Replication stress in hematopoietic stem cells in mouse and man

Accepted Manuscript Title: Replication stress in hematopoietic stem cells in mouse and man Authors: Johanna Flach, Michael Milyavsky PII: DOI: Reference:

S0027-5107(17)30033-7 https://doi.org/10.1016/j.mrfmmm.2017.10.001 MUT 11624

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Mutation Research

Received date: Revised date: Accepted date:

24-2-2017 31-8-2017 12-10-2017

Please cite this article as: Johanna Flach, Michael Milyavsky, Replication stress in hematopoietic stem cells in mouse and man, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis https://doi.org/10.1016/j.mrfmmm.2017.10.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Replication stress in hematopoietic stem cells in mouse and man Johanna Flach1,#, Michael Milyavsky2,# 1

Department of Hematology and Medical Oncology & Institute of Molecular Oncology, University Medical Center Goettingen, Germany 2

Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel #correspondence: [email protected] or [email protected] Abstract Life-long blood regeneration relies on a rare population of self-renewing hematopoietic stem cells (HSCs). These cells’ nearly unlimited self-renewal potential and lifetime persistence in the body signifies the need for tight control of their genome integrity. Their quiescent state, tightly linked with low metabolic activity, is one of the main strategies employed by HSCs to preserve an intact genome. On the other hand, HSCs need to be able to quickly respond to increased blood demands and rapidly increase their cellular output in order to fight infection-associated inflammation or extensive blood loss. This increase in proliferation rate, however, comes at the price of exposing HSCs to DNA damage inevitably associated with the process of DNA replication. Any interference with normal replication fork progression leads to a specialized molecular response termed replication stress (RS). Importantly, increased levels of RS are a hallmark feature of aged HSCs, where an accumulating body of evidence points to causative relationships between RS and the aging-associated impairment of the blood system’s functional capacity. In this review, we present an overview of RS in HSCs focusing on its causes and consequences for the blood system of mice and men. RS - definition and pathways Broadly, the term replication stress (RS) defines all obstacles that occur during DNA replication causing the replication fork to stall [1, 2] (Figure 1). Accurate genome duplication is by its nature a very complex and challenging process. It requires the action of many molecular players that need to exert their functions in a perfectly concerted manner. In order to preserve the integrity of the DNA and to replicate DNA with high accuracy and efficiency, sophisticated mechanisms have evolved. As detailed below, these are mainly mediated by checkpoint kinases with the overall goal to stabilize stalled replication forks and to block origin firing [3]. Their actions help the cell to survive and faithfully complete replication under conditions of stress. When the replication machinery encounters “simple” problems, such as barriers caused by DNA lesions, insufficient nucleotides, or unusual DNA structures, its activity is temporarily ceased with the overall structure of the replisome remaining intact. This process is called fork stalling. Once the problem responsible for stalling is resolved, DNA replication can resume [4]. Alternatively, if the forks are unable to overcome the damage, they collapse. Fork collapse is characterized by a disruption of replisome integrity with dissociation of involved proteins from the template. It is important to note that although stalled and collapsed forks are formally distinct entities, forks that have stalled for more than a few hours tend to collapse [5]. Collapsed forks often undergo

