WO2004039980A1 - Agents modulating mal activity - Google Patents

Abstract

Agents that modulate the Rho-dependent SRF pathway by modulating a MAL activity through modulating MAL-SRF interactions; translocation of MAL to and/or from the nucleus; MAL C-terminal phosphorylation; MAL-actin interactions; MAL dimerisation; or MAL gene expression; pharmaceutical compositions containing these agents, and methods of treatment using them.

Description

TREATMENT METHODS AND COMPOSITIONS

This invention relates to treatment methods, agents and compositions, and in particular relates to methods, agents and compositions for treating various disorders such as cancer, wounds and myopathies.

Serum response factor (SRF) is an essential regulator of many genes through binding to the sequence CC(A/T)₆GG (SEQ ID No: 1) termed the CArG box (Treisman, 1986). SRF has been shown to be involved in cell processes like immediate early and tissue-specific gene expression, cell proliferation, cell migration, and apoptotic pathways (Schratt et al, 2001; Camoretti-Mercado, et al, 2000; Ding, et al, 2001; Bertolotto et al, 2000; Zhang et al, 2001). Deletion of the SRF gene causes embryonic lethality due to failure of gastrulation and the failure of muscle-specific and inducible gene expression (Arsenian et al, 1998; Schratt et al, 2001). Deletion of SRF is also associated with aberrant adhesive properties and defects in expression of a number of cytoskeletal proteins, some of which are known SRF target genes (Schratt et

Analysis of a set of growth factor-inducible SRF targets surprisingly shows that the genes fall into two classes, responsive either to RhoA-actin or to ERK signalling (Gineitis and Treisman, 2001). Accordingly, the activity of SRF has been shown to be regulated in two ways: (i) at certain promoter sequences it recruits a second Ets-domain transcription factor, Ternary Complex Factor (TCF) which is regulated by ERK / MAP kinase signalling (Treisman, 1994). The TCF family of transcription factors is composed of three members, Elk-1, Sap la, and Sap-2-Erp-Net, that bind DNA through their Ets domain and are recruited to the SRF binding site by direct protein-protein interaction between the TCF "B-box" motif and the SRF DNA binding domain, also named Serum Response Element (SRE) (Hassler and Richmond, 2001 ; Ling et al, 1997). One of the best examples of SRF-mediated gene activation regulated through the TCF and ERK/c-Jun NH2-terminal kinase/p38 pathways pathway is the immediate early proto-oncogene c-fos (Yang et al, 1998; Ducret et al, 2000). (ii) At others, SRF activity is controlled independently of TCF by a second pathway controlled by Rho-family small GTPases (Hill et al, 1995).

Amongst the Rho responsive genes are included those encoding SRF itself, the focal adhesion protein vinculin, cytoskeletal actins, and the secreted adhesion factors CTGF and cyrόl (Gineitis and Treisman, 2001; Sotiropoulos et al, 1999). The Rho GTPases control SRF activity by altering actin dynamics, and such alterations are both necessary for the activation of SRF by extracellular signals and sufficient for its activation in the absence of signal (Sotiropoulos et al, 1999). Rho family members regulate diverse processes including cytoskeletal rearrangements, gene transcription, cell-cycle progression, cell transformation, and cytokinesis. These GTPases control SRF activity by altering actin dynamics, and such alterations are both necessary for the activation of SRF by extracellular signals and sufficient for its activation in the absence of signal (Sotiropoulos et al, 1999). Although TCF binding is not required for regulation of SRF by the actin pathway, the presence of inactive TCF associated with SRF inhibits its operation, suggesting a model in which TCF competes for a binding site on SRF with an unidentified factor (Hill et al, 1994).

Functional significance of Rho-regulated SRF activity

Rho signalling is increasingly being recognised as of significance for the control of cell adhesion, mobility and cytoskeletal dynamics (Evers et al, 2000; Schmitz et al, 2000). These processes are likely to play important roles in invasion and metastasis: recent reports indicate that disregulation of RhoC signalling is associated with metastasis (Clark et al, 2000), and both RhoA and SRF are implicated in the epithelial-mesenchymal transition associated with tumour progression (Psichari et al, 2002; Schratt et al, 2002). In addition, the transformation by oncogenic Ras is accompanied by dramatic alterations in actin cytoskeleton, and it is known that GTPases of the Ras subfamily can activate cascades of the Rho family members (Qiu et al, 1995).

Consistent with this, SRF null cells have defects in adhesion consistent with aberrant expression of the cytoskeletal genes under Rho-actin control (Schratt et al, 2002). The RhoA-actin signalling pathway is likely to be important in cell-cell interactions, especially tumour-stromal interactions. The growth factor-induced immediate-early gene program constitutes part of the wound healing response, which changes the spectrum of transcriptional regulators expressed in the cell and induces cell-cycle re-entry, secretion of cytokines, cytoskeletal changes, and the production of chemotactic, anti-apoptotic and matrix remodelling proteins (Iyer et al, 1999). The CTGF and cyrόl genes form part of this program and both are targets for the Rho-actin signalling pathway and are intimately involved in these processes. Expression of Cyrόl mediates cell adhesion and induces adhesive signalling, stimulates cell migration and proliferation, and promotes cell survival in both fibroblasts and endothelial cells (Chen et al, 2001a; Chen et al, 2001b; Grzeszkiewicz et al, 2001), while CTGF has additionally been implicated as a secondary mediator of the response to TGFβ-family ligands (Abreu et al, 2002; Grotendorst, 1997).

Signalling from Rho to SRF

We have previously studied the signalling pathways leading from Rho via actin dynamics to SRF. Two Rho effector pathways are involved. The ROCK-LIMK pathway stabilises F-actin by inhibiting the activity of the depolymerising/severing factor cofilin (Maekawa et al, 1999), while the diaphanous pathway stimulates F-actin assembly by promoting filament nucleation (Pruyne et al, 2002; Sagot et al, 2002; Watanabe et al, 1997). In each pathway the protein domains or activities required for F-actin assembly are identical to those which mediate SRF activation. Interfering and activated forms of pathway components, constructed in the laboratory, affect both processes (Copeland and Treisman, 2002; Geneste et al, 2002; Sotiropoulos et al, 1999).

SRF activation can also be affected by direct interference with actin dynamics. SRF activation appears to be a consequence of the depletion of either the bulk G-actin pool or a G-actin subpopulation, since it can be inhibited by over- expression either of actin itself or its nonpolymerisable derivatives (β-actins G13R, R62D or VP16; (Posern et al, 2002)), or by blockade of actin polymerisation using drugs or toxins (Sotiropoulos et al, 1999). In general, signals which promote F-actin accumulation (or G-actin depletion) activate SRF; however, certain actin binding proteins and drugs, including cytochalasin D and profilin, can activate SRF without promoting F-actin assembly (Sotiropoulos et al, 1999). We have previously proposed that these agents activate SRF because they compete for a binding site on actin with a presumed SRF cofactor which mediates actin signalling (Sotiropoulos et al, 1999). SRF can also be activated upon expression of certain actin mutants which generate F-actin of increased stability (β-actins N159N, S14C and G15S; (Posern et al, 2002)). Posern et al. (2002) and Sotiropoulos et al (1999) have previously proposed that actin regulates SRF activity either by direct repression or by inhibiting the activity of an SRF co-activator.

We now present evidence that the MAL gene product, also known as MKL1, (Ma et al 2001 and Mercher et al 2001) represents an actin-regulated SRF co- activator controlled by Rho signalling.

MAL is a member of the MAL/myocardin family of SRF co-activators, which contains, at least, myocardin (MC), MAL and MAL 16. The MC protein was previously identified as an SRF co-activator (Wang et al, 2001). As originally reported, MC comprises an 807 residue open-reading frame with short basic and glutamine-rich boxes, a SAP (SAF-A/B, Acinus and PIAS) domain, and a potential leucine zipper region, but no other obvious structural domains or sequence motifs. MC was shown to be a heart-specific nuclear protein which, when expressed in tissue culture cells, could generate an activity capable of interacting with SRF in DNA binding assays. The basic and glutamine-rich domains were identified as essential for interaction with SRF in biochemical assays using MC expressed in extracts. The C-terminus of MC contains an extremely potent transcriptional activation domain. MC was shown to be active on heart-specific SRF binding sites and promoters but to leave the c-fos IE gene promoter unaffected in gene expression assays.

Database searches revealed that MAL is related to MC, maintaining the conserved domains and additional short homology patches towards the C- terminus. As shown in Figures 1 and 2, MAL also contains an approximately 120 residue "MAL" domain at its N-terminus. The MAL domain is the region of maximum homology between MAL and related genes MAL 16, MC and DMAL (Drosophila homologue) as defined by Mercher et al. (2001). (The MAL domain was missed in the original MC cDNA but has subsequently been submitted to the Genbank database). The MAL domain is present in MAL, MC, and MALI 6, which also contains the conserved domains present in the other two proteins. No biochemical studies of MAL have been reported. Recently, a mouse homologue of MAL, termed BSAC, was reported (Sasazuki et al, 2002) and was shown to activate c-fos, a Rho-independent SRF target gene and evidence was provided that the protein was nuclear.

The MAL domain of the MAL protein contains two sequence motifs known as RPEL motifs, the consensus sequence of which is: dvLkrKLsqRPtreELvernlLkees (SEQ ID No: 2; Pfam protein families database: accession number PF02755, Bateman et al, Nucleic Acids Research, (2002), 30: 276-280). The highly conserved residues are in upper case letters.

As shown in Figure 2, the RPEL domains of both human MAL and mouse MAL are at amino acid residues 24 to 49 and 68 to 93. The first RPEL domain in human MAL (residues 24 to 49) has the amino acid sequence DYLKRKIRSRPERSELNRMHILEETS (SEQ ID No: 3), while this domain in mouse has one amino acid difference DYLKRKIRSRPERAELNRMHILEETS (SEQ ID No: 4). The second RPEL domain in both humans and mouse (residues 68 to 93) is identical, with the amino acid sequence DDLNEKIAQRPGPMELVEKNILPNES (SEQ ID No: 5).

BSAC has an additional 35 amino acids N-terminal to those of mouse MAL as defined in Figure 1 which contain a third N-terminal RPEL-like motif, SVLQLKLQQRRTREELVSQGIMPPLK (SEQ ID No: 6) at residues 15-40 of BSAC (Genbank Accession no. AF385582).

Other domains within MAL include the basic-rich region 2 (B2 box) at residues 57 to 65 in both human and mouse MAL; the basic-rich region 1 (Bl box) at residues 224 to 250 in both human and mouse MAL; the glutamine- rich region (Q box) at residues 264 to 281 in human MAL and at residues 264 to 285 in mouse MAL; a SAP domain at residues 347 to 381 in human MAL and at residues 350 to 384 in mouse MAL; and a leucine-zipper-like domain (LZL) at residues 518 to 558 in human MAL and at residues 520 to 560 in mouse MAL.

Several other genes in mammals contain RPEL motifs, although none contains close matches to other sequences in the MAL domain. Intriguingly, the RPEL motifs in the MC MAL domain (RPEL MC 18-43: SVLQLRLQQRRTQEQLANQGLIPPLK (SEQ ID No: 7); RPEL MC 62-67: DSLRRKGRNRSDRASLVTMHILQAST (SEQ ID No: 8); and RPEL MC 106-131: DDLNEKIALRQGPLELVEKNILPMDS (SEQ ID No: 9)), and in the N-terminal RPEL motif in BSAC (RPEL BSAC 15-40; SVLQLKLQQRRTREELVSQGIMPPLK (SEQ ID No: 10)), contain point changes at a subset of the conserved positions. These RPEL motifs are classified as RPEL motifs by homology, but are believed not to be functional. For example, the highly conserved Pro is mutated in all these motifs.

