Early warning signals of ageing in human stem cells and age-related disorders

Final Report Summary - SYSTEMAGE (Early warning signals of ageing in human stem cells and age-related disorders)

Executive Summary:
Besides loss of functions, reduced capacity to regenerate tissues or organs upon injury and a propensity to development of cancers are prominent hallmarks of senescence. The regenerative power of a living organism is closely linked to the potential of its stem cells to replace the corresponding damaged tissue1. A living organism is therefore as old as its stem cells. Whereas the vulnerability to cancer and chronic inflammation is associated with a decline of the immune system, the latter is in turn a sum product of interactions among haematopoietic stem cells (HSC) and the cellular determinants in the bone marrow niche, which include mesenchymal stromal cells (MSC), endothelial cells (EC) of the vascular system, as well as macrophages/monocytes, lymphocytes, etc. Hence loss of regenerative function and propensity to contract cancers can be interpreted as harbingers of ageing at the level of somatic stem cells3. The ultimate goal of SyStemAge is to understand age-associated functional alterations in somatic stem cells and their microenvironment, to identify molecular targets and to develop small molecule inhibitors/activators of pathways associated with normal and pathologic senescence.
Project Context and Objectives:
Using HSC and their microenvironment as a model, the principal goal of SyStemAge is to develop a systems-level understanding of the molecular mechanisms of ageing in somatic stem cells, the consequences and means to correct these age-related alterations and diseases.

Specifically, the missions of SyStemAge are:
1. To define the fundamental molecular and cell biological mechanisms of ageing in models of somatic stem cells, in order to comprehend the roles of genetic and proteomics alterations on the process of ageing.
2. To determine the molecular and cellular alterations of HSC and other cellular determinants that constitute the marrow niche (e.g. MSC, macrophages and lymphocytes) with ageing, and the impact of ageing of these cellular determinants on their respective supportive function for HSC.
3. To combine the cell biological, “-omics” and genetic studies to build models of age-related diseases such as myelodysplastic syndromes (MDS) and B-cell chronic lymphocytic leukemia (B-CLL).
4. To translate the systems understanding of stem cell ageing into novel drug discovery strategies.

HSC and cellular determinants of the marrow niche, e.g. MSC, macrophages, lymphocytes, etc., are ideal models for studies on the influence of age and environment on somatic stem cells as these cell types can be readily harvested from the bone marrow without undue
complications to human subjects.
We applied integrative systems biology approaches to characterize the molecular players, genes and pathways that are associated with physiological processes of ageing and with two examples of age-related disorders, MDS and B-CLL. We will then exploit the resulting models of ageing to develop novel, molecular targeted strategies to treat age- related diseases.
To achieve these goals, SyStemAge brings together a multidisciplinary consortium that builds on the most recent scientific achievements. It integrates a wide-range of technologies and expertise: (1) a strong link to the clinics with well established, ethical access to healthy donors and patient samples with well documented clinical history, (2) an international network of experts in somatic stem cell biology, providing a battery of cellular and animal models for the study of senescence and assays for defining the activity of somatic stem cells (i.e. proliferative and differentiation potential), (3) access to state-of-the-art transcriptomics, proteomics and metabolomics for systems-wide and time-lapse quantification of the molecular changes associated with ageing as well as with the clinical course of age-related diseases, (4) access to advanced resources in bioinformatics required to refine the biocomputational models and predictions: existing collections of large comparative genomics, epigenetics and transcriptomics datasets and expertise in the biology, (5) access to the clinical expertise related to the treatment of MDS and B-CLL, as well as biological samples combined with records of the clinical courses of diseases from these patients, (6) strong multidisciplinary computational and systems modelling approaches with proven record of successes in translational research, and (7) state-of-the-art biochemistry and phenotypic screening platforms based on innovative cellular co-culture assays together with libraries of bioactive compounds.

The project comprises 5 scientific work packages and one WP for coordination and management.