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endonuclease-mediated DNA cleavage, which results in double-strand breaks (DSBs) and launches a classic DNA damage response (DDR) [4]. When DNA synthesis is blocked due to replication fork stalling, long stretches of single-stranded DNA (ssDNA) are generated by the continued action of the mini-chromosome maintenance (MCM) helicases triggering the recruitment of replication protein A (RPA). Through the action of ATR interacting protein (ATRIP) this leads to activation of ATR kinase. ATR is regarded as the central RS response mediator and phosphorylates numerous proteins, including RPA, RAD17, histone H2AX, as well as its most prominent target, checkpoint kinase 1 (CHK1). CHK1 delays cell cycle progression and downregulates origin firing mainly through inactivation of its downstream target CDC25 [6]. This gives the cell additional time to resolve the damage and to successfully finish DNA synthesis in the vicinity of the stalled forks. In addition, it helps stabilize and restart stalled replication forks and suppress local DNA recombination [4, 7, 8]. DNA helicases, in particular those belonging to the RecQ family, are major players required for the efficient rescue of replication fork stalling [9, 10]. If the RS response fails, activation of dormant origins takes place. Dormant origins are replication start sites that remain inactive in an unperturbed S-phase, but can be activated following RS [11]. Alternatively, the replication machinery can re-start downstream of a lesion that cannot be removed, leaving behind a ssDNA gap [12]. These gaps can then later be filled using a special DNA repair process called DNA damage tolerance (DDT) allowing the cell to complete replication without prolonged fork stalling [13]. However, in cases of a prolonged RS response or when some of its components are lost, forks may eventually collapse resulting in the formation of a DSB. DSBs are generated from collapsed replication forks via the action of topoisomerases or structure-specific endonuclease complexes such as MUS81–EME1 and SLX1–SLX4, and activate a DSB response that relies on ATM and DNAPK kinases [4]. When the cell cycle resumes, unresolved RS is reflected in gaps and breaks on mitotic chromosomes [14]. Recent work has revealed that during anaphase, a subset of under-replicated sites caused by RS remains connected by so-called ultrafine DNA bridges, coated by BLM, BLM-associated proteins and Polo-like kinase 1 (PLK1)-interacting checkpoint helicase (PICH) [15, 16]. After subsequent entry into mitosis, DNA lesions caused by RS are converted to chromatin lesions, which are then transmitted to daughter cells where they become apparent in G1 as very distinct molecular structures called 53BP1 bodies [17]. Alternatively, sites of underreplicated DNA trigger the recruitment of site-specific endonucleases that activate mitotic DNA synthesis by a POLD3dependent mechanism thereby trying to counteract any deleterious effects on DNA integrity [18]. Next, we shall discuss the various origins of RS as well as their relevance to HSC physiology. Sources of replication stress During S-phase, a variety of cellular disturbances can lead to RS (Figure 2). One of the most common sources are unrepaired DNA lesions, which inevitably impose a barrier on the replication machinery causing it to stall [19]. ssDNA at forks can also be generated in response to replication inhibitors such as aphidicolin, which targets DNA polymerase alpha [20]. Alkylating agents and platinum derivatives, which are frequently used in cancer therapy, induce RS by directly modifying DNA leading to the formation of intrastrand or interstrand crosslinks between bases. These crosslinks form a barrier against DNA replication and stop the progression of

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replication forks [21]. Insufficiency or depletion of deoxynucleotide triphosphates (dNTPs) is another way to induce fork stalling [22]. By inhibiting ribonucleotide reductase (e.g. by gemcitabine or hydroxyurea) or thymidylate synthase (e.g. by 5-fluoruracil (5-FU)), the lack of nucleotides results in RS by decreasing the speed of DNA synthesis at replication forks [23]. In addition, RS can be caused by DNA sequences that are challenging to replicate, such as trinucleotide repeats, highly repetitive DNA sequences or complex secondary structures, such as DNA-RNA- structures (R-loops) [24]. Intramolecular G-quadruplexes that preferentially form in guanine/cytosine (GC)-rich DNA sequences or DNA-RNA-hybrids caused by interference of replication with transcription may also impose a problem on the replication machinery. Human telomeres with their repetitive GC-rich sequences are particularly affected by this phenomenon [25] as are genomic regions referred to as common fragile sites (CFSs) [24]. HSC protective mechanisms against RS Tissue-specific stem cells are at particularly high risk for the deleterious effects of RS as they carry the largest regenerative burden for life-long maintenance of tissue homeostasis. They are responsible for the formation, maintenance and regeneration of most tissues in adult organisms due to their unique ability to self-renew and produce all types of differentiated mature cells [26]. The blood system provides an especially good example, as mature blood cells constantly need to be replenished. The integrity of our blood system is maintained by a small number of hematopoietic stem cells (HSCs), whose tight balance between self-renewal and differentiation is crucial to ensure life-long blood production. One of the most important, if not the main strategy to protect HSCs from functional exhaustion is their quiescent cell cycle state. It has been calculated that at steady state, most mouse HSCs enter the cell cycle only once a month [27-29]. Similarly, studies in humans have shown that HSCs divide every 25–50 weeks indicating that the numbers of HSC replications per lifetime are comparable between mouse and human [30]. Quiescence in HSCs is tightly regulated by a complex network of both cell-intrinsic and extrinsic factors that govern the activities of quiescence-promoting cyclin-dependent kinase inhibitors (CKIs) and replication-promoting cyclin-dependent kinases (CDKs) (as reviewed in [31]). Given the importance of quiescence for the maintenance of HSC functionality it is not surprising that loss of quiescence and associated increase in proliferation have been correlated with premature exhaustion of both murine and human HSCs, especially under strong RS triggers such as transplantation [32] or chronic inflammatory conditions [33]. Furthermore, quiescence in HSCs is associated with their preferential use of anaerobic glycolysis and low metabolic activity thereby protecting HSCs from the potentially dangerous side effects of metabolic reactive oxygen species (ROS) as byproducts of activated mitochondria in association with oxidative phosphorylation [34]. What causes RS in HSCs? Despite these protective means RS in HSCs becomes critical under conditions of hematopoietic stress where HSC exit from quiescence and rapid entry into the cell cycle is required to ensure fast recovery and regeneration of the hematopoietic system (Figure 2). Without doubt, HSCs play a pivotal role in hematopoietic reconstitution after repetitive blood loss [35] or myeloablative stress induced by chemotherapy such as 5-FU [36]. The ultimate challenge for HSCs in this context is the transplantation of small numbers of HSCs into lethally irradiated

