ES2970019A1 - Compounds for the treatment of pancreatic ductal adenocarcinoma (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

The invention describes compounds for the treatment of pancreatic ductal adenocarcinoma that act as agents that modulate the activity of EIF4A3, compositions that include these compounds and combined preparations, together with other active ingredients for use as medications. Also described are methods of diagnosis and for the selection of therapeutic agents useful in the prevention, improvement, alleviation, and/or treatment of pancreatic ductal adenocarcinoma. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Compounds for the treatment of pancreatic ductal adenocarcinoma

FIELD OF TECHNIQUE

The present invention is within the field of medicine, and more specifically cancer treatments, and refers to compounds for the treatment of pancreatic ductal adenocarcinoma, specifically to the use of an EIF4A3-specific siRNA as a modulating agent. the activity of EIF4A3.

STATE OF THE ART

Pancreatic cancer is the most lethal and aggressive of common cancers, because it is usually diagnosed in advanced stages and barely responds to available treatments. In general, it is a relatively rare tumor that represents 2.1% of all cancers, but its incidence and mortality are increasing. In Western societies it is already the fourth cause of death from cancer and it is estimated that in just a decade it will become the second. Every year about 233,000 new cases are diagnosed worldwide (125,000 men, 108,000 women) and, in Spain, about 4,000 cases are registered annually (2.2% of tumors in men, 2.7% in women). Pancreatic ductal adenocarcinoma (PDA), derived from the exocrine pancreas, accounts for 90% of pancreatic tumors, and is what we commonly refer to as pancreatic cancer, while only 5% of them are pancreatic neuroendocrine tumors (pNETs). ), derived from the endocrine pancreas. Unfortunately, scientific and medical advances in the study of ADP and, especially, its application, are still very limited, which has made it the most lethal type of common cancer, with 5-year survival rates of less than 7%. and with an average survival after diagnosis of 4-6 months. This makes it essential and urgent to generate tools to early identify individuals at high risk, detect pre-cancerous lesions early, and discover new biomarkers, which facilitate a more personalized and precise knowledge of patients' prognosis and help predict their outcome. response to available treatments (gemcitabine, folfirinox or gemcitabine/nabpaclitaxel) and, above all, to develop new, more effective therapeutic targets for their treatment.

Currently, there are several biomarkers used in clinical practice for the diagnosis of pancreatic ductal adenocarcinoma (PDA), such as the sialylated Lewis antigen, or CA 19-9, which recognizes glycoproteins in blood circulation, and which is elevated in ADP, colon, esophageal and liver cancer, as well as in some benign pathologies such as cholangitis and pancreatitis; or carcinoembryonic antigen (CEA), a glycoprotein that is produced during fetal development, but which can appear elevated in ADP, but also in colorectal, gastric, lung, breast and thyroid cancer, as well as in non-neoplastic diseases such as ulcerative colitis, pancreatitis , cirrhosis, COPD, Crohn's disease, hypothyroidism, as well as in smokers. Therefore, although they may be useful, their low specificity, both because they occur in other tumor and non-tumor pathologies, make them far from being considered a gold standard for the diagnosis of ADP.

The development of new diagnostic markers that could represent an alternative to the evaluation of CA 19-9 and CEA, and that could discriminate between control samples or those with pancreatic tumor with high sensitivity and specificity, would be a great advance in the fight against this pathology. which is generally diagnosed when it is in an advanced state due to the lack of effective early specific markers. Furthermore, its study as a therapeutic target could improve treatment, which is not effective or safe enough for the patient, since current targets have important limitations.

The limited knowledge available of the genetic, molecular and cellular events underlying the development and progression of ADP remains one of the main barriers in its study. This explains why biomarkers are not yet available, not only for the early diagnosis of these tumors, but even to unequivocally discriminate between cancer and an inflammatory lesion of the pancreas.

In this sense, distinctive features of cancer appear to be associated with specific alterations in the unfolding process, which in turn leads to a more aggressive and invasive cancer phenotype. Therefore, the deployment process has emerged as a promising alternative for the identification of new biomarkers for the diagnosis and prognosis of various metabolic pathologies and numerous types of cancer, as well as a possible source for the identification of possible therapeutic targets for the treatment of cancer.

Splicing is the process by which the coding and non-coding regions of a gene are restructured and rearranged to generate a variety of RNA transcripts, which determines the appearance of different protein variants and isoforms that, in many cases, exert different biological functions. and even opposites. The alteration of the machinery that carries it out can lead to the generation of aberrant unfolding variants, which have been related to the development and/or progression of various diseases, including cancer.

A splicing unfolding factor is a protein involved in removing introns from messenger RNA strands so that exons can be spliced together; the process takes place in particles known as a spliceosome

EIF4A3 is an ATP-dependent RNA helicase, a member of the EIF4A family of transcription initiation factors, which in turn belongs to the Asp-Glu-Ala-Asp (DEAD) protein superfamily, “DEAD box” family. DEAD. EIF4A transcription initiation factors are involved in various aspects of RNA biology, from transcription and translation, to mRNA decay. Specifically, EIF4A3 is involved in pre-mRNA splicing as a component. of the spliceosome. It is a central component of the multiprotein exon junction complex ("EJC"). The EJC is a dynamic structure consisting of central proteins and several associated cytoplasmic, nuclear and peripheral factors. complex only transiently during EJC assembly or during subsequent mRNA metabolism. The EJC core consists of four proteins, EIF4A3 (DDX48), MAGOH, Y14 (RBM8A), and Barentsz (BTZ, also called CASC3 or MLN51), that bind stably to spliced mRNA, very close to exon-exon junctions. The EJC marks the position of the exonexon junction on the mature mRNA for the gene expression machinery and the core components remain bound to spliced mRNAs at all stages of mRNA metabolism, influencing downstream processes, including the export of Nuclear mRNA, subcellular mRNA localization and the efficiency of translation and mRNA decay mediated by terminator mutations (NMD -Nonsense Mediated Decay).

