Emmanuel Puig

Christophe Desmarets

Geoffrey Gontard

Marie Noelle Rager

An- Drew L Cooksy

Hani Amouri

Capturing a Square Planar Gold(III) Complex Inside a Platinum Nanocage: A Combined Experimental and Theoretical Study

A novel synthetic procedure was set up to gain access to platinum coordination cages Pt2L4, which are less investigated compared to their palladium counterparts. This Pt2L4 nanocage exhibits an adequate cavity for guest encapsulation. Indeed the Au(III) metal complex [Au(bdt)2] -(bdt = benzene-1,2dithiolate) was successfully captured inside the cavity, in contrast to the analogous palladium cage which failed to host the gold complex. This result represents a rare example where a metal complex with thioligands can be encapsulated in a coordination cage. Moreover it highlights the role of the metal center and the robustness of the platinum cage for host-guest chemistry. This discovery will inspire researchers in this area to pay more attention to Pt-cages. The host-guest system was fully characterized by NMR techniques and X-ray crystallographic analysis. Moreover the nature of the host-guest interaction in this unique example was investigated and rationalized by DFT computational studies.

n INTRODUCTION

Host-guest chemistry of self-assembled coordination cages has attracted much attention in the last two decades. [START_REF] Fujita | Molecular paneling via coordination[END_REF][START_REF] Caulder | Supermolecules by design[END_REF][START_REF] Ward | Polynuclear coordination cages[END_REF][START_REF] Severin | Self-assembled organometallic receptors for small ions[END_REF][START_REF] Fujita | Coordination Assemblies from a Pd(II)-Cornered Square Complex[END_REF][START_REF] Chakrabarty | Supramolecular Coordination: Self-Assembly of Finite Twoand Three-Dimensional Ensembles[END_REF] These cages can now be obtained with different shapes and display cavities with controllable sizes. [START_REF] Mirtschin | A Coordination Cage with an Adaptable Cavity Size[END_REF][START_REF] Amouri | Confined Nanospaces in Metallocages: Guest Molecules, Weakly Encapsulated Anions, and Catalyst Sequestration[END_REF][START_REF] Smulders | Building on architectural principles for threedimensional metallosupramolecular construction[END_REF][START_REF] Han | Selfassembled coordination cages based on banana-shaped ligands[END_REF][START_REF] Li | Multi-component coordination-driven self-assembly toward heterometallic macrocycles and cages[END_REF][START_REF] Freye | Template Control over Dimerization and Guest Selectivity of Interpenetrated Coordination Cages[END_REF][START_REF] Harris | Giant hollow MnL2n spherical complexes: structure, functionalisation and applications[END_REF][START_REF] Szaloki | Controlling the Host-Guest Interaction Mode through a Redox Stimulus[END_REF][START_REF] Turega | Mapping the Internal Recognition Surface of an Octanuclear Coordination Cage Using Guest Libraries[END_REF] As a consequence, a wide range of applications have been investigated including molecular sensing, chiral separation, drug delivery and catalytic properties. [START_REF] Wang | Selfassembled nanospheres with multiple endohedral binding sites pre-organize catalysts and substrates for highly efficient reactions[END_REF][17][START_REF] Therrien | Complex-in-a-Complex" cations [(acac)(2)M subset of RU6(p-iPrC(6)H(4)Me)(6)(tpt)(2)(dhbq)(3)](6+): A Trojan horse for cancer cells[END_REF][START_REF] Casini | The Promise of Self-Assembled 3D Supramolecular Coordination Complexes for Biomedical Applications[END_REF][START_REF] Zheng | Encapsulation of Pt(IV) prodrugs within a Pt(II) cage for drug delivery[END_REF] Among these cages, particular interest has been devoted to the formation of the M2L4 cage-type. [START_REF] Schmidt | Self-assembled M2L4 coordination cages: Synthesis and potential applications[END_REF][START_REF] Lewis | Stimuli-responsive Pd2L4 metallosupramolecular cages: towards targeted cisplatin drug delivery[END_REF][START_REF] Yamashina | Anisotropic Expansion of an M2L4 Coordination Capsule: Host Capability and Frame Rearrangement[END_REF][START_REF] Croue | A selfassembled M2L4 cage incorporating electron-rich 9-(1,3dithiol-2-ylidene)fluorene units[END_REF][START_REF] Preston | Controlled Formation of Heteroleptic [Pd2(La)2(Lb)2]4+ Cages[END_REF][START_REF] August | Maximizing Coordination Capsule-Guest Polar Interactions in Apolar Solvents Reveals Significant Binding[END_REF] These architectures display high symmetry and are capable of encapsulating guests such as anions or organic molecules but to a lesser extent metal compounds. [START_REF] Clever | Stacked Platinum Complexes of the Magnus' Salt Type Inside a Coordination Cage[END_REF][START_REF] Yoshizawa | Metal-Metal d-d Interaction through the Discrete Stacking of Mononuclear M(II) Complexes (M = Pt, Pd, and Cu) within an Organic-Pillared Coordination Cage[END_REF][START_REF] Desmarets | Encapsulation of a Metal Complex within a Self-Assembled Nanocage: Synergy Effects, Molecular Structures, and Density Functional Theory Calculations[END_REF][START_REF] Johnstone | Desymmetrization of an Octahedral Coordination Complex Inside a Self-Assembled Exoskeleton[END_REF] They are usually obtained by mixing metal precursors displaying four available coordination sites and bidentate ligands. While Pd2L4 cages have been intensively described, surprisingly so far only a few platinum analogues have been reported. [START_REF] Chand | A new class of self-assembly multinuclear Pt(II) coordination cages by a modular approach[END_REF][START_REF] Kaiser | Self-assembled palladium and platinum coordination cages: Photophysical studies and anticancer activity[END_REF] Our group have developed a variety of metallacages of type M2(L 1 )4 based on Co(II) and Cu(II) using a semi-rigid bidentate ligand where L 1 = 1,3-bis(benzimidazol-1-ylmethyl)-2,5-dimethoxy-4,6-dimethylbenzene. [START_REF] Amouri | Host-guest interactions: Design strategy and structure of an unusual cobalt cage that encapsulates a tetrafluoroborate anion[END_REF][START_REF] Amouri | Supramolecular cobalt cages and coordination polymers templated by anion guests: self-assembly, structures, and magnetic properties[END_REF][START_REF] Desmarets | A unique type of a dicobalt cage templated by a weakly coordinated hexafluorophosphate anion: design, structure and solid-state NMR investigations[END_REF][START_REF] Desmarets | Self-Assembly, and Molecular Structures of 3D Copper(II) Capsules Templated by BF4-Guest Anions[END_REF] We also demonstrated that these systems are capable of selectively encapsulating weakly coordinated anions such as BF4 and PF6 anions. [START_REF] Desmarets | A unique type of a dicobalt cage templated by a weakly coordinated hexafluorophosphate anion: design, structure and solid-state NMR investigations[END_REF] However in the presence of strongly coordinated anions the related coordination polymers were obtained instead, highlighting the lability of metal-ligand (L 1 ) interaction in these metallacages. [START_REF] Amouri | Supramolecular cobalt cages and coordination polymers templated by anion guests: self-assembly, structures, and magnetic properties[END_REF] Subsequently, we shifted our efforts towards the design of another kind of M2L4 metallacage but using the more rigid pyridine-based ligands. [START_REF] Desmarets | Self-assembled M2L4 nanocapsules: synthesis, structure and host-guest recognition toward square planar metal complexes[END_REF] The latter are more strongly bound to the metal centers. Indeed we demonstrated that the metallacage Pd2L4 successfully accommodates anionic organometallic species [Pt(NO2)4] 2-inside the cavity. [START_REF] Desmarets | Encapsulation of a Metal Complex within a Self-Assembled Nanocage: Synergy Effects, Molecular Structures, and Density Functional Theory Calculations[END_REF] 1 H-NMR studies and DFT calculations allowed us to rationalize the nature of non-covalent interactions occurring between the guest complex and the Pd2L4 cage. It is worth mentioning that encapsulating kinetically labile metal complexes inside self-assembled cages is a difficult task and only few examples have been reported. [START_REF] Therrien | Complex-in-a-Complex" cations [(acac)(2)M subset of RU6(p-iPrC(6)H(4)Me)(6)(tpt)(2)(dhbq)(3)](6+): A Trojan horse for cancer cells[END_REF][START_REF] Clever | Stacked Platinum Complexes of the Magnus' Salt Type Inside a Coordination Cage[END_REF][START_REF] Osuga | Metal Ion Arrays Templated by Coordination Cages[END_REF] n RESULTS AND DISCUSSION Pursuing our investigations in this area we sought again to accommodate metal complexes, but now displaying thio-ligands such as

[n- Bu4N][Au(bdt)2] (bdt = benzene-1,2-dithiolate).
This type of molecule is highly challenging because it contains strongly coordinating sulfur centres and displays interesting magnetic and redox properties. [START_REF] Ray | S'-Coordinated o-Dithiobenzosemiquinonate(1-) Radicals Exist in Coordination Compounds? The [AuIII(1,2-C6H4S2)2]1-/0 Couple[END_REF][START_REF] Murata | Efficient Synthesis of One-and Two-Dimensional Multimetallic Gold-Bis(dithiolene) Complexes[END_REF] In this paper we report the synthesis of nanocages of the type M2L4 (M = Pd, Pt) (Figure 1) and our investigations towards the encapsulation of a square planar Au(III) guest molecule.

Our first approach was to examine whether our Pd2L4 cage available in hand, (L = 1,3bis(pyridin-3-ylethynyl)-5-methoxybenzene) is adequate to host the gold(III) complex since we demonstrated that such a metallacage was able to accommodate [PtCl4] 2-species inside the cavity. [START_REF] Desmarets | Self-assembled M2L4 nanocapsules: synthesis, structure and host-guest recognition toward square planar metal complexes[END_REF] We reasoned that recognition could be enhanced through electrostatic interactions between the positively charged Pd(II) cations and the anionic nature of the guest but also by hydrogen bonding provided by the endohedral hydrogens of ligand L pointing towards the interior of the cavity. The host-guest studies were carried out in CD3CN/DMSO-d6 (4:1) mixture due to solubility limitations. Thus, when one equivalent of [n-Bu4N][Au(bdt)2] was added to a solution of cage [Pd2(L)4][OTf]4 (1a), the 1 H NMR spectrum showed the formation of multiple peaks relative to the starting material 1a and the appearance of the free ligand L and unidentified materials (Figure S1). Further addition of another equivalent of [Au(bdt)2] -to the reaction mixture provided L in equal amount to the metallacage 1a, while addition of a third equivalent led to the decomposition of the metallacage and formation of the free ligand L almost quantitatively along with free [Au(bdt)2] -as well . We reasoned that the analogous Pt2L4 cage should display stronger Pt-ligand interaction and hence should be more appropriate to study this type of host-guest interaction with the challenging Au(III) guest [n-Bu4N][Au(bdt)2].

