WO2008080956A1

WO2008080956A1 - Prognostic molecular markers for the cancer therapy with aplidine

Info

Publication number: WO2008080956A1
Application number: PCT/EP2007/064592
Authority: WO
Inventors: Amancio Carnero Moya; Victoria MONEO OCAÑA
Original assignee: Pharma Mar, S.A.
Priority date: 2006-12-29
Filing date: 2007-12-27
Publication date: 2008-07-10

Abstract

The present invention relates to the use of aplidine in patients having certain levels of molecular markers which can predict the outcome of chemotherapy, in particular in patients having low levels of p27 protein.

Description

PROGNOSTIC MOLECULAR MARKERS FOR THE CANCER THERAPY WITH APLIDINE

FIELD OF THE INVENTION

The present invention relates to the use of aplidine, and more especially to the use of aplidine in patients having certain levels of molecular markers, in particular having low levels of p27^kiP¹ (p27) expression.

BACKGROUND OF THE INVENTION

Cancer comprises a group of malignant neoplasms that can be divided into two categories: carcinoma, comprising a majority of the cases observed in the clinics, and other less frequent cancers, which include leukemia, lymphoma, central nervous system tumors and sarcoma. Carcinomas have their origin in epithelial tissues while sarcomas develop from connective tissues and those structures that had their origin in mesoderm tissues. Sarcomas can affect, for instance, muscle or bone and occur in the bones, bladder, kidneys, liver, lung, parotid, spleen, etc.

Cancer is invasive and tends to metastasise to new sites. It spreads directly into surrounding tissues and also may be disseminated through the lymphatic and circulatory systems.

Many treatments are available for cancer, including surgery and radiation, for localised disease, and drugs. However, the efficacy of available treatments on many cancer types is limited and new improved forms of treatment showing clinical benefit are needed. This is especially true for those patients that present the disease in advanced and /or metastatic state. It is also true for patients relapsing with progressive disease after having been previously treated with established therapies for which further treatment with the same therapy is mostly ineffective due to the acquisition of resistance or to limitations in the administration of the therapies because of associated toxicities.

Chemotherapy plays a significant part in cancer treatment, as it is required for treatment of advanced cancers with distant metastasis and often helpful for tumor reduction before surgery. Many anti-cancer drugs have been developed based on various modes of action.

The most commonly used types of anticancer agents include: DNA-alkylating agents (for example, cyclophosphamide, ifosfamide), antimetabolites (for example, methotrexate, a folate antagonist, and 5- fluorouracil, a pyrimidine antagonist), microtubule disrupters (for example, vincristine, vinblastine, paclitaxel), DNA intercalators (for example, doxorubicin, daunomycin, cisplatin), and hormone therapy (for example, tamoxifen, flutamide). The ideal antineoplastic drug would kill cancer cells selectively, with a wide therapeutic index relative to its toxicity towards non-malignant cells. It would also retain its efficacy against malignant cells, even after prolonged exposure to the drug. Unfortunately, none of the current chemotherapies possess an ideal profile. Most possess very narrow therapeutic indexes and, in practically every instance, cancerous cells exposed to slightly sublethal concentrations of a chemotherapeutic agent will develop resistance to such an agent, and quite often cross-resistance to several other antineoplastic agents.

Since cancer is a leading cause of death in animals and humans, several efforts have been and are still being undertaken in order to obtain an antitumor therapy active and safe to be administered to patients suffering from a cancer. The problem to be solved by the present invention is to provide antitumor therapies that are useful in the treatment of cancer.

Aplidine (Dehydrodidemnin B) is a cyclic depsipeptide that was isolated from a Mediterranean marine tunicate, Aplidium albicans, and it is the subject of WO 91 09485. It is related to compounds known as didemnins, and has the following structure:

More information on aplidine, aplidine analogues, its uses, formulations and synthesis can be found in patent applications WO 99 42125, WO 01 35974, WO 01 76616, WO 02 30441, WO 02 02596, WO 03 33013, WO 2004 080421 and WO 2004 080477. We incorporate by specific reference the content of each of these PCT texts.

Aplidine is being developed as an antineoplastic agent because of its potent antitumour activity in preclinical models against a wide variety of human tumours (Faircloth et al., Proc. Am. Assoc. Cancer Res. 1997, 38: Abst. 692; Depenbrock et al., Br. J. Cancer, 1998, 78, 739- 744). Results from phase I clinical trials showed signs of clinical benefit, in terms of stabilisation of the disease, and a favourable tolerability profile, with mild and relatively infrequent side effects (Anthoney et al., Proc. Am. Soc. Clin. Oncol. 2000, 19: 189a: Abst. 734; Bowman et al., Proc. Am. Soc. Clin. Oncol. 2000, 20: 120a: Abst. 476; Mauroun et al., Proc. Am. Soc. Clin. Oncol. 2001 , 20:83b: Abst. 2082; Ciruelos et al., Proc. Am. Soc. Clin. Oncol. 2002, 21 : 106a: Abst. 422; Faivre et al., J. Clin. Oncol. 2005, 23, 7871-7880).

Aplidine has selective cytotoxicity in vitro towards childhood leukaemia cells and generally lacks crossresitance with other known cytotoxic drugs (Bresters et al., Leukemia, 2003, 17, 1338- 1343; Erba et al., Br. J. Cancer, 2002, 86, 1510- 1517).

Aplidine is currently in phase II clinical trials in a variety of solid tumors and haematological malignancies. Moreover, clinical studies of aplidine in combination with other agents are ongoing. Recently, both the European Agency for the Evaluation of Medicinal Products (EMEA) and the American Food and Drug Administration (FDA) awarded aplidine Orphan drug' status for the treatment of acute lymphoblastic leukaemia (ALL) and multiple myeloma.

