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A CellAge epigenetic clock for expedited discovery of anti-ageing compounds in vitro

cellage epigenetic clock anti-ageing

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#1 Engadin

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Posted 16 October 2019 - 05:39 PM


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F U L L   T E X T   S O U R C E :    bioRxv

 

 

 

 

Abstract

 

We aim to improve anti-ageing drug discovery, currently achieved through laborious and lengthy longevity analysis. Recent studies demonstrated that the most accurate molecular method to measure human age is based on CpG methylation profiles, as exemplified by several epigenetics clocks that can accurately predict an individual’s age. Here, we developed CellAge, a new epigenetic clock that measures subtle ageing changes in primary human cells in vitro. As such, it provides a unique tool to measure the effects of relatively short pharmacological treatments on ageing. We validated our CellAge clock against known longevity drugs such as rapamycin and trametinib. Moreover, we uncovered novel anti-ageing drugs, torin2 and Dactolisib (BEZ-235), demonstrating the value of our approach as a screening and discovery platform for anti-ageing strategies. CellAge outperforms other epigenetic clocks in measuring subtle ageing changes in primary human cells in culture. The tested drug treatments reduced senescence and other ageing markers, further consolidating our approach as a screening platform. Finally, we showed that the novel anti-ageing drugs we uncovered in vitro, indeed increased longevity in vivo. Our method expands the scope of CpG methylation profiling from measuring human chronological and biological age from human samples in years, to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, providing a novel accelerated discovery platform to test sought after geroprotectors.

 

One of the remarkable achievements of developed countries is a continuous increase in life expectancy at birth, leading to greater longevity. However, a higher proportion of elderly in modern societies is accompanied by a steep increase in people suffering from age-related diseases. For example, cancer incidence rates, currently at 17 million worldwide, are expected to increase to 26 million in 20401, and a similar rise is expected for Alzheimer’s and Parkinson’s disease2. Compression of late-life morbidity is, therefore, a priority to alleviate suffering in the elderly3 and to reduce a growing economic burden to society4.

 

Critically, seminal discoveries in the biology of ageing showed that ageing is a malleable process and that down-regulation of major cellular nutrient signalling pathways, either glucose-sensing insulin signalling or amino acid-sensing target-of-rapamycin signalling, results in longevity and health improvement in all model organisms tested from yeast to mammals5. For instance, the long-lived mutants in C. elegans are protected from tumorous cell proliferation6 and have reduced toxic protein aggregation7, while Drosophila show less deterioration in their hearts8. Long-lived mouse mutants are protected from osteoporosis, cataracts and skin pathology, as well as decline in glucose homeostasis, immune and motor function9. The effect of these mutations is conserved from yeast to mammals, and it is, therefore, expected that if drugs replicate the biological impact of these changes, this could improve health in the elderly and prevent age-related disease. It is increasingly recognised that directly targeting ageing through pharmacological interventions, as opposed to specific age-related diseases, is a highly promising strategy for broad-spectrum disease protection10. However, at present, there are only a handful of reliable anti-ageing drugs whose effects have been confirmed in mammals, such as rapamycin11 and metformin12. Crucially, there are currently no sufficiently reliable ageing biomarkers for testing drugs on human cells in vitro, and the development of a specialised epigenetic clock seems the most promising current approach13,14,15.

 

To accelerate the discovery workflow for anti-ageing drugs, we took advantage of the breakthrough in the ageing field which showed that epigenetic clocks provide the most accurate measurements of human age, for instance, the approximate error rate for the Skin and Blood clock is ±2.5 years (maximal correlation coefficient 0,98)13. Epigenetic clocks surpass the accuracy of other ageing biomarkers such as telomere length and those based on transcriptomic, metabolomics or proteomic approaches, potentially because the latter approaches detect more transient and less stable cellular changes16. Ageing is accompanied by overall CpG hypomethylation, whilst some CpG islands and gene regions become hypermethylated17. Remarkably, only a small selection of the 56 million CpG sites in the diploid human genome, coupled with computational algorithms, is sufficient to provide an accurate readout of human age. One of the first epigenetic clocks was developed by Hannum using just 71 CpG sites to estimate age from blood samples18, while Horvath’s multi-tissue age estimator16 and Skin and Blood clock13 use 353 and 391 CpG sites, respectively19,20. Even a single CpG site in the ELOVL2 gene is sufficient to determine age21, albeit clocks using only a few CpG sites are less accurate and less applicable to different tissues19. The epigenetic clocks measure the ageing process inherent to all our cells and tissues, irrespective of their proliferation rate14. As the human epigenome reflects physiological changes, epigenetic clocks cannot only predict chronological age from a human sample but also give an estimate of biological age as has widely been demonstrated by the associations of epigenetic age with morbidity and mortality19,22. Recently, valuable predictors focussing on this aspect have been developed: PhenoAge23 and GrimAge24, which form the best epigenetic morbidity and mortality predictors available to date.