Our first step was to find a rational synthetic approach to this kind of platinum nanocage Pt2L4. Gratifyingly after several attempts we found that treatment of ligand L with freshly prepared [Pt(EtCN)4][OTf]2 precursor in acetonitrile solution at 80°C for several days afforded the desired nanocage complex [Pt2L4][OTf]4 [1b] in 80% yield (figure 1). The infrared spectrum of 1b showed the presence of triflate anions bands at 1256 cm -1 and displayed the alkyne stretching band at 2219 cm -1 . Moreover the integrity of the nanocage [Pt2L4][OTf]4 (1b) was demonstrated by electrospray spectrometry, in which the [Pt2L4] 4+ fragment with varying numbers of triflate counterions was observed. The 1 H NMR spectrum of 1b was recorded in CD3CN due to solubility limitations. The spectrum shows that the coordinated ligands in 1b display a symmetric pattern close to the free ligand L. A downfield shift relative to free ligand was observed, particularly for the Ha and He protons (Figure S2). Full characterization of 1b is given in the supporting information.

We then investigated the host-guest properties of the metallacage 1b towards the metal complex guest [n-Bu4N][Au(bdt)2] under similar conditions to those used for the palladium cage [Pd2L4][OTf]4 (1a). It is worth mentioning that examples where a square metal complex is encapsulated within a metallacage, especially in the case of a platinum capsule, are limited 27, 28 and more scarce when the complex displays thioligands because they are also able to bind the metal centre. [START_REF] Moussa | Induced phosphorescence from Pt -> Ag and Ag(I)center dot center dot center dot Ag(I) metallophilic interactions in benzenedithiolatodiimine-Pt-2/Ag-2 clusters: a combined experimental and theoretical investigation[END_REF] When the host molecule [Pt2L4][OTf]4 (1b) was treated by one equivalent of the guest complex [n-Bu4N][Au(bdt)2] in a mixture of CD3CN/DMSO-d6 (4:1), pronounced chemical shift changes were observed for the anionic complex and the cage as well, suggesting the encapsulation of the guest. For instance the signals attributed to hydrogens He and Ha for 1b moved from δ 9.34 ppm and δ 9.06 ppm to δ 9.92 ppm and δ 9.39 ppm while the NMR resonances of the aromatic protons H2 and H3 for the guest changed from δ 7.09 ppm and δ 6.78 ppm to δ 7.21 ppm and δ 6.31 ppm (Figure 2c, Figure S3). Addition of another equivalent of guest molecule led to two novel signals at δ 7.09 ppm and δ 6.78 ppm, which correspond to the signals of free Au(III) guest (Figure 2d). No changes in the 1 H NMR spectrum occurred in the presence of an excess of guest molecules. Moreover upon leaving the solution to stand over time no evolution occurred, suggesting that this host-guest system is kinetically and thermodynamically stable. These results contradict completely those obtained for the analogous and more labile Pd2L4 metallacage 1a, highlighting the robust nature of our platinum cage 1b.

We then attempted to isolate the host-guest assembly. Thus, treatment of 1b with excess of [n-Bu4N][Au(bdt)2] in CH3CN, afforded a green precipitate which was isolated and characterized by NMR spectroscopy in DMSO ( 1 H-, [START_REF] Harris | Giant hollow MnL2n spherical complexes: structure, functionalisation and applications[END_REF] The 1 H-NMR spectrum displayed two series of resonances for the Au(III) complex : two doublets of doublets at δ 7.08 ppm and δ 6.80 ppm corresponding to H2 and H3 protons of free Au(bdt)2 molecules and two other doublets of doublets at δ7.13 ppm and δ 6.34 ppm corresponding to H2 and H3 protons of encapsulated Au(III) complex, respectively in 3/1 ratio. 2D NOESY experiment showed correlation between He proton of Pt2L4 host and H2 proton from [Au(bdt)2] -guest, proving the encapsulation of the latter. In addition Hk protons which point to the interior of the cage moved up-field (from δ 7.56 ppm to δ 6.22 ppm) and are perpendicular to the Au(III) guest since no NOE correlation was observed between Hk and H2 or H3. Finally, NOESY spectra showed exchange between the encapsulated Au(III) complex and the free [Au(bdt)2] -molecule is occurring (Figure S5). Interestingly the 195 Pt-H couplings are initially visible for complex 1b but disappear just after guest addition confirming this dynamic exchange. As shown in Figure S4 in the supporting information, both free cage and complexed-cage protons were seen in the spectrum, during sequential guest additions, indicating a slow exchange of the host-guest complex. 42 . In all cages the X-ray structure showed the presence of one encapsulated molecule of the anionic [Au(bdt)2] -complex inside the cavity of Pt2L4 cage and the presence of free [Au(bdt)2] -in accord with the 1 H-NMR solution studies (Figure 4). The Pt-N bond lengths lie in the range of 2.01-2.03 Å. The Pt---Pt distance is 11.880(2) Å and the average distance between two facing phenyl rings is around 10.7 Å so that the cavity size is comparable to that observed for cage 1a. [START_REF] Desmarets | Self-assembled M2L4 nanocapsules: synthesis, structure and host-guest recognition toward square planar metal complexes[END_REF] The anionic [Au(bdt)2] -guest is located inside the cavity and adopts a perpendicular orientation relative to planes containing the square platinum centers. The three metal centers Pt---Au---Pt are not perfectly aligned, and the Pt---Au distances are 5.962(1) and 5.999(1) Å, precluding any direct metal-metal interaction. However, the guest interacts with the frame of the cage via hydrogen bonding interactions between each sulfur atom of the [Au(bdt)2] -and two ortho aromatic protons from two different coordinated pyridines. Indeed, the C---S and H---S distances lie respectively in the range of 3.65-3.89 Å and 2.76-2.99 Å, with C-H---S bond angles of ca. 139 to 171°. These values are comparable to those reported for complexes displaying C-H---S bonding interactions. [START_REF] Krepps | Hydrogen bonding with sulfur[END_REF] On the other hand the structure reveals the presence of other anionic molecules [Au(bdt)2] -located outside the assembly, but stacking perfectly over the platinum centres of the cage (Figure 3). The Au---Pt distances are 4.626 (1) and 4.698 (1) Å, larger than the sum of van der Waals radii (3.73 Å) precluding any metal-metal interaction. 44 To shed light on the nature of the host-guest interactions in the supramolecular assembly, properties of the host-guest complex were calculated by density functional theory (DFT), using both the B3LYP-D3 [START_REF] Becke | Density-Functional Thermochemistry .3. The Role of Exact Exchange[END_REF][START_REF] Lee | Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron-Density[END_REF][START_REF] Schwabe | Double-hybrid density functionals with long-range dispersion corrections: higher accuracy and extended applicability[END_REF] and M06 48 methods, and several basis sets for comparison. [START_REF] Binkley | Self-Consistent Molecular-Orbital Methods .21. Small Split-Valence Basis-Sets for 1st-Row Elements[END_REF][START_REF] Dunning | Gaussian-Basis Sets for Use in Correlated Molecular Calculations .1. The Atoms Boron through Neon and Hydrogen[END_REF][START_REF] Stevens | Compact Effective Potentials and Efficient Shared-Exponent Basis-Sets for the 1st-Row and 2nd-Row Atoms[END_REF][START_REF] Stevens | Relativistic Compact Effective Potentials and Efficient, Shared-Exponent Basis-Sets for the 3rd-Row, 4th-Row, and 5th-Row Atoms[END_REF][START_REF] Hay | Abinitio Effective Core Potentials for Molecular Calculations -Potentials for the Transition-Metal Atoms Sc to Hg[END_REF] Additional details are provided in the Supplemental Information.

The gas-phase host-guest complexation energies were calculated to be quite similar among the different basis sets and DFT methods, suggesting that these methods are adequate for estimating the energetics. The interior gold guest is predicted to be more strongly bound than the exterior guest, by 12-18 kcal/mol in the absence of dispersion and by 42-47 kcal/mol when dispersion is included. This makes sense, given that the interior guest has more near-atom interactions available and also the possibility of π-stacking interactions between the central methoxyphenyl ring of each arm and the phenyl rings of the guest. Furthermore, the overall binding energy of the two guests with the host is greater than the sum of the individual binding energies, indicating that each guest activates the host towards accepting a second guest. The donor-acceptor interaction energies, including charge-transfer contributions, were estimated by NBO analysis. For both the interior and exterior guests, the dominant contribution arises from the guest acting as a donor to the organic component of the host cage. The Pt atoms are too distant from the guest molecules for any metal-metal host-guest interaction to account for more than about a quarter of the total binding energy. Looking specifically for Pt-Au interactions, we find that in the case of the exterior guest, most of the energy contribution involving the neighbouring Pt atom is ascribed to direct Pt-Au interaction, whereas for the interior guest the Pt atoms interact roughly equally with the Au atom and with the associated S atoms. In either case, however, Pt-Au bonding appears to be a small contribution to the stabilization of these guests. Instead, most of the donor-acceptor stabilization is provided by the nearest-neighbour interactions, especially the electropositive hydrogen atoms on the host aryl groups as the acceptors and the guest sulfur atoms and aryl carbons as donors. This is consistent in part with the NBO orbital analysis, which is further discussed in the SI. Unfortunately, a full-scale energy decomposition analysis is prohibitive for a system of this size. However, the present DFT calculations strongly indicate that both guest molecules are stabilized by a combination of dispersion, donor-acceptor interaction, and electrostatics. The interior guest appears to be stabilized largely by the dispersion interaction, whereas electrostatic interactions may play a larger role in stabilizing the exterior guest. Overall, the interior guest is predicted to be more strongly bound to the cage by roughly 40-50 kcal/mol.

n CONCLUDING REMARKS

In this work we have investigated the encapsulation of square planar gold complex [n-Bu4N][Au(bdt)2] containing thioligands using metallacages of type M2L4 M = Pd (1a), M = Pt (1b). The latter was prepared using a novel synthetic procedure. While the Pd2L4 cage decomposed in the presence of the guest molecule [Au(bdt)2] -, in stark contrast the platinum cage successfully accommodated an anionic square planar gold(III) complex as demonstrated by NMR techniques (2D NOESY). This result constitutes a rare example in the literature. Furthermore, single crystal X-ray diffraction studies confirm the presence of the gold guest inside the cavity stabilised through hydrogen-bonding with the ligands of the cage Pt 2 L 4 . The X-ray structure also revealed the presence of a weak interaction between the platinum centre of the assembly and another anionic gold complex located outside the cavity. DFT calculations have been advanced to rationalize the nature of host-guest interactions in this rare and unique assembly n EXPERIMENTAL SECTION General synthetic methods All solvents used were reagent grade or better. Commercially available reagents were used as received. The ligand L, the solvated platinum building block [Pt(EtCN)4][OTf]2, and the goldbis(dithiolene) complex were prepared according to published methods. [START_REF] Murata | Efficient Synthesis of One-and Two-Dimensional Multimetallic Gold-Bis(dithiolene) Complexes[END_REF][START_REF] Desmarets | A Rigid Angular Bidentate Ligand for the Design of a New Class of Coordination Polymers Based on Silver(I) Salts -Influence of the Anion on Coordination Assemblies[END_REF][START_REF] Kukushkin | Tetrakis(propanenitrile)platinum(II) trifluoromethanesulfonate as a suitable intermediate in synthetic Pt(II) chemistry[END_REF] All experimental manipulations were carried out under argon using Schlenk techniques. IR spectra were recorded on a Bruker Tensor 27 (Bruker Corp., Rheinstetten, Germany) equipped with a Harrick ATR. Elemental analyses were performed at the Microanalytical Laboratory of the Sorbonne-université. Positive ESI mass spectra were obtained using a triple quadruple mass spectrometer (Quattro II Micromass, Waters, UK). Automatic data acquisition was processed using the software Masslynx V4.0. NMR experiments were carried out on a Bruker Avance II 300 MHz, or Bruker Neo 500 MHz spectrometers operating at 300 K with chemical shifts referenced to residual solvent peaks. Chemical shifts are reported as parts per million (ppm) and coupling constants (J) in hertz (Hz). Standard abbreviations indicating multiplicity were used as follows: m = multiplet; t = triplet; d = doublet; s = singlet and b = broad.

