Myc induced replicative stress response: How to cope with it and exploit it.

BBAGRM-00707; No. of pages: 8; 4C: 3 Biochimica et Biophysica Acta xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagrm

Review

Myc induced replicative stress response: How to cope with it and exploit it☆ Sara Rohban, Stefano Campaner ⁎ Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia (IIT), Via Adamello 16, 20139 Milan, Italy

a r t i c l e

i n f o

Article history: Received 19 January 2014 Received in revised form 7 April 2014 Accepted 8 April 2014 Available online xxxx Keywords: Myc Replicative stress DNA replication ATR Chk1

a b s t r a c t Myc is a cellular oncogene frequently deregulated in cancer that has the ability to stimulate cellular growth by promoting a number of proliferative and pro-survival pathways. Here we will focus on how Myc controls a number of diverse cellular processes that converge to ensure processivity and robustness of DNA synthesis, thus preventing the inherent replicative stress responses usually evoked by oncogenic lesions. While these processes provide cancer cells with a long-term proliferative advantage, they also represent cancer liabilities that can be exploited to devise innovative therapeutic approaches to target Myc overexpressing tumors. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology. © 2014 Published by Elsevier B.V.

1. Introduction c-Myc is a transcription factor acting as a master regulator of genes involved in cell cycle progression, cell growth, differentiation, metabolism and apoptosis. It is also a potent cellular oncogene that is found frequently deregulated in human cancers. This pathological upregulation is frequently due to chromosomal translocations leading to promoter rearrangement [1–5], gene amplification [6,7] or viral mediated insertional mutagenesis [8,9]. Alternative mechanisms of deregulation found in cancers are represented by point mutations in the coding region, potentially leading to gain of function [10,11] and genetic polymorphisms identified in distant regulatory region(s) which are activated in a tissue and cancer specific fashion [12]. c-Myc belongs to the basic Helix-Loop-Helix Leucine Zipper (bHLHZip) transcription factor family. These domains are located at the C-terminus where the basic region, needed for DNA interaction, is followed by the Helix-Loop-Helix [13] and the Leucine Zipper domains [14]. The latter domains are both required for the interaction with Max, the obligatory Myc partner needed for the formation of the transcriptionally active heterodimers [15,16]. The N-terminal domain possesses transactivating properties [17] and contains four regions called Myc homology boxes which are highly conserved within the other members of the Myc family (i.e. N-Myc ☆ This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology. ⁎ Corresponding author at: Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia (IIT), Via Adamello 16, 20139 Milan, Italy. Tel.: + 39 0294372407. E-mail address: [email protected] (S. Campaner).

and L-Myc). Myc can also repress gene transcription by tethering other transcription factors like Miz-1 at the promoter of selected genes [18–20]. Recent genome wide studies have highlighted the pervasive nature of c-Myc dependent transcriptional programs with an estimated 25,000 genomic sites bound by Myc which include RNA pol III and pol I dependent genes as well as non coding RNAs [21–24]. This global transcriptional regulatory role of Myc accounts for its pleiotropic activity in regulating cellular processes. In this review we will concentrate on c-Myc ability to regulate DNA replication by providing an overview of how DNA synthesis is controlled during S-phase, describing how the overexpression of c-Myc can ensure a productive DNA synthesis avoiding the pitfalls of replication stress responses and we will conclude by highlighting how this knowledge could be exploited for therapeutic intervention in cancer. 2. Chapter 1. DNA replication in mammals Faithful cell cycle progression requires that DNA is precisely and efficiently duplicated during S-phase. Given the large size of the metazoan genome, higher eukaryotes had to evolve mechanisms to allow DNA synthesis from a sufficient number of independent replication origins to ensure timely and co-ordinated DNA duplication. Yet, the activity of DNA replication origins has to be controlled in order to ensure that each replication origin would fire only once every cell cycle thus avoiding the re-replication of the genome, which would lead to regional genome amplification. The correct regulation of DNA synthesis is achieved by a process called replication origin licensing, which is established in late mitosis and early G1, well before cells enter S-phase.

http://dx.doi.org/10.1016/j.bbagrm.2014.04.008 1874-9399/© 2014 Published by Elsevier B.V.

Please cite this article as: S. Rohban, S. Campaner, Myc induced replicative stress response: How to cope with it and exploit it, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbagrm.2014.04.008

2

S. Rohban, S. Campaner / Biochimica et Biophysica Acta xxx (2014) xxx–xxx

Origin licensing begins with the recruitment of the Origin Recognition Complex (ORC) onto DNA replication origins along with the two licensing factors Cdc6 and Cdt1. Once this complex is loaded it allows the recruitment of the minichromosome maintenance (MCM) complex which is composed of six essential replication proteins (MCM2-7) that are though to act as helicases needed to unwind the template DNA ahead of the replication fork. Overall, this multi-protein complex, termed the prereplicative complex (pre-RC), is loaded onto DNA before S-phase entry [25,26]. Replication is then initiated by the action of S-phase cyclin dependent kinases and Cdc7, which promotes the binding of Cdc45 and GINS to the pre-RC [27,28]. DNA is then unwound by the Cdc45-MCM2-7GINS (CMG) complex [29–32], which also allows the loading of DNA polymerase-α to initiate DNA synthesis [33–35]. In order to avoid rereplication the licensing system has to be shut down before entry and during S-phase progression, therefore a tight regulatory mechanism is set in place to prevent intra S-phase re-licensing. This is based on the efficient proteasomal degradation of Cdt1 which is regulated by the Sphase specific activity of the cyclin A/Cdk2 complex and the recruitment of the processivity factor PCNA on the replicative fork. In addition, the expression of Geminin and its nuclear targeting provide an efficient mechanism to inactivate Cdc6 during S-phase. Consistent with this model, the deregulation of either Cdt1 or Cdc6 has been shown to cause DNA re-replication [36]. Given the crucial role of replication forks during DNA replication, eukaryotes have devised a number of safeguarding mechanisms to improve the robustness of the system: not only replication origins are fired in excess over the minimum number required to complete S-phase in a timely manner, but also cells, during G1, load onto DNA an excess of MCM molecules relative to the number of replication origins that will be used during DNA synthesis [37–39]. This renders cells relatively resistant to the fluctuation of the MCM levels [38–40] and provides a reservoir of dormant origins that may be used by cells as a back-up in the event of irreversible stalling of the active forks. Experimental evidences suggest that excess of MCM2–7 is required for cells to properly cope with replicative stress [41–43].

suggesting an important role for D-type cyclins in c-Myc-mediated transformation [63]. On the other hand, c-Myc represses antiproliferative genes like the Cdk inhibitors p21 [64–66] and p15INK4A [67,68], through an interaction with the Miz-1 protein at the core promoter, and several other genes involved in growth arrest, such as Gadd45 [69] and Gas1 [70]. Thus c-Myc regulates a variety of genes directly involved in cell cycle regulation. Complementary to this, c-Myc manages the supply chain of metabolites needed for DNA replication by transcriptionally regulating the majority of the genes involved in purine and pyrimidine biosynthesis pathways, these genes not only are bound and transcriptionally regulated in cell lines but also are responsive to c-Myc activation in the liver of transgenic mice [71,72]. Of note, chemical inhibition of the c-Myc targets inosine monophosphate dehydrogenases (IMPDH1 and IMPDH2) results in S-phase arrest and apoptosis suggesting that balancing the nucleotide pool is essential for c-Myc's orchestration of DNA replication, to the point that the uncoupling of these two processes creates DNA replication stress and apoptosis [72]. Rather unexpectedly, c-Myc can also promote Sphase via a transcription independent function. Indeed the c-Myc protein was shown to localize at nuclear sites coinciding with active ongoing DNA replication, possibly representing early replicating origins. This was supported by the identification of high molecular weight complexes containing c-Myc and the pre-RC complex but devoid of proteins involved in DNA replication elongation (MCM10, RPA and PCNA). The use of Xenopus extracts which are transcriptionally incompetent, suggested a transcriptional independent function of c-Myc in controlling S-phase progression possibly by directly participating in licensing or assembly of (pre)-replicative complexes [73]. Thus, the S-phase promoting activity of c-Myc relies on its direct activation of the basic cell cycle machinery (cyclins and Cdks), the transcriptional regulation of pathways directly linked to DNA metabolism and direct regulation of the firing of early replisomes. The combined action of these different processes results in a harmonic acceleration of DNA synthesis where the increased number of highly processive replisomes is fuelled by a robust supply of nucleotides.

