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WO2025114512A1 - Utilisation thérapeutique de modulateurs de surf2 - Google Patents

Utilisation thérapeutique de modulateurs de surf2 Download PDF

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Publication number
WO2025114512A1
WO2025114512A1 PCT/EP2024/084049 EP2024084049W WO2025114512A1 WO 2025114512 A1 WO2025114512 A1 WO 2025114512A1 EP 2024084049 W EP2024084049 W EP 2024084049W WO 2025114512 A1 WO2025114512 A1 WO 2025114512A1
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Prior art keywords
surf2
cells
cell
protein
cancer
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Inventor
Simon LEBARON
Sophie TAGNERES
Pierre-Emmanuel GLEIZES
Célia PLISSON-CHASTANG
Virginie MARCEL
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Cnrs Dsi
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Leon Berard
Universite Claude Bernard Lyon 1
Universite de Toulouse
Original Assignee
Cnrs Dsi
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Leon Berard
Universite Toulouse III Paul Sabatier
Universite Claude Bernard Lyon 1
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Publication of WO2025114512A1 publication Critical patent/WO2025114512A1/fr
Pending legal-status Critical Current
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    • A—HUMAN NECESSITIES
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
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    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to SURF2 modulators and its therapeutic use in a patient in need thereof.
  • 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.
  • Nucleolar stress 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.
  • 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)).
  • 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)).
  • 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.
  • 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)).
  • 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)).
  • Free 5S RNP homeostasis is also key in an ensemble of diseases originating from ribosome production defects and regrouped as ribosomopathies.
  • DBA Diamond-Blackfan Anemia syndrome
  • 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)).
  • the patients suffering from these diseases show a higher cancer incidence compared to the general population.
  • 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.
  • SURF2 to impede p53 activation following nucleolar stress
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a chemotherapeutic agent preferably selected from the group consisting of: Actinomycine D, 5 Fluoro-Uracil (5-FU), doxorubicin (DRB), Etoposide (ETO), cyclophosphamide and cisplatin.
  • the present disclosure also relates to a pharmaceutical composition
  • 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.
  • a chemotherapeutic agent preferably selected from the group consisting of: Actinomycine D, 5 Fluoro- Uracil (5-FU), doxorubicin (DRB), Etoposide (ETO), cisplatin and cyclophosphamide.
  • 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.
  • a cancer preferably hormonal cancer, such as adrenocortical carcinoma and prostate cancer
  • hormonal independent cancers such as head and neck cancer or liver cancers
  • 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.
  • DBA Diamond-Blackfan anemia syndrome
  • DBA Diamond-Blackfan anemia syndrome
  • 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 viral vector selected from the
  • 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.
  • DBA Diamond-Blackfan anemia syndrome
  • 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.
  • the present disclosure also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the activator as defined above or isolated cells as defined above and a pharmaceutical acceptable carrier.
  • 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.
  • 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.
  • 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.
  • transgene refers to exogenous DNA or cDNA encoding a gene product.
  • the gene product may be an RNA, peptide or protein.
  • 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.
  • a promoter 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.
  • nucleic acid construct 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.
  • 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.
  • amino acid and “amino acid residue” are used interchangeably throughout.
  • 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.
  • 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.
  • 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.
  • sequence identity refers to the number (%) of matches (identical amino acid residues) in positions from an alignment of two polypeptide or nucleic acid sequences.
  • sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • 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.
  • 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.
  • substituted or “modified” the present invention includes those amino acids that have been altered or modified from naturally occurring amino acids.
  • conservative substitution 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).
  • 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).
  • inverted terminal repeat 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • AAV viral particles 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.
  • 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.
  • 5S RNPs or 5S ribonucleoproteins refers to the association of 5S ribosomal RNA (rRNA) with the ribosomal proteins RPL5 and RPL11.
  • 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.
  • 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.
  • 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.
  • the present disclosure relates to SURF2 inhibitor, in particular for use in treatment of a cancer in a subject in need thereof.
  • 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).
  • 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).
  • Human SURF2 protein comprises, or consists of, the amino acid sequence of SEQ ID NO: 1.
  • SURF2 inhibitor 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.
  • 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 inhibitor according to the present disclosure having such properties can be screened using for examples the following assays.
  • 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.
