Therapeutic use of surf2 modulators

Abstract

The present invention relates to SURF2 modulators and its therapeutic use in a patient in need thereof. In particular, the present invention relates to a SURF2 inhibitor or a mean of its depletion for use in the treatment of a cancer in a subject in need thereof and a SURF2 activator or a mean of its overexpression for use in the treatment of a ribosomopathy in a subject in need thereof.

Description

[0001] THERAPEUTIC USE OF SURF2 MODUEATORS

[0002] TECHNICAE FIELD

[0003] The present invention relates to SURF2 modulators and its therapeutic use in a patient in need thereof. In particular, the present invention relates to a SURF2 inhibitor or a mean of depletion for use in the treatment of a cancer in a subject in need thereof and a SURF2 activator or a mean of overexpression for use in the treatment of a ribosomopathy in a subject in need thereof.

[0004] BACKGROUND

[0005] Nucleolar stress (NS) can be defined as any stress resulting from the impairment of ribosome synthesis, a complex and energy-consuming process that begins with transcription of rDNA by the specific RNA polymerase I (Pol I) in the nucleolus (A. K. Henras, et al. Cell. Mol. Life Sci. 65, 2334-2359 (2008)). Under stress, cells must reduce their production of ribosomes to avoid wasting energy and to stop cell proliferation (B. Albert, et al. eLife 8, e45002 (2019)). These regulations of ribosome synthesis are thus essential to the control of cell growth in all living cells. Nevertheless, mature ribosomal subunits are stable entities, so the inhibition of their synthesis only affects cellular protein homeostasis after longterm exposure to stress. In addition to these long-term regulations, ribosome synthesis shutdown upon stress signaling promotes direct and fast cell cycle arrest (C. Mayer, et al. Cell Cycle 4, 1036-1038 (2005); M. O. J. Olson, et al. Sci. STKE 2004 (2004)).

[0006] In human, ribosome synthesis shutdown promotes cell cycle arrest by stabilization and activation of the well-known tumor suppressor p53 (C. Mayer, I. et al. Cell Cycle 4, 1036-1038 (2005), E. Nicolas, et al. Nature Communications 7, 11390 (2016), L. Golomb, et al. FEBS Lett., (2014)). This pathway is prevalent in the regulation of p53 under normal and pathological conditions (K. M. Hannan, et al. Cell Rep 41, 111571 (2022); M. S. Lindstrom, et al. Cell Death Differ 29, 972-982 (2022); C. P. Rubbi, et al. The EMBO Journal 22, 6068-6077 (2003)).

[0007] Several ribosomal proteins are able to mediate such p53 regulation in response to stress. The general idea is that ribosomal proteins that are released from or not integrated to ribosomes can accumulate as free form in the nucleoplasm. There, they can directly bind MDM2 and inhibit its E3 -ubiquitin ligase activity (X. Zhou, et al. Oncogene 32, 388-396 (2013); X. Zhou, et al. Journal of Molecular Cell Biology 7, 92-104 (2015); S. Yadavilli, et al. DNA Repair (Amst.) 8, 1215-1224 (2009);X. Zhang, et al. Oncogene 32, 2782-2791 (2013); V. Marechai, et al. Mol. Cell. Biol. 14, 7414-7420 (1994)). Such role for ribosomal proteins has been generally termed as extra-ribosomal function. However, among the different ribosomal proteins to support such regulation of p53 activation only RPL5 and RPL11 are instrumental to this response. Interestingly, both proteins are components of the same complex, the 5S ribonucleoprotein (RNP) particle (K. M. Hannan, et al. Cell Rep 41, 111571 (2022); K. E. Sloan, et al. Cell Rep 5, 237-247 (2013); K. Nishimura, et al. Cell Reports 10, 1310-1323 (2015); S. Bursae, et al. Proc. Natl. Acad. Sci. U.S.A. 109, 20467-20472 (2012)).

[0008] The 5S RNPs consist in the association of 5S ribosomal RNA (rRNA) with the ribosomal proteins RPL5 and RPL11. These particles are largely incorporated into nascent large / 60S ribosomal subunits. Disruption of ribosome synthesis results in the accumulation of so-called free 5S RNPs in the nucleoplasm. Once released, free 5S RNPs interact with the E3 -ubiquitin ligase MDM2, that normally targets p53 to the proteasome for degradation, and inhibit its activity, which thus promotes stabilization and activation of p53 (K. M. Hannan, et al. Cell Rep 41, 111571 (2022), K. E. Sloan, et al. Cell Rep 5, 237-247 (2013)).

[0009] Importantly, NS does not result in p53 stabilization and activation in the absence of 5S RNPs components, demonstrating that 5S RNPs play a critical role in the NS response (K. M. Hannan, et al. Cell Rep 41, 111571 (2022), K. E. Sloan, et al. Cell Rep 5, 237-247 (2013)). It appears that altering the balance in favor of 5S RNP integration into ribosomes contributes to cancer development and therapeutic resistance (P. Cao, et al. Sci Adv 7, eabf4304 (2021)). Thus, promoting the extra-ribosomal activity of free 5S RNPs in wild -type TP53 cancers may improve the p53 -dependent anticancer effects of therapeutic agents such as chemotherapy used in most poor-prognosis cancers. On the other hand, little is known about the putative existence of a free 5S RNP pool under basal conditions. Given that 5S rRNA is independently transcribed by RNA polymerase III instead of RNA polymerase I for other rRNAs, a logical hypothesis would be that 5S rRNA production is not in complete stoichiometry with other ribosomal RNAs, even under basal conditions.

[0010] Many cancer treatments that are not designed to target ribosome synthesis induce a NS response, including chemotherapies (K. M. Hannan, et al. Cell Rep 41, 111571 (2022), C. P. Rubbi. The EMBO Journal 22, 6068-6077 (2003), K. Burger, et al. Journal of Biological Chemistry 285, 12416-12425 (2010)). Even though ribosome synthesis is essential in all living cells, cancer cells are more sensitive to its inhibition. Indeed, ribosome synthesis is strongly increased in cancer cells, which require a greater synthesis of all its components including 5S RNPs. When this process is altered a greater amount of free 5S RNPs accumulates and subsequently mediates a stronger p53 activation (E. Derenzini, et al. J Hematol Oncol 11, 75 (2018)). This phenomenon partly explains why cancer cells are more sensitive to nucleolar stress than healthy ones (D. Drygin, et al. Cancer Res. 71, 1418-1430 (2011); R. Ferreira, et al. Cells 9, 266 (2020)). Therefore, activation of the NS response by well-described anticancer drugs such as 5 -fluorouracil (5-FU) or doxorubicin contribute to their therapeutic benefit (K. Burger, et al. Journal of Biological Chemistry 285, 12416-12425 (2010)). However, as frequently observed for many cancer treatments, in clinical trials using new class of Pol I inhibitors, half of the patients showed progressive disease, indicating that resistance mechanisms may be occurring (A. Khot, N et al. Cancer Discov 9, 1036-1049 (2019)). Thus, there remains a need to discover new therapeutic targets of 5S RNP pathway to improve cancer treatment.

[0011] Free 5S RNP homeostasis is also key in an ensemble of diseases originating from ribosome production defects and regrouped as ribosomopathies. In Diamond-Blackfan Anemia syndrome (DBA), a well characterized ribosomopathy, activation of p53 by free-5S particles is at the core of the etiology of these diseases, since some symptoms are linked to early p53 activation such as growth retardation, developmental problems and even erythropoiesis (A. Aspesi, et al. Sci Rep 7, 12010 (2017) ; S. Le Goff, et al. Blood 137, 89-102 (2021); N. C. Jones, et al. Nat. Med. 14, 125-133 (2008)). In addition, the patients suffering from these diseases show a higher cancer incidence compared to the general population.

[0012] The current treatment options for diseases belonging to the ribosomopathy family are far from optimal, especially for Diamond Blackfan anemia syndrome (DBA). Therefore, there is a need to discover novel, effective, and targeted therapies for these diseases associated with ribosomopathy.

[0013] SUMMARY

[0014] The inventors in the present application identified a new partner, the SURF2 protein, that regulates the activity of free 5 S RNP under nucleolar stress conditions . They demonstrated that SURF2 acts as a buffer for free-5S RNP particles in control cells to avoid unnecessary activation of p53. SURF2 inhibitor can therefore be used to enhance p53 activation, in particular after NS in a cancer treatment. They also showed that SURF2 expression is upregulated in cancers and negatively correlates with overall survival.

[0015] In another hand, the use of SURF2 to impede p53 activation following nucleolar stress represents an interesting therapeutic target. Indeed, mimicking SURF2 or part of it using small peptides or compound drug approaches could inactivate p53 through blocking free 5S RNPS - MDM2 interactions, and could alleviate symptoms of ribosomopathy patients and prevent their risk of developing cancers for these patients.

[0016] The present disclosure relates to a SURF2 inhibitor for use in the treatment of a cancer in a patient in need thereof, preferably wherein said SURF2 inhibitor activates p53 by free 5S RNP. In a preferred embodiment, said inhibitor is a fragment of SURF2 protein comprising no more than an amino acid sequence between residue position 1 to 136 of SEQ ID NO: 1, a functional fragment or a functional variant thereof having at least 80, 85, 90 or 95% identity to the amino acid sequence between residue position 1 to 136 of SEQ ID NO: 1, preferably a peptide comprising or consisting of an amino acid sequence of SEQ ID NO: 6, a functional fragment or a functional variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 6. In another particular embodiment, the inhibitor is at least one interfering RNA molecule, preferably comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2 to 5, more preferably comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7 to 10.

[0017] In a specific embodiment, said inhibitor is administered in combination with a chemotherapeutic agent, preferably selected from the group consisting of: Actinomycine D, 5 Fluoro-Uracil (5-FU), doxorubicin (DRB), Etoposide (ETO), cyclophosphamide and cisplatin.

[0018] The present disclosure also relates to a pharmaceutical composition comprising a SURF2 inhibitor as defined above and a pharmaceutical acceptable carrier, and preferably further comprising a chemotherapeutic agent, preferably selected from the group consisting of: Actinomycine D, 5 Fluoro- Uracil (5-FU), doxorubicin (DRB), Etoposide (ETO), cisplatin and cyclophosphamide.

[0019] The present disclosure also relates to an in vitro method for diagnosis a cancer or the prognosis of survival outcome of a patient suffering from a cancer, preferably hormonal cancer, such as adrenocortical carcinoma and prostate cancer, but also hormonal independent cancers such as head and neck cancer or liver cancers comprising the steps of determining SURF2 gene expression level in a patient sample, preferably tumor patient sample, wherein a higher SURF2 gene expression level in a patient sample compared to a control value is indicative that said patient has a lower survival time and a lower SURF2 gene expression level in a patient sample compared to a control value is indicative that said patient has a higher survival time.

[0020] In another aspect, the present disclosure relates to a SURF2 activator for use in the treatment of a ribosomopathy, preferably selected from the group consisting of: Diamond-Blackfan anemia syndrome (DBA), 5q-syndrome, Schwachman-Diamond syndrome, X-linked dyskeratosis congenita, cartilagehair hypoplasia, Treacher-Collins syndrome, Bowen-Conradi syndrome, North American Indian childhood cirrhosis, more preferably Diamond-Blackfan anemia syndrome (DBA) in a patient in need thereof, preferably wherein said SURF2 activators impedes activation of p53 by free 5S RNP.

[0021] In a particular embodiment said activator is a nucleic acid construct comprising a transgene encoding a SURF2 protein, preferably a human SURF2 protein comprising or consisting of SEQ ID NO: 1 or a function variant thereof having at least 80, 85, 90 or 95% identity to SEQ ID NO: 1, preferably comprised in an expression vector, more preferably a viral vector selected from the group consisting of: Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV or SNV, lentiviral vectors, adenoviral (Ad) vectors, adeno-associated viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors, again more preferably a lentiviral vector. In particular, said nucleic acid construct or expression vector is comprised in a viral particle, preferably a lentiviral particle. The present disclosure also relates to an isolated cell for use in the treatment of a ribosomopathy in a patient in need thereof, preferably selected from the group consisting of: Diamond-Blackfan anemia syndrome (DBA), 5q-syndrome, Schwachman-Diamond syndrome, X-linked dyskeratosis congenita, cartilage-hair hypoplasia, Treacher-Collins syndrome, Bowen-Conradi syndrome, North American Indian childhood cirrhosis, more preferably Diamond-Blackfan anemia syndrome (DBA), wherein said cell comprises a nucleic acid construct as defined above, preferably wherein said cells is transduced with a viral particle comprising a nucleic acid construct as defined above. In a preferred embodiment, said cell is a hematopoietic cell selected from the group consisting of hematopoietic progenitor or stem cells, preferably selected from the group consisting of: bone-marrow derived cells, peripheral blood cells, and umbilical cord blood cells.

[0022] The present disclosure also relates to a pharmaceutical composition comprising the activator as defined above or isolated cells as defined above and a pharmaceutical acceptable carrier.

[0023] DETAILED DESCRIPTION

[0024] The inventors in the present application have identified a new partner of free 5S RNP particles that acts as a buffer for free-5S RNP particles to avoid unnecessary activation of p53. The activity regulation of this protein allows to modulate the response to nucleolar stress involved in several pathologies.

[0025] Definitions

[0026] The terms “nucleic acid sequence” and “nucleotide sequence” may be used interchangeably to refer to any molecule composed of or comprising monomeric nucleotides. A nucleic acid may be an oligonucleotide or a polynucleotide. A nucleotide sequence may be a DNA or RNA. A nucleotide sequence may be chemically modified or artificial. Nucleotide sequences include peptide nucleic acids (PNA), morpholines and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acid (TNA). Each of these sequences is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule. Also, phosphorothioate nucleotides may be used. Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'- phosphoramidates and oligoribonucleotide phosphorothioates and their 2'-0-allyl analogs and 2'-0- methylribonucleotide methylphosphonates which may be used in a nucleotide of the disclosure.

[0027] As used herein, the term "transgene" refers to exogenous DNA or cDNA encoding a gene product. The gene product may be an RNA, peptide or protein. In addition to the coding region for the gene product, the transgene may include or be associated with one or more elements to facilitate or enhance expression, such as a promoter, enhancer(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s) and / or other functional elements. Embodiments of the disclosure may utilize any known suitable promoter, enhancer(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s) and / or other functional elements. Suitable elements and sequences will be well known to those skilled in the art.

[0028] The term “nucleic acid construct” as used herein refers to a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. A nucleic acid construct is a nucleic acid molecule, either single- or double -stranded, which has been modified to contain segments of nucleic acids sequences, which are combined and juxtaposed in a manner, which would not otherwise exist in nature. A nucleic acid construct usually is a “vector”, i.e. a nucleic acid molecule which is used to deliver exogenously created DNA into a host cell.

[0029] The term "amino acid" refers to naturally occurring and unnatural amino acids (also referred to herein as "non-naturally occurring amino acids"), e.g., amino acid analogues and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogues refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogues can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid. The terms "amino acid" and "amino acid residue" are used interchangeably throughout.

[0030] As used herein, the term “protein” refers to any organic compounds made of amino acids arranged in one or more linear chains (also referred as “polypeptide chains”) and folded into a globular form. It includes proteinaceous materials or fusion proteins. The amino acids in such polypeptide chain may be joined together by the peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The term “protein” further includes, without limitation, peptides, single chain polypeptide or any complex proteins consisting primarily of two or more chains of amino acids. It further includes, without limitation, glycoproteins or other known post-translational modifications. It further includes known natural or artificial chemical modifications of natural proteins, such as without limitation, glycoengineering, pegylation, hesylation, PASylation and the like, incorporation of non-natural amino acids, amino acid modification for chemical conjugation or other molecule, etc. . .

[0031] As used herein, the term “fusion protein” refers to a recombinant protein comprising at least one polypeptide chain which is obtained or obtainable by genetic fusion, for example by genetic fusion of at least two gene fragments encoding separate functional domains of distinct proteins. A protein fusion of the present disclosure thus includes at least one of SURF2 polypeptide, a fragment or variant thereof as described below, and at least one other moiety, the other moiety being a polypeptide other than a SURF2 polypeptide, fragment or a variant thereof as described below.

[0032] As used herein, the term "sequence identity" or "identity" refers to the number (%) of matches (identical amino acid residues) in positions from an alignment of two polypeptide or nucleic acid sequences. The sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al, 1997; Altschul et al., 2005). Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software available on internet web sites such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, % amino acid sequence identity values refers to values generated using the pair wise sequence alignment program EMBOSS Needle that creates an optimal global alignment of two sequences using the Needleman- Wunsch algorithm, wherein all search parameters are set to default values, i.e. Scoring matrix = BLOSUM62, Gap open = 10, Gap extend = 0.5, End gap penalty = false, End gap open = 10 and End gap extend = 0.5.

[0033] By "substituted" or "modified" the present invention includes those amino acids that have been altered or modified from naturally occurring amino acids.

[0034] The term "conservative substitution" as used herein denotes the replacement of an amino acid residue by another, without altering the overall conformation and function of the peptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, shape, hydrophobic, aromatic, and the like).

[0035] Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (methionine, leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). As used herein the term “inverted terminal repeat (ITR)” refers to a nucleotide sequence located at the 5 ’-end (5 ’ITR) and a nucleotide sequence located at the 3 ’-end (3 ’ITR) of a virus, that contain palindromic sequences and that can fold over to form T-shaped hairpin structures that function as primers during initiation of DNA replication. They are also needed for viral genome integration into the host genome; for the rescue from the host genome; and for the encapsidation of viral nucleic acid into mature virions. The ITRs are required in cis for the vector genome replication and its packaging into the viral particles.

[0036] AAV ITRs for use in the viral vector of the disclosure may have a wild-type nucleotide sequence or may be altered by the insertion, deletion or substitution. The serotype of the inverted terminal repeats (ITRs) of the AAV may be selected from any known human or nonhuman AAV serotype. In specific embodiments, the nucleic acid construct or viral expression vector may be carried out by using ITRs of any AAV serotype, including AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV serotype or engineered AAV now known or later discovered.

[0037] The term AAV vector particle encompasses any recombinant AAV vector particle or mutant AAV vector particle, genetically engineered. A recombinant AAV particle may be prepared by encapsidating the nucleic acid construct or viral expression vector including ITR(s) derived from a particular AAV serotype on a viral particle formed by natural or mutant Cap proteins corresponding to an AAV of the same or different serotype.

[0038] The construction of recombinant AAV viral particles is generally known in the art and has been described for instance in US 5,173,414 and US5, 139,941; WO 92 / 01070, WO 93 / 03769, Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol, and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793- 801.

[0039] Proteins of the viral capsid of an adeno-associated virus include the capsid proteins VP1, VP2, and VP3. Differences among the capsid protein sequences of the various AAV serotypes result in the use of different cell surface receptors for cell entry. In combination with alternative intracellular processing pathways, this gives rise to distinct tissue tropisms for each AAV serotype.

[0040] Several techniques have been developed to modify and improve the structural and functional properties of naturally occurring AAV viral particles (Bunning H et al. J Gene Med, 2008; 10: 717-733; Paulk et al. Mol then 2018; 26(l):289-303; Wang L et al. Mol Then 2015; 23(12): 1877-87; Vercauteren et al. Mol Then 2016; 24(6): 1042-1049; Zinn E et al., Cell Rep. 2015; 12(6): 1056-68). As used herein, the term “5S RNPs” or 5S ribonucleoproteins” refers to the association of 5S ribosomal RNA (rRNA) with the ribosomal proteins RPL5 and RPL11.

[0041] SURF2 inhibitor

[0042] Free-5 S RP particles are key in the response to nucleolar stress, a cellular mechanism that is instrumental to cancer treatment by chemotherapeutic drugs. Thus, promoting the extra-ribosomal activity of free 5S RNPs may improve the p53 -dependent anticancer effects of therapeutic agents such as chemotherapy used in most poor-prognosis cancers.

[0043] The inventors in the present application showed that inhibition of SURF2 induces increase of p53 and p21 in a free-5S RNP dependent manner, with or without inducing nucleolar stress. Moreover, in response to nucleolar stress induced by chemotherapeutic agent, inhibition of SURF2 induces a greater accumulation of p53 and p21 and a stronger arrest in G1 cell cycle with associated cell death compared to control cell.

[0044] The present disclosure relates to SURF2 inhibitor, in particular for use in treatment of a cancer in a subject in need thereof.

[0045] The gene Surfeit 2 (SURF2) (Gene ID: 6835, updated on November 6, 2023), also known as SURF -2 encodes SURF-2 protein (UniProtKB: Q15527, updated on November 08, 2023). In particular, the gene encodes two human SURF2 protein isoforms, isoform 1 (accession number: NP_059973.4, updated on December 24, 2022) and isoform 2 (accession number: NP_001265857.1, updated on December 25, 2022). Preferably, Human SURF2 protein comprises, or consists of, the amino acid sequence of SEQ ID NO: 1.

[0046] By “SURF2 inhibitor” is meant any agent able to decrease specifically SURF2 expression and / or biological activity, in particular that results in an increase of nucleolar stress response, more specifically results in a decrease of the binding of SURF2 protein to free-5S RNPs, cause an increased ratio of free 5S RNP particles associated with MDM2 and p53, an increased activation of p53 by free 5S particles, in particular an increased level of p53, p21 and / or MDM2, preferably during nucleolar stress response (e.g. in cells treated with chemotherapeutic agent such as Actinomycin D), a stronger arrest in G1 cell cycle, preferably during nucleolar stress response (e.g. in cells treated with chemotherapeutic agent such as Actinomycin D), a decrease of cell proliferation and / or an increase of cell apoptosis, preferably during nucleolar stress response (e.g. in cells treated with chemotherapeutic agent such as Actinomycin D).

[0047] The SURF2 inhibitor according to the present disclosure having such properties can be screened using for examples the following assays.

[0048] The decrease of the binding of SURF2 protein to free-5S RNP can be determined by enriching 5S RNP partners by immunoprecipitation with for example anti-RPE5 antibody or anti-flag (or other markers such as HA, fluorescent marker) antibody in cells expressing RPL5-flag (or other markers such as HA, fluorescent marker) transgene in presence of said SURF2 inhibitor as described in example 1.1 of the present application with the corresponding material and methods 2.2 and 2.11 and comparing the quantity of SURF2 protein with a negative control obtained in same experimental condition without said inhibitor or with a compound known to have no effect. It may be considered that a compound decreases the binding of SURF2 protein to free-5S RNP when quantity of SURF 2 protein co-immunoprecipitated with 5S RNP particles is lower than the negative control (e.g. at least 1.2, 1.3, 1.4, 1.5, preferably 1.6 or even lower fold than negative control).

[0049] The increased ratio of free 5S RNP particles associated with MDM2 and / or p53 can be determined by enriching 5S RNP partners by immunoprecipitation with for example anti-RPL5 antibody or anti -flag (or other markers such as HA, fluorescent marker) antibody in cells expressing RPL5-flag (or other markers such as HA, fluorescent marker) transgene as described in example 1.5 of the present application with the corresponding material and methods 2.4 and 2.12 in presence of SURF2 inhibitor and comparing the quantity of MDM2 and / or p53 with a negative control obtained in same experimental condition without said inhibitor or with a compound known to have no effect. It may be considered that a compound increases ratio of free 5S RNP particle associated with MDM2 and / or p53 when quantity of p53 and / or MDM2 co-immunoprecipitated with 5S RNP particles is higher than the negative control (e.g. at least 1.2, 1.3, 1.4, 1.5, preferably 1.6 or even higher fold than negative control).

