Development of a screen to identify senotherapeutics

In mice and humans, reduced expression of the DNA repair endonuclease ERCC1-XPF, which is required for the repair of bulky DNA lesions, interstrand crosslinks and some double-strand breaks, causes accelerated aging38. Primary MEFs from Ercc1 −/− mice undergo premature senescence if grown at atmospheric oxygen38, presumably as a consequence of unrepaired DNA damage. Thus, we reasoned that these murine cells would be useful for identifying senotherapeutic drugs that modulate senescence driven by physiologically relevant processes that can affect all cell types. MEFs isolated from pregnant females 13 days post-coitus were incubated at 3% O 2 followed by a shift to 20% O 2 for three passages to induce senescence (Fig. 1a). To quantify senescence, SA-ß-Gal activity was measured using the colorimetric substrate X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) and the fluorescent substrate C 12 FDG (5-dodecanoylaminofluorescein-di-b-D-galactopyranoside)39. X-gal-positive cells were counted using a light microscope (Fig. 1b), whereas C 12 FDG-positive cells were quantified via flow analysis (Fig. 1c) and with an IN Cell Analyzer 6000 confocal imager (Fig. 1d). Passaging of the Ercc1-deficient MEFs at 20% O 2 resulted in ~50% of the cells being senescent (S) at passage 5 (p5), as determined by using three different methods for measuring SA-ß-Gal (Fig. 1e). In addition, we demonstrated that different ratios of co-plated senescent and non-senescent cells could be measured accurately (Supplementary Fig. 1). Cell senescence also was confirmed by measuring other markers of senescence including decreased proliferation (Fig. 2a), increased expression of the cell cycle inhibitors p21Cip1 and p16Ink4a (Fig. 2b, c), increased expression of the DNA damage marker γ-H2AX (Fig. 2d) increased cell size and volume (Fig. 2e, f), and increased percent of cells positive for expression of p16Ink4a (Fig. 2h) and the SASP factor IL-6 (Fig. 2h) by fluorescence in situ hybridization (FISH). Together, these results demonstrate a robust increase in the fraction of senescent Ercc1-deficient MEFs by passage 5 at 20% O 2 (Supplementary Table 1).

Fig. 1 Development of a novel assay to screen for senotherapeutics. a Schematic diagram of the assay. Passage 2 primary mouse embryonic fibroblasts MEFs from DNA repair-deficient Ercc1 −/− mice are passaged at 20% O 2 to induce oxidative DNA damage. After 3 passages, 50% of the cells are senescent (red) and 50% remain non-senescent (yellow). Drugs are tested on these mixed cultures to determine if they affect senescent (C 12 FDG positive) or non-senescent (C 12 FDG negative) cells. b Representative images derived from three replicate experiments of p5 Ercc1 −/− MEF cultures measuring senescence-associated b-gal (SA-β-Gal) activity using colorimetric X-gal staining. Scale bar, 50 μm. c Representative flow cytometric histogram detecting SA-β-Gal activity using C 12 FDG in senescent and non-senescent Ercc1 −/− MEF populations. The dotted line indicates the cut-off in intensity levels used to define senescent cells. d Representative image from IN Cell Analyzer 6000 to detect SA-β-Gal in p5 Ercc1 −/− MEFs using C 12 FDG. Senescent cells are outlined in red (S), non-senescent cells are outlined in yellow (NS) and blue fluorescence indicates Hoechst-stained DNA in nuclei (N) used to obtain a total cell count. Scale bar, 50 μm. e Quantification of the senescent cell population in non-senescent (NS) and senescent (S) p5 Ercc1 −/− MEF cell cultures detected with X-gal and with C 12 FDG by flow cytometry and INCell 6000 analyzer. Error bars indicate SD for n = 3. *p < 0.05, two-tailed Student’s t-test Full size image

Fig. 2 Detection of senescence markers in Ercc1 −/− MEFs. a Cell proliferation was measured in congenic WT and Ercc1 −/− MEFs from p2 through p5. n = 2. b Relative expression of p21Cip1 and c p16INK4a in p2 non-senescent (NS) and p5 senescent (S) Ercc1 −/− MEFs determined by qRT-PCR. Error bars indicate SD for n = 3. *p < 0.05, two-tailed Student’s t-test. d Protein expression levels of γH2AX in p2 NS and p5 S Ercc1 −/− MEFs. Changes in diameter e and volume f in p2 NS and p5 S Ercc1 −/− MEFs. Error bars indicate SD for n=3. * p < 0.05, two-tailed Student’s t-test. Ratio of cells expressing p16INK4a g and IL-6 h in p2 NS and p5 S cultures determined by ViewRNA fluorescence in situ hybridization. Error bars indicate SD for n ⩾ =2. * p < 0.05, two-tailed Student’s t-test. i Representative FISH image of p16INK4a and IL-6 of passage 5 S Ercc1 −/− MEFs Full size image

