Denis Velic

Cathy Charlier

Milena Popova

Titouan Jaunet-Lahary

Zakaria Bouchouireb

Sebastien Henry

Pierre Weigel

Jean-Yves Masson

Adele D Laurent

Igor Nabiev

Sébastien Henry

Fabrice Fleury
email: fleury-f@univ-nantes.fr

Interactions of the Rad51 inhibitor DIDS with human and bovine serum albumins: Optical spectroscopy and isothermal calorimetry approaches

Keywords: repair, protein-ligand interaction warfarin, Ibu, ibuprofen, BR, bilirubin, BV, biliverdin

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Since the early 1970s 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, known as DIDS (Figure 1), has been used as a one of the few efficient chloride channel blockers [START_REF] Wulff | New light on the "old" chloride channel blocker DIDS[END_REF]. Although DIDS may not be used as a drug for treatments of bone and kidney diseases provoked by mutations of these channels, a study has shown that DIDS also acts as an inhibitor of Rad51mediated processes [START_REF] Ishida | DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange[END_REF]. Rad51 is an eukaryotic protein with a key role in the homologous recombination repair pathway, which is an accurate pathway for DNA double-strand break repair. Rad51 binds to the single-stranded tails of the break and forms a helical nucleofilament which allows homologous pairing and strand exchange with a homologous sequence in order to carry out DNA repair [START_REF] Nomme | Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide[END_REF]. Overexpression of Rad51 is frequently found in tumor cells, which suggests that this protein promotes tumorigenesis and cell resistance to treatment. In the past decade, a large number of publications have proposed this protein as a new therapeutic target [START_REF] Huang | Systematic screen identifies miRNAs that target RAD51 and RAD51D to enhance chemosensitivity[END_REF][START_REF] Nagathihalli | RAD51 as a potential biomarker and therapeutic target for pancreatic cancer[END_REF][START_REF] Scott | Small-molecule inhibitors that target protein-protein interactions in the RAD51 family of recombinases[END_REF][7][START_REF] Martinez | Targeting human Rad51 by specific DNA aptamers induces inhibition of homologous recombination[END_REF]. Several studies were focused on inhibiting different steps of Rad51mediated homologous recombination by small molecules [START_REF] Ishida | DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange[END_REF][START_REF] Budke | Optimization of Drug Candidates That Inhibit the D-Loop Activity of RAD51[END_REF][START_REF] Hengel | Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy[END_REF][START_REF] Lamont | Attenuating homologous recombination stimulates an AID-induced antileukemic effect[END_REF]. Recently a phase 1/2 clinical trial has been proposed in the last AACR meeting using a Rad51 inhibitor associated with PARP inhibitor [START_REF] Maclay | CYT01B, a novel RAD51 inhibitor, acts synergistically with PARP inhibitors[END_REF].

Prior to in vivo assays or therapeutic use, it would be interesting to assess the behavior of DIDS in the presence of the most abundant protein in blood plasma, human serum albumin (HSA). Indeed, it has been shown that the distribution, the available concentration and the metabolism of numerous drugs can be significantly altered by their binding to HSA [START_REF] Cui | Binding of daunorubicin to human serum albumin using molecular modeling and its analytical application[END_REF].

HSA shares about 80 percent of sequence similarity with bovine serum albumin (BSA). Both proteins have three domains (I, II and III), each composed of two subdomains, A and B [START_REF] Cui | Binding of daunorubicin to human serum albumin using molecular modeling and its analytical application[END_REF].

HSA has at least three main binding pockets located in subdomains IB, IIA, and IIIA. Most compounds bind to a single site, however, some have two or more binding sites. Carter et al [START_REF] Carter | Crystallographic Survey of Albumin Drug Interaction and Preliminary Applications in Cancer Chemotherapy[END_REF] have estimated that 39%, 19%, and 27% of the compounds are found at three major sites IB, IIA, and IIIA, respectively. However, the contribution of each site to ligand transport is not clearly established.

The distribution, available concentration, and elimination rate of many drugs can be significantly altered by their binding to HSA [START_REF] Cui | Binding of daunorubicin to human serum albumin using molecular modeling and its analytical application[END_REF]. Using different biophysical techniques, including circular dichroism (CD), fluorimetry, UV-vis spectrophotometry, and isothermal titration calorimetry (ITC), we have obtained evidence for the interaction between DIDS and human and bovine albumins. We have also identified and characterized the binding sites for DIDS on these proteins. Our data suggests that DIDS may bind efficiently to serum albumin proteins. This interaction may change the balance between the free and bound DIDS forms, thus affecting its bioavailability and inhibitor efficiency against the Rad51 protein.

Materials and Methods

Chemicals and proteins

DIDS, its derivatives, and human and bovine serum albumins were purchased from Sigma-Aldrich Chemical Co. Both proteins (>99% purity) are essentially fatty-acid-and globulinfree. Stock solutions of DIDS and its analogs were prepared in water at a concentration of 10 mM and then diluted in PBS.

UV-Vis. absorption spectroscopy

UV-vis spectra (from 300 to 450 nm) were recorded with a Jasco V-530 UV-visible scanning spectrophotometer equipped with a Peltier temperature controller. The measurements were performed using quartz cells with a path length of 1 cm. The experimental conditions are described in the figure legends.

Fluorescence spectroscopy

A Jasco spectrofluorometer, model FP-6500, was used for fluorescence measurements. The slit widths used for excitation (350 nm) and emission (380-600 nm) were 1 and 3 nm, respectively. The measurements were made using a 1 cm × 0.2 cm quartz cell at 25°C. Fluorescence emission intensity data were collected directly on a computer connected to the spectrofluorometer and were analyzed using the Jasco Spectra Manager software. A 10-µM DIDS solution was titrated successively by HSA and BSA solutions.

ANS was used to probe the hydrophobic surfaces of HSA and BSA. The fluorescence emission was measured from 400 to 600 nm for λ ex =370 nm. The data were corrected for the effect of solvent on the intrinsic ANS fluorescence by subtracting the ANS fluorescence spectra in the absence of protein for each DIDS concentration.

Isothermal titration calorimetry

Automated titrations of BSA and HSA with different ligands were conducted by means of isothermal titration calorimetry (iTC) using an Auto-iTC 200 microcalorimeter (MicroCal, Northampton, MA, USA). The experiments were carried out at 22°C in PBS. The reference cell was filled with deionized water, and the BSA or HSA protein solution (70 µM) was loaded into the sample cell. The syringe was filled with a solution of DIDS and its derivatives (2 mM). Titrations were performed with 20 successive injections of 2 µL of the ligand in the sample cell. A 150-s time interval between the injections was used in order to ensure complete thermal equilibration after each injection. The same titration was used for site competition experiments, but HSA was preincubated with one of the three competitor ligands (warfarin, ibuprofen, or bilirubin) at concentrations of 50 or 500 µM. The titration experiments were made at least in triplicate.

