Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice

Abstract

Senescent cells (SCs) accumulate with age and after genotoxic stress, such as total-body irradiation (TBI). Clearance of SCs in a progeroid mouse model using a transgenic approach delays several age-associated disorders, suggesting that SCs play a causative role in certain age-related pathologies. Thus, a 'senolytic' pharmacological agent that can selectively kill SCs holds promise for rejuvenating tissue stem cells and extending health span. To test this idea, we screened a collection of compounds and identified ABT263 (a specific inhibitor of the anti-apoptotic proteins BCL-2 and BCL-xL) as a potent senolytic drug. We show that ABT263 selectively kills SCs in culture in a cell type- and species-independent manner by inducing apoptosis. Oral administration of ABT263 to either sublethally irradiated or normally aged mice effectively depleted SCs, including senescent bone marrow hematopoietic stem cells (HSCs) and senescent muscle stem cells (MuSCs). Notably, this depletion mitigated TBI-induced premature aging of the hematopoietic system and rejuvenated the aged HSCs and MuSCs in normally aged mice. Our results demonstrate that selective clearance of SCs by a pharmacological agent is beneficial in part through its rejuvenation of aged tissue stem cells. Thus, senolytic drugs may represent a new class of radiation mitigators and anti-aging agents.

Figure 1

ABT263 has senolytic activity in cell culture and mice. (a) Quantification of viable WI-38 non-senescent cells (NC), IR-induced senescent cells (IR-SC), replication-exhausted senescent cells (Rep-SC) or Ras-induced senescent cells (Ras-SC; in which oncogenic Ras is ectopically expressed) 72 h after treatment with increasing concentrations of ABT263 (n = 3–6 for NC and IR-SC; n = 3 for Rep-SC; n = 4 for Ras-SC). (b) Quantification of viable IR-SCs at the indicated times after treatment of the IR-SCs with 1.25 µM ABT263 (left) or after the cells had been incubated with 1.25 µM ABT263 for the indicated amounts of time followed by removal of the drug and a further culture period of 72 h (right) (n = 3). (c) Quantification of viable non-senescent (NC) and IR-induced senescent (IR-SC) human IMR-90 fibroblasts (IMR-90), human renal epithelial cells (REC) and mouse embryonic fibroblasts (MEF) 72 h after treatment with increasing concentrations of ABT263 (n = 3 per group). (d) Experimental design for e–g. Sham-irradiated (Ctl) and TBI-treated young male p16-3MR mice were administered vehicle (Veh), ganciclovir (GCV) or ABT263 (ABT) and analyzed as indicated. I.p., intraperitoneal injection; p.o., oral administration. (e) Left, representative luminescence images of Ctl and TBI mice after treatment with vehicle, GCV or ABT263. Right, quantification (in arbitrary units, a.u.) of whole-body luminescence (Ctl mice: vehicle-treated, n = 6; GCV-treated, n = 4; ABT263-treated, n = 6; TBI mice: vehicle-treated, n = 8; GCV-treated, n = 4; ABT263-treated, n = 7). A wild-type C57BL/6 mouse (WT) was included as a negative control. The vertical color bar indicates luminescence-signal strength. Scale bars, 15 mm. (f) Quantification of luminescence in lungs of Ctl or TBI mice treated as indicated (n = 5 per group). (g) Quantification of mRNA expression for Cdkn2a, Il1a, Tnfa, Ccl5 and Cxcl10 in lungs from Ctl or TBI mice treated as indicated (n = 4 per group). Throughout, data are means ± s.e.m. **P < 0.01, ***P < 0.001 and ****P < 0.0001 versus without ABT263 for a (one-way analysis of variance (ANOVA)); versus NC treated with the same concentrations of ABT263 for c; versus Ctl for e–g; two-way ANOVA for c–g.