During the past 30 years medical oncologists have focused to optimise the outcome of cancer patients and it is just now that the new technologies available are allowing to investigate polymorphisms, gene expression levels and gene mutations aimed to predict the impact of a given therapy in different groups of cancer patients to tailor chemotherapy. Representative examples include the relation between the TS niRNA expression and the response and the survival with antifolates, beta tubulin III mRNA levels and response to tubulin interacting agents, PTEN methylation and resistance to CPT-I l and STAT3 over expression and resistance to EGF interacting agents. The mode of action of aplidine in tumour cells is only partially understood. Aplidine induces an early oxidative stress response, which results in a rapid and sustained activation of the epidermal growth factor receptor (EGFR), the non-receptor protein tyrosine kinase Src, and the serine threonine kinases c-jun N-terminal kinase (JNK) and p38 MAPK. These early events rapidly trigger the induction of the mitochondrial apoptotic pathway via cytochrome c release, activation of the caspase cascade and activation of protein kinase C (PKC) -δ, which seems to exert an important effector role in mediating the cellular death induced by the drug (Garcia- Fernandez et al., Oncogene, 2002, 21, 7533-7544; Cuadrado et al., J. Biol. Chem., 2003, 278, 241-250). Aplidine action in leukaemic cells is mediated, at least in part, through Fas CD95 cell death receptor, a member of the tumour necrosis factor (TNF) receptor family (Gajate et al., Clin. Cancer Res. 2003, 9, 1535- 1545). Depending on the cell system, aplidine induces either a very rapid apoptotic death without previous cell cycle arrest, or causes a block in Gl and/or a delay in the progression from S to G2 phases of the cell cycle (Erba et al., Br. J. Cancer, 2002, 86, 1510-1517; Garcϊa- Fernandez et al., Oncogene, 2002, 21, 7533-7544). Further studies have shown that aplidine induces apoptosis by altering glutathione homeostasis, thereby increasing the levels of reactive oxygen species and inducing Racl GTPase activation and MKP-I phosphatase downregulation (Gonzalez- Santiago et al., CeM Death and Differentiation, 2006, 13, 1968-1981). As part of its antitumoral action in leukaemic cells, aplidine has been shown to reduce the secretion of vascular endothelial growth factor (VEGF) secretion and downregulate its receptor, VEGFR-I (fit- 1 ) (Taraboletti et al., Br. J. Cancer, 2004, 90, 2418-2424; Broggini et al., Leukemia, 2003, 17, 52-59; Biscardi et al., Annals Oncology, 2005, 16, 1667-1674).

Gene expression profiling of leukemic blasts with different sensitivity to aplidine has allowed the identification of a minimum set of genes that represent the molecular signature of aplidine sensitivity/ resistance in acute myeloid and acute lymphoid leukemic blasts (Martinez et al., Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, 2004, 15: Abst. 37; Martinez et al., Proceedings of the 6th International Symposium and Expert Workshops on Leukemia and Lymphoma, 2005, 92: Abst. P- 14).

SUMMARY OF THE INVENTION

It is an object of the invention to provide an efficacious use of aplidine for the treatment of cancer. More particularly, an object of this invention is to provide an effective use of aplidine in patients having certain levels of molecular markers, and in particular having low levels of p27^klP^χ (p27) protein expression when normalized with the expression level of a reference protein in the same sample. In a particular embodiment said reference protein is a housekeeping protein (e.g., alpha-tubulin, actin, etc.)

Therefore, in an aspect the invention is directed to the use of aplidine in the manufacture of a medicament for the treatment of cancer in a subject having low levels of p27 protein when normalized with the expression level of a reference protein in the same sample.

In another aspect the invention is directed to the use of p27^klP^J (p27) as a marker for the selection of cancer patients to be efficaciously treated with aplidine.

In a further aspect the invention is directed to an in vitro method for designing an individual chemotherapy for a subject suffering from cancer, comprising: a) assaying p27 protein expression level in a biological sample from said subject; b) assaying the expression level of a reference protein in the same sample as in a); c) comparing said p27 protein expression level obtained in a) with the expression level of the reference protein obtained in b); and d) selecting a chemotherapy treatment based on aplidine when said p27 protein expression level is below a determined value of the expression level of the reference protein obtained in b) .

In another aspect the invention is directed to a method of treating cancer in a patient, the method comprising the steps of: assaying a biological sample from said patient for p27^kiP¹ (p27) protein expression level, comparing this level with a determined value of the expression level of a reference protein in the same sample, and when the expression level of p27 protein is below said determined value, treating the patient with aplidine.

In an additional aspect, the invention is directed to a screening method for selecting a patient suffering from cancer for a treatment with aplidine, comprising the steps: a) determining p27 protein levels from a tissue sample of the patient; b) comparing the p27 protein level with those of α- tubulin in the same sample; c) classifying the patient in one of the 3 groups defined as "low level" when p27 protein levels are lower than 50% of those of α- tubulin, "moderate level" when p27 protein levels vary from 50% to 150% of those of α-tubulin, and "high level" when p27 protein levels are higher than 150% of those of α-tubulin; and d) selecting said patient classified in the "low level" group for a chemotherapy treatment based on aplidine. In a further aspect, the invention relates to a method of predicting the clinical response of a cancer patient to the treatment with aplidine comprising a) assaying p27 protein expression level in a biological sample from said cancer patient; b) assaying the expression level of a reference protein in the same sample as in a); and c) comparing said p27 protein expression level obtained in a) with the expression level of the reference protein obtained in b); wherein a p27 protein expression level is below a determined value of the expression level of the reference protein obtained in b) is indicative of a positive clinical response of the cancer patient to the treatment with aplidine.

BRIEF DESCRIPTION OF THE FIGURES

Fig. IA. Expression of p27 in a panel of sarcoma cell lines analyzed by

RT-PCR and Western blot.

Fig. IB. Expression levels of p27 protein were quantified by densitometry and normalized by α-tubulin (aplidine ICso is shown above each bar).

Fig. 2. Correlation between aplidine ICso and p27 protein expression in a panel of sarcoma cell lines.

Fig. 3A. Increase in apoptotic nucleus in p27^~/^~ and wild type MEFs treated with different concentrations of aplidine at different times.