WP1's ( Systems biology of somatic stem cell ageing in the bone marrow") main scientific focus is to define the molecular determinants and alterations associated with the physiology of somatic stem cell ageing in the bone marrow, using HSC as a model for somatic stem cells, and elements that constitute the bone marrow niche, comprising MSC, macrophages/monocytes, lymphocytes, and granulocytic as well as erythroid precursors. These subpopulations will be derived from healthy human subjects from different age groups. In addition to transcriptomics studies, WP1 will put special emphasis on novel proteomics analyses. We concentrate on the influence of environmental cues on the regulation of cellular ageing in adult stem cells.

WP2 (Animal models) is complementing the work on human cells, the overall objective of this WP is to establish and use various manipulatory and genetic animal models to prototypically study ageing of HSC and endothelial cells in vivo. The experiments are aimed at:
(1) Determining the role of environmental cues, such as DNA damage and oxidative stress on cellular senescence and subsequently of organismal ageing.
(2) Defining the molecular mechanisms and functional consequences of vascular ageing.
(3) Providing a series of established models systems for WP5 and the development of screening assays.

WP3 (Systems biology of age-related disorders of the bone marrow) focuses on the systems biology of age-related disorders with emphasis on the bone marrow microenvironment. We will concentrate on two diseases, MDS and B-CLL, as models of age-related disorders. The overall objectives of this WP are:
(1) To define the molecules that are commonly deregulated in MDS and B-CLL versus normal bone marrow of comparable age groups, as well as molecules specifically associated with the early stages of MDS such as refractory anaemia.
(2) To determine the common signalling motives in malignant and non-malignant bone marrow together with the cell populations involved.
Pathways specifically deregulated in the malignant cell populations (HSC for MDS, B-cells for CLL) will guide our search for pathway inhibitors that can interfere or revert disease phenotype.

WP4 (Data integration and systems modeling) main goal is to integrate all the data generated within the consortium with those available in the literature in order to generate complex computational models that will provide a global view of the processes involved in HSC ageing, MDS and B-CLL. The main objectives are:
(1) Elucidation of the basic wiring of cellular processes related to somatic stem cell ageing.
(2) Transformation of the cell networks involved in HSC ageing and associated diseases into mathematical models (i.e. systems biology) in order to provide mechanistic descriptions.
(3) Identification of points of intervention with potential therapeutic value.

WP5 (Translation, therapy and drug discovery) - the overall objective of this WP is to translate the knowledge into guiding principles for new therapeutic strategies and drug discovery:
(1) Identification and preliminary characterization of hit compounds targeting age-related diseases by combining biochemical and cellular assay approaches.
(2) Establishment of novel 3D co-culture phenotypic assays for age-related diseases B-CLL and MDS.
(3) Bioinformatics prediction of mechanism of action, adverse effects and synergistic co-therapies of hit compounds.

Project Results:
The main S&T results and foreground could be summarised as following as per each corresponding Work Package. For more details please consult the previous periodic reports.

Work Package 1

All the goals and deliverables as delineated in the WP1 “Systems biology of somatic stem cell ageing in the bone marrow” have been achieved. The targeted sampling of bone marrow (BM) material from human subjects as originally described was successfully accomplished well ahead of schedule. The protocols for sample acquisition, for labelling, for proteomics analysis and RNA-sequencing of human BM samples were well established for the proteomic analysis of the 270 healthy samples, and RNA-Sequencing has been completed in 139 samples from the 59 human subjects recruited into the original study. The quality of the material and the data proved to be outstanding. In collaborations with the Beneficiaries of WP4, the proteomics and transcriptomics data have been thoroughly analysed, statistical and informatics analyses performed as planned. After extensive analyses, a major and surprising finding is the reorganisation of central carbon metabolism in the human hematopoietic stem cells upon ageing, as well as many other alterations in the mesenchymal stem cells that are complementary to the alterations in HSC.