Figure 2

ABT263 kills SCs by apoptosis. (a) Representative flow cytometric plots to measure apoptosis (left) and quantitation of the percentage of viable (gate II: PI⁻annexin V⁻) and apoptotic (gates III and IV: PI⁻annexin V⁺ and PI⁺annexin V⁺) (right) cells of WI-38 IR-SC 24 h after treatment with vehicle (Veh), 1.25 µM ABT263, 20 µM Q-VD-Oph (QVD), or the combination of ABT263 and QVD. (b) Quantification of the percentage of viable IR-SC 72 h after treatment with vehicle or ABT263 ± QVD as in a. (c) Quantification of SA–β-gal⁺ cells (white bars) 10 d after exposure to increasing doses of IR (left) or after increasing periods of time after treatment with 10 Gy IR (right), and the viability of the irradiated cells under these conditions after an additional incubation with 1.25 µM ABT263 for 72 h (gray bars). (d) Representative western blot analysis of procaspase-8 (Procasp-8), cleaved caspase-8 (cCasp-8), RIP1, cleaved RIP1 (cRIP1) and β-actin in NC and IR-SC 24 h after incubation with vehicle or 1.25 µM ABT263 (ABT). (e) Top, representative western blot analysis of procaspase-3 (Procasp-3), cleaved caspase-3 (cCasp-3) and β-actin in NC and IR-SC from d. Bottom, normalized expression of Procasp-3 and cCasp-3 in NC and IR-SC. Because the samples used for the western blots in d,e were the same, the same β-actin blot was used as a control for both panels. A mixture of cell lysates from etoposide- or cytochrome c–treated Jurkat cells was used as a positive control (Ctl) for detection of cCasp-3 and cCasp-8. (f) Normalized expression of BCL-2, BCL-xL, BAK and BAX in WI-38 cells by western blot analysis at increasing times after treatment with IR (10 Gy). (g) Quantification of NC (left) and IR-SC (right) viability 72 h after incubation with vehicle, 2.5 µM ABT199, 0.625 µM WEHI539, or the combination of ABT199 and WEHI539. (h) Quantification of NC and IR-SC viability 72 h after transfection with a control shRNA (Ctl) or shRNAs specific for BCL2, BCL2L1, or both BCL2 and BCL2L1. Throughout, data are means ± s.e.m. of three experiments, except for h (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; one-way ANOVA for c,f; two-way ANOVA for b,e,g,h.

Figure 3

SC clearance by treatment with ABT263 or GCV rejuvenates HSCs after TBI. (a) Experimental design for b–f. Sham-irradiated (Ctl) and TBI-treated young male C57BL/6 mice were administered vehicle (Veh) or ABT263 (ABT) and analyzed as indicated. (b,c) Quantification of changes in Cdkn2a mRNA levels (b, left; n = 3 biological replicates) and the percentage of SA–β-gal⁺ cells (b, right; n = 4 mice per group) in sorted HSCs, and the number of day 35 cobblestone area–forming cells (CAFCs) in bone marrow cells (BMCs; c; n = 4 mice per group). ^#P < 0.05 versus Ctl mice; ^&P < 0.05 versus vehicle-treated TBI mice; two-way ANOVA; n.d., not detectable. (d) Scheme for competitive and serial bone marrow transplantation (BMT; see Online Methods for details). (e,f) Representative flow cytometry plots (e) and quantification (f) of donor-derived total white blood cells (CD45.2⁺; e, top; f, top left), and T cells (T; CD45.2⁺Thy-1.2⁺), B cells (B; CD45.2⁺B220⁺) and myeloid cells (M cells; CD45.2⁺CD11b/Gr-1⁺) (e, bottom; f) in the peripheral blood (PB) of recipient mice after primary BMT (n = 6 recipients per group). Donor-cell engraftment in the PB of secondary recipients is shown in Supplementary Figure 6. (g) Experimental design for h–j. Sham-irradiated (Ctl) and TBI-exposed young male p16–3MR mice were treated with vehicle or GCV and analyzed as indicated. (h) Quantification of the percentages of SA–β-gal⁺ cells in sorted HSCs from mice treated as outlined in g (Ctl mice: vehicle-treated, n = 7; GCV-treated, n = 7; TBI mice: vehicle-treated, n = 11; GCV-treated, n = 12). (i) Quantification of the number of day 35 CAFCs in BMCs (Ctl mice: vehicle-treated, n = 3; GCV-treated, n = 3; TBI mice: vehicle-treated, n = 7; GCV-treated, n = 6). (j) Quantification of the percentages of donor-derived total cells, T cells, B cells and M cells in the PB of primary recipients (Ctl donors: vehicle-treated, n = 3 recipients; GCV-treated, n = 5 recipients; TBI donors: vehicle-treated, n = 7 recipients; GCV-treated, n = 9 recipients). Throughout, data are means ± s.e.m. In f,j, ^#P < 0.05 versus recipients of donor-derived cells from Ctl mice; ^&P < 0.05 versus recipients of donor-derived cells from vehicle-treated TBI mice; in h,i, ^#P < 0.05 versus Ctl mice; ^&P < 0.05 versus vehicle-treated TBI mice; two-way ANOVA; n.d., not detectable.

