WO2012113965A1 - Method for obtaining data that can be used to evaluate, predict and/or prognosticate response to treatment with pyrimidine analogues - Google Patents

Abstract

The invention relates to an in vitro method for obtaining data that can be used to predict and prognosticate response to treatment with pyrimidine analogues and, preferably, with 5-fluorouracil (5-FU). The invention also relates to a kit and to the uses thereof.

Description

 Method of obtaining useful data to evaluate, predict and / or predict the response to treatment with pyrimidine analogues.

The present invention is within medicine and molecular biology, and refers to an in vitro method of obtaining useful data to predict and predict the response to treatment with pyrimidine analogs, and more preferably with 5-fluorouracil (5 -FU), particularly in patients with colon and breast cancer, which allows the establishment of a specific individual (qualitative and quantitative) recognition pattern, establishing different groups of patients

STATE OF THE PREVIOUS TECHNIQUE

Interferon PKR-induced cellular protein is a kinase that plays a fundamental role in the antitumor and antiviral activity that Interferon type I plays.

The best characterized PKR activator is the double stranded RNA that occurs during the infection of various viruses, but is also activated by different types of cellular stress such as polyols such as heparin, and other compounds such as ethanol, arsenic, ceramide, ect .., Its fundamental mechanism of action is to cause the cell protein synthesis to stop and activate the NFkB transcription factor that allows the cell to continue living if the problem that caused its activation is resolved. If cell stress continues, PKR eventually causes cell death in a controlled manner by apoptosis. PKR, therefore, can act as a tumor suppressor and has recently been shown to be critical for tumor suppressor activity of the known p53 protein. One of the main mechanisms through which, 5-fluorouracil (5-FU) exerts its antitumor activity is through induction of apoptosis. The protein involved in this phenomenon is the p53 tumor suppressor. But it is known that more than 50% of tumors have mutations in p53, and that tumor cells with p53 Inactive can die by apoptosis in response to 5-FU chemotherapeutic. Therefore it is unknown how 5FU induces the death of tumor cells in the absence of p53. We have discovered that it is precisely through the activation of the PKR protein that this process is carried out. PKR is activated in response to 5-FU even in the absence of p53, inducing the stop in the synthesis of cellular proteins and finally inducing cell death by apoptosis.

Therefore, p53 together with PKR are key cell markers in the effect of tumor cell death due to apoptosis caused by therapy based on the use of 5-FU or other pyrimidine analogues.

There are several compounds that have been combined with pyrimidine analogs, and especially with 5-FU to increase its effectiveness, such as oxaliplatin and irinotecan. In addition, numerous clinical trials are demonstrating that the combined use of drugs with natural cytokines is increasing the antitumor efficacy of chemotherapeutics used in clinical practice. Specifically, IFN alpha has been shown to improve the efficacy of 5-FU both in in vitro trials and in clinical trials with patients. Although the mechanism of this increase in efficacy is not yet fully known, and therefore it is not known why there are patients who do not respond to this combination, we propose that the increase that IFNalpha exerts on PKR may be one of the phenomena involved in such effectiveness. Therefore, knowing if PKR and p53 are mutated in tumors can be very useful for the prognosis of response to therapies based on the use of pyrimidine analogues, preferably in 5-FU, as well as their combination with other drugs or cytokines like the IFN. It is known that there is a very considerable percentage of patients who do not respond to chemotherapy based on the use of pyrimidine analogues, and preferably on 5-FU. Given the role that p53 plays in death induced by 5-FU, numerous studies have attempted to demonstrate that the lack of response is due to mutations in said tumor suppressor. Unfortunately, most of the works have failed to demonstrate that relationship, concluding that there must be more cell markers involved in the response to chemotherapy. The alternative therapies discussed above, such as the combination of IFNalpha and 5-FU, have been useful in some carcinomas. However, these therapies have aggressive side effects such as the appearance of fever, chills, general aches and pains, headache (headache). ), poor appetite, temporary decrease in the levels of white and red blood cells and platelets, which increases the risk of infection, anemia and / or bleeding, etc.

It is necessary, therefore, to find a method of obtaining useful data to evaluate the response to treatment with the pyrimidine analogues, and preferably with 5-FU, and its different combinations, which minimizes the iatrogenesis of the use of combination therapies of drugs

DESCRIPTION OF THE INVENTION The authors of the present invention have identified PKR as a new evaluation marker for cancer treatment with pyrimidine analogs, and preferably with 5-FU. The results of the studies in cell lines and samples of cancer patients show mutations in the genetic sequence of the PKR protein, and alterations in the activity of this protein, which can be used as a marker of the response the pyrimidine analogues, and in particular to 5-FU.

Therefore, a first aspect of the invention relates to the use of the PKR protein (or the pkr gene that encodes it) for obtaining useful data to predict the response to treatment with pyrimidine analogs. In a preferred embodiment of this aspect of the invention, the pyrimidine analogs are selected from the list consisting of cytarabine, fluoracil (5-fluoracil or 5- FU), tegafur, carmofur, gemcitabine, capecitabine, azacitidine, decitabine, or any of its salts, isomers, prodrugs, derivatives or corresponding analogs, and combinations thereof. In a particular embodiment of the invention, the pyrimidine analog is 5-fluorouracil (5-FU), or any of its salts, isomers, prodrugs, derivatives or the like. Treatment with pyrimidine analogues, and in particular with 5-FU, may be alone, or in combination with other drugs, preferably with IFN alpha.