3. Chapter 2. Myc and DNA replication 4. Chapter 3. Origin of DNA damage in Myc overexpressing cells Since its early identification as an avian oncogene, several observations linked c-Myc to cellular proliferation. Its mRNA was shown to be quickly expressed upon mitogenic stimulation of quiescent cells [44] and kept to steady levels during cell cycle progression [45]. Conversely, anti-proliferative signals were shown to quickly downregulate c-Myc mRNA abundance [46,47]. Moreover, c-Myc expression was not only associated with cellular growth but also with allowed cellular proliferation in reduced serum conditions, in tissue culture cells [48]. In addition, the conditional activation of c-Myc in quiescent cells induced cell cycle entry and promoted cellular proliferation in vitro [49,50] and in vivo [51,52]. c-Myc ability to directly control DNA synthesis is integral to its role in supporting cellular growth. Indeed, overexpression studies have evidenced how ectopic expression or conditional activation of c-Myc triggers an increase in the percentage of S-phase cells in asynchronous populations. This increase is frequently matched by an overall acceleration of the S-phase which is achieved by increasing the number of firing replication origins [53–55]. This ability of c-Myc to promote cellular proliferation stems from its proficiency, as a transcription factor, to directly control the expression of a large number of genes implicated in S-phase entry and progression [56]. First, elevated c-Myc accelerates progression through G1 by positively regulating the expression of several cyclins and cyclin dependent kinases (Cdks) like cyclins D1 and D2, Cdk4, and cyclin B1 [57–60] and many others, as suggested by the recent genome wide studies [21]. This regulation is not only relevant in physiological conditions but also linked to the oncogenic function of c-Myc: in vivo genetic studies have suggested a role for Cdk4 in c-Myc mediated transformation [61], while Cdk2 is required to repress Myc induced senescence [62]. In addition, fibroblasts lacking cyclins D1–3 fail to be transformed by c-Myc,

The overexpression of c-Myc in a number of in vitro cellular systems has been associated with the activation of a DNA damage response (DDR) [74–76] and increased genomic instability [77–81] thus suggesting that elevated c-Myc levels lead to the accumulation of DNA damage. These observations have been complemented by in vivo studies with c-Myc transgenic mouse models showing that tissue specific deregulation of c-Myc is associated with a DNA damage response [74–76]. Although the molecular details are ill defined, it is likely that c-Myc deregulation affects DNA stability in different ways thus possibly leading to the rampant genomic instability observed in some c-Myc dependent tumor models [77–80,82]. Several evidences suggest that c-Myc induced genomic instability is multifactorial. First Myc over-expression can cause DNA oxidative damage by enhancing mitochondrial biogenesis and cellular metabolism since it may boost cellular metabolism in the absence of the appropriate compensatory mechanisms that normally scavenge free oxygen radicals, thereby exposing cells to elevated ROS which will oxidatively damage DNA [83–85]. This is supported by the observation that the accumulation of reactive oxygen species (ROS) is frequently associated with c-Myc activation [83,86,87]. Another source of genome instability may be related to c-Myc ability to regulate telomere biology. Although, a mechanistic insight is still missing, there is evidence that conditional activation of c-Myc affects the spatial distribution of telomeres in the interphase nuclei leading to a strong aggregation of telomeres. This in turn associates with an increase in the frequencies of end-to-end chromosomes fusions suggesting that this nuclear reorganization affects telomere function by promoting breakage-fusion-bridge cycles [88].



3

5. Chapter 4. The Myc induced DNA damage response and the control of replication dynamics

Yet the strongest and most vicious type of damage induced by c-Myc is generated during DNA replication and is linked to the control of replication forks dynamics, which may become difficult to manage when cells overexpress c-Myc. A first hint connecting c-Myc induced DNA damage to replication forks came from studies showing that ectopic c-Myc activation in primary cells led to cell cycle progression up to the G2 phase, where cells arrested due to the activation of a p53 dependent checkpoint, thus suggesting that unrepaired DNA damage, possibly generated in Sphase, triggered the activation of a DNA damage checkpoint [89]. Direct evidences linking c-Myc induced DDR to S-phase, came from work in Xenopus extracts where the lack of transcriptional activity and possibly other protective factors (discussed later) exacerbates c-Myc induced DDR. Indeed, while the overexpression of c-Myc in Xenopus extract triggered DNA synthesis, kinetic analysis revealed that after an initial phase of efficient synthesis there was an arrest coinciding with the DDR dependent activation of an ATM/ATR checkpoint [73]. These observations are supported by reports showing that in normal human cells the overexpression of c-Myc directly increases S-phase rate by enhancing the number of replisomes actively firing and it also triggers a replicative stress response characterized by the increase in RPA coated single stranded DNA enriched for γH2Ax and BrdU [54,73]. More recently, using biochemical experiments with frog extracts combined with DNA combing assay, Gautier and colleagues, analyzed c-Myc induced replication forks dynamics and elegantly showed that elevated levels of c-Myc enhanced the number of replication origins. These forks were highly asymmetric suggesting uneven replication processivity on either side of the replication bubble, which was indicative of replication stress and fork stalling events. Importantly, supplementing the cellular extracts with CDC45 or GINS phenocopied c-Myc overexpression, while partial CDC45 or GINS depletion abrogated c-Myc induced H2AX phosphorylation, thus suggesting that the c-Myc induced replicative stress responses require the downstream activity of the CMG complex [55]. Thus, there is an emerging body of evidences suggesting that a major component of c-Myc induced DNA damaged is generated during S-phase at the sites of replication forks.

The observations that DNA damage and the DNA damage response (DDR) generated in c-Myc overexpressing cells co-localize with replisomes raise some mechanistic questions. Here we propose some explanations that may help in rationalizing why controlling replication forks dynamics may become critical in c-Myc overexpressing cells (Fig. 1). A first consideration is that the matter here may simply be probabilistic: if a single cell is firing more replication origins the chances of accumulating DNA damage will be the product of the risk of the single replicative fork to undergo replicative stress multiplied by the number of active forks. Thus a cell, simply by firing more origins will have higher chances to trigger a DDR. Also, given the way DNA replication is regulated in higher eukaryotes, the use of more replication origins implies that fewer “dormant origins” will remain available to backup DNA synthesis in case of fork stalling or premature termination. Although this per se may not trigger an intra S-phase DDR, it will certainly increase the risk of leaving partially unreplicated DNA thus leading to uneven DNA segregation and genome instability. In addition a high number of replication origins firing at once may pose a problem for a cell in terms of dealing with the supply of all the protein components and metabolites needed for faithful DNA replication. Indeed c-Myc, by increasing origin firing, may overwhelm cellular processes required for proper fork progression or needed to restart of DNA synthesis at stalled forks. In these instances enzymes as DNA topoisomerases, nucleases (MRN complex), or DNA helicases which are involved in the resolution of aberrant replication intermediates may become limiting, thus leading to the accumulation of unresolved topologically active DNA structures. These will have to be resolved by recombinatorial DNA repair processes that will trigger a DDR. The same line of reasoning can also be extended to nucleotides, which will have to be available in a sufficient amount to fuel highly processive DNA synthesis. In the absence of a proper supply of nucleotides, replicative forks may stall and collapse. The issue of quantitatively managing a high number of firing replisomes and the impact on the cellular mechanism actively controlling S-phase progression by buffering

