ES2347619A1 - PROCEDURE TO DESIGN AN ANTITUMORAL INDIVIDUAL THERAPY BASED ON THE DETECTION OF HSP90 PROTEIN LEVELS, THE USE OF HSP90 PROTEIN INHIBITORS FOR THE PREPARATION OF PHARMACEUTICAL COMPOSITIONS, THE COMPOSITIONS AS OBTAINED AND THEIR APPLICATIONS. - Google Patents

Abstract

Procedure to design an individual antitumor therapy based on the detection of HSP90 protein levels, the use of HSP90 protein inhibitors for the preparation of pharmaceutical compositions, the compositions thus obtained and their applications. The present invention describes a procedure for designing an individual antitumor therapy for a subject based on the detection of levels of the HSP90 protein responsible for resistance to a drug or a mixture of antitumor drugs that inhibit IGF1R/KIT receptors. Likewise, it describes the use of HSP90 protein inhibitors for the preparation of drugs for the treatment of tumors resistant to IGF1R/KIT inhibitors, the pharmaceutical compositions thus obtained and their applications in tumors such as Ewing's sarcoma.

Description

Procedure to design a therapy individual antitumor based on the detection of levels of HSP90 protein, the use of HSP90 protein inhibitors for the preparation of pharmaceutical compositions, compositions as well obtained and its applications.

Technical sector

Biomedicine, tumor treatment, more specifically treatment of Ewing Sarcomas. Selection of specific therapies

State of the art

Ewing's sarcoma is a neoplasm that affects children and young adults, with a five-year survival that remains around 50% (1, 2). This sarcoma expresses in a de-regulated manner several autocrine loops that contribute to its pathogenesis. The Insulin-like growth factor 1 (IGF1R) and KIT receptors are transmembrane receptors that mediate two of these loops (3-13). In vitro studies have shown promising results by inhibiting IGF1R, and, it could be said, inhibition of KIT, (5, 9, 12-14), suggesting potential uses in clinical trials.

Drug sensitivity depends on several molecular mechanisms, including the stress response. The molecules involved in the stress response are the proteins of heat shock (Heat Shock Proteins, HSP). These proteins are chaperones that act, in a global way, as "guardians" of the proteome, facilitating the correct protein folding, maintaining the stability of your client proteins (PC), renaturing the deployed proteins or inducing their degradation (15-17). Many of them are involved in signaling pathways that control growth, cell survival and differentiation, thus being related with the pathogenesis of various human tumors (18, 19) and so therefore, inhibition of HSP proteins has become a new therapeutic strategy to inhibit multiple pathways of receivers HSP proteins that protect against stress are often found overexpressed in tumors and cell lines carcinogenic (18-22). In addition to its activity of protein re-assembly, they are also involved in the regulation of apoptosis, growth cellular and drug resistance anti-carcinogens, having demonstrated a direct correlation between HSP levels and progression tumor (19). Among HSPs, HSP90 protein is one of the main chaperones whose expression is increased by stress situations, having among your clients / target proteins to the following: AKT, HIF, p53, ErbB2 and Raf-1 (20-22). Currently, several are available specific HSP90 inhibitors, such as Geldanamicin or its derivatives, such as 17-allylamino-17-demethoxyigeldanamicin (17-AAG), which is being used in trials clinicians with good results as antitumor (23-27).

The search for new ways to treat sarcomas is an urgent need. IGF1R blockade (12, 13) is a promising possibility in SE therapy (14, 32-38). Based on this, and taking into account our previous experience with the synergistic effects of the in vitro combination of blocking IGF1R / KIT pathways with vincristine or doxorubicin (14), we have studied the global proteomic changes induced by treatment with these drugs and performed a molecular validation of the possible treatment targets.

Description of the invention brief description

An aspect of the present invention is it constitutes a procedure to design an individual therapy antitumor to a subject, hereinafter method of the invention, based on the detection of responsible HSP90 protein levels of resistance to a medication or a mixture of medications anti-tumor inhibitors of IGF1R / KIT receptors and that understands:

to)

Ex vivo determination of the expression levels of the HSP90 protein gene in a tumor biological sample,

b)

comparison of the levels of a) with with respect to a biological control sample,

C)

resistance determination to IGF1R / KIT receptor inhibitors when levels of a) are greater than those obtained in b), and

d)

selection of an inhibitor of the HSP90 protein as an adjuvant anti-tumor drug of a anti-tumor inhibitor of IGF1R / KIT receptors.

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A particular aspect of the invention is constitutes the process of the invention in which the sample biological tumor of a) belongs, by way of illustration and without limit the scope of the invention to the following group: sarcoma Ewing gastrointestinal stromal tumor (GIST) and dermatofibrosarcoma protuberans.

Another particular aspect of the invention is it constitutes the process of the invention in which the determination of protein gene expression levels HSP90 are carried out with or without the induction of a stress of the cells of the biological tumor sample using a compound anti-tumor inhibitor of IGF1R / KIT receptors.

Another particular aspect of the invention is it constitutes the process of the invention in which the determination of protein gene expression levels HSP90 as mentioned in step a) is carried out by means of determination of mRNA levels of the Hsp90 gene and that understands:

i)

the mRNA extraction from the tumor biological sample,

ii)

mRNA amplification of the Hsp90 gene, Y

iii)

mRNA levels determination of the Hsp90 gene.

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Another particular aspect of the invention is it constitutes the process of the invention in which the determination of protein gene expression levels HSP90 as mentioned in step a) is carried out by means of determination of HSP90 protein levels and that understands:

i)

the extraction of proteins from the biological tumor sample, Y

ii)

determination of the levels of the HSP90 protein, either by an immune method (antibodies, already whether monoclonal or not) or nonimmune.

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Another aspect of the invention is the use of an HSP90 protein activity inhibitor compound, either human or animal, hereinafter use of a compound inhibitor of the present invention, in the preparation of a medicament or Pharmaceutical composition useful for the treatment of a tumor, human or animal, resistant to treatment with inhibitors of IGF1 / KIT receivers.

Another more particular aspect of the invention is constitutes the use of a compound of the invention in which the HSP90 protein inhibitor belongs, by way of illustration and without that limits the scope of the invention, to the following group: geldanamicin, 17-AAG, 17-DMAG, Retaspimycin (IPI-504), ansamycin Non-benzoquinone, diarylpyrazole compounds (CCT018159 and CCT0129397 / VER49009) and novobiocin.

Another more particular aspect of the invention is constitutes the use of an HSP90 inhibitor compound of the invention wherein the inhibitor compound is a nucleic acid or polynucleotide that prevents or decreases gene expression encoder of the HSP90 protein, either human or animal, and that includes at least one selected nucleotide sequence between:

to)

a antisense nucleotide sequence specific to the sequence of the HSP90 protein mRNA or gene,

b)

a HSP90 protein mRNA specific ribozyme,

C)

a HSP90 protein mRNA specific aptamer,

d)

a RNA interference (siRNA or shRNA) specific to the mRNA of the HSP90 protein, and

and)

a microRNA (miRNA) specific for the HSP90 protein mRNA.

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Another particular embodiment of the invention is the use of an RNAi as mentioned in step d) in which the siRNA is constituted by the pair of specific nucleotide sequences of the siRNA of the following beta form: sense sequence: GGUGACAAUCUCCAAUAGAtt ( SEQ ID NO1) and antisense sequence:
UCUAUUGGAGAUUGUCACCttde (SEQ ID NO2) (sequences 5 '-> 3, see RefSeq: NM_007355-siRNA ID: 119760; Ambion, Austin, TX).

Another more particular aspect of the invention is constitutes the use of an HSP90 inhibitor compound of the invention which comprises the use in combination of a protein inhibitor IGFR1 / KIT, and more specifically in combination with at least the compound ADW742 (ADW), AEW541 (AEW) and Imatinib (IMA).

Another aspect of the present invention is it constitutes a pharmaceutical composition or a medicament useful for the treatment of a tumor process resistant to inhibitors of IGF1 / KIT, hereinafter pharmaceutical composition of the present invention, which comprises a therapeutically effective amount of an HSP90 protein inhibitor compound or agent, together with, optionally, one or more pharmaceutical adjuvants and / or vehicles acceptable.

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Another more particular aspect of the invention is constitutes the pharmaceutical composition of the invention comprising a HSP90 protein inhibitor belonging, by way of illustrative and without limiting the scope of the invention, to next group: geldanamicin, 17-AAG, 17-DMAG, retaspimycin (IPI-504), Non-benzoquinone ansamycin compounds diarylpyrazoles (CCT018159 and CCT0129397 / VER49009) and novobiocin.

Another more particular aspect of the invention is constitutes the pharmaceutical composition of the invention that it comprises, in addition to the HSP90 protein inhibitor, an inhibitor of the IGFR1 / KIT proteins, and more specifically, at least one inhibitor belonging to the following group: ADW742 (ADW), AEW541 (AEW) and Imatinib (IMA).

Another more particular aspect of the invention is constitutes the pharmaceutical composition of the invention in which the inhibitor compound is a nucleic acid or polynucleotide that prevents or decreases the expression of the HSP90 protein coding gene human and that includes at least one nucleotide sequence selected from:

to)

a antisense nucleotide sequence specific to the sequence of the HSP90 protein mRNA or gene,

b)

a HSP90 protein mRNA specific ribozyme,

C)

a HSP90 protein mRNA specific aptamer,

d)

a Interference RNA (iRNA) specific to the protein mRNA HSP90, and

and)

a microRNA (miRNA) specific for the HSP90 protein mRNA.

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Another particular embodiment of the invention is constitutes the pharmaceutical composition of the invention in which the HSP90 inhibitor is a siRNA constituted by the pair of specific nucleotide sequences of the beta form siRNA next: sequence meaning: GGUGACAAUCUCCAAUAGAtt (SEQ ID NO1) and antisense sequence: UCUAUUGGAGAUUGUCACCttde (SEQ ID NO2) (sequences 5 '-> 3, see RefSeq: NM_007355-siRNA ID: 119760; Ambion, Austin, TX).

Another object of the present invention is constitutes the use of the pharmaceutical composition of the invention in a method of treating a mammal, preferably a being human, affected by a tumor process or disease resistant to IGF1 / KIT receptor inhibitors, hereinafter use of the pharmaceutical composition of the present invention, consisting of the administration of said therapeutic composition that inhibits the tumor process

Detailed description

The present invention is based on the fact that the inventors have observed, by proteomic analysis, that surprisingly the HSP90 protein is expressed differently in Ewing tumor cells sensitive and resistant to specific inhibitors of IGF1R / KIT receptors (ADW / IMA) . More specifically, HSP90 protein expression increased dramatically in cell lines A673 and TTC466 (both resistant to ADW / IMA, (14)) at 24 hours of treatment, while it was slightly reduced in the other cell lines studied.
(Example 1).

