Targeting the c-MYC / MDM2 pathway for the treatment of proliferative disorders

Abstract

Using a Burkitt's lymphoma (BL) cell model, the present inventors have shown that it is possible to suppress the synthesis of c-Myc by using specific compounds that bind to MDM2. The present inventors have shown that under low-cell proliferation conditions, MDM2 binds to the 5' untranslated region (UTR) of the c-Myc mRNA and suppresses its synthesis in a p53-independent manner, but 5 that this interaction does not take place under under medium / high cell proliferation conditions due to a conformational change in MDM2. The inventors have shown that under such conditions, it is possible to use specific MDM2-binding agents that induce an allosteric transition of MDM2 to promote its ability to interact with c-Myc mRNA, thereby suppressing its expression. The present invention therefore pertains to an MDM2-binding agent for use in the treatment of a proliferative disorder, 10 wherein said agent restores the c-Myc binding conformation of MDM2 and wherein said proliferative disorder is a p53-mutated disorder.

Description

Title: Targeting the c-Myc / MDM2 pathway for the treatment of proliferative disordersTechnical Field

[0001] The present invention pertains to novel therapeutic ways for the treatment of proliferative disorders such as cancer. The invention particularly relates to the use of specific MDM2-binding agents for the treatment of proliferative disorders.Background Art

[0002] C-Myc is a proto-oncogene that plays a critical role in regulating cell growth, proliferation, and apoptosis. Studies show that c-Myc regulates the expression of 15% of all genes (Gearhart et al., The New England Journal of Medicine. 357 (15): 1469-72). Dysregulations in c-Myc expression, in particular those leading to its overexpression, lead to an increase in the expression of multiple genes, including genes involved in cell proliferation, thereby contributing to neoplastic cell growth and proliferation. C-Myc was found to be constitutively expressed in no less than 70% of human cancers. Its inhibition has been shown to induce tumor regression in mice with only mild and fully reversible side effects, suggesting that c-Myc could represent an extremely promising therapeutic target for the treatment of cancer.

[0003] Unfortunately, the development of potent and promising c-Myc inhibitors faces major challenges (Soucek et al., Nature. 2008;455:679-83). Firstly, c-Myc exerts its activity through protein-protein and protein-DNA interactions that have proven difficult to disrupt with small molecules. Secondly, c-Myc overexpression in some human cancers does not result from mutations in the Myc gene itself, but rather from its induction by ‘upstream’ oncogenic signals. Developing c- Myc inhibitors having potent neoplastic properties in this context might therefore be challenging. Thirdly, c-Myc is essential in multiple biological pathways, including proliferation and stem cell compartment maintenance of regenerative adult tissues. C-Myc inhibition might therefore induce devastating and irreversible side effects.

[0004] Many inhibition strategies using genetic knockout, siRNA or indirect mechanisms that impede c-Myc function have been tested in the last few years (Madden et al., Mol Cancer 20, 3 (2021). For instance, lipid nanoparticle-based formulations (DCR-MYC) have been used to deliver siRNA into tumor cells, leading to inhibition of translation and expression of the c-Myc protein (Tolcher et al., J Clin Oncol. 2015;33(15_suppl):11006). Unfortunately, these inhibitors have not proven satisfactory.

[0005] More recently, a c-Myc dominant negative protein referred to as the Omomyc inhibitor was tested (Sodir et al., Genes Dev. 2011 ;25:907-16; Soucek et al., Genes Dev. 2013;27:504-13; Soucek et al., Nature. 2008). This protein provided interesting results: Omomyc has been shown to induce significant tumor regression in various cancers, even those in which c-Myc is not the driver oncogene, thereby validating c-Myc as a potential drug target in cancer.

[0006] However, no c-Myc inhibitor has yet been approved in cancer treatment, and there is therefore a major need for the development of new c-Myc-targeting compounds that would meet the therapeutic expectations and allow for a potent and safe treatment of cancer.Summary of the Invention

[0007] The invention is defined by the claims.

[0008] Using a Burkitt’s lymphoma (BL) cell model, a cancer characterized by c-Myc translocation and high expression, the present inventors have shown that it is possible to suppress the synthesis of c-Myc by using specific compounds that bind to the MDM2 (murine double minute 2) protein.

[0009] The results obtained by the present inventors show that under low-cell proliferation conditions, MDM2 binds to the 5’ untranslated region (UTR) of the c-Myc mRNA and suppresses its synthesis. However, this interaction does not take place under medium / high cell proliferation conditions due to a conformational change in MDM2. The inventors have shown that under such conditions, it is possible to use specific MDM2-binding agents that induce an allosteric transition of MDM2 to promote its ability to interact with c-Myc mRNA, thereby suppressing its expression. The inventors have shown that such a suppression allows stopping cell proliferation in BL cells.

[0010] This specific interaction between 5’ untranslated region (UTR) of the c-Myc mRNA and MDM2 was not known from the prior art. Faruq et al. (Cancers 14.6 (2022): 1592) discloses an oncogenic positive regulatory loop between MDM2 and c-Myc which would rely on a stabilizing effect of MDM2 on c-Myc mRNA (via an alleged interaction between MDM2 and the 3’ UTR of c-Myc RNA) to upregulate its expression, and on a transcriptional induction of MDM2 by c-Myc. These results suggest that inhibiting MDM2 would be useful for reducing c-Myc expression.

[0011] The results provided herein demonstrate, on the contrary, that a specific allosteric conformation of MDM2 suppresses c-Myc expression by interacting with the 5’ UTR of the c-myc RNA, and that under proliferative conditions, it is possible to inhibit c-Myc by restoring this specific conformation.

[0012] The present invention therefore relates to the use of MDM2-binding agents which allow restoring the c-Myc binding conformation of MDM2 for the treatment of proliferative disorders.

[0013] Most importantly, the inventors have shown that the MDM2-mediated suppression of c-myc synthesis is p53 independent.

[0014] Indeed, MDM2 is a protein known as being an important regulator of the p53 tumor suppressor. MDM2 is overexpressed in many cancers and disruptions in the p53-MDM2 pathway have been shown to be correlated with tumorigenesis (see e.g. Oliner et al., Cold Spring Harbor perspectives in medicine 6.6 (2016)). The development and use of MDM2 inhibitors for restoring p53 expression / activity is considered as a promising strategy for the treatment of cancer.

[0015] To date, p53 status is the major determinant of response to MDM2 treatment (see e.g. Haronikova, Lucia, et al (Cellular & Molecular Biology Letters 26 (2021): 1-33). MDM2 inhibitors are indeed deemed useful in conditions wherein lifting MDM2 repression on p53 would effectively allow restoring p53 activity, i.e. in conditions wherein p53 is not mutated and is functional.

[0016] However, as discussed above, the MDM2-mediated c-Myc regulation highlighted by the present inventors is p53-independent. This therefore offers promising new therapeutic perspectivesfor the treatment of p53-mutated disorders - corresponding to more than 50% of all cancers - wherein MDM2 inhibitors were considered as ineffective.

[0017] Therefore, according to a first embodiment, the present invention pertains to an MDM2- binding agent for use in the treatment of a proliferative disorder, wherein said agent restores the c- Myc binding conformation of MDM2 and wherein said proliferative disorder is a p53-mutated disorder.

[0018] According to a specific embodiment, said proliferative disorder is cancer, more particularly a c-Myc-overexpressing cancer.

[0019] According to a further embodiment, said MDM2-binding agent is selected from the group consisting of milademetan-like agents, nutlins-like agents such as idasanutlin or nutlin-3a and MDM2-targeted stapled peptides such as ALRN-6924 or ATSP-7041.Brief Description of Drawings:

[0020] Figure 1 - MDM2 regulates c-MYC in a p53 independent manner:

[0021] A) WB showing c-MYC levels in p53 null H1299 cells treated with increased concentration of Nut-3a (0.6, 1.25, 2.5, 5 and 10uM) during 8h. B) WB showing c-MYC levels in Burkitt’s lymphoma Ramos cells treated with increased concentration of DS-3032 (0.6, 1 .25, 2.5, 5 and 10uM) during 8h C) WB showing c-MYC levels in HCT116 cells expressing p53 wild type (WT) and HCT116 cells in which p53 was deleted (KO). Cells were treated with DS-3032 10uM during 8h or 24h. D) WB showing c-MYC levels in Ramos treated with DS-3032 10uM during increasing time (0.5, 1 , 2, 4 and 8 hours) E) H1299 cells treated with 5 mM DS-3032 in the presence of the proteasome inhibitor MG132 (25 mM) or the protein synthesis inhibitor cycloheximide (CHX; 20mg / ml) for indicated time points. F) RT-qPCR analysis of c-MYC mRNA in Ramos treated with DS-3032 5uM up to 8 hours. G) RT-qPCR analysis of c-MYC mRNA in Ramos treated with the RNA synthesis inhibitor Actinomycin D in the presence, or not, of DS-3032. H) Autoradiograph on 35S-Met pulse cell labelling followed by immunoprecipitation using c-MYC antibody from cells treated with DS-3032 (10 uM) during 8h.

