The CKS1/CKS2 Proteostasis Axis Is Crucial to Maintain Hematopoietic Stem Cell Function

Abstract

Long-term hematopoietic stem cells are rare, highly quiescent stem cells of the hematopoietic system with life-long self-renewal potential and the ability to transplant and reconstitute the entire hematopoietic system of conditioned recipients. Most of our understanding of these rare cells has relied on cell surface identification, epigenetic, and transcriptomic analyses. Our knowledge of protein synthesis, folding, modification, and degradation-broadly termed protein homeostasis or "proteostasis"-in these cells is still in its infancy, with very little known about how the functional state of the proteome is maintained in hematopoietic stem cells. We investigated the requirement of the small phospho-binding adaptor proteins, the cyclin-dependent kinase subunits (CKS1 and CKS2), for maintaining ordered hematopoiesis and long-term hematopoietic stem cell reconstitution. CKS1 and CKS2 are best known for their roles in p27 degradation and cell cycle regulation, and by studying the transcriptome and proteome of Cks1 ^-/- and Cks2 ^-/- mice, we demonstrate regulation of key signaling pathways that govern hematopoietic stem cell biology including AKT, FOXO1, and NFκB, together balancing protein homeostasis and restraining reactive oxygen species to ensure healthy hematopoietic stem cell function.

Figure 1.

Hematopoietic profile of Cks1⁻/⁻ and Cks2⁻/⁻ mice. (A) Color coding for mouse models (white = WT, red = Cks1^−/−, blue = Cks2^−/−). (B) Total cell number per femur. (C) Percentage LSK cells of total bone marrow. (D) Example flow plot for LSK cells. Percentage (E) LT-HSCs, (F) MPP1, (G) MPP2, (H) MPP3, (I) MPP4, (J) GMP, (K) MEP of total bone marrow. (L) Number of colony-forming units per 10⁴ lineage negative cells seeded. For all graphs, a one-way ANOVA was used to calculate significance of differences. *P < 0.05, **P < 0.005, ***P < 0.0005. ANOVA = analysis of variance; GMP = granulocyte myeloid progenitor; LT-HSC = long-term hematopoietic stem cell; LSK = lineage marker negative, Sca1⁺, cKit⁺; MEP = megakaryocyte erythroid progenitor; MPP = multipotent progenitor; WT = wild type.

Figure 2.

Serial transplantation of LT-HSCs from WT, Cks1⁻/⁻ and Cks2⁻/⁻ mice. (A) Schematic of transplantation in primary and secondary recipient mice (white/black line = WT, red = Cks1^−/−, blue = Cks2^−/−). (B) Example flow plot for CD45.1 vs CD45.2 staining to demonstrate chimerism differences between WT, Cks1^−/− and Cks2^−/− donors after 24 wk in a primary recipient. Percentage of total, myeloid, B and T donor derived cells in the peripheral blood of (C) primary and (D) secondary recipient mice. (E) Percentage donor-derived CD45.2 cells of total CD45-positive cells in the bone marrow of primary recipient mice. Percentage (F) LSK, (G) LT-HSC, and (H) MPP1 cells of total CD45.2 donor-derived cells in primary recipients. (I) Number of colony-forming units per 10⁴ lineage negative CD45.2-positive cells seeded from primary recipients. (J) Percentage donor-derived CD45.2 cells of total CD45-positive cells in the bone marrow of secondary recipient mice. Percentage (K) LSK, (L) MPP1, (M) MPP2, (N) MPP3, and (O) MPP4 cells of total CD45.2 donor-derived cells in secondary recipients. (P) Number of colony-forming units per 10⁴ lineage negative CD45.2-positive cells seeded from secondary recipients. For all graphs, a 1-way ANOVA was used to calculate significance of differences. *P < 0.05, **P < 0.005, ***P < 0.0005. ****P < 0.0001. ANOVA = analysis of variance; LT-HSC = long-term hematopoietic stem cell; LSK = lineage marker negative, Sca1+, cKit+; MPP = multipotent progenitor; WT = wild type.

Figure 3.