  • anti-RPE5 antibody or anti-flag or other markers such as HA, fluorescent marker
  • RPL5-flag or other markers such as HA, fluorescent marker
  • 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).
  • 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.
  • anti-RPL5 antibody or anti -flag or other markers such as HA, fluorescent marker
  • RPL5-flag or other markers such as HA, fluorescent marker
  • 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).
  • 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 MDM2 C305F mutant, 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 MDM2 C305F mutant.
  • 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).
  • the increase of p53 pathway is determined during nucleolar stress response, for example in cells treated with chemotherapeutic agent such as Actinomycin D.
  • 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.
  • 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).
  • 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.
  • 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.
  • the decrease of cell proliferation is determined during nucleolar stress response, for example in cells treated with chemotherapeutic agent such as Actinomycin D.
  • 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.
  • the expression level of SURF2 mRNA may be determined by any suitable methods known by skilled persons.
  • 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.
  • enzyme -labelled and mediated immunoassays such as ELISAs, biotin/avidin type assays, radioimmunoassay, immunoelectrophoresis, mass spectrometry or immunoprecipitation or by protein or antibody arrays.
  • 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.
  • 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.
  • said SURF2 inhibitor may be an interfering nucleic acid which specifically decreases SURF2 expression.
  • iRNA Ribonucleic acid
  • RNAi Ribonucleic acid
  • 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).
  • 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).
  • Said interfering nucleic acid can be as non-limiting examples anti-sense oligonucleotide constructs, small inhibitory RNAs (siRNAs) or short hairpin RNA.
  • siRNAs small inhibitory RNAs
  • short hairpin RNA short hairpin RNA
  • 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.
  • 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).
  • small inhibitory RNAs 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).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • 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.
  • 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.
  • short hairpin RNA can also be used to decrease the SURF2 expression level in the present disclosure.
  • a short hairpin RNA is a sequence of RNA that makes a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi).
  • RNAi RNA interference
  • 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.
  • 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.
  • the interfering nucleic acid is a siRNA of at least about 10-40 nucleotides in length, preferably about 15-30 base nucleotides.
  • interfering nucleic acid according to the disclosure comprises at least one sequence selected from the group consisting of:
  • up to four interfering nucleic acids comprising each a sequence SEQ ID NO: 2 to 5 are used concomitantly.
  • said interfering nucleic acid is at least one duplex siRNA selected from the group consisting of: SEQ ID NO: 7 to 10.
  • duplex siRNA selected from the group consisting of: SEQ ID NO: 7 to 10.
  • up to four duplex siRNA comprising each a sequence SEQ ID NO: 7 to 10 are used concomitantly.
  • 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.
  • interfering nucleic acid for use in the disclosure can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • 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.
  • RNA molecules thymine is replaced by uracil (U), which, similar to thymine (T), forms a complementary base pair with adenine (A).
  • Interfering nucleic acid may be modified to have enhanced stability, nuclease resistance, target specificity and improved pharmacological properties.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the other moiety may also be a non-protein moiety, such as, for example, a polyethyleneglycol (PEG) moiety or other chemical moiety or conjugates.
  • the second moiety can be a Fc region of an antibody, a reporter, a nuclear localization signal or a tag protein.
  • 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.
  • the NLS sequence is KKKRK (SEQ ID NO: 22).
  • the polypeptide cargo may comprise its natural NLS.
  • said SURF2 peptide inhibitor comprises or consists of the amino sequence of SEQ ID NO: 21, or a functional variant thereof.
  • the SURF2 peptide inhibitor acts as a dominant negative peptide by binding to free-5S RNPs, and inhibiting native SURF2 protein biological activity.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Peptides described herein can be synthesized using standard synthetic methods known to those skilled in the art, for example chemical synthesis or genetic recombination.
  • N- and C-termini of the peptides described herein may be optionally protected against proteolysis.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • PROTAC PROteolysis Targeting Chimera
  • 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.
  • 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.
  • An antibody or antigen-binding fragment thereof of the present antagonizing SURF2 biological activity can be screened using the assays as described above.
  • antibody 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.
  • 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.
  • antibody fragment 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.
  • 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.
  • 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).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody.
  • 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.
  • SURF2 inhibitor can be a small molecule inhibiting the SURF2 expression, activity or function.
  • 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.