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hosts, where the need to reconstitute the entire blood system puts them under enormous RS. As a result, after serial rounds of transplantation, HSCs gradually decline in their function and are eventually exhausted [37-40]. As detailed below, any proliferative stress exposes HSCs to RS, which becomes particularly dangerous if repair mechanisms fail. Eventually, this may result in HSC functional impairment reflected in BM failure, aging, or malignant transformation (Figure 2). Inflammatory signaling/infection (acute versus chronic) In response to a depletion of mature blood cells caused by bacterial or viral infection, HSCs exit quiescence and enter a proliferative program. This model of inflammation-driven RS is supported by a number of studies. Treatment of mice with polyinosinic:polycytidylic acid (pI:C), a synthetic double stranded RNA, which mimics a viral infection, pushes HSCs into the cell cycle by inducing pro-inflammatory signaling pathways [41]. Similarly, IFN that is produced in response to a bacterial infection leads to HSC activation [42]. Inflammation is communicated to HSCs either by direct sensing via Toll-like receptors (TLRs) or indirectly via a series of proinflammatory cytokines [43, 44]. These include interferons (IFN), tumor necrosis factor alpha (TNFα), interleukin (IL)-1, IL-6 and transforming growth factor (TGFβ) that all directly impact HSC fate by inducing and accelerating the differentiation of HSCs into cells required to fight the threat [45, 46]. While the short-term integration of inflammatory signals with hematopoietic output may promote effective innate immune host defense in acute conditions, their prolonged exposure can be detrimental to HSCs function. As a result, chronic, unabrogated IFNα signaling leads to HSCs exhaustion [47]. Along this line, chronic systemic exposure to TLR ligands has myelosuppressive effects, as supported by HSC exhaustion seen in mice exposed to repeated administrations of low-dose lipopolysaccharide (LPS) [48]. How inflammatory signaling is communicated to the molecular players of the RS response is not yet understood, but may very well be a side effect of the corresponding proliferative stress. Supporting this hypothesis, type I IFNs (IFNα and β) have been shown to exert their pro-proliferative program through direct inhibition of key mediators of HSC quiescence, including the CKI p57, transcription factor FOXO3A as well as components of the Notch and TGFβ pathways [49]. Very recently, it has been demonstrated that HSCs respond to bacterial infection by activating the ROS/p38 pathway, which could at least partly explain the proliferation-associated HSC functional decline [50]. Furthermore, MYC with its pro-proliferative transcriptional activity is induced in HSCs in response to inflammatory stress [51]. Several clinical scenarios in the hematopoietic system have been linked to excessive pro-inflammatory signaling. CD34+ cells from patients with bone marrow failure syndromes, including aplastic anemia, paroxysmal nocturnal hemoglobinuria and myelodysplastic syndromes (MDS), have been associated with heightened IFN levels [52]. Furthermore, evidence for a direct link between chronic inflammation and the promotion of mutagenesis exists through constitutively activated JAK-STAT signaling in BCR-ABL-negative myeloproliferative neoplasms, which is why these disorders have recently been termed “human inflammation models for cancer development” [53]. It is believed that JAK-STAT signaling not only causes uncontrolled myelopoiesis but also results in aberrant cytokine secretion. The resulting pro-inflammatory milieu in the BM further promotes the acquisition of genetic instability thereby facilitating clonal evolution and disease progression [54]. Metabolism