[0050] The increase of the activation of p53 by free 5S RNP in a cell can be defined as an increase of p53 that is reduced in presence of MDM2C305Fmutant, that impedes free5S p53 pathway, in the cell. Such activation of p53, will be determined for example, by assessing the level of p53, MDM2 and / or p21 for example by Western Blot in a cell treated with said inhibitor, as exemplified in examples 1.4 of the present disclosure with the corresponding material and method 2.4 and comparing the level with a negative control obtained in same experimental condition without said inhibitor or with a compound known to have no effect and in cell expressing MDM2C305Fmutant. It may be considered that a compound increases p53 pathway activation when level of p53, MDM2 and / or p21 is higher than the negative control (e.g. at least 1.2, 1.3, 1.4, 1.5, preferably 1.6 fold higher than negative control). In a preferred embodiment the increase of p53 pathway is determined during nucleolar stress response, for example in cells treated with chemotherapeutic agent such as Actinomycin D.

[0051] The arrest in G1 cell cycle can be determined by any well-known method in the art, in particular by staining the cell DNA with a fluorescent dye in presence of SURF2 inhibitor and measuring, for example by flow cytometry, the fluorescent intensity allowing differentiation of cells in G / Gl, S phase and G2 / M as exemplified in example 1.6 of the present application with corresponding material and method 2.5 and 2.7 and comparing the percentage of cells in G1 phase with a negative control obtained in same experimental condition without said inhibitor or with a compound known to have no effect. It may be considered that a compound triggers the arrest of cell cycle in G1 phase when the percentage of cells in G1 phase is higher than the negative control (e.g., at least 1.2, preferably 1.3, 1.4, preferably 1.5-fold higher than negative control). In a preferred embodiment the percentage of cells in G1 phase is determined during nucleolar stress response, for example in cells treated with chemotherapeutic agent such as Actinomycin D.

[0052] The decrease of cell proliferation can be determined for example by incubated the cells treated with a SURF2 inhibitor with a viability dye such as crystal violet that binds to DNA of viable cells in presence of a SURF2 inhibitor and by measuring the optical density at 595 nm as exemplified in Example 1.6 with the corresponding material and methods 2.5 or 2.7. In a preferred embodiment the decrease of cell proliferation is determined during nucleolar stress response, for example in cells treated with chemotherapeutic agent such as Actinomycin D.

[0053] SURF2 inhibitor can also be identified by measuring the decrease of SURF2 expression, in particular by measuring the expression level of SURF2 in a cell treated with said SURF2 inhibitor. The SURF2 activity is decreased in cells when the expression level of SURF2 is at least 1.5 -fold lower, or 2, 3, 4, 5- fold lower than in non-treated cells.

[0054] The expression level of SURF2 mRNA may be determined by any suitable methods known by skilled persons. For example, the nucleic acid contained in the sample is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and / or amplification (e.g., RT-PCR). The expression level of SURF2 protein may also be determined by any suitable methods known by skilled persons. The quantity of the protein may be measured, for example, by semi -quantitative Western blots, enzyme -labelled and mediated immunoassays, such as ELISAs, biotin / avidin type assays, radioimmunoassay, immunoelectrophoresis, mass spectrometry or immunoprecipitation or by protein or antibody arrays.

[0055] The SURF2 expression and / or biological activity can be decreased by agents including, but are not limited to, chemicals, compounds known to modify gene expression, modified or unmodified polynucleotides (including oligonucleotides), polypeptides, peptides, small molecules and interfering nucleic acid molecule.

[0056] SURF2 interfering nucleic acid

[0057] In a particular embodiment, said SURF2 inhibitor may be an interfering nucleic acid which specifically decreases SURF2 expression. As used herein, the term "iRNA", "RNAi", “interfering nucleic acid” or "interfering RNA" means any nucleic acid, preferably RNA which is capable of down-regulating the expression of the targeted protein. Nucleic acid molecule interference designates a phenomenon by which dsRNA specifically suppresses expression of a target gene at post-transcriptional level. In normal conditions, RNA interference is initiated by double -stranded RNA molecules (dsRNA) of several thousands of base pair length. In vivo, dsRNA introduced into a cell is cleaved into a mixture of short dsRNA molecules called siRNA. The enzyme that catalyzes the cleavage, Dicer, is an endo-RNase that contains RNase III domains (Bernstein, Caudy et al. 2001 Nature. 2001 Jan 18;409(6818):363-6). In mammalian cells, the siRNAs produced by Dicer are 21-23 bp in length, with a 19 or 20 nucleotides duplex sequence, two-nucleotide 3' overhangs and 5 '-triphosphate extremities (Zamore, Tuschl et al. Cell. 2000 Mar31;101(l):25-33; Elbashir, Lendeckel et al. Genes Dev. 2001 Jan 15; 15(2): 188-200; Elbashir, Martinez et al. EMBO J. 2001 Dec 3;20(23):6877-88).

[0058] Said interfering nucleic acid can be as non-limiting examples anti-sense oligonucleotide constructs, small inhibitory RNAs (siRNAs) or short hairpin RNA.

[0059] Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of SURF2 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of SURF2, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

[0060] In another embodiment, small inhibitory RNAs (siRNAs) can also be used to decrease the SURF2 expression level in the present disclosure. SURF2 gene expression can be reduced by administrating into a subject a small double stranded RNA (dsRNA) also named duplex siRNA, or a vector or construct causing the production of a small double stranded RNA, such that SURF2 expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA- encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01 / 36646, WO 99 / 32619, and WO 01 / 68836).

[0061] Most commonly, synthetic siRNA duplexes are designed such as a stretch of 19 contiguous ribonucleotide base-pairs is flanked with 2-3 unpaired nucleotides at the 3'-end of each strand ("overhangs"). This 21 -nt siRNA species has been found to be generated during DICER-mediated cleavage of long ds-RNA in mammalian and non-mammalian systems.

[0062] In a preferred embodiment, short hairpin RNA (shRNA) can also be used to decrease the SURF2 expression level in the present disclosure. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. The promoter choice is essential to achieve robust shRNA expression. At first, polymerase III promoters such as U6 and HI were used; however, these promoters lack spatial and temporal control. As such, there has been a shift to using polymerase II promoters to regulate expression of shRNA.

[0063] Interfering nucleic acid are usually designed against a region 19-50 nucleotides downstream the translation initiator codon, whereas 5'UTR (untranslated region) and 3'UTR are usually avoided. The chosen interfering nucleic acid target sequence should be subjected to a BLAST search against EST database to ensure that the only desired gene is targeted. Various products are commercially available to aid in the preparation and use of interfering nucleic acid.

[0064] In a particular embodiment, the interfering nucleic acid is a siRNA of at least about 10-40 nucleotides in length, preferably about 15-30 base nucleotides. In particular, interfering nucleic acid according to the disclosure comprises at least one sequence selected from the group consisting of:

[0065] - 5 ’ -ATGC TGT CAT CAC TTG CAG- 3 ’ (SEQ ID NO: 2)

[0066] - 5 -TCC ATC TGG TCC TCC CTC C- 3’ (SEQ ID NO: 3)

[0067] - 5’-AAC TTC ACG GCA TGT GCT T- 3’ (SEQ ID NO: 4)

[0068] - 5’-TTT ACA CAG AGC TCG CTG G- 3’ (SEQ ID NO: 5)

[0069] In a more preferred embodiment, up to four interfering nucleic acids comprising each a sequence SEQ ID NO: 2 to 5 are used concomitantly.

[0070] In a particular embodiment, said interfering nucleic acid is at least one duplex siRNA selected from the group consisting of: SEQ ID NO: 7 to 10. In a more preferred embodiment, up to four duplex siRNA comprising each a sequence SEQ ID NO: 7 to 10 are used concomitantly.

[0071] In a preferred embodiment, said interfering nucleic acid is a shRNA comprising at least one sequence selected from the group consisting of SEQ ID NO: 2 to 5, preferably comprising all the sequences SEQ ID NO: 2 to 5.

[0072] An interfering nucleic acid for use in the disclosure can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. Particularly, interfering RNA can be chemically synthesized, produced by in vitro transcription from linear (e.g. PCR products) or circular templates (e.g., viral or non- viral vectors), or produced by in vivo transcription from viral or non- viral vectors.

[0073] Depending on whether the interfering nucleic acid is chemically synthesized, produced in vitro or by in vivo transcription from viral or non-viral vectors, the sequence is adapted to the application. In RNA molecules, thymine is replaced by uracil (U), which, similar to thymine (T), forms a complementary base pair with adenine (A).

[0074] Interfering nucleic acid may be modified to have enhanced stability, nuclease resistance, target specificity and improved pharmacological properties. For example, antisense nucleic acid may include modified nucleotides or / and backbone designed to increase the physical stability of the duplex formed between the antisense and sense nucleic acids.

[0075] SURF2 Peptide inhibitor

[0076] The inventors have characterized the structure of SURF2 protein that comprises a structural domain (SD) in N-terminal and a disordered domain in C-terminal. The structural domain comprises the amino acid sequence between 1 to 136 of SEQ ID NO: 1 (SEQ ID NO: 6). A SURF2 peptide fragment consisting of an amino acid sequence of SEQ ID NO: 6, preferably comprising the RPL5-binding domain acts as a dominant negative peptide.

[0077] Therefore, according to the present disclosure, said SURF2 inhibitor can be a SURF2 peptide fragment comprising no more than the amino acid sequence between 1 to 136 of SEQ ID NO: 1 or consisting of the amino acid sequence between 1 to 136 of SEQ ID NO: 1 (SEQ ID NO: 6), a functional fragment thereof or a functional variant thereof.

[0078] It is intended that said SURF2 peptide inhibitor comprises no more than the amino acid sequence between 1 to 136 of SEQ ID NO: 1 without the C-terminal part of SURF2 protein from amino acid 137 to the end of SEQ ID NO: 1.

[0079] However, said SURF2 peptide inhibitor fragment may be comprised in a fusion protein with one other moiety polypeptide other than SURF2 protein, functional fragment or variant thereof as described above. In certain embodiments, the other moiety may also be a non-protein moiety, such as, for example, a polyethyleneglycol (PEG) moiety or other chemical moiety or conjugates. In preferred embodiments, the second moiety can be a Fc region of an antibody, a reporter, a nuclear localization signal or a tag protein.

[0080] In a preferred embodiment, said SURF2 inhibitor comprises the amino acid sequence between 1 to 136 of SEQ ID NO: 1 and a nuclear localization signal (NLS) sequence. A NLS is an amino acid sequence that address a protein into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. In a preferred embodiment, the NLS sequence is KKKRK (SEQ ID NO: 22). Of course, it is understood that, in certain embodiments, the polypeptide cargo may comprise its natural NLS. In a particular embodiment, that said SURF2 peptide inhibitor comprises or consists of the amino sequence of SEQ ID NO: 21, or a functional variant thereof.

[0081] The SURF2 peptide inhibitor acts as a dominant negative peptide by binding to free-5S RNPs, and inhibiting native SURF2 protein biological activity. In particular, SURF2 peptide inhibitor causes a decrease of the binding of wild-type SURF2 protein to free-5S RNPs, an increased ratio of free 5S RNP particles associated with MDM2 and / or p53, an increased activation of p53 pathway, in particular an increased level of p53, p21 or MDM2, preferably during nucleolar stress response (e.g. in cells treated with chemotherapeutic agent such as Actinomycin D), a stronger arrest in G1 cell cycle, preferably during nucleolar stress response (e.g. in cells treated with chemotherapeutic agent such as Actinomycin D), a decrease of cell proliferation and / or an increase of cell apoptosis, preferably during nucleolar stress response (e.g. in cells treated with chemotherapeutic agent such as Actinomycin D). The SURF2 peptide inhibitor according to the present disclosure having such properties can be screened using the assays as in the previous section.

[0082] As used herein, the term “SURF2 peptide inhibitor functional variant” refers to a polypeptide sequence that is derived from SURF2 peptide inhibitor comprising or consisting of SEQ ID NO: 6 or 21 as described above and comprises an alteration, i.e., a substitution, insertion, and / or deletion, at one or more (e.g., several) positions, but retains the capacity to inhibit SURF2 protein biological activity. The variant may be obtained by various techniques well known in the art. Examples of techniques for altering the DNA sequence encoding the native protein, include, but are not limited to, site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction.

[0083] Preferably, as used herein, the term “variant” or “functional variant” refers to a polypeptide having an amino acid sequence having at least 70, 75, 80, 85, 90, 95 or 99% sequence identity to the native amino acid sequence (e.g., SEQ ID NO: 6 or 21).

[0084] Preferably, the term “variant” or “functional variant” refers to a polypeptide having an amino acid sequence that differs from a sequence of SEQ ID NO: 6 or 21 by less than 10, 9, 8, 7, 6, 5, 4, 3 or 2 substitutions, insertions and / or deletions.

[0085] In particular, the functional variant is substantially homologous to amino acid sequence SEQ ID NO: 6 or 21. Two amino acid sequences are “homologous”, “substantially homologous” or “substantially similar” when one or more amino acid residues are replaced by a biologically similar residue, i.e. conservative substitution.

[0086] In a preferred embodiment, the functional variant differs from the amino acid sequence of SEQ ID NO: 6 or 21 by one or more conservative substitutions, preferably by less than 10, 9, 8, 7, 6, 5, 4, 3 or 2 conservative substitutions.

[0087] As used herein, the term " SURF2 peptide inhibitor functional fragment" refers to a polypeptide sequence that is a fragment of SURF2 peptide inhibitor comprising or consisting of SEQ ID NO: 6 or 21 as described above, but retains the capacity to inhibit SURF2 protein biological activity.

[0088] Preferably, the SURF2 peptide inhibitor as described above is a functional fragment of 8 to 200 amino acids, 8 to 180 amino acids, 8 to 150 amino acids, 8 to 120 amino acids residues, preferably 8 to 100 amino acids residues, more preferably 8 to 80 amino acids residues, again more preferably 8 to 50 amino acids residues within an amino acid sequence of SEQ ID NO: 6 or 21 or an amino acid sequence having at least 70, 75, 80, 85, 90, 95 or 99% sequence identity to amino acid sequence of SEQ ID NO: 6 or 21 or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 6 or 21 by one or more conservative substitutions, preferably by less than 6, 5, 4, 3, or 2 conservative substitutions.

[0089] Peptides described herein can be synthesized using standard synthetic methods known to those skilled in the art, for example chemical synthesis or genetic recombination.

[0090] The N- and C-termini of the peptides described herein may be optionally protected against proteolysis. For instance, the N-terminus may be in the form of an acetyl group, and / or the C-terminus may be in the form of an amide group. Internal modifications of the peptides to be resistant to proteolysis are also envisioned, e.g. wherein at least a -CONH- peptide bond is modified and replaced by a (CH2NH) reduced bond, a (NHCO) retro- inverse bond, a (CH2-0) methylene-oxy bond, a (CH2-S) thiomethylene bond, a (CH2CH2) carba bond, a (CO-CH2) cetomethylene bond, a (CHOH-CH2) hydroxyethylene bond), a (N-N) bound, a E-alcene bond or also a -CH=CH-bond.

[0091] For instance, the peptide may be modified by acetylation, acylation, amidation, cross- linking, cyclization, disulfide bond formation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, phosphorylation, and the like.

[0092] The peptides of the disclosure may be composed of amino acid(s) in D configuration, which render the peptides resistant to proteolysis. They may also be stabilized by intramolecular crosslinking, e.g. by modifying at least two amino acid residues with olefinic side chains, preferably C3-C8 alkenyl chains, preferably penten-2-yl chains) followed by chemical crosslinking of the chains, according to the so- called "staple" technology described in Walensky et al, 2004. For instance, amino acids at position i and i+4 to i+7 can be substituted by non-natural amino acids that show reactive olefinic residues. All these proteolysis-resistant chemically modified peptides are encompassed in the present disclosure.

[0093] In another aspect of the disclosure, peptides are covalently bound to a polyethylene glycol (PEG) molecule by their C-terminal terminus or a lysine residue, notably a PEG of 1500 or 4000 MW, for a decrease in urinary clearance and in therapeutic doses used and for an increase of the half-life in blood plasma. In yet another embodiment, peptide half- life is increased by including the peptide in a biodegradable and biocompatible polymer material for drug delivery system forming microspheres. Polymers and copolymers are, for instance, poly(D,L-lactide-co-glycolide) (PLGA) (as illustrated in US2007 / 0184015, SoonKap Hahn et al).

[0094] Other SURF2 inhibitors

[0095] In another particular embodiment, said SURF2 inhibitor according to the present disclosure can be a PROteolysis Targeting Chimera (PROTAC) that binds SURF2 protein at one end while binding an E3 ligase at the other end, and forms a ternary complex to hijack the cellular ubiquitin -proteasome system for proteasomal degradation of SURF2 protein. According to the present disclosure, SURF2 PROTAC may have three components - an E3 ubiquitin ligase binding group (E3LB), a linker, and a protein binding domain that binds to SURF2 protein. PROTACs and PROTAC binding domains are known to the skilled person (see e.g., An et al, EBioMedicine. 2018 Oct; 36: 553-562). The SURF2 PROTAC inhibitor according to the present disclosure able to inhibit the SURF2 expression or biological activity can be screened using the assays as described in the previous section.

[0096] In some embodiments, SURF2 inhibitor is an anti-SURF2 antibody or antigen binding region thereof that specifically bind to SURF2 protein, preferably to free 5S RNP binding domain of SURF2 protein and antagonizes the SURF2 biological activity as described above, for example by preventing the interaction between SURF2 and free 5S RNP.

[0097] An antibody or antigen-binding fragment thereof of the present antagonizing SURF2 biological activity can be screened using the assays as described above.

[0098] The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses whole antibody molecules such as four-chain antibodies comprising 2 heavy chains and 2 light chains, such as polyclonal antibodies, monoclonal antibodies or recombinant antibodies.

[0099] The term "antigen-binding fragment" of an antibody (or simply "antibody fragment"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., SURF2). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341:544- 546), which consists of a VH domain, or any fusion proteins comprising such antigen-binding fragments. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single chain protein in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0100] Typically, binding affinity may be measured by methods known in the art like but not limited to Biacore analysis, Blitz analysis, ELISA assay or Scatchard plot.

[0101] In another particular embodiment, SURF2 inhibitor can be a small molecule inhibiting the SURF2 expression, activity or function.

[0102] As used herein, the term "small molecule inhibiting SURF2 activity, expression or function" refers to small molecule that can be an organic or inorganic compound, usually less than 1000 daltons, with the ability to inhibit or reduce the activity, expression or function of SURF2 protein. This small molecule can be derived from any known organism (including, but not limited to, animals, plants, bacteria, fungi and viruses) or from a library of synthetic molecules.

[0103] According to the present disclosure, small molecules able to inhibit the SURF2 expression or biological activity can be screened using the assays as described in the previous section.

[0104] Nucleic acid construct, expression vector and viral particle

[0105] In a preferred embodiment, transgene encoded SURF2 inhibitor such as peptide, PROTAC, anti-SURF2 antibody or antigen-binding fragment thereof, anti-sense oligonucleotide constructs, small inhibitory RNAs (siRNAs) or short hairpin RNA is included in a nucleic acid construct coding for them.

[0106] Preferably, the transgene encoding peptide, PROTAC, anti-SURF2 antibody or antigen-binding fragment thereof, anti-sense oligonucleotide constructs, small inhibitory RNAs (siRNAs) or short hairpin RNA as described above is included in a nucleic acid construct coding for them operably linked to one or more control sequences that direct the expression in host cells, preferably tumoral cells. In a preferred embodiment, said nucleic acid construct comprises an interfering nucleic acid able to repress SURF2 gene expression comprising at least one sequence selected from sequences SEQ ID NO: 2 to 5. More preferably, said nucleic acid construct comprises four interfering nucleic acid of sequences SEQ ID NO: 2 to 5.

[0107] In another preferred embodiment, said nucleic acid construct comprises a transgene encoding a SURF2 peptide inhibitor as described above, preferably comprising or consisting of SEQ ID NO:6 or 21, a functional fragment or variant thereof as described above.

[0108] The nucleic acid construct as described above may be contained in an expression vector. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.

[0109] Examples of appropriate vectors include, but are not limited to, recombinant integrating or nonintegrating viral vectors and vectors derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. Preferably, the vector is a recombinant integrating or non -integrating viral vector. Examples of recombinant viral vectors include, but not limited to, vectors derived from herpes virus, retroviruses, lentivirus, vaccinia viruses, adenoviruses, adeno-associated viruses or bovine papilloma virus.

[0110] In one embodiment, the nucleic acid construct or expression vector comprising transgene as described above further comprises a 5’ITR and a 3 TR sequences, preferably a 5’ITR and a 3’ ITR sequences of an adeno-associated virus. In one embodiment, the nucleic acid construct further comprises a 5’ITR and a 3 TR of the corresponding capsid, or preferably 5’ITR and a 3 ’ITR of a serotype AAV-2.

[0111] In one embodiment, the nucleic acid construct or viral vector of the disclosure comprises a 5’ITR, a y packaging signal, and a 3 ’ITR of a virus. “y packaging signal” is a c / .s-acting nucleotide sequence of the virus genome, which in some viruses (e.g. adenoviruses, lentiviruses ...) is essential for the process of packaging the virus genome into the viral capsid during replication.

[0112] In a preferred embodiment, the present disclosure relates to viral particles including a nucleic acid construct or expression vector as described above.

[0113] The nucleic acid construct or the expression vector of the disclosure may be packaged into a virus capsid to generate a "viral particle", also named “viral vector particle”. In a particular embodiment, the nucleic acid construct or the expression vector as described above is packaged into an AAV-derived capsid to generate an "adeno-associated viral particle" or "AAV particle". The present disclosure relates to a viral particle comprising a nucleic acid construct or an expression vector of the disclosure and preferably comprising capsid proteins of adeno-associated virus.

[0114] Thus, in AAV viral particle according to the present disclosure, the nucleic acid construct or viral expression vector including ITR(s) of a given AAV serotype can be packaged, for example, into: a) a viral particle constituted of capsid proteins derived from the same or different AAV serotype ; b) a mosaic viral particle constituted of a mixture of capsid proteins from different AAV serotypes or mutants; c) a chimeric viral particle constituted of capsid proteins that have been truncated by domain swapping between different AAV serotypes or variants.

[0115] The skilled person will appreciate that the AAV viral particle for use according to the present disclosure may comprise capsid proteins from any AAV serotype including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2i8, AAVrhlO, AAVrh39, AAVrh43, AAVrh74, AAV-LK03, AAV2G9, AAV.PHP, AAV-Anc80, AAV3B and AAV9.rh74.

[0116] Pharmaceutical composition

[0117] The SURF2 inhibitor, nucleic acid construct, expression vector or viral particle according to the present disclosure is preferably used in the form of a pharmaceutical composition comprising a therapeutically effective amount of the product according to the present disclosure.

[0118] In the context of the disclosure, a therapeutically effective amount refers to a dose sufficient for reversing, alleviating or inhibiting the progress of the disorder or condition to which such term applies, or reversing, alleviating or inhibiting the progress of one or more symptoms of the disorder or condition to which such term applies.

[0119] The effective dose is determined and adjusted depending on factors such as the composition used, the route of administration, the physical characteristics of the individual under consideration such as sex, age and weight, concurrent medication, and other factors, that those skilled in the medical arts will recognize.

[0120] In the various embodiments of the present disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or vehicle.