Since detection of senescence using the IN Cell Analyzer 6000 was consistent with other methods (Supplementary Table 1) and offered a semi-automated approach, we used it for further drug screening. Since ~50% of the Ercc1 −/− MEFs were senescent under our conditions, drugs that affect only senescent cells without affecting non-senescent neighborhood cells can be identified (Fig. 3a). Detectable outcomes for this screen include toxicity to all cells, non-senescent cells only or senescent cells only. A reduction in the ratio of senescent to non-senescent cells could be achieved by three drug activities: (1) specifically killing senescent cells, termed senolytics29, (2) suppressing cell senescence phenotypes, termed senomorphics, or (3) increasing proliferation of the non-senescent cells. Drugs that only increase proliferation are not considered senotherapeutic. The percentage of senescent MEFs relative to the total cell number was calculated for cultures treated with drugs relative to untreated controls.

Fig. 3 Characterization of a novel, C 12 FDG single-cell SA-ß-gal drug screening assay. a Scheme of possible outcomes after treating a senescent p5 Ercc1 −/− MEF culture with a potential senotherapeutic. Red=senescent cells, yellow=non-senescent cells. Senotherapeutics are characterized by either killing of senescent cells (senolytics) or by altering the senescent state of cells otherwise (senomorphics). After 48 h treating p5 senescent Ercc1 −/− MEFs with a drug, the relative number of senescent cells b and total cells c remaining is quantitated and plotted relative to untreated control cultures. Red bars indicate relative number of senescent cells, grey bars indicate relative number of total cells. Error bars indicate SD for n = 3. *p < 0.05 for senescent cells, two-tailed Student’s t-test Full size image

To validate the assay, we used select compounds previously tested by the NIA-sponsored Interventions Testing Program (ITP) for their effect on lifespan of mice. Two drugs that extend the lifespan of mice, rapamycin and NDGA8, 9, significantly reduced senescence of Ercc1 −/− MEFs (Fig. 3b). Curcumin, an active ingredient of turmeric, and resveratrol, a sirtuin agonist, neither of which extend the lifespan of mice40, had no effect on senescent cells (Fig. 3b). In contrast, two established senolytics, a combination of dasatinib plus quercetin (D/Q) and navitoclax29, 32, significantly reduced senescent and total cell counts relative to untreated cultures. Of note, rapamycin and NDGA reduced the number of senescent cells, but not the total number of cells, suggesting they are senomorphic compounds. In contrast, D/Q and navitoclax reduced both the number of senescent cells and the total number of cells, consistent with senolytic activity. These data demonstrate that the assay can be used to identify senolytics and senomorphics regardless of their molecular target.

Other aspects of the assay were validated, including measuring the consistency of inducing cell senescence with atmospheric oxygen, repeatability and reproducibility as well as Z′-factors for the controls (rapamycin and non-senescent wild-type MEFs) (Table 1). Given that rapamycin had a favorable Z′ score of >0.5 when 50% of cells were senescent, similar cell growth and passage conditions were then used for all subsequent experiments with rapamycin used as a positive control in every screen. These data demonstrate that the assay has characteristics amenable to transferable and reproducible drug discovery.