Events leading to replicative stress Legend:

shortage of replication factors

topological stress

T

C

A

C

replication-transcription interfearence A T

A T

G

T A

T

C

C

A

C

A

T T

A

A

G

C

A

A

T

A

C

T

G

A

C

C

A primase

A RNA pol

A

T

C A

C

G

T

T

C

C A

G

A

C

G T

A

G

T

C A

C

A

DNA pol primase

C

A T

C

A

C

C G

A

DNA-resolving enzymes

G T

A

G

A

C

T

T C

A T

G

A

G

C

A

G C

po

primase

A

A

A G

RPA

PCNA

DN

C G

C

MCMs

DNA pol

pol

l

A

A T

C

DNA

DNA pol

DNA pol

PCNA A

A

A

A

DN

G

A

C

T C

C G

A

nucleases attack

C

G T

C

C

A

DNA pol

PCNA

G

C

C

T

A

C

C G

C

C

G A C

A

A

]

primase

G

G C

C

A

A A

C G

C

po l

T

G

G

A A

primase

C

C

T

DNA pol

A

C

T C

A T

C

G A

C

T

A

A

C

C

G

A T

C

G T

C

C G

C

A A

nucleotide starvation

A

C

C A

Topological enzymes (i.e.nucleases, topoisomerases)

Nucleotide Biosynthesis

RNA pol A,T Nucleotides C,G

nascent RNA

Synthesis of replication factors (i.e. MCMs, RPA...) ATR/Chk1 pathway (prevents excessive origin firing, provides intrinsic fork stability, regulates biosinthesis)

Myc controlled pathways (safegarding against replicative stress) Fig. 1. Myc and replicative stress. Schematic representation of the critical events that can lead to the development of replicative stress (top). A pivotal property of Myc is that once overexpressed, is able to positively regulate a number of genes and pathways that collectively safeguard DNA synthesis by counteracting these intrinsic replicative stress responses (bottom part of the cartoon).


4


limiting factors has been recently highlighted by the report that an excess of RPA proteins is required to provide a protection of ssDNA generated during replicative stress. Indeed lowering RPA levels unleashed the generation of DSBs at the stalled replication fork [90]. Apart from this, there are also peculiar aspects in c-Myc induced S-phase progression that are deeply connected to c-Myc biological activity. c-Myc not only can accelerate S-phase by promoting DNA synthesis but also is a pervasive transcription factor that transactivates a large number of genes. This poses an intrinsic conundrum since DNA replication and RNA transcription are two processes involving the same template molecule, single stranded DNA, whose accessibility will need to be co-ordinated and regulated. In fact, intra S-phase transcription may lead to the head-on collision of the converging replication bubble and the transcribing RNA polymerase that, if not properly handled, may result in either the formation of R-loops or the insurgence of topological tension, both of which will be resolved by the formation of DSBs. R-loops consist of RNA–DNA hybrid structures arising at the sites of RNA-polymerase pausing due to the presence of secondary structures in DNA [91] or trinucleotide repeats [92–94] or more frequently by collision with the replication fork [95,96]. If not removed by the RNase H, these DNA–RNA hybrids lead to DSBs and genome instability [92,97–99]. Topological tension may instead occur when the transcription and replication machineries encounter at loci that are tethered to the nuclear periphery. In this instance, while DNA supercoiling resulting from this collision is usually resolved by the ATR pathway, in some occasions, when unresolved, may trigger the convergence of topological stress leading to the generation of DSBs [100]. The association of DNA replication and transcription is particularly relevant in the case of c-Myc, a transcription factor that when overexpressed, not only can regulate a wide fraction of genes but also leads to a sharp increase in the number of active replication origins, thus inherently sensitizing cells to transcription dependent DNA replication interference. While to date there is no direct evidence linking c-Myc activation to transcription dependent replication stress responses, it is interesting to note that the overexpression of cyclin E1, a downstream target of c-Myc [101] results in a marked decrease of replication rates matched by an increase in replication–transcription interference. Thus, replication stress in cyclin E-overexpressing cells seems to result from increased replication initiation events coupled to replication and transcription conflicts [102]. Given that many of the S-phase related phenotypes resulting from cyclin E upregulation phenocopy the effects of c-Myc overexpression, it seems plausible that cMyc dependent transcription per se may derange S-phase progression by a similar mechanism. The clash of replication and transcription is probably at the heart of the genomic instability that characterizes fragile sites (FSs). Indeed common fragile sites (CFSs) are enriched in late replicating origins and are characterized by extremely long genes whose transcription kinetics exceed cell cycle timing, thus these regions have high chances of being replicated while being transcriptionally active [99]. A second class of fragile sites is represented by the Early Replicating Fragile Sites (ERFSs), which have been recently identified as early replicating DNA origins [103]. These fragile sites are characterized by high gene density, the presence of repetitive elements and for being genomic loci particularly enriched for the localization of factors like BRCA1, SMC5 and RPA, as demonstrated by ChIP-seq experiments. ERFSs are also extremely sensitive to mild replicative stress responses and to oncogene induced replication stress, indeed early S-phase arrest by hydroxyurea induces DNA damage at ERFSs but not at known fragile sites [103]. This is particularly interesting considering that the DDR observed upon ATR/Chk1 inhibition in c-Myc overexpressing cells occurs at the early S-phase [104] suggesting that perhaps c-Myc upregulation might selectively augment the number of ERFS thus accounting for c-Myc induced DDR. Despite these diametrically opposite properties, both CFS and ERFS fragility are increased by ATR inhibition, oncogenic stress and deficiencies in homologous recombination [103,105–107]. In summary we propose two key aspects that characterize the c-Myc induced instability of replication forks: one is related to the consequences

of dealing with the high number of replicative forks, which lowers the amount of protein factors and metabolites needed for faithful DNA synthesis; the second aspect relates to the intrinsic peculiarity of the S-phase progression, where c-Myc overexpressing cells will have to cope robust transcriptional activity with the high density of processive forks.

6. Chapter 5. Myc restrains intrinsic replicative stress As recently shown, the DDR response generally associated with overexpression of oncogenes constitutes an efficient tumor suppressor mechanism and barrier to cancer progression [108,109]. A prototypic example is represented by oncogenic Ras, that when expressed in primary or immortalized cells generates a potent DDR leading to a senescent arrest [110]. A prominent role in regulating this tumor suppressive DDR is exerted by the ATM/Chk2 pathway which is activated by double stranded DNA breaks and is often engaged by oncogenes at the early steps during oncogenic transformation [108,109]. Ample experimental evidence supports the role of the ATM/Chk2 pathway in tumor suppression also in Myc dependent tumors, where mutations of ATM and other modulators of the DDR, like Tip60 and WIP1, clearly affect Myc dependent tumorigenesis in mouse models [74–76,111,112]. Interestingly, the related ATR/Chk1 pathway, which responds to single stranded DNA breaks appears to play an opposite role since, as it will be discussed later, it is necessary to support clonal propagation of cancer cells. Thus whereas the ATM/Chk2 pathway functions as a barrier to tumor development in pre-malignant lesions and therefore is frequently mutated in cancer, a functional ATR/Chk1 pathway is integral to tumor survival and propagation [113]. Contrary to other oncogenes, c-Myc overexpression provides a long-term growth advantage, thus suggesting that the anti-proliferative effects triggered by RS and DDR, while still contributing to tumor suppression, may be mitigated by c-Myc itself, thus preventing cells from reaching a signaling threshold needed for a checkpoint activation. In fact several evidences that will be presented below suggest that while unrestricted activation of c-Myc might generate robust replication stress, this is prevented by c-Myc ability to trigger several pathways that actively restrain the extent of replication stress to allow proficient cellular proliferation (fig. 1). A good example is provided by the WRN helicase, a gene found mutated in Werner syndrome, an autosomal recessive human progeroid syndrome characterized by cellular senescence, increased chromosomal instability characterized by frequent chromatid breaks, premature aging and increased incidence of rare cancers [114]. The WRN gene encodes for a RecQ-helicase resolving topologically unfavorable DNA structures, such as those arising at stalled replication forks during DNA replication, therefore representing a bona fide protective factor endowed with anti-replication stress activity [115]. This activity becomes particularly relevant when cells experience elevated c-Myc level, as suggested by the observation that ectopic c-Myc activation in WRN-null cells triggers the accumulation of substantial DNA damage at the site of DNA replication [54]. This elevated DNA damage engages the ATR-Chk1 replication checkpoint, resulting in a strong antiproliferative response driving cells into cellular senescence. The observation that WRN is transcriptionally regulated by c-Myc suggests the existence of a positive feed-forward regulatory mechanism that is controlled by c-Myc and that, by raising the levels of this helicase grants extra protection against S-phase topological stress, thus ensuring proficient cellular proliferation [53]. This concept met further validation in vivo, where tissue specific transgenic mice expressing a c-Myc in B-lymphocytes showed a significantly delayed tumor onset when homozygous for the Wrn Δhel allele, a loss of function allele of WRN with compromised helicase activity. Accordingly, xenograft models using human carcinoma cell lines expressing high levels of c-Myc, displayed marked growth inhibition upon WRN silencing [116].



