ES2674176B1 - Use of the Prkaca gene to predict the response of a subject to treatment with a purine analogue - Google Patents

Description

Use of the PRKACA gene to predict the response of a subject to treatment with a purine analogue

DESCRIPTION

The present invention falls within the scope of biotechnology and relates to a method for predicting response to treatment with a purine analogue in a subject suffering from low-grade lymphoproliferative syndrome, secondary acute leukemias, high-grade myelodysclerosis syndromes or will receive the transplant of an organ, which includes the determination of expression levels of the PRKACA gene.

STATE OF THE ART

The nucleoside anblogos are compounds with a structure similar to that of the monomers that constitute the nucleic acids, widely used as antiviral or anticancer agents. These compounds are activated within the cell by phosphorylation of their triphosphate forms and integrated into the DNA. Its formulas include modifications with respect to the natural nucleotides that poison the DNA replication machinery, which leads to premature termination of nascent chains and cell death.

A nucleoside is a molbular formed by the union of a nitrogenous base with a pentose. The nitrogenous bases can be of two types: the purine type has a structure formed by two fused rings and its derivatives are adenine and guanine and the pyrimidine type, whose structure is constituted by a single ring and its derivatives are thymine, uracil and the cytosine.

Fludarabine (9-beta-D-arabinofuranosyl-2-fluoroadenine 5-phosphate 6 F-Ara-AMP) is the most effective purine ani-root in the treatment of indolent lymphoproliferative disorders, including chronic lymphocytic leukemia ( LLC) and certain low-grade lymphomas. Likewise, it has been shown effective in the treatment of myelodysplastic syndromes with rapid evolution to acute leukemias due to the synergic effect with the cytosine arabinbsido and in the conditioning of allogeneic transplantation due to its immunosuppressive effect.

Fludarabine is administered in its monophosphate form, whose electronegative charge makes it necessary to be dephosphorylated before entering the cells by facilitated transport. Once inside the cell, it is activated by re-phosphorylation to its triphosphate form (F-Ara-ATP) by deoxycytidine kinase (dCK). Several enzymes involved in the synthesis of DNA are targets of the action of fludarabine, among which are DNA polymerase and DNA primase, since the F-Ara-ATP competes with the natural nucleotide dATP. Furthermore, fludarabine is incorporated into RNA and DNA, which leads to the termination of the synthesis of the chains (5 '-> 3'), and its location at the 3 'end prevents ligase I from binding it to the chain. of adjacent DNA. In addition, in non-dividing cells, fludarabine is incorporated into DNA using the mechanisms of DNA repair, but inhibits the process of repair by nucleotide excision (NER), causing irreparable damage.

The cytotoxicity of this agent is radically dependent on the intracellular concentration of the active triphosphate form of the drug that can be reached during treatment. A deficiency in dCK has been related to the generation of resistance to nelarabine, another purine anvil (Yamauchi et al., BMC Cancer, 2014 vol 14 p.547).

The patent application US2013344168A1 describes a method to determine the clinical results of a treatment against cancer comprising the determination of a glicic marker of expression of the CREB pathway and, additionally, of the APC pathway of the cell cycle.

Patent application W02009117153A1 discloses a method for identifying subjects with acute myeloid leukemia sensitive to treatment comprising measuring the level of phosphorylation of a member of the signaling pathway of the AMP-dependent protein kinase (AMPK) in the sample. However, it does not refer to the validity of this method as an indicator for the treatment and prevention of other diseases.

The patent application US2015141470A1 describes a method to identify subjects with resistance to therapies against cancer or at risk of suffering it, by means of the evaluation of certain genetic markers that activate the production of CAMP, including PRKACA. The treatments with guanine nucleotide exchange factor inhibitors (GEFs) and histone deacetylase inhibitors (HDACs) are mentioned, but not treatments with purine analogues.

However, none of these methods allows us to find out whether treatment with purine analogues will be effective when administered to an individual. Therefore, there exists in the state of the art the need to provide a method that allows to find out if a subject will respond positively to treatment with purine analogues, avoiding the loss of time with ineffective treatments that delay the cure of the subject .

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have observed that the determination of the expression levels of the cAMP-dependent protein kinase gene (PRKACA gene) in a subject allows the prediction of the response of said subject to treatment with a purine analogue. This is very beneficial in those situations in which the administration of purine anblogs is advisable, as is the case, for example, of the treatment of lymphoproliferative diseases or the prevention of a graft-hubs rejection.

During the course of experiments with cell lines, the inventors observed that stable transfection of fibroblasts with plasmids containing the prokaryotic NeoR gene encoding the aminoglycoside-3'-phosphatase-lla protein (APH (3 ') - lla), provided a Increase in resistance to the fludarabine purine anchor (Example 1). Crystallographic studies revealed a structural relationship of said protein with some eukaryotic kinases such as ERK and cAMPK (encoded by the PRKACA gene), so the inventors carried out additional experiments which confirmed that the transfection of fibroblasts with the PRKACA gene conferred resistance to the purine anblogs (Figure 11). Based on this discovery, a series of inventive aspects have been developed that will be described in detail below.

Mbtodo of the invention

As previously mentioned, the determination of the expression levels of the cAMP-dependent protein kinase gene (PRKACA gene) in a subject makes it possible to determine whether said subject will respond positively to treatment with purine anblogs, that is, if the treatment will be effective, or find out if said subject is susceptible to being treated with a purine analogue.

Therefore, in a first aspect, the present invention relates to an in vitro method to predict the response of a subject to treatment with a compound of formula (I), or to select a subject capable of being treated with a compound of fbrmula (I),

where:

R ₁ is selected from NH 2 or OCH 3,

R2 is selected from a halogen or NH2 ⁱ

R3 is selected from H or P03H2,

R4 is selected from OH or a halogen,

or any of its isomers or pharmaceutically acceptable salts thereof, wherein the method comprises the following steps:

(a) quantifying the expression levels of the cAMP-dependent protein kinase gene (PRKACA gene) in a biological sample isolated from said subject, and

(b) comparing the expression levels of said gene with a reference level, in which a level of expression of said gene less than a reference level is indicative that the treatment with a compound of formula (I) is going to be effective, or that the subject is susceptible to being treated with a compound of formula (I).

In the present invention, "predicting the response of a subject to treatment with a compound of formula (I)" to be known from a parameter (in the present context, the expression levels of the PRKACA gene) if the treatment with said compound of formula (I) will be effective, that is, if the response of the subject to the treatment is positive. It is considered that a treatment is effective, or that the response of the subject is positive, when as a consequence of the administration of the compound of formula (I) the subject either ceases to suffer the disease or the symptoms associated with said disease disappear or decrease, or avoid a disease, such as graft-hubs rejection. Thus, in the present invention "treatment" or "treatment" is understood to mean both therapeutic and prophylactic administration (preventive measure) of the compound of formula (I), in which the object is to prevent or slow down (reduce) a physiological change unwanted or upset As a consequence of the administration of the compound of formula (I), the subject will obtain beneficial clinical results.

Beneficial or desired clinical outcomes include, but are not limited to, refusal to refuse a transplant of tissue or tissue, avoidance of immunological attack of the organ or tissue transplanted to the recipient, alleviation of symptoms, reduction of the extent of the disease, stabilized disease status ( concretely not worsened), delay or brake of the progression of the disease, improvement or palliation of the pathological state and remission (both partial and total), both detectable and undetectable. Subjects in need of treatment include both those subjects already suffering from the condition or disorder, those who are prone to the condition or disorder, or those in whom the condition or disorder is to be prevented.

