OF ACUTE MYELOID LEUKEMIA

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of acute myeloid leukemia.

BACKGROUND OF THE INVENTION:

Acute myeloid leukemia (AML) arise from the malignant clonal expansion of undifferentiated myeloid precursors, resulting in bone marrow hematopoiesis failure. Although many AML patients initially respond to conventional induction therapy, relapses are common and carry a very poor prognosis (1). The standard treatments for AML have remained almost unchanged over the past 40 years, demonstrating that despite improvements in our understanding of AML pathogenesis, a more in-depth knowledge of the biology of AML is still required in order to rationally design more efficient therapies. Over the past few decades, extensive molecular characterization studies have highlighted the complex heterogeneity of AML (2). The most common genetic abnormalities, which occur in about 30% of AML patients, lie within the Fms-Like Tyrosine kinase 3 (FLT3) gene which encodes a receptor tyrosine kinase (RTK). The most frequent mutations in this gene occur via internal tandem duplication (FLT3-ITD) in the juxta-membrane domain, or through point mutations, usually of the Asp835 residue within the activation loop (FLT3-TKD). Both types of mutations result in constitutive activation of FLT3 and promote leukemic cell proliferation and survival. The poor outcome associated with FLT3-ITD mutations (3) has generated great interest in the development of specific tyrosine kinase inhibitors for more than 10 years. Of the new generation of FLT3 inhibitors developed, quizartinib (AC-220) is considered the most potent and selective, but resistance still commonly arises due in particular to secondary mutations (e.g. FLT3-TKD). Recently, midostaurin (PKC-412) has shown efficiency against both FLT3-ITD and FLT3- TKD, and early clinical trials are currently underway (for review, see 4). Although important efforts are aimed at developing more efficient tyrosine kinase inhibitors, novel approaches to eradicate FLT3 -mutated leukemic cells must also be developed.

Autophagy is a highly-conserved catabolic process used by the cell to degrade and recycle damaged cellular components in response to adverse environmental stimuli. It relies on the formation of the autophagosome, a double-membraned vesicle that sequesters cytoplasmic bulk, proteins and organelles before fusing with lysosomes to degrade the engulfed material. The role of autophagy in cancer is complex since it plays a tumor suppressor role during the early phases of tumor initiation through the prevention of genomic instability yet promotes tumor development in established tumors by promoting cancer cell survival (5). Interestingly, oncogenes such as K-RASG12D, BRAFV600E and the fusion protein BCR-ABL1 were reported to support a high level of basal autophagy in different types of cancers which is required for cell survival and proliferation (6-8). Moreover, autophagy has also been described to be a mechanism of resistance to different types of drugs or treatments (9,10), and inhibiting autophagy in this context was shown to enhance treatment efficiency. The publication of these studies has instigated several clinical trials combining the only available in vivo autophagy inhibitor, hydroxychloroquine (HCQ), with conventional treatments in different cancers (1 1).

A few studies have sought to understand the role of autophagy in AML, and suggest that inhibiting autophagy sensitizes particular subgroups of AML cells to chemotherapies (12,13) or to small molecules inhibitors (e.g. histone deacetylase inhibitor) (14,15). However, the potential role of autophagy in AML cell biology as a mechanism of progression in FLT3- mutated AML remains to be clarified.

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the treatment of acute myeloid leukemia. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors recently identified the ATF4 transcription factor as an important regulator of autophagy and proliferation in acute myeloid leukemia expressing the FLT3-ITD mutation. Here they demonstrate that ATF4 is tightly regulated by a molecular process involving the EIF6 translation initiation factor. They found that FLT3-ITD activates protein kinase C isoforms, which in turn phosphorylate the EIF6 factor on Ser235 residue, and finally activate ATF4 translation. Inhibiting PKC recapitulates the effects of FLT3-ITD inhibition or ATF4 down- regulation on cell proliferation and autophagy. Most importantly, the inventors demonstrate that R A interference down-regulation of EIF6 induces massive apoptotic cell death in FLT3-ITD cells, while not influencing the behaviour of leukemic cells expressing wild type FLT3. These data suggest that FLT3-ITD positive AML are highly dependent on the EIF6 factor for their survival, identifying this protein as a possible therapeutic target in these pathologies.

Accordingly, the first object of the present invention relates to a method of treating acute myeloid leukemia expressing the FLT3-ITD mutation in a patient in need thereof comprising administering to the patient a therapeutically effective amount of EIF6 inhibitor. As used herein, the term "acute myeloid leukemia" or "AML", also known as "acute myelogenous leukemia", has its general meaning in the art and refers to a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. AML may be classified using either the World Health Organization classification (Vardiman J W, Harris N L, Brunning R D (2002). "The World Health Organization (WHO) classification of the myeloid neoplasms". Blood 100 (7): 2292-302); or the FAB classification (Bennett J, Catovsky D, Daniel M, Flandrin G, Galton D, Gralnick H, Sultan C (1976). "Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group". Br J Haematol 33 (4): 451-8.).