Control experiments were performed by injection of the buffer into the protein solution under identical conditions and yielded insignificant heats of dilution.

Data analysis was carried out using the Microcal Origin 7.0 software; the data were fitted with the one-or two-set of site model. Experimental data yielded the association constant (Ka), stoechiometry (n), and molar enthalpy binding (ΔH) values. The reaction entropy was calculated using the equations ΔG = ΔH -TΔS = -RT lnK a , where T is the absolute temperature and R = 1.9872 cal mol -1 K -1 .

Circular dichroism measurements

The circular dichroism (CD) spectra of small molecules were measured using a J-810 spectropolarimeter (Jasco, Japan) and a mini-quartz cell with a path length of 1 cm. The spectra were recorded in the range from 385 to 525 nm and averaged over three scans to increase the signal-to-noise ratio [START_REF]Schrödinger Release 2014-1[END_REF] (response time, 0.125 s; resolution, 0.1 nm). The final concentrations of DIDS and HSA were 0-300 µM and 70 µM, respectively, in phosphate buffer saline (PBS), pH 7.4. Warfarin, ibuprofen, bilirubin, and biliverdin were used as competitor ligands at concentrations of 50 µM.

Molecular modeling study

The standard docking protocol was employed to dock DIDS into different binding pockets of HSA. Since no crystallographic data exist for DIDS embedded into HSA, three potential binding sites (IB, IIA, and IIIA) were considered, as in the experiments. Starting from the following crystallized structure of BR, War, or Ibu ligand embedded into HSA (e.g., 2VUE, 2BXD and 2BXG pdb code). For the ligand, a set of initial ligand conformations were produced using the Confgen module [START_REF][END_REF] of the Schrödinger suite 2015-1 [START_REF]Schrödinger Release 2014-1[END_REF]. The Glide Glide v6.6 module [START_REF] Wells | Structural answers and persistent questions about how nicotinic receptors work[END_REF] was t en em lo ed to doc eac conformation into eac binding site e multiste Sc r dinger's rotein re aration Wizard was employed to prepare the HSA protein. 578 residues (residues 5-582) were considered, and the terminal N (C) was replaced with the NH 3 + (COO -) chemical function. PROPKA 3.0 was used to set up all protonation states. A pH of 7.4 was used to ensure correspondence with biological conditions, the lysine and arginine (aspartate and glutamate) residues were (de)protonated, except for Lys199, which was deprotonated, and Glu244 and His9, which were protonated, as described in the literature [START_REF] Carter | Structure of serum albumin[END_REF][START_REF] Diaz | Molecular dynamics study of the IIA binding site in human serum albumin: influence of the protonation state of Lys195 and Lys199[END_REF][START_REF] Walker | Lysine residue 199 of human serum albumin is modified by acetylsalicyclic acid[END_REF]. For the docking, a grid generation was used to prepare structural inputs and to define all binding sites. The protein residues inside the 10(30) Å 3 box, centered in the ligand, were included to define the inner (outer) box. The extra-precision (XP) [START_REF] Friesner | Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes[END_REF] mode of the docking algorithm was employed to dock DIDS. All docking simulations were conducted with a maximum of 10 000 poses per ligand, whose 1000 best poses were chosen for energy minimization. All other parameters were maintained at their default settings, except of an aromatic hydrogens which were considered as the donors. The OPLS-AA force field was used to estimate the ligand-HSA minimization energies [START_REF] Jorgensen | Development and testing of the OPLS all-atom force field on conformational energetics and properties of organic liquids[END_REF]. For each binding site, clustering steps have been performed using the linkage average algorithm to facilitate the pose selection.

Results and Discussion

3.1 Effect of solvent polarity on the physico-chemical proprieties of DIDS.

The physico-chemical characteristics of DIDS were investigated by UV-vis spectrophotometry and fluorescence spectroscopy. The DIDS absorption spectra in different solvents were recorded. Figure 2A shows that the position and shape of the DIDS absorption maximum strongly depend on the solvent polarity. Indeed, the absorption maximum position shifts from 350 nm in water to 372 nm in DMSO, which corresponds to a bathochromic shift with a decrease in solvent polarity.

We have further measured the fluorescence emission spectra of DIDS as a function of the ethanol content in the aqueous buffer. The data showed that, although the positions of the maxima of the fluorescence emission spectra of DIDS were only slightly shifted, the fluorescence emission intensity of DIDS in an aqueous buffer was twofold higher than that in ethanol. Therefore, changes of the solvent polarity strongly modify the spectral characteristics of DIDS. These results confirm our recent theoretical and experimental data on the structural and optical properties of DIDS [START_REF] Jaunet-Lahary | A Joint Theoretical and Experimental Study of the Behavior of the DIDS Inhibitor and its Derivatives[END_REF].

3.2 Evidence of the interaction of DIDS with human and bovine serum albumins.

Figure 3 shows the results of titration of DIDS by BSA and HSA in a buffer solution using the UV-vis and fluorescence spectroscopic approaches. An increase in the concentration of BSA or HSA caused considerable changes in the spectra of DIDS. Upon addition of albumins, we observed a decrease in UV-vis DIDS absorbance. These spectral modifications appear to have been similar for BSA and HSA (Figures 3A and3B). Thus, our study is the first to show that DIDS interacts with both proteins, HSA and BSA.

The interaction between DIDS and HSA or BSA was further confirmed using the fluorescence approach. We performed titration analysis as described above. The results clearly show a diminution of the DIDS signal with an increase in protein concentration. Figure 3 shows that the fluorescence emission intensity of DIDS decreased by a factor of 2-3 in the presence of 4.5 µM of HSA and BSA. The protein mediated-diminutions of fluorescence intensity of DIDS spectra were similar to those observed upon a decrease in the polarity of the DIDS solvent (Figure 2B) suggesting that the residues in the vicinity of bound DIDS are likely in buried hydrophobic/apolar site.

