Methods for inducing cell death in a population of solid tumor cells - Patents.com

Abstract

It has been previously demonstrated that the RAC2 G12R mutation rapidly induced cell death and hematopoietic control of HSPCs. Now, the present inventors have evaluated the effect of said mutation on three tumor cell lines: MDA-MB-231 (breast adenocarcinoma), HT29 (colon adenocarcinoma) and HepG2 (hepatocellular carcinoma). Briefly, cells were transduced with lentiviral vectors containing a green fluorescent protein (GFP) reporter cDNA (WPI) or a wild-type form of RAC2 cDNA (WT) or a RAC2 mutant cDNA form (G12R). The inventors showed that the number of GFP+ cells was dramatically reduced in the G12R condition compared to the WT and WPI conditions. The morphology and contents of the cells were particularly disrupted. These observations were confirmed in a time-course proliferation assay performed on MDA-MB-231 and HT29 cell lines. In summary, these data provide the basis for the deleterious effect of the RAC2 G12R mutation on the proliferation of tumor cell lines.

Description

[Technical field]

[0001] Field of the invention: The present invention is an invention in the field of medicine, in particular oncology. [Background technology]

[0002] Background of the invention: Cancer is one of the leading causes of death. For example, in 2019, there were 599,601 cancer deaths; of which, 283,725 were women and 315,876 were men (Centers for Disease Control and Prevention. An Update on Cancer Deaths in the United States. Atlanta, GA: US Department of Health and Human Services, Centers for Disease Control and Prevention, Division of Cancer Prevention and Control; 2021). Lung cancer was the leading cause of cancer death, accounting for 23% of all cancer deaths in the United States in 2019. Other common causes of cancer death were cancer of the colon and rectum (9%), pancreas (8%), female breast (7%), prostate (5%), and liver and intrahepatic bile duct (5%). Other cancers each accounted for less than 5% of cancer deaths. Therefore, new cancer treatments are needed.

[0003] Recently, an autosomal dominant (AD) missense mutation (i.e., p.G12R) in the RAC2 gene (encoding Ras-associated C3 botulinum toxin substrate 2 (RAC2)) was identified in three severe combined immunodeficiency (SCID) patients whose clinical symptoms overlap with the RD SCID form but lacked AK2 mutations and hearing loss (Lagresle-Peyrou C, Olichon A, Sadek H, Roche P, Tardy C, Da Silva C, Garrigue A, Fischer A, Moshous D, Collette Y, Picard C, Casanova JL, Andre I, Cavazzana M. A gain-of-function RAC2 mutation is associated with bone-marrow hypoplasia and an autosomal dominant form of severe combined immunodeficiency. Haematologica. 2021 Feb 1;106(2):404-411). RAC2 mutations were closely associated with impaired cell differentiation potential and defects in cellular and mitochondrial networks. Consequently, WO 2021 / 009336 teaches a method of inducing cell death of a population of malignant hematopoietic cells, comprising contacting the population with an effective amount of i) a RAC2 polypeptide comprising the p.G12R mutation, or ii) a polynucleotide encoding such a polypeptide. Summary of the Invention [Problem to be solved by the invention]

[0004] Summary of the invention: The invention is defined by the claims. In particular, the invention relates to a method of inducing cell death in a population of solid tumor cells. [Means for solving the problem]

[0005] Detailed description of the invention: A first object of the present invention relates to a method for inducing cell death in a population of solid tumor cells, comprising contacting said population with an effective amount of i) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, wherein the amino acid residue at position 12 (G) is mutated, or ii) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, wherein the amino acid residue at position 12 (G) is mutated.

[0006] A further object of the present invention relates to a method for treating a solid tumor in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of i) a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:1, wherein the amino acid residue at position 12 (G) is mutated, or ii) a polynucleotide encoding a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:1, wherein the amino acid residue at position 12 (G) is mutated. [Brief description of the drawings]