Two groups have reported the identification of genes involved in the rare childhood leukaemia AML-M7, which features a t(l ;22)(pl3;ql3) translocation (Ma et al, 2001 and Mercher et al, 2001). These genes, MAL/MKL1 (hereafter MAL - megakaryocytic acute leukaemia) and OTT/RBM15, are fused so as to join the entire ORFs together. Both Ma et al. (2001) and Mercher et al. (2001) identified a SAP domain within the MAL gene, which is a putative DNA-binding motif involved in chromosomal organization (see Aravind L. & Koonin E.V. (2000) SAP-a putative DNA- binding motif involved in chromosomal organization. Trends Biochem. Sci., 25: 112-114). This led Ma et al to believe that the MKLl SAP domain would be expected to aberrantly relocalise the RRM and SPOC motifs of RBM15 to sites of transcriptionally active chromatin, deregulating RNA processing and/or Hox and Ras/MAP kinase signalling and altering the normal proliferation or differentiation of megakaryoblasts. Mercher et al also speculated that the fusion oncoprotein is expected to participate in chromatin organisation through the binding of AT-rich DNA sequences, recognised by the SAP box in the OTT-MAL fusion.

Neither Ma et al nor Mercher et al mention or suggest that MAL is involved in the SRF-Rho dependent pathway. Furthermore, neither Ma et al. nor Mercher et al mention or suggest modulating the SRF-Rho dependent pathway by modulating a MAL activity.

We have now shown that: 1. MAL potentiates SRF reporter activity in a Rho dependent manner.

2. MAL is an SRF co-activator which forms a direct complex with the SRF DNA binding domain.

3. MAL forms an active dimer through the LZL motif.

4. Regulation of MAL involves nuclear-cytoplasmic shuttling controlled by Rho via its ability to induce alterations in actin dynamics.

5. MAL translocation is also induced by the WASP, N-WASP and VASP actin regulators, implicating Cdc42 and Rac GTPases in its regulation, presumably in response to other stimuli.

6. Regulation of MAL shuttling to the nucleus requires sequences at the N- terminus and C-terminus of the protein. At the N-terminus the MAL domain is required, specifically the Bl and B2 box basic-rich regions and the Q box glutamine rich region, and the two RPEL motifs.

7. MAL is phosphorylated in response to all signals which induce changes in actin dynamics, suggesting that phosphorylation may be essential for activation in response to signals.

8. Actin physically interacts with MAL. Interaction requires the N-terminal MAL domain, specifically the RPEL motifs. Sequences in the basic region, the glutamine rich region, and in the MAL domain (distinct from the RPEL motifs) affect the distribution of MAL in the absence of signalling.

9. Rho-mediated gene activation of SRF can be inhibited through the expression of a dominant-negative MAL mutant, for example MAL with the activator domain corresponding to the C-terminal 200 amino-acid residues deleted or MAL deleted of both Bl and B2 box basic-rich regions.

Without being bound by theory, we believe that disruption of Rho-actin signalling and SRF target gene expression due to MAL abnormality might be a causal factor in leukaemia in general, and particularly in AML-M7 due to the MAL rearrangement and gene fusion. Furthermore, the mouse homologue of MAL has been identified as an anti-apoptotic factor, suggesting that SRF targets may be important for maintaining cell survival (Sasazuki et al, 2002). Many SRF target genes in muscle are up-regulated as part of the response to hypertrophy and SRF is required for the hypertrophic response (Paradis et al, 1996; Reecy et al, 1996), and least some of these genes are likely targets for the Rho-actin pathway (Mack et al, 2001). Together our new observations suggest that manipulation of the Rho-actin signalling pathway to SRF by modulation of a MAL activity may have important consequences for a number of disorders including cancer (including invasion and metastasis), cell survival, wound healing, angiogenesis, and myopathies.

A first aspect of the present invention provides an agent that modulates a MAL activity.

By "MAL" we include the gene product of the human MAL gene and naturally occurring variants thereof. The sequence of the human MAL gene is found in Genbank Accession No. AJ297258, and the human MAL cDNA sequence is listed in Figure 1 (SEQ ID No: 11). Human MAL includes the amino acid sequence listed in Figure 1 (SEQ ID No: 12), the sequence found in Genbank Accession No. CAC38827.1, and naturally occurring variants thereof.

By MAL we also include a homologous gene product from MAL genes from other species, including MAL from the mouse. The cDNA and amino acid sequence of mouse MAL includes the sequences listed in Figure 1 (SEQ ID Nos: 13 and 14, amplified from a mouse fibroblast NIH3T3 cDNA library), the BSAC sequences found in Genbank Accession No. AF385582, and naturally occurring variants thereof, including those containing additional or alternative protein sequences N-terminal to those encoded by the sequence in Figure 1. At the protein level, the mouse MAL amino acid sequence in Figure 1 is 99% homologous to the BSAC amino acid sequence in AF385582, which corresponds to a 9 amino acid difference.

By "homologous gene product" we include a MAL polypeptide having at least 80% sequence identity with the human MAL amino acid sequence in Figure 1. More preferably, a homologous gene product includes a MAL polypeptide having at least 84% sequence identity with human MAL. Yet more preferably, a homologous gene product includes a MAL polypeptide having at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98% sequence identity with human MAL. Most preferably, a homologous gene product includes a MAL polypeptide having at least 99% sequence identity with the human MAL amino acid sequence.

By a "MAL activity" we include the meaning of any activity, function or interaction of MAL, or any process performed on, by, or involving MAL, that occurs within a cell. Thus MAL activity includes, but is not limited to, the interaction of MAL with actin, the phosphorylation and dephosphorylation of MAL, the translocation of MAL to and/or from the nucleus, the dimerisation of MAL, the interaction of MAL with SRF, and the expression of MAL from the gene encoding it. Most preferably, the MAL activity which is modulated is an activity of MAL within the cell.

By "modulates" we include the meaning of inhibiting or stimulating an activity of MAL. By "inhibiting" we include the meaning of reducing the rate or level of an activity of MAL. The reduction can be a low level reduction of about 10%, or about 20%, or about 30%, or about 40% of an activity of MAL. The reduction can be a medium level reduction of about 50%, or about 60%, or about 70%, or about 80% reduction of an activity of MAL. The reduction can also be a high level reduction of about 90%, or about 95%, or about 99%, or about 99.9%, or about 99.99% of an activity of MAL. Inhibition can also include the elimination of an activity of MAL or its reduction to an undetectable level.

By "stimulating" we include the meaning of enhancing the rate or level of an activity of MAL. The enhancement can be a low level increase of about 1.5- fold, or about a 2-fold, or about a 3-fold, or about a 4-fold, or about a 5-fold increase of an activity of MAL. The enhancement can be a medium level increase of about a 10-fold, or about a 20-fold, or about a 30-fold, or about a 50-fold increase of an activity of MAL. The enhancement can also be a high level increase of about a 100-fold, or about a 200-fold, or about a 500-fold or about a 1, 000-fold or about a 10,000-fold increase, or more, of an activity of MAL. By "stimulating" we also include the meaning of de novo initiation of an activity of MAL.

Interaction of MAL with SRF

In a preferred embodiment, the agent modulates an interaction of MAL with SRF.

By "SRF" we include the gene product of the human SRF gene and naturally occurring variants thereof. The nucleotide sequence of human SRF is found in Genbank Accession No. J03161, and the amino acid sequence of human SRF is found in Genbank Accession No. AAA36647.1.

By SRF we also include the gene product from homologous SRF genes from other species, including the mouse. The cDNA and amino acid sequence of mouse SRF is found in Genbank Accession No. AB038376 and BAA92314.1, respectively.

Preferably, the invention includes an agent which inhibits an interaction of MAL with SRF. Alternatively, the invention includes an agent which stimulates an interaction of MAL with SRF.

Agents that inhibit an interaction of MAL with SRF include an anti-MAL antibody that inhibits SRF binding to MAL; an anti-SRF antibody that inhibits MAL binding to SRF; a MAL fragment or mutant that binds to SRF; an SRF fragment or mutant that binds to MAL; or an inhibitor of MAL-SRF interaction such as a small-molecule inhibitor. The inhibitor of MAL-SRF interaction may be identified by the screening methods described in later aspects of the invention.

An agent that selectively binds to the Bl domain (amino acid residues 224 to 250 of the human MAL sequence in Figure 1) and/or the Q domain of MAL (amino acid residues 264 to 281 of the human MAL sequence in Figure 1) would be expected to inhibit binding of SRF to MAL.

In one embodiment, the agent is an antibody. In another embodiment, the agent may be a polypeptide consisting of or comprising the DNA binding domain (residues 133-222) of SRF.

An agent that selectively binds to the DNA binding domain of SRF (residues 133 to 222) and in particular to the hydrophobic pocket within the DNA binding domain of SRF would also be expected to inhibit binding of SRF to MAL.

The hydrophobic pocket on the SRF DNA binding domain includes the amino acid residues His at position 193, Nal at position 194, Thr at position 196, He at position 206 and He at position 215. In one embodiment, the agent is an antibody. In another embodiment, the agent may be a mutant of MAL. For example, MAL 1-471 binds to SRF (Figure 16B) and inhibits the Rho-SRF signalling pathway (Example NIII).

The invention includes an agent that selectively binds the Bl domain and/or the Q domain of MAL.

The invention also includes an agent that selectively binds the DΝA binding domain of SRF at residues 133-222.

The invention also includes an agent that selectively binds to the hydrophobic pocket within the DΝA binding domain of SRF.

In an embodiment, the agent is an antibody.

As shown in Figures 19 and 20, the Bl and Q domains of MAL bind to the SRF DΝA binding domain, and the Bl domain alone has been shown to be sufficient to inhibit complex formation between MAL and the SRF DΝA binding domain. The invention also includes a polypeptide consisting of or comprising the Bl domain of MAL, or the Q domain of MAL or both the Bl and Q domains.

As used herein, reference to a polypeptide comprising a specific fragment, domain, region or sequence of a protein does not include the full-length protein sequence. The polypeptide can comprise the specific fragment, domain, region or sequence and at least 1, or at least 2, or at least 5, or at least 10, or at least 20, or at least 50, or at least 100, or at least 200 or more amino acids from the full-length protein C and/or Ν terminal or the specific fragment, domain, region or sequence, providing that the polypeptide does not comprise the full-length protein. Thus, for example, the polypeptide can comprise a deletion mutant of the full-length protein. Alternatively, the polypeptide can comprise the specific fragment, domain, region or sequence and exogenous C and/or N terminal amino acid sequences of any length. By "exogenous" we include the meaning that the C and/or N terminal amino acid sequences are not found in the full-length protein.

By an agent "selectively binding" a specified domain of a target protein, we include the meaning that the agent binds the specific domain with a greater affinity than for any other region of the target protein. Preferably, the agent binds the specific domain with at least 2, or at least 5, or at least 10 or at least 50 times greater affinity than any other region of the target protein. More preferably, the agent binds the specific domain with at least 100, or at least 1,000, or at least 10,000 times greater affinity than any other region of the target protein.

