WO2018060452A1 - Therapeutic approaches to cancer - Google Patents

Abstract

The present invention provides a phosphorylated TAK1 polypeptide selected from the group consisting of: (a) the amino acid sequence SEQ ID NO:1 wherein one or more of the residues at positions 344, 389, 444, and T51 1 are phosphorylated, (b) a functional fragment of SEQ ID NO: 1 which comprises one or more of the amino acid residues identified in sequence SEQ ID NO:1 as 344, 389, 444, and T511, phosphorylated; and (c) an amino acid sequence with an identity degree with SEQ ID NO:1 of at least 85%, provided that this amino acid sequence comprises one or more of the amino acid residues identified in sequence SEQ ID NO:1 as 344, 389, 444, and 511, phosphorylated. The present invention further provides antibodies or fragments thereof able to bind to the phosphorylated TAK1 polypeptide of the invention, as well as kits comprising either the polypeptide or the antibody and methods for predicting the response to a TAK1-based therapy by a patient suffering from cancer.

Description

Therapeutic approaches to cancer

This application claims the benefit of European Patent Application

EP16382449.3 filed on September 30^th, 2016.

The present invention is related to the field of medicine, more particularly of oncology. Concretely, the present invention provides new therapeutic uses of TAK1 inhibitors, a combination of ROCK and β-catenin inhibitors and the therapeutic uses thereof and methods for identifying a patient having cancer who would be responsive to treatment with a TAK1 inhibitor.

BACKGROUND ART

Cutaneous T-cell lymphomas (CTCL) are lymphoid malignant neoplasms included as peripheral T-cell non-Hodgkin's lymphomas that primarily manifest in the skin. The most frequent CTCL, mycosis fungoides (MF) and the leukemic variant Sezary syndrome (SS), are characterized by a

proliferation of T-helper cells with a mature phenotype (CD3+, CD4+,

CD45RO+). MF is characterized by a clinical multistage development starting with erythematous scaly patches that are followed years later by infiltrated plaques, and final transformation into the tumor stage. In SS, the disease is clinically characterized by erythroderma associated with peripheral blood involvement manifested by circulating malignant lymphoid cells with

cerebriform nuclei (Sezary cells). Tumor-stage MF and SS are considered aggressive forms of the disease and usually have an unfavorable prognosis.

Although the pathogenic mechanisms implicated in CTCL progression are fairly unknown, several reports have suggested a relevant role of STAT3, Notch and β-catenin pathways in this group of disorders (Bellei et al., 2006; Gallardo et al., 2015; Kamstrup et al., 2010; McKenzie et al., 2012; van der Fits et al., 2012; Vieyra-Garcia et al., 2016). Recently, massive DNA and RNA sequencing of tumor-stage MF and SS samples has clearly demonstrated a pivotal role for the NF-κΒ signaling pathway in CTCL (Braun et al., 201 1 ; Choi et al., 2015; da Silva Almeida et al., 2015; Ungewickell et al., 2015), which is not surprising since NF-κΒ had been previously identified as an essential regulator of normal T-cell homeostasis and function (Chang et al., 2012; Li et al., 201 1 ; Ren et al., 2002). Moreover, specific inactivation of the pathway leads to a blockage in the differentiation and survival of the mature T cell compartment (Schmidt-Supprian et al., 2003; Senftleben et al., 2001 ; Voll et al., 2000) and precludes tumor progression in a mouse model of Notch- induced Acute T-cell Leukemia (Espinosa et al., 2010).

Phosphorylation of ΙκΒα by ΙΚΚβ is the critical step on NF-κΒ activation, which is initiated, in a stimulus-dependent manner, by the TAK1 kinase downstream of the ubiquitin-ligase elements TRAF6 and Ubc13.

Nevertheless, treatment of primary and transformed T cells with PP2A or PP1 inhibitors has been found to increase the amount of phosphorylated ΙκΒα leading to NF-κΒ activation (Menon et al., 1993; Sun et al., 1995), thus indicating the existence of constitutive IKK activity that is counteracted by phosphatase activity in this particular cell lineage. Various phosphatases have been identified that negatively regulate the IKK complex, thus guaranteeing precise and transient cellular responses to extracellular stimuli in particular cell types. That is the case of the CUEDC2/PP1 (Li et al., 2008) and the PP4R1 (Brechmann et al., 2012) phosphatase complexes. One step upstream in the pathway, PP1 through GADD34 was found to repress the IKK kinase TAK1 in macrophages (Gu et al., 2014) by dephosphorylation of its regulatory S412 residue (Ouyang et al., 2014), thus preventing excessive activation of the TLR pathway during inflammatory immune responses.

However, the impact of NF-κΒ regulation by specific phosphatase elements on cancer has remained mostly unexplored. Interferon, oral retinoids (bexarotene) and non-specific histone deacetylase inhibitors are currently prescribed as therapeutic options, but in most cases achieve response rates of about 30%.

Up to date there are no targeted therapies that provide a curative alternative for advanced CTCL patients.

SUMMARY OF THE INVENTION

The present inventors have studied the contribution of the MYPT1/PP1 phosphatase complex to the regulation of NF-κΒ through transforming growth factor activated kinase-1 (TAK1 ) in CTCL cells, both in primary tumor cells and cell lines, and its potential therapeutic relevance. As it is illustrated below, the inventors found that the inhibition of PP1 activity promotes NF-κΒ activation, being detected a massive accumulation of phosphorylated IKK and ΙκΒα. This hyperphosphorilzation was reverted when a TAK1 inhibitor was administered (FIG. 1 D). In an attempt to elucidate the PP1 's modulation effect on TAK1 phosphorylation, the present inventors identified 4 positions which resulted to be critical for inducing IKK and ΙκΒα phosphorilization: T344, S389, T444, and T51 1 , the numbering position being provided with respect to the murine sequence for TAK1 isoform A. The results obtained can be extrapolated to human TAK1 isoform B (Uniprot reference 043318-1 , June 1 , 1998 - v1 ) because, in addition to the high identity sequence between human and murine sequences (99% identity), human sequence also comprises the same amino acid residues at the same positions as those identified by the present inventors in the murine model as relevant.

From the results provided below, the present inventors have found new versions of phosphorylated TAK1 enzyme, which can provide valuable information for the development and management of anticancer therapies.

Therefore, in a first aspect, the present invention provides a phosphorylated TAK1 polypeptide selected from the group consisting of:

(a) the amino acid sequence SEQ ID NO: 1 , corresponding to human TAK1 isoform B amino acid sequence with Uniprot reference 043318-1 (June 1 ,

1998 - v1 ), wherein one or more of the residues at positions 344, 389, 444, and 51 1 are phosphorylated,

(b) a functional fragment of SEQ ID NO: 1 which comprises one or more of the amino acid residues identified in sequence SEQ ID NO: 1 as 344, 389,

444, and 51 1 , phosphorylated; and

(c) an amino acid sequence with an identity degree with SEQ ID NO:1 of at least 85%, provided that this amino acid sequence comprises one or more of the amino acid residues identified in sequence SEQ ID NO: 1 as 344, 389, 444, and 51 1 , phosphorylated. In a second aspect, the present invention provides an antibody or a functional fragment thereof which binds to the phosphorylated TAK1 polypeptide as defined in the first aspect of the invention, wherein the antibody or fragment thereof specifically binds to the one or more phosphorylated amino acid residues referred in the first aspect of the invention (i.e., 344, 389, 444, and T51 1 ).

In a third aspect, the present invention provides a kit comprising either the polypeptide as defined in the first aspect of the invention or the antibody as defined in the second aspect of the invention.