As used herein, the term "FLT3" has its general meaning in the art and refers to a receptor tyrosine kinase that plays a role in regulating hematopoiesis. "FLT3" is also known as CD135, stem cell tyrosine kinase 1 (STK1), or fetal liver kinase 2 (FLK2). The receptor has an extracellular domain that includes five immunoglobulin-like domains, a transmembrane domain and an intracellular domain that includes a kinase domain. A FLT3 receptor is activated by binding of the FMS-related tyrosine kinase 3 ligand to the extracellular domain, which induces homodimer formation in the plasma membrane leading to autophosphorylation of the receptor. Human FLT3 protein sequence has the UniProtKB accession number P36888. An example of a human FLT3 polypeptide sequences is available under the reference sequences NP— 004110.2 in the NCBI polypeptide sequence database. Example of a representative FLT3 polynucleotide sequence is available in the NCBI database under accession number NM— 004119.2. Mutations that result in the constitutive activation of this receptor result in leukemia, e.g., acute myeloid leukemia and acute lymphoblastic leukemia. The most common FLT3 mutations are internal tandem duplications (ITDs) that lead to in-frame insertions within the juxtamembrane domain of the FLT3 receptor. FLT3-ITD mutations have been reported in 15- 35% of adult AML patients. See Nakao M, S Yokota and T Iwai. Internal tandem duplication of the FLT3 gene found in acute myeloid leukemia. Leukemia. 1996; 10: 191 1-1918; H Kiyoi, M Towatari and S Yokota. Internal Tandem duplication of the FLT3 gene is a novel modality of elongation mutation, which causes constitutive activation of the product. Leukemia. 1998; 12: 1333-1337; H Kiyoi, T Naoe and S Yokota. Internal tandem duplication of FLT3 associated with leukocytosis in acute promyelocytic leukemia. Leukemia Study Group of the Ministry of Health and Welfare (Kohseisho). Leukemia. 1997; 11 : 1447-1452; S Schnittger, C Schoch and M Duga. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood. 2002; 100:59-66. A FLT3- ITD mutation is an independent predictor of poor patient prognosis and is associated with increased relapse risk after standard chemotherapy, and decreased disease free and overall survival. See FM Abu-Duhier, Goodeve AC, Wilson GA, et al. FLT3 internal tandem duplication mutations in adult acute myeloid leukemia define a high risk group. British Journal of Haematology. 2000; 11 1 : 190-195; H Kiyoi, T Naoe, Y Nakano, et al. Prognostic implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia. Blood. 1999;93:3074-3080.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient 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. By "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. The phrase "maintenance regimen" or "maintenance period" 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 a 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., pain, disease manifestation, etc.]). As used herein, the term "EIF6" has its general meaning in the art and refers to the eukaryotic translation initiation factor 6 (Gene ID: 3692). The term EIF6 is also known as CAB; EIF3A; eIF-6; p27BBP; ITGB4BP; b(2)gcn; and p27(BBP). An exemplary human amino acid sequence is represented by the NCBI reference sequence NP_001254739.1. An exemplary human nucleic acid sequence (mRNA) is represented by the NCBI reference sequence NP 001254739.1 (SEQ ID NO: l).

SEQ ID NO:l (NM 001267810.1):

1 gtgtgagggg aacctgggag ggcctcatgg cggtccgagc ttcgttcgag aacaactgtg

61 agatcggctg ctttgccaag ctcaccaaca cctactgtct ggtagcgatc ggaggctcag

121 agaacttcta cagtgtgttc gagggcgagc tctccgatac catccccgtg gtgcacgcgt

181 ctatcgccgg ctgccgcatc atcgggcgca tgtgtgtggg gaacaggcac ggtctcctgg

241 tacccaacaa taccaccgac caggagctgc aacacattcg caacagcctc ccagacacag

301 tgcagattag gcgggtggag gagcggctct cagccttggg caatgtcacc acctgcaatg

361 actacgtggc cttggtccac ccagacttgg acagggagac agaagaaatt ctggcagatg

421 tgctcaaggt ggaagtcttc agacagacag tggccgacca ggtgctagta ggaagctact

481 gtgtcttcag caatcaggga gggctggtgc atcccaagac ttcaattgaa gaccaggatg

541 agctgtcctc tcttcttcaa gtcccccttg tggcggggac tgtgaaccga ggcagtgagg

601 tgattgctgc tgggatggtg gtgaatgact ggtgtgcctt ctgtggcctg gacacaacca

661 gcacagagct gtcagtggtg gagagtgtct tcaagctgaa tgaagcccag cctagcacca

721 ttgccaccag catgcgggat tccctcattg acagcctcac ctgagtcacc ttccaagttg

781 ttccatgggc tcctggctct ggactgtggc caaccttctc cacattccgc ccaatctgta

841 ccggatgctg gcagggaggt ggcagagagc tcactgggac tgaggggctg ggcacccaac

901 ccttttccac ctgtgcttat cgcctggatc tatcattact gcaaaaacct gctctgttgt

961 gctggctggc aggccctgtg gctgctggct gagggttctg ctgtcctgtg ccaccccatt

1021 aaagtgcagt tccctccggg ccattctgaa tgtgaaaaaa aaaaa

As used herein, the term "EIF6" inhibitor refers to any compound capable of inhibiting the activity or expression of EIF6. In particular, the EIF6 inhibitor of the present invention is capable of inducing massive apoptotic cell death. The EIF6 inhibition may be determined by any assay well known in the art and typically the assay as described in the EXAMPLE section of the present specification. Typically, said inhibitor is a small organic molecule or a biological molecule (e.g. nucleic acid, peptides, lipid, antibody, aptamer...).