In order to analyze the environment of protein-bound DIDS, we used 8-anilino-1naphthalenesulfonic acid (ANS), a fluorescent compound known as a probe for hydrophobic binding sites of proteins [START_REF] De | The use of the fluorescent probe 8-anilinonaphthalene sulfate (ANS) for collagen and elastin histochemistry[END_REF][START_REF] Essassi | Use of 1-anilino-8-naphthalene sulfonate as a fluorescent probe in the investigation of drug interactions with human alpha-1-acid glycoprotein and serum albumin[END_REF][START_REF] Gasymov | ANS fluorescence: potential to augment the identification of the external binding sites of proteins[END_REF]. Free ANS fluoresces weakly in an aqueous solution, and its fluorescence at about 470 nm strongly increases when it binds to buried hydrophobic sites [START_REF] Slavik | Anilinonaphthalene sulfonate as a probe of membrane composition and function[END_REF]. The fluorescence of ANS in the presence of HSA and BSA provides information on all the hydrophobic binding sites of these proteins. As shown in Figure 4, ANS fluorescence strongly increases in the presence of HSA or BSA. When DIDS was added to the albumin/ANS complex, ANS fluorescence emission decreased with increasing DIDS concentration from 0 to 20 µM.

This finding provides clear evidence that DIDS replaces ANS from its albumin-binding site(s), suggesting that DIDS and ANS bind to the same site or in vicinity binding site. The data also indicate that the DIDS-binding site has a hydrophobic environment for both HSA and BSA proteins. It is noteworthy that ANS and DIDS have two negatively charged sulfonate groups that may strongly bind to positively charged residues within the albumin binding sites [START_REF] Gasymov | ANS fluorescence: potential to augment the identification of the external binding sites of proteins[END_REF].

Identification of structural determinants of DIDS involved in the interaction

ITC titration allows the measurement of heat exchange induced by the interaction between a protein and a ligand. In our case, we performed titrations of HSA and BSA with DIDS and its derivatives.

First, the ITC titrations of HSA and BSA with DIDS (Figures 5A,5B) have shown considerable heat exchange, confirming the interaction between DIDS and both serum albumins. Initially positive, the enthalpy becomes negative when the ligand-to-protein molar ratio rises above two unity. This result indicates that the association reaction is endothermic at two molar ratio below unity and becomes exothermic above this value. This enthalpy variation demonstrates that there are at least two types of binding sites for DIDS in both proteins. As described in "Materials and Methods", we fitted all data points and calculated several binding parameters, including the stoichiometric number N and binding constant Ka (Table 1). We found that, for both HSA and BSA proteins, there are high-affinity and lowaffinity DIDS-binding sites. The Ka values of DIDS for the high-affinity sites were 3.37×10 6

M -1 and 2.08×10 6 M -1 for HSA and BSA, respectively, whereas for the low-affinity sites, the Ka were 100-fold lower. Regarding the stoichiometry of interaction, it seems that only one DIDS ligand binds to the high-affinity site, while two or three ligands interact with the lowaffinity binding site of either HSA or BSA. Hence, DIDS behaves similarly with regard to HSA and BSA, which suggests that the binding sites of both proteins are identical or similar.

The same analysis is performed with DIDS analog, DAZDS, and the binding parameters are presented in the table 2. It note that the affinity constant Ka of DAZDS for HSA (4.12 10 5 M - 1 ) or BSA (9.12 10 5 M -1 ) were 10-fold lower than Ka of DIDS demonstrating a weak affinity of both proteins for DAZDS. Like the high affinity interaction site for DIDS, the stoichiometric number N was one, suggesting one bound DAZDS ligand in this site for both HSA and BSA proteins (Table 2).However ITC titration does not allow the characterization of a second interaction site (Not Determined in the Table 2) for DAZDS, likely because of the very low affinity. This affinity difference between DIDS and DAZDS provides evidence that the substituted chemical groups are involved in the HSA and BSA interaction.

Second, in order to identify the chemical groups of DIDS involved in these protein-ligand interactions, we used two other DIDS analogs where the isothiocyanate group was replaced by an azido (DAZDS) or an amino (DADS) group (Figure 1).

The same experiments have been performed with the DIDS derivatives. ITC titration with DAZDS results in a response similar to that observed with DIDS: the association reaction is first endothermic and then exothermic, when the ligand-to-protein molar ratio rises above unity (Figures 5C,5D). However, the association enthalpy is slightly smaller in the case of DIDS. As with DIDS, the ligand behavior was found to be the same for DAZDS interactions with HSA and BSA.

The titrations with the DADS ligand are very interesting, since there is almost no ITC signal.

This shows that DADS does not interact with HSA or BSA, which is likely to result from the presence of an amino group. Thus, the position of this chemical group of stilbene is important for the interaction.

Localization of DIDS-binding sites in HSA

HSA has three main binding sites, 1, 2, and 3, located in subdomains IIA, IIIA, and IB, respectively [START_REF] Carter | Structure of serum albumin[END_REF]. X-ray crystallography has shown that a large number of ligands are specific for each binding site. For example, warfarin (War), ibuprofen (Ibu), and bilirubin (BR) have been reported to bind to subdomains IIA, IIIA, and IB, respectively [START_REF] Sudlow | The characterization of two specific drug binding sites on human serum albumin[END_REF][START_REF] He | Atomic structure and chemistry of human serum albumin[END_REF]. In order to identify the binding sites for DIDS, we probed the ability of DIDS to displace these albuminbinding drugs from their respective binding sites. Therefore, we preincubated HSA with the above-mentioned competitor ligands before carrying out ITC and CD titrations with DIDS.

Figure 6 shows the ITC titrations with DIDS of HSA alone and HSA complexed with War, Ibu, or BR. Preincubation with Ibu induced a slight signal variation in comparison with HSA alone at HSA-to-Ibu ratios of 2:1 and 1:5, whereas no variation was observed in the case of HSA complexed with War at the same ratios (Figure 6A). This result provides evidence that the War-binding site is not involved in the interaction of DIDS with HSA. The most significant variations in the obtained profiles were observed when HSA was preincubated with BR and especially when the BR concentration was five times higher than the HSA one (Figure 6C).

We hypothesized that the primary binding site for DIDS is located in subdomain IIIA (Ibu site). The secondary binding site could involve subdomain IB (BR site), while site IIA is certainly not involved in the interaction.

To test this hypothesis, we preincubated HSA with bilirubin and titrated the resultant HSA/BR complex with DIDS in CD experiments. In the presence of HSA, BR displays a high induced circular dichroism (ICD) signal caused by its binding to HSA. This optical property is described in detail in many publications and is often used to prove the involvement of subdomain IB in protein-ligand interactions [START_REF] Bouvier | The induced circular dichroism of bilirubin complexed with the alpha-helix form of poly(L-lysine)[END_REF][START_REF] Goncharova | The location of the high-and low-affinity bilirubin-binding sites on serum albumin: ligand-competition analysis investigated by circular dichroism[END_REF][START_REF] Lighnter | Understanding bilirubin conformation and binding. Circular dichroism of human serum albumin complexes with bilirubin and its esters[END_REF].