[0121] A "pharmaceutically acceptable carrier” refers to a vehicle that does not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid fdler, diluent, encapsulating material or formulation auxiliary of any type. Preferably, the pharmaceutical composition contains vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

[0122] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or suspensions. The solution or suspension may comprise additives which are compatible with viral vectors and do not prevent viral vector particle entry into target cells. In all cases, the form must be sterile and must be fluid to the extent that easy syringe ability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. An example of an appropriate solution is a buffer, such as phosphate buffered saline (PBS) or Ringer lactate.

[0123] Therapeutic use

[0124] The SURF2 inhibitor, nucleic acid construct, expression vector, viral particle or pharmaceutical composition according to the present disclosure may be used as a medicament, in particular for use in the treatment of cancer in a subject in need thereof.

[0125] As used herein, the term “subject”, “patient” or “individual” denotes a mammal. Preferably, a patient, a subject or individual according to the disclosure is a human, in particular a human subject or patient, more preferably suffering from a cancer.

[0126] The terms “cancer”, “tumor”, are used interchangeably herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation.

[0127] As used herein, the term “treatment”, “treat” or “treating” refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease. In certain embodiments, such term refers to the amelioration or eradication of a disease or symptoms associated with a disease. In other embodiments, this term refers to minimizing the spread or worsening of the disease resulting from the administration of one or more therapeutic agents to a subject with such a disease.

[0128] "Treating cancer" includes, without limitation, reducing the number of cancer cells or the size of a tumor in the patient, reducing progression of a cancer to a more aggressive form (i.e. maintaining the cancer in a form that is susceptible to a therapeutic agent), reducing proliferation of cancer cells or reducing the speed of tumor growth, killing of cancer cells, reducing metastasis of cancer cells or reducing the likelihood of recurrence of a cancer in a subject. Treating a subject as used herein refers to any type of treatment that imparts a benefit to a subject afflicted with cancer or at risk of developing cancer or facing a cancer recurrence. Treatment includes improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disease, delay in the onset of symptoms or slowing the progression of symptoms, etc.

[0129] Since, SURF2 inhibitor acts on tumor cell growth by enhancing the activation of p53 pathway, in a preferred embodiment, the patient suffering from a cancer is a patient having a non-mutated p53, also referred herein as a wild-type p53 cancer. Therefore, according to the present disclosure, said cancer is preferably a wild-type p53 cancer.

[0130] According to the present disclosure, the cancer is any cancer type as disclosed above. In some embodiments said cancer is selected from the group consisting of both adult and paediatric cancers with both haematological and solid tumours such as: haematologic cancer, in particular acute myelogenous leukaemia (AML), chronic lymphocytic leukaemia (CLL), multiple myeloma, Hodgkin's disease, nonHodgkin's lymphoma, B cell, cutaneous T cell lymphoma, or a non-haematologic cancer, for instance melanoma, sarcoma, brain, epidermoid (in particular lung, breast, ovarian), head and neck (squamous cell), adrenocortical carcinoma, bladder, gastric, pancreatic, head, neck, renal, colon, prostate, cervical, testicular, liver, colorectal, oesophageal or thyroid cancer. In a preferred embodiment, said cancer is an endocrine cancer including as non limiting examples thyroid cancer, adrenal gland cancer such as adrenocortical carcinoma, parathyroid gland cancer, pituitary gland cancer, hypothalamus, and pancreas cancer. In a preferred embodiment, the cancer is an adrenocortical carcinoma, head or neck cancer, hepatocarcinomas and osteosarcomas. In a preferred embodiment, said cancer is an hormonal cancer, such as adrenocortical carcinoma and prostate cancer, but also hormonal independent cancers such as head and neck cancer or liver cancers.

[0131] The disclosure also provides a method for treating a cancer in a subject in need thereof comprising administering to said subject a therapeutically effective amount of the SURF2 inhibitor, nucleic acid construct or expression vector encoding said SURF2 inhibitor or pharmaceutical composition as described above.

[0132] By “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of the product of the disclosure or pharmaceutical composition that comprises it may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the product or pharmaceutical composition to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also typically one in which any toxic or detrimental effect of the product or pharmaceutical composition is outweighed by the therapeutically beneficial effects. In certain aspects, the disclosure provides to the use of a SURF2 inhibitor, nucleic acid construct, expression vector and / or a pharmaceutical composition according to any one of the preceding embodiments for the manufacture of a medicament for treating a cancer in a subject in need thereof.

[0133] In a particular embodiment, SURF2 inhibitor is advantageously administered in combination with a chemotherapeutic agent that stimulates nucleolar stress response.

[0134] Chemotherapeutic agents include cytotoxic anti-neoplastic agents, such as alkylating agents, antimetabolites, anti-microtubule agents, Topoisomerase inhibitors, cytotoxic antibiotics and others. Examples of chemotherapeutic drugs that enhance nucleolar stress response include with no limitations: Actinomycin D, Doxurubicin, Cyclophospahime, Capecitabine, Mitomycin C, Iriotecan (Topotecan), Etoposide, 5-FU, Roscovitine, Flavopereirine (PB-100), Nanoparticles (Ti02 or gold), DNA aptamers Naphthalene diimides, CX-3543, CX-5461, Rapamycin, Everolimus, AKti-1 / 2, MK-2206, Nutilin- 1 / 2 / 3, RG7112, RG7388, PXN727, PXN822, MI-77301, MI-219, MK-8242, AMG 232, CGM097, DS3032b, JNJ-26854165, RITA, and p53-SLP (for review see Pietri Carotenuto et al. Cell. 2019; 8(9): 1090), preferably Actinomycine D, 5 Fluoro-Uracil (5-FU), doxorubicin (DRB), Etoposide (ETO), or cisplatin.

[0135] The administration of the chemotherapeutic agent can be simultaneous, prior to or after the administration of the SURF2 inhibitor. Depending on the nature of the chemotherapeutic agent, a co- administration can be prepared in the form of a combination drug (product), also known as a “combo”. A combo is a fixed-dose combination that includes two or more active pharmaceutical ingredients combined in a single dosage form, which is manufactured and distributed in fixed doses. But the dose regimen and / or the administration route can also differ. In particular, the present disclosure also relates to a pharmaceutical composition comprising a SURF2 inhibitor as described above, a chemotherapeutic agent and preferably a pharmaceutically acceptable carrier and / or vehicle.

[0136] In one embodiment the product of the disclosure is administered to the subject or patient by a parenteral route, in particularly by intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular route.

[0137] The amount of product of the disclosure that is administered to the subject or patient may vary depending on the particular circumstances of the individual subject or patient including, age, sex, and weight of the individual; the nature and stage of the disease, the aggressiveness of the disease; the route of administration; and / or concomitant medication that has been prescribed to the subject or patient. Dosage regimens may be adjusted to provide the optimum therapeutic response.

[0138] For any particular subject, specific dosage regimens may be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners. Method for prognostic of survival outcome of a patient suffering from a cancer

[0139] In the present application, the inventors showed that SURF2 expression in cancer is upregulated in cancers and negatively correlates with overall survival.

[0140] Therefore, the present disclosure also relates to the use of SURF2 mRNA and protein as a biomarker for diagnosing a patient suffering from a cancer or for the prognosis of survival outcome of a patient suffering from a cancer. According to the method of the present disclosure, the analysis of gene expression level SURF2 genes in a patient sample, preferably tumor patient sample can be used as a biomarker to diagnostic a cancer or prognostic the survival outcome of a patient suffering from a cancer.

[0141] The present disclosure relates to an in vitro or ex vivo method for the prognosis of survival outcome of a patient suffering from a cancer, comprising the steps of determining SURF2 gene expression level in a patient sample, preferably tumor patient sample, wherein a higher SURF2 gene expression level in a patient sample compared to a control value is indicative that said patient has a lower survival time and a lower SURF2 gene expression level in a patient sample compared to a control value is indicative that said patient has a higher survival time.

[0142] The gene expression level of said genes may be determined by any suitable methods known by the person skilled in the art in a patient sample.

[0143] Usually, these methods comprise measuring the quantity of mRNA or protein as described above in a patient sample. Methods for determining the quantity of mRNA are well known in the art. For example, the mRNA contained in the sample is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and / or amplification (e.g., RT-PCR) by using primer pairs and probes specific to said genes as described in the examples of the present disclosure. Quantitative or semi -quantitative RT-PCR is preferred. In another particular embodiment, the mRNA expression level is measured by RNA seq method. Methods for determining the quantity of proteins are well-known in the art. For example, the quantity of the protein may be measured, for example, by semi -quantitative Western blots, enzyme- labelled and mediated immunoassays, such as ELISAs, biotin / avidin type assays, radioimmunoassay, immunoelectrophoresis, flow cytometry, mass spectrometry or immunoprecipitation or by protein or antibody arrays.

[0144] In some embodiments, the expression level of SURF2 gene measured for example by quantitative RT- PCR is normalized by subtracting the expression levels of housekeeping genes determined in the same experiment and the gene expression level may correspond to the normalized gene expression level of one gene or to the sum of normalized gene expression level of the set of genes as described above. In another embodiments, determining the gene expression level comprises determining the expression level of said gene in a patient sample, in particular by RNA-seq or DNA microarray.

[0145] The terms "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman animals. As used herein, the term “patient” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a patient according to the invention is a human. According to the present method, said patient or subject is a cancer patient, i.e., a patient having a tumor as described in the previous section. In a preferred embodiment, said cancer is an endocrine cancer including as non limiting examples thyroid cancer, adrenal gland cancer such as adrenocortical carcinoma, parathyroid gland cancer, pituitary gland cancer, hypothalamus, and pancreas cancer. In a more preferred embodiment, said patient suffers from adrenocortical carcinoma, head or neck cancer, hepatocarcinomas and osteosarcomas. In a preferred embodiment, said cancer is an hormonal cancer, such as adrenocortical carcinoma and prostate cancer, but also hormonal independent cancers such as head and neck cancer or liver cancers.

[0146] The term “patient sample” means any biological sample derived from a patient. Examples of such samples include tissue sample, cell samples, organs, biopsies, preferably tumor sample.

[0147] The tumor “sample” used in the context of the present disclosure is typically obtained from a tumor biopsy. It can e.g. be a fresh or a preserved sample such as a frozen sample, or any tumor sample preserved by other means.

[0148] Said patient sample may also be any biological fluid such as blood sample comprising cell-free circulating tumor DNA (cfcDNA) or circulating tumor DNA (ctDNA). In some embodiments of the present disclosure, the DNA can be obtained from reverse transcription of an RNA sample.

[0149] As used herein, the term "control value " may refer to the gene as described above in biological sample obtained from a general population or from a selected population of subjects. For example, the general population may comprise apparently healthy subjects, such as individuals who have not previously had any sign or symptoms indicating the presence of cancer. The term "healthy subjects" as used herein refers to a population of subjects who do not suffer from any known condition, and in particular, who are not affected with any cancer. In another embodiment, the control value refers to the gene expression level of each gene in a biological sample obtained from cancer patients known to not have a high survival outcome.

[0150] In another particular embodiment, said control value may be a “threshold value” or “cut-off value” determined experimentally, empirically, or theoretically. The threshold value may be established based upon comparative measurements between patients having a tumor with high or low survival outcome. Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the gene expression level in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured values in samples to be tested, and thus obtain a classification standard having significance for sample classification. In a particular embodiment, Receiver operating characteristic (ROC) analysis was performed to calculate the gene expression level cut-off value of SURF2 gene using tumor samples high and low survival outcome. The gene expression level values offering the highest sensitivity and specificity were selected as cut-off points. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STATVIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

[0151] Typically, the gene expression level of SURF2 gene in a patient sample is deemed to be higher than the control value if the gene expression level as described above in said patient sample to that of said control value is higher than at least 0.1, preferably 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, more preferably 1, 2, 3, 4 again more preferably 5 and the gene expression level of SURF2 gene in a patient sample is deemed to be lower than the control value if the gene expression level as described above in said patient sample to that of said control value is lower than at least 0.1, preferably 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, more preferably 1, 2, 3, 4 again more preferably 5.

[0152] SURF2 inhibitor of the present disclosure can be administered to improve the survival outcome of the patient. Therefore, in a preferred embodiment, SURF2 inhibitor is administered, preferably in combination with a chemotherapeutic agent as described above in patient previously identified as having a lower survival time.

[0153] Therefore, the present disclosure relates to a SURF2 inhibitor as described above for use in the treatment of a cancer in a patient in need thereof wherein said SURF2 inhibitor is administered in a patient previously identified as having a low survival outcome with a method as previously described.

[0154] The present invention relates also to a method for treating a cancer in a patient in need thereof comprising: i) determining the gene expression level of SURF2 in a patient sample, wherein a higher gene expression level of the gene in comparison to a control value is indicative that the patient has a lower survival time, ii) administering a therapeutically effective amount of said SURF2 inhibitor, preferably in combination with a chemotherapeutic agent to said patient previously identified as having a lower survival time.

[0155] In a preferred embodiment, the administration of the chemotherapeutic agent can be simultaneous, prior to or after the administration of the SURF2 inhibitor. 1

[0156] SURF2 activator

[0157] In the present application, the inventors also showed that the activation of SURF2 can impede p53 activation following nucleolar stress by inactivating p53 pathway through blocking free 5S RNPs- MDM2 interactions and therefore alleviate symptoms for ribosomopathy patients.

[0158] Therefore, the present disclosure also relates to a SURF2 activator, in particular for use as a medicament, more particularly in the treatment of ribosomopathy in a subject in need thereof.

[0159] By “SURF2 activator” is meant any agent that increases SURF2 expression and / or biological activity, in particular that result in an increase of the binding of SURF2 protein to free-5S RNPs, cause a decreased ratio of free 5S RNP particles associated with MDM2 and p53, impedes the activation of p53 by free 5S RNP particles, in particular impedes the increase of p53, p21 and / or MDM2 level, impedes an arrest in G1 cell cycle, induces an increase of cell proliferation and / or a decrease of cell apoptosis, preferably in CD34+ cells of DBA patients or siRPS19 transfected CD34+ cells (Rio S et al. Blood 2019, 133(12): 1358-1370).

[0160] The SURF2 activator according to the present disclosure having such properties can be screened using for examples the following assays.

[0161] The increase of the binding of SURF2 protein to free-5S RNP can be determined by enriching 5S RNP partners by immunoprecipitation with for example anti-RPL5 antibody or anti -flag (or other markers such as HA, fluorescent marker) antibody in cells expressing RPL5-flag (or other markers such as HA, fluorescent marker) transgene as described in example 1.1 of the present application with the corresponding material and methods 2.2 and 2.11 in presence of SURF2 activator and comparing the quantity of SURF2 with a negative control obtained in same experimental condition without said activator or with a compound known to have no effect. It may be considered that a compound increases the binding of SURF2 protein to free-5S RNP when quantity of SURF 2 protein is higher than the negative control (e.g. at least 1.2, 1.3, 1.4, 1.5, preferably 1.6 or even higher fold than negative control).

[0162] The decreased ratio of free 5S RNP particles associated with MDM2 and / or p53 can be determined by enriching 5S RNP partners by immunoprecipitation with for example anti-RPL5 antibody or anti -flag (or other markers such as HA, fluorescent marker) antibody in cells expressing RPL5-flag (or other markers such as HA, fluorescent marker) transgene as described in example 1.5 of the present application with the corresponding material and methods 2.4 and 2.12 in presence of SURF2 activator and comparing the quantity of MDM2 and / or p53 with a negative control obtained in same experimental condition without said activator or with a compound known to have no effect. It may be considered that a compound decreases ratio of free 5S RNP particle associated with MDM2 and / or p53 when quantity of p53 and / or MDM2 co-immunoprecipitated with 5S RNP particles is lower than the negative control (e.g. at least 1.2, 1.3, 1.4, 1.5, 1.6 preferably 1.7 or even lower fold than negative control).

[0163] The activation of p53 by free 5S RNP in a cell can be defined as an increase of p53 that is reduced in presence of MDM2C305Fmutant, that impedes free5S p53 pathway, in the cell. The activation of p53 can be determined for example, by determining the level of p53, MDM2 and / or p21 in a cell treated with said activator as exemplified in examples 1.4 of the present disclosure with the corresponding material and method 2.4 and comparing the level with a negative control obtained in same experimental condition without said activator or with a compound known to have no effect and in cell expressing MDM2C305Fmutant. It may be considered that a compound impedes p53 pathway activation when level of p53, MDM2 and / or p21 is lower or similar than the negative control (e.g., less than 1.1 higher than the negative control or lower than the negative control). In a preferred embodiment the increase of p53 pathway is determined in CD34+ cells of DBA patients or siRPS19 CD34+ transfected cells (Rio S et al. Blood 2019, 133(12): 1358-1370).

[0164] The decrease of cell apoptosis can be measured by any well-known methods in the art, for example measured with for example Annexin V, preferably in combination with 7AAD as exemplified in examples 1.6 of the present disclosure with the corresponding material and method 2.6. In a preferred embodiment, cells are incubated with a SURF2 activator according to the present disclosure and stained with annexin-V, preferably in combination with 7AAD or Propidium Iodide (PI). The percentage of apoptotic Annexin-V+, preferably Annexin-V+7AAD- early apoptotic cells is determined for example by flow cytometry. The SURF2 activator according to the present disclosure induces a decrease of apoptosis in a cell when the percentage of apoptotic cells is decreased, preferably at least 1.5, 1.8, 2.0, 2.5 -fold in comparison to a control value such as not treated cells or cells treated with an agent known not to induce apoptosis. In a preferred embodiment the decrease of cell apoptosis is determined in CD34+ cells of DBA patients or siRPS19 transfected CD34+ cells (Rio S et al. Blood 2019, 133(12): 1358-1370).

[0165] The arrest in G1 cell cycle can be determined by any well-known method in the art, in particular by staining the DNA of a cell with a fluorescent dye and measuring, for example by flow cytometry the fluorescent intensity allowing differentiation of cells in G / Gl, S phase and G2 / M as exemplified in example 1.6 of the present application with corresponding material and method 2.5 or 2.7 in presence of SURF2 activator and comparing the percentage of cells in G1 phase with a negative control obtained in same experimental condition without said activator or with a compound known to have no effect. It may be considered that a compound does not trigger (i.e., impedes) the arrest of cell cycle in G1 phase when the percentage of cells in G1 phase is similar or lower than the negative control (e.g., less than 1. 1-fold higher than the negative control or lower than the negative control). In a preferred embodiment the arrest in G1 cell cycle is determined in CD34+ cells of DBA patients or siRPS19 transfected CD34+ cells (Rio S et al. Blood 2019, 133(12): 1358-1370).

[0166] The increase of cell proliferation can be determined for example by incubated the cells treated with a SURF2 activator with a viability dye such as crystal violet that binds to DNA of viable cells and by measuring the optical density at 595 nm as exemplified in Example 1.6 with the corresponding material and methods 2.7. In a preferred embodiment the cell proliferation is determined in CD34+ cells of DBA patients or siRPS19 transfected CD34+ cells (Rio S et al. Blood 2019, 133(12): 1358-1370)..

[0167] SURF2 activator can also be identified by measuring the increase of SURF2 expression level, in particular by measuring the expression level of SURF2 in a cell treated with said SURF2 activator. The SURF2 activity is increased in cells when the expression level of SURF2 is at least 1.5-fold higher, or 2, 3, 4, 5-fold higher than in non-treated cells.

[0168] The expression level of SURF2 mRNA may be determined by any suitable methods known by skilled persons. For example, the nucleic acid contained in the sample is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and / or amplification (e.g., RT-PCR). The expression level of SURF2 protein may also be determined by any suitable methods known by skilled persons. The quantity of the protein may be measured, for example, by semi -quantitative Western blots, enzyme -labelled and mediated immunoassays, such as ELISAs, biotin / avidin type assays, radioimmunoassay, immunoelectrophoresis, mass spectrometry or immunoprecipitation or by protein or antibody arrays.

[0169] The SURF2 expression and / or activity can be increased by agents including, but are not limited to, chemicals, compounds known to modify gene expression, modified or unmodified polynucleotides (including oligonucleotides), polypeptides, peptides, small RNA molecules, nucleic acid construct or viral vector. Such agents are well-known in the art.

[0170] SURF2 protein

[0171] In a particular embodiment, said SURF2 activators are human SURF2 protein, preferably comprising amino acid sequence SEQ ID NO: 1, functional fragment or functional variant thereof that maintain the biological activity of native SURF2 protein as described above.

[0172] Preferably, as used herein, the term "variant" or “functional variant” refers to a polypeptide having an amino acid sequence having at least 70, 75, 80, 85, 90, 95 or 99% sequence identity to the native amino acid sequence (e.g., SEQ ID NO: 1). Preferably, the term "variant" or “functional variant” refers to a polypeptide having an amino acid sequence that differs from a sequence of SEQ ID NO: 1 by less than 20, 15, 10, 8, 7, 6, 5, 4, 3 or 2 substitutions, insertions and / or deletions.

[0173] In particular, the functional variant is substantially homologous to amino acid sequence SEQ ID NO: 1. Two amino acid sequences are “homologous”, “substantially homologous” or “substantially similar” when one or more amino acid residues are replaced by a biologically similar residue, i.e. conservative substitution.

[0174] In a preferred embodiment, the functional variant differs from the amino acid sequence of SEQ ID NO: 1 by one or more conservative substitutions, preferably by less than 20, 15, 10, 8, 7, 6, 5, 4, 3 or 2 conservative substitutions.

[0175] As used herein, the term "SURF2 protein functional fragment" refers to a polypeptide sequence that is a fragment of SURF2 protein, preferably human SURF2 protein comprising or consisting of SEQ ID NO: 1 as described above, but retains the capacity to maintain SURF2 protein biological activity.

[0176] Preferably, the SURF2 protein functional fragment as described above is a functional fragment of 8 to 200 amino acids, 8 to 180 amino acids, 8 to 150 amino acids, 8 to 120 amino acids residues, preferably 8 to 100 amino acids residues within an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70, 75, 80, 85, 90, 95 or 99% sequence identity to amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 1 by one or more conservative substitutions, preferably by less than 20, 15, 10, 8, 7, 6, 5, 4, 3 or 2 conservative substitutions.

[0177] Peptides or proteins described herein can be synthesized using standard synthetic methods known to those skilled in the art, for example chemical synthesis or genetic recombination.

[0178] The N- and C-termini of the proteins described herein may be optionally protected against proteolysis. For instance, the N-terminus may be in the form of an acetyl group, and / or the C-terminus may be in the form of an amide group. Internal modifications of the peptides to be resistant to proteolysis are also envisioned, e.g. wherein at least a -CONH- peptide bond is modified and replaced by a (CH2NH) reduced bond, a (NHCO) retro- inverse bond, a (CH2-0) methylene-oxy bond, a (CH2-S) thiomethylene bond, a (CH2CH2) carba bond, a (CO-CH2) cetomethylene bond, a (CHOH-CH2) hydroxyethylene bond), a (N-N) bound, a E-alcene bond or also a -CH=CH-bond.

[0179] For instance, the protein may be modified by acetylation, acylation, amidation, cross- linking, cyclization, disulfide bond formation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, phosphorylation, and the like. The proteins of the disclosure may be composed of amino acid(s) in D configuration, which render the peptides resistant to proteolysis. They may also be stabilized by intramolecular crosslinking, e.g. by modifying at least two amino acid residues with olefinic side chains, preferably C3-C8 alkenyl chains, preferably penten-2-yl chains) followed by chemical crosslinking of the chains, according to the so- called "staple" technology described in Walensky et al, 2004. For instance, amino acids at position i and i+4 to i+7 can be substituted by non-natural amino acids that show reactive olefinic residues. All these proteolysis-resistant chemically modified peptides are encompassed in the present disclosure.