Fig. 3B. Increase in Annexin V staining in p27^~/^~ and wild type MEFs treated with different concentrations of aplidine at different times.

Fig. 4A and 4B. Cell cycle analysis of p27^*/- and wild type MEFs treated with different concentrations of aplidine. Fig. 5A. Levels of p27 protein in A673 cells expressing different siRNAs against p27.

Fig. 5B. Aplidine induces p27 expression in A673 cells. Fig. 5C. Aplidine induction of p27 is inhibited by antioxidant treatments (10 mM GSH).

DETAILED DESCRIPTION OF THE INVENTION

Aplidine is a natural compound represented by the following formula:

As used herein, the term "aplidine" also covers any pharmaceutically acceptable salt, ester, solvate, hydrate or a prodrug compound which, upon administration to the patient is capable of providing (directly or indirectly) the compound aplidine. The preparation of salts and other derivatives, and prodrugs, can be carried out by methods known in the art.

Now, we have found that p27^klP¹ (p27) expression can also play an important role in predicting differential chemotherapy sensitivity in cancer patients treated with aplidine.

Thus, in an aspect, the invention is directed to the use of aplidine in the treatment of cancer patients having low levels of p27^klP¹ (p27) protein.

Accordingly, in an aspect, the invention is directed to the use of aplidine in the manufacture of a medicament for the treatment of cancer in a patient having low levels of p27 protein when normalized with the expression level of a reference protein in the same sample. In another aspect, the invention relates to aplidine for the treatment of cancer in a subject having low levels of p27 protein when normalized with the expression level of a reference protein.

The values for "low", "moderate", or "high" levels of p27^klp! (p27) expression are determined by comparison with the expression levels of a reference protein in the same sample. In an embodiment, said reference protein is a housekeeping protein (i.e., a protein involved in the basic functioning of a cell or the set of cells in an organism, for example, alpha- tubulin, actin, etc.). In a particular embodiment, said reference protein is alpha- tubulin (α-tubulin), and, accordingly, "low" levels of p27 protein expression correspond to those when p27 protein levels are lower than 50% of those of the α-tubulin protein, "moderate" levels of p27 protein expression correspond to those when p27 protein levels vary from 50% to 150% of those of the α-tubulin protein, and "high" levels of p27 protein expression correspond to those when p27 protein levels are higher than 150% of those of α-tubulin.

Preferably the protein levels are measured by taking a biological sample from the patient, lysating the cells and determining the amount of protein through Western blot immunodetection. Other methods known to the person skilled in the art can be used, as long as the relative levels of p27 protein and reference protein (e.g. , α-tubulin) can be obtained with enough accuracy.

Aplidine effectively inhibits cell viability by triggering a canonical apoptotic program resulting in alterations in cell morphology, caspase activation and chromatin fragmentation. Pro-apoptotic concentrations of aplidine induce early oxidative stress, which results in a rapid and persistent activation of both JNK and p38 MAPK and a biphasic activation of ERK (Cuadrado et al., J. Biol. Chem. 2003, 278, 241-250). Inhibition of JNK and p38 MAPK blocks the apoptotic program induced by aplidine demonstrating its central role in the integration of the cellular stress induced by the drug. JNK and p38 MAPK activation results in downstream cytochrome c release and activation of caspases- 9 and -3 and PARP cleavage, demonstrating the mediation of the mitochondrial apoptotic pathway in this process. There appear to be a series of pathways that lead to this phosphorylation. It has been demonstrated that aplidine acts both at the cell membrane and intracellularly. At the membrane, aplidine caused direct oxidative damage and Rac- 1 activation. Inside the cell, the molecule reduces levels of GSH, thus increasing oxidative stress. The addition of antioxidants attenuates aplidine response (Garcia- Fernandez et al., Oncogene, 2002, 21 , 7533-7544). Aplidine was also found to be cytostatic at nanomolar concentrations inducing both a Gi arrest and a G2 blockade. The drug- induced cell cycle perturbations and subsequent cell death do not appear to be related to macromolecular synthesis (protein, RNA, DNA) since the effects occur at concentrations (e.g. 10 nM) in which macromolecule synthesis was not markedly affected (Erba et al., Br. J. Cancer, 2002, 86, 1510- 1517).

We have found, that the levels of p27^klP^χ determine aplidine sensitivity in human tumor cells and that the elimination of p27 in these cells (by siRNA) or in MEFs (p27 KO) increases sensitivity to aplidine. We have also found that the increase in p27 levels is a component of the aplidine response. Therefore, p27 might act as a hinge protein determining the cytostatic or cytotoxic response to aplidine.

Cell-cycle progression depends on the regulated expression of cyclins, which affect the activation of Cdks. Members of the Cdk- inhibitor CIP/KIP family (including p21^wafl, p27^klP¹, and p57^klP²) promote cell-cycle arrest by binding and inhibiting cyclin-Cdk protein complexes during differentiation, in response to either mitogen deprivation or toxic stress (reviewed in Malumbres and Carnero, Prog, cell cycle res. 2003, 5, 5- 18; Carnero, Encyclopedia of Respiratory Medicine, 2006, 347-355, Elsevier Ltd). These proteins preferentially associate with the cyclin-CDK complex rather than with the individual CDK subunits. The overexpression of these proteins arrest cells in different stages of the cell cycle according to their biochemical function. p27ki_Pi _can bind and inactivate CDKl, CDK2, CDK4 and CDK6 providing the basis for Gl and G2/M arrest.