Thus, based on the expression levels of the PRKACA gene, the method of the invention allows selecting a subject capable of being treated with a compound of formula (I). The subject selected by the method of the invention will be one that presumably responds positively to treatment with a compound of formula (I).

In the present invention, "subject" or "individual" is understood as any human, male or female, of any race or age. In the context of the present invention, the subject suffers from a disease, in which case the treatment with the compound of formula (I) would be profilbctico, or the subject is going to receive a transplant, in which case the treatment would be preventive, that is to say, the disease will be prevented graft against hubsped. In a particular embodiment, the subject suffers from a disease whose treatment and / or prevention comprises the administration of purine anblogues, more particularly, purine analogues comprising the formula (I).

Examples of diseases treated with purine anblogos include, but are not limited to, myeloid hematolgic neoplasias (chronic myeloid leukemia, positive BCR-ABL, chronic neutrophilic leukemia, polycythemia vera, primary myelofibrosis, essential thrombocytopenia, chronic eosinophilic leukemia, systolic mastocytosis, unclassifiable myeloproliferative neoplasms. , refractory cytopenia with unilineal dysplasia, sideroblbstic refractory anemia, refractory cytopenia with multiline dysplasia, refractory anemia with excess blasts, myelodisplastic syndrome with isolated (5q), unclassifiable myelodysloblasic syndrome, childhood myelodyscleptic syndrome, chronic myelomonocytic leukemia, atyclic chronic myeloid leukemia, BCR-ABL negative, juvenile myelomonocytic leukemia, myelodiplasic neoplasm / unclassifiable myeloproliferative, acute myeloma leukemia (AML) or acute myeloid leukemia (AML) with recurrent genetic alterations, AML with changes related to myelodyspla sia, AML related to previous therapies, AML without characteristics of the previous categories, myeloid sarcoma, myeloid proliferations related to Down syndrome, neoplasm of plasma dendritic plasmocytic cells and acute leukemias of ambiguous lineage) lymphoid hematolgy neoplasms (lymphoid neoplasms of cells) precursors, neoplasms of mature B cells, neoplasms of T cells and mature NK cells and Hodgkin's lymphoma) and in the prevention of graft versus hub disease.

In a particular embodiment, the subject suffers from a disease selected from the group consisting of low-grade lymphoproliferative syndrome, secondary acute leukaemias and high-grade myelodysplastic syndromes, or the subject is going to receive a transplant from a organ or tissue. In a more particular embodiment, the low-grade lymphoproliferative syndrome is chronic lymphocytic leukemia.

The compounds of formula (I) are included in the compounds considered purine anblogs, insofar as all the purine anblogs comprise a chemical structure similar to that of a purine. Thus, it is understood by "anblogo de purina" to a chemical compound with a structure similar to purine that can be inserted into the DNA substituting bsta. "Purine" means an aromatic heterocyclic organic compound Purine is a nitrogenous base, precursor of the bases adenine and guanine, which are part of the DNA structure Examples of purine analogs include, but are not limited to, fludarabine, cladribine , clofarabine, mercaptopurine, nelarabine and thioguanine.

The purine analogs of the invention are selected from the compounds of the formula (I)

where:

R ₁ is selected from NH 2 or OCH 3,

R2 is selected from a halogen or NH2,

R3 is selected from H or P03H2,

R4 is selected from OH or a halogen,

or any of its isomers or pharmaceutically acceptable salts thereof.

In the present invention, "halogen" means an atom of bromine (Br), chlorine (Cl), iodine (I) or fluorine (F).

In a particular embodiment of the method of the invention, R ^t of the compound of formula (I) is an NH 2 group.

In another particular embodiment, R 2 of the compound of formula (I) is a group NH 2 1 a F or a Cl.

In another particular embodiment, R, of the compound of formula (I) is an OH group or an F.

In another still more particular embodiment, the compound of formula (I) is fludarabin phosphate of formula (la) or clofarabine of formula (lb):

In a first step [step (a)], the method of the invention comprises quantifying the expression levels of the PRKACA gene in a biological sample of said subject.

The PRKACA gene, also known as the PKACA gene or PPNAD4 gene of the inglbs "protein kinase cAMP-activated catalytic subunit alpha" is a human gene located on chromosome 19. This gene encodes one of the catalytic subunits of protein kinase A cAMPK-dependent protein (cAMPK protein) that exists as a holoenzyme The cAMPK protein-dependent phosphorylation of cAMPK protein plays an important role in many cellular processes, including cellular differentiation, proliferation and apoptosis. Instead of multiple transcripts encoding different isoforms, in a particular embodiment, the PRKACA gene comprises a nucleotide sequence with a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% 0 100% with the sequence SEQ ID NO: 1 (access number to GeneBank NG_029699.1).

In the present invention, "sequence identity" is understood to be the degree of similarity between two nucleotide (or amino acid) sequences obtained by the alignment of the two sequences. Depending on the number of common residues between the aligned sequences, a degree of identity expressed as a percentage will be obtained. The degree of identity between two nucleotide (or amino acid) sequences can be determined by conventional methods, for example, by standard algorithms of sequence alignment known in the state of the art, such as BLAST [Altschul SF et al. Basic local alignment search tool. J Mol Biol.

1990 Oct 5; 215 (3): 403-10]. The BLAST programs, for example, BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, are public domain on the National Center for Biotechnology Information (NCBI) website.

The person skilled in the art understands that mutations in the nucleotide sequence of the genes that give rise to conservative amino acid substitutions in non-critical positions for the functionality of the protein are evolutionarily neutral mutations that do not affect its overall structure or functionality , resulting in protein variants. Said variants fall within the scope of the present invention, that is, those variants of the cAMPK protein that present insertions, deletions or modifications of one or more amino acids with respect to the sequence SEQ ID NO: 2. Thus, in a particular embodiment, the protein encoded by the PRKACA gene, ie the cAMPK protein, comprises a sequence of amino acids with an identity sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99% or 100% with the amino acid sequence SEQ ID NO: 2 (GenBank NP_002721.1).

SEQ ID NO: 2

MGNAAAAKKG SEQESVKEFLAKAKEDFLKKWESPAQNTAH LDQFERIKTL GTGSFGRVML

VKHKETGNHYAMKILDKQKVVKLKQIEHTLNEKRILQAVN FPFLVKLEFS FKDNSNLYMV

MEYVPGGEMF SHLRRIGRFS EPHARFYAAQ IVLTFEYLHS LDLIYRDLKP ENLLIDQQGY

IQVTDFGFAK RVKGRTWTLC GTPEYLAPEI ILSKGYNKAVDWWALGVLIY EMAAGYPPFF

ADQPIQIYEK IVSGKVRFPS HFSSDLKDLL RNLLQVDLTK RFGNLKNGVN DIKNHKWFAT

TDWIAIYQRK VEAPFIPKFKGPGDTSNFDD YEEEEIRVSI NEKCGKEFSE F

In the present description, the terms "expression" and "genetic expression" include the transcription and / or translation of the nucleic acid, therefore, the quantification of the expression level of the PRKACA gene can be made from the RNA resulting from the transcription of the nucleic acid. said gene (messenger RNA or mRNA) through the synthesis of a complementary DNA (cDNA) thereof, therefore, in a particular embodiment of In the invention, the quantification of expression levels of the PRKACA gene comprises the quantification of the messenger RNA of the PRKACA gene, or a fragment of said mRNA, of the DNA complementary to the PRKACA gene, or a fragment of said cDNA, or their mixtures.