The experimental data provided below further supports the fact that CTCL patients can be stratified based on the activation status of β-catenin and NF- KB: patients carrying NF-κΒ and high β-catenin active (determined by the detection of Ρ-ΙκΒα and nuclear β-catenin, respectively) would be indicative that they can benefit from treatments targeting TAK1 kinase activity.

Therefore, in a fourth aspect, the present invention provides a method for predicting the response to TAK1 inhibitor-based therapy by a patient suffering from cancer, the method comprising the step of determining, in an isolated sample of the patient, whether one or more amino acid residues 344, 389, 444, and 51 1 of TAK1 protein is/are phosphorylated, wherein if one or more residues are phosphorylated in TAK1 protein, this is indicative that the patient will positively respond to TAK1 inhibitor-based therapy.

The results provided herein also show that the phosphatase PP1 and its regulator MYPT1 directly bind TAK1 in CTCL cells. Blocking PP1 activity, both pharmacologically and genetically, resulted in TAK1

hyperphosphorylation, leading to canonical NF-κΒ signaling. Conversely, inhibition of TAK1 induced apoptosis of both CTCL cell lines and primary human Sezary cells.

As it can be seen in FIG. 4B, a reduction in tumor cell growth of about 70% was detected when cells were treated with a TAK1 inhibitor ((5Z)-7- oxozeaenol). This meant a significant improvement when compared with the about 27% reduction in cell growth observed when tumor cells were treated with inhibitors of other proteins forming part of the NF-κΒ pathway, such as ROCK1 or IKK (FIG. 4B). FIG. 6 also illustrates that only 36h with TAK1 inhibitor treatment was enough to preclude tumor cell growth (FIG. 4E).

A more detailed analysis demonstrated that TAK1 inhibition specifically induced a massive apoptosis in CTCL cells, as determined by the

accumulation of active caspase 3, associated with a strong reduction in the amount of total β-catenin (FIG. 4C).

This result supports the use of TAK1 inhibitors in the treatment of cancer.

Thus, in a fifth aspect the present invention provides an inhibitor compound of TAK1 for use in the treatment of T-cell lymphoma.

The present inventors have also surprisingly found that the combined use of a ROCK1 and β-catenin inhibitors also imposed a higher growth arrest on all tested CTCL cell lines when compared with either treatment alone (FIG. 4D). These results suggest that TAK1 exerts two complementary prosurvival activities in CTCL cells, one downstream of ROCK1 , that is IKK- and N F-KB- related and a second that links to β-catenin signaling pathway.

Therefore, in a sixth aspect the present invention provides a combination of a ROCK1 inhibitor and a β-catenin inhibitor as a potential cancer therapy.

In a seventh aspect, the present invention provides a pharmaceutical or veterinary composition comprising a therapeutically effective amount of the combination as defined in the sixth aspect of the invention together with one or more pharmaceutically or veterinary acceptable excipients or carriers.

As it is further illustrated in FIG. 4D, 36h of treatment with a combination of a ROCK1 and a β-catenin inhibitors were sufficient to preclude tumor cell growth, which is indicative of the high efficacy that this kind of therapy can provide.

In an eighth aspect the present invention provides a combination as defined in the sixth aspect of the invention for use in the treatment of cancer.

In a ninth aspect the present invention provides a pharmaceutical or veterinary product comprising a ROCK inhibitor and a β-catenin inhibitor as a combined preparation for simultaneous, sequential or separate use in therapy. In a tenth aspect the present invention provides an inhibitor compound of ROCK for use in combination therapy with β-catenin in the treatment of cancer.

In a final aspect the present invention provides an inhibitor compound of β- catenin for use in combination therapy with a ROCK inhibitor in the treatment of cancer.

DRAWINGS FIG. 1 . (A) Western blot (WB) analysis of different CTCL cells treated with calyculin A1 (CALY) for 40 minutes, (B) WB analysis of CTCL cells treated with 10 nM of calyculin A1 and collected at the indicated time-points. (C-E) Western blot of the indicated CTCL cells transduced with specific shRNA against PP1 (C), or treated with the TAK1 (D) or ΙΚΚβ inhibitors (E) for 16 hours. (F) Immunofluorescence analysis to measure the levels of active p65- N F-KB in control and calyculin A1 -treated CTCL cells.

FIG. 2. (A) Western blot (WB) of CTCL cell lysates separated in a Superdex S200 column. (B) WB analysis of MYLA cell lysates immunoprecipitated (IP) with antibodies against active TAK1 (P-S412) or non-related control IgG. 1/10 of the total lysate is shown as input control. (C) WB analysis of the indicated CTCL cells left untreated (UNT), or treated for 40 minutes with vehicle (DMSO) or the PP1 inhibitors calyculin A1 (CALY) and okadaic acid (OKA). (D) Lysates from calyculin A1 -treated CTCL cells where left untreated or incubated with calf intestinal phosphatase (CIP) and analyzed by WB. (E)

Mass-spectrometry analysis of anti-P-TAK1 precipitates to identify TAK1 peptides that were differentially phosphorylated following calyculin treatment; SEQ ID NO: 2= RRSIQDLTVTGTEPGQVSSR; SEQ ID NO: 3=

VQTEIALLLQR; SEQ ID NO: 4= MITTSGPTSEKPTR; SEQ ID NO: 5=

RMSADMSEIEAR; SEQ ID NO: 6= SDTNMEQVPATNDTIK. (F) WB analysis of HEK-293T cells transiently expressing the indicated TAK1 kinase mutants. FIG. 3. (A) Mass-spectrometry analysis of anti-P-TAK1 precipitates to identify elements associated with TAK1 complex in CTCL. (B) Western blot (WB) analysis of lysates from TNFa-treated MYLA cells immunoprecipitated with antibodies against the IKK subunit NEMO. (C) WB analysis of total cell lysates from TNFa-treated MYLA cells. (D) WB of untreated or TNFa-treated CTCL cell lysates separated in a Superdex S200 columns. (E) WB analysis of MYLA cells treated with TNFa at different time-points in the absence or presence of ROCK inhibitor. (F) Relative levels of ΙκΒα protein from 3 independent experiments performed as in E. The average values and standard deviation of the media are shown. (G) Same experiment as in E from cells pretreated 16h with the proteasome inhibitor MG132. Average and standard deviation of the data obtained by densitometry analysis of 3 independent experiments performed. In the different experiments n.s.= nonsignificant; ^* p <0.05; ^** p< 0.01 and ^*** p<0.001 obtained by T-test.

FIG. 4. (A) Western blot (WB) analysis of CTCL cells dissociated and replated under non-adherent (N) or adherent (A) conditions. As it is derived from the experimental data, the process of cellular resuspension in nonadherent conditions induced phosphorylation of all relevant elements in the pathway including MYPT1 , TAK1 , IKK and ΙκΒα. (B) Quantification of the number of cells obtained after culturing them in the indicated conditions (upper panels) and WB analysis (lower panels) to evaluate the efficacy of the treatments. (C) WB of the same cells shown in B with the indicated

antibodies. (D) Quantification of the number of CTCL cells produced after 36 hours of culture with the indicated inhibitors.