As shown in Figure 7, the CD signal of BR alone was almost zero at 50 µM. The addition of HSA induces the ICD signal of BR in the form of a positive band at 475 nm and a negative band at 420 nm. The titration of the HSA/BR complex by DIDS leads to a decrease in the intensity of the ICD signal, confirming that DIDS and BR compete for binding to the same site. The same experiment was performed with DAZDS, which resulted in a similar competition with BR, whereas the titration with DADS had a clearly less pronounced effect.

The interpretation of these competition assays is complicated by the presence of more than one binding site for the displaced molecule. This is the case for BR. HSA has one highaffinity (Ka = 10 8 M -1 ) [START_REF] Knudsen | Spectroscopic properties of bilirubinhuman serum albumin complexes: a stoichiometric analysis[END_REF][START_REF] Jacobsen | Albumin-bilirubin binding mechanism[END_REF] and one or two low-affinity binding sites for bilirubin [START_REF] Goncharova | The location of the high-and low-affinity bilirubin-binding sites on serum albumin: ligand-competition analysis investigated by circular dichroism[END_REF][START_REF] Zunszain | Crystallographic analysis of human serum albumin complexed with 4Z,15E-bilirubin-IXalpha[END_REF]. The IIIA site has been suggested to be of low affinity [START_REF] Goncharova | The location of the high-and low-affinity bilirubin-binding sites on serum albumin: ligand-competition analysis investigated by circular dichroism[END_REF]. A number of studies have been conducted in order to determine the location of the primary binding site; however, the results are contradictory. Some authors have presented evidence that BR can bind to the IIA site with a high affinity [START_REF] Goncharova | The location of the high-and low-affinity bilirubin-binding sites on serum albumin: ligand-competition analysis investigated by circular dichroism[END_REF][START_REF] Sochacka | Application of phenol red as a marker ligand for bilirubin binding site at subdomain IIA on human serum albumin[END_REF][START_REF] Minomo | Biological characteristics of two lysines on human serum albumin in the high-affinity binding of 4Z,15Z-bilirubin-IXalpha revealed by phage display[END_REF]. However, many studies have shown the IB site to be the primary binding site [START_REF] Zunszain | Crystallographic analysis of human serum albumin complexed with 4Z,15E-bilirubin-IXalpha[END_REF][START_REF] Zsila | Subdomain IB is the third major drug binding region of human serum albumin: toward the three-sites model[END_REF]. We have probed the ability of War to displace pre-bound BR from the HSA molecule in order to determine whether the two molecules bind to the same site. War reduced the BR CD signal by 50% at much higher concentrations than DIDS. This demonstrates that the IIA site is not the primary binding site for BR. Thus, our results suggest that DIDS binds with a high affinity to the IB site.

To confirm this result and to eliminate any doubt related to the presence of secondary binding sites for bilirubin on HSA, we conducted the same CD experiments using biliverdin.

Biliverdin is known to bind to only one site on HSA, IB [START_REF] Zsila | Subdomain IB is the third major drug binding region of human serum albumin: toward the three-sites model[END_REF]. As shown in Figure 8, DIDS and DAZDS displaced BV from its binding site, whereas DADS had no effect. This confirms that DIDS and DAZDS bind primarily to the IB site.

As in the experiments, the IB and IIIA sites are the most favorable pockets to bind the ligand using the docking procedure. The IIIA binding site is known to be a hydrophobic pocket mainly consisting of apolar residues (Leu, Ala, Gly, and Ile). The entrance of the binding site is, however, characterized by polar and charged amino acids, including Asn391, Arg410, Lys414, and Ser489. The IIIA site is known to efficiently bind non-steroidal aromatic carboxylate (e.g., Ibu). DIDS, containing two sulfonate groups, is undoubtedly a candidate ligand for this binding site [START_REF] Ghuman | Structural basis of the drug-binding specificity of human serum albumin[END_REF][START_REF] Curry | Lessons from the crystallographic analysis of small molecule binding to human serum albumin[END_REF]. The obtained best pose revealed that four specific interactions are establis ed wit t e ligand's sulfonate grou s wo drogen bonds (hereinafter, H-bonds) are formed with Asn391 and Tyr411, and a salt bridge is formed between Arg410 and one of the sulfonate groups. Arg410 is also involved into a -cation interaction with one of the aromatic cycles of DIDS (Figure 9, right). The IB binding site is known to be hydrophobic, quite large, and flexible: it binds, e.g., heme, bilirubin, and biliverdin [START_REF] He | Atomic structure and chemistry of human serum albumin[END_REF]. First, the best pose for a single DIDS was determined; it exhibited numerous strong interaction between the ligand and its neighbors: specific H-bonds with His146 and Arg117 and a salt bridge between Lys190 and one of the sulfonate group. Since the binding pocket was large, it was shown that a second DIDS can easily be bound within the 1B site, as the experimental data suggested. The second DIDS is stabilized by strong salt bridges with Lys137 and forms H-bonds with Thr133, Asn120, and Phe134 (see Figure S2). Note that docking was also performed within the IIA site, but only a limited number of poses of DIDS were located within the hydrophobic pocket, while the majority of them were outside the pocket, which may be explained by the small size of the IIA pocket.

Conclusion

In this study, we demonstrated that the primary, high-affinity binding site of DIDS in HSA is located in subdomain IIIA, while the secondary, low-affinity binding site could be located in subdomain IB.

In the past decade, resurgence of interest has occurred in the development of inhibitors targeting DNA repair pathways [START_REF] Helleday | Homologous recombination in cancer development, treatment and development of drug resistance[END_REF]. Since 2009, when DIDS was discovered as a potential

Rad51 inhibitor, at least seven other small molecules have been described as inhibitors of this human recombinase. These molecules, coupled with anti-cancer drugs, could improve chemotherapy by killing more effectively Rad51-mediated resistant cancer cells [START_REF] Huang | A small molecule inhibitor of human RAD51 potentiates breast cancer cell killing by therapeutic agents in mouse xenografts[END_REF].

Understanding the interaction between Rad51 inhibitors and HSA is a pivotal element for understanding the pharmacokinetics and pharmacodynamics of these inhibitors. Indeed, the interaction with HSA could play the dominant role in the bioavailability of anticancer drugs such as DIDS.

Our data show that the potential interaction with blood proteins should be taken into consideration when developing more effective DIDS derivatives against Rad51. DIDS derivatives not interacting with these circulating proteins while remaining effective against Rad51 may become the serious candidates to sensitize resistant cancers with altered Rad51 activity.