With this finding one could explain, at least in part, the resistance of lines A673 and TTC466 to drugs KIT / IGF1R inhibitors, suggesting that these cells have developed a stress-induced response mechanism that It helps them to counteract the effect of treatment with these drugs, which is activated in the initial stages of treatment (before 24 h) and that makes them more resistant than other lines cell phones. In order to corroborate the above, the effects of specific inhibition of HSP90 using a derivative of Geldanamicin, 17-AAG in a 5-line panel of SE. This drug inhibited proliferation in a way dose-dependent on all lines of SE and caused an increase in apoptosis, especially in those treatment resistant lines (A673 and TTC466) (Figure 2), data which confirmed the previous hypothesis.

You could argue that the differences in sensitivity to treatment should be due to the fact that example, A673 has a mutation in the BRAF gene (39); what you It would make more sensitive to HSP90 inhibition. However, TTC466 has a similar response and has neither that mutation nor any other type change (such as p53 mutation (40) or loss of p16 / INK4 (41)) that can explain these results and have not been found also in the bibliography other types of changes that may explain these results, suggesting independence of genetic changes of these cell lines. These results are also considered they are independent of the type of transcript, since TTC466 has a type of fusion different from that of A673, and has behavior similar, while TC-71 has the same type of fusion than A673 and has different behavior from A673.

On the other hand, when HSP90 protein is inhibited with 17-AAG and with siRNA it was observed that the growth and survival of the Ewing lines, especially in the resistant cell line and that the observed data initially with 17-AAG they were really due to their action on HSP90, transforming cells into sensitive treatment. On the contrary, the induced overexpression of HSP90 provided resistance in treatment sensitive cell line anti-IGF1R / KIT (ADW / IMA compounds), reaching levels of complete apoptosis block, and high stimulation of proliferation, confirming again the hypothesis that the development of the stress response is a resistance mechanism to the treatment, something that until now had not been described in the Ewing's sarcoma.

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In the present invention it is noted that the 17-AAG treatment induced the degradation of HSP90 client proteins, including IGF1R, KIT and AKT through the reduction of your physical interactions with HSP90. It has also reduced phosphorylation of AKT, mTOR and MAPK42 / 44 and, therefore, proliferation inhibition and induction of apoptosis, it have given, at least in part, for the inhibition of these routes Signaling. The physical interaction of HSP90 with KIT and AKT has been described in detail above (22, 43), so they were expect the results obtained (AKT is a client protein of HSP90 and 17-AAG decreases expression levels of KIT) (44). However, although many receptors have been described hormonal or TK as HSP90 client proteins (19, 21-23, 45, 46) and inhibition has been demonstrated of IGF1R after treatment with HSP90 inhibitors (47-49), as far as it is known, has not been described until the present invention that IGF1R was a client protein of HSP90. In the present invention, physical interaction is described. by immunoprecipitation between HSP90 and IGF1R. This finding is of special interest since the IGF1R signaling path is great relevance in the pathogenesis of sarcomas (6-10, 14). This may suggest two possible ways of resistance mechanism:

one)

the IGF1R function is maintained after treatment by its interaction with HSP90, and / or

2)

the HSP90 protein confers resistance to sarcoma cells from Ewing by other routes related to proliferation and apoptosis by stabilizing client proteins such as AKT or KIT; Y

therefore, a new treatment strategy of these tumors or others in which the biological action of IGF1R participate in their pathogenesis.

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The fact that the malignant tumors are the result of a set of multiple genetic changes, makes the mono-therapy is ineffective due to the presence of routes of collateral signaling. In this way it is interesting to use the HSP inhibition combined with other drugs, so that reduces the resistance acquired by tumor cells at along the malignant progression to conventional treatment. Thus considered that treatment with 17-AAG could improve the susceptibility of SE cells to treatment with ADW / IMA, by inhibiting various proteins involved in signaling of survival and resistance to treatment, especially in lines A673 and TTC466, widely insensitive to both drugs ADW / IMA.

For this reason, it was decided to analyze the effect of combine 17-AAG with ADW or with IMA in the proliferation and induction of cell line apoptosis of sarcoma. The results presented in the present invention confirm that the design of combinations of different therapies is more efficient, and that this combination must be carried out in a Rational and specific tissue / cell form (Figure 2).

Although, the relevance of KIT is questionable in the SE (5), the results obtained suggest that a possible combination of IMA with 17-AAG could have a Interesting clinical application in these cases. In addition, it may be also applicable beyond the SE, especially in cases of gastrointestinal stromal tumor (GIST) or dermatofibrosarcoma IMA-resistant protuberans, inhibiting the action of HSP90, which which gives even more strength to the results obtained.

Xenotransplant models developed with the A673 cell line (resistant to ADW / IMA treatment) confirmed that HSP90 inhibition by 17-AAG, alone or in combination with IGF1R inhibition via AEW541 (an analogue of ADW742, more specific and less toxic), significantly reduced tumor growth and Expression of client proteins.

Immunohistochemical studies demonstrated a possible degradation of HSP90 client proteins after treatment, which explains the effect of stunting tumor, as described previously (50). In the case of combination, not only the tumor growth rate was reduced it also reduced the size of the tumors during experiment, confirming the suggestion of a possible application of the combination of HSP90 + IGF1R inhibition in the treatment of BE.

It should be noted that using two groups of independent clinical samples, it was found that about half of IGF1R positive tumors also showed overexpression diffuse of HSP90. This is obviously not related to any type. of response to stress since these biopsies have been taken before treatment. Anyway, this data is important as it suggests that the stress response mechanisms are already active in some patients of SE at the time of diagnosis. In those patients, the overexpression of HSP90 could be a treatment resistance marker to those same agents blockers All this delimits a subgroup of patients who could benefit from the combination of agents anti-HSP90 when considering therapies aimed at the IGF1R target and underlines the importance of studying the levels of HSP90 expression in patient samples included in the trials clinicians using blocking agents or IGF1R inhibitors.

It should be noted that the sensitivity to ADW / IMA drugs depend not only on expression levels and on the basal activation of IGF1R / KIT, but also, and for the first time described in Ewing's sarcoma, of the development of the mechanism of stress response through HSP90 protein that directly interacts with IGF1R.

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In summary, new mechanisms of resistance to treatment not yet described in the SE., so that the synergy between HSP90 inhibition with treatment with ADW / IMA would have therapeutic value in the SE. Finally, in view of Recent clinical trials using IGF1R inhibitors, the HSP90 expression could be evaluated as a predictive factor of the treatment response

Therefore, an aspect of the present invention constitutes a procedure to design a therapy individual antitumor to a human subject, hereinafter procedure of the invention, based on the detection of protein levels HSP90 responsible for resistance to a drug or mixture of receptor inhibitor antitumor drugs IGF1R / KIT and comprising:

to)

Ex vivo determination of the expression levels of the HSP90 protein gene in a tumor biological sample,

b)

comparison of the levels of a) with with respect to a biological control sample,

C)

resistance determination to IGF1R / KIT receptor inhibitors when levels of a) are greater than those obtained in b), and

d)

selection of an inhibitor of the HSP90 protein as an adjuvant anti-tumor drug of a anti-tumor inhibitor of IGF1R / KIT receptors.

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In mammalian cells there are 2 genes that encode cytosolic homologs of the HSP90 protein, resulting that human α-HSP90 has 85% of sequence homology with β-HSP90. It is believed that the α- and β- forms are due to a duplication gene that will have occurred millions of years ago. Both proteins, the α-HSP90 protein and / or the β-HSP90 form dimers for constitution posterior of the protein complex with other proteins which is what it is associated with its client proteins, being able to form in the cell dimers α-HSP90 / α-HSP90, α-HSP90 / β-HSP90 or β-HSP90 / β-HSP90, of such . so that the study of the expression or inhibition of anyone of both proteins is part of the object of the present invention, although preferably the study of the expression or inhibition of the β-HSP90 form.

As used in the present invention the term "HSP90 protein" or "protein coding gene HSP90 "refers to both the α-HSP90 form or? -HSP90, either in the protein form or DNA / mRNA, respectively.

A particular aspect of the invention is constitutes the process of the invention in which the sample biological tumor as mentioned in step a) belongs, to illustrative title and without limiting the scope of the invention, to Next group: Swing sarcoma, stromal tumor gastrointestinal (GIST) and dermatofibrosarcoma protuberans.

Another particular aspect of the invention is it constitutes the process of the invention in which the determination of protein gene expression levels HSP90 are carried out with or without the induction of a stress of the cells of the biological tumor sample using a compound anti-tumor inhibitor of IGF1R / KIT receptors.

Another particular aspect of the invention is it constitutes the process of the invention in which the determination of protein gene expression levels HSP90 as mentioned in step a) is carried out by the determination of mRNA levels of the Hsp90 gene, either from the beta or alpha form, and comprising:

i)

the mRNA extraction from the tumor biological sample,

ii)

mRNA amplification of the Hsp90 gene, Y

iii)

mRNA levels determination of the Hsp90 gene.

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Another more particular embodiment of the invention it is constituted by the process of the invention in which the mRNA amplification of the Hsp90 gene consists of amplification of the beta (β) form of said gene.

Another more particular embodiment of the invention it is the process of the invention in which the mRNA amplification of the Hsp90 gene consists of amplification of the alpha (α) form of said gene.

Another particular aspect of the invention is it constitutes the process of the invention in which the determination of protein gene expression levels HSP90 of a) is carried out by determining the levels of the HSP90 protein and comprising:

i)

the extraction of proteins from the biological tumor sample, Y

iii)

determination of the levels of the HSP90 protein, either by an immune method (antibodies, already whether monoclonal or not) or nonimmune.

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Another more particular embodiment of the invention it is constituted by the process of the invention in which the HSP90 protein levels determination consists of the determination of the beta (β) form of said protein.

Another more particular embodiment of the invention it is constituted by the process of the invention in which the HSP90 protein levels determination consists of the determination of the alpha (α) form of said protein.

Another more particular aspect of the invention is constitutes the process of the invention in which the inhibitor of the selected HSP90 protein belongs, by way of illustration, to the following group: geldanamicin, 17-AAG, 17-DMAG, retaspimycin (IPI-504), Non-benzoquinone ansamycin compounds diarylpyrazoles (CCT018159 and CCT0129397 / VER49009) and novobiocin.

Another more particular aspect of the invention is constitutes the process of the invention in which the inhibitor of the IGF1R / KIT receivers is selected as an illustration of the Next group: ADW742 (ADW), AEW541 (AEW) and Imatinib (IMA).

Another more particular embodiment of the invention it is constituted by the process of the invention in which the HSP90 protein inhibitor selected is the compound 17-AAG adjuvant of a compound inhibitor of IGF1R / KIT receivers belonging, by way of illustration and without limit the scope of the invention to the following group: ADW742 (ADW),  AEW541 (AEW) and Imatinib (IMA).