Aplidine triggered a very early and pronounced increase in ROS production. In this context, it is well known that disruption of the mitochondrial function under conditions of oxidative stress is an important contributor to the apoptotic response (Kannan and Jain, Pathophysiol. 2000, 7, 153- 163; Freytag, MoI. Cell Biol 1988, 8, 1614- 1624; Zamzami et al., J. Exp. Med. 1995, 182, 367-377). Significantly, blockade of ROS production by the free radical scavengers inhibited both mitochondrial damage and apoptosis (Garcia-Fernandez et al., Oncogene, 2002, 21, 7533-7544; Cuadrado et al., J. Biol. Chem. 2003, 278, 241-250). On the other hand, ROS might trigger the accumulation of p27 through p38MAPK (Faust et al., Oncogene, 2005, 24, 7941-7945) or JNK, two kinases previously described involved in the sensitivity to aplidine. p27 phosphorylation triggers the inhibition of its ubiquitination and degradation through Skp2 (Koff, Cancer Cell, 2006, 9, 75-76). An accumulation of p27 will inhibit cyclin-CDK complexes acting at Gl and G2/M boundaries (Malumbres and Carnero, Prog, cell cycle res. 2003, 5, 5-18; Carnero, Encyclopedia of Respiratory Medicine, 2006, 347-355, Elsevier Ltd). Inhibition of cell cycle will result in decreased aplidine sensitivity as has been broadly reported in similar contexts for p27 and p21 (Brugarolas et al., Nature, 1995, 377, 552- 557; Bissonnette et al., Oncogene, 1998, 16, 3461-3469; Burgess et al, MoI. Pharmacol. 2001, 60, 828-837; Kim et al., Biochem. Biophys. Res. Commun. 2001 , 289, 34-38; Rosato et al., Cancer Res. 2003, 63, 3637- 3645; Miura et al, Atherosclerosis, 2005, 182, 267-275) and correlating with previous works reporting that aplidine has higher cytotoxic activity against proliferating than quiescent cells (Garcϊa-Fernandez et al., Oncogene, 2002, 21, 7533-7544; Cuadrado et al., J. Biol Chem. 2003, 278, 241-250). Therefore, absence of p27 will result in increased sensitivity to aplidine, and can be used as a marker for treatment in vivo.

As a member of the CIP/KIP family of CDK inhibitors, p27^klP¹ is also a potential tumor suppressor. The levels of p27^klP¹ protein decrease during tumor development and progression in some epithelial, lymphoid and endocrine tissues. This decrease occurs mainly at the post- translational level with protein degradation by the ubiquitin-proteasorne pathway. A large number of studies have characterized p27^klP¹ as an independent prognostic factor in various human cancers including leukemia, breast, colon and prostate adenocarcinomas (reviewed in Malumbres and Carnero, Prog, cell cycle res. 2003, 5, 5-18). Therefore these indications could be a tumor target for aplidine response.

Accordingly, the present invention relates to the use of aplidine for the treatment of cancer in patients having low levels of p27 protein when normalized with the expression level of a reference protein in the same sample. Treatment of cancer in patients with a p27 protein level which is lower than 50% of those of the α-tubulin protein is preferred.

Aplidine is typically supplied and stored as a sterile lyophilized product which comprises aplidine and pharmaceutically acceptable excipients in a formulation adequate for therapeutic use. Examples of pharmaceutical compositions containing aplidine include liquid (solutions, suspensions or emulsions) with suitable composition for intravenous administration, and they may contain the pure compound or in combination with any carrier or other pharmacologically active compounds. Solubilised aplidine shows substantial degradation under heat and light stress testing conditions, and a lyophilized dosage form was developed, see WO 99 42125 incorporated herein by reference.

Administration of aplidine or compositions of the present invention is based on a Dosing Protocol preferably by intravenous infusion. We prefer that infusion times of up to 72 hours are used, more preferably 1 to 24 hours, with about 1 , about 3 or about 24 hours most preferred. Short infusion times which allow treatment to be carried out without an overnight stay in hospital are especially desirable. However, infusion may be around 24 hours or even longer if required. Infusion may be carried out at suitable intervals with varying patterns, illustratively once a week, twice a week, or more frequently per week, repeated each week optionally with gaps of typically one or several weeks.

Representative schedules and dosages of aplidine are for example: a) about 5 mg/m² body surface area, administered as an intravenous infusion over 3 hours /weekly every two weeks; b) about 3.2 mg/m² body surface area, administered as an intravenous infusion over 1 hour/weekly during 3 weeks and one week rest; c) about 5 mg/m² body surface area, administered weekly as an intravenous infusion over 24 hours/weekly every two weeks; d) about 3.75 mg/m² body surface area, administered as an intravenous infusion over 24 hour/weekly during 3 weeks and one week rest; e) about 1.2 mg/m² body surface area, administered as an intravenous infusion over 1 hour/ daily x 5 days every 3 weeks.

Depending on the type of tumor and the developmental stage of the disease, the treatments of the invention are useful in preventing the risk of developing tumors, in promoting tumor regression, in stopping tumor growth and/or in preventing metastasis. In particular, the method of the invention is suited for human patients, especially those who are relapsing or refractory to previous chemotherapy. First line therapy is also envisaged.

Although guidance for the dosage is given above, the correct dosage of the compound will vary according to the particular formulation, the mode of application, and the particular situs, host and tumor being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Administration can be carried out continuously or periodically within the maximum tolerated dose.

The use of aplidine according to the invention is particularly preferred for the treatment of acute lymphoblastic leukaemia, multiple myeloma, NHL, melanoma, kidney cancer, colon cancer, renal cancer, medullary thyroid cancer, pancreas cancer, lung cancer, sarcoma, prostate cancer, urothelial cancer, head & neck cancer, and breast cancer.

According to the invention, p27^klP¹ (p27) can be used as a marker for the selection of cancer patients to be efficaciously treated with aplidine.

Thus, in further aspect, the invention is directed to an in vitro method for designing an individual chemotherapy for a subject suffering from cancer, comprising: a) assaying p27 protein expression level in a biological sample from said subject; b) assaying the expression level of a reference protein in the same sample as in a); c) comparing said p27 protein expression level obtained in a) with the expression level of the reference protein obtained in b) ; and d) selecting a chemotherapy treatment based on aplidine when said p27 protein expression level is below a determined value of the expression level of the reference protein obtained in b).

Protein levels for p27 and for the reference protein can be measured by conventional means, for example, by taking a biological sample from the subject suffering from cancer, Iy sating the cells and determining the amount of protein through Western blot immunodetection. Other methods known to the person skilled in the art can be used, as long as the relative levels of p27 protein and reference protein can be obtained with enough accuracy.