DADS

DAZDSFigure 1 .

 1 Figure 1. Chemical structures of DIDS and its amino and azido derivatives.

FigFigure 2 .

 2 Fig 2A : Effect of solvent polarity (Assay 566 DIDS_Dmso_solvants_2)

Figure 3 .Figure 4 .Figure 5 .

 345 Figure3A : BSA+DiDS (Absorbance assay 549)

Figure 6 .AFigure 7 .Figure 8 .Figure 9 .

 6789 Figure 6. Identification of DIDS binding sites in HSA by binding competition. HSA at a concentration of 70 µM was preincubated with competing ligands for one hour in PBS at 22°C. Warfarine and bilirubin were at a concentration of 50 (blue) or 500 µM (green) and ibuprofen was at a concentration of 35 (blue) or 350 µM (green). Preincubation of HSA with (A) warfarin, (B) ibuprofen, or (C) bilirubin was performed to occupy their respective sites in subdomains IIA, IIIA, or IB. HSA alone (red) or competitor-bound HSA were titrated by DIDS at increasing concentrations from a DIDS solution at 2mM. ITC titrations without protein are shown in black.

Figure

  Figure S1. Left, schematic representation the IB binding site with the predicted poses of two DIDS in the IB binding site. Right, the main residues interacting with the second DIDS in IB.

Table 1 .

 1 Binding and thermodynamic parameters for DIDS interactions with HSA or BSA. HSA and BSA at the concentrations of 70 µM were titrated by DIDS. The data are derived from at least four ITC experiments performed in PBS at pH 7.5 and 22°C.

			Interaction Site 1: high Affinity		Interaction Site 2: low Affinity
		N 1	Ka 1 (M -1 )	H 1	S 1	N 2	Ka 2 (M -1 )	H 2	S 2
				(cal/mol)	(cal/mol/deg)			(cal/mol)	(cal/mol/de
									g)
	HSA	0.93	3.37 10 6	4310	42.8	3.73	3.16 10 4	-4988.3	3.47
		+/-0.05	+/-1.79 10 6	+/-922	+/-2	+/-0.61	+/-1.50 10 4	+/-1360	+/-6
	BSA	1.04	2.08 10 6	3827.3	42.1	3.23	1.71 10 4	-4237.0	5.31
		+/-0.25	+/-0.59 10 6	+/-675	+/-2	+/-0.76	+/-0.26 10 3	+/-512	+/-2

Table 2 .

 2 Binding and thermodynamic parameters for DAZDS interactions with HSA or BSA. HSA and BSA at the concentrations of 70 µM were titrated by DIDS. The data are derived from at least four ITC experiments performed in PBS at pH 7.5 and 22°C.

Acknowledgments

This study was supported by grants from the Ligue Contre le Cancer (Comité 44) and from Program Samuel-De-Champlain grant (n°63/104). SH and DV are supported by a fellowship from the Region Pays de la Loire (CIMATH2). T. J.-L. thanks the Région Pays de la Loire (MES-TransMED) for his PhD grant. A.D.L. and F. F. acknowledge the Région des Pays de la Loire for financial support within the framework of MES-TransMed Pari Scientifique. This research used resources of (1) the GENCI-CINES/IDRIS (Grants c2016085117) and (2) CCIPL (Centre de Calcul Intensif des Pays de Loire). This study was also supported by the Ministry of Education and Science of the Russian Federation, State Contract no. 16.1034.2017/ПЧ (to I.N.).


Audrey Griveau

Clotilde Wiel

Dorian V Ziegler

Martin O Bergo

David Bernard
email: david.bernard@lyon.unicancer.fr

The JAK1/2 inhibitor ruxolitinib delays premature aging phenotypes

Keywords: pathway, progeria, ruxolitinib

.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway regulates cellular senescence and some age-associated alterations, as evidenced by the use of the JAK1/2 inhibitor ruxolitinib, an FDA-approved drug used in the management of myeloproliferative disorders, which decreased bone loss and adipose tissue inflammation and improved physical condition during aging in mice (Farr et al.,

the pro-senescent effects of PLA2R1 [START_REF] Augert | The M-type receptor PLA2R regulates senescence through the p53 pathway[END_REF][START_REF] Bernard | PLA2R1: Expression and function in cancer[END_REF][START_REF] Griveau | Targeting the phospholipase A2 receptor ameliorates premature aging phenotypes[END_REF][START_REF] Vindrieux | PLA2R1 mediates tumor suppression by activating JAK2[END_REF].

Consistently, the JAK/STAT pathway drives the cell cycle arrest associated with PLA2R1-induced senescence, thus affecting senescence beyond the sole inhibition of the SASP as described by others [START_REF] Farr | Targeting cellular senescence prevents age-related bone loss in mice[END_REF][START_REF] Xu | JAK inhibition alleviates the cellular senescence-associated secretory phenotype and frailty in old age[END_REF][START_REF] Xu | Perspective: Targeting the JAK/STAT pathway to fight age-related dysfunction[END_REF] or/and because the SASP can be also a critical mediator of the full senescence [START_REF] Acosta | Chemokine signaling via the CXCR2 receptor reinforces senescence[END_REF]. Moreover, we recently observed that PLA2R1 contributes to progeria phenotypes in vitro in progerin-expressing cells and in vivo in the Zmpste24-/-murine model of progeria [START_REF] Griveau | Targeting the phospholipase A2 receptor ameliorates premature aging phenotypes[END_REF].

These results raise the interesting possibility that targeting JAK/ STAT signaling directly might overcome senescence and reduce disease phenotypes in progeria. In this study, we defined the impact of ruxolitinib, an FDA-approved JAK1/2-competitive inhibitor that binds to and inhibit their kinase activities [START_REF] Mascarenhas | Ruxolitinib: The first FDA approved therapy for the treatment of myelofibrosis[END_REF], on the development of senescence and premature aging phenotypes induced by mutant prelamin A.

To define the ability of ruxolitinib to influence progerin-induced senescence, we used normal human fibroblasts (MRC5) constitutively expressing progerin or control cells expressing lamin A [START_REF] Griveau | Targeting the phospholipase A2 receptor ameliorates premature aging phenotypes[END_REF]. The expression of progerin strongly inhibited cell proliferation when compared to lamin A-expressing cells (Figure 1a,b). The addition of ruxolitinib prevented the progerin-associated growth inhibitory effect (Figure 1a,b). As expected, the constitutive expression of progerin induced premature senescence, as evidenced by an increase in senescence markers, such as senescence-associated β -galactosidase staining (SA-β-Gal), increased expression of the cyclin-dependent kinase inhibitors (CKI) CDKN1A and CDKN2A, and increased expression of the IL-8 SASP component (Figure 1c-f).