Preferably, when the target protein is present in a cell, the agent binds the target protein at the specific domain with a greater affinity than for any other molecule in the cell. Preferably, the agent binds the target protein at the specific domain with at least 2, or at least 5, or at least 10 or at least 50 times greater affinity than for any other molecule in the cell. More preferably, the agent binds the target protein at the specific domain with at least 100, or at least 1 ,000, or at least 10,000 times greater affinity than any other molecule in the cell.

Translocation of MAL to and/or from the nucleus

In another preferred embodiment, the agent modulates translocation (shuttling) of MAL to and/or from the nucleus.

Preferably, the invention includes an agent which inhibits translocation of MAL to and/or from the nucleus. Alternatively, the invention includes an agent which stimulates translocation of MAL to and/or from the nucleus. Agents that inhibit translocation of MAL to and/or from the nucleus include an anti-MAL antibody that inhibits MAL shuttling to and/or from the nucleus; a MAL fragment or mutant that inhibits MAL shuttling to and/or from the nucleus; or a MAL binding compound such as a small-molecule. The MAL binding compound may be identified by the screening methods described in later aspects of the invention.

An agent that selectively binds to the Bl domain (amino acid residues 224 to 250 of the human MAL sequence in Figure 1) or the Bl and the B2 domain of MAL (amino acid residues 57 to 65 of the human MAL sequence in Figure 1) would be expected to inhibit MAL shuttling to the nucleus.

An agent that selectively binds to the LZL domain (amino acid residues 518 to 558 of the human MAL sequence in Figure 1) would also be expected to inhibit the translocation of MAL to the nucleus.

In an embodiment the agent is an antibody.

In another embodiment, an agent which inhibits MAL shuttling to the nucleus can be a MAL deletion mutant, for example, MAL ΔB1ΔB2 which complexes the wild-type MAL and prevents it from entering the nucleus (see Example NIII).

An agent that selectively binds to the Q domain (amino acid residues 264 to 281 of the human MAL sequence in Figure 1) would be expected to inhibit MAL shuttling from the nucleus.

The invention thus includes an agent that selectively binds the nuclear import signal of MAL, including the Bl and B2 box domains.

The invention also includes an agent that selectively binds the nuclear export signal of MAL including the Q box glutamine-rich domain. The invention further includes an agent that selectively binds the LZL domain of MAL.

Preferably, the agent is an antibody.

The invention also includes MAL ΔB1ΔB2.

C-terminal phosphorylation of MAL

In another preferred embodiment, the agent modulates C-terminal phosphorylation of MAL.

Preferably, the method includes an agent which inhibits C-terminal phosphorylation of MAL. Alternatively, the invention includes an agent which stimulates C-terminal phosphorylation of MAL.

Agents that inhibit C-terminal phosphorylation of MAL include an antibody or other compounds, such as small molecules, that bind to dephosphorylated C- terminal serine or threonine residue(s) of MAL, thus inhibiting the phosphorylation of these residue(s). Such compounds may be identified by the screening methods described in later aspects of the invention.

Suitable serine or threonine residues can easily be determined by a person of skill in the art with reference to the amino acid sequence of MAL in Figure 1.

Peptides which encompass C-terminal serine or threonine residue(s) of MAL are useful in both their phosphorylated and unphosphorylated form, for example in preparing reagents which are useful in raising such antibodies.

Measurement of phosphorylation of these specific residues may be carried out by any suitable means. The invention thus includes an agent which binds to MAL which is not phosphorylated at at least one C-terminal serine or threonine residue with a greater affinity than it binds to MAL which is specifically phosphorylated at the at least one C-terminal serine or threonine residue.

The invention also includes an agent which binds to MAL which is specifically phosphorylated at at least one C-terminal serine or threonine residue with a greater affinity than it binds to MAL which is not phosphorylated at the specific at least one C-terminal serine or threonine residue.

In an embodiment the agent is an antibody.

Interaction of actin with MAL.

In a preferred embodiment, the agent modulates an interaction of actin with MAL.

Preferably, the agent modulates an interaction of β-actin with MAL.

By "β-actin" we include the gene product of the human β-actin gene and naturally occurring variants thereof. The nucleotide sequence of human β- actin is found in Genbank Accession No. X00351, and the amino acid sequence of human β-actin is found in Genbank Accession No. CAA25099.1.

By β-actin we also include the gene product from homologous β-actin genes from other species, including the mouse. The cDNA and amino acid sequence of mouse β-actin is found in Genbank Accession No NM_007393 and NP_031419, respectively.

Preferably, the invention includes an agent which inhibits an interaction of actin with MAL. More preferably, the agent inhibits an interaction of β-actin with MAL.

Agents that inhibit an interaction of actin with MAL include an anti-MAL antibody that inhibits actin binding to MAL; an anti-actin antibody that inhibits MAL binding to actin; a MAL fragment that binds to actin; an actin fragment that binds to MAL; and an inhibitor of MAL-actin interaction such as a small-molecule inhibitor of MAL-actin interaction. The inhibitor may be identified by the screening methods described in later aspects of the invention.

Alternatively, the invention includes an agent which stimulates an interaction of actin with MAL, thus stimulating a MAL activity. Agents that stimulate an interaction of actin with MAL include molecules identified by the screening methods described in later aspects of the invention.

An agent that selectively binds to one or both of the RPEL motifs (amino acid residues 24 to 49 and 68 to 93 of the human MAL sequence in Figure 1) would be expected to inhibit binding of MAL to actin.

The invention thus includes an agent that selectively binds one or both of the RPEL domains of MAL.

In an embodiment the agent is an antibody.

The invention also includes a polypeptide fragment of MAL comprising one or both of the RPEL motifs.

Antibodies

The term "antibody" as used herein includes but is not limited to polyclonal, monoclonal, chimaeric, single chain, Fab fragments and fragments produced by a Fab expression library. Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody. Furthermore, the antibodies and fragments thereof may be humanised antibodies, for example as described in US-A-239400. Neutralising antibodies, ie, those which inhibit biological activity of the substance polypeptides, are especially preferred for diagnostics and therapeutics.

Antibodies may be produced by standard techniques, for example by immunisation with the appropriate fragment of MAL, or by using a phage display library.

If polyclonal antibodies are desired, a selected mammal (eg, mouse, rabbit, goat, horse, etc) is immunised with an immunogenic polypeptide bearing a epitope(s) such as the particular MAL domains described herein. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (Bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants which may be employed if purified the substance polypeptide is administered to immunologically compromised individuals for the purpose of stimulating systemic defence.

Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an epitope obtainable from an identified agent and/or substance of the present invention contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in animals or humans.

Monoclonal antibodies directed against particular epitopes, such as the particular domains or fragments of MAL described herein, can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody- producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against orbit epitopes can be screened for various properties; ie, for isotype and epitope affinity.

Monoclonal antibodies may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256: 495-497), the human B- cell hybridoma technique (Kosbor et al. (1983) Immunol Today 4: 72; Cote et al. (1983) Proc Natl Acad Sci 80: 2026-2030) and the EBV-hybridoma technique (Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, pp 77-96). In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison et al. (1984) Proc Natl Acad Sci 81: 6851-6855; Neuberger et al (1984) Nature 312: 604-608; Takeda et al. (1985) Nature 314: 452-454). Alternatively, techniques described for the production of single chain antibodies (US Patent No. 4,946,779) can be adapted to produce the single chain antibodies specific to, for example, a particular MAL domain. Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al. (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991; Nature 349: 293-299).

Antibody fragments which contain specific binding sites for the substance may also be generated. For example, such fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulphide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al (1989) Science 256: 1275-1281).

Fragments and Mutants

As described herein, the agent may comprise a MAL fragment, derivative or mutant that binds to SRF, or an SRF fragment, derivative or mutant that binds to MAL, that inhibits an interaction of MAL with SRF. The agent may also comprise a MAL fragment, derivative or mutant that binds to actin, or an actin fragment, derivative or mutant that binds to MAL, that inhibits an interaction of actin with MAL.

A fragment or mutant of MAL is typically one which has at least one functional area removed or mutated to inhibit or prevent at least one activity of MAL.

Agents of the present invention may include the MAL mutants that are described and shown in the Examples and the Figures, particularly in Example

V and Figure 11. Additional MAL mutants can be designed based upon the function and significance of sections of the MAL protein as described and shown in the Examples and in the Figures.

The invention includes the mouse MAL mutants described in Figure 11. The invention also includes the human equivalent of each of the mouse MAL mutants described in Figure 11. The human equivalents can readily be determined by a person of skill in the art by reference to the human and mouse MAL amino acid sequence and structure shown in Figure 1 and Figure 2.

In particular, Rho-mediated gene activation of SRF can be inhibited through the expression of a dominant-negative MAL mutant, for example with the activator domain corresponding to the C-terminal 200 amino acids residues deleted.

A polypeptide agent may be isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.

Synthesis of peptide agents can be performed using various solid-phase techniques (Roberge JY et al. (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequences comprising the agent or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant agent.

Alternatively, the coding sequence of a peptide agent (or variants, homologues, derivatives, fragments or mimetics thereof) may be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al. (1980) Nuc Acids Res Symp Ser 215-23, Horn T et al. (1980) Nuc Acids Res Symp Ser 225-232). Examples of suitable expression hosts for expressing the peptide agents for use in the invention are include bacteria such as E. coli, fungi such as Aspergillus species (such as those described in EP-A-0184438 and EP-A-0284603) and Trichoderma species; bacteria such as Bacillus species (such as those described in EP-A-0134048 and EP-A-0253455), Streptomyces species and Pseudomonas species; and yeasts such as Kluyveromyces species (such as those described in EP-A-0096430 and EP-A-0301670) and Saccharomyces species. By way of example, typical expression hosts may be selected from Aspergillus niger, Aspergillus niger var. tubigenis, Aspergillus niger var. awamori, Aspergillus aculeatis, Aspergillus nidulans, Aspergillus orvzae, Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis , Bacillus amyloliquefaciens, Kluyveromyces lactis and Saccharomyces cerevisiae.

The use of suitable eukaryotic host cells, such as yeast, fungal and plant host cells, may provide for post-translational modifications (eg myristoylation, glycosylation, truncation, lipidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products, such as MAL variants, for use in the present invention.

Typically, the agents described above modulate an activity of MAL inside the cell. The agents may therefore be fused to peptides or other molecules that carry the agent into a cell. Furthermore, MAL functions both in the nucleus and cytoplasm of the cell. The agents may therefore be fused to peptides or other molecules that target the agent to the nucleus or the cytoplasm of the cell, once it has been delivered intracellularly.

Suitable carrier and targeting peptides and molecules are known to a person of skill in the art, and the choice thereof depends upon the mode of administration of the agent. For example, transducing peptides, such as human immunodeficiency virus (HIN)-l Tat-(48-60) and Drosophila Antennapedia (Antp)-(43-58) (penetratin), can carry large biomolecules from the extracellular environment directly into the cytoplasm and the nucleus of cells (Futaki. (2002); Morris et al. (2001); and Schwarze et al. (1999)).

Polynucleotides encoding the agents

The invention also includes polynucleotides encoding the polypeptide agents described above, or the nucleic acid agents described below, for example those that modulate MAL gene expression.