  • small molecules able to inhibit the SURF2 expression or biological activity can be screened using the assays as described in the previous section.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • vectors examples include, but are not limited to, recombinant integrating or nonintegrating viral vectors and vectors derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA.
  • the vector is a recombinant integrating or non -integrating viral vector.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present disclosure relates to viral particles including a nucleic acid construct or expression vector as described above.
  • 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”.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and/or vehicle.
  • 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.
  • the pharmaceutical composition contains vehicles, which are pharmaceutically acceptable for a formulation capable of being injected.
  • saline solutions monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts
  • 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.
  • 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.
  • 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.
  • a patient denotes a mammal.
  • a subject or individual is a human, in particular a human subject or patient, more preferably suffering from a cancer.
  • cancer refers 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.
  • treatment refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease.
  • such term refers to the amelioration or eradication of a disease or symptoms associated with a disease.
  • 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.
  • 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.
  • 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.
  • the cancer is any cancer type as disclosed above.
  • 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.
  • haematologic cancer in particular acute myelogenous leukaemia (AML), chronic lymphocytic le
  • 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.
  • the cancer is an adrenocortical carcinoma, head or neck cancer, hepatocarcinomas and osteosarcomas.
  • 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.
  • 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.
  • 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.
  • 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.
  • SURF2 inhibitor is advantageously administered in combination with a chemotherapeutic agent that stimulates nucleolar stress response.
  • Chemotherapeutic agents include cytotoxic anti-neoplastic agents, such as alkylating agents, antimetabolites, anti-microtubule agents, Topoisomerase inhibitors, cytotoxic antibiotics and others.
  • 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
  • the administration of the chemotherapeutic agent can be simultaneous, prior to or after the administration of the SURF2 inhibitor.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the inventors showed that SURF2 expression in cancer is upregulated in cancers and negatively correlates with overall survival.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the mRNA expression level is measured by RNA seq method.
  • 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.
  • 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.
  • 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.
  • 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.
  • a patient denotes a mammal, such as a rodent, a feline, a canine, and a primate.
  • a patient according to the invention is a human.
  • said patient or subject is a cancer patient, i.e., a patient having a tumor as described in the previous section.
  • 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.
  • said patient suffers from adrenocortical carcinoma, head or neck cancer, hepatocarcinomas and osteosarcomas.
  • 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.
  • patient sample means any biological sample derived from a patient. Examples of such samples include tissue sample, cell samples, organs, biopsies, preferably tumor sample.
  • 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.
  • 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).
  • cfcDNA cell-free circulating tumor DNA
  • ctDNA circulating tumor DNA
  • the DNA can be obtained from reverse transcription of an RNA sample.
  • 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.
  • 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.
  • health subjects refers to a population of subjects who do not suffer from any known condition, and in particular, who are not affected with any cancer.
  • 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.
  • 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.
  • the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the administration of the chemotherapeutic agent can be simultaneous, prior to or after the administration of the SURF2 inhibitor.
  • 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.
  • 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.
  • SURF2 activator 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).
  • the SURF2 activator according to the present disclosure having such properties can be screened using for examples the following assays.
  • 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.
  • anti-RPL5 antibody or anti -flag or other markers such as HA, fluorescent marker
  • RPL5-flag or other markers such as HA, fluorescent marker
  • 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).
  • 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.
  • anti-RPL5 antibody or anti -flag or other markers such as HA, fluorescent marker
  • RPL5-flag or other markers such as HA, fluorescent marker
  • 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).
  • 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 MDM2 C305F mutant, 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 MDM2 C305F mutant.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • the expression level of SURF2 mRNA may be determined by any suitable methods known by skilled persons.
  • 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.
  • enzyme -labelled and mediated immunoassays such as ELISAs, biotin/avidin type assays, radioimmunoassay, immunoelectrophoresis, mass spectrometry or immunoprecipitation or by protein or antibody arrays.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • N- and C-termini of the proteins described herein may be optionally protected against proteolysis.
  • 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.
  • 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.
  • 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.
  • PEG polyethylene glycol
  • 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).
  • the SURF2 activator is a nucleic acid construct comprising a transgene encoding SURF2 protein, functional fragment or a functional variant thereof as described above.
  • 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.
  • 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.
  • 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).