For each cell type, the alterations in abundance of proteins detected (range ~7,000 to ~12,000 per cell type) with ageing were assessed. The age range of the 59 human subjects was 20 to 60 years. As the HPCs became older, pathways in central carbon metabolism exhibited features reminiscent of the Warburg effect where glycolytic intermediates are rerouted towards anabolism. Simultaneously, altered abundance of early regulators of HPC differentiation revealed a reduced functionality and a bias towards myeloid differentiation at the expense of lymphoid development. Ageing caused significant alterations in the bone marrow niche simultaneously, such as functionality of the pathways involved in HPC homing and levels of soluble factors influencing lineage differentiation. The data represents a valuable resource for further in-depth mechanistic analyses, and for validation of knowledge gained from animal models. A manuscript submitted to Nature Communications in August 2017 is being revised and validation experiments are underway.

After recruitment of the originally targeted number of 59 healthy human subjects, we discovered a disproportionate distribution of male subjects (n = 45) in our pipeline. In addition, no subjects over 60 years could be recruited into the study. As deliberated in our amendment submitted in May 2017, we have modified our collections to recruit more female subjects from all ages, and, in addition, 15 to 20 additional human subjects above 60 years. The approval to collect BM samples by the Ethics Committee of the University of Heidelberg has in the meantime been expanded and approved for subjects beyond the age of 70. The respective re-distribution of financial resources within the budget of SyStemAge has also been approved by the European Commission and implemented.

By expanding the scope, we will steadily improve the depth and foundation of our analysis, thus enhancing the validity and significance of our datasets. Approval of the Amendments has been granted since August 2017 and proteomics and transcriptomics analyses of these samples are still ongoing and will be completed within the budgeted resources. These additional datasets will enhance the validity and relevance of the comparisons of ageing versus young HSC, of age-matched healthy versus MDS human subjects, or the possibility of gender differences.

In addition to the novel knowledge as deliberated above, our datasets represent a valuable resource and basis for development of treatment strategies targeting metabolic alterations and pharmacologic manipulations to enhance HPC regeneration. Thus, inhibition of specific enzymes and pathways, through nutritional measures or drug targeting, might be able to influence the ageing process and thus of enormous potential for an ageing society as in the European Community.

Work Package 2

Avoiding accumulation of mutations as causes of hematological diseases is substantive matter for hematopoietic stem cell (HSC) homeostasis. In Task 2.1 Suda and co-workers demonstrated by using genetically altered mice, that apoptosis-stimulating protein of p53 1 (Aspp1) is a critical regulator of HSCs. In coordination with p53 Aspp1-deficiency provides HSCs sustained self-renewal and enhanced quiescence after serial transplantation, suggesting that Aspp1 counteracts stem cell ability in stressed condition. Furthermore, Aspp1 sensitize HSCs to apoptosis after genotoxic stress, resulting in elimination of HSCs with persistent DNA damage. Enhanced HSC capacities and persistent DNA damage by Aspp1 deficiency still remained in a p53-deficient background, suggesting that Aspp1 has some contributions besides p53 to the regulation of the cellular stress response. Finally, concomitant loss of Aspp1 and p53 led HSCs to hematological malignancies. These results uncover coordinated function of Aspp1 and p53, which regulates HSC self-renewal, contributes to maintain soundness of HSC pool and further prevents development of hematological malignancies.

The population of the developed world is ageing with major implications for health care resources and the productive workforce. Hence, it is of general interest to gain a better understanding of the molecular mechanisms of aging and to develop strategies to improve healthy lifespan. One major factor contributing to aging and malfunctioning of an organism is accumulation of oxidative damage of macromolecules (e.g. DNA). In Task 2.2 we show that Vitamin-D3-Upregulated Protein 1 (VDUP1) negatively influences the redox balance and cellular oxidative defense capacity during aging. We elucidate a novel mechanism conserved from fly (Drosophila melanogaster) to man showing that age-dependent upregulation of VDUP1 induces a perturbation of the intracellular redox equilibrium. VDUP1 upregulation leads to an increase in reactive oxygen species (ROS) and accumulation of oxidative DNA damage, both hallmarks of aging. Drosophila melanogaster is a widely accepted aging model comparable to humans. We demonstrate that increased VDUP1 expression leads to induction of DNA damage and, therefore, to a significant reduction in healthy (median) lifespan. Accordingly, decreased VDUP1 expression results in prolonged healthy (median) lifespan. Thus, manipulation of VDUP1 expression/activity could be a promising option to prolong healthy aging. The data is submitted for publication (Oberacker et al. “Vitamin-D3-Upregulated-Protein 1 (VDUP1) - A key regulator of healthy aging”).

