M Zeniou

L Nguekeu-Zebaze

F Dantzer

Therapeutic considerations of PARP in stem cell biology: relevance in cancer and beyond

Keywords: PARP, cancer stem cell, PARP inhibitors

Therapeutic considerations of PARP in stem cell

biology: Relevance in cancer and beyond

Introduction

Nowadays, tumors are thought to correspond to heterogeneous ecosystems combining multiple tumor cell subpopulations and several types of stromal cells. Moreover, tumor cell subpopulations within single cancers have been shown to be functionally diverse. Tumor cell subclones differ with respect to genetic and epigenetic modifications resulting in distinct proliferative and stemness abilities [START_REF] Kreso | Evolution of the cancer stem cell model[END_REF]. This functional diversity of tumor cells is further modulated by microenvironmental and/or niche-derived signaling molecules as well as by antitumor therapeutic intervention strategies [START_REF] Chen | Cancer Stem Cell Quiescence and Plasticity as Major Challenges in Cancer Therapy[END_REF]. Importantly, only a subpopulation of tumor cells is able to drive tumorigenesis and initiate the formation of similarly heterogeneous tumors in vivo [START_REF] Quintana | Phenotypic heterogeneity among tumorigenic melanoma cells from patients that is reversible and not hierarchically organized[END_REF]. Cancer cells with tumor initiation ability, designated as cancer stem or stem-like cells (CSCs), were first identified in hematopoietic malignancies [START_REF] Bonnet | Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell[END_REF]. Subsequently, many solid tumors were shown to harbor cancer cells with stem-like properties. These include breast carcinoma, hepatocellular carcinoma, pancreatic, colon, prostate, ovarian, pediatric and adult brain malignancies as well as melanoma [START_REF] Al-Hajj | Prospective identification of tumorigenic breast cancer cells[END_REF][START_REF] Collins | Prospective identification of tumorigenic prostate cancer stem cells[END_REF][START_REF] Dalerba | Phenotypic characterization of human colorectal cancer stem cells[END_REF][START_REF] Fang | A tumorigenic subpopulation with stem cell properties in melanomas[END_REF][START_REF] Galli | Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma[END_REF][START_REF] Hemmati | Cancerous stem cells can arise from pediatric brain tumors[END_REF][START_REF] Li | Identification of pancreatic cancer stem cells[END_REF][START_REF] O'brien | A human colon cancer cell capable of initiating tumour growth in immunodeficient mice[END_REF][START_REF] Quintana | Efficient tumour formation by single human melanoma cells[END_REF][START_REF] Ricci-Vitiani | Identification and expansion of human colon-cancerinitiating cells[END_REF][START_REF] Schatton | Identification of cells initiating human melanomas[END_REF][START_REF] Singh | Identification of a cancer stem cell in human brain tumors[END_REF][START_REF] Singh | Identification of human brain tumour initiating cells[END_REF][START_REF] Szotek | Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness[END_REF]. It is however important to note that any cell within a tumor can acquire or lose tumor-initiation ability as a function of environmental cues and/or therapeutic intervention. Thus, cancer stemness should rather be considered as a state cells can acquire, rather than as a static subpopulation of cancer cells [START_REF] Kreso | Evolution of the cancer stem cell model[END_REF][START_REF] Chen | Cancer Stem Cell Quiescence and Plasticity as Major Challenges in Cancer Therapy[END_REF].

In addition to their tumor initiation/propagation properties, and the expression of specific stem-cell markers, CSCs are endowed with long-term self-renewal and proliferation abilities, differentiation potential and high efflux capacity underlined by overexpression of ATPbinding (ABC) cassette transporters or pathways which export or metabolize drugs [START_REF] Turdo | Meeting the Challenge of Targeting Cancer Stem Cells[END_REF].

CSCs are also proposed to be major determinants of conventional treatment resistance that results in tumor recurrence, treatment failure and metastatic disease. The difficulty to target CSCs relies both on inherent characteristics of these cells and on acquired phenotypes following therapeutic intervention. One of the major characteristics of CSCs is their resistance to radiation therapy. Indeed, like their embryonic and adult normal counterparts [START_REF] Soteriou | A matter of life and death: stem cell survival in tissue regeneration and tumour formation[END_REF], CSCs have developed a constitutive upregulation of the DNA damage response (DDR) exemplified by a higher capacity to arrest at specific cell cycle checkpoints and an increased ability to repair DNA damage [START_REF] Turdo | Meeting the Challenge of Targeting Cancer Stem Cells[END_REF][START_REF] Manic | Replication stress response in cancer stem cells as a target for chemotherapy[END_REF]. The therapeutic relevance of this phenotype has been demonstrated in numerous reports showing that the genetic or chemical inhibition of checkpoint proteins or of repair factors efficiently revert the radioresistant phenotype in various models of CSCs including the ones from brain, lung and head and neck malignancies [START_REF] Bao | Glioma stem cells promote radioresistance by preferential activation of the DNA damage response[END_REF][START_REF] Chen | Inhibition of tumorigenicity and enhancement of radiochemosensitivity in head and neck squamous cell cancer-derived ALDH1-positive cells by knockdown of Bmi-1[END_REF][START_REF] Desai | CD133+ cells contribute to radioresistance via altered regulation of DNA repair genes in human lung cancer cells[END_REF]. Additional mechanisms underlying therapeutic resistance of CSCs include preferential activation of signaling modules favoring stemness, namely in hypoxic and/or acidic tumor microenvironments [START_REF] Borah | Targeting self-renewal pathways in cancer stem cells: clinical implications for cancer therapy[END_REF][START_REF] Heddleston | Hypoxia inducible factors in cancer stem cells[END_REF][START_REF] Heddleston | The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype[END_REF][START_REF] Kim | Hypoxic tumor microenvironment and cancer cell differentiation[END_REF][START_REF] Philip | HIF expression and the role of hypoxic microenvironments within primary tumours as protective sites driving cancer stem cell renewal and metastatic progression[END_REF]. To this respect, Bone Morphogenic proteins (BMP), inducing stem cell differentiation, were shown to counteract, in some cases, the tumorigenic potential of human glioblastoma CSCs [START_REF] Chapman | Modeling differentiation-state transitions linked to therapeutic escape in triplenegative breast cancer[END_REF][START_REF] Piccirillo | Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells[END_REF][START_REF] Porcu | BMP9 counteracts the tumorigenic and pro-angiogenic potential of glioblastoma[END_REF]. However, resistance to differentiation due to epigenetic regulation of the expression of BMPs was also reported in CSCs from glioblastoma [START_REF] Lee | Epigenetic-mediated dysfunction of the bone morphogenetic protein pathway inhibits differentiation of glioblastomainitiating cells[END_REF] and clinically relevant doses of temozolomide, the standard of care for the treatment of these highly aggressive brain tumors were shown to increase the glioblastoma CSC pool in part by inducing dedifferentiation of non-stem cancer cells [START_REF] Auffinger | Conversion of differentiated cancer cells into cancer stem-like cells in a glioblastoma model after primary chemotherapy[END_REF]. Increased resistance to apoptotic signals through downregulation of caspases or upregulation of antiapoptotic molecules such as BCL2, BCLXL and survivin was also described in these cells [START_REF] Capper | Stem-cell-like glioma cells are resistant to TRAIL/Apo2L and exhibit down-regulation of caspase-8 by promoter methylation[END_REF][START_REF] Hata | The BCL2 Family: Key Mediators of the Apoptotic Response to Targeted Anticancer Therapeutics[END_REF][START_REF] Jin | Comparison between cells and cancer stem-like cells isolated from glioblastoma and astrocytoma on expression of anti-apoptotic and multidrug resistance-associated protein genes[END_REF]. In addition, their ability to reside within tumors in vivo in an actively regulated reversible quiescent state protecting them from compounds which target the proliferative state, is another major determinant of CSC resistance to treatment [START_REF] Chen | Cancer Stem Cell Quiescence and Plasticity as Major Challenges in Cancer Therapy[END_REF]. As a consequence, many cancer stem-like cell targeted therapies are currently under investigation in preclinical studies and in the clinic with the aim to reduce recurrence and metastatic spread [START_REF] Turdo | Meeting the Challenge of Targeting Cancer Stem Cells[END_REF][START_REF] Marcucci | Anti-Cancer Stem-like Cell Compounds in Clinical Development -An Overview and Critical Appraisal[END_REF]. In this review we highlight the newest findings demonstrating the potential therapeutic relevance of PARP inhibitors in this highly aggressive sub-population of cancer cells (Table 1) (Figure 1) and we summarize the major known roles of PARP1 in the non-cancer counterparts (Figure 1).