FIG. 5. (A-B) Immunofluorescence analysis of the indicated proteins in different human primary CTCL frozen samples. Representative images from 8 different samples analyzed. (C) Immunohistochemistry analysis of P-TAK1 (T444) in two representative (1 MF and 1 SS) paraffin-embedded human primary CTCL samples. (D-E) Tables showing the distribution of samples that were positive for P-TAK1 and/or Ρ-ΙκΒα in the indicated groups. Statistical significance of the correlations was obtained by chi-square test. (F) Number of primary Sezary syndrome cells produced after 36 hours of culture with the indicated inhibitors. In all experiments n.s.= non-significant; ^* p <0.05; ^** p< 0.01 and ^*** p<0.001 by T-test. FIG. 6 (A) Immunofluorescence analysis of total P65-NF-KB in control and 40 minutes calyculin A1 -treated CTCL cells. (B) Western blot (WB) analysis of control and calyculin A1 -treated human cancer cells from breast (MDA435), colon (LIM1215, DLD1 , LS174, WiDr) and CTCL (SeAX, HH and MYLA). (C) WB analysis of control and calyculin A1 -treated (40 minutes) wildtype (WT) and ΙΚΚβ-deficient Mouse Embryonic Fibroblast (MEF) cultures.

FIG. 7 (A) Western blot (WB) analysis of HEK-293T cells transfected with HA- TAK1 and treated for 40 minutes as indicated, 48 hours after transfection. (B) WB analysis of TAK1 (P-S412) precipitates from untreated, vehicle-treated and calyculin A1 -treated (40 minutes) MYLA cells.

FIG. 8 (A) Western blot (WB) analysis of HH cells treated for 5 or 40 minutes with TNFa, or 40 minutes with calyculin A1. In these experiments, the expected molecular weight for P-TAK1 was 75 kDa and higher. (B) WB analysis of anti-P-TAK1 (S412) precipitates obtained from untreated or 40 minutes-treated HH cells. The WB for P-TAK1 (S412) is shown as positive control of the experiment. FIG. 9 (A-C) Immunofluorescence (IF) analysis of the indicated proteins in frozen CTCL biopsy samples. (D) Representative image of the

immunohistochemical analysis for β-catenin and Ρ-ΙκΒα in paraffin embedded SS biopsies. (E) Table showing the number and distribution of samples that were categorized as negative or positive for Ρ-ΙκΒα and P-TAK1 in the analysis of human CTCL samples included in Table 1. A chi-square test was used to determine the significance of the correlation between both

parameters.

DETAILED DESCRIPTION OF THE INVENTION

All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition. In a first aspect, the present invention provides phosphorylated TAK1 polypeptides.

"Transforming growth factor activated kinase-1 " and "TAK1 " are used interchangeably. It is a protein kinase of the MLK family that mediates signal transduction induced by TGF beta and morphogenetic protein (BMP), and controls a variety of cell functions including transcription regulation and apoptosis. Illustrative non-limitative examples of TAK1 are the human

(Uniprot database accession number 043318), mouse (Uniprot database accession number Q62073), among others. For the particular case of human TAK1 , 4 isoforms have been disclosed: isoform 1 B, which has been chosen by the skilled person as the 'canonical' sequence (043318-1 ); isoform 1A (043318-2) which differs from canonical isoform 1 B in that it lacks residues 404-430; isoform 1 C (043318-3), which differs from the canonical sequence in a change of amino acid portion from 509 to 518 and the deletion of residues 519-606; and isoform 1 D (043318-4), which lacks the consensus amino acid residues 404-430 and 519 to 606 and differs in the sequence in the portion defined by consensus amino acids 509-518. All four human TAK1 isoforms comprise the consensus amino acid of SEQ ID NO: 1 at positions 344, 389, and 444. And, with the exception of isoform D, all TAK1 isoforms also comprises the consensus amino acid at position 51 1 .

In one embodiment of the first aspect of the invention, the phosphorylated TAK1 polypeptide comprises the amino acid sequence SEQ ID NO: 1 , corresponding to human TAK1 isoform A amino acid sequence, wherein one or more of the residues at positions 344, 389, 444, and T51 1 are

phosphorylated. In another embodiment of the first aspect of the invention, the phosphorylated TAK1 polypeptide consists of the amino acid sequence SEQ ID NO: 1 , corresponding to human TAK1 isoform A amino acid sequence, wherein one or more of the residues at positions 344, 389, 444, and T51 1 are phosphorylated

In an embodiment of the first aspect, the phosphorylated TAK1 polypeptide of the invention comprises the amino acid sequence SEQ ID NO: 1 which comprises one of the residues at position 344, 389, 444, and 51 1 ,

phosphorylated. In another embodiment of the first aspect of the invention, the phosphorylated TAK1 polypeptide of the invention comprises the amino acid sequence SEQ ID NO: 1 with two of the residues at positions 344, 389, 444, and 51 1 phosphorylated. In another embodiment of the first aspect of the invention, the phosphorylated TAK1 polypeptide of the invention comprises the amino acid sequence SEQ ID NO: 1 with three of the residues at positions 344, 389, 444, and 51 1 phosphorylated. In another embodiment of the first aspect of the invention, the phosphorylated TAK1 polypeptide of the invention comprises the amino acid sequence SEQ ID NO: 1 with all four residues 344, 389, 444, and 51 1 , phosphorylated. In still another embodiment, the phosphorylated TAK1 polypeptide of the invention comprises the amino acid sequence SEQ ID NO: 1 with the following amino acid residues

phosphorylated: residues at positions 344 and 389; residues at positions 344, 444; residues at positions 344 and 51 1 ; residues at positions 389 and 444; residues at positions 389 and 51 1 ; residues at positions 444 51 1 ; residues at positions 344, 389, and 444; residues at positions 344, 389, and 51 1 ; residues at positions 344, 444, and 51 1 ; or residues at positions 389, 444, and 51 1 .

In the present invention, the positions referred as "344", "389", "444" and

"51 1 " correspond to the amino acid residue positions in the sequence referred in the present application as sequence SEQ ID NO:1 .

In the present invention, the expression "functional fragment of SEQ ID NO: 1 " has to be understood as any fragment of the sequence SEQ ID NO: 1 which comprises one or more of the residues identified in SEQ ID NO: 1 as amino acid residue number 344, number 389, number 444 and/or number 51 1 , phosphorylated, and which maintain the characteristic kinase activity of the phosphorylated TAK1 . In view of this definition, human isoforms A, C, and D are encompassed within this term. The kinase activity of TAK1 can be determined by routine methods such western blot analysis, mass

spectrometry for particular phosphorylated residues, kinase assays or IHC analysis.

In the present invention the term "identity" refers to the percentage of residues or bases that are identical in the two sequences when the

sequences are optimally aligned. If, in the optimal alignment, a position in a first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the sequences exhibit identity with respect to that position. The level of identity between two sequences (or "percent sequence identity") is measured as a ratio of the number of identical positions shared by the sequences with respect to the size of the sequences (i.e., percent sequence identity = (number of identical positions/total number of positions) x 100).

A number of mathematical algorithms for rapidly obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include the MATCH-BOX, MULTAIN, GCG, FASTA, and ROBUST programs for amino acid sequence analysis, among others.

Preferred software analysis programs include the ALIGN, CLUSTAL W, and BLAST programs (e.g., BLAST 2.1 , BL2SEQ, and later versions thereof). For amino acid sequence analysis, a weight matrix, such as the BLOSUM matrixes (e.g., the BLOSUM45, BLOSUM50, BLOSUM62, and BLOSUM80 matrixes), Gonnet matrixes, or PAM matrixes (e.g., the PAM30, PAM70,

PAM120, PAM160, PAM250, and PAM350 matrixes), are used in determining identity. When the alignment is performed it is also checked whether the particular sequence has an amino acid residue capable of being

phosphorylated (i.e., a Thr or Ser residue) at positions equivalent to positions 344, 389, 444 and/or 51 1 of sequence SEQ ID NO: 1 .