[0022] Figure 2 - C-MYC IRES is the MDM2 inhibitors-response domain:

[0023] A) Agarose gel showing 5’ (1-406) c-MYC mRNA motility in presence of increased amount of MDM2 protein (1.25, 2.5, 5, 10, 20, 40, 80pmol) B) Schema illustrating Ha-MYC CDS construct and MYC-IRES-Ha-MYC CDS construct C) RT-qPCR analysis of c-MYC and Ha-MYC expression in H1299 transfected with Ha-MYC construct (left panel) and IRES-Ha-MYC (right panel) treated with Nutlin-3a and DS-3032 10uM during 8h D) WB showing Ha levels in H1299 transfected with Ha- MYC construct and IRES-Ha-MYC treated with DS-3032 10uM during 8h.

[0024] Figure 3 - Growth inhibition signaling induce MDM2 166A / C-MYC mRNA interaction :

[0025] (A) Graph showing the fold enrichment of c-MYC mRNA co-immunoprecipitated (RNA co-IP) with MDM2 166A and MDM2 166D from Ramos cells treated with DS-3032 (10 pM) for 8 hours. DS- 3032 treatment led to a significant (~30-fold) increase in the interaction between c-MYC mRNA and MDM2 166A, while MDM2 166D showed no significant interaction with c-MYC mRNA.(B) Westernblot (WB) showing c-MYC protein levels in Ramos cells subjected to different growth conditions: J2 (proliferative, post-passage) and J5 (high confluence). c-MYC protein levels were markedly reduced in the J5 (high confluence) condition. (C) Graph showing the reduction in c-MYC mRNA levels (~25%) in Ramos cells subjected to low serum conditions (0.2% fetal calf serum) for 8 and 15 hours, demonstrating only a modest decrease in mRNA levels despite significant suppression of c-MYC protein expression. (D) Graph showing the fold enrichment of c-MYC mRNA co-immunoprecipitated with MDM2 166A after Ramos cells were cultured in 0.2% fetal calf serum for 8 or 15 hours. A 2.5- fold increase in c-MYC mRNA interaction with MDM2 166A was observed after 8 hours of serum starvation, which further increased to over 10-fold after 15 hours. (E) Western blot (WB) showing c- MYC protein levels in Ramos cells under serum starvation (0.2%) for 8 and 15 hours. c-MYC protein expression was significantly suppressed in both conditions. (F) Graph showing an ~8-fold increase in c-MYC mRNA binding to MDM2 166A in Ramos cells grown to high confluence, indicating enhanced interaction under non-proliferative conditions.

[0026] Figure 4 - MDM2 inhibitors induce conformational changes leading to a second interaction interface between p53 and MDM2:

[0027] (A) Proximity ligation assay (PLA) showing an increased number of interactions between MDM2 and p53 proteins in cells expressing wild-type p53 treated with MDM2 inhibitors, Nutlin-3a or DS-3032 (10 pM, 8 hours). (B) Quantification of the interactions from the PLA shows that DS-3032 is more effective than Nutlin-3a in promoting the formation of the second interaction interface between p53 and MDM2 (left and right graphs). (C) Western blot (WB) showing that DS-3032 is more efficient in suppressing c-MYC expression compared to Nutlin-3a in Ramos cells. (D) Deuterium exchange assays (DHX) reveal structural changes in MDM2 following ligand binding, as indicated by altered deuteration patterns. Both Nutlin-3a and DS-3032 induce expected changes in the N-terminal hydrophobic pocket, but differ in their allosteric effects on the N-terminal lid domain and regions further towards the C-terminus. (E) In silico docking model based on the crystal structure of the MDM2 N-terminus. This model indicates that in addition to the main hydrophobic pocket, DS-3032 also interacts with a second binding pocket near the lid domain, explaining the distinct allosteric effects of Nutlin-3a and DS-3032 on MDM2.

[0028] Figure 5 - MDM2 inhibitors induce c-MYC mRNA allosteric changes :

[0029] RNA SHAPEmap analysis showing the structural changes in the 5' UTR of c-MYC mRNA following treatment with DS-3032. Under normal conditions, the GC-rich 5' UTR region, which forms G4 structures, was not detectable due to the lack of sequenced RNA fragments (upper panel). However, treatment with DS-3032 rendered this region "visible" (lower panel), indicating that the inhibitor affected the structure of the 5' UTR.

[0030] Figure 6 - DS-3032 inhibits protein synthesis and induces differential cell cycle effects in Burkitt’s lymphoma cells:

[0031] (A) Autoradiograph showing a significant (~50%) overall reduction in protein synthesis in p53- mutant Ramos cells treated with DS-3032 (10 pM) for 8 hours. Differential effects on various proteinswere observed. (B) Dose-response curve showing the inhibition of proliferation in Ramos (EBV- negative) and Raji (EBV-positive) Burkitt’s lymphoma cells treated with DS-3032 for 8, 16, and 24 hours. Both cell lines responded similarly with an IC50 of approximately 8 pM. Cell viability remained unaffected, indicating no induction of apoptosis. (C) Cell cycle analysis of Raji cells treated with DS- 3032 for 8, 16, and 24 hours, showing a gradual increase in the G1 phase and a corresponding decrease in the S phase, with minimal effect on the G2 phase. (D) Cell cycle analysis of Ramos cells treated with DS-3032 for 8, 16, and 24 hours, showing a decrease in the G1 phase and an increase in the G2 phase, with little effect on the S phase. These results highlight the cell type-specific effects of c-MYC on gene expression and cell cycle progression.

[0032] Figure 7 - MDM2 inhibitors inhibit tumor growth independently of p53:

[0033] NSG mice bearing Raji xenografts were treated daily by oral gavage (p.o) with 20mg / kg DS- 3032 for 15 days. (A) Tumor volumes were measured on the indicated days using calipers (n=5). (B) Tumors were collected and weighted at the endpoint of experiment (n-5). One-way ANOVA was used to test for statistical significance between the two groups and the difference was considered significant for P value <0.05 (**P <0.01).Detailed description of the invention

[0034] The present invention relies on the identification of a new regulatory pathway between c-Myc, a well-known protooncogene, and MDM2. The present invention indeed shows that MDM2 suppresses c-Myc expression under low proliferative conditions and loses this ability under medium / high proliferative conditions due to a conformational change. The present invention pertains to the use of specific MDM2 binding agents that restore the c-Myc-binding conformation of MDM2 for treating p53-mutated proliferative disorders.

[0035] Thus, a first aspect of the present invention relates to an MDM2-binding agent for use in the treatment of a proliferative disorder, wherein said agent restores the c-Myc binding conformation of MDM2 and wherein said proliferative disorder is a p53-mutated disorder.

[0036] “C-Myc” is a member of the Myc family of regulator genes. It is a transcription factor that plays a critical role in regulating cell growth, proliferation, and apoptosis. Dysregulation of the c-Myc gene can lead to its overexpression and contribute to the development of various cancers. Activation of c- Myc requires its heterodimerization with the Max protein. Once dimerized, the c-Myc / Max complex acts as a master transcriptional regulator by binding to a specific DNA consensus sequence known as the Enhancer-box. C-Myc targets as many as 15% of all genes (Dang et al., Semin Cancer Biol. 2006;16:253-64) and is therefore involved in the regulation of multiple cellular processes. C-Myc regulated genes include those involved in cell cycle regulation, such as cyclins D1 , D2, B1 and cyclin- dependent kinase 4 (CDK4), metabolism-associated genes such as enolase A, hexokinase II, lactate dehydrogenase A, phosphofructokinase, and glucose transporter I. Other target genes include those involved in protein synthesis, ribosome biogenesis and cell adhesion (Dang et al., (2006)). The amino acid sequence of human c-Myc is available under reference P01106 in the Uniprot database.

