Kruppel-like factor 7 overexpression suppresses hematopoietic stem and progenitor cell function

Abstract

Increased expression of Kruppel-like factor 7 (KLF7) is an independent predictor of poor outcome in pediatric acute lymphoblastic leukemia. The contribution of KLF7 to hematopoiesis has not been previously described. Herein, we characterized the effect on murine hematopoiesis of the loss of KLF7 and enforced expression of KLF7. Long-term multilineage engraftment of Klf7(-/-) cells was comparable with control cells, and self-renewal, as assessed by serial transplantation, was not affected. Enforced expression of KLF7 results in a marked suppression of myeloid progenitor cell growth and a loss of short- and long-term repopulating activity. Interestingly, enforced expression of KLF7, although resulting in multilineage growth suppression that extended to hematopoietic stem cells and common lymphoid progenitors, spared T cells and enhanced the survival of early thymocytes. RNA expression profiling of KLF7-overexpressing hematopoietic progenitors identified several potential target genes mediating these effects. Notably, the known KLF7 target Cdkn1a (p21(Cip1/Waf1)) was not induced by KLF7, and loss of CDKN1A does not rescue the repopulating defect. These results suggest that KLF7 is not required for normal hematopoietic stem and progenitor function, but increased expression, as seen in a subset of lymphoid leukemia, inhibits myeloid cell proliferation and promotes early thymocyte survival.

Figure 1

Loss of KLF7 does not affect bone marrow repopulating activity. The frequency of KLS (A) and colony-forming cells (B) in the fetal liver from 13.5-14.5-dpc Klf7^−/− and Klf7^+/+ embryos is shown. Data represent 2 or 3 independent experiments with 2 or 3 livers per genotype. Fetal liver chimeras were generated by transplanting Klf7^+/+, Klf7^−/+, or Klf7^−/− fetal liver (Ly5.2) cells with an equal number of WT competitor fetal liver (Ly5.1) cells into irradiated congenic (Ly5.1/5.2) mice. (C) The percentage of donor leukocytes at the indicated times. (D) The contribution of donor cells to B cells (B220⁺), T cells (CD3e⁺), neutrophils (Gr-1 high), and monocytes (Gr-1⁺ CD115⁺) assessed 12 weeks after transplantation is shown for 1 representative experiment. Bone marrow was harvested from primary recipients at 12 weeks and serially transplanted. Shown is donor contribution in secondary (E) or tertiary (F) recipients. Data represent 2 or 3 independent experiments for each genotype, with 4 or 5 mice per genotype per experiment.

Figure 2

KLF7 overexpression inhibits in vitro myeloid colony formation. WT bone marrow cells from 5-fluorouracil–treated donors were transduced with MSCV-IRES-GFP expressing KLF7 or vector alone. (A) Transduced (GFP⁺) cells were isolated 72 hours after transduction, and the expression of KLF7 (relative to β-actin) was measured by real-time RT-PCR. Data represent 2 independent experiments. *P = .033. Unsorted bone marrow cells exposed to control or KLF7 retrovirus were plated in methylcellulose and cultured for 7 days. (B) Representative photomicrograph showing the GFP⁺ colonies (top panel) and total colonies (bottom panel). Original magnification ×10. (C) The percentage of GFP⁺ colonies relative to the input percentage of GFP⁺ cells. *P = .026. Data represent the average ± SEM of 2 independent experiments, each plated in duplicate. (D) GFP⁺ sorted cells from lentivirally transduced c-Kit⁺ cells were plated in complete methylcellulose, and colonies were scored by morphology after 7 days of growth.