The relevance of PARP inhibition in cancer stemness

PARP1 inhibition and standard-of-care therapies in CSCs

Poly(ADP-ribose polymerase 1 (PARP1) is the founding member of a family of 17 enzymes that regulate a variety of biological processes by catalyzing the covalent addition of ADP-ribose polymers onto acceptor proteins including PARP1 itself, using NAD + as substrate.

Numerous studies describe the prime role of PARP1 in various DNA repair pathways and in the maintenance of genome integrity. In these contexts, the catalytic activity of PARP1 mediates chromatin remodeling, single-strand break repair, double strand break repair, nucleotide excision repair and the stabilization of DNA replication forks [START_REF] Lin | PARP Inhibitors for Cancer Therapy[END_REF][START_REF] Gupte | PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes[END_REF]. Hence, PARP inhibitors are now considered as among the most exciting new treatments in cancer therapy to potentialize the cytotoxic actions of anti-tumoral drugs and radiation therapy and in targeted therapies to selectively kill tumor cells presenting homologous recombination deficiency i.e BRCA1 or BRCA2 mutations [START_REF] Gibson | New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs[END_REF][START_REF] Pilie | PARP Inhibitors: Extending Benefit Beyond BRCA-Mutant Cancers[END_REF]. Olaparib (KuDOS/AstraZeneca) was the first small molecule PARP inhibitor to enter the clinic in 2014 as an FDA and EMA-approved compound for use in the treatment of advanced-stage BRCA1/2-mutated ovarian cancers.

Rucaparib (Pfizer/Clovis) and Niraparib (Janssen/Merck/Tesaro), together with Olaparib were subsequently approved for ovarian cancer treatment in various settings not only restricted to homologous recombination repair mutant tumors. PARP inhibitor clinical use was extended more recently to the treatment of other types of malignancies with Olaparib and Talazoparib (Pfizer) applied to the treatment of advanced or metastatic breast cancers presenting BRCA1/2 mutations. In 2019, Olaparib yielded positive results in progression-free survival in a phase III trial in pancreatic cancer patients suggesting that it could also obtain approval in this indication [START_REF] Pilie | PARP Inhibitors: Extending Benefit Beyond BRCA-Mutant Cancers[END_REF][START_REF] Lord | PARP inhibitors: Synthetic lethality in the clinic[END_REF][START_REF] Pilie | State-of-the-art strategies for targeting the DNA damage response in cancer[END_REF].

Numerous discoveries acquired up to date suggest that PARP1 also meets the criteria to be considered as an attractive therapeutic target to attack CSCs as we outline below.