In some embodiments, the EIF6 inhibitor is an inhibitor of EIF6 expression. An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In a preferred embodiment of the invention, said inhibitor of gene expression is a siR A, an antisense oligonucleotide or a ribozyme.

In some embodiments, the inhibitor of expression is a siRNA. Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. EIF6 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 EIF6 gene expression is specifically inhibited (i.e. RNA interference or RNAi).

In some embodiments, the inhibitor of expression is an endonuclease. The term "endonuclease" refers to enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as Deoxyribonuc lease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases or restriction enzymes, cleave only at very specific nucleotide sequences. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the error prone non homologous end-joining (NHEJ) and the high-fidelity homo logy-directed repair (HDR). In a particular embodiment, the endonuclease is CRISPR- Cas. As used herein, the term "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. In some embodiment, 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. In some embodiment, 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).

In some embodiments, the inhibitor of expression is an antisense oligonucleotide. The term "antisense oligonucleotide" refers to an oligonucleotide sequence that is inverted relative to its normal orientation for transcription and so expresses an RNA transcript that is complementary to a target gene mRNA molecule expressed within the host cell (e.g., it can hybridize to the target gene mRNA molecule through Watson-Crick base pairing). An antisense strand may be constructed in a number of different ways, provided that it is capable of interfering with the expression of a target gene. For example, the antisense strand can be constructed by inverting the coding region (or a portion thereof) of the target gene relative to its normal orientation for transcription to allow the transcription of its complement, (e.g., RNAs encoded by the antisense and sense gene may be complementary). Furthermore, the antisense oligonucleotide strand need not have the same intron or exon pattern as the target gene, and noncoding segments of the target gene may be equally effective in achieving antisense suppression of target gene expression as coding segments.

As used herein, the term "oligonucleotide" refers to a nucleic acid sequence, 3'-5' or 5'- 3' oriented, which may be single- or double-stranded. The antisense oligonucleotide used in the context of the invention may in particular be DNA or R A.

According to the invention, the antisense oligonucleotide of the present invention targets an mRNA encoding EIF6 (e.g. SEQ ID NO: l), and is capable of reducing the amount of EIF6 in cells. As used herein, an oligonucleotide that "targets" an mRNA refers to an oligonucleotide that is capable of specifically binding to said mRNA. That is to say, the antisense oligonucleotide comprises a sequence that is at least partially complementary, preferably perfectly complementary, to a region of the sequence of said mRNA, said complementarity being sufficient to yield specific binding under intra-cellular conditions. As immediately apparent to the skilled in the art, by a sequence that is "perfectly complementary to" a second sequence is meant the reverse complement counterpart of the second sequence, either under the form of a DNA molecule or under the form of a RNA molecule. A sequence is "partially complementary to" a second sequence if there are one or more mismatches. The antisense oligonucleotide of the present invention that target an mRNA encoding EIF6 may be designed by using the sequence of said mRNA as a basis, e.g. using bioinformatic tools. For example, the sequence of SEQ ID NO: 1 can be used as a basis for designing nucleic acids that target an mRNA encoding EIF6. Preferably, the antisense oligonucleotide according to the invention is capable of reducing the amount of EIF6 in cells, e.g. in cancerous cells. Methods for determining whether an oligonucleotide is capable of reducing the amount of EIF6 in cells are known to the skilled in the art. This may for example be done by analyzing EIF6 protein expression by Western blot, and by comparing EIF6 protein expression in the presence and in the absence of the antisense oligonucleotide to be tested.

