the project

AGE-RELATED CHANGES IN HEMATOPOIESIS

The aim of ARCH is to understand the causal relationship between the physiological changes in the hematopoietic system during lifespan and the occurrence of specific age-related hematological diseases, towards the development of new therapeutic treatments.

To reach this goal, the ARCH network brings together 15 beneficiaries (12 academic, 2 SMEs and 1 private research institute) and 5 Partner organisations (3 academic, 1 SME and 1 patients’ association); 2 European Consortia are represented through their italian nodes.

The ARCH network, through its training programme, will provide 15 highly qualified European experts in the field of Molecular Hematology needed to address the increasing medical and socio-economical burden inflicted by hematological diseases to the European population.

The clinical management of hematological diseases represents for the European Healthcare a serious medical challenge and an important socio-economical burden, which is expected to increase in the next few years, because of the increased longevity of the European population. An estimated 80 millions people are currently affected by blood disorders in Europe. Despite the enormous progress in diagnosis and therapy in the last decades, about 115.000 patients still die each year from these disorders, with a cost to the Healthcare in Europe of more than 20 billions Euros per year[1]. Even more important, hematological disorders have a high impact on the quality of life of patients and their families.

Ageing is associated with an increased risk of acquiring various blood disorders, either malignant (myelodysplastic syndromes, myeloma, lymphomas) or non-malignant (such as anemias, primary immunodeficiency and coagulation disorders). On the other hand, pediatric blood disorders also represent a serious medical challenge. The different incidence of different blood disorders at different ages clearly suggests that they might reflect the physiological changes at the DNA or protein levels in hematopoietic cells and in the environment where they mature (the niche) in response to different stimuli throughout life.

Despite this evidence, the knowledge of these molecular mechanisms, indispensable for a more accurate patients’ stratification and for the design of innovative treatments targeting specific pathways/mutations (“personalized medicine”), is still poor. Moreover, although gender differences in incidences and outcome of hematological diseases have been described, at present, they are poorly understood.


Scientific background

The hematopoietic system: Hematopoietic stem cells (HSCs) represent a rare population, residing in the Bone Marrow (BM), at the top of a hierarchy of progenitors that become progressively restricted to multiple and ultimately single, specific lineages to generate all mature blood cells. In turn, mature blood cells can signal back to the BM niche to respond throughout life to both physiological homeostatic demand and to stress conditions (Fig1)[2]. In particular, stress signals such as interferons, lipopolysaccharide or chemotherapy activate dormant HSCs, a sub-population within the most refined HSCs. These highly quiescent cells are metabolically inactive, harbor the highest long-term reconstitution potential within the hematopoietic system and serve as a reserve pool for situations of emergency. This fine forward-and-feedback crosstalk is regulated by both extrinsic and intrinsic factors[3]. Extrinsic factors mostly derive from the BM microenvironment – composed of different cell types, extracellular matrix, growth factors and chemokines – which provides support to hematopoietic stem/progenitors cells (HSPCs)[4]. Intrinsic factors entail the expression of a repertoire of specific transcription factors, epigenetic modifiers, microRNA, long-noncoding-RNA and splicing factors governing lineage specification and differentiation[5].

Hematopoiesis throughout life (Fig. 2): Ageing, as a natural process, undergoes the same major rules of biology, namely natural selection and evolution: HSPCs change their properties during lifespan, as they undergo selective pressure and repeated evolutionary bottlenecks in response to environmental and developmental cues. With ageing, this process promotes the progressive emergence and expansion of selected HSPCs clones carrying potentially detrimental genetic variants. The resulting reduced genetic diversity of HSPCs outcomes in deteriorate regenerative potency, the major cause of anemias and primary immunodeficiency, a common and very serious comorbidity factor in the elderly[6]. Clonal hematopoiesis is associated with pre-leukemia and a high risk of developing blood cancer[7]. Recently, the clear link between the accumulation of DNA damage in HSPCs and ageing has emerged as major contributing factor in age-related clonal expansion, tissue degeneration and malignant transformation[8]. Leukemias are classified based on the affected cell type. Interestingly, Acute Lymphoblastic Leukemias (ALL) have a more severe prognosis in adults than in pediatric cases, whereas Acute Myeloid Leukemia (AML) is prevalent in adults. Moreover, Chronic Leukemias are more common in people between ages 40 and 70. Finally, Myelodysplastic Syndromes (MDS), in which the bone marrow dysfunction is coupled with the expansion of specific hematopoietic progenitors, often at the expenses of other cell types, are often found in patients approaching their 60s and 70s. These observations suggest that these cancers might reflect and undergo, in their onset and evolution, age-related changes in hematopoiesis, in a so far largely unknown manner[9]. It is thus important to understand the causal relationship between the genetic lesions causing the different types of leukemia and the physiological age-related changes in hematopoiesis. This opens many potential opportunities to understand key factors triggering leukemia in different age groups and to find options to postpone or to prevent it.

Research methodology and approach

ARCH will integrate genetic, epigenetic, metabolic, molecular, biochemical, cellular and genomic approaches, including known and recently developed functional assays, such as lentiviral-mediated overexpression, RNAi knockdowns, CRISPR/Cas9 knock out and genome editing, in vitro and in vivo hematopoietic stem cell assays, transplantation studies, transgenic and knockout mouse models, organotypic cultures, single cell analysis, functional genomics, epigenomics and bioinformatics data analysis (as detailed here below and outlined in Fig. 3).

ARCH is organized in three coordinated scientific WPs, focused on strictly interconnected aspects.