Table 1 Bioassay qualification parameter Full size table

Screening a library of small molecule autophagy regulators

Rapamycin, which regulates mTOR activity and autophagy, attenuates expression of SASP factors and extends healthspan in vivo9, 41, 42. Therefore, initially we screened the Enzo Screen-well Autophagy library containing 97 drugs in 35 different functional classes with defined autophagy-inducing or autophagy-inhibitory activity (Fig. 4a and Supplementary Table 2). The primary screen was performed at 1 μM concentration. Most of the drugs had no effect on cellular senescence (Fig. 4b, grey dots), but 15 compounds significantly reduced the fraction of senescent cells to below 50% (Fig. 4b, blue shaded area). These 15 drugs are in 11 different functional classes (Fig. 4c) and all have been reported to induce autophagy (Supplementary Table 2). Seven of these drugs, including rapamycin and its homologs, did not significantly change the total cell number and were therefore considered senomorphic (Fig. 4b, blue dots), whereas six drugs significantly reduced senescent cells and total cell number and were therefore considered to have senolytic potential (Fig. 4b, red dots). Also, due to the fact that lysosomal lumen alkalizers like chloroquine could give false-positive results, we excluded drugs known to change the lysosomal pH from our analyses43 (Supplementary Table 2). Staurosporine and Verapamil were highly toxic to all cells and therefore excluded from further analyses (Fig. 4b). To validate the results of the primary screen, all 13 of the potential senotherapeutic drugs were tested again in triplicate at 1 μM (Fig. 4d). All drugs significantly reduced senescence (potential senotherapeutics), but only six drugs also significantly reduced the total cell number (potential senolytics). However, we cannot rule out that some of these drugs might have toxicity on both non-senescent and senescent cells.

Fig. 4 Screening of a library of autophagy regulators yields senolytics. a Pie chart indicating the different functional groups of drugs in the autophagy library used in the screen. b The primary screen of all 97 autophagy regulators at 1 mM concentration. Plotted on the x-axis is the number of senescent cells in the drug-treated cultures relative to cells treated with vehicle only. On the y-axis is the fraction of total cells remaining after drug treatment relative to vehicle treated controls. Drugs that reduce the number of senescent cells > 50% can be found in the blue shaded area. Drugs that cause no change in cell number (senomorphics) are indicated by blue dots. Drugs that caused a decrease in total cell number to < 75% (potential senolytics) are indicated in red. Grey dots indicate drugs that lead to no significant change in cell senescence at the concentration used. c Pie chart indicating the functional groups of potential senescence-modulating drugs identified in the autophagy library. d Independent validation of the primary screen expressed as cell senescence and cell number relative to untreated control cultures (UT) of senescent cells. Known lysosomal inhibitors (lysosomal pH changing compounds, Fig. 4C) were excluded. All drugs were used at 1 μM, n = 3, graphed + SD. *p < 0.05, two-tailed Student’s t-test Full size image

HSP90 inhibitors as a novel class of senolytics

To examine the selectivity of the compounds, confluent, non-senescent wild-type MEFs and senescent Ercc1 −/− MEFs were treated with four different concentrations of each drug for 48 h and their viability measured using a CellTox Green assay. All but two drugs had the same or higher toxicity to non-senescent cells at the concentrations used (Fig. 5a,b). Plotting the viability of confluent, non-senescent cells vs. senescent cells after drug treatment clearly shows that only two drugs, geldanamycin and 17-AAG (tanespimycin), were able to reduce the viability of senescent cells specifically at a concentration of 1 μM without significantly affecting the viability of healthy cells (Fig. 5c). Both of these drugs are N-terminal ansamycin-derived heat shock protein (HSP90) inhibitors44. HSP90 is a ubiquitously expressed molecular chaperone, which plays an important role in protein stabilization and degradation45. It is upregulated in many cancers, stabilizing otherwise unstable oncogenic drivers such as mutant EGFR46, mutant BRAF47, 48, wild-type and mutant HER2, as well as certain anti-apoptotic factors49. In addition to geldanamycin, a benzoquinone ansamycin antibiotic original discovered in the bacterium Streptomyces hygroscopicus, and its first synthetic derivate 17 AAD, an improved, more water soluble geldanamycin-derived HSP90 inhibitor 17-DMAG (alvespimycin) also has been tested in clinical trials50. We used 17-DMAG for all subsequent studies as it showed a very promising profile with an almost 10-fold lower EC 50 values on senescent cells compared to overall cell death (Fig. 5d).

