Inhibitors of SWI / SNF enzyme activity for the treatment of nut carcinoma

Abstract

The present invention proposes the inhibition of the catalytic function of the SWI / SNF complex in patients with NUT carcinoma, to promote the differentiation and specific death of tumour cells. Specifically, inhibitors of the activity of the SMARCA4 and / or SMARCA2 catalytic subunits of the SWI / SNF complex, for the use thereof in the treatment of NUT carcinoma, are described.

Description

[0001] SWI / SNF ENZYMATIC ACTIVITY INHIBITORS FOR

[0002] TREATMENT OF NUT CARCINOMA

[0003] DESCRIPTION

[0004] TECHNICAL FIELD

[0005] The present invention falls within the pharmaceutical sector and is intended for the clinical area of ​​oncology. Specifically, it focuses on the use of SWI / SNF enzymatic activity inhibitors in the treatment of NUT carcinoma.

[0006] STATE OF THE PRIOR ART

[0007] NUT carcinoma (NC, also known as midline NUT carcinoma) is a rare but extremely aggressive form of squamous cell carcinoma. Squamous cells are epithelial cells that form part of the skin and the lining of some organs, such as the lungs and stomach. Although NUT carcinoma usually originates in midline structures, such as the chest or head and neck, it can actually develop in almost any part of the body, such as the kidneys or pancreas. This cancer can affect people of all ages, but is most common in adolescents and young adults. NUT carcinoma accounts for a significant proportion of poorly differentiated carcinomas, and its diagnosis is increasing thanks to greater awareness and improved diagnostic testing. Among solid tumors in humans, NUT carcinoma is the second most aggressive, with a median survival of only 6.5 months.Unfortunately, there are currently no systemic treatments for NUT carcinoma. The main therapeutic strategies include standard treatments such as radiotherapy or chemotherapy, which have a very poor response in patients, highlighting the urgent need for therapeutic options [1].

[0008] The entire oncogenic process of NUT carcinoma appears to be driven exclusively by the effect of NUT fusion proteins [2], NUT fusion proteins are generated by chromosomal rearrangement in the NUTM1 gene, the testicular nuclear protein gene. This gene encodes a NUTM1 protein, also known as NUT midline carcinoma family member 1, which is expressed almost exclusively in testes, ovaries and ciliary ganglia. The NUTM1 coding sequence on band 14 of the long arm of chromosome 15 is fused with the coding sequence of BRD4 (bromodomain protein 4) located on band 13 of the short arm of chromosome 19, creating a chimeric gene encoding the BRD4-NUT fusion protein. The BRD4-NUT chimeric protein, resulting from this t(15;19) genetic rearrangement (chromosome 15-19 translocation), is the most common NUT fusion protein [3],

[0009] BRD4 is a member of the BET (Bromodomain and Extra Terminal) protein family. The BET protein family is acetyl-lysine reader that binds to these proteins, promoting transcription by interacting with and recruiting pTEFb (positive transcription elongation factor) and chromatin remodeling complexes. This group includes proteins with BRD4 or BRD3 bromodomains. Bromodomains (BRDs) are reader domains that recognize and bind to acetylated histone regions.

[0010] As previously stated, BRD4 is known to bind to acetylated histones via its bromodomains. Under normal conditions, recruitment of BRD4 to areas enriched with histone 3 acetylated at lysine 27 (H3K27ac) promotes gene transcription by activating RNA polymerase II at those specific locations. H3K27ac is an epigenetic modification of the DNA packaging protein histone H3 by acetylation of the N-terminal lysine residue 27 [4], This regulated process helps maintain the keys necessary for the differentiation of healthy squamous cells.

[0011] However, when BRD4 is fused to NUT, the BRD4-NUT chimeric protein can recruit and stimulate the histone acetyltransferase p300, which in turn has the ability to acetylate other histones (HAT activity), initiating a positive feedback loop of histone acetylation [4,5]. In this loop, acetylated histones recruit BRD4-NUT through their binding to the BRD4 moiety. In turn, BRD4-NUT mediates the acetylation of nearby histones through its interaction with p300, which in turn recruits more BRD4-NUT, restarting the cycle (Fig. 1; Bottom). This chain reaction ultimately generates large domains of acetylated histones in the chromatin of tumor cells, also known as “megadomains”. "Megadomains" are large super-enhancers or "super-enhancers" enriched in H3K27ac.

[0012] Thanks to histone acetylation in the "megadomains," they are recognized by chromatin remodeling complexes that mediate chromatin unwinding, thus facilitating the expression of various genes with oncogenic potential. Chromatin is a highly organized, condensed structure composed of DNA, RNA, and histone and non-histone proteins that gives rise to chromosomes. Long chains of DNA molecules wrap around histone complexes to form nucleosomes, which in turn fold together to form a chromatin fiber that condenses and gives rise to chromosomes. Chromatin enables many important cellular processes, such as DNA replication, transcription, DNA repair, genetic recombination, and cell division.

[0013] Chromatin remodelers are protein complexes that, depending on ATP hydrolysis, modify the interaction between histones and DNA, generating a restructuring of nucleosomes so that the transcription machinery can access target genes. They can act through different mechanisms, such as the destructuring of the histone octamer and transient DNA unwinding, the formation of DNA loops, or the repositioning of nucleosomes. This leads to variations, and transcription factors can access previously unreachable areas of DNA.

[0014] Therefore, megadomains drive the expression of crucial oncogenic genes, such as MYC, S0X2, and TP63, by enhancing the action of chromatin remodeling complexes and thus promoting chromatin unwinding. These oncogenes play a pivotal role in maintaining the undifferentiated state of NUT carcinoma [8] (Fig. 1; Bottom).

[0015] BRD4-NUT mediated megadomains and chromatin interactions can be visualized by immunohistochemistry or immunofluorescence microscopy [4]. The functional outcome of these processes is the blockage of NUT carcinoma cell differentiation and sustained growth. Therefore, inhibition of BRD4-NUT or of the vahantes NUT fusion proteins would result in rapid growth arrest of NUT carcinoma cells and their terminal squamous differentiation [3].

[0016] Currently, several clinical trials are underway with BET bromodomain inhibitors (BETi) as a promising therapeutic strategy for the treatment of NUT carcinoma. Molecularly, inhibiting the BET bromodomains of proteins such as BRD3 or BRD4 has a repressive effect on the chromatin opening necessary for the tumor development of NUT carcinoma. BETi can prevent BRD4-NUT from binding to chromatin, leading to growth arrest and induction of differentiation [9,10]. However, there are alternative processes, such as the effect of other proteins with bromodomains such as BRD1 or BRD2, etc., that may allow this tumor process at residual levels and therefore generate some resistance to treatment. Furthermore, BETi monotherapy has other limitations, such as short-lived (1-3 months) and only partial responses in a subset of patients (20-30%) in clinical trials [11,12].BETis appear to have a greater effect than cycles of radiotherapy and chemotherapy, but in any case, their effect remains very limited.

[0017] Therefore, the identification of new therapeutic targets within the biology of NUT carcinoma is crucial to address the extremely low patient survival rates.

[0018] As mentioned above, histone acetylation is mediated by histone acetyltransferases (HATs) and is associated with gene activation (Fig. 2 ). However, the link between histone acetylation and gene transcription involves the aforementioned downstream players known as chromatin remodelers

[0013] . These remodelers can read H3K27ac epigenetic marks and mediate chromatin relaxation by displacing nucleosomes along DNA in an ATP-dependent manner, allowing the transcription machinery to access specific loci to ultimately promote gene expression. These protein complexes are divided into four families: the SWI / SNF family which includes ARID1 A, ARID1 B, BRG1 (also called SMARCA4), SNF5 (also known as SMARCB1, IN111 or BAF47) and PBRM1; the ISWI family ; the NuRD / M¡2 / chromodomain DNA helicase-binding (CHD) family; and the INO80 family.