Synthesis of [PtL4][OTf]4 (1b)

To an oven dried Schlenk flask equipped with a magnetic stirrer was added [Pt(EtCN)4][OTf]4 (126 mg, 0.176 mmol, 1 equiv.), 5-Methoxy-1,3bis(pyridin-3-ylethynyl)benzene (L) (140 mg, 0,451 mmol, 2 equiv.) which were solubilized in argondegassed MeCN (30 ml) freshly distilled over CaH2. The solution was heated to reflux and maintained under argon with stirring for four days at 80°C. The solution was then allowed to cool at room temperature and concentrated under vacuum to 3 ml. The mixture was precipitated by slow addition of freshly distilled and degassed diethylether (20 ml). The white solid was filtered using a filtrating cannula, washed three times with diethylether (10 ml) and recrystallized from a mixture of CH2Cl2, DMSO/Et2O. Dried under vacuum to afford the desired product as air-stable white solid. 170 mg (86 %). [START_REF] Fujita | Molecular paneling via coordination[END_REF] H NMR (300 MHz, CD3CN, 298K): δ 9.34 (s, 8H, He); 9.06 (dd, 8H, J=6.0, 0.5 Hz, Ha); 8.05 (dt, 8H, J=9.0, 0.5 Hz, Hc); 7.61 (ddd, 8H, J=9.0, 6.0, 0.5Hz, Hb); 7.56 (t, 4H, J=1.3Hz, Hk); 7.24 (d, 8H, J=1.3Hz, Hi), 3.82(s, 12H, OMe). IR (KBr): : 1 H NMR (500 MHz, DMSO-d6): δ 9.88 (s, 8H, He), 9.45 (d, J = 6.0 Hz, 8H, Ha), 8.23 (dt, J = 7.9 Hz, J = 1.4 Hz, 8H, Hc), 7.87 (dd, J = 7.9 Hz, J = 6.0 Hz, 8H, Hb), 7.13 (dd, J = 5.9 Hz, J = 3.1 Hz, 4H, H2int), 7.08 (dd, J = 5.9 Hz, J = 3.2 Hz, 12H, H2ext), 7.02 (d, J = 1.2 Hz, 8H, Hi), 6.80 (dd, J = 5.9 Hz, J = 3.2 Hz, 12H, H3ext), 6.34 (dd, J = 5.9 Hz, J = 3.1 Hz, 4H, H3int), 6.22 (t, J = 1.2 Hz, 4H, Hk), 3.72 (s, 12H, OMe).). [START_REF] Harris | Giant hollow MnL2n spherical complexes: structure, functionalisation and applications[END_REF] provided suitable crystals. A single crystal was selected, mounted and transferred into a cold nitrogen gas stream. Intensity data was collected with a Bruker Kappa-APEX2 system using finefocus sealed tube Mo-Kα radiation. Unit-cell parameters determination, data collection strategy, integration and absorption correction were carried out with the Bruker APEX2 suite of programs. The structure was solved with SHELXT-2014 56 and refined anisotropically by full-matrix least-squares methods with SHELXL-2014 56 using the WinGX suite. [START_REF] Farrugia | WinGX suite for small-molecule singlecrystal crystallography[END_REF] Significant electron density remains near gold and platinum atoms despite our repeated attempts at improving absorption correction. The crystal contains severely disordered and partially missing molecules of volatile solvent (diethyl ether, dimethyl sulfoxide and/or acetonitrile). PLATON SQUEEZE 58 was used for the final refinement cycles to account for the resulting voids. The structure was deposited at the Cambridge Crystallographic Data Centre with number CCDC 1841449 and can be obtained free of charge via www.ccdc.cam.ac.uk.

(ν, cm -1 ) ν(C≡C) = 2219, ν(C-F) =1256, ν(S=O) =1231, ν(S-O) =1029.ES-MS (m/

Crystal data for 3

Yellow plate, C134.5 H104 Au3.5 F1.5 N8 O7.5 Pt2 S14.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website. CCDC reference number 1841449 contains crystallographic data for complexes 3 presented in this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Additional figures and tables (PDF)

n AUTHOR INFORMATION Corresponding Author *E-mail: hani.amouri@sorbonne-universite.fr. Phone: +33-1-44273083. *E-mail:Christophe.desmarets@sorbonne-universite.fr.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources

n ACKNOWLEDGMENT Sorbonne Université-Campus Pierre et Marie Curie, CNRS and Ile de France region for financial support of the 500 MHz NMR spectrometer to Chimie Paristech are gratefully acknowledged. Financial support from the National FT-ICR network (FR 3624 CNRS) for conducting the research is gratefully acknowledged SYNOPSIS TOC. A novel platinum-based metallacage Pt2L4 has been designed and self-assembled. This nanocage displays a cavity for guest encapsulation. Indeed the Au(III) metal complex [Au(bdt)2] -(bdt = benzene-1,2-dithiolate) was successfully captured inside the cavity of the Pt2L4 and in contrast to that observed for the analogous Pd2L4 metallacage.

Figure 1 .

 1 Schematic representation of the ligand L and the metallacages [M2L4][OTf]4. M = Pd (1a); M = Pt (1b).

M

  = Pd (1a); M = Pt (1b)

  Figure 2. 1 H NMR host-guest studies with metallacage 1b in the presence of [n-Bu4N][Au(bdt)2] in CD3CN/DMSO-d6 (4:1) a) free [n-Bu4N][Au(bdt)2] guest b) the cage complex 1b c) 1 eq. of [n-Bu4N][Au(bdt)2] d) excess of [n-Bu4N][Au(bdt)2]. (Hint = H interior Hext = H exterior).

Figure 3 .

 3 Figure 3. Experimental and theoretical isotope distribution of a) {[Au(bdt)2⊂Pt2L4]} and b) {[Au(bdt)2⊂Pt2L4][Au(bdt)2]}

  Figure 4. a) X-ray crystal structure of [Au(bdt)2]⊂[Pt2L4][(Au(bdt)2)] 2+ . For clarity purposes only the encapsulated Au(III) guest and one Au(bdt)2 outside the cavity perched over the Pt(II) centre are shown. b) 1D chain made of host-guest assemblies interacting with the outside Au(bdt)2 -1 species. All hydrogen omitted, thermal displacement ellipsoids are shown at 30% probability.

  5, triclinic P -1, a = 12.8265(9) Å, b = 21.1589(15) Å, c 9767(21) Å, α = 107.374(3)°, β = 94.244(3)°, γ = 98.877(2)°, V = 7101.2(9) Å 3 , Z = 4, T= 200(1) K, λ = 0.71073 Å, μ = 5.834 mm -1 , min / max transmission = 0.24 / 0.90, θ range = 0.77° to 30.55°, 209447 reflections measured, 43422 independent (Rint = 0.0384), 30862 observed [I>2σ(I)], completeness = 0.997, 1661 parameters, 334 restraints, final R indices R1 [I>2σ(I)] = 0.0643 and wR2 (all data) = 0.1750, GOF on F 2 = 1.054, largest difference peak / hole = 7.82 / -6.15 e.Å -3 .

Synthesis of [(Au(bdt)2⊂ Pt2L4][Au(bdt)2]3 (

  2)[n-Bu4N][Au(bdt)2] (40 mg, 0.056 mmol) was added to a solution of [PtL4][OTf]4 (31 mg, 0.014 mmol) in CH3CN (5 mL). The solution was stirred at room temperature for 30 min, during which time a green precipitate formed. The solid was collected by filtration, washed with diethyl ether (3 × 5 mL), and dried under vacuum: 42 mg of green solid (87%); This metallacage was isolated and character-

				ized as [Au(bdt)2⊂Pt2(L)4][Au(bdt)2]3
			z): [Pt2L4] 4+ :
	407.59; found: 407.84, [Pt2L4(CF3SO3)] 3+ : 593.10;
	found: 593.84, [Pt2L4(CF3SO3)] 2+ : 964.13; found:
	964.63;	Anal.	calcd	for
	[Pt2L4][OTf]4.DMSO.CH2Cl2: C 45.72, H 2.70, N
	4.69; Found: C 45.90, H 1.93, N 4.71.


Vincent & Van Seuningen

Dr Nicolas Jonckheere
email: nicolas.jonckheere@inserm.fr

Audrey Vincent

Isabelle Van Seuningen

Of autophagy and in vivo pancreatic carcinogenesis: the p53 status matters!

Summary:

Autophagy is a lysosomal recycling process essential for tissue or cell homeostasis. The role of autophagy in cancer is complex with either tumor suppressive or pro-carcinogenesis activities. This question has been addressed by Kevin Ryan's laboratory by using Kras-driven genetic engineering mouse models in order to decipher the involvement of essential Atg5/7 autophagy genes and p53 status in pancreatic homeostasis and carcinogenetic progression. The authors show that combined loss of autophagy and p53 dramatically promotes progression from early PanIN lesions toward adenocarcinoma and alters the cellular metabolism with an enrichment of anabolic pathway that can fuel the tumor growth.

Comment on:

Rosenfeldt, [START_REF] Rosenfeldt | p53 status determines the role of autophagy in pancreatic tumour development[END_REF] p53 status determines the role of autophagy in pancreatic tumour development. Nature 504, 296-300.

Autophagy is a lysosomal catabolic pathway used to degrade and recycle components of the cytoplasm (organelles and macromolecules). This cellular process is essential for survival, differentiation, development, and homeostasis [START_REF] Levine | Autophagy in the pathogenesis of disease[END_REF]. Autophagy occurs when cells need to generate intracellular nutrients and energy. Among genes that are essential for the execution of autophagy, Atg5 and Atg7 are key elements involved in the vesicle elongation forming the phagosome.

The role of autophagy in cancer is complex. Reports indicate that autophagy promotes either tumor suppressive or pro-carcinogenesis activities. Notably, the in vivo involvement of autophagy in pancreatic carcinogenesis remains to be elucidated. This question has been addressed by the findings of Kevin Ryan's laboratory. In the 2013 December issue of Nature, The authors use genetic engineered mouse models (preclinical pancreatic cancer mouse model Pdx1-Cre; LstopL-K-ras G12D ; p53 -/-and conditional invalidation of Atg5 flox/flox or Atg7 flox/flox ) to decipher the involvement of essential autophagy genes in pancreatic homeostasis and carcinogenetic progression [START_REF] Rosenfeldt | p53 status determines the role of autophagy in pancreatic tumour development[END_REF].

First, Rosenfeldt et al demonstrate that the mosaic losses of Atg5 and Atg7 lead to the pancreas destruction independently of Kras and reduce the overall survival of mice. Both Atg5-and Atg7invalidated mice harbor a significant decrease of autophagosome markers (LC3 puncta and p62 aggregates), significant accumulation of p53 and activated caspase-3. These alterations of pancreatic functions subsequently drive to a diabetic phenotype (increased glucose and fructosamine).