A polynucleotide encoding a polypeptide agent, for example a single chain antibody that binds to a specific region of MAL, or a MAL fragment or mutant, may be administered to a target cell as described herein. Expression of the agent from the polynucleotide thus results in intra-cellular administration of the polypeptide agent. Similarly, a polynucleotide encoding a nucleic acid agent, for example an antisense agent that modulates MAL gene expression, can be administered intra-cellularly. Depending on the mode of administration, the polynucleotide can be administered into the nucleus or the cytoplasm of the target cell as desired. Suitable vectors include both viral and non-viral vectors, such as those described herein, and are well known to a person of skill in the art

Typically, a polynucleotide encoding an agent is operably linked to a regulatory sequence which is capable of providing for the expression of the polynucleotide, in or by a chosen host cell. The invention includes a genetic construct, such as a vector, comprising the polynucleotide of the present invention operably linked to such a regulatory sequence.

The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner eg to express the polypeptide agent. The term "regulatory sequences" includes promoters and enhancers and other expression regulation signals. The term "promoter" is used in the normal sense of the art, eg an RNA polymerase binding site.

As used herein, the term "nucleotide sequence" is synonymous with the term "polynucleotide" and "nucleic acid". The nucleotide sequence may be DNA or RNA of genomic or synthetic or of recombinant origin. The nucleotide sequence may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.

For some applications, preferably, the nucleotide sequence is DNA. For some applications, preferably, the nucleotide sequence is prepared by use of recombinant DNA techniques (eg recombinant DNA). For some applications, preferably, the nucleotide sequence is cDNA. For some applications, preferably, the nucleotide sequence may be the same as the naturally occurring form.

Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al, Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al, Short Protocols in Molecular Biology (1999) 4^th Ed, John Wiley & Sons, Inc. PCR is described in US Patents Nos. 4,683,195, 4,800,195 and 4,965,188.

MAL gene expression

In yet another preferred embodiment, the agent modulates MAL gene expression.

Preferably, the invention includes an agent which inhibits MAL gene expression. Alternatively, the invention includes an agent which stimulates MAL gene expression. Agents that inhibit MAL gene expression include antisense RNA, small interfering RNAs (such as described in Hannon et al. Nature, 418 (6894): 244- 51 (2002); Brummelkamp et al, Science 21, 21 (2002); and Sui et al, Proc. Natl Acad. Sci. USA 99, 5515-5520 (2002), and described below), and ribozyme molecules which selectively cleave polynucleotides encoding MAL.

Agents that inhibit or stimulate MAL gene transcription can be designed, for example using an engineered transcription repressor described in Isalan et al. Nat Biotechnol, 19(7): 656-60 (2001) and in Urnov F. Biochem Pharmacol, 64 (5-6) :919 (2002), or they can be selected, for example using the screening methods described in later aspects of the invention.

siRNA

RNA interference (RNAi) is the process of sequence-specific post- transcriptional gene silencing in animals initiated by double-stranded (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific mRNA degradation are typically 21- and 22-nucleotide small interfering RNAs (siRNAs) which, in vivo, may be generated by ribonuclease III cleavage from longer dsRNAs. Elbashir et al. (2001, Nature 411, 494-498) has shown that 21-nucleotide siRNA duplexes specifically suppress expression of both endogenous and heterologous genes in, for example, mammalian cells. In mammalian cells it is believed that the siRNA has to be comprised of two complementary 21mers as described below since longer double-stranded (ds) RNAs will activate PKR (dsRNA-dependent protein kinase) and inhibit overall protein synthesis.

Duplex siRNA molecules selective for MAL can readily be designed by reference to the MAL cDNA sequence. For example, they can be designed by reference to the human MAL cDNA sequence shown in Figure 1 , or naturally occurring variants thereof. Typically, the first 21-mer sequence that begins with an AA dinucleotide which is at least 120 nucleotides downstream from the initiator methionine codon is selected. The RNA sequence perfectly complementary to this becomes the first RNA oligonucleotide. The second RNA sequence should be perfectly complementary to the first 19 residues of the first, with an additional UU dinucleotide at its 3' end. Once designed, the synthetic RNA molecules can be synthesised using methods well known in the art.

siRNAs may be introduced into cells in the patient using any suitable method. Typically, the RNA is protected from the extracellular environment, for example by being contained within a suitable carrier or vehicle. Liposome- mediated transfer is preferred. Liposomes are described in more detail with respect to antisense nucleic acids below. It is particularly preferred if the oligofectamine method is used.

Antisense and triplet-forming nucleic acid.

Antisense nucleic acid molecules selective for MAL can be designed by reference to the cDNA or gene sequence, as is known in the art.

Antisense nucleic acids, such as oligonucleotides, are single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed. These nucleic acids are often termed "antisense" because they are complementary to the sense or coding strand of the gene. Recently, formation of a triple helix has proven possible where the oligonucleotide is bound to a DNA duplex. It was found that oligonucleotides could recognise sequences in the major groove of the DNA double helix. A triple helix was formed thereby. This suggests that it is possible to synthesise a sequence-specific molecules which specifically bind double-stranded DNA via recognition of major groove hydrogen binding sites. By binding to the target nucleic acid, the above oligonucleotides can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking the transcription, processing, poly(A)addition, replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradations.

Antisense oligonucleotides are prepared in the laboratory and then introduced into cells, for example by microinjection or uptake from the cell culture medium into the cells, or they are expressed in cells after transfection with plasmids or retroviruses or other vectors carrying an antisense gene. Antisense oligonucleotides were first discovered to inhibit viral replication or expression in cell culture for Rous sarcoma virus, vesicular stomatitis virus, herpes simplex virus type 1, simian virus and influenza virus. Since then, inhibition of mRNA translation by antisense oligonucleotides has been studied extensively in cell-free systems including rabbit reticulocyte lysates and wheat germ extracts. Inhibition of viral function by antisense oligonucleotides has been demonstrated in vitro using oligonucleotides which were complementary to the AIDS HIV retrovirus RNA (Goodchild, J. 1988 "Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides", Proc. Natl. Acad. Sci. (USA) 85(15), 5507-11). The Goodchild study showed that oligonucleotides that were most effective were complementary to the poly(A) signal; also effective were those targeted at the 5' end of the RNA, particularly the cap and 5 ' untranslated region, next to the primer binding site and at the primer binding site. The cap, 5' untranslated region, and poly(A) signal lie within the sequence repeated at the ends of retrovirus RNA (R region) and the oligonucleotides complementary to these may bind twice to the RNA.

Typically, antisense oligonucleotides are 15 to 35 bases in length. For example, 20-mer oligonucleotides have been shown to inhibit the expression of the epidermal growth factor receptor mRNA (Witters et al. , Breast Cancer Res Treat 53:41-50 (1999)) and 25-mer oligonucleotides have been shown to decrease the expression of adrenocorticotropic hormone by greater than 90% (Frankel et al, J Neurosurg 91 :261-7 (1999)). However, it is appreciated that it may be desirable to use oligonucleotides with lengths outside this range, for example 10, 11, 12, 13, or 14 bases, or 36, 37, 38, 39 or 40 bases.

Antisense polynucleotides may be administered systemically. Alternatively the inherent binding specificity of polynucleotides characteristic of base pairing is enhanced by limiting the availability of the polynucleotide to its intended locus in vivo, permitting lower dosages to be used and minimising systemic effects. Thus, polynucleotides may be applied locally to achieve the desired effect. The concentration of the polynucleotides at the desired locus is much higher than if the polynucleotides were administered systemically, and the therapeutic effect can be achieved using a significantly lower total amount. The local high concentration of polynucleotides enhances penetration of the targeted cells and effectively blocks translation of the target nucleic acid sequences.

It will be appreciated that antisense agents also include larger molecules which bind to MAL mRNA or genes and substantially prevent expression of MAL mRNA or genes and substantially prevent expression of the MAL protein. Thus, an antisense molecule which is substantially complementary to MAL mRNA is envisaged as part of the invention.

The larger molecules may be expressed from any suitable genetic construct and delivered to the patient. Typically, the genetic construct which expresses the antisense molecule comprises at least a portion of the MAL cDNA or gene operatively linked to a promoter which can express the antisense molecule in the cell.

Although genetic constructs for delivery of polynucleotides can be DNA or RNA it is preferred if it is DNA. Equivalent genetic constructs can be used to deliver antisense polynucleotides to a patient as described above in relation to the delivery of polynucleotides encoding MAL.

Preferably, the genetic construct is adapted for delivery to a human cell.

In a preferred embodiment, the polynucleotide which is antisense further comprises a vector which is designed to express antisense DNA. Hence, the invention further provides a polynucleotide comprising a nucleic acid sequence which is antisense to a polynucleotide encoding the MAL polypeptide for use in medicine, especially in the manufacture of a medicament for treating cancer.

Ribozymes

Ribozymes are RNA or RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity. For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate. This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids. For example, US Patent No 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme- mediated inhibition of gene expression may be particularly suited to therapeutic applications, and may be designed by reference to the cDNA which is a copy of the mRNA to be cleaved (eg the human MAL cDNA shown in Figure 1 , or naturally occurring variants thereof). The invention also includes variants of the polynucleotides encoding the agents described above, or variants of the antisense polynucleotides, or variants of the siRNAs.

The term "variant" includes polynucleotides having at least 90%, preferably at least 91%, or at least 92%, or more preferably at least 93%, or at least 94%, or at least 95%, or at least 96%, or yet more preferably at least 97%, or at least 98%, or most preferably at least 99% sequence identity with the polynucleotides encoding the agents described above, or the antisense polynucleotides, or the siRNAs.

The term "variant" also encompasses sequences that are complementary to sequences that are capable of hybridising under highly stringent conditions (eg 65°C and O.lxSSC { lxSSC = 0.15 M NaCl, 0.015 M Na₃citrate pH 7.0}) to polynucleotides encoding the agents described above, or to the polynucleotide agents.

A second aspect of the present invention provides a pharmaceutical composition comprising an agent which modulates a MAL activity and a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).

The invention includes a pharmaceutical composition comprising a polynucleotide that encodes an agent which modulates a MAL activity and a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).

Preferably and typically, agents that modulate a MAL activity are as described above in the first aspect of the invention. The pharmaceutical compositions may be for human or veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient.

Acceptable earners or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).

Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

Formulations and Routes of Administration

There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be administered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be administered by a number of routes.

The routes for administration (delivery) include, but are not limited to, one or more of: oral (eg as a tablet, capsule, or as an ingestable solution), topical, mucosal (eg as a nasal spray or aerosol for inhalation), nasal, parenteral (eg by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, vaginal, epidural, sublingual.

Where the composition is to be administered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects ofbile.

Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

If the pharmaceutical is a tablet, then the tablet may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmefhylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

If a component of the present invention is administered parenterally, then examples of such administration include one or more of: intravenously, intra- arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the component; and/or by using infusion techniques.

For parenteral administration, the component is best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

As indicated, the component(s) of the present invention can be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, eg dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A™) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA™), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, eg using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, eg sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the agent and a suitable powder base such as lactose or starch.

Alternatively, the component(s) of the present invention can be administered in the form of a suppository or pessary, or it may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. The component(s) of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes. They may also be administered by the ocular route. For ophthalmic use, the compounds can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the component(s) of the present invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, it can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.

The component(s) of the present invention may be formulated into a pharmaceutical composition, such as by mixing with one or more of a suitable carrier, diluent or excipient, by using techniques that are known in the art.

The composition may also be administered via the peripheral blood, for example by using skin patches.