METHOD OF OBTAINING USEFUL DATA TO PREACH AND / OR FORECAST THE RESPONSE TO TREATMENT WITH PYRIMIDINE ANALOGS Another aspect of the invention relates to a method of obtaining useful data to predict the response to treatment with pyrimidine analogs, or any of its salts, isomers, prodrugs, derivatives or the like, hereinafter method of the invention, comprising:

 a) obtain an isolated biological sample from an individual, and

 b) determine the activity of the PKR protein, in the biological sample isolated from (a), or the sequence of the PKR protein or the p / r gene that encodes it.

In a preferred embodiment of this aspect of the invention, the pyrimidine analogs are selected from the list consisting of cytarabine, fluoracil (5- fluoracil or 5-FU), tegafur, carmofur, gemcitabine, capecitabine, azacitidine, decitabine, or any of its corresponding salts, isomers, prodrugs, derivatives or analogs, and combinations thereof.

Treatment with the pyrimidine analogs, and in particular with 5-FU, may be alone, or in combination with other drugs, preferably with IFN alpha. In a preferred embodiment of the invention, the activity of the PKR protein is determined by detecting the amount of expression of the pkr gene. In another preferred embodiment, the activity of the PKR protein is determined by the sequence of the PKR gene. In another preferred embodiment, mutations of the sequence encoding PKR are detected. Mutations in said sequence may be indicative of a different activity.

In another preferred embodiment, the activity of the PKR protein is determined by detecting the amount of PKR protein, and more preferably, by the amount of phosphorylated PKR protein. More preferably, the biological sample of the individual from step (a) are tumor cells.

In another preferred embodiment, the method of the invention further comprises: c) comparing the amounts obtained in step (b) with a reference amount, or with a reference sequence.

If the amount or activity of the PKR protein, or the expression of the pkr gene is not sufficient, or the gene has certain polymorphisms, then it can be determined that the patient is not responding to the 5-FU treatment.

Steps (b) and / or (c) of the method described above can be fully or partially automated, for example, by means of a robotic sensor device for the determination of PKR activity or the detection of pkr expression levels. , or the identification of several polymorphisms in step (b) or the computerized comparison in step (c).

The determination of the activity of the PKR protein, for example, by detecting the amount of expression of the pkr gene, or of certain polymorphisms in the gene sequence, allows discriminating between responding individuals and non-responding individuals, thus facilitating the choice and establishment of appropriate therapeutic regimens. This discrimination as understood by an expert in the field is not intended to be correct. in 100% of the samples analyzed. However, it requires that a statistically significant amount of the analyzed samples be classified correctly. The amount that is significantly statistical can be established by one skilled in the art by using different statistical tools, for example, but not limited, by determining confidence intervals, determining the p-value, Student's test or discriminant functions of Fisher Preferably, the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. Preferably, the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001. Preferably, the present invention makes it possible to correctly detect the responding individuals differentially by at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a certain group or population analyzed.

An "isolated biological sample" includes, but is not limited to, cells, tissues and / or biological fluids of an organism, obtained by any method known to a person skilled in the art. Preferably, the isolated biological sample are cells, and more preferably tumor cells.

The term "individual", as used in the description, refers to animals, preferably mammals, and more preferably, humans. The term "individual" is not intended to be limiting in any aspect, and may be of any age, sex and physical condition. In a particular embodiment of the invention the individual has previously been diagnosed with cancer. More preferably, the individual has been diagnosed with colon cancer and / or breast cancer.

The PKR protein, or "RNA-dependent protein kinase", also referred to as "eukaryotic translation initiation factor 2-alpha kinase 2" (EIF2AK2). Also called EIF2AK1, MGC126524, PKR, PRKR, OTTHUMP00000201320; P1 / elF-2A protein kinase; double stranded RNA activated protein kinase; elF-2A protein kinase 2; interferon-induced, double-stranded RNA-activated protein kinase; interferon-inducible RNA-dependent protein kinase; interferon-inducible elF2alpha kinase; p68 kinase; RNA-activated protein kinase; protein kinase, interferon-inducible double stranded RNA dependent. It is encoded by a gene found on chromosome 2 (2p22-p21). Its amino acid sequence is found in SEQ ID NO: 1, (nucleotide sequence SEQ ID NO: 2 with access number in GenBank (NCBI) NM_002759.2). It also refers to any of its variants (currently 3 variants are known).

In the context of the present invention, PKR is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 1, and which would comprise various variants from:

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

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

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

 d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 1, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the PKR protein.

The PKR protein activity can be measured by any means known in the state of the art. In addition, the presence of polymorphisms or mutations can be determined by DNA sequencing.

Thus, the amount or concentration of the pkr gene expression product, preferably semi-quantitatively or quantitatively, can be brought to out directly or indirectly. Direct measurement refers to the measure of the quantity or concentration of the gene expression product, based on a signal that is obtained directly from the transcript of said gene, or from the protein (PKR), and that is directly correlated with the number of RNA molecules or proteins produced by the gene. Said signal - which we can also refer to as an intensity signal - can be obtained, for example, by measuring an intensity value of a chemical or physical property of said products. The indirect measurement includes the measurement obtained from a secondary component or a biological measurement system (for example the measurement of cellular responses, ligands, "tags" or enzymatic reaction products).

The term "quantity", as used in the description, refers to, but is not limited to, the absolute or relative quantity of the gene expression product, as well as any other value or parameter related to it or that may be derived. of this one. Said values or parameters comprise values of signal strength obtained from any of the physical or chemical properties of said expression product obtained by direct measurement. Additionally, said values or parameters include all those obtained by indirect measurement, for example, any of the measurement systems described elsewhere in this document.