Because of the high levels of PARP1 and/or high PARP1-dependent PARylation activity in these cells and their radio-and chemo-resistant nature, the inhibition of PARP1 in combination to radiation or other chemotherapeutic agents has shown a significant advantage in targeting CSCs from several tumor types. For example, markedly higher levels of PARP1 activity were reported in glioblastoma CSCs from xenografted patient specimens compared to non-CSC cells in basal conditions. PARP1 inhibition by Olaparib reduced stem cell survival, expansion and in vivo tumor initiation properties of these cells revealing their dependence on PARP1 function and preferential sensitivity to the compound. PARP1 was also shown to contribute to survival of these cells following irradiation since pretreatment with Olaparib resulted in the elimination of radioresistant glioblastoma CSCs after a single exposure to ionizing radiation [START_REF] Venere | Therapeutic targeting of constitutive PARP activation compromises stem cell phenotype and survival of glioblastoma-initiating cells[END_REF]. The PARP inhibitor Talazoparib, associated to radiation therapy, was also effective on CSCs from human glioblastoma [START_REF] Venere | Therapeutic targeting of constitutive PARP activation compromises stem cell phenotype and survival of glioblastoma-initiating cells[END_REF][START_REF] Lesueur | Radiosensitization Effect of Talazoparib, a Parp Inhibitor, on Glioblastoma Stem Cells Exposed to Low and High Linear Energy Transfer Radiation[END_REF]. PARP1 was also shown to participate to outgrowth, increased cell motility, invasion and self-renewal of neuroblastoma cells by binding to plant homeodomain finger-containing protein 20 (PHF20). When bound to PARP1, PHF20 is able to modulate histone marks epigenetically promoting stem-cell associated SOX2 (sex-determining region Y-box 2) and OCT4 (octamer-binding transcription factor 4) gene expression. ShRNA-mediated inhibition of PARP1 resulted in a significant decrease in the expression of these pluripotency genes and in reduced neurosphere formation ability of neuroblastoma cells [START_REF] Long | PHF20 collaborates with PARP1 to promote stemness and aggressiveness of neuroblastoma cells through activation of SOX2 and OCT4[END_REF]. Furthermore, PARP1 inhibition by Olaparib or Veliparib sensitized colorectal cancer-initiating cells to 5-fluorouracil (5-FU) chemotherapy by synergistically reducing CSC viability, self-renewal, DNA repair efficiency and in vivo tumor maintenance [START_REF] Jarrar | Poly(ADP-Ribose) Polymerase Inhibition Sensitizes Colorectal Cancer-Initiating Cells to Chemotherapy[END_REF]. Again, higher PARP1 expression and activity levels have been associated to the stem-cell phenotype of colorectal cancer cells and to advanced and or metastatic stages of colorectal cancer disease [START_REF] Jarrar | Poly(ADP-Ribose) Polymerase Inhibition Sensitizes Colorectal Cancer-Initiating Cells to Chemotherapy[END_REF]. PARP1 has also been found highly expressed in CSCs of hepatocellular carcinoma (HCC) and its chemical inhibition revealed promises to inhibit their resistance and recurrence to the DNA-damage inducing drug hydroxycamptothecin [START_REF] Xu | The properties of tumor-initiating cells from a hepatocellular carcinoma patient's primary and recurrent tumor[END_REF].

A number of other studies revealed synthetic lethality between PARP1 inhibition and various cell signaling pathways. As an example, glioblastoma CSCs were sensitive to PARP1 inhibition combined to ataxia telangiectasia -and rad3-related protein (ATR) targeting [START_REF] Ahmed | Selective Inhibition of Parallel DNA Damage Response Pathways Optimizes Radiosensitization of Glioblastoma Stem-like Cells[END_REF][START_REF] Carruthers | Replication Stress Drives Constitutive Activation of the DNA Damage Response and Radioresistance in Glioblastoma Stem-like Cells[END_REF].

In colon cancer, ectopic overexpression of nicotinamide phosphoribosyl transferase (NAMPT), the rate-limiting enzyme of the NAD salvage pathway, conferred cancer stem-cell properties mediated by PARP1 and SIRT1. The combined treatment of colon cancer tumor models with the NAMPT inhibitor FK866 and Olaparib was effective against the colon cancer-initiating population [START_REF] Lucena-Cacace | NAMPT Is a Potent Oncogene in Colon Cancer Progression that Modulates Cancer Stem Cell Properties and Resistance to Therapy through Sirt1 and PARP[END_REF]. In breast cancer, the PARP inhibitor Niraparib in combination with the cyclin-dependent kinase inhibitor dinaciclib, which downregulates MYC expression and MYC-driven homologous recombination, increased DNA damage and reduced epithelial to mesenchymal transition (EMT) and cancer stem-like cell phenotypes [START_REF] Carey | Synthetic Lethality of PARP Inhibitors in Combination with MYC Blockade Is Independent of BRCA Status in Triple-Negative Breast Cancer[END_REF]. Finally, it has previously been shown that CSCs from HCC express the stem cell marker EpCAM [START_REF] Yamashita | EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features[END_REF]. Chemoresistance and stemness in EpCAM-positive cells is partly mediated by the PARP1-dependent recruitment of the chromodomain-helicase-DNA binding protein (CHD4), a component of the nucleosome remodeling and histone deacetylase (NuRD) complex. Hence, the combined inhibition of HDAC and PARP1 impaired the functions of CHD4 and successfully restored sensitivity to chemotherapy in EpCAM positive liver CSCs [START_REF] Nio | Defeating EpCAM(+) liver cancer stem cells by targeting chromatin remodeling enzyme CHD4 in human hepatocellular carcinoma[END_REF].

Collectively, these data indicate that PARP inhibition serves as a point of fragility for

CSCs by augmenting therapeutic efficacy of radio and/or chemotherapy. However, in some contexts, CSCs displayed resistance to PARP1 inhibition. Indeed, the ovarian CSC population was enriched in vitro and in vivo following Olaparib or Rucaparib treatment. Increased resistance of ovarian CSCs to these inhibitors was attributed to the activation of additional homologous-recombination related DNA repair mechanisms in these cells and was proposed to be at the origin of ovarian cancer PARP inhibitor resistance and relapse. This observation has important implications for ovarian cancer patient management since, as mentioned above, Olaparib and Rucaparib, together with Niraparib, are FDA-approved chemotherapies for the treatment of advanced ovarian cancer (56).

PARP1 inhibition to target replication stress in CSCs

Recently, elevated levels of DNA replication stress were identified as another determinant of upregulated DDR and subsequent radiation resistance in glioblastoma CSCs [START_REF] Carruthers | Replication Stress Drives Constitutive Activation of the DNA Damage Response and Radioresistance in Glioblastoma Stem-like Cells[END_REF]. Replication stress is a condition characterized by inefficient DNA replication resulting in long stretches of single-stranded DNA adjacent to double-stranded DNA, stalling and collapse of the replication fork or slow replication fork progression [START_REF] Puigvert | Targeting DNA repair, DNA metabolism and replication stress as anti-cancer strategies[END_REF]. Stalled or slowly progressing replication forks may result in disassembly or displacement of the replication machinery and/or in passive or endonuclease-mediated DNA breakage and generation of double-strand breaks (DSB) [START_REF] Manic | Replication stress response in cancer stem cells as a target for chemotherapy[END_REF]. Replication stress is recognized as a general feature of cancer cells [START_REF] Manic | Replication stress response in cancer stem cells as a target for chemotherapy[END_REF]. In their report, Carruthers and collaborators [START_REF] Carruthers | Replication Stress Drives Constitutive Activation of the DNA Damage Response and Radioresistance in Glioblastoma Stem-like Cells[END_REF] demonstrate that glioblastoma CSCs exhibit reduced DNA replication velocity and higher frequency of stalled replication forks as well as increased expression of long neural genes containing common fragile sites which are more prone to replication stress, supporting the idea that conflicts between replication and transcription processes may underlie replication stress in these cells.