In some embodiments, the antisense oligonucleotide of the present invention has a length of from 12 to 50 nucleotides, e.g. 12 to 35 nucleotides, from 12 to 30, from 12 to 25, from 12 to 22, from 15 to 35, from 15 to 30, from 15 to 25, from 15 to 22, from 18 to 22, or about 19, 20 or 21 nucleotides. The antisense oligonucleotide according to the invention may for example comprise or consist of 12 to 50 consecutive nucleotides, e.g. 12 to 35, from 12 to 30, from 12 to 25, from 12 to 22, from 15 to 35, from 15 to 30, from 15 to 25, from 15 to 22, from 18 to 22, or about 19, 20 or 21 consecutive nucleotides of a sequence complementary to the mRNA of SEQ ID NO : 1. In some embodiments, the antisense oligonucleotide of the present invention is further modified, preferably chemically modified, in order to increase the stability and/or therapeutic efficiency of the antisense oligonucleotide in vivo. In particular, the antisense oligonucleotide used in the context of the invention may comprise modified nucleotides. Chemical modifications may occur at three different sites: (i) at phosphate groups, (ii) on the sugar moiety, and/or (iii) on the entire backbone structure of the antisense oligonucleotide. For example, the antisense oligonucleotide may be employed as phosphorothioate derivatives (replacement of a non-bridging phosphoryl oxygen atom with a sulfur atom) which have increased resistance to nuclease digestion. 2'-methoxyethyl (MOE) modification (such as the modified backbone commercialized by ISIS Pharmaceuticals) is also effective. Additionally or alternatively, the antisense oligonucleotide of the present invention may comprise completely, partially or in combination, modified nucleotides which are derivatives with substitutions at the 2' position of the sugar, in particular with the following chemical modifications: O-methyl group (2'-0-Me) substitution, 2-methoxyethyl group (2'-0-MOE) substitution, fluoro group (2'- fluoro) substitution, chloro group (2'-Cl) substitution, bromo group (2'-Br) substitution, cyanide group (2'-CN) substitution, trifluoromethyl group (2'-CF3) substitution, OCF3 group (2'-OCF3) substitution, OCN group (2'-OCN) substitution, O-alkyl group (2'-0-alkyl) substitution, S-alkyl group (2'-S-alkyl) substitution, N-alkyl group (2'-N-akyl) substitution, O-alkenyl group (2'-0- alkenyl) substitution, S-alkenyl group (2'-S-alkenyl) substitution, N-alkenyl group (2'-N- alkenyl) substitution, SOCH3 group (2 ^* -SOCH3) substitution, S02CH3 group (2 ^* -S02CH3) substitution, ON02 group (2'-ON02) substitution, N02 group (2'-N02) substitution, N3 group (2'-N3) substitution and/or NH2 group (2 -NH2) substitution. Additionally or alternatively, the antisense oligonucleotide of the present invention may comprise completely or partially modified nucleotides wherein the ribose moiety is used to produce locked nucleic acid (LNA), in which a covalent bridge is formed between the 2' oxygen and the 4' carbon of the ribose, fixing it in the 3'-endo configuration. These constructs are extremely stable in biological medium, able to activate RNase H and form tight hybrids with complementary R A and DNA. Accordingly, in a preferred embodiment, the antisense oligonucleotide used in the context of the invention comprises modified nucleotides selected from the group consisting of LNA, 2'- OMe analogs, 2 '-phosphorothioate analogs, 2'-fluoro analogs, 2'-Cl analogs, 2'-Br analogs, 2'- CN analogs, 2'-CF3 analogs, 2'-OCF3 analogs, 2'-OCN analogs, 2'-0-alkyl analogs, 2'-S- alkyl analogs, 2 '-N-alkyl analogs, 2 '-O-alkenyl analogs, 2 '-S-alkenyl analogs, 2 '-N-alkenyl analogs, 2'-SOCH3 analogs, 2'-S02CH3 analogs, 2'-ON02 analogs, 2'-N02 analogs, 2'-N3 analogs, 2'-NH2 analogs and combinations thereof. More preferably, the modified nucleotides are selected from the group consisting of LNA, 2'-OMe analogs, 2'-phosphorothioate analogs and 2'-fluoro analogs. In some embodiments, the antisense is a Tricyclo-DNA antisense. The term "tricyclo-DNA (tc-DNA)" refers to a class of constrained oligodeoxyribonucleotide analogs in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict conformational flexibility of the backbone and to optimize the backbone geometry of the torsion angle γ as (Ittig D, et al, Nucleic Acids Res, 2004, 32:346-353; Ittig D, et al, Prague, Academy of Sciences of the Czech Republic. 1 :21-26 (Coll. Symp. Series, Hocec, M., 2005); Ivanova et al, Oligonucleotides 2007, 17:54-65; Renneberg D, et al, Nucleic Acids Res, 2002, 15 30:2751-2757; Renneberg D, et al, Chembiochem, 2004, 5: 1114-1118; and Renneberg D, et al, JACS, 2002, 124:5993-6002). In detail, the tc-DNA differs structurally from DNA by an additional ethylene bridge between the centers C(3' ) and C(5' ) of the nucleosides, to which a cyclopropane unit is fused for further enhancement of structural rigidity. See e.g. WO2010115993 for examples of tricyclo- DNA (tc-DNA) antisense oligonucleotides. The advantage of the tricyclo-DNA chemistry is that the structural properties of its backbone allow a reduction in the length of an AON while retaining high affinity and highly specific hybridization with a complementary nucleotide sequence. Unexpectedly, tc-DNA AON may be advantageously used in microgram dosages in the in vivo setting using intramuscular application, which are at least 10-fold less than the dosages required for conventional antisense oligonucleotide technologies. In addition, tc-DNA retains full activity with reduced antisense lengths. Thus, for example, tc-DNA AON of 13 to 15 nucleotides are highly effective in the ex vivo and in vivo applications exemplified by the present disclosure.

The antisense oligonucleotide of the invention can be synthesized de novo using any of a number of procedures well known in the art. These chemistries can be performed by a variety of automated nucleic acid synthesizers available in the market. These nucleic acids may be referred to as synthetic nucleic acids. Alternatively, antisense oligonucleotide can be produced on a large scale in plasmids (see Sambrook, et al, 1989). The antisense oligonucleotide can be prepared from existing nucleic acid sequences using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases.