Several recent studies suggest that EIF4A3 promotes tumor growth in multiple human cancers. Molecular biology studies showed that EIF4A3 is recruited by long non-coding RNAs to regulate the expression of certain proteins in tumors. Since EIF4A3 plays a key role in NMD, strategies targeting EIF4A3 may open a new avenue for treating cancer. However, the tumor-related functions of EIF4A3 and the underlying mechanisms are not well understood, nor has the protein's potential utility as a diagnostic biomarker, therapeutic target or prognostic indicator, nor its potential effectiveness been evaluated to date [ 1].

Some studies have proposed EIF4A3 as a serum diagnostic marker for patients with pancreatic cancer, as it is expressed more strongly in most pancreatic cancer tissues than in normal tissues, with reactivity observed in 20 of 60 patients with pancreatic cancer. pancreas (33.33%). Furthermore, this reactivity has not been observed in patients with other pathologies such as chronic pancreatitis, nor in healthy individuals [2].

DESCRIPTION OF THE INVENTION

As shown in the examples of the present invention, the unfolding factor EIF4A3 is clearly deregulated in pancreatic ductal adenocarcinoma (PDA) samples, compared to adjacent control tissue (Figure 1).

This molecule, EIF4A3, is capable of distinguishing, according to its expression, between a control sample and a tumor sample with high sensitivity and specificity (Figure 2) far beyond what could be predicted based on what was noted in previous studies [2]. where the relationship between EIF4A3 overexpression and ADP was quite weak, limited to 33% of the cases analyzed.

Furthermore, its expression correlates with characteristics of tumor malignancy, such as the presence of metastasis (Figure 3).

When this factor is silenced with a specific siRNA in ADP-derived cell lines, it is observed that they reduce their tumor characteristics, such as cell proliferation, migration, and the ability to form spheres and colonies (Figures 6 and 7).

The authors of the present invention have demonstrated that the expression levels of the unfolding factor EIF4A3 are overexpressed in samples of pancreatic ductal adenocarcinoma tumors and that its silencing in cell lines derived from ADP is capable of reducing tumor characteristics such as cell proliferation or migration. , as well as the ability to form spheres or colonies. The overexpression of this gene, which clearly discriminates between a control sample and a tumor sample, could be used as a new diagnostic and prognostic marker, as well as a new therapeutic target for the treatment of pancreatic cancer.

Therefore, a first aspect of the invention relates to an agent that modulates the activity of EIF4A3, hereinafter modulating agent of the invention, for use in the treatment of pancreatic ductal adenocarcinoma.

The EIF4A3 activity modulating agent is preferably an inhibitor of EIF4A3 activity.

Herein is understood by EIF4A3 or eukaryotic transcription initiation factor 4A3, also called Fal1; RCPS; DDX48; MUK34; NUK34; NMP265; eIF4AIII; eIF4A-III; eIF-4A-NI, to a gene that encodes a member of the “DEAD boXo DEAD box” protein family. DEAD box proteins, characterized by the conserved Asp-Glu-Ala-Asp (DEAD) motif, are putative RNA helicases. They are involved in a series of cellular processes that involve the alteration of RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and the assembly of ribosomes and spliceosomes. Based on their distribution patterns, some members of this family are believed to be involved in embryogenesis, spermatogenesis, and cell growth and division. The protein encoded by this gene is a nuclear matrix protein. Its amino acid sequence is very similar to the amino acid sequences of the translation initiation factors EIF4AI and EIF4AII, two other members of the “DEAD box” protein family.

In the context of the present invention, EIF4A3 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the EIF4A3 protein, and which would comprise various variants from:

a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 1,

b) nucleic acid molecules whose complementary chain hybridizes with the polynucleotide sequence of a),

c) nucleic acid molecules whose sequence differs from a) and/or b) due to the degeneration of the genetic code,

d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 1. in the that the polypeptide encoded by said nucleic acids has the activity and structural characteristics of the EIF4A3 protein. Preferably, it is SEQ ID NO: 2

SEQ ID NO: 1

SEQ ID NO: 2

The term "activity modulating" as used here primarily refers to inhibiting (decreasing) the activity level of EIF4A3. The activity of EIF4A3 can be modulated by modifying the levels and/or activity of EIF4A3, or by modifying the levels at which the genes encoding EIF4A3 are transcribed such that the activity levels of the EIF4A3 protein in the cell is modulated. Modulatory agents can also be agonists (substances that are capable of binding to a receptor and causing a response in the cell, preferably a decrease in the activity of EIF4A3, or antagonists (substances that not only do not activate the receptor, but actually blocks its activation by agonists). In the context of the present invention, inhibition is the preferred form of modulation.

In a preferred embodiment of this aspect of the invention, the modulating agent of the invention is selected from a list consisting of:

a) an organic molecule,

b) an RNA molecule,

c) an antisense oligonucleotide,

d) an antibody,

e) a ribozyme,

or any of its combinations.

Organic molecules that act as EIF4A3 modulating agents are described in the state of the art, with various examples of these compounds. Specifically, numerous EIF4A3 inhibitors are described that can be used to carry out the object of the present invention. Some of them, as a non-exhaustive list of possible embodiments of the invention, are those described in US9957277B2, US8008346B2, EA201891229A1 among other patent documents.

Nucleotide sequences specifically complementary to a given DNA or RNA sequence could form complexes and block transcription or translation. Thus, with the progress of post-transcriptional gene silencing, and in particular RNA interference (RNA interference or RNAi), tools have been developed that allow the specific inhibition of the expression of a gene. The inhibition of EIF4A3 expression would therefore constitute the inhibition of its biological activity, and specifically, it would be useful in the treatment of pancreatic ductal adenocarcinoma.

