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WO2023111173A1 - Agent de dégradation ou inhibiteur de l'ezh2 destiné à être utilisé dans le traitement de la leucémie aiguë myéloïde résistante - Google Patents

Agent de dégradation ou inhibiteur de l'ezh2 destiné à être utilisé dans le traitement de la leucémie aiguë myéloïde résistante Download PDF

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WO2023111173A1
WO2023111173A1 PCT/EP2022/086142 EP2022086142W WO2023111173A1 WO 2023111173 A1 WO2023111173 A1 WO 2023111173A1 EP 2022086142 W EP2022086142 W EP 2022086142W WO 2023111173 A1 WO2023111173 A1 WO 2023111173A1
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Prior art keywords
ezh2
leukemia
resistant
cells
degrader
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Estelle DUPREZ
Mathilde POPLINEAU
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Aix Marseille Universite
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Jean Paoli and Irene Calmettes
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Aix Marseille Universite
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Jean Paoli and Irene Calmettes
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Publication of WO2023111173A1 publication Critical patent/WO2023111173A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4155—1,2-Diazoles non condensed and containing further heterocyclic rings
    • 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
    • A61K31/33—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
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form

Definitions

  • the present invention relates to an EZH2 degrader or an inhibitor of the EZH2 gene expression for use in the treatment of resistant leukemia in a patient in need thereof.
  • AML Acute myeloid leukemia
  • APL Acute promyelocytic leukemia
  • RARA Retinoic Acid Receptor alpha
  • the resulting fusion proteins behave as RARA signaling repressors due to their ability to oligomerize and to recruit epigenetic repressors at cis-regulatory DNA regions of RARA target genes and initiate oncogenic gene expression signatures (Grignani et al., 1993) and X-RARA fusion proteins were among the first transcription factors to be identified as drivers of cancer (Di Croce, 2005).
  • RA resistance To advance in the understanding of RA resistance, the inventor studied APL cellular heterogeneity by integrating scRNA-seq and scATAC-seq data obtained from PLZF-RARA transformed promyelocytes treated with RA. Establishing cellular clusters and arranging them in hierarchies helped them to identify a subpopulation of transformed promyelocytes that were insensitive to RA-induced differentiation and characterized by a DNA repair gene expression signature and high expression of Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of Polycomb Repressive Complex 2 (PRC2).
  • EZH2 Enhancer of Zeste Homolog 2
  • PRC2 Polycomb Repressive Complex 2
  • the present invention relates to an EZH2 degrader or an inhibitor of the EZH2 gene expression for use in the treatment of resistant leukemia in a patient in need thereof.
  • the invention is defined by its claims.
  • the invention relates to an EZH2 degrader or an inhibitor of the EZH2 gene expression for use in the treatment of resistant leukemia in a patient in need thereof.
  • the invention relates to an EZH2 degrader or an inhibitor of the EZH2 gene expression for use to sensitize resistant cancerous cells to therapeutic compounds used to treat leukemia like RA.
  • the invention relates to an EZH2 degrader or an inhibitor of the EZH2 gene expression to sensitize resistant leukemia cancerous cells to therapeutic compounds used to treat leukemia.
  • the invention relates to an i) EZH2 degrader or an inhibitor of the EZH2 gene expression and a ii) therapeutic compound already used to treat leukemia according to the invention as a combined preparation for simultaneous, separate or sequential use in the treatment of resistant leukemia or use in the sensitization of resistant leukemia cancerous cells.
  • the resistant leukemia is a resistant acute lymphocytic leukemia (ALL) or a resistant acute myeloid leukemia (AML).
  • ALL acute lymphocytic leukemia
  • AML resistant acute myeloid leukemia
  • the resistant leukemia is a resistant acute promyelocytic leukemia (APL), a resistant PLZF-RARA acute promyelocytic leukemia (APL), a resistant cytogenetically normal AML (CN-AML), a resistant acute myeloid leukemia with trisomy 8 or a resistant acute leukemia with MLL translocations.
  • APL acute promyelocytic leukemia
  • APL resistant PLZF-RARA acute promyelocytic leukemia
  • CN-AML resistant cytogenetically normal AML
  • a resistant acute myeloid leukemia with trisomy 8 or a resistant acute leukemia with MLL translocations a resistant acute promyelocytic leukemia with MLL translocations.