Figure 3

KLF7 overexpression inhibits bone marrow reconstitution. (A) Bone marrow cells transduced with MSCV expressing KLF7 or vector alone were transplanted into lethally irradiated recipients, and peripheral blood was analyzed 6 and 12 weeks after transplantation. Transduction efficiency was determined by measuring the percentage of GFP⁺ KLS cells just before transplantation (72 hours after viral exposure, time = 0). Data represent 2 individual experiments with 3-5 mice per genotype per experiment. *P < .001. (B) Shown is the percentage of donor B cells (B220⁺), T cells (CD3e⁺), and neutrophils (Gr-1 high) that were GFP⁺ from 1 representative experiment. *P < .01. (C) Bone marrow cells were transduced with HIV-MND–expressing KLF7 or vector alone at a low multiplicity of infection were transplanted into irradiated congenic recipients and the percentage GFP⁺ leukocytes in the blood measured. Data represent 3 independent experiments. *P < .001. **P < .05. (D) The percentage of donor B cells (B220⁺), T cells (CD3e⁺), and neutrophils (Gr-1 high) that were GFP⁺ at 6 weeks after transplantation. *P < .05. **P < .01. ***P < .0001. (E) GFP⁺ cells were sorted 72 hours after viral transduction and RNA analyzed by real-time RT-PCR for KLF7 expression. Data are normalized to β-actin and represent 2 independent experiments. *P = .043. (F) To assess homing, GFP⁺ KLS cells were transplanted into irradiated recipients and the number of GFP⁺ cells recovered from the bone marrow 20 hours later determined. Shown are the number of GFP⁺ cells per 10⁶ whole bone marrow cells, corrected for the number of cells injected. Data represent 3 individual experiments, with 3-6 mice per condition.

Figure 4

KLF7 overexpression suppresses hematopoietic stem and progenitors. Bone marrow stem and progenitor populations were analyzed by flow cytometry 6 weeks after transplantation with control or KLF7 lentivirally transduced cells. (A) Gating strategy used to identify HSCs (KLS CD34⁻), common myeloid progenitors (CMP, Lin⁻ c-Kit⁺ Sca-1⁻ CD16/32^lo CD34⁺), granulocyte-monocyte progenitors (GMPs, Lin⁻ C-Kit⁺ Sca-1⁻ CD16/32^hi CD34⁺), and megakaryocytic-erythroid progenitors (MEP, Lin⁻ c-Kit⁺ Sca-1⁻ CD16/32^lo CD34⁻; top panel) and common lymphoid progenitors (CLP, Lin⁻ CD27⁺ IL7R⁺ Flk-2⁺ Ly6D⁻; bottom panel). Cells are first gated on live, donor, and lineage-negative populations. (B) Representative histograms showing GFP expression in the indicated progenitor cell population. (C) A summary of the percentages of donor cells that are GFP⁺ for the indicated progenitor population is provided. Input refers the percentage of bone marrow cells that were GFP⁺ at the time of transplantation. Data represent the mean ± SEM of 2 independent experiments. ***P < .001.

Figure 5

KLF7 suppression of HSPC function is not mediated by CDKN1A. (A) Cdkn1a mRNA expression relative to β-actin in GFP⁺ KLS cells 72 hours after transduction with lentivirus expressing KLF7 or vector alone. (B) Bone marrow cells exposed to KLF7 or control lentivirus were plated in methylcellulose and colonies scored on day 7. The percentage of GFP⁺ colonies is shown, normalized for the transduction efficiency. Data represent 2 independent experiments, plated in duplicate. *P = .0002. Cdkn1a^−/− bone marrow was transduced with KLF7 or control lentivirus and transplanted into irradiated congenic recipients. Six weeks after transplantation, the percentage of donor leukocytes (C), cells of the indicated hematopoietic lineage (D), or indicated progenitor cell population (E) that was GFP⁺ was determined. Time zero in panel C refers to the transduction efficiency. Input indicates the percentage of GFP⁺ cells at the time of transplantation. Data represent 2 individual experiments, each with 5 or 6 mice per genotype. *P < .01. **P < .05. ***P < .0001.

Figure 6

KLF7 overexpression supports early thymocyte development. Blood, spleen, and thymic T-cell populations were analyzed by flow cytometry 6 weeks after transplantation with control or KLF7 lentivirally transduced cells. (A) Representative flow plot for the thymus, with each of the 4 major thymic populations (CD4⁺ CD8⁺ [DP], CD4⁻ CD8⁻ [DN], CD4⁺ CD8⁻ [CD4 SP], and CD4⁻ CD8⁺ [CD8 SP]) subsequently gated for GFP. (B) CD4 SP and CD8 SP T-cell subsets in the blood and spleen and DN, DP, CD4 SP, and CD8 SP subsets in the thymus as gated in the representative flow plot (A) were analyzed, and the percentage of GFP⁺ for each population from each tissue is shown. Input refers to the percentage GFP⁺ cells at the time of transplantation. *P < .05 compared with input values. (C) GFP⁺ thymic populations as sorted in panel A were analyzed for annexin V and DAPI staining. The results are summarized in panel D, showing the percentage of the GFP⁺ fraction of the indicated thymic population that were annexin V⁺. Data represent 2 independent experiments with 4 mice per condition. *P < .05.