  • the transgene according to the disclosure may be any nucleic acid sequence encoding an SURF2 protein functional fragment or variant as described above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the transgene encoding SURF2 is operably linked to a transcriptional promoter and a transcription terminator.
  • nucleic acid constructs 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.
  • 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.
  • 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.
  • vectors examples include, but are not limited to, recombinant integrating or nonintegrating viral vectors and vectors derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA.
  • the vector is a recombinant integrating or non-integrating viral vector.
  • 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.
  • said vector is a lentiviral vector.
  • lentiviral vector 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.
  • lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface.
  • 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.
  • integrative lentiviral vectors or LV
  • NILV non-integrative lentiviral vectors
  • HIV human immunodeficiency virus
  • HIV type 1 HIV type 2
  • 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
  • BIV bovine immune deficiency virus
  • SIV simian immunodeficiency virus
  • 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.
  • 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.
  • 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.
  • an envelope protein is the vesicular stomatitius virus G (VSV G) protein or from measles virus.
  • viruses may alternatively be pseudotyped with ecotropic envelope protein that limit infection to a specific species, such as mice or birds.
  • 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).
  • the viral virus vectors may also be self-inactivating.
  • 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).
  • said lentivirus vector can be the lentivirus vector as disclosed in Gimenez Y. et al. JCI Insight. 2024 May 22;9(10):el71650.
  • nucleic acid construct or the expression vector of the present disclosure can be packaged into a “viral particle”.
  • 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.
  • 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.
  • a nucleic acid construct e.g., a nucleic acid construct encoding a human SURF2 protein
  • a nucleic acid construct is delivered to a cell by contacting the cell with a virion, preferably containing a lentiviral vector described herein.
  • 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).
  • said viral vector e.g. lentiviral vector
  • said 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).
  • the host cell of the disclosure may be used for ex vivo gene therapy purposes.
  • 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.
  • the host cell is used for ex vivo gene therapy into hematopoietic cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • hematopoietic cells are CD34+ cells.
  • ISCs induced pluripotent stem cells
  • the virion can be directly administered in vivo to a subject or a localized area of a subject (e.g., bone marrow).
  • 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.
  • hematopoietic cell e.g., hematopoietic stem cell
  • 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.
  • composition comprising a SURF2 activator
  • 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.
  • 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.
  • 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.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and/or vehicle.
  • 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.
  • the pharmaceutical composition contains vehicles, which are pharmaceutically acceptable for a formulation capable of being injected.
  • 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.
  • the form 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.
  • 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.
  • a patient denotes a mammal.
  • 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.
  • 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.
  • 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).
  • DBA Diamond-Blackfan anemia syndrome
  • 5q-syndrome X-linked dyskeratosis congenita
  • cartilage-hair hypoplasia cartilage-hair hypoplasia
  • myelodysplasia myelodysplasia
  • Shwachman-Diamond Syndrome SDS
  • Bowen-Conradi syndrome North American Indian childhood cirrhosis and Treachers Collins Syndrome (TCS).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a ribosomopathy e.g. DBA
  • 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.
  • 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.
  • a ribosomopathy e.g. DBA
  • 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.
  • 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.
  • an isolated cell e.g., hematopoietic cell
  • 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.
  • 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.
  • a cell e.g., hematopoietic cell
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 1 Label-free quantitative proteomics analysis of RPL5-FLAG co-purified proteins.
  • 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.
  • FIG. 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.
  • FC log2-transformed fold-change
  • p-value -value
  • RNAs associated with HEXIM 1 and SURF2 were 3’-labelled and separated on a 6% acrylamide gel. Control IP reaction performed without anti-bodies (beads) is shown.
  • U2OS cell extracts were fractionated using the PSE method.
  • FIG. 3 SURF2 is overexpressed in adrenocortical carcinomas and its knock-out negatively affects cancer cells similarly to MDM2.
  • 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.
  • IQR interquartile range
  • b-c Association between SURF2 mRNA levels and patient survivals.
  • 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.
  • Figure 5 SURF2 depletion promotes cell cycle arrest and apoptosis upon nucleolar stress. Codepletion experiment.
  • C Cell apoptosis assays with an annexin-V-FITC and propidium iodide of differentially treated U2OS cells.
  • FIG. 7 SURF2 expression level affect U2OS phenotypic traits. Analysis of U2OS KO SURF2 or U2OS overexpressing SURF2-Flag cell migration by wound healing assay.