  • the resistant APL cells express the PLZF-RARA fusion protein due to the translocation t(l I;17)(q23;q21).
  • the invention also relates to an EZH2 degrader or an inhibitor of the EZH2 gene expression for use in the treatment of a resistant acute promyelocytic leukemia (APL) in a patient in need thereof.
  • APL acute promyelocytic leukemia
  • the invention relates to an EZH2 degrader or an inhibitor of the EZH2 gene expression for use in the treatment of resistant acute promyelocytic leukemia (APL) in a patient in need thereof.
  • APL acute promyelocytic leukemia
  • leukemia of the invention like AML or APL are resistant to Retinoic Acid (RA) and for example to the all-trans retinoic acid (ATRA) or to the tretinoin.
  • RA Retinoic Acid
  • ATRA all-trans retinoic acid
  • resistant leukemia denotes a leukemia which will resist or not respond to classical treatment or therapeutic compounds already used to treat such diseases.
  • classical treatment or therapeutic compounds already used to treat leukemia can be for example all-trans retinoic acid (ATRA; tretinoin), gemtuzumab ozogamicin, the combination of methotrexate, mercaptopurine and ATRA, demethylating agent, or chemotherapy such as cytarabine (araC), docetaxel, etoposide, idarubicin, volasertib, tozasertib (VX-680), nutlin 3, Olaparib or allograft.
  • ATRA all-trans retinoic acid
  • gemtuzumab ozogamicin the combination of methotrexate, mercaptopurine and ATRA
  • demethylating agent or chemotherapy such as cytarabine (araC), docetaxel, etoposide, idarubicin, volasertib, tozasertib (VX-680), nutlin 3, Olaparib or allograft.
  • chemotherapy refers to use of chemotherapeutic agents to treat a subject.
  • chemotherapeutic agent or “anti-cancer agents” refers to chemical compounds that are effective in inhibiting tumor growth.
  • the therapeutic compounds used to treat leukemia are chemotherapeutic agents.
  • the therapeutic compounds used to treat leukemia is selected from cytarabine (araC), volasertib, tozasertib (VX-680), nutlin 3 or olaparib.
  • EZH2 denotes a histone-lysine N-m ethyltransferase enzyme (EC 2.1.1.43) encoded by EZH2 gene, that participates in histone methylation and, ultimately, transcriptional repression.
  • EZH2 catalyzes the addition of methyl groups to histone H3 at lysine 27, by using the cofactor S-adenosyl-L-methionine. Methylation activity of EZH2 facilitates heterochromatin formation thereby silences gene function. Remodeling of chromosomal heterochromatin by EZH2 is also required during cell mitosis. Its Entrez access gene number is 2146.
  • the invention relates to an EZH2 degrader for use in the treatment of resistant leukemia in a patient in need thereof. In some embodiments, the invention relates to an EZH2 degrader for use in the treatment of resistant acute promyelocytic leukemia in a patient in need thereof.
  • the term “degrader” denotes a molecule capable of degrading another one by using the proteasome system of the cells. It results that the molecule targeted by the degrader is not visible.
  • a degrader according to the invention can also be a PROTAC (proteolysis targeting chimeras).
  • EZH2 degrader denotes a molecule capable of degrading EZH2 in a cell and particularly in resistant leukemia cells according to the invention.
  • an enzyme inhibiting the activity of EZH2 is not considered as an EHZ2 degrader since it is not a molecule capable of degrading EZH2 by using the proteasome system of the cells.
  • the oncogenic activity of EZH2 is not solely based on its catalytic activity and that EZH2 degraders are relevant for the treatment of cancers dependent on non-catalytic (non-canonical) activity of EZH2.
  • the term “patient” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the patient according to the invention is a human.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patients at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • the degrader according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).
  • small organic molecule refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.
  • the EZH2 degrader is the molecule MS 1943 of formula I (see also Ma et al., 2020):
  • the EZH2 degrader (EZH2 PROTAC) can be a molecule as described in Liu, Z. et 2021.
  • the degrader according to the invention is a polypeptide.