The BLAST programs provide analysis of at least two amino acid sequences, either by aligning a selected sequence against multiple sequences in a database (e.g., GenSeq), or, with BL2SEQ, between two selected sequences. BLAST programs are preferably modified by low complexity filtering programs such as the DUST or SEG programs, which are preferably integrated into the BLAST program operations. If gap existence costs (or gap scores) are used, the gap existence cost preferably is set between about -5 and -15. Similar gap parameters can be used with other programs as appropriate. The BLAST programs and principles underlying them are further described in, e.g.,

Altschul et al., "Basic local alignment search tool", 1990, J. Mol. Biol, v. 215, pages 403-410.

For multiple sequence analysis, the CLUSTAL W program can be used. The CLUSTAL W program desirably is run using "dynamic" (versus "fast") settings. Amino acid sequences are evaluated using a variable set of

BLOSUM matrixes depending on the level of identity between the sequences. The CLUSTAL W program and underlying principles of operation are further described in, e.g., Higgins et al., "CLUSTAL V: improved software for multiple sequence alignment", 1992, CABIOS, 8(2), pages 189-191 . In one embodiment, the sequence of the first aspect of the invention has an identity of 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,1 %, 99,2%, 99,3%, 99,4%, 99,5%, 99,6%, 99,7%, 99,8%, 99,9% or 100% with respect to sequence SEQ ID NO: 1 . In a second aspect, the present invention provides an antibody or a fragment thereof, which binds to the phosphorylated TAK1 polypeptide as defined in the first aspect of the invention.

The term "antibody or a fragment thereof able to bind to the phosphorylated TAK1 polypeptide" is to be understood as any immunoglobulin or fragment thereof able to selectively bind the phosphorylated TAK1 polypeptide via one or more of the phosphorylated residues at positions 344, 389, 444 and 51 1 , the numbering position being in respect of the sequence numbering of sequence SEQ ID NO: 1 . It includes monoclonal and polyclonal antibodies. The term "fragment thereof encompasses any part of an antibody having the size and conformation suitable to bind an epitope of the target protein.

Suitable fragments include F(ab), F(ab') and Fv.

There are well known means in the state of the art for preparing and characterizing antibodies. Methods for generating polyclonal antibodies are well known in the prior art. Briefly, one prepares polyclonal antibodies by immunizing an animal with the polypeptide of the eighth aspect of the invention; then, serum from the immunized animal is collected and the antibodies isolated. A wide range of animal species can be used for the production of the antiserum. Typically the animal used for production of antisera can be a rabbit, mouse, rat, hamster, guinea pig or goat.

Moreover, monoclonal antibodies (MAbs) can be prepared using well-known techniques. Typically, the procedure involves immunizing a suitable animal with the polypeptide of the eighth aspect of the invention. The immunizing composition can be administered in an amount effective to stimulate antibody producing cells. Methods for preparing monoclonal antibodies are initiated generally following the same lines as the polyclonal antibody preparation. The polypeptide is injected into animals as antigen. The antigen may be mixed with adjuvants such as complete or incomplete Freund's adjuvant. At intervals of two weeks, approximately, the immunization is repeated with the same antigen.

In a third aspect, the present invention provides a kit comprising the phosphorylated TAK1 polypeptide, as defined in the first aspect of the invention, or the antibody or fragment thereof, as defined in the second aspect of the invention .

The kit may additionally comprise means (additives, solvents) to visualize the antibody-protein interactions. The kit may also comprise instructions for its use.

These antibodies can be used as "means" for determining the expression of the target proteins in the fifth aspect of the invention. In a fourth aspect the present invention provides a method for identifying a patient having cancer who can be responsive to treatment with a TAK1 inhibitor

There are well-known methods in the state of the art for determining the phosphorylation state of an amino acid residue forming part of TAK1 , β- catenin or NF-κΒ activation, such as mass spectrometry analysis of tissue lysates or detection by specific antibodies in western blot or immuno- histochemistry assays. The skilled person in the art, making use of the general knowledge, can optimize the parameters of the selected method for determining the phosphorylation status of an amino acid residue (i.e., whether the amino is phosphorylated).

In view of the above, in one embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above, it is determined whether one or more of the amino acid residues at positions 344, 389, 444, and 51 1 with respect to SEQ ID NO: 1 are phosphorylated. In another embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above, the determining step is performed by contacting the enzyme with an antibody as defined in the second aspect of the invention, wherein if it is detected the binding of the antibody to the enzyme, it will be indicative that one or more residue(s) identified as positions 344, 389, 444, and 51 1 in sequence SEQ ID NO: 1 are phosphorylated and, therefore, the subject will positively respond to a TAK1 inhibitor-based therapy. In one embodiment of the method of the fourth aspect of the invention, the cancer is a lymphoma.

In the present invention the term "lymphoma" refers to a group of blood cells tumors that develop from lymphocytes. It is the most common blood cancer. Lymphoma occurs when cells of the immune system called lymphocytes, a type of white blood cell, grow and multiply uncontrollably. Cancerous lymphocytes can travel to many parts of the body, including the lymph nodes, spleen, bone marrow, blood, or other organs, and form a mass called a tumor. The body has two main types of lymphocytes that can develop into

lymphomas: B-lymphocytes (B-cells) and T-lymphocytes (T-cells).

In one embodiment of the method of the fourth aspect of the invention, the lymphoma is a T-cell lymphoma. There are many different forms of T-cell lymphomas, some of which are extremely rare. T-cell lymphomas can be aggressive (fast-growing) or indolent (slow-growing). Illustrative non-limitative examples of T-cell lymphomas are:

peripheral T-cell lymphoma, anaplastic large cell lymphoma,

angioimmunoblastic Lymphoma, cutaneous T-cell lymphoma, adult T-cell

Leukemia/Lymphoma, blastic NK-cell Lymphoma, enteropathy-type T-cell lymphoma, hematosplenic gamma-delta T-cell lymphoma, lymphoblastic lymphoma, and nasal NK/T-cell lymphomas. In one embodiment of the fourth aspect of the invention, the cancer is a cutaneous T-cell lymphoma. In another embodiment of the method of the fourth aspect of the invention, the isolated sample is selected from peripheral blood and tumor biopsies.

The present invention further provides in a fifth aspect the use of TAK1 inhibitors in the treatment of cancer.

In one embodiment of the fifth aspect of the invention, the cancer is a lymphoma. In the present invention, the expression "inhibitor compound" either when the target protein is TAK1 , ROCK, β-catenin or IKK, encompasses any compound known in the state of the art as being inhibiting the activity of the target protein. For example, if the target protein is TAK1 , the inhibitor compound will inhibit the activity of the target kinase. Of course, the inhibitor compound can show an inhibitory effect on the activity of the target protein (e.g. TAK1 ), which can be of any source, such as human or mouse. The inhibition effect can be due to a direct interaction/binding with the target protein or due to the modification of the activity of any of the components forming part of the cascade route to which target protein belongs, and which act prior the target protein: so taking advantage of the "cascade" effect, affecting any of such prior components, target protein's activity can be inhibited.

In one embodiment of the fifth aspect, the inhibitor binds TAK1 . In one embodiment of the fifth aspect, the TAK1 inhibitor compound is a small molecule, an antibody or a fragment thereof which retain the ability to bind TAK1 . Illustrative non-limitative examples of TAK1 inhibitor compounds, which are commercially available, are: 5Z-oxozeaenol, hypothemycin, epoxyquinol B and N-Des(aminocarbonyl). In one embodiment, the inhibitor compound is 5Z-oxozeaenol.