Fig. 5 HSP90 inhibitors selectively kill senescent cells. a, b Celltox Green cytotoxicity assay. All potential senolytic drugs were added to cultures of a non-senescent (confluent) wild-type and b senescent Ercc1 −/− primary MEFs at 4 concentrations (0.03–1.00 μM). Error bars indicate SD for n = 3. c Graph depicting drugs that specifically kill senescent cells. Plotted is the viability of non-senescent WT vs. senescent Ercc1 −/− cells after treatment with each drug for 48 h at a 1 μM concentration. Cell toxicity was defined as cell viability < 75%. Only cells that kill senescent cells, without significant toxicity to quiescent, non-senescent cells, are considered as senolytics (indicated in yellow shaded area). d Dose response analysis of senolytic activity of 17-DMAG. Increasing concentrations of 17-DMAG (0.1–1000 nM) were tested and plotted against the fraction of remaining senescent (red) and non-senescent cells (yellow) after 48 h treatment. The EC 50 values of their senolytic potential were determined from their dose response curves using a 4-parameter curve fit analysis (graphpad). Error bars indicate SD for n = 3. e Flow cytometric analysis of cell death of senescent Ercc1 −/− MEF cell cultures treated with 17-DMAG via AnnexinV/7-AAD staining. MEF cells are either senescent (C 12 FDG+; Top) or non-senescent (C 12 FDG−, Bottom). Live cells were double negative for 7-AAD and AnnexinV (bottom left quadrant), early apoptotic cells were positive for Annexin V (bottom right quadrant), late apoptotic cells were positive for AnnexinV and 7-AAD (top left quadrant) and dead cells were positive for 7-AAD only (bottom right quadrant). f Quantification of the flow cytometry data. Apoptosis of senescent (red) and non-senescent (yellow) cells was calculated by summing up all AnnexinV-positive cells. Error bars indicate SD for n = 3, *p < 0.05, two-tailed Student’s t-test Full size image

To determine whether HSP90 inhibition preferentially triggers apoptosis of senescent cells, cultures of senescent Ercc1 −/− MEFs grown at 20% O 2 were stained for AnnexinV/7-AAD and C 12 FDG, and analyzed by flow cytometry (Fig. 5e, Supplementary Fig. 2). Using a combination of AnnexinV, an early apoptosis phosphatidylserine binding compound, and 7-aminoactinomycin D (7-AAD), a membrane impermeable dye that is generally excluded from viable cells, together with C 12 FDG enables the detection of cells in different stages of cell death as well as senescence51. Treating the cells with low concentrations (50–200 nM) of 17-DMAG (17D) caused a dose-dependent, increase in C 12 FDG and AnnexinV double-positive cells compared to untreated cells. In contrast, there was no significant increase in apoptosis of C 12 FDG-negative cells, supporting the conclusion that 17-DMAG selectively kills senescent cells (Fig. 5f).

HSP90 inhibitors are senolytic in different cell types and species

To demonstrate that this senolytic activity of 17-DMAG was not due to an off-target effect, seven HSP90 inhibitors from diverse classes, including ansamycin, resorcinol, and purine and pyrimidine-like N-terminal HSP90 inhibitors, were tested in the assay. All of the inhibitors showed a dose-dependent reduction of senescent, C 12 FDG+MEFs (Fig. 6a) followed by a significantly delayed reduction in the number of passage 5, ERCC1-deficient, non-senescent MEFs (Fig. 6b). Similar to 17-DMAG, EC 50 values for the senolytic activity of the inhibitors exceeded their non-senescent cell killing potential and corresponded well with their previously determined IC 50 values for HSP90 inhibition (Fig. 6c). The one exception was NVP-BEP800, the only pyrimidine-like inhibitor tested, which had an EC 50 10-fold higher than its IC 50 , possibly due to its intracellular concentration or localization and did not kill non-senescent cells at all at the concentrations used. These results support the conclusion that HSP90 is a valid molecular target for killing senescent cells.

Fig. 6 HSP90 inhibitors (HSP90inhs) are senolytic in two species and multiple cell types. a, b Representative dose response curves of seven HSP90 inhibitors. Eight concentrations (0.1–1000 nM) of each drug were tested and plotted against the relative fraction of remaining senescent a and non-senescent passage 5 ERCC-deficient MEFs b to determine their senolytic and cytotoxic potential. c Structure, origin, and IC 50 (HSP90a/b inhibition) of the HSP90 inhibitors tested in (A and B). EC 50 values of their cytotoxic potential for senescent (sen) and non-senescent (non-sen) cells were determined from their dose response curves in A and B using a 3-parameter curve fit analysis (graphpad), n = 2. d Effect of 17-DMAG on multiple cell types. Cell senescence was measured after treatment of senescent cultures of two different types of mouse cell (MEFs and mesenchymal stem cells) and two different human cell types (IMR90 primary myofibroblasts and WI38 primary lung fibroblasts) with 100 nM 17-DMAG. Senescence was induced by three methods: oxidative stress (murine cells), genotoxic stress (etoposide IMR90), and replicative stress (WI38, passage 30). All experiments were performed in triplicate. Error bars indicate SD, *p < 0.05, two-tailed Student’s t-test. e Viability of HUVECs (human umbilical vein endothelial cells) treated with the HSP90 inhibitor ganetespib. Proliferating and senescent HUVECs were exposed to different concentrations of ganetespib (5–800 nM). After 72 h, the number of viable cells was measured. The red line denotes plating densities on day 0 of non-dividing senescent (set to 100%) as well as proliferating, non-senescent cells (also set to 100%). Plotted are the means ± SEM of five replicates at each concentration. Senescence was induced by 10 Gy ionizing radiation Full size image