  • polypeptide is a decoy receptor of EZH2 and is capable to capture the enzyme EZH2.
  • polypeptide of the invention may be linked to a cell-penetrating peptide” to allow the penetration of the polypeptide in the cell.
  • cell-penetrating peptides are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).
  • polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art.
  • expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention.
  • the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.
  • the polypeptide When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell.
  • a host cell Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.
  • polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy.
  • modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution.
  • the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.
  • adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.
  • a strategy for improving drug viability is the utilization of water-soluble polymers.
  • Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body.
  • water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.
  • PEG Polyethylene glycol
  • Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity.
  • PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule.
  • copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.
  • PEGylation techniques for the effective modification of drugs.
  • drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.).
  • the PEG chains typically 2000 daltons or less
  • Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain.
  • the reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules.
  • These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer.
  • the molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading).
  • increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory halflife of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa).
  • linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue.
  • a specific trigger typically enzyme activity in the targeted tissue.
  • tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology.
  • Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.
  • the invention also relates to an inhibitor of the EZH2 gene expression.
  • inhibitor of the EZH2 gene expression denotes inhibitor of the expression of the gene coding for the EZH2 protein like for example siRNA or shRNA.
  • Small inhibitory RNAs can also function as inhibitors of EZH2 gene expression for use in the present invention.
  • EZH2 gene expression can be reduced by contacting a patient or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that EZH2 gene 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 for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ.
  • Ribozymes can also function as inhibitors of EZH2 gene expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of EZH2 mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as inhibitors of EZH2 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing EZH2.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno
  • Non-cytopathic viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest.
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • adeno-viruses and adeno-associated viruses are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • wildtype adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencap sul ati on .
  • the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • the promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes
  • a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable.
  • the promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
  • an endonuclease can be used to abolish the expression of the gene, transcript or protein variants of EZH2.
  • the endonuclease is CRISPR-cas.
  • CRISPR-cas has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.
  • the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes.
  • the CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol.
  • the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).
  • the invention in another embodiment, relates to a method for treating a resistant leukemia comprising administering to a patient in need thereof a therapeutically effective amount of an EZH2 degrader or an inhibitor of the EZH2 gene expression.
  • the resistant leukemic cells of the invention have a high level of H3K27me3 at specific genes compared to non-resistant leukemic cells.
  • level of H3K27me3 at these genes can be used as biomarker of resistant leukemia cells.
  • the invention also relates to a method to diagnose or predict a resistant leukemia of a subject suffering from a leukemia comprising determining, in a biological sample from the patient the level of H3K27me3.
  • another object of the invention relates to an in vitro method for diagnosing or predicting a resistant leukemia in a patient suffering a leukemia comprising i) determining in a sample obtained from the subject the histone methylation profile level of H3K27 ii) comparing the histone methylation profile level of H3K27 at step i) with its predetermined reference value and iii) providing a bad diagnosis or prognosis when the histone methylation profile level determined at step i) is higher than its predetermined reference value, or providing a good diagnosis or prognosis when the histone methylation profile level determined at step i) is lower than its predetermined reference value.
  • the term “bad diagnosis or prognosis” denotes that the patient has a resistant leukemia.
  • the histone methylation profile level of H3K27 denotes methylation profile of H3K27 and notably the profile of H3K27 tri -methylated (H3K27m3).
  • H3K27m3 the histone methylation profile level of H3K27 denotes methylation profile of H3K27 and notably the profile of H3K27 tri -methylated (H3K27m3).
  • chromatin isolation procedures comprise lysis of cells after one step of crosslink that will fix proteins that are associated with DNA. After cell lysis, Chromatin is fragmented, immunoprecipitated and DNA is recovered. DNA is then extracted with phenol, precipitated in alcohol, and dissolved in an aqueous solution.
  • the H3K27 methylation level can be determined by chromatin IP (see for example Boukarlessness H., et al, 2009) ChlP-chip or by ChlP-qPCR (see for example the materiel and methods part and Wu J. et al., 2006).
  • the "reference value” is the histone methylation level of H3K27 determined in a biological sample of a subject not afflicted by a leukemia or by a resistant leukemia.