The determined value of the expression level of the reference protein will depend on the reference protein to be assayed and can be established following a methodology as that disclosed in Example 1. As mentioned therein, different groups of p27 protein levels can be established when said levels are compared with the expression levels of the reference protein and then correlate the normalized p27 protein levels with sensitivity to aplidine. The skilled person will appreciate that many different antibodies specific for p27 can be used in the context of the present invention. Preferably, the antibody is from a commercial source (e.g. Transduction Laboratories) and can be used at any suitable dilution, in particular, 1 :2000.

In a particular embodiment, said reference protein is a housekeeping protein such as α-tubulin and patients are selected for treatment with aplidine when the level of p27 protein is lower than 50% of the level of the α-tubulin protein, said value being considered as the determined value to be compared with in this case. The skilled person will appreciate that many different antibodies specific for a-tunulin can be used in the context of the present invention. Preferrably, the antibody is from a commercial source (e.g. Sigma) and can be used at any suitable dilution, in particular, 1 : 10000.

In another aspect the invention is directed to a method of treating cancer in a patient, the method comprising the steps of: assaying a biological sample from said patient for p27^klP¹ (p27) protein expression level, comparing this level with a determined value of the expression level of a reference protein in the same sample, and when the expression level of p27 protein is below said determined value, treating the patient with aplidine. These steps can be carried out as previously mentioned. In a particular embodiment, said reference protein is α- tubulin and patients are selected for treatment with aplidine when the level of p27 protein is lower than 50% of the level of the α-tubulin protein.

In a further aspect, the invention is directed to a screening method for selecting a patient suffering from cancer for a treatment with aplidine, comprising the steps: a) determining p27 protein levels from a tissue sample of the patient; b) comparing the p27 protein level with those of α-tubulin in the same sample; c) classifying the patient in one of the 3 groups defined as "low level" when p27 protein levels are lower than 50% of those of α- tubulin, "moderate level" when p27 protein levels vary from 50% to 150% of those of α-tubulin, and "high level" when p27 protein levels are higher than 150% of those of α-tubulin; and d) selecting said patient classified in the "low level" group for a chemotherapy treatment based on aplidine. These steps can be carried out as previously mentioned. In a particular embodiment, the tissue sample is a tumour biopsy. In another particular embodiment, the cancer is selected from the group consisting of acute lymphoblastic leukaemia, multiple myeloma, NHL, melanoma, kidney cancer, colon cancer, renal cancer, medullary thyroid cancer, pancreas cancer, lung cancer, sarcoma, prostate cancer, urothelial cancer, head & neck cancer and breast cancer.

In another aspect, the invention relates to a method for predicting the clinical response of a cancer patient to the treatment with aplidine comprising

(a) assaying p27 protein expression level in a biological sample from said cancer patient;

(b) assaying the expression level of a reference protein in the same sample as in (a); and

(c) comparing said p27 protein expression level obtained in a) with the expression level of the reference protein obtained in b); wherein a p27 protein expression level is below a determined value of the expression level of the reference protein obtained in b) is indicative of a positive clinical response of the cancer patient to the treatment with aplidine.

In a preferred embodiment, the method for predicting the clinical response of a cancer patient to the treatment with aplidine uses α- tubulin as reference protein.

These steps can be carried out as previously mentioned. In a particular embodiment, the tissue sample is a tumour biopsy. In another particular embodiment, the cancer is selected from the group consisting of acute lymphoblastic leukaemia, multiple myeloma, NHL, melanoma, kidney cancer, colon cancer, renal cancer, medullary thyroid cancer, pancreas cancer, lung cancer, sarcoma, prostate cancer, urothelial cancer, head & neck cancer and breast cancer.

The invention being thus described, the practice of the invention is illustrated by the experimental examples provided below. These examples should not be interpreted as limiting the scope of the claims.

EXAMPLE 1 Response to aplidine of different low passaged cell lines

With the aim of identifying new markers of sensitivity and resistance to aplidine response, a panel of low passaged human tumor cell lines mainly from mesenchimal origin, was treated with aplidine.

Sterile fragments from resected tumors were minced in culture medium and then disaggregated by 1-2 h incubation in collagenase (100 U/ml) at 37°C. 24 h later medium was changed to F- 10 Ham (Gibco) supplemented with 1% Ultroser G (Biosepra). Cell lines generated were cultured in F- 10 Ham supplemented with 1% Ultroser G. A673 cells were cultured in RPMI (Sigma) and SW872 in Leibovitz L- 15 (Sigma). All media were supplemented with 10% FBS, fungizone and penicillin /streptomycin. Once cells became confluent, adherent cells were removed by trypsin treatment and seeded at 1/2 or 1/3 ratio with medium. Throughout the establishment of these cell lines, phenotypic features were followed. Additionally, they were routinely checked for mycoplasma contamination (INVIVOGEN). All cell lines used in the study were obtained following the above described method, excepting SW-872, A-673 and Saos-2 cell lines that were obtained from the ATCC.

Aplidine was tested on 96-well trays. Cells growing in a flask were harvested just before they became confluent, counted using a haemocytometer and diluted down with media adjusting the concentration to the required number of cells per 0.2 ml (volume for each well). Cells were then seeded in 96-well trays at a density between 1000 and 4000 cells /well, depending of the cell size. Cells were left to plate down and grow for 24 hours before adding the drug.

Aplidine was weighed out and diluted with DMSO to get it into solution to a concentration of 1OmM. From here a "mother plate" with serial dilutions was prepared at 200X the final concentration in the culture. The final concentration of DMSO in the tissue culture media should not exceed 0.5%. The appropriate volume of the compound solution (usually 2 microlitres) was added automatically (Beckman FX 96 tip) to media to make it up to the final concentration for each drug.