In the present invention, "mRNA fragment" or "cDNA fragment" is understood as the nucleotide sequence of the PRKACA gene comprising one or more nuclebots absent from the 3 'and / or 5' ends. In a particular embodiment, the PRKACA gene fragment is a fragment of the sequence SEQ ID NO: 1.

If the quantification of PRKACA gene expression is to be performed from the cDNA or mRNA, the nucleic acid extraction of the biological sample isolated from the subject is first extracted. For this purpose, the biological sample can be treated to physically or mechanically disgregate the structure of the tissue or cell by releasing the intracellular components in an aqueous or organic solution to isolate and prepare the nucleic acids. The nucleic acids are extracted by methods known to the person skilled in the art (Sambrook et al., "Molecular cloning: a Laboratory Manual", 2012; Vol. 1-3) and commercially available. Once the nucleic acid was extracted, the quantification of PRKACA gene expression was carried out.

Virtually any conventional method can be used within the scope of the invention to detect and quantify the levels of mRNA encoded by the PRKACA gene or its corresponding cDNA. By way of illustration, not limitation, the levels of mRNA encoded by said gene can be quantified by the use of conventional methods, for example, methods comprising the amplification of the mRNA and the quantification of the product of the amplification of said mRNA, such as electrophoresis. and tincibn, or alternatively, by Southern blot and use of appropriate probes, northern blot and use of specific probes of the mRNA of the gene of interest (PRKACA gene) or of its corresponding cDNA, mapping with nuclease S1, RT-PCR, hybridization, microarrays, etc. Preferably, the method of choice for quantifying the expression levels of the PRKACA gene is an RT-qPCR (from the English quantitative reverse transcriptase-polymerase chain reaction), in which after obtaining the cDNA from the mRNA by random hexagonal oligonucleotides, the PRKACA gene is amplified with the use of the primers comprising the sequences SEQ ID NO: 3 and SEQ ID NO: 4.

SEQ ID NO: 3 - 5 ' - GAGCAGGAGAGCGTGAAAGA- 3' and

SEQ ID NO: 4 - 5 ' - TCATGGCATAGTGGTTCCCG-3' .

Conventional methods of quantifying expression levels can be found, for example, in Sambrook et al. cited ad supra. Thus, in a particular embodiment, the quantification of the expression levels of the PRKACA gene is carried out by means of a quantitative polymerase chain reaction or an array of DNA or RNA.

Alternatively, the expression levels of the PRKACA gene can also be quantified through the levels of cAMPK protein or a fragment thereof. Thus, in another particular embodiment, the quantification of the expression levels of the PRKACA gene comprises the quantification of the levels of protein encoded by said gene (cAMPK protein) or of a fragment thereof.

In the present invention, "cAMPK protein fragment" is understood to mean the amino acid sequence of the cAMPK protein comprising one or more amino acids absent from its terminal amino or carboxyl terminus. In particular, the fragment of the cAMPK protein is a fragment of the sequence SEQ ID NO: 2.

If the quantification of PRKACA gene expression levels is to be performed from the cAMPK protein (or a fragment thereof), then the biological sample isolated from the subject has to be treated to extract the proteins. Methods for extracting and isolating the proteins are known to those skilled in the art (Sambrook et al., Supra) and are commercially available.

The level of expression of the cAMPK protein can be quantified by any conventional method that allows to detect and quantify said protein in a sample of a subject. By way of illustration, not limitation, the levels of said protein can be quantified, for example, by the use of antibodies capable of binding to the cAMPK protein and the subsequent quantification of the complexes formed.

"Antibody" means a glycoprotein of the gamma globulin type that is part of the humoral immune system that binds specifically to an antigen.

The term antibody as used herein includes polyclonal sera, supernatants of hybridomas or monoclonal antibodies, antibody fragments, Fv, Fab, Fab 'and F (ab') 2, scFv, diabodies, triabodies, tetrabodies and humanized antibodies. Examples of specific antibodies of the commercially available protein encoded by the PRKACA gene include, but are not limited to, PKAa cat Antibody (A-2): sc-28315 from Santa Cruz Biotechnology and Anti-PKAc alpha / beta / gamma antibody-C-terminal (ab211265) by ABcam. The antibodies can be labeled to allow their detection after their union with the genic product (antigen). The terms "mark" or "mark" refer to a composition capable of producing a detectable signal indicative of the presence of the labeled molecule. Suitable labels include radioisotopes, nucleotide chromphors, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a brand is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, bptical or chemical means.

There is a wide variety of known assays that can be used in the present invention, which utilize unlabeled antibodies (primary antibody) and labeled antibodies (secondary antibody); these techniques include Western-blot or Western blot, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (sandwich ELISA with double antibody), immunocytochemical and immunohistochemical techniques , techniques based on the use of biochips or protein microarrays that include specific antibodies or tests based on colloidal precipitation in formats such as dipsticks. Other ways to detect and quantify said cAMPK protein include affinity chromatography techniques, ligand binding assays, etc. However, in a particular embodiment, the quantification of protein levels is carried out by western blot, ELISA or an array of proteins. When an immunological method is used, any antibody or reagent known to bind to the cAMPK protein with high affinity can be used to detect the amount thereof.

To carry out the first stage of the method of the invention, it is necessary to have a biological sample isolated from the subject under study. The term "sample" or "biological sample" as used herein, refers to any sample that can be obtained from the subject, such as a tissue, a cell or a fluid (serum, saliva, semen, sputum, cerebrospinal fluid (CSF ), Ibgrimas, mucus, sweat, milk and the like), and which can hold information about the characteristic genetic endowment of a person In a particular embodiment, the biological sample is selected from the group consisting of peripheral blood, serum, plasma and tissue In the context of the present invention, the term "isolated" implies that the biological sample has been separated or extracted from the rest of the components that naturally accompany it.

In a second step [step (b)], the method of the invention comprises comparing the expression levels of the PRKACA gene obtained in step (a) with a reference level.

In this step (b), the determination of PRKACA gene expression levels needs to be correlated with a reference value corresponding to the level of PRKACA gene expression measured in biological samples of a reference population. In general, said biological samples will be obtained from subjects who are clinically well documented, wherein said subjects are healthy subjects or subjects who show a corroborated sensitivity to fludarabine. In such samples, the normal (reference) concentrations of the biomarker (PRKACA gene) can be determined, for example, by providing the mean concentration over the reference population. Several considerations are taken into account when determining the reference concentration of the marker. Among such considerations are age, weight, sex, general physical state of the subject and the like. For example, equal amounts of a group of at least 2, at least 10, at least 100 to preferably more than 1,000 subjects are taken as reference group, preferably classified according to the above considerations, for example of various age categories.

After comparing PRKACA gene expression levels with the reference value, if the level of PRKACA expression in the subject is lower than the reference level, it can be concluded that treatment with a compound of formula (I) is to be effective or that the subject is susceptible to being treated with said compound of formula (I).

In the present invention, "lower expression level" or "lower expression levels" means a decrease in the level of expression of the gene in question with respect to the reference value. Said decrease in the level of expression can be at least 1.1 times, 1.5 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or even higher compared to the reference value.

Uses of the invention

The invention contemplates as a second inventive aspect the in vitro use of the expression levels of the PRKACA gene to predict the response of a subject to treatment with a compound of formula (I) described above in the present description, or to select a subject susceptible to be treated with said compound of formula (I).