[0180] In another aspect of the disclosure, proteins are covalently bound to a polyethylene glycol (PEG) molecule by their C-terminal terminus or a lysine residue, notably a PEG of 1500 or 4000 MW, for a decrease in urinary clearance and in therapeutic doses used and for an increase of the half-life in blood plasma. In yet another embodiment, peptide half- life is increased by including the peptide in a biodegradable and biocompatible polymer material for drug delivery system forming microspheres. Polymers and copolymers are, for instance, poly(D,L-lactide-co-glycolide) (PLGA) (as illustrated in US2007 / 0184015, SoonKap Hahn et al).

[0181] Nucleic acid construct encoding SURF2 protein

[0182] In a preferred embodiment, the SURF2 activator is a nucleic acid construct comprising a transgene encoding SURF2 protein, functional fragment or a functional variant thereof as described above.

[0183] The transgene according to the disclosure may be any nucleic acid sequence encoding an SURF2 protein, in particular a native mammalian, preferably human SURF2 protein comprising or consisting of SEQ ID NO: 1, a functional fragment or a functional variant thereof.

[0184] The coding sequences of a number of different mammalian SURF2 proteins are known including, but being not limited to, human, pig, chimpanzee, dog, cow, mouse, rabbit or rat, and can be easily found in sequence databases. Alternatively, the coding sequence may be easily determined by the skilled person based on the polypeptide sequence.

[0185] In a preferred embodiment, said transgene comprises coding sequence for SURF2 protein which can be selected from the group consisting of the reference sequences of the human SURF2 transcript variant 1 (accession number: NM_017503.5, updated on December 24, 2022) and transcript variant 2 (accession number: NM_001278928, updated on December 25, 2022).

[0186] In a particular embodiment, the transgene according to the disclosure may be any nucleic acid sequence encoding an SURF2 protein functional fragment or variant as described above.

[0187] In a particular embodiment said transgene may be an optimized sequence encoding SURF2 protein, functional fragment or functional variant thereof. The term "codon optimized" means that a codon that expresses a bias for human (i.e. is common in human genes but uncommon in other mammalian genes or non-mammalian genes) is changed to a synonymous codon (a codon that codes for the same amino acid) that does not express a bias for human. Thus, the change in codon does not result in any amino acid change in the encoded protein.

[0188] Preferably, the nucleic acid construct comprises the transgene operably linked to one or more control sequences that direct the expression of said transgene in host cells, preferably hematopoietic cells, more preferably hematopoietic stem cells.

[0189] The promoter contains transcriptional control sequences that mediate the expression of SURF2 protein upon introduction into a host cell. The promoter may be any polynucleotide that shows transcriptional activity in cells including mutant, truncated, and hybrid promoters. The promoter may be a constitutive or inducible promoter, preferably a constitutive promoter, and more preferably a strong constitutive promoter. The promoter may also be tissue-specific, in particular specific of hematopoietic cells, more particularly hematopoietic stem cell.

[0190] The control sequence may also include appropriate transcription initiation, termination, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); and / or sequences that enhance protein stability. A great number of expression control sequences, e.g., native, constitutive, inducible and / or tissue- specific, are known in the art and may be utilized to drive expression of the nucleic acid sequence encoding SURF2. Typically, the transgene encoding SURF2 is operably linked to a transcriptional promoter and a transcription terminator.

[0191] Apart from the specific delivery systems embodied below in the examples, various delivery systems are known and can be used to administer the nucleic acid construct as described above, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the SURF2 coding sequence, receptor-mediated endocytosis, construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc.

[0192] The nucleic acid construct as described above may be contained in an expression vector. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Examples of appropriate vectors include, but are not limited to, recombinant integrating or nonintegrating viral vectors and vectors derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. Preferably, the vector is a recombinant integrating or non-integrating viral vector. Examples of recombinant viral vectors include, but not limited to, vectors derived from herpes virus, retroviruses, lentivirus, vaccinia viruses, adenoviruses, adeno-associated viruses or bovine papilloma virus. In a preferred embodiment, said vector is a lentiviral vector.

[0193] By "lentiviral vector" is meant HIV -Based lentiviral vectors that are very promising for gene delivery because of their relatively large packaging capacity, reduced immunogenicity and their ability to stably transduce with high efficiency a large range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double -stranded linear viral DNA, which is the substrate for viral integration in the DNA of infected cells. By "integrative lentiviral vectors (or LV)", is meant such vectors as nonlimiting example, that are able to integrate the genome of a target cell. At the opposite by "non-integrative lentiviral vectors (or NILV)" is meant efficient gene delivery vectors that do not integrate the genome of a target cell through the action of the virus integrase.

[0194] Several examples of lentiviruses include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2), the etiologic agent of the human acquired immunodeficiency syndrome (AIDS); visna- maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritis - encephalitis virus, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immune deficiency in cats; bovine immune deficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which cause immune deficiency and encephalopathy in sub-human primates.

[0195] A lentiviral genome is generally organized into a 5' long terminal repeat (LTR), the gag gene, the pol gene, the env gene, the accessory genes (nef, vif, vpr, vpu) and a 3' LTR. The viral LTR is divided into three regions called U3, R and U5. The U3 region contains the enhancer and promoter elements. The U5 region contains the polyadenylation signals. The R (repeat) region separates the U3 and U5 regions and transcribed sequences of the R region appear at both the 5' and 3' ends of the viral RNA. The 5' and 3' LTRs serve to promote transcription and polyadenylation of the virion RNAs. The LTR contains all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef and vpx. Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site). If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of genomic RNA. However, the resulting mutant remains capable of directing the synthesis of all virion proteins.

[0196] Engineered lentiviral vectors are also known that may transduce hematopoietic stem cells and HSC lineages (see, for example, “RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., Oxford University Press, (2000); O Narayan and Clements (1989) J. Gen. Virol. 70: 1617-1639; Fields et al. (1990) Fundamental Virology, Raven Press.; Miyoshi et al. (1998) J. Virol. 72:8150-8157; U.S. Pat. Nos. 5,994,136, 6,013,516, 8,551,773, and 8,361,787; Evans et al. (1999) Hum. Gene Ther. 10: 1479-1489; Case et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:2988-2993; Uchida et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95: 11939-11944; Miyoshi et al. (1999) Science 283:682-686; Sutton et al. (1998) J. Virol. 72:5781-5788).

[0197] The viral virus vectors may be pseudotyped. A “pseudotyped” virus is a viral particle having an envelope protein that is from a virus other than the virus from which the RNA genome is derived. The envelope protein may be from a different virus. For example, an envelope protein is the vesicular stomatitius virus G (VSV G) protein or from measles virus. However, to eliminate the possibility of human infection, viruses may alternatively be pseudotyped with ecotropic envelope protein that limit infection to a specific species, such as mice or birds. For example, in one embodiment, a mutant ecotropic envelope protein is used, such as the ecotropic envelope protein 4.17 (see, for example, Powell et al. (2000) Nat. Biotech. 18: 1279-1282).

[0198] The viral virus vectors may also be self-inactivating. For example, a “self-inactivating 3' LTR” is a 3' long terminal repeat (LTR) that contains a mutation, substitution or deletion that prevents the LTR sequences from driving expression of a downstream gene. Self-inactivating 3' LTRs and other viral selfinactivating methods and reagents are well known in the art (see, for example, Zuffrey et al. (1998) J. Virol. 72:9873-9880; Miyoshi et al. (1998) J. Virol. 72:8150-8157; and Iwakuma et al. (1999) Virol. 261: 120-132). In a particular embodiment, said lentivirus vector can be the lentivirus vector as disclosed in Gimenez Y. et al. JCI Insight. 2024 May 22;9(10):el71650.

[0199] The nucleic acid construct or the expression vector of the present disclosure can be packaged into a “viral particle”.

[0200] In a particular embodiment, the nucleic acid construct or the expression vector of the disclosure is packaged into a lentiviral particle. The present disclosure relates to a viral particle (e.g., lentiviral particle) comprising a nucleic acid construct or an expression vector as described above. Methods for transfection are well known by those of skill in the art. After cotransfection of the packaging vectors and the transfer vector to the packaging cell line, the recombinant virus is recovered from the culture media and titered by standard methods used by those of skill in the art.

[0201] The lentiviral vector and lentiviral particle described herein are capable of transferring a nucleic acid construct (e.g., a nucleic acid construct encoding a human SURF2 protein) into a mammalian cell.

[0202] Accordingly, in certain embodiments, a nucleic acid construct is delivered to a cell by contacting the cell with a virion, preferably containing a lentiviral vector described herein.

[0203] In some embodiments, the present disclosure relates to an isolated cell, preferably transduced with said viral vector (e.g. lentiviral vector) comprising a nucleic acid construct comprising a transgene encoding a SURF2 activator (e.g., human SURF2 protein comprising or consisting of SEQ ID NO: 1, functional fragment or functional variant thereof).

[0204] The host cell of the disclosure may be used for ex vivo gene therapy purposes. In such embodiments, the cells are transduced with the viral particle of the disclosure and subsequently transplanted to the patient or subject. Transplanted cells can have an autologous, allogenic or heterologous origin. In a particular embodiment, the host cell is used for ex vivo gene therapy into hematopoietic cell.

[0205] In general, the therapeutic use according to the present disclosure methods involve use of the lentiviral vectors described herein comprising a transgene encoding a SURF2 protein as described above, to introduce these sequences into host cells, preferably hematopoietic cells. The transduced cells are reintroduced into the subject where they restore the nucleolar stress response and thereby ameliorate and / or cure the ribosomopathy, in particular Diamond Blackfan disease. The method that can be used to transduce hematopoietic cell with lentiviral vector before administering said cells into the subject is known in the art and is disclosed for example in Gimenez Y. et al. JCI Insight. 2024 May 22;9(10):el71650.

[0206] Preferably, said cells are eukaryotic cells such as mammalian cells, these include, but are not limited to, humans, non-human primates such as apes; chimpanzees; monkeys, and orangutans, domesticated animals, including dogs and cats, as well as livestock such as horses, cattle, pigs, sheep, and goats, or other mammalian species including, without limitation, mice, rats, guinea pigs, rabbits, hamsters, and the like. A person skilled in the art will choose the more appropriate cells according to the patient or subject to be transplanted.

[0207] In a preferred embodiment, said cell is a hematopoietic cell, preferably hematopoietic progenitor cell or hematopoietic stem cell (HSCs), obtained either from the bone marrow, the peripheral blood or umbilical cord blood. In a more preferred embodiment, hematopoietic cells are CD34+ cells. In certain embodiments the use of induced pluripotent stem cells (IPSCs) is contemplated. Alternatively, the virion can be directly administered in vivo to a subject or a localized area of a subject (e.g., bone marrow).

[0208] In a preferred embodiment, the present disclosure relates to a host cell, preferably hematopoietic cell (e.g., hematopoietic stem cell) transduced with a lentiviral particle comprising a nucleic acid construct comprising a transgene encoding a SURF2 protein, preferably a human SURF2 protein comprising or consisting of SEQ ID NO: 1, functional fragment or functional variant thereof, for use in a ribosomopathy, preferably a Diamon Blackfan disease in a subject in need thereof.

[0209] Pharmaceutical composition comprising a SURF2 activator

[0210] The SURF2 activator, protein, nucleic acid construct, expression vector, viral particle or host cell according to the present disclosure is preferably used in the form of a pharmaceutical composition comprising a therapeutically effective amount of SURF2 activator, nucleic acid construct, expression vector, viral particle or host cell according to the present disclosure.

[0211] In the context of the disclosure, a therapeutically effective amount refers to a dose sufficient for reversing, alleviating or inhibiting the progress of the disorder or condition to which such term applies, or reversing, alleviating or inhibiting the progress of one or more symptoms of the disorder or condition to which such term applies.

[0212] The effective dose is determined and adjusted depending on factors such as the composition used, the route of administration, the physical characteristics of the individual under consideration such as sex, age and weight, concurrent medication, and other factors, that those skilled in the medical arts will recognize.

[0213] In the various embodiments of the present disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or vehicle.

[0214] A "pharmaceutically acceptable carrier” refers to a vehicle that does not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid fdler, diluent, encapsulating material or formulation auxiliary of any type.

[0215] Preferably, the pharmaceutical composition contains vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or suspensions. The solution or suspension may comprise additives which are compatible with viral vectors and do not prevent viral vector particle entry into target cells. In all cases, the form must be sterile and must be fluid to the extent that easy syringe ability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. An example of an appropriate solution is a buffer, such as phosphate buffered saline (PBS) or Ringer lactate.

[0216] Therapeutic use of SURF2 activator

[0217] The SURF2 activator, protein, nucleic acid construct, expression vector, viral particle, host cell or pharmaceutical composition according to the present disclosure may be used as a medicament, in particular for use in the treatment of a ribosomopathy in a subject in need thereof.

[0218] As used herein, the term “subject”, “patient” or “individual” denotes a mammal. Preferably, a patient, a subject or individual according to the disclosure is a human, in particular a human subject or patient, more preferably suffering from a ribosomopathy such as Diamond Blackfan anemia.

[0219] The terms “Ribosomopathy” “ribosomal disorder” or “ribosomal protein disorder”, is used herein to refer to a disease or disorder linked to a mutated and / or abnormal function of a ribosomal biogenesis proteins, small nucleolar ribonucleoproteins or ribosome protein. It can include a disease due to mutation in a ribosomal biogenesis proteins, small nucleolar ribonucleoproteins or ribosomal protein, or a disease due to a decreased level, or partial loss of function, of a ribosomal biogenesis proteins, small nucleolar ribonucleoproteins or ribosomal protein, or alternatively, a disease due to an increased level of a ribosomal biogenesis proteins, small nucleolar ribonucleoproteins or ribosomal protein, as compared to a normal healthy control subject. The term ribosomal disorder includes diseases including but not limited to, Diamond-Blackfan anemia syndrome (DBA), 5q-syndrome, X-linked dyskeratosis congenita, cartilage-hair hypoplasia, myelodysplasia, Shwachman-Diamond Syndrome (SDS), Bowen-Conradi syndrome, North American Indian childhood cirrhosis and Treachers Collins Syndrome (TCS).

[0220] In a preferred embodiment, said ribosomopathy is Diamond-Blackfan anemia syndrome (DBA). Diamond-Blackfan anemia syndrome is a congenital erythroid aplasia that usually presents in infancy, characterized by anemia (low red blood cell counts) with decreased erythroid progenitors in the bone marrow. The rest of their blood cells (the platelets and the white blood cells) are normal. This is in contrast to Shwachman-Bodian-Diamond syndrome, in which the bone marrow defect results primarily in neutropenia, and Fanconi anemia, where all cell lines are affected resulting in pancytopenia. This usually develops during the neonatal period. About 47% of affected individuals also have a variety of congenital abnormalities, including craniofacial malformations, thumb or upper limb abnormalities, cardiac defects, urogenital malformations, and cleft palate. Low birth weight and generalized growth delay are sometimes observed. “Treating ribosomopathies” includes, without limitation, reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with inappropriate ribosomal protein function. As used herein with respect to a ribosomal protein disorder, the term treating is used to refer to the reduction of a symptom and / or a biochemical marker of a ribosomal protein disorder, for example a reduction ofp21 and / or p53 levels in CD34+ cells in the subject, a return of hemoglobin back to normal levels, or a restoration or prevention of craniofacial deformities.

[0221] In a preferred embodiment, said SURF2 activator, protein, nucleic acid construct, expression vector, viral particle, host cell or pharmaceutical composition according to the present disclosure is used to prevent risk of developing a cancer in a patient suffering from ribosomopathy.

[0222] The disclosure also provides a method for treating a ribosomopathy (e.g. DBA) in a subject in need thereof comprising administering to said subject a therapeutically effective amount of the SURF2 activator, protein, nucleic acid construct, expression vector, viral vector encoding said SURF2 activator, viral particle, host cell or pharmaceutical composition as described above.

[0223] By “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of the product of the disclosure or pharmaceutical composition that comprises it may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the product or pharmaceutical composition to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also typically one in which any toxic or detrimental effect of the product or pharmaceutical composition is outweighed by the therapeutically beneficial effects.

[0224] In certain aspects, the disclosure provides to the use of a SURF2 activator, protein, nucleic acid construct, expression vector, viral particle, host cell and / or a pharmaceutical composition according to any one of the preceding embodiments for the manufacture of a medicament for treating a ribosomopathy (e.g. DBA) in a subject in need thereof.

[0225] In a more specific embodiment, the present disclosure relates to a hematopoietic cell transduced with a lentiviral particle comprising a nucleic acid construct comprising a transgene encoding a human SURF2 protein comprising or consisting of SEQ ID NO: 1, functional fragment or functional variant thereof for use in the treatment of DBA in a subject in need thereof.

[0226] In a specific embodiment, the present disclosure relates to a method of treating DBA comprising administering a therapeutically efficient amount of an isolated cell (e.g., hematopoietic cell) comprising a nucleic acid construct comprising or consisting of SEQ ID NO: 1, functional fragment or functional variant thereof for use in the treatment of DBA in a subject in need thereof. In a preferred embodiment, the present disclosure relates to a method of treating DBA comprising administering a therapeutically efficient amount of a cell (e.g., hematopoietic cell) transduced with a lentiviral particle comprising a nucleic acid construct comprising or consisting of SEQ ID NO: 1, functional fragment or functional variant thereof for use in the treatment of DBA in a subject in need thereof into a subject in need thereof.

[0227] In one embodiment the product of the disclosure is administered to the subject or patient by a parenteral route, in particularly by intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular route.

[0228] The amount of product of the disclosure that is administered to the subject or patient may vary depending on the particular circumstances of the individual subject or patient including, age, sex, and weight of the individual; the nature and stage of the disease, the aggressiveness of the disease; the route of administration; and / or concomitant medication that has been prescribed to the subject or patient. Dosage regimens may be adjusted to provide the optimum therapeutic response.

[0229] For any particular subject, specific dosage regimens may be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.

[0230] The invention will now be exemplified with the following examples, which are not limitative, with reference to the attached drawings in which:

[0231] FIGURE LEGENDS

[0232] Figure 1: Label-free quantitative proteomics analysis of RPL5-FLAG co-purified proteins. Nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS / MS) analysis of trypsin digested proteins retained on flag coated beads issued from U2OS control cells (LEFT quadrant) or cell expressing RPL5-FLAG (RIGHT quadrant). At least three independent experimental replicates were performed. Volcano plots showing proteins significantly enriched for control cells (dark grey) versus cells expressing RPL5-FLAG (A) in untreated cells (B) in cells treated with actinomycin D (ACTD, 10 ng / ml) for 24 h. An unpaired bilateral Student t-test with equal variance was used. Enrichment significance thresholds are represented by an absolute log2 -transformed fold-change (FC) greater than 1 and a -loglO-transformed (p-value) greater than 1.3. The iBAQ (intensity-Based Absolute Quantification) values which are relevant to rank the absolute abundance of different proteins within a single sample are represented by the diameter of each dot.

[0233] Figure 2: SURF2 is a new partner of free-5S particles and is involved in p53 regulation. (A) Label- free quantitative proteomics analysis of trypsin digested proteins retained on beads coated by anti- GAPDH or anti-SURF2 antibodies. At least three independent experimental replicates were performed. (A) Volcano plot showing proteins significantly enriched in the GAPDH IP versus the SURF2 IP. An unpaired bilateral Student t-test with equal variance was used. Enrichment significance thresholds are represented by an absolute log2-transformed fold-change (FC) greater than 1 and a -loglO-transformed (p-value) greater than 1.3. (B) Focus on the iBAQ values, represented by the diameter of each dot, for the proteins significantly enriched in the SURF2 IP. (C) Detection of RNAs associated with HEXIM 1 and SURF2. U2OS RNAs co-immunoprecipitated with endogenous HEXIM1 (a-HEXIMl) or endogenous SURF2 (a-SURF2) were 3’-labelled and separated on a 6% acrylamide gel. Control IP reaction performed without anti-bodies (beads) is shown. (D) U2OS cell extracts were fractionated using the PSE method. Western-blot analyses showing the contents of different factors (indicated at the left of the gel) in the SN1, SN2 and SN3 fractions obtained with the PSE method. (E) Microscopy analyses of U2OS cells over-expressing SURF2-GFP from TET inducible promoter. Cells were induced with tetracycline to obtain a similar expression as the endogenous copy (tetracycline at 5 ng / mL) and SURF2- GFP signal detected. The same cells were also probed for fibrillarin to stain nucleoli. DAPI coloration is used to localize nuclear compartment. (F) Western-blot analyses showing the accumulation of different proteins in 20 pg of cellular extracts produced from U2OS cells treated with scrambled siRNAs (siSCR) or with siRNAs directed against SURF2 (siSurf2). (G) Quantification of signals obtained by western-blot analysis and normalized to actin signals in each condition (f) (n=3). t-test analysis was used for statistics. Significant differences are indicated by stars (p-value <0.05*; <0.01** ; <0.001*** and < 0.0001****). (H) Co-depletion experiment. Western-blot analyses showing the accumulation of different proteins in 20 pg of cellular extracts issued from U2OS cells treated with a combination of different siRNAs: scrambled (siSCR); RPL5 (siRPL5); RPL11 (siRPLl l) or SURF2 (siSurf2). (I) Quantification of signals obtained by western-blot analysis and normalized to GAPDH signals in each condition (h) (n=3). Anova test was used for statistics. Significant differences are indicated by stars (p- value <0.05*; <0.01** and <0.001***).

[0234] Figure 3: SURF2 is overexpressed in adrenocortical carcinomas and its knock-out negatively affects cancer cells similarly to MDM2. a, Comparison of SURF2 mRNA levels in non-tumoral (left panel, grey values) and tumoral tissues (right panel, blue values). Data were extracted from the standardized Xena database to compare healthy adrenal gland tissues (n=128, origin GTEx dataset) and adrenocortical carcinoma tissues (n=77, origin TCGA dataset). Compared to healthy adrenal gland tissues, SURF2 mRNA levels are higher in adrenocortical carcinoma tissue. ****: p < 0.0001 (Wilcoxon Test). Data are presented as violin plot. It comprises a density plot, the width of which indicates the frequency, and a box plot, where the extreme points reflect the minimum (Q0) and maximum (Q4), the length of the box represents the interquartile range (IQR: percentile QI to Q3) and the center represents the median, b-c, Association between SURF2 mRNA levels and patient survivals. Using the standardized Xena dataset, associations between SURF2 mRNA levels (median as cut-off value) and overall survival (b) or progression-free survival (c) were determined using Kaplan-Meier curves. A significant association was observed between high levels of SURF2 mRNA levels and poor patient survivals. *: p < 0.05 ; **: p < 0.01 (Log -rank), d-e, Association between SURF2 mRNA levels and patient survivals after adjustment on clinical parameters. Using the standardized Xena dataset, multivariate Cox regression models for overall survival (Low n=39, High n=38) (d) or progression-free survival (e) were performed on clinical parameters significantly associated with patient survivals (z.e., TP 53 mutation status and tumor stage, See Supplementary Table S 6) . High mRNA levels of SURF2 and stage IV are independent markers of poor survivals of patients carrying adrenocortical carcinoma. * : p < 0.05; **: p < 0.01 (Cox Proportional -Hazards Model, two-sided hypothesis test). Error bars show 95%CI (confidence interval) and square HR (hazard ratio), p- value (0.032 and 0.003 respectively), source data are provided as a Source Data file, f, Top SURF2 correlated genes in project Achilles. Pearson correlation score on the right indicates the strength of co-dependencies between SURF2 and the indicated genes, g, SURF2 knock-out chronos effect on different cancer cells classified in three groups depending on their TP53 status: wild-type (blue, n=386), hot-spot mutations (brown, n=545), damaging mutations (yellow, n=169). The data are presented as the mean ± s.d. Statistical analysis by two-way ANOVA with Turkey’s multiple comparisons test. Significant differences are indicated by p value <0.0001 (****) (ns= no significance), h, Classification of more than 1000 cancer cells based on their associated chronos effect of SURF2 knock-out and MDM2 knock-out. Pearson correlation between the two chronos effects is indicated depending on TP53 status, i, Same as in (h) but focusing on bones cancers cell lines. Source data are provided as a Source Data file.