In the Kras-driven PanIN model (Pdx1-Cre; LstopL-K-ras G12D ), the mosaic loss of Atg5 or Atg7 expression leads to an enhanced accumulation of early PanIN (PanIN1A/B) that scarcely progress to later-grade PanIN (PanIN2/3) during the entire lifespan of the mouse (up to 500 days). This could be explained by the sustained expression of p53 that induces growth arrest and cellular senescence and acts as a barrier to the progression of PanINs to PDAC.

It is estimated that about 50% of PDAC cases are associated with the loss of p53. It was previously shown that concomitant endogenous expression of Trp53 R172H and Kras G12D leads to development of invasive and widely metastatic adenocarcinoma and thus to a dramatically shortened median survival [START_REF] Hingorani | Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice[END_REF].

Complex transgenic mice displaying Pdx1-Cre; LstopL-K-ras G12D ; p53 -/-; Atg7 flox/flox orAtg5 flox/flox were generated. The genetic conditional invalidation of Atg5 or Atg7 and the following autophagy inhibition accelerates PDAC formation in the absence of p53 suggesting that loss of p53 is crucial and dramatically promotes progression from early PanIN lesions toward adenocarcinoma. The inhibition of autophagy via hydroxychloroquine, initially reported as beneficial in cancer treatment, promotes similar PDAC acceleration in the absence of p53.

Finally the authors showed that the combined loss of p53 and Atg7 leads to a decreased oxygen consumption, typical of reduced oxygen metabolism. Indeed the loss of autophagy is associated with acidification of extracellular environment. Cell lines derived from p53 -/-; Atg7 KO pancreatic tumors revealed an increase of intracellular glucose, glycolytic and pentose phosphate pathway intermediates indicating an enrichment of anabolic pathway that can fuel the tumor growth.

This outstanding analysis is contradictory to previous work performed in vitro by Yang et al who showed that autophagy inhibition in PDAC cell lines attenuates growth and tumorigenicity of xenografted tumors [START_REF] Yang | Pancreatic cancers require autophagy for tumor growth[END_REF]. In this particular study, the same K-ras driven mouse model was treated with chloroquinine, blocking lysosomal acidification and autophagosome degradation, following the establishment of advanced PanINs or focal PDAC. The authors observed a significant improvement of overall survival. This observation was consistent with Rosenfeldt's findings showing that loss of autophagy leads to accumulation of low grade PanIN and slows down the carcinogenetic progression but did not take into account the extreme importance of p53 status.

Pancreatic adenocarcinoma (PDAC) is one of the most deadly cancers with an extremely poor prognosis notably because of a lack of efficient therapeutic tools [START_REF] Vincent | Pancreatic cancer[END_REF]. Autophagy inhibition that is commonly used in other pathologies (as anti-malarial therapies or for rheumatologic conditions) was thought as a promising therapeutic strategy. However, the discrepancy between these two studies suggests that caution shall be kept regarding autophagy inhibition in pancreatic cancer. There is an urgent need to further explain the dichotomy between observations in human cancer cell lines and mouse preclinical model before even considering hydroxychloroquine or similar molecules as a bona fide therapeutic tool for pancreatic cancer patients.

Figure legend

Figure 1 :

 1 Figure 1: Loss of autophagy profoundly alters pancreatic homeostasis (A) or carcinogenesis (B) in genetically modified mouse model of pancreatic ductal adenocarcinoma (Pdx1-Cre; LstopL-Kras G12D ; p53 -/-; Atg5 flox/flox or Atg7 flox/flox )

Acknowledgments

This work is supported by Inserm and by SIRIC ONCOLille, Grant INCaDGOS-Inserm 6041. Dr Vincent is the recipient of a postdoctoral fellowship from the Fondation ARC.


PhD Nicolas Jonckheere
email: nicolas.jonckheere@inserm.fr

PhD Romain Vasseur

PhD Isabelle Van Seuningen

Keywords: K-RAS, pancreatic cancer, cell signaling, metabolism reprogramming, therapy network, target genes, biological processes to therapeutic targeting K-RAS, pancreatic cancer, cell signaling, metabolism reprogramming, therapy

RAS belongs to the super family of small G proteins and plays crucial roles in signal transduction from membrane receptors in the cell. Mutations of K-RAS oncogene lead to an accumulation of GTP-bound proteins that maintains an active conformation. In the pancreatic ductal adenocarcinoma (PDAC), one of the most deadly cancers in occidental countries, mutations of the K-RAS oncogene are nearly systematic (>90 %). Moreover, K-RAS mutation is the earliest genetic alteration occurring during pancreatic carcinogenetic sequence. In this review, we discuss the central role of K-RAS mutations and their tremendous diversity of biological properties by the interconnected regulation of signaling pathways (MAPKs, NF-κB, PI3K, Ral...). In pancreatic ductal adenocarcinoma, transcriptome analysis and preclinical animal models showed that K-RAS mutation alters biological behavior of PDAC cells (promoting proliferation, migration and invasion, evading growth suppressors, regulating mucin pattern, and miRNA expression). K-RAS also impacts tumor microenvironment and PDAC metabolism reprogramming. Finally we discuss therapeutic targeting strategies of K-RAS that have been developed without significant clinical success so far. As K-RAS is considered as the undruggable target, targeting its multiple effectors and target genes should be considered as potential alternatives.

Introduction: RAS GTPase and mutations

RAS super family belongs to the small G protein class that includes more than 150 guanosine tri-phosphate hydrolases (GTPase) [START_REF] Wennerberg | The Ras superfamily at a glance[END_REF]. This highly conserved protein family plays major roles in signal transduction from membrane receptors within the cell [START_REF] Saad | The RAS subfamily Evolution -tracing evolution for its utmost exploitation[END_REF][START_REF] Van Dam | Evolution of the Ras-like small GTPases and their regulators[END_REF]. The family is divided in 5 main sub-families: RAS, Rho, Rab, ARF and Ran [START_REF] Saad | The RAS subfamily Evolution -tracing evolution for its utmost exploitation[END_REF].

Among them, RAS proteins are mainly involved in the regulation of proliferation, differentiation, metabolism and survival genes. Among this family, the most characterized are H-RAS, N-RAS, and two isoforms K-RAS4A and K-RAS4B that originate from alternate splicing of the exon 4 of the K-RAS gene. Mostly, K-RAS designates K-RAS4B which expression is ubiquitous whereas K-RAS4A expression is more variable in the different tissues [START_REF] Tsai | K-Ras4A splice variant is widely expressed in cancer and uses a hybrid membrane-targeting motif[END_REF].

The RAS-family proteins are small 188-189 aa-21kDa GTPases with a very similar three dimensional structure. These proteins harbor two domains and a central region [START_REF] Schubbert | Hyperactive Ras in developmental disorders and cancer[END_REF]: The Nterminal G domain (aa 1-165) is highly conserved and includes a P-loop that binds phosphate, an effector binding domain (aa [START_REF] Vincent | Tif1gamma suppresses murine pancreatic tumoral transformation by a Smad4-independent pathway[END_REF][START_REF] Bryant | KRAS: feeding pancreatic cancer proliferation[END_REF][START_REF] Rejiba | K-ras oncogene silencing strategy reduces tumor growth and enhances gemcitabine chemotherapy efficacy for pancreatic cancer treatment[END_REF][START_REF] Singh | A gene expression signature associated with "K-Ras addiction" reveals regulators of EMT and tumor cell survival[END_REF][START_REF] Viale | Oncogene ablationresistant pancreatic cancer cells depend on mitochondrial function[END_REF][START_REF] Baker | Modeling pancreatic cancer with organoids[END_REF][START_REF] Huang | Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell-and patient-derived tumor organoids[END_REF][START_REF] Herrmann | Ras-effector interactions: after one decade[END_REF][START_REF] Herrmann | Ras and its effectors[END_REF] and two switch regions (aa 32-38 and aa [START_REF] Okada | Targeting the K-Ras--JNK axis eliminates cancer stem-like cells and prevents pancreatic tumor formation[END_REF][START_REF] Kang | Autophagy in pancreatic cancer pathogenesis and treatment[END_REF][START_REF] Liu | DUSP1 is a novel target for enhancing pancreatic cancer cell sensitivity to gemcitabine[END_REF][START_REF] Sclabas | NF-kappaB in pancreatic cancer[END_REF][START_REF] Joyce | NF-kappaB and cell-cycle regulation: the cyclin connection[END_REF][START_REF] Viatour | Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation[END_REF][START_REF] Daniluk | An NF-kappaB pathway-mediated positive feedback loop amplifies Ras activity to pathological levels in mice[END_REF][START_REF] Ling | KrasG12D-induced IKK2/beta/NF-kappaB activation by IL-1alpha and p62 feedforward loops is required for development of pancreatic ductal adenocarcinoma[END_REF][START_REF] Prabhu | Critical role of NF-kappaB in pancreatic cancer[END_REF][START_REF] Rachagani | Activated KrasG(1)(2)D is associated with invasion and metastasis of pancreatic cancer cells through inhibition of E-cadherin[END_REF][START_REF] Engelman | Targeting PI3K signalling in cancer: opportunities, challenges and limitations[END_REF][START_REF] Eser | Selective requirement of PI3K/PDK1 signaling for Kras oncogene-driven pancreatic cell plasticity and cancer[END_REF][START_REF] Baer | Implication of PI3K/Akt pathway in pancreatic cancer: When PI3K isoforms matter?[END_REF][START_REF] Baer | Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110alpha[END_REF][START_REF] Nussinov | The Key Role of Calmodulin in KRAS-Driven Adenocarcinomas[END_REF][START_REF] Hamad | Distinct requirements for Ras oncogenesis in human versus mouse cells[END_REF][START_REF] Lim | Activation of RalA is critical for Ras-induced tumorigenesis of human cells[END_REF][START_REF] Lim | Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells[END_REF] that are responsible for conformation changes (Figure 1A). The central region (aa 85-165) displays 85-90 % homology across the RAS GTPase super family. The C-terminal hyper variable region (HVR) (aa 165-185) controls the membrane fixation and acts as autoinhibitory domain. The CAAX final sequence is farnesylated on the cysteine residue allowing membrane anchoring. In addition, K-RAS4B HVR domain also contains a lysine-rich sequence that stabilizes the protein at the membrane.

In its inactivated state; K-RAS protein is located at the cell membrane internal surface and is bound to a guanosine-di-phosphate (GDP) (Figure 1B-C). Activation of a membrane receptor leads to the recruitment of the RAS Guanidine Exchange Factor (RASGEF) SOS1 via SH2 and SH3 domains of adaptors. This allows SOS1 interaction with RAS and induces GDP dissociation. The free RAS protein then quickly binds to a guanosine-tri-phosphate (GTP) molecule, propagating the signal in the cell activating K-RAS target genes [START_REF] Feig | Tools of the trade: use of dominant-inhibitory mutants of Ras-family GTPases[END_REF].

However, RAS activation is transient because its GTPase activity hydrolyses GTP to GDP and goes back to the inactivated state. The GTP hydrolysis is promoted by a GTPase activating protein (GAP) that allows normal K-RAS activation/inactivation cycle (1,6) (Figure 1C). In the GDP-bound state, the catalytic domain of K-Ras4B interacts with HVR maintaining auto-inhibitory state that will be reverted upon activation [START_REF] Chavan | High-Affinity Interaction of the K-Ras4B Hypervariable Region with the Ras Active Site[END_REF].