Strikingly, all of these senescent characteristics were abolished upon ruxolitinib treatment (Figure 1c-f). Moreover, similar results were obtained using HGPS-derived fibroblasts in which ruxolitinib increased the replicative potential and decreased several senescence markers (Figure S1). Hence, the JAK1/2 inhibitor ruxolitinib inhibits progerin-induced senescence in normal human fibroblasts.

Earlier studies revealed that PLA2R1 contributes to progerin-induced senescence by stimulating farnesyl diphosphate synthase (FDPS) expression and misshapen nuclei formation [START_REF] Augert | The M-type receptor PLA2R regulates senescence through the p53 pathway[END_REF][START_REF] Griveau | Targeting the phospholipase A2 receptor ameliorates premature aging phenotypes[END_REF][START_REF] Vindrieux | PLA2R1 mediates tumor suppression by activating JAK2[END_REF]. FDPS controls the production of a farnesyl lipid, which is attached to the carboxyl terminus of progerin and which is required for its ability to cause disease and induce the characteristic nuclear shape abnormalities observed in HGPS cells [START_REF] Varela | Combined treatment with statins and aminobisphosphonates extends longevity in a mouse model of human premature aging[END_REF]. We argued that if JAK/ STAT signaling mediates PLA2R1-induced senescence in progerin-expressing cells, ruxolitinib should prevent those phenotypes.

Indeed, that was the case: Ruxolitinib reduced the high level of FDPS expression and misshapen nuclei in progerin-expressing cells (Figure 1g,h), without changing GFP-progerin protein level (Figure 1h and Figure S2). Overall, these results confirm that JAK1/2 inhibition by ruxolitinib impacts not only the secretome of senescent cells, as already suggested by others [START_REF] Farr | Targeting cellular senescence prevents age-related bone loss in mice[END_REF][START_REF] Xu | JAK inhibition alleviates the cellular senescence-associated secretory phenotype and frailty in old age[END_REF][START_REF] Xu | Perspective: Targeting the JAK/STAT pathway to fight age-related dysfunction[END_REF], but also the cell cycle arrest, which can itself be induced by the SASP [START_REF] Acosta | Chemokine signaling via the CXCR2 receptor reinforces senescence[END_REF].

Next, we determined whether ruxolitinib treatment could affect progeria-like phenotypes in vivo. Full-length prelamin A accumulates in cells of Zmpste24-deficient mice, which triggers nuclear shape abnormalities and several hallmarks of progeria, including bone abnormalities, rib fractures, muscle weakness, low body weight, and premature death [START_REF] Bergo | Zmpste24 deficiency in mice causes spontaneous bone fractures, muscle weakness, and a prelamin A processing defect[END_REF]. Ruxolitinib administration reduced the number of rib fractures and increased grip strength, bone mineral content (BMC), and survival (Figure 2a-d). These results represent the first proof of concept in cells and mice for the use of ruxolitinib in the treatment of premature aging syndromes.

The effect of ruxolitinib on progeria phenotypes is a phenocopy of that observed in Pla2r1 knockout mice, that is, reduced numbers of rib fractures, increased BMC, and improved grip strength [START_REF] Griveau | Targeting the phospholipase A2 receptor ameliorates premature aging phenotypes[END_REF]. Moreover, similar to Pla2r1-/-mice, ruxolitinib did not affect body weight or bone mineral density (BMD) (Figure S3a,b).

These results support the idea that JAK1/2 mediates PLA2R1induced cellular senescence corroborating our previous findings [START_REF] Griveau | The PLA2R1-JAK2 pathway upregulates ERRalpha and its mitochondrial program to exert tumor-suppressive action[END_REF][START_REF] Vindrieux | PLA2R1 mediates tumor suppression by activating JAK2[END_REF]. Even if unusual, increased BMC without increased BMD has already been reported in human and mouse studies [START_REF] Andreassen | The effects of growth hormone on cortical and cancellous bone[END_REF][START_REF] Khamoui | Bone mineral density and content are differentially impacted by aerobic and resistance training in the colon-26 mouse model of cancer cachexia[END_REF][START_REF] Van Loan | Effect of weight loss on bone mineral content and bone mineral density in obese women[END_REF][START_REF] Windahl | Increased cortical bone mineral content but unchanged trabecular bone mineral density in female ERbeta(-/-) mice[END_REF], and this can eventually occur in the context of weight loss [START_REF] Khamoui | Bone mineral density and content are differentially impacted by aerobic and resistance training in the colon-26 mouse model of cancer cachexia[END_REF][START_REF] Van Loan | Effect of weight loss on bone mineral content and bone mineral density in obese women[END_REF], the murine model of progeria used in our study losing weight. This increase in BMC without increase in BMD was explained by increased bone area (Figure S3c).

We administered ruxolitinib in subcutaneously implanted slow-release pellets. Advantages of this system are that it delivers drug continuously over a long period of time and it reduces the daily stress associated with traditional drug delivery approaches. A potential disadvantage is that drug doses will likely be lower than with traditional methods. Hence, it is possible that the effects of ruxolitinib observed in the current study are underestimated. This argument F I G U R E 1 Ruxolitinib prevents progerin-induced senescence. MRC5 cells were infected with retroviral vectors encoding lamin A or progerin, and subsequently, puromycin was selected. Cells were then treated every 2 days with ruxolitinib at 0.1 µM. (a) Growth curves were performed by counting and seeding the same number of cells at each passage. Cell population doubling was calculated. (b-c) Twelve days after the beginning of the treatment, the same number of cells was seeded, (b) fixed and stained by crystal violet, or (c) assessed for SA-β-Gal activity. (d-g) RNAs were isolated 15 days after the beginning of the treatment and reverse-transcribed, and RT-qPCR was performed for CDKN2A, CDKN1A, IL-8, or FDPS. Results were normalized against ACTB. as the Lmna G609G/G609G mouse model, which more faithfully recapitulates phenotypes of HGPS, alone or in combination with compounds such as farnesyl transferase inhibitor (FTI) [START_REF] Gordon | Clinical trial of the protein farnesylation inhibitors lonafarnib, pravastatin, and zoledronic acid in children with hutchinson-gilford progeria syndrome[END_REF][START_REF] Gordon | Clinical trial of a farnesyltransferase inhibitor in children with Hutchinson-Gilford progeria syndrome[END_REF]. Indeed, our results support that ruxolitinib, by inhibiting FDPS expression that is upstream of FT, impacts the same pathway than FTI. They may then cooperate to revert progerin effects.