Another strategy deployed by c-Myc to counteract replication stress is increasing the rate of nucleotide synthesis. As already discussed, while oncogenes can enforce cell proliferation their activation is also associated with replication perturbation, DNA damage accumulation and genome instability. This replication stress is at least in part due to the high rate of DNA synthesis which, if not properly supplied by the biosynthetic pathways that provide purine and pyrimidine nucleotides results in replication fork collapse. Indeed the expression of viral and human oncogenes leads to a sharp decrease in the intra-cellular nucleotides with the concomitant induction of replication-induced DNA damage. In this context, c-Myc overexpression can rescue the biosynthetic deficiency and prevent replication stress. A similar result is achieved by supplementing cells with an excess of exogenous nucleosides. Thus, c-Myc has the unique ability to safeguard the highly demanding oncogene driven DNA synthesis by controlling the nucleotide pool size [117]. A key pathway that is engaged by c-Myc to control the replication stress response is the ATR/Chk1 pathway. This DNA damage response pathway is activated by single stranded DNA (ssDNA) arising during replication stress or in DNA repair processes like in double strand breaks resection, repair of DNA base adducts or DNA crosslinks [118,119]. It has also a housekeeping function during DNA replication probably related to its role in preventing replication fork collapse, which may account for the observation that homozygous deletion of the ATR kinase and Chk1 compromise cellular viability and embryonic development [119–121]. The replication stress response is apically controlled by the activation of the ATR kinase, a large ATM-related protein kinase, which is recruited at the sites of ssDNA along with ATRIP and other essential accessory factors such as Claspin and TopBP1 [122]. Hypomorphic homozygous mutations affecting the splicing of the ATR mRNA have been identified in patients with the rare Seckel syndrome, a disease characterized by microcephaly and growth retardation [123]. A direct prove causally linking the ATR mutations found in Seckel to the human pathology comes from mouse genetics, where mice carrying homozygous ATR alleles recapitulate all the hallmarks of these genetic syndromes, such as accelerated aging, microcephaly, growth retardation and pervasive replication stress during embryonic development [124]. The other key component of this pathway is Chk1, a soluble protein kinase activated downstream of ATR, which contributes to the transduction of the DDR signals to activate effector functions of the pathway, such as the S and the G2 checkpoints and involved in the regulation of replication fork progression, nucleotide synthesis and anti-apoptotic signaling [118,125]. This pathway is implicated in supporting c-Myc dependent oncogenic transformation since hypomorphic ATR mutations or sublethal doses of ATR or Chk1 inhibitors, while causing mild replicative stress in normal cells, show strong synthetic lethality in c-Myc overexpressing cells that associates with a potent genotoxic response [104,126–130]. Indeed, as part of a transcriptional program aimed at providing robust DNA replication the levels Chk1 [126], as well as those of other components of the pathway, are directly controlled by c-Myc [21]. The concept that high levels of RS components may provide extra protection toward oncogene induced RS is experimentally supported by the observations that supra-physiological expression of Chk1 limits the extent of DDR and DNA damage in cells challenged with RS inducers and effectively cooperates with oncogenes in cellular transformation assays [131]. Interestingly this housekeeping function of ATR and Chk1 in providing RFs stability may be also related to the role of this pathway in regulating nucleotide biosynthesis through the regulation of the ribonucleotide reductase (RNR) activity. This has been shown to be relevant in yeast where Mec1 and Rad53 (the yeast orthologs of ATM/ATR and Chk1/ Chk2, respectively) were found to regulate the RNR expression [132–135]. This appears to be conserved in higher eukaryotes where ATR/Chk1 has been reported to trigger the activation of RNR during S-phase progression and DNA damage response [136–138]. Relevant to this, the suppression of RNR activity thought the inhibition of the RRM2 subunit leads to cell cycle

5

arrest and cellular senescence [139]. Overall, by inducing the ATR/Chk1 pathway, Myc may achieve a pleiotropic control of DNA synthesis where stability and processivity of the replicative forks are coordinated with an appropriate rate of nucleotide synthesis. Thus coopting the ATR/Chk1 pathway appears an effective strategy adopted by c-Myc to enhance the reliability of DNA synthesis. 7. Chapter 6. Unleashing replicative stress to devise cancer specific therapies The concept that c-Myc itself may actively restrain intrinsic replicative stress by engaging several pathways bears direct consequences relevant to the therapeutic targeting of c-Myc dependent tumors, since we may use this knowledge to tailor cancer specific pharmacological treatments. This idea is supported by several recent findings showing how specific targeting of the ATR/Chk1 pathway [104,126–130] has a strong anticancer activity. For instance, mice bearing the Eμ-Myc transgene showed a strong genetic interaction with the ATR Seckel allele: they displayed a widespread replicative stress response detectable in many different tissues, due to low levels of expression of the Eμ-Myc transgene in non-lymphoid tissue, and most strickingly in Blymphocytes, where the Eμ enhancer drives high Myc activity, there was complete protection against lymphoma development. Accordingly, pharmacological studies using Chk1 inhibitors, confirmed that shortterm treatment of full-blown Eμ-Myc lymphomas triggered a potent DDR response, cell cycle arrest and widespread apoptosis, while continuous administration caused fast tumor regression [104]. A similar therapeutic effect was reported in a mouse model of mature B-cell lymphomas where RNAi experiments and pharmacological inhibition of Chk1 effectively triggered a DDR response in c-Myc overexpressing cells and led to improved disease free survival in mice [126]. Of note, cell lines derived from human B-cell lymphomas with elevated c-Myc were extremely sensitive to Chk1 inhibitors [104,126]. These observations are not limited to lymphoid tissues since chemical inhibition of Chk1 provoked a strong replicative stress response and widespread apoptosis in c-Myc induced pancreatic tumors [104]. Similarly, an RNAi screen performed on neuroblastoma cell lines that over-express N-Myc has led to the identification of Chk1 as a promising therapeutic target [130]. In this study, cellular sensitivity to Chk1 inhibition or knock-down positively correlated with N-Myc protein levels. In addition, while Chk1 inhibition had negligible effects on primary nontransformed cells, the same cells, when engineered to express high levels of N-Myc, became highly sensitive to the treatment [130]. Of note, a replication stress therapy may also prove effective on “non-Myc driven cancer”, as is the case for some models of Ras driven tumorigenesis where ATR knockdown [128] or tissue specific hypomorphism [140] was synthetic lethal with RAS. 8. Conclusion and remarks There is raising awareness of the opportunity offered by the targeting of Myc induced replicative stress responses. While the role of ATR and Chk1 is now fully appreciated we do think that there is still an ample opportunity for identifying new key mediators of RS that could be successfully exploited in cancer treatment. Only to cite a few, the identification of novel downstream effectors of the ATR/Chk1 pathway, like NEK8 [141], the definition of new RS regulators mutated in chromosomally unstable colon cancers [142] and the identification of other PI3K family members (besides ATM and ATR) implicated in the regulation of DNA damage responses [143–145] already provide an experimental avenue worthwhile exploring. We believe and hope that this renewed interest in Myc induced DNA replication will stimulate the quest to the identification of novel components and pathways, which hold the promise of prospective clinical applications in cancer treatment.


