The Lkb1 metabolic sensor maintains haematopoietic stem cell survival

Abstract

Haematopoietic stem cells (HSCs) can convert between growth states that have marked differences in bioenergetic needs. Although often quiescent in adults, these cells become proliferative upon physiological demand. Balancing HSC energetics in response to nutrient availability and growth state is poorly understood, yet essential for the dynamism of the haematopoietic system. Here we show that the Lkb1 tumour suppressor is critical for the maintenance of energy homeostasis in haematopoietic cells. Lkb1 inactivation in adult mice causes loss of HSC quiescence followed by rapid depletion of all haematopoietic subpopulations. Lkb1-deficient bone marrow cells exhibit mitochondrial defects, alterations in lipid and nucleotide metabolism, and depletion of cellular ATP. The haematopoietic effects are largely independent of Lkb1 regulation of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling. Instead, these data define a central role for Lkb1 in restricting HSC entry into cell cycle and in broadly maintaining energy homeostasis in haematopoietic cells through a novel metabolic checkpoint.

Figure 1. Lkb1 is required for haematopoiesis

Mutant (Mx1-cre; Lkb1^L/L) and control (Mx1-cre; Lkb1^L/+ or Lkb1^L/L) mice were injected with pIpC every second day over 7 days. a, Survival analysis; n >10 mice per genotype; P<0.001. b, Mononuclear bone marrow cellularity (n=4). c–f, Analysis at day 5 for the indicated subpopulations (n = 6 mice per genotype). CLP, common lymphoid progenitors; CMP, common myeloid progenitors; GMP, granulocyte-macrophage progenitors; MEP, megakaryocyte-erythrocyte progenitors; NEU, neutrophil; RBC, red blood cell. g, CFU-C assay for myeloid progenitors. *P < 0.01; **P < 0.001. Error bars in b–f indicate mean ± s.d.

Figure 2. Cell-autonomous role of Lkb1 in haematopoiesis

a–c, After pIpC induction, transplanted mice were analysed for survival (P < 0.005) (a), bone marrow cellularity (left) and donor chimaerism (right) on day 5 (b), and total HSC numbers and donor contribution at day 18 (c). d, e, Competitively transplanted mice were induced with pIpC and analysed for CD45.1 status in peripheral blood (d), and per cent CD45.1 HSCs at 4 weeks (e). n > 6 mice per genotype in a–e. *P < 0.05, **P < 0.01, all error bars indicate mean ± s.d.

Figure 3. Impact of Lkb1 inactivation on proliferation and apoptosis

a, Quantification of HSCs in the Mx1-cre model at day −3 and day +2 after pIpC treatment. n = 3 mice per genotype; P < 0.01, error bars indicate mean ± s.d. b, Cell-cycle analysis of HSCs (Ki-67/propidium iodide (PI) staining) at day −3 (n = 3; P < 0.01). c, Analysis viability in HSC and progenitor cells and Lin⁺ cells at day 5 by 7AAD staining (*P < 0.001, error bars indicate mean ± s.d.). d–f, Immunoblot of control (C) and Lkb1 mutant (M) mice for cleaved caspase-3 (Cl.Casp3) in bone marrow (BM) (d), LC3 in the indicated tissues at day 5 after pIpC (e), and phospho-H2AX levels in bone marrow (f).

Figure 4. mTORC1 inhibition and AMPK activation do not rescue bone marrow failure in Lkb1 mutants

a, Phospho(Ser 235/236)-S6 expression in bone marrow subpopulations at day 5 after pIpC treatment. b, c, Rapamycin (Rapa.) treatment does not rescue the drop in bone marrow cellularity (b) or HSCs (c) in Lkb1 mutants at day 5 (n = 4 mice per genotype). d–f, The AMPK activator A-769662 restores phospho(Ser 79)-ACC levels in Lkb1 mutant bone marrow cells (d), yet does not rescue loss of bone marrow cellularity (e), or HSCs (f) at day 5 (n = 4). * P < 0.001, all error bars indicate mean ± s.d.

Figure 5. Inactivation of Lkb1 alters mitochondrial function of bone marrow cells

a, Mitochondrial membrane potential of control and mutant cells at day 3 after pIpC assayed by DilC5 staining. b, Oxygen consumption rates (OCR) in control and mutant bone marrow cells under basal conditions and in response to 0.25 µM oligomycin, 5 µM fluoro-carbonyl cyanide phenylhydrazone (FCCP) or 1 µM antimycin + rotenone at day 1 in the Rosa26-creERt2 (top) and Mx1-cre (bottom) models. c, ATP levels of bone marrow cells at day 5 after pIpC. d, Glucose uptake in bone marrow at day 1. NBDG, fluorescent d-glucose analogue. e, Quantification of relative mitochondrial mass by Mitotracker staining (Mx1-cre model). n = 3 mice per genotype; P < 0.001; all error bars indicate mean ± s.d.