[0037] “MDM2” (Murine Double Minute 2 homolog), also referred to as E3 ubiquitin-protein ligase Mdm2, is encoded by the MDM2 gene and is mainly known for its role as an important negative regulator of the p53 tumor suppressor. MDM2 functions both as an E3 ubiquitin ligase that binds to the N-terminal transcriptional activation domain (TAD) of the p53 tumor suppressor and as an inhibitor of p53 transcriptional activation. The full-length transcript of the MDM2 gene encodes a protein of 491 amino acids. The p53-binding pocket is located in the N-terminal 100-residue of MDM2. The MDM2 protein also contains a central acidic domain (residues 230-300) which is involved in the regulation of MDM2 function, and a C-terminal RING domain (residues 430-480), whose proper folding requires zinc binding. The complete amino acid sequence of the human MDM2 protein is available under reference Q00987 in the Uniprot database. The human MDM2 amino acid sequence is shown in SEQ ID No. 1 .SEQ ID No.1 :MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLFYLGQYIMTKRL YDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSENRCHLEG GSDQKDLVQELQEEKPSSSHLVSRPSTSSRRRAISETEENSDELSGERQRKRHKSDSISLSFDESL ALCVIREICCERSSSSESTGTPSNPDLDAGVSEHSGDWLDQDSVSDQFSVEFEVESLDSEDYSLS EEGQELSDEDDEVYQVTVYQAGESDTDSFEEDPEISLADYWKCTSCNEMNPPLPSHCNRCWALR ENWLPEDKGKDKGEISEKAKLENSTQAEEGFDVPDCKKTIVNDSRESCVEENDDKITQASQSQES EDYSQPSTSSSIIYSSQEDVKEFEREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHL MACFTCAKKLKKRNKPCPVCRQPIQMIVLTYFP

[0038] The present invention comprises using an MDM2 binding agent. In the context of the present invention an “MDM2 binding agent” refers to a compound that has the ability to target and associate with the MDM2 protein through molecular binding. The nature of the interaction between MDM2 and the binding agent can be a non-covalent, reversible covalent or irreversible covalent binding. The skilled person knows which compounds can be qualified as MDM2 binding agents and may therefore be useful in the context of the present invention. MDM2 binding agents can e.g. be selected from polyclonal or monoclonal antibodies, antibody fragments, nucleic acids, peptides, aptamers or small molecules.

[0039] The MDM2 binding agent used in the context of the present invention “restores the c-Myc binding conformation of MDM2”. As explained above, the present inventors have shown that MDM2 has two distinct conformations, or 3-dimensional shapes. According to one of these conformations which is that observed under low-proliferative conditions, MDM2 binds the 5’ UTR of the c-Myc RNA, thereby suppressing its synthesis. Under mild / high proliferative conditions, MDM2 undergoes a conformational change which prevents it from binding to c-Myc, thereby allowing for c- Myc synthesis. In the context of the present invention, the MDM2 binding agent restores the c-Myc binding conformation of MDM2, meaning that its binding to MDM2 induces a conformational change in the MDM2 protein which, as a result, has the conformation observed under low-proliferative conditions, i.e. has a conformation that allows MDM2 to bind to the 5’ extremity of the c-Myc RNA. Therefore, in the context of the present invention, the binding of the MDM2 binding agent to MDM2induces a significant decrease in the level of c-Myc protein expression. The skilled person knows several techniques that allow determining whether an MDM2 binding agent allows restoring the c- Myc binding conformation of MDM2. The simplest technique consists in evaluating whether the binding of the agent to MDM2 allows suppressing / significantly reducing the c-Myc protein synthesis. C-Myc being a well-known protein, the skilled person can easily determine its concentration in a sample. A simple technique for measuring the level of c-Myc is e.g. disclosed in the experimental section of the present application. Typically, c-Myc synthesis in a cell can be measured by submitting said cell to a lysis and analyzing its protein content by Western blot or other protein expression assays. In this context, c-Myc can advantageously be probed with antibodies. This procedure is routine to the skilled person. The 5’ UTR of c-myc RNA can also be fused to a reporter construct and the effect of compounds on synthesis can be tested by analyzing the effect on the expression of the reporter protein. MDM2 binding agents that restore the c-Myc binding conformation of MDM2 as provided in the present invention allow reducing the levels of c-Myc of at least 30%, preferably 40%, more preferably 50%, even more preferably 60% as compared to controls (not treated with said MDM2 binding agent).

[0040] Alternatively, the skilled person can determine whether an MDM2 binding agent allows restoring the c-Myc binding conformation of MDM2 by analyzing its 3D structure, e.g. by crystallography such as Cryogenic electron microscopy or by in silico modelling, upon binding with said agent. Crystallography and in silico modelling allow obtaining a 3D image of the protein, which can then be compared to controls. Such methods can therefore allow comparing the conformation of MDM2 upon binding with the agent to that of MDM2 under low proliferative conditions (i.e. the c-Myc binding conformation of MDM2) so as to evaluate whether these conformations are similar (which would mean that the agent allows restoring the c-Myc binding conformation of MDM2) or not.

[0041] The present inventors have shown that the most potent MDM2 binding agents that allow restoring the c-Myc binding conformation of c-Myc are those which specifically bind to a specific binding pocket located in the N-terminus of the MDM2 protein. This binding pocket comprises amino acids 28-31 , 33-34, 50, 53-54, 57, 84-86, 99-101 of the MDM2 amino acid sequence as shown in SEQ ID No. 1 , The MDM2 binding agents according to the present invention can, in addition to this specific binding pocket, bind to the well -known p53 binding pocket as well (see for review Wang et al. Protein-protein interactions (2012): 57-79),

[0042] In the context of the present invention, the MDM2-binding agent can be selected from the group consisting of milademetan-like agents, nutlins-like agents such as idasanutlin or nutlin-3a and MDM2-targeted stapled peptides such as ALRN-6924 or ATSP-7041.

[0043] “Milademetan” or “DS-3032” is a synthetic compound qualified as an MDM2 inhibitor. It is being tested in clinical trials for use in the treatment of patients with advanced solid tumors or lymphomas (Gounder et al., J Clin Oncol / ''\ 7'\ -'\ 724 (2023)). Milademetan binds to MDM2 thereby preventing its binding to the TAD of p53. By preventing the MDM2-p53 interaction, Milademetan inhibits the proteosome-mediated enzymatic degradation of p53. This results in the restoration of p53 signaling and leads to the p53-mediated induction of tumor cell apoptosis.Milademetan is therefore used for tumors having a functional p53. By “Milademetan-like” agents, it is herein referred to compounds that have the same mechanism of action as Milademetan, in particular that bind to the same binding pocket within MDM2 (see da Mota et al. World Journal of Clinical Oncology 13.6 (2022): 496). Milademetan-like agents include Milademetan.

[0044] “Nutlins” are cis-imidazoline analogs which act as an MDM2 antagonist: they inhibit the MDM2-p53 axis by mimicking the interaction of critical amino acid residues with the hydrophobic activity of MDM2 (see for review Azer, Samy A. Journal of Clinical Medicine 7.4 (2018): 64). By binding with MDM2 in the p53-binding pocket, Nutlins activate the p53 pathway in cancer cells, resulting in cell cycle arrest, apoptosis, and tumor growth inhibition. These compounds used on tumors that present a functional p53. Nutlins include Nutlins- 1 , -2, and -3 as well as Nutlin derivatives such as RG7112 and RG7388 (idasanutlin). By “Nutlins-like” agents, it is herein referred to compounds that have the same mechanism of action as Nutlins, in particular that bind to the same binding pocket as Nutlins (see Azer (2018)). Nutlins -like agents include Nutlins such as Nutlins- 1 , - 2, and -3 as well as Nutlin derivatives such as RG7112 and RG7388 (idasanutlin).

[0045] “Stapled peptides” are synthetic small peptides that are constrained by a synthetic brace ("staple"). The staple is formed by a covalent linkage between two amino acid side-chains, forming a peptide macrocycle. “MDM2-targeted stapled peptides” are stapled peptides that target and bind to MDM2. Several MDM2-targeted stapled peptides have been synthesized and used as MDM2 antagonists (Chee et al. PLoS One 9.8 (2014): e104914). As the MDM2 inhibitors discussed above, they are used to re-instate p53 effector functions in tumors with functional p53. MDM2-targeted stapled peptides can e.g. be selected from ALRN-6924 or ATSP-7041 .

