0345; wild-type, n = 20; Phf6 KO, n = 21). (D) Aged Phf6 KO mice had lower CD4+ T-cell counts in their peripheral blood (P = .0126; wild-type, n = 20; Phf6 KO, n = 21). (E) Aged Phf6 KO mice had lower CD8+ T-cell counts in their peripheral blood (P = .0258; wild-type, n = 20; Phf6 KO, n = 21). (F) Aged Phf6 KO mice had larger spleens (P = .014; wild-type, n = 7; Phf6 KO, n = 10). (G) Tissue sections showed increased extramedullary hematopoiesis in the red pulp of spleens from Phf6 KO mice (inset 20×). (H) Bone marrow sections showed increased megakaryocyte number, decreased cell size, and nuclear lobation of megakaryocytes (indicated by white arrows) in Phf6 KO mice (inset 1000×; scale bar, 10 μm). (I) Megakaryocytes of aged Phf6 KO mice had decreased cell size (P = .0007; both, n = 3). (J) Aged Phf6 KO mice had increased megakaryocyte number (P = .0004; both, n = 3). (K) Aged Phf6 KO mice had higher percentages of CD4+ cells (left) and lower percentages of CD8+ cells (right) in the spleens (P = .0105 and .0247, respectively; wild-type, n = 5; Phf6 KO, n = 9). (L) In the fraction of CD4+ splenic T cells, Phf6 KO mice had higher percentages of naive regulatory T cells (left panel) and lower percentages of effector T cells (right panel) (P = .018 and .0205; wild-type, n = 5; Phf6 KO, n = 9). (M) In the bone marrow, aged Phf6 KO mice had decreased LT-HSCs (P = .0424; wild-type, n = 7; Phf6 KO, n = 10). *P < .05; ***P < .001. HPF, high-power field; TCRβ+, T-cell receptor-β+.

  • Figure 5.

    Transplantation assays of Phf6 knockout (KO) BMCs. (A) Chimerism was higher in the marrow of recipients transplanted with bulk Phf6 KO BMCs (P = .0312; wild-type, n = 7; Phf6 KO, n = 7). The recipients transplanted with sorted LSK cells from bone marrow of Phf6 KO mice also had significantly higher chimerism compared with wild-type (P = .0478; wild-type, n = 9; Phf6 KO, n = 8). (B) In a competitive repopulation unit assay, the recipients of Phf6 KO BMCs had higher percentages of Lin, LK, GMP, and megakaryocyte-erythroid progenitor (MEP) cells (P = .0065, .000169, .0037, and .0032, respectively; wild-type, n = 7; Phf6 KO, n = 7) among the donor-derived cells. (C) Recipients of Phf6 KO BMCs had higher WBC and neutrophil (NEUT) counts but lower lymphocyte (LYMPH) counts than those of wild-type BMCs. (D) Within donor-derived blood cells, recipients of Phf6 KO BMCs had a lower proportion of CD4+ and CD8+ cells but higher CD11b+Gr1 cells in the blood compared with wild-type BMCs. (E) The chimerism in blood (left panel) and marrow (right panel) of the recipients of Phf6 KO BMCs was further enhanced in the secondary transplantation. Representative FACS plots of bone marrow are shown. *P < .05; **P < .01; ***P < .001; ****P < .0001. CMP, fluorescence-activated cell sorting analysis and cell sorting; PB, peripheral blood.

  • Figure 6.

    The synergistic effects of Phf6 deletion on ICN1 overexpression. (A) Six weeks after transplantation, the recipients of Phf6 KO+ICN1 cells had lower hemoglobin (HGB) levels. (B) The recipients of Phf6 KO+ICN1 cells had higher percentages of CD4+CD8 cells within the ICN1 overexpressing BMCs (P = .0325). Representative FACS plots of bone marrow are shown. (C) The recipients of Phf6 KO+ICN1 cells had larger spleens. (D) The recipients of Phf6 KO+ICN1 cells had a shorter overall survival (P < .0001). (E) The survival disadvantages of Phf6 KO+ICN1 mice held true with different cell doses. (F) There were more LICs (CD4CD8CD25+CD127+) in Phf6 KO+ICN1 mice marrow. (G) Phf6 KO+ICN1 mice had more LIC (P < .0001) according to limiting dilution assays. *P < .05.

  • Figure 7.

    Phf6 deletion enriched differentiation and cell cycle–associated functions in HSPCs. (A) Enrichment Map showing the significantly perturbed functions in Phf6-null compared with wild-type HSPCs nodes are Gene Ontology gene sets, and edges indicate shared genes. (B) GSEA plot for representative gene sets enriched in Phf6 KO vs wild-type cells. (C) Heatmap representation of oncogenic signatures significantly enriched or depleted (P < .05) in Phf6 KO HSPCs. (D) GSEA plot for representative oncogenic signatures enriched in Phf6 KO cells vs wild-type cells. E2F1, Myc oncogene (MYC), and MTOR signatures were positively enriched in Phf6 KO LT-HSCs, MPP2, and MPP3. ER, endoplasmic reticulum.