Another aspect of the invention is the use of an HSP90 protein activity inhibitor compound, either human or animal, hereinafter use of a compound inhibitor of the present invention, in the preparation of a medicament or Pharmaceutical composition useful for the treatment of a tumor, human or animal, resistant to treatment with inhibitors of IGF1 / KIT receivers.

As used in the present invention the term "inhibitor compound" refers to a molecule that when it binds or interacts with the HSP90 protein, it is either human or another mammal, decreases or eliminates the intensity or duration of the biological activity of said protein. This definition includes in addition those compounds that prevent or reduce the expression of HSP90 protein coding gene, that is, that prevent or decrease gene transcription, mRNA maturation, mRNA translation, and even prevent modification adequate post-translational protein. A inhibitor compound may be constituted by a peptide, a protein, a nucleic acid or polynucleotide, a carbohydrate, a antibody, a chemical compound or any other type of molecule that diminishes or eliminates the effect and / or function of the protein HSP90.

Another more particular aspect of the invention is the use of a compound of the invention in which the HSP90 protein inhibitor belongs, by way of illustration and without limiting the scope of the invention, to the following group: geldanamicin, 17-AAG , 17-DMAG, retaspimycin (IPI-504), non-benzoquinone ansamycin, diarylpyrazole compounds (CCT018159 and CCT0129397 / VER49009) and novobiocin (for details and examples of compounds see: Swee Sharp and Paul Workman (2006). Inhibitors of the HSP90 Molecular Chaperone: Current Status ADVANCES IN CANCER RESEARCH 95: 323-348; Solit DB, Chiosis G (2008). Development and application of Hsp90 inhibitors. DRUG DISCOVERY TODAY 13. 1-2: 38-43; Brough PA et al (2008) 4,5-diarylisoxazole HSP90 chaperone inhibitors: Potential therapeutic agents for the treatment of cancer JOURNAL OF MEDICINAL CHEMISTRY 51, 2: 196-218; Menzella H et al (2007) Potent non-benzoquinone ansamycin Hsp90 inhibitors from mutasynthesis, MOLECULAR CANCER THERAPEUTICS 6 (12): 3602S-3602S).

HSP90 protein inhibitors are being used in different tumors, more specifically the Retaspimycin (Infinity) is being used for treatment of gastrointestinal stromal tumors and tissue sarcomas soft; in patients in non-small cell lung tumors (NSCLC) in and patients with prostate cancer resistant to hormonal treatment On the other hand, tanespimycin has shown activity in HER2 positive metastatic breast tumors, and even in the case of Non-Benzoquinone Ansamycin It has been used for treatment in animal models of leukemia acute myelodes, multiple myeloma and colon cancer.

On the other hand, and by way of illustration, the mentioned polynucleotide can be a polynucleotide that encodes a sequence specific antisense nucleotide sequence of the HSP90 protein mRNA or gene, or a polynucleotide encoding a specific mRNA ribozyme of the HSP90 protein, or a polynucleotide encoding a specific aptamer of the HSP90 protein mRNA, either polynucleotide encoding an RNA of interference ("small interference RNA" or siRNA or a shRNA) specific for the HSP90 protein mRNA or a microRNA (miRNA).

These mentioned polynucleotides can be used in a gene therapy process in which by any technique or procedure is allowed to integrate themselves in the cells of a human or animal patient. This goal can be achieved by administration to these cells tumor of a gene construct that comprises at least one of the aforementioned polynucleotides in order to transform said cells allowing their expression inside them of so that the expression of the HSP90 protein gene is inhibited. Advantageously, said gene construct may be included. within a vector, such as, for example, an expression vector or A transfer vector.

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As used in the present invention the term "vector" refers to systems used in the process of transfer of an exogenous gene or of a gene construct exogenous inside a cell, thus allowing the Gene vehicle and exogenous gene constructs. Sayings vectors can be non-viral vectors or viral vectors (Pfeifer and Verma, 2001) and its administration can be prepared by a subject matter expert based on needs and specificities of each case.

Therefore, another more particular aspect of the invention is constituted by the use of an HSP90 inhibitor compound of the invention in which the inhibitor compound is a nucleic acid or polynucleotide that prevents or decreases gene expression encoder of the HSP90 protein, either human or animal, and that includes at least one selected nucleotide sequence between:

to)

a antisense nucleotide sequence specific to the sequence of the HSP90 protein mRNA or gene,

b)

a HSP90 protein mRNA specific ribozyme,

C)

a HSP90 protein mRNA specific aptamer,

d)

a RNA interference (siRNA or shRNA) specific to the mRNA of the HSP90 protein, and

and)

a microRNA (miRNA) specific for the HSP90 protein mRNA.

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Previously siRNAs have been described that inhibit the expression of HSP90 (Chatterjee M, Jain S, StühmerT, Andrulis M, Ungethüm U, Kuban RJ, Lorentz H, Bommert K, Topp M, Krämer D, Müller-Hermelink HK, Einsele H, Greiner A, Bargou RC. (2007) Heat shock protein 90 alpha und beta are overexpressed in multiple myeloma cells and critically contribute to survival '' 1: Blood. fifteen; 109 (2): 720-8; Håvik B, Bramham CR. (2007) Additive viability-loss following hsp70 / hsc70 double interference and Hsp90 inhibition in two breast cancer cell lines. Oncol Rep. 17 (6): 1501-10; Didelot C, Lanneau D, Brunet M, Bouchot A, Cartier J, Jacquel A, Ducoroy P, Cathelin S, Decologne N, Chiosis G, Dubrez-Daloz L, Solary E, Garrido C. (2008) Interaction of heat-shock protein 90beta isoform (HSP90beta) with cellular inhibitor of apoptosis 1 (c-IAP1) is required for cell differentiation. Cell Death Differ 2008 15 (5): 859-66; John H. Connor, Margie O. McKenzie, Griffith D. Parks, and Douglas S. Lyles. (2007) Published online 2007 January 26. doi: 10.1016 / J. virol Antiviral activity and RNA polymerase degradation following Hsp90 inhibition in a range of negative strand viruses. Virology May 25; 362 (1): 109-119; Fang He, Zhuan-Hong Qiao, Jian Cai, William Pierce, Da-Cheng He, and Zhao-Hui Song (2006) Involvement of the 90-kDa heat shock protein (Hsp-90) in CB2 cannabinoid receptor-mediated cell migration: a new role of Hsp-90 in migration signaling of a G protein-coupled receptor. Nan Fang Yi Ke Da Xue Xue Beam. 26 (8): 1118-20; Chen XM, Zou F. (2006) Stress response changes of NIH-3T3 cells with HSP90alpha expression inhibition by RNA interference. 1: Nan Fang Yi Ke Da Xue Xue Bao. 26 (8): 1118-20; Zaarur N, Gabai VL, Porco JA Jr, Calderwood S, Sherman MY. (2006) Targeting heat shock response to sensitize cancer cells to proteasome and Hsp90 inhibitors. Cancer Res. 66 (3): 1783-91). Any of these sequences from nucleotides described in the state of the art as inhibitors of HSP90 expression are incorporated as embodiments of the present invention as potential therapeutic compounds useful for the manufacture of medicines for the treatment of a process tumor resistant to IGF1 / KIT inhibitors. On the other hand, these techniques of gene inhibition, and more specifically the vehiculization of the compounds - antisense oligonucleotides, iRNA, ribozymes or aptamers - can be carried out by use of liposomes, nanoparticles or other vehiculizers that increase the success of said transfer into the cell, preferably to the cell nucleus (Lu and Woodle, 2005; Hawker and Wooley, 2005). In principle, inhibitors of the translation of HSP90 mRNA that bind so much to your region coding as the non-coding, for example in front of the zone 3 'non-coding.

Thus, another particular embodiment of the invention it is constituted by the use of an RNAi as mentioned in step d) in which the siRNA is constituted by the sequence pair of siRNA specific nucleotides of the following beta form: Sequence sequence: GGUGACAAUCUCCAAUAGAtt (SEQ ID NO1) and sequence antisense: UCUAUUGGAGAUUGUCACCttde (SEQ ID NO2) (sequences 5 '-> 3, see RefSeq: NM_007355-siRNA ID: 119760; Ambion, Austin, TX), and which has been used herein invention, and directed against the target exons of siRNA: NM_007355: exon 10.11.

Cornos has been commented previously, in the human mammalian cells, there are 2 genes the gene α-HSP90 and the gene β-HSP90 with a high homology of sequence. In this regard, and in order to take advantage of the strategy of inhibiting the expression of both genes can, of a preferably, use gene silencing systems that specifically bind to mRNA regions conserved in both forms α-HSP90 and β-HSP90, creating greater synergy in inhibition of both HSP90 proteins, which can no longer participate in the formation of dimers (this strategy is what has led the selection of the above siRNA (SEQ ID NO1 and 2), which recognize a region common to forms α- and β-).

The nucleotide sequences mentioned previously in sections a), b), c), d) and e), prevent gene expression in mRNA or mRNA in HSP90 protein, and, by therefore, they cancel their biological function, and can be developed by an expert in the genetic engineering sector based on existing knowledge in the state of the art about transgenesis and cancellation of gene expression. On the other hand, the inhibitors of HSP90 can be used in isolation in the preparation of antitumor drug described in the present invention or in combination with other antitumor drugs to take advantage of the synergies of different mechanisms of action, and especially in combination with IGFR1 / KIT protein inhibitors, and more specifically with described in the present invention.

Therefore, another more particular aspect of the invention is constituted by the use of an HSP90 inhibitor compound of the invention comprising the use in combination of an inhibitor of IGFR1 / KIT proteins, and more specifically in combination with, at less, the compound ADW742 (ADW), AEW541 (AEW) and Imatinib (IMA).

Another aspect of the present invention is it constitutes a pharmaceutical composition or a medicament useful for the treatment of a tumor process resistant to inhibitors of IGF1 / KIT, hereinafter pharmaceutical composition of the present invention, which comprises a therapeutically effective amount of an HSP90 protein inhibitor compound or agent, together with, optionally, one or more pharmaceutical adjuvants and / or vehicles acceptable.

Another more particular aspect of the invention is constitutes the pharmaceutical composition of the invention comprising a HSP90 protein inhibitor belonging, by way of illustrative and without limiting the scope of the invention, to next group: geldanamicin, 17-AAG, 17-DMAG, retaspimycin (IPI-504), Non-benzoquinone ansamycin compounds diarylpyrazoles (CCT018159 and CCT0129397 / VER49009) and novobiocin.

Another more particular aspect of the invention is constitutes the pharmaceutical composition of the invention that it comprises, in addition to the HSP90 protein inhibitor, an inhibitor of the IGFR1 / KIT proteins, and more specifically, at least one inhibitor belonging to the following group: ADW742 (ADW), AEW541 (AEW) and Imatinib (IMA).