[0046] According to a specific embodiment, the MDM2 binding agent is Milademetan.

[0047] In the context of the present invention, the MDM2 binding agent is used for the treatment of proliferative disorders. “Proliferative disorders” are a group of diseases that are characterized by abnormal and excessive cell proliferation. In the context of the present invention, the proliferative disorder is typically a cancer. The term "cancer" includes solid tumors and blood born tumors. Typically, the cancer can be of various origin (such as breast, colon, gastric, rectum, pancreatic, lung, skin, head and neck, bladder, ovary, prostate etc....), and also involve various cancer cell types (adenocarcinoma, squamous cell carcinoma, large cell cancer, melanoma, etc).

[0048] The cancer can be a disseminated cancer, that is to say one which has metastases (patients classified in the NxMx category according to the TNM classification).

[0049] In a particular embodiment, the cancer can be a solid cancer selected from the group consisting of skin cancer (e.g. melanoma, nonmelanoma skin cancer), colorectal cancer, adrenal cortical cancer, anal cancer, bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma), sarcomas such as liposarcoma and soft-tissue sarcoma, brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma,germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ), cervical cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adnocarcinoma), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hepatic adenoma, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma).

[0050] In another embodiment, the cancer can be a hematological cancer such as leukemia, lymphoma (including Burkitt’s lymphoma, Hodgkin lymphoma or non-Hodgkin lymphoma) and myeloma.

[0051] According to a specific embodiment, said cancer is a “c-Myc overexpressing cancer”. Indeed, as explained above, the MDM2 binding agent according to the present invention advantageously allows inducing a MDM2-mediated suppression of c-Myc synthesis. It therefore finds a particular interest in cancers wherein c-Myc has been reported to be overexpressed. As mentioned above, C-Myc was found to be constitutively expressed in no less than 70% of human cancers. These cancers include (see for review Madden et al., 2021): Burkitt Lymphoma (BL) (a B-cell lymphoma which is often associated with chromosomal translocations involving the c-Myc gene, leading to its overexpression), Diffuse Large B-cell Lymphoma (DLBCL) (a common type of non-Hodgkin lymphoma where c-Myc overexpression can occur due to various mechanisms, including translocations and gene amplification), multiple myeloma (a cancer of plasma cells, exhibiting c-Myc dysregulation due to translocations or other genetic alterations), leukemia, neuroblastoma (a pediatric cancer that affects nerve cells, c-Myc amplification is associated with high-risk neuroblastoma cases), lung cancer including Small Cell Lung Cancer (SCLC) (an aggressive lung cancer which frequently exhibits c-Myc overexpression) and non-small cell lung cancer (NSCLC), prostate cancer, breast cancer (subtypes of breast cancer, including some triple-negative breast cancers, can display c-Myc overexpression), colorectal cancer, gastric cancer, ovarian cancer, pancreatic cancer (c-Myc overexpression has e.g. been reported in pancreatic ductal adenocarcinoma), esophageal cancer (c-Myc is frequently overexpressed in both esophageal squamous cell carcinoma and esophageal adenocarcinoma) and melanoma.

[0052] Therefore, according to a specific embodiment, said cancer is selected from the group consisting of BL, DLBCL, multiple myeloma, leukemia, neuroblastoma, lung cancer (including SCLCand NSCLC), prostate cancer, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, pancreatic cancer, esophageal cancer and melanoma.

[0053] According to a particular embodiment, said cancer is Burkitt’s lymphoma.

[0054] As explained above, the MDM2-mediated c-Myc regulation highlighted by the present inventors is p53-independent. This therefore offers promising new therapeutic perspectives for the treatment of p53-mutated disorders - corresponding to more than 50% of all cancers - wherein MDM2 inhibitors were considered as ineffective. Therefore, in the context of the present invention, the proliferative disorder, in particular cancer, is a p53-mutated disorder. This therefore means that the patient suffering from said disorder has been identified as displaying a p53 mutation / a non-functional p53 protein.

[0055] By “p53-mutated disorder” it is herein referred to a condition wherein the cells, in particular cancer cells, display a mutation in the p53 protein and / or the corresponding TP53 gene which results in a suppression of the synthesis of p53 or in the synthesis of a non-functional p53 protein in these cells.

[0056] “p53”, also known as “tumor protein P53”, “cellular tumor antigen p53” or “transformation- related protein 53” is a regulatory protein that acts as a tumor suppressor in many tumor types. It induces growth arrest or apoptosis depending on the physiological circumstances and cell type. P53 is involved in cell cycle regulation as a trans-activator that acts to negatively regulate cell division by controlling a set of genes required for this process. P53 is often qualified as a tumor suppressor. The TP53 gene, which encodes the p53 protein, is the most frequently mutated gene (>50%) in human cancer. It is now well established that TP53 plays a crucial role in preventing cancer formation. The sequence of the human p53 is available under reference P04637 in the Uniprot database.

[0057] A "mutation" refers to any detectable change in genetic material, e.g. DNA, RNA, cDNA, or in an amino acid sequence encoded by such a genetic material. This includes gene mutations, in which the structure (e.g. DNA sequence) of a gene is altered any gene as well as protein mutations, in which the amino-acid structure of the protein is altered. Generally, a mutation is identified in a by comparing the sequence of a nucleic acid or of a polypeptide expressed with the corresponding nucleic acid or polypeptide expressed in a control. When the mutation to be detected is a somatic mutation, i.e. a mutation that occurs in a specific cell line other than the germline, it can also be detected by comparing the sequence of a nucleic acid or of a polypeptide expressed by said cell line (e.g. tumor cells) with the corresponding nucleic acid or polypeptide expressed by another cell line (e.g. a non-cancerous cell line).

[0058] In the context of the present invention, the mutation of P53 refers to a mutation of one or more nucleotide in the TP53 gene / mRNA sequence. Such a mutation can be a deletion (i.e. removal of at least one nucleotide in the gene sequence), an insertion (i.e. addition of at least one nucleotide in the gene sequence) or a substitution (i.e. replacement of one nucleotide with another). In the context of the present invention, the mutation is preferably located in the exons (i.e. in the coding parts of the gene) and has an effect on the p53 protein encoded by the TP53 gene such as missensemutations (which result in a codon that codes for a different amino acid), nonsense mutations (which result in a premature stop codon or a nonsense codon in the transcribed mRNA and thus in a truncated, and usually non-functional protein) or frameshift mutations (which result from the insertion or deletion of a number of nucleotides in a DNA sequence that is not divisible by three and induce a change the reading frame resulting in a completely different translation from the original).

[0059] A person skilled in the art knows a great many techniques allowing him to detect a mutation within a gene.

[0060] The presence of a mutation in TP53 genes according to the present invention may be detected by analysing TP53 nucleic acid molecules. In the context of the invention, nucleic acid molecules include mRNA, genomic DNA and cDNA derived from mRNA. DNA or RNA can be single stranded or double stranded. These may be utilized for detection by amplification and / or hybridization with a probe, for instance. The nucleotide sequence can be obtained from a genomic DNA sample isolated from the biological sample. In this case, any biological sample containing genomic DNA can be used.

[0061] TP53 mutations may be detected in a RNA or DNA sample, preferably after amplification. For instance, the isolated RNA may be subjected to coupled reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for the mutated site or that enable amplification of a the region containing the mutated site. Conditions for primer annealing may be chosen to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of a mutation according to the invention. Otherwise, RNA may be reverse-transcribed and amplified, or DNA may be amplified, after which the mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art. For instance, a cDNA obtained from RNA may be cloned and sequenced to identify the mutated sequence of TP53.

[0062] As discussed above, the mutation detected in the TP53 gene results in a mutated mature p53 protein. Thus, the mutation of TP53 can be detected at the protein level by detecting the mutated form of p53. In this case, any biological sample wherein the mature protein is present may be used for detecting mutated forms of this protein. In this context, a sample, such as a cancer cell sample, obtained from a patient may be contacted with antibodies specific of the mutated form or the protein, i.e. antibodies that are capable of distinguishing between the mutated form and the wild-type protein (or any other protein), to determine the presence or absence of the mutation specified by the antibody.