In some embodiments, the antisense oligonucleotide of the present invention is modified by substitution at the 3' or the 5' end by a moiety comprising at least three saturated or unsaturated, preferably saturated, linear or branched, preferably linear, hydrocarbon chains comprising from 2 to 30 carbon atoms, preferably from 5 to 20 carbon atoms, more preferably from 10 to 18 carbon atoms as described in WO2014195432. In some embodiments, the modified antisense oligonucleotide is of the general formula

(I):

wherein:

Oligo represents the antisense oligonucleotide sequence of the present invention oriented 3 '-5' or 5 '-3',

X represents a divalent linker moiety selected from ether -0-, thio -S-, amino -NH-, and methylene -CH ₂ - ;

Ri and R ₂ may be identical or different and represent:

i hydrogen atom,

a halogen, in particular fluorine atom,

(iii) a hydroxy 1 group,

(iv) an alkyl group comprising from 1 to 12 carbon atoms ;

Mi, M ₂ and M ₃ may be identical or different and represent:

- a saturated or unsaturated, preferably saturated, linear or branched, preferably linear, hydrocarbon chain comprising from 2 to 30 carbon atoms, preferably from 6 to 22 carbon atoms, more preferably from 12 to 20 carbon atoms, which may be substituted by one or more halogen atoms, notably be fluorinated or perfluorinated and/or be interrupted by one or more groups selected from ether -0-, thio -S-, amino -NH-, oxycarbonyl -O-C(O)-, thiocarbamate - 0-C(S)-NH-, carbonate -0-C(0)-0-, carbamate -0-C(0)-NH-, phosphate -0-P(0)(0)-0- and phosphonate -P-0(0)(0)- groups; and/or be substituted at the terminal carbon atom by an aliphatic or aromatic, notably benzylic or naphtylic ester or ether group;

- an acyl radical with 2 to 30 carbon atoms, preferably with 6 to 22 carbon atoms, more preferably with 12 to 20 carbon atoms, or

- an acylglycerol, sphingosine or ceramide group.

In the context of the invention, the term "alkyl" refers to a hydrocarbon chain that may be a linear or branched chain, containing the indicated number of carbon atoms. For example, Ci-Ci ₂ alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. In the context of the invention, the term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl or heteroarylcarbonyl substituent. Typically, the antisense oligonucleotide sequence "Oligo-" is connected to the divalent linker moiety X via a phosphate moiety -0-P(=0)(0 ^" )-, at its 3' or 5' end, advantageously at its 5' end.

In some embodiments, the antisense modified oligonucleotide is of the general formula

(Γ):

wherein:

X, Ri, R ₂ , Mi, M ₂ and M ₃ are as defined above in formula (I),

[3'— 5'] represents, along with the P0 ₃ ^" residue, the antisense oligonucleotide of the present invention, and

A ⁺ represents a cation, preferably H ⁺ , Na ⁺ , K ⁺ or NH ₄ ⁺ .

In the formulae (I) and (Γ), the divalent linker moiety is preferably ether -0-.

In the formulae (I) and (Γ), Ri and R ₂ are preferably hydrogen atoms.

In some embodiments, the antisense modified oligonucleotide is of the formula (!"):

wherein A ⁺ , Mi, M ₂ and M ₃ are as defined above in formula (I) and [3'— 5'] represents, along with the P0 ₃ ^" residue, the antisense oligonucleotide of the present invention.

In the formulae (I), (Γ) and (I"), Mi, M ₂ and M ₃ preferably represent a hydrocarbon chain, preferably a linear hydrocarbon chain, comprising from 6 to 22 carbon atoms, preferably from 12 to 20 carbon atoms, more preferably 18 carbon atoms.

In some embodiments, the antisense modified oligonucleotide is of the formula (!" '):

wherein A ⁺ is as defined above in formula (I) and [3'— 5'] represents, along with the PO3 ^" residue, the antisense oligonucleotide of the present invention.

In formula (Γ "), the chains -C18H37 are preferably straight alkyl chains.

In some embodiments, the antisense oligonucleotide of the present invention is modified by substitution at the 3 ' or the 5 ' end by a moiety comprising at least one ketal functional group, wherein the ketal carbon of said ketal functional group bears two saturated or unsaturated, preferably saturated, linear or branched, preferably linear, hydrocarbon chains comprising from 1 to 22 carbon atoms, preferably from 6 to 20 carbon atoms, in particular 10 to 19 carbon atoms, and even more preferably from 12 to 18 carbon atoms as described in WO2014195430.

In some embodiments, the modified antisense oligonucleotide is of the general formula

(I):

(I)

wherein:

Oligo represents the antisense oligonucleotide of the present invention;

X represents a divalent linker moiety selected from ether -0-, thio -S-, amino -NH-, and methylene -CH2-;

Ri and R2 may be identical or different and represent:

(i) a hydrogen atom,

(ii) a halogen atom, in particular fluorine atom,

(iii) a hydroxy 1 group,

(iv) an alkyl group comprising from 1 to 12 carbon atoms ;

Li and L2 may be identical or different and represent a saturated or unsaturated, preferably saturated, linear or branched, preferably linear, hydrocarbon chain comprising from 1 to 22 carbon atoms, preferably from 6 to 20 carbon atoms, more preferably from 12 to 18 carbon atoms,

B is an optionally substituted nucleobase, selected from the group consisting of purine nucleobases, pyrimidine nucleobases, and non-natural monocyclic or bicyclic heterocyclic nucleobases wherein each cycle comprises from 4 to 7 atoms.

Typically, the antisense oligonucleotide sequence "Oligo-" is connected to the divalent linker moiety X via a phosphate moiety -0-P(=0)(0 ^" )-, at its 3' or 5' end, advantageously at its 5' end.