[0235] Figure 4: SURF2 depletion increases free-5S binding to MDM2 and increase cell sensitivity to nucleolar stress. (A) Detection of proteins associated with RPL5-Flag. Cell extracts produced from U2OS cells overexpressing RPL5-FLAG or not (control) and differentially treated by actinomycin D addition for 24 h (at 10 ng / mL) or not (ACTD + or -) and transfected with scrambled siRNAs (siSCR) or directed against SURF2 (siSurf2) were used to perform immuno-precipitation on beads coated with anti-flag. 10% of the inputs were loaded (In) aside IPs (IP) on gel to perform western-blots using antibodies directed against the indicated proteins to analyze co -purification efficiency of the different factors. (B) Quantification of the co-purification efficiency with RPL5-Flag observed (A) for the indicated proteins (n=3). Results are represented as a comparison of the enrichments observed in cells treated with siRNAs directed against SURF2 (siSurf2) normalized to the ones observed in cell treated with scrambled siRNAs (siSCR). t-test analysis was used for statistics. Significant differences are indicated by stars (p-value < 0.05*; < 0.01** and < 0.001***). (C) Quantification of the signals corresponding to the indicated proteins in the different inputs of panel (A) n=3. t-test analysis was used for statistics. Significant differences are indicated by stars (p-value <0.05*). (d) DNA content analysis of U2OS cells treated as indicated (siSCR: scrambled siRNAs; siSurf2: siRNAs against SURF2; ACTD: actinomycin D at 10 ng / mL for 24 h) by FACS. (E) Quantification of U2OS cells repartition in the different phase of cell cycle following different treatments (n=3). Anova Test was used for statistics. Significant differences are indicated by stars (p-value <0.05*; <0.01** and <0.001***). (F and G) Proliferation analysis of differentially treated U2OS cells. Cells are treated as indicated (siSCR: scrambled siRNAs; siSurf2: siRNAs against SURF2; ACTD: actinomycin D at 10 ng / mL for 24 h). (F) Cells are platted on 6 well plate after being transfected with the indicated siRNAs, 24 h before being analyzed cells were treated with actinomycin D (10 ng / mL) or H2O. Cells were stained by crystal violet to take picture. (G) Fixed crystal violet was resolubilized and quantified by absorbance for each condition after different time exposure to treatments. (H) Cell apoptosis assays with an annexin-V-FITC and propidium iodide of differentially treated U2OS cells. Both apoptosis and necrosis are regrouped as dead cells (n=3). Data are presented as mean values + / - SD. The One-way Anova Test was used for statistics. Significant differences are indicated by stars (p-value <0.05*; <0.01**; <0.001*** and <0.0001****) or with the precise p value on the graph (p). Source data are provided as a Source Data file.

[0236] Figure 5: SURF2 depletion promotes cell cycle arrest and apoptosis upon nucleolar stress. Codepletion experiment. (A and B): DNA content analysis of U2OS cells treated as indicated (siSCR: scrambled siRNAs; siSurf2: siRNAs against SURF2; 5-Fu: 5 -fluorouracil (50pM); BMH-21 (150nM) for 24h) by FACS. Quantification of U2OS cells repartition in the different phase of cell cycle following different treatments (n=3). (C) Cell apoptosis assays with an annexin-V-FITC and propidium iodide of differentially treated U2OS cells. Cells are treated as indicated (siSCR: scrambled siRNAs; siSurf2: siRNAs against SURF2; 5-Fu: 5 -fluorouracil (50pM); BMH-21 (150nM) for 24h), both apoptosis and necrosis are regrouped as dead cells. (n=3). (D) DNA content analysis of U2OS cells treated as indicated (siSCR: scrambled siRNAs; siSurf2: siRNAs against SURF2; siP53: siRNAs against p53; for 96h) by FACS. Quantification of U2OS cells repartition in the different phase of cell cycle following different treatments (n=3). (E) DNA content analysis of HCT116, p53+ / + or HCT116, p53- / - cells treated as indicated (siSCR: scrambled siRNAs; siSurf2: siRNAs against SURF2 for 96h) by FACS. Quantification of HCT116 cells repartition in the different phase of cell cycle following different treatments (n=3). Anova test was used for statistics. Significant differences are indicated by stars (p- value 0.05*, 0.01**, and 0.001***). (F) Western-blot analyses showing the accumulation of different proteins, in 20pg of cellular extracts produced from from U2OS cells treated as indicated (ko Ctrl: scrambled siRNAs; koSurf2: siRNAs against SURF2; Doxo: Doxorubicin). (B) Quantification of the signals observed in independent experiment (A). Anova Tests were used for statistics. Significant differences are indicated by stars (p-value <0.05*; <0.01** and <0.001***).

[0237] Figure 6: SURF2 overexpression impedes p53 activation and cell cycle arrest following nucleolar stress. (A) Western-blot analyses showing the accumulation of different proteins, in 20 pg of cellular extracts produced from control U2OS cells or from U2OS cells that overexpress SURF2-Flag after Tetracycline induction (1 pg / ml) from their T-Rex locus for 24 hours. Cells were also treated differentially with or without addition of actinomycin D (10 ng / ml) for the same period. (B) Quantification of the signals observed in independent experiment (A) (n=3). Anova Tests were used for statistics. Significant differences are indicated by stars (p-value <0.05*; <0.01** and <0.001***). (C) DNA content analysis of U2OS cells treated as indicated (Ctrl: U2OS control cells; OE SURF2-Flag: U2OS cells that overexpress SURF2-Flag; ACTD: Treated with actinomycin D) by FACS. Quantification of different U2OS cells repartition in the different phase of cell cycle following different treatments is represented as histograms (n=3). Anova Tests were used for statistics. Significant differences are indicated by stars (p-value <0.05*; <0.01** and <0.001***). Source data are provided as a Source Datafile. (D) Comparison ofp53 and p21 stabilization, assessed by western-blots, following drugs treatment (5-Fu : 50uM; MG-132: lOuM; staurosporine: 25nM for 24 hours) in U2OS control cells (uninduced) or in cells overexpressing SURF2-Flag (Tetracycline Img / ml). P53 and p21 signal were normalized to 1 in the control experiment and relative stabilization in cell overexpressing SURF2- Flag was quantified from three independent biological replicates (n=3). Data are presented as mean values + / - SD. Two-tailed paired T-test was used for statistics. Significant differences are indicated by stars (p-value <0.05*; <0.01** and <0.001***).

[0238] Figure 7: SURF2 expression level affect U2OS phenotypic traits. Analysis of U2OS KO SURF2 or U2OS overexpressing SURF2-Flag cell migration by wound healing assay. A and C, Images were taken with Cell Imaging EVOS (Gx40) at Oh and 24h (n=3), (scale bar = 0.1mm). B and D, Quantification of wound healing by measuring the percentage of persistent scar area after 24h from three independent biological replicates (n=3). Use of an unpaired two-tailed t-test for statistical tests Data are presented as mean values + / - SD, and significant differences are indicated by stars as follows p-value < 0.05*; 0.01** 0.001*** and 0.0001****) or with the precise p value on the graph (p). Source data are provided as a Source Data file.

[0239] Figure 8: SURF2 is able to directly interact with both RPL5 and RPL11 and competes for their binding with MDM2 in vivo. (A) Extracts from U2OS cells that overexpress SURF2 (OE SURF2-Flag) or not (control) and treated by actinomycin D (ACTD) are used to perform IPs using beads only, beads coupled to anti-SURF2 or beads coupled to anti-MDM2. After washes, remaining proteins are resuspended in loading dye and analyzed by western-blots using the indicated antibodies. (B) GST- pulldown assays. Extracts of BL21 that overexpress recombinant SURF2-HIS were mixed with extracts that overexpress either GST alone, GST-RPL5 or GST-RPL11. Proteins specifically retained on glutatione-sepharose beads were analyzed both by Coomassie staining and western-blot analysis (WB). 10% of the extracts were used for inputs. (C) Secondary structure of SURF2 and MDM2 proteins. Functional domains are indicated. SURF2 3D structure modelisation by Alphafold software is represented (D) Same experiments as in (B) but replacing extract with SURF2-HIS by extracts that overexpress structural domain of SURF2 (SURF2-SD-HIS) as prey. Proteins specifically retained on glutatione-sepharose beads were analyzed both by western-blots with indicated antibodies. Figure 9: SURF2-SD structural domain overexpression promotes cell cycle arrest following nucleolar stress. A. DNA content analysis of U2OS cells treated as indicated (Ctrl: U2OS control cells; OE SURF2-Flag: U2OS cells that overexpress SURF2-Flag; OE SURF2-SD-Flag: U2OS cells that overexpress peptide SURF2(1-136)-Flag, ACTD: Treated with actinomycin D) by FACS. B. Cell apoptosis assays with an annexin-V-FITC and propidium iodide of differentially treated U2OS cells. Both apoptosis and necrosis are regrouped as dead cells.

[0240] Figure 10: Model of SURF2 function in free 5S RNP regulation. Schematic representation of free- 5S regulation by SURF2 in different conditions. In normal cells, both 5S and 47S rDNAs are transcribed by RNA polymerase III and I respectively. Ribosomes synthesis is producing pre-60S ribosomes and 5S particles constituted by the association of RPL5 and RPL11 to 5S rRNA are incorporated into these large pre-ribosomes. The remaining overproduced free 5S are bound by SURF2 to avoid MDM2-Free 5S interaction, which would induce p53 stabilization and cell cycle arrest. At the same time, p53 is constantly ubiquitinated by MDM2 to promote its degradation by the proteasome. After nucleolar stress (drug-induced or caused by genetic mutations / ribosomopathies), ribosome synthesis is impaired and a larger amount of free 5S particles accumulate in the nucleoplasm. The extra free 5S can then be recognized by MDM2, which can no longer ubiquitinylate p53, thereby stabilizing p53 and promoting cell cycle arrest. In cells lacking SURF2, nucleolar stress still impairs ribosome synthesis, but this time even more free 5S are able to bind to MDM2, inducing stronger stabilization and activation of p53, followed by more cell cycle arrest. In contrast, in cells overexpressing SURF2, nucleolar stress promotes free-5S accumulation in the nucleoplasm, all of which are recognized by SURF2, which competes with MDM2 for binding. As a result, MDM2 is free and ubiquitinylate s p53, conferring to these cells a capacity to resist to nucleolar stress.

[0241] EXAMPLES

[0242] 1. Results

[0243] 1.1 SURF2 is a new binding partner of free 5S RNP particles

[0244] To identify potential regulators of free 5S RNPs, the inventors set out to purify these particles outside the ribosomes. 5S particles are formed by the association of the 5S rRNA, with the ribosomal proteins RPL5 and RPL11, whether they are integrated into the ribosome or accumulate as independent particles.

[0245] The inventors therefore developed a novel U2OS cell line, known to respond well to nucleolar stress (NS), that inducibly overexpresses RPL5-Flag using Flip-In T-REx constructs. First, the inventors verified the expression of RPL5-Flag, and used sucrose gradient fractionation to show that this protein was able to accumulate in both ribosomal and free fractions without altering ribosomal subunits production. The process of ribosome assembly consists of the maturation of a large primary transcript that contains mature rRNA sequences (18S, 5.8S, and 28S) separated by spacers that are removed after exo and endo-nucleolytic steps. rRNA processing occurs inside pre-ribosomal particles constituted of pre-rRNAs associated to both ribosomal proteins and transiently associated factors, the latter being absent from mature ribosomal subunits. Disruption of ribosome assembly will lead to abnormal accumulation of rRNA precursors and mature rRNAs. To confirm that ectopic expression of RPL5-Flag did not affect ribosome synthesis and maturation, the inventors assessed pre-rRNA processing using northern blots. No changes in accumulation of rRNAs precursors nor mature forms were observed, indicating that RPL5-Flag expression does not perturb ribosome assembly.

[0246] Then, in order to enrich free 5S RNP partners, that accumulate outside the ribosome, the inventors performed a sucrose cushion step to pellet both mature and precursors of ribosomes, before further RPL5-flag purification of free 5S RNP particles on beads coupled with anti-Flag antibodies to remove both matures and precursors ribosomes. Immunopurified interacting proteins were then digested with trypsin and analyzed by nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS / MS) to identify enriched free 5S RNP partners using a differential label-free quantitative proteomics approach (Fig 1). To evaluate interaction changes, pairwise comparison based on MS intensity values were performed for each quantified protein, first, between RPL5 -Flag -expressing cells and control U2OS cells only expressing the Flag -tag (Fig 1A). Enriched proteins were selected based on their significant protein abundance variations between the two compared conditions (fold-change (FC) > 2 and < 0.5, and Student t-test p-value < 0.05) (see Mat&Meth for details). The volcano plot presented in Figure 1A shows that several proteins were significantly co-purified with RPL5-Flag, indicating them as partners of free-5S (Fig 1A). RPL11, the other component of the 5S particles was found with a similar fold-change (FC = 33.34) compared to the RPL5-Flag bait (FC = 59.3), confirming the efficient purification of the 5S particles and that RPL5-Flag is mainly in complex with RPL11. No other ribosomal proteins were found specifically enriched in this purification, attesting the efficiency of the ribosomal fraction elimination step.

[0247] The inventors also found MDM2 (FC = 58.01), already described as a major free-5S partner (19, 33- 35). SSB / LA-protein, a known chaperone of 5S rRNA, is also enriched as well as HEATR3, a chaperone of RPL5 (11, 36-40). Next, the inventors found p53, which was not anticipated but might correspond to an indirect interaction via MDM2. The inventors decided to test this hypothesis, by using Nutlin-3a, a p53 interaction inhibitor that targets MDM2 binding pocket. During these experiments, the inventors could reproduce the previous results showing an interaction between p53 and free-5S using IPs followed by western-blot analysis. However, this association is lost in presence of Nutlin-3a, both added in cell medium and in IP buffer. This observation demonstrates that binding of MDM2 to free-5S and p53 are not mutually exclusive in U2OS cells.

[0248] HEXIM1 was also significantly enriched (FC= 8.75) during this experiment, but the inventors could not reproduce this interaction using HEXIM 1 as a bait during reverse IP experiment (data not shown). Of interest, SURF2, a previously uncharacterized protein, was significantly and highly enriched (FC= 45.69) during RPL5-Flag purification (Fig 1A). Other proteins such as mitochondrial ribosomal proteins (MRPS14, MRPS15, MRPS16, MRPS18B, MRPS22, MRPS25 and MRPS26); UBE2O (a ubiquitinligase proteins) and EIF2B1 (translation initiation factor) were also specifically found albeit with reduced fold-changes compared to other partners. To investigate how the panel of free 5S RNP binding partners changes during stress, the inventors used the same purification strategy but this time after inducing NS with a low dose of actinomycin D (ACTD) (10 ng / mL for 24 h) (Fig IB). Surprisingly, the repertoire of potential free RPL5-flag partners did not change much, only showing an increased enrichment of MDM2 compared to RPL5 (Fig IB).

[0249] Among the different partners of RPL5-Flag outside the ribosome found during these experiments, SURF2 really stands out, both as an abundant (areas of circles is proportional to the IBAQ abundance of each protein) and strongly enriched (FC = 45.69 and 14.42, before or following NS induction respectively) partner. Indeed, it is the second-best enriched partner of RPL5-Flag in both normal and NS conditions. Surprisingly, nothing was known about SURF2 except that its promoter, shared with SURF1, is controlled by c-Myc and that this gene is located in a crowded and conserved locus, hence its name Surfeit locus (T. Duhig, et al. Genomics 52, 72-78 (1998); K. Gaston, et al. Nucleic Acids Res 23, 901— 909 (1995); E. G. Vernon, et al. Biochim Biophys Acta 1492, 172-179 (2000)). Interestingly, this genomic locus is constituted by other genes encoding for factors involved in ribosome production (SURF3 / RPL7A and SURF6 / RRP14), a transmembrane receptor involved in endoplasmic reticulum export (SURF4), a component of the mediator complex (SURF5 / MED22) and a factor involved in cytochrome-c synthesis (SURF1 / SHY1), all of which promote cell growth and proliferation. To understand what role SURF2 might play in relation to free 5S particles, the inventors undertook its functional characterization in U2OS cells.

[0250] 1.2 SURF2 binds 5S RNP particles but is not involved in ribosome assembly

[0251] As free 5S RNP particles are known to bind different partners, such as RPF2, RRS1, HEATRIII and LA- protein, which ensure their synthesis and incorporation to ribosomes, the inventors wanted to test whether SURF2 was involved in such regulation. SURF2 is described in the human protein atlas as a nucleoplasmic and nucleolar component suggesting a potential role of this factor in ribosome assembly (https: / / www.proteinatlas.org / ENSG00000148291-SURF2). To test this hypothesis, the inventors first purified this factor from a U2OS whole cell extract, after immunoprecipitation using anti-SURF2 or anti-GAPDH antibodies as control antibodies, the inventors searched for potential protein partners involved in ribosome assembly by a similar differential label-free quantitative proteomics approach. The volcano plot presented in Figure 2A shows that, among the factors significantly enriched with SURF2 and most abundant in the sample, the inventors found TRIM21, PRDX1, DLST, RPL5, and RPL11 (Fig 2A and 2B). TRIM21 and PRDX1 are common contaminants from U2OS extracts (see CRAPome database) (D. Mellacheruvu, et al. Nat Methods 10, 730-736 (2013)). DLST, dihydrolipoamide S- succinyltransferase, is a mitochondrial protein, as the inventors focus on nuclear components, they did not take this protein into account for the rest of the study. Interestingly, among the different ribosomal proteins or ribosome assembly factors (RAFs) found during this experiment, only RPL5, RPL11, RPF2 / BXDC1 and RRS1 were specifically co-purified with SURF2 (Fig 2A). BXDC1 and RRS1 mediate proper integration of 5S RNP particles to pre-60S pre-ribosomal subunits (C. Madru, et al. Genes & Development 29, 1432-1446 (2015) ; S. Kharde, et al. Nucleic Acids Research, doi: 10. 1093 / nar / gkv640 (2015)). Their low abundance could reflect an indirect interaction of these proteins with free 5S RNP particles before being integrated in ribosomes (Fig 2B). On the other hand, the highly significant enrichment of RPL5 and RPL11 confirm their specific interaction with SURF2.

[0252] To test whether SURF2 interaction with RPL5 and RPL11 occurs inside or outside of free 5S RNP particles, the inventors tested the interaction of SURF2 with RNAs in the cell by performing IP using either beads coupled to anti-SURF2, anti-HEXIMl (HEXIM1 being found associated with RPL5-Flag, see Figi above) or beads not coupled with antibodies (termed “beads” in Fig 2) as control experiment. RNAs enriched during the IPs in the different conditions were labelled with P32using pCp labelling and separated on a polyacrylamide gel (Fig 2C). During this experiment, a specific binding of HEXIM 1 to its known partner 7SK RNA was noticed both in normal and NS conditions following actinomycin D treatment, but no other RNAs, including rRNAs were specifically enriched compared to control (“beads”). On the contrary, SURF2 retains significantly more 5S rRNA than control beads, indicating that on top of RPL5 and RPL11, SURF2 specifically interacts with 5S rRNA. To confirm that SURF2 only interacts with 5S RNP particles in their free form, the inventors performed a cell fractionation analysis using the recently developed PSE method (B. Nieto et al. RNA Biol 18, 182-197 (2021)) (see material and methods) (Fig 2D). There, localization of SURF2 in the different cell fractions (cytoplasmic / nuclear and nucleolar fractions) was tested by western-blots and compared to other protein signals. SURF2 was preferentially enriched in the cytoplasmic / nuclear fraction as observed for p53, while RPL5, RPL11 and RPL17 are equivalently present in nucleolar fractions. This indicates that SURF2 preferentially localizes outside the nucleoli. To reinforce this analysis, the inventors set-out to localize SURF2 using microscopy. Despite being efficient for western-blot and IPs, anti-SURF2 antibodies did not work for immune -fluorescence experiments (data not shown). Therefore, the inventors developed a new U2OS cell line expressing a SURF2-eGFP tagged version in the presence of tetracycline. When expressed in the same range as the endogenous one, SURF2-GFP localizes mainly in the nucleoplasm with some signal found in the nucleolar fraction, reflecting what was found during the PSE method (Figure 2E).

[0253] Since an absence of direct interaction between SURF2 and pre-ribosomal components cannot entirely exclude an indirect role of this factor in ribosome assembly, and because the inventors observed a nucleolar localization of SURF2, the inventors wanted to assess whether SURF2 could influence ribosome assembly. The inventors thus used siRNAs to extinct SURF2 expression in U2OS cells. First, the inventors confirmed an 85% reduction in SURF2 protein accumulation by western-blot analysis after 96 hours of treatment by two successive transfections with siRNAs (Fig 2F and 2G). As a consequence, the inventors kept this treatment through the remaining part of this study. After SURF2 depletion, the inventors analyzed pre-rRNAs maturation process by northern-blots and quantified it by RAMP analysis (M. Wang, et al. Nucleic Acids Res 42, 11180-11191 (2014)). This analysis allows to detect alterations of rRNA maturation by displaying the ratios between one pre-RNAand its immediate processing product (data not shown). First, no obvious changes in the accumulation of mature rRNAs, nor of their precursors were observed after SURF2 depletion. Only a sporadic and insignificant accumulation of 41S could be observed following SURF2 depletion in some experiments, without affecting downstream precursors levels such as 32S, 21S, 12S or 18S-E (data not shown). This absence of pre-rRNAs processing defects was confirmed by sucrose gradient analyses, showing no depletion of the small or large ribosomal subunit (data not shown). Indeed, sucrose profile were not affected by SURF2 depletion; moreover, this protein was only found in the lighter, so-called free fractions of the gradient, suggesting that it is not associated with (pre-)ribosomal particles. To confirm this observation, the inventors also tested whether SURF2 depletion could affect RPL5 or RPL11 association to ribosomal particles using sucrose gradient followed by western-blot analysis. Indeed, the presence of RPL5 or RPL11 in different gradient fractions were not affected by SURF2 depletion, compared to RPL17. Altogether, the present data support the idea that SURF2 does not intervene in ribosome assembly, and that it interacts only with the free form of 5S RNP particles.

[0254] 1.3 SURF2 is overexpressed in several cancers and is linked to MDM2-p53 pathway

[0255] SURF2 expression level seem to regulate cell reaction to nucleolar stress. In cancer, NS stress can both participate in tumor development and drug response. Therefore, the inventors sought to characterize whether SURF2 expression level could be associated with cancer patient outcome. The inventors thus analyzed SURF2 expression in PanCancer patient cohort using TGCA resource (https: / / portal.gdc.cancer.gov). First, the inventors compared expression of SURF2 in tumors from different cancer types relative to their associated healthy tissues (GTex datasets) (Fig 3A).