Another more particular aspect of the invention is constitutes the pharmaceutical composition of the invention in which the inhibitor compound is a nucleic acid or polynucleotide that prevents or decreases the expression of the HSP90 protein coding gene human and that includes at least one nucleotide sequence selected from:

to)

a antisense nucleotide sequence specific to the sequence of the HSP90 protein mRNA or gene,

b)

a HSP90 protein mRNA specific ribozyme,

C)

a HSP90 protein mRNA specific aptamer,

d)

a Interference RNA (iRNA) specific to the protein mRNA HSP90, and

and)

a microRNA (miRNA) specific for the HSP90 protein mRNA.

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Another particular embodiment of the invention is constitutes the pharmaceutical composition of the invention in which the HSP90 inhibitor is a siRNA constituted by the pair of specific nucleotide sequences of the beta form siRNA next: sequence meaning: GGUGACAAUCUCCAAUAGAtt (SEQ ID NO1) and antisense sequence: UCUAUUGGAGAUUGUCACCtt of (SEQ ID NO2) (sequences 5 '-> 3, see RefSeq: NM_007355-siRNA ID: 119760; Ambion, Austin, TX), and that has been used in the present invention, and directed against the target exons of siRNA: NM_007355: exon 10.11.

Pharmaceutical adjuvants and vehicles Acceptable that can be used in such compositions are the adjuvants and vehicles known to those skilled in the art and commonly used in the preparation of compositions therapeutic

In the sense used in this description, the expression "therapeutically effective amount" refers to the amount of agent or compound inhibiting the activity of the HSP90 protein, calculated to produce the desired effect and, in In general, it will be determined, among other causes, by own characteristics of the compounds, including age, patient's condition, severity of the disorder or disorder, and of the route and frequency of administration.

In a particular embodiment, said composition Therapeutic is prepared in the form of a solid form or suspension aqueous, in a pharmaceutically acceptable diluent. The composition Therapeutic provided by this invention can be administered by any appropriate route of administration, for which said Composition will be formulated in the appropriate pharmaceutical form to the route of chosen administration. In a particular embodiment, the administration of the therapeutic composition provided by this invention is carried out parenterally, orally, orally intraperitoneal, subcutaneous, etc. A review of the different pharmaceutical forms of drug administration and of excipients necessary to obtain them can found, for example, in Faulí i Trillo, 1993.

Another object of the present invention is constitutes the use of the pharmaceutical composition of the invention in a method of treating a mammal, preferably a being human, affected by a tumor process or disease resistant to IGF1 / KIT receptor inhibitors, hereinafter use of the pharmaceutical composition of the present invention, consisting of the administration of said therapeutic composition that inhibits the tumor process

Description of the figures

Figure 1.- Results of 2D gels and validation of HSP90 expression . Lines A673 and A4573 were treated with 0.1 µM of ADW742 and / or 10 µM of Imatinib, for 24 and 72 hours, and subsequently the protein was extracted. A) 2D gels of the A673 line. The panel on the left presents a control situation and the one on the right one of the ADW treatment. After the second dimension, a manual analysis of the protein points with more relevant changes was made and these were analyzed by MALDI-ToF. The proteins highlighted in the image are: 1 and 3- not determined by having a low score; 2- PRDX1, 4- NASP, 5-HSP90. The close-up image shows the expression changes of the HSP90 protein. B) Function of proteins that showed changes in expression after treatment with ADW and / or IMA. The largest group is that of the proteins involved in the response to stress (21%) and apoptosis (17%). C) Densitometric analysis of the study by WB of the effects of treatment with ADW and / or IMA on HSP90 levels. HSP90 expression increased at 24 hours on lines A673 and TTC466 and decreased on the other lines. At 72 hours the expression of HSP90 decreased in all cell lines, especially in A673. *: p <0.05, determined with the ANOVA One-Way analysis of the Variance for Independent Samples.

Figure 2.- Effects of treatment with 17-AAG on the proliferation and apoptosis of the SE lines . Sub-confluent cells have been treated with 17-AAG (0.01-25 µM) alone or in combination with ADW742 (0.1-0.5 µM) or Imatinib (5-15 µM), grown by 24 or 72 hours, at different combinations, and the proliferation measured by the MTT assay and apoptosis analyzed by flow cytometry, with detection with Annexin-V / PI. A) IC50 proliferation (µM) of the SE lines treated at 24 and 72 hours with 17-AAG. All lines have had similar results, with a dose-dependent growth inhibition. The IC 50 has depended on each cell line. B) Representation of apoptosis levels obtained in different treatments of the A673 line treated with 17-AAG, ADW and / or IMA for 72 hours. While with monotreatment apoptosis was not induced, with combinations if induction of apoptosis was obtained. A greater impact was obtained in the levels of late apoptosis / necrosis than in those of early apoptosis. C) Graphical representation of all treatment conditions. The higher the concentration of the drugs used, the higher the ratio between apoptotic / necrotic cells and living cells, indicating an increase in the induction of cell death. The effect of combining 17AAG with ADW742 or Imatinib resulted in an additional 10-50% increase in apoptosis, depending on the cell line studied. Additive effects of the combinations have been observed mainly but in the case of line A673 synergistic effects have been observed. A / N: apoptosis / necrosis; A: ADW and I: IMA.

Figure 3.- Effects of treatment with 17-AAG on the expression and phosphorylation of some HSP90 client proteins in SE cell lines . A) Lines A673 and A4573 were treated with 0.25 µM 17-AAG for 24 and 72 hours, stimulated with IGF1 for 10 minutes and the extracted protein. Treatment with 17AAG resulted in a degradation of IGF1R, c-kit and AKT, with a decrease in phosphorylation of AKT, mTOR and MAPK42 / 44. Degradation levels have long depended on the cell line used, the A673 line being more sensitive to this type of treatment than A4573, in which there is almost no degradation of IGF1R or KIT. B) Effects of treatment with 17-AAG on the physical interaction of HSP90 and some of its client proteins. Line A673 was treated with 0.1 µM 17-AAG for 72 hours, immunoprecipitation made against HSP90, c-kit, AKT and IGF1R and the extracted proteins. All the proteins studied were in the total extract (TE; first band on the left) and c-KIT, AKT and IGF1R were co-immunoprecipitated with HSP90 (eighth band on the left). Treatment with 17-AAG reduced the interaction of IGF1R, AKT and c-KIT with HSP90 (ninth band). The client proteins did not immunoprecipitate each other (in each blot no signal is observed for any other client protein but only the specifically immunoprecipitated one, together with the HSP90 signal), since HSP90 can only interact with one PC at a time, being observable just an IP signal for the PC that is "chaperoning".

Figure 4.- Effects of induction of HSP90 expression in the SE lines . A) Effects of HSP90-siRNA on HSP90 expression of lines A673 and TTC466. Sub-confluent cells have been treated with HSP90-siRNA (75 nM), grown for 24 hours and HSP90 expression analyzed. HSP90-siRNA reduced HSP90 expression by 35-65% while neg.-siRNA had no effect. B) Antiproliferative effects of blocking HSP90 expression with HSP90-siRNA combined with ADW / IMA. Subconfluent cells were transfected with HSP90-siRNA (75 nM), grown for 24 hours and treated with ADW742 (0.1-0.5 µM) or Imatinib (5-15 µM Imatinib), for an additional 72 hours, and the Proliferation was analyzed by the MTT assay. The results obtained with the HSP90-siRNA were very similar to those of the 17-AAG treatment, with a decrease in the proliferation of the order of 20-30%. C) "Time-course" of the transfection effects of the HSP90 clone on the expression of HSP90. Sub-confluent cells were transfected with a clone of HSP90 (15 µg), grown for 96 hours and the expression of HSP90 analyzed. The HSP90 clone effectively induced an overexpression of HSP90, especially in the A4573 line. D) Effects of the combination of clone HSP90 with ADW / IMA at the level of proliferation of SE lines. Subconfluent A4573, SK-ES-1 and TC-71 cells were transfected with the HSP90 clone (15 µg), grown for 24 hours and treated with ADW742 (0.1-0.5 µM) or Imatinib (5- 15 µM Imatinib), for an additional 72 hours, and proliferation was analyzed by the MTT assay. Over-expression of HSP90 induced an increase in proliferation, obtaining values above 75%, even after treatment with ADW / IMA. *: p <0.05, determined with the ANOVA One-Way analysis of the Variance for Independent Samples.

Figure 5.- In vivo studies of treatment with 17-AAG, alone or in combination with the AEW541 and its correlation with clinical samples . NOD-Scid mice were injected sc with 5x106 A673 cells. They were randomized in groups treated with vehicle (25 mM tartaric acid or PBSTween 80 0.05% DMSO 10%), 17-AAG only (80 mg / kg / day), AEW only (50 mg / kg / day) or 17-AAG + AEW (same conditions), for 15 days. A) Inhibition of tumor growth of mice treated with 17-AAG alone or in combination with AEW541. Tumors in the control group showed an increase of 340% and those in the group treated with AEW had an increase of 270%. The mice in the group treated with 17AAG only had an increase of 72% and those in the group of the combination 17-AAG + AEW -38%. Animals treated with 17-AAG showed a delay in tumor growth while those in the combination had a reduction in tumor size., About 40% *: p <0.05, determined with the ANOVA One-Way Analysis of Variance for Samples Independent. *: 17-AAG alone or 17-AAG + AEW vs control; *: 17-AAG + AEW vs 17-AAG alone. B) Morphological and visual evaluation of animal tumors. The H&E stained sections of the tumors of the animals treated with 17AAG + AEW (lower panel) indicated a marked increase in the neurotized area and a drastic reduction in size, with respect to the tumors of the control group (upper panel). Bar: 1cm C) ICQ evaluation of the expression levels of HSP90, IGF1R, AKT, m-TOR and c-kit of animal tumors after treatment with 17-AAG and / or AEW, for two weeks. The expression of IGF1R, c-kit and AKT was almost completely eliminated in animals treated with 17-AAG + AEW (right), with respect to the control group (left). However, the expression of m-TOR had no significant changes. (400x). D) ICQ evaluation of HSP90 expression in clinical SE samples. The expression of HSP90 and IGF1R was cytoplasmic and uniform in the tumor. The intensity was low to moderate for HSP90 and moderate to intense for IGF1R. KIT expression was detected in the cell membrane, with a heterogeneous signal and moderate to intense intensity. An image of low magnification (4x), an intermediate view (200x) and a view of greater magnification (1000x) in positive cases and an intermediate one for negative cases are presented.