Proteomic analysis of Cks1⁻/⁻ and Cks2⁻/⁻ hematopoietic stem and progenitor cells. (A) Volcano plots for differentially abundant proteins measured by mass spectrometry between Cks1/2^−/− LSK cells and WT controls (red circles are significantly upregulated proteins, blue circles are significantly down regulated proteins). Gene ontology analysis of differentially abundant proteins in (B) Cks1^−/− and (C) Cks2^−/− LSK cells vs WT controls. (D) Venn diagram illustrating overlap of proteins differentially abundant between Cks1/2^−/− LSK cells and WT controls. (E) Annotated biological process for differentially abundant proteins in Cks1^−/− (red) and Cks2^−/− (blue) LSK cells. Protein abundance of known CKS1 interactors (F) XAF1 and (G) p27 from mass spectrometry analyses. (H) String network analysis of transcription associated proteins differentially abundant in either Cks1^−/− or Cks2^−/− LSK cells compared with WT controls. Protein abundance of (I) Paf1, (J) Med12, and (K) Ctr9 from mass spectrometry analyses. (L) Venn diagram depicting overlap of genes bound by Paf1, Med12, and Ctr9 from Chip-atlas.org. Gene ontology analysis of (M) all genes bound by either Paf1, Med12, or Ctr9 and (N) genes bound by all 3 proteins (Paf1, Med12, and Ctr9). (O) MFI of nonphosphorylated-β-catenin in LT-HSCs from WT and Cks1/2^−/− mice. (P) Relative TCF/LEF activity in LSK cells transfected with TOPFlash constructs and cultured for 48 h. For all graphs, a 1-way ANOVA was used to calculate significance of differences. *P < 0.05, **P < 0.005, ***P < 0.0005. ****P < 0.0001. ANOVA = analysis of variance; LT-HSC = long-term hematopoietic stem cell; LSK = lineage marker negative, Sca1⁺, cKit⁺; MFI = mean fluorescence intensity; TCF/LEF = T cell factor/lymphoid enhancer factor; WT = wild type.

Figure 4.

The transcriptomic landscape of Cks1⁻/⁻ and Cks2⁻/⁻ hematopoietic stem and progenitor cells. (A) Unsupervised clustering of differentially abundant genes between Cks1/2^−/− LSK cells and WT controls from RNA sequencing analysis. Gene ontology analysis of differentially abundant genes between (B) Cks1^−/− and (C) Cks2^−/− LSK cells vs WT controls. GSEA analysis of transcriptomic data from (D) Cks1^−/− and (E) Cks2^−/− LSK cells vs WT controls demonstrating downregulation of HSC signatures. (F) Individual differentially expressed genes from the Ivanova GSEA module. (G) Integrated analysis of transcriptomic and proteomic data using cytoscape.org. For all graphs a 1-way ANOVA was used to calculate significance of differences. *P < 0.05, **P < 0.005, ***P < 0.0005. ANOVA = analysis of variance; HSC = hematopoietic stem cell; LSK = lineage marker negative, Sca1⁺, cKit⁺; WT = wild type.

Figure 5.

Deficiency of Cks1 or Cks2 leads to unbalanced intracellular signaling pathway and ROS accumulation, resulting in poor LT-HSC function. (A) MFI of p27 in LSK cells. MFI of Akt phosphorylated at Serine 473 in (B) LSK and (C) LT-HSC cells. (D) Diagram depicting Akt regulation of Foxo1 and Foxo1 interaction with NFκB for antioxidant gene expression. (E) MFI of Foxo1 phosphorylated at Serine 256 in LT-HSCs. (F–L) Individual differentially expressed genes from the Foxo1_01 GSEA module. (M) MFI of NFκB phosphorylated at Serine 536 in LT-HSCs. (N–Q). Individual differentially expressed genes from the NFKB_c GSEA module. (R) Flow cytometry histogram and (S) MFI of ROS in LSK cells. For all graphs, a 1-way ANOVA was used to calculate significance of differences. *P < 0.05, **P < 0.005, ***P < 0.0005. ANOVA = analysis of variance; LT-HSC = long-term hematopoietic stem cell; LSK = lineage marker negative, Sca1⁺, cKit⁺; MFI = mean fluorescence intensity; ROS = reactive oxygen species.