[0019] The most important and best studied chromatin remodeler is the SWI / SNF complex [13,14] (Fig. 2). The SWI / SNF (SWItch / Sucrose Non-Fermentable) complex is the main chromatin remodeling complex responsible for reading the aforementioned epigenetic marks (acetylated histones) and the subsequent opening of chromatin. This complex is able to recognize and bind these acetylated histones through bromodiminogens present in their subunits and induce nucleosome ejection and chromatin unwinding in a process dependent on ATP hydrolysis. They act by mobilizing nucleosomes by sliding them or inserting histone octamers, such that the SWI / SNF complexes bind to DNA at a specific position, interact with the DNA-histone junction and separate it, translocate the DNA by means of an ATPase and form a DNA loop.This enzymatic process is carried out by the complex's catalytic subunits: SMARCA2 and SMARCA4. These two subunits are homologous, mutually exclusive, and essential for the complex's proper function [15,16].

[0020] Apart from the general way of recruitment of the SWI / SNF complex on specific epigenetic marks, it has been seen that in the context of NUT carcinoma, the acetyl transferase p300 is able to interact and recruit SMARCA4 to the megadomains of NUT carcinoma

[0017] ,

[0021] DESCRIPTION OF THE INVENTION

[0022] Since NUT carcinoma biology requires the formation of H3K27ac-enriched megadomains and subsequent transcription of NUT carcinoma-associated oncogenes (MYC, S0X2, and TP63), it is hypothesized that NUT carcinoma must therefore be highly dependent on the activity of the SWI / SNF complex. In particular, SMARCA4 has been reported to interact with and be recruited by acetylated histones, BRD4 and p300, which are key players in NUT carcinoma [17–20].

[0023] The present invention proposes the inhibition of the catalytic function of the SWI / SNF complex (in different ways; PROTACS, CRISPR, enzyme inhibitors...) in patients with NUT carcinoma to promote the differentiation and specific death of tumor cells. It should be taken into account that the effect of the BRD4-NUT protein requires chromatin remodeling to promote oncogenic events associated with NUT carcinoma, and that the SWI / SNF protein complex is involved in chromatin opening.

[0024] Eliminating the enzymatic activity of SWI / SNF represents a therapeutic strategy in patients with NUT carcinoma with greater therapeutic efficacy and a lower probability of generating resistance.

[0025] This is a specific treatment since tumor cells are strongly dependent on the function of this chromatin remodeling complex, while normal cells are not as dependent.

[0026] The in vitro studies with NUT carcinoma models shown below prove that genetic or pharmacological inhibition of the SWI / SNF catalytic function by inhibiting the SMARCA2 and SMARCA4 subunits causes the death of NUT carcinoma tumor cells without affecting the viability of other related cell types. Within the framework of the present invention, "inhibitor" or "inhibitory agent" is understood to mean a compound or methodology capable of partially or completely decreasing the enzymatic activity, in this case catalytic, of the SMARCA4 and / or SMARCA2 subunits of the SWI / SNF complex and therefore, of decreasing the level of SWI / SNF activity.

[0027] The terms "inhibit" and "inhibition" refer to slowing, stopping, or reversing the growth or progression of a disease, infection, condition, metabolic pathway, or group of cells. Inhibition may be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of treatment or contact.

[0028] Inhibition of the catalytic function of the SWI / SNF complex can be achieved through a variety of pathways. Among these, the present invention proposes the use of PROTACS, CRISPR, enzyme inhibitors, etc.

[0029] Therefore, a first aspect of the invention relates to an inhibitory agent of the catalytic subunits SMARCA4 and / or SMARCA2 of the SWI / SNF complex for use in the treatment and / or prevention of NUT carcinoma.

[0030] The SMARCA2 and SMARCA4 subunits of SWI / SNF can be inhibited primarily by three pathways: a) inhibition of the enzymatic activity of SMARCA2 and SMARCA4 subunits; b) reduction in the levels at which the genes encoding SMARCA2 and SMARCA4 subunits are transcribed; and c) the intervention of suppressive agents that cause proteolysis of the expressed SMARCA2 and SMARCA4 proteins.

[0031] The inhibitory agent of the invention can act through any of these three pathways to reduce the enzymatic activity of SWI / SNF.

[0032] The inhibitory agent of the invention is selected from the list comprising, among others, organic molecules, RNA molecules, antisense oligonucleotides, antibodies, or ribozymes, capable of inhibiting the activity of the SMARCA2 and SMARCA4 subunits, or of inhibiting the expression of said subunits, or of causing their reduction by proteolysis subsequent to their expression.

[0033] Agents that inhibit the activity of the SMARCA2 and SMARCA4 subunits can be either agonists (substances that are capable of binding to a receptor and eliciting a response in the cell, preferably a decrease in SWI / SNF activity) or antagonists (substances that not only do not activate the receptor, but actually block its activation by agonists). They primarily fall into the categories of organic molecules or antibodies.

[0034] Therefore, in a preferred embodiment of this aspect of the invention, the agent that inhibits the activity of the SMARCA4 and SMARCA2 subunits is selected from the list consisting of PFI-3, BRM / BRG1 ATP Inhibitor 1, BRM / BRG1 ATP Inhibitor 2, BRM / BRG1 ATP Inhibitor 3, BRM014, FHD-286, and any combination thereof. More preferably, it is PFI-3.

[0035] PFI-3 (CAS: 1819363-80-8) stands out for being a selective, potent and cell-permeable commercial inhibitor of the SMARCA2 and SMARCA4 bromodomains.

[0036] PFI-3, like other organic molecules such as GNE-064 (CAS: 1997321 -20-6), is capable of inhibiting the bromodomains of the two SWI / SNF ATPases: SMARCA2 and SMARCA4. -064

[0037] In addition to inhibiting SMARCA2 and / or SMARCA4 by attacking their bromodomains, there are other compounds that inhibit the ATPase catalytic capacity of these subunits. Among these compounds are BRM / BRG1 ATP Inhibitor 1 (CAS:2270879-17-7), BRM / BRG1 ATP Inhibitor 2 (CAS:2270879-17-7), BRM / BRG1 ATP Inhibitor 3 (CAS:2270879-17-7), BRM014 (CAS:2270879-17-7) and FHD-286 (CAS:2671128-05-3), all of them capable of inhibiting SMARCA2 and SMARCA4.

[0038]

[0039] Agents that reduce the levels at which genes encoding SMARCA2 and / or SMARCA4 subunits are transcribed can act both as inhibitors of gene expression or "silencers." They primarily fall into the categories of RNA molecules and antisense oligonucleotides. Within the scope of the present invention, an agent that inhibits gene expression or "transcription silencer" includes RNA interference or RNAi, RNA molecules involved in the sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression (also known as co-suppression, post-transcriptional gene silencing (PTGS), or smothering). Once the mRNA is degraded, post-transcriptional silencing occurs by preventing protein translation, thus allowing the suppression of desired genes thanks to its high degree of efficacy and specificity.RNAi include microRNAs (miRNAs), small interfering RNAs or silencing RNAs (siRNAs), and RNAis that can be introduced into target cells by the exogenous introduction of double-stranded RNA (dsRNA) or constructs that express small hairpin RNAs (shRNAs).

[0040] Therefore, in another preferred embodiment of this aspect of the invention, the agent that inhibits the activity of the SMARCA4 and / or SMARCA2 subunits is an agent that reduces the levels at which the SMARCA2 and / or SMARCA4 genes are transcribed, specifically, it is a microRNA (miRNA) or a small interfering RNA (siRNA) or a short hairpin RNA or small hairpin RNA (shRNA), or any combination thereof, capable of silencing the SMARCA2 and / or SMARCA4 genes.