In an analogous manner to the method of the invention, a PRKACA gene expression level lower than a reference level is indicative that the treatment with said compound of formula (I) is going to be effective, or that the subject is likely to be treated with said compound of formula (I).

The terms and expressions used in the present inventive aspect have been explained or defined in previous paragraphs.

In a particular embodiment, the subject suffers from a disease selected from the group consisting of low-grade lymphoproliferative syndrome, secondary acute leukaemias and high-grade myelodysplastic syndromes, or the subject is going to receive a transplant of an organ or tissue. In another more particular embodiment, the subject suffers from chronic lymphocytic leukemia.

Since the expression levels of the PRKACA gene are indicative of the efficacy of the treatment with a compound of formula (I), the use of the formula (I) compounds for treating patients with certain types of compounds is also included among the uses of the present invention. PRKACA gene expression levels lower than a reference level. Therefore, in another aspect the invention relates to the use of a compound of formula (I), in the preparation of a pharmaceutical composition for the treatment of a subject suffering from a disease selected from the group consisting of low-grade lymphoproliferative syndrome, acute secondary leukemias and high-grade myelodysplastic syndromes, or going to receive a transplant of a organ or tissue, wherein the expression levels of the PRKACA gene in said subject are less than a reference level. In a more particular embodiment, the low-grade lymphoproliferative syndrome suffered by the subject is chronic lymphocytic leukemia.

In the preparation of the pharmaceutical composition, the compound of formula (I) may be accompanied by other components such as excipients, adjuvants and / or pharmaceutically acceptable vehicles, as well as other compounds used in the treatment of the above mentioned diseases. Said excipients, adjuvants and pharmaceutically acceptable vehicles, as well as the compounds used in the treatment of the mentioned diseases, are widely known in the state of the art and any of them can be used in the context of the invention.

In the present invention, the term "excipient" refers to a substance that aids the absorption of any of the components of the composition of the present invention, stabilizes said components or assists in the preparation of the composition in the sense of providing consistency. or to contribute flavors that make it more pleasant. Thus, the excipients may have the function of keeping the components together, such as starches, sugars or celluloses, a sweetening function, a coloring function, a protection function against drugs such as air and / or humidity isolation, the function of filling a tablet, capsule or other form of presentation such as, for example, dibasic calcium phosphate, a function of disintegration to facilitate the dissolution of the components and their absorption in the intestine, without excluding any other type of excipients. mentioned in this paragraph. Therefore, the term "excipient" is defined as the material included in the galenic forms, is added to the active ingredients or their associations to facilitate their preparation and stability, modify their organoleptic properties or determine the physicochemical properties of the pharmaceutical composition and its bioavailability Preferred excipients for use in the present invention include sugars, starches, celluloses, gums and proteins.

As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, in terms of compositions, carriers, diluents and reagents, are used interchangeably and indicate that the materials they are susceptible to be administered to a subject without the production of undesirable physiological effects. Examples of pharmaceutically acceptable vehicles are known in the state of the art and include phosphate-buffered saline solutions, water, emulsions, such as oil / water emulsions, different types of wetting agents, sterile solutions, etc. The compositions comprising said vehicles can be formulated by conventional methods known in the state of the art.

The subject to be treated with the pharmaceutical composition is characterized in that it comprises PRKACA gene expression levels lower than a reference level. As explained in previous inventive aspects, the expression levels of the PRKACA gene can be quantified from the RNA, cDNA or protein encoded by said gene. Thus, in a particular embodiment, the expression levels of the PRKACA gene comprise the expression levels of the messenger RNA of said gene, or a fragment of said mRNA, of the DNA complementary to said gene, or a fragment of said cDNA, or of its mixtures, or protein levels encoded by the PRKACA gene.

The implementation of both the method and the uses described above requires the use of a kit comprising the minimum components necessary to carry out the invention. Therefore, in another aspect the present invention relates to the in vitro use of a kit, hereinafter "kit of the invention", which comprises

(a) a pair of primers comprising a nucleotide sequence that hybridizes specifically to the nucleotide sequence of the PRKACA gene,

(b) a probe that hybridizes specifically with the nucleotide sequence of the PRKACA gene, and / or

(c) an antibody that specifically recognizes the protein encoded by the PRKACA gene,

to predict the response of a subject to treatment with a compound of formula (I), or to select a subject capable of being treated with a compound of formula (I).

In the present invention "kit" is understood as that product that contains the different components or active principles necessary to implement the invention, that is, those components necessary to quantify the expression levels of the PRKACA gene and, if applicable, administer to the subject the compound of formula (I) described previously: The components necessary to quantify the expression levels of the PRKACA gene include, but are not limited to, primers, probes and / or antibodies.

In the present invention, "primer", "primer" or " primer * " is understood to mean the nucleic acid chain that allows the DNA polymerase to begin the synthesis of the new DNA strand. In most DNA replications, the main primer for DNA synthesis is a short chain of RNA. This RNA is produced by an RNA polymerase (primase) and then a DNA polymerase removes it and replaces it by DNA. As understood by the person skilled in the art, the primers comprised within the kit of the invention will comprise a sequence of nucleotides that hybridizes specifically with the nucleotide sequence of the PRKACA gene. In a particular embodiment, the PRKACA gene comprises a nucleotide sequence with a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% with the sequence SEQ ID NO: 1.

In another particular embodiment, the primers of the kit of the invention comprise a sequence of nucleotides that hybridizes specifically with the sequence SEQ ID NO: 1, that in another particular embodiment, at least one of the primers comprises the sequence SEQ ID NO. : 3 b SEQ ID NO: 4. In another still more particular embodiment, the pair of primers comprises a first primer comprising the sequence SEQ ID NO: 3 and a second primer comprising the sequence SEQ ID NO: 4.

In the present invention it is understood that a given nucleotide sequence hybridizes specifically with another nucleotide sequence when both sequences share a degree of complementarity under conditions of moderate or high stringency. The exact conditions that determine the astringency of hybridization depend not only on the biological strength, temperature and concentration of destabilizing agents such as formamide, but also on factors such as the length of the nucleic acid sequence, the base composition, the percentage of inequality between the hybridization sequences and the frequency of the presence of subseries of that sequence within other non-identical sequences. By varying the hybridization conditions from a level of stringency in which hybridization does not occur at a level at which hybridization is first observed, conditions can be determined that will allow a given sequence to hybridize with another sequence. Thus the conditions of high or moderate astringency can be determined empirically, which is routine practice for the person skilled in the art. In general, the higher the hybridization temperature and the lower the concentration of salts in the hybridization buffer, the higher the astringency and only hybridization between sequences of very similar nucleotides, that is, with a high degree of complementarity. . In general, it is considered that any primers or probe whose nucleotide sequence has a sequence identity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99% or 100% with the sequence SEQ ID NO: 1, hybridized in a specific manner with SEQ ID NO: 1.

In the present invention, "probe" is understood as a fragment of small nucleic acid used as a tool to detect a complementary sequence of nucleic acid.

As understood by the person skilled in the art, both the probes and the primers can be marked at their ends to facilitate their location. Examples of markings have been explained above in the present description.