In a sixth aspect, the present invention provides a combination of a ROCK kinase inhibitor and a β-catenin inhibitor. "Rho-associated protein kinase" and "ROCK" are used interchangeably and refers to a kinase belonging to the AGC (PKA PKG/PKC) family of serine- threonine kinases. It is involved mainly in regulating the shape and movement of cells by acting on the cytoskeleton. ROCK1 (and its homologous ROCK2) is expressed in mammals (human Q13464, chimpanzee P61584, rat Q63644, mouse P70335), chicken F1 NRH6, zebrafish E7F9X9 and also invertebrates such as Drosophila Q9VXE3.

In one embodiment of the sixth aspect of the invention, optionally in

combination with any of the embodiments provided above or below ROCK inhibitor binds to ROCK protein. In another embodiment of the sixth aspect of the invention, optionally in combination with any of the embodiments provided above or below, ROCK inhibitor is a ROCK1 inhibitor.

There are well-known ROCK inhibitors under commercialization, either as small molecules or antibodies or fragments of suh antibodies with the ability of binding to ROCK protein. Illustrative non-limitative examples are Y27632, Thiazovivin™, GSK429286A, RKI-1447, KD025 (SLx-21 19), KD025 (SLx- 21 19), GSK429286A, Ripasudil (K-1 15), and Fasudil™. In one embodiment, ROCK1 inhibitor is Y27632.

"β-catenin" protein (Uniprot P352222 in humans, Q9WU82 in rat and Q02248 in mouse) is a dual function protein, regulating the coordination of cell-cell adhesion and gene transcription. In humans, this protein is encoded by the ctnnbl gene. Beta-catenin is widely expressed in many tissues. In cardiac muscle, beta-catenin localizes to adherens junctions in intercalated disc structures, which are critical for electrical and mechanical coupling between adjacent cardiomyocyte.

In one embodiment of the sixth aspect of the invention, optionally in

combination with any of the embodiments provided above or below, the β- catenin inhibitor binds to β-catenin.

There are well-known β-catenin inhibitors in the state of the art that are commercially available, either as small molecules, antibodies or fragments of such antibodies with the ability of binding to β-catenin protein. Illustrative non- limitative examples are: PKF1 15-584 from Sigma or IWP4 and JW67 from R&D systems. In one embodiment of the sixth aspect of the invention, the combination comprises PKF1 15-584 (β-catenin inhibitor) and Y-27632 (ROCK inhibitor), both of Sigma:

In a seventh aspect, the present invention provides a pharmaceutical or veterinary composition comprising a therapeutically effective amount of the combination of the sixth aspect of the invention together with one or more pharmaceutically or veterinary acceptable excipients or carriers.

The expression "therapeutically effective amount" as used herein, refers to the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed. The particular dose of compound

administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations.

The expression "pharmaceutically acceptable excipients or carriers" refers to pharmaceutically acceptable materials, compositions or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio. Likewise, the term "veterinary acceptable" means suitable for use in contact with a non-human animal. Examples of suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. The relative amounts of the combination, the pharmaceutically or veterinary acceptable excipient, and/or any additional ingredients in a pharmaceutical or veterinary composition of the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.

In an ninth aspect, the present invention provides a pharmaceutical or veterinary product.

In one embodiment of the ninth aspect of the invention, the product is in the form of a pharmaceutical or veterinary composition comprising one or more pharmaceutically or veterinary acceptable excipients or carriers.

In one embodiment of the combination as defined in the sixth aspect of the invention, the pharmaceutical composition as defined in the seventh aspect of the invention, and the pharmaceutical product as defined in the ninth aspect of the invention, a IKB quinase (IKK) inhibitor is further included.

IKK is an enzyme complex that is involved in propagating the cellular response to inflammation. The ΙκΒ kinase enzyme complex and more specifically the IKKbeta subunit (Uniprot 014920 in human) is part of the upstream NF-κΒ signal transduction cascade. The ΙκΒα (inhibitor of kappa B) protein inactivates the NF-κΒ transcription factor by masking the nuclear localization signals (NLS) of NF-κΒ proteins and keeping them sequestered in an inactive state in the cytoplasm.

In one embodiment, optionally in combination with any of the embodiments provided above or below, IKK inhibitor binds IKK. In another embodiment, optionally in combination with any of the

embodiments provided above or below, IKK inhibitor is a small molecule, an antibody or fragment thereof with the ability of binding IKK.

Illustrative non-limitative examples of IKK inhibitors available in the market are: BAY65-581 1 from Bayer, IKK2 inhibitor XI and CAM 0657 from Santa Cruz. In one embodiment IKK inhibitor is BAY65-581 1 (Margalef et al., 2015).

IKK inhibitor can be forming part of the combination as defined above, as a combined preparation for simultaneous, sequential or separate use in therapy.

In one embodiment of the therapeutic uses defined in the fifth, eighth, ninth, tenth, and eleventh aspects of the invention, IKK inhibitor is also used in combination. In another embodiment of the therapeutic uses defined in the fifth, eighth, ninth, tenth, and eleventh aspects of the invention, the cancer is a lymphoma. In another embodiment of the therapeutic uses defined in the fifth, eighth, ninth, tenth, and eleventh of the invention, the cancer is a T-cell lymphoma. In another embodiment of the therapeutic uses defined in the fifth, eighth, ninth, tenth, and eleventh aspects of the invention, the cancer is a cutaneous T-cell lymphoma.

Throughout the description and claims the word "comprise" and variations of the word are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" and its variations encompasses the term "consisting of. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein. EXAMPLES

Material and Methods Cell cultures and cell lines.

MDA435 (breast cancer), LIM1215, DLD1 , Ls174T, WiDr (colon cancer) and HEK-293T (human embryonic kidney) cells were cultured in DMEM 10% FBS. Cutaneous T-cell Lymphoma (CTCL) cell lines (HH, HUT78, MYLA and SeAx) and primary Sezary cells were grown in RPMI 10% FBS.

Antibodies and chemicals

For Western Blot ("WB") and Immunohystochemistry ("IHC") we used the following antibodies: a-PP1 (sc-7482), α-ΙκΒα (sc-1643), a-p65 (sc-109), a- NEMO (sc-8330), a-P-IKKa-S180/IKK -S181 (sc-23470) were from Santa Cruz, α-ΙΚΚα (OP133) was purchased from Oncogen (La Jolla, CA). a-P- IKKa-S176,180/IKKp-S177,181 (2697S), a-P-kBa-S32,36 (92465), a-P- MYPT1 -T696 (5163), a-MYPT1 (2634), a-TAK1 (4505), a-P-TAK1 -S412 (9339), a-P-TAK1 -T184,187 (4508), a-cleaved caspase3 (9661 ) were from Cell Signaling. a-Active-p-catenin clone 8E7 (05-665) was from Millipore, and a-tubulin and α-β-catenin (c2206) were from Sigma (St. Louis, MO). a-Ki67 was from Novocastra (MM1 ). Secondary antibodies conjugated with

horseradish peroxidase (HRP) were from DAKO (Glostrup, Denmark, and AlexaFluor488 (A-1 1055) was from Molecular Probes (Invitrogen, Carlsbad, CA) and Cy3-coupled tyramide was from PerkinElmer (Wellesley, MA).

ΙΚΚβ, P-p65 (S536), Lentiviral vectors (pLKO.1 -Puro) containing the shRNA against PP1 were from Sigma (MISSION sh-RNA) and used following manufacturer instructions.