[0063] The invention will now be further illustrated by means of the following examples.Examples

[0064] MDM2 inhibitors regulate NMYC protein levels in a p53-dependent manner.

[0065] MDM2 has been suggested to control the stability of the N-Myc mRNA in a p53-dependent fashion. The treatment of the neuroblastoma cell lines IMR-32 carrying wild type p53 and SK-N-FI (p53 mutant (M246R)) with the MDM2 inhibitor Nutlin-3a at different concentrations (0.5uM, 1 ,25uM,2.5uM, 5uM and 10uM) for 8h resulted in a decrease in NMYC protein levels in the IMR-32, but not SK-N-FI, cells. The same treatment using the MDM2 inhibitor DS-3032 showed similar, but more potent, effect. These results are in line with previous observations showing that MDM2- inhibitors controls NMYC expression dependent on p53 status. Neither drug showed any significant effect on N-Myc mRNA levels in either cell line.

[0066] MDM2 inhibitors suppress c-MYC protein synthesis independently ofp53.

[0067] Treating the p53 null H1299 cells with increasing concentrations of Nutlin-3a 8 hours resulted in a reduction in c-Myc expression starting at 5 mM (Fig. 1A). c-Myc levels started to decrease in the Burkitt’s lymphoma (BL) cell line Ramos that carry a mutant p53 with DS-3032 starting at 1 mM (Fig. 1B). p53 wild type HCT cells, or HCT p53 knock-out cells, were also treated and a similar reduction in c-Myc expression was observed in both cells types. An induction of MDM2 expression was also oberved in the HCT p53 WT cells due to the effect of the compound on activating the p53 pathway and the consequent induction of MDM2 expression (Fig. 1C). A time course showed reduced c-Myc expression in Ramos cells starting at 1 hour with 10 mM DS-3032 treatment (Fig. 1D). To determine at what level of c-Myc expression MDM2 inhibitors control c-Myc expression cells were treated with the proteasome inhibitor MG132 or with the protein synthesis inhibitor cycloheximide (CHX). Figure 1 E shows that MG132 induces the expression of c-Myc but does not affect the relative suppression of expression caused by DS-3032 treatment (10 mM), indicating that MDM2 inhibitors do not affect c-Myc protein stability. Cycloheximide (CHX) treatment knocked down expression of c-Myc independently of the presence of MDM2 inhibitor. c-Myc RNA levels are sensitive to growth conditions but when we treated Ramos cells with 10 mM of DS-3032 for 8 hours we observed no significant change in c-Myc RNA levels (Fig. 1F). Furthermore, when cells were treated cells with the RNA polymerase inhibitor actinomycin D we observed an approximately 75 % reduction in c-Myc RNA levels after one hour treatment, independently if the cells were treated, or not, with DS-3032 (Fig. 1G). Finally, when carried out a 60 minutes35S metabolic pule labeling followed by a c-Myc immune-precipitation (IP) a reduced rate of c-MYC synthesis was observed (Fig. 1H). Taken together, these results show that MDM2 inhibitors suppress c-Myc protein synthesis in a p53-independent way.

[0068] MDM2 interacts with the 5’ untranslated region (UTR) of the c-MYC mRNA.

[0069] It was next determined how MDM2 affects c-Myc synthesis. The 5’ untranslated region (UTR) of the c-Myc RNA contains a G-quadruple (G4) structure with mRNA translation regulatory properties and with the capacity to promote cap-independent mRNA translation via an internal ribosome entry site (IRES). To test the potential role of this regulatory domain in MDM2-mediated suppression of c- Myc synthesis two c-Myc cDNA constructs were generated carrying an HA-tag in the N-terminus with, or without, the c-Myc 5’ UTR (5’ UTR-HA-MYC and HA-MYC) (Fig. 2A). Using anti-HA antibodies it was observed that the presence of the 5’UTR reduced HA-c-Myc expression in H1299 cells, illustrating its mRNA translation regulatory activity. Importantly, the MDM2 inhibitor only suppressed expression of the HA-c-Myc expression from construct carrying the c-Myc 5’ UTR (Fig. 2B). RT- qPCR confirmed that MDM2 inhibitor treatment did not affect respective RNA levels (Fig. 2C). To test if MDM2 interacts directly with the c-Myc 5’ UTR the inventors carried out gel shifts using a fixedconcentration (1 pmol) of transcribed and purified c-MYC 5’UTR RNA and increasing concentrations of different recombinant MDM2 proteins (Fig. 2D). At a ratio of 1 :20 RNA:MDM2 protein it was observed a shift in c-MYC 5’ UTR RNA migration with most RNA shifted at 1 :80 (Fig. 2E). Deletion of the RING domain (MDM2(D432-491)) that is required for the interaction with the p53 mRNA reduced the binding efficacy but did not prevent it. A further deletion including the intrinsic disordered domain located between the RING and the Zn domain (MDM2(D354-491)) did not affect the interaction. However, a further deletion (MDM2(D299-491)) prevented the interaction. The RING domain alone (MDM2(396-491)) resulted in a smear and a larger shift at the highest concentration, indicative of unspecific binding. This interaction pattern resembles MDM2’s interaction with the coding sequence of the E2F1 message in which the RING domain does not interact with the RNA but plays a regulatory role. These results show that MDM2 interacts directly with the 5’ UTR of the c-Myc mRNA and that this interaction is required for the suppression of c-Myc protein synthesis by MDM2-inhibitory compounds.

[0070] MDM2 interacts with the c-MYC mRNA following treatment with MDM2 inhibitors or at non-proliferative growth conditions.

[0071] The inventors next wanted to test if MDM2 also interacts with the endogenous c-Myc mRNA. Ramos cells were treated with DS-3032 and the total RNA was extracted and separated from cellular proteins followed by incubation with recombinant MDM2 proteins. The MDM2 protein is immunoprecipitated (IPed) and the bound RNA quantified using RT-qPCR (RNA-ColP). It has previously been shown that the interaction between MDM2 with the E2F1 mRNA is regulated by the serine 166 residue which is a phosphorylation site for the Akt growth stimulatory pathway and we could show that an MDM2 carrying a phosphomimetic aspartic acid substitution at 166 (MDM2S166D) binds to the E2F1 mRNA but not the c-Myc RNA. However, treatment of cells with DS-3032 resulted in an approximately 30-fold increase in the MDM2(166A) - c-Myc mRNA interaction. An MDM2 carrying a phosphomimetic mutation at serine 395 (MDM2(395D)) binds the p53 mRNA but does not interact with the E2F1 orthe c-Myc mRNAs, showing the selection of mRNAs interacting with MDM2 depending on MDM2 modifications (Fig. 3A).

[0072] The inventors wanted to know if the interaction between endogenous MDM2 and the c-Myc mRNA is limited to the effect of MDM2 inhibitors, or if this also reflects a physiological event where MDM2 controls c-Myc expression. It was tested if growth conditions influence the interactions and Ramos cells were subjected to a low (0,2%) foetal calf serum concentration for 8 and 15 hours. This resulted in an important suppression of c-Myc protein expression, as expected (Fig. 3B). However, the c-Myc RNA levels were reduced by only an approximate 25% (Fig. 3C). Under these conditions we observed an estimated 2,5-fold increase in the interaction between the c-Myc RNA to MDM2(166A) after 8 hours and over 10-fold increase after 15 hours while there was no, or weak, interaction with the MDM2(166D) (Fig. 3D). To test the idea that non-growth conditions stimulates the c-Myc RNA - MDM2 interaction Ramos cells were allowed to grow at high confluence which also resulted in a suppression of c-MYC protein expression (Fig. 3E) and a nearly 8-fold induction in c- Myc mRNA binding to MDM2(S166A), but not MDM2(166D) (Fig. 3F). These results show thatMDM2 interacts with the c-Myc mRNA and suppresses c-Myc protein expression in cells treated with MDM2 inhibitors or under non cell-proliferative conditions.

[0073] MDM2 inhibitors induce MDM2 allosteric changes.

[0074] The RNA coIP was carried out using recombinant MDM2 proteins and observed an increase in the binding of MDM2 to c-Myc RNA isolated from cells was observed. This indicates that the treatment with MDM2 inhibitors or non-growth conditions affects the conformation of MDM2 as well as the structure of the c-Myc mRNA. If so, this would resemble the interaction between MDM2 - p53 mRNA taking place following DNA damage in which both the protein and the RNA change structures.