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Examples of realization Example 1 HSP90 protein levels are determinants in the Ewing Sarcoma cell line response to treatment with ADW and / or IMA and its inhibition sensitizes cell lines resistant to these treatments 1.1.- Treatment with ADW742 (ADW) and / or Imatinib (IMA) induces proteomic changes in sarcoma cell lines of Ewing

Since with previous studies of inhibition of IGF1R / KIT routes were verified that the combination of inhibition of IGF1R with that of c-kit had synergistic effects at level of proliferation inhibition, induction of apoptosis and blocking the signaling route (14), it was decided to do a study proteomic to be able to characterize the changes more broadly of protein expression induced in SE cells after ADW and / or IMA treatment. This study was also intended identify relevant proteins that could be used as possible targets / markers of the treatment response in the BE.

Initially, line A673 and A4573 (the more resistant and treatment sensitive lines, respectively) with 0.1 µM of ADW and / or 10 µM of IMA, for 24 and 72 hours, then making the respective protein extracts and analyzing the proteome with a two-dimensional electrophoresis (Figure 1A). For each of the gels obtained an analysis was executed comparative manual of the changes of expression of the points most relevant proteins and these were analyzed by MALDI-TOF mass spectroscopy.

Thus, a large number of proteins were detected whose expression was altered under treatment (in these analyzes a level of " minimal store " of identity of 65 was chosen), which have been summarized in Table 1. The detected proteins are involved in numerous important cellular processes, such as proliferation regulation (EBP1, RhoA), apoptosis (Set, 14-3-3) and stress response (GRP75, HSP90, HSP70)
(Figure 1B).

With the data obtained, an analysis of the interaction of signaling routes with the Ingenuity Pathway Analysis software was performed, in order to detect the most important signaling routes / networks altered with the treatment, selecting a smaller panel of relevant proteins (marked with * in the table), which were subsequently studied at the level of mRNA and protein.

TABLE 1 Proteins identified with 2D / MALDI-ToF electrophoresis studies. Each protein was identified by " matching peptide-mass fingerprínting " against a non-redundant Swiss-Prot human database. The proteins marked with * were characterized at the level of mRNA and protein

100

200

With the studies of qRT-PCR and WB it was shown that almost all the proteins chosen were really altered, both at the level of mRNA and protein expression, and that the results were not due to the unspecificity of the cell background , typical of each line mobile. Significant differences have been obtained between the studies at 24 and 72 hours of treatment, mainly demonstrating an initial activation of the stress response at 24 hours of treatment and obtaining more consistent effects and cell / drug-specific line at 72 hours. Significant differences have been verified between the two cell lines studied.

Globally it was observed that the mono-treatment (ADW or IMA) had similar effects and that it was less effective than its combination (ADW + IMA). Proteins overexpressed were related to the apoptosis and under-expressed were related with the proliferation and response to stress.

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1.2.- HSP90 protein expression levels are modify dramatically with treatment ADW742 / Imatinib

Seen one of the most cellular processes changed with ADW and / or IMA treatment was the answer to stress, and since specific inhibitors of HSP90, being already characterized its function in cancer, it was decided study the role of HSP90 in the stress response of the lines of SE.

It started by checking the expression changes of HSP90 in the panel of 5 SE cell lines with different Fusion types EWS-ETS (28-31), in control conditions and after treatment with ADW and / or IMA, for 24 or 72 hours

As can be verified by Figure 1C, the HSP90 expression changed in all cell lines, depending on the effects of the line in question. In a global way, at 24 hours of treatment, the expression of HSP90 had a increase in lines A673 and TTC466 and a decrease in other lines At 72 hours, HSP90 expression decreased by all cell lines, especially in the case of A673.

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1.3.- The HSP90 block using 17-AAG inhibits cell proliferation and survival by reducing HSP90 key client protein activation

After confirming the expression changes of HSP90 in the SE lines, we proceeded to study its role in the proliferation and apoptosis of the same cell lines, inhibiting specifically its function when treating cell lines with 17-AAG.

All cell lines have had answers similar to HSP90 inhibition, obtaining an inhibition of proliferation in a dose-dependent manner, at 24 and 72 hours of treatment, with an IC 50 at 24 hours of 1.01-1.62 µM and after 72 hours of treatment of 0.42-0.72 µM (Figure 2A).

It was verified that the effects of treatment with 17-AAG in the induction of apoptosis of the lines of SE specifically depended on each line in question. How I know check in Figure 2B and C, the induction of apoptosis varied between 20-30% on lines TTC466 and A673 and the 8-20% in the other cell lines, with an impact more pronounced in the levels of late apoptosis / necrosis than in the of early apoptosis.

The results obtained have been explained, at least partially, when the expression levels of some HSP90 client proteins were analyzed after treatment with 17-AAG. It was observed that 17-AAG per se (Figure 3A) inhibited the expression of IGF1R, c-kit and AKT and that, as a consequence, a decrease in phosphorylation levels of AKT, m-TOR and MAPK42 / was also obtained. 44. These results were more evident in line A673 than in A4573, in which no significant degradation of IGF1R or c-kit was detected.

Immunoprecipitation studies have demonstrated that IGF1R, KIT and AKT had their physical interaction with HSP90 reduced after treatment with 17-AAG. From Again, the interaction between HSP90 and KIT or IGF1R was much more weak in ADW / IMA sensitive lines (A4573, SK-ES-1 and TC-71) which in the resistant J (A673 and TTC466) (Figure 3B).

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1.4.- The HSP90 block using 17-AAG sensitizes cell lines resistant to treatment with ADW742 or Imatinib

How it was verified that treatment with 17-AAG was effective on lines A673 and TTC466 (which had proved to be the most insensitive to ADW / IMA (14)) and that reduced p-AKT, p-m-TOR and p-MAPK, se decided to study the combination of this drug with ADW or IMA, to level of proliferation and apoptosis of the SE lines.

It was observed that the combination of 17-AAG with ADW / IMA caused an additional decrease of the proliferation of the order of 15-50%, depending of the sensitivity of each cell line. Combining 17-AAG with ADW was synergistic in almost every cell lines (an IC range of 0.35-0.9). In the case of the combination with IMA, observed synergistic effects on lines A673 and TTC466 (in the line A673 the range of IC was 0.53-0.8 and in that of TTC466 line was 0.44-0.796).

In apoptosis studies, a dose-dependent induction of apoptosis after treatment with ADW / IMA. The effect of combining 17-AAG with ADW or IMA caused an additional 10-50% increase in apoptosis, depending on the cell line (Figure 2B and C). Be mainly observed additive effects but, again, of detected synergy (CI 0.5-0.7) in the case of lines A673 and TTC466, reaching levels of just 25% of survival.

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1.5.- HSP90 protein levels are determinants of Ewing's sarcoma cell line response to ADW / IMA treatment

To verify that the results obtained with 17-AAG were due to the specific HSP90 block, He decided to do two additional studies. One of the studies was based in the technique of RNA interference (in which it has been reduced HSP90 expression in ADW / IMA resistant cell lines) and the other was based on induction of overexpression of HSP90 (on ADW / IMA sensitive lines) to prove high HSP90 expression conferred resistance to ADW treatment or IMA.

As suspected, the HSP90-siRNA (which reduced the expression of HSP90 in a 35-65%, as shown in Figure 4A) improved the Effects of ADW or IMA treatment on inhibition of proliferation and induction of apoptosis, the results being very similar to those obtained with 17-AAG (Figure 4B). Be observed a 20-30% decrease in proliferation and 15-40% in the induction of apoptosis of the cells treated with the HSP90-siRNA (when compared to the control situations of non-treatment or neg.-siRNA).

These results confirmed that the results obtained with 17-AAG were specific to the HSP90 inhibition and not due to effects Alternative / nonspecific of this drug. With these results the hypothesis that the expression levels of HSP90 are determinants for the response of SE cells to ADW / IMA treatment.

The proposed hypothesis was also proven. in HSP90 overexpression studies, although by an opposite approach. One was obtained HSP90 overexpression of 30-73% on lines A4573, SK-ES-1 and TTC466, which increased after treatment with ADW / IMA (figure 4C).

As can be verified in Figure 4D, the overexpression of HSP90 induced an increase in proliferation levels of the SE lines, giving them resistance to the pro-apoptotic effects of ADW / IMA. The induction of apoptosis was drastically blocked (not more than 20% induction of apoptosis was obtained under the conditions under treatment, values close to the control situation). All the conditions had values of more than 75%, higher than in non-transfected conditions. These results have not been due, just, for transfection purposes anymore that it was verified that the mock (empty vector) did not affect the proliferation or apoptosis of the studied lines.

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Example 2 Inhibition of HSP90 protein, alone or in combination with IGF1R inhibitors, reduces tumor growth in vivo

After checking in vitro the effects of treatment with 17-AAG, alone or in combination with ADW / IMA, it was decided to conduct an in vivo study, with a xenograph model using the A673 line, resistant to ADW / IMA treatment, assessing the efficacy antitumoral treatment with 17-AAG, alone or in combination with AEW541 (analogue of ADW, which has the same mechanism of action but is more specific for IGF1R and less toxic (32)).

This study confirmed the results obtained in the in vitro studies. While in the final part of the study, in the control situation, an increase in the average tumor size of 340% was detected and in the group treated with AEW of 270%, this increase was only 74% in the group treated with 17AAG and -38% in the combination group (Figure 5A). Animals treated with AEW monotreatment had a similar behavior to those in the control group, while animals treated with 17AAG had a delay in tumor growth during the two weeks of treatment. In the case of the animals of the group treated with the combination of 17-AAG + AEW, these had not only a delay in tumor growth, but also a marked reduction in the average tumor size, approximately 40%. The effect of the combination was even more marked in those animals that initially had lower tumors of medium size, in which the reduction became 66%.

Histopathological studies done with tumor samples demonstrated that tumors treated with the combination were not just smaller, but if they had more areas extensive necrosis, as can be seen in 5B. It was verified that the expression of IGF1R, AKT and c-c-KIT was decreased in tumors treated with 17-AAG, AEW and 17-AAG + AEW, regarding the samples of the control group, which is in agreement with the fact that are HSP90 client proteins. On the contrary, the m-TOR expression did not change significantly with the treatment (except in the case of treatment with AEW).

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Example 3 A subset of positive clinical samples for IGF1R also has HSP90 expression

The expression of HSP90 proteins was analyzed, IGF1R and KIT in two groups of clinical samples of patients with Ewing's sarcoma (independent analysis). The prevalence of expression of these three molecules was similar between both series of samples.

Regarding the expression of IGF1R, a cytoplasmic and relatively uniform marking was detected in the tumors, in both groups of clinical samples of SE (5/10, 50% - in the case of the group of samples of Dr. Enrique of Álava (EAC) and 33/44, 75% - in the case of TMA ). The signal intensity was moderate to intense. The expression of HSP90 was cytoplasmic, in certain cases with a dotted tide, and uniform in the tumors (3/10, 30% - in the EAC samples and 19/44, 43% - in the case of TMA ). The intensity was weak to moderate. The expression of c-kit was membrane, having a heterogeneous tide in tumors, with 64% positivity in cases of CAD (7/11) and 48% in TMA (21/44). The signal intensity was moderate to intense.