For Task 2.3 “Functional Consequences of Vascular Ageing” the transcriptome and methylome data of infant, young, mature and old lung EC have been generated and analyzed and a manuscript entitled “Genome-wide profiling of endothelial cells identifies TGFß family signaling as key regulator of vascular quiescence” by Schlereth et al. is submitted. Maintenance of a quiescent and organotypically-differentiated layer of blood vessel-lining endothelial cells (EC) is vital for human health. Yet, the molecular mechanisms of vascular quiescence remain largely elusive. Here we identify the genome-wide transcriptomic program controlling the acquisition of quiescence by comparing lung EC of infant and adult mice, revealing a prominent regulation of TGFß family members. These transcriptomic changes are distinctly accompanied by epigenetic modifications, measured at single CpG resolution. Gain of DNA methylation affects developmental pathways, including NOTCH signaling. Conversely, loss of DNA methylation preferentially occurs in intragenic clusters affecting intronic enhancer regions of genes involved in TGFβ family signaling. Functional experiments prototypically validated the strongly epigenetically regulated inhibitors of TGFβ family signaling SMAD6 and SMAD7 as regulators of EC quiescence. These data establish the transcriptional and epigenetic landscape of vascular quiescence that will serve as a foundation for further mechanistic studies of vascular homeostasis and disease-associated activation.

For Task 2.4 mice with YFP-labeled EC compartment are available. Rag2-/-Il2rg-/-KitW/Wv mouse strain was utilized for forward fate mapping experiments. By analyzing the regeneration of liver vasculature in irradiation-based myeloablative and non-myeloablative bone marrow transplantation mouse models, we found that neoangiogenesis in livers with intact endothelium is solely mediated by proliferation of resident EC. However, following irradiation-induced EC damage, bone marrow-derived endothelial progenitor cells were recruited and incorporated into the vasculature. Further experiments with direct bone marrow infusion or granulocyte colony stimulating factor (G-CSF) mediated progenitor cell mobilization, which resemble clinical conditions, demonstrated that bone marrow-derived cells did not contribute to the regeneration of liver vasculature post partial hepatectomy. A manuscript entiteled “Endothelial cell fitness dictates the source of regenerative liver vasculature” by Singhal et al. is under revision.

Work Package 3

Proteomic and transcriptomic profiling has been performed on bone marrow cell populations of patients with myelodysplasia (MDS).
For proteomics, the bone marrow cellular constituents of 34 MDS patients were analyzed. Hematopoietic stem cells (HSC), macrophages/monocytes (MON), lymphocytes (LYM), granulocytic (GRA) and erythroid precursors (ERP) were successfully analyzed for 11 MDS patients and all cell types but one were analyzed for additional 14 MDS patients. HSC were analyzed for 22 MDS patients and for all other cell types at least 24 MDS patients could be evaluated.

Over 500 individual LC-MS runs were performed in total. Proteins resulting from the transcription of more than 9600 genes could be detected and 6494 could be quantified. In MON, LYM and HSC populations, more than 7000 proteins (>7000 in each population) were identified. In the ERP and GRA populations, >5500 and >6300 proteins could be identified respectively.
Transcriptomic data were obtained using RNA sequencing on a total of 160 samples from 36 MDS patients. 32 HPC, 34 LYM, 32 MON, 30 GRA and 32 ERP samples were analyzed. Generally the number of genes detected per sample was between 8,000 and 16,000, with fewer genes expressed in ERP and the highest number of genes expressed in HSC. Principal component analysis (PCA) and t-distributed stochastic neighbour embedding (tSNE) were able to separate the samples by cell type on the basis of their expression profile.
Downstream integrative analysis of the proteomic and transcriptomic data has been performed by Ben 6.