[0075] MDM2 inhibitors such Nutlin-3a induces conformational changes to produce and a second interphase between p53 and MDM2. In line with this, the proximity ligation assay (PLA) shows that treating cells expressing wild type p53 (MV-4-11) with MDM2 inhibitors actually results in an increase in the number of interactions between the two proteins (Fig. 4A). Quantification of interactions shows that DS-3032 is more efficient in promoting the second interphase (Fig 4B, left and right graphs). We also observed that DS-3032 is more efficient in suppressing c-MYC expression as compared to Nutlin-3a in Ramos cells (Fig. 4C). To test which domains of MDM2 that are structurally affected by MDM2 inhibitors a deuterium exchange assays (DHX) in which the structural changes in MDM2 in solution can be indirectly determined following addition of ligands was carried out. The recombinant p53 protein, Nutlin-3a or DS-3032 gave the expected changes in the DHX pattern at the N-terminal hydrophobic pocket where they bind (Fig. 4D). However, the exchange pattern differs from each drug and they have also different allosteric effects on the N-terminal lid domain as well as domains further towards the C-terminus, indicating that the two compounds interact differently with MDM2. An in silico docking model based on the crystal structure of MDM2 N-terminus indicates that in addition to a common interaction site, DS-3032 also interacts with a second pocket that is in close proximity to the main hydrophobic pocket. NMR models show that this second docking site is in proximity to the predicted location of the lid domain, offering an explanation to the differences of the two compounds and their respective allosteric effects on MDM2 (Fig. 4E). Using PDBePISA (an interactive tool for the exploration of macromolecular interfaces from EMBL-EBI), it was shown that the interface amino acids of MDM2 for interaction with DS-3032 are amino acids 28-31 , 33-34, 50, 53-54, 57, 84-86, 99-101 of MDM2.

[0076] MDM2 inhibitors induce c-Myc mRNA allosteric changes.

[0077] These results illustrate the allosteric effects of MDM2 inhibitors on the MDM2 protein. The next step was to address changes in c-Myc mRNA structure following treatment with MDM2 inhibitors and we carried out RNA SHAPEmap which is a highly reproducible technique to determine changes in RNA structures in live cells. The 5’ UTR of the c-Myc mRNA is GC rich and forms G4 structures that are difficult to resolve using SHAPEmap. In line with this, the structure of the full 5’ UTR under normal conditions could not be determined due to the lack of sequenced RNA fragments but, interestingly, in the presence of DS-3032 this region became “visible”, showing that the presence of DS-3032 affected the structure of this region (Fig. 5). When the regions surrounding the first AUG (+1) that were visible under both normal and drug treatment conditions were compared usingdeltaSHAPE, changes in the labeling of nucleotides in this region imposed by DS-3032 could be observed, further demonstrating changes in RNA structure following treatment with MDM2 inhibitors.

[0078] Physiological consequences ofc-MYC translation downregulation

[0079] C-Myc plays a key role in promoting cell growth by stimulating ribosomal biogenesis and cell cycle progression and in accordance, the treatment with DS-3032 resulted in an approximately 50% overall reduction in protein synthesis in the p53 mutant Ramos cells 8 hours after treatment, with the notable differences between different proteins (Fig. 6A). The effect of DS-3032 on the rate of proliferation of Ramos and Raji cells following treatment for 24 hours was studied. Both cell lines are Burkitt’s lymphomas with mutant p53 and c-Myc translocation but while Ramos is Epstein-Barr virus (EBV) negative Raji cells are EBV positive. Both cells responded with a similar inhibition of proliferation following DS-3032 treatment with an IC 50 of approximately 8 pM (Fig. 6B). Upon study of the cell cycle profile, it was observed a time-dependent increase in the G1 phase with a corresponding decrease in the S phase and little effect on the G2 (Fig. 6C and 6D).

[0080] MDM2 inhibitors inhibit tumor growth independently ofp53.

[0081] To investigate the therapeutic efficacy of DS-3032 in a p53-independent model, a Raji-derived xenograft model was used. The Raji cells were implanted subcutaneously in NSG mice, and once tumors reached an average volume of 100 mm3, the mice were randomly assigned to receive daily treatments. The treatment groups were vehicle or DS-3032 administered orally at 20 mg / kg. DS- 3032 treatment led to a notable reduction in tumor volume compared to vehicle (Fig. 7A). Differences were confirmed at the endpoint of the experiment by measuring tumor weights (Fig. 7B). No significant changes in body weight were observed, indicating that the reduction in tumor size was not due to general toxicity.

[0082] Discussion

[0083] A drug that targets c-Myc has been highly thought after and these results indicate that c-Myc mRNA translation is druggable by ligands binding the N-terminus of MDM2. The fact that MDM2 inhibitors can suppress c-Myc synthesis in cancer cells independently of p53 status will lead to a new understanding of how these compounds can best be used in clinical practice. Since these compounds can easily be used to knock out c-Myc expression, it provides an important step forward for studying the effects of targeting c-Myc in vivo and in vitro. Since they also target p53 activity one can foresee that using the combination of p53 wild type and p53 mutant cells will shed light on the different and combined effects of these pathways on cell biology and cancer development. An interesting observation with regard to how MDM2 inhibitors inducing conformational changes in MDM2 promote MDM2’s interaction with the c-Myc mRNA comes from the deutrium exchange (DHX) and RNA SHAPE studies. The DHX shows that DS-3032 and Nutlin-3a binds MDM2 differently with one common site at the hydrophobic pocket and a predicted additional adjacent second interaction site for DS-3032. It can be speculated that a new category of compounds can be developed that preferentially targets this second interaction site on MDM2 and preferentially target c-MYC expression while having less effect on the interaction with p53. The observation that non-growthconditions, such as serum starvation or over confluence also stimulates MDM2 binding to the c-MYC mRNA, suggests that the conformational changes imposed by the MDM2 inhibitor also take place during non-growth conditions via cellular pathways. The c-MYC mRNA - MDM2 interactions were carried out using recombinant MDM2 proteins and, hence, the stimulatory effect of the MDM2 inhibitors on the RNA - protein interaction is also related to the structure of the c-MYC mRNA. Previous studies on the interaction between MDM2 and the p53 mRNA showed that both the MDM2 protein and the p53 mRNA underwent ATM-mediated structural changes in order for the interaction to take place. The data presented here suggest something similar for the c-MYC mRNA - MDM2 interaction. The serine 166 on MDM2 is phosphorylation target for the Akt pathway that promotes cell growth and proliferation. An MDM2 with a phosphomimetic aspartic acid at this position does not bind the c-MYC mRNA, indicating that when cells are proliferating MDM2 will not suppress c-MYC expression. However, the same MDM2(S166D) protein binds strongly to the E2F1 mRNA and stimulates E2F1 synthesis. These data support the notion that MDM2 harbors two RNA binding domains that are mutually exclusive: one responsive to the growth conditions of the cell and one to genotoxic stress. The interaction with the c-MYC mRNA is the only example that results in a suppression of synthesis. It is, however, also the only example of MDM2 binding to the 5’ UTR of an mRNA.

[0084] One of the difficulties with targeting c-MYC for therapeutic intervention has been that it is an intrinsic disorder protein that lacks druggable pockets. The interaction with its co-regulatory factor Max, or other c-MYC interacting factors, have been exploited to prevent its activity but so far the different approaches to target c-MYC have not resulted in new clinical practices. Targeting the expression of c-Myc offers a different approach to treat cancers in which c-MYC overexpression is believed to be a cause. The synthesis of c-MYC is different in the sense in that the 5’ UTR provides a highly structural region that is adapted to provide regulated control of c-MYC mRNA translation initiation that can respond to changes in cellular conditions and thereby rapidly regulate c-MYC expression. In line with this notion, it was observed that under serum starvation the c-MYC RNA levels are reduced with approximately 25% but the expression of the c-MYC protein was further suppressed, suggesting that during non-proliferative conditions reduced levels of c-MYC RNA and a reduced translation rate both play a role in controlling c-MYC protein levels.