To determine whether the senolytic effect of the HSP90 inhibitors is cell-type or species specific, we tested 17-DMAG on senescent cultures of primary murine mesenchymal stem cells (MSCs) isolated from Ercc1-deficient mice, human IMR90 fibroblasts, and human WI38 cells (Fig. 6d). 17-DMAG significantly reduced the fraction of senescent human fibroblasts and mouse stem cells. Senescence was induced by oxidative stress in murine cells, genotoxic stress in IMR90 (etoposide), and telomere shortening in WI38. A second HSP90 inhibitor, ganetespib, had senolytic activity in human umbilical vein endothelial cells (HUVECs) induced to senesce with ionizing radiation (Fig. 6e), but not in pre-adipocytes (Supplementary Fig. 3) indicating that although very potent, not all HSP90 inhibitors work on all cell types33. These data demonstrate that HSP90 inhibitors have senolytic activity in five cell types from two species when senescence is caused by a variety of mechanisms.

Reduction of other senescence markers by HSP90 inhibitors

To confirm the results obtained by measuring SA-ß-Gal activity, we incubated senescent Ercc1 −/− MEF cells grown at 20% O 2 with 100 nM 17-DMAG, then examined their cell size (Fig. 7a), measured expression of p16Ink4a and the SASP factor IL-6 via qPCR (Fig. 7b) and fluorescent in situ hybridization (Fig. 7c–d) and collected cell lysates at 6 or 24 h post-treatment to measure γH2AX by immunoblot. All of these markers of senescence were reduced by treatment with 17-DMAG.

Fig. 7 Multiple senescence markers are reduced in Ercc1 −/− MEFs after treatment with HSP90 inhibitors. a Diameter and volume of senescent Ercc1 −/− MEF cells were measured before (red) and after (orange) 1 µM 17-DMAG (17D) treatment for 24 h. Error bars indicate SD for n = 3, *p < 0.05, two-tailed Student’s t-test. b Relative mRNA expression of IL-6 and p16INK4a determined by qRT-PCR was measured in untreated (UT) and 17-DMAG (17D) treated cells for 24 h. Error bars indicate SD for n = 3. * p < 0.05, two-tailed Student’s t-test. c Representative FISH images of p16INK4a (green) and IL-6 (red) in senescent Ercc1 −/− MEFs with (17D) and without (UT) 17-DMAG treatment. d Quantification of the fraction of cells expressing p16INK4a and IL-6 in p5 Ercc1 −/− MEF cells with (orange) and without (red) 17-DMAG treatment for 24 h determined by ViewRNA FISH. Error bars indicate SD for n = 2. *p < 0.05, two-tailed Student’s t-test. e Immunoblot detection of the DNA damage/senescence marker γH2AX in p5 Ercc1 −/− MEF cultures at several time points following exposure to 100 nM 17-DMAG. Semi-quantitative analysis of γH2AX expression relative to β-actin. Western blots were quantified with ImageJ Full size image

HSP90 inhibitors downregulate the anti-apoptotic PI3K/AKT pathway

Senescent cells, like cancer cells, have upregulated pro-survival and anti-apoptotic signaling that confers resistance to apoptotic signals29, 52. Numerous studies on tumor cells demonstrate that cell survival can be mediated by an HSP90-dependent stabilization of factors such as AKT or ERK53. These suppress apoptosis via impacting mTOR, NF-κB, FOXO3A, and other signaling pathways (Fig. 8a). Inhibition of HSP90 leads to a destabilization of AKT and ERK and increased apoptosis, making HSP90 inhibitors useful for cancer treatment either alone or in combination with other cytotoxic or cytostatic compounds such as borzetomib, rapamycin, or tyrosine kinase inhibitors54,55,56. AKT and its activated form p-AKT (Ser473) were upregulated in late passage, senescent Ercc1-deficient MEFs compared to early passage proliferating cells (Fig. 8b). A low concentration of the HSP90 inhibitor 17-DMAG (100 nM) was sufficient to reduce the level of p-AKT in senescent Ercc1 −/− cells by 6 h and the reduction lasted for at least 24 h (Fig. 8c). In contrast, the expression of total AKT was not altered by 17-DMAG. These results demonstrate that HSP90 inhibitors are functional in senescent cells to block the stabilization of p-AKT, consistent with a model in which p-AKT plays a role inducing cell senescence57. Disruption of the HSP90-AKT interaction leads to a destabilization of the active, phosphorylated form of AKT, resulting in apoptosis of senescent cells.