Alternatively, and in additional embodiments of the invention, the genetic expression can be quantified by protein analysis encoded by the PRKACA gene by the use of one or more specific antibodies for one or more eplotypes of individual genetic products (proteins), or fragments. proteolytics thereof, in the biological sample from a subject. Thus, in a particular embodiment of the kit of the invention, the protein encoded by the PRKACA gene is the protein comprising a sequence of amino acids with a sequence identity of at least 80%, 85%, 90%, 95% , 96%, 97%, 98%, 99% or 100% with the amino acid sequence SEQ ID NO: 2.

The term "antibody" has been previously defined Examples of specific antibodies of the protelna encoded by the commercially available PRKACA gene include, but are not limited to, PKAa cat Antibody (A-2): sc-28315 from Santa Cruz Biotechnology and Anti-PKAc alpha / beta / gamma antibody-C * terminal (ab211265) from ABcam. The antibodies can be labeled to allow their detection after their binding to the genic product (antigen).

Some detection methodologies suitable for use in the practice of the invention include, but are not limited to, immunohistochemistry of samples containing cells or tissues, enzyme-linked immunosorbent assays (ELISAs) that include antibody sandwich assays of tissue samples. containing cells or blood samples, mass spectroscopy and immuno-PCR. Preferably, the methodology employed includes western blot, ELISA or array of proteins.

In another particular embodiment, the kit of the invention further comprises a composition comprising a compound of formula (I) described above inventive aspects.

The terms and expressions used in the present inventive aspect have already been defined in previous inventive aspects.

Components useful for the implementation of the invention and which may be included in the kit include, but are not limited to, tampon solution, lysis solution, sterile material (syringes, swabs, swabs, tweezers, etc.), distilled water , alcohols (ethanol), etc. Additionally, the kit may contain instructions or indications that guide the person skilled in the art in the administration of the peptide 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 be apparent in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. NeoR provides resistance to Fludarabine. Feasibility curves of fibroblasts stably transfected with an expression plasmid of the Neomycin resistance gene (NeoR) in increasing concentrations of G418 or Fludarabine, compared with those of controls without transfecting.

Figure 2. Resistance of NeoR transfectant fibroblasts to different nucleoside analogues. Feasibility curves of fibroblasts transfected or not with NeoR in increasing concentrations of the indicated nuclebidos anbics.

Figure 3. Combination with Cytarabine does not increase the effect of Fludarabine on cells transfected with APH (3 ') - lla. A. Viability analysis by XTT of fibroblasts transfected with the NeoR gene or untransfected, treated or not with 0.1 pM Citarabine (AraC) and increasing doses of Fludarabine. B. Feasibility of the same cells as in A, treated with a fixed concentration of Fludarabine (0.1 pM) and increasing doses of Cytarabine.

Figure 4. Mutational analysis of resistance to Fludarabine by APH (3 ') - lla (NeoR). A. Viability curves of fibroblasts transfected with wild-type NeoR or various mutants in response to increasing doses of Geneticin (G418) or Fludarabine. B. Effect of the addition of the prokaryotic antibiotic Kanamycin on the viability curve of fibroblasts transfected with wild NeoR (left) or the mutant K50R (right), growing in increasing concentrations of Fludarabine.

Figure 5. Protection against Fludarabine mediated by NeoR and PRKACA in different cell lines. Representative viability curves of cell lines of human fibroblasts, 293FT and MCF7, transfected with the indicated NeoR gene mutants or with the ANDc of PRKACA, in response to increasing doses of Fludarabine. The expression of the mRNA of the transfected genes is shown in the panel on the right.

Figure 6. Nuclei encoding of purines at the unibn site for APH (3 ') nuclei.

The ATP to APH (3 ') - lla (1nd4.pdb) unit (dark gray) was modeled using the APH (2') - lla (2hav.pdb) structure (light gray) (A) as a model. The residues involved in the ATP unit are highlighted. The nuclei unit of purines was modeled using the coordinates of the ATP relative to APH (3 ') - lla. Fludarabine (B) and Clofarabine (C) present a hydroxyl group and fluoride at the C2 'position of the ribose ring, respectively. Cladribine (D) lacks any radical in that position. The distance to the carboxyl group of Asp208 from APH (3> lla (gray dashes) of the hydroxyl group of Fludarabine and the fluoride ion of Clofarabine is 2.7.

Figure 7. Effect of eukaryotic kinases on resistance to Fludarabine.

A. Viability curves of a cell line of transfectant fibroblasts for ERK2 growing in increasing concentrations of Fludarabine, compared with non-transfected control (UT) cells. A western blot showing the overexpression of ERK2 is presented on the right. B. Viability of 293FT and MCF7 cells stably transfected with PRKACA, NeoR or untransfected, in increasing concentrations of Fludarabine. The expression of the NeoR mRNA and PRKACA in each case is shown below.

Figure 8. Cells transfected with NeoR do not incorporate Fludarabine.

Feasibility curves of fibdablasts sensitive to Fludarabine growing in medium conditioned by the indicated cell lines grown for 24 hours in 1 pM Fludarabine. The curve of the fibroblasts incubated in fresh Fludarabine is shown, as a reference.

Figure 9. Fludarabine induces the monoubiquitination of FANCD2 and apoptosis, and NeoR prevents it. A. Analysis of the mRNA expression of genes regulated by Fludarabine (10 pM, 16 h) in transfectant fibroblasts for different mutations of APH (3 ') - lla, its wild type (wt) or non transfected (UT). B. Analysis of the cell cycle of cells transfected with NeoR or untransfected in the presence or absence of 10 pM Fludabine. C. Western blot showing the change in mobility of FANCD2 and the digestion of PARP induced by 10 pM Fludarabine in cells transfected with the indicated NeoR mutants, their wild type (wt) or untransfected (UT). D. Comparison between mobility change of the band of FANCD2 induced by 10 pM Fludarabine (left) or by 100 pM Citarabine (right) in fibroblasts transfected with NeoR or non-transfected (UT). The amounts of the GAPDH and p53 proteins are shown as control.

Figure 10. APH (3 ') - lla possesses ATPase activity and F-Ara-AMP inhibits it.

A. Hydrolysis of ATP in vitro catalyzed by APH (3 ') - lla (1.5 pM) in the absence (light gray) or presence (dark gray) of 200 pM Kanamycin. The activity is shown in the presence of 200 pM Kanamycin but without enzyme as control (black). B. Effect of Fluradabine monophosphate (F-Ara-AMP) on the ATPase activity of APH (3 ') - lla. The figure shows the levels of relative ATPase activity in the presence of different nucleotide derivatives: Fludarabine monophosphate (FAP), adenosine monophosphate (AMP) or adenosine 5 '- ((3, Y-imido) triphosphate (AMP-PNP). of nucledtids (control sample), the protein presents a degree of ATP hydrolysis of 2100 nmol ATP / min / mg protein, which was considered as 100% activity.The data represent the average of ten experiments (SD-bars). error).

Figure 11. cAMPK also protects against Fludarabine. A. Viability of stably transfected cells with PRKACA or an empty vector, in increasing concentrations of Fludarabine. The analysis of PRKACA mRNA expression is shown on the right. B. Lace F-Ara-ATP in the pocket of the cAMPK ATP.