  • said normal level of histone methylation is assessed in a control sample (e.g., sample from a healthy patient, which is not afflicted by a resistant leukemia) and preferably, the average of histone methylation profile level in several control samples.
  • Another object of the invention relates to a therapeutic composition comprising an EZH2 degrader or an inhibitor of the EZH2 gene expression according to the invention for use in the treatment of a resistant leukemia in a patient in need thereof.
  • the invention relates to a therapeutic composition
  • a therapeutic composition comprising an EZH2 degrader or an inhibitor of the EZH2 gene expression according to the invention to sensitive resistant cancerous cells to therapeutic compounds used to treat leukemia.
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do 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 filler, diluent, encapsulating material or formulation auxiliary of any type.
  • compositions for example, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
  • compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.
  • the pharmaceutical compositions contain 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 doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • compositions include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
  • compositions of the present invention may comprise a further therapeutic active agent.
  • the present invention also relates to a kit comprising an agonist, antagonist or inhibitor of the expression according to the invention and a further therapeutic active agent.
  • anti-cancer agents may be added to the pharmaceutical composition as described below.
  • Anti-cancer agents may be Melphalan, Vincristine (Oncovin), Cyclophosphamide (Cytoxan), Etoposide (VP- 16), Doxorubicin (Adriamycin), Liposomal doxorubicin (Doxil) and Bendamustine (Treanda).
  • Others anti-cancer agents may be for example cytarabine (AraC), anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5 -fluorouracil, taxanes such as docetaxel and paclitaxel, leuco
  • additional anti cancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.
  • Additional anti-cancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.
  • Additional anti-cancer agents may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.
  • the further therapeutic active agent can be an antiemetic agent.
  • Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiefhylperazine, thioproperazine and tropisetron.
  • the further therapeutic active agent can be an hematopoietic colony stimulating factor.
  • Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.
  • the other therapeutic active agent can be an opioid or nonopioid analgesic agent.
  • opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene.
  • Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.
  • the further therapeutic active agent can be an anxiolytic agent.
  • Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.
  • the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent.
  • the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin- like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).
  • CTL4 cytotoxic T lymphocyte-associated protein 4
  • PDCD1 programmed cell death 1
  • NK cells like various members of the killer cell immunoglobulin- like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).
  • the checkpoint blockade cancer immunotherapy agent is an antibody.
  • the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti -IDO 1 antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti- BTLA antibodies, and anti-B7H6 antibodies.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 Impact of targeting Ezh2 on APL Progression. Experimental scheme of the analysis of RA, GSK or combo treated bone marrow.
  • B In vitro efficiency of MS1943 on EZH2 protein level. PLZF-RARA TG cells are treated for 96H with increasing doses of MS1943 (MS) (NT: 0, 1.25, 2.5, 5 pM).
  • PLZF-RARA TG BM cells were grown in Iscove’s Modified Dulbecco’s Medium (Gibco) containing 20% of FCS and 1% penicillin/streptomycin and supplemented with 20 ng/ml IL-6, 20 ng/ml IL-3, 50 ng/ml SCF and 10 ng/ml GM-CSF (PreproTech).
  • PLZF-RARA and PML- RARA expression was achieved by treating U937 cell lines with 0.1 mM of ZnSO4 for at least 48 hours.
  • 293T cells were transfected with PLZF-RARA and/or Ezh2-Flag constructs by CaC12 coprecipitation.
  • 20,000 PLZF-RARA TG BM cells or 10,000 Fdcpl and 416b cells were cultured for 96 hours with GSK126 or MS1943 (MedChemExpress). Cell viability was evaluated using the CellTiter-Glo® Luminescent Cell Viability Assay according to the manufacturer's instructions.
  • the PLZF-RARA/RARA-PLZF-induced APL mouse model (called here PLZF-RARA model) was previously described by Pandolfi and colleagues (Cheng et al., 2000).
  • PLZF-RARA model For transplantation, 0.5 millions of Cd45.2 leukemic cells were transplanted in sub-lethally irradiated (1.5 Gy) NSG mice.
  • APL development was monitored by standard hematological procedures and mice were sacrificed for promyelocyte purification at 2-2.5-weeks post transplantation.