It was interesting to verify that, when comparing the expression of IGF1R and HSP90, almost half of the samples have to be IGF1R + were also HSP90 +. However, almost 80% of the samples HSP90 + also had expression of IGF1R. KIT's expression had a behavior very similar to IGF1R.

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Materials and methods Cell lines

The A673 line was obtained from the ECACC ( European Collection of Animal Culture Cells , #cat. 85111504) and the SK-ES-1 was obtained from the ATCC ( American Type Culture Collection , #cat. HTB-86) .The TC line -71 was a gift from Dr. Iranzu González, from the University of Navarra and line A4573 from Dr. Samuel Navarro, from the University of Valencia. The TCC466 line was a gift from Dr. Jaume Mora, from the Hospital Sant Joan de Déu, in Barcelona.

The A673 line has been grown in DMEM medium (Invitrogen / Life Technologies, #cat. 14167) supplemented with 10% of SBF The SK-ES-1 line has grown in between McCoy's (Invitrogen / Life Technologies, #cat. 26600-023) supplemented with 15% of SBF and line TC-71 in IMDM medium; (Invitrogen / Life Technologies, #cat 21980-032) supplemented with 20% SBF. The lines A4573 and TTC466 have been grown in RPMI medium (Invitrogen, Life Technologies, #cat. 21875-034) supplemented with 15 and 10% of SBF, respectively.

All cell lines were analyzed to rule out the presence of mycoplasma, with the VenorGeM kit (Minerva Biolabs, #cat. 11-1050). These lines They have different types of EWS-ETS translocation. Lines A673 and TC-71 have a translocation EWS-FLI1 of tipol, the SK-ES-1 has a translocation EWS-FLI1 type2, the A4573 has a EWS-FLI1 of type3 and the TTC466 has a EWSERG type translocation, as described above (28-31).

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Clinical samples

A total of 62 samples of clinical biopsies of Ewing's sarcoma and 11 other tumors (used as positive phosphorylation or negative controls of translocation), belonging to 23 men and 18 women (32 patients without information), with an average age of 26 years in the moment of diagnosis (18 in the case of SE patients). fifty% of the cases of SE were located in the lower limbs (20.8% in the tibia, 16% in the femur), 12.5% in the upper limbs and 21% in the pelvic region, having 42% of them metastatic in the lungs, bone and marrow at the time of diagnosis.

23 samples were included in paraffin blocks and the remaining 50 were obtained in collaboration with Dr. Christopher Poremba, Heinrich-Heine University, Dusseldorf, Germany, and were in two TMAs ( Tissue Microarray ). The use of this material was approved by the ethical committees of both institutions.

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Pharmaceutical compounds

ADW742 (ADW), AEW541 (AEW) and Imatinib (IMA) have ceded by Novartis Pharma AG, Basel, Switzerland. He 17-AAG was obtained from Alexis Biochemicals, Switzerland (#cat. 380-091-M001). All these drugs have been resuspended in DMSO (10 mM) (with the exception of VCR and DXR that have been resuspended in MiliQ water) and aliquoted in the desired concentrations.

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2D electrophoresis

Initially the cell lines were treated with 0.1 µM of ADW742 and / or 10 µM of Imatinib, for 24 and 72 hours. The respective cell extracts were obtained by scraping of the cells with a handle "Scrapper", on ice, with 500 µl of lysis buffer ((1% NP40, 150 mM NaCl, 50 mM EDTA, 10% glycerol, 20 mM Tris-HCl pH 7, protease inhibitor cocktail (Roche, Switzerland), 50 mM NaF and 2 mM Na_ {3} VO_ {4}). The proteins precipitated with trichloroacetic acid / acetone solubilized in 7 M Urea, 2 M Thiourea, 2% CHAPS and inhibitors of proteases

100 µg of starting protein (determined using the PlusOne Quant Kit (GE Healthcare, Sweden)) 250 µl of rehydration solution (8M have been added urea, 0.5% CHAPS, 0.2% DTT, 0.5% IPG buffer and 0.002% blue bromophenol), placing them in 13 cm strips (pH 3-11), leaving the samples hydrating, for adsorption, for 12 hours.

The isoelectric focusing was done on an Ettan device IPGphor (GE Healthcare, Sweden) at 30,000V.h at 20 ° C. The reduction and the alkylation made according to the manufacturer's instructions (Bio-Rad, USA). SDS-PAGE gels of the second dimension (10 or 12%) have been stained with nitrate of silver.

Differentially expressed protein points have been trypsinized and processed for spectrometry analysis MALDI-ToF mass. Proteins were identified by comparison of the data obtained from the mass of the peptides identified with the human databases of Swiss-Prot (minimum score of 65). It has been realized At least 3 independent experiments. An analysis of interaction of signaling pathways with proteins identified, to highlight the most relevant that changed with the treatment, using the Ingenuity Pathway Analysis Software (Ingenuity® Systems, Redwood City, CA).

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Western Blotting (WB) and Immunoprecipitation (IP)

WB studies have been conducted to study the inhibition of HSP90 PC expression after treatment with 17-AAG.

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Initially the extracts were obtained cell, by scraping the cells on ice, with a handle Scrapper, with 500 µl of lysis buffer ([1% NP40, 150 mmol / L NaCl, 50 mmol / L EDTA, 10% glycerol, 20 mmol / L Tris-HCI (pH 7), inhibitor coktail tablets of proteases (Roche), 50 mmol / L NaF, and 2 mmol / L Na3V04], Protein concentration was determined using the kit BCA (Bicinchoninic Acid Protein) protein determination Assay Reagent (Pierce, Rockford, IL)). 50 \ mug of have been loaded protein per well, have been run on 10% gels and have transferred to PVDF membranes. The blots have been blocked with a solution of TBS-T with 5% BSA, incubated with the specific antibodies and revealed with the ECL kit, from Amersham. To quantitatively analyze the results obtained, we proceeded to a densitometric analysis, with the Quantity One program 4.3.1 (Biorad), of the GELDoc 2000 apparatus, in which the amount of pixels that each band contained, being able to compare and Normalize each band with the control situation.

Immunoprecipitation studies have been performed to detect the physical interaction of HSP90 with its PC

1.5 mg of protein have been immunoprecipitated total with anti-HSP90 antibodies, anti-AKT, anti-c-kit and anti-IGF1R and g-sepharose protein, for 6 hours, at 4 ° C, subsequently washing 4x with a buffer of lysis, and following, from this point, with the WB protocol. 2-3 independent experiments have been performed For each case.

The antibodies used have been: anti-HSP90 (Alexis Biochemicals, Lausen, Switzerland), anti-GAPDH (Abcam, Cambridge UK), anti-IGF1R (Santa Cruz Biotech, Santa Cruz, CA), anti-CD117 (Dako, Carpinteria, CA) and anti-AKT, anti-pAKT (Ser473), anti-mTOR, anti-p-mTOR (Ser2448), anti-MAPK42 / 44, anti-p-MAPK42 / 44 (Thr202 / Tyr204) (all from Cell Signaling, Danvers CA). All these antibodies have have been optimized so as not to have non-specificity at the time of immunodetection

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Proliferation Studies

First, proliferation has been studied of cell lines after treatment with 17-AAG to determine the proliferation IC50. Secondly it has studied the percentage of proliferation inhibition after treatment with 17 AAG combined with ADW / IMA.

4-6 have been made independent experiments

To determine proliferation levels, we has performed the MTT test) 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide).

In summary, 200,000 cells were seeded per well, in 24-well plates and treated with 17-AAG, for 72 hours. After that time of treatment, the culture medium was aspirated from the plates and added to each well 300 µl of a mixture of 1:10 MTT (dissolved in PBS1X, at a final concentration of 5 \ mug / \ mul, protected from light): cellular medium. The plates were incubated for 1 hour at 37 ° C and after that time the mixture was aspirated from the wells and DMSO added (250-500 µl). The plates were shaken for 10-15 minutes at room temperature (TA) and the absorbance at 570 nm was read.

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Apoptosis

Apoptosis has been measured after treatment with 17-AAG alone or in combination with ADW or IMA, for 72 hours. The rates of apoptosis and necrosis have been analyzed by flow cytometry, with the AnnexinV-FITC Apoptosis Detection kit (Immunostep, Spain), according to the manufacturer's instructions. The data was acquired on a FACSort cytometer (Becton Dickinson, Franklin Lakes, NJ) using the CellQuest and Paint-a-Gate software. For each analysis, 20,000 events have been acquired.

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siRNA

The HSP90 validated siRNA and Silencer negative control siRNA have been purchased from Ambion (Austin, TX) (RefSeq: NM_007355-siRNA ID: 119760 and Cat # AM4611, respectively). In summary, lines A673 and TTC466 have been maintained in culture as described above, centrifuged at 1500 g for 5 minutes and resuspended in 200 µl of medium with 75 nM HSP90siRNA, 5 µL 1.5 M NaCl and 20 µg of DNA carrier, and transferred to electroporation cuvettes of Bio-Rad 0.4-cm. Electroporation was done in a Gene Pulser II (Bio-Rad) at 126 V / 800 µF. The cells were transferred to 10 ml of culture medium and grown for 24 hours before treatment. Proliferation and apoptosis have been analyzed with the methods described above.

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HSP90 Transfection

The HSP90 clone clone was obtained from Invitrogen Ultímate ORF clones resource (Carlsbad, CA), cloneID:
IOH61079, and efficiently transferred to a Gateway Destination Vector (Invitrogen PCDNA3.1 (+), catalog No. V790-20), previously linked with an RfcassetteA of the Invitrogen Gateway Vector Conversion System (catalog No. 11828-029), using the Gateway LR Clonase Enzyme mix (catalog nº 11791-019), according to the manufacturer's instructions. The Gateway Destination Vector containing the HSP90 clone or the empty vector (Mock) (15 µg) has been transfected into lines A4573, SK-ES-1 and TC71 by electroporation, as described above. Proliferation and apoptosis have also been analyzed with the methods described above.

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In vivo studies

Young NOD / SCID mice of 4-5 weeks (NOD / CrCrl-Prkdc scid, provided by Charles River laboratories), under the Spanish guidelines and the European Union ((RD 1201/05 and 86/609 / EEC, respectively).