Ruxolitinib is currently used as an antitumor agent in the context of myeloproliferative disorders and is proposed to decreased inflammation [START_REF] Bose | JAK2 inhibitors for myeloproliferative neoplasms: What is next?[END_REF][START_REF] Ghoreschi | Jakpot! New small molecules in autoimmune and inflammatory diseases[END_REF][START_REF] Lussana | Inflammation and myeloproliferative neoplasms[END_REF], in agreement with previous observations that ruxolitinib represses pro-inflammatory SASP production [START_REF] Farr | Targeting cellular senescence prevents age-related bone loss in mice[END_REF][START_REF] Xu | JAK inhibition alleviates the cellular senescence-associated secretory phenotype and frailty in old age[END_REF][START_REF] Xu | Perspective: Targeting the JAK/STAT pathway to fight age-related dysfunction[END_REF]. Although the use of ruxolitinib is relatively safe, there are reports of reduced blood cell counts and increased risk of infection and nonmelanoma skin cancer, which would be important to consider before testing in HGPS patients [START_REF] Fabiano | Eruptive squamous cell carcinomas with keratoacanthoma-like features in a patient treated with ruxolitinib[END_REF][START_REF] Saeed | Managing side effects of JAK inhibitors for myelofibrosis in clinical practice[END_REF]. Beyond ruxolitinib and JAK inhibition, another key mediator of the pro-inflammatory SASP, namely the NF-kB transcription factor, is also an important mediator of HGPS [START_REF] Osorio | Nuclear lamina defects cause ATM-dependent NF-kappaB activation and link accelerated aging to a systemic inflammatory response[END_REF]. Taking into account the lack of current therapeutic oppor- new strategy [START_REF] Gordon | Clinical trial of the protein farnesylation inhibitors lonafarnib, pravastatin, and zoledronic acid in children with hutchinson-gilford progeria syndrome[END_REF][START_REF] Gordon | Clinical trial of a farnesyltransferase inhibitor in children with Hutchinson-Gilford progeria syndrome[END_REF] to improve the health of children with HGPS.

ACK N OWLED G M ENTS

We thank the laboratory members and Brigitte Manship for helpful discussions. We also thank Elin Tüksammel for her technical help

  (h) Fifteen days after the beginning of the treatment, cells were analyzed by confocal microscopy. Representative pictures are shown, and counting of normal, mildly misshapen, and severely misshapen nuclei was performed. Nd means not detected. Results presented in this figure are representative of at least two independent experiments. Error bars indicate SD of measurements taken in triplicate. Statistical analysis was performed with Student's t test (*p < .05; **p < .01; ***p < .005) the robust effects of ruxolitinib on cultured cells and the relatively modest effects in vivo. Regardless, some improvements in disease phenotypes in vivo were statistically significant and reproducible. Overall, we believe our study should encourage the design of comprehensive preclinical trials to adjust dosage and routes of administration in progerin-knock-in mice, such

  tunities for the devastating HGPS disease, ruxolitinib, possibly in combination with other validated drugs, could thus constitute a F I G U R E 2 Ruxolitinib delays premature aging phenotypes. (a) Left, number of rib fractures in Zmpste24-/mice treated with vehicle (n = 22) or ruxolitinib (n = 15) at time of death. Right, plot showing the number of rib fractures (y-axis) and survival (x-axis) and the linear regressions obtained for each condition. (b) Dual-energy X-ray absorptiometry (DEXA) analyses of the bone mineral content (BMC) from mice at time of death are shown. (n = 8, five females and three males; per group, average age of mice analyzed is 26.75 weeks and 27.875 weeks for vehicle-or ruxolitinib-treated groups, respectively). (c) Kaplan-Meier plot showing the percentage of Zmpste24-/vehicle-treated (n = 24) and Zmpste24-/ruxolitinib-treated (n = 16) mice with normal grip strength. (d) Kaplan-Meier curves showing survival of vehicle-treated and ruxolitinib-treated mice (n = 24 and n = 16, respectively). Errors bars indicate SEM. Statistical analyses were performed using the two-sided Student's t test for (a) and (b) and the log-rank test

  with mouse experiments. This work was carried out with the support of ANR (ANR-14-CE12-0003). CW is the recipient of a Marie Sklodowska-Curie Individual Fellowship and a Postdoctoral grant from the Swedish Cancer Society. DZ is supported by the Fondation pour la Recherche Médicale (FRM) (FDT201904008259). , and DVZ performed experiments. AG, CW, and DVZ analyzed experiments. MOB and DB designed the study. CW and DB wrote the manuscript with the contribution of all coauthors. MOB and DB supervised the study.

Funding information

ANR, Grant/Award Number: ANR-14-CE12-0003; Fondation pour la Recherche Médicale, Grant/Award Number: FDT201904008259

S U PP O RTI N G I N FO R M ATI O N

Additional supporting information may be found online in the Supporting Information section.


MAIN TEXT

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and one of the main causes of cancer-related deaths. HCC is mainly promoted by chronic viral hepatitis B and C, alcohol, non-alcoholic fatty liver disease (NAFLD) induced by obesity, as well as several genetic diseases of the liver. Due to the intricacy and heterogeneity of risk factors, microenvironment and genetic alterations associated with HCC, the dissection of molecular players and cellular processes underlying tumor development is still challenging [START_REF] Villanueva | Hepatocellular Carcinoma[END_REF]. In a recent paper ( Moreover, this loss triggered fibrosis and accelerated tumor initiation and progression in three distinct murine models of liver tumors: (i) HCC induced by DEN treatment or by a combination of western diet and CCl4 treatment and (ii) intrahepatic cholangiocarcinoma induced by hepatic p53 deletion. As fibrosis is known to result from activation of HSCs, the authors then focused on investigating their role in the observed pro-tumoral effect of FBP1 deletion. Hepatocytic loss of FBP1 induced HSC activation coupled to an initial pro-fibrotic effect, followed by their senescence and their switch to a pro-inflammatory senescence-associated secretory phenotype (SASP). Dasatinib and Quercetin combination or Navitoclax senolytics were then used to explore the potential benefit of killing senescent HSCs. After validating in vitro the toxicity of these compounds towards senescent HSCs, Li et al observed that these treatments dampened tumor progression induced by hepatocytic FBP1 deletion. Finally, they proposed high mobility group protein 1 (HMGB1) as a mediator of the crosstalk between FBP1-deficient hepatocytes and HSCs.