The term "comparison", as used in the description, refers to, but is not limited to, the comparison of the quantity of the expression product of the pkr gene of the biological sample to be analyzed, also called the biological problem sample, with a amount of expression product of one or more desirable reference samples described elsewhere in the present description. The reference sample can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample. The comparison described in section (c) of the method of the present invention can be performed manually or assisted by a computer. The term "reference amount", as used in the description, refers to the absolute or relative amount of expression product of the pkr gene that allows discriminating individuals responding to 5-FU treatment of non-responders. Suitable reference amounts can be determined by the method of the present invention from a reference sample that can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample. Particularly, the reference amount. The reference sequences are SEQ ID NO: 1 for protein, and SEQ ID NO; 2 for the nuecleotidic sequence.

In the sense used in this description, the term "variant" refers to a protein substantially homologous to the PKR protein. In general, a variant includes additions, deletions or substitutions of amino acids. The term "variant" also includes the proteins resulting from posttranslational modifications, such as, but not limited to, glycosylation phosphorylation, sumoylation, methylation or acylation.

As used herein, a protein is "substantially homologous" to the PKR protein when its amino acid sequence has a good alignment with the amino acid sequence SEQ ID NO: 1, that is, when its amino acid sequence has a degree of identity with respect to the amino acid sequence SEQ ID NO: 1, of at least 50%, typically of at least 80%, advantageously of at least 85%, preferably of at least 90%, more preferably at least 95%, and even more preferably at least 99%. The homologous sequences to the PKR protein can be easily identified by one skilled in the art, for example, with the help of an appropriate computer program to compare sequences. The term "functionally equivalent", as used herein, means that the proteins or fragment / s of the protein / s in question essentially maintain the biological or immunological properties described herein. document. Said capacity can be determined by conventional methods.

The "pyrimidine analogues" constitute a well defined group in the ATC code or System of Anatomical, Therapeutic, Chemical Classification (instituted by the World Health Organization, and adopted in Europe). They belong to section ATC L01, section of the ATC classification system within group L, corresponding to antineoplastic and immunomodulating agents. Specifically they are classified as L01 BC Pyrimidine analogues, and in the latest update of 201 1 includes:

 - L01 BC01 cytarabine

 - L01 BC02 Fluoracil

 - L01 BC03 tegafur

 - L01 BC04 carmofur

- L01 BC05 gemcitabine

 - L01 BC06 Capecitabine

 - L01 BC07 azacitidine

 - L01 BC08 decitabine

 - L01 BC52 fluoracil, combinations

- L01 BC53 tegafur, combinations

In this specification, fluorouracil, 5-fluorouracil (5-FU), or 5- Fluoropyrimidine-2,4-dione, a compound with CAS number 51-21-8, of formula (I)

 Formula (I), or any of its salts, isomers, prodrugs, derivatives or the like.

In this specification, cytarabine, Ara-C, arabinofuranosylcytosine arabinosylcytosine, cytosine arabinoside, or 4-amino-1 - [(2R, 3S, 4R, 5R) -3.4 dihydroxy-5- (hydroxymethyl) oxolan-2-yl] pyrimidin-2-one, a compound with CAS number 147-94-4, of formula (II)

Formula (II), or any of its salts, isomers, prodrugs, derivatives or the like.

Tegafur, or (RS) -5-fluoro-1 - (tetrahydrofuran-2- yl) pyrimidine-2,4 (1 H, 3H) -dione, a compound with CAS number 17902-23-7 , of formula (III)

o cualquiera de sus sales, isómeros, profármacos, derivados o análogos.

or any of its salts, isomers, prodrugs, derivatives or the like.

In this specification, carmofur, HCFU or 5-fluoro-N-hexyl-2,4-dioxo-pyrimidine-1-carboxamide, a compound with CAS number 61422-45-5, of formula (IV)

 Formula (IV), or any of its salts, isomers, prodrugs, derivatives or the like.

Gemcitabine, or 4-amino-1 - (2-deoxy-2,2-difluoro- -D-erythro-pentofuranosyl) pyrimidin-2 (1 H) -on, a compound with CAS number 95058- is understood herein. 81 -4, of formula (V)

Formula (V),

or any of its salts, isomers, prodrugs, derivatives or the like.

In this specification capecitabine, or pentyl [1 - (3,4-dihydroxy-5-methyltetrahydrofuran-2-yl) -5-fluoro-2-oxo-1 H- pyrimidin-4-yl] carbamate, is understood as a compound with CAS number 154361 -50-9, of formula (VI)

 Formula (VI),

or any of its salts, isomers, prodrugs, derivatives or the like. Azacitidine, or 4-amino-1 - -D-ribofuranosyl- 1, 3,5-triazin-2 (1 7) -one, a compound with CAS number 320-67-2, of formula

(VII)

Formula (VII),

or any of its salts, isomers, prodrugs, derivatives or the like.

In this specification, decitabine is understood as, or 4-amino-1- (2-deoxy-bD-erythropentofuranosyl) -1, 3,5-triazin-2 (1 H) -one, a compound with CAS number 2353- 33-5, of formula (VIII)

Formula (VIII),

or any of its salts, isomers, prodrugs, derivatives or the like.

As used herein, the term "derivative" includes both pharmaceutically acceptable compounds, including derivatives of the compound of formula (I), (II), (III), (IV), (V), (VI), (VII), and / or (VIII), which may be used in the manufacture of a medicament, as pharmaceutically acceptable derivatives, since these may be useful in the preparation of pharmaceutically acceptable derivatives.