 

DNA methylation also captures information on the approximate number of cell divisions a stem cell has been through, as has been shown by epiTOC25, a mitotic clock that approximates stem cell divisions and correlates with cancer risk26. The biology underlying CpG methylation alterations at the sites linked to ageing clocks is not well understood. Horvath suggests that it is linked to epigenetic maintenance programmes being reflected in DNA methylation alterations16,19. Some recent findings implicate loss of H3K36 histone methyltransferase NSD1 in epigenetic ageing clock acceleration 27. Despite the enigma regarding epigenetic clock mechanism, these clocks are extremely useful and reliable predictors of age. However, little is known so far about the performance of these clocks in in vitro ageing experiments. It has recently been shown that the rate of epigenetic ageing in cultured cells is significantly faster than in the human body14,28 and that epigenetic age is retarded by rapamycin in vitro14, but neither of the clocks specialised for in vitro drug discovery nor were they tested on multiple anti-ageing drugs.

 

Therefore, we aimed to exploit the exceptional accuracy of CpG methylation clocks to uncover new anti-ageing pharmacological treatments. The current gold standard for discovering novel anti-ageing drugs are longevity experiments, which are laborious, lengthy and expensive. For instance, in mice, they take three years, thereby precluding any large scale drug screens. Existing screens in C. elegans commonly use live E. coli as food29,30, which is a disadvantage as drugs are metabolised first by the bacteria making their effect on worms secondary, which may lead to confounded results31,32. Yeast drug screens lack the crucial aspect of tissue toxicity33. In addition, all longevity assays require constant supply of the drug, making them highly expensive. Other attempts to uncover anti-ageing effects of drugs are based on computational analysis using existing transcriptomic information on the ageing process combined with drug characteristics34. However, transcriptomic changes are more transient and noisy when compared to DNA methylation and are, therefore, a less consistent ageing marker19.

 

We tested if existing epigenetic clocks could be used to measure anti-ageing drug potential in human primary cells in vitro and if we could build a new clock specialised for this purpose. Senescence is tightly associated with ageing of the organism, and because of the pronounced resemblance of ageing in primary cells in vitro to ageing in vivo, together with the evidence that human DNA methylation signatures are conserved and accelerated in cultured fibroblasts28, we used cultured human cells as a proxy for human ageing14,35. The ability to test anti-ageing drug properties directly on human cells in vitro could considerably accelerate the discovery of new compounds promoting healthy ageing. To this end, we used normal human mammary fibroblasts (HMFs) from a healthy 21-year old donor that we cultured from passage 10 to passage 20, which is before these cells reach senescence at passage 29 (Supplementary Fig. 1a-d). To measure CpG methylation, we used EPIC Arrays (Illumina) that measure methylation at 850,000 sites.

 

First, we tested the three most suitable existing epigenetic clocks, to determine if they could detect weekly and monthly ageing differences occurring during serial passaging of HMFs (Fig. 1a). The Multi-tissue clock16 consistently predicted a higher epigenetic age, and at passage ten this was 43.6±1.0 years (Fig. 1a), consistent with what was recently reported28. This increased age estimate, compared to 16 years of the donor, is in accordance with published data demonstrating that this epigenetic clock overestimates the age of mammary tissue samples16. The PhenoAge clock23, developed to predict mortality and morbidity risks, reported the epigenetic age of the donor to be 3.5±1.1 years younger (Fig. 1a). The most accurate age estimate, predicting the age of the donor at 23.2±0.87 years, was obtained using the Skin and Blood clock, which is specialised for determining donor age of the cells in culture and of easily accessible human tissues (Fig. 1a). The Multi-tissue clock and Skin and Blood clock showed a small increase in age with progressive passaging (from passage 10 to 20, age estimate increased from 43.6±1.0 to 53.9±1.7 and from 23.2±0.87 to 31.6±1.2 years, respectively), whilst this increase was greater for the PhenoAge clock (from 3.5±1.1 to 26.6±9.7 years). This suggests that, of the tested clocks, the PhenoAge clock is most suitable to measure ageing in vitro (Fig. 1a). However, the PhenoAge clock showed substantial variability in predictions for higher passages, which would obstruct the detection of subtle ageing differences upon anti-ageing drug treatments. In conclusion, while the Skin and Blood clock13 measures fibroblast ageing in culture, none of the existing clocks was ideally suited to accurately measure subtle anti-ageing drug potential in human primary cells in vitro, and similar comparisons have recently been reported by others14,28.