6


Acknowledgements Research in S.C.'s laboratory is funded by Associazione Italiana per la Ricerca sul Cancro (AIRC, IG 13135). We apologize to those whose work could not be cited due to space limitations. References [1] G.L. Shen-Ong, E.J. Keath, S.P. Piccoli, M.D. Cole, Novel Myc oncogene RNA from abortive immunoglobulin-gene recombination in mouse plasmacytomas, Cell 31 (1982) 443–452. [2] S. Crews, R. Barth, L. Hood, J. Prehn, K. Calame, Mouse c-Myc oncogene is located on chromosome 15 and translocated to chromosome 12 in plasmacytomas, Science 218 (1982) 1319–1321. [3] R. Dalla-Favera, M. Bregni, J. Erikson, D. Patterson, R.C. Gallo, C.M. Croce, Human cMyc onc gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells, Proc. Natl. Acad. Sci. U. S. A. 79 (1982) 7824–7827. [4] B.G. Neel, S.C. Jhanwar, R.S. Chaganti, W.S. Hayward, Two human c-onc genes are located on the long arm of chromosome 8, Proc. Natl. Acad. Sci. U. S. A. 79 (1982) 7842–7846. [5] R. Taub, I. Kirsch, C. Morton, G. Lenoir, D. Swan, S. Tronick, et al., Translocation of the c-Myc gene into the immunoglobulin heavy chain locus in human Burkitt lymphoma and murine plasmacytoma cells, Proc. Natl. Acad. Sci. U. S. A. 79 (1982) 7837–7841. [6] R. Dalla-Favera, F. Wong-Staal, R.C. Gallo, Onc gene amplification in promyelocytic leukaemia cell line HL-60 and primary leukaemic cells of the same patient, Nature 299 (1982) 61–63. [7] S. Collins, M. Groudine, Amplification of endogenous Myc-related DNA sequences in a human myeloid leukaemia cell line, Nature 298 (1982) 679–681. [8] G.S. Payne, S.A. Courtneidge, L.B. Crittenden, A.M. Fadly, J.M. Bishop, H.E. Varmus, Analysis of avian leukosis virus DNA and RNA in bursal tumours: viral gene expression is not required for maintenance of the tumor state, Cell 23 (1981) 311–322. [9] W.S. Hayward, B.G. Neel, S.M. Astrin, Activation of a cellular onc gene by promoter insertion in ALV-induced lymphoid leukosis, Nature 290 (1981) 475–480. [10] M.T. Hemann, A. Bric, J. Teruya-Feldstein, A. Herbst, J.A. Nilsson, C. Cordon-Cardo, et al., Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants, Nature 436 (2005) 807–811. [11] R. Schmitz, R.M. Young, M. Ceribelli, S. Jhavar, W. Xiao, M. Zhang, et al., Burkitt lymphoma pathogenesis and therapeutic targets from structural and functional genomics, Nature 490 (2012) 116–120. [12] I. Sur, S. Tuupanen, T. Whitington, L.A. Aaltonen, J. Taipale, Lessons from functional analysis of genome-wide association studies, Cancer Res. 73 (2013) 4180–4184. [13] C. Murre, P.S. McCaw, D. Baltimore, A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and Myc proteins, Cell 56 (1989) 777–783. [14] W.H. Landschulz, P.F. Johnson, S.L. McKnight, The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins, Science 240 (1988) 1759–1764. [15] E.M. Blackwood, R.N. Eisenman, Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc, Science 251 (1991) 1211–1217. [16] B. Amati, S. Dalton, M.W. Brooks, T.D. Littlewood, G.I. Evan, H. Land, Transcriptional activation by the human c-Myc oncoprotein in yeast requires interaction with Max, Nature 359 (1992) 423–426. [17] J. Stone, T. de Lange, G. Ramsay, E. Jakobovits, J.M. Bishop, H. Varmus, et al., Definition of regions in human c-Myc that are involved in transformation and nuclear localization, Mol. Cell. Biol. 7 (1987) 1697–1709. [18] L.H. Li, C. Nerlov, G. Prendergast, D. MacGregor, E.B. Ziff, c-Myc represses transcription in vivo by a novel mechanism dependent on the initiator element and Myc box II, Embo J. 13 (1994) 4070–4079. [19] D.Y.L. Mao, J.D. Watson, P.S. Yan, D. Barsyte-Lovejoy, F. Khosravi, W.W.-L. Wong, et al., Analysis of Myc bound loci identified by CpG island arrays shows that Max is essential for Myc-dependent repression, Curbio 13 (2003) 882–886. [20] D. Kleine-Kohlbrecher, S. Adhikary, M. Eilers, Mechanisms of transcriptional repression by Myc, Curr. Top. Microbiol. Immunol. 302 (2006) 51–62. [21] D. Perna, G. Faga, A. Verrecchia, M.M. Gorski, I. Barozzi, V. Narang, et al., Genomewide mapping of Myc binding and gene regulation in serum-stimulated fibroblasts, Oncogene 31 (2012) 1695–1709. [22] C.Y. Lin, J. Lovén, P.B. Rahl, R.M. Paranal, C.B. Burge, J.E. Bradner, et al., Transcriptional amplification in tumor cells with elevated c-Myc, Cell 151 (2012) 56–67. [23] Z. Nie, G. Hu, G. Wei, K. Cui, A. Yamane, W. Resch, et al., c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells, Cell 151 (2012) 68–79. [24] V. Seitz, P. Butzhammer, B. Hirsch, J. Hecht, I. Gütgemann, A. Ehlers, et al., Deep sequencing of MYC DNA-binding sites in Burkitt lymphoma, PLoS ONE 6 (2011) e26837. [25] H. Nishitani, Z. Lygerou, DNA replication licensing, Front. Biosci. 9 (2004) 2115–2132. [26] J.J. Blow, A. Dutta, Preventing re-replication of chromosomal DNA, Nat. Rev. Mol. Cell Biol. 6 (2005) 476–486. [27] A. Costa, I. Ilves, N. Tamberg, T. Petojevic, E. Nogales, M.R. Botchan, et al., The structural basis for MCM2-7 helicase activation by GINS and Cdc45, Nat. Struct. Mol. Biol. 18 (2011) 471–477.