"Antisense polynucleotides" refer to chains of ribonucleotides or deoxyribonucleotides that can inhibit the production of EIF4A3 by one of three mechanisms:

1- Interfering with transcription, by hybridizing in the structural gene or in a regulatory region of the gene that codes for EIF4A3. Since transcription or expression is effectively blocked by hybridization of the antisense oligonucleotide to DNA, the production of EIF4A3 is decreased.

2- The binding of the antisense oligonucleotide in the cytoplasm with the mRNA, interfering with the formation of the translation construct itself, inhibiting the translation of mRNA to the protein.

3- The formation of an mRNA - antisense duplex that allows rapid degradation of the duplex mRNA by RNases (such as RNase H). This results in lower production of EIF4A3.

Antisense oligonucleotides capable of inhibiting EIF4A3 are known in the state of the art. For example, and without limitation, it could be a sequence of ribonucleotides or RNA that belongs to the so-called siRNA (small interfering RNA), small interfering RNA or silencing RNA, capable of inhibiting the genetic expression of EIF4A3. In the context of the present memory, "siRNA" (small interfering RNA) is understood to be a class of double-stranded RNA of 19 to 25 nucleotides long, and more preferably between 21 and 23 nucleotides, which is involved in the RNA interference pathway, where siRNA interferes with the expression of a specific gene. In the present invention, this specific gene is EIF4A3.

There are currently numerous commercial EIF4A3 siRNAs on the market, among which those marketed by Thermo Fisher stand out, among which is the EIF4A3 siRNA that is used in the embodiments of the present invention (ID 138378).

They could also be an RNA construct that contains at least any of the possible nucleotide sequences of siRNA capable of inhibiting the expression of EIF4A3, and without prejudice to the fact that any of the RNA sequences and constructs of the invention described above that are subject to modifications, preferably chemical, that lead to greater stability against the action of ribonucleases and therefore greater efficiency, without said modifications involving the alteration of its mechanism of action, which is the union specific to the RISC (RNA-induced silencing complex) complex, activating it and manifesting a helicase activity that separates the two strands, leaving only the antisense strand associated with the complex. The resulting ribonucleoprotein complex binds to the target mRNA (EIF4A3 messenger RNA, which is set forth in SEQ ID NO: 2). If the complementarity is not perfect, RISC remains associated with the messenger and the translation is attenuated. But if it is perfect, RISC acts as RNase, cutting off the messenger and leaving it free to repeat the process.

Additionally, it is evident to a person skilled in the art that a large amount of mRNA polynucleotides can be translated into EIF4A3 as a consequence, for example, of the genetic code being degenerate. Any siRNA capable of inhibiting the translation of these mRNAs is also part of the invention.

The preparation of the siRNA sequence of the invention or the RNA construction of the invention would be evident to a person skilled in the art, and could be carried out by chemical synthesis, which also allows the incorporation of chemical modifications in both the different nucleotides of the product as well as the incorporation of other chemical compounds at either end. On the other hand, the synthesis could also be carried out enzymatically using any of the available RNA polymerases. Enzymatic synthesis also allows for some chemical modification of sRNA products or inhibitors.

The design of the nucleotide sequence of the siRNA of the invention would also be apparent to one skilled in the art. Thus, it could be carried out using a random design in which 19-25 bases of the target mRNA are selected without taking into account the sequence or positional information it has in the transcript. Another non-limiting alternative to the present invention would be the conventional design using simple parameters developed by the pioneers of the technique [3] completed with a nucleotide BLAST analysis. Another possibility could be a rational design, in which a computational procedure is used to identify the optimal siRNA targets in an mRNA. Target sequences are analyzed in groups of 19 nucleotides at a time and those with the best characteristics are identified based on an algorithm that incorporates a large number of thermodynamic and sequence parameters.

A DNA genetic construct could also form part of the composition of the invention, which would direct the in vitro or intracellular transcription of the siRNA sequence or RNA construct of the invention, and which comprises at least one of the following types of sequences:

a) DNA nucleotide sequence, preferably double-stranded, comprising at least the coding sequence of the siRNA of the invention or the RNA construct of the invention for transcription, or,

b) DNA nucleotide sequence, preferably double-stranded, corresponding to a gene expression system or vector that comprises the coding sequence of the RNA sequence of the invention operatively linked with, at least, one promoter that directs the transcription of said nucleotide sequence of interest, and with other sequences necessary or appropriate for transcription and its proper regulation in time and place, for example, start and stop signals, cutting sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers). ), transcriptional silencers (silencers), etc. for use in those pathological contexts in which EIF4A3 is contributing to the aggravation of sympathetic overactivation. Multiple of these constructions, systems or expression vectors can be obtained by conventional methods known to experts in the field [4].

The compositions of the present invention allow the transfection of the siRNA of the invention into a cell, in vivo or in vitro. The transfection could be carried out, but not limited to, direct transfection or vectors that facilitate access of the siRNA to the interior of the cell. Thus, examples of these vectors are, but are not limited to, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, Herpes simplex viruses, non-viral DNA plasmids, cationic liposomes and molecular conjugates. Thus, for example, the siRNAs of the present invention, as well as RNA or DNA precursors of these siRNAs, can be conjugated with release peptides or other compounds to promote the transport of these siRNAs into the cell.

The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts with) the protein. EIF4A3. Examples of portions of immunologically active immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. It can be a monoclonal or polyclonal antibody.