  • Source data are provided as a Source Data file.
  • FIG. 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.
  • 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.
  • 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.
  • MDM2 can no longer ubiquitinylate p53, thereby stabilizing p53 and promoting cell cycle arrest.
  • 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.
  • nucleolar stress promotes free-5S accumulation in the nucleoplasm, all of which are recognized by SURF2, which competes with MDM2 for binding.
  • MDM2 is free and ubiquitinylate s p53, conferring to these cells a capacity to resist to nucleolar stress.
  • 1.1 SURF2 is a new binding partner of free 5S RNP particles
  • 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.
  • 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.
  • NS nucleolar stress
  • 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.
  • 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).
  • SSB/LA-protein a known chaperone of 5S rRNA
  • HEATR3 a chaperone of RPL5 (11, 36-40).
  • 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.
  • 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.
  • 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.
  • SURF1/RPL7A and SURF6/RRP14 a transmembrane receptor involved in endoplasmic reticulum export
  • SURF5/MED22 a component of the mediator complex
  • SURF1/SHY1 factor involved in cytochrome-c synthesis
  • SURF2 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).
  • TRIM21, PRDX1, DLST, RPL5, and RPL11 are common contaminants from U2OS extracts (see CRAPome database) (D. Mellacheruvu, et al. Nat Methods 10, 730-736 (2013)).
  • DLST dihydrolipoamide S- succinyltransferase
  • RAFs ribosome assembly factors
  • RNAs enriched during the IPs in the different conditions were labelled with P 32 using pCp labelling and separated on a polyacrylamide gel (Fig 2C).
  • SURF2 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.
  • 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).
  • 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).
  • the inventors wanted to assess whether SURF2 could influence ribosome assembly.
  • the inventors thus used siRNAs to extinct SURF2 expression in U2OS cells.
  • 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.
  • SURF2 expression level seem to regulate cell reaction to nucleolar stress.
  • 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).
  • TGCA resource https://portal.gdc.cancer.gov.
  • the inventors compared expression of SURF2 in tumors from different cancer types relative to their associated healthy tissues (GTex datasets) (Fig 3A).
  • the inventors found an increased level of SURF2 mRNA compared to healthy tissue (p-value inferior to 2 e 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).
  • 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.
  • the inventors set out to identify how SURF2 mediates its function.
  • 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.
  • 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* 10 A -14) and 0.738 (p value 1.26* 10 A -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.
  • SURF2 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.
  • 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.
  • DEGs differentially expressed genes
  • GSEA Gene Set Enrichment Analysis
  • 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.
  • NS Nucleolar Stress
  • 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).
  • free-5S RNP promotes p53 activation through sequestration of MDM2, its primary negative regulator.
  • 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.
  • the inventors thus then tested how SURF2 might affect the binding of free 5S RNP particles to MDM2.
  • 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.
  • 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).
  • p53, MDM2 and p21 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.
  • actinomycin D that promote more apoptosis
  • siRNAs actinomycin D or SURF2 depletion by siRNAs induce some apoptosis compared to control experiments (10% and 8% of dead cells, respectively).
  • the two treatments together significantly increase apoptosis (20%).
  • the inventors propose that the combination of both treatments promotes apoptosis, in addition to slowing proliferation.
  • 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).
  • 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.
  • HCC hepatocellular carcinoma
  • the inventors then compared the effect of SURF2 overexpression on both p53 and p21 stabilization promoted by these different drugs.
  • SURF2 overexpression is not able to compensate for p53 nor p21 stabilization induced by MG-132 or staurosporine exposure.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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)).
  • 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.
  • 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.
  • 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.
  • therapeutic agents such as chemotherapy used in most poor-prognosis cancers.
  • 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)).
  • RPL5-Flag 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.
  • 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.
  • 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.
  • SURF2 mainly interacts with free-5S particles through an interaction with RPL11 that involves SURF2’s structural domain (Fig 8).
  • 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 .
  • free 5S RNP particles can bind MDM2 to promote p53 activation and cell cycle arrest.
  • any small amount of free-5S can induce p53 activation and cell cycle slow-down.
  • 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 (2016).
  • DBA Diamond- Blackfan Anemia
  • 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.
  • 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.