5x10 6 A673 cells in exponential growth phase were injected subcutaneously into the right flank of the mice in a Matrigel cell suspension (0.2 ml of cells resuspended in 1: 1 Matrigel Matrix cell media (BD Bioscience , #cat. 35624) Mice were randomized in control and treatment groups (2-3 weeks after injection, zero day of treatment) (mice without tumor and those whose volume exceeded 1 cm3 were not included in the analysis) The treatments were as follows: vehicle 1 (25 mM tartaric acid, orally, 2 times per day, 7 days / week, for 2 weeks); vehicle 2 (PBSTween 80 0.05% DMSO 10%, ip, twice per day, 5 days / week, for 2 weeks), 17-AAG only (ip, twice a day, 80 mg / kg / day on days 0 to 5 and 8 to 12); AEW only (50 mg / kg / day, orally, twice a day, 7 days / week, for 2 weeks) or 17-AAG + AEW (same conditions) The evolution of the tumors and the effect of the tumor were monitored. The treatments, assessing the condition of the animals daily. The tumors were measured every two-three days with a caliber, and the diameters noted. Tumor volumes were calculated according to the following formula: 300 where a is the smallest diameter and b the largest.

At the end of the study the mice were sacrificed for overexposure to Isofluoran and tumors extracted for subsequent histopathological analysis.

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Histopathological Analysis

In the case of the animal model, at the time of animal sacrifice, tumors were placed directly in formalin and processed as quickly as possible (in less than 15 minutes) and were included in paraffin. In the case of samples clinics, formalin fixed preparations were used, included in paraffin. All sections have been stained with hematoxylin-eosin, and prepared for studies of ICQ.

Two sets of clinical samples of SE have been used, obtained before treatment to verify the results obtained in vitro / in vivo .

For the ICQ, two periods of treatment for 8 minutes at 95 ° C and 4 minutes at 100 ° C, in a pot at pressure, using Tris-EDTA buffer, pH8.0 (except for IGF1R, in which the citrate buffer pH6.0 was used) before dewaxing the sections. These were incubated at then at 37 ° C, for 1 hour, with the desired antibodies (anti-HSP90 (Alexis Biochemicals ALX 804, dilution 1/500), anti-IGF1R (Santa Cruz Biotechnologies, 1/50 dilution), anti-anti AKT (Cell Signaling) Technologies # 9272, dilution 1/500), anti-m-TOR (Cell Signaling) Technologies # 2972, 1/50 dilution) and anti-c-kit (Dako Cytomation A4502, dilution 1/200). The marking was done with the IHC DAB MAP system (Ventana Medical Systems, Tucson, AR). The results were evaluated at blind for molecular and clinicopathological data. The number and The intensity of the immunoreactive cells were analyzed in 3 randomly chosen fields. The signal strength was classified as + a +++ (+: weak intensity signal, ++: intensity signal moderate and +++: strong intensity signal).

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Statistic analysis

To study the statistical significance of all the results obtained, an ANOVA One-Way analysis of the Variance for Independent Samples was performed, using the VassarStats for Statistical Computation website .

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References

1. Bacci G, Ferrari S, Rosito P, et al . [Ewing's sarcoma of the bone. Anatomoclinical study of 424 cases]. Minerva Pediatric 1992 ; 44: 345-59.

2. Koscielniak E, Jurgens H, Winkler K, et al . Treatment of soft tissue sarcoma in childhood and adolescence. A report of the German Cooperative Soft Tissue Sarcoma Study. Cancer 1992 ; 70: 2557-67.

3. Cooper R, Kaanders JH. Biological surrogate end-points in cancer triáis: potential uses, benefits and pitfalls. Eur J Cancer 2005 ; 41: 1261-6.

4. Furukawa M, Raffeld M, Matthew C, et al . Increased expression of insulin-like growth factor I and / or its receptor in gastrinomas is associated with low curability, increased growth, and development of metastases. Clin Cancer Res 2005 ; 11: 3233-42.

5. González I, Andreu EJ, Panizo A, et al . Imatinib inhibits proliferation of Ewing tumor cells mediated by the stem cell factor / KIT receptor pathway, and sensitizes cells to vincristine and doxorubicin-induced apoptosis. Clin Cancer Res 2004 ; 10: 751-61.

6. Heinrich MC, Blanke CD, Druker BJ, Corles s CL. Inhibition of KIT tyrosine kinase activity: a novel molecular approach to the treatment of KIT-positive malignancies. J Clin Oncol 2002 ; 20: 1692-703.

7. Landuzzi L, De Giovanni C, Nicoletti G, et al . The metastatic ability of Ewing's sarcoma cells is modulated by stem cell factor and by its receptor c-kit. Am J Pathol 2000 ; 157: 2123-31.

8. Mateo-Lozano S, Tirado OM, Notary V. Rapamycin induces the fusion-type independent downregulation of the EWS / FLI-1 proteins and inhibits Ewing's sarcoma cell proliferation. Oncogene 2003 ; 22: 9282-7.

9. Merchant MS, Woo CW, Mackall CL, Thiele CJ. Potential use of imatinib in Ewing's Sarcoma: evidence for in vitro and in vivo activity. J Natl Cancer Inst 2002 ; 94: 1673-9.

10. Pollak MN, Schernhammer ES, Hankinson SE. Insulin-like growth factors and neoplasia. Nat Rev Cancer 2004 ; 4: 505-18.

11. Samani AA, Chevet E, Fallavollita L, Galipeau J, Brodt P. Loss of tumorigenicity and metastatic potential in carcinoma cells expressing the extracellular domain of the type 1 insulin-like growth factor receptor. Cancer Res 2004 ; 64: 3380-5.

12. Scotlandi K, Benini S, Sarti M, et al . Insulin-like growth factor I receptor-mediated circuit in Ewing's sarcoma / peripheral neuroectodermal tumor: a possible therapeutic target. Cancer Res 1996 ; 56: 4570-4.

13. Strammiello R, Benini S, Manara MC, et al . Impact of IGF-I / IGF-IR circuit on the angiogenetic properties of Ewing's sarcoma cells. Horm Metab Res 2003 ; 35: 675-84.

14. Martins AS, Mackintosh C, Martin DH, et al . Insulin-like growth factor I receptor pathway inhibition by ADW742, alone or in combination with imatinib, doxorubicin, or vincristine, is a novel therapeutic approach in Ewing tumor. Clin Cancer Res 2006 ; 12: 3532-40.

15. Buchner J. Hsp90 & Co. - a holding for folding. Trends Biochem Sci 1999 ; 24: 136-41.

16. Leppa S, Sistonen L. Heat shock response-pathophysiological implications. Ann Med 1997 ; 29: 73-8.

17. Smith DF, Whitesell L, Katsanis E. Molecular chaperones: biology and prospects for pharmacological intervention. Pharmacol Rev 1998 ; 50: 493-514.

18. Thomas X, Campos L, Le QH, Guyotat D. Heat shock proteins and acute leukemias. Hematology 2005 ; 10: 225-35.

19. Zhang H, Burrows F. Targeting multiple signal transduction pathways through inhibition of Hsp90. J Mol Med 2004 ; 82: 488-99.

20. Jackson SE, Queitsch C, Toft D. Hsp90: from structure to phenotype. Nat Struct Mol Biol 2004 ; 11: 1152-5.

21. Solit DB, Rosen N. Hsp90: a novel target for cancer therapy. Curr Top Med Chem 2006 ; 6: 1205-14.

22. Whitesell L, Lindquist SL. HSP90 and the chaperoning of cancer. Nat Rev Cancer 2005 ; 5: 761-72.

23. Ge J, Normant E, Porter JR, et al . Design, synthesis, and biological evaluation of hydroquinone derivatives of 17-amino-17-demethoxygeldanamycin as potent, water-soluble inhibitors of Hsp90. J Med Chem 2006 ; 49: 4606-15.

24. Neckers L. Using natural product inhibitors to validate Hsp90 as a molecular target in cancer. Curr Top Med Chem 2006 ; 6: 1163-71.

25. Pacey S, Banerji U, Judson I, Workman P. Hsp90 inhibitors in the clinic. Handb Exp Pharmacol 2006 : 331-58.

26. Sharp S, Workman P. Inhibitors of the HSP90 molecular chaperone: current status. Adv Cancer Res 2006 ; 95: 323-48.

27. Zhang H, Chung D, Yang YC, et al . Identification of new biomarkers for clinical triáis of Hsp90 inhibitors. Mol Cancer Ther 2006 ; 5: 1256-64.

28. Martínez-Ramírez A, Rodríguez-Perales S, Melendez B, et al . Characterization of the A673 cell line (Ewing tumor) by molecular cytogenetic techniques. Cancer Genet Cytogenet 2003 ; 141: 138-42.

29. Pagani A, Fischer-Colbrie R, Eder U, Pellín A, Llombart-Bosch A, Bussolati G. Neural and mesenchymal differentiations in Ewing's sarcoma cell lines. Morphological, immunophenotypic, molecular biological and cytogenetic evidence. Int J Cancer 1995 ; 63: 738-43.

30. Selleri L, Hermanson GG, Eubanks JH, Lewis KA, Evans GA. Molecular localization of the t (11; 22)
(q24; q12) translocation of Ewing sarcoma by chromosomal in situ suppression hybridization. Proc Natl Acad Sci USA 1991 ; 88: 887-91.

31. Szuhai K, Ijszenga M, Tanke HJ, Rosenberg C, Hogendoorn PC. Molecular cytogenetic characterization of four previously established and two newly established Ewing sarcoma cell lines. Cancer Genet Cytogenet 2006 ; 166: 173-9.

32. Manara MC, Landuzzi L, Nanni P, et al . Preclinical in vivo study of new insulin-like growth factor-I receptor-specific inhibitor in Ewing's sarcoma. Clin Cancer Res 2007; 13: 1322-30.

33. Benini S, Manara MC, Baldini N, et al . Inhibition of insulin-like growth factor I receptor increases the antitumor activity of doxorubicin and vincristine against Ewing's sarcoma cells. Clin Cancer Res 2001 ; 7: 1790-7.

34. Garcia-Echeverria C, Pearson MA, Marti A, et al. In vivo antitumor activity of NVP-AEW541 -A novel, potent, and selective inhibitor of the IGF-IR kinase. Cancer cell 2004 ; 5: 231-9.

35. Maloney EK, McLaughlin JL, Dagdigian NE, et al . An anti-insulin-like growth factor I receptor antibody that is a potent inhibitor of cancer cell proliferation. Cancer Res 2003 ; 63: 5073-83.

36. Scotlandi K, Avnet S, Benini S, et al . Expression of an IGF-I receptor negative negative mutant induces apoptosis, inhibits tumorigenesis and enhances chemosensitivity in Ewing's sarcoma cells. Int J Cancer 2002 ; 101: 11-6.

37. Warshamana-Greene GS, Litz J, Buchdunger E, Garcia-Echeverria C, Hofmann F, Krystal GW. The insulin-like growth factor-I receptor kinase inhibitor, NVP-ADW742, sensitizes small cell lung cancer cell lines to the effects of chemotherapy. Clin Cancer Res 2005 ; 11: 1563-71.