[0007] [Figure 1A] The RAC2 G12R mutation impaired the proliferation of solid tumor cell lines. Evaluation of the proliferation potential of three solid tumor cell lines (MDA-MB-231, HT29 and HepG2) after 5 and 8 days of in vitro culture (grey and black histograms, respectively). The number of viable GFP+ cells was assessed after lentiviral transduction with a fluorescent protein (GFP) reporter cDNA (WPI), a wild-type form of RAC2 cDNA (WT) or a RAC2 mutant cDNA form (G12R). For each cell line, the histogram is representative of three independent experiments. [Figure 1B] RAC2 G12R mutation impaired the proliferation of solid tumor cell lines. In vitro kinetic proliferation assay of MDA-MB-231 (left panel) and HT29 (right panel) cell lines non-transduced (NT) or transduced with WPI, WT or G12R lentiviral constructs. Growth of GFP+ cells (measured as % of confluence using a green filter) was assessed every 3 hours for 14 days. For each cell line, curves are representative of three independent experiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] As used herein, the term "solid cancer" has its general meaning in the art and refers to one or more cells that are growing or have grown to form cancerous tissue in an uncontrolled manner. As used herein, the term "solid cancer" includes, but is not limited to, "carcinoma," "adenocarcinoma," and "sarcoma." "Sarcoma" is a cancer of connective tissue, cartilage, bone, muscle, etc. "Carcinoma" is a cancer of epithelial (surface) cells. "Adenocarcinoma" refers to a carcinoma derived from cells of glandular origin. The terms "cancer" and "tumor" are used interchangeably throughout the subject specification. The term "cancer" is not limited to any stage, grade, histomorphological features, invasiveness, aggressiveness, or grade of malignancy of the affected tissue or cell population. In particular, it includes stage 0 cancer, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, grade I cancer, grade II cancer, grade III cancer, malignant cancer, and primary carcinoma.

[0009] Typically, patients undergoing the above methods are those with adrenal cortical carcinoma, anal cancer, bile duct cancer (e.g., periphilar carcinoma, distal bile duct carcinoma, intrahepatic cholangiocarcinoma), bladder cancer, bone cancer (e.g., osteoblastoma, osteochondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, multiple myeloma), brain and central nervous system cancer (e.g., meningioma, astrocytoma, oligodendroglioma, ependymoma, glioma, medulloblastoma, , ganglioglioma, schwannoma, germinoma, craniopharyngioma), breast cancer (e.g., ductal carcinoma in situ, invasive ductal carcinoma, invasive lobular carcinoma, lobular carcinoma in situ, gynecomastia), cervical cancer, colorectal cancer, endometrial cancer (e.g., endometrial adenocarcinoma, adenoid carcinoma (adenocanthoma), papillary serous adenocarcinoma, clear cell), esophageal cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g., choriocarcinoma, destructive villous adenoma), ka Posiform sarcoma, kidney cancer (e.g., renal cell carcinoma), cancer of the pharynx and hypopharynx, liver cancer (e.g., hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, cancer of the nasal cavity and paranasal sinuses (e.g., olfactory neuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, cancer of the oral cavity and oropharynx, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g., The patient may be suffering from a solid cancer selected from the group consisting of: ovarian cancer, ...

[0010] As used herein, the term "treatment" or "treating" refers to both preventative or prophylactic treatments and curative or disease-curative treatments, including treatments of patients at risk of or suspected of having a disease, as well as patients who are ill or have been diagnosed with a disease or medical condition, including suppression of clinical recurrence. Treatments may be administered to patients with a medical disorder or those at risk of eventually acquiring a disorder, to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to prolong the survival of the patient beyond that expected in the absence of such treatment. "Therapeutic regimen" refers to a pattern of treatment of a disease, e.g., a pattern of dosing used during treatment. The therapeutic regimen may include induction regimens and maintenance regimens. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to the patient during the initial period of the treatment regimen. The induction regimen may employ (partially or entirely) a "loading regimen", which may involve administering a higher dose of the drug than the physician would employ during a maintenance regimen, administering the drug more frequently than the physician would administer during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used to maintain a patient during disease treatment, for example, to keep the patient in remission for an extended period of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering the drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., discontinued treatment, intermittent treatment, treatment upon relapse, or treatment upon reaching a certain predetermined criterion (e.g., symptoms of disease, etc.).

[0011] As used herein, the term "Rac2" has its general meaning in the art and refers to Ras-related C3 botulinum toxin substrate 2. An exemplary amino acid sequence for human Rac2 is represented by SEQ ID NO:1. [ka]

[0012] As used herein, the term "mutation" has its general meaning in the art and refers to substitution, deletion or insertion. The term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted at the same position. The term "deletion" means that a specific amino acid residue is removed. The term "insertion" means that one or more amino acid residues are inserted before or after a specific amino acid residue, more specifically, that one or more, preferably one or several amino acid residues are attached to the a-carboxyl group or a-amino group of a specific amino acid residue.

[0013] In some embodiments, the amino acid residue at position 12 (G) is substituted. In some embodiments, the amino acid residue at position 12 (G) is substituted with an amino acid residue (R).

[0014] As used herein, the term "polypeptide" has its general meaning in the art and refers to a polymer of amino acids of any length. A polymer can contain modified amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component). Also included within this definition are polypeptides that contain, for example, one or more analogs of amino acids (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.