The medium was removed from the cells and replaced with 0.2 ml of medium dosed with drug. Each concentration was assayed in triplicate. Two sets of control wells were left on each plate, containing either medium without drug or medium with the same concentration of

DMSO. A third control set was obtained with the cells untreated just before adding the drug (seeding control, number of cells starting the culture).

Cells were exposed to the drug for 96 hours and then washed twice with phosphate buffered saline before being fixed with 10% glutaraldehyde. Cells were washed twice and fixed with crystal violet 0.5% during 30 minutes. Then washed extensively, solubilized with 15% acetic acid and absorbance measured at 595 nm.

All cell lines were treated under similar conditions and ICsos were calculated as an average of 3 independent experiments performed in triplicate (Table 1).

Table 1 Aplidine sensitivity of a panel of low-passaged sarcoma cell lines

MPNST: Malignant peripheral nerve sheath tumour GIST: Gastrointestinal stromal tumour

Response to aplidine varied from < 1 nM, such as in CNIO BG, 1455 and CNIO AA cell lines, to > 30 nM, such as in CNIO BC and SW872 cell lines, more than a 100-fold difference between the most sensitive and the most resistant cell lines. The response was independent of the tumor type.

In addition, expression of protein and mRNA levels from different genes were analyzed and correlated with the in vitro sensitivity to aplidine. Total RNA was collected using the TRI-REAGENT (Molecular Research Center, Inc). RT was performed (Promega) with 1 μg of Rnasy following the manufacturer's protocol. The following primers were used to amplify regions: β-actin, forward primer 5'- AGGCCAACCGCGAGAAGATGAC-3', and reverse primer 5'- GAAGTCCAGGGCGACGTAGCA-3'. cDNA was subjected to PCR, and products were analyzed by electrophoresis on a 1% agarose gel.

The following genes involved in tumour progression, cell adhesion, cell cycle control and cell signalling were analyzed: Apaf-1, APC, cdk4, c-kit, cyclin Dl, E-cadherin, MDM2, MLH- I, MSH-2, pl4^ARF, pl5^INK4b, _{p l 6}iNK4a_{) p}2 lcipi_; p27^klP¹, p53, p73, p85, PDGFR, p60^src, PTEN and β- catenin.

Expression of p27 protein and sensitivity to aplidine were found to be correlated. Levels of p27 protein were analyzed by Western blot with cells actively proliferating under the same conditions. Cells were seeded at 40% saturation and grown until 80% confluence was reached, then harvested and total protein extracted; p27 was detected in total lysates by Western blot immunodetection (Figure IA). To prepare the whole-cell extract, cells were washed once in cold phosphate-buffered saline (PBS) and suspended in 1 ml lysis-buffer (50 mM Tris-HCl pH 7.5, 1% NP-40, 10% glycerol, 150 mM NaCl, 2 mM, Complete protease inhibitor cocktail -Roche-). The protein content of the lysates was determined by the modified method of Bradford. Samples were separated on 7.5% SDS-PAGE gels, transferred onto Immobilon-P membrane (Millipore) and immunostained. To identify p27 we used a 1:2000 dilution of an anti p27 (Transduction laboratories) as primary antibody and a 1:2500 dilution of a HRP-conjugated anti-mouse (Promega) as secondary antibody. Proteins were visualized using the ECL detection system (Amersham). The experiment was repeated three independent times with similar results.

p27 levels were quantified by densitometry and normalized with α-tubulin levels in the same membrane. Three different p27 levels were selected: low levels, when p27 levels are lower than 50% of those of α- tubulin; moderate levels, when they vary from 50% to 150%; and high levels, when p27 levels are higher than 150% of those of α-tubulin. α- tubulin was quantified using a 1 : 10000 dilution of an anti- α-tubulin antibody (Sigma). The different expression levels of p27 protein were correlated with the sensitivity to aplidine in the panel of sarcoma cell lines (Figure IB).

The mean IC 50 in each of the three subgroups of cell lines was calculated and correlated with aplidine sensitivity (Figure 2). Cell lines with high levels of p27 (SW872, CNIO BC, A673 and CNIO BP) showed higher IC50. Cell lines with moderate levels of p27 (CNIO AX, CNIO AZ, CNIO BB, CNIO BM, CNIO BN, CNIO BI, CNIO BJ, SAOS2 and CNIO CE) showed intermediate IC50 with a significant statistical difference when compared to cells with high levels of p27 (p=0.0031). Cells with lower levels of p27 (CNIO AA, CNIO AW, CNIO AY, CNIO BF, CNIO BG and 1455) showed the highest sensitivity to aplidine (p=0.0072 when compared to cells with high levels of p27; p=0.13 when compared to cells with medium levels of p27). In summary, cell lines with high levels of p27 were more resistant to aplidine treatment, whereas cell lines with lower levels of p27 were more sensitive to aplidine.

EXAMPLE 2 Absence ofp27 increases sensitivity to aplidine in MEFs

Mouse embryonic fibroblasts (MEFs) were prepared from day 13.5 embryos derived from KO mice (p21-/- or p27-/-). Head and blood organs were removed, and the torso was minced and dispersed in 0.1% trypsin (45 min at 37°C). The cells were grown for two population doublings and then frozen. MEFs were subcultured 1 :4 upon reaching confluence; each passage was considered to be two population doubling levels (PDLs). The p21-/- MEFs were kindly provided by M. Serrano

(CNIO) from KO mice published in: - Brugarolas J, Chandrasekaran C, Gordon JI, Beach D, Jacks T, Hannon GJ, Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature. 1995 Oct 12; 377(6549):552-7;

- Efeyan A, Collado M, Velasco-Miguel S, Serrano M. Oncogene. 2006 Sep 1 1 ; [Epub ahead of print];

- Martin-Caballero J, Flo res JM, Garcia- Palencia P, Collado M, Serrano M. Different cooperating effect of p21 or p27 deficiency in combination with INK4a/ARF deletion in mice. Oncogene. 2004 Oct 28; 23(50):8231-7; - Martin-Caballero J, Flores JM, Garcia- Palencia P, Serrano M.