In some embodiments, the antisense modified oligonucleotide is of the general formula

wherein:

wherein X, Ri, R ₂ , Li, L ₂ and B are as defined above in formula (I),

[3'— 5'] represents, along with the PO3 ^" residue, the antisense oligonucleotide of the present invention, and

A ⁺ represents a cation, preferably H ⁺ , Na ⁺ , K ⁺ or NH ₄ ⁺ .

In the formulae (I) and (Γ), the divalent linker moiety is preferably ether -0-.

In the formulae (I) and (Γ), Ri and R ₂ are preferably hydrogen atoms.

In some embodiments, the antisense modified oligonucleotide is of the formula (!"):

wherein A ⁺ , X, Li, L ₂ and B are as defined above in formula (I) and [3 '— 5 '] represents, along with the PO3 ^" residue, the antisense oligonucleotide of the present invention.

In the formulae (I), (Γ) and (I"), Li and L ₂ preferably represent a hydrocarbon chain, preferably a linear hydrocarbon chain, comprising from 6 to 22 carbon atoms, preferably from 8 to 18 carbon atoms, advantageously from 12 to 16 carbon atoms, more advantageously 15 carbon atoms.

In the formulae (I), (Γ) and (I"), B preferably represents a non-substituted nucleobase selected from the group consisting of uracil, thymine, adenine, guanine, cytosine, 6- methoxypurine, 7-methylguanine, xanthine, 5,6-dihydrouracil, 5-methylcytosine, 5- hydroxymethylcytosine and hypoxanthine. Preferably, in the formulae (I), (Γ) and (I"), B represents a non-substituted nucleobase selected from the group consisting of uracil, thymine, adenine, cytosine, 6-methoxypurine and hypoxanthine. More preferably, in the formulae (I), (Γ) and (I"), B represents uracil.

In some embodiments, the antisense modified oligonucleotide is of the formula (!" '):

wherein A ⁺ is as defined above in formula (I) and [3'— 5'] represents, along with the PO3 ^" residue, the antisense oligonucleotide of the present invention.

In some embodiments, the antisense oligonucleotide of the present invention is associated with a carrier or vehicle, e.g., liposomes or micelles, although other carriers could be used, as would be appreciated by one skilled in the art. Liposomes are vesicles made of a lipid bilayer having a structure similar to biological membranes. Such carriers are used to facilitate the cellular uptake or targeting of the antisense oligonucleotide, or improve the antisense oligonucleotide's pharmacokinetic or toxicologic properties. For example, the antisense oligonucleotide of the present invention may also be administered encapsulated in liposomes, pharmaceutical compositions wherein the active ingredient is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers. The antisense oligonucleotide, depending upon solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension. The hydrophobic layer, generally but not exclusively, comprises phopholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, or other materials of a hydrophobic nature. The diameters of the liposomes generally range from about 15 nm to about 5 microns. The use of liposomes as drug delivery vehicles offers several advantages. Liposomes increase intracellular stability, increase uptake efficiency and improve biological activity. Liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids, which make up the cell membrane. They have an internal aqueous space for entrapping water soluble compounds and range in size from 0.05 to several microns in diameter. Several studies have shown that liposomes can deliver nucleic acids to cells and that the nucleic acids remain biologically active. Finally, cost-effective manufacture of liposome -based pharmaceuticals, either in a liquid suspension or lyophilized product, has demonstrated the viability of this technology as an acceptable drug delivery system.

In some embodiments, the antisense oligonucleotide of the present invention is complexed with a complexing agent to increase cellular uptake of oligonucleotides. An example of a complexing agent includes cationic lipids. Cationic lipids can be used to deliver oligonucleotides to cells. The term "cationic lipid" includes lipids and synthetic lipids having both polar and non-polar domains and which are capable of being positively charged at or around physiological pH and which bind to polyanions, such as nucleic acids, and facilitate the delivery of nucleic acids into cells. In general cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides, or derivatives thereof. Straight-chain and branched alkyl and alkenyl groups of cationic lipids can contain, e.g., from 1 to about 25 carbon atoms. Preferred straight chain or branched alkyl or alkene groups have six or more carbon atoms. Alicyclic groups include cholesterol and other steroid groups. Cationic lipids can be prepared with a variety of counterions (anions) including, e.g., C1-, Br-, I-, F-, acetate, trifluoroacetate, sulfate, nitrite, and nitrate. Examples of cationic lipids include: polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, Lipofectamine, DOPE, Cytofectin (Gilead Sciences, Foster City, Calif), and Eufectins (JBL, San Luis Obispo, Calif). Cationic liposomes may comprise the following: N-[l-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[l-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3p-[N-(N' ,Ν' -dimethylaminoethane)carbamoyl]cholesterol (DC-Choi), 2,3,- dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-pr opanaminium

trifluoroacetate (DOSPA), l,2-dimyristyloxypropyl-3-dimethy-l -hydroxy ethyl ammonium bromide; and dimethyldioctadecylammonium bromide (DDAB). The cationic lipid N-(l-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), for example, was found to increase 1000-fold the antisense effect of a phosphorothioate oligonucleotide. (Vlassov et al., 1994, Biochimica et Biophysica Acta 1197:95-108). Oligonucleotides can also be complexed with, e.g., poly(L-lysine) or avidin and lipids may, or may not, be included in this mixture (e.g., steryl-poly(L-lysine). Cationic lipids have been used in the art to deliver oligonucleotides to cells (see, e.g., U.S. Pat. Nos. 5,855,910; 5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al. 1996. Proc. Natl. Acad. Sci. USA 93:3176; Hope et al. 1998. Molecular Membrane Biology 15: 1). Other lipid compositions which can be used to facilitate uptake of the instant oligonucleotides can be used in connection with the claimed methods. In addition to those listed supra, other lipid compositions are also known in the art and include, e.g., those taught in U.S. Pat. No. 4,235,871; U.S. Pat. Nos. 4,501,728; 4,837,028; 4,737,323.