Figure 4

SC clearance by ABT263 treatment rejuvenates HSCs and MuSCs in normally aged mice. (a) Representative whole-body luminescent images (left) and quantification of luminescence (right) 1 d after 21- to 22-month-old male p16-3MR mice (aged) received vehicle (Veh; n = 7) or ABT263 (ABT; n = 8), according to the scheme shown in Figure 3a. 2-month-old male p16-3MR mice (young; n = 5) without treatment and a WT C57BL/6 mouse were used as controls. The vertical color bar indicates luminescence-signal strength. ^#P < 0.05 versus young mice, ^&P < 0.05 versus vehicle-treated aged mice; unpaired Student’s t-test. Scale bars, 15 mm. (b–j) Aged (21- to 22-month-old) male C57BL/6 mice received vehicle (Veh) or ABT263 (ABT) according to the scheme shown in Figure 3a and were analyzed 1 week after the treatment; 2-month-old male C57BL/6 mice without treatment (young) were used as controls. (b) Quantification of changes in mRNA levels of Cdkn2a, Tnfa and Ccl5 in the lungs of aged (Veh, n = 11; ABT, n = 10) and young (n = 9) mice. (c) Scheme for competitive and serial BMT using sorted long-term HSCs (LT-HSCs; CD34⁻CD48⁻CD150⁺Lin⁻Sca1⁺c-Kit⁺). (d) Quantification of the percentages of donor-derived total cells, T cells, B cells and M cells in the PB of primary recipients (recipients of donor cells from: young, n = 11; vehicle-treated aged, n = 15; ABT263-treated aged, n = 13; from two independent BMT experiments). Donor cell engraftment in the PB of secondary recipients is shown in Supplementary Figure 10c. (e) Gating strategy for analysis and isolation of MuSCs by flow cytometry (top) and representative micrographs showing immunostaining for the myogenic transcription factor Pax7 (a biomarker for MuSCs) (bottom). IF, immunofluorescence; MCFC, myogenic colony-forming cell. Scale bars, 20 µm. (f) Quantification of MuSCs in muscle tissue. (g) Quantification of primary (left) and secondary (right) MCFCs in sorted MuSCs. (h,i) Quantification of p16⁺ cells (h) and phosphorylated p38⁺ (p-p38⁺) (i) cells in MuSCs. (j) Quantification of the average number of γ-H2AX foci in MuSCs. n = 5, 6, and 6 mice per group for Young, Aged + Veh, and Aged + ABT, respectively, for f,h,i,j; 8, 12, and 13 mice per group for g. Throughout, data are means ± s.e.m. In d, ^#P < 0.05 versus recipients of donor cells from young mice, ^&P < 0.05 versus recipients of donor cells from vehicle-treated aged mice; in g–j, ^#P < 0.05 versus young mice, ^&P < 0.05 versus vehicle-treated aged mice; unpaired Student’s t-test.