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Active cell cycling requires increased metabolic activity in HSCs, which comes at the price of elevated ROS production due to activation of mitochondrial oxidative phosphorylation [34]. ROS are a group of oxygen-containing molecules that are involved in both normal cellular signaling processes as well as immune defense. The maintenance of low intracellular ROS levels is vital for HSC function. Excessive ROS (also termed oxidative stress) have been shown to mediate HSC functional decline associated with loss of self-renewal potential, loss of quiescence and increased apoptosis in Atm-/- HSCs [55] and in mice deficient in all three FOXO transcription factors (FOXO1, FOXO3 and FOXO4) [56]. The damaging effects on HSC functional capacity can be rescued by treatment with N-acetylcysteine or inhibition of the downstream signaling molecule p38 [56, 57]. High ROS levels have been shown to cause DNA damage through the generation of highly mutagenic 8-oxo-Guanine (8-oxo-G) adducts that by pairing equally well with the correct cysteine and the incorrect adenine base causes elevated levels of guanine to cytosine and thymine to adenine transversions [58]. 8-oxo-G adducts cause the replication machinery to stall and are therefore a dangerous source of RS [59]. This is further increased by fork collisions with the single-strand breaks caused by the AP endonuclease during the base excision repair process [59]. If not properly repaired, for example due to impaired function of the Fanconi anemia (FA) pathway (Fanca-/-), elevated ROS levels can lead to loss of HSC function resulting in accumulation of DNA damage and ultimately the acquisition of mutations [60, 61]. Along this line, irradiation has also been shown to cause oxidative stress, which continuously rises for days or months after the initial exposure [62]. Through elevated and persistent production of intracellular ROS this serves as a strong inducer of RS further enhancing the genetic instability associated with irradiation [63]. Although ROS are perhaps the most ubiquitous and well known metabolically derived molecules, endogenous aldehydes are another form of highly reactive molecules that can damage the genome. Endogenous formaldehyde is constantly produced within the cell as a byproduct of enzymatic oxidative demethylation reactions [64]. It has potent DNA crosslinker function, which causes replication forks to stall and induce RS. This is greatly enhanced in the absence of functional detoxifying mechanisms by alcohol dehydrogenase 5 (Adh5-/-) or deficiencies in repair activity by the FA pathway (Fancd2-/-), leading to accumulation of DNA damage in HSCs and ultimately their functional exhaustion [65, 66]. Oncogene activation Overexpression or constitutive activity of oncogenes is another source of RS. Although the exact mechanisms are not entirely clear, oncogene activation causes an increase in the fraction of terminated forks, a decrease in replication fork speed, an increase in fork asymmetry, and a decrease in inter-origin distance (as reviewed in [67]). Accelerated cell cycle progression with insufficient supply of dNTPs may promote RS, as does an increased likelihood of interference between replication and transcription machineries [22, 24]. By enhancing transcriptional programs related to cell cycle entry and progression, oncogenic MYC triggers collisions between replication forks and transcribing RNA polymerases [68], a finding that may also be relevant for other oncogenic transcription factors. Likewise, reduced functionality of certain tumor suppressor genes can induce RS. Impaired activity of p53, for example, has been shown to cause RS by impairing replication fork processivity [69], although the cell type as well as the source of RS may be relevant in this context [69-71]. Tumor cells often also have enhanced metabolic

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activity, which indirectly promotes RS through the accumulation of ROS. Not too much is known about oncogene-induced RS specifically in blood cancers. However, upregulation of BCL6, which frequently occurs as a result of translocation in certain forms of lymphoma, inhibits ATR function [72]. This allows B cells to proliferate rapidly and - coupled with mutagenic activity of AID - promotes the acquisition of additional lesions [73, 74]. Furthermore, it has been demonstrated that depletion of SRSF1 or SRSF2, two splicing factor frequently mutated in myeloid neoplasms, gives rise to DNA damage and genomic instability by promoting the formation of R-loops [75]. The presence of certain mutations may also enhance the acquisition of further alterations and perpetuate clonal evolution. Along this line, premalignant mutations in HSCs including TET2/3 and DNMT3A have recently been shown to be associated with increased RS and proposed to promote the accumulation of mutations and chromosomal aberrations that drive progression to malignancy [76, 77]. Consequences of RS on hematopoiesis Under physiologic conditions HSCs are able to cope with RS and associated DNA damage through their DNA damage repair machinery. However, continuous activation of the RS response or failure of any of its players may cause HSCs to functionally exhaust. HSC functional exhaustion is reflected in senescence or increased apoptosis leading to BM failure or the accumulation of mutations that may eventually result in the development of malignancies. Loss of hematopoietic function with time is also a hallmark of physiological aging, where RS has been shown to be a major driver [78]. Senescence and bone marrow failure As soon as HSCs are induced to proliferate in response to hematopoietic stress conditions, intact DDR mechanisms are crucial in order to prevent the emergence of mutations characteristic of human hematopoietic malignancies [79]. These are mediated by cell cycle checkpoints, whose engagements prevent cell cycle progression in response to RS. However, persistent cell cycle checkpoint activation can result in a state of irreversible cell cycle arrest, a phenomenon known as senescence. Senescence is characterized by significant changes in cell morphology and function coupled with widespread changes in chromatin organization and gene expression [80]. The progressive shortening of telomeres after serial rounds of replication is one example that irreversibly leads to senescence [81, 82]. Active telomerase activity - although required to ensure the long replicative lifespan of stem and progenitor cells - is apparently not sufficient to prevent replication-associated shortening of telomeres in HSCs. This is particularly relevant for human HSCs as their telomeres are about 5-10 times shorter than that of mice [83]. However, the effects of telomere shortening in mice can be recapitulated by using successive generations of telomerase knockout mice (Terc-/-) with critically short, dysfunctional telomeres emerging in generation three [84]. Studies on these animals strongly support the idea that checkpoints activated by telomere erosion drive HSC functional decline and bone marrow failure [85, 86]. On the molecular level, senescence is mediated by activation of the p16/RB and p53/p21 pathways, which results in inhibition of Cyclin-CDK complexes [87]. This imposes a brake on the cell cycle, eventually leading to an irreversible cell cycle arrest. A crucial component responsible for the implementation of senescence is the hypo-phosphorylation of the retinoblastoma protein [88]. The exact players that ultimately lead to senescence may vary depending on the