EXAMPLES

Example 1

The prokaryotic aminoglycoside-3 '* phosphotransferase and the structurally related kinase dependent cyclic eukaryotic AMP confer resistance to Fludarabine

I. MATERIALS AND METHODS

Cell culture and cell viability assay

Fibroblasts and 293FT embryonic kidney cells, both transformed with the SV40 large T antigen, were maintained in DMEM medium (BioWest, Nuailld, France) supplemented with 10% fetal calf serum inactivated by heat in an atmosphere with a 5% C02 at 37 ° C. MCF7 breast cancer cells were maintained similarly in RPMI medium with 10% fetal calf jurisdiction. To determine the resistance of the cells to the treatments, 3,000 cells were seeded per well in 96-well plates and incubated 3 days in the presence of the indicated drugs. Cell viability was determined using the XTT Cell Proliferation Kit II (Roche, Basel, Switzerland), following the manufacturer's instructions.

Analysis by RT-PCR

The TRIZOL reagent (Invitrogen, Carlsbad, USA) was used to prepare total RNA. To determine the expression of mRNA, a reverse transcription and semiquantitative PCR (RT-PCR) method was used. For the RT reaction, the RNA (5pg) was primed with random hexameric oligonucleotides and reverse transcribed with the Superscript MMLV reverse transcriptase (Invitrogen, Carlsbad, USA) in a volume of 20pl, following the manufacturer's instructions. The cDNA was used as template in amplifications with primers for the genes shown in Table 1.

Table 1.

The amplifications were carried out following the instructions of the manufacturer of the thermoresistant polymerase during 32 cycles, except in the case of GAPDH, which used 25 cycles.

Analysis by Western Blot

Complete cell lysates were obtained as previously described (Sbnchez-Carrera D, et al., Biosci Rep. 2015 Jun 11; 35 (3)). The protein concentration was determined by BCA following the manufacturer's instructions (G Biosciences, St. Louis, USA). The proteins (25pg) were resolved by SDS-PAGE and transferred to PVDF filters. The blots were incubated with antibodies against FANCD2 (H-300), p53 (FL-393), PARP (H-250), ERK2 (C-14), GAPDH (FL-335) or alpha-tubulin (B-5-) 1) (Santa Cruz Biotechnology, Santa Cruz, USA), and then incubated with a secondary antibody conjugated with peroxidase (Santa Cruz Biotechnology, Santa Cruz, USA). The bound antibody was detected by a chemiluminescent assay (Thermo Scientific, Rockford, USA) in a mini LAS4000 camera (GE Healthcare, Little Chalfont Buckinghamshire, UK).

Molecular modeling and ligand binding

The atomic model of APH (3 ') was used (Access number to Protein Data Bank 1nd4.pdb, described in Nurizzo et al., J.Mol.Biol 2003, 327 (2): 491-506) as a mold . The structural coordinates of fludarabine, clofarabine and cladribine were obtained from the PubChem database. The molecules were prepared for their fitting with the DockPrep tool of the UCSF Chimera informatics package. This process included the addition of hydrogens, the replacement of incomplete side chains with the Dunbrack rotamer library, the elimination of water molecules as a solvent and the inclusion of partial charges using AMBERff12SB force field. The files containing the atomic coordinates of the target protein and fatty acids were sent to the SwissDock server, which uses the EADock dihedral spacing sampling (DSS) engine to fit drug-type ligands onto macromolecules. The fitting tests were carried out blindly on the entire molecule, without defining any specific region of the protein to avoid a bias. The results were examined with UCSF Chimera and the pairings were classified with respect to the Full-Fitness value (FF) of Swissdock. The poses with the best FF rating and with the lowest energy were finally selected. Further refinement was carried out using the ATP coordinates linked to APH (2 ') - IVa (Accession Data Protein Data Bank 3hav.pdb; Young PG et al., 2009. J. Bacteriol., 191 (13): 4133- 4143 after alignment with APH (3 ') - lla (access number of Protein Data Bank 1nd4.pdb; Nurizzo et al., Cited ad supra.) Representations of molecular structures are generated with PyMOL. A similar procedure was followed to fit Fludarabine into cAMPK. In this case the coordinates of the catalytic domain of the muscle-dependent cAMP protein kinase of Mus musculus were used (access number Protein Data Bank laTP.pdb; Zheng J, et al., Protein Sci. Cold Spring Harbor Laboratory Press; 1993, 2 ( 10): 1559-73) as a target.

Cloning of NeoR and PRKACA

The NeoR gene was amplified from the vector pCR3.1 (Invitrogen, Carlsbad, USA), cloned into the vector pGEX 2T and subsequently subcloned into the expression vector pET28a (Novagen, Madison, USA). The resulting protein contained a tag of 34 His residues in the N-terminal domain. NeoR was also cloned into an expression vector with a eukaryotic selection marker of Puromycin resistance. The PRKACA and ERK2 cDNAs were amplified by RT-PCR and cloned in the same expression vector. The different APH (3 ') - lla mutants were obtained by PCR-directed mutagenesis on the NeoR gene and the products were cloned in the same vector as the wild-type cDNA. All constructions were confirmed by sequencing.

Overexpression and purification of APHfSJ-lla

Overexpression of the protein was induced in strain C41 (DE3) of Escherichia coli (Miroux and Walker, 1996; J Mol Biol, 1996 vol 260 (3) pp. 289-298) by addition of 1mM of IPTG (isopropyl). beta-D-thiogalactopyranbsido). Bacteria were grown in 1 liter of LB medium and, after 6 hours of induction at 25 ° C, the cells were harvested and stored at -80 ° C. The thawed cells were resuspended in 40 ml of buffer A (100 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.001% PMSF) and lysed by sonication. The lysates were collected by centrifugation and the supernatants were loaded onto a HisTrap HP column (1 ml) (GE Healthcare). The protein was eluted from the column in a linear imidazole gradient using buffer B (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 500 mM imidazole and 0.001% PMSF). Fractions containing APH (3 ') - lla were pooled and loaded in a Superdex 7510/300 GL (GE Healthcare) column equilibrated with a 100 mM Tris-Hcl pH 7.5 buffer, 150 mM NaCl, 5% (w / v) glycerol and 0.001% PMSF. The protein is stored at -80 ° C.

Assay of hydrolysis of ATP

The hydrolysis of ATP was analyzed by a coupled enzyme assay, as previously described (Kreuzery Jongeneel, 1983 Meth Enzymol, 1983 vol 100 pp.

144-160). The APH protein (3 ') - lla (1.5 pM) was pre-incubated with the substrates AMP, Fludarabine monophosphate or AMP-PNP (10 mM) for 5 minutes at 37 ° C. Reactions were initiated by addition of this sample to a mixture for assay of ATPase (150 pi) consisting of 50 mM Pipes-NaOH pH 7.0, 35 mM NaCl, 5 mM MgCl 2, 0.05 mM ATP, 225 pM Kanamycin, 5 % (w / v) glycerol, 0.5 mM phosphoenolpyruvate, 0.25 mM NADH, 60 pg / ml pyruvate kinase and 60 pg / ml lactate dehydrogenase (Roche Applied Science). The activity was measured through the decrease in absorbance of NADH at 340 nm for 5 minutes at 37 ° C on a UV-1800 spectrophotometer (Shimadzu).

II. RESULTS

Transfection with a Neomycin resistance gene provides resistance to Fludarabine.

The neomycin resistance gene included in most commercial pldsmidos encodes an aminoglycoside-3'-phosphatase-lla (APH (3 ') - lla) that inactivates different aminoglycoside antibiotics by transferring a phosphate group from an ATP mold . During the development of our experiments it was observed that the stable transfection of fibroblasts with pldsmides containing this resistance gene provided an increase in resistance to the analogous purine fludarabine. This effect was not due to the specific transfected gene, since the empty vector generated a similar resistance (Figure 1). However, those same cells transfected with a puromycin resistance gene, another widely used gene, remained as sensitive to fludarabine as their untransfected parental cells (Figure 1). In this way, it could be concluded that the resistance to fludarabine observed is due to the APH protein (3 ') - lla encoded by the plasmid.