[0256] In a vast majority of evaluated cancers, the inventors found an increased level of SURF2 mRNA compared to healthy tissue (p-value inferior to 2e l6), indicating that SURF2 is over-expressed in several cancer types. The inventors then analyzed the association between SURF2 mRNA expression and the overall or progression-free survival in several cancer types (Fig 3B, 3C, 3D and 3E). The inventors found that in adrenocortical carcinoma, a pediatric cancer, high level of SURF2 expression significantly correlates with poor overall survival compared to low expression of SURF2 (25% at 6 years, p adj value= 0.01) (Fig 3B). Interestingly, this difference and trend is conserved in progression-free survival in wild-type (WT), but not in mutant (MT) TP53 tumors, suggesting that in WT TP53 adrenocortical carcinoma, SURF2 expression would distinguish patient survival (Fig 3C). A similar trend was also observed in head and neck carcinomas, in which a better progression free survival correlates with low SURF2 expression in WT TP53 tumors compared to high SURF2 expression (Fig 3D and E). This is coherent with the previous biochemical data, that show opposite levels of SURF2 and p53 accumulation, and support the idea of an antagonist role of SURF2 and p53 in response to NS.

[0257] All these data indicate that SURF2 could indeed be used as prognostic marker in specific cancers, but could also represent a new therapeutic target to treat patients suffering from some cancers expressing wild-type TP53. In order to design new molecules that could affect SURF2 in vivo, the inventors set out to identify how SURF2 mediates its function.

[0258] Considering that SURF2 expression is increased in cancer and that it is able to bind to free 5S RNP particles, the inventors tested whether this increase in SURF2 level was linked to the function of free 5S RNP particles on the regulation of the MDM2-p53 pathway. To this end, they took an unbiased approach by analyzing datasets from the Cancer Dependency Map project (DepMap portalhttps: / / depmap.org / portal). The DepMap is an ongoing project that systematically assesses the effect of single-gene inactivation on cell proliferation by genome-wide CRISPR screens across a large panel of well-characterized human cancer cell lines p53 (>1000). The dependency of cancer cells to a gene is indicated by its Chronos score (Proj ectAchilles) (M. S. Lindstrom, et al. Mol. Cell. Biol. 27, 1056-1068 (2007)). Positive or negative Chronos scores suggest either increased or decreased proliferation, respectively, upon gene KO (a score of -1 indicates the gene as essential in the associated cell line). On average, SURF2 was indicated as a strongly selective gene that affects proliferation but not as essential (Chronos score around -0.25). Then, the inventors analyzed the effects on SURF2 knockout on more than 1,000 cancer cell lines. As a first analysis they looked for co -dependency of cancer cells to knockout of other genes as it is often indicative of a role in similar pathways (Fig 3F). Interestingly, the top 10 co-dependencies were found with genes involved in p53 pathway and with MDM2 in particular, suggesting again a strong relation between SURF2 and the MDM2-p53 pathway. To go further, they compared the Chronos score on cells harboring wild-type TP53 genes to mutated genes defined as damaging or as hotspot mutation (Fig 3G). As seen on our violin plot, SURF2 depletion is more deleterious in cancer cells that express wild-type p53, confirming the strong functional correlation between SURF2 and p53. Nevertheless, in TP53 mutant cells SURF2 depletion still negatively impacts cancer cells, suggesting that in addition to promoting p53 activation, SURF2 also induces p53 -independent negative effects. Mdm2 is the most strongly co-dependent genes found by DepMap analysis and is required for p53 activation by free 5S RNPs upon nucleolar stress. Therefore, they wanted to test how these genes correlated in different cancers. The inventors thus analyzed the correlation between knockouts effect of SURF2 and MDM2 on all cancer cell lines in the dataset or on bone cancer cell lines in which the correlation was stronger (Fig 3H and 31). In this different analysis, they observed a Pearson correlation of 0.368 (p value 7.57* 10A-14) and 0.738 (p value 1.26* 10A-4) in all cancers or in bone cancers respectively for cancer cell lines harboring a wild-type p53. This correlation was strongly reduced or lost in cells harboring TP53 mutations.

[0259] 1.4 SURF2 depletion promotes p53 activation independently of NS induction

[0260] In view of SURF2's role in the regulation of p53 in cancer, its strong connection with MDM2 highlighted by the DepMap data and its association with free 5S particles, the inventors wished to initiate the functional characterization of this factor using U2OS cells. To characterize the functions of SURF2, the inventors used the siRNAs strategy to perform an RNA-seq analysis (GSE267134). They analyzed differentially expressed genes (DEGs) from three independent experiments in which U2OS cells were treated either by scrambled (siSCR) or by siRNAs targeting SURF2. SURF2 depletion significantly affected gene expression with 588 down-regulated genes and 727 up-regulated ones. Gene Set Enrichment Analysis (GSEA) was conducted to identify pathways affected by SURF2 depletion among all Hallmarks pathways. Interestingly, the most significantly up-regulated pathway was the p53 pathway whereas the most significantly down -regulated ones were E2F, MYC and G2 / M checkpoint, all supporting a function of SURF2 in regulating cell proliferation, including cell. Moreover, the data reinforce the functional relationship between SURF2 and p53 pathways.

[0261] Since SURF2 is not directly involved in ribosome synthesis, the inventors therefore sought to characterize the role of SURF2 relative to free -5 S RNP These particles are known to induce Nucleolar Stress (NS) response, by promoting p53 stabilization and expression of its target gene p21 / CDKNlA, which in turn triggers G1 arrest and / or apoptosis. Using siRNAs and western-blots, the inventors first investigated whether SURF2 depletion could promote activation of p53 pathway in absence of NS exposure by western blots. As a control, the inventors used cells treated with scrambled siRNAs. After SURF2 depletion alone in U2OS cells, the inventors observed increased protein levels of p53, MDM2 and p21 compared to control conditions (Fig 2F and 2G).

[0262] To determine whether the activation of p53 induced by SURF2 depletion was related to or independent from free 5S RNP function, the inventors repeated SURF2 depletion in the absence of RPL5 or RPL11, conditions known to abolish p53 stabilization (K. E. Sloan, et al. Cell Rep 5, 237-247 (2013)) (Fig 2H and 21). Following the treatments with various combination of siRNAs, accumulation of NS-related proteins involved in p53 regulation was examined by western-blots (Fig 2H). As already shown elsewhere, both depletion of RPL5 or RPL11 affect the stability of RPL11 and RPL5, respectively (S. Bursae, et al. Proc. Natl. Acad. Sci. U.S.A. 109, 20467-20472 (2012)) (Fig 2H, lane 5 and 6 compare to 2 and 3). After U2OS cells treatment with siRPL5 or siRPLll, mild accumulations of p53 and p21 were observed (Fig 2H and 21). A greater accumulation of p53 was observed after RPL11 depletion, but without correlated p21 induction. This confirms previous results showing that although RPL5 and RPL11 depletion hinders ribosome assembly, their depletion impedes p53 and p21 accumulation by free - 5S particles (K. M. Hannan, et al. Cell Rep 41, 111571 (2022) ; K. E. Sloan, et al. Cell Rep 5, 237-247 (2013)). In this new set of experiments, the inventors were able to confirm by western-blots that SURF2 depletion significantly promotes both p53 and p21 accumulation (Fig 2H and 21). However, when SURF2 is co-depleted with RPL5 or with RPL11, the inventors could no longer observe any p21 stabilization and a strong reduction in p53 stabilization observed when the inventors only deplete for SURF2 (Fig 2H, lanes 5 and 6 and 21). This result indicates that SURF2 depletion effect on p53 activation is mediated by free-5S particles, independently of NS induction. Interestingly, depletion of either RPL5, RPL11 and SURF2 proteins affects the stability of the two other members of this group, strongly supporting a direct interaction between these three components (Fig 2H and 21).

[0263] 1.5 SURF2 depletion increases MDM2 binding to free-5S particles

[0264] As previously described, free-5S RNP promotes p53 activation through sequestration of MDM2, its primary negative regulator. To decipher how SURF2 might regulate free-5S RNP function, the inventors investigated whether and how SURF2 was related to MDM2. While studying SURF2 interactome, the inventors could not find any interaction of SURF2 with MDM2 nor with p53 (Fig 2A). This data indicates that the regulation of free 5S RNP function might not occur through a direct interaction between SURF2 and MDM2.

[0265] The inventors thus then tested how SURF2 might affect the binding of free 5S RNP particles to MDM2. First, the inventors performed IPs of free-5S RNP particles in the presence or absence of SURF2, using RPL5-Flag as bait from cellular extracts devoid of ribosomes to only focus on free-5S RNP particles. As the interaction between free-5 S RNP and MDM2 is enhanced after NS, the inventors performed these purifications before or after NS exposure by treating U2OS cells with low dose of actinomycin D (ACTD, as for Fig 1). The inventors used U2OS that do not express RPL5-Flag as a control (Figure 4A). As expected, only a mild but specific retention of MDM2 and p53 is observed in cells expressing RPL5- Flag under normal conditions. Importantly, no interaction between RPL5-Flag and p21 is noticed, confirming that p21 accumulation only reflects p53 activity. Under the same conditions, depletion of SURF2 specifically increases both interactions of MDM2 and p53 with RPL5-Flag (Fig 4A and 4B). Following NS induction by actinomycin D, both interactions of MDM2 and p53 with RPL5-Flag are also increased (Fig 4A and 4B). Remarkably, a further increase, statistically significant, of p53 retention with RPL5-Flag is observed when cells are both exposed to NS and depleted of SURF2 (Fig 4A and 4B) A similar trend was also observed for MDM2, although the variability in signal quantification between experiments could not support such statistical significance. These data indicate that SURF2 depletion promotes both MDM2 and p53 binding to free 5S RNP particles under normal conditions or upon nucleolar stress.

[0266] 1.6 SURF2 depletion increases cells sensitivity to NS

[0267] The inventors next wondered if SURF2 depletion could somehow modulate free-5 S RNP function in NS response. The inventors thus analyzed the levels of p53, MDM2 and p21 in the precedent IPs (Fig 4A). As the present quantification demonstrates, p53, p21 and MDM2 are all stabilized both after NS exposure (ACTD) or following SURF2 depletion. More importantly, depleting SURF2 in cell exposed to actinomycin D promotes a statistically significant increase in the accumulation of p53 and p21 compared to each condition independently (Figure 4C). This result indicates that the depletion of SURF2 increases the response to nucleolar stress in U2OS cells. It is well documented that in response to NS, free-5S promote p53 activity and consequently a cell cycle arrest in G1 (A. Domostegui, S. et al. Blood 137, 3351-3364 (2021); G. Donati, et al. Cell Rep 4, 87-98 (2013); J. Pelletier, et al. EMBO J 39, e 103838 (2020)). To confirm that SURF2 depletion increases U2OS cells response to NS, the inventors analyzed the effect of such treatments on cell cycle by flow-cytometry analysis (Fig 4D and 4E). As expected, exposure of cells to actinomycin D induced an accumulation of cells in G1 phase, reflecting an arrest in this phase. This arrest is also observed following SURF2 depletion alone (Fig 4D). Interestingly, inducing NS in cells depleted of SURF2 promotes an even stronger and statistically significant G1 arrest (Fig 4D and 4E). These data reinforce the idea that depletion of SURF2 increases cell response to NS. To confirm this observation, the inventors performed cell proliferation assays on the same number of U2OS cells for 36 h (Fig 4F and 4G). These cells were either depleted of SURF2 with siRNAs, exposed to actinomycin D or receiving both treatments. Cell proliferation was followed at different timepoints using crystal violet staining, and repeated three times. Both treatments with anti- SURF2 siRNAs and actinomycin D are reducing cell proliferation compared to scramble siRNAs (si SCR). Strikingly, addition of both treatments has the strongest effect on cell proliferation, and is accompanied with a loss of cellular material between 24 and 36 h of treatment (Fig 4G). This last observation suggests that on top of slowing down proliferation, the combination of both treatments promotes apoptosis, which was not obvious in cells treated with only one of these treatments independently. These results point towards the idea that inhibition of SURF2 can potentialize cell response to nucleolar stress.

[0268] To test this possibility, the inventors used higher concentrations of actinomycin D that promote more apoptosis, and then tested for the effect of SURF2 depletion on apoptosis induction using annexin V labelling using flow cytometry (Fig 4H). In these assays, actinomycin D or SURF2 depletion by siRNAs induce some apoptosis compared to control experiments (10% and 8% of dead cells, respectively). But the two treatments together significantly increase apoptosis (20%). The inventors propose that the combination of both treatments promotes apoptosis, in addition to slowing proliferation. To test if this effect of SURF2 depletion on drug sensitivity was only observed with actinomycin D, the inventors also tested other drugs known to induce nucleolar stress, namely 5-FU and BMH-21 (Fig 5A, 5B and 5C). There again, the inventors were able to observe an increased sensitivity to ribosome synthesis inhibition, with more G1 arrest and an increase in apoptosis in cells treated both with the drugs and siRNAs targeted towards SURF2 compared to control cells or cells receiving individual treatments (Fig 5A, 5B and 5C). Then, to assess if the negative effect of SURF2 depletion on cell cycle progression was linked to Mdm2 / p53 pathways, the inventors performed the same analyses in cell either depleted of p53 (siP53) or mutated for this gene (HCT116, TP53+ / + compared to HCT116, TP53- / -) (Fig 5D and 5E). In both cases, G1 arrest was significantly reduced and the inventors could observe more cells in S phase, strongly supporting a role of p53 in SURF2 depletion -mediated cell cycle defects. However, the absence of p53 does not fully compensate for SURF2 depletion effects on cell cycle, with some G1 arrest still visible, suggesting additional p53 independent effects of SURF2 depletion on cell cycle progression (Fig 5D and 5E).

[0269] Then, the inventors also showed that the combination of Doxorubicine treatment, another chemotherapeutic drug treatment and SURF2 depletion by siRNAs significantly increases together the accumulation of p53 (Fig 5F), confirming that SURF2 depletion increases apoptosis mediated by various chemotherapies through p53.

[0270] To confirm what the inventors observed in U2OS cells, the inventors repeated these experiments using another cancer cell line. The inventors chose the hepatocellular carcinoma (HCC) HepG2 cell line carrying a wild-type TP53 gene. Indeed, a recent study underlined the link between free-5S RNPs homeostasis and HCC (P. Cao, et al. Sci Adv 7, eabf4304 (2021)). There again, the inventors were able to show that SURF2 depletion promotes p53 activation and cell cycle arrest (data not shown). Moreover, combination of low dose of actinomycin D and SURF2 depletion increases cell proliferation defects and cell death (sub-Gl), that is not observed when cells are exposed to only one of these treatments (SURF2 depletion or actinomycin D) (data not shown).

[0271] 1.7 SURF2 overexpression impedes p53 activation following NS

[0272] As SURF2 depletion increases cell response to NS, its overexpression could inversely, inhibit cell response to NS. To test this hypothesis, the inventors developed a new U2OS Flip-In T-rex cell line with a tetracycline -induced overexpression of SURF2-Flag. To avoid misinterpretation due to side effects of SURF2 overexpression, the inventors verified that this induction did not alter ribosome assembly (data not shown). The inventors then compared by western-blot analysis the level of p53 and p21 in control cells treated or not with actinomycin D, with or without overexpression of SURF2-flag for 24 hours (Fig 6A). Quantification of these signals confirms that cells exposure to actinomycin D promotes p53 stabilization and activity, reflected by p21 increased level (Fig 6A). In stark contrast, in cells overexpressing SURF2, only a mild increase in p53 and p21 levels were observed (Fig 6A and 6B). These data indicate that overexpression of SURF2-Flag impedes free 5S RNP capacity to activate p53. To confirm this observation, the inventors performed cell cycle analysis by flow cytometry using the same cells and treatments, and quantified the percentage of cells in each phase (Fig 6C). While in normal cells, exposure to actinomycin D promotes G1 arrest, in cells that overexpress SURF2-Flag, this arrest is fully lost when cells are exposed to the same treatment (Fig 6C). This striking result, indicates that overexpression of SURF2 inhibits NS response in U2OS cells in this time frame. To test if the compensatory effect of SURF2 over-expression on p53 stabilization upon drug treatment, was related to its role nucleolar stress regulation, or was related to a more general p53 regulation pathway, the inventors repeated these experiments using either another drug (5-FU) that induce nucleolar stress and requires the free 5S RNPs, or drugs that regulate p53, independently of nucleolar stress : the proteasome inhibitor MG-132, or the broad-spectrum protein kinase inhibitor staurosporine (K. M. Hannan, et al. Cell Rep 41, 111571 (2022)). The inventors then compared the effect of SURF2 overexpression on both p53 and p21 stabilization promoted by these different drugs. The quantification of the p53 and p21 levels upon drug treatments, from three independent experiments, clearly shows that SURF2 overexpression compensates for p53 and p21 levels when cells are treated by 5-FU, as for actinomycin D. In contrast, SURF2 overexpression is not able to compensate for p53 nor p21 stabilization induced by MG-132 or staurosporine exposure. These data, strongly argue for a specific role of SURF2 overexpression on impairing both p53 and p21 stabilization upon drugs that induce nucleolar stress.

[0273] 1.8 SURF2 expression levels affect U2OS capacity to migrate.

[0274] The precedent data on SURF2 depletion or sur-expression that modulate NS stress sensitivity, clearly places SURF2 as a key factor able to regulate nucleolar stress response. Furthermore, the inventors have demonstrated that SURF2 is overexpressed in most cancers. In order to test how SURF2 expression could affect phenotypic traits of cancer cells, they developed knock-out SURF2 cell lines using CRISPR technology. To obtain these cell lines clearly demonstrates that this factor is not essential in U2OS, confirming DepMap data. The inventors then compared the capacity of control and of KO SURF2 cells to migrate in a wound healing assay (Fig 7A and 7B). The present results demonstrate that deletion of SURF2 impedes cells capacity to migrate. The inventors also used the same approach to assess the effect of SURF2 overexpression on wound healing assays (Fig 7C and 7D). During these experiments the inventors could observe that, conversely to its deletion, overexpressing SURF2 increases cancer cells capacity to migrate upon nucleolar stress induction (Actinomycin D treatment).

[0275] 1.9 SURF2 competes with MDM2 for free 5S RNP binding in vivo

[0276] The present results demonstrate that SURF2 expression modulates NS response in cancer cells by affecting MDM2 binding to free-5S RNP particles, potentially through a binding competition between these two factors. To test such mechanism of action, the inventors compared both MDM2 and SURF2 binding to free-5S RNP particles in control cells or in cells overexpressing SURF2-Flag from the U2OS Flip-In T-rex locus following exposure to NS. This time, to assess the binding of free 5S RNP to MDM2 or SURF2, the inventors used beads coupled with either anti-MDM2 or anti-SURF2 antibodies, and the same beads devoid of antibodies for control experiments (Fig 8A). In control cells (not overexpressing SURF2-Flag), the inventors can clearly observe specific retention of RPL5 and RPL11 on beads coupled to either MDM2 or SURF2 antibodies. This demonstrates that free-5S RNP particles can interact with these two factors. Interestingly, SURF2 was not retained by MDM2 immunoprecipitates, nor MDM2 on SURF2 ones, confirming that these proteins do not interact in cellulo, as suggested by the prior interactome analysis of SURF2 (Figure 2A and 2B).

[0277] Interestingly, in cell overexpressing SURF2-Flag, RPL5 and RPL11 are enriched in total cell extracts (see inputs Fig 8A). This enrichment must correspond to a stabilization of RPL5 and RPL11 in the complex formed by SURF2, RPL5, RPL11 and 5S rRNA (Fig 8A). Moreover, in cells overexpressing SURF2-Flag, despite the numerous repeats the inventors were not able to detect any signal for RPL5 nor RPL11 in the IPs using anti-MDM2 coupled beads. This observation demonstrates that overexpression of SURF2 impedes free-5S binding to MDM2 following treatment with actinomycin D. Suggesting that SURF2 and MDM2 associations to free-5S RNPS might be mutually exclusive.

[0278] 1.9 SURF2 interacts directly with RPL5 and RPL11 in vitro

[0279] Following nucleolar stress, MDM2 binds to free-5S RNP particles through direct contacts with RPL11 (J. Zheng, Y et al. Genes & Development 29, 1524-1534 (2015); N. M. Castillo Duque de Estrada, et al. Nat Struct Mol Biol 30, 1119-1131 (2023)). Furthermore, SURF2 and MDM2 do not interact with each other, and compete for free-5S RNP binding. From these two observations, the inventors postulated that SURF2 competes with MDM2 for direct contacts with free-5S particles. To test such hypothesis, the inventors performed GST pull-downs experiments from recombinants protein expressed in E. coli (Figure 8B). The inventors first overexpressed and purified recombinant RPL5-GST, RPL11-GST, or GST alone as a negative control. The inventors then mixed these purified proteins with same amount of recombinant SURF2-HIS and performed pull-down assays (Fig 8B). The inventors can observe both by Coomassie staining and western-blot, a weak but specific retention of recombinant SURF2 on beads coated with RPL5-GST or RPL 11 -GST but not with GST alone. Comparison of the SURF2-HIS signals allow to conclude that although SURF2 binds with both RPL5-GST and RPL 11 -GST, RPL 11 -GST retains more SURF2 than RPL5-GST (Fig 8B).

[0280] SURF2 and MDM2 are proteins that contain intrinsically disordered domains (Fig 8C). MDM2 binds to free-5S particles mainly through a direct interaction with RPL11, mediated by its zinc-finger domain (J. Zheng, et al. Genes & Development 29, 1524-1534 (2015)). A mutation in this domain (MDM2C305F) fully abolishes its interaction with free-5S particles (M. S. Lindstrom, et al. Mol. Cell. Biol. 27, 1056-1068 (2007)). To decipher by which domain SURF2 interacts with RPL5 and / or RPL11, the inventors performed a new set of GST pull-down experiment with truncated version of SURF2 (comprising amino acids 1-136), corresponding to its structured domain (here after termed SURF2-SD) (Fig 8C and 8D). Here again, the inventors observed a specific retention of SURF2-SD with beads coated with both RPL5-GST and RPL11-GST. Again, the binding of SURF2-SD seems stronger for RPL11 than for RPL5, which is reminiscent of what is observed for MDM2 zinc finger domain. The present results nonetheless indicate that by interacting preferentially with RPL11, both MDM2 and SURF2 compete for binding with free-5S RNP particles and that the SD domain of SURF2 is sufficient for its association to free 5S particles.

[0281] Morevover, the inventors showed that SURF2-SD structural domain overexpression, by competing with endogenous full-length SURF2 for binding with free-5S RNA particle acts as a repressor of endogenous SURF2 protein and promotes cell cycle arrest following nucleolar stress as observed during is knockdown by siRNAs treatment (Fig 9 A and B). These results present SURF2-SD as a potential inhibitor of endogenous SURF2 function.