As used herein, the term "therapeutically effective amount" as used herein refers to an amount or dose of the inhibitor of the present invention that is sufficient to treat the patient. The amount of the inhibitor in a given therapeutically effective combination may be different for different individuals and different tumor types, and will be dependent upon the one or more additional agents or treatments included in the combination. The "therapeutically effective amount" is determined using procedures routinely employed by those of skill in the art such that an "improved therapeutic outcome" results. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. The useful dosage to be administered and the particular mode of administration will also vary depending upon the form of the formulation, for example, suspension, emulsion, micelle or liposome, as will be readily apparent to those skilled in the art.

In some embodiments, the EIF6 inhibitor of the present invention is administered to the patient in combination with at least one FLT3 inhibitor. Exemplary FLT3 inhibitors that are contemplated by the invention include but are not limited to those described in Sternberg et al. 2004 and in International Patent Application Nos WO 2002032861, WO 2002092599, WO 2003035009, WO 2003024931, WO 2003037347, WO 2003057690, WO 2003099771, WO 2004005281, WO 2004016597, WO 2004018419, WO 2004039782, WO 2004043389, WO 2004046120, WO 2004058749, WO 2004058749, WO 2003024969, WO 2006/138155, WO 2007/048088 and WO 2009/095399 which are incorporated herein by reference. More particularly, FLT3 inhibitors may consist in FLT3 kinase inhibitors. Exemplary of FLT3 kinase inhibitors that are contemplated include Quizartinib (AC220), AG 1295 and AG 1296; Lestaurtinib (also known as CEP-701, formerly KT-5555, Kyowa Hakko, licensed to Cephalon); CEP-5214 and CEP-7055 (Cephalon); CHIR-258 (Chiron Corp.); GTP 14564 (Merk Biosciences UK). Midostaurin (also known as PKC 412 Novartis AG); MLN-608 (Millennium USA); MLN-518 (formerly CT53518, COR Therapeutics Inc., licensed to Millennium Pharmaceuticals Inc.); MLN-608 (Millennium Pharmaceuticals Inc.); SU- 11248 (Pfizer USA); SU-11657 (Pfizer USA); SU-5416 and SU-5614; THRX-165724 (Theravance Inc.); AMI- 10706 (Theravance Inc.); VX-528 and VX-680 (Vertex Pharmaceuticals USA, licensed to Novartis (Switzerland), Merck & Co USA); and XL 999 (Exelixis USA).

The EIF6 inhibitor 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. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Galenic adaptations may be done for specific delivery in the small intestine or colon. Preferably, the pharmaceutical compositions contain 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 dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol ; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability 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. Solutions comprising EIF6 inhibitors of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The EIF6 inhibitor of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifusoluble agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. The EIF6 inhibitor of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered. In addition to the EIF6 inhibitors of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations ; time release capsules ; and any other form currently used.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Impact of EIF6 down regulation on ATF4 protein expression and on autophagy. MOLM-14 (FLT3-ITD) (A), MV4-11 (FLT3-ITD) (B) or OCI-AML3 (FLT3wt) (C) cell lines expressing doxycycline-inducible shRNA against EIF6 were generated. The impact of EIF6 down regulation on the ATF4 protein level and on the autophagy process was followed by western blot analysis of LC3b and ATF4 in these 3 cell lines. Figure 2: Importance of EIF6 for FLT3-ITD AML cells proliferation and survival.

MOLM-14 shEIF6 (A-B), MV4-11 shEIF6 (C) and OCI-AML3 shEIF6 (D) cell lines were induced with doxycycline to down-regulate EIF6, and counted each day (A, C and D respectively) to estimate the importance of EIF6 in the proliferation process of these cells. For MOLM14 cells, the impact of EIF6 down-regulation on cell death (apoptosis) was evaluated by Annexin V labelling (C).

Figure 3: PKC regulates ATF4 protein level and autophagy downstream of FLT3- ITD. A. Protein kinase C βΙΙ phosphorylation on Ser 266 was followed by western blot in MOLM-14 cells treated with FLT3-ITD inhibitor. B. MOLM-14 cells were treated with the PKC inhibitors Bisindolylmaleimide (Bis) or enzastaurin (Enza) for 2 hours, and ATF4 protein level was estimated by western blot. C. MOLM-14 cells were treated with Bisindolylmaleimide (Bis) or enzastaurin (Enza) for 16 hours in the presence or the absence of chloroquine, and the autophagy level was estimated by western blot analysis of LC3.

Figure 4: Importance of EIF6 for the survival of mice engrafted with FLT3-ITD AML cells. NSG mice (n=15) engrafted with MOLM-14 cells stably expressing the eIF6 inducible shR As by IV injection, were treated with sucrose (10 μg/ml) with or without doxycycline (200 μg/ml) via their drinking water and their overall survival was analyzed. The graph represents the Kaplan-Meier survival curves.