In addition to glioblastoma stem-like cells, CSCs from colorectal and breast cancers also exhibit higher levels of replicative stress. Thus, targeting the replicative stress responses in this context appears as a promising strategy to eliminate such cells [START_REF] Manic | Replication stress response in cancer stem cells as a target for chemotherapy[END_REF][START_REF] Morgan | Replication Stress: An Achilles' Heel of Glioma Cancer Stem-like Cells[END_REF]. In addition to its involvement in the DNA damage response, PARP1 was described to play key functions in cell response to replication stress. PARP1 participates to replication fork restart by recruiting Mre11 thus promoting DNA resection [START_REF] Bryant | PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination[END_REF]. PARP1 together with PARP2 were shown to stabilize the Rad51 protein at sites of damaged and/or stalled replication forks [START_REF] Ronson | PARP1 and PARP2 stabilise replication forks at base excision repair intermediates through Fbh1-dependent Rad51 regulation[END_REF].

Stabilization of stalled replication forks by PARP1 involving alternative mechanisms was also reported [START_REF] Ying | Mre11-dependent degradation of stalled DNA replication forks is prevented by BRCA2 and PARP1[END_REF]. In view of these properties, PARP1 shows up as an attractive target to inhibit replication stress in glioblastoma CSCs. Accordingly, the combined inhibition of PARP1 and ATR abrogated radiation resistance and reduced the proliferation and self-renewal capacity of glioblastoma CSCs (51).

From PARP1 to PARP3 and TNKS1 in breast cancer stemness

In breast cancer, the triple negative breast cancer (TNBC) subtype is recognized as one of the most aggressive breast malignancies with an incidence of approximately 10-15% of all breast cancers. Despite the expansion of new therapeutic options for these cancers, the development of resistance to standard or targeted therapies remains a challenging question.

There is increasing evidence that CSCs drive this resistance. Breast CSCs display increased expression of some DNA repair genes and increased efficiency in DNA damage repair, suggesting that breast CSCs may be particularly resistant to PARP inhibitors. Accordingly, an unbiased proteomic approach identified PARP1 as overexpressed in breast CSCs [START_REF] Gilabert | Poly(ADP-ribose) polymerase 1 (PARP1) overexpression in human breast cancer stem cells and resistance to olaparib[END_REF]. A recent study showed that in both BRCA1-mutated and BRCA1-wild-type TNBC, the resistance of CSCs to Olaparib-mediated PARP1 inhibition is mediated by RAD51 [START_REF] Liu | RAD51 Mediates Resistance of Cancer Stem Cells to PARP Inhibition in Triple-Negative Breast Cancer[END_REF]. Consequently, the depletion of RAD51 sensitized both CSCs and bulk cells to PARP1 inhibition in these tumors [START_REF] Liu | RAD51 Mediates Resistance of Cancer Stem Cells to PARP Inhibition in Triple-Negative Breast Cancer[END_REF].

In addition to PARP1, recent results also revealed the importance of PARP3 in breast CSCs [START_REF] Karicheva | PARP3 controls TGFbeta and ROS driven epithelial-to-mesenchymal transition and stemness by stimulating a TG2-Snail-E-cadherin axis[END_REF]. PARP3 was shown to promote stem-like cell features by inducing the expression of the stem cell markers SOX2 and OCT4, by increasing the proportion of the tumor initiating CD44 high /CD24 low cell population and their capacity to form mammospheres, and by fostering stem cell renewal. In spite of a tight link between the Epithelial-mesenchymal transition (EMT) program and cancer stemness, the action of PARP3 in EMT is strictly dependent on the cytokine TGFβ, while the importance of TGFβ in the modulation of stem-like properties is less pronounced in accordance with a broader role of PARP3 in stem cell development that remains to be identified.

Of additional relevance, the aberrant activation of the Wnt/β-catenin signaling pathway enhanced by Tankyrase (TNKS) is associated with breast cancer stemness. TNKS1 directly binds and ADP-ribosylates Axin, thus promoting its ubiquitination-mediated degradation. This impairs the activity of the β-catenin destruction complex, contributes to the nuclear translocation of β-catenin and enhancement of the Wnt signaling pathway. Therefore, down-regulating this pathway may be decisive to inhibit breast cancer stemness and activity.

Few studies have addressed this hypothesis. The Tankyrase inhibitor IWR-1 was found to be specifically cytotoxic to osteosarcoma CSCs and elicited synergistic cytotoxicity when combined with doxorubicin [START_REF] Martins-Neves | IWR-1, a tankyrase inhibitor, attenuates Wnt/beta-catenin signaling in cancer stem-like cells and inhibits in vivo the growth of a subcutaneous human osteosarcoma xenograft[END_REF]. Among the natural inhibitors, the miRNA miR-1 was described to reduce the Frizzled 7 and TNKS2 expression leading to downregulation of the Wnt/β-catenin signaling and stemness of breast CSCs [START_REF] Liu | MicroRNA-1 down-regulates proliferation and migration of breast cancer stem cells by inhibiting the Wnt/beta-catenin pathway[END_REF]. Another report identified the Angiomotin isoform p130 (Amot-p130), an angiostatin-binding protein, as an inhibitor of breast CSCs presumably by competing with Axin for binding to TNKS1 [START_REF] Yang | Angiomotin-p130 inhibits beta-catenin stability by competing with Axin for binding to tankyrase in breast cancer[END_REF]. By binding to TNKS1, Amot-p130 allows Axin to form the β -catenin destruction complexes leading to its degradation.