WP1 will study physiological changes in ageing HSCs and the impact of the impairment of DNA damage response (DDR) on HSCs ageing. The aim of WP1 is to pinpoint common genetic changes (in gene expression, metabolic changes and/or in epigenetic regulation) of particular mutations and/or genetic variants that influence the physiological behavior (repopulation capability in transplantation/serial transplantation assays, differentiation potential, stress-related function) of aged HSCs (ESR2) and of the dormant HSCs compartment (ESR8). ESR2 (UMCG) will exploit a specific experimental design in which single cells HSCs of different age/genetic background, are labeled by using cellular barcoding tools and traced in vivo. ESR3 (CERBM) and ESR4 (KCL) will study the effect of the perturbation of genes crucial for genome integrity, such as Helios and ERCC1 on ageing HSCs. These studies will mainly rely on mouse models, so far considered the most informative model system for the in vivo study of hematopoiesis[1]. Primary human cells from peripheral blood from donors of different ages will be carefully phenotyped by Flow Cytometry (ESR5, FME) to set reliable diagnostic reference panels (based on B-cells, T-cells and monocytes markers) that reflect phenotypical differences in functional potential between subpopulations, from HSCs to multipotent progenitors and differentiated populations (WP1, WP3).

WP2 is focused on the genetic and epigenetic control of transcription in normal and leukemic cells. The aim of WP2 is to elucidate the molecular basis of the function of some key TFs (MML fusion proteins: ESR7, VETMEDUNI; Sox6 and NR2F2: ESR1, UNIMIB), the contribution of epigenetic mechanisms (Polycomb repressive complexes: ESRs 9-10, CRCM and CSIC) and of the epitranscriptome (ESR6, UNIROMA1) in regulating lineage specification in normal and aberrant leukemic differentiation.

WP3 explores the role of intrinsic and niche-dependent signaling in acute leukemias. The aim of WP3 is to investigate specific aspects of the cross-talk between intrinsic and extrinsic signaling regulating HSCs proliferation/differentiation to identify novel molecular pathways amenable to therapeutic intervention. In particular, ESR11 (IC) will study the biological/therapeutic response of T-ALL xenografts in immunodeficient (NSG) mice to monotherapy using CXCR4 and CXCL12 inhibitors. ESR13 (FT) and ESR14 (DIAGENODE) will develop epigenomics tools to identify variable regions that could account for MLL-rearranged leukemia specificity in order to implement diagnostic/ prognostic protocols. ESR15 (BRFAA) will investigate STAT5 signaling in AML and the role of STAT5 target gene networks in leukemia progression. Finally, ESR12 (SCHNEIDER CHILDREN’S) will explore the role of the thymic stromal lymphopoietin TSLP/TSLPR axis in Acute Lymphoblastic Leukemia.

Originality

The original approach of ARCH is to center its research and training programme on the characterization of hematological diseases in the light of the corresponding physiological age-related evolution of hematopoiesis throughout life.

We believe that this parallel study of normal and pathological hematopoiesis is totally novel and will result: i) in the development of novel treatments to postpone the physiological age-related deterioration of hematopoiesis ii) in the improvement of therapeutic strategies for treating pre-leukemic conditions and leukemias iii) in the development of diagnostic markers for a better stratification of patients and a consequent better management of hematological diseases.

[1] Engert A et al 2016 Haematologica 101:115-208 and references therein; http://www.who.int/topics/global_burden_of_disease/en/; http://www.leukemia-et.org/content/home/eln_meetings/eln_symposia/previous_meetings/e8206/infoboxContent8207/ELNHealthEconomicsBJ_pdf.pdf.

[2] Orkin&Zon Cell 2008, 132:613; Wilkinson&Göttgens, Adv. Exp. Med. Biol. 2013, 786:187; Orkin  Nat. Rev. Genet. 2000; Fig 1 from Marciniak-Czochra et al. Aging 2009, 723-732.

[3] Baldridge et al. Nature 2010; Essers et al. Nature 2009; Walter et al. Nature 2015, Cabezas-Walscheid et al. Cell 2017).

[4] Orkin&Zon Cell 2008, 132:613;Regan&Rosen Nat. Rev. Rheum. 2015 doi:10.1038/nrrheum.2015; Scadden Nature 2006 441:1075; Mendelson & Frenette2015 Nat. Med. 20:833.

[5] Orkin&Zon Cell 2008, 132:613;Wilkinson& Göttgens, Adv. Exp. Med. Biol. 2013, 786:187; Orkin Nat. Rev. Genet. 2000, 1:57; Cabezas-Wallscheid  et al. Cell Stem Cell 2014, 15:507; Cullen et al Curr. Top. Dev. Biol. 2014;107:39; Shivasdani Blood 2006 108:3646; Lazare   Exp. Cell Res. 2014, 329:234; Morlando et al. Front. Med.  2015 2:23.

[6] Rossi et al. PNAS 2005,102:9194; Shlush, Int. J. Hematol. 2015,102:513; Artandi et al. Cell Stem Cell. 2015 16:578; Fukada Front. Cell. Dev. Biol. 2014; 2:10.

[7] Liran Shlush, Blood, 2017; Cooper & Young, Blood, 2017, 130:2363.

[8] Rossi DJ et al. Cell 2008, 132:681–696; Walter D et al. Nature 2015, 520:549-564.

[9] Wahlestedt et al. Stem Cells Transl Med. 2015, 4:186; Kikushige et al. Int J Hematoll. 2014,100:335; Klepin et al. J Clin.Oncol.2014 2532:2541;Woolthuis et al. Curr Opin Immunol. 2011 23:512.

[10] Sykes SM, Scadden DT Semin Hematol. 2013 Apr;50(2):92-100.