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cell type, which may explain conflicting results being published on p16 with regard to its role in senescent HSCs [89, 90]. As an alternative to senescence, chronic RS may induce HSCs to switch on pro-apoptotic pathways. In response to heightened levels of DNA damage HSCs isolated from human cord blood switch on a pro-apoptotic pathway mediated by p53 in order to maintain homeostasis [91]. A similar function has recently been attributed to ASPP1, which induces apoptosis in HSCs upon increased RS [92]. Heightened levels of apoptosis in HSCs have been shown to be counteracted by the BH3-only protein BID, which serves to enable HSC long-term regenerative capacity. In response to replicative DNA damage, BID acts by amplifying the ATRdirected cellular response and thereby promotes HSC survival [93]. Altogether, these data demonstrate the need for a tight regulation of RS-associated cell fate decisions in order to maintain blood homeostasis and to prevent the emergence of bone marrow failure and hematological diseases. Aging As stem cells are responsible for life-long maintenance of tissue homeostasis, the agedependent decline in regenerative capacity has been directly attributed to their functional decline with limited possibility to replace these cells. During aging, HSCs become functionally impaired. This loss of HSC functionality is reflected in many well-characterized age-associated changes of the blood system, including a decline of erythroid and lymphoid potential, a bias towards myeloid lineage differentiation, and, most strikingly, a decreased HSC repopulating ability upon transplantation [94]. Accumulation of DNA damage associated with heightened levels of H2AX within the HSC compartment has long been regarded as a major driver for the aging-induced impairment of the hematopoietic system in both mice and humans [86, 95]. Mice deficient in components of several DNA repair pathways have recapitulated the agingassociated decline in blood function. These include double-strand repair (Rad50S/S [96]), nonhomologous end joining (Ku80-/- [86], Lig4Y288C [97]), telomere maintenance (mTR-/- [86]), mismatch repair (Msh2-/- [98]), nucleotide excision repair (XPDTTD [86]) and the FA pathway (Fancd2/Brca2Δ27/Δ27 [99]). At least in mice, RS has been identified as a major driver of the ageassociated accumulation of DNA damage, and directly been linked to the deterioration of HSCs with increasing age [78]. In addition, the authors found an association of RS with ribosomal stress pointing to a functional crosstalk between the replication and translation machineries in response to stress. On the molecular level, insufficient replication origin licensing through diminished expression of MCM helicase components has been found to drive the RS features in aged HSCs. The aging-associated HSCs functional decline could also be recapitulated in mice with elevated levels of RS induced by hypomorphic expression of MCM3 [100]. These data suggest that the MCM helicase is a key player in the aging-associated functional decline of HSCs. However, it cannot explain the loss of lymphoid potential, another important hallmark of aging hematopoiesis. A possible answer to this question was recently provided by a study on the protein period circadian clock 2 (PER2), which has been found to promote RS by limiting replication factors [101]. In response to aging, PER2 is activated predominantly in HSCs that are biased towards the lymphoid lineage resulting in apoptosis of these cells [101]. Thus, deletion of PER2 may be a potential future strategy to a potential promising target for the rejuvenation of the innate immune system. Malignant Transformation