Metabolome analysis was performed by comparing CD34+ hematopoietic stem/progenitor cells (HSPCs) from 5 healthy controls and CD34+ cells from 15 AML patients. AML CD34+ cells had significantly high pyruvate/lactate ratio compared to CD34+ HSPCs. The O2 consumption rate (OCR) and extracellular acidification rate (ECAR) were directly measured, and CD34+ AML cells had significantly higher OCR/ECAR ratio. These results are suggestive of increase aerobic respiration in CD34+ AML cells. AML CD34+ cells also showed higher levels of the primary intracellular antioxidant glutathione (GSH).

The optimization of cell culture systems to reflect the in vivo behaviour of cells in the human environment is important. Protocols were developed for functional tests for validation of hypotheses and small molecules.
The activity of leflunomide (an approved immunosuppressive agent with indirect NF-κB inhibitory activity) in primary CLL cells supplemented with human serum (HS) was strongly diminished as compared to cell systems supplemented with fetal calf serum (FCS), i.e. HS exerts protective effects. These protective anti-apoptotic effects could also be observed with two other NF-κB inhibitors (BAY11-7082 and EF24).
This inhibitory activity of HS could be reproduced in the CLL cell line MEC1 and in several myeloid cell lines (MOLM13, HL60, KG1a, SKM1, K562 and LAMA84) and could also be observed in the context of different tyrosine kinase inhibitors (sorafenib, ruxolitinib and imatinib) shown to involve indirect inhibitory activity on the NF-κB signaling pathway.
The activity of NF-κB signaling pathway modulators involves and depends on cellular reactive oxygen species (ROS) homeostasis, and in MEC1 and MOLM13 cells the basal ROS levels were significantly decreased in HS versus FCS supplemented cell systems.
Serum albumin is known to exhibit antioxidant properties. In contrast to bovine serum albumin (BSA), addition of human serum albumin (HSA) to serum free culture medium antagonized the action of NF-κB inhibitors in MEC1 and MOLM13 cell lines.

The efficiency of inhibitors of the NF-κB signaling pathway in HS or in presence of HSA could be restored by by the inhibition of γ-glutamyltransferase (γ-GT) and/or by the inhibition of the glutathione catabolism. Addition of non-toxic doses of the γ-GT-inhibitor OU749 was able to enhance/restore the activity of NF-κB inhibitors in HS supplemented cell systems in vitro. Accordingly, treatment with non-toxic doses of OU749 resulted in enhancement/restoration of cellular ROS content in HS supplemented cell systems.

Work Package 4

The main goal of WP4 was to integrate all the data generated within the consortium with those available in the literature in order to generate complex computational models that will provide a global view of the processes involved in HSC ageing, MDS and B-CLL. As reported, we have largely fulfilled the objectives. Please, find below an executive summary of the most interesting (and unexpected) findings and developments.

1. By integrating high-throughput proteomics and transcriptomics human data, we have seen a rather poor correlation between the differential changes indicated by each type of data. After through analyses, we have seen that the correlation is significantly higher, although not outstanding, when we only consider those genes/proteins that do change in the different conditions considered. On the one hand, this poor correlation is a bit disturbing, but it also strongly supports the use of more costly techniques, such as proteomics, that are closer to the biological reality studied.

2. When considering healthy ageing, and using standard methodologies, we have observed very few significant changes at transcript or protein levels. Thus, we developed specific strategies to monitor those changes that are maintained through time, even if the amount of change is insignificant. This helped identifying relevant processes (i.e. glycolysis) likely involved in hematopoietic ageing.

3. Network-based strategies have proved useful to increase the significance and coherence of subtle changes observed in individual gene-protein analyses that, when integrated with partners, have shown unexpected robustness.

4. Our analyses have revealed molecular signatures characteristic of each defined MDS stage (i.e. RA(RS)/RCMD/RCUD, RAEB and AML), and it remains to be seen if they have sufficient power to predict the advancement to the next stage.

5. Lineage skewing upon ageing, firstly observed in mice, also occur in human.

Work Package 5

The main goal of WP5 was to translate the knowledge into guiding principles for new therapeutic strategies and drug discovery. We were aiming at identifying and characterizing pharmacologically active small molecules which could interfere with age-associated diseases MDS and B-CLL with subsequent analysis of mechanism of action based on bioinformatics modeling. The most interesting (and unexpected) findings and developments are summarized below.