[0282] Discussion

[0283] Free-5 S RP particles are key in the response to nucleolar stress, a cellular mechanism that is instrumental to cancer treatment by chemotherapeutic drugs. Thus, promoting the extra-ribosomal activity of free 5S RNPs in wild-type TP53 cancers may improve the p53 -dependent anticancer effects of therapeutic agents such as chemotherapy used in most poor-prognosis cancers. In order to understand how the function of these particles could be regulated at basal level and under nucleolar stress conditions, the inventors set out a characterization of these particles in U2OS cells. The inventors retrieved well-known partners of free 5S RNP particles, such as SSB / La and MDM2 (Fig 1A). SSB / La, a chaperone of 5S rRNA, binds this RNAjust after its transcription (J. Rinke, et al. Cell 29, 149-159 (1982)). The specific enrichment of SSB / La protein with RPL5-Flag suggests that during 5S particles biogenesis there is a transient interaction / exchange between 5S rRNA-La and 5S rRNA-RPL5 complex. Other known partners such as HEATR3 and BXDC1 / RPF2 were also found associated to RPL5-Flag but with a statistic significance below the threshold (-loglO(p-value)< 1) of 0.974 and 0.929 reciprocally. In addition, the inventors identified the tumor suppressor p53 as a partner of free 5S RNP particles, and were able to show that this interaction is mediated through MDM2 binding. So, how free 5S RNP particles fully inhibit MDM2 catalytic activity and promote p53 function remains elusive and will need further examination. Interestingly, the fact that the inventors can purify free 5S RNP particles even in absence of stress demonstrates that a portion of these particles are not incorporated into ribosomes even in proliferative cell state. This over production of free 5S RNP particles observed during the present experiment even in absence of stress finds its origin in the genomic and transcriptional independence of 5S rRNA compared to other rRNAs, that most likely fail to warrant a fully equilibrated production of rRNAs. Interestingly, 5S rRNA both genomic and transcriptional independence of 5S rRNA have been reinforced during evolution, going from an extra-copy of 5S rDNA in some bacteria such as E. coli. to a transcriptional independence in yeast .S', cerevisiae to a fully genomic separation in Humans. Therefore, having an imbalance in the production of 5 S rRNA compared to other rRNAs seems to give an advantage to the cells, but how this occurs remains an open question. In addition to these factors, the inventors found a potential new partner and precedingly uncharacterized factor: SURF2 (Figure 1A). SURF2 is a member of the surfeit genomic locus that contains 6 genes, all involved in promoting cell proliferation. Furthermore, it shares a bidirectional promoter with SURF1 that is positively regulated by the oncogene c-Myc, which highly supports cell proliferation (E. G. Vernon, et al. Biochim Biophys Acta 1492, 172-179 (2000); J. van Riggelen, et al. Nature Reviews Cancer 10, 301-309 (2010)). All these data indicate this factor as a potential regulator of free 5S RNP particles. In the course of its characterization, the inventors were able to show that this mainly nucleoplasmic factor is not directly involved in pre-ribosomes processing and only interacts with 5S RNP particles independently of ribosomes in their free state (Fig 2). The inventors then showed that in absence of SURF2, cells accumulate p53 and p21 in a free -5 S RNP dependent manner, with or without inducing nucleolar stress (Fig 2H, 21 and Fig 3A, 3C). Moreover, in response to nucleolar stress, induced by actinomycin D, cells depleted of SURF2 showed a greater accumulation of p53 and p21 (Fig 4 C) and a stronger arrest in G1 cell cycle with associated cell death compared to control cells (Fig 4D- H). This greater cell sensitivity to nucleolar stress in absence of SURF2 is associated with an increased ratio of free 5S RNP particles associated with MDM2 and p53 (Fig 4A and 4B). Reciprocally, an overexpression of SURF2 impedes p53 activation and cell cycle arrest which are normally induced by NS, and abolishes the interaction between free 5S RNP particles and MDM2 (Fig 6 and 8A). This strong correlation between NS response and SURF2 level is also supported by PanCancer analysis performed using TCGA data (Fig 3). The inventors found that high levels of SURF2 associate with poor overall survival in specific cancers such as Head and Neck squamous cell carcinomas, and adrenocortical carcinomas, as well as allowing to distinguish the outcome of patients displaying WT TP53 tumors. This position SURF2 both as a good anti -cancer therapeutic target and a prognostic marker, at least in these cancer types. To further characterize SURF2 and identify its mode of action, the inventors performed both in cellulo and in vitro analysis on SURF2, free 5S RNP particles and MDM2. Here, the inventors could show that in cellulo interaction of SURF2 or MDM2 with free-5S particles are mutually exclusive and that in vitro, SURF2 mainly interacts with free-5S particles through an interaction with RPL11 that involves SURF2’s structural domain (Fig 8).

[0284] All these observations support a functional model presented in Fig 10. In normal conditions (ie, without ribosomal stress), SURF2 acts as a buffer of free 5S RNP particles that could accumulate due to an imbalance in 5S rRNA production compared to other ribosomal components . However, when cells are stressed due to chemotherapeutic drugs for example, a greater portion of free-5S RNP particles accumulates in the nucleoplasm, and cannot be buffered by SURF2. There, free 5S RNP particles can bind MDM2 to promote p53 activation and cell cycle arrest. In absence of SURF2, in normal conditions, any small amount of free-5S can induce p53 activation and cell cycle slow-down. But upon nucleolar stress, a greater portion of free-5S RNP accumulates in nucleoplasm compared to normal cells, that promotes an even stronger response to NS. Finally, when SURF2 is over-expressed, free-5S particles released upon nucleolar stress are sequestered by SURF2, thus inhibiting their interaction with MDM2, which prevents p53 activation. This free 5S RNP buffering capacity of SURF2 could be hijacked by cancer cells to resist to p53 activation upon nucleolar stress, hence its overexpression found in many cancers compared to healthy tissues (Fig 3A).

[0285] Such regulation of free-5S RNPs by SURF2 represents a highly innovative therapeutic strategy in order to increase cancer cell sensitivity to chemotherapeutic drugs, since most of them rely on NS to activate p53 and induce cell death. This is supported by the negative correlation between overall survival and SURF2 expression level in some cancers. It is of particular interest to notice that co-treatment of U2OS cell by Actinomycin D and SURF2 siRNAs induce cell death whereas these treatments taken individually only promote cell cycle arrest. This suggests a synergistic effect of these two treatments. Furthermore, typical drawbacks of chemotherapies to treat cancer are the associated secondary effects. Hence, finding a way to reduce drug dosage by specifically increase cancerous cell sensitivity to stress (without affecting pathways equally important for healthy cells, such as ribosome production), could represent a mean to increase therapeutic window and to reduce such associated effects. Of note, another study underlines the link between 5S RNPs homeostasis and HCC cancers, suggesting a particular link between some cancers and 5S RNPs metabolism, which needs to be studied in depth (P. Cao, et al. Sci Adv 7, eabf4304 (2021)).

[0286] Free 5 S RNP homeostasis is also key in a group of diseases originating from ribosome production defects and regrouped as ribosomopathies (M. Aubert, et al. Biomolecules 2018 8(4):123 (2018). In Diamond- Blackfan Anemia (DBA), a well characterized ribosomopathy, activation of p53 by free -5 S particles is at the core of the etiology of these diseases, since some symptoms are linked to early p53 activation such as growth retardation, developmental problems and even erythropoiesis (A. Aspesi, V et al. Sci Rep 7, 12010 (2017); S. Le Goff, et al. Blood 137, 89-102 (2021); N. C. Jones, et al. Nat. Med. 14, 125- 133 (2008)). In addition, the patients suffering from these diseases show a higher cancer incidence compared to general population. Hence, DBA is associated with a 30-fold increased risk to develop acute myeloid leukemia (AML), osteosarcoma or colon cancer (A. Vlachos, et al. Blood 119, 3815— 3819 (2012)). Recently, two different studies on patients suffering from Shwachman -Diamond syndrome (SBDS), another ribosomopathy, nicely demonstrate how patients first harbor hypo- proliferative patterns due to SBDS mutations, then acquire compensatory mutations on TP53 to relieve from this growth selective pressure, and thus develop a pre-malignant cancer state (A. L. Kennedy, et al. Nat Commun 12, 1334 (2021); S. Tan, et al. Nat Commun 12, 5044 (2021); C. R. Reilly, et al. Blood 141, 1513-1523 (2023)). As a consequence, SURF2 being able to impede p53 activation following nucleolar stress represents an interesting therapeutic target. Indeed, mimicking SURF2 or part of it using small peptides or compound drug approaches could inactivate p53 through blocking free 5S RNPS - MDM2 interactions, and maybe alleviate symptoms and risk of developing cancers for ribosomopathy patients. It would also be interesting to investigate if there is any correlation between SURF2 expression level and the variation of symptoms severity observed between patients from the same family.

[0287] Of note, over-expression of SURF2 in U2OS cells does not seem to alter ribosome synthesis nor cell fitness as seen by cell cycle analysis (data not shown). First, this observation validates this strategy although testing this over-expression in more relevant cellular models will be needed in the future. Second, this implies that 5S RNP integration into ribosome is not affected by SURF2 over-expression, suggesting that SURF2 only interacts with free 5S RNPs that are released from or not integrated into pre-ribosomes. How free 5S RNP particles interact with their different partners and how this is regulated in cells remains to be elucidated.

[0288] In summary the work presented here, demonstrates how free 5S are key in cell stress response, and highlight SURF2 as a therapeutic target to either increase cancer cells sensitivity to chemotherapeutic drugs or impede free 5S RNP function to reduce p53 -associated symptoms in genetic diseases such as ribosomopathies.

[0289] 2. Materials and Methods

[0290] 2.1 Cell lines

[0291] The U-2OS Flp-In T-Rex cell lines were produced according manufacturer’s instruction (Invitrogen / Thermo Fisher). The cDNAs of RPL5 or SURF2 were cloned into a pcDNA5-FRT-TO vector to enable expression of the protein with a C-terminal 2xFLAG-Pre Scission protease site-His6 (FLAG) tag. Alternatively, the cDNA of SURF2 was fused to EGFP tag and cloned into pcDNA5-FRT- TO plasmid. These plasmids or the empty plasmids (control) were co-transfected with a pOG44 plasmid into Flp-In T-Rex U-2OS or HepG2 cells and cells that had stably integrated the plasmid into their genome were selected using Hygromycin B, according to the manufacturer’s instructions. Expression of tagged proteins was induced by addition of Ipg / mL of tetracycline for 24h prior harvesting.

[0292] All the cell lines were maintained in high -glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and ImM sodium pyruvate. The cells were incubated at 37°C in a humidified incubator containing 5% CO2. Flip-In T-Rex cells were maintained using hygromycin B (30 pg / mL, InvivoGen, ant-hg-5) and blasticidin (30 pg / mL, InvivoGen, ant-bl-1: 1). The expression of proteins was induced with tetracyclin (1 pg / mL; Sigma- Aldrich, T7660). Cells were treated with actinomycin D (10 ng / mL, Sigma-Aldrich, A9415).

[0293] A pool of four siRNA duplexes from eurogentech were used to target SURF2 mRNA

[0294] (CUG-CAA-GUG-AUG-ACA-GCA-U / AUG-CUG-UCA-UCA-CUU-GCA-G,

[0295] GGA-GGG-AGG-ACC-AGA-UGG-A / UCC-AUC-UGG-UCC-UCC-CUC-C,

[0296] AAG-CAC-AUG-CCG-UGA-AGU-U / AAC-UUC-ACG-GCA-UGU-GCU-U and CCA-GCG-AGC-UCU-GUG-UAA-A / UUU-ACA-CAG-AGC-UCG-CUG-G, SEQ ID NO: 7 to 10). On duplex for RPL11 mRNA (AAG-GUG-CGG-GAG-UAU-GAG-UUA / UAA-CUC-AUA-CUC- CCG-CAC-CUU, SEQ ID NO: 11) and on duplex for RPL5 mRNA (GCC-ACA-AUG-UUG-CAG- AUU-A / UAA-UCU-GCA-ACA-UUG-UGG-C, SEQ ID NO: 12) were used. Each siRNA solution was added at a final concentration of 5 pM to 106cells diluted in ZAP buffer (lOmM sodium phosphate buffer, pH 7.25, containing 250 mM sucrose and 1 mM MgC12). Electroporation was performed at 240 V with a Gene Pulser (Bio-Rad). After 5 min incubation at RT, cells were plated and grown at 37°C for 48h. Depletion of SURF2 was completed with a second round of siRNA treatment (96h total). Control cells were electroporated with a scramble siRNA (siRNA-negative control duplex; Eurogentec).

[0297] 2.2 Label-free quantitative proteomics

[0298] Protein preparation for 5S particle partners identification

[0299] For the proteome analysis, cells were prepared in quadruple biological replicates for four conditions: i) U2OS cells (control); ii) U2OS cells overexpressing RPL5-flag; iii) control cells treated with Actinomycin D; iv) U2OS cells overexpressing RPL5-flag and treated with actinomycin D (10 ng / mL, Sigma-Aldrich A9415). RPL5-Flag proteins and associated complexes were immunoprecipitated using the same protocol as “proteins immunoprecipitation after sucrose cushion” until the elution step. Trapped proteins on anti-Flag beads were eluted using Flag buffer (20 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM MgC12) supplemented with 0.4 mg / mL 2xflag peptide (H- MDYKDDDDKGTDYKDDDDKG-OH, Schafer, SEQ ID NO: 13), precipitated by TCA (Sigma- Aldrich, T9159) and glycogen (Thermo Scientific, R0551) and re-suspended in a buffer containing 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM MgC12, 5% glycerol, 5 % SDS. Then the protocol “Trypsin digestion and mass spectrometry analysis ” was followed.

[0300] Protein preparation for SURF2 partners identification

[0301] For the proteomic analysis, U2OS control cells were prepared in quadruple biological replicates for two conditions: i) GAPDH immunoprecipitation and ii) SURF2 immunoprecipitation. Cells were harvested, washed with PBS with ImM EDTA, resuspended in buffer E (20 mM Tris-HCl pH 7,5, 200 mM NaCl, 5 mM MgC12, 0.5 mM EDTA, 0.2% Triton, ImM DTT, complete protease inhibitor cocktail (Roche), RNase ribonuclease inhibitor (Promega, N261B)) and disrupted with a Bioruptor Sonicator by sonication (2 min, 5 s / 5 s on / off, 20% amplitude). Cell debris were removed by centrifugation (10 min, 14,000 g, 4 °C). Protein concentrations of the extracts were determined using a Bio-Rad protein assay kit (Biorad, 5000006). The same amounts of proteins were incubated with antibodies (anti-SURF2 or anti-GAPDH antibodies) coupled to protein G sepharose beads (Cytiva, 17061801) for 2 h at 4 °C. After immunoprecipitation, beads were washed tree times with buffer E (20 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM MgC12, 0.5 mM EDTA, 0.2% Triton, 1 mM DTT) and the associated proteins were eluted with 2X buffer (100 mM Tris-HCl pH 7.5, 10% glycerol, 10 % SDS). Then the protocol "Trypsin digestion and mass spectrometry analysis ” was followed.

[0302] Trypsin digestion and mass spectrometry analysis

[0303] Disulfide bonds were reduced with 25 mM DTT for 5 min at 95°C under agitation followed by an alkylation of cysteine residues in 60 mM iodoacetamide for 30 min in the dark at room temperature. Each reduced / alkylated protein sample was then digested using the S-Trap™ Mini spin column protocol (63). Briefly, undissolved matter was removed by centrifugation for 8 min at 13,000g. 12% aqueous phosphoric acid was added at 1: 10 to the protein sample for a final concentration of -1.2% phosphoric acid followed by seven volumes of S-Trap binding buffer (90% methanol, 100 mM TEAB, pH 7.1). After gentle mixing, the protein solution was loaded onto an S-Trap filter by centrifugation at 4,000 g. Afterwards, the captured proteins were washed six times with 400 pL S-Trap binding buffer. Digestion was performed over-night at 37°C by addition of 20 pL of trypsin (Sequencing Grade Modified Trypsin, Promega) at 37.5 ng / pL in 50 mM ammonium bicarbonate. The digested peptides were eluted by addition of 40 pL of 50 mM ammonium bicarbonate, followed by 40 pL of 0.2% formic acid (FA), and finally 35 pL of 50% aqueous acetonitrile containing (ACN) 0.2 % FA. Each elution was performed at 4,000 g during 1 min. The different eluates were pooled together, dried down, resuspended in 20 pl of 0.05% trifluoroacetic acid (TFA) in 2% ACN, and sonicated for 10 min before analysing by online nanoLC using an UltiMate® 3000 RSLCnano LC system (ThermoScientific, Dionex) coupled to an Orbitrap Fusion™ Tribrid™ mass spectrometer (Thermo Scientific, Bremen, Germany) operating in positive mode. 200 ng of each sample were loaded onto a 300 pm ID x 5mm PepMap C18 pre-column (Thermo Scientific, Dionex) at 20 pL / min in 2% ACN, 0.05% TFA. After 3 min of desalting, peptides were on-line separated on a 75 pm ID x 50 cm C18 column (in-house packed with Reprosil C18-AQ Pur 3 pm resin, Dr. Maisch; Proxeon Biosystems, Odense, Denmark) equilibrated in 90% of buffer A (0.2% FA), with a gradient of 10 to 30% of buffer B (80% ACN, 0.2% FA) for 100 min then 30% to 45% for 20 min at a flow rate of 300 nL / min. The instrument was operated in data-dependent acquisition (DDA) mode using a top-speed approach (cycle time of 3 s). Survey scans MS were acquired in the Orbitrap over 350-1400 m / z with a resolution of 120,000 (at 200 m / z), an automatic gain control (AGC) target value of 4e5, and a maximum injection time of 60 ms. Most intense multiply charged ions (2+ to 6+) per survey scan were selected at 1.7 m / z with quadrupole and fragmented by Higher Energy Collisional Dissociation (HCD). The monoisotopic precursor selection was turned on, the intensity threshold for fragmentation was set to 25,000 and the normalized collision energy was set to 28%. The resulting fragments were analyzed in the Orbitrap with a resolution of 30,000 (at 200 m / z), an automatic gain control (AGC) target value of 5e4, and a maximum injection time of 54 ms. Dynamic exclusion was used within 60 s with a 10 ppm tolerance, to prevent repetitive selection of the same peptide. For internal calibration the 445.120025 ion was used as lock mass. MS-based protein identification

[0304] Acquired MS and MS / MS data as raw MS files were converted to the mzDB format using the pwiz- mzdb converter (version 0.9.10, https: / / github.com / mzdb / pwiz-mzdb) executed with its default parameters. Generated mzDB files were processed with the mzdb-access library (version 0.7, https: / / github.com / mzdb / mzdb-access) to generate peaklists. Peak lists were searched against UniProtKB / Swiss-Prot protein database with homo sapiens taxonomy in Mascot search engine (version 2.6.2, Matrix Science, London, UK). Cysteine carbamidomethylation was set as a fixed modification and methionine oxidation as variable modification. Up to two missed trypsin / P cleavages were allowed. Mass tolerances in MS and MS / MS were set to 10 ppm and 0.6 Da, respectively. Validation of identifications was performed through a false -discovery rate set to 1% at protein and peptide -sequence match level, determined by target-decoy search using the in-house -developed software Proline software version 1.6.

[0305] 2.3 RNA-seq analysis

[0306] Total RNA extraction

[0307] U2OS cells were treated with either siSCR or siSURF2 siRNAs for 96 hours in 10 cm dishes, with three independent replicates. Total RNAs were extracted with TRizol following manufacturer procedure. Total RNA concentration and RNA integrity of each sample were determined with NanoDrop and Qubit 4 Fluorometer (Thermo Fisher Scientific). RNASeq library preparations and sequencing reactions were conducted by GENEWIZ.

[0308] Bioinformatics

[0309] Initial bioinformatics analysis of the RNASeq was conducted by GENEWIZ. Briefly, data was generated with an Illumina HiSeq 2 x 150 PE HO configuration. Sequence reads were trimmed to remove adapter sequences and nucleotides with poor quality (Trimmomatic v.0.36). Using the STAR aligner v.2.5.2b the trimmed reads were mapped to the Homo sapiens GRCh38pl3 reference genome available on ENSEMBL. Gene expression between distinct groups were compared using DESeq2 package. The Wald test was used to generate P-values and log2 fold changes. Genes with an adjusted P-value < 0.05 and absolute log2 fold change > 1 were called as differentially expressed genes for each comparison. Raw data are deposited on the GEO service (GSE267134).

[0310] For the GSEA analysis, the GSEA software package (Desktop v4.3.3) developed by the MIT / BROAD Institute was used. Gene sets H.all.v2023.2.Hs.symbols.gmt was used. All gene set files for this analysis were obtained from GSEA website www.broadinstitute.org / gsea / . Enrichment map was used for visualization of the GSEA results. GSEA computes four key statistics for the gene set enrichment analysis. GSEA P-values were derived from permutation testing and corrected for multiple testing using the False discovery rate (FDR) method. Enrichment score (ES) and FDR value were applied to sort SURF2 depleted and control cells genes-enriched after gene set permutations were performed 1000 times for the analysis. Gene Network Analysis was based on DEGs (log2foldchange > 1).

[0311] 2.4 Immunoblotting assays

[0312] Protein extraction was done in ice-cold lysis buffer (1% Triton, 50 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM EDTA and complete protease inhibitor cocktail (Roche)). Protein concentrations of the extracts were determined using a Bio-Rad protein assay kit (Biorad, 5000006). Proteins were diluted in Invitrogen 2X sample buffer (NuPAGE™ LDS Sample Buffer (4X) (NP0007) and NuPAGE™ Sample Reducing Agent (1 OX) (NP0009)) and 20 pg / lane of protein were loaded on NuPAGE™ 4 to 12 %, BisTris protein gels (Invitrogen, NP0321BOX) and transferred to nitrocellulose membrane using the TransBlot Turbo Transfer System from Biorad. Membranes were immunoblotted with a primary antibody, followed by incubation with a secondary antibody coupled with HRP. The blots were visualized using the Clarity Western ECL kit from Biorad. The following antibodies were used: p53 (Invitrogen, MAS- 12557 (DO7)), p21 (CDKNlA / p21CIPl AB clonal, A2691), SURF2 (Bethyl lab, A304-611A), Flag (MERCK, F3165), GAPDH (Genetex, GTX627408), RPL11 (Invitrogen, 37-3000), RPL5 (Invitrogen, PA5-102539), RPL17 (Gentech, GTX111934), Fibrilarin (collaborators), MDM2 (SMP14 Santa Cruz, SC965), Actin (Sigma, A4700).

[0313] 2.5 Cell cycle assays

[0314] The cell cycle was analyzed by flow cytometry. Cells were harvested by trypsinization, fixed in 70% ethanol, and stained with DAPI (1 pg / ml; Sigma, D9542) in PBS completed with RNase A (100 pg / ml, Thermo Scientific, EN0531) for 30 min at RT. Samples were then analyzed for their DNA content using CytoFLEX S Flow Cytometer and CytExpert software.

[0315] 2.6 Apoptosis assays

[0316] Cell death was quantified by using the Annexin V-FITC & Propidium Iodide (PI) Dead Cell Apoptosis kit from Invitrogen (V13242). In brief, cells depleted for SURF2 and treated with the indicated drugs were harvested and washed in cold PBS. Then, 1.105cells were resuspended in 100 pL of incubation buffer containing Annexin V-FITC and propidium iodide (PI) and incubated in the dark for 15 min. Data were acquired on a CytoFLEX S Flow Cytometer and CytExpert software.

[0317] 2.7 Viability assays

[0318] Cells depleted for SURF2 were plated in 6 well culture dishes. After 12h, cells were treated with actinomycin D (10 ng / mL, Sigma- Aldrich A9415) then, at each time point, cells were incubated with 400 pL of 1% crystal violet staining solution (Sigma- Aldrich, V5265) for 20 min at RT. Cells were washed 2 times with PBS and 3 pictures were taken under microscope. Then, crystal violet was resolubilized with 33% acid acetic and diluted into 96 well plate to read optical density OD595 with a plate reader.