38. Warshamana-Greene GS, Litz J, Buchdunger E, Hofmann F, Garcia-Echeverria C, Krystal GW. The insulin-like growth factor-I (IGF-I) kinase inhibitor receptor NVP-ADW742, in combination with STI571, delineates a spectrum of dependence of small cell lung cancer on IGF-I and stem cell factor signaling. Mol Cancer Ther 2004 ; 3: 527-35.

39. Davies H, Bignell GR, Cox C, et al . Mutations of the BRAF gene in human cancer. Nature 2002 ; 417: 949-54.

40. from Alava E, Antonescu CR, Panizo A, et al . Prognostic impact of P53 status in Ewing sarcoma. Cancer 2000 ; 89: 783-92.

41. Wei G, Antonescu CR, from Alava E, et al . Prognostic impact of INK4A deletion in Ewing sarcoma. Cancer 2000 ; 89: 793-9.

42. Manara MC, Perdichizzi S, Serra M, et al . The molecular mechanisms responsible for resistance to ET-743 (Trabectidin; Yondelis) in the Ewing's sarcoma cell line, TC-71. Int J Oncol 2005 ; 27: 1605-16.

43. Basso AD, Solit DB, Chiosis G, Giri B, Tsichlis P, Rosen N. Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function. J Biol Chem 2002 ; 277: 39858-66.

44. Yu W, Rao Q, Wang M, et al . The Hsp90 inhibitor 17-allylamide-17-demethoxygeldanamycin induces apoptosis and differentiation of Kasumi-1 harboring the Asn822Lys KIT mutation and down-regulates KIT protein level. Leuk Res 2006 ; 30: 575-82.

45. Georgakis GV, Li Y, Rassidakis GZ, Martinez-Valdez H, Medeiros LJ, Younes A. Inhibition of heat shock protein 90 function by 17-allylamino-17-demethoxygeldanamycin in Hodgkin's lymphoma cells down-regulates Akt kinase, dephosphorylates extracellular signal -regulated kinase, and induces cell cycle arrest and cell death. Clin Cancer Res 2006 ; 12: 584-90.

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46. Munster PN, Marchion DC, Basso AD, Rosen N. Degradation of HER2 by ansamycins induces growth arrest and apoptosis in cells with HER2 overexpression via a HER3, phosphatidylinositol 3'-kinase-AKT-dependent pathway. Cancer Res 2002 ; 62: 3132-7.

47. Bagatell R, Beliakoff J, David CL, Marron MT, Whitesell L. Hsp90 inhibitors deplete key anti-apoptotic proteins in pediatric solid tumor cells and demonstrate synergistic anticancer activity with cisplatin. Int J Cancer 2005 ; 113: 179-88.

48. Mitsiades CS, Mitsiades NS, McMullan CJ, et al . Antimyeloma activity of heat shock protein-90 inhibition. Blood 2006 ; 107: 1092-100.

49. Zheng FF, Kuduk SD, Chiosis G, et al . Identification of a geldanamycin dimer that induces the selective degradation of HER-family tyrosine kinases. Cancer Res 2000 ; 60: 2090-4.

50. Banerji U, Walton M, Raynaud F, et al . Pharmacokinetic-pharmacodynamic relationships for the heat shock protein 90 molecular chaperone inhibitor 17-allylamino, 17-demethoxygeldanamycin in human ovarían cancer xenograft models. Clin Cancer Res 2005 ; 11: 7023-32.

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<110> SUPERIOR INVESTIGATION COUNCIL SCIENTISTS UNIVERSITY OF SALAMANCA

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<120> PROCEDURE TO DESIGN A INDIVIDUAL ANTITUMORAL THERAPY BASED ON THE DETECTION OF LEVELS OF HSP90 PROTEIN, THE USE OF HSP90 PROTEIN INHIBITORS FOR THE ELABORATION OF PHARMACEUTICAL COMPOSITIONS, THE COMPOSITIONS SO OBTAINED AND ITS APPLICATIONS

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Claims (28)

1. Procedure for the selection of a HSP90 protein inhibitor expressed in at least one tumor resistant to anti-tumor inhibitors of IGF1R / KIT receptors, which includes:

to.

ex vivo determination of the expression levels of the HSP90 protein gene in a tumor biological sample,

b.

comparison of expression levels from step a) with respect to a biological control sample,

C.

resistance determination to IGF1R / KIT receptor inhibitors when levels of expression of step a) are greater than those obtained in step b), Y

d.

selection of an inhibitor of the HSP90 protein adjuvant to an antitumor inhibitor of IGF1R / KIT receivers.

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2. Method according to claim 1 characterized in that the tumor biological sample from step a) belongs to the following group: Ewing's sarcoma, gastrointestinal stromal tumor (GIST) and dermatofibrosarcoma protuberans. 3. Method according to claim 1, characterized in that the determination of the expression levels of the HSP90 protein gene is carried out with or without the induction of a stress of the cells of the tumor biological sample by means of an antitumor compound inhibitor of the receptors. IGF1R / KIT. 4. Method according to claim 3 characterized in that the IGF1R / KIT receptor inhibitor is selected from the following group: ADW742, AEW541 and Imatinib. 5. Method according to claim 1 characterized in that the determination of the expression levels of the HSP90 protein gene in step a) is carried out by determining the mRNA levels of the Hsp90 gene and because it comprises:

i)

the mRNA extraction from the tumor biological sample,

ii)

mRNA amplification of the Hsp90 gene, Y

iii)

mRNA levels determination of the Hsp90 gene.

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Method according to claim 5, characterized in that the amplification of the mRNA of the Hsp90 gene consists in the amplification of the beta (?) Form of said gene. Method according to claim 5, characterized in that the amplification of the mRNA of the Hsp90 gene consists in the amplification of the alpha (α) form of said gene. Method according to claim 1, characterized in that the determination of the expression levels of the HSP90 protein gene in step a) is carried out by determining the levels of the HSP90 protein and comprising:

i)

the extraction of proteins from the biological tumor sample, Y

ii)

determination of the levels of the HSP90 protein, either by an immune method or not immunological

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9. Method according to claim 8, characterized in that the determination of the levels of the HSP90 protein consists in the determination of the beta (?) Form of said protein. Method according to claim 8, characterized in that the determination of the levels of the HSP90 protein consists in the determination of the alpha (α) form of said protein. 11. Method according to claim 1 characterized in that the HSP90 protein inhibitor selected in step d) belongs to the following group: geldanamicin, non-benzoquinone ansamycin, diarylpyrazole compounds and novobiocin. 12. Method according to claim 11, where the geldanamicin compound belongs to the following group: 17-AAG, 17-DMAG and retaspimycin, and The diarylpyrazole compounds belong to the following group: CCT018159 and CCT0129397 / VER49009. 13. The method according to claim 1, characterized in that the inhibitor of the HSP90 protein selected is the compound 17-AAG adjuvant of an inhibitor compound of the IGF1R / KIT receptors belonging to the following group: ADW742, AEW541 and Imatinib. 14. Use of a compound that inhibits HSP90 protein activity expressed in at least one tumor antitumor-resistant receptor inhibitors IGF1R / KIT for the preparation of a drug or pharmaceutical composition for the treatment of said tumor. 15. Use according to claim 14 characterized in that the HSP90 protein inhibitor belongs to the following group: geldanamicin, non-benzoquinone ansamycin, diarylpyrazole compounds and novobiocin. 16. Use according to claim 15, wherein the geldanamicin compound belongs to the following group: 17-AAG, 17-DMAG and retaspimycin, and the diarylpyrazole compounds belong to the following group: CCT018159 and CCT0129397 /
VER49009. 17. Use according to claim 14 characterized in that the inhibitor compound is a nucleic acid or polynucleotide that prevents or decreases the expression of the HSP90 protein coding gene and because it includes at least one nucleotide sequence selected from:

to)

a sequence specific antisense nucleotide sequence HSP90 protein mRNA or gene,

b)

a HSP90 protein mRNA specific ribozyme,

C)

a HSP90 protein mRNA specific aptamer,

d)

a RNA interference (siRNA or shRNA) specific to the mRNA of the HSP90 protein, and

and)

a microRNA (miRNA) specific for the HSP90 protein mRNA.

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18. Use according to claim 17 characterized in that the siRNA of step d) is constituted by the pair of specific nucleotide sequences of a siRNA of the beta form of the HSP90 protein: sense sequence SEQ ID NO1 and antisense sequence SEQ ID NO2. 19. Use according to claim 14 characterized in that it comprises the use in combination of an inhibitor of IGFR1 / KIT proteins. 20. Use according to claim 19, characterized in that the IGF1R / KIT protein inhibitor belongs to the following group: ADW742, AEW541 and Imatinib. 21. Pharmaceutical composition or medicament characterized in that it comprises a compound or agent inhibiting the HSP90 protein expressed in at least one tumor resistant to anti-tumor inhibitors of the IGF1R / KIT receptors. 22. Pharmaceutical composition or medication according to claim 21, further comprising one or more adjuvants and / or pharmaceutically acceptable vehicles. 23. Pharmaceutical composition according to any of claims 21 or 22 characterized in that it comprises an HSP90 protein inhibitor belonging to the following group: geldanamicin, non-benzoquinone ansamycin, diarylpyrazole compounds and novobiocin. 24. Composition according to claim 23, where the geldanamicin compound belongs to the following group: 17-AAG, 17-DMAG and retaspimycin, and The diarylpyrazole compounds belong to the following group: CCT018159 and CCT0129397 / VER49009. 25. Pharmaceutical composition according to any of claims 21 or 22 characterized in that it comprises, in addition to the HSP90 protein inhibitor, an inhibitor of the IGFR1 / KIT proteins. 26. Pharmaceutical composition according to claim 25 characterized in that the IGF1R / KIT protein inhibitor belongs to the following group: ADW742, AEW541 and Imatinib. 27. Pharmaceutical composition according to any of claims 21 or 22 characterized in that the inhibitor compound is a nucleic acid or polynucleotide that prevents or decreases the expression of the gene encoding the human HSP90 protein and that includes at least one nucleotide sequence selected from :

to)

a sequence specific antisense nucleotide sequence HSP90 protein mRNA or gene,

b)

a HSP90 protein mRNA specific ribozyme,

C)

a HSP90 protein mRNA specific aptamer,

d)

a Interference RNA (iRNA) specific to the protein mRNA HSP90, and

and)

a microRNA (miRNA) specific for the HSP90 protein mRNA.

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28. Pharmaceutical composition according to claim 27 characterized in that the HSP90 inhibitor is a siRNA constituted by the pair of specific nucleotide sequences of a siRNA of the beta form of the HSP90 protein: sense sequence SEQ ID NO1 and antisense sequence SEQ ID2 .