Antibodies capable of binding to the EIF4A3 protein can be used to inhibit the activity of said protein. Such antibodies are commercially available and/or are easily obtainable by those skilled in the art by known methods. The antibodies, or fragments thereof, may be able to inhibit the activity of the EIF4A3 protein that contributes to pancreatic ductal adenocarcinoma. Antibodies can be polyclonal (typically include different antibodies directed against different determinants or epitopes) or monoclonal (directed against a single determinant on the antigen). The monoclonal antibody can be altered biochemically, by genetic manipulation, or it can be synthetic, possibly lacking the antibody in its entirety or in parts, of portions that are not necessary for the recognition of EIF4A3 and being replaced by others that communicate to the antibody additional advantageous properties. The antibody may also be recombinant, chimeric, humanized, synthetic or a combination of any of the above.

A "recombinant antibody or polypeptide" (rAC) is one that has been produced in a host cell that has been transformed or transfected with the nucleic acid encoding the polypeptide, or produces the polypeptide as a result of homologous recombination.

These rACs can be expressed and directed to specific cellular subcompartments when appropriate sequences are incorporated for intracellular trafficking. These antibodies are called “intrabodies”, and have demonstrated their effectiveness not only in diverting proteins from their usual compartment or blocking interactions between proteins involved in signaling pathways, but also in activating intracellular proteins.

Also part of the invention are genetic DNA constructions capable of being transcribed into a peptide, antibody or antibody fragment, for use against EIF4A3, and in the treatment of pathologies that cause sympathetic overactivation. Said DNA genetic construction would direct the in vitro or intracellular transcription of the antibody sequence or fragment thereof, and comprises, at least, one of the following types of sequences: a) DNA nucleotide sequence, preferably double-stranded, which comprises, at least, the coding sequence of the antibody of the invention or the antibody fragment of the invention for its in vitro, or intracellular transcription, b) DNA nucleotide sequence, preferably double-stranded, corresponding to a gene expression system or vector that comprises the coding sequence of the antibody sequence or antibody fragment of the invention operatively linked with at least one promoter that directs the transcription of said nucleotide sequence of interest, and with other sequences necessary or appropriate for transcription and its proper regulation in time and place, for example, start and end signals, cutting sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc. for use in those pathological contexts that occur with infection.

A "ribozyme" as understood herein refers to a catalytic polynucleotide (typically RNA), which can be constructed to specifically recognize, by hybridization, an mRNA and cleave it or eliminate its expression. Ribozymes can be introduced into the cell as catalytic RNA molecules or as genetic constructs that are expressed as catalytic RNA molecules.

One skilled in the art could prepare organic molecules that can specifically bind to EIF4A3 without binding to other polypeptides or proteins. The organic molecules will preferably have a weight of 100 to 20,000 daltons, more preferably 500 to 15,000 daltons, and more preferably 1,000 to 10,000 daltons. Libraries of organic molecules are commercially available. The route of administration can be, but is not limited to, intraperitoneal, intrathecal, intravenous, intramuscular, subcutaneous, intraventricular, oral, enteral, parenteral, intranasal or dermal.

Therefore, in a preferred embodiment of this aspect of the invention, the EIF4A3 modulating agent of the invention is the EIF4A3 siRNA which causes its elimination through the decay of the mRNA mediated by terminator mutations ("NMD -Nonsense Mediated Decay). ').

Pharmaceutical composition and pharmaceutical form of the invention

Another aspect of the invention relates to a composition comprising a modulating agent of the invention, hereinafter composition of the invention, for use in the treatment of pancreatic ductal adenocarcinoma.

In a preferred embodiment of this aspect of the invention, the composition is a pharmaceutical composition. More preferably, the composition further comprises another active ingredient.

Another aspect of the invention relates to a pharmaceutical form, hereinafter pharmaceutical form of the invention, comprising an EIF4A3 modulating agent of the invention, or the composition of the invention.

In this report, "pharmaceutical form" is understood as the mixture of one or more active ingredients with or without additives that have physical characteristics for their adequate dosage, conservation, administration and bioavailability.

In another preferred embodiment of the present invention, the compositions and dosage forms of the invention are suitable for oral administration, in solid or liquid form. The possible forms for oral administration are tablets, capsules, syrups or solutions and may contain conventional excipients known in the pharmaceutical field, such as aggregating agents (e.g. syrup, acacia, gelatin, sorbitol, tragacanth or polyvinyl pyrrolidone), fillers (e.g. lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine), disintegrants (e.g. starch, polyvinyl pyrrolidone or microcrystalline cellulose) or a pharmaceutically acceptable surfactant such as sodium lauryl sulfate. Other pharmaceutical forms can be colloidal systems, which include nanoemulsions, nanocapsules and polymeric nanoparticles.

Compositions for oral administration can be prepared by conventional Galenic Pharmacy methods, such as mixing and dispersion. The tablets can be coated following methods known in the pharmaceutical industry.

The compositions and pharmaceutical forms can be adapted for parenteral administration, as sterile solutions, suspensions, or lyophilisates of the products of the invention, using the appropriate dosage. Suitable excipients, such as pH buffering agents or surfactants, may be used.

The aforementioned formulations can be prepared using conventional methods, such as those described in the Pharmacopoeias of different countries and in other reference texts.

In this report, “active ingredient” is understood as a substance or set of substances that have the capacity to exert a pharmacological, immunological or metabolic action in order to restore, correct or modify physiological functions, or to establish a diagnosis.

The term "medicine", as used herein, refers to any substance used for the prevention, diagnosis, relief, treatment or cure of diseases in man and animals.

Administration of the compounds, compositions or dosage forms of the present invention can be carried out by any suitable method, such as intravenous infusion and oral, topical or parenteral routes. Oral administration is preferred for patient convenience.

The amount administered of a compound of the present invention will depend on the relative effectiveness of the compound chosen, the severity of the disease to be treated and the weight of the patient. However, the compounds of this invention will be administered one or more times a day, for example 1, 2, 3 or 4 times daily, with a total dose between 0.1 and 1000 mg/Kg/day. It is important to keep in mind that it may be necessary to introduce variations in the dose, depending on the age and condition of the patient, as well as modifications in the route of administration.