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As mentioned above, RS as a source of genomic instability is a major cause of malignant transformation and cancer development [67]. This is most evident in patients with cancer predisposition syndromes, such as Fanconi anemia, which often show defects in RS response genes [102]. At stalled forks, RS can give rise to mutations by an error-prone DDT pathway activity that recruits a specialized low fidelity translesion synthesis (TLS) polymerase [103]. Moreover, recombinogenic processes, which are activated in response to fork collapse following RS, may lead to loss of heterozygosity (LOH) or cause deletions, duplications or translocations [104]. Furthermore, secondary structures resulting from the collision of transcription and replication machineries recruit nucleases that in turn promote chromosome breakage and misrejoining. The MLL locus, which is altered in certain forms of acute myeloid leukemia, has been shown to be particularly affected by this mechanism [105]. This may explain why leukemogenic MLL rearrangements frequently occur in response to therapy with RS-inducing topoisomerase II inhibitors. In addition to problems during S-phase, RS can trigger genome instability by impacting chromosome segregation. Following RS, underreplicated regions of DNA or unresolved replication intermediates may be mis-cleaved by structure-specific nucleases [106]. This may cause problems during chromosome segregation leading to chromosome breakage and non-disjunction. Similarly, aneuploidy has been shown to make cells more susceptible to RS and could thus serve as a driver for clonal evolution. This mechanism may provide a partial answer to the question of why patients with trisomy 21 show an increase in hematological malignancies [107]. In light of these data it is tempting to speculate that by fostering the gradual acquisition of mutations in the HSC pool RS is responsible for the heightened incidences of myeloid malignancies that are seen with advanced age [79, 108-112]. However, recent data revealed only a two to three-fold increase in mutation frequency in murine HSCs upon physiological aging [113], which on its own is not enough to induce leukemia [114]. In line with this, mice deficient in major players of the RS response such as ATR do not show an increase in leukemogenesis [115]. In contrast, RS plays a critical role in the etiology of therapy-induced leukemia, whereby HSCs are forced into rapid replication as a result of chemotherapy-associated cytopenia [116]. Altogether, it appears that additional triggers are needed, such as failure in DDR system, to allow the shift to a malignant phenotype. Cancer cells with high levels of RS become dependent on the ATR-CHK1-mediated RS response for their survival [117]. This has led to numerous in vitro and in vivo studies, which aim at exploiting the dependency on RS signaling therapeutically by selectively impairing the repair of tumor-specific DNA damage. Promising results from preclinical studies have led to the development of an array of compounds targeting mediators of RS signaling both in solid tumors and leukemias [21, 118, 119]. Inhibitors of ATR and CHK1 are two classes of compounds that have so far entered clinical trials either as single agents or in combination with other chemotherapeutic agents. While initial candidate drugs including UCN-01, AR458323, PF00477736 and AZD7762 lacked efficacy and showed rather non-specific effects accompanied by serious side effects, promising results are being reported with the newest generation of CHK1 inhibitors, including MK8776 [120]. However, larger clinical trials have not been conducted yet. As the major indication of these novel agents have so far been solid tumors, limited information exists on their clinical relevance in hematological malignancies.