Administration of proteins

Proteins and peptides may be delivered to a patient using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections. An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.

The protein and peptide can be administered by a surgically implanted device that releases the drug directly to the required site. For example, Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis. The direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.

Electroporation therapy (EPT) systems can also be employed for the administration of proteins and peptides. A device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.

Proteins and peptides can be delivered by electroincorporation (El). El occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In El, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.

An alternative method of protein and peptide delivery is the ReGel injectable system that is thermo-sensitive. Below body temperature, ReGel is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.

Protein and peptide pharmaceuticals can also be delivered orally. The process employs a natural process for oral uptake of vitamin B₁₂ in the body to co- deliver proteins and peptides. By riding the vitamin B₁₂ uptake system, the protein or peptide can move through the intestinal wall. Complexes are synthesised between vitamin B]₂ analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin B₁₂ portion of the complex and significant bioactivity of the drug portion of the complex.

Proteins and polypeptides can be introduced to cells by "Trojan peptides". These are a class of polypeptides called penetratins which have translocating properties and are capable of carrying hydrophilic compounds across the plasma membrane. This system allows direct targetting of oligopeptides to the cytoplasm and nucleus, and may be non-cell type specific and highly efficient. See Derossi et al (1998), Trends Cell Biol 8, 84-87.

Administration of polynucleotides

If the agent is a protein, the protein may be prepared in situ in the subject being treated. In this respect, a polynucleotide encoding the agent may be delivered by use of non-viral techniques and/or viral techniques (both of which are described below) such that the protein is expressed from the polynucleotide. Similarly, if the agent itself is a polynucleotide, it may be administered using any suitable technique. The term "administered" includes delivery by viral or non- viral techniques. Viral delivery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors. Non- viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.

Polynucleotides may be administered systemically. Alternatively the inherent binding specificity of polynucleotides characteristic of base pairing is enhanced by limiting the availability of the polynucleotide to its intended locus in vivo, permitting lower dosages to be used and minimising systemic effects. Thus, polynucleotides may be applied locally to achieve the desired effect. The concentration of the polynucleotides at the desired locus is much higher than if the polynucleotides were administered systemically, and the therapeutic effect can be achieved using a significantly lower total amount. The local high concentration of polynucleotides enhances penetration of the targeted cells and effectively blocks translation of the target nucleic acid sequences. The polynucleotides can be delivered to the locus by any means appropriate for localised administration of a drug. For example, a solution of the polynucleotides can be injected directly to the site or can be delivered by infusion using an infusion pump. The polynucleotides also can be incorporated into an implantable device which when placed adjacent to the desired site, to permit the polynucleotides to be released into the surrounding locus.

The polynucleotides may be administered via a hydrogel material. The hydrogel is non-inflammatory and biodegradable. Many such materials now are known, including those made from natural and synthetic polymers. In a preferred embodiment, the method exploits a hydrogel which is liquid below body temperature but gels to form a shape-retaining semisolid hydrogel at or near body temperature. Prefened hydrogel are polymers of ethylene oxide- propylene oxide repeating units. The properties of the polymer are dependent on the molecular weight of the polymer and the relative percentage of polyethylene oxide and polypropylene oxide in the polymer. Prefened hydrogels contain from about 10% to about 80% by weight ethylene oxide and from about 20% to about 90% by weight propylene oxide. A particularly prefened hydrogel contains about 70% polyethylene oxide and 30% polypropylene oxide. Hydrogels which can be used are available, for example, from BASF Corp., Parsippany, NJ, under the tradename Pluronic^R.

In this embodiment, the hydrogel is cooled to a liquid state and the oligonucleotides are admixed into the liquid to a concentration of about 1 mg polynucleotides per gram of hydrogel. The resulting mixture then is applied onto the surface to be treated, for example by spraying or painting during surgery or using a catheter or endoscopic procedures. As the polymer warms, it solidifies to form a gel, and the polynucleotides diffuse out of the gel into the sunounding cells over a period of time defined by the exact composition of the gel. The polynucleotides can be administered by means of other implants that are commercially available or described in the scientific literature, including liposomes, microcapsules and implantable devices. For example, implants made of biodegradable materials such as polyanhydrides, polyorthoesters, polylactic acid and polyglycolic acid and copolymers thereof, collagen, and protein polymers, or non-biodegradable materials such as ethylenevinyl acetate (EVAc), polyvinyl acetate, ethylene vinyl alcohol, and derivatives thereof can be used to locally deliver the polynucleotides. The polynucleotides can be incorporated into the material as it is polymerised or solidified, using melt or solvent evaporation techniques, or mechanically mixed with the material. In one embodiment, the polynucleotides are mixed into or applied onto coatings for implantable devices such as dextran coated silica beads, stents, or catheters.

The dose of polynucleotides is dependent on the size of the polynucleotides and the purpose for which is it administered. In general, the range is calculated based on the surface area of tissue to be treated. The effective dose of polynucleotide is somewhat dependent on the length and chemical composition of the polynucleotides but is generally in the range of about 30 to 3000 μg per square centimetre of tissue surface area.

The polynucleotides may be administered by any effective method, for example, parenterally (eg intravenously, subcutaneously, intramuscularly) or by oral, nasal or other means which permit the oligonucleotides to access and circulate in the patient's bloodstream. Polynucleotides administered systemically preferably are given in addition to locally administered polynucleotides, but also have utility in the absence of local administration. A dosage in the range of from about 0.1 to about 10 grams per administration to an adult human generally will be effective for this purpose.

Preferably, the genetic construct is adapted for delivery to a human cell. Means and methods of introducing a genetic construct into a cell in an animal body are known in the art. For example, the constructs of the invention may be introduced into cells by any convenient method, for example methods involving retroviruses, so that the construct is inserted into the genome of the cell. For example, in Kuriyama et al. (1991) Cell Struc. and Func. 16, 503- 510 purified retroviruses are administered. Retroviral DNA constructs comprising a polynucleotide as described above may be made using methods well known in the art. To produce active retrovirus from such a construct it is usual to use an ecotropic psi2 packaging cell line grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal calf serum (FCS). Transfection of the cell line is conveniently by calcium phosphate coprecipitation, and stable transformants are selected by addition of G418 to a final concentration of 1 mg/ml (assuming the retroviral construct contains a neo^κ gene). Independent colonies are isolated and expanded and the culture supernatant removed, filtered through a 0.45 μm pore-size filter and stored at - 70°C.

Alternatively, as described in Culver et al. (1992) Science 256, 1550-1552, cells which produce retroviruses are injected. The retrovirus-producing cells so introduced are engineered to actively produce retroviral vector particles so that continuous productions of the vector occuned within the tumour mass in situ. Thus, proliferating epidermal cells can be successfully transduced in vivo if mixed with retroviral vector-producing cells.

Targeted retroviruses are also available for use in the invention; for example, sequences conferring specific binding affinities may be engineered into pre- existing viral env genes (see Miller & Vile (1995) Faseb J. 9, 190-199 for a review of this and other targeted vectors for gene therapy).

Other methods involve simple delivery of the construct into the cell for expression therein either for a limited time or, following integration into the genome, for a longer time. An example of the latter approach includes liposomes (Nassander et al. (1992) Cancer Res. 52, 646-653).

For the preparation of immuno-liposomes MPB-PE (N-[4-(p- maleimidophenyl)butyryl]-phosphatidylethanolamine) is synthesised according to the method of Martin & Papahadjopoulos (1982) J. Biol Chem. 257, 286-288. MPB-PE is incorporated into the liposomal bilayers to allow a covalent coupling of the antibody, or fragment thereof, to the liposomal surface. The liposome is conveniently loaded with the DNA or other genetic construct of the invention for delivery to the target cells, for example, by forming the liposomes in a solution of the DNA or other genetic construct, followed by sequential extrusion through polycarbonate membrane filters with 0.6 μm and 0.2 μm pore size under nitrogen pressures up to 0.8 MPa. After extrusion, entrapped DNA construct is separated from free DNA construct by ultracentrifugation at 80 000 x g for 45 min. Freshly prepared MPB-PE- liposomes in deoxygenated buffer are mixed with freshly prepared antibody (or fragment thereof) and the coupling reactions are carried out in a nitrogen atmosphere at 4°C under constant end over end rotation overnight. The immunoliposomes are separated from unconjugated antibodies by ultracentrifugation at 80,000 x g for 45 minutes. Immuno-liposomes may be injected intraperitoneally or directly into the site where they are required, eg a tumour.

Other methods of delivery include adenoviruses canying external DNA via an antibody-polylysine bridge (see Curiel Prog. Med. Virol. 40, 1-18) and transferrin-polycation conjugates as carriers (Wagner et al (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414). In the first of these methods a polycation- antibody complex is formed with the DNA construct or other genetic construct of the invention, wherein the antibody is specific for either wild-type adenovirus or a variant adenovirus in which a new epitope has been introduced which binds the antibody. The polycation moiety binds the DNA via electrostatic interactions with the phosphate backbone. The adenovirus, because it contains unaltered fibre and penton proteins, is internalised into the cell and carries into the cell with it the DNA construct of the invention. It is prefened if the polycation is polylysine.

The DNA may also be delivered by adenovirus wherein it is present within the adenovirus particle, for example, as described below.

In an alternative method, a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to cany DNA macromolecules into cells is employed. This is accomplished by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, or the chicken homologue conalbumin, or combinations thereof is covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with DNA constructs or other genetic constructs of the invention independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and the DNA constructs or other genetic constructs of the invention are supplied to the tumour cells, a high level of expression from the construct in the cells is expected.

High-efficiency receptor-mediated delivery of the DNA constructs or other genetic constructs of the invention using the endosome-disruption activity of defective or chemically inactivated adenovirus particles produced by the methods of Cotten et al. (1992) Proc. Natl. Acad. Sci. USA 89, 6094-6098 may also be used. This approach appears to rely on the fact that adenoviruses are adapted to allow release of their DNA from an endosome without passage through the lysosome, and in the presence of, for example transferrin linked to the DNA construct or other genetic construct of the invention, the construct is taken up by the cell by the same route as the adenovirus particle.

It will be appreciated that "naked DNA" and DNA complexed with cationic and neutral lipids may also be useful in introducing the DNA of the invention into cells of the individual to be treated. Non- viral approaches to gene therapy are described in Ledley (1995) Human Gene Therapy 6, 1129-1144.

Alternative targeted delivery systems are also known such as the modified adenovirus system described in WO 94/10323 wherein, typically, the DNA is carried within the adenovirus, or adenovirus-like, particle. Michael et al (1995) Gene Therapy 2, 660-668 describes modification of adenovirus to add a cell- selective moiety into a fibre protein. Mutant adenoviruses which replicate selectively in p53-deficient human tumour cells, such as those described in Bischoff et al (1996) Science 274, 373-376 are also useful for delivering the genetic construct of the invention to a cell. Thus, it will be appreciated that a further aspect of the invention provides a virus or virus-like particle comprising a genetic construct of the invention. Other suitable viruses or virus-like particles include HSV, AAV, vaccinia and parvovirus.

A third aspect of the present invention provides a method of combating a disorder in an individual, the method comprising modulating a MAL activity in the individual.

Typically, the disorder is one in which it is beneficial to modulate an activity of MAL, and in particular a MAL activity involved in rho-dependent SRF signalling in a cell.