  • All-Trans Retinoic Acid (RA) (MedChemExpress) was reconstituted in 90% com oil and 10% DMSO and was intraperitoneally administered at a dose of 0.8-1 mg per mice for 3 or 7 consecutive days.
  • GSK126 (MedChemExpress) was reconstituted in 20% SBE-P-CD adjusted to pH 4-4.5 with 1 N acetic, supplemented with 10% DMSO, and was intraperitoneally injected at a dose of 1.25 mg per mice for 10 consecutive days.
  • Ezh2fl/fl mice were crossed with Rosa26::Cre-ERT mice (Taconic Artemis GmbH) to achieve the conditional deletion of Ezh2 (Mochizuki-Kashio et al., 2011).
  • Rosa26::Cre-ERT mice Teconic Artemis GmbH
  • Cre-ERT activity for in vivo Ezh2 deletion mice were intraperitoneally injected with 100 pl of tamoxifen dissolved in com oil at a concentration of 10 mg/ml for 5 consecutive days.
  • APL models were bred and maintained in the CRCM mouse facility (Marseille, France) in accordance with institutional guidelines for the use of laboratory animals and approved by the French authority (authorization number: 23893) and Ezh2 models were bred and maintained in the animal research facility of the graduate School of Medicine, Chiba University (Chiba, Japan) in accordance with institutional guidelines.
  • Leukemic myeloid progenitors (Promyelocytes: Cd45.2+, C-Kit+, Grl+; ProReP : Cd45.2+, C-Kit+, Grl+ , Cd48+, Cdl lb- ; NeuRA : Cd45.2+, C-Kit+, Grl+ , Cd48-, Cdl lb+ ) were purified using the FACS Aria III cell sorter (Beckman Dickinson).
  • pMy-IRES-PLZF-RARA-GFP plasmid or the pMy-IRES-GFP empty vector were transfected into PlatE packaging cells by CaC12 coprecipitation.
  • Lin- cells were pre-cultured for 24 hours in S-Clone SF- 03 (Sanko Junyaku) with 0.2 % BSA (Stemcell technologies), 1% penicillin/streptomycin, 50pM P-mercapto ethanol and supplemented with 20 ng/ml IL-6, 20 ng/ml IL-3, 50 ng/ml SCF and 10 ng/ml GM-CSF (PreproTech).
  • GFP positive cells (PLZF-RARA or IRES transduced Lin- cells) were cultured 48 hours in the same S-Clone SF-03 complete medium than described above and 1 x 104 cells were seeded into M3234 methylcellulose (Stemcell technologies). Cells were replated every 1-2 weeks. For Ezh2 deletion (data not shown), 150nM 4-OHT was directly added into the methylcellulose medium. For the experiment described in Figure 1, 2.5 pM of GSK126 and/or 1 pM of RA were added into the methylcellulose. At each replating, cells were cytospun and their morphology was analyzed by May-Griinwald staining. Immunophenotypic analysis (C- Kit, Cdl lb, Gr, FceRl, Cd45.2) were performed by FACS.
  • Co-IP co-immunoprecipitation
  • nuclear proteins were extracted using the dounce homogenizer with high salt concentration according to Dignam and Roeder, https://www.ncbi.nlm.nih.gov/pubmed/20150077.
  • 100 pg of nuclear extracts were diluted in isotonic buffer, precleared with Protein A or G agarose beads (Thermo Scientific) and incubated overnight at 4°C with Rabbit IgG (Cell Signaling Technology), EZH2 (Active Motif), SUZ12 (Active Motif), FLAG (Sigma Aldrich) or PLZF 2A9 (Active Motif) antibodies.
  • Protein A or G agarose beads were added the next day for 2 hours and the resulting complexes were washed, denatured and eluted in the Laemmli sample buffer.
  • Lysis Buffer A 0.25% Triton XI 00, 10 mM Tris-HCl pH8, 10 mM EDTA, 0.5 mM EGTA
  • Lysis Buffer B 250 mM NaCl, 50 mM Tris-HCl pH8, 1 mM EDTA, 0.5 mMEGTA
  • Nuclei were suspended in Buffer C (0.5% SDS, 10 mM Tris-HCl pH8, ImM EDTA, 0.5 mM EGTA) and chromatin was sonicated using the Bioruptor® Pico (Diagenode) in order to obtain DNA fragments with an average size of 300 bp.