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RS in human HSCs: the long road ahead Research into RS in human HSCs has lagged behind that of murine counterparts due to our inability until recently to isolate and genetically manipulate candidate human HSCs and obtain durable engraftment in the immunodeficient animals. Fortunately, in recent years, markers that permit isolation and maintenance of the relatively homogenous population of human HSCs capable of multi-lineage, long-term engraftment and serial transplantation in highly immunodeficient mice have been described [121-123]. In this context, Ando and colleagues found incremental increase of ROS and oxidative DNA damage in human umbilical cord derived HSCs in which RS was induced by serial transplantation [124]. Similarly to the transplantationinduced elevation of RS markers in the newborn HSCs, they also detected induction of ROS and γH2AX foci in HSCs isolated from elderly individuals and transplant patients. Statistically significant inverse correlation was observed between the number of γH2AX foci in HSCs and their engraftment potential in the immunodeficient murine recipients. Serial transplantation experiments as well as in vitro induction of intracellular ROS levels convincingly demonstrated that HSCs, but not progenitors, specifically predisposed to oxidative DNA damage that restricts their self-renewal capacity [124]. Interestingly, the source of elevated ROS in human HSCs triggered to undergo intensive replication as in the case of transplantation remains unknown and may be linked to recipients’ bone marrow microenvironment [39]. Significant improvement of human HSCs serial transplantability via antioxidant treatment of the recipient animals provides strong support for the idea that elevated ROS levels are the primary cause of HSC deterioration upon RS [124]. Although many factors regulating ROS levels and protecting human HSCs from endogenous and exogenous RS remain unknown, p53 tumor suppressor was implicated in reducing γH2AX foci levels in the immature human hematopoietic cells isolated from secondary recipients [91]. Moreover, wild type p53 facilitated resolution of stalled replication forks in CD34+ human cells undergoing cross-linker-induced RS [70]. Importantly, HSCs from older individuals (>60 years) had inferior recovery of multilineage hematopoiesis at one year post-transplantation relatively to the young (<50 years) HSCs even at the context of autologous transplantation further supporting the notion that aging impairs HSCs capacity to withstand RS associated with transplantation [125]. Of note, decreased quiescence status rather than elevated ROS levels characterized human HSCs in the context of autologous transplantation [40]. In the future, toxic conditioning regiments, prior chemotherapy and host immunity status (CMV reactivation, graft versus host disease) that can profoundly affect the HSC niche should be evaluated as important predictors for human HSC function under transplantation induced RS. In the last several years, with the advent of next-generation sequencing, a number of breakthroughs were made in the attempt to quantitate the number of DNA mutations accumulated in human HSCs, as well as, to determine clinically relevant associations between particular mutations and leukemia / mortality rate [126]. Firstly, age-dependent increase at the rate of 1.3 +/- 0.2 somatic exonic mutations per decade in long-lived self-renewing HSCs was estimated, supporting the notion that HSCs are repositories of both neutral and potentially hazardous changes [79, 108]. Strikingly, these mutant HSCs recurrently containing mutations in DNMT3A, TET2, JAK2, ASXL1, SF3B1 and SRSF2 genes (now named pre-L-HSCs) are capable of normal differentiation, but also undergo clonal expansion, persist and further expand following induction chemotherapy and can account for 10-50% of normal hematopoietic cells during the

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remission period [112]. Secondly, deep sequencing of blood cells from multiple lineages obtained from healthy adults reported clonal hematopoiesis (defined by unusual variant allele frequency) that was very rare (<1% of individuals) before the age of 60 and rising dramatically (up to 20-95%) in individuals > 90 years old [108-112]. The presence of pre-L-HSCs in frequencies that meet the definition of clonal hematopoiesis significantly increased the risk for leukemia development (hazard ratio, 12.9) [109] and all-cause mortality (hazard ratio, 1.4) [111]. Although the clear reasons for old age-associated drastic increase in clonal hematopoiesis is currently unknown potential links to the age-associated RS in HSCs should be considered. For instance, increase in oxidative DNA damage load with each cell division, decrease in telomere length, increased inflammation in the bone marrow microenvironment and changes in DNA repair capacity as discussed earlier capable to induce RS in HSCs [127]. In many cases such RS would inactivate HSC self-renewal capacity by one of the earlier discussed mechanisms (apoptosis, differentation or senescence) and thus RS-associated DDR will play a tumor suppressive function. However, it is possible that certain mutations in HSCs acquired as a result of replication errors would confer selective clonal advantage to HSCs. Indeed, when transplanted into immunodeficient mice pre-L-HSCs (DNMT3Amut) outcompeted non-mutated HSCs from the same patient, consistent with their clonal expansion in the elderly [112, 128]. Moreover, CRISPR/Cas9 mediated genome targeting of TET2 and DNMT3A loci in normal human HSCs led to the clonal expansion of the mutant HSC clones upon xenotransplantation over the course of 5 month [129]. These results further strengthen the link between certain pre-leukemic mutations, clonal hematopoiesis and RS. Taking into account that clonal hematopoieisis represents a human-specific aspect of HSCs aging that is not observed in laboratory mouse strains, this powerful genome engineering strategy followed by xenotransplantation allows accurate modeling and identification of RS related mediators in human HSCs. Concluding remarks As we tried to emphasize in this review, genome replication represents an extremely complex stage in the life of HSCs with important implications for the process of hematopoiesis. Inevitable replication-associated DNA damage or RS lead to replicative exhaustion and degeneration of blood tissue with age. Moreover, a recent though-provoking study emphasized that the total number of stem cell divisions over the life time of the individual heavily influence the chances of cancer development, including leukemia [130]. Intensive research of RS associated biology in the last half-century has identified hundreds of genes that form countless intracellular and extracellular functional interactions to accomplish RS provoked responses, such as resolution of stalled replication forks, S-phase arrest and reinitiation of DNA replication, cell cycle arrest, apoptosis and others (Figures 1, 2). Although just at the beginning, research into RS of the self-renewing HSCs already contributed to the growing knowledge of the molecular origins of hematopoietic tissue aging, regeneration and leukemia susceptibility. In particular, insights into the HSC-specific DDR have revealed principles utilized by highly regenerative tissues to cope with DNA damage. Based on the available experimental evidences, some of which we outlined in this review, a number of principles utilized by HSCs to cope with RS can be drawn. First, HSCs minimize their encounter with RS by virtue of infrequent divisions during the life span of the host and tight preservation of quiescence. Second, HSCs, as opposed to progenitors, are hypersensitive to the elevated ROS levels that can impair DNA