RAS is also regulated by its cellular distribution and notably by its clustering in lipid rafts called nanoclusters [START_REF] Prior | Direct visualization of Ras proteins in spatially distinct cell surface microdomains[END_REF]. This modulation of the local phospho-lipid content subsequently alters RAS/RAF/MAPK signaling. According to its activation state, RAS forms nanocluster and allows efficient recruitment of effectors. The number of nanoclusters is proportional to the activation signal intensity. RAS conformation changes during GDP/GTP cycling: GDPbound K-RAS is associated with the membrane via its HVR domain whereas GTP-bound is partially detached. Furthermore, GTP or GDP-bound RAS associates with different kind of phospholipids and therefore alter lipid content of the nanocluster [START_REF] Zhou | Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling[END_REF][START_REF] Tian | Plasma membrane nanoswitches generate high-fidelity Ras signal transduction[END_REF]. Interestingly, H-and N-RAS isoforms each form nanoclusters that do not non-overlap. This distribution could explain differential activation of signaling pathways despite their high homologies [START_REF] Zhou | Ras nanoclusters: Versatile lipid-based signaling platforms[END_REF]. While being mainly localized at the plasma membrane, K-RAS can also be phosphorylated on S181 by PKC leading to its delocalization on the mitochondrial membrane where it interacts with Bcl XL and induces apoptosis [START_REF] Bivona | PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis[END_REF]. This Ser181 phosphorylation is inhibited by calmodulin [START_REF] Alvarez-Moya | K-Ras4B phosphorylation at Ser181 is inhibited by calmodulin and modulates K-Ras activity and function[END_REF].

K-RAS mutation and PDAC: So it begins.

Mutations of K-RAS gene are detected in 27% of cancers (COSMICv78 database http://www.sanger.ac.uk/genetics/CGP/cosmic/). These mutations (97-99 %) affect glycine12 (G12), glycine13 (G13) or glutamine61 (Q61) residues (Figure 1D). G12 residue mutation sterically blocks the orientation of the Q61 that is essential for RAS/GAP interaction, leading to an accumulation of GTP-bound RAS unable to hydrolyze the GTP and hence maintaining the active conformation [START_REF] Bos | GEFs and GAPs: critical elements in the control of small G proteins[END_REF][START_REF] Khrenova | Modeling the role of G12V and G13V Ras mutations in the Ras-GAP-catalyzed hydrolysis reaction of guanosine triphosphate[END_REF]. G13 amino acid mutation enhances the flexibility of GTP binding area and leads to faster GTP/GDP cycle and impaired sustaining of activation [START_REF] Khrenova | Modeling the role of G12V and G13V Ras mutations in the Ras-GAP-catalyzed hydrolysis reaction of guanosine triphosphate[END_REF]. The Q61L mutation interferes with the nucleophilic attack on the γ-phosphate of GTP and impairs its hydrolysis [START_REF] Buhrman | Allosteric modulation of Ras positions Q61 for a direct role in catalysis[END_REF].

In pancreatic adenocarcinomas (PDAC), one of the most deadly cancers in occidental countries [START_REF] Vincent | Pancreatic cancer[END_REF], mutations of K-RAS gene are nearly systematic and are associated with a bad prognosis [START_REF] Bournet | KRAS G12D Mutation Subtype Is A Prognostic Factor for Advanced Pancreatic Adenocarcinoma[END_REF][START_REF] Bournet | KRAS mutations and their correlation with survival of patients with advanced pancreatic cancer[END_REF]. Analysis of cBioPortal pancreatic adenocarcinoma samples confirmed the alteration of K-RAS in 89.8-94.9% of 740 cases out of 4 independent studies (ICGC, UTSW, TCGA and QCMG 2016). Recently, Bailey and colleagues confirmed that K-RAS activation occurs in more than 90% of PDAC in a wide transcriptional analysis clustering 96 tumors with high epithelial content [START_REF] Bailey | Genomic analyses identify molecular subtypes of pancreatic cancer[END_REF]. Interestingly, genes defined as "upregulated in overexpressing oncogenic K-RAS cells" were enriched in the aberrantly differentiated endocrine/exocrine subtype (ADEX) cells whereas squamous cells, previously named quasimesenchymal, harbor lesser enrichment of those genes [START_REF] Bailey | Genomic analyses identify molecular subtypes of pancreatic cancer[END_REF][START_REF] Collisson | Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy[END_REF].

The mouse model developed by D. Tuveson using the Cre-Lox strategy using Pdx1 (key transcription factor in pancreatic fate) promoter-driven, displays the pancreatic tissue specific expression of a constitutively active mutant K-Ras G12D [START_REF] Hingorani | Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse[END_REF][START_REF] Kim | Signaling and transcriptional control of pancreatic organogenesis[END_REF][START_REF] Herreros-Villanueva | Mouse models of pancreatic cancer[END_REF]. In this model that harbors Cre-recombination in all pancreatic cell lineages, expression of the K-Ras G12D is sufficient to induce occurrence of Pancreatic Intraepithelial Neoplasia (PanINs) highlighting its major role in the initiation of pancreatic carcinogenesis. Ptf1/P48-Cre (exocrine Cre expression), LoxStopLox(LSL)-K-ras G12D mice develop a similar spectrum of ductal and PanIN lesions [START_REF] Hingorani | Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse[END_REF]. These K-ras G12D mice are usually referred as KC mice. Using elastase promoter-driven recombinase Cre (acinar cells), Ji and colleagues showed that an increase above threshold of Ras activity intensity leads to senescence of acinar cells, promotes inflammation and induces fibrosis mimicking histologic features of human chronic pancreatitis (CP), acinarductal metaplasia and PanIN lesions [START_REF] Ji | Ras activity levels control the development of pancreatic diseases[END_REF]. Once these lesions appeared, K-RAS remains an essential actor to their survival and progress although a slow evolution that requires additional genetic events such as Trp53, Ink4a/ARF, Smad4 tumor suppressor mutations to form invasive adenocarcinoma [START_REF] Ji | Ras activity levels control the development of pancreatic diseases[END_REF][START_REF] Aguirre | Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma[END_REF][START_REF] Collins | Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice[END_REF][START_REF] Collins | Metastatic pancreatic cancer is dependent on oncogenic Kras in mice[END_REF][START_REF] Bardeesy | Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer[END_REF]. Characterization of an inducible oncogenic K-Ras model (iK-ras p53 L/+ ) showed that K-Ras G12D is required for PDAC maintenance as K-Ras extinction (by doxycycline removal) led to rapid tumor regression and degeneration of stromal compartment [START_REF] Ying | Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism[END_REF]. Other genetic events such as GNAS R201H or loss of TIF1γ lead to distinct cancer phenotypes and notably promote the intraductal papillary mucinous neoplasm (IPMN)-to-PDAC progression [START_REF] Taki | GNAS(R201H) and Kras(G12D) cooperate to promote murine pancreatic tumorigenesis recapitulating human intraductal papillary mucinous neoplasm[END_REF][START_REF] Vincent | Tif1gamma suppresses murine pancreatic tumoral transformation by a Smad4-independent pathway[END_REF]. Once pancreatic adenocarcinoma is developed, K-RAS still participates in the tumor cell-proliferation, -survival and -migration as well as in chemoresistance abilities [START_REF] Collins | Metastatic pancreatic cancer is dependent on oncogenic Kras in mice[END_REF][START_REF] Bryant | KRAS: feeding pancreatic cancer proliferation[END_REF][START_REF] Rejiba | K-ras oncogene silencing strategy reduces tumor growth and enhances gemcitabine chemotherapy efficacy for pancreatic cancer treatment[END_REF][START_REF] Singh | A gene expression signature associated with "K-Ras addiction" reveals regulators of EMT and tumor cell survival[END_REF]. Signature of K-RAS dependency/addiction genes was determined and was shown to be associated with epithelial differentiation (e.g. integrin-β6) [START_REF] Singh | A gene expression signature associated with "K-Ras addiction" reveals regulators of EMT and tumor cell survival[END_REF] and corresponds to classical subtype described by Collisson (21). On the contrary, K-RAS independent phenotype, occurring in later stages of PDAC, is linked to a metabolic adaptation relying on increase mitochondrial adaptation [START_REF] Viale | Oncogene ablationresistant pancreatic cancer cells depend on mitochondrial function[END_REF].

Recently, the use of PDAC organoids cultures expanded and allowed new avenues in fundamental and clinical research regarding pancreatic cancer [START_REF] Baker | Modeling pancreatic cancer with organoids[END_REF]. Expression of mutant K-RAS protein in progenitor organoids induces morphological changes such as cystic organization with apically positioned nuclei that are consistent with early pancreatic tumor lesions [START_REF] Huang | Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell-and patient-derived tumor organoids[END_REF]. Therefore, these progenitor organoids are suggested as models to investigate early stages of cancer transformation. Organoids culture also allows study of normal pancreatic cells without immortalization and thus enables comparisons of normal and tumor cells retaining patient-specific traits. Organoids may be used to predict clinical responses and are essential for drug screening [START_REF] Huang | Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell-and patient-derived tumor organoids[END_REF].

K-RAS is central in cellular signaling network

Activated K-RAS mutants participate in a tremendous diversity of biological events by interconnecting regulation of several signaling pathways [START_REF] Herrmann | Ras-effector interactions: after one decade[END_REF][START_REF] Herrmann | Ras and its effectors[END_REF] (Figure 2). The best characterized direct effectors lead to the activation of the Mitogen-Activated Protein Kinase (MAPK), Phosphoinositide 3-Kinase (PI3K) and RalGEF (Ral Guanidine exchange factor) pathways [START_REF] Rajalingam | Ras oncogenes and their downstream targets[END_REF][START_REF] Feig | Evidence for a Ras/Ral signaling cascade[END_REF][START_REF] Shields | Understanding Ras: 'it ain't over 'til it's over[END_REF][START_REF] Moodie | Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase[END_REF][START_REF] Rodriguez-Viciana | Phosphatidylinositol-3-OH kinase as a direct target of Ras[END_REF][START_REF] Sjolander | Association of p21ras with phosphatidylinositol 3-kinase[END_REF][START_REF] Peterson | Identification of a novel RalGDS-related protein as a candidate effector for Ras and Rap1[END_REF][START_REF] White | A role for the Ral guanine nucleotide dissociation stimulator in mediating Ras-induced transformation[END_REF]. Stress response (p38 and c-Jun N-terminal Kinase (JNK)) and Nuclear Factor-kappa B (NF-κB) pathways are also induced by K-RAS activation, either by interconnection with other activated signaling pathways or by oncogenic stress signals such as production of reactive oxygen species (5,41,49) (Figure 2).

In pancreatic cancer, activation of the MAPK pathway is associated with a bad prognosis [START_REF] Handra-Luca | Biological and prognostic relevance of mitogen-activated protein kinases in pancreatic adenocarcinoma[END_REF]. Recruitment of RAF effector and subsequent activation of the ERKs pathway by K-RAS promotes proliferation and independent anchorage-growth of pancreatic tumor cells [START_REF] Ji | Ras activity levels control the development of pancreatic diseases[END_REF][START_REF] Tuveson | Endogenous oncogenic K-ras(G12D) stimulates proliferation and widespread neoplastic and developmental defects[END_REF].