Also, within the scope of this invention are the prodrugs of the compound of formula (I), (II), (III), (IV), (V), (VI), (VII), and / or (VIII) . The term "prodrug" as used herein includes any compound derived from the compound of formula (I), (II), (III), (IV), (V), (VI), (VII), and / or (VIII), for example, esters, including esters of carboxylic acids, amino acid esters, phosphate esters, sulphonate esters of metal salts, carbamates, amides, etc., which, when administered to an individual, are capable of providing, directly or indirectly, the effect of the compound of formula (I) on said individual. Advantageously, said derivative is a compound that increases the bioavailability of the compound of formula (I), (II), (III), (IV), (V), (VI), (VII), and / or (VIII), when administered to an individual or that enhances the release of the compound of formula (I) (II), (III), (IV), (V), (VI), (VII), and / or (VIII), in a biological compartment The nature of said derivative is not critical as long as it can be administered to an individual and provides the compound of formula (I) (II), (III), (IV), (V), (VI), (VII), and / or (VIII), in a biological compartment of an individual. The preparation of said prodrug can be carried out by conventional methods known to those skilled in the art. In another preferred embodiment, the detection of the amount of PKR protein is performed by an immunoassay. The term "immunoassay," as used herein, refers to any analytical technique that is based on the reaction of conjugation of an antibody with an antigen. Examples of immunoassays known in the state of the art are, for example, but not limited to: immunoblot, enzyme-linked immunosorbent assay (ELISA), linear immunoassay (LIA), radioimmunoassay (RIA), immunofluorescence, x-map or protein chips .

In another preferred embodiment, the immunoassay is an enzyme-linked immunosorbent assay or ELISA (Enzyme-Linked ImmunoSorbent Assay). The ELISA is based on the premise that an immunoreactive (antigen or antibody) can be immobilized on a solid support, then bringing that system into contact with a fluid phase containing the complementary reagent that can bind to a marker compound. There are different types of ELISA: direct ELISA, indirect ELISA or sandwich ELISA.

The term "marker compound", as used herein, refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive and / or chemiluminescent signal that allows the detection and quantification of the amount of antibodies against to the PKR protein. The marker compound is selected from the list comprising radioisotopes, enzymes, fluorophors or any molecule capable of being conjugated with another molecule or detected and / or quantified directly. This marker compound can bind to the protein directly, or through another compound. Some examples of directly binding marker compounds are, but are not limited to, enzymes such as alkaline phosphatase or peroxidase, radioactive isotopes such as ³² P or ³⁵ S, fluorochromes such as fluorescein or metal particles, for direct detection by colorimetry, autoradiography, fluorimetry, or metallography respectively.

"Gene expression profile" means the gene profile obtained after quantification of mRNA and / or protein produced by the genes of interest or biomarkers, that is, by the pkr gene, in an isolated biological sample. The expression profile of the genes is preferably performed by determining the level of mRNA derived from its transcription, after extracting the total RNA present in the isolated biological sample, which can be performed by protocols known in the state of the art. The mRNA level derived from pkr gene transcription can be determined, for example, but not limited to, by amplification by polymerase chain reaction (PCR), back transcription in combination with polymerase chain reaction (RT). -PCR), quantitative RT-PCR, back transcription in combination with the ligase chain reaction (RT-LCR), or any other nucleic acid amplification method; serial analysis of gene expression (SAGE, SuperSAGE); DNA chips made with oligonucleotides deposited by any mechanism; DNA microarrays made with oligonucleotides synthesized in situ by photolithography or by any other mechanism; in situ hybridization using specific probes labeled with any method of marking; by electrophoresis gels; by membrane transfer and hybridization with a specific probe; by nuclear magnetic resonance or any other diagnostic imaging technique using paramagnetic nanoparticles or any other type of detectable nanoparticles functionalized with antibodies or by any other means. The gene expression profile could also be obtained by the detection and / or quantification of the proteins resulting from the translation of the mRNA derived from the transcription of the pkr gene, for example, but not limited to, western blot immunodetection. Quantitative detection of pkr gene expression can be performed more preferably by real-time PCR (RT-PCR or RTqPCR). Real-time detection of amplified products can be carried out through the use of molecules fluorescents that are embedded in the double stranded DNA or by hybridization with different types of probes. Thus, in another preferred embodiment, the expression of the pkr gene is detected by RTqPCR using primers SEQ ID NO: 3 - SEQ ID NO: 10, indicated in Table 2.

Table 2. Primers.

Another aspect of the present invention relates to a kit or device comprising the elements necessary to analyze PKR activity, or sequence the pkr gene.

More preferably it comprises the means necessary to compare the amount (of expression of the pkr gene and / or amount of PKR protein) detected in step (b) with a reference amount, or the sequence of the PKR protein or gene, with the reference sequences (SEQ ID NO: 1 and / or SEQ ID NO: 2).

Even more preferably, the kit of the present invention comprises the means necessary to obtain the sequence encoding the PKR protein, and preferably any of the primers collected in the sequences SEQ ID NO: 3 to S EQ ID NO: 1 0 to carry out the method of the present invention, or any combination thereof. Said kit may comprise all those reagents necessary to analyze the activity of the PKR by means of any of the methods described above in this document. It can also comprise any means to obtain the sequence encoding the PKR protein. Thus, it may include without limitation, any of the primers collected in the sequences SEQ D NO: 3 to SEQ ID NO: 10. Preferably, it comprises the pair of primers selected from the list comprising: SEQ ID NO: 3 and SEQ ID NO: 4, the pair of primers SEQ ID NO: 5 and EQ ID NO: 6, the pair of primers SEQ ID NO: 7 and EQ ID NO: 8 and the pair of primers SEQ ID NO: 9 and EQ ID NO : 10, or any of its combinations.