APH (3) - it confers resistance to Fludarabina and Clofarabina, but not to other analogous of nucledsidos.

Given that there are other analogous nucledsides commonly used in the treatment of different tumors, we wanted to know if APH (3 ') - lla confers resistance to other structurally related drugs. Cells transfected with APH (3 ') - lla showed resistance to clofarabine, another andlogo of nucledsidos (Figure 2), although to a lesser extent. As a general observation, the efficacy of fludarabine as cytotoxic in the experiments with fibroblasts was much greater than with any of the other drugs tested that required higher doses to observe a reduction in cell viability.

In clinical practice, a previous treatment with fludarabine often improves the response to cytarabine, so! that it was decided to investigate the response of cells transfected with APH (3 ') to a combination of both drugs. The cells were treated with a fixed concentration of one of the drugs and with increasing concentrations of the other. However, the combination fludarabine + clofarabine did not modify the response of the cells (Figure 3).

Resistance to geneticin (G418) and to fludarabine require different domains of the protein.

Fludarabine is rapidly phosphorylated within the cells to its active triphosphate (F-Ara-ATP) form, thus becoming an ATP analogue, so it is likely that its direct interaction with APH (3 ') can catalyze its deactivation. To test this hypothesis, the mutations of the enzyme previously described were studied to study its influence on resistance to fludarabine. Stable cell lines carrying H188Y, R211H, K50R and D190A mutations of APH (3 ') were generated. H188Y and R211H are mutations that alter the pocket for the aminoglycoside of the protein and, consequently, interfere with their ability to deactivate G418 (Figure 4A). However, these two mutations did not significantly alter the resistance to Fludarabine mediated by APH (3 ') - lla (Figure 4A). Lysine 50 is found at the binding site of ATP, where it has been proposed to interact with phosphate (1) of the nucleotide.The introduction of the K50R mutation in APH (3 ') greatly affects the induction of resistance to both G418 as a fludarabine in fibroblasts (Figure 4A) Something similar happens when Asp190 is mutated to Ala.Asp190 is considered the catalytic residue of the enzyme and its mutation completely inactivates it.As shown in Figure 4A, the transfected cells with mutant D190A-APH (3 ') - lla show a sensitivity to fludarabine similar to that of nontransfected parental cells.

To rule out a possible cell specific effect, the protection to fludarabine mediated by APH (3 ') was analyzed in other cell lines. The transfection of the gene NeoR in 293FT cells of embryonic renal carcinoma, or in MCF7 breast cancer cells also provided protection against fludarabine (Figure 5). The introduction of the previously described mutations in the APH protein (3 ') had similar effects on the protection against fludarabine.

These observations suggest that fludarabine and aminoglybides bind to different sites of APH (3 ') - lla, as! It was decided to try without any synergic effect when using a combination treatment. Treatment of the cells with a fixed dose of kanamycin, a prokaryotic antibiotic, and increasing doses of fludarabine improved the viability of transfected fibroblasts either with the wild type of APH (3 ') or with its K50R mutant (Figure 4B). .

Taken together, these results suggest that fludarabine binds to the pocket of the APH ATP (3 ') - lla and is deactivated by an unknown new mechanism.

Lace of the purine analogues in the structure of APH (3) -lla.

The resistance to the purine anblogs of the cells transfected with APH (3 ') depends on the nucleoside used, which suggests a different mode of binding of the nucleids to the protein. In order to explore possible binding sites for purine nuclei in APH (3 '), a computer-aided analysis was carried out. The blind blind predictions using the EADock dihedral spacing sampling engine from the Swiss-dock server (http://www.swissdock.ch/) showed the ATP unibn site as the most likely unibn point of the purine anblogs. Most of the unibn poses coincide on this site. Since no APH (3 ') structure bound to ATP or to any other nucleotide is available, the coordinates of APH (2') - lla (Protein Dta Bank access number 3hav.pdb; Young et al. 2009 cited ad supra) to model the interactions of ATP with APH (3 ') - lla. The residues involved in the interactions with the adenine ring (for example, Y87 and D219 in APH (2 ') - lla; F48 and D208 in APH (3') - lla) and with the phosphate chain (K42 and E56 in APH (2 ') - lla; K50 and E63 in APH (3') - lla) are shown in Figure 6A. The unibn models for fludarabine and clofarabine were refined using the coordinates of the ATP in the APH (3 ') model as a model. Fludarabine and clofarabine have a hydroxyl group and a fluoride ion at the C2 'position of the ribose ring, respectively. As seen in Figures 6B and 6C, the distances of these radicals from fludarabine and clofarabine to the carboxyl group of Asp208 is less than 3A. In support of this idea, it could be shown that the Asp208 mutation (D208A) has a profound effect on the resistance to fludarabine mediated by APH (3 ') - lla (Figure 5).

APHfiJ-lla blocks the incorporation of fludarabine into the cells.

To try to clarify the mechanism by which APH (3 ') - lla protects the cells from the deleterious effect of fludarabine, the inventors conducted an experiment to find out if it was capable of preventing their incorporation into the nucleic acids of the cells or it simply interfered with the mechanisms of apoptosis activated after the incorporation of fludarabine. To this end, cells transfected with APH (3 ') or carrier cells of a mutant form of the enzyme were grown in the presence of fludarabine. After 24 hours of incubation, the remaining fludarabine was assayed in those conditioned media by culturing fludarabine-sensitive cells therein. The experiments showed that fibroblasts that grew in medium conditioned by cells transfected with APH (3 ') - lla experienced a reduction in their viability similar to those that grew in fresh fludabine (Figure 7). On the contrary, the conditioned medium from cells not transfected or transfected with the APH (3 ') mutant K50R hardly affected the viability of the receptor cells. Taken together, these experiments suggest that APH (3 ') - lla prevents Fludarabine from being incorporated by the cells.

Fludarabine induces monoubiquitination of FANCD2 but APHfSJ-lla prevents it.

Fludarabine is a purine angogue that is incorporated into the newly synthesized DNA, giving rise to the termination of the chain synthesis and the inhibition of the enzymes involved in the generation of nuclebtids and the synthesis of RNA, leading to cell death. According to previously published data, we observed that fludarabine induces genes such as c-jun and represses the expression of others such as Bcl-XL and MMP-9, involved in the modulation of apoptosis pathways (Figure 8A). Certainly, fludarabine induced apoptosis in fibroblasts, as it is derived from the analysis of its cell cycle and the digestion of PARP (Figures 8B and 8C). These effects were completely blocked in cells transfected with APH (3 ') wild or that harbored the mutations H188Y or R211H. However, the K50R and D190A mutations undermined the ability of APH (3 ') to block PARP digestion and cellular fragmentation (Figures 8A and 8C).

Interestingly, we observed that treatment with Fludarabine induced the monoubiquitination of FANCD2 (Figures 8A and 8D), an indication that it was being incorporated into DNA and clogging its replication. The transfection of APH (3 ') in the fibroblasts blocked the monoubiquitination of FANCD2, but the mutations that affected its induction of resistance to Fludarabine restored it (Figure 8C).