[0085] The 5’ UTR of MYCN is similar to that of c-MYC and a suppression of MYCN in the neuroblastoma cell line IMR32 that carries a wild type p53 was also observed, but no effect in the p53 mutant SK-N-FI cells was shown. MYCN has been shown to regulate MDM2 expression and it has also been suggested that MDM2 can control the growth of neuroblastoma cells and that MDM2 promotes the stabilisation of NMYC RNA levels and synthesis, independently of p53. However, in this study Nutlin-3a had no effect on NMYC expression despite the cells being p53 positive. MYCN and c-MYC have indistinguishable biological activities and it is interesting to note the apparent differences in the regulation of their respective synthesis.

[0086] An important aspect of this study is that it provides a link between two main cell growth regulatory pathways: the c-MYC and p53. Both are essential for the development of cancer, and it isestimated that together, these two pathways are dysregulated in an absolute majority of all human cancers. To have MDM2 as a link between these pathways is very interesting and should help shed light on the coordinated control of cell cycle progression and arrest under different cellular conditions. MDM2’s capacity to regulate p53 has overshadowed its other functions. Apart from its interaction with different mRNA to control synthesis of growth regulatory factors it also interacts with both oncogenic factors such as E2F1 , but also tumour suppressors like p53, p14Arf and the retinoblastoma protein as well as interacting with chromatin. This multifunctional activity of MDM2 illustrates why it is rarely mutated in cancers.Materials and Methods

[0087] Cell culture, transfection and drug treatments

[0088] Experiments were performed using Ramos cells (Burkitt’s lymphoma human cell line) (ATCC® CRL-1596)], Raji cells (Burkitt’s Lymphoma cell line (ATCC® CCL-86), H1299 (Non-small- cell lung carcinoma human cell line) (ATCC® CRL-5803), IMR-32 (Neuroblastoma human cell line) (ATCC® CRL5803), SK-N-FI (Neuroblastoma human cell line) (ATCC® CRL-2142), MV-4-11 (Biphenotypic B-myelomonocytic leukemia) (ATCC® CRL-9591). All cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U / ml penicillin, 100 pg / ml streptomycin (Invitrogen) and 5 pg / ml Plasmocin prophylactic (Invivogen). Cell lines were routinely checked for mycoplasma contamination using PlasmoTest kit (Invivogen). Drugs: MG132 (474790-5, Calbiochem), Nutlin-3a (HY-10029, MedChemExpress), DS-3032 (1398568-47-2), Chemietek), Actonomycin (50-76-0, Sigma-Aldrich), Cycloheximide (C4859, Sigma-Aldrich).

[0089] Plasmid constructs

[0090] Plasmids constructs were created using the eukaryotic expression vector pcDNA3 (pcDNA3- HA-MYC CDS, pcDNA3-IRES-HA-MYC CDS). pGL2-basic-XNM Luc 447 (35157, Addgene) has been purchased. Using site directed mutagenesis pGL2-basic-XNM Luc 468 was created. For transfection of plasmid DNA, Genejuice (EMD chemicals) reagents was used respectively according to the manufacturer’s instructions.

[0091] Western blot analysis

[0092] Cells were lysed using BC200 lysis buffer (200 mM NaCI, 0.2% NP-40, 10% (v / v) glycerol, 1.0 mM dithiothreitol (DTT), 1.0 mM EDTA, and 25 mM Tris-HCI, pH 7.8) containing 1 % (v / v) eukaryotic protease inhibitor cocktail (Calbiochem). Equal protein amounts were loaded and resolved in 10% Bis-Tris Plus Gels (Thermo Fisher), transferred on BioTrace NT pure nitrocellulose blotting membrane (PALL Corporation) and blocked with 5% non-fat dry milk in Tris-buffered saline pH 7.6 containing 0.1 % Tween-20. Proteins were then probed with corresponding antibodies; anti-MDM2 mouse mAbs [1 :500] (4B2-Recamo), anti-Ha rabbit pAbs [1 :1000] (#3724, Cell Signaling), anti-c- MYC mouse mAbs [1 :100] (sc-40, Santacruz), and anti- Actin mouse pAbs [1 :2000] (AC-15, Sigma- Aldrich).

[0093] RNA extraction and RT-PCR, qRT-PCR

[0094] Cells were washed with cold PBS and total RNA was purified using the RNeasy Mini Kit and on-column DNase treatment (74104, Qiagen) following the manufacturer's protocol. RT was carried out using Moloney Murine Leukaemia Virus Reverse Transcriptase and random hexamers or oligo (dT) primers (Invitrogen). RT-qPCR was performed on StepOne real- time PCR system (Applied Biosystems) using Perfecta SYBR Green FastMix, ROX (Quanta Biosciences)

[0095] Electrophoretic Shift Assay

[0096] For MDM2 WT protein-c / WyC IRES RNA gel shift, cMyC IRES RNA was refolded by heating at 90°C for 3 minutes and kept on ice for 5 minutes, added to the tube containing RNA folding buffer (20 mM Tris-HCI, 10 mM ZnSO4, 150 mM NaCI, 2 mM MgCh) and indicated purified recombinant MDM2 proteins. After a 30 minutes incubation at room temperature, the samples were loaded into a 0.5 % agarose gel and run at 95 V for 1 hour.

[0097] Proliferation assay

[0098] The following concentrations were used for Nutlin-3a and DS-3032 : 0,6uM, 1 ,25uM, 2,5uM, 5uM, 10uM and 20uM. Proliferating cells were plated, incubated for three days, and then counted. Experiments were performed at least in triplicate.

[0099] Flow cytometry

[0100] Cells expressing were fixed by overnight incubation in cold 70% ethanol. After incubation with 100 U / ml RNase A (Sigma-Aldrich) and propidium iodide (421301 , Biolegend) according to the manufacturer's recommendations, stained cells were analysed with a Canto II flow cytometer and Flowjow software.

[0101] Proximity ligation assays

[0102] Cells were fixed with 4% paraformaldehyde (PFA) for 20 min, permeabilized with PBS 0.4% Triton X-100, 0.05% CHAPS for 10 min at room temperature and saturated with PBS 3% BSA for 30 min. Samples were saturated with PBS 3% BSA, 0.1 % saponine and incubated for 2 h with anti- MDM2 mouse mAbs (4B2) and anti-p53 rabbit mAbs (#9282, Cell Signaling technology) primary antibodies diluted in blocking solution. The proximity ligation assay (PLA) was carried out using the Duolink PLA in situ kit (Sigma) following the manufacturer's protocol. Cover slips were mounted in DAKO medium and image acquired using Carl-Zeiss Axiovert inverted microscope.

[0103] Metabolic pulse labelling

[0104] Pulse labelling was performed as described previously (Gnanasundram et al., 2017). Briefly, 8h post treatment, cells were cultured for 1 h in Dulbecco’s modified Eagle’s starvation medium (Sigma-Aldrich) (without methionine, cysteine and L-glutamine supplemented with 2% dialysed foetal bovine serum together with 20 pM of proteasome inhibitor MG132. Cells were metabolically labelled for 1 h with 45 pCi / ml of EasyTag Express 35S-methionine Protein Labelling Mix (Perkin-Elmer). C- MYC proteins were then immunoprecipitated with corresponding antibodies anti-c-MYC mouse mAbs [1 :100] (sc-40, Santacruz) using DynabeadsTM Protein G Immunoprecipitation Kit (10007D,ThermoFischer) and eluted proteins were resolved in 4-12% Bis-Tris Plus Gels and visualized on autoradiograph.

[0105] In-cell RNA SHAPE-MaP

[0106] The RNA SHAPE-MaP was performed with the protocol adapted from the methods published by the Weeks group (19, 20). Briefly, Ramos were treated 8h (DMSO / DS-3032). Cells were washed with PBS and added 900 pl of RPMI media. The SHAPE reagent 1-Methyl-7-nitroisatoic anhydride (1 M7) (Sigma-Aldrich) was added to a final concentration of 10 mM by adding 100 pL of 100 mM 1 M7 to 900 pL of RPMI media and treated for ~90 seconds at 37°C. The same volume of DMSO was added to the untreated samples. Cells were then washed with PBS and harvested. RNA purification was carried out using the RNeasy kit (Qiagen), followed by DNAse I digestion for 30 minutes at 37°C. Reverse-transcription of purified RNA was carried out with the primers indicated in the Supplementary Table. S1 , using the MaP buffer and Superscript II Reverse Transcriptase (Thermo Fisher Scientific). Synthesized cDNAs were then purified and amplified using Q5 DNA polymerase (NEB) using the indicated p53 primers. The PCR product was purified and quantified with a Qubit fluorometer and diluted to 0.2 ng I pL. Purified amplicons were then tagmented and a library was created using the llumina Nextera PCR library kit. Products from library PCR were then purified with the Agencourt AMPure XP beads (Beckman Coulter Life Sciences) as described in (20). Library concentration was measured with a Qubit fluorometer and the size distribution was measured using an Agilent 2100 Bioanalyzer according to the manufacturer’s instructions. Libraries were then sequenced with the Paired-end 150 NovaSeq System (NovaSeq PE150, Novogene, UK). SHAPE reactivity profiles and comparisons were generated using the ShapeMapper 2 and deltaSHAPE scripts, using default settings as described in (20) and aligned to indicated c-MYC coding sequences (CDS).