Another aspect of the invention relates to the sequence primer SEQ ID NO: 3. Another aspect of the invention relates to the sequence primer SEQ ID NO: 4. Another aspect of the invention relates to the sequence primer SEQ ID NO: 5 Another aspect of the invention relates to the sequence primer S EQ IDNO: 6. Another aspect of the invention relates to the sequence primer SEQ ID NO: 7. Another aspect of the invention relates to the sequence primer SEQ ID NO: 8. Another aspect of the invention relates to the sequence primer SEQ ID NO: 9. Another aspect of the invention relates to the sequence primer SEQ ID NO: 10.

The kit can also include, without any limitation, buffers, agents to prevent contamination, inhibitors of protein degradation, etc. On the other hand, the kit can include all the supports and containers necessary for commissioning and optimization. Preferably, the kit further comprises instructions for carrying out any of the methods of the invention. Another aspect of the invention relates to the use of the kit or device of the invention for obtaining useful data in the evaluation of the response to treatment with 5-FU, and preferably in individuals diagnosed with cancer. Preferably, the kit of the invention comprises any of the primers collected in the sequences SEQ D NO: 3 to SEQ ID NO: 10. Preferably, it comprises the pair of primers selected from the list comprising: SEQ ID NO: 3 and EQ ID NO: 4, the primer pair SEQ ID NO: 5 and EQ ID NO: 6, the primer pair SEQ ID NO: 7 and EQ ID NO: 8 and the primer pair SEQ ID NO: 9 and EQ ID NO: 10, or any of its combinations.

The terms "polynucleotide" and "nucleic acid" are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA).

The terms "amino acid sequence", "peptide", "oligopeptide", "polypeptide" and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may be coding or non-coding, Chemically or biochemically modified.

Throughout the description and the 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 derived 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

Fig 1. Activation of PKR during 5-FU treatment.

(A) SW480, T84, MCF-7 and T47D cells were treated with 1 0μΜ of 5-FU at 4, 8, 16 and 24 hours. (B) PKR ^{+ +} and PKR ^" '^" MEFs and (C) HCT-1 16 colon cancer cells expressing shRNA versus PKR (shRNA-PKR) or expressing the shRNA control (shRNAc) were treated with 500 Ul / ml of IFNa for 16 hours or treated with 10μΜ of 5-FU for 4, 8, 16 and 24 hours. Proteins were extracted and analyzed using the following antibodies: anti-phospho PKR, anti-whole PKR, anti-phospho elF2aanti-whole elF2a and anti- -actin.

Fig. 2. Implication of PKR in cell cycle arrest and 5-FU induced apoptosis. (A) PKR ^{+ +} and PKR ^" '^" MEFs were treated with 5 μΜ of 5- FU for 48 hours. The cell cycle was analyzed by flow cytometry after staining the cells with Pl. (B) PKR ^{+ +} and PKR ^" '^" MEFs were treated with 5 μΜ, 1 0 μΜ and 1 00 μΜ were 5-FU or 1 00 μg nnl of Irinotecan for 48 hours. Subsequently the cells were analyzed with Annexin V detection kit. (C) MCF-7 breast tumor cells expressing shRNA against PKR (shRNA-PKR) or the shRNA control system (shRNAc) and (D) colon and breast tumor cells SW-480, T84, MCF-7 and T47D, were treated with 10 μΜ of 5-FU for 48 hours. Subsequently the cells were analyzed with Annexin V detection kit.

Fig. 3. Regulation of PKR by 5-FU in HCT-116 p53 ^{+ / +} and HCT-116 p53 ^{_ / _} cells. (A) HCT-1 16 p53 ^{+ +} cells, ^and HCT-1 16 p53 ^" '^" were treated with 500 IU / ml of IFNa for 1 6 hours or treated with 1 0μΜ of 5-FU for 4, 8, 1 6 and 24 hours . Proteins were extracted and analyzed using the following antibodies: whole p53, PKR, anti phospho PKR, anti whole PKR, anti phospho elF2a, anti whole elF2a and anti b-actin. (B) HCT-1 16 p53 ^{+ +} , HCT-1 16 p53 ^" '^" cells were treated with 10 μΜ, όβ 5-FU or 500UI / ml IFNa for 16 hours, PKR mRNA expression was analyzed by real -time RT-PCR (means ± SEM, n = 3). (C) HCT-1 16 p53 ^{+ +} cells, HCT-1 16 p53 ^" '^" which expressing shRNA against PKR or the shRNA control system were treated with 10 μΜ of 5-FU for 48 hours. Subsequently the cells were analyzed with Annexin V detection kit. Fig. 4. Cytotoxic effect of 5-FU and the combined 5-FU / IFNa therapy

(A) PKR ^" '^" and PKR ^{+ +} MEFs, (B) human colon cancer T84 and SW-480 cell lines, and (C) human breast cancer cell lines, T47D and MCF-7 were treated with increasing amounts of 5-FU alone (triangles) or in combination with 50 U l / ml of I FNa (squares) for 6 days. The cell survival curve is represented as a percentage referred to untreated cells.

Hear

 Read phonetically Fig. 5. Apoptosis induced in MEFs and human cell lines of colon and breast cancer during the combined treatment of 5-FU / IFNa.