Tumor susceptibility of p21(Wafl/Cipl) -deficient mice. Cancer Res. 2001 Aug 15; 61 (16):6234-8;

- Carnero A, Beach DH. Absence of p2 IWAFl cooperates with c- myc in bypassing Ras-induced senescence and enhances oncogenic cooperation. Oncogene. 2004 Aug 5; 23(35):6006- l l.

Following the same procedure P27-/- MEFs were generated in Dr. Carnero's lab from KO mice published in:

- Sotillo R, Renner O, Dubus P, Ruiz-Cabello J, Martin-Caballero J, Barbacid M, Carnero A, Malumbres M. Cooperation between Cdk4 and p27kip l in tumor development: a preclinical model to evaluate cell cycle inhibitors with therapeutic activity. Cancer Res. 2005 May 1 ; 65(9):3846-52.

- Martin-Caballero J, Flores JM, Garcia-Palencia P, Collado M, Serrano M. Different cooperating effect of p21 or p27 deficiency in combination with INK4a/ARF deletion in mice. Oncogene. 2004 Oct 28; 23(50):8231-7.

Mouse embryonic fibroblasts (MEFs) lacking p27 gene were then treated with aplidine and their sensitivity was compared with that of the corresponding wild type MEFs. In the cytotoxicity assessment, same procedures were used as those already disclosed in example 1. Sustaining the correlation between p27 levels and aplidine sensitivity found in the panel of sarcoma cell lines, p27^~/^~ MEFs were more sensitive to aplidine as compared to isogenic MEFs from wild type littermates. When wild type and p27^~/- MEFs were treated with other drugs, such as vinblastine and flavopiridol, p27-/- cells were equal or even less sensitive to the antitumor treatment than the corresponding wild type cells (Table 2).

Table 2

Sensitivity to aplidine, vinblastin and flavopiridol of wild type and p27 -/- MEFs

Results are shown as the mean ± SD of 5 independent experiments.

It is possible to argue that the effect of aplidine is due to absence of cell cycle inhibition, making p27^~/^~ cells more sensitive to aplidine treatment. To study this specificity p2W^~ cells from p21 knock-out mice were treated with aplidine under the same conditions as above. In this case p21^~/^~ cells were even less sensitive to aplidine than wild type cells (Table 3). These results confirm that the correlation between aplidine sensitivity and p27 expression is not due to an absence of cell cycle inhibition.

Table 3

Sensitivity to aplidine, vinblastin and flavopiridol of wild type and p21 ^■/- MEFs

Results are shown as the mean ± SD of 5 independent experiments.

Additionally, the induction of apoptosis by aplidine in p27^~/- cells was analyzed as apoptotic nucleous and by Annexin Staining:

Apoptotic nucleous: Apoptosis was visually assessed by staining cells with Hoechst 33258 pentathydrate (Molecular-Probes) for 5 minutes. The cells were then examined with a Leica fluorescent microscope and apoptotic cells were distinguished by condensed fragmented nuclear regions. We analyzed a total of 400 cells per treatment and results are given as percentage.

Apoptosis assessment by Annexin-V/Propidium iodide staining: The effect of aplidine on MEFs wt and p27-/- was assessed using flow cytometry. Cells were grown up to about 70% confluence and treated with aplidine (1-1OnM) for 6h. Briefly, after treatment cells were harvested and cell concentration adjusted to 3x10⁶ cells /ml with binding buffer (BD), stained with 5 μl of Annexin V (BD) and 10 μl of PI (Sigma) and incubated in the dark for 15 min. A total of 10000 size gated cells were analyzed by FACSCalibur (BD).

It was found that treatment of p27"/- MEFs with different concentrations of aplidine (1, 5 or 10 nM) induced an increase of apoptosis measured as apoptotic nucleus (Figure 3A) and by Annexin V staining (Figure 3B).

Finally, the effect of aplidine on the cell cycle was evaluated by measuring the DNA content (Figure 4A y 4B) . Cellular DNA content was determined by flow cytometric analysis of Pi-labeled cells. MEFs wt and

MEFs p27^~/^~ were grown to exponential phase, seeded at a density of 2xlO⁶ cells/ 10 cm dish, and treated with the indicated concentrations (1- 10 nM) of aplidine for 6 h. Cells were harvested, fixed in ice cold 70% ethanol, stored at 4°C, washed with phosphate-buffered saline, treated with 25 μg/ml Rnase A at 37°C for 15 min, and stained with 50 μg/ml propidium iodide (PI) for 10 min. For flow cytometric analysis, we used a FACSCalibur flow cytometer (Becton Dickinson, NJ). The excitation wavelength was 488 nm. Forward light scatter and right-angle light scatter, were used to establish size gates and exclude cellular debris from the analysis. A minimum of 10000 cells per sample were used for the analysis performed using CellQuest software.

Neither a cell cycle arrest nor a sub-Gl population was induced after treatment of wild type MEFs during 24 hrs with different concentrations of aplidine. However, p27 / cells showed a clear G2 arrest with a concomitant induction of a sub-Gl population which increased with the concentration of aplidine. The results confirm the sensitivity of p27^~/^~ cells to aplidine treatment and its induction of apoptosis with a concomitant G2/M arrest.

EXAMPLE 3

Functional relationship between p27 levels and response to aplidine

To evaluate the correlation between sensitivity to aplidine and p27 levels, human isogenic cell lines differing only in the levels of p27 were analyzed. Three different siRNA against p27 were selected and analyzed for their ability to produce a significant reduction of p27 protein levels.

To target human p27 we used shRNAs supplied by CNIO shRNA library. Three different small interference RNAs were selected against p27 sequences by the bioinformatics unit; Hs-604, GCA CTG CAG AGA

CAT GGA A ( 122 bases downstream the start codon); Hs-933, CCG ACG ATT CTT CTA CTC A (451 bases downstream the start codon); and Hs- 960, GAG CCA ACA GAA CAG AAG A (477 bases downstream the start codon). As vector we used pA70 Retro derived from pSuperRetro. Cells from lines A673, CNIO AA and CNIO AW were plated in 10 cm dishes at 50% confluency, and transfected 24 h later by calcium phosphate precipitation using 20 μg of pA70 Retro vector. The clones were selected in puromycin (lμg/ml) (Sigma).