Antibodies targeting phosphorylated TAK1 (at residues 344, 389, 444 and 51 1 corresponding to the mouse and human isoform A) were generated by Abyntek (Bizkaia, Spain) by immunizing rabbits with the corresponding phosphor-peptides and then depleting the serum from antibodies recognizing the corresponding non-phosphorylated peptides.

Human TNFa is from Preprotech and was used at 40 ng/ml. Calyculin A1 and Okadaic Acid were from Cell Signaling and used at 10 nM and 2 nM, respectively. MG132 was fr4om Calbiochem and used at 10 μΜ, ROCK inhibitor (Y-27632) from Sigma was used at 4 μΜ. The specific ΙΚΚβ inhibitor BAY65-581 1 from Bayer was used at 5 μΜ as previously validated in

(Margalef et al., 2015). PKF-1 15-584 was used at 100 nM and the TAK1 inhibitor (5Z-7-oxozeanol) from Sigma at 10 μΜ. All commercial drugs were prepared as specified by the manufacturer and used at the indicated concentrations Generation of TAK1 point mutants and cell transfection

The inventors introduced the following specific point mutations in the

TAK1 protein individually: threonine 344 was changed to alanine; serine 389 was changed to alanine; threonine 444 was changed to alanine and threonine 51 1 was changed to alanine. The change of threonine or serine to alanine prevents these amino acid positions from being phosphorylated. TAK1 point mutants were generated using sequence overlap extension with specific mutagenic oligonucleotides and the mouse TAK1 cDNA as template. For sequence overlap extension a primer annealing 100% to the 5' end of the cDNA, that is100% identical to the coding strand, was used as forward primer in the PCR reaction. The reverse primer was centered to the codon (3 bases) encoding the amino acid to be changed and contained 18 extra nucleotides 5' to the desired change and 18 extra nucleotides 3' to the desired change. These 18 plus 18 nucleotide sequences were 100% complementary to the coding sequence. To proceed with the primer overlap extension approach, the inventors used a primer 100% complementary to the just described reverse primer (that is, a long primer (39 bases=18+3+18) 100% identical to the coding sequence except for the three bases in the middle that contained the desired change so that an alanine was introduced in the resulting protein when the sequence got translated). In addition the inventors designed a primer 100% complementary to the 3' end of the TAK1 coding sequence, including the stop codon.

Star-Prime polymerase from Takara was used following the manufacturer's instructions. The PCR products obtained using the TAK1 cDNA and the two primer pairs just described were mixed together (just 0.1 % of each reaction volume) and used to generate the final mutated cDNA or insert using again PCR with the non mutagenic primers described, the 5' end and 3' end primers. Mutated Tak1 inserts, generated by primer overlap extension as just described above, were sequenced using an automatic sequencing instrument at the CRG-genomics facility to ascertain that they contain the mutations of interest with no additional undesired changes. HEK-293T Cells were transfected using polyethylenimine (PEI) at a DNA reagent ratio of 1 to 5 in serum-free medium.

Immunofluorescence of cell cultures

Cells were grown in coverslips, then fixed in 4% paraformaldehid for 20 minutes at 4°C, washed in phosphate buffered saline (PBS) and

permeabilized for 2 hours in PBS 0.3% Triton X-100 (Pierce) plus dehydrated skim milk. Primary antibodies were incubated overnight at 4°C, then

extensively washed and incubated for 2 h at room temperature with

secondary AlexaFluor 488 (Molecular Probes) at a 1 :1000 dilution. Slides were mounted in VectaShield with DAPI (Vector).

Gel Filtration Assay on Superdex200 Columns

CTCL cells were lysed in 100 μΙ of PBS containing 0.5% Triton X-100, 1 mM EDTA, 100 mM Na-orthovanadate, 0.25 mM phenylmethanesulfonylfluoride (PMSF), and complete protease inhibitor cocktail (Roche, Basel, Switzerland), centrifuged at 13.000 r.p.m. and the supernatant loaded on Superdex200 gel filtration column (GE Healthcare). One drop (about 40 μΙ) per fraction was collected and analyzed by western blot.

Western blot analysis and immunoprecipitation assays Cells were lysed 30 minutes at 4°C in 300μΙ PBS plus 0.5% Triton X-100,

1 mM EDTA, 100 mM Na-orthovanadate, 0.25 mM PMSF and complete protease inhibitor cocktail (Roche). For immunoprecipitation, supernatants were pre-cleared 2h with 1 % of BSA, ^g IgGs and 50μΙ_ Sepharose Protein A (SPA) beads. Precleared lysates were incubated O/N with 3 μg of the indicated antibodies. Antibody-Protein complexes were captured with 30μΙ_ SPA beads for 2h. After washing, precipitates were analyzed by western blot. In most of the experiments, we used the Clean-Blot IP Detection Reagent (Thermo Fisher Scientific) instead of standard secondary antibodies, which is optimized for postimmunoprecipitation WB.

Mass spectrometry analysis

The beads with the immuno-precipitated proteins were reduced (10 mM dithiothreitol (DTT), 1 h, 37 °C), alkylated (20 mM iodoacetamide (IAA), 30 min, room temperature) and digested with trypsin (o/n, 37 °C) prior being analyzed in a LTQ-Orbitrap Velos Pro mass spectrometer (see

Supplementary materials for details). Raw data were analyzed using the Proteome Discoverer software suite (v1 .4) and the Mascot search engine (v2.5). Data were searched against the human protein database derived from SwissProt with a precursor ion mass tolerance of 7 ppm, and up to three missed cleavages. The fragment ion mass tolerance was set to 0.5 Da.

Oxidation (M), Phosphorylation (STY), Acetylation (Protein N-term) and Methylation (K), were defined as variable modifications, whereas

carbamidomethylation (C) was set as fixed modification. The identified peptides were filtered by False Discovery Rate (FDR) < 5%. Tissue samples, preparation and Immunohistochemistry

All biological samples used in this study were obtained from Pare de Salut MAR Biobank (MARBiobanc, Barcelona) with the consent of its ethical Committee, and following all Spanish Ethical regulations and the guidelines of the Declaration of Helsinki. Patient identity remained anonymous.

Formalin-fixed, paraffin-embedded tissue blocks of primary human cutaneous T-cell lymphomas were retrieved from MARBiobanc archives. Areas of invasive lymphoma lesions were identified on the corresponding hematoxylin- eosin-stained slides. Paraffin sections of 4μηη were de-waxed, rehydrated, and endogenous peroxidase activity was quenched (20 min, 1 .5% H₂O₂),

Antigen retrieval was performed depending on the antibody, and primary antibodies were incubated overnight at 4°C. After extensive washing in PBS, samples were incubated with HRP-linked secondary antibodies for 2 hours and then developed with the tyramide amplification system from PerkinElmer (Wellesley, MA). Image analysis

IHC of cutaneous lymphoma sections was observed and evaluated by two investigators in an Olympus BX61 microscope. Immunofluorescence images of the samples were taken by using confocal Leica SP5 TCS upright microscope and the Leica Application Suite Advanced Fluorescence software.