The contribution of PARP1 in pluripotency, differentiation and reprogramming in a physiological context

Beside the importance of PARPs in stemness activity of CSCs providing the therapeutic advantage of their inhibition as stated above, several reports reveal the engagement of PARP1 in the stem cell properties of the non-CSC counterparts announcing possible adverse effects of PARP inhibitors or relevance in non-cancer diseases. In this section, we outline the major discoveries describing the chromatin-associated functions of PARP1 in stem cell biology in a more physiological context.

PARP1 functions in Neural Stem/Progenitor Cells

Neural stem/progenitor cells (NSPCs) express high levels of PARP1. Its activation has important functions in neural lineage commitment as outlined here. In the developing mammalian central nervous system, NSPC located in the subventricular zone (SVZ) proliferate and differentiate into neurons or astrocytes while migrating to dedicated areas for regeneration in response to various stimuli. These events involve the opposing activities of basic helix-loop-helix (bHLH)-containing transcription factors, the inhibitory Hairy/Enhancer of split (HES) family of proteins and the bHLH proneural proteins. HES associates with the corepressor groucho (Gro)/transducing like enhancer of split (TLE) to inhibit the transcription of the proneural transcription factor MASH1 and to prevent neuronal differentiation. PARP1 has been identified as a component of the groucho/TLE1corepressor complex by promoting a switch in the function of the HES transcription factor from a TLE-dependent repressor to an activator necessary for neuronal differentiation. Specifically, the induction of the calciumdependent protein kinase CaMKIIδ causes a phosphorylation-dependent activation of PARP1, the poly(ADP-ribosyl)ation and consequent dissociation of components from the corepressor complex and consequently the HES-dependent recruitment of coactivators that together mediate the transcriptional activation of the neurogenic program [START_REF] Ju | Activating the PARP-1 sensor component of the groucho/ TLE1 corepressor complex mediates a CaMKinase IIdelta-dependent neurogenic gene activation pathway[END_REF]. Complementing this mechanism, another study revealed an additional sequence of events involving a distinct couple of homeodomain-containing transcription factors: the myeloid ecotropic viral integration site (MEIS) in association with the pre-B cell leukemia homeobox (PBX). PBX and MEIS are required for adult neurogenesis in the SVZ. In NSPCs, PBX acts as a pioneer transcription factor and binds H1-compacted chromatin. Using the promoter/proximal enhancer of the neuron-specific gene doublecortin (Dcx) as a model, the authors showed that in the first hours of neuronal differentiation, MEIS associates with PBX1 on chromatin, recruits PARP1 and initiates the PARP1-dependent eviction of histone H1 from chromatin to activate the neuron-specific Dcx gene [START_REF] Hau | MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1[END_REF]. Together, these results demonstrate that SVZderived NSPCs need PARP1 to initiate neurogenic programs.

PARP1 maintains stemness to prevent from differentiation to fat cells

PARP1 maintains resting preadipocytes and inhibits the formation of fat cells. The group of Lee Kraus identified the transcription factor C-EBPβ, a key element of fat cell formation, as a prime target of PARP1's enzymatic activity. The PARP1-mediated poly(ADPribosyl)ation of C-EBPβ interferes with its DNA binding and transcriptional activities onto adipogenic target genes and thus inhibits the differentiation of stem cells to fat cells [START_REF] Luo | PARP-1 Controls the Adipogenic Transcriptional Program by PARylating C/EBPbeta and Modulating Its Transcriptional Activity[END_REF]. In their recent report, the same group uncovered the importance of compartmentalized NAD + synthesis by NMNATs as a key mediator in this process [START_REF] Ryu | Metabolic regulation of transcription through compartmentalized NAD(+) biosynthesis[END_REF]. In response to adipogenic signaling and enhanced glucose metabolism, cytoplasmic NMNAT2 is activated and competes with nuclear NMNAT1 for the use of their common substrate nicotinamide mononucleotide. The consequent sudden drop in nuclear NAD + compromises PARP1catalysed PARylation of C-EBPβ and adipogenic transcription. Accordingly, the specific deletion of PARP1 in adipose tissues in mice or the genetic or chemical inhibition of PARP1 in various cell models promotes adipogenesis of primary preadipocytes (70).

PARP1 modulates pluripotency and reprogramming

PARP1 and its activity also play central roles in various aspects of embryonic stem cell (ESC) biology including stem cell maintenance, lineage-specific differentiation and the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). ESC gene analysis of PARP1 knockout mice revealed a significant shift in the expression of stemness genes indicating that PARP1 modulates ESC gene activity [START_REF] Ogino | Loss of Parp-1 affects gene expression profile in a genome-wide manner in ES cells and liver cells[END_REF]. PARP1 acts as a cofactor of the stem cell marker and pioneer transcription factor SOX2 in ESCs. SOX2 functions with OCT4, Nanog and Klf4 to promote the transcription of key pluripotency target genes. In this situation, PARP1 facilitates the binding of SOX2 onto a subset of its recognition motifs located within intractable regions of euchromatin that are characterized by high nucleosome occupancy [START_REF] Liu | Catalytic-Independent Functions of PARP-1 Determine Sox2 Pioneer Activity at Intractable Genomic Loci[END_REF]. When ESCs' differentiation starts, PARP1 poly(ADP-ribosyl)ates SOX2 to restrain its expression and promote the expression of the FGF4 enhancer [START_REF] Gao | PARP1 poly(ADP-ribosyl)ates Sox2 to control Sox2 protein levels and FGF4 expression during embryonic stem cell differentiation[END_REF][START_REF] Weber | Artd1/Parp1 regulates reprogramming by transcriptional regulation of Fgf4 via Sox2 ADPribosylation[END_REF]. Of additional relevance, during the postnatal period, when oligodendrogliogenesis peaks, the absence of PARP1 enhanced SOX2-positive cells directed towards an oligodendroglial fate at the expense of neuroblast production in the SVZ, presumably to compensate the deficiency in myelinization in the corpus callosum [START_REF] Plane | PARP-1 deletion promotes subventricular zone neural stem cells toward a glial fate[END_REF]. The first evidence implying a role of PARPs in genome reprogramming has been reported during primordial germ cell development. During this process, DNA methylation is erased upon oxidation of the 5-methylcytosine residues by TET hydroxylase enzymes for replacement by unmethylated cytosine by the TDG-initiated base excision repair (BER) process. Concomitantly, PARP1 activity is induced [START_REF] Ciccarone | 5mC-hydroxylase activity is influenced by the PARylation of TET1 enzyme[END_REF]. In the context of somatic cell reprogramming into IPSC, PARP1 cooperates with the methylcytosine dioxygenase Tet2 to establish an activated chromatin state at the Nanog and Esrrb pluripotency loci to maintain a pluripotent cell state. Moreover, PARP1 induction further promotes accessibility to the Oct4 reprogramming factor (78).