[0319] 2.8 Northern-blot assays

[0320] Total RNAs were extracted with Trizol from cell pellets containing 20x106cells. The aqueous phase was extracted with phenol -chloroform-isoamylic alcohol (25:24: 1; Sigma), then with chloroform. Total RNAs were recovered after precipitation with 2-propanol. For northern blot analyses, 3pg / lane of total RNAs were separated on two types of gels. Long RNAs were separated on a 1.2% agarose gel containing 1.2 % formaldehyde and Tri / Tri buffer (30 mM triethanolamine, 30 mM tricine, pH 7.9). Small RNAs were separated on 6% polyacrylamide gel containing 7% urea and TBE buffer (90 mM Trizma base, 90mM Boric acid, 2mM EDTA). Then, RNAs were transferred to a Hybond N+ nylon membrane by a passive transfer overnight. Pre -hybridization was performed for 1 h at 45° C in a buffer containing 6x SSC, 5 Denhardfs solution, 0.5 % SDS and 0.9 g / mL tRNAs). The 5 '-radiolabeled oligonucleotide probe were incubated overnight. The sequences of the probes were: ITS1: CCT-CGC-CCT-CCG-GGC- TCC-GTT-AAT-GAT-C (SEQ ID NO: 14), ITS2: GCG-CGA-CGG-CGG-ACG-ACA-CCG-CGG-CGT (SEQ ID NO: 15), + CTG-CGA-GGG-AAC-CCC-CAG-CCG-CGC-A (SEQ ID NO: 16), 18S: CCG- GCC-GTC-CCT-CTT-AAT-CAT-GGC (SEQ ID NO: 17), 28S: CCC-GTT-CCC-TTG-GCT-GTG-GTT- TCG-CTG-GAT-A (SEQ ID NO: 18), 5.8S: GGG-GCG-ATT-GAT-CGG-CAA-GCG-ACG-CTC (SEQ ID NO: 19), 5S: CCU-CGC-CCU-CCG-GGC-UCC-GUU-AAU-GAU-C (SEQ ID NO: 20). Membranes were washed twice for 10 min in 2 SSC, 0.1% SDS and once in l x SSC, 0.1% SDS, and then exposed. Signals were acquired with a Typhoon Trio PhosphoImager (GE Healthcare) and quantified using the MultiGauge software.

[0321] 2.9 Immunofluorescence assays

[0322] The expression of SURF2-GFP was induced to the same level of the endogenous SURF2 protein (tetracycline at 5 ng / mL for 24h). Cells were seeded in 12-well plates on microscope cover glasses and grown for 24h. Cells were fixed with 4% paraformaldehyde for 5 min, permeabilized with (0.1% Triton X-100 and 0.02% SDS in PBS). Fixed cells were incubated in blocking solution 2% BSA in PBS for 30 min and incubated overnight at 4°C with anti-fibrillarin antibodies at 1:200. Cells were washed 3 times for 5 min with (2% BSA in PBS), and subsequently incubated for 30 min with secondary antibodies (Alexa Fluor goat anti -mousse IgG (H+L) / Alexa Fluor 647 secondary antibody (Invitrogen, A21236) at 1: 1000. After 3 washes, cells were incubated briefly in 0.1% Triton X-100, 0.02% SDS in PBS, and post-fixed with 4% PFA in PBS. Cells were incorporated with DAPI (Ipg / mL, Sigma, D9542) for 10 minutes. After washes with PBS, coverslips were mounted in Mowiol. Imaging was performed on a Leica TCS SP8 MP Multiphoton microscope. Images were captured in confocal mode using x63 objective. Image analyses were performed using Image J software.

[0323] 2.10 Sucrose sedimentation profiling Buffers contains cycloheximide (10 pg / mL, Merck, C7698) at each step of this protocol. 50xl06cells were harvested and resuspended in a lysis buffer (10 mM Hepes KOH, pH 7.9, 1.5 mM MgC12, 10 mM KC1, 1 mM DTT, 10 pg / mL cycloheximide). Then cells were homogenized with aDounce tissue grinder on ice with atight pestle and centrifuged at 1000g for 10 min at 4 °C. The top soluble phase was clarified through two centrifugations at 10 000g for 15 min at 4 °C. Supernatants were collected and protein concentrations were determined using a Bio-Rad protein assay kit (Biorad 5000006). 750 pg of extracts were loaded on a 10-50% sucrose gradient. Gradients were centrifuged at 36,000 rpm for 2 h at 4 °C in an Optima L-100XP ultracentrifuge (Beckman-Coulter). Following centrifugation, the fractions were collected using a Foxy Jr fraction collector (Teledyne ISCO) and the absorbance at 254 nm was measured with a UA-6 device (Teledyne ISCO). For protein analyses, 250 pL of each fraction were precipitated with TCA (Sigma- Aldrich, T9159) and glycogen (Thermo Scientific, R0551) and protein pellets were resuspended in Invitrogen 2X sample buffer (NuPAGE™ LDS Sample Buffer (4X) (NP0007) and NuPAGE™ Sample Reducing Agent (10X) (NP0009)).

[0324] 2.11 Cell fractionation following PSE method

[0325] Cells were harvested by scrapping with PBS and pelleted by centrifugation. Cells were gently resuspended in SN1 buffer (20 mM HEPES-NaOH (pH 7.5), 130 mM KC1, 10 mM MgC12, 0.05% Igepal, 600U / mL RNasin ribonuclease inhibitor (Promega, N261B)) completed with cOmplete protease inhibitor (1 / 30) (Roche) and centrifugated (3,800 rpm, 3 minutes, 4°C). SN 1 supernatants were collected and proteins were diluted in Invitrogen 2X sample buffer (NuPAGE™ LDS Sample Buffer (4X) (NP0007) and NuPAGE™ Sample Reducing Agent (10X) (NP0009)). SN1 was then store at -80°C. The previous pellets of cellular extracts were vortexed with SN1 buffer and spined down (3,800 rpm, 3 min, 4°C). The supernatants were eliminated and the pellets were resuspended in SN2 buffer (10 mM HEPES-NaOH (pH 7.5), 10 mM NaCl, 5 mM MgC12, 0.1% Igepal, 0.5 mg / mL heparin, 600 U / ml RNasin ribonuclease inhibitor (Promega, N261B)) completed with Dnase I (Invitrogen, 18068015), then incubated 10 min at RT with gentle mixing. Extracts were centrifugated (11,500 rpm, 10 min, 4 °C). SN2 supernatants were collected and proteins were diluted in Invitrogen 2X sample buffer (NuPAGE™ LDS Sample Buffer (4X) (NP0007) and NuPAGE™ Sample Reducing Agent (10X) (NP0009)). SN2 was then store at -80°C. Finally, remaining pellets of cellular extracts were resuspended with SN3 buffer (20 mM HEPES-NaOH (pH 7.5), 200 mM NaCl, 4 mM EDTA, 0.1% Igepal, 0.04% sodium deoxycholate, 4 mM imidazole, 0.1 mg / ml heparin, ImM DTT, cOmplete protease inhibitor (1 / 100), 600 U / ml RNasin ribonuclease inhibitor (Promega, N261B)) and mixed for 20 minutes at RT. Extracts were centrifugated (11,500 rpm, 10 min, 4°C). SN3 supernatants were collected and proteins were diluted in Invitrogen 2X sample buffer (NuPAGE™ LDS Sample Buffer (4X) (NP0007) and NuPAGE™ Sample Reducing Agent (10X) (NP0009)). SN3 was then store at -80°C. Proteins were analyzed by western blots. 2.12 Proteins immunoprecipitation after sucrose cushion

[0326] Cells were harvested, washed with ImM EDTA in PBS, resuspended in buffer E (20mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM MgC12, 0.5 mM EDTA, 0.2% Triton, 1 mM DTT, complete protease inhibitor cocktail (Roche), RNasin ribonuclease inhibitor (Promega, N261B)) and disrupted with a Bioruptor Sonicator by sonication (2 min, 5 s / 5 s on / off, 20% amplitude). Cell debris were removed by centrifugation (10 min, 14,000 g, 4 °C). Protein concentrations of the extracts were determined using a Bio-Rad protein assay kit (Biorad, 5000006). The same amounts of proteins were loaded on a double sucrose cushion (20% and 30% sucrose) and centrifuged at 190,000 g for 2 h at 4 °C in an Optima L- 100XP ultracentrifuge (Beckman-Coulter). Extracts were incubated with pre-washed anti-Flag beads (Sigma, A2220) or with antibodies (anti-SURF2 or anti-MDM2 antibodies) coupled to protein G sepharose beads (Cytiva, 17061801) for 2 h at 4 °C. 10 % of inputs were conserved, precipitated with TCA (Sigma- Aldrich, T9159) and glycogen (Thermo Scientific, R0551), pellets were resuspended with Invitrogen 2X sample buffer (NuPAGE™ LDS Sample Buffer (4X) (NP0007) and NuPAGE™ Sample Reducing Agent (10X) (NP0009)). After immunoprecipitation, beads were washed tree times with Flag buffer (20 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM MgC12) and the associated proteins were eluted with Invitrogen 2X sample buffer (NuPAGE™ LDS Sample Buffer (4X) (NP0007) and NuPAGE™ Sample Reducing Agent (10X) (NP0009)).

[0327] 2.13[5’32P1pCp labelling of immunoprecipitated RNAs

[0328] Cells were harvested in TBS buffer (150 mM NaCl, 40 mM Tris-HCl pH 7.4), resuspended in lysis buffer (150 mM NaCl, 0.05% Igepal, 50 mM Tris-HCl pH 7.4, 5 mM MgC12) completed with complete protease inhibitor cocktail (Roche) and disrupted with a Bioruptor Sonicator by sonication (2 min, 5 s / 5 s on / off, 20% amplitude). Cell debris were removed by centrifugation at 16,000 g for 10 min and the clarified extracts were incubated with antibodies (anti-SURF2 or anthi-HEXIMl antibodies) coupled to protein G sepharose beads (Cytiva, 17061801) for 2 h at 4 °C. Then, beads were washed 3 times and immunoprecipitated RNAs were extracted using phenol -chloroform protocol. RNAs were incubated with t5’-32p] pCp and T4 RNA ligase (Promega, M1051) O / N at 4 °C. Then RNAs were precipitated with ammonium acetate and ethanol for 10 min at -70 °C and pelleted by centrifugation (10 min, 13,000 g). RNA pellets were washed with 70 % ethanol and recovered with a formamide loading buffer. Then, a loading dye buffer was added and RNA were analyzed on a 12 % acrylamide gel.

[0329] 2.14 In vitro pull-down assays

[0330] The cDNAs of the SURF2 or SURF2(1-136) were cloned into pScodon plasmid (open biosystem) in translational fusion with HIS6(His tag). RPL5 or RPL11 cDNAs were synthetized by Genscript and cloned into pGEX-6T. The expression and purification were essentially as described (66). Briefly, the proteins were expressed in the BL21 strain from Escherichia coli at 37°C in LB medium (Sigma) supplemented with 100 pg / mL ampicillin until ODeoo between 0.4 and 0.5. Recombinant protein expression was induced by adding 1 mM isopropyl-P-D-1 -thiogalactopyranoside, incubating overnight at 20°C, harvesting by centrifugation and cell pellets were frozen at -20°C. Cells pellets were resuspended in buffer A (300 mM NaCl, 20 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 10 mM B- mercaptonethanol, 10% glycerol, tablet roche, 5mM Imidazole) supplemented with complete EDTA- free protease inhibitors (Roche). Cells were lysed by sonication, and lysate was centrifuged at 20,000 rpm for 30 min. The cleared lysates were mixed as indicated for 2 hours at 4 °C. The mix was then loaded on gluthatione-sepharose beads pre -equilibrated with buffer A (300 mM NaCl, 20 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 10 mM B-mercaptonethanol, 10% glycerol, tablet roche, 5mM Imidazole) and mixed for 2 h at 4 °C. Beads were then washed 4 times with buffer A and resuspended in loading dye (IX LDS with denaturing reagent from Invitrogen) and loaded on Nu-PAGE 4-12% gels using IX MOPS as running buffer.

[0331] 2.15 Wound-healing assays

[0332] 4.5 to 5xl05cells were seeded in 6-well plates. After 48 h of growth, the confluent cell monolayer was ablated with a 20 pL tip. The wells were then rinsed 3x with PBS IX and incubated in DMEM supplemented with 3% SVF at 37°C. When indicated cells were then treated with 0.5 pg / mL tetracycline per well. Images were taken at the EVOS Floid cell imaging station at TOh and T24h. Scar size was analyzed by Image J software using the Wound Healing size tool.

[0333] 2.16 Pancancer analysis

[0334] Different databases were used to export data of interest, namely transcriptome quantification, clinical and genomic data.

[0335] GTEx: (https: / / gtexportal.org / home / ) Genotype-Tissue Expression is a public platform containing molecular data of healthy tissues derived from people of all age gender and ethnicity.

[0336] TCGA: (https: / / www.cancer.gov / ccg / research / genome-sequencing / tcga) The Cancer Genome Atlas is a public databased compiled from the National Cancer Institute’s CDM portal. It includes 33 cancers from 11,000 patient samples over 12 years and contains annotated clinical data and molecular data.

[0337] TARGET:(https: / / www.cancer.gov / ccg / research / genome-sequencing / target / about) therapeutically Applicable Research to Generate Effective Treatments is a public platform dedicated to molecular characterization of paediatric cancers with available clinical, genomic and transcriptomic data. cBioPortal: (https: / / www.cbioportal.org / ) This public portal contains data from over 300 multidimensional studies in open-access. It includes genomic, transcriptomic, molecular and clinical data from multiple datasets. It allows exploratory analysis and corresponds to a global visualization webtool, which includes exportation of data from TCGA, TARGET, ICGC and other individual datasets. The raw data are not directly downloadable on the portal. XENA UCSC: (htps: / / xena.ucsc.edu / ) The University of California Santa Cruz (UCSC) Xena browser. This public data portal contains over 1,500 datasets and 50 different types of cancers with clinical, genomic, transcriptomic and other type of data. It enables interactive exploratory analysis and exportation of accurate data of interest from TCGA, ICGC, GDC, TARGET, GTEx and other databases.

[0338] 2.17 Statistical Analysis

[0339] Label-free quantitative proteomics analysis

[0340] Proteins enriched over 2-fold with a p-value below 0.05 were considered significantly enriched. For label-free relative quantification across samples, raw MS signal extraction of identified peptides was performed using Proline. The cross-assignment of MS / MS information between runs was enabled, allowing to assign peptide sequences to detected but non -identified features. Each protein intensity was based on the sum of unique peptide intensities and was normalized across all samples by the median intensity. Missing values were independently replaced for each run by its 5% quantile. For each pairwise comparison, an unpaired two-tailed Student’s t-test was performed and proteins were considered significantly enriched when their absolute log2-transformed fold change was higher than 1 and their p- value lower than 0.05. To eliminate false-positive hits from quantitation of low intensity signals, two additional criteria were applied: only the proteins identified with a total number of averaged peptide spectrum match (PSM) counts>4 and quantified in a minimum of two biological replicates, before missing value replacement, for at least one of the two compared conditions were selected. The p-value and fold change were calculated between the IP groups from RPL5_Flag overexpressing cells and the control IP groups (IPs with the a-flag antibody). The same statistical analysis was performed on equivalent IP groups resulting from cells incubated with Actinomycin D during 24 h. Volcano plots were drawn to visualize significant protein abundance variations between conditions in the presence or absence of drogue. They represent -log 10 (p-value) according to the log2 ratio. The complete list of proteins identified and quantified in immunopurified samples and analysed according to this statistical procedure is described in Supplementary file.

[0341] General Data Analysis

[0342] Data are expressed as means ± SD. All statistical data (n>3) were calculated using GraphPad Prism. Statistical details and significance reports can be found in the corresponding figure legends.

[0343] PanCancer analysis

[0344] Datasets were uploaded from XENA UCSC portal (htps: / / xena.ucsc.edu / ) that compiled normalized gene expression levels from normal (GTEX) and tumoral (TCGA) tissues, as well as related clinical and genomic data of tumoral tissues (Goldman et al, Nat Biotechnol 2020; GTEx consortium, Nat Genet 2013; Grossman et al, New England J Medecine 2016). After data export and transformation, tumor datasets were built by conserving only tumoral tissues collected at diagnosis from non -metastatic patients. A quality control was performed to verify normal distribution of gene expression dataset (Shapiro test) or cohort consistency (follow-up median, survival association with gold-standards such as metastatic relapse). Comparison of gene expression between two conditions was assessed using nonparametric test (Wilcoxon -test). Survival was investigated by plotting Kaplan -Meier survival curves and log-rank tests using median as cut-off of gene expression. Overall survival (OS) corresponds to the length of time from the date of cancer diagnosis to the date of death or censoring, while Progression- Free survival (PFS) corresponds to the length of time from the date of cancer diagnosis to the date of progression (i.e., death or local / distant relapse) or censoring. The statistical significance was based on p-value < 0.05 where the HO hypothesis is rejected. Statistics and visualization were performed using R studio (version 4.2.2).

[0345] SEQUENCES FOR USE IN PRACTICING THE INVENTION

[0346] The first methionine allowing to initiate protein synthesis, it is understood that the first methionine of the following sequences can be removed, for example when said sequences are comprised in a fusion protein.

[0347] SEQ ID NO: 1: human SURF2 protein:

[0348] MSELPGDVRAFLREHPSLRLQTDARKVRCILTGHELPCRLPELQVYTRGKKYQRLVRASPAFD YAEFEPHIVPSTKNPHQLFCKLTLRHINKCPEHVLRHTQGRRYQRALCKYEECQKQGVEYVP ACLVHRRRRREDQMDGDGPRPREAFWEPTSSDEGGAASDDSMTDLYPPELFTRKDLGSTED GDGTDDFLTDKEDEKAKPPREKATDEGRRETTVYRGLVQKRGKKQLGSLKKKFKSHHRKPK SFSSCKQPG

[0349] SEQ ID NO: 6: SD domain:

[0350] MSELPGDVRAFLREHPSLRLQTDARKVRCILTGHELPCRLPELQVYTRGKKYQRLVRASPAFD YAEFEPHIVPSTKNPHQLFCKLTLRHINKCPEHVLRHTQGRRYQRALCKYEECQKQGVEYVP ACLVHRRRRRE

[0351] SEQ ID NO: 21: SD domain - NLS

[0352] MSELPGDVRAFLREHPSLRLQTDARKVRCILTGHELPCRLPELQVYTRGKKYQRLVRASPAFD YAEFEPHIVPSTKNPHQLFCKLTLRHINKCPEHVLRHTQGRRYQRALCKYEECQKQGVEYVP ACLVHRRRRREKKKRK

Claims

CLAIMS1. A SURF2 activator for use in the treatment of ribosomopathy in a patient in need thereof, preferably wherein said SURF2 activators impedes activation of p53 by free 5S ribonucleoprotein.

2. The activator for use of claim 1 wherein said ribosomopathy is selected from the group consisting of: Diamond Blackfan anemia syndrome (DBA), 5q-syndrome, Schwachman-Diamond syndrome, X-linked dyskeratosis congenita, cartilage -hair hypoplasia, Treacher-Collins syndrome, Bowen- Conradi syndrome, North American Indian childhood cirrhosis, preferably Diamond Blackfan anemia syndrome (DBA).

3. The activator for use according to claim 1 or 2 wherein said activator is a human SURF2 protein comprising or consisting of SEQ ID NO: 1, or a functional variant thereof having at least 70, 75, 80, 85, 90 or 95% identity to SEQ ID NO: 1, preferably said activator is a nucleic acid encoding a human SURF2 protein comprising or consisting of SEQ ID NO: 1, or a function variant thereof having at least 70, 75, 80, 85, 90 or 95% identity to SEQ ID NO: 1.

4. The activator for use according to claim 3 wherein said nucleic acid construct is comprised in an expression vector, preferably a viral vector selected from the group consisting of: Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV or SNV, lentiviral vectors, adenoviral (Ad) vectors, adeno-associated viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors, more preferably a lentiviral vector.

5. The activator for use according to claim 3 or 4 wherein said nucleic acid construct or expression vector is comprised in a viral particle, preferably a lentiviral particle.

6. An isolated cell for use in the treatment of a ribosomopathy in a patient in need thereof, preferably selected from the group consisting of: Diamond-Blackfan anemia syndrome (DBA), 5q-syndrome, Schwachman-Diamond syndrome, X-linked dyskeratosis congenita, cartilage -hair hypoplasia, Treacher-Collins syndrome, Bowen-Conradi syndrome, North American Indian childhood cirrhosis, more preferably Diamond-Blackfan anemia syndrome (DBA), wherein said cell comprises a nucleic acid construct as defined in any one of claims 3 to 5, preferably wherein said cells is transduced with a viral particle comprising a nucleic acid construct as defined in any one of claims 3 to 5.

7. An isolated cell for use according to claim 6 wherein said cell is a hematopoietic cell selected from the group consisting of hematopoietic progenitor or stem cells, preferably selected from the group consisting of: bone-marrow derived cells, peripheral blood cells, and umbilical cord blood cells.

8. A pharmaceutical composition comprising the activator as defined in any one of claims 1 to 7 or isolated cells according to claim 6 or 7 and a pharmaceutical acceptable carrier.

9. A SURF2 inhibitor for use in the treatment of a cancer in a patient in need thereof, preferably wherein said SURF2 inhibitor activates p53 by free 5S ribonucleoprotein.

10. The inhibitor for use according to claim 9 wherein said inhibitor is a fragment of SURF2 protein comprising no more than an amino acid sequence between residue position 1 to 136 of SEQ ID NO: 1, a functional fragment or a functional variant thereof having at least 70, 75, 80, 85, 90 or 95% identity to the amino acid sequence between residue position 1 to 136 of SEQ ID NO: 1 .

11. The inhibitor for use according to claim 10 wherein said inhibitor is a peptide consisting of an amino acid sequence of SEQ ID NO: 6 or 21, a functional fragment or a functional variant thereof having at least 70, 75, 80, 85, 90 or 95% identity to SEQ ID NO: 6 or 21.

12. The inhibitor for use according to claim 9 wherein said inhibitor is at least one interfering RNA molecule, preferably comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2 to 5, more preferably comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7 to 10.

13. The inhibitor for use according to any one of claims 9 to 12 wherein said inhibitor is administered in combination with a chemotherapeutic agent, preferably selected from the group consisting of: Actinomycine D, 5 Fluoro-Uracil (5-FU), doxorubicin (DRB), Etoposide (ETO), cyclophosphamide and cisplatin.

14. A pharmaceutical composition comprising a SURF2 inhibitor as defined in any one of claims 9 to 13 and a pharmaceutical acceptable carrier, and preferably further comprising a chemotherapeutic agent, preferably selected from the group consisting of: Actinomycine D, 5 Fluoro-Uracil (5-FU), doxorubicin (DRB), Etoposide (ETO), cyclophosphamide and cisplatin.

15. An in vitro method for the diagnosis of a cancer or prognosis of survival outcome of a patient suffering from a cancer, preferably endocrine cancer, more preferably adrenocortical carcinoma, head and neck cancer, hepatocarcinomas or osteosarcomas, comprising the steps of determining SURF2 gene expression level in a patient sample, preferably tumor patient sample, wherein ahigher SURF2 gene expression level in a patient sample compared to a control value is indicative that said patient has a lower survival time and a lower SURF2 gene expression level in a patient sample compared to a control value is indicative that said patient has a higher survival time.

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