ERKs activation is also involved in the survival/apoptosis balance [START_REF] Neuzillet | Targeting the Ras-ERK pathway in pancreatic adenocarcinoma[END_REF] and in migration and invasion properties of pancreatic tumors [START_REF] Endo | Activation of two distinct MAPK pathways governs constitutive expression of matrix metalloproteinase-1 in human pancreatic cancer cell lines[END_REF][START_REF] Zhao | Expression of oncogenic K-ras and loss of Smad4 cooperate to induce the expression of EGFR and to promote invasion of immortalized human pancreas ductal cells[END_REF]. Finally, the activation of ERK1/2 MAPK pathway contributes to tumor cell chemoresistance [START_REF] Zheng | ERK1/2 activity contributes to gemcitabine resistance in pancreatic cancer cells[END_REF] and to inflammation [START_REF] Shin | Transcriptional regulation of the interleukin-11 gene by oncogenic Ras[END_REF].

The implication of p38-MAPKs pathway is controversial in carcinogenesis. Although, this pathway promotes invasive abilities of pancreatic tumors [START_REF] Taniuchi | Peroxiredoxin 1 promotes pancreatic cancer cell invasion by modulating p38 MAPK activity[END_REF], the detection of its activation is associated with a good prognosis [START_REF] Korc | p38 MAPK in pancreatic cancer: finding a protective needle in the haystack[END_REF]. The effect of JNK activation is opposed to that of p38 MAPK pathway. The activation of JNK pathway by K-RAS promotes pancreatic tumor formation and cancer stem cell maintenance [START_REF] Okada | Targeting the K-Ras--JNK axis eliminates cancer stem-like cells and prevents pancreatic tumor formation[END_REF]. The pathway is also involved in autophagic processes increasing survival of tumor cells [START_REF] Kang | Autophagy in pancreatic cancer pathogenesis and treatment[END_REF]. Finally, JNK pathway is involved in the occurrence of chemoresistance and invasion of pancreatic tumor cells [START_REF] Endo | Activation of two distinct MAPK pathways governs constitutive expression of matrix metalloproteinase-1 in human pancreatic cancer cell lines[END_REF][START_REF] Liu | DUSP1 is a novel target for enhancing pancreatic cancer cell sensitivity to gemcitabine[END_REF].

The NF-κB pathway, which is indirectly activated by K-RAS, is constitutively activated in pancreatic cancer [START_REF] Sclabas | NF-kappaB in pancreatic cancer[END_REF]. The canonical NF-κB signaling leads to activation of p65/p50 complex and involves IKK complex (IκB kinase). This cytoplasmic complex consists in IKKα and IKKß kinases associated with the protein NEMO (=IKKε) [START_REF] Joyce | NF-kappaB and cell-cycle regulation: the cyclin connection[END_REF][START_REF] Viatour | Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation[END_REF]. NF-κB is mainly described as activated by stress and pro-inflammatory signals but also displays important interconnections with other pathways. The NF-κB pathway plays an essential role in carcinogenesis and inflammation through an amplification loop of K-RAS activity [START_REF] Daniluk | An NF-kappaB pathway-mediated positive feedback loop amplifies Ras activity to pathological levels in mice[END_REF][START_REF] Ling | KrasG12D-induced IKK2/beta/NF-kappaB activation by IL-1alpha and p62 feedforward loops is required for development of pancreatic ductal adenocarcinoma[END_REF][START_REF] Prabhu | Critical role of NF-kappaB in pancreatic cancer[END_REF]. The NF-κB is also involved in invasion and metastases properties of the pancreatic tumor cells [START_REF] Rachagani | Activated KrasG(1)(2)D is associated with invasion and metastasis of pancreatic cancer cells through inhibition of E-cadherin[END_REF].

PI3Ks are a family of heterodimeric kinases composed of p110 catalytic and regulatory subunits that lead to phosphatidylinositol triphosphate (PIP3) production and activation of downstream signals and biological processes [START_REF] Engelman | Targeting PI3K signalling in cancer: opportunities, challenges and limitations[END_REF]. The key downstream effectors of PI3K signaling such as Akt or GSK3 are activated in KC mice [START_REF] Eser | Selective requirement of PI3K/PDK1 signaling for Kras oncogene-driven pancreatic cell plasticity and cancer[END_REF]. PI3K signaling enhances acinar-ductal metaplasia (ADM) that generate duct from acinar cells. These ADM lesions will evolve towards PanIN lesions [START_REF] Baer | Implication of PI3K/Akt pathway in pancreatic cancer: When PI3K isoforms matter?[END_REF]. Pancreas-specific invalidation of p110α isoform (kinase dead) prevent occurrence of all type of lesions. Interestingly, pancreatic invalidation of the other p110β isoform did not affect PanIN formation in an activated K-RAS context [START_REF] Baer | Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110alpha[END_REF]. K-RAS4B isoform can also interact with calcium modulator protein calmodulin via HVR and modulates preferentially PI3K/Akt rather than RAF/MEK/ERK pathway [START_REF] Nussinov | The Key Role of Calmodulin in KRAS-Driven Adenocarcinomas[END_REF].

The RalA/B pathway, initially described as a minor actor in transformation in rodents, is an essential component of RAS transformation in human cells [START_REF] Rajalingam | Ras oncogenes and their downstream targets[END_REF][START_REF] Hamad | Distinct requirements for Ras oncogenesis in human versus mouse cells[END_REF][START_REF] Lim | Activation of RalA is critical for Ras-induced tumorigenesis of human cells[END_REF]. In pancreatic cancer, Ral GTPases are activated by K-RAS and are involved in anchorage-independent growth and survival [START_REF] Lim | Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells[END_REF][START_REF] Vigil | Aberrant overexpression of the Rgl2 Ral small GTPase-specific guanine nucleotide exchange factor promotes pancreatic cancer growth through Ral-dependent and Ral-independent mechanisms[END_REF]. RalA promotes tumor initiation whereas RalB is essential for invasion and metastasis [START_REF] Lim | Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells[END_REF]. Ral GTPase pathway is also involved in pancreatic tumor chemo-and radio-resistance [START_REF] Kidd | Ras-related small GTPases RalA and RalB regulate cellular survival after ionizing radiation[END_REF][START_REF] Seguin | An integrin beta(3)-KRAS-RalB complex drives tumour stemness and resistance to EGFR inhibition[END_REF].

Overall, more than 100 proteins containing putative RAS-association (RA) or RAS binding domains (RBDs) were described (41,80) (Figure 2): T-lymphoma invasion and metastasis protein-1 (Tiam1) is a Rac-GEF harboring a RBD and activates JNK pathways. RAS association domain family (RASSF) contains a RAS domain lacking catalytic function.

RASSFs act as tumor suppressors and are involved in apoptotic processes. Nore1 binds to RAS-like GTPases and heterodimerizes with RASSFs [START_REF] Ortiz-Vega | The putative tumor suppressor RASSF1A homodimerizes and heterodimerizes with the Ras-GTP binding protein Nore1[END_REF]. The RAS inhibition (RINs) GEF links RAS signaling and receptor-mediated endocytosis via the activation of Rab5 [START_REF] Tall | Ras-activated endocytosis is mediated by the Rab5 guanine nucleotide exchange activity of RIN1[END_REF]. RAS was also proposed to allow convergence of signals from PLC, AF6 and PKC [START_REF] Rajalingam | Ras oncogenes and their downstream targets[END_REF].

Among pathways undergoing genetic alterations in PDAC, K-RAS was also linked to Wnt and Hippo pathways. K-RAS is able to repress the Frizzled 8 (Frz8) and CaMKII-mediated non-canonical Wnt/Ca2+ signaling pathway via direct calmodulin binding (83) thereby relaxing the suppression of the canonical Wnt signaling, which occurs by blocking βcatenin/TCF4 interaction [START_REF] Macdonald | SnapShot: Wnt/beta-catenin signaling[END_REF][START_REF] Semenov | SnapShot: Noncanonical Wnt Signaling Pathways[END_REF]. Indeed, enhanced Wnt/β-catenin signaling has been observed in human PDAC tissues. The Hippo/MST1/2 pathway includes the coactivator Yesassociated protein (YAP) which is abundantly expressed in the PanIN lesions of p48-Cre; LSL-K-ras G12D mice. The Hippo/MST1/2 pathway plays a crucial role in regulating tissue homeostasis and organ size [START_REF] Yu | Hippo Pathway in Organ Size Control, Tissue Homeostasis, and Cancer[END_REF]. The oncogenic K-Ras-MAPK pathway induces posttranscriptional modification of YAP that inactivates YAP-mediated signaling [START_REF] Zhang | Downstream of mutant KRAS, the transcription regulator YAP is essential for neoplastic progression to pancreatic ductal adenocarcinoma[END_REF]. However, YAP is also associated with K-Ras G12D -independent tumor maintenance (88).

K-RAS regulates a great diversity of biological processes

Understanding K-RAS and intrinsic biological properties of PDAC cells: the contribution of transcriptomes

As K-RAS mutations control a wide array of cell signaling pathways (Figure 2), it was expected that these mutations might regulate a tremendous diversity of genes involved in every biological property that defines hallmarks of cancer (proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis) [START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF]. As previously described, the K-Ras G12D mutant mouse model is associated with 100 % occurrence of PanINs highlighting the mandatory role of K-Ras in the initiation of pancreatic carcinogenesis [START_REF] Hingorani | Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse[END_REF]. Characterization of mice expressing reversible iKras G12D indicates that K-Ras is also mandatory for tumor survival and maintenance [START_REF] Collins | Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice[END_REF].

Moreover, K-Ras regulates the abilities of tumor cell chemoresistance [START_REF] Perez-Mancera | What we have learned about pancreatic cancer from mouse models[END_REF]. Indeed, K-Ras is described as central in the proliferation regulation circuit but is also connected with motility and viability regulatory circuits. At this stage, microarray-based transcriptome analysis allowed a global view of K-ras target genes in pancreatic cancer. These genes are involved in functions such as transcription, proteolysis, cell proliferation, cell death, adhesion, cell surface receptor or intracellular signaling [START_REF] Qian | In vitro modeling of human pancreatic duct epithelial cell transformation defines gene expression changes induced by K-ras oncogenic activation in pancreatic carcinogenesis[END_REF]. In Table 1, we present the gene ontology clustering obtained by analyzing three available PDAC expression data sets retrieved from Gene Omnibus Expression database and compared the mutated vs wild type K-ras transcriptomes. PDAC bearing Pdx1-Cre ; K-ras G12D mice show a strong enrichment in gene clusters involved in protein glycosylation, inflammatory response, extracellular matrix or protease activity, (GSE53695, ( 92)). Ablation of K-Ras in mouse pancreas, using a tetracycline-inducible K-Ras allele, leads to an important enrichment of intracellular organelle genes and a mild enrichment of genes involved in non-coding or ribosomal RNA processing and protein trafficking (GSE58307, [START_REF] Viale | Oncogene ablationresistant pancreatic cancer cells depend on mitochondrial function[END_REF]). The succession of K-ras activation-inactivationreactivation in pancreatic tumor spheres also revealed an enrichment of genes involved in metabolic pathways and notably highlighted an increased mitochondrial activity [START_REF] Viale | Oncogene ablationresistant pancreatic cancer cells depend on mitochondrial function[END_REF]. The surviving cells following K-Ras-ablation harbor an increase oxidative phosphorylation (OXPHOS) similarly to what is observed in patients resistant to MEK or PI3K targeting highlighting the promise of combined K-Ras and mitochondrial respiration targeting [START_REF] Viale | Oncogene ablationresistant pancreatic cancer cells depend on mitochondrial function[END_REF].