[0041] More preferably, it is a SMARCA4 siRNA or siSMARCA4 and / or a SMARCA4 or siSMARCA2 siRNA. The siRNAs used in this particular invention are commercially available: s¡SMARCA4 (Horizon, J-010431-06-0005), SÍSMARCA2 (Horizon J-010431-07-0005) and the negative control Scramble (Horizon, D-001810-10-05 [si-NT]). Any siRNA design capable of silencing SMARCA2 and / or SMARCA4 can be used for this same purpose.

[0042] Any small hairpin RNA (shRNA) design capable of silencing SMARCA2 and / or SMARCA4 can also be used, such as those offered by the commercial houses Thermo Fisher (Cat#106957 [SMARCA2], Cat#106960 [SMARCA4]), Sigma Aldrich (EsiRNA EHU135151 [SMARCA2], esiRNA EHU076601 [SMARCA4]), OriGene (Cat#SR510178 [SMARCA2], Cat#SR509768 [SMARCA4]) or Santa Cruz (sc-29831 [SMARCA2], sc-29827 [SMARCA4]).

[0043] Within the scope of the present invention, reducing the levels at which the SMARCA4 and / or SMARCA2 genes are transcribed also includes the use of the technology known as CRISPR-Cas9 or “molecular scissors,” used to edit genes within organisms.

[0044] CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences originate from DNA fragments of bacteriophages that previously infected the prokaryote. They are used to detect and destroy the DNA of similar bacteriophages during subsequent infections. Therefore, these sequences play a key role in the antiviral (i.e., antiphage) defense system of prokaryotes and provide a form of acquired immunity.

[0045] Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and open specific DNA strands complementary to the CRISPR sequence.

[0046] This technology can be used in vitro and in vivo (using viral vectors) and suppresses the genetic expression of target genes through the use of "molecular scissors" called CRISPR Cas9. The Cas9 protein is capable of cutting and excising a specific part of the genome thanks to the sequence specificity conferred by an RNA molecule (guide RNA) previously designed for a specific target gene.

[0047] Therefore, in another preferred embodiment of this aspect of the invention, the elimination of SMARCA2 and / or SMARCA4 is carried out by CRISPR Cas9 technology, for example, as previously described in the state of the art for SMARCA2

[0021] and for SMARCA4

[0022] ,

[0048] Thus, the present invention also relates to a method of inhibiting the activity of the SMARCA4 and / or SMARCA2 catalytic subunits of the SWI / SNF complex using CRISPR Cas9 technology for use in the treatment of NUT carcinoma.

[0049] Suppressor agents cause proteolysis of SMARCA2 and SMARCA4 proteins once expressed, and mainly fall into the categories of organic molecules and β-enzymes.

[0050] For the purposes of the present invention, "suppressor" refers to a compound capable of decreasing, reducing, or completely eliminating the amount of intracellular SMARCA4 and / or SMARCA2 protein by degrading the same once expressed. Therefore, a suppressor agent ultimately causes the same final effect as an agent that inhibits SMARCA4 and / or SMARCA2 activity or as an agent that reduces the levels at which the SMARCA2 and / or SMARCA4 genes are transcribed, i.e., total or partial inhibition of the catalytic activity of the SWI / SNF complex.

[0051] Within the scope of the present invention, a suppressor compound includes degrading compounds capable of inducing selective intracellular proteolysis or degradation of the target protein SMARCA4 and / or SMARCA2. Prominent among these suppressor agents are, but are not limited to, proteolysis-targeting chimeras or PROTACs. A PROTAC is a heterobifunctional molecule composed of two active domains and a linker, capable of eliminating specific unwanted proteins. Rather than acting as a conventional enzyme inhibitor, a PROTAC acts by inducing selective intracellular proteolysis. PROTACs consist of two covalent protein-binding molecules: one capable of attracting an E3 ubiquitin ligase and another that binds to a target protein destined for degradation. Recruitment of the E3 ligase to the target protein results in ubiquitination and subsequent degradation of the target protein via the proteasome (ubiquitin-proteasome system).The selection PROTAC in the present invention is preferably ACBI1.

[0052] Thus, in another preferred embodiment of this aspect of the invention, the agent that inhibits the activity of the SMARCA4 and / or SMARCA2 subunits is a suppressor agent, specifically a proteolysis-targeting chimera or PROTAC. Preferably, the PROTAC is selected from the list consisting of ACBI1, AU-15330, A947, YD23, PRT3789, SMD-3040, and ACBI2. More preferably, the PROTAC is ACBI1 (CAS:2375564-55-7), a compound capable of mediating the degradation of SMARCA2 and SMARCA4. AU-15330 (CAS:2380274-50-8) is another PROTAC capable of mediating the degradation of SMARCA2 and SMARCA4.

[0053] ACBI1 AU-15330

[0054] In addition to these two PROTACs with action on the two ATPase subunits of SWI / SNF (such as ACBI1 and AU-15330), other PROTACs are available that promote the specific elimination of SMARCA2: A947 (CAS:2378056-80-3), YD23, PRT3789, SMD-3040 and ACBI2 (CAS:2913161-19-8), which according to the results set forth below in the present invention, could also be used for the treatment of NUT carcinoma. A second aspect of the invention relates to a pharmaceutical composition, or “composition of the invention”, comprising at least one inhibitor of the activity of the SMARCA4 and / or SMARCA2 catalytic subunits of the SWI / SNF complex for use as a medicament in the treatment and / or prevention of NUT carcinoma.

[0055] As used herein, the term "pharmaceutical composition" refers to any substance used for the diagnosis, prevention, alleviation, treatment, or cure of a disease in humans or animals. The pharmaceutical composition of the invention may be used alone or in combination with other pharmaceutical compositions.

[0056] In a particular embodiment, the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier or excipient.

[0057] The term "pharmaceutically acceptable excipient" refers to a substance that aids in the absorption of the pharmaceutical composition comprising the composition of the invention, stabilizes said pharmaceutical composition, or aids in its manufacture by giving it consistency, shape, flavor, or any other specific functional characteristic. Thus, excipients could have the function of holding the ingredients together, such as starches, sugars, or cellulose, a sweetening function, a coloring function, a protective function, such as isolating it from air and / or moisture, a filling function for a tablet, capsule, or any other form of formulation, such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption, without excluding other types of excipients not mentioned in this paragraph.

[0058] A "pharmaceutically acceptable carrier" (or "pharmacologically acceptable carrier") refers to any substance, or combination of substances, known in the pharmaceutical industry, used in the manufacture of pharmaceutical dosage forms and includes, but is not limited to, solids, liquids, solvents, or surfactants. The carrier may be an inert substance or have a similar action to any of the compounds of the present invention, serving to facilitate the incorporation of the drug as well as other compounds, allowing for improved dosage and administration, or providing consistency and shape to the pharmaceutical composition. When the pharmaceutical form is liquid, the carrier is the diluent. The term "pharmacologically acceptable" refers to the fact that the compound it refers to is permitted and evaluated to not cause harm to the organisms to which it is administered.

[0059] The pharmaceutical composition of the invention can be administered via any route of administration, and as such, said composition will be formulated in the pharmaceutical form appropriate to the chosen route of administration. Thus, the pharmaceutical composition of the invention can be administered orally, nasally, ocularly, topically, intradermally, intracranially, intravenously, or intraperitoneally.

[0060] The pharmaceutical composition may also comprise another compound useful in the treatment and / or prevention of cancer.

[0061] The pharmaceutical composition may include a single composition or separate compositions.

[0062] In a preferred embodiment, the composition of the invention comprises an activity inhibitor, preferably PFI-3; an siRNA, preferably s¡SMARCA4 or ¡SMARCA2; a chimera targeting proteolysis or PROTAC, preferably ACBI1, or any combination thereof.