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replication, induce RS and suppress their regeneration. Accordingly, antioxidant treatment and or quiescence restoration are effective in reversing ROS-induced decline in HSCs self-renewal [124]. Third, physiological aging, chronic inflammation or experimental/clinical transplantation represent strong triggers for HSC-associated RS that profoundly affect their function. Finally, regulators of RS, including ATR and CHK1 kinases represent promising targets to treat hematological malignancies, whereas MCM helicases activators might improve aged HSC functionality. Exciting discoveries of recent years regarding HSC replication regulation under normal and pathological conditions have challenged the scientific community with many unresolved questions. For example, how replication history and associated RS is recorded by HSCs and whether this mechanism can be reset or modulated? Epigenetic configuration of the individual murine HSC was recently proposed to account for their behaviors under steady state as well as various stress conditions [131]. Whether these epigenetic changes can account for aging associated changes in MCM levels in HSC remains to be determined [78]. Finding direct marker/s suitable for measuring RS in HSCs or their microenvironment in vivo represent an additional area of active investigation. Having such a marker in our disposal will allow monitoring and predicting HSC functionality before their use in bone marrow transplantation setting. New strategies to manage RS in HSCs are most needed. For example, reducing RS during normal HSC aging might well counteract human age-associated hematopoietic decline. Antioxidants and fasting based strategies both demonstrated promising results [124, 132] although further research is required to substantiate their roles in HSC functional decline [133]. On the contrary, reducing RS by overexpressing CHK1 in mice promotes malignant transformation [134] pointing to the need of better characterization of RS in the context of carcinogenesis. Niche-dependent factors that induce RS in HSCs have recently emerged and suggest that reducing mesenchymal inflammation [135] or inhibiting TGFβ axis [136] therapeutically improve HSC regeneration. Last, but not least, whether age associated pre-L-HSC evolvement and striving can be prevented or attenuated by modulating RS? Modeling of human clonal hematopoiesis using CRISPR/Cas9 genome engineering technologies coupled with robust xenotransplantation hold the potential to solve this question. In this review, we tried to summarize the current knowledge on RS and its consequences on HSC function in model organism, such as laboratory mouse, and humans. Given the vast differences in the life span, body size, microorganisms exposure preclude direct extrapolation of findings from one species to the other. Future collaborative research into RS signaling and its consequences for blood regeneration of rodents and humans holds the promise to better manage the risk involved in HSCs’ DNA replication journey.

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Acknowledgments Research in the MM laboratory is supported in part by grants from Israel Science Foundation (ISF), Varda and Boaz Dotan Research Center in Hemato-Oncology, Israel Cancer Research Fund (ICRF) and The German-Israeli Foundation for Scientific Research and Development. JF is supported by a scholarship from the K.H. Bauer-Program of the Goettingen Comprehensive Cancer Center (G-CCC) of the University Medical Center Goettingen. The authors apologize to their colleagues whose important work was not cited due to space limitations.

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Figure Legends

Figure 1. Molecular translation of replication stress (RS) onto major players of the RS response in HSCs. Shown here are the main mechanisms by which RS is induced in HSCs on the molecular level. They result in activation of the ATR-CHK1 pathway, whose cellular responses are depicted as well. ROS, reactive oxygen species; dNTPs, deoxynucleotide triphosphates

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Figure 2. Causes and consequences of Replication Stress (RS) on HSCs. The main mechanisms inducing HSC replication are summarized as well as some of the known additional triggers necessary to cause RS-associated damage to the blood system. IL, interleukin; LPS, lipopolysaccharide; IFN, interferon; pI:C, polyinosinic:polycytidylic acid; 5-FU, 5-fluoruracil; DDT, DNA damage tolerance; TLS, translesion synthesis

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