(A) 5 x 10 ⁵ human colon and breast cancer cells, (B) 5 χ 10 ⁵ of PKR ^{+ +} or PKR ^" '^" MEF cells and (C) 5 10 ⁵ human colon cancer cells HCT-1 16 p53 ^{+ +} or HCT-1 16 p53 ^" '^" expressing shRNA against PKR or the shRNA control, were treated with 500 Ul / ml of IFNa, treated with 5μΜ of 5- FU or treated with the combination of IFNa plus 5 -FU for 48 hours. Subsequently the cells were analyzed with Annexin V detection kit.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which demonstrates the specificity and effectiveness of the method of the invention. PKR plays an important role in translation inhibition, cell cycle arrest and 5-FU induced apoptosis. PKR protein activation during 5-FU treatment was demonstrated in several tumor cell lines of breast and colon cancer through the detection of phosphorylated PKR protein and phosphorylation of its main substrate elF2a (Figl A) . In order to analyze the impact of PKR activation by 5-FU treatment, MEF cells derived from wild-type (PKR ^{+ +} ) and knockout (PKR ^" '^" ) mice were analyzed for various biological functions. As shown in Fig. 1 B, in the PKR ^{+ +} cells treated with 5-FU elF2a was phosphorylated, whereas in the ^" "^" treated PKR cells no phosphorylation was detected. Similarly, in human colon cancer HCT-1 1 6 cells, treatment with 5-FU did not increase phosphorylation of elF2a when PKR was interfered with specific shRNA. Cells that expressed an unrelated control shRNA also showed an increase in phosphorylation of elF2a after 16 and 24 hours post-treatment (Fig. 1C). These results suggest that 5-FU could induce inhibition of protein synthesis through phosphorylation of elF2a, and this effect is mediated by PKR. Since phosphorylation of elF2a correlates with the arrest of the cell cycle, the effect of 5-FU on the cell cycle of PKR ^{+ +} and PKR ^" '^" MEFs cells was characterized at 48 hours post-treatment. As shown in Flg. 2A, in wild-type MEFs, 5-FU produced the accumulation of both the S phase and the G2 / M phase (18.2% and 24.8% respectively) as described to occur. However, the 5-FU treatment of PKR ^" '^" MEFs cells did not produce accumulation in either the S phase or the G2 / M phase (3.9% and 10.6% respectively. These results correlate well with the significant decrease in apoptosis in cells lacking PKR or with interfering PKR (Fig. 2B, C and D) as explained below.

In order to analyze the implication of PKR activation in apoptosis induced by 5-FU, PKR ^{+ +} and PKR ^" '^" MEFs cells were treated with increasing amounts of 5-FU. As shown in Fig 2B, in PKR ^{+ +} cells, 10 μΜ of 5-FU were able to significantly induce apoptosis levels that were slightly increased with the high dose of 100 μΜ of 5-FU. However, apoptosis did not occur in PKR cells ^" '^" treated. Irinotecam treatment was used as a positive control of the induction of apoptosis in both cell lines. Cell lines of colon cancer HCT-1 16 and breast cancer MCF-7 that expressed shRNA for PKR also showed reduced levels of apoptosis after 5-FU treatment compared to cells expressing control shRNA (Fig. 2C and D).

We have identified two cell lines, one breast and one colon (T47D and T84) that curiously do not express detectable levels of PKR protein (Fig 1 A). This finding is of great importance, since although they are lines established in the laboratory, they initially come directly from patient biopsies. Therefore, these data suggest the importance and the need to analyze PKR status in patients with colon and breast cancer who will receive chemotherapy based on the use of 5-FU. Overall, the results shown in Fig. 2 demonstrate that PKR is a molecular target of 5-FU.

PKR activation by 5-FU occurs independently of p53 It has been previously described that induction of apoptosis in response to 5- FU was mediated by p53. On the other hand, it has been recently suggested that one of the functions of p53 is modular PKR. In order to determine whether PKR activation and induction of apoptosis in response to 5-FU depend on p53, the HCT1 16 p53 ^{+ +} and HCT1 16 p53 cell lines were analyzed As shown in Figure 3A, PKR and elF2a were phosphorylated during treatment with 5-FU in both lines. In addition, the total PKR level was also increased in the absence of the p53 protein. To determine whether regulation of PKR is at the transcriptional level, mRNA PKR PCR was measured in real time in HCT1 cells 16 p53 ^{+ +} and p53 ^{_. "(Figure} 3B). As previously shown, p53 it is involved in apoptosis induced by 5-FU as shown in HCT1 16 cells in p53 ⁺ p53 ⁺ and ^{_ ~.} However, by decreasing evels n PKR interference occurred by reduction significant in 5-FU induced apoptosis in the two cell lines (Figure 3C). These results suggest the importance of PKR activation in the induction of apoptosis by 5-FU, even in the absence of p53. PKR is involved in the cytotoxic effect of 5-FU as well as in the efficacy of the combined 5-FU / IFNa therapy.

Taking into account the role of PKR in the cellular response to 5-FU, we hypothesize that the increase in the level of PKR produced by the IFN could be one of the mechanisms by which this cytokine improves the antitumor activity of the chemotherapeutic drug. 5-FU. To test this hypothesis, cell survival was determined in PKR ^{+ 1 +} and PKR ^'1' MEFs treated with increasing amounts of 5-FU, either alone or in combination with 50 Ul / mi IFNa. Our results showed that the cytotoxic effect of 5-FU was higher in PKR ⁺ ' ⁺ MEFs compared to PKR ^" '^" MEFs (Figure 4). The concentration of 5-FU needed to produce 50% cell death (IC50 values) is shown in Table I. On the other hand, IFN only improved the cytotoxic effect of 5-FU in PKR ^{+ +} MEFs cells. Similar experiments were carried out on human cell lines of colon and breast cancer (Figure 4 B and C). The cytotoxic effect of 5-FU was higher in cell lines expressing PKR protein compared to cells in which PKR was not detected. In fact, the IC50 value of 5-FU was approximately 2 times higher in T84 cells than in SW-480 cells. On the other hand, the IC50 value of 5-FU in T47D cells was about 4 times higher than in MCF-7 cells (Table I). In addition, cells lacking PKR, such as T84 and T47D, were not affected by the addition of I FNα to the 5-FU treatment. In contrast, IFN increased the cytotoxic effect of 5-FU by reducing IC ₅ or 5-FU in SW-480 and MCF-7 cells (Figure 4 B and C, Table I).