[0063] The pharmaceutical composition may include an effective amount of the SMARCA4 and / or SMARCA2 inhibitor. The term "effective amount" as used herein refers to an amount sufficient to inhibit the activity of the catalytic subunits of the SWI / SNF SMARCA4 and / or SMARCA2 complex or suppress the catalytic subunits of the SWI / SNF SMARCA4 and / or SMARCA2 complex and thereby prevent or treat cancer in an individual in need of such prevention or treatment. The effective amount may be appropriately selected based on a cell or individual selected by one of skill in the art.For example, the effective amount may be determined based on the severity of the disease, the patient's age, body weight, health conditions, sex, patient sensitivity to the drug, duration of administration, route of administration, excretion rate, duration of treatment, and other factors, including the use of a drug in combination with or concurrently with the pharmaceutical composition, and other factors known in the field of medicine. Therapy is considered "personalized" when the compound administered to the individual to treat a disease (cancer) is specifically tailored to both the genotypic and phenotypic characteristics of the individual to be treated, thus avoiding wasted time with ineffective therapies.

[0064] The term "prevention", as used herein, refers to the ability of the pharmaceutical composition of the invention to avoid, minimize or hinder the progression of a cancer.

[0065] The term "patient" or "subject," as used herein, refers to any animal, preferably a mammal, and includes, but is not limited to, domestic and farm animals, primates, and humans, e.g., humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents such as rats and mice. In a preferred embodiment, the subject is a human of any age or race.

[0066] In a particular embodiment, the subject suffers from cancer, preferably NUT carcinoma. The term "cancer," as used herein, refers to a disease characterized by uncontrolled cell division (or by increased resistance to survival or apoptosis) and by the ability of said cells to invade other neighboring tissues (invasion) and spread to other areas of the body where cells are not normally found (metastasis) through the lymphatic and blood vessels, circulate through the bloodstream, and then invade normal tissues in other parts of the body. Depending on whether or not they can spread by invasion and metastasis, tumors are classified as benign or malignant: benign tumors are tumors that cannot spread by invasion or metastasis, that is, they only grow locally; while malignant tumors are tumors capable of spreading by invasion and metastasis.Biological processes known to be related to cancer include angiogenesis, immune cell infiltration, cell migration, and metastasis.

[0067] A third aspect of the invention relates to a method for selecting therapeutic agents useful in the prevention, improvement, alleviation, and / or treatment of NUT carcinoma, comprising determining the activity of SMARCA4 and / or SMARCA2 at an established concentration of the compound to be tested or in the absence of said compound, and determining the activity of SMARCA4 and / or SMARCA2 at a concentration of the compound to be tested different from that of a), such that compounds capable of inhibiting the activity of SMARCA4 and / or SMARCA2 are identified as potential therapeutic agents against NUT carcinoma.

[0068] Determining the ability of a compound to inhibit SMARCA4 and / or SMARCA2 activity can be carried out, for example, by determining the ability of SMARCA4 and / or SMARCA2 to bind or interact with a target molecule of said compound, directly or indirectly. They can also be activity assays, directly or indirectly measuring SMARCA4 and / or SMARCA2 activity. It can also be an expression assay, directly or indirectly determining the expression of SMARCA4 and / or SMARCA2 mRNA or SMARCA4 and / or SMARCA2 proteins. These assays can also be combined with an in vivo assay measuring the effect of a test compound on the symptoms of SMARCA4 and / or SMARCA2-related diseases, and in particular NUT carcinoma (for example, but not limited to, in animal models or other model systems known in the art).

[0069] The compounds to be tested used in the therapeutic agent selection method are not limited to low molecular weight organic molecules, proteins (including antibodies), peptides, oligonucleotides, etc. They can be natural and / or synthetic compounds.

[0070] For example, antibodies capable of binding to SMARCA4 and / or SMARCA2 that can be used therapeutically can also be used in immunohistochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunoprecipitation assays, or other immunohistochemical assays known in the art. SMARCA4 and / or SMARCA2 polypeptides can be used to immunize an animal to obtain polyclonal antibodies. Monoclonal antibodies can also be prepared by techniques that allow for the production of antibodies by cell lines in culture, including, but not limited to, hybridomas and human B-cell hybridomas. Techniques for producing chimeric, humanized, or synthetic antibodies are known.

[0071] The therapeutic agents identified by the screening method described herein can be used in an animal or other model to determine the agent's mechanism of action. Furthermore, the therapeutic agents selected by the method described herein could be used to treat diseases that involve the catalytic activity of the SWI / SNF complex, specifically, NUT carcinoma.

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning commonly understood by one skilled in the art to which this invention pertains. Methods and materials similar or equivalent to those described herein can be utilized in the practice of the present invention. Throughout the description and claims, the word "comprises" and variations thereof are not intended to exclude other technical features, additives, components, or steps. Other objects, advantages, and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples, drawings, and sequence listing are provided by way of illustration and are not intended to be limiting of the present invention.

[0073] DESCRIPTION OF FIGURES Figure 1. Oncogenic mechanism of BRD4-NUT. Top, normal BRD4 activity activating transcription and differentiation signals. Bottom, when BRD4 fuses to NUT, recruitment of p300 leads to the formation of hyperacetylated megadomains and associated transcriptional activation of the oncogenic target genes MYC, S0X2, and TP63.

[0074] Figure 2. Sequential mechanism of action of histone acetyltransferases and the SWI / SNF complex. To promote gene transcription, SWI / SNF hydrolyzes ATP to slide nucleosomes along the DNA helix and expose the DNA to transcription factors and the transcription machinery.

[0075] Figure 3. Representation of the dependency effect of a set of 17,000 genes represented in the DepMap database; highly dependent genes are associated with lower Z-scores. The level of dependency was assessed using CRISPR and RNAi for NUT carcinoma cells (A) HCC2429 and by CRISPR for (B) RPMI 2650. The relative position of the SWI / SNF ATPase SMARCA4 in the dependency ranking is highlighted in all graphs.

[0076] Figure 4. (A) Western blot of SMARCA4 protein expression levels in the NUT carcinoma cell line HCC2429 after 18 h of treatment with increasing concentrations (0, 1, 5, 20, 40, 80, 125, 250, 500, 1000 nM) of ACBI1 or the inactive compound cis-ACBH; α-tubulin used as a loading control. (B) Percentage viability of HCC2429 cells after treatment for 5 days with different concentrations of ACBI1 or the inactive compound cis-ACBI1 (0.1, 2, 4, 5, 8, 10, 16, 20, 32, 40, 64, 80, 125, 160, 250 nM).

[0077] Figure 5. Percentage viability of HCC2429 (NUT carcinoma cell line) and HPF (NUT non-carcinoma cell line; control) cells at increasing concentrations of ACBI1 (A) or PFI-3 (B) after 5 days of treatment. (C) Percentage cell viability of 2 NUT carcinoma cell lines (HCC2429 and RPMI 2650) and 4 control cell lines (H1395, HPF, MCF7, HEK293) after 5 days of treatment with 14nM ACBI1 and its negative control cis-ACBH. (D) Western blot showing the expression levels of various NUT carcinoma markers (SMARCA4, SMARCA2, BRD4-NUT, c-Myc and SOX2), using actin as a positive control, in HCC2429 cells treated with ACBI1 at a concentration of 15 nM (+) or the negative control c¡s-ACBI1 (-) for 5 days. Figure 6. Effect of SMARCA4 silencing on carcinoma cell lines.

[0078] NUT HCC2429 (A) and RPMI 2650 (B) cell viability 5 days after treatment with SMARCA4-specific siRNA (s¡SMARCA4). (C, D) Western blot showing the expression levels of NUT carcinoma markers (SMARCA4, BRD4-NUT, c-Myc, p63, SOX2) using actin as control, in HCC2429 and RPMI 2650 after treatment for 48h with the s¡SMARCA4 silencer or the scramble negative control (SCR).