  • the cDNA of SURF2 was fused to EGFP tag and cloned into pcDNA5-FRT- TO plasmid.
  • plasmids or the empty plasmids 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.
  • a pool of four siRNA duplexes from eurogentech were used to target SURF2 mRNA
  • RPL11 mRNA AAG-GUG-CGG-GAG-UAU-GAG-UUA/UAA-CUC-AUA-CUC- CCG-CAC-CUU, SEQ ID NO: 11
  • RPL5 mRNA GCC-ACA-AUG-UUG-CAG- AUU-A/UAA-UCU-GCA-ACA-UUG-UGG-C, SEQ ID NO: 12
  • 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).
  • 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.
  • 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).
  • 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.
  • 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-TrapTM 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).
  • 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.
  • trypsin Sequencing Grade Modified Trypsin, Promega
  • the instrument was operated in data-dependent acquisition (DDA) mode using a top-speed approach (cycle time of 3 s).
  • DDA data-dependent acquisition
  • 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.
  • AGC automatic gain control
  • 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).
  • HCD Higher Energy Collisional Dissociation
  • 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
  • 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.
  • 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.
  • GSEA 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).
  • 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 (NuPAGETM LDS Sample Buffer (4X) (NP0007) and NuPAGETM Sample Reducing Agent (1 OX) (NP0009)) and 20 pg/lane of protein were loaded on NuPAGETM 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).
  • 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.
  • 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.
  • actinomycin D 10 ng/mL, Sigma- Aldrich A9415
  • crystal violet staining solution Sigma- Aldrich, V5265
  • RNAs were extracted with Trizol from cell pellets containing 20x10 6 cells. 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).
  • 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.
  • ITS1 CCT-CGC-CCT-CCG-GGC- TCC-GTT-AAT-GAT-C
  • 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
  • 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.
  • SURF2-GFP 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.
  • Sucrose sedimentation profiling Buffers contains cycloheximide (10 pg/mL, Merck, C7698) at each step of this protocol.
  • 50xl0 6 cells 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.
  • SN 1 supernatants were collected and proteins were diluted in Invitrogen 2X sample buffer (NuPAGETM LDS Sample Buffer (4X) (NP0007) and NuPAGETM 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).
  • SN2 supernatants were collected and proteins were diluted in Invitrogen 2X sample buffer (NuPAGETM LDS Sample Buffer (4X) (NP0007) and NuPAGETM Sample Reducing Agent (10X) (NP0009)). SN2 was then store at -80°C.
  • 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 (NuPAGETM LDS Sample Buffer (4X) (NP0007) and NuPAGETM 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
  • 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.
  • 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).
  • TBS buffer 150 mM NaCl, 40 mM Tris-HCl pH 7.4
  • lysis buffer 150 mM NaCl, 0.05% Igepal, 50 mM Tris-HCl pH 7.4, 5 mM MgC12
  • RNAs were incubated with t 5 ’- 32p ] pCp and T4 RNA ligase (Promega, M1051) O/N at 4 °C.
  • 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.
  • the cDNAs of the SURF2 or SURF2(1-136) were cloned into pScodon plasmid (open biosystem) in translational fusion with HIS 6 (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).
  • Genotype-Tissue Expression is a public platform containing molecular data of healthy tissues derived from people of all age gender and ethnicity.
  • 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.
  • 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.
  • Proteins enriched over 2-fold with a p-value below 0.05 were considered significantly enriched.
  • 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.
  • 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.
  • 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.
  • SEQ ID NO: 1 human SURF2 protein:
  • SEQ ID NO: 6 SD domain:
  • SEQ ID NO: 21 SD domain - NLS

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Abstract

La présente invention concerne des modulateurs de SURF2 et leur utilisation thérapeutique chez un patient en ayant besoin. En particulier, la présente invention concerne un inhibiteur de SURF2 ou un moyen d'appauvrissement de celui-ci, destiné à être utilisé dans le traitement d'un cancer chez un sujet en ayant besoin et un activateur de SURF2 ou un moyen de surexpression de celui-ci pour une utilisation dans le traitement d'une ribosomopathie chez un sujet en ayant besoin.
PCT/EP2024/084049 2023-11-29 2024-11-29 Utilisation thérapeutique de modulateurs de surf2 Pending WO2025114512A1 (fr)

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