[28] D. Remus, J.F.X. Diffley, Eukaryotic DNA replication control: lock and load, then fire, Curr. Opin. Cell Biol. 21 (2009) 771–777. [29] Y.V. Fu, H. Yardimci, D.T. Long, T.V. Ho, A. Guainazzi, V.P. Bermudez, et al., Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase, Cell 146 (2011) 931–941. [30] A. Gambus, R.C. Jones, A. Sanchez-Diaz, M. Kanemaki, F. van Deursen, R.D. Edmondson, et al., GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks, Nat. Cell Biol. 8 (2006) 358–366. [31] I. Ilves, T. Petojevic, J.J. Pesavento, M.R. Botchan, Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins, Mol. Cell 37 (2010) 247–258. [32] M. Pacek, A.V. Tutter, Y. Kubota, H. Takisawa, J.C. Walter, Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication, Mol. Cell 21 (2006) 581–587. [33] R.C. Heller, S. Kang, W.M. Lam, S. Chen, C.S. Chan, S.P. Bell, Eukaryotic origindependent DNA replication in vitro reveals sequential action of DDK and S-CDK kinases, Cell 146 (2011) 80–91. [34] K. Labib, How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells? Genes Dev. 24 (2010) 1208–1219. [35] S. Mimura, H. Takisawa, Xenopus Cdc45-dependent loading of DNA polymerase alpha onto chromatin under the control of S-phase Cdk, Embo J. 17 (1998) 5699–5707. [36] J.J. Blow, P.J. Gillespie, Replication licensing and cancer—a fatal entanglement? Nat. Rev. Cancer 8 (2008) 799–806. [37] M. Lei, Y. Kawasaki, B.K. Tye, Physical interactions among Mcm proteins and effects of Mcm dosage on DNA replication in Saccharomyces cerevisiae, Mol. Cell. Biol. 16 (1996) 5081–5090. [38] H.M. Mahbubani, J.P. Chong, S. Chevalier, P. Thommes, J.J. Blow, Cell cycle regulation of the replication licensing system: involvement of a Cdk-dependent inhibitor, J. Cell Biol. 136 (1997) 125–135. [39] M.C. Edwards, A.V. Tutter, C. Cvetic, C.H. Gilbert, T.A. Prokhorova, J.C. Walter, MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts, J. Biol. Chem. 277 (2002) 33049–33057. [40] M. Oehlmann, A.J. Score, J.J. Blow, The role of Cdc6 in ensuring complete genome licensing and S phase checkpoint activation, J. Cell Biol. 165 (2004) 181–190. [41] X.Q. Ge, D.A. Jackson, J.J. Blow, Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress, Genes Dev. 21 (2007) 3331–3341. [42] A. Ibarra, E. Schwob, J. Mendez, Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 8956–8961. [43] A.M. Woodward, T. Gohler, M.G. Luciani, M. Oehlmann, X. Ge, A. Gartner, et al., Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress, J. Cell Biol. 173 (2006) 673–683. [44] K. Kelly, B.H. Cochran, C.D. Stiles, P. Leder, Cell-specific regulation of the c-Myc gene by lymphocyte mitogens and platelet-derived growth factor, Cell 35 (1983) 603–610. [45] C.B. Thompson, P.B. Challoner, P.E. Neiman, M. Groudine, Levels of c-Myc oncogene mRNA are invariant throughout the cell cycle, Nature 314 (1985) 363–366. [46] T.J. Gonda, D. Metcalf, Expression of Myb, Myc and Fos proto-oncogenes during the differentiation of a murine myeloid leukaemia, Nature 310 (1984) 249–251. [47] H.M. Lachman, A.I. Skoultchi, Expression of c-Myc changes during differentiation of mouse erythroleukaemia cells, Nature 310 (1984) 592–594. [48] H.A. Armelin, M.C. Armelin, K. Kelly, T. Stewart, P. Leder, B.H. Cochran, et al., Functional role for c-Myc in mitogenic response to platelet-derived growth factor, Nature 310 (1984) 655–660. [49] M. Eilers, D. Picard, K.R. Yamamoto, J.M. Bishop, Chimaeras of Myc oncoprotein and steroid receptors cause hormone-dependent transformation of cells, Nature 340 (1989) 66–68. [50] M. Eilers, S. Schirm, J.M. Bishop, The MYC protein activates transcription of the alpha-prothymosin gene, Embo J. 10 (1991) 133–141. [51] S. Pelengaris, T. Littlewood, M. Khan, G. Elia, G. Evan, Reversible activation of c-Myc in skin: induction of a complex neoplastic phenotype by a single oncogenic lesion, Mol. Cell 3 (1999) 565–577. [52] S. Pelengaris, M. Khan, G.I. Evan, Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression, Cell 109 (2002) 321–334. [53] C. Grandori, K.-J. Wu, P. Fernandez, C. Ngouenet, J. Grim, B.E. Clurman, et al., Werner syndrome protein limits MYC-induced cellular senescence, Genes Dev. 17 (2003) 1569–1574. [54] K. Robinson, N. Asawachaicharn, D.A. Galloway, C. Grandori, c-Myc accelerates S-phase and requires WRN to avoid replication stress, PLoS ONE 4 (2009) e5951. [55] S.V. Srinivasan, D. Dominguez-Sola, L.C. Wang, O. Hyrien, J. Gautier, Cdc45 is a critical effector of Myc-dependent DNA replication stress, Cell Rep. 3 (2013) 1629–1639. [56] B. Amati, K. Alevizopoulos, J. Vlach, Myc and the cell cycle, Front. Biosci. 3 (1998) d250–d268. [57] P.C. Fernandez, S.R. Frank, L. Wang, M. Schroeder, S. Liu, J. Greene, et al., Genomic targets of the human c-Myc protein, Genes Dev. 17 (2003) 1115–1129. [58] C. Bouchard, K. Thieke, A. Maier, R. Saffrich, J. Hanley-Hyde, W. Ansorge, et al., Direct induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27, Embo J. 18 (1999) 5321–5333. [59] A. Menssen, H. Hermeking, Characterization of the c-MYC-regulated transcriptome by SAGE: identification and analysis of c-MYC target genes, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 6274–6279.


S. Rohban, S. Campaner / Biochimica et Biophysica Acta xxx (2014) xxx–xxx [60] H. Hermeking, C. Rago, M. Schuhmacher, Q. Li, J.F. Barrett, A.J. Obaya, et al., Identification of CDK4 as a target of c-MYC, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 2229–2234. [61] P.L. Miliani de Marval, E. Macias, R. Rounbehler, P. Sicinski, H. Kiyokawa, D.G. Johnson, et al., Lack of cyclin-dependent kinase 4 inhibits c-Myc tumorigenic activities in epithelial tissues, Mol. Cell. Biol. 24 (2004) 7538–7547. [62] S. Campaner, M. Doni, P. Hydbring, A. Verrecchia, L. Bianchi, D. Sardella, et al., Cdk2 suppresses cellular senescence induced by the c-Myc oncogene, Nat. Cell Biol. 12 (2010) 54–59 (sup pp 1–14). [63] K. Kozar, M.A. Ciemerych, V.I. Rebel, H. Shigematsu, A. Zagozdzon, E. Sicinska, et al., Mouse development and cell proliferation in the absence of D-cyclins, Cell 118 (2004) 477–491. [64] G.F. Claassen, S.R. Hann, A role for transcriptional repression of p21CIP1 by c-Myc in overcoming transforming growth factor beta-induced cell-cycle arrest, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 9498–9503. [65] J. Seoane, H.-V. Le, J. MassaguE, Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage, Nature 419 (2002) 729–734. [66] A.L. Gartel, X. Ye, E. Goufman, P. Shianov, N. Hay, F. Najmabadi, et al., Myc represses the p21(WAF1/CIP1) promoter and interacts with Sp1/Sp3, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 4510–4515. [67] P. Staller, K. Peukert, A. Kiermaier, J. Seoane, J. Lukas, H. Karsunky, et al., Repression of p15INK4b expression by Myc through association with Miz-1, Nat. Cell Biol. 3 (2001) 392–399. [68] J. Seoane, C. Pouponnot, P. Staller, M. Schader, M. Eilers, J. Massague, TGFbeta influences Myc, Miz-1 and Smad to control the CDK inhibitor p15INK4b, Nat. Cell Biol. 3 (2001) 400–408. [69] W.W. Marhin, S. Chen, L.M. Facchini, A.J.J. Fornace, L.Z. Penn, Myc represses the growth arrest gene gadd45, Oncogene 14 (1997) 2825–2834. [70] T.C. Lee, L. Li, L. Philipson, E.B. Ziff, Myc represses transcription of the growth arrest gene gas1, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 12886–12891. [71] D. Perna, G. Faga, A. Verrecchia, M.M. Gorski, I. Barozzi, V. Narang, et al., Genomewide mapping of Myc binding and gene regulation in serum-stimulated fibroblasts, Oncogene 31 (2012) 1695–1709. [72] Y.-C. Liu, F. Li, J. Handler, C.R.L. Huang, Y. Xiang, N. Neretti, et al., Global regulation of nucleotide biosynthetic genes by c-Myc, PLoS ONE 3 (2008) e2722. [73] D. Dominguez-Sola, C.Y. Ying, C. Grandori, L. Ruggiero, B. Chen, M. Li, et al., Non-transcriptional control of DNA replication by c-Myc, Nature 448 (2007) 445–451. [74] R.V. Pusapati, R.J. Rounbehler, S. Hong, J.T. Powers, M. Yan, K. Kiguchi, et al., ATM promotes apoptosis and suppresses tumorigenesis in response to Myc, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 1446–1451. [75] M. Reimann, C. Loddenkemper, C. Rudolph, I. Schildhauer, B. Teichmann, H. Stein, et al., The Myc-evoked DNA damage response accounts for treatment resistance in primary lymphomas in vivo, Blood 110 (2007) 2996–3004. [76] C. Gorrini, M. Squatrito, C. Luise, N. Syed, D. Perna, L. Wark, et al., Tip60 is a haploinsufficient tumour suppressor required for an oncogene-induced DNA damage response, Nature 448 (2007) 1063–1067. [77] D.W. Felsher, J.M. Bishop, Transient excess of MYC activity can elicit genomic instability and tumorigenesis, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 3940–3944. [78] A. Karlsson, D. Deb-Basu, A. Cherry, S. Turner, J. Ford, D.W. Felsher, Defective double-strand DNA break repair and chromosomal translocations by MYC overexpression, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 9974–9979. [79] P.E. Neiman, R. Kimmel, A. Icreverzi, K. Elsaesser, S.-J. Bowers, J. Burnside, et al., Genomic instability during Myc-induced lymphomagenesis in the bursa of Fabricius, Oncogene 25 (2006) 6325–6335. [80] E.V. Prochownik, c-Myc: linking transformation and genomic instability, Curr. Mol. Med. 8 (2008) 446–458. [81] S. Ray, MYC can induce DNA breaks in vivo and in vitro independent of reactive oxygen species, Cancer Res. 66 (2006) 6598–6605. [82] A. Kuzyk, S. Mai, c-MYC-induced genomic instability, Cold Spring Harb. Perspect. Med. 4 (2014). [83] R.A. Egler, E. Fernandes, K. Rothermund, S. Sereika, N. de Souza-Pinto, P. Jaruga, et al., Regulation of reactive oxygen species, DNA damage, and c-Myc function by peroxiredoxin 1, Oncogene 24 (2005) 8038–8050. [84] J.A. Graves, M. Metukuri, D. Scott, K. Rothermund, E.V. Prochownik, Regulation of reactive oxygen species homeostasis by peroxiredoxins and c-Myc, J. Biol. Chem. 284 (2009) 6520–6529. [85] D.R. Wonsey, K.I. Zeller, C.V. Dang, The c-Myc target gene PRDX3 is required for mitochondrial homeostasis and neoplastic transformation, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 6649–6654. [86] P. Gao, H. Zhang, R. Dinavahi, F. Li, Y. Xiang, V. Raman, et al., HIF-dependent antitumorigenic effect of antioxidants in vivo, Cancer Cell 12 (2007) 230–238. [87] H. Zhang, P. Gao, R. Fukuda, G. Kumar, B. Krishnamachary, K.I. Zeller, et al., HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHL-deficient renal cell carcinoma by repression of C-MYC activity, Cell 11 (2007) 407–420. [88] S.F. Louis, B.J. Vermolen, Y. Garini, I.T. Young, A. Guffei, Z. Lichtensztejn, et al., c-Myc induces chromosomal rearrangements through telomere and chromosome remodeling in the interphase nucleus, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 9613–9618. [89] D.W. Felsher, A. Zetterberg, J. Zhu, T. Tlsty, J.M. Bishop, Overexpression of MYC causes p53-dependent G2 arrest of normal fibroblasts, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 10544–10548. [90] L.I. Toledo, M. Altmeyer, M.-B. Rask, C. Lukas, D.H. Larsen, Lou Klitgaard Povlsen, et al., ATR prohibits replication catastrophe by preventing global exhaustion of RPA, Cell 155 (2013) 1088–1103.