[0015] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA"). It also includes modified, for example, by alkylation and / or by capping, as well as unmodified forms of the polynucleotide. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribonucleotides), polyribonucleotides (containing D-ribonucleotides), including tRNA, rRNA, hRNA, siRNA, and mRNA (whether spliced ​​or unspliced), any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing normucleotidic backbones, such as polyamides (e.g., peptide nucleic acids, "PNAs") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in an arrangement that allows for base pairing and base stacking, as found in DNA and RNA. In some embodiments, the polynucleotide comprises an mRNA. In other aspects, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA comprises at least one unnatural nucleobase. In some embodiments, all nucleobases of a particular class are substituted with a non-natural nucleobase (e.g., all uridines in a polynucleotide disclosed herein can be substituted with a non-natural nucleobase, such as 5-methoxyuridine). In some embodiments, a polynucleotide (e.g., synthetic RNA or DNA) contains only natural nucleobases, i.e., A, C, T, and G in the case of synthetic DNA, or A, C, T, and U in the case of synthetic RNA.

[0016] In some embodiments, a polynucleotide of the present invention is messenger RNA (mRNA).

[0017] In some embodiments, the polynucleotide is inserted into a vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector. Typically, the vector is a viral vector, such as an adeno-associated virus (AAV), a retrovirus, a bovine papilloma virus, an adenovirus vector, a lentivirus vector, a vaccinia virus, a polyoma virus, or an infectious virus. Typically, the vector of the present invention includes "control sequences", which collectively refer to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, etc., which collectively provide for the replication, transcription, and translation of the coding sequence in the recipient cell. Not all of these control sequences need to be present, so long as the selected coding sequence is capable of being replicated, transcribed, and translated in a suitable host cell. Another nucleic acid sequence is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region that contains DNA regulatory sequences, where the regulatory sequences are derived from a gene that are capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. Transcriptional promoters can include "inducible promoters" (wherein expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (wherein expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters."

[0018] In some embodiments, the polypeptide or polynucleotide of the present invention can be conjugated to at least one other molecule, typically selected from the group consisting of a polynucleotide, a polypeptide, a lipid, a lectin, a carbohydrate, a vitamin, a cofactor, and a drug.

[0019] By "therapeutically effective amount" is meant an amount of active ingredient sufficient to treat or reduce symptoms at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a wide variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed, the age, weight, general health, sex, and diet of the subject; the time of administration, the route of administration, and the rate of excretion of the specific compound employed; the duration of treatment; drugs used in combination with the active ingredient; and similar 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 gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the drug may vary over a wide range, from 0.01 to 1,000 mg per adult per day. Typically, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of active ingredient for symptomatic adjustment of dosage to the subject to be treated. The medicament typically contains about 0.01 mg to about 500 mg of active ingredient, typically 1 mg to about 100 mg of active ingredient. An effective amount of the drug is usually supplied at a dosage level of 0.0002 mg / kg to about 20 mg / kg (body weight) per day, in particular about 0.001 mg / kg to 7 mg / kg (body weight) per day.

[0020] Typically, the active ingredient (i.e., polypeptide or polynucleotide) of the present invention is combined with a pharma- ceutically acceptable excipient and, optionally, a sustained release matrix, such as a biodegradable polymer, to form a pharmaceutical composition. The term "pharmaceutical" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to a mammal, particularly a human, as appropriate. A pharma-ceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0021] In some embodiments, the polypeptide or polynucleotide of the present invention is formulated with a lipidoid, the synthesis of which has been extensively described (see Mahon et al., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart et al., Proc Natl Acad Sci US A. 2011 108:12996-3001). While these lipidoids have been used to effectively deliver double-stranded small interfering RNA molecules in rodents and non-human primates (see Akinc et al., Nat Biotechnol. 2008 26:561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920; Akinc et al., Mol Ther. 2009 17:872-879; Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Leuschner et al., Nat Biotechnol. 2011 29:1005-1010), the present disclosure describes their formulation and use in the delivery of polynucleotides.

[0022] In some embodiments, the polypeptides or polynucleotides of the invention are formulated using one or more lipid-based structures, including but not limited to liposomes, lipoplexes, or lipid nanoparticles (Paunovska, Kalina, David Loughrey, and James E. Dahlman. "Drug delivery systems for RNA therapeutics." Nature Reviews Genetics (2022): 1-16).