The compounds and compositions of the present invention can be used together with other medications in combination therapies. The other drugs may be part of the same composition or a different composition, for administration at the same time or at different times.

Diagnostic and/or prognostic method of the invention

Diseases in which altered EIF4A3 activity can be diagnostic, and specifically pancreatic ductal adenocarcinoma, can be detected by measuring the amount of nucleic acids (DNA and/or RNA and/or mRNA) that code for EIF4A3, or the amount of EIF4A3 protein that is expressed, compared to normal cells. The detection of oligonucleotides can be done by methods well known in the state of the art (such as, but not limited to, probes with labeled nucleotides, DNA-DNA or DNA-RNA hybridization, PCR amplification using labeled nucleotides, RT- PCR).

Procedures to detect the expression of the EIF4A3 protein are also well known in the state of the art, such as poly or monoclonal antibodies, ELISA, radioimmunoassay (RIA), and FACS (fluorescence activated cell sorting).

Therefore, another aspect of the invention refers to the in vitro use of EIF4A3 expression levels in pancreatic tissue as a biomarker to diagnose and/or predict pancreatic ductal adenocarcinoma.

Another aspect of the invention is a method for obtaining data useful in the diagnosis and/or prognosis of pancreatic ductal adenocarcinoma, which comprises:

a) determine the expression of EIF4A3 in a sample previously extracted from a mammal, and preferably, in addition

b) compare the EIF4A3 expression values obtained in a) with the standard values in healthy or diseased mammals.

Another aspect of the invention is an in vitro method to diagnose and/or predict pancreatic ductal adenocarcinoma that includes the method for obtaining useful data described above and that also comprises: assigning the individual who presents the expression levels of EIF4A3 overexpressed with respect to the values means of a normal individual, to the group of patients suffering from pancreatic ductal adenocarcinoma.

Another aspect of the invention is a method for determining the malignancy of pancreatic ductal adenocarcinoma, which comprises determining the expression levels of EIF4A3 in a sample of pancreatic tissue previously isolated from an individual; compare the EIF4A3 expression values obtained from the sample to be evaluated with the standard values of tissues obtained from individuals who present pancreatic ductal adenocarcinoma; and classify the tumor as malignant when the expression levels of EIF4A3 are overexpressed with respect to the average values of individuals who present pancreatic ductal adenocarcinoma.

Another aspect of the invention refers to the in vitro use of EIF4A3 expression levels as an early biomarker of the presence of KRAS mutations and/or overexpression of KRAS, to plan the treatment of pancreatic ductal adenocarcinoma.

Screening method of the invention

Another aspect of the invention relates to a method of selecting therapeutic agents useful in the prevention, improvement, alleviation, and/or treatment of pancreatic ductal adenocarcinoma, comprising:

a) determine the activity of EIF4A3 at an established concentration of the compound to be analyzed or in the absence of said compound and

b) determine the activity of EIF4A3 at a concentration of the compound to be analyzed different from that of a).

Compounds that bind to the EIF4A3 polypeptide would be identified as potential therapeutic agents against pancreatic ductal adenocarcinoma.

As stated, these assays may involve the complete EIF4A3 polypeptide, a biologically active fragment thereof, or a fusion protein involving all or a portion of the EIF4A3 polypeptide. Determining the ability of a compound to modulate the activity of EIF4A3 can be done, for example, by determining the ability of EIF4A3 to bind or interact with a target molecule of said compound, directly or indirectly. They can also be activity assays, directly or indirectly measuring the activity of EIF4A3. It can also be an expression assay, directly or indirectly determining the expression of the EIF4A3 mRNA or the EIF4A3 protein. These assays can also be combined with an in vivo assay measuring the effect of a test compound on the symptoms of EIF4A3-related diseases, and specifically pancreatic ductal adenocarcinoma (for example, but not limited to, on animal models or other model systems known in the art. technique).

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.

For example, antibodies capable of binding to an epitope of EIF4A3, which can be used therapeutically, as set out above, can also be used in immunohistochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunoprecipitation assays, or other immunohistochemical assays known in the art. the state of the art. The EIF4A3 polypeptides can be used to immunize an animal, to obtain polyclonal antibodies. Monoclonal antibodies can also be prepared by techniques that allow the production of antibodies by cell lines in culture, including, but not limited to, hybridomas, human B cell hybridomas. Techniques for producing chimeric, humanized or synthetic antibodies are known.

Therapeutic agents identified by the screening method described herein may be used in an animal or other model to determine the mechanism of action of said agent. Furthermore, the therapeutic agents selected by the method described here would be used in the treatment of diseases that cause alteration of EIF4A3 and, specifically, pancreatic ductal adenocarcinoma.

Invention Kit

Another aspect of the invention refers to a kit or device, hereinafter kit or device of the invention, which comprises the elements necessary to quantify the expression level of EIF4A3.

In a preferred embodiment, the kit or device of the invention comprises:

(a) means for detecting in a biological sample obtained from the subject the expression levels of EIF4A3, and

(b) Means for comparing the expression level of EIF4A3 in (a) with a reference sample.

In a preferred embodiment, the kit or device comprises at least one oligonucleotide capable of hybridizing with EIF4A3.

It is preferred that said oligonucleotide(s) be capable of doing so under stringent conditions. In a preferred embodiment, one or more of said one or more oligonucleotides (preferably DNA) are further defined by the following probes or primers: hsa-miRXX-miRCURY LNA miRNA measured with syber, LNA™ PCR specific primers.