Analysis of deregulated genes in K-Ras addicted pancreatic cancer cell lines revealed enrichment of gene expression signature involved in various biological processes such as metal and ion bindings, apical and basal plasma membrane cytoskeletal protein binding, fatty acid metabolic process and vesicular cell systems (GSE15126, [START_REF] Singh | A gene expression signature associated with "K-Ras addiction" reveals regulators of EMT and tumor cell survival[END_REF]). K-Ras dependency signature genes such as SYK, integrin-β6 (ITGB6) and MST1R are related with both epithelial differentiation state and well-to moderately-differentiated tumor phenotype [START_REF] Singh | A gene expression signature associated with "K-Ras addiction" reveals regulators of EMT and tumor cell survival[END_REF].

Altogether this highlights the intrinsic pivotal role of K-Ras activation in pancreatic cancer cells and how it is linked to tumor growth and metastasis (Figure 3).

Cell metabolism reprogramming

A common feature of cancer cells is described as the Warburg effect which is an increase in glucose uptake and a shift from mitochondrial oxidative phosphorylation to aerobic glycolysis [START_REF] Vander Heiden | Understanding the Warburg effect: the metabolic requirements of cell proliferation[END_REF]. This metabolic shift allows generating energy and nutrients in order to survive in a generally hypoxic and energetic inhospitable environment. Indeed, K-RAS-transformed human cells show glucose and glutamine metabolism alteration [START_REF] Gaglio | Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth[END_REF][START_REF] Yun | Glucose deprivation contributes to the development of KRAS pathway mutations in tumor cells[END_REF][START_REF] Perera | Pancreatic Cancer Metabolism: Breaking It Down to Build It Back Up[END_REF]. K-Ras G12D extinction alters multiple metabolic pathways. Notably, K-Ras G12D promotes the glycolytic flux by increasing glucose uptake and lactate production through increase of glucose transporters (glut1/Slc2a1), crucial glycolytic enzymes (Hk1, Hk2, PfkI, Ldha), enzyme of the hexosamine pathway (Gfpt1) and non-oxidative pentose phosphate pathway (PPP) enzymes (Rpia and Rpe). These genes are regulated at the transcriptional level via MAPK and Myc signaling pathways, both affected by K-Ras activation. Moreover, K-ras G12D activates the hexosamine biosynthesis pathway (HBP) affecting protein O-glycosylation and non-oxydative PPP generation for ribose production [START_REF] Ying | Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism[END_REF]. Interestingly, lactate that is produced by glycolytic cells in hypoxic area is used for normoxic cells growth in a paracrine manner [START_REF] Guillaumond | Strengthened glycolysis under hypoxia supports tumor symbiosis and hexosamine biosynthesis in pancreatic adenocarcinoma[END_REF]. PDAC cells harbor an increased expression of lactate transporter MCT4 that reflects glycolytic activity and promotes cell survival and tumorigenic growth [START_REF] Baek | MCT4 defines a glycolytic subtype of pancreatic cancer with poor prognosis and unique metabolic dependencies[END_REF]. K-Ras activation also contributes to stimulating glutamine (Gln) metabolism by transcriptional repression of glutamate dehydrogenase (GLUD1) and induction of aspartate aminotransferase (GOT1) expression and thus coordinates the switch to Gln metabolism that is critical to maintain tumor growth and survival probably via the redox balance [START_REF] Son | Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway[END_REF]. The oncogenic Reactive Oxygen Species

Mucins

Mucins belong to a heterogeneous group of large O-glycoproteins (secreted or membrane bound) that are expressed by epithelial cells. The extended structure of mucins extracellular domains confer them a role of molecular sensors and in cell-cell, cell-extracellular matrix interactions and in cell signaling. MUC1 and MUC4 were extensively described as key promoters of pancreatic carcinogenesis (126-129). Mucus-producing cells normally restricted to respiratory and intestinal epithelia are observed in PanINs of the K-Ras G12D mouse model [START_REF] Hingorani | Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse[END_REF]. Indeed, membrane-bound Muc1 and Muc4 and secreted Muc5ac are aberrantly expressed during pancreatic cancer progression (PanINs, primary tumors and metastases) (130). Interestingly, mucin expression is correlated with an increase of inflammatory cytokines IFN-γ, CXCL1 and CXCL2. Recently, we also showed that MUC4 is a target of K-Ras G12D mutation via both transcriptional (p42/44 MAPK and p65 NF-κB) and posttranscriptional (RalB) mechanisms (131). Therefore, we hypothesize that mucins, as K-RAS target genes, could be promising therapeutic tools for gene therapy and immunotherapeutic approaches in pancreatic cancer (126).

miRNA-mediated gene regulation

MicroRNAs (miRNA) are 22-24 nucleotides-long non coding RNAs that regulate expression of mRNA mostly by binding their 3'-untranslated region (UTR) and emerge as major posttranscriptional regulatory mechanisms with potential therapeutic interest. MiRNA expression pattern is profoundly altered in carcinogenesis where they can act as tumor suppressor or oncomiR depending on the panel of their targets (132-135). Laser capture microdissection of PanIN tissues of K-Ras G12D mice led to the identification of miR-21, miR-205 and miR-200 as all induced by K-Ras (136). In addition, miR-155, that is also induced by K-Ras, mediates cell proliferation through ROS accumulation [START_REF] Wang | Micro-RNA-155 is induced by K-Ras oncogenic signal and promotes ROS stress in pancreatic cancer[END_REF]. On the contrary, the tumor-suppressors miR-29a, miR-330-5p and miR-219-1-3p are decreased in PanIN lesions (137,138). Finally, some miRNA, such as miR-96, miR-217 and Let7 were shown to directly regulate K-Ras expression or alter its associated signaling pathways and could be considered as potential therapeutic strategies (139-141).

K-RAS as a therapeutic target

As a central point of pancreatic carcinogenesis, K-RAS is an obvious and bona fide therapeutic target. In the next section, we describe strategies that were designed at various levels: (i) direct targeting of the K-RAS protein, (ii) indirect K-RAS targeting of the protein location and (iii) targeting K-RAS oncogenic activity by interfering with its downstream effectors (142) (Figure 4).

Direct targeting of K-RAS activity

The first strategy to inhibit K-RAS expression was the development of small antisense nucleotides sequences directly targeting K-RAS mRNA. The ISI6957 oligonucleotide showed efficient inhibition of K-RAS expression in vitro (143). However, its stability and the strong toxicity did not allow a clinical use.

Despite of this failure, the strategy was optimized and Khvalevsky and colleagues developed a local prolonged siRNA delivery system (Local Drug EluteR, LODER) (144). Use of a biodegradable polymer, allowing RNA protection from degradation, reduces the toxicity.

Although encouraging, this strategy needs to be further improved and therefore is still not clinically usable (145).

A specific and irreversible inhibitor of the K-RAS G12C mutant induces an internal steric bulk blocking the conformational change necessary for activation (146,147). However, the G12C mutation remains rare in pancreatic cancer (3 % of K-RAS mutations) and other compounds targeting the G12D and G12V main mutants remain to be developed. The compound ARS-853 reacts with GDP-bound K-RAS G12C isoform, and alters downstream signaling, and cell survival in K-RAS G12C cell lines (148). ARS-853 is thought to impair normal SOS recruitment and strengthens the SOS targeting strategy that was previously developed thanks to the knowledge of well-defined binding pockets (149,150).

Targeting K-RAS-GTP interaction is considered as not feasible because of the high affinity of K-RAS for the GTP (pico molar range) and the strong intracellular concentration of GTP (milli molar range). An alternative strategy was to modify the GTP in order to make it hydrolysable by the mutant. The GTP analog DABP-GTP (Diamino-benzophenone-phosphoroamidate-GTP) can be hydrolyzed by G12 mutants more effectively than by wild type K-RAS (151).

However, no compound derived from this strategy has passed beyond the in vitro studies.

Indirect targeting of K-RAS

Icmt1 inhibitors

The K-RAS protein undergoes a cleavage of the AAX terminal sequence followed by a methylation of the formed isoprenylcysteine. These two stages are successively catalysed by two enzymes: The endoprotease RAS converting enzyme 1 (Rce1) and Isoprenylcysteine carboxymethyl transferase 1 (Icmt1), respectively. The targeting of these two key enzymes was investigated. Since Rce1 inhibition induces severe cardiomyopathies, the efforts focused on Icmt1 inhibition (159,160). Icmt1 targeting was developed in cancers and in particular in pancreatic cancer. However, Icmt1 inhibition dramatically accelerated the development of K-Ras-driven pancreatic neoplasia because of its requirement for Notch1 signaling (161).

Deltarasin, the PDEδ inhibitor

K-RAS addressing to the plasma membrane involves the hydrophobic prenylation trafficking process by the prenyl-binding chaperon protein: the phospho diesterase 6δ (PDE6δ/ PDEδ).

Deltarasin, an inhibitor of the PDEδ, showed promising results. Indeed, Deltarasin blocks PDEδ-K-RAS interaction preventing membrane localization in human K-RAS mutated pancreatic tumor cells, hence inducing a fast decrease of proliferation and increased apoptosis. In vivo, pancreatic tumor cells xenografts showed a dose dependent reduction of tumor growth following Deltarasin regimen (162,163). Clinical use of this recent strategy remains to be proven.

K-RAS downstream pathway targeting

A large number of inhibitors targeting components of K-RAS oncogene downstream signaling pathways were developed (164-166). Targeted therapies were focused on the MAPK pathway and led to the development of RAF and MEK inhibitors (167,168). In the context of pancreatic cancer, RAF inhibitors were ineffective. This may be related to the paradoxical induction of increased MAPK activity via a decreased feedback regulation as it was demonstrated with PLX4032 in melanoma (169). RAF inhibitors lack of effectiveness could be related to the binary recruitment of RAF by activated K-RAS in nanoclusters [START_REF] Tian | Plasma membrane nanoswitches generate high-fidelity Ras signal transduction[END_REF]. MEK kinases were also targeted by compounds such as CI-1040 but induced only partial response in pancreatic cancers independently of the K-ras mutation status (170). Another MEK inhibitor, Selumetinib (AZD6244), led to promising results on preclinical models but eventually failed in the clinical trial as it showed not significant benefit as second line treatment (171-173). Pimasertib is associated with sensitivity to gemcitabine and is currently under clinical evaluation (174). These failures are probably due to strong interconnections between signaling pathways (166,167). As MAPK and PI3K seem to be the major deregulated signaling pathways, combined MEK-PI3K inhibition is under evaluation in a cellular and mouse models of pancreatic cancer (173,175). PI3K targeting alone by using Evolorimus (mTOR inhibitor) has been evaluated in phase 2 clinical trial but only showed minimal clinical benefit (176). Very recently, Rigosertib, a RAS mimetic that is evaluated in clinical trials for myelodisplastic syndrome associated with acute myeloid leukemia, has been proposed for PDAC treatment. Rigosertib binds to RBD and thus inhibits the interaction of RAS-RAF, Ral and PI3K, blocks the activation of the RAS-RAF-MEK pathway and impairs tumor growth in K-Ras G12D mice (177). PKC inhibitor bryostatin-1 blocks K-RAS-S181

phosphorylation by PKC and induces apoptosis by inducing the K-RAS delocalization onto the mitochondrial membrane (178). Inhibition of other pathways, such as JNK and NF-κB was also investigated. However, most of these treatments are still either in experimental stages and are not yet used routinely in clinics (59,164,168,179-183).