RESULTS Inhibition of PP1 activity promotes TAK1 -dependent activation of the IKK complex

The present inventors found that treatment of CTCL cells, both MF and SS, with the PP1/PP2 inhibitor calyculin A1 leads to a massive accumulation of phosphorylated IKK and ΙκΒα in the absence of cytokine-mediated

stimulation, as determined by WB analysis (FIG. 1A) and

immunofluorescence (not depicted). In this set of experiments, it was detected a shift in the molecular weight of TAK1 that was not linked to an increase in the amount of phosphorylated serine 412 (FIG. 1A), previously reported as a substrate for PP1 activity (Gu et al., 2014). Phosphorylation of IKK and ΙκΒα in response to calyculin A1 was robustly detected after 15-30 minutes of treatment (FIG. 1 B) suggesting that it did not require additional events upstream of TAK1 and IKK. Further demonstrating that IKK activation induced by calyculin A1 treatment was the result of PP1 inhibition, there was found a comparable increase in IKK phosphorylation after shRNA-mediated PP1 knock down in CTCL cells (FIG. 1 C). Phosphorylation of IKK and ΙκΒα following calyculin A1 treatment was abrogated by 16 hours of pre-incubation with the TAK1 inhibitor (5Z)-7-oxozeaenol (FIG. 1 D) or the ΙΚΚβ-specific inhibitor BAY65-8072 (FIG. 1 E), further suggesting that PP1 is a negative regulator of canonical ΤΑΚΙ -ΙΚΚβ-dependent NF-κΒ pathway in CTCL cells.

In agreement with this notion, 40 minutes of calyculin A1 treatment led to a massive nuclear accumulation of total p65 (FIG. 7A) and activated

(phosphorylated) p65 in CTCL cell lines (FIG. 1 F). Then, it was tested the possibility that calyculin A1 treatment similarly induced IKK phosphorylation in other cancer cell types. The results indicated that canonical IKK activation following PP1 inhibition (as denoted by the detection of a P-IKK band at 85- 87 kDa or higher) was not restricted to CTCL cells, although it was cell type specific (FIG. 7B). Interestingly, calyculin A1 treatment in CRC cells led to the accumulation of a distinct P-IKK isoform that is compatible with the previously identified p45-IKKa (Margalef et al., 2015; Margalef et al., 2012). To a minor extent, it was also detected NF-κΒ activation induced by calyculin A1 in mouse embryonic fibroblasts that was prevented in the absence of the canonical ΙΚΚβ kinase (FIG. 7C).

PP1 binds TAK1 and modulates TAK1 phosphorylation at non-canonical regulatory sites

As a first approach, freshly isolated CTCL cellular extracts were separated on Superdex S200 gel filtration columns, which are commonly used to identify components of specific multi-protein complexes. As shown in FIG. 2A, canonical IKK subunits including ΙΚΚα, ΙΚΚβ and ΙΚΚγ/ΝΕΜΟ were found to coelute in fractions 71 to 81 , showing a partial overlap with their upstream kinase TAK1 (eluting from fraction 79 to 91 ). Interestingly, PP1 that was mostly restricted to fraction 105 (corresponding to low molecular weight complexes) was also detected in small amounts ranging from fractions 73 to 83 (co-distributing with IKK and TAK1 ). To test whether PP1 physically interacted with elements of the NF-κΒ pathway in these cells, coprecipitation assays were performed. PP1 was detected in the precipitates from active P- TAK1 (S412) together with IKK (FIG. 2B). Data in FIG. 1 A showed that PP1 inhibition induces a shift in the apparent molecular weight of TAK1 protein, which was compatible with TAK1 hyperphosphorylation. Treatment of different CTCL cell lines with the PP1 inhibitors calyculin A1 and okadaic acid confirmed this result (FIG. 2C). The electrophoretic mobility shift of TAK1 imposed by calyculin A1 was reversed by incubation with alkaline

phosphatase but did not correlate with differences in phosphorylation of TAK1 at S412 (FIG. 2D). Similar results were obtained by calyculin A1 treatment of HEK293T cells transfected with wild type HA-TAK1 or the kinase inactive HA-

TAK1 T184/187A mutant (FIG. 9A). To directly investigate whether PP1 activity counteracted TAK1 phosphorylation at specific non-canonical sites, P- TAK1 (S412) was precipitated from CTCL cells untreated or treated with calyculin A1 for 40 minutes (FIG. 9B) and determined the TAK1 phospho- peptides that were differentially present in either condition by mass

spectrometry analysis. The results indicated that PP1 inhibition increased TAK1 phosphorylation at residues T344, S389, T444 and T51 1 (FIG. 2E), whereas we consistently failed to detect any increase in the canonical phosphorylation sites T184 or T187, in three independent experiments performed. To study the functional impact of PP1 -regulated phosphorylation of TAK1 , different HA-TAK1 point mutants of the residues identified by mass- spectrometry were generated. Mutation of T344, S389, T444 and T51 1 into A reduced the capacity of exogenous HA-TAK1 to induce IKK phosphorylation and to promote ΙκΒα phosphorylation and degradation when compared to wild type TAK1 , and comparable to the T184/187A mutant (FIG. 2F).

Together these results indicate that PP1 dephosphorylates TAK1 at previously unidentified sites to modulate its kinase activity on canonical IKK signaling. Regulation of canonical NF-κΒ by PP1 in CTCL lays downstream of the ROCK/MYPT1 pathway.

To further identify candidate regulators of NF-κΒ activation in CTCL, searches in the mass spectrometry data generated from TAK1 precipitates were performed. Canonical elements of the TAK1 complex including TAB1 , TAB2 and TAB3 were consistently recognized in our analysis (FIG. 3A).

Importantly, TAK1 precipitates also contained peptides corresponding to the PP1 phosphatase itself and the PP1 regulatory subunit PPP1 E12A MYPT1 (FIG. 3A). By coprecipitation experiments from CTCL cell lysates, we found that MYPT1 was binding not only TAK1 but also the IKK complex and this interaction was lost following TNFa stimulation (FIG. 3B). Interestingly, 5 minutes of TNFa treatment were sufficient to induce MYPT1 phosphorylation at its specific inhibitory residue T696 (FIG. 3C). In agreement with the idea that MYPT1 phosphorylation at T696 might counteract the inactivation of canonical NF-κΒ by PP1/MYPT1 , no PP1 was detected that coeluted with IKK and TAK1 in the Superdex S200 columns from CTCL cells treated with TNFa. In addition the amount of MYPT1 in the IKK-containing fractions was also severely reduced, and there was no detection of P-MYPT1 coeluting with IKK or TAK1 neither in the control nor in TNFa-treated cells (FIG. 3D). Then we tested the possible effect of inhibiting ROCK1 , which is the principal upstream kinase for MYPT1 , on TNFa-induced NF-κΒ signaling in CTCL cells.

Preincubation with the ROCK1 kinase inhibitor Y-27632 suppressed MYPT1 phosphorylation at T696 in these cells and greatly reduced ΙκΒα

phosphorylation and degradation after TNFa treatment (FIG. 3E and 3F). ROCK1 kinase inhibitor treatment also reduced the amounts of

polyubiquitinated Ρ-ΙκΒα that was detected in CTCL cells preincubated with the proteasome inhibitor MG132 and then stimulated with TNFa (FIG. 3G).

These results indicate that the MYPT1/PP1 complex and its upstream kinase ROCK1 modulate cytokine-dependent activation of canonical NF-κΒ in CTCL. Inhibition of ROCK, TAK1 and IKK impairs CTCL cell growth

The ROCK1 kinase is responsible for the inhibitory phosphorylation at T696 of MYPT1 leading to MYPT1/PP1 complex inactivation. This inhibition results in increased myosin phosphorylation and cytoskeleton reorganization in the context of cell shape regulation.