Astringency is a term used in hybridization experiments. Astringency reflects the degree of complementarity between the oligonucleotide and the nucleic acid (which in this case is EIF4A3 mRNA); The greater the astringency, the greater the percentage of homology between the probe and the nucleic acid bound to the filter. The person skilled in the art knows well that temperature and salt concentrations have a direct effect on the results obtained. It is recognized that the results of hybridization are related to the number of degrees below the Tm (melting temperature) of the DNA at which the experiment is performed. Often, stringent conditions are defined as a wash with 0.1X SSC (sodium saline citrate (SSC) buffer at 65 °C. (SSC is usually provided as a 20X stock solution, consisting of 3 M sodium chloride and citrate of 300 mM trisodium (adjusted to pH 7.0 with HCl)).

In particular embodiments, the kit is selected from (a) a kit suitable for PCR, (b) a kit suitable for Northern Blot, (c) a kit suitable for microarray analysis and (d) an immunoassay. Two or more of these embodiments may also be combined, so that the kit may comprise, for example, both (a) and (c).

In the case of (a) a kit suitable for PCR, this PCR is typically a real-time quantitative PCR (RQ-PCR), a sensitive and reproducible gene expression quantification technique.

A Northern blot involves the use of electrophoresis to separate RNA samples by size and subsequent detection with an oligonucleotide(s) (hybridization probe) complementary to (part of) the target RNA sequence of interest.

It is also possible that the oligonucleotide(s) is immobilized in spots on a (preferably solid) surface. In one embodiment thereof, the kit comprises a microarray. An RNA microarray is an array on a solid substrate (usually a glass slide or a silicon thin film cell) that analyzes large quantities of different RNAs, which can be detected by specific probes immobilized on spots on the solid substrate. Each spot contains a specific nucleic acid sequence, typically a DNA sequence, known as probes (or reporters). While the number of spots is not limited, there is a preferred embodiment in which the microarray is adapted to the methods of the invention.

The kit or device of the invention can be used and the use is not particularly limited, although use in the method of the invention in any of its embodiments is preferred.

In another preferred embodiment of this aspect of the invention, the oligonucleotides, primers, probes or antibodies are modified or labeled, for example, but not limited to, by radioactive or immunological labeling. Thus, preferably, the oligonucleotides present modifications in some of their nucleotides, such as, but not limited to, nucleotides that have some of their atoms with a radioactive isotope, usually 32P or tritium, immunologically labeled nucleotides, such as with a molecule of digoxigenin, and/or immobilized in a membrane. Several possibilities are known in the state of the art.

The kit or device of the invention may comprise controls, program instructions and information necessary to carry out any of the methods of the invention.

Another aspect of the invention refers to the use of any of the kits or devices of the present invention, in obtaining useful data for the diagnosis and/or prognosis of pancreatic ductal adenocarcinoma.

Automation of the method of the invention by implementing it in a computer program

Another aspect of the invention relates to a computer program comprising instructions for carrying out the procedure according to any of the methods of the invention.

In particular, the invention encompasses computer programs arranged on or in a carrier. The carrier can be any entity or device capable of supporting the program. When the program is incorporated into a signal that can be transported directly by a cable or other device or medium, the carrier may be constituted by said cable or other device or medium. As a variant, the carrier could be an integrated circuit in which the program is included and which has been adapted to execute, or to be used in the execution of, the corresponding processes.

For example, the programs could be embedded in a storage medium, such as a ROM memory, a CD ROM memory or a semiconductor ROM memory, a USB memory, or a magnetic recording medium, for example, a floppy disk or a disk. hard. Alternatively, the programs could be supported on a transmissible carrier signal; For example, it could be an electrical or optical signal that could be transported via electrical or optical cable, by radio or by any other means.

The invention also extends to computer programs adapted so that any processing means can implement the methods of the invention. Such programs may be in the form of source code, object code, an intermediate source code and object code, for example, as in partially compiled form, or in any other form suitable for use in implementing the processes according to the invention. . Computer programs also include cloud applications based on this procedure.

Other aspects of the invention relate to the readable storage medium and the transmissible signal comprising program instructions necessary for the execution of the inventive method by a computer.

Therefore, another aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of causing a computer to carry out the steps of any of the methods of the invention.

Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1.-Expression levels of EIF4A3, mRNA adjusted by Normalization Factor (calculated from the House keeping genes B-actin, HPRT and GAPDH,), in tumor tissue samples from patients with ADP and control. Data represent the mean ± standard deviation. Asterisks indicate significant differences (****p < 0.0001).

Figure 2.-. ROC curve. p value 0.0004, area under the curve (AUC) 0.6844.

Figure 3.-EIF4A3 expression data, mRNA adjusted by Normalization Factor, in different stages of tumor development, I, II, III and IV (A), tumor size T1-T4 (B), and in relation to the presence (M1) or absence (M0) of metastasis (C). Data represent the mean ± standard deviation. Asterisks indicate significant differences (*p < 0.05; **p < 0.01).

Figure 4.-Probability of disease-free progression (A), probability of overall survival (B) and probability of disease-specific survival (C) in patients with ADP (sample with 177 patients) (Time expressed in days).

Figure 5.-Mutations (A) and expression level (B) of KRAS in patients with ADP in relation to the expression levels of EIF4A3. (**p < 0.01).

Figure 6.-Validation of silencing of EIF4A3 with siEIF4A3 in cell lines derived from ADP Capan-2; Capan-2 mRNA expression levels adjusted by ACTB (A); cell proliferation at 24, 48 and 72 hours after silencing compared to the control (B); and colony (C) and sphere (D) formation capacity. Data represent mean ± standard deviation. Asterisks indicate significant differences (***p < 0.001).l

Figure 7.-Validation of EIF4A3 silencing with a specific siRNA (siEIF4A3) in the ADP MIA PaCa-2 derived cell lines: MIA PaCa-2 mRNA expression levels. adjusted for ACTB (A); cell proliferation at 24, 48 and 72 hours after silencing compared to the control (B); colony (C) and sphere (D) formation capacity; evaluation of cell migration after 24 hours of EIF4A3 silencing compared to its control (E). Data represent mean ± standard deviation. Asterisks indicate significant differences (**p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 8.-Monitoring of the tumor volume obtained from the inoculation of MIA PaCa-2 cells in immunodepressed mice. Inoculation of an EIF4A3-specific siRNA is indicated with an arrow (day 15) (A); photos of the tumors generated (B). Data represent mean ± standard deviation. Asterisks indicate significant differences (*p < 0.05).