Synthetic lethal interaction with K-RAS

An alternative strategy that has been developed is to identify new therapeutic target linked to K-RAS status: RNAi screening were performed in cell lines from different organs that were either K-RAS wild-type or K-RAS mutated in order to select genes that specifically impair viability of K-RAS mutated cells (153). Hit-genes involved in different biological processes were identified such as genes involved in survival signals (TAK1, TBK1), transcriptional program (GATA2, SNAIL2), chromosomal stability (Survivin, TPX2, PLK1, APC/C, proteasome) and apoptosis/senescence (WT1, Bcl XL ). Future research will be necessary to validate those targets in PDAC. Following the initial screening, the top candidates including GATA2 and PLK1 were further evaluated as new therapeutic targets (184,185). Very few analyses were focused on pancreatic cancer. 187 genes were identified and were enriched with the immune system, the ETS and the LAIR pathways (186).

Conclusions

More than 50 years after the inception of RAS research in 1964 showing that a retroviral isolate induced sarcoma; and the beginning of RAS characterization in the early 80s; the scientific community has produced huge efforts in deciphering RAS structure, biological functions, cell-signaling mechanisms and identifying target genes in patho-physiological conditions (187). K-RAS oncogene has been proven to be a central driver in cancers. K-RAS mutation, as the earliest event in pancreatic cancer, could be seen as the "starting pistol" of carcinogenesis sequence. Targeting K-RAS is an obvious goal but it has been an unfortunate clinic failure despite many different strategies (153) highlighting the urgent need for new approaches. Targeting cell adaptative metabolic mechanisms occurring after K-RAS activation via KPM2 or the non-specific OXPHOS inhibitor Metformin is promising (188).

Recently, checkpoint inhibitors/immune modulators such as antibodies targeting the programmed cell death protein 1 (PD-1) are promising targets in gastrointestinal cancer, including pancreatic cancer, and are a hot topic in the field (189). Based on this literature review, one can suggest that targeting K-RAS effectors (cell signaling or miRNA) or some crucial target genes may be a potential alternative strategy. Lately, FOLFIRINOX and Nab-Paclitaxel emerge as new standard of care but the overall survival still does not exceed one year (190,191) and pancreatic cancer remains one of the deadliest cancers; Therefore, no option should be excluded. Table1: Available PDAC data set retrieved from Gene Omnibus Expression comparing mutated K-RAS vs wild type K-RAS. GSE15126, GSE53659, GSE58307 transcriptome were analysed with with GEO2R. Gene ontology clustering was subsequently performed using David Functional Annotation Tool (https://david.ncifcrf.gov/). positive regulation of immune system process, negative regulation of lymphocyte activation, immune response, negative regulation of cell activation, negative regulation of leukocyte activation, immunoglobulin mediated immune response, B cell mediated immunity, lymphocyte mediated immunity, negative regulation of immune system process, immune effector process, adaptive immune response, adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains, immune response, leukocyte mediated immunity, Systemic lupus erythematosus

K-RAS-GTP

Tumor growth Metastasis Angiogenesis

Inflammatory reaction

(

  ROS) levels are tightly regulated by Nrf2 transcription factor (100). K-ras G12D drives the increase of Nrf2 transcription via RAF/MEK/ERK/Jun pathway. This Nrf2 increase promotes the antioxidant program (Hmox1, Nqo1, Gclc, Gclm, Ggt1) and decreases the intracellular ROS, thus favors a reduced intracellular redox microenvironment. The characterization of Nrf2 KO mice showed that activation of ROS by K-Ras/Nrf2 axis contributes to pancreatic carcinogenesis initiation (101). K-Ras-activated organoids also exhibit an increased activation of translation machinery. Combined K-Ras activation and loss of Nrf2 led to oxidation of factors involved in eIF4F complex (involved in cap-dependent mRNA translation), altered EGF and Akt signaling pathways and consequently impaired protein synthesis and maintenance of pancreatic tumors (102). This work highlights the promise of synthetic lethality of combined AKT inhibitors with oxidizing agents in PDAC. K-Ras also stimulates recycling process such as macrospinocytosis or autophagy.Macrospinocytosis is the uptake of nutrients via endocytosis leading to accumulation of catabolic intermediates that stimulates carbon metabolism and sustains cell proliferation (103). Autophagy is a process addressing cytoplasmic constituents to lysosomes for degradation and is critical for the maintenance of late stages of PDAC. K-ras G12D mice develop PanIN lesions harboring markers of autophagosomes (LC3 puncta) in an Atg5/Atg7 dependent manner (104). Therapeutic targeting of autophagy by chloroquinine could be promising. It was initially debated because homozygous Trp53 deletion led to paradoxical pro-carcinogenic effect following autophagy inhibition suggesting p53 status dependency (104-106). However, autophagy inhibition is in fact not affected when using the more clinically relevant model harboring Trp53 loss of heterozygosity as usually observed in patients (107).

4. 3 .

 3 Tumor microenvironment and immune systemOncogenic RAS alters the tumor microenvironment by promoting angiogenesis and by modulating immune responses. Immune cell infiltration is classically observed in the tumor stroma of PDAC patients. LSL-K-RasG12D immune compartment was characterized and a high fibro-inflammatory reaction containing both stromal and immune cells was thus shown to emerge within the tumor. Regulatory T cells infiltrate early in disease progression of pancreatic cancer, notably before invasive stage. Disease progression was also accompanied by infiltration of macrophages (early) and Myeloid-derived Suppressor Cells (MDSC) (late) (108). K-Ras activation was previously shown to foster inflammation through chemokine production (CXCL1/KC, CXCL2/MIP2, CXCL5/LIX and CXCL8/IL8) in lung or breast cancer cells (109,110). IL6 secretion is the most characterized cytokine in PDAC as it is induced both by the tumor cells and the myeloid cells from the surrounding stroma and is associated with tumor survival (111-114). Different fibroblastic and epithelial cell types transfected with oncogenic K-RAS G12D had an increased secretion of IL6 which is required for human tumor cell growth in vivo (115). However, among the different cell types, the macrophages were the principal source of IL-6 in PDAC. Paracrine IL6 induces strong phosphorylation of signal transducer and activator of transcription 3 (STAT3) that promotes PanIN-PDAC progression in K-Ras G12D mice (111). In acute pancreatitis, treatment with a RAS inhibitor (farnesylthiosalicylic acid) decreases levels of CXCL1, CXCL2 and IL6 and regulates neutrophil recruitment (116). Zhang and colleagues showed that the K-Ras secretome (CTGF, Cyr61, Cox2, mmp7, IL1A and IL6) of pancreatic cancer cells is regulated by YAP mediated transcriptional activity[START_REF] Zhang | Downstream of mutant KRAS, the transcription regulator YAP is essential for neoplastic progression to pancreatic ductal adenocarcinoma[END_REF]. Its invalidation in Yap flox/flox mice leads to a lack of CD45 lymphocyte infiltration, compromises the activation of stromal fibroblasts and their collagen secretion.PanINs also produce granulocyte-macrophage colony-stimulating factor (GM-CSF) which subsequently leads to recruitment of Gr1(+)CD11b(+) myeloid cells suppressing anti-tumor CD8+ T-cell immunity (117,118). These mice also harbor a high expression of receptorinteracting-1 (RIP1) and RIP3 kinases that are the main components of a macromolecular signaling complex called the necrosomes mediating the programmed necrose (necroptosis).Necroptotic tumor cells release soluble factors that induce peri-tumoral immune suppression.This immune-suppressive microenvironment relies on the RIP1/3 signaling via CXCL1 and the C-type lectin receptor Mincle signaling (activating NF-κB) to favor tumor progression (119).Angiogenesis occurs in response to tumor hypoxia and lack of nutrients and is essential for tumor growth. Angiogenesis is defined as the formation of new blood vessel and involves an equilibrium of angiogenic and anti-angiogenic factors (120). VEGF which appears to be critical in pancreatic angiogenesis (121) is correlated with detection of K-RAS mutation and is associated with a poorer prognosis (122). The oncogenic K-RAS G12V induces VEGF, CXC chemokines and COX2 promoting human umbilical vein endothelial cells (HUVEC) invasion and tumor formation via MEK/c-jun pathway (123). Similarly, in colorectal cancer cells, disruption of mutant K-RAS led to a reduced VEGF production (124). In addition, chemokine CXCL8/IL8 was also shown to drive the RAS-induced tumor angiogenesis (110).Oncogenic K-RAS is crucial in the crosstalk with stroma and notably pancreatic stellate cells (PSC). Additionally to K-RAS cell autonomous effect, both cell types set up a reciprocal signaling involving Sonic hedgehog (SHH) mediated activation of PSC and reciprocal signals via IGFR1/AXL and Akt activity that leads to increased mitochondrial performance, proliferation and resistance to apoptosis (125).

oncogene by altering its localization 5 . 2 . 1 .

 521 Prenylation inhibitorOther therapeutic strategies target post-translational modifications of the K-RAS protein, necessary for its membrane location. The first post-translational modification of K-RAS proteins is a modification of its HVR region. The farnesyl transferase enzyme (FTase) mediate farnesylation/prenylation on the cysteine of the terminal CAAX sequence adding a hydrophobic chain allowing membrane stabilization (152). Numerous inhibitors of FTase were developed to target this modification and inhibit K-RAS addressing to the membrane (153,154). The non-peptidomimetic competitive FTI, Tipifarnib (R115777), was tested in phase III trials in pancreatic cancer (155,156) but was ineffective because of an unexpected alternative prenylation mechanism (geranylgeranylation) involving geranylgeranyl transferase (GGTase) (157). GGT-I can recognize the CAAL motif and geranylates K-RAS4B. Combined therapies using inhibitors of FTase and GGT-I display high toxicity precluding their use in clinic (158).

Figure 1 :

 1 Figure 1: Schematic representation of K-RAS GDP/GTP cycling and its mutations (A) Schematic K-RAS4B protein. β: β-strand, α: helix-α. HVR: hypervariable region, KKKK: lysine rich region. * indicates the cysteine residue that is farnesylated. CAAX represents a sequence in which C= cysteine, A= aliphatic amino acid (Leu, Ileu or Val), X= Met, Ser, Leu or Gln. (B) 3D structure of GDP-bound Human K-RAS (4OBE) from protein data bank (http://www.rcsb.org/pdb) (C) K-RAS GTPase cycle. K-RASG12 mutation induces an accumulation of GTP-bound proteins unable to hydrolyze GTP and maintains the active conformation. (D) Frequency of K-RAS G12, G13, Q61 and other mutations in pancreatic cancer.

Figure 2 :Figure 3 :Figure 4 :

 234 Figure 2: K-RAS effectors and signaling pathways

2 Table1:

 2 Figure1

  Figure 3

Figure 4 Highlights

 4 Figure 4

Table hydroxylation ,

 hydroxylation C1Q, Collagen triple helix repeat, collagen, domain: Collagen-like Cluster 8

Acknowledgements:

Romain Vasseur was the recipient of a doctoral fellowship of Lille2 University. This work is supported by grants from la Ligue Nationale Contre le Cancer (Ligue 62, NJ), from SIRIC ONCOLille, Grant INCaDGOS-Inserm 6041 (IVS, NJ) and from region Nord-Pas de Calais.

We are grateful to Dr B. Neve and Dr A. Vincent (Inserm UMR-S1172, Lille) for their critical reading of the manuscript.

The study sponsors have no involvement in the writing or in the decision to submit the manuscript for publication.

Acknowledgements

Conflicts of interest: Authors declare no conflict of interest