[0079] Figure 7. Fluorescence microscopy images of HCC2429 cells transfected for 72 hours with s¡SMARCA4 or the negative control SCR. H3K27ac is shown in green and nuclei in blue.

[0080] Figure 8. Effect of SMARCA4 silencing with S¡SMARCA4 in HCC2429 cells. (A) Western blot showing the expression levels of SMARCA4 and the differentiation markers involucrin and b-catenin, using tubulin as a loading control. (B) Fluorescence microscopy, showing b-catenin in green and nuclei in blue. (C) Distribution of HCC2429 cells in the different phases of the cell cycle (G1 phase, S phase and G2 phase) and statistical analyses of three biological replicates transfected with s¡SMARCA4 (red) or the negative control SCR (blue). Two-tailed Student’s t test of unpaired samples. (*P < 0.05; **P < 0.01; ***P < 0.001).

[0081] Figure 9. (A) Fluorescence values ​​of HCC2429 cells stained with Cell Trace Violet taken at day 0 and 5 days after treatment with s¡SMARCA4 or the SCR negative control and comparison between treatments after normalizing the mean fluorescence with respect to day 0. (B) Fluorescence microscopy images of HCC2429 cells after 72 h of treatment with s¡SMARCA4 or the SCR negative control, treated with immunostaining for the proliferation marker K¡67 (K¡67 in green) and DAPI (nuclei in blue) and comparison of the percentage of K¡67 positive cells between treatments. Two-tailed Student’s t test of unpaired samples. (*P < 0.05; **P < 0.01; ***P < 0.001).

[0082] Figure 10. Percentage viability of NUT carcinoma cells (HCC2429, RPMI 2650 and NMC1015) 5 days after transfection with siRNAs targeting SWI / SNF ATPases (s¡SMARCA2 and SÍSMARCA4) or the negative control SCR. Two-tailed Student’s t test for unpaired samples. (*P < 0.05; **P < 0.01; ***P < 0.001).

[0083] DETAILED DESCRIPTION OF THE INVENTION In silico studies on the relevance of SMARCA4 in NUT carcinoma

[0084] An analysis of the DepMap cancer dependency database (depmap.org) revealed that NUT carcinoma cell lines showed a high dependence on the SWI / SNF ATPase subunit SMARCA4.

[0085] The DepMap database is based on empirical analyses in which gene expression is eliminated and a relative score (Z-score) is assigned based on the effect of this elimination on cell viability compared to a control. Z-score values ​​below 0 imply a decrease in cell viability following inhibition of gene expression, while a value above 0 implies a benefit based on said viability. Genes can then be ranked based on their Z-score, or the effect of gene inhibition on the viability of the different cell lines. The analysis was performed on two cell lines represented in this database: HCC2429, a cell line derived from NUT lung carcinoma, and RPMI2650, a cell line with epithelial morphology from the nasal septum of a patient with squamous cell carcinoma.They were analyzed using two alternative approaches: CRISPR and RNAi, using either CRISPR technology or RNA interference to extract information from the database on the effect of inhibiting each of the 17,000 genes in the panel. The results were ranked by relevance and are shown in Figure 3. In the graphs, the essential genes, which are associated with relatively lower Z-scores, are located on the left side of the graph, following the dependency ranking from left to right.

[0086] In the HCC2429 cell line, SMARCA4 was ranked among the top 10 most essential genes using both CRISPR and RNAi approaches (Figs. 3A and 3B). In the RPMI 2650 cell line, for which only CRISPR data were available, SMARCA4 ranked 200 among the most essential genes out of over 17,000 genes, underscoring the importance of SMARCA4 in these NUT carcinoma cell lines (Fig. 3C).

[0087] In vitro study of the effect of SMARCA4 degradation on the viability of NUT carcinoma cells

[0088] To confirm these findings in vitro and explore the essentiality of SMARCA4 for NUT carcinoma cells, the HCC2429 cell line was obtained from Dr. French's laboratory (where NUT carcinoma was discovered over two decades ago) and the PROTAC (PROteolysis TArgeting Chimera) ACBI1 was used to induce inhibition of SMARCA4 activity.

[0089] ACBI1 is a potent commercial PROTAC degrader (CAS: 2375564-55-7) that promotes the specific degradation of SMARCA4 and its paralog SMARCA2. Its negative control is the c¡s-ACBI1 molecule, where the hydroxyproline of the binding moiety is in the inactive cis conformation, preventing binding and, therefore, protein degradation.

[0090] The NUT carcinoma cell line HCC2429 was incubated for 18 h with increasing concentrations (0, 1, 5, 20, 40, 80, 125, 250, 500, 1000 nM) of ACBI1 or the inactive compound cis-ACBH, after which SMARCA4 protein expression levels were measured by Western blotting using tubulin as a loading control. After 18 h of treatment, cells were lysed with lysis buffer supplemented with protease inhibitors and phosphatase inhibitors. After mixing with loading buffer, samples were boiled for 5 min. Proteins were separated by electrophoresis in 8% SDS-PAGE polyacrylamide gels and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking the membranes for 1 hour in blocking buffer at room temperature, incubation with primary antibodies 1:1000 was carried out overnight at 4°C.The following day, the membranes were washed 3 times in PBS-tween for 5 minutes at room temperature before being incubated for 1 hour with the HPR peroxidase-conjugated secondary antibody solution at a dilution of 1:2,000. After incubation with the secondary antibodies, the membranes were washed an additional 3 times in PBS-tween and developed using the SuperSignal West Femto maximum sensitivity substrate and photographed using an ImageQuant LAS 4000 viewer. This method was used for all Western blots described herein.

[0091] ACBI1 efficiently induced SMARCA4 degradation, as opposed to the lack of effect observed in cells treated with the negative control compound cis-ACBI1 (Fig. 4A).

[0092] On the other hand, the NUT carcinoma cell line HCC2429 was incubated for 5 days with different concentrations (0.1, 2, 4, 5, 8, 10, 16, 20, 32, 40, 64, 80, 125, 160, 250 nM) of ACBI1 or the inactive compound c¡s-ACBI1 and the percentage of viability of HCC2429 cells was measured after treatment. For this type of viability assays 10 3Cells were seeded in 96-well plates. The following day, the culture medium (RPMI with 5% FBS) was changed for medium supplemented with PROTAC or the inactive compound cis-ACBI1 at the different concentrations. This medium change was also performed at 72 h. Five days after the first treatment, cells were incubated with 0.12 mM resazurin sodium salt for 4 h before the addition of 3% SDS. Fluorescence was then measured in a Glomax® Discover Multimode microplate reader (Promega, Madison, WI, USA) (excitation fluorescence at 520 nm and emission fluorescence at 580-640 nm). Relative viability (%) calculations for the two treatments (ACBI1 and cis-ACBI1) were performed by comparing the signal obtained for each concentration compared to the signal obtained for untreated cells.

[0093] Significantly, SMARCA4 degradation caused a dose-dependent reduction in the viability of NUT carcinoma cells (Fig. 4B).

[0094] Therefore, it is concluded that PROTAC ACBI1 promotes the degradation of SMARCA4, which in turn reduces the viability of NUT carcinoma cells.

[0095] In vitro study of the specificity of NUT carcinoma dependence on SMARCA2

[0096] To determine whether this dependence was specific to NUT carcinoma cells, the normal lung cell line HPF (human lung fibroblast) was included in the analysis.

[0097] In addition to ACBI1, PFI-3, a commercial selective, potent, and cell-permeable inhibitor of the SMARCA2 and SMARCA4 bromodomains (CAS: 1819363-80-8), was used.