Disorders that can be treated by the methods, agents and compositions described herein include cancer, such as leukaemia, particularly the childhood leukaemia AML-M7; wounds; myopathies such as muscle hypertrophy; and any disorder that would benefit from enhanced angiogenesis. Although in the treatment of some disorders it may be beneficial to enhance the activity of MAL, it is typically prefened if a MAL activity is inhibited.

The invention thus provides a method of combating a disorder in an individual by modulating the SRF Rho-dependent pathway in the individual. Details of the SRF Rho-dependent pathway are provided above, and in the publications referenced therein, and in WO 02/20092 Al, incorporated herein by reference. Typically, the SRF Rho-dependent pathway is modulated at any of the following stages: the interaction of MAL with SRF; translocation of MAL to and/or from the nucleus; MAL C-terminal phosphorylation; dimerisation of MAL; the interaction of actin with MAL interaction, or by modulating MAL gene expression.

By "combating" we include the meaning that the method can be used to alleviate symptoms of the disorder (ie the method is used palliatively), or to treat the disorder, or to prevent the disorder (ie the method is used prophylactically).

The therapy (treatment) may be on humans or animals. Preferably, the methods of the inventions are used to treat humans.

The invention includes a method of combating a disorder in an individual comprising administering to the individual an agent which modulates an interaction of MAL with SRF, or a polynucleotide which encodes such an agent, or a pharmaceutical composition comprising the agent or the polynucleotide. Suitable agents and compositions include those described above with respect to the first and second aspects of the invention.

The invention also includes a method of combating a disorder in an individual comprising administering to the individual an agent which modulates translocation of MAL to and/or from the nucleus, or a polynucleotide which encodes such an agent, or a pharmaceutical composition comprising the agent or the polynucleotide. Suitable agents and compositions include those described above with respect to the first and second aspects of the invention.

The invention also includes a method of combating a disorder in an individual comprising administering to the individual an agent which modulates C- terminal phosphorylation of MAL, or a polynucleotide which encodes such an agent, or a pharmaceutical composition comprising the agent or the polynucleotide. Suitable agents and compositions include those described above with respect to the first and second aspects of the invention.

The invention also includes a method of combating a disorder in an individual comprising administering to the individual an agent which modulates dimerisation of MAL, or a polynucleotide which encodes such an agent, or a pharmaceutical composition comprising the agent or the polynucleotide. Suitable agents and compositions include those described above with respect to the first and second aspects of the invention.

The invention also includes a method of combating a disorder in an individual comprising administering to the individual an agent which modulates an interaction of actin with MAL, or a polynucleotide which encodes such an agent, or a pharmaceutical composition comprising the agent or the polynucleotide. Suitable agents and compositions include those described above with respect to the first and second aspects of the invention.

The invention also includes a method of combating a disorder in an individual comprising administering to the individual an agent which modulates MAL gene expression, or a polynucleotide which encodes such an agent, or a pharmaceutical composition comprising the agent or the polynucleotide. Suitable agents and compositions include those described above with respect to the first and second aspects of the invention. The disorder to be combated can be cancer, and the method can be used to combat aspects of cancer including tumour cell growth, adhesion, cellular mobility, invasion and metastasis by modulating a MAL activity at the site of the cancer in the individual.

As the Rho pathway is involved in cell adhesion and mobility, agents inhibiting MAL might be of use for the treatment of a broad range of cancers and particularly cancer with metastasis.

The disorder to be combated can be a wound, and the method can be used to enhance wound healing by modulating a MAL activity in the individual at the site of the wound.

The disorder to be combated can be a myopathy such as muscle hypertrophy, and the method can be used to combat the myopathy by modulating a MAL activity at the site of the myopathy in the individual.

The disorder to be combated can be any disorder that would benefit from enhanced angiogenesis, and the method can be used to enhance angiogenesis enhance by modulating a MAL activity in the individual at region of the individual requiring enhanced angiogenesis.

A fourth aspect of the invention provides an agent which modulates a MAL activity for use in medicine.

The invention includes a polynucleotide that encodes an agent, for example a polypeptide or polynucleotide agent, that modulates a MAL activity for use in medicine.

Preferably, and in particular, the agent modulates a MAL activity involved in rho-dependent SRF signalling in a cell. Thus the agent or polynucleotide is packaged and presented for use in medicine.

Preferred agents that modulate a MAL activity are as described above in the first aspect of the invention. Suitable polynucleotides that that encode an agent which modulates a MAL activity may be as described above in the first aspect of the invention.

A fifth aspect of the invention provides the use of an agent which modulates a MAL activity in the manufacture of a medicament for combating a disorder that would benefit from a modulation of MAL activity.

The invention includes the use of a polynucleotide that encodes an agent which modulates a MAL activity in the manufacture of a medicament for combating a disorder that would benefit from a modulation of MAL activity.

Preferably, and in particular, the medicament modulates a MAL activity involved in rho-dependent SRF signalling in a cell.

Preferred agents that modulate a MAL activity are as described above in the first aspect of the invention. Suitable polynucleotides that that encode an agent which modulates a MAL activity may be as described above in the first aspect of the invention.

Disorders that can be combated by the medicaments include cancer, such as leukaemia, particularly the childhood leukaemia AML-M7; wounds; myopathies such as muscle hypertrophy; and any disorder that would benefit from enhanced angiogenesis, as described above in the third aspect of the invention.

Preferably, the medicament for combating cancer combats at least one of tumour cell growth, adhesion, cellular mobility, invasion or metastasis. Thus the medicament preferably modulates an interaction of MAL with SRF; translocation of MAL to and/or from the nucleus; C-terminal phosphorylation of MAL; dimerisation of MAL; or an interaction of actin with MAL, or MAL gene expression.

Preferably, the medicament inhibits a MAL activity in the individual. Alternatively, the medicament may stimulate a MAL activity in the individual.

A yet further aspect of the invention provides the use of an agent that modulates a MAL activity as described above, or a polynucleotide encoding the agent, or a genetic construct comprising the polynucleotide, for modulating an activity of MAL in vitro. Typically, the MAL activity is modulated in cells in culture.

Further aspects of the present invention relate to screening methods for agents such as drugs for modulating a MAL activity, or lead compounds for the development of drugs that modulate a MAL activity, in particular a MAL activity involved in rho-dependent SRF signalling in a cell.

The MAL used in the screening methods and assays may be MAL as defined above in the first aspect of the invention. However, it is appreciated that the MAL used in the screening methods may be a fragment, variant, derivative or fusion of MAL, providing that it retains an activity of MAL, particularly a MAL activity involved in rho-dependent SRF signalling in a cell.

In a further aspect, the invention provides a method of screening for an agent that modulates the MAL/SRF interaction, comprising

(a) providing a composition comprising SRF and MAL;

(b) providing a test agent; and (c) assessing the interaction of MAL with SRF in the composition, wherein a change in the interaction of MAL with SRF in the presence of the test agent indicates that the test agent may modulate the MAL/SRF interaction.

It is appreciated that in this aspect the MAL may comprise a fragment, variant, derivative or fusion of MAL which binds to SRF, and/or the SRF may comprise a fragment, variant, derivative or fusion of SRF which binds to MAL. When the composition comprises both a fragment, variant, derivative or fusion of SRF and a fragment, variant, derivative or fusion of MAL, the respective SRF and MAL fragments, variants, derivatives or fusions bind together. Suitable fragments of MAL include the Bl domain and the Bl and Q domains, and suitable fragments of SRF include the DNA binding domain.

Preferably, the composition is a cell culture. In an embodiment, the interaction of MAL with SRF is measured using an SRF reporter gene such as that described in Example 1 and Figure 3. It is appreciated that Rho does not have to be present, it can be replaced by a drug which activates the pathway, for example a drug as described in Example 4.

Typically, a decrease in the interaction of MAL with SRF indicates that the compound may be an inhibitor of the MAL/SRF interaction. Alternatively, an increase in the interaction of MAL with SRF indicates that the compound may be an enhancer of the MAL/SRF interaction.

Preferably, the composition may comprise a cell culture, and either or both of MAL and SRF may be expressed by a cell in the culture. In one prefened embodiment, the cell endogenously expresses SRF, and MAL is expressed from an exogenous genetic construct. The timing and extent of MAL expression can be controlled through the use of appropriate promoters in the genetic construct. In an alternative prefened embodiment, the cell endogenously expresses MAL, and SRF is expressed from an exogenous genetic construct. The timing and extent of SRF expression can be controlled through the use of appropriate promoters in the genetic construct. Suitable promoters are well known in the art.

In yet a further aspect, the invention provides a method of screening for an agent that modulates the MAL/actin interaction, comprising

(a) providing a composition comprising MAL and actin;

(b) providing a test agent; and

(c) assessing the interaction of MAL with actin in the composition, wherein a change in the interaction of MAL with actin in the presence of the test agent indicates that the test agent may modulate the MAL/actin interaction.

Typically, a decrease in the interaction of MAL with actin indicates that the compound may be an inhibitor of the MAL/actin interaction. Alternatively, an increase in the interaction of MAL with actin indicates that the compound may be an enhancer of the MAL/actin interaction.

It is appreciated that in this aspect the MAL may comprise a fragment, variant, derivative or fusion of MAL which binds to actin.

Preferably, the composition may comprise a cell culture, and either or both of MAL and actin may be expressed by a cell in the culture. In one prefened embodiment, the cell endogenously expresses actin, and MAL is expressed from an exogenous genetic construct. The timing and extent of MAL expression can be controlled through the use of appropriate promoters in the genetic construct. In an alternative and less prefened embodiment, the cell endogenously expresses MAL, and actin is expressed from an exogenous genetic construct. The timing and extent of actin expression can be controlled through the use of appropriate promoters in the genetic construct. Suitable promoters are well known in the art. In an additional aspect, the invention provides a method of screening for an agent that modulates MAL dimerisation, comprising

(a) providing a composition comprising MAL;

(b) providing a test agent; and (c) assessing the dimerisation of MAL in the composition, wherein a change in the level, extent or rate of dimerisation MAL in the presence of the test agent indicates that the test agent may modulate dimerisation of MAL.

In an embodiment, the composition may comprise a cell culture, and MAL may be expressed by a cell in the culture. The MAL may be expressed from an exogenous genetic construct, allowing the timing and extent of MAL expression to be controlled through the use of appropriate promoters in the genetic construct. Suitable promoters are well known in the art.

In a further aspect, the invention provides a method of screening for an agent that modulates the translocation of MAL to and/or from the nucleus, comprising

(a) providing a cell culture comprising MAL;

(b) providing a test agent; and

(c) assessing the translocation of MAL to and/or from the nucleus in the cells, wherein a change in the cellular localisation of MAL in the presence of the test agent indicates that the test agent may modulate MAL nuclear translocation.

In an embodiment, a cell culture comprising MAL includes a cell culture in which at least some cells in the culture endogenously express MAL.

More preferably, a cell culture comprising MAL includes a cell culture in which at least some cells in the culture express MAL from an exogenous genetic construct to allow the timing and extent of MAL expression to be controlled through the use of appropriate promoters in the genetic construct.

Alternatively, a cell culture comprising MAL includes a cell culture to which recombinant MAL has been added.

In a further aspect, the invention provides a method of screening for an agent that modulates the phosphorylation of specific C-terminal residue(s) of residues of MAL, comprising

(a) providing a composition comprising MAL;

(b) providing a protein kinase; (c) providing a source of phosphate;

(d) providing a test agent; and

(e) assessing the phosphorylation of specific MAL residue(s), wherein a change in phosphorylation status of the specific MAL residue(s) in the presence of the test agent indicates that the test agent may modulate MAL phosphorylation.