A673 cells expressing either the parental vector or constitutively expressing each of the different siRNAs were generated as mentioned.

One of them (Hs-960) reduced the levels of p27 in 80-90% (Figure 5A).

This siRNA was transfected into cell lines expressing different p27 levels. Cells selected for siRNA expression were tested for their sensitivity to aplidine and compared to cells expressing the parental vector (Table 4).

Table 4 Aplidine sensitivity of different sarcoma cell lines transfected with a siRNA (Hs-960) against ρ27 as compared to their wild type counterparts

Results are shown as the mean ± SD of 4 (for A673 and CNIO AW cells) or 3 (for CNIO AA cells) independent experiments

Reduction of p27 levels yielded cells more sensitive to aplidine in cell lines with high (A673) and moderate (CNIO AW) levels of p27; on the contrary, the sensitivity of the CNIO AA cell, with low p27 levels, was not affected by the siRNA. Since aplidine had an anti-proliferative effect depending on the p27 levels, this effect should be through cell- cycle regulation. We examined the levels of the CDK inhibitor p27 after aplidine treatment in A673 cell line. Aplidine induced the upregulation of p27 (Figure 5B). This induction of p27 occurs through an early oxidative stress response since treatment of cells with antioxidants, such as 10 mM GSH, inhibits this response (Figure 5C).

In all experiments reported, Prisma 4 statistical software was used for the statistical analysis. Determination of statistical significance was performed by analysis of variance (one-way ANOVA). Post hoc comparison was completed using Bonferroni^'s Multiple Comparison

Test. All data are reported as the mean ± standard error of the mean.

Statistical significance was considered as *p<0.05, **p<0.01, ***p<0.001.

In conclusion, in these examples we have demonstrated that p27 protein is a putative marker of aplidine sensitivity.

Claims

1. Use of aplidine in the manufacture of a medicament for the treatment of cancer in a subject having low levels of p27 protein when normalized with the expression level of a reference protein in the same sample.

2. Use according to claim 1, wherein said reference protein is the α- tubulin protein.

3. Use according to claim 2, wherein the level of p27 protein in said patient is lower than 50% of those of α-tubulin.

4. Use of aplidine according to any of the preceding claim, wherein the cancer to be treated is selected from the group consisting of acute lymphoblastic leukaemia, multiple myeloma, NHL, melanoma, kidney cancer, colon cancer, renal cancer, medullary thyroid cancer, pancreas cancer, lung cancer, sarcoma, prostate cancer, urothelial cancer, head & neck cancer, and breast cancer.

5. Use of p27 protein as a marker for the selection of cancer patients to be treated with aplidine.

6. A method of treating cancer in a patient, comprising: assaying a biological sample from said patient for p27^klP¹ (p27) protein expression level, comparing this level with a determined value of the expression level of a reference protein in the same sample, and when the expression level of p27 protein is below said determined value, treating the patient with aplidine.

7. An in vitro method for designing an individual chemotherapy for a subject suffering from cancer, comprising: a) assaying p27 protein expression level in a biological sample from said subject; b) assaying the expression level of a reference protein in the same sample as in a); c) comparing said p27 protein expression level obtained in a) with the expression level of the reference protein obtained in b); and d) selecting a chemotherapy treatment based on aplidine when said p27 protein expression level is below a determined value of the expression level of the reference protein obtained in b).

8. Method according to claim 6 or 7, wherein the cancer is selected from the group consisting of acute lymphoblastic leukaemia, multiple myeloma, NHL, melanoma, kidney cancer, colon cancer, renal cancer, medullary thyroid cancer, pancreas cancer, lung cancer, sarcoma, prostate cancer, urothelial cancer, head & neck cancer and breast cancer.

9. A method according to any of claims from 6 to 8, wherein the biological sample is a tumour biopsy.

10. A screening method for selecting a patient suffering from cancer for a treatment with aplidine, comprising the steps: a) determining p27 protein levels from a tissue sample of the patient; b) comparing the p27 protein level with those of α-tubulin in the same sample; c) classifying the patient in one of the 3 groups defined as "low level" when p27 protein levels are lower than 50% of those of α-tubulin, "moderate level" when p27 protein levels vary from 50% to 150% of those of α-tubulin, and "high level" when p27 protein levels are higher than 150% of those of α-tubulin; and d) selecting said patient classified in the "low level" group for a chemotherapy treatment based on aplidine.

11. Method according to claim 10, wherein the tissue sample is a tumour biopsy.

12. Method according to claim 10 or 11, wherein the cancer is selected from the group consisting of acute lymphoblastic leukaemia, multiple myeloma, NHL, melanoma, kidney cancer, colon cancer, renal cancer, medullary thyroid cancer, pancreas cancer, lung cancer, sarcoma, prostate cancer, urothelial cancer, head & neck cancer and breast cancer.

13. A method of predicting the clinical response of a cancer patient to the treatment with aplidine comprising a) assaying p27 protein expression level in a biological sample from said cancer patient; b) assaying the expression level of a reference protein in the same sample as in a); and c) comparing said p27 protein expression level obtained in a) with the expression level of the reference protein obtained in b); wherein a p27 protein expression level is below a determined value of the expression level of the reference protein obtained in b) is indicative of a positive clinical response of the cancer patient to the treatment with aplidine.

14. Method according to claim 13, wherein the reference protein is the α-tubulin protein.

15. Method according to claims 13 and /or 12, wherein the biological sample is a tumour biopsy.

16. Method according to anyone of claims 13 to 15, wherein the cancer is selected from the group consisting of acute lymphoblastic leukaemia, multiple myeloma, NHL, melanoma, kidney cancer, colon cancer, renal cancer, medullary thyroid cancer, pancreas cancer, lung cancer, sarcoma, prostate cancer, urothelial cancer, head & neck cancer and breast cancer.