Conclusion, remaining challenges and future directions

Strong evidence supports that the cancer stem cells (CSCs), the self-renewing subpopulation of tumors cells, drive tumor progression and relapse. These cells are thus prime targets for therapeutic intervention. While PARP inhibitors are now broadly used in clinical trials, a better understanding of the function of PARP family members and of the effects of their inhibition in CSCs is required in order to provide the scientific foundations supporting further development of clinical trials utilizing PARP inhibitors to target cancer stemness. A number of obstacles remains in the present context, although sometimes opening the way to promising future directions. Key among these, is the specific targeting of CSCs versus normal cells. An interesting strategy would be the use of conjugated monoclonal antibodies in which the antibody is used as a homing device to guide the PARP inhibitor directly to the CSCs by binding to an antigen specifically expressed on the surface of the cells. This antibody-drug conjugate (ADC)-based approach has already proven its efficiency to target among others leukemic stem cells [START_REF] Jiang | CLT030, a leukemic stem cell-targeting CLL1 antibody-drug conjugate for treatment of acute myeloid leukemia[END_REF] or CSCs of small cell lung cancers (SCLC) [START_REF] Marcucci | Antibody-Drug Conjugates (ADC) Against Cancer Stem-Like Cells (CSC)-Is There Still Room for Optimism?[END_REF]. Another important question to consider using the PARP inhibitors relates to the metabolic profile of CSCs. A number of studies suggest that CSCs largely rely on the NAD + salvage pathway because elevated NAD + levels boost glycolysis, a prominent feature of cancer stemness [START_REF] Jung | Nicotinamide metabolism regulates glioblastoma stem cell maintenance[END_REF][START_REF] Lucena-Cacace | NAMPT overexpression induces cancer stemness and defines a novel tumor signature for glioma prognosis[END_REF][START_REF] Yaku | NAD Metabolism in Cancer Therapeutics[END_REF].

Owing to the fact that PARP1 constitutes a major NAD + consumer, its inhibition could induce an unfortunate accumulation of NAD + beneficial to CSCs that needs to be carefully considered. Thus, targeting these pathways in combination with PARP inhibition may constitute an interesting therapeutic strategy to efficiently eradicate CSCs. Finally, the emerging roles of PARP proteins in non-cancer stem cell biology, require sound considerations not only to ensure that patients meet augmented clinical benefit but also to exploit the potential use of PARP inhibitors in other non-cancer pathologies.

In conclusion, it is important to focus future investigations on unraveling the specific functions of PARP members in stemness that will certainly help to guide the use of selective PARP inhibitors to eliminate cancer stem cells of various origins. described in [START_REF] Lotti | Chemotherapy activates cancerassociated fibroblasts to maintain colorectal cancer-initiating cells by IL-17A[END_REF]. Note 6: Hep-11 and Hep-12 were established from primary and recurrent tumor specimens from a patient with hepatocellular carcinoma as described in [START_REF] Xu | Recurrent hepatocellular carcinoma cells with stem cell-like properties: possible targets for immunotherapy[END_REF]. When (1) In adult preadipocytes, PARP1 PARylates the pro-adipogenic transcription factor C/EBPβ to modulate its DNA binding and transcriptional activities and inhibit adipogenesis. In ESCs, PARP1 acts as a cofactor of the pioneer transcription factor SOX2 and facilitates it binding at intractable genomic loci [START_REF] Luo | PARP-1 Controls the Adipogenic Transcriptional Program by PARylating C/EBPbeta and Modulating Its Transcriptional Activity[END_REF]. [START_REF] Chen | Cancer Stem Cell Quiescence and Plasticity as Major Challenges in Cancer Therapy[END_REF] In adult neural stem cells, the contribution of PARP1 in neuronal differentiation involves two sequence of events: (i) the induction of a specific calcium-dependent protein kinase, CaMKIIδ, causes a phosphorylation-dependent activation of PARP1 resulting in dissociation of PARylated components from the groucho/TLE1 corepressor complex, while simultaneous site-specific phosphorylation of the transcription factor HES permits recruitment of coactivators and subsequent neuronal differentiation (68);

(ii) when neural progenitor cells begin to differentiate into neurons, the transcription factors PBX and MEIS cooperate to induce chromatin relaxation, recruitment of PARP1 and subsequent eviction of the linker histone H1 [START_REF] Hau | MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1[END_REF]. When ESCs start to differentiate, PARP1 PARylates SOX2 and limits its expression while expression of the FGF4 enhancer is increased [START_REF] Gao | PARP1 poly(ADP-ribosyl)ates Sox2 to control Sox2 protein levels and FGF4 expression during embryonic stem cell differentiation[END_REF][START_REF] Weber | Artd1/Parp1 regulates reprogramming by transcriptional regulation of Fgf4 via Sox2 ADPribosylation[END_REF]. ( 3) During somatic cell reprogramming into IPSC, PARP1 plays two roles:

(i) it cooperates with the methylcytosine dioxygenase Tet2 to establish histone modification marks that characterize an active chromatin state at pluripotency loci; (ii) it promotes OCT4 binding to pluripotency factors necessary for reprogramming [START_REF] Doege | Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2[END_REF]. (B) PARP1, PARP3 or TNKS1 have been found to contribute to key features characterizing cancer stem cells and driving tumor recurrence including unlimited self-renewal capacity, the expression of EMT markers promoting metastasis, resistance to DNA damaging therapies and enhanced replication stress. (1) In breast CSCs, PARP3 promotes the expression of the stem cell markers Sox2 and Oct4, an increase in the proportion of tumor initiating CD44 high /CD24 low population, the formation of tumor spheroid bodies, and stem cell self-renewal, while TNKS1 induces an aberrant activation of the Wnt/β-catenin signaling pathway [START_REF] Karicheva | PARP3 controls TGFbeta and ROS driven epithelial-to-mesenchymal transition and stemness by stimulating a TG2-Snail-E-cadherin axis[END_REF][START_REF] Liu | MicroRNA-1 down-regulates proliferation and migration of breast cancer stem cells by inhibiting the Wnt/beta-catenin pathway[END_REF]. In glioblastoma and neuroblastoma CSCs, PARP1 has been shown to contribute to stem cell survival, renewal, and in vivo tumor initiating properties [START_REF] Venere | Therapeutic targeting of constitutive PARP activation compromises stem cell phenotype and survival of glioblastoma-initiating cells[END_REF][START_REF] Long | PHF20 collaborates with PARP1 to promote stemness and aggressiveness of neuroblastoma cells through activation of SOX2 and OCT4[END_REF]. [START_REF] Chen | Cancer Stem Cell Quiescence and Plasticity as Major Challenges in Cancer Therapy[END_REF] In breast cancer cells, PARP3 responds to TGFβ-induced ROS to promote a TG2-Snail-E-cadherin axis during 32 EMT (64). (3) Owing their DNA repair activities, PARP1 promotes resistance to radiation therapy, 5-FU, hydroxycamptothecin in CSCs of glioblastoma, colorectal cancer and hepatocellular carcinoma respectively, while PARP3 promotes resistance to etoposide in breast cancer cells [START_REF] Venere | Therapeutic targeting of constitutive PARP activation compromises stem cell phenotype and survival of glioblastoma-initiating cells[END_REF][START_REF] Lesueur | Radiosensitization Effect of Talazoparib, a Parp Inhibitor, on Glioblastoma Stem Cells Exposed to Low and High Linear Energy Transfer Radiation[END_REF][START_REF] Jarrar | Poly(ADP-Ribose) Polymerase Inhibition Sensitizes Colorectal Cancer-Initiating Cells to Chemotherapy[END_REF][START_REF] Xu | The properties of tumor-initiating cells from a hepatocellular carcinoma patient's primary and recurrent tumor[END_REF]. ( 4) Various models of CSCs harbor high but heterogeneous levels of replication stress that drive constitutive activation of the DDR. In view of its several contributions in cell response to replication stress, PARP1 appears as an interesting therapeutic target to deplete DNA replication-stressed CSCs.

1

  described in the litterature, surface markers for CSCs are indicated. No indication is given otherwise. GIC: glioblastoma initiating cell; IR: ionizing radiation; ATRi: ataxia telangiectasia -and rad3-related protein (ATR) inhibitor; 5-FU: 5-fluorouracil; NAMPTi: nicotinamide phosphoribosyl transferase (NAMPT) inhibitor; BRCA: Breast Cancer gene; CSC: cancer stem cells; TGFβ: Transforming Growth Factor β; HDACi: histone deacetylase (HDAC) inhibitor; CHD4: chromodomain-helicase-DNA binding protein 4; TNKS1i: Tankyrase 1 (TNKS1) inhibitor; CD: cluster of differentiation; NCAM: neural cell adhesion molecule; VLA-2: integrin subunit alpha 2 also known as CD49B; CD133 is also known as prominin-1; ICAM-1: intercellular adhesion molecule also known as CD54; LFA-3: lymphocyte function-associated antigen 3 also known as CD58; HLA-ABC: MHC (major histocompatibility complex) class I antigen; CDCP1: CUB domain-containing protein 1 also known as membrane glycoprotein gp140; CEA: carcinoembryonic tumor associated antigen; CA 19-9 is a tumor associated antigen; Ncadherin: neural cadherine; EGFR: epidermal growth factor receptor; EpCAM: epithelial cell adhesion molecule; Ecadherin is also known as E324; ESA: epithelial specific antigen; CD338 or ABCG2: ATP Binding Cassette Subfamily G Member 2; SSEA-4: stage-specific embryonic antigen-4; OV6: oval cell marker; NI: not identified.

Figure 1 :

 1 Figure 1: PARPs in normal and cancer stem cells. (A) PARP1 contributes to pluripotency,

  Figure1

Figure 1 .

 1 Figure 1. Localisation du gisement de Revelles en région Hauts-de-France et dans le département de la Somme aux côtés des principaux gisements cités dans le texte (source des fonds de carte : BD Carthage, réalisation P.G. Salvador, modifié).Figure 1. Location of Revelles in Hauts-de-France region and in the Somme administrative region associated with the main settlements described in the text (map from Carthage BD source, realization P.G. Salvador, modified).

Figure 1 .

 1 Figure 1. Localisation du gisement de Revelles en région Hauts-de-France et dans le département de la Somme aux côtés des principaux gisements cités dans le texte (source des fonds de carte : BD Carthage, réalisation P.G. Salvador, modifié).Figure 1. Location of Revelles in Hauts-de-France region and in the Somme administrative region associated with the main settlements described in the text (map from Carthage BD source, realization P.G. Salvador, modified).

Figure 2 .

 2 Figure 2. Revelles, Terres-Sellier -A : coupe détaillée de la doline ; B : log de la doline (d'après P. Antoine).Figure 2. Revelles, Terres-Sellier -A : detailled cross section of the dolina ; B : dolina log (after P. Antoine).

Figure 2 . 3 Figure 3 .

 233 Figure 2. Revelles, Terres-Sellier -A : coupe détaillée de la doline ; B : log de la doline (d'après P. Antoine).Figure 2. Revelles, Terres-Sellier -A : detailled cross section of the dolina ; B : dolina log (after P. Antoine).

Figure 3 .

 3 Figure 3. Revelles, Terres-Sellier -1 à 3 : nucleus à unique surface de débitage à méthode unipolaire ; 4 : nucleus discoïde (dessins : A. Lamotte).Figure 3. Revelles, Terres-Sellier -1 to 3: core with main debitage surface and with unipolar method ; 4 : discoïd core (drawings : A. Lamotte).

Figure 4 .

 4 Figure 4. Revelles -typologie des talons selon la catégorie d'éclat.Figure 4. Revelles -typology of flake heel.

Figure 4 .Figure 5 .

 45 Figure 4. Revelles -typologie des talons selon la catégorie d'éclat.Figure 4. Revelles -typology of flake heel.

Figure 5 . 6 Figure 6 .