1. The establishment of cell-line-based B-CLL 3D co-culture assay has been successfully accomplished by Ben 8 (ProQinase). Unfortunately Ben 8 was not be able to remain a partner in the SyStemAge consortium due to an insolvency proceeding. The fact that resources had already been used, and in order to avoid future competition with ProQinase, the consortium agreed that the establishment of 3D spheroid co-culture assays was not going be continued.

2. A compound screening has been performed following a phenotype-driven approach. A B-CLL/MSC co-culture model to mimic the influence of the protective microenvironment has been used to screen a microenvironment-focus compound library. The best compounds in terms of proliferation, cytotoxicity and dose-response in co-culture and in primary cell lines turn out to be compounds showing promiscuous binding. Two bioactive compounds (C7 and D1) and a repurpose drug (Simvastatin) have been selected for further analysis.

3. Target-specific analysis in cell culture have been done for the some of the known targets, NOD1 and NOD2, of the small molecule hits. But results were not conclusive and the underlying molecular targets of the small molecule hits have not been fully unveiled. Their effects may arise from a pleiotropic effect on different targets. Some of the effects seem to go through signaling pathways leading to STAT3 inhibition. Further analysis of these compounds will proceed outside the project.

4. Candidate compounds for drug repositioning have been identified either with the phenotypic screen or with target-based drug repurposing using the network model built in WP4. The repurposed drug identified in the phenotypic screen, Simvastatin a lipid-lowering agent, has been further screened in dose-response assays and in primary cell lines giving good specificity and similar effects as standard of care treatment, Ibrutinib. Other statins (lipid-lowering agents) have been screened and we could observe that statins with a lipophilic profile like simvastatin were performing best in the model system used probably due to a better diffusion into the cells. Network analysis provided addition drugs with a potential to be repurposed. Rifaximin (an antibiotic with an off-target effect on NR1I2) and Rilpivirine (a NNRTI non-nucleoside reverse transcriptase inhibitor used for the treatment of HIV-1 infections), have been identified to have a potential for interfering the network around NOD1 and NOD2 two of the highlighted protein targets. There are already some reports reporting a potential role in cancer for both drugs.

5. The Mode-of-Action of these compounds has been further evaluated by relating input (i.e. targets) and output (i.e. effector proteins related to a phenotype) network nodes through system biology models. C7 is predicted to have a stronger effect over B-CLL microenvironment over D1. When comparing the action of NOD1 and NOD2 alone the models predict a stronger effect of NOD1 over the B-CLL microenvironment. In addition, we have further analysed the models to select the compounds with highest level of efficacy and safety profile. Sinvastatin has not been evaluated in this respect as the adverse drug reactions are already known. For NOD1 and NO2 only a few adverse drug reactions based on target have been identified mainly related with infections but many potential indications have been highlighted making boosting the potential for drugs directed to those targets. These are estimates based on target, with the idea of guiding any possible further drug development by carefully monitoring the potential ADRs and /or to identifying the most suitable patients to be treated.

6. The network models have been further analyzed to identify possible synergistic co-therapies with existing treatments and compounds identified within the project. The three main targets already give a high prediction score for affecting B-CLL and its microenvironment for this reason it has been more difficult to identify compounds that would increase their action. For NOD1 several antineoplastic therapies were identified with a potential for synergy, between them Lenalidomide a treatment in use for B-CLL. For NOD2 a fewer combinations were identified but also included Lenalidomide. For Simvastatin the best combination therapy with an approved drug was with Ibrutinib, a standard of care for B-CLL. These combination therapies are planned to be further screened in cell cultures.

Potential Impact:
Dissemination activities during the project duration included, but not limited to scientific publications, presentations, various scientific meetings, conferences, website and website updates and changes. For a more detailed description of the dissemination activities please check the Deliverable 6.3.

Website

The project website is the main communication tool for the project, where all the dissemination materials are timely published. A continuous exchange of information between the partners of the project and other key players (e.g. SAB members) is one of the most important conditions for functioning of the network.