[0023] Liposomes are artificially prepared vesicles that may be composed primarily of lipid bilayers and can be used as delivery vehicles for administration of pharmaceutical formulations. Liposomes can be of various sizes, including but not limited to multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments, small unilamellar vesicles (SUVs), which can be less than 50 nm in diameter, and large unilamellar vesicles (LUVs), which can be 50-500 nm in diameter. Liposome designs can include, but are not limited to, opsonins or ligands to improve attachment of liposomes to unhealthy tissues or to activate events such as, but not limited to, endocytosis. Liposomes can include low or high pH to improve delivery of pharmaceutical formulations. As non-limiting examples, liposomes such as synthetic membrane vesicles are prepared by the methods, apparatus and devices described in U.S. Patent Application Publication No. 20130177638, U.S. Patent Application Publication No. 20130177637, U.S. Patent Application Publication No. 20130177636, U.S. Patent Application Publication No. 20130177635, U.S. Patent Application Publication No. 20130177634, U.S. Patent Application Publication No. 20130177633, U.S. Patent Application Publication No. 20130183375, U.S. Patent Application Publication No. 20130183373 and U.S. Patent Application Publication No. 20130183372. In some embodiments, the liposomes are formed from liposomes capable of delivering small molecule drugs such as 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (as described in U.S. Patent Application Publication No. 20100324120), and without limitation, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).The polypeptides of the polynucleotides of the invention can be encapsulated by liposomes and / or can be contained in an aqueous core which can then be encapsulated by liposomes (see WO2012031046, WO2012031043, WO2012030901 and WO2012006378, and U.S. Patent Application Publication Nos. 20130189351, 20130195969 and 20130202684).

[0024] In some embodiments, the polynucleotides of the invention are formulated using stabilized plasmid-lipid particles (SPLPs) or stabilized nucleic acid lipid particles (SNALPs), which have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (Wheeler et al. Gene Therapy. 1999 6:271-281; ​​Zhang et al. Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010 441:111-114; 28:172-176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132; see US Patent Publication No. 20130122104). The original preparation method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is called the spontaneous vesicle formation method. The liposome formulation is composed of three to four lipid components in addition to the polynucleotide. As an example, the liposome can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidylcholine (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) as described by Jeffs et al.As another example, one particular liposomal formulation contains, but is not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, which can be 1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA) as described by Heyes et al.

[0025] In some embodiments, the polynucleotides of the invention are formulated in lipid nanoparticles, such as those described in WO2012170930. Lipid nanoparticle formulations typically include lipids, particularly ionizable cationic lipids, and further include neutral lipids, sterols, and molecules capable of reducing particle aggregation, such as PEG or PEG-modified lipids. The lipids can be selected from, but are not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids, and aminoalcohol lipids. In some embodiments, the lipids are cationic lipids, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and aminoalcohol lipids. The aminoalcohol cationic lipid can be a lipid described in U.S. Patent Publication No. 20130150625 and / or can be produced by the methods described therein.As non-limiting examples, cationic lipids include 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 1 in U.S. Patent Application Publication No. 20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (Compound 2 in U.S. Patent Application Publication No. 20130150625); 2-amino-3-[ (9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (Compound 3 in US Patent Publication No. 20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 4 in US Patent Publication No. 20130150625); or any pharma- ceutically acceptable salt or stereoisomer thereof. The nanoparticle formulations of the present disclosure can be coated with a surfactant or polymer to improve the delivery of the particles. In some embodiments, the nanoparticles are coated with a hydrophilic coating, such as, but not limited to, a PEG coating and / or a coating with a neutral surface charge. Hydrophilic coatings can aid in the delivery of nanoparticles with larger payloads, such as, but not limited to, polynucleotides, into the central nervous system. By way of non-limiting example, nanoparticles including hydrophilic coatings and methods for making such nanoparticles are described in U.S. Patent Application Publication No. 20130183244.

[0026] The present invention will be further illustrated by the following figures and examples, which should not, however, be construed as limiting the scope of the present invention in any way. EXAMPLES

[0027] Example: RAC2 G12R mutation impaired the proliferation of tumor solid cell lines. Severe combined immunodeficiency (SCID) is an inherited disorder characterized by a disturbance in T lymphocyte differentiation accompanied by an absence or functional defect of the B NK cell or neutrophil lineages. 1 In three newborns with frequent infections and severe leukopenia, the present inventors identified the RAC2 gene ( Ra Related C 3. Botulinum Toxin Substrates 2 We identified a private heterozygous mutation in RAC2, which encodes for RAC2-GTP-binding domain. The RAC2 protein belongs to the Rac subfamily of RHO small GTPases and is tightly regulated. In the inactive GDP-bound state, RAC2 is localized in the cytosol, and upon stimulation, the active RAC2-GTP-bound form translocates to the plasma membrane. 2 Unlike other members of the Rac subfamily (RAC1 and RAC3), RAC2 is expressed predominantly on hematopoietic cells. 3,4 .