In a still further aspect, the invention provides a method of screening for an agent that modulates the expression of the MAL gene, comprising

(a) providing a cell culture in which at least some cells in the culture comprise a polynucleotide having a MAL regulatory sequence fused to a polynucleotide encoding a detectable product;

(b) providing a test agent; and

(c) assessing the expression of the detectable product in the cells, wherein a change in the expression of the detectable product in the presence of the test agent indicates that the test agent may modulate MAL gene expression. Preferably, the MAL regulatory sequence includes the promoter sequence of the MAL gene, more preferably, the promoter sequence of the human MAL gene.

The detectable product could be an RNA or polypeptide product. Suitable detectable products are well known in the art.

Preferably, the cells in the culture express the detectable product in the absence of the agent, as this allows a decrease or increase in expression to be detected in the presence of the agent. Alternatively, the cells in the culture do not express the detectable product in the absence of the agent, which only allows an increase in expression to be detected in the presence of the agent. Typically, a decrease in expression of the detectable product indicates that the agent may be an inhibitor of MAL gene expression. Alternatively, an increase in expression of the detectable product indicates that the agent may be a promoter or enhancer of MAL gene expression. Suitable techniques for measuring levels of RNA or polypeptides products are well known in the art.

In one embodiment, the detectable product may be MAL RNA or polypeptide.

An agent tested in these screening methods may be an organic compound or another chemical. The agent includes, but is not limited to, a compound which may be obtainable from or produced by any suitable source, whether natural or not. The agent can be a peptide or polypeptide, or a chemical derivative thereof, or a combination thereof. The agent may even be a nucleotide sequence - which may be a sense sequence or an anti-sense sequence.

The agent may be designed or obtained from a library of compounds which may comprise peptides, as well as other compounds, such as small organic molecules, such as lead compounds. By way of example, the agent may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic agent, a semi-synthetic agent, a structural or functional mimetic, a peptide, a peptidomimetics, a derivatised agent, a peptide cleaved from a whole protein, or a peptides synthesised synthetically (such as, by way of example, either using a peptide synthesiser or by recombinant techniques or combinations thereof, a recombinant agent, an antibody, a natural or a non-natural agent, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof.

It is contemplated that in future further compounds which are able to modulate a MAL activity will be discovered, such as low molecular weight organic molecules. These are considered to be within the scope of the present invention.

The agent may be in the form of a pharmaceutically acceptable salt - such as an acid addition salt or a base salt - or a solvate thereof, including a hydrate thereof. For a review on suitable salts see Berge et al, J. Pharm. Sci., 1977, 66, 1-19.

The agents may exist as stereoisomers and/or geometric isomers - eg they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present invention contemplates the use of all the individual stereoisomers and geometric isomers of those agents, and mixtures thereof. The terms used in the claims encompass these forms, provided they retain the appropriate functional activity (though not necessarily to the same degree).

The agent for use in the present invention may exist in polymorphic form. It will be appreciated by those skilled in the art that the agent for use in the present invention may be derived from a prodrug. Examples of prodrugs include entities that have certain protected group(s) and which may not possess pharmacological activity as such, but may, in certain instances, be administered (such as orally or parenterally) and thereafter metabolised in the body to form the agent of the present invention which are pharmacologically active.

It will be further appreciated that certain moieties known as "pro-moieties", for example as described in "Design of Prodrugs" by H. Bundgaard, Elsevier, 1985 (the disclosure of which is hereby incorporated by reference), may be placed on appropriate functionalities of the agents. Such prodrugs are also included within the scope of the invention.

The present invention also includes the use of zwitterionic forms of the agent for use in the present invention. The terms used in the claims encompass one or more of the forms just mentioned.

The present invention also includes the use of solvate forms of the agent for use in the present invention. The terms used in the claims encompass these forms.

The term "derivative" or "derivatised" as used herein includes chemical modification of an agent. Illustrative of such chemical modifications would be replacement of hydrogen by a halo group, an alkyl group, an acyl group or an amino group.

In one embodiment of the present invention, the agent may be a chemically modified agent. The chemical modification of an agent of the present invention may either enhance or reduce hydrogen bonding interaction, charge interaction, hydrophobic interaction, Van Der Waals interaction or dipole interaction between the agent and the target. It will be appreciated that in the screening methods described herein, the agent identified may be a drug-like compound or lead compound for the development of a drug-like compound.

The term "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons and which may be water- soluble. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes, but it will be appreciated that these features are not essential.

The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.

Alternatively, the methods may be used as "library screening" methods, a term well known to those skilled in the art. Thus, for example, the methods of the invention may be used to detect (and optionally identify) a polynucleotide capable of expressing a polypeptide activator of MAL. Aliquots of an expression library in a suitable vector may be tested for the ability to give the required result. It will be appreciated that several cycles of identifying pools of polynucleotides comprising a polynucleotide having the required property and then rescreening those polynucleotides may be required in order to identify a single species of polynucleotide with the required property. Methods of preparing a suitable expression library for screening are well known to those skilled in the art.

A further aspect of the invention provides a method of identifying a drug-like compound or lead compound for the development of a drug-like compound that modulates the activity of MAL, the method comprising contacting a compound with MAL or a suitable variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof and determining whether, an activity of MAL (or variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof) is changed compared to the its activity in the absence of the compound.

It will be understood that it will be desirable to identify compounds that may modulate the activity of MAL in vivo. Thus it will be understood that reagents and conditions used in the method may be chosen such that the interactions between MAL and SRF and/or actin are substantially the same as in vivo.

In one embodiment, the compound decreases the activity of MAL. For example, the compound may bind substantially reversibly or substantially ineversibly to the site at which MAL binds or interacts with SRF or actin. In a further example, the compound may bind to a portion of MAL that is not the site at which MAL binds or interacts with SRF or actin, but nevertheless interferes with the binding or interaction of Mai with SRF or actin. In a still further example, the compound may bind to a portion of MAL so as to decrease its activity by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of MAL's activity, for example in the activation of MAL by an "upstream activator". In a further embodiment, the compound increases the activity of MAL. For example, the compound may bind to a portion of MAL that is not the site at which MAL binds or interacts with SRF or actin, but which enhances with the binding or interaction of Mai with SRF or actin. In a still further example, the compound may bind to a portion of MAL so as to enhance MAL's activity by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of MAL's activity for example in the activation of MAL by an "upstream activator".

A change in the activity of SRF may be measured. This may be done in a whole cell system or using purified or partially purified components.

Expression of an protein encoded by an RNA transcribed from a promoter regulated by SRF may be measured. The protein may be one that is physiologically regulated by SRF or may be a "reporter" protein, as well known to those skilled in the art (ie a recombinant construct may be used). A reporter protein may be one whose activity may easily be assayed, for example β-galactosidase, chloramphenicol acetyltransferase or luciferase (see, for example, Tan et al. (1996)). In a further example, the reporter gene may be fatal to the cells, or alternatively may allow cells to survive under otherwise fatal conditions. Cell survival can then be measured, for example using colourimetric assays for mitochondrial activity, such as reduction of WST- 1

(Boehringer). WST-1 is a formosan dye that undergoes a change in absorbance on receiving electrons via succinate dehydrogenase.

It will be appreciated that screening assays which are capable of high throughput operation will be particularly prefened. Examples may include cell based assays and protein-protein binding assays. An SPA-based (Scintillation Proximity Assay; Amersham International) system may be used. For example, an assay for identifying a compound capable of modulating the activity of a protein kinase may be performed as follows. Beads comprising scintillant and a polypeptide that may be phosphorylated may be prepared. The beads may be mixed with a sample comprising the protein kinase and P- ATP or ³³P-ATP and with the test compound. Conveniently this is done in a 96- well or 384-well format. The plate is then counted using a suitable scintillation counter, using known parameters for P or ³³P SPA assays. Only ³²P or ³³P that is in proximity to the scintillant, i.e. only that bound to the polypeptide, is detected. Variants of such an assay, for example in which MAL is immobilised on the scintillant beads via binding to an antibody, may also be used.

Other methods of detecting polypeptide/polypeptide interactions include ultrafϊltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods. Fluorescence Energy Resonance Transfer (FRET) methods, for example, well known to those skilled in the art, may be used, in which binding of two fluorescent labelled entities may be measured by measuring the interaction of the fluorescent labels when in close proximity to each other.

Alternative methods of detecting binding of a polypeptide to macromolecules, for example DNA, RNA, proteins and phospholipids, include a surface plasmon resonance assay, for example as described in Plant et al (1995) Analyt Biochem 226(2), 342-348. Methods may make use of a polypeptide that is labelled, for example with a radioactive or fluorescent label.

A further method of identifying a compound that is capable of binding to MAL is one where the polypeptide is exposed to the compound and any binding of the compound to MAL is detected and/or measured. The binding constant for the binding of the compound to the polypeptide may be determined. Suitable methods for detecting and/or measuring (quantifying) the binding of a compound to a polypeptide are well known to those skilled in the art and may be performed, for example, using a method capable of high throughput operation, for example a chip-based method. Technology called VLSIPS™, has enabled the production of extremely small chips that contain hundreds of thousands or more of different molecular probes. These biological chips or anays have probes ananged in arrays, each probe assigned a specific location. Biological chips have been produced in which each location has a scale of, for example, ten microns. The chips can be used to determine whether target molecules interact with any of the probes on the chip. After exposing the anay to target molecules under selected test conditions, scanning devices can examine each location in the anay and determine whether a target molecule has interacted with the probe at that location.

Biological chips or anays are useful in a variety of screening techniques for obtaining information about either the probes or the target molecules. For example, a library of peptides can be used as probes to screen for drugs. The peptides can be exposed to a receptor, and those probes that bind to the receptor can be identified. See US Patent No. 5,874,219 issued 23 February 1999 to Rava et /.

Another method of targeting proteins that modulate the activity of MAL is the yeast two-hybrid system, where the polypeptides of the invention can be used to "capture" MAL binding proteins. The yeast two-hybrid system is described in Fields & Song, Nature 340:245-246 (1989).

An example of the use of the yeast two-hybrid system is the use of two compounds, MAL and SRF, or MAL and actin, which interact to form a complex involved in the Rho-dependent SRF pathway, to facilitate the identification of compounds that modulate this pathway. These compounds are detected by adapting yeast two-hybrid expression systems known in the art for use as described herein. These systems which allow detection of protein interactions via a transcriptional activation assay, are generally described by Gyuris et al, Cell 75:791-803 (1993) and Fields & Song, Nature 340:245-246 (1989), and are commercially available from Clontech (Palo Alto, CA).

In this approach, a region of MAL which interacts with SRF, or a region of MAL which interacts with actin, is fused to the GAL4-DNA-binding domain by subcloning a DNA fragment encoding this into the expression vector, pGBT9, provided in the MATCHMAKER Two-Hybrid System kit commercially available from Clontech (catalogue number Kl 605-1). A fusion of the GAL4 activation domain with at least one of the various MAL domains, as described herein in the Examples, is generated by subcloning the domain- encoding DNA fragment into the expression vector, PGAD424, also provided in the Clontech kit. Analogous expression vectors may also be used. Yeast transformations and colony lift filter assays are carried out according to the methods of MATCHMAKER Two-Hybrid System and various methods known in the art. Prior to the colony filter assay, transformed yeast may be treated with candidate compounds being screened for the ability to modulate a MAL activity. The interaction results obtained using the candidate compound in combination with the yeast system may then be compared to those results observed with the yeast system not treated with the candidate compound, all other factors (eg. cell type and culture conditions) being equal. A compound capable of altering the interaction between MAL and SRF or between MAL and actin may be capable of modulating the Rho-dependent SRF pathway.