To confirm that PKR activation is involved in the synergistic effect on apoptosis exerted by the combined treatment, the 5-FU / IFN-induced apoptosis. Such combined induced a considerable level of apoptosis in colon and breast cell lines that express the PKR protein. In contrast, cells lacking PKR (T84 and T47D) were not affected (Figure 5). When the PKR ^{+ 1 +} and PKR ^" '^" MEFs, and p53 HCT1 16 ^{+ 1 +} and HCT1 16 p53 ^" '^" intervening lines were analyzed, apoptosis induced by the combination of 5-FU / IFNa was significantly reduced in PKR ^" '^" MEFs compared to PKR ⁺ ' ⁺ MEFs. PKR interference in p53 HCT1 16 ^{+ / +} and HCT1 16 p53 ^" '^" cells also caused a significant reduction in apoptosis levels compared to cells expressing control hRNA (Figure 5C). These results demonstrate the main role of PKR in the cytotoxic effect of 5- FU. Furthermore, this effect is increased by the IFN, in part, through the increase in the level of PKR that this cytokine produces. Table 1. IC ₅₀ values of 5-FU derived from the curves represented in Fig 4.

In summary, the results show that PKR is a 5- FU molecular target, which plays an important role in its cytotoxic effect at least, in part, at through the inhibition of translation and through the induction of death by apoptosis of tumor cells in an independent form of p53. These results indicate the clinical importance that PKR status can play in response to 5-FU-based chemotherapy. On the other hand, the efficacy of the cytotoxic effect of IFN-induced 5-FU, especially in tumors that express a mutated form or that lack p53 (more than 50% of tumors), but with functional PKR, could have a relevant clinical application in patients.

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Materials and methods

Cell culture and reagents

Mouse embryonic fibroblasts PKR + / + and PKR - / -, and human colon cells HCT1 16 p53 + / + and p53 - / - were supplied by M Esteban (National Center for Biotechnology, Spain) and B. Vogelstein (Johns Hopkins Oncology Center , USA), respectively. Human cell lines from colon cancer SW-480, T84, RKO, and human cell lines from breast cancer MCF-7, MDA-MB-231, T47D were obtained from the American Type Culture Collection (ATCC) . Interferred cells for PKR (shRNA-PKR) were prepared as indicated below. Most cells were maintained in DMEM medium supplemented with 10% FBS, 1% penicillin-streptomycin, and penicillins, and 1% solution of nonessential amino acids. The MDA-MB-231 cell line was cultured in DMEM / F1 2- medium. All cells were used in exponentially growing experiments. 5-Fluorouracil (Sigma-Aldrich, F6627-1 G) was dissolved in DMSO and stored at -20 ° C. IFNalpha 2b (Intron A) was supplied by Schering-Plow.

Cell Viability Assay The effect of 5-FU on cell viability was evaluated with a colorimetric assay based on the use of sulforhodamine-B (SRB). 3 10 ³ cells per well were treated with different concentrations of 5-FU in the presence or absence of IFNa (50 lU / ml for six days having replaced the medium with the reagents at three days. Finally the cells were processed as described in Villalobos et al. Using the Titertek Multiscan luminometer (Flow, Irvine, CA, 6 USA) at 492 nm. The IC ₅ value was calculated by interpolating in the semilogarithmic curve generated from the dose response considering the untreated cells as the 100% feasibility All experiments were carried out in triplicate and repeated at least twice.

Apoptosis analysis Cells were incubated for 48 hours with 5-FU alone or in combination with IFN (500IU / ml). IFIa was added 16 hours before 5-FU. After 72 hours, the cells were analyzed using the TACSTM Annexin V-FITC kits (R&D System, TA4638), and Annexin V-APC (ebioscence, 88-8007-74) for the interfered cells. Samples were processed using the FACScan cytometer (Becton Dickson, San José, CA, USA) of the instrumentation service of the University of Granada.

Analysis of the cell cycle Exponentially growing cells treated for 24 and 48 hours with 10 □ M of 5-FU were fixed with 70% (vol / vol) cold ethanol for 12 hours. Finally, they were resuspended in 250 μΙ of a suspension of propidium lodide (Pl) (100 μΙ / ml RNAse, 40 μΙ / ml Pl in PBS) for 30 minutes at 37 ° C in the dark. Samples were processed immediately after using the FACScan cytometer (Becton Dickson, San José, CA, USA) of the instrumentation service of the University of Granada. Immunoassay (Western Blot)

The cells treated during the established times are lysed in Laemmli buffer and after having undergone SDS-PGE electrophoresis, they are transferred to nitrocellulose membranes (Biorad, 162-01 15), blocked with PBS -5% skimmed milk for 1 hour at room temperature . The primary antibodies used are: total anti-PKR supplied by M Esteban (National Biotechnology Center, Spain), anti-phospho-PKR (Thr 451) (Calbiochem, 527460), anti-phosphoelF2D (Ser 51) (Invitrogen, 44- 728, G), anti-phospho-p53 (Ser15) (Cell signaling, 92845), anti-actin (Sigma, A2228). Secondary antibodies are: anti-rabbit IgG peroxidase conjugate (Sigma, A0545) and anti-mouse IgG peroxidase conjugate (Sigma, A9044). Bands were visualized using ECL reagent (Amersham Pharmacia Biotech, UK). PKR interference in colon and breast cell lines through the use of shRNA

To interfere with PKR, shRNAmir pGIPZ-lentiviral vectors containing non-silencing sequences (NS) or three specific "hairpin" sequences against PKR (Open Biosystems, RHS4430) were used. The pGIPZ vectors express GFP that allows to identify the transfected cells and puromycin resistance gene that allows to create stable lines. The MCF-7, and HCT1 16 p53 + / + and p53 - / - cell lines were transfected with said vectors using the Amaxa Cell line nucleofector kit V (Lonza Cologne AG, VCA-1003) following the commercial house protocol. To generate stable interfered lines, the cells were selected with puromycin (Invivogen, 58-58-2) at two days post-transfection at a concentration of i Dg / ml for HCT1 16 and 3 μς / ιτιΙ for MCF- cells 7. RNA extraction and real-time qRT-PCR analysis.