The SyStemAge website features the following functionalities:

• Overview of the concept, objectives, the partnership and the activities during the project
• News and information on SyStemAge activities plus important calendar events
• Network information: List of all project leaders per each WP and other researches/collaborators/students and information on each partner
• Access to a password protected (consortium members only) collaborative space for sharing info and documents. It is also a place that helps the Project Manager to keep track of the activities of all partners.

Newsletter

Another vital tool to keep in touch with the stakeholders and the public interested in the project is the implementation of a project’s newsletter. Newsletter is a part of the project website and informs all interested stakeholders during the project twice/once a year during the project lifetime.

The newsletter is implemented in form of a pdf file on the SyStemAge website and tries to promote the following info:
• Project development, progress and achievements
• Words from the Coordinator Dr. Anne-Claude Gavin
• New publications
• Introduction to project leaders, Scientific Advisory Board and other project members
• Various announcements (e.g. General Assembly, Upcoming Events etc.)

Publications

Many publications have appeared in peer-review international journals during the execution of the project. The latest important publications appeared in “Advances in Biological Regulations” by A. Pellagatti (OUXF, Ben10) with the title “Splicing factor gene mutations in the myelodysplastic syndromes: Impact on disease phenotype and therapeutic applications.” and in the journal “Annals of the New York Academy of Science” by T. Suda et al. (NUS, Ben11) with the title “Regulation of hematopoietic stem cell integrity through p53 and its related factors.”

A complete list of publications is visible on the official SyStemAge website . Several other publications have been submitted recently and are currently under review.

Press releases

During the project, several beneficiaries used their own communication portals to point out SyStemAge project and to announce some interesting results and project’s progress. Thus, two press releases were published via UKL-HD (Ben9) in the German language “Wie altern Stammzellen?” and in addition, on BioPro website . Another two press release appeared written in the Spanish language on the IRB (Ben6) website with the title: “A 6-million-euro European project to study stem cell aging” and similar press release on AX (Ben3) official website.

Events and conferences

To increase networking opportunities, the SyStemAge consortium tried to seek a presence at some major events in the relevant areas. Events included academic lectures and academic conferences, forums and other research day initiatives. The consortium (EMBL as coordinators) also organized some work group meetings and seminars regarding the SyStemAge project. For example, every year EMBL researchers presented the results of the SyStemAge project during Molecular Medicine Partnership Unit (MMPU) Research Day, which aims at discussing the diseases studied in different EMBL groups with wider scientific and medical communities. In 2016, EMBL has organized a conference: “Hematopoietic stem Cells from Embryo to the Aging organism”, which brought together recognized experts and outstanding young team leaders around the central theme of hematopoietic stem cells production and regulation throughout life.

Dutch Honors Students from the University of Utrecht visited EMBL on 3d of July to have a half-day scientific event with presentations and discussions on SyStemAge project.

The main exploitation of the results are included in the Deliverable 6.3 and include for most partners general advancement of knowledge, exploitation of the results through innovation, in further research activities and commercial exploitation of the results, such as developing new tools (AX).

Very important to mention that during the project, DKFZ (Ben5) has applied for a patent on “Inhibitors of thioredoxin-interacting protein (TXNIP) for therapy”. Its compound capable of reducing or inhibiting (a) the biological activity of thioredoxin-interacting protein (TXNIP) or (b) the expression of the gene encoding TXNIP for treating a condition, where improving the resistance towards oxidative stress has a beneficial effect, e.g. for improving female fertility or extending healthy lifespan. Please consult here to get detailed information on the patent: https://www.google.com/patents/EP2657340A1?hl=de&cl=zh

On 16th of May 2017 UKL-HD (Ben 9b), namely Aleksandar Radujkovic and Thomas Luft, applied for a European patent on “Inhibition of y-glutamyltransferase and glutathione catabolism to enhance the efficacy of NF-kB signaling pathway inhibitors”. The identification number of the patent application is the following: 17 171 283.9

The potential impact (including socio-economic and wider societal implications) could be summarised as following:

The systematic transcriptomics, proteomics and metabolomics experiments to be pursued within the SyStemAge consortium yield important insights into the fundamental molecular mechanisms associated with physiological and pathological (MDS and B-CLL) human ageing processes. We systematically characterized the molecular factors, signatures or the signalling routes specifically deregulated during the progression of MDS or B-CLL. Further expanding the study beyond these cell intrinsic factors, the SyStemAge consortium conducted unique sets of experiments to monitor the impact of the environment on cellular senescence and provide a systematic survey of the interactions and early ageing signals taking place between the HSC and their environment, i.e. a variety of somatic stem cells that constitute the haematopoietic niche. The models we generated are based on data essentially derived from human healthy donors or patients; they are directly be relevant to human biology. The SyStemAge consortium was committed to translate the cell biological, the “-omics” and the current knowledge into innovative strategies to treat age-related pathologies in human. The pursued computational and systems modelling experiments within the SyStemAge consortium directly delivered series of tools and opportunities to detect, monitor and treat age-related diseases. The SyStemAge consortium activity immediately contributed to bio-gerontology.

For example, in WP1, in defining the proteomics landscapes of the ageing process of human HPCs and five other cellular components of the bone marrow niche, the most prominent, novel finding is a re-routing of the pathways in central carbon metabolism towards anabolism in the HPC. This alteration was reminiscent of the Warburg effect described in cancer cells. Recent evidence has indicated a strong mechanistic link between cell growth and metabolic pathways, and that enhanced activities of enzymes and pathways in glycolysis is intimately associated with development of malignant growth. One example is phosphoinositide 3 kinase kinase (PI3K), increased activity of which has been shown to function as the key intermediate that allows growth factor receptors to direct glucose uptake. Other examples are mutations in the metabolic enzymes IDH1 and IDH2 that has led to the production of onco-metabolite 2- hydroxyglutarate (2-HG) in leukemia and gliomas. Specific inhibitors of these enzymes, i.e. Idelalisib, a PI3K-inhibitor, and Enasidenib, an IDH2-inhibitor, have recently been approved for treatment of refractory lymphomas (Idelalisib), or refractory acute myeloid leukemia (Enasidenib).

Our datasets also represent a valuable resource and basis for development of treatment strategies targeting metabolic alterations and pharmacologic manipulations to enhance HPC regeneration. Thus inhibition of specific enzymes and pathways, through nutritional measures or drug targeting might be able to influence the ageing process and thus of enormous potential for an ageing society as in the European Community.

In WP2, for example, animal models were employed to study hematopoietic and somatic stem cell ageing. These models permit studying the role of environmental cues, including DNA damage and oxidative stress, on cellular senescence and subsequently on ageing of the organism. This information cannot be achieved to this extend in human. The results will contribute to defining the molecular mechanisms and functional consequences of vascular ageing. WP2 also provides established model systems for validation. These animal models will also represent valuable resources for translation into the clinic, therapy and drug discovery.

The main impact of WP4 (and, arguably, the whole project) is the discovery that ageing in HSCs is very subtle, and the observed skewing towards different lineages is likely due to other factors (i.e. changes in the niche). From a socio-economic perspective, the identification of biomarkers of disease progression in both MDS and B-CLL could also have a significant impact.

In WP5, the drugs identified in this project although need further development have shown very promising results in treating B-CLL. This is particularly relevant for the drugs identified that have a potential to be repurposed as they can reach the patient in a shorter time period. Simvastatin has been evaluated during the course of the project with positive results for safety and efficacy in cell culture. The evaluation of Simvastatin in combination with Ibrutinib and other antineoplastic agents will be pursued further and we will evaluate the possibility of doing a meta-analysis of clinical trials to try to gain further support for a new application for the drug.
Ben 3 by consolidating the collaboration with a clinical partner with extensive know-how on B-CLL and drug testing has strengthened its business position, ensuring that the modeling tools are validated for clinical applications. The collaboration will be extended beyond the project specially aiming to validate the results of the bioactive compounds identified in the project and to move further the potential repurposed drugs.

List of Websites:
http://systemage.eu/

Final Report Summary - SYSTEMAGE (Early warning signals of ageing in human stem cells and age-related disorders)

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