It was found that cellular clump disaggregation and mechanical detachment from the culture plates induced MYPT1 and TAK1 phosphorylation and N F-KB activation, which was favored when cells were seeded again in non-adherent polyHEME-coated dishes (FIG. 4A). Pharmacologic inhibition of ROCK1 in these conditions reduced CTCL cell growth about 30-35%, associated with a reduction of MYPT1 phosphorylation and accumulation of ΙκΒα, as

determined by WB analysis (FIG. 4B). Treatment of CTCL cells with different inhibitors of the proposed ROCK1 -TAK1 -IKK pathway led to variable results, with TAK1 inhibition showing a significantly higher effect on cell growth (an average of 70% reduction at 36 hours considering all three cell lines) compared to the ROCK1 and IKK (about 27% reduction) inhibitors, while they all showed a comparable effect on ΙκΒα stabilization (FIG. 4B). A more detailed analysis demonstrated that TAK1 inhibition specifically induced a massive apoptosis in CTCL cells, as determined by the accumulation of active caspase 3, associated with a strong reduction in the amount of total β- catenin (FIG. 4C), which was previously identified as a target of TAK1 in KRAS-dependent CRC cells (Singh et al., 2012). In contrast, TAK1 or IKK inhibitors similarly repressed the MAPK/ERK pathway in these experiments (FIG. 4C). To further study the possibility that NF-κΒ and β-catenin cooperate to promote CTCL cell survival, the effects of simultaneous targeting both pathways were determined. Our results demonstrated that simultaneous inhibition of ROCK1 or IKK and β-catenin (by PKF1 15-584) imposed a higher growth arrest on all tested CTCL cell lines when compared with either treatment alone (FIG. 4D). These results suggest that TAK1 exerts two complementary prosurvival activities in CTCL cells, one downstream of ROCK1 that is IKK-related and a second that links to the β-catenin signaling pathway.

The ROCK-MYPT1 -TAK1 pathway is activated in human CTCL

Finally, the inventors studied the relative contribution of this mechanism to human CTCL lesions. With this aim, polyclonal antibodies to the specific phosphor-residues of TAK1 identified in the mass-spectrometry analysis were prepared. Specificity of antibodies against phosphorylated T344, S389, T444 and T51 1 of TAK1 (see above) was determined by WB analysis of TNFa or calyculin A1 -treated HH cell lysates (FIG. 8A) and P-TAK1 (S412)

precipitates from control or calyculin A1 -treated HH cells (FIG. 8B).

Antibodies against phosphorylated T444 and T51 1 recognized bands compatible with activated TAK1 that were slightly increased following TNFa and massively augmented in calyculin A1 -treated cells (FIG. 9A), whereas all P-TAK1 antibodies recognized phosphorylated TAK1 from calyculin A1 - treated precipitates (FIG.s 8A and B).

Anti-P-TAK1 (T444) was selected for being tested by IF in human frozen biopsies, a specific staining pattern was obtained in the lymphocytic component of MF samples and adjacent keratinocytes (FIG. 5A). In the tumor tissue of these same samples P-MYPT1 (T696) (FIG. 5B), Ρ-ΙκΒα and active β-catenin were also detected by the antibody recognizing the non- phosphorylated protein (FIG.s 9A and B). Moreover, cells carrying P-MYPT1 mainly corresponded to CD3-positive tumor T-cells (FIG. 9), as expected.

By IHC analysis, the prevalence of P-TAK1 (T444), Ρ-ΙκΒα, and total and nuclear β-catenin in a cohort of 59 human samples obtained from

MARbiobanc that included CTCL lesions (MF and SS) (n= 25), 2 CD30+ and 1 uncharacterized (UN) lymphoma samples, and several inflammatory diseases such as psoriasis (PSO), pseudopsoriasis (PP) and lupus (LE) (n= 31 ) (FIG.s 5C, 9 and Table 1 ). The results demonstrated that 16 out of 25 lymphoma samples analyzed (64%) were positive for anti-P-T444 staining compared with 12 out of 31 non-lymphoma samples (38%) (p=0.059).

Notably, P-T444 detection positively and significantly correlated with the presence of Ρ-ΙκΒα in both the total cohort analyzed (p=0.013) and the lymphoma samples (p=0.005) (FIG.s 5D and 9E). Moreover, 56 out of the 59 samples that we were able to analyze showed variable levels of β-catenin (both in the cytoplasm and nucleus) and/or Ρ-ΙκΒα, and 40 of them were annotated as double positive (Table 1 ). Of note that the only 3 samples that were negative for both β-catenin and Ρ-ΙκΒα were also negative for P-TAK1 staining. Importantly, high nuclear active β-catenin levels (≥2+) was detected in 9 out of 28 lymphoma samples (32%) compared with 5 out of 31 (16%) non-tumor samples (FIG. 8), although this differential distribution did not reach statistical significance (p=0.148). A better characterization of the control group showed that nuclear β-catenin accumulated in the lupus erythematosus lesions similar to the CTCL samples (p=0.5) and different from the psoriasis and parapsoriasis group (p= 0.072).

Finally, the therapeutic potential of TAK1 inhibition or double inhibition of ROCK and β-catenin signaling in primary human SS cells was evaluated. It was found that 36 hours of treatment with TAK1 or the double treatment with ROCK and β-catenin inhibitors were sufficient to preclude tumor cell growth (FIG. 4E), indicative of the efficacy of the treatment.

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Claims

1 . A phosphorylated TAK1 polypeptide selected from the group consisting of: (a) the amino acid sequence SEQ ID NO: 1 wherein one or more of the residues at positions 344, 389, 444, and 51 1 are phosphorylated,

(b) a functional fragment of SEQ ID NO: 1 which comprises one or more of the amino acid residues identified in sequence SEQ ID NO: 1 as 344, 389, 444, and 51 1 , phosphorylated; and

(c) an amino acid sequence with an identity degree with SEQ ID NO:1 of at least 85%, provided that this amino acid sequence comprises one or more of the amino acid residues identified in sequence SEQ ID NO: 1 as 344, 389, 444, and 51 1 , phosphorylated.

2. The phosphorylated TAK1 polypeptide according to claim 1 , which comprises the amino acid sequence SEQ ID NO: 1 wherein one or more of the residues at positions 344, 389, 444, and T51 1 are phosphorylated.

3. The phosphorylated TAK1 polypeptide according to any of the claims 1 -2, which consists of the amino acid sequence SEQ ID NO: 1 wherein one or more of the residues at positions 344, 389, 444, and T51 1 are

phosphorylated

4. An antibody or a functional fragment thereof which binds to the

phosphorylated TAK1 polypeptide as defined in any of the claims 1 -3, wherein the antibody or fragment thereof specifically binds to the one or more phosphorylated amino acid residues referred in claim 1 .

5. The antibody according to claim 4, which is a polyclonal antibody.

6. The antibody according to claim 4, which is a polyclonal antibody.

7. A kit comprising either the polypeptide as defined in any of the claims 1 -3 or the antibody as defined in any of the claims 4-6.

8. A method for predicting the response to TAK1 inhibitor-based therapy by a patient suffering from cancer, the method comprising the step of determining, in an isolated sample of the patient, whether one or more amino acid residues of TAK1 protein at positions 344, 389, 444, and 51 1 is/are phosphorylated, wherein if one or more of the amino acids forming TAK1 protein is/are phosphorylated, it is indicative that the patient will positively respond to TAK1 inhibitor-based therapy.

9. The method according to claim 8, wherein the determining step comprises contacting the isolated sample with an antibody or fragment thereof as defined in any of the claims 4-6, wherein if it is detected the binding of the antibody or fragment thereof, it will be indicative that the patient will positively respond to TAK1 inhibitor-based therapy.

10. The method according to any of the preceding claims 8-9, wherein the patient is suffering from a lymphoma.

1 1 . The method according to claim 10, wherein the lymphoma is a T-cell lymphoma.

12. The method according to claims 1 1 , wherein the lymphoma is a cutaneous T-cell lymphoma.