7

[91] K. Skourti-Stathaki, N.J. Proudfoot, N. Gromak, Human senataxin resolves RNA/ DNA hybrids formed at transcriptional pause sites to promote Xrn2-dependent termination, Mol. Cell 42 (2011) 794–805. [92] Y. Lin, S.Y.R. Dent, J.H. Wilson, R.D. Wells, M. Napierala, R loops stimulate genetic instability of CTG.CAG repeats, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 692–697. [93] K. Reddy, M. Tam, R.P. Bowater, M. Barber, M. Tomlinson, K. Nichol Edamura, et al., Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats, Nucleic Acids Res. 39 (2011) 1749–1762. [94] A. Helmrich, M. Ballarino, E. Nudler, L. Tora, Transcription-replication encounters, consequences and genomic instability, Nat. Struct. Mol. Biol. 20 (2013) 412–418. [95] H.E. Mischo, B. Gomez-Gonzalez, P. Grzechnik, A.G. Rondón, W. Wei, L. Steinmetz, et al., Yeast Sen1 helicase protects the genome from transcription-associated instability, Mol. Cell 41 (2011) 21–32. [96] P. Huertas, A. Aguilera, Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination, Mol. Cell 12 (2003) 711–721. [97] D. Dutta, K. Shatalin, V. Epshtein, M.E. Gottesman, E. Nudler, Linking RNA polymerase backtracking to genome instability in E. coli, Cell 146 (2011) 533–543. [98] S. Tuduri, L. Crabbe, C. Conti, H. Tourriere, H. Holtgreve-Grez, A. Jauch, et al., Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription, Nat. Cell Biol. 11 (2009) 1315–1324. [99] A. Helmrich, M. Ballarino, L. Tora, Collisions between replication and transcription complexes cause common fragile site instability at the longest human genes, Mol. Cell 44 (2011) 966–977. [100] R. Bermejo, M.S. Lai, M. Foiani, Preventing replication stress to maintain genome stability: resolving conflicts between replication and transcription, Mol. Cell 45 (2012) 710–718. [101] R. Beier, A. Bürgin, A. Kiermaier, M. Fero, H. Karsunky, R. Saffrich, et al., Induction of cyclin E-cdk2 kinase activity, E2F-dependent transcription and cell growth by Myc are genetically separable events, Embo J. 19 (2000) 5813–5823. [102] R.M. Jones, O. Mortusewicz, I. Afzal, M. Lorvellec, P. Garcia, T. Helleday, et al., Increased replication initiation and conflicts with transcription underlie Cyclin E-induced replication stress, Oncogene 32 (2013) 3744–3753. [103] J.H. Barlow, R.B. Faryabi, E. Callén, N. Wong, A. Malhowski, H.T. Chen, et al., Identification of early replicating fragile sites that contribute to genome instability, Cell 152 (2013) 620–632. [104] M. Murga, S. Campaner, A.J. Lopez-Contreras, L.I. Toledo, R. Soria, M.F. Montana, et al., Exploiting oncogene-induced replicative stress for the selective killing of Myc-driven tumors, Nat. Struct. Mol. Biol. 18 (2011) 1331–1335. [105] J. Bartek, J. Bartkova, J. Lukas, DNA damage signalling guards against activated oncogenes and tumour progression, Oncogene 26 (2007) 7773–7779. [106] S.G. Durkin, T.W. Glover, Chromosome fragile sites, Annu. Rev. Genet. 41 (2007) 169–192. [107] T.D. Halazonetis, V.G. Gorgoulis, J. Bartek, An oncogene-induced DNA damage model for cancer development, Science 319 (2008) 1352–1355. [108] V.G. Gorgoulis, L.-V.F. Vassiliou, P. Karakaidos, P. Zacharatos, A. Kotsinas, T. Liloglou, et al., Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions, Nature 434 (2005) 907–913. [109] J. Bartkova, Z. Horejsi, K. Koed, A. Kramer, F. Tort, K. Zieger, et al., DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis, Nature 434 (2005) 864–870. [110] R. Di Micco, M. Fumagalli, A. Cicalese, S. Piccinin, P. Gasparini, C. Luise, et al., Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication, Nature 444 (2006) 638–642. [111] S. Shreeram, W.K. Hee, O.N. Demidov, C. Kek, H. Yamaguchi, A.J.J. Fornace, et al., Regulation of ATM/p53-dependent suppression of Myc-induced lymphomas by Wip1 phosphatase, J. Exp. Med. 203 (2006) 2793–2799. [112] K.H. Maclean, M.B. Kastan, J.L. Cleveland, Atm deficiency affects both apoptosis and proliferation to augment Myc-induced lymphomagenesis, Mol. Cancer Res. 5 (2007) 705–711. [113] S. Campaner, B. Amati, Two sides of the Myc-induced DNA damage response: from tumor suppression to tumor maintenance, Cell Div. 7 (2012) 6. [114] P. Pichierri, F. Ammazzalorso, M. Bignami, A. Franchitto, The Werner syndrome protein: linking the replication checkpoint response to genome stability, Aging 3 (2011) 311–318 (Albany NY). [115] J.M. Sidorova, N. Li, A. Folch, R.J.J. Monnat, The RecQ helicase WRN is required for normal replication fork progression after DNA damage or replication fork arrest, Cell Cycle 7 (2008) 796–807. [116] R. Moser, M. Toyoshima, K. Robinson, K.E. Gurley, H.L. Howie, J. Davison, et al., MYC-driven tumorigenesis is inhibited by WRN syndrome gene deficiency, Mol Cancer Res. 10 (2012) 535–545. [117] A.C. Bester, M. Roniger, Y.S. Oren, M.M. Im, D. Sarni, M. Chaoat, et al., Nucleotide deficiency promotes genomic instability in early stages of cancer development, Cell 145 (2011) 435–446. [118] A.J. Lopez-Contreras, O. Fernandez-Capetillo, The ATR barrier to replication-born DNA damage, DNA Repair (Amst) 9 (2010) 1249–1255. [119] E.J. Brown, D. Baltimore, Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance, Genes Dev. 17 (2003) 615–628. [120] A. de Klein, M. Muijtjens, R. van Os, Y. Verhoeven, B. Smit, A.M. Carr, et al., Targeted disruption of the cell-cycle checkpoint gene ATR leads to early embryonic lethality in mice, Curbio 10 (2000) 479–482. [121] H. Takai, K. Tominaga, N. Motoyama, Y.A. Minamishima, H. Nagahama, T. Tsukiyama, et al., Aberrant cell cycle checkpoint function and early embryonic death in Chk1(−/−) mice, Genes Dev. 14 (2000) 1439–1447. [122] E.A. Nam, D. Cortez, ATR signalling: more than meeting at the fork, Biochem. J. 436 (2011) 527–536.