  • the soluble chromatin was diluted in Buffer D (0.6% Triton XI 00, 0.06% NaDOC, 150 mM NaCl, 12 mM Tris-HCl pH8, 1 mM EDTA, 0.5 mM EGTA) and immunoprecipitated overnight at 4°C with magnetic beads (Active Motif) pre-incubated with the following antibodies: H3K27me3 (Cell Signalling), H3K27ac (Active Motif), H3K4mel (Abeam), H3K4me3 (Diagenode).
  • Buffer D 0.6% Triton XI 00, 0.06% NaDOC, 150 mM NaCl, 12 mM Tris-HCl pH8, 1 mM EDTA, 0.5 mM EGTA
  • H3K27me3 Cell Signalling
  • H3K27ac Active Motif
  • H3K4mel Abeam
  • H3K4me3 Diagenode
  • A, B, C, D buffers were supplemented with IX protease inhibitor cocktail (PIC) (Life Technology # 11873580001) and 0.5 mM PMSF. ChIP were washed with the following combinations of wash buffers: W1 (1% Triton X100, 0.1% NaDOC, 150 mM NaCl, 10 mM Tris-HCL pH8), W2 (0.5% NP40, 0.5% Triton X100, 0.5% NaDOC, 150 mM NaCl, 10 mM Tris-HCL pH8), W3 (0.7% Triton X100, 0.1% NaDOC, 250 mM NaCl, 10 mM Tris-HCL pH8), W4 (0.5% NP40, 0.5% NaDOC, 250 mM LiCl, 20mM Tris-HCL pH8, ImM EDTA, W5 (0.1% NP40, 150 mM NaCl, 20 mM Tris-HCL pH8, 1 mM EDTA), W6 (20 m
  • Immunoprecipitated DNA was purified with LPure Kit (Diagenode). ChlP-seq libraries were generated using the MicroPlex Library Preparation Kit (Diagenode) following the manufacturer's instructions and analyzed on a 2100 Bioanalyzer system (Agilent) prior sequencing. Sequencing was performed with a Next-seq500 sequencer (Illumina) using a 75-nt single-end protocol, at the Paoli Calmettes Institute Sequencing Facility (IPC, Marseille).
  • Sequencing quality control was determined using the FastQC tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads with a Phred quality score less than 30 were filtered out.
  • a scaling factor was calculated using dm6 mapped tags (H2Av-bound regions) as previously described (Egan et al., 2016) and mm 10 mapped tags were normalized according to this scaling factor (Spike-in correction). Normalized-mapped reads were converted into BigWig using the deepTools suite (v2.2.4) (Ramirez et al., 2016) / bamCoverage -bs 20 — scaleFactor, multiBigwigSummary. Fragment length prediction was performed using MACS2 predictd and later applied for peak calling High confidence binding sites were determined using MACS2 callpeak broad mode / - -broad-cutoff 0.05 -q 0.01 — extsize. Inputs were used as controls.
  • Bedtools intersect (v2.17.0) (Quinlan and Hall, 2010) with a minimum overlap of 1-base was used to determine enhancer coordinates.
  • FACS purified promyelocytes were loaded 30 min after the sorting onto a Chromium Single Cell Chip and processed with the Chromium Controller (lOx Genomics) according to the manufacturer’s instructions for single cell barcoding at a target capture rate of 6000 individual cells per sample.
  • Libraries were prepared using Chromium Single-Cell 3' Reagent Kits v2 (lOx Genomics). Sequencing was performed with a Next-seq500 sequencer (Illumina) using a 75-nt single-end protocol, at the Paoli Calmettes Institute Sequencing Facility (IPC, Marseille).
  • the 6 obtained clusters were manually annotated using Differentially Expressed Genes (DEG) and gene list enrichments.
  • SASP Senescence Associated Secretory Phenotype
  • Nuclei from FACS purified promyelocytes were purified according lOx Genomics instructions and processed using the Chromium Next GEM Single Cell ATAC Reagent Kits vl .1 and Single Index Kit N Set A according to the manufacturer’s instructions for single cell barcoding at a target capture rate of 10,000 nuclei per sample. Sequencing was performed with a HiSeq2500 sequencer (Illumina) using a 150 nt paired-end protocol (Read IN: 50 cycles, i7 Index: 8 cycles, i5 Index : 16 cycles, Read 2N: 50 cycles), at the Institute of Medical Science, Tokyo University.