EXAMPLES OF THE INVENTION

Determination of EIF4A3 expression levels and their relationship with patients' clinical information

To determine the expression level of EIF4A3, a total of 150 samples from patients diagnosed with ADP from the cohort of formalin-fixed paraffin-embedded samples (FFPE) obtained from the Biobank of Andalusia were used; Of these 150 samples, 75 corresponded to a tumor sample and 75 to a sample of adjacent non-tumor tissue, used as a control. The Ethics Committee of the Reina Sofía University Hospital (Córdoba, Spain) approved the study, which was carried out in accordance with the Declaration of Helsinki.

Total RNA from FFPE samples was extracted using Maxwell MDx 16 Instrument (Promega, Madrid, Spain) with the Maxwell 16 LEV RNA FFPE kit (Promega, Madison, USA) according to the manufacturer's instructions. The amount of RNA recovered and its purity was determined (before and after DNase treatment) using the NanoDrop2000 (Thermo Fisher). The expression levels of EIF4A3 mRNA were determined by qPCR in the RNA isolated from the FFPE samples and its quantification was adjusted by Normalization Factor (calculated from the House keeping genes B-actin, HPRT and GAPDH).

Clinical data relating to tumor stage (I, II, III, IV), size (T1-T4) and the presence or absence of metastasis were obtained from these same patients.

Figure 1 shows the different expression levels of EIF4A3 in each group of samples and a clear overexpression of EIF4A3 is observed in the tumor tissue (****p < 0.0001). The overexpression of EIF4A3 in tumor tissues compared to adjacent non-tumor tissues obtained from the same patients provides proof of the effectiveness of the use of EIF4A3 as a biomarker for pancreatic ductal adenocarcinoma.

The ROC curve of EIF4A3, obtained from these same data, presents an area under curve of 0.688, thus showing its high sensitivity and specificity (Figure 2), which makes EIF4A3 an excellent biomarker.

EIF4A3 expression data are significantly correlated (**p < 0.01) with tumor development stages II and III (Figure 3A), T3 and T4 tumor size (*p < 0.05); (Figure 3B), and is associated (*p <0.05) with the presence (M1) of metastasis (Figure 3C). Thus, EIF4A3 expression data correlate with characteristics of tumor malignancy, being useful to discriminate between different stages of tumor development and constituting in turn an indicator to predict tumor size and the presence of metastasis.

Study of survival of patients with ADP

Data belonging to the PanCancer cohort (accessible data from the Cancer Genome Atlas Study - TCGA) of 177 patients with ADP were used, classified between patients who presented high and low EIF4A3 expression levels using the computer tool “mclust”.

Based on the data collected from TCGA, the following parameters were studied: disease-free progression, overall survival, and disease-specific survival. We define disease-free progression as the time that passes from the beginning of ADP treatment during which the patient lives with the disease, but it does not worsen. We define overall survival as the percentage of patients who are still alive after a defined period of time since treatment began. We defined disease-specific survival as the time from the date of diagnosis or initiation of ADP treatment to the date of death from the disease. This measure does not include patients who die from other causes unrelated to the disease.

It was observed that patients with higher expression levels of EIF4A3 have lower probabilities of disease-free progression (p=0.00027) (Figure 4A), overall survival (p=0.031) (Figure 4B), as well as disease-specific survival. the disease (p=0.02) (Figure 4C). Therefore, the expression levels of EIF4A3 would not only constitute a biomarker for the diagnosis of pancreatic ductal adenocarcinoma, but also for prognosis of the evolution of the disease.

Study of KRAS mutations and expression in relation to EIF4A3 expression levels in patients with ADP

Data from the PanCancer cohort (accessible data from the Cancer Genome Atlas Study - TCGA) of 177 patients with ADP were used.

The presence/absence of KRAS mutations was first analyzed in relation to EIF4A3 expression levels. The KRAS gene is a gene that gives rise to a protein that participates in cell signaling pathways that control the multiplication, maturation and destruction of cells. The unaltered natural form of the gene is called natural KRAS. Mutated forms of the KRAS gene have been found in some types of cancer, such as non-small cell lung cancer, colorectal cancer, and pancreatic cancer. These changes may stimulate cells to multiply and spread throughout the body. Knowing whether a patient's tumor has a mutated or wild-type form of the KRAS gene can help plan cancer treatment. The results indicated show that there is a significant relationship (**p < 0.01) between the overexpressed levels of EIF4A3 and the presence of mutations in the KRAS gene (Figure 5A), the gene most frequently mutated in PDAC and is considered the main driver of tumorigenesis. On the other hand, the expression levels of KRAS were analyzed in relation to the expression levels of EIF4A3 (Figure 5B), which has been shown to be related to a higher degree of dysplasia, appearing even in early stages of the lesion. Thus, EIF4A3 expression levels would act as a biomarker for the presence of KRAS mutations, which would allow cancer treatment to be planned.

Modulation of EIF4A3 expression in ADP-derived cell lines.