RNA was isolated directly from cell lines using the RNeasy mini kit (Qiagen74106). The complementary DNA chain was generated using the reverse transcription system (Promega A3500), following the protocol of the commercial house. The quantitative PCR reaction was carried out in 96-well plates using TaqMan Gene Expression Assays (Applied Biosystems) and Applied Biosystems 7500 Real Time PCR System that quantifies the amplicons. The taqman probes used were: PKR (Hs00169345_ml) and GAPDH (Hs 99999905_ml) and the reaction was carried out according to the specific Applied Biosystems protocol.

Claims

The use of the PKR protein or the pkr gene that encodes it, to obtain useful data to predict and predict the response to treatment with pyrimidine analogues.  The use of the PKR protein or the pkr gene that encodes it according to the preceding claim, wherein the pyrimidine analogs are selected from the list consisting of cytarabine, fluoracil (5-fluoracil or 5-FU), tegafur, carmofur, gemcitabine , capecitabine, azacitidine, decitabine, or any of its corresponding salts, isomers, prodrugs, derivatives or analogs, and combinations thereof  The use of the PKR protein or the pkr gene that encodes it according to any of claims 1-2, wherein the pyrimidine analog is 5-fluorouracil (5-FU), or any of its salts, isomers, prodrugs, derivatives or analogues  The use of the PKR protein or the pkr gene that encodes it according to any one of claims 1-3, wherein the 5-FU treatment is a combination treatment with IFN alpha.  A method of obtaining useful data for predicting or forecasting the response to treatment with pyrimidine analogs, or any of its salts, isomers, prodrugs, derivatives or analogs, comprising: a) obtaining an isolated biological sample from an individual, and b) determine the activity of the PKR protein, in the biological sample isolated from (a), or the sequence of the PKR protein or the pkr gene that encodes it. The method according to claim 5, wherein the pyrimidine analogs are selected from the list consisting of cytarabine, fluoracil (5-fluoracil or 5-FU), tegafur, carmofur, gemcitabine, capecitabine, azacitidine, decitabine, or any of their salts , isomers, prodrugs, derivatives or corresponding analogs, and combinations thereof 7- The method according to any of claims 5-6, wherein the pyrimidine analog is 5-fluorouracil (5-FU), or any of its salts, isomers, prodrugs, derivatives or the like. 8- The method according to any of claims 5-7, wherein the treatment with 5-FU is a combined treatment with IFN alpha. 9- The method according to any of claims 5-8, further comprising:  c) compare the quantities obtained in step (b) with a reference quantity or with a reference sequence. 10-EI method according to any of claims 5-9, wherein the biological sample of the individual of step (a) are tumor cells. 1 - The method according to any of claims 5-10, wherein the activity of the PKR protein is determined by detecting the amount of expression of the pkr gene. 12-EI method according to any of claims 5-1 1, wherein the activity of the PKR protein is determined by detecting the amount of PKR protein. 13- The method according to any of claims 5-12, wherein the detection of the amount of expression of the pkr gene is performed by PCR. 14- The method according to any of claims 5-13, wherein the detection of mutations and polymorphisms of the pkr gene or PKR protein is determined by DNA sequencing. 15- The method according to any of claims 5-14, wherein the detection of PKR activity is determined by an immunoassay. 16- A kit or device that includes the elements necessary to analyze PKR activity, or sequence the pkr gene. 17- The kit or device according to the preceding claim, comprising the elements necessary to analyze the amount of expression products of the pkr gene 18- The kit or device according to any of claims 16-17, comprising any of the primers collected in the sequences SEQ D NO: 3 to SEQ ID NO: 10. 19- Nucleotide sequence primer SEQ ID NO: 3. 20- Nucleotide sequence primer SEQ ID NO: 4. 21 - Nucleotide sequence primer SEQ ID NO: 5. 22-Nucleotide sequence primer SEQ ID NO: 6. 23- Nucleotide sequence primer SEQ ID NO: 7. 24- Nucleotide sequence primer SEQ ID NO: 8. 25- Nucleotide sequence primer SEQ ID NO: 9. 26- Nucleotide sequence primer SEQ ID NO: 10. 27- The use of the kit or device according to any of claims 16-18, for obtaining useful data to predict and predict the response to treatment with pyrimidine analogs. 28- The use of the kit or device according to the preceding claim, wherein the pyrimidine analogs are selected from the list consisting of cytarabine, fluoracil (5-fluoracil or 5-FU), tegafur, carmofur, gemcitabine, capecitabine, azacitidine, decitabine , or any of its corresponding salts, isomers, prodrugs, derivatives or analogs, and combinations thereof 29- The use of the kit or device according to any of claims 27-28, wherein the pyrimidine analog is 5-fluorouracil (5-FU), or any of its salts, isomers, prodrugs, derivatives or the like. 30- The use of the kit or device according to the preceding claim, wherein the 5-FU treatment is a combined treatment with IFN alpha.