  • TF regulons were found using pySCENIC (1.10.0) (Aibar et al., 2017).
  • a total of 9 APL patient (3 PLZF-RARA; 6 PML-RARA) affymetrix RNA microarrays were analyzed (GSM211183, GSM211184, GSM211185, GSM1057933, GSM1057934, GSM1057935, GSM1057936, GSM1057937, GSM1057938). Each group was processed separately using the following steps. Briefly, CEL files were read using ReadAffy function from affy package (vl.66.0) and normalization steps (background adjustment, log transformation) were carried out using germa package (v2.60.0).
  • Probes were annotated using annotationDbi (vl.50.0) and hgul33a2.db (v3.2.3) packages, and probes from the same transcript were merged and median value was kept. PLZF-RARA and PML-RARA groups were then merged together and potential batch effects were removed using quantile normalization. Finally, DEG analysis was performed using linear model fit and Empirical Bayes statistics from the limma package (v3.44.1).
  • GSEA gene set enrichment analyses
  • the “Methyl, targeting” signature consisted of a list of differentially expressed genes in GSK-treated versus untreated cells.
  • the “Non methyl, targeting” signature was obtained by subtracted the previous gene list to the list of differentially expressed genes in MS 1943 -treated versus untreated cells.
  • TFs myeloid transcription factors
  • ReP cluster was characterized by the expression of genes involved in homologous recombination and cell-cycle such as Pena, Mcm3 and Rad51 (data not shown) and was equally composed of Ctrl and RA-treated cells (data not shown) suggesting that RA treatment did not impact on the cell identity of these promyelocytes.
  • Trajectories 1 (states A-C-D) and 2 (states A-C-E) ended towards RA differentiated cells, since their final states C, D and E were largely composed RA-treated cells grouped in NeuRAl and NeuRA2, respectively (data not shown), confirming a pronounced differentiating effect of RA on a portion of PLZF-RARA expressing cells and consistent with a stronger expression of differentiation marker in NeuRA2 (data not shown).
  • the third trajectory (state B), which went far into the pseudotime was composed mostly of Ctrl cells grouped in Prom2 and Prom3 (data not shown), suggesting a spontaneous differentiation program in leukemic cells. This analysis showed a pronounced but partial differentiating effect of RA on PLZF-RARA expressing cells and designated cells in the ReP cluster as the treatment-persistent cells.
  • ReP cluster unlike the Prom2, Prom3 and NeuRAl clusters, lacked the Secretory Associated Signature Phenotype (SASP) (Reactome; GSEA), suggesting the absence of a senescence program in this cluster (data not shown).
  • SASP Secretory Associated Signature Phenotype
  • GSEA Secretory Associated Signature Phenotype
  • Integrative single-cell multi-omics analysis highlights chromatin genes as potential actors of RA resistance of PLZF-RARA expressing cells
  • scRNA-seq and scATAC-seq data are uniquely valuable to define gene regulatory networks at the cellular scale.
  • TF activity that might be associated with RA resistance
  • ReP TF activity was selected by considering, in our scATAC-seq data, the accessibility of DNA motifs of TFs in the ReP cluster using the Signac chromVAR package (Stuart et al., 2020) (data not shown).
  • Selected ReP TFs were cross- referenced with master transcriptional regulators identified from scRNA-seq data using SingleCell Regulatory Network Inference and Clustering (SCENIC), which measures TF regulon activity (Aibar et al., 2017) (data not shown). Doing so and in line with our previous annotation, we identified 3 TFs, linked to E2F (E2F1, E2F4, TFDP1) with high transcriptional activity in the ReP cluster (data not shown). Interestingly E2fl and Tfdpl regulon activity were increased upon RA treatment while E2f4 activity was diminished (data not shown).
  • SCENIC SingleCell Regulatory Network Inference and Clustering
  • RA transcriptional resistance can be defined at the chromatin level and is dependent on E2F transcriptional activity and chromatin accessibility differences mainly at enhancer regions of the genome of a subset of promyelocytes.