[0098] The percentage viability of HCC2429 (NUT carcinoma cell line) and HPF (NUT non-carcinoma cell line; control) cells was measured at increasing concentrations of ACBI1 (Fig. 5A) or PFI-3 (Fig. 5B) after 5 days of treatment. This assay was performed similarly to that described for Figure 4 regarding the cell treatment methodology. In this case the concentrations used for the PROTAC were 0, 0.5, 1, 2, 4, 8, 16, 32, 40, 64, 80, 125, 160, 250, 500, 1000 nM while for the PFI-3 inhibitor they were 0, 0.32, 0.64, 1.25, 2.5, 5, 10, 20, 40, 80, 16, 32, 64, 125, 250, 500 |iM. Once the relative viability for each treatment was obtained in each of the cellular contexts, the necessary concentration of each compound at which cell viability is reduced to 50% (IC50) was calculated using Graph Pad Prism version 9.5.1. This measure reflects the relative sensitivity of a cell type to a certain treatment.HPF demonstrated increased resistance to ACBI1 treatment compared to the NUT carcinoma cell line (Fig. 5A). Treatment with the PFI-3 inhibitor further confirmed these results (Fig. 5B).

[0099] After deducing the IC50 of ACBI1 for HCC2429 from the data in Figure 5B, which for 5-day treatments was found to be 14 nM, a set of relevant cell lines were evaluated to determine their relative sensitivity to this drug.

[0100] Two NUT carcinoma cell lines were included in this trial: HCC2429 and RPMI 2650, and four control cell lines: HPF, H1395 (a non-NUT carcinoma lung carcinoma cell line), MCF7 (breast carcinoma, a non-NUT carcinoma and non-lung carcinoma cell line), and HEK293 (embryonic kidney cell, a non-lung cancer cell line).

[0101] After 5 days of treatment with 14 nM ACBI1 and its negative control c¡s-ACBI1, cell viability was measured (Fig 5C). For these assays, the different cell lines were seeded in 96-well plates, as described above for Figure 4, and after 5 days in the presence of ACBI1 or c¡s-ACBI1 at a concentration of 14 nM, cell viability was measured using resazurin sodium salt and the Glomax® plate reader. The relative viability (%) was calculated in each case for treatments with ACBI1 compared to the negative control (c¡s-ACBI1).

[0102] Treatment with ACBI1 only affected cell viability of the NUT carcinoma cell lines included in this analysis, as opposed to having no effect on the various control cells.

[0103] Expression levels of various NUT carcinoma markers such as SMARCA4, SMARCA 2, BRD4-NUT, c-Myc, SOX2 were also measured by Western Blot (as previously described), using actin as a loading control, in HCC2429 cells treated for 5 days with ACBI1 at a concentration of 15nM and with its negative control c¡s-ACBI1. The use of actin or tubulin as loading control is interchangeable and is decided depending on the availability of membrane based on the molecular size of the rest of the proteins to be revealed.

[0104] Western blotting results show that degradation of SWI / SNIF ATPases caused a significant decrease in the levels of NUT carcinoma-associated markers (SOX2 and c-Myc) in NUT carcinoma cells (Fig. 5D). These data confirm the specific sensitivity of NUT carcinoma cells to degradation and / or inhibition of the activity of the SWI / SNF ATPases SMARCA2 and SMARCA4.

[0105] In vitro study of the effect of SMARCA4 silencing in NUT carcinoma cells

[0106] The effect of SMARCA4 silencing on the viability of two NUT carcinoma cell lines, HCC2429 and RPMI 2650, was studied after 5 days of treatment with specific siRNAs (siRNAs, silencing RNA or small interfering RNA) that interfere with the expression of SMARCA4 and SMARCA2. Commercial siRNAs were used, specifically: s¡SMARCA4 (Horizon, J-010431 -06-0005), s¡SMARCA2 (Horizon J-010431 -07-0005) and the negative control Scramble (Horizon, D-001810-10-05 [s¡- NT]).

[0107] Silencing of SMARCA2 and SMARCA4 was achieved by transfection of siRNAs into 10 6 cells (HCC2429, RPMI or NMC1015), using the Lipofectamine™ RNAiMAX transfection reagent following the manufacturer's instructions. Silencing of SMARCA2 or SMARCA4 was evident by Western blotting from 24h post-transfection.

[0108] In line with the data in Figure 3, it was confirmed in in vitro models that silencing SMARCA4 using specific siRNAs was sufficient on its own to dramatically reduce cell viability in NUT carcinoma cells (HCC2429 and RPMI 2650) (Fig. 6, A and B).

[0109] The expression levels of NUT carcinoma markers, namely SMARCA4, BRD4-NUT, c-Myc, p63 and SOX2, were measured, using actin as a positive control, in NUT carcinoma cell lines HCC2429 and RPMI 2650 after silencing SMARCA4 using the SMARCA4 siRNA s¡SMARCA4 (Horizon, J-010431-06-0005) and the negative control Scramble (Horizon, D-001810-10-05 [si-NT]).

[0110] Western blotting showed significantly reduced expression levels of all markers in s¡SMARCA4-treated lines. Again, this reduction in cell viability was associated with a significant reduction in NUT carcinoma-associated markers such as c-Myc, SOX2, and p63 (Fig. 6, C and D).

[0111] Thus, SMARCA4 silencing reduces cell viability and levels of NUT carcinoma markers in NUT carcinoma cells. In vitro study of the effect of SMARCA4 silencing on meqadomain formation

[0112] To study the effect of the absence of SMARCA4 on the formation of megadomains, HCC2429 cells were transfected with s¡SMARCA4 or with the negative control SCR for 72 hours.

[0113] For immunofluorescence assays, cells were grown on glass coverslips. After siRNA silencing, cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature. Cells were washed three times with PBS containing and permeabilized in 0.25% Triton X-100 for 5 minutes, and washed three times with PBS. Cells were blocked with 1% BSA in PBS for 30 minutes, followed by a 2-hour incubation with primary antibody (1:100) at room temperature. Cells were subsequently washed three times with PBS and incubated with Alexa Fluor 488-conjugated secondary antibodies for 30 minutes at room temperature. Cells were then washed three times with PBS and mounted on slides using Mowiol medium. During the last wash, cells were incubated with DAPI (1:1000) for 5 minutes to stain the nuclei.Images were taken with a Zeiss LSM 710 confocal microscope using lasers 405 and 488 for DAPI and Alexa488, respectively. Images were subsequently processed using Imaged software (NIH). Cells were stained for histone 3 acetylated at lysine 27 (H3K27ac in green) and DAPI (nuclei in blue). Fluorescence microscopy images were obtained for both treatment conditions (Fig. 7).

[0114] Surprisingly, the absence of SMARCA4 had no effect on the normal formation of acetylated (H3K27ac) histone megadomains associated with NUT carcinoma (Fig. 7). Thus, in line with our hypothesis, although it does not reverse the formation of NUT carcinoma megadomains, SMARCA4 is required for the expression derived from these megadomains (SOX2, p63, and c-Myc), and is therefore essential for the survival of NUT carcinoma cells.

[0115] In vitro study of the effect of silencing SMARCA4 expression on squamous cell differentiation markers

[0116] Next, the role of SMARCA4 in the biology of NUT carcinoma was studied in more detail. As already mentioned, the NUT carcinoma markers SOX2, c-Myc and p63 are often associated with an undifferentiated state and an elevated proliferation profile of squamous cells [1, 6- 8],

[0117] To study the effect of SMARCA4 depletion in HCC2429 cells, SMARCA4 silencing was induced with s¡SMARCA4. Expression levels of SMARCA4 and the differentiation markers involucrin and b-catenin were measured, using tubulin as a loading control, by Western blotting as previously described.