Senescence is a cellular response to stresses (telomere shortening, oxidative stress…) which can be induced in an autocrine or paracrine manner. In addition of being cell cycle arrested, senescent cells secrete numerous factors, including pro-inflammatory factors, which impact the microenvironment (3). In the context of liver cancer, HSC senescence was previously shown to promote obesity-associated HCC development through the SASP, using mice harboring a knockout for the SASP component and inducer IL-1β or depleted in HSCs (4). Li et al further highlighted the functional role for HSC senescence in HCC development using other experimental models (in the context of loss of FBP1) and demonstrated it for the first time using senolytic compounds. Of note, Wakita et al also showed that a newly identified senolytic compound (BET family protein degrader) reduces obesity-associated HCC development in mice [START_REF] Wakita | A BET family protein degrader provokes senolysis by targeting NHEJ and autophagy in senescent cells[END_REF]. Altogether, these articles support a key role of HSC senescence in promoting liver tumorigenesis. Still, as senolytics used in these studies are not specific of senescent HSCs and depletion of p53 in HSCs was reported to decrease HSC senescence and increase liver tumorigenesis [START_REF] Lujambio | Noncell-autonomous tumor suppression by p53[END_REF], the role of HSC senescence in hepatocarcinogenesis remains controversial.

Li et al documented senescence of activated HSCs with several senescence markers, some of which were shown to colocalize with markers of HSCs (CD140B) or activated HSCs (α-SMA). Initially fibrogenic, activated HSCs were previously reported to acquire proinflammatory properties by entering senescence during liver fibrosis. However, the identification of components and regulators of the SASP in the last years unraveled that this secretome is multifaceted and triggers other effects aside from the pro-inflammatory ones, including cell plasticity, regeneration and stemness [START_REF] Gorgoulis | Cellular Senescence: Defining a Path Forward[END_REF]. In this context, a more detailed characterization of the SASP of senescent HSCs and of its functional impact could improve our understanding of their role in liver tumorigenesis.

In an effort to understand the molecular mechanisms underlying the effects of hepatic FBP1 depletion, the authors performed secretome profiling of FBP1-deleted hepatocytes and focused on the observed increased release of HMGB1. They used inflachromene (ICM), a molecule shown to block this secretion, to assess the functional role of HMGB1 and observed an inhibition of HSC activation, subsequent HSC senescence and tumorigenesis. ICM can impact all cell types and is known to also impair autophagy and in another context, HMGB1 deletion in liver had no impact on inflammation and fibrosis [START_REF] Khambu | HMGB1 promotes ductular reaction and tumorigenesis in autophagy-deficient livers[END_REF]. Hence, further specific assessment of the role of hepatocyte-secreted HMGB1 would be needed, using genetic tools.

Moreover, the mechanisms downstream of HMGB1 as well as the involvement of other components of the hepatocytic secretome remain to be explored to fully understand the crosstalk between FBP1-deficient hepatocytes and HSCs. Interestingly, several proteins identified in the secretome of FBP1-deleted hepatocytes, including HMGB1, CCN1, CCL2, CCL7, IGFBP1 and GDF15, have previously been described as SASP components (3). Moreover, FBP1-deleted hepatocytes display marks of unfolded protein response and endoplasmic reticulum (ER) stress, evidenced by transcriptomic profiling and ER morphology, which may be associated with senescence. Altogether, these findings suggest that FBP1 loss in hepatocytes could induce senescence in these cells, resulting in a SASP-dependent communication with HSCs involving HMGB1 and other factors as mentioned above. The potential induction of senescence in hepatocytes and its functional implication in liver tumorigenesis remains an open question.

Hepatic fibrosis is considered as a key contributor to HCC, and activated HSCs as critical mediators by promoting fibrosis and inflammation [START_REF] Villanueva | Hepatocellular Carcinoma[END_REF][START_REF] Coulouarn | Stellate cells and the development of liver cancer: therapeutic potential of targeting the stroma[END_REF]. Interestingly, Li et al by administrating a combination of Dasatinib and Quercetin suppressed progression of liver tumors in DEN-treated mice without improving liver fibrosis, supporting the idea that liver fibrosis is not sufficient to drive HCC and that entry of activated HSCs into senescence is needed [START_REF] Li | FBP1 loss disrupts liver metabolism and promotes tumorigenesis through a hepatic stellate cell senescence secretome[END_REF]. A question raised by these findings is whether fibrosis is a required step to promote HCC formation and progression or whether it is a mark of HSC activation, persistent HSC activation being required for their entry into senescence and the promotion of inflammation by the SASP, which could then promote HCC. This pro-inflammatory SASP could also reinforce senescence in its environment and especially in hepatocytes, potentially creating a vicious circle between senescent hepatocytes and senescent HSCs and long-term tumor promotion.

Additional experiments using single cell analysis at early and late time points in the tumorigenesis process will be helpful to prove the existence of this feedforward loop.

To conclude, Li et al present novel insights into the interplay between hepatocytes and HSCs (Fig. 1), which improve our understanding of the biology of HCC and particularly of the role of senescence in this context. Moreover, although the identification of senolytic drugs displaying high specificity and efficacy remains a major challenge, the authors provide evidence that targeting senescent HSCs could be a promising therapeutic approach for HCC.

More generally, this study reinforces the idea that targeting senescent cells in the tumor microenvironment could be in some contexts a valuable therapeutic strategy. Of importance, eliminating senescent cells could also be a relevant preventive approach in cirrhotic patients to decrease inflammation and prevent development of HCC.

  Li F, Huangyang P, Burrows M et al. FBP1 loss disrupts liver metabolism and promotes tumorigenesis through a hepatic stellate cell senescence secretome. Nat Cell Biol. 2020 Nat Cell Biol 2020;22(6):728-739), Li et al identified the gluconeogenic enzyme fructose 1,6-bisphosphatase 1 (FBP1) as a tumor suppressor in the liver and present evidences using senolytic compounds (compounds inducing death of senescent cells) that senescence of hepatic stellate cells (HSCs) induced by loss of FBP1 promotes HCC in mice (2). Li et al first observed that FBP1 expression is silenced during HCC progression, both in human tumors from the TCGA database and murine tumors from Trp53 flox/flox mice, diethylnitrosamine (DEN)-treated mice and NAFLD-HCC murine models. The authors then took advantage of a conditional Fbp1 flox/flox allele to study the effects of specific FBP1 deletion in hepatocytes. FBP1 loss disturbed liver metabolism, as evidenced by abolished gluconeogenesis, defective fatty acid oxidation, hepatomegaly and mild hepatic steatosis.

ACKNOWLEDGEMENTS

We thank INCA, INSERM and CNRS for their support.

CONFLICT OF INTEREST

None declared.

Role of senescence in the crosstalk between hepatocytes and HSCs in liver tumorigenesis