8


[123] M. O'Driscoll, V.L. Ruiz-Perez, C.G. Woods, P.A. Jeggo, J.A. Goodship, A splicing mutation affecting expression of ataxia–telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome, Nat. Genet. 33 (2003) 497–501. [124] M. Murga, S. Bunting, M.F. Montana, R. Soria, F. Mulero, M. Canamero, et al., A mouse model of ATR-Seckel shows embryonic replicative stress and accelerated aging, Nat. Genet. 41 (2009) 891–898. [125] K.A. Cimprich, D. Cortez, ATR: an essential regulator of genome integrity, Nat. Rev. Mol. Cell Biol. 9 (2008) 616–627. [126] A. Hoglund, L.M. Nilsson, S.V. Muralidharan, L.A. Hasvold, P. Merta, M. Rudelius, et al., Therapeutic implications for the induced levels of Chk1 in Myc-expressing cancer cells, Clin. Cancer Res. 17 (2011) 7067–7079. [127] D.W. Schoppy, R.L. Ragland, O. Gilad, N. Shastri, A.A. Peters, M. Murga, et al., Oncogenic stress sensitizes murine cancers to hypomorphic suppression of ATR, J. Clin. Invest. 122 (2012) 241–252. [128] O. Gilad, B.Y. Nabet, R.L. Ragland, D.W. Schoppy, K.D. Smith, A.C. Durham, et al., Combining ATR suppression with oncogenic Ras synergistically increases genomic instability, causing synthetic lethality or tumorigenesis in a dosage-dependent manner, Cancer Res. 70 (2010) 9693–9702. [129] P.T. Ferrao, E.P. Bukczynska, R.W. Johnstone, G.A. McArthur, Efficacy of CHK inhibitors as single agents in MYC-driven lymphoma cells, Oncogene 31 (2012) 1661–1672. [130] K.A. Cole, J. Huggins, M. Laquaglia, C.E. Hulderman, M.R. Russell, K. Bosse, et al., RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma, Proceedings of the National Academy of Sciences 108 (2011) 3336–3341. [131] A.J. Lopez-Contreras, P. Gutierrez-Martinez, J. Specks, S. Rodrigo-Perez, O. Fernandez-Capetillo, An extra allele of Chk1 limits oncogene-induced replicative stress and promotes transformation, J. Exp. Med. 209 (2012) 455–461. [132] Z. Zhou, S.J. Elledge, DUN1 encodes a protein kinase that controls the DNA damage response in yeast, Cell 75 (1993) 1119–1127. [133] M. Huang, Z. Zhou, S.J. Elledge, The DNA replication and damage checkpoint pathways induce transcription by inhibition of the Crt1 repressor, Cell 94 (1998) 595–605. [134] X. Zhao, R. Rothstein, The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 3746–3751.

[135] X. Zhao, E.G. Muller, R. Rothstein, A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools, Mol. Cell 2 (1998) 329–340. [136] Y.W. Zhang, T.L. Jones, S.E. Martin, N.J. Caplen, Y. Pommier, Implication of checkpoint kinase-dependent up-regulation of ribonucleotide reductase R2 in DNA damage response, J. Biol. Chem. 284 (2009) 18085–18095. [137] V. D'Angiolella, V. Donato, F.M. Forrester, Y.-T. Jeong, C. Pellacani, Y. Kudo, et al., Cyclin F-mediated degradation of ribonucleotide reductase M2 controls genome integrity and DNA repair, Cell 149 (2012) 1023–1034. [138] H. Naruyama, M. Shimada, H. Niida, D.H. Zineldeen, Y. Hashimoto, K. Kohri, et al., Essential role of Chk1 in S phase progression through regulation of RNR2 expression, Biochem. Biophys. Res. Commun. 374 (2008) 79–83. [139] K.M. Aird, G. Zhang, H. Li, Z. Tu, B.G. Bitler, A. Garipov, et al., Suppression of nucleotide metabolism underlies the establishment and maintenance of oncogeneinduced senescence, Cell Rep. 3 (2013) 1252–1265. [140] D.W. Schoppy, R.L. Ragland, O. Gilad, N. Shastri, A.A. Peters, M. Murga, et al., Oncogenic stress sensitizes murine cancers to hypomorphic suppression of ATR, J. Clin. Invest. 122 (2012) 241–252. [141] H.J.C. Choi, J.-R. Lin, J.-B. Vannier, G.G. Slaats, A.C. Kile, R.D. Paulsen, et al., NEK8 links the ATR-regulated replication stress response and S phase CDK activity to renal ciliopathies, Mol. Cell 51 (2013) 423–439. [142] R.A. Burrell, S.E. McClelland, D. Endesfelder, P. Groth, M.-C. Weller, N. Shaikh, et al., Replication stress links structural and numerical cancer chromosomal instability, Nature 494 (2013) 492–496. [143] E. Fokas, M. Yoshimura, R. Prevo, G. Higgins, W. Hackl, S.-M. Maira, et al., NVPBEZ235 and NVP-BGT226, dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitors, enhance tumor and endothelial cell radiosensitivity, Radiat. Oncol. 7 (2012) 48. [144] M. Schonbrun, D. Laor, L. Lopez-Maury, J. Bahler, M. Kupiec, R. Weisman, TOR complex 2 controls gene silencing, telomere length maintenance, and survival under DNA-damaging conditions, Mol. Cell. Biol. 29 (2009) 4584–4594. [145] K. Shimada, I. Filipuzzi, M. Stahl, S.B. Helliwell, C. Studer, D. Hoepfner, et al., Short article, Mol. Cell 51 (2013) 829–839


The electronic information revolution and how to exploit it.

Parental stress in pediatric intensive care unit: how do we cope with it?

ER stress: how trypanosomes deal with it.

Iterative reconstruction: how it works, how to apply it.

Implementation research: what it is and how to do it.

Food reward. What it is and how to measure it.

Lymphadenectomy: how to do it?

Pericardiocentesis – How to do it.

Quality of life in patients with atrial fibrillation: how to assess it and how to improve it.

The detectability half-life in arthropod predator-prey research: what it is, why we need it, how to measure it, and how to use it.

CONSORT: when and how to use it.

How nurses can cope with stress and avoid burnout.

Medical device-induced thrombosis: what causes it and how can we prevent it?

The CA3 region of the hippocampus: how is it? What is it for? How does it do it?

SELEX: How It Happened and Where It will Go.

How to do it . . . Appear on television.

How to do it. Conduct an interview.

How to do it. Attend an inquest.

How I Do It.

Micromanagement: When to avoid it and how to use it effectively.

Cerebellar tDCS: how to do it.

How to do it. Give a reference.

Lung donor shortage - how to overcome it?

Innominate artery aneurysm, how to solve it?