The ATPase activity of APH (3) -lla is inhibited by Fludarabine monophosphate. Mutational analysis and predictions of interaction suggested that the pocket of ATP acts as the binding site for fludarabine in APH (3 '). Therefore, it was hypothesized that in the presence of fludarabine triphosphate (F-Ara-ATP), APH (3 ') - lla could hydrolyze this substrate instead of ATP, thus preventing its incorporation into DNA. In order to obtain direct evidence of this, it was probed whether fludarabine was a substrate of the enzyme. APH (3 ') was purified to measure its ATPase activity in vitro. The experiments demonstrated a discrete activity of ATP hydrolysis of the enzyme (-17 nmol ATP / minute / mg), although it was strongly enhanced when adding kanamycin to the reaction (-2100 nmol ATP / minute / mg) (Figure 9A). Since there is no commercially available fludarabine triphosphate (in clinical practice patients are administered the monophosphate isoform, which is subsequently phosphorylated within the cells), it was decided to use the monophosphate form in the in vitro assays. It is conjecturized that if Fludarabine triphosphate is a genuine APH (3 ') substrate, its monophosphate form would act as an inhibitor of the ATP hydrolysis reaction, similar to how AMP does with other ATPases. Certainly, when Fludarabine monophosphate (F-Ara-AMP) was added to the reaction, a 35% reduction in ATPase activity of APH (3 ') was observed, whereas AMP induced a 50% inhibition ( Figure 9B). These results indicate that fludarabine monophosphate binds to APH (3 ') - lla and suggests that dephosphorylation of the active form of fludarabine is the mechanism of resistance mediated by the enzyme.

Analysis of eukaryotic kinases with structural similarity to APH (3) -lla.

The crystallographic studies of APH (3 ') and of some of the members of their family revealed a structural relationship with some eukaryotic kinases, so we wonder if an increase in the expression of these kinases could generate refractoriness of the patients. treatment with fludarabine. Two of the eukaryotic kinases with high structural similarity to APH (3 ') - lla are ERK (extracellular regulated MAP kinase) and cAMPK (the catalytic subunit of the activated kinase). cAMP, encoded by the PRKACA gene). While the overexpression of ERK2 did not induce any change in the viability of human fibroblasts in response to fludarabine (Figure 10B), transfection of PRKACA conferred resistance to the drug similar to that obtained with APH (3 ') - lla (Figure 11). TO). Similar results were obtained when PRKACA was overexpressed in other cell lines (Supplementary Figure 10A). A blind simulation indicated that fludarabine binds to cAMPK in the ATP pocket (Figure 11B).

Claims (23)

1. In vitro method to predict the response of a subject to treatment with a compound of formula (I), or to select a subject capable of being treated with a compound of formula (I)

where: R ₁ is selected from NH 2 or OCH 3, R2 is selected from a halogen or NH2 R3 is selected from H or PO3H2. R4 is selected from OH or a hagen, or any of its pharmaceutically acceptable isomers or salts, wherein the method comprises the following steps (a) quantifying the expression levels of the cAMP-dependent protein kinase gene (PRKACA gene) in a biological sample isolated from said subject, and (b) compare the expression levels of said gene with a reference level, wherein a level of expression of said gene less than a reference level is indicative that treatment with a compound of formula (I) is going to be effective, or that the subject is susceptible to being treated with a compound of formula (I) 2. Method according to claim 1, wherein R is NH2. 3. Method according to claim 1 or 2, wherein R2 is F, Cl or NH2. 4 The method according to any one of claims 1 to 3, wherein R4 is F 0 OH. Method according to any one of claims 1 to 4, wherein the compound of formula (I) is fludarabin phosphate or clofarabine. Method according to any one of claims 1 to 5. wherein the subject suffers from a disease selected from the group consisting of low-grade lymphoproliferative syndrome, secondary acute leukaemias and high-grade myelodyspetic syndromes, or will receive a transplant an organ or tissue. 7. Method according to claim 6, wherein the low-grade lymphoproliferative syndrome is chronic lymphocytic leukemia. The method according to any one of claims 1 to 7, wherein the quantification of the expression levels of the PRKACA gene comprises the quantification of the messenger RNA of said gene, or a fragment of said mRNA, the complementary DNA of said gene, or a fragment of said cDNA, or mixtures thereof. The method according to any one of claims 1 to 8, wherein the quantification of expression levels of the PRKACA gene is performed by a quantitative polymerase chain reaction or an array of DNA or RNA. 10. Method according to any one of claims 1 to 7, wherein the quantification of the expression levels of the PRKACA gene comprises the quantification of protein levels encoded by said gene or a fragment thereof. 11. Method according to claim 10, wherein the protein encoded by the PRKACA gene is the protein comprising a sequence of amino acids with an identity sequence of at least 80%, 85%, 90%, 95 %, 96%, 97%. 98%, 99% or 100% with the amino acid sequence SEQ ID NO: 2 (GenBank NP_002721.1). 12. Method according to claim 10 or 11, wherein the quantification of protein levels is carried out by means of western blot, ELISA or an array of proteins. The method according to any one of claims 1 to 12, wherein the PRKACA gene comprises a sequence of nucleotides with a sequence identity of at least 80%. 85%, 90%, 95%. 96%, 97%, 98%, 99% or 100% with the sequence SEQ ID NO: 1 (GenBank NG_029699.1). Method according to any one of claims 1 to 13, wherein the biological sample is selected from the group consisting of peripheral blood, serum, plasma and tissue. 15. Use of the in vitro expression of the PRKACA gene to predict the response of a subject to treatment with a compound of formula (I) described in any of claims 1 to 5, or to select a subject capable of being treated with a composed of the formula (I) described in any of claims 1 to 5. 16. Use according to claim 15, in which the subject suffers from a disease selected from the group consisting of low-grade lymphoproliferative syndrome, secondary acute leukaemias and high-grade myelodysplastic slundromes, or the subject is going to receive a transplant from a donor or tissue. 17. Use according to claim 16. wherein the low-grade lymphoproliferative syndrome is chronic lymphocytic leukemia. 18. In vitro use of a kit comprising (a) a pair of primers comprising a nucleotide sequence that hybridizes specifically to the nucleotide sequence of the PRKACA gene, (b) a probe that hybridizes specifically with the nucleotide sequence of the PRKACA gene, and / or (c) an antibody that specifically recognizes the protein encoded by the PRKACA gene, to predict the response of a subject to treatment with a compound of formula (I) described according to any of claims 1 to 5. or to select a subject susceptible of being treated with a compound of formula (I) described according to any of claims 1 to 5. 19. Use of a kit according to claim 18, wherein at least one of the primers comprises the sequence SEQ ID NO: 36 SEQ ID NO: 4. 20. Use of a kit according to claim 186 19, wherein the pair of primers comprises a first primer comprising the sequence SEQ ID NO: 3 and a second primer comprising the sequence SEQ ID NO: 4. 21. Use of a kit according to any one of claims 18 to 20, wherein the protein encoded by the PRKACA gene is the protein comprising an amino acid sequence with a sequence identity of at least 80%. %, 90%, 95%. 96%, 97%, 98%, 99% or 100% with the amino acid sequence SEQ ID NO: 2 (GenBank NP_002721.1). 22. Use of a kit according to any one of claims 18 to 21, wherein the PRKACA gene comprises a sequence of nucleotides with a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% with the sequence SEQ ID NO: 1 (GenBank NG_029699.1). 23. Use of a kit according to any of claims 18 to 22, wherein the kit further comprises a composition comprising a compound of formula (I) described in any of claims 1 to 5.