[0118] Consistent with the data in Figure 6, SMARCA4 silencing induced the expression of the squamous cell-specific differentiation markers b-catenin and involucrin (Fig. 8A).

[0119] Cells were stained for b-catenin (green) and nuclei (blue) by DAPI staining, respectively. Immunofluorescence staining protocols for b-catenin and DAPI were performed as previously described. Fluorescence microscopy of stained cells confirmed that the increased levels of b-catenin occurred at the cell membrane, which is the specific subcellular location where b-catenin should be found in the context of squamous cell differentiation [23,24](Fig. 8B).

[0120] A study was also performed on the distribution of HCC2429 cells in the different phases of the cell cycle (G1 phase, S phase, and G2 phase). 10 6Cells previously treated with siRNA were washed with PBS, stained with 7-AAD at a dilution of 1:400, and incubated for 20 minutes at room temperature. The 7-AAD dye gives a different signal per cell depending on the cell cycle in which it is (G1, S, or G2). Finally, the cells were washed and fixed with 2% paraformaldehyde. The 7-AAD signal was evaluated by flow cytometry using the BD FACSVerse™ cytometer, and the data were analyzed using the cell cycle tool of the FlowJo software (version 10.7.1).

[0121] Statistical analyses compared the percentage of cells in each phase of the cell cycle for three replicates of HCC2429 cells transfected with s¡SMARCA4 (red) versus their negative control treated with SCR (blue). Statistical calculations were performed using an unpaired, two-tailed Student's t test using Graph Pad Prism version 9.5.1.

[0122] As expected, differentiation signals induced by SMARCA4 depletion caused a subsequent cell cycle arrest in NUT carcinoma cells, increasing the proportion of cells in G1 phase to the detriment of those in G2 phase (actively dividing) (Fig. 8C).

[0123] In vitro study of the effect of silencing SMARCA4 expression on the proliferation of NUT carcinoma cells

[0124] Cell proliferation was measured by flow cytometry using CelITrace™ Violet (CTV) dye to evaluate the effect of silencing SMARCA4 expression on cell proliferation. HCC2429 cells were treated with either s¡SMARCA4 or the negative control SCR. The Cell trace Violet (CTV) dye facilitates analysis of cell proliferation by flow cytometry. 10 6Cells pretreated with siRNAs were washed with PBS and stained with CTV following the manufacturer's instructions (Life Technologies). CTV fluorescence was evaluated on day 0 and day 5 by flow cytometry using the BD FACSVerse™ cytometer, and data were analyzed using FlowJo software (version 10.7.1). The mean fluorescence was then normalized to day 0 and compared between treatments. Higher CTV intensities after 5 days imply a lower proliferation rate.

[0125] Cell cycle arrest can be seen to reduce the overall cell proliferation rate of NUT carcinoma cells (Fig. 9). Higher levels are associated with fewer divisions, as CTV dye is inversely correlated with cell division rate (Fig. 9A). Statistical calculations were performed using a two-tailed, unpaired Student's t test using Graph Pad Prism version 9.5.1. P values ​​< 0.05 were considered statistically significant.

[0126] Cell proliferation was also measured by immunofluorescence using the proliferation marker K¡67 (Fig. 9B). For this purpose, cells were immunostained for the proliferation marker K¡67 (green) and DAPI (blue; nuclei) after 72 h of s¡SMARCA4 treatment and images were obtained by fluorescence microscopy. Immunofluorescence staining protocols were performed as previously described. The percentage of K¡67 positive cells (in 2 fields of 25 cells each) from 2 independent replicates was subsequently measured using ImageJ software. Statistical calculations were performed using an unpaired, two-tailed Student t test using Graph Pad Prism version 9.5.1. P values ​​< 0.05 were considered statistically significant. It can be clearly seen how silencing the SWI / SNF ATPase SMARCA4 successfully reduced the growth of NUT carcinoma cells.

[0127] In vitro study of the effect of silencing SMARCA2 and SMARCA4 expression on the viability of NUT carcinoma cells

[0128] To study the effect of SMARCA2 silencing compared to SMARCA4 on the viability of NUT carcinoma cells, some knockdown analyses for SMARCA2 (SMARCA4 paralog and SWI / SNF alternative ATPase) were performed in order to establish the exclusive or rather shared role of SMARCA4 in the development of NUT carcinoma.

[0129] Three NUT carcinoma cell lines were used: HCC2429, RPMI 2650 and NMC1015, which were transfected with siRNAs for the SWI / SNF ATPases SMARCA2, SMARCA4: SÍSMARCA4 (Horizon, J-010431 -06-0005), SÍSMARCA2 (Horizon J-010431 -07-0005) and the negative control Scramble (Horizon, D-001810-10-05 [si-NT]).

[0130] 5 days after transfection, the percentage of cell viability was measured in each of the cell lines and treatments as described above.

[0131] Silencing of SMARCA2 and SMARCA4 reduces the viability of NUT carcinoma cells. Although knockdown of SMARCA2 using specific siRNAs also resulted in a reduction in NUT carcinoma cell viability, the therapeutic potential of targeting SMARCA2 in NUT carcinoma appears to be slightly lower than that of targeting the SMARCA4 ATPase (Fig. 10). These results were confirmed in three independent NUT carcinoma cell lines (HCC2429, RPMI 2650, and NMC 1015), underlining their relevance (Fig. 10).

[0132] It can be concluded from these data that SWI / SNF plays a very important role in the development of NUT carcinoma, and, in particular, the in silico and in vitro results confirm that the ATPase SMARCA4 is essential for NUT carcinoma.

[0133] Although depletion of SWI / SNF ATPases in NUT carcinoma does not prevent the formation of megadomains (it is located upstream of SWI / SNF activity), it severely limits the expression of NUT carcinoma markers derived from those megadomains. Consequently, the reduction in NUT carcinoma marker levels induces differentiation of NUT carcinoma cells, which in turn causes cell cycle arrest in G1 and, consequently, a reduction in cell proliferation.

[0134] Thus, targeting SWI / SNF ATPases independently can control the growth of NUT carcinoma cells.

[0135] Furthermore, NUT carcinoma cells are particularly sensitive to treatments with drugs targeting both SMARCA2 and SMARCA4, as opposed to the effect on other cells / tissues.

[0136] References

[0137] 1 French, C. A. et al. Report of the First International Symposium on NUT Carcinoma. Clin Cancer Res 28, 2493-2505, doi:10.1158 / 1078-0432.CCR-22-0591 (2022).

[0138] 2 Durall, R. T. et al. The BRD4-NUT fusion alone drives malignant transformation of NUT carcinoma. Cancer Res, doi:10.1158 / 0008-5472.CAN-23-2545 (2023).

[0139] 3 French, C. A. et al. BRD-NUT oncoproteins: a family of closely related nuclear proteins that block epithelial differentiation and maintain the growth of carcinoma cells. Oncogene 27, 2237-2242, doi:10.1038 / sj.onc.1210852 (2008).

[0140] 4 Alekseyenko, A. A. et al. Ectopic protein interactions within BRD4-chromatin complexes drive oncogenic megadomain formation in NUT midline carcinoma. Proc Natl Acad Sci U S A 114, E4184-E4192, doi:10.1073 / pnas.1702086114 (2017).

[0141] 5 Ibrahim, Z. et al. Structural insights into p300 regulation and acetylation-dependent genome organisation. Nat Commun 13, 7759, doi:10.1038 / s41467-022-35375-2 (2022).

[0142] 6 Grayson, A. R. et al. MYC, a downstream target of BRD-NUT, is necessary and sufficient for the blockade of differentiation in NUT midline carcinoma. Oncogene 33, 1736-1742, doi:10.1038 / onc.2013.126 (2014).