To validate the silencing of EIF4A3 with a specific siRNA (siEIF4A3), the ADP-derived cell lines were used: Capan-2 (Figure 6) and MIA PaCa-2 (Figure 7). Capan-2 is a cell line with polygonal morphology that was isolated in 1975 from the pancreas of a 56-year-old white male patient with pancreatic adenocarcinoma. MIA PaCa-2 is an epithelial cell line that was derived from pancreatic tumor tissue obtained from a 65-year-old white man.

Silencing was performed with EIF4A3 siRNA (ID 138378, Thermo Fisher) at 30 nM.

To carry it out, 150,000 cells per well were transfected in 6-well plates, with Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher), according to the commercial company's instructions. After 48 hours, the transfection was validated by qPCR to carry out the different assays.

Gene expression values were normalized to beta-actin (ACTB) mRNA levels, where ACTB showed no significant differences between conditions.

The expression levels of EIF4A3 in cells treated with the siEIF4A3 inhibitor, Capan-2 and MIA PaCa-2, were significantly lower than in the control (Figure 7A). With these same cell lines, Capan-2 and MIA PaCa-2, a study of the cell proliferation of the cells treated with the inhibitor with respect to the control was carried out at 24, 48 and 72 hours after their silencing, observing a gradual decrease over time in the proliferation rate (Figures 6B and 7B).

The ability to form colonies and spheres in response to EIF4A3 silencing was also affected in cells treated with the inhibitor (Figures 6C, 6D, 7C and 7D) compared to the control.

In the case of MIA PaCa-2, an evaluation of cell migration was additionally carried out 24 hours after EIF4A3 silencing compared to its control, decreasing its migration capacity by almost 50% compared to the control (Figure 7E). .

These results support the ability of the siEIF4A3 inhibitor not only to modulate the expression of EIF4A3, but through said inhibition, reduce the ability of proliferation, formation of colonies and spheres and cell migration in ADP tumor cells, which makes it an excellent candidate for use in the treatment of ADP.

Monitoring of the tumor volume obtained from the inoculation of MIA PaCa-2 cells in immunodepressed mice.

MIA PaCa-2 cells were inoculated by xenograft into 8 immunocompromised mice. To do this, 2 million cells were inoculated on both flanks of the 7-week-old mouse. Tumor growth was monitored twice a week throughout the experiment. When the size of the tumor was necessary to carry out the silencing (day 15), the EIF4A3-specific siRNA inoculation was carried out, observing a significant decrease in tumor growth of the mice that were treated with the siRNA compared to their control (Figure 8A).

Representative photos of the tumors generated are shown (Figure 8B). These show how the tumor size decreased in all the tumors treated with the EIF4A3 siRNA compared to the tumors treated with the control (scramble). Specifically, a significant decrease in the tumor was achieved starting 5 days after inoculation of the compound and maintained until the day of sacrifice (14 days later).

References

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Claims (17)

1. - An agent that modulates the activity of EIF4A3 for use in the treatment of pancreatic ductal adenocarcinoma. 2. - The modulating agent according to claim 1, which is selected from a list consisting of: a) an organic molecule, b) an RNA molecule, c) an antisense oligonucleotide, d) an antibody, e) a ribozyme, or any of its combinations. 3. - The modulating agent according to any of claims 1-2, wherein the modulating agent is an EIF4A3 siRNA. 4. - A composition comprising a modulating agent according to any of claims 1-3, for use in the treatment of pancreatic ductal adenocarcinoma. 5. - The composition according to the previous claim, which is a pharmaceutical composition. 6. - In vitro use of EIF4A3 expression levels in pancreatic tissue as a biomarker to diagnose and/or predict pancreatic ductal adenocarcinoma. 7. - A method for obtaining useful data in the diagnosis and/or prognosis of pancreatic ductal adenocarcinoma, which includes a) determine the expression levels of EIF4A3 in an isolated sample previously extracted from an individual: b) compare the EIF4A3 expression values obtained from the sample to be evaluated with the standard values in healthy individuals. 8.- An in vitro method to diagnose and/or predict pancreatic ductal adenocarcinoma that includes the method for obtaining useful data according to the previous claim and that also comprises: assigning the individual who presents the expression levels of EIF4A3 overexpressed with respect to the average values of a normal individual, to the group of patients suffering from pancreatic ductal adenocarcinoma. 9. - A method to determine the malignancy of pancreatic ductal adenocarcinoma, which includes: a) determine the expression levels of EIF4A3 in a pancreatic tissue sample previously isolated from an individual, b) compare the EIF4A3 expression values obtained from the sample to be evaluated with the standard values of tissues obtained from individuals who present pancreatic ductal adenocarcinoma, and c) classify the tumor as malignant when the expression levels of EIF4A3 are overexpressed with respect to the average values of individuals who present pancreatic ductal adenocarcinoma. 10. - A method of selecting therapeutic agents useful in the prevention, improvement, relief, and/or treatment of pancreatic ductal adenocarcinoma, comprising: a) determine the activity of EIF4A3 at an established concentration of the compound to be analyzed or in the absence of said compound and b) determine the activity of EIF4A3 at a concentration of the compound to be analyzed different from that of a). 11. - In vitro use of EIF4A3 expression levels as an early biomarker of the presence of KRAS mutations and/or KRAS overexpression, to plan the treatment of pancreatic ductal adenocarcinoma. 12. - A kit that includes the elements necessary to quantify the level of expression of EIF4A3 in a biological sample previously obtained from the subject 13. - The kit according to claim 12 which also comprises means for comparing the expression level of EIF4A3 with a reference sample. 14. - Use of the kit according to claims 12 to 13, to carry out the methods of claims 7 to 10. 15. - A computer program adapted so that any processing means can implement the method according to any of claims 7 to 10. 16. - A computer-readable storage medium comprising program instructions capable of causing a computer to carry out the steps of the method according to any of claims 7 to 10. 17. - A transmissible signal comprising program instructions capable of causing a computer to carry out the steps of the method according to any of claims 7 to 10.