[0107] Ex vivo and in vitro RNA co-immunoprecipitation

[0108] Ex vivo MDM2 RNA co-immunoprecipitation (RNA co-IP) was carried out using DynabeadsTM Protein G Immunoprecipitation Kit (ThermoFischer) following the manufacturer’s instructions, with anti-MDM2 mouse antibody (4B2). Briefly, 48 h post-transfection cells were lysed using IP lysis buffer (100 mM KCI, 50mM HEPES-KOH, 5mM MgCI2, 1 mM DTT, 0.5% NP-40, Proteasome inhibitor cocktail). Cleared lysate was used for MDM2-IP with Protein G dynabeads. RNA was purified from input and IP- elute samples using TRIzol (Life Technologies) and analyzed by RT- qPCR for E2F1 mRNA enrichment. Percentage of enrichment of E2F1 mRNA from the input normalized with actin was plotted for both EV and GAr.

[0109] In vitro RNA co-IP was carried out as described using monoclonal MDM2 antibody (4B2). Briefly, 1 pg of total RNA from transfected cells were co-incubated under agitation with 100 ng of recombinant MDM2 in binding buffer (50 mM Tris pH 7.5, 150 mM NaCI, 0.02 mg / ml yeast tRNA, 0.2 mg / ml BSA) at 4°C. After incubation, MDM2 - RNA complexes were pulled down using protein G- coated sepharose beads (Sigma-Aldrich) according to standard conditions and purified using the TRIzol (Life Technologies). Precipitated RNAs were analyzed by RT-qPCR for mRNA enrichment. Fold enrichment of mRNA levels between cells expressing EV and GAr was plotted in graph.

[0110] Hydrogen / Deuterium exchange

[0111] Deuteration of the N-terminal domain of MDM2, either free or in complex with Nutlin-3 or DS- 3032, was followed on intact protein (global kinetics) or on peptides generated after pepsin di- gestion (local kinetics). The exchange was initiated by a ten- fold dilution into a deuterated buffer (25 mM Hepes, pD 7.5 (defined previously (Glasoe and Long, I 960)) and 150 mM NaCI). The molar ra- tios between the / V-term MDM2 and Nutlin-3 were 1 :1 and 1 :4 and the protein concentration during H / D exchange was 1.5 uM. Protein was incubated with Nutlin-3 and DS-3032 for 30 min prior to the exchange. Aliquots (10 uL) were taken after 40 s, 2, 5, 10, 30 min, 1 , 3, and 5 h either for global or local kinetics. The exchange was done at 21 °C and was quenched by the addition of 10 uL of 40 mM HCI (pH 2.3) and rapid freezing in a liquid nitrogen. A temperature of 21 °C was chosen as the temperature for ligand binding since this is the normal reference temperature for all ELISA assays using MDM2 (for MDM2-peptide binding (Picksley et al., 1994); and for Nutlin-3 / MDM2 competition (Worrall et al., 2009) that measure ligand binding.

[0112] Digestion and HPLC separation

[0113] Each sample for the local kinetics was quickly thawed and injected onto an immobilized pepsin column (15 uL bed volume, flow rate 20 uL / min, 0.4% formic acid in water, di- gestion time 45 s). Peptides were trapped and desalted online on a peptide microtrap (Michrom Bioresources) for 1 min at a flow rate 20 uL / min. Next, the peptides were eluted onto an analytical column (Halo C18, 0.3 x 50 mm, 2.7 urn, 90 A° , Michrom Bioresources) and separated by a linear gradient elution 10- 25% B in 1 min, followed by 5 min isocratic elution at 40% B at a flow rate 9 uL / min. Solvents were: A — 0.4% formic acid in water, B — 95% acetonitrile / 0.4% formic acid. Samples for the global kinetics were analyzed directly with- out digestion. They were thawed, quickly desalted on a protein microtrap (Michrom Bioresources) and eluted with 50% acetonitrile / 0.4% formic acid. In all analyses, injection and switching valves, immobilized pepsin column, trap cartridge, and the analytical column were kept at 0°C (immersed into an ice-water bath) to minimize back-exchange. Outlet of the LC system was interfaced to ESI source of a mass spectrometer.

[0114] MS and data analysis

[0115] Mass spectrometric analysis was done on ESI-FT-ICR MS (9.4T Apex-Qe, Bruker Daltonics). For peptide map- ping (HPLC-MS / MS) the instrument was operated in a data-dependent mode. Each MS scan was followed by MS / MS scans of the top three most intense ions. Tandem mass spectra were searched using MASCOT against a database containing sequence of the N-terminal MDM-2 con- struct. Sequence coverage was visualized using Draw Map script — http: / / ms.biomed.cas.cz / MSTools

[0024] , Analysis of deuterated samples (with or without digestion — local and global kinetics, respectively) was done in HPLC-MS mode and the data were processed in DataAnalysis 4.0. For global kinetics, the average mass of the intact protein at different deuteration times was read out using charge deconvolution. Local kinetics data were processed by ExPro script provided by Gary H. Kruppa (personal communication) and transferred to DataAnalysis. Deuteration percentages were calculated as follows: % D = 100*((Mpd-Mnd) / A / ) where Mpd is mass of partially deuterated peptide / protein, Mnd is mass of nondeuterated and N is total number of exchangeableamide hydrogen within the peptide / protein. No correction for back- exchange was done. The figures showing deuteration kinetics for all peptides used in this study are included in the supplementary material.

[0116] Mouse Xenograft Studies

[0117] Raji cells (5 x 1 OA6) were combined with an equal volume of Matrigel (50 pL) and injected subcutaneously into the right flank of 6- to 8-week-old male NOD SCID gamma (NSG) mice. Once tumors reached an average volume of 100 mm3, the mice were randomly assigned to one group (n=5 per group). The groups were treated daily for 15 days by oral administration of vehicle or DS-3032 at a dose of 20 mg / kg. Tumor volume and body weight were monitored every 4-5 days until sacrifice. The mice were housed in a pathogen-free facility, and all experimental procedures were conducted in accordance with the guidelines approved under the agreement #C75-10-08. Every effort was made to ensure the ethical treatment and well-being of the animals throughout the study.

Claims

Claims

1. An MDM2-binding agent for use in the treatment of a proliferative disorder, wherein said agent restores the c-Myc binding conformation of MDM2 and wherein said proliferative disorder is a p53-mutated disorder.

2. The MDM2-binding agent for use according to claim 1 , wherein said proliferative disorder is cancer.

3. The MDM2-binding agent for use according to claim 1 or 2, wherein said proliferative disorders is a c-Myc overexpressing cancer.

4. The MDM2-binding agent for use according to any one of claims 1 to 3, wherein said proliferative disorder is a cancer selected from Burkitt Lymphoma (BL), Diffuse Large B-cell Lymphoma (DLBCL), multiple myeloma, leukemia, neuroblastoma, lung cancer including Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, pancreatic, esophageal cancer and melanoma.

5. The MDM2-binding agent for use according to any one of claims 1 to 4, wherein said proliferative disorder is Burkitt lymphoma.

6. The MDM2-binding agent for use according to any one of claims 1 to 5, wherein said agent is binds with MDM2 in the binding pocket comprising amino acids 28-31 , 33-34, 50, 53-54, 57, 84-86, 99-101 of SEQ ID No.1.

7. The MDM2-binding agent for use according to any one of claims 1 to 5, wherein said agent is selected from the group consisting of milademetan-like agents, nutlins-like agents such as idasanutlin or nutlin-3a and MDM2-targeted stapled peptides such as ALRN-6924 or ATSP-7041 .

8. The MDM2-binding agent for use according to claim 7, wherein said agent is milademetan.

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