[0028] The missense mutation (p.G12R) we identified is located at position 12 of the protein, in a highly conserved guanine nucleotide-binding region required for GTP hydrolysis and the RAC2 signaling pathway. We showed that the mutation affects the catalytic domain of the protein and induces abnormally high and sustained levels of GTP-bound RAC2. Furthermore, this gain-of-function mutation rapidly blocks the proliferation and differentiation of cord blood hematopoietic stem / progenitor cells (HSPCs) into T lymphocyte, neutrophil and monocyte lineages. Such observations are associated with defective mitochondrial function, disrupted reactive oxygen species (ROS) production and hyperapoptosis. Overall, the RAC2 G12R mutation has a strong impact on the homeostatic control of hematopoiesis, which may explain the severity of the clinical and immunological phenotype of patients. 5 This study is the first to describe an autosomal form of SCID, and RAC2 gene sequence analysis is now integrated into the newborn screening program for SCID detection at Necker Hospital (Paris, France). 6Of note, all other RAC2 mutations previously described in the literature did not have such a dramatic effect on the fate of HSPCs. 7 .

[0029] We have described above that RAC2 G12R mutation rapidly induced cell death and hematopoietic control of HSPCs. Using the same lentiviral vector approach, we evaluated the impact of RAC2 G12R mutation on three tumor cell lines: MDA-MB-231 (breast adenocarcinoma), HT29 (colon adenocarcinoma) and HepG2 (hepatocellular carcinoma). Briefly, cells were transduced with lentiviral vectors containing green fluorescent protein (GFP) reporter cDNA (WPI) or wild-type form of RAC2 cDNA (WT) or RAC2 mutant cDNA form (G12R). After the transduction step, cells were cultured in complete medium for up to 8 days (Figure 1A). On days 5 and 8, the number of GFP-positive (GFP+) cells was assessed after total cell count (trypan blue staining to remove dead cells) and immunofluorescence analysis. Whatever the day and cell line, the number of GFP+ cells is dramatically reduced in G12R condition compared to WT and WPI conditions. As observed by live cell imaging microscopy, which measures the quantitative refractive index of cells, the morphology and contents of the cells are particularly disrupted (data not shown). To confirm these observations, a time-course proliferation assay was performed on MDA-MB-231 and HT29 cell lines. Using an Incucyte live cell analysis system, the growth of GFP+ transduced cells (expressed as % of confluence observed with a green filter) was assessed every 3 hours for 12 days. For the MDA-MB-231 cell line, we observed exponential GFP+ cell growth in WPI and WT conditions and a flat curve in G12R condition (Figure 1B, upper panel). For the HT29 cell line, exponential GFP+ cell growth is higher in the WT condition compared to WPI and G12R conditions. Notably, from day 8 onwards, the curve reached a plateau (10% confluence) in the G12R condition, suggesting that most of the GFP-transduced cells had died. In WT and WPI conditions, the % confluence increased over time, reaching 65% and 25%, respectively, from day 12 onwards (Figure 1B, lower panel).

[0030] Altogether, these data provide a basis for the deleterious effect of the RAC2 G12R mutation on the proliferation of tumor cell lines.

[0031] References: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are incorporated by reference into this disclosure. [Table 1]

Claims

1. A pharmaceutical composition for inducing cell death in a population of solid tumor cells, comprising: i) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in which the amino acid residue at position 12 (G) is mutated, or ii) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in which the amino acid residue at position 12 (G) is mutated.

2. A pharmaceutical composition for treating a solid tumor, comprising: i) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in which the amino acid residue (G) at position 12 is mutated; or ii) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in which the amino acid residue (G) at position 12 is mutated.

3. 3. The pharmaceutical composition according to claim 1, wherein the amino acid residue (G) at position 12 is substituted.

4. 4. The pharmaceutical composition of claim 3, wherein the amino acid residue (G) at position 12 is replaced by an amino acid residue (R).

5. The pharmaceutical composition of claim 1 or 2, wherein the polynucleotide is messenger RNA (mRNA).

6. The pharmaceutical composition of claim 1 or 2, wherein the polynucleotide is inserted into a vector.

7. 3. The pharmaceutical composition of claim 1 or 2, wherein the polypeptide or the polynucleotide is conjugated to at least one other molecule selected from the group consisting of polynucleotides, polypeptides, lipids, lectins, carbohydrates, vitamins, cofactors and drugs.

8. 3. The pharmaceutical composition of claim 1 or 2, wherein the polypeptide or the polynucleotide is formulated using one or more lipid-based structures, including liposomes, lipoplexes, or lipid nanoparticles.

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