In another embodiment of this approach, a compound capable of inhibiting the Rho-dependent SRF pathway by disrupting the binding or interaction of MAL with SRF, or of MAL with actin, may be isolated using the modified yeast two-hybrid system described above, in which the reporter gene encodes a protein, such as ricin, that is toxic to yeast. Yeast cells containing such a ricin reporter die unless the binding or interaction of MAL with SRF, or of MAL with actin, is disrupted. Yeast cells treated with a compound that disrupts the MAL/SRF or MAL/actin interaction form viable colonies, and from this result it may be infened that the compound is capable of decreasing, and possibly inhibiting, this pathway.

The agent identified by the screening methods described above may not itself be optimal for use in a pharmaceutical or medical context. The identified agent may be a lead-compound for the identification of further agents that would be more suitable for such uses. The invention therefore includes modifying an agent identified as a result of the screening methods described above, or taking a further compound having or expected to have similar properties to an agent identified as a result of the screening methods, and screening the modified agent or further compound as described above.

Typically, the test agents which have the desired effects in the above assays are selected for further investigation. Preferably, they are screened further, for example in a cell and/or animal model of a disorder and test agents are selected from these assays for further study if they are seen to have a desirable effect in the further screen.

Suitable disorders include cancer, such as leukaemia, particularly the childhood leukaemia AML-M7; wounds; myopathies such as muscle hypertrophy; and any disorder that would benefit from enhanced angiogenesis.

The invention also includes holding pre-clinical and clinical trials of an agent identified as a result of any of the above screening methods.

The invention further includes packaging and presenting an agent identified as a result of any of the above screening methods for use in medicine.

A further aspect of the invention is an agent identifiable by any of the screening methods described herein. Typically, the agent is a compound such as a polypeptide, polynucleotide, or a small molecule, preferably an organic molecule. Preferably, small molecules are of less than 5000 daltons, and may be water-soluble.

A further aspect of the invention is an agent identified by any of the screening methods described herein. Typically, the agent is a compound such as a polypeptide, polynucleotide, or small molecule, preferably an organic small molecule.

A still further aspect of the invention includes SRF mutants, and fragments thereof, and fusions thereof, that are useful in assessing the interaction between SRF and MAL. We have shown that sequences within a hydrophobic pocket on the SRF DNA binding domain are required for interaction with MAL and that point mutations within this pocket which decrease its depth such as I206F and I215F, or that introduce charge such as V194E or T196E, inhibit binding of MAL to SRF (Figure 18).

The invention thus includes a mutant SRF, or a fragment thereof, or a fusion thereof, having a mutation at one or more of positions within a hydrophobic pocket on the SRF DNA binding domain, and in particular at one or more of positions 194, 196, 206 and 215, and which inhibits binding of SRF to MAL. Preferably, the mutant SRF, or fragment thereof, or fusion thereof, has one or more of the following mutations I206F, I215F, V194E or T196E.

All of the documents refened to herein are incorporated herein, in their entirety, by reference.

The invention will now be described in more detail with the aid of the following Figures and Examples.

Figure 1. Mouse Mai 22 is the homologue of Human MAL. A) DNA sequence alignment of mouse MAL and human MAL. B) Protein sequence alignment of mouse MAL and human MAL. Figure 2. Mouse MAL and Human MAL share conserved sequence domains. Domain structure of mouse and human MAL proteins. The amino-acid residues spanning the different domains are shown.

Figure 3. MAL mediates Rho-signalling to SRF. A) MAL potentiates SRF reporter gene activation. NIH3T3 cells were transfected with SRF reporter and the indicated amount of plasmid expressing MAL, maintained in 0.5% serum for 18 hours, then stimulated with 15% serum for 7 hours. Reporter activation is presented as mean ± SEM of three independent experiments. (B) MAL activation of SRF is dependent of RhoA activity and of its N-terminal region. NIH3T3 cells were transfected with SRF reporter and increasing amounts (5ng or 50ng) of plasmids expressing MAL, MAL(Δl-80) and myocardin in the presence or absence of expression of the Rho-inactivating C3 transferase. Reporter activation is as in (A). (C) MAL does not activate the altered specificity SRF-M2 reporter system. NIH3T3 cells were transfected with 50ng, of SRE.L2 and SRE.LM2 reporter plasmids and increasing amounts (0, 5, 50, 100, or 250ng) of MAL expression plasmid in the absence or presence of MLVSRF or MLVSRF-M2 as indicated. Cells were maintained in 0.5% serum for 18 hours, then stimulated with 15% serum for 7 hours. Reporter activation is presented as mean ± SEM of 3 independent experiments.

Figure 4. MAL interacts with SRF. (A) Formation of a Ternary Complex between SRF and MAL and effects of MAL deletion mutants. Gel Mobility Shift Assays were performed using whole cell extracts prepared from cells expressing WT MAL or MAL deletion mutants. Binding reactions included recombinant SRF (133-265) and a probe from the c-fos SRE.L mutant. (B) MAL does not interact with altered specificity SRF-M2. Whole cell extracts were made from cells transfected with MLV.plink vector or ΔN MAL. Binding assays in lanes 1-4 were performed in the presence or absence of SRF (1-265) whole-cell extract as indicated, using the SRE.L probe. Binding assays in lanes 5-8 were performed in the presence or absence of SRF-M2 (1- 265) whole-cell extract as indicated, using probe SRE.LM. (C) Effects of point mutations in the Bl box in ternary complex formation. GMSAs were performed using extracts from cells expressing MAL point mutants. Binding assays included recombinant SRF (133-265) and probe SRE.L.

Figure 5. TCF Elkl competes with MAL for interaction with SRF in vivo. NIH3T3 cells were transfected with lOng of ΔSIFΔTCF.luc reporter gene in the presence or absence of MAL (5ng) and with increasing amounts (0, 3, 10, 30, 100, 300ng) of GAL4. Δ33NA (an Elkl derivative which cannot be activated by ERK signalling) or GAL4.Y159A NA (a GAL4. Δ33NA derivative which cannot bind SRF). Reporter activation is presented as mean ± SEM of 3 independent experiments

Figure 6. MAL translocates into the nucleus in response to extracellular signals. In the upper panel, NIH3T3 cells were plated onto coverslips, maintained in 1 mg/ml BSA for 24 hours, then stimulated with 15% serum for 1 hour before fixation and immunostaining with rabbit anti-MAL antibodies (Lower panel). Immunostaining of cells transfected with 50ng of Flag-MAL expression plasmid using rabbit anti-Flag antibodies. Left panels show cytoplasmic staining of MAL in unstimulated cells, whereas Mai immunostaining is found in the nucleus after serum stimulation (right panels).

Figure 7. Inhibitors of Rho signalling block signal-induced accumulation of nuclear Mai. Inhibition of nuclear shuttling by RhoA pathway and actin polymerisation inhibitors. NIH3T3 cells were transfected with 50ng of plasmid expressing Flag-MAL and maintained in 1 mg/ml BSA for 18 hours, then stimulated with 15% serum for 1 hour. When indicated, C3-transfecrase, Dial- and VaspΔB expression plasmids (5ng, 200ng and 200ng, respectively) were cotransfected. Toxin B (TB) and Latruculin B (LB) were added to the cells for one hour prior to serum stimulation. Immunostaining was performed with rabbit-anti Flag and mouse 9E10 antibodies. F-actin was stained with phalloidin. The merge panels conespond to MAL and F-actin co-staining. Upon expression in NIH3T3 cells, BSAC also migrated to the nucleus upon serum stimulation, and this was inhibited by C3-transferase co-expression (not shown).

Figure 8 (A) Wild type and non-polymerisable actins inhibit nuclear shuttling of transfected MAL. NIH3T3 cells were cotransfected with 50 ng of plasmid expressing MAL-HA and different Flag- Actin expression plasmids (lμg for Wild type-actin and G13R derivative mutant; and 200 ng for Actin-R62D). Cells were maintained in 1 mg/ml BSA for 36 hours, then stimulated with 15% serum for 1 hour. Immunostaining was carried out with rabbit anti-Flag (Actin staining) and mouse 12CA5 (MAL staining) antibodies. Actin R62D expression also inhibits BSAC shuttling. (B) Interfering Dial, wild type and non-polymerisable actins inhibit nuclear shuttling of endogenous MAL. NIH3T3 cells were transfected with plasmids expressing Flag- Actin, Flag- ActinG13R and Myc-Dial(-), maintained in 1 mg/ml BSA for 36 hours, then stimulated with 15% serum for 1 hour. Immunostaining was carried out with rabbit anti- Mai antibodies and mouse monoclonal 9E10 and M2 antibodies. Anows indicate transfected cells.

Figure 9. Activation of RhoA-Actin signalling induces nuclear MAL accumulation. Proteins that activate SRF induce nuclear accumulation of MAL. NIH3T3 cells were cotransfected with 50ng of plasmid expressing either MAL-HA or Flag-MAL and 100 ng of various expression plasmids as indicated. Cells were maintained in 1 mg/ml BSA for 18 hours. MAL immunostaining was performed with rabbit-anti Flag and mouse 9E10, M2 and 12CA5 antibodies.

Figure 10. Nuclear MAL translocation is induced by an actin-binding drug or mutant actins that activate SRF. NIH3T3 cells were transfected with 50ng of plasmid expressing either MAL-HA Flag-MAL or cotransfected with various Flag- Actin mutants expression plasmids (200-lOOOng). Cells were maintained in 1 mg/ml BSA for 18 hours before fixation. Jasplakinolide, cytochalasin D (CD) and Swinholide A(SwA) were added to the cells for an hour before fixation. For drug treatments, cells were also stained with phalloidin.

Figure 11. Cellular localisation of MAL mutants. NIH3T3 cells were transfected with 50ng of plasmids expressing the indicated MAL derivatives, maintained in 1 mg/ml BSA for 18 hours, and then stimulated with 15% serum for one hour before fixation and staining for Flag-tag. N and C represent nuclear and cytoplasmic localisation, respectively. N+C indicates localisation all over the cell.

Figure 12. MAL interacts with actin in vitro. NIH3T3 cells were cotransfected with plasmids expressing different derivatives of MAL-HA and Flag-Actin as indicated. Immunoprecipitation was performed as described in the Materials and Methods.

Figure 13. MAL interacts with actin in vitro. 1- MAL associates with unpolymerised actin. Targeting of Actin to different cellular compartments results in recolocalisation of MAL. NIH3T3 cells were cotransfected with 50ng of plasmid expressing MAL-HA and 1 μg of Flag-Actin derivatives. Cells were maintained in 1 mg/ml BSA for 18 hours before fixing and subsequent immunostaining with rabbit-anti Flag and mouse 12CA5 antibodies. Top and bottom panels show membrane targeted actin and nuclear targeted nls actin, respectively

Figure 14. MAL associates with unpolymerised actin. 2- The N-terminal domain of MAL mediates interaction with actin. NIH3T3 cells were cotransfected with 50ng of plasmids expressing different MAL-HA derivatives and 1 μg