  • heterogeneity in terms of target regulation inside of a regulon.
  • Ezh2 deletion at these steps of replating decreased the proliferation of cells and prevented them to form colonies at the next round of replating (data not shown). These assays suggest that Ezh2 is required for the initiation and maintenance of PLZF-RARA clonogenic activity.
  • PLZF-RARA modulates chromatin landscape and EZH2 methyltransferase activity at pro-apoptotic genes
  • Bini the genes pertinent for differentiation and leukemia we found Bini, P2rx4, Dusp6 and Ly86 that presented an H3K27ac/H3K27me3 enhancer switch (data not shown). Consistently, the expression of these genes was lower in the presence of PLZF-RARA than in GMPs (data not shown).
  • H3K27me3 did not follow the level of EZH2 in RA-treated BM, suggesting a contrasting effect of RA on EZH2: an increase of its level but an inhibition of its histone methyltransferase activity, while leukemia relapse was characterized with a restoration of high levels of H3K27me3 level in the BM (data not shown).
  • H3K27me3 signal at poised enhancers was decreased resulting in a diminution in numbers of these enhancers after 3 or 7 days of RA treatment (data not shown).
  • PLZF-RARA transduced cells exhibited some differentiated cell features upon GSK that were more pronounced upon RA or RA plus GSK treatments, evidenced by the presence of granules into their cytoplasm and loss of the KIT stem-progenitor marker (after exclusion of Kit+, FcsRl+ mast cells) (data not shown).
  • Kit+, FcsRl+ mast cells KIT stem-progenitor marker
  • JASPAR 2020 update of the open-access database of transcription factor binding profdes. Nucleic Acids Res 48, D87-D92. 10.1093/nar/gkzl001.
  • APL Acute promyelocytic leukemia
  • the acetyltransferase GCN5 maintains ATRA-resi stance in non-APL AML. Leukemia 33, 2628-2639. 10.1038/s41375-019-0581-y.
  • SWI/SNF -mutant cancers depend on catalytic and non-catalytic activity of EZH2. Nat Med 21, 1491-1496. 10.1038/nm.3968.
  • Acute Myeloid Leukaemia in Its Niche the Bone Marrow Microenvironment in Acute Myeloid Leukaemia. Curr Oncol Rep 22, 27. 10.1007/sl 1912-020-0885-0.
  • chromVAR inferring transcription-factor-associated accessibility from single-cell epigenomic data. Nat Methods 14, 975-978. 10.1038/nmeth.4401.

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Abstract

La présente invention concerne le traitement de la leucémie résistante. Les inventeurs ont étudié l'hétérogénéité cellulaire de la LPA en intégrant des données scRNA-seq et scATAC-seq obtenues à partir de promyélocytes transformés par PLZF-RARA et traités par RA. L'établissement de groupes cellulaires et leur hiérarchisation ont permis d'identifier une sous-population de promyélocytes transformés insensibles à la différenciation induite par la RA et caractérisés par une signature d'expression des gènes de réparation de l'ADN et une forte expression de l'EZH2 (Enhancer of Zeste Homolog 2), la sous-unité catalytique du complexe PRC2 (Polycomb Repressive Complex 2). La contribution d'EZH2 à la pathogenèse de la leucémie étant variable, la fonction d'EZH2 dans le développement de la LPA et la réponse au traitement de la RA ont été approfondies. Les chercheurs ont découvert un double rôle d'EZH2 dans l'apparition de la LPA et la réponse à la RA, suggérant la nécessité de cibler l'activité méthyltransférase non-histone d'EZH2 pour lutter contre la leucémie. En particulier, ils ont montré que l'utilisation d'un agent de dégradation sélectif d'EZH2 réduisait de manière significative la croissance et la viabilité des cellules leucémiques exprimant le PLZF-RARA et augmentait la survie des souris transplantées avec une leucémie prétraitée. La présente invention concerne donc un agent de dégradation d'EZH2 ou un inhibiteur de l'expression du gène EZH2 destiné à être utilisé dans le traitement d'une leucémie résistante chez un patient dont l'état nécessite un tel traitement.
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