[0143] 7 Wang, R. et al. Activation of SOX2 expression by BRD4-NUT oncogenic fusion drives neoplastic transformation in NUT midline carcinoma. Cancer Res 74, 3332-3343, doi: 10.1158 / 0008-5472.CAN-13-2658 (2014).

[0144] 8 Sholl, L. M. et al. Primary Pulmonary NUT Midline Carcinoma: Clinical, Radiographic, and Pathologic Characterizations. J Thorac Oncol 10, 951-959, doi : 10.1097 / JTO .0000000000000545 (2015). 9 Stathis, A. et al. Clinical Response of Carcinomas Harboring the BRD4-NUT Oncoprotein to the Targeted Bromodomain Inhibitor OTX015 / MK-8628. Cancer Discov 6, 492-500, doi:10.1158 / 2159-8290. CD-15-1335 (2016).

[0145] 10 Tontsch-Grunt, II. et al. Therapeutic impact of BET inhibitor Bl 894999 treatment: backtranslation from the clinic. Br J Cancer 127, 577-586, doi:10.1038 / s41416-022- 01815-5 (2022).

[0146] 11 Shapiro, G. I. et al. A Phase 1 study of R06870810, a novel bromodomain and extra-terminal protein inhibitor, in patients with NUT carcinoma, other solid tumours, or diffuse large B-cell lymphoma. Br J Cancer 124, 744-753, doi: 10.1038 / s41416-020-01180- 1 (2021).

[0147] 12 Lewin, J. et al. Phase lb Trial With Birabresib, a Small-Molecule Inhibitor of Bromodomain and Extraterminal Proteins, in Patients With Selected Advanced Solid Tumors. J Clin Oncol 36, 3007-3014, doi:10.1200 / JC0.2018.78.2292 (2018).

[0148] 13 Clapier, C. R., Iwasa, J., Cairns, B. R. & Peterson, C. L. Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes. Nat Rev Mol Cell Biol 18, 407-422, doi: 10.1038 / nrm.2017.26 (2017).

[0149] 14 Lu, C. & Allis, C. D. SWI / SNF complex in cancer. Nat Genet 49, 178-179, doi: 10.1038 / ng.3779 (2017).

[0150] 15 Mashtalir N, D'Avino AR, Michel BC, Luo J, Pan J, Otto JE, Zullow HJ, McKenzie ZM, Kubiak RL, St Pierre R, Valencia AM, Poynter SJ, Cassel SH, Ranish JA, Kadoch C. Modular Organization and Assembly of SWI / SNF Family Chromatin Remodeling Complexes. Cell. 2018 Nov 15;175(5):1272-1288.e20.

[0151] 16 Kwon H, Imbalzano AN, Khavari PA, Kingston RE, Green MR. Nucleosome disruption and enhancement of activator binding by a human SW1 / SNF complex. Nature. 1994 Aug 11 ;370(6489):477-81 .

[0152] 17 . Wu T, Kamikawa YF, Donohoe ME. Brd4's Bromodomains Mediate Histone H3 Acetylation and Chromatin Remodeling in Pluripotent Cells through P300 and Brg1. Cell Rep. 2018 Nov 13;25(7):1756-1771.

[0153] 18 Enriquez, P. et al. Binding specificity and function of the SWI / SNF subunit SMARCA4 bromodomain interaction with acetylated histone H3K14. J Biol Chem 297, 101145, doi:10.1016 / j.jbc.2021.101145 (2021). 19 Alver, B. H. et al. The SWI / SNF chromatin remodelling complex is required for maintenance of lineage specific enhancers. Nat Commun 8, 14648, doi:10.1038 / ncomms14648 (2017).

[0154] 20 Naidu, S. R., Love, I. M., Imbalzano, A. N., Grossman, S. R. & Androphy, E. J. The SWI / SNF chromatin remodeling subunit BRG1 is a critical regulator of p53 necessary for proliferation of malignant cells. Oncogene 28, 2492-2501, doi:10.1038 / onc.2009.121 (2009).

[0155] 21 - Maher J, Stagg N, Cain G, Andaya R, Katavolos P, Gallardo-Chang F, Pham A, Ye X, Januario T, Alcantar T, Caothien R, Roose-Girma M, Zhang D, Li R, Chen S, Yauch RL. Smarca2 genetic ablation is phenotypically benign in a safety assessment of tamoxifen- inducible conditional knockout rats. Toxicol Appl Pharmacol. 2023 Sep 15;475:116627. doi: 10.1016 / j.taap.2O23.116627. Epub 2023 Jul 14. PMID: 37453479.

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[0158] 24 Kaur, J. et al. Clinical significance of altered expression of beta-catenin and E- cadherin in oral dysplasia and cancer: potential link with ALCAM expression. PLoS One 8, e67361 , doi:10.1371 / journal. pone.0067361 (2013).

Claims

CLAIMS 1. Inhibitory agent of the catalytic subunits SMARCA4 and / or SMARCA2 of the SWI / SNF complex for use in the treatment and / or prevention of NUT carcinoma.

2. The inhibitory agent for use according to the preceding claim characterized in that it is capable of inhibiting the activity of the SMARCA2 and SMARCA4 subunits.

3. The inhibitory agent for use according to the preceding claim characterized in that it is selected from the list consisting of: PFI-3, GNE-064, BRM / BRG1 ATP Inhibitor 1, BRM / BRG1 ATP Inhibitor 2, BRM / BRG1 ATP Inhibitor 3, BRM014, FHD-286 and any combination thereof.

4. The inhibitory agent for use according to claim 1 characterized in that it is capable of reducing the levels at which the genes encoding the SMARCA2 and / or SMARCA4 subunits are transcribed.

5. The inhibitory agent for use according to the preceding claim characterized in that it is selected from the list consisting of: a) a microRNA, b) a small interfering RNA, and c) a short hairpin RNA, or any combination thereof.

6. The inhibitory agent for use according to the preceding claim characterized in that the SISMARCA4 is J-010431-06-0005 of Horizon and the SISMARCA2 is J-010431-07-0005 of Horizon.

7. The inhibitory agent for use according to claim 5 characterized in that it is a SMARCA4 specific short hairpin RNA that is selected from the list consisting of: Cat#106960 from Thermo Fisher, esiRNA EHU076601 from Sigma Aldrich, Cat#SR509768 from OriGene and sc-29827 from Santa Cruz, and / or a SMARCA2 specific short hairpin RNA that is selected from the list consisting of: Cat#106957 from Thermo Fisher, EsiRNA EHU135151 from Sigma Aldrich, Cat#SR510178 from OriGene and sc-29831 from Santa Cruz.

8. The inhibitory agent for use according to claim 1 characterized in that it is capable of causing proteolysis of the SMARCA2 and / or SMARCA4 proteins once expressed.

9. The inhibitory agent for use according to the preceding claim characterized in that it is a chimera directed at proteolysis.

10. The inhibitory agent for use according to the preceding claim characterized in that the chimera directed at proteolysis is selected from the list consisting of: ACBI1, AU-15330, A947, YD23, PRT3789, SMD-3040 and ACBI2.

11. Pharmaceutical composition comprising at least one inhibitory agent of the SMARCA4 and / or SMARCA2 catalytic subunits of the SWI / SNF complex according to any of claims 1 to 10 for use as a medicament in the treatment and / or prevention of NUT carcinoma.

12. Method for selecting therapeutic agents useful in the treatment and / or prevention of NUT carcinoma, comprising: a) determining the activity of SMARCA4 and / or SMARCA2 at an established concentration of the compound to be analyzed or in the absence of said compound, and b) determining the activity of SMARCA4 and / or SMARCA2 at a concentration of the compound to be analyzed different from that of a), such that compounds capable of inhibiting the activity of SMARCA4 and / or SMARCA2 are identified as therapeutic agents against NUT carcinoma.

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