 566 Figure 5. Revelles, Terres-Sellier -1 à 4 : couteaux à dos naturels (dessins : A. Lamotte).Figure 5. Revelles, Terres-Sellier -1 to 4 : back knives (drawings : A. Lamotte).

Figure 6 .

 6 Figure 6. Revelles, Terres-Sellier -1 à 4 : éclats laminaires ; 5 : pointe Levallois ; 6 à 8 : éclats Levallois (dessins : A. Lamotte).Figure 6. Revelles, Terres-Sellier -1 to 4 : Levallois blade flakes ; 5 : Levallois point ; 6 to 8 : Levallois flakes (drawings : A. Lamotte).

Figure 7 .

 7 Figure 7. Revelles, Terres-Sellier -1 à 4 : éclats kombewa (dessins : A. Lamotte).Figure 7. Revelles, Terres-Sellier -1 to 4 : kombewa flakes (drawings : A. Lamotte).

Figure 7 .

 7 Figure 7. Revelles, Terres-Sellier -1 à 4 : éclats kombewa (dessins : A. Lamotte).Figure 7. Revelles, Terres-Sellier -1 to 4 : kombewa flakes (drawings : A. Lamotte).

Figure 8 .

 8 Figure 8. Revelles, Terres-Sellier -1 : biface à base corticale et bords réservés ; 2 : rare biface quasi-entièrement façonné (dessins : A. Lamotte).Figure 8. Relles, Terres-Sellier -1 : handaxe with cortical basis and edges ; 2 : rare well shaped handaxe (drawings : A. Lamotte).

Figure 8 .

 8 Figure 8. Revelles, Terres-Sellier -1 : biface à base corticale et bords réservés ; 2 : rare biface quasi-entièrement façonné (dessins : A. Lamotte).Figure 8. Relles, Terres-Sellier -1 : handaxe with cortical basis and edges ; 2 : rare well shaped handaxe (drawings : A. Lamotte).

Figure 9 .

 9 Figure 9. Revelles, Terres-Sellier -Analyse métrique des bifaces et des fragments (Triangle noir : biface entiers, carré : fragments de bifaces).Figure 9. Revelles, Terres-Sellier -metric analysis of handaxes (black triangle : whole handaxe ; white square : broken handaxes).

Figure 9 .Figure 10 .

 910 Figure 9. Revelles, Terres-Sellier -Analyse métrique des bifaces et des fragments (Triangle noir : biface entiers, carré : fragments de bifaces).Figure 9. Revelles, Terres-Sellier -metric analysis of handaxes (black triangle : whole handaxe ; white square : broken handaxes).

Figure 10 .

 10 Figure 10. Revelles, Terres-Sellier -1 : fracture liée au gel ; 2 et 3 : flexion ; 4 et 5 : coup porté en pleine face ; 6 : coup de tranchet latéral (dessins : A. Lamotte).Figure 10. Revelles, Terres-Sellier -1 : break due to bad raw materials ; 2 and 3 : flexion ; 4 and 5 : knock in the main face ; 6 : lateral tranchet blow technique (drawing : A. Lamotte).

Figure 11 .

 11 Figure 11. Revelles, Terres-Sellier -modes de fracturation des apex des bifaces (dessins : A. Lamotte).Figure 11. Revelles, Terres-Sellier -way to break handaxes tip (drawings : A. Lamotte).

Figure 11 .

 11 Figure 11. Revelles, Terres-Sellier -modes de fracturation des apex des bifaces (dessins : A. Lamotte).Figure 11. Revelles, Terres-Sellier -way to break handaxes tip (drawings : A. Lamotte).

Figure 12 .

 12 Figure 12. Revelles, Terres-Sellier : 1, 2, 4 : racloirs sur couteau à dos naturel ; 3 : racloir sur éclat Levallois (dessins : A. Lamotte).Figure 12. Revelles, Terres-Sellier : 1,2, 4 : scrapers with back-knife blank ; 3 : scraper on Levallois flake (drawings : A. Lamotte).

Figure 12 .

 12 Figure 12. Revelles, Terres-Sellier : 1, 2, 4 : racloirs sur couteau à dos naturel ; 3 : racloir sur éclat Levallois (dessins : A. Lamotte).Figure 12. Revelles, Terres-Sellier : 1,2, 4 : scrapers with back-knife blank ; 3 : scraper on Levallois flake (drawings : A. Lamotte).

Acknowledgements

This work was supported by Ligue Nationale Contre le Cancer (LNCC), Ligue Contre Le Cancer Grand Est-Bourgogne-Franche Comté (CCIR-Grand Est), Université de Strasbourg, Région Grand Est. This work has been published within the LABEX ANR-10-LABX-0034_Medalis.

98.

Qu, Y., Han

Legends to figures

Table 1 : Scientific foundations for the relevance of PARPs inhibition to target CSCs.

The findings describing the impact of PARPs inhibition in various models of CSCs are summarized. Note 1: R633 and TG1 cells are glioblastoma stem cells isolated from neurosurgical biopsy samples of human glioblastoma [START_REF] Patru | CD133, CD15/SSEA-1, CD34 or side populations do not resume tumor-initiating properties of long-term cultured cancer stem cells from human malignant glio-neuronal tumors[END_REF][START_REF] El-Habr | A driver role for GABA metabolism in controlling stem and proliferative cell state through GHB production in glioma[END_REF][START_REF] Assad Kahn | The antihypertensive drug prazosin inhibits glioblastoma growth via the PKCdelta-dependent inhibition of the AKT pathway[END_REF][START_REF] Wilm | LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from highthroughput sequencing datasets[END_REF]. Note 2: CD133-positive Glioblastoma initiating cells (GICs) were isolated from subcutaneous xenografts originally derived from primary human brain tumor patients including GBM patient specimen 3691.

Note 3: Xenografts of freshly dissociated glioblastoma cells from a xenograft originally derived from the primary glioblastoma patient specimen 3691. Note 4: Primary glioblastoma cell lines E2, G7, R10 were derived from resected human tumors [START_REF] Al-Mayhani | An efficient method for derivation and propagation of glioblastoma cell lines that conserves the molecular profile of their original tumours[END_REF]. Note 5: CD44 + colon adenocarcinoma cells enriched in colon cancer CSCs were isolated and characterized as