EXAMPLE:

In a recent work, we found that FLT3-ITD mutations are able to induce an increase in basal autophagy in leukemic cell lines as well as in primary cells from patients (data not shown). We found that this occurs through a previously uncharacterised signalling cascade involving the transcription factor ATF4 which is tightly regulated by FLT3-ITD activity and is involved in the autophagy induced by this receptor (data not shown). Moreover, inhibiting autophagy or ATF4 significantly impaired FLT3-ITD leukemic cell proliferation (data not shown) as well as tumour burden in murine xenograft models (data not shown). Importantly, autophagy inhibition also overcame FLT3 inhibitor resistance due to FLT3-TKD mutation both in vitro and in vivo (data not shown). These results suggest that targeting ATF4 or autophagy in AML patients carrying FLT3 mutations may represent a promising alternative therapeutic strategy.

To further precise the molecular pathways working in this process, we investigated how the transcription factor ATF4 is regulated downstream of FLT3-ITD. By inhibiting either translation (cycloheximide) or degradation (proteasome inhibitor) of the proteins in FLT3-ITD AML cells, we could establish that FLT3-ITD receptor activity regulates the translation of ATF4. Indeed, FLT3-ITD inhibition did not affect the mRNA level of ATF4, as determined by RT-QPCR experiments, nor its stability (data not shown). Instead, we found that inhibiting FLT3 did not modify the half-life of the protein, but rather reduced its synthesis and re- accumulation following its previous down-regulation by cycloheximide treatment of the cells.

Since ATF4 translation has been extensively described downstream of the EIF2alpha integrated stress pathway (for a review see 16) we asked whether this pathway could be involved in the regulation of ATF4 in the AML model. Although we observed increased phosphorylation of EIF2alpha in response to FLT3-ITD inhibition, this was correlated with quick down-regulation of ATF4, at the opposite/contrary of the process described in the literature, indicating that ATF4 is regulated though another translation pathway downstream of FLT3-ITD (data not shown). In a recent work, ATF4 was described as a target of the initiation translation factor EIF6 for the regulation of lipid metabolisms and glycolysis in the context of insulin sensitivity (17). In consequence, we investigated the status of the EIF6 protein in AML cell lines, and whether this factor could be involved in ATF4 protein regulation. As shown in Figure 1, the EIF6 factor is well expressed in AML cell lines, and its down-regulation by doxycycline-induced RNA interference completely switched off the protein expression of ATF4. Since we previously identified ATF4 as a master regulator of autophagy in these cells, we looked whether EIF6 down-regulation also had an impact on the autophagy process. In good agreement with the precedent data, EIF6 down-regulation indeed reduced the autophagy level in FLT3-ITD positive AML cell lines (Figure 1). Interestingly, EIF6 down regulation did not impact ATF4 level nor autophagy in the OCI-AML3 cell line expressing wild type FLT3 (Figure 1). More importantly, when we investigated the impact of EIF6 down regulation on the proliferation, we found that FLT3-ITD cells were highly dependent of EIF6 not only for their proliferation, but also for their survival. Indeed, as shown in Figure 2 down-regulating EIF6 induced a complete arrest of cell proliferation and massive apoptotic cell death in MOLM-14 and MV4-11 cells, while similar shRNA-induced down-regulation had almost no impact on the proliferation and the survival of OCI-AML3 cells, which express the wild type FLT3 receptor. These data strongly suggest that EIF6 is essential for survival of FLT3-ITD positive cells, in addition to being important for the autophagy process mediated by FLT3-ITD.

We did not detect any variation of EIF6 protein level when we treated MOLM-14 cells with the FLT3 inhibitor AC -220 (data not shown), suggesting that EIF6 regulation by FLT3- ITD occurred at the post-translational level.

Although there are only few information concerning post-translational regulations of EIF6, phosphorylation of EIF6 by protein kinase C leading to its activation has been described as a major mechanism of regulation of this factor (18). In consequence, we decided to investigate whether PKCs are involved in EIF6-dependant regulation of autophagy and proliferation in this model. First we monitored PKC activation level in response to AC-220, and found that FLT3 inhibition decreased the activating phosphorylation of PKC iI on Ser 660, indicating that PKCs are indeed activated down-stream of FLT3-ITD (Figure 3). We then treated MOLM-14 cells with two independent inhibitors of PKC, Bisindolylmaleimide, and enzastaurine. We observed that ATF4 was quickly down-regulated in these conditions, and that the autophagy level of the cells was consequently decreased, recapitulating the effects observed with AC-220 (Figure 3). We then tested whether FLT3 inhibition modifies the phosphorylation status of EIF6. Phospho-specific antibodies against Ser235 phosphorylated EIF6 are commercially available, but we were unable to detect a specific signal efficiently in our cell models (data not shown).

Finally, we investigated the impact of EIF6 down regulation in the survival of mice engrafted with a FLT3-ITD positive AML cell line (i.e. MOLM-14). We showed that the survival is significantly increased when the EIF6 down regulation is induced by doxycycline (Figure 4).

In conclusion, down regulating the expression or activity of EIF6 is of a particular interest for the treatment of acute myeloid leukemia expressing the FLT3-ITD mutation.

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