Fig. 8 Expression of heat shock proteins and HSP90 client proteins in senescent and non-senescent Ercc1 -/- MEFs. a Proposed model for how HSP90 promotes resistance to apoptosis in senescent, p5 Ercc1 −/− MEFs. b Immunoblot detection of HSP90, pAKT (Ser473), AKT, and actin in early passage, non-senescent cells (NS), intermediate passage (I), and late passage, senescent Ercc1-deficient MEFs (S) grown at 20% O 2 . c Immunoblot detection of the same proteins in senescent, p5 Ercc1 −/− MEFs treated with 100 nM 17-DMAG at 6 and 24 h post-treatment Full size image

HSP90 inhibitor delays the onset of several age-related symptoms in Ercc1 −/Δ mice

To examine the effects of HSP90 inhibitors on the healthspan of aged mice, the Ercc1 −/∆ mouse model of a human progeroid syndrome was used. The mice spontaneously develop age-related degenerative diseases and have a maximum lifespan of 7 months58. 17-DMAG was administered three times per week every 3 weeks at a relatively high concentration (10 mg/kg) to Ercc1 −/∆ mice beginning at 6 weeks of age (Fig. 9a). Phenotypes associated with aging were measured three times per week by an investigator blinded as to treatment groups29. Treatment with 17-DMAG resulted in a significant reduction in a composite score of age-related symptoms including kyphosis, dystonia, tremor, loss of forelimb grip strength, coat condition, ataxia, gait disorder, and overall body condition, as shown in detail for a sex-matched and age-matched sibling mouse pair (Fig. 9b) and all the treated mice (Fig. 9c, Supplementary Fig. 4). The significant therapeutic effect of 17-DMAG on healthspan by intermittent treatment was confirmed in a second, short term treatment cohort (Supplementary Fig. 5).

Fig. 9 Intermittent treatment of progeroid Ercc1 −/Δ mice with the senolytic HSP90 inhibitor 17-DMAG extends healthspan. a Schematic diagram of the in vivo treatment regiment. Animals were treated with 10 mg/kg 17-DMAG by oral gavage 3 times per week, every 3 weeks. Eight symptoms associated with frailty and aging were measured in the mice, 3 times each week. b Graphed is the age at onset of each symptom (appearance of a colored bar) and severity (height of the bar) for a sex-matched sibling pair of Ercc1 −/Δ mice treated with or without 17-DMAG. The composite height of the bar is an indication of the overall health (i.e., body condition score with a higher value being worse). c Comparison of age-related symptoms between cohorts of mice treated with the HSP90 inhibitor or vehicle only over time. The average fraction of total symptoms appearing in each age group is plotted. An increase in the percent of symptoms indicates a decrease in health. n = 6 mice per treatment group, error bars indicate SEM, *p < 0.05, **p < 0.01, ***p < 0.001. Relative expression levels of p16INK4a in kidney d and liver e of Ercc1 −/Δ mice treated for 1 week by oral gavage with HSP90 inhibitor (10 mg/kg 17-DMAG oral gavage per treatment, 3×) compared to vehicle only. p16INK4a was measured by qRT-PCR. n = 4 mice per group; error bars indicate SD, *p < 0.05, two tailed Student t-test Full size image

To determine if the delay of age-related diseases in HSP90 inhibitor treated mice is due to its senolytic effect, Ercc1 −/∆ mice were treated three times over a 1 week period with 10 mg/kg of 17-DMAG and sacrificed 4 days after the last treatment. qPCR analyses showed a significant reduction of p16INK4A expression in the kidneys of treated mice compared to vehicle treated mice (Fig. 9d), but no significant changes in the liver (Fig. 9e). Taken together, these data demonstrate that periodic treatment with senolytic HSP90 inhibitors is sufficient to reduce senescent cell markers in vivo and delay the onset of age-related phenotypes, indicating a health span extension. These results also demonstrate that senolytic compounds identified in the senescent MEF assay indeed can reduce the level of senescent markers in vivo and extend healthspan in a mouse model of accelerated aging, validating the screening platform.