 

This prompted us to develop a new clock that, rather than predicting donor age in years, specialises in measuring methylation changes occurring during ageing of primary cells in culture and could differentiate DNA methylation state between each passage. To this end, we developed a clock using two different cell types, the above-mentioned HMFs and human dermal fibroblasts (HDFs), which were obtained from a different donor, have a different proliferative lifespan in vitro, and a different rate of DNA methylation change. Like the HMFs, the HDFs were serially passaged and sampled every other passage for DNA methylation analysis (Fig. 1b). We filtered for significant probes using a p-value threshold of 1×10−11, which gave the lowest error under our experimental setup, leaving 2,543 probes to build the clock using lasso regression, similar to the method used by Horvath16. We then tested our novel epigenetic clock, which we named the CellAge clock, using an entirely different set of samples, and we observed accurate prediction of passage number for both HMFs and HDFs, with a Root Mean Square Error (RMSE) of 0.37 (Fig. 1c and Supplementary Fig. 2a).

 

F1.large.jpg

 

FIGURE 1.
 
Development of CellAge clock for monitoring subtle ageing difference in cells in culture.
 
A) Predicted age of control samples using three existing epigenetic clocks. Predicted epigenetic age for control samples across all experiments as estimated by the Multi-tissue clock (green), the Skin and Blood clock (orange) and the PhenoAge clock (yellow). All three clocks show a trend to increase in predicted age with progressing passage, however there is a lot of variability in predictions, particularly for the PhenoAge clock. The Multi-tissue clock consistently predicted cells to have the highest epigenetic age, while the PhenoAge clock consistently predicted cells to have the lowest epigenetic age, which even reached below zero for several samples at various passages.
 
B) Genome-wide methylation changes upon cell passages of primary human mammary fibroblasts (HMF) and primary human dermal fibroblast (HDF).
 
C) Testing the CellAge clock on HMF and HDF samples that were not used to train the clock, demonstrates accurate prediction of the cell passage.
 
 
Having built a precise epigenetic clock that measures methylation changes during replicative ageing of human primary cells in vitro, we tested if anti-ageing drug treatment of HMFs and HDFs decelerated the CellAge clock. We chose an mTOR inhibitor, rapamycin, which is one of the most robust and evolutionarily conserved anti-ageing drug targets36, and which mediates its effect through down-regulation of S6K and Pol III, and up-regulation of autophagy37,38. We chose relatively low rapamycin concentration of 5nM that did not inhibit cell growth (Supplementary Fig. 1a) but moderately downregulated mTOR signalling, as evidenced by decreased pS6K and p4E-BP phosphorylation (Supplementary Fig. 3). This setup mimics the pro-longevity effects of rapamycin in vivo where it is well accepted that only mild nutrient sensing pathway inhibition increases life- and healthspan5,39. DNA methylation profiles from HMFs collected following four, six and eight weeks of rapamycin treatment (passage 16, 18 and 20; Fig. 2) were analysed using the CellAge clock and clearly demonstrated that rapamycin slows down methylation changes associated with replicative ageing. Interestingly, this clock deceleration was more pronounced upon longer treatment as shown by the gradual decrease of predicted-actual passage from 16 to 20 weeks. The low dose rapamycin treatment did not affect population doublings, confirming that the methylation changes were not a reflection of proliferation inhibition or slowing of the cell cycle (Supplementary Fig. 1). This is further evidenced by comparing the predicted passage from the CellAge clock against cumulative population doubling, showing rapamycin samples lie on a separate line to that of the control samples (Supplementary Fig. 2b,c). Contrarily, rapamycin samples and controls differed to considerably lesser extent when actual passage and cumulative population doublings are compared (Supplementary Fig. 2b,c). Importantly, we observed a similar pattern for HMFs and HDFs (Fig. 2), suggesting that the CellAge clock could be applicable to different cells, albeit calibration is required for cells that reach senescence at different rates.
 
 
F2.large.jpg
 
 
FIGURE 2.
 
Using the CellAge clock for the detection of anti-ageing drugs.
 
A) The methylome of Human Dermal Fibroblasts (HDF) and
 
B) Human Mammary Fibroblasts (HMF) was analysed using the CellAge clock. Represented is Predicted-Actual Passage for Passage 16, 18 and 20, showing deceleration of CellAge upon treatment with anti-ageing drugs rapamycin (5nM), Dactolisib/BEZ235 (10nM), torin2 (5nM) and Trametinib (0.1nM).
 

 

We then focused on HMFs to test another anti-ageing drug, trametinib40, an inhibitor of the MEK/ERK signalling pathway, which we also applied in low concentration to avoid any effect on growth and population doubling (Supplementary Fig. 1 and Fig. 3). The CellAge clock analysis of trametinib treatment showed clock deceleration for all three passages tested (Fig. 2), thereby confirming previous results in Drosophila in vivo that trametinib extends lifespan40. Next, we examined the effect of two other inhibitors of nutrient-sensing pathways as mutations in these pathways in model organisms represent the most evolutionary conserved anti-ageing interventions5.
 
We tested Dactolisib/BEZ235, a dual ATP competitive PI3K and mTOR inhibitor, for which we again optimised the dose of the treatment to obtain a reduction in signalling without significant proliferation impairment, as shown by pS6K downstream target 4E-BP (Supplementary Fig. 3). Dactolisib/BEZ235 slowed down the DNA methylation changes similar to rapamycin, suggesting that Dactolisib/BEZ235 could be a new anti-ageing drug according to the output of the CellAge clock (Fig. 2). We also tested torin2, which is a selective inhibitor of the mTOR pathway that inhibits both mTORC1 and mTORC2, unlike rapamycin, which targets solely mTORC1. Owing to its more complete inhibition of the mTOR pathway, we were interested in examining its effect on replicative ageing, especially as the role of mTORC2 in ageing is less well established. The impact of mTORC2 inhibition on lifespan can be positive or negative depending on which of the mTORC2 downstream effectors is affected, in which tissue, and whether females or male mice are used for the experiment41. Some of the negative effects of mTOR pathway inhibition, such as insulin resistance and hyperlipidemia, are attributed to the mTORC2 branch of the pathway and may arise under certain conditions of prolonged and/or high dose rapamycin treatment41. Interestingly, while our CellAge clock suggests that torin2 is indeed a novel anti-ageing drug (Fig. 2), its effect on ageing in mammalian cell culture appears to be less pronounced than that of rapamycin. This is in line with literature suggesting that a promising strategy to improve healthy ageing is the development of inhibitors that are highly specific for mTORC1 or that target mTORC1 downstream effectors separately41.
 
Next, we compared our anti-ageing drug screening results obtained by the CellAge clock with analyses using Horvath’s Multi-tissue and Skin and Blood clock, as well as the PhenoAge clock. The clocks did not detect any significant effect of anti-ageing drug treatment (Supplementary Fig. 4). The Skin and Blood clock26 was used recently to measure deceleration of ageing in primary fibroblasts14,28, however the concentration of rapamycin used in our conditions was five times lower without effect on cell growth, highlighting the sensitivity of our epigenetic clock to detect age-related methylation changes at very low drug concentrations. Under our conditions, the only epigenetic clock that detected gradual methylation changes from passage 10 to passage 20 was the PhenoAge clock (Supplementary Fig. 4). However, its output was more variable between samples and inconsistent for anti-ageing drug treatments, reporting both clock acceleration and deceleration. For instance, rapamycin, Dactolisib/BEZ235 and torin2 treated cells appeared slightly younger compared to controls, whereas trametinib treated cells were estimated older to some extent (Supplementary Fig. 4), unlike the results we obtained with our CellAge clock (Fig. 2). Overall, the CellAge clock that we developed here was more consistent and performed significantly better on ageing cells in culture and following known anti-ageing drug treatments compared to existing clocks, as evidenced by its ability to detect subtle ageing differences. Our results are supportive of clocks being highly specialised for a certain task, and suggests that while other popular epigenetic clocks perform remarkably on determining donor’s age in years and their health status, they were not able to robustly detect slight ageing changes in human primary cells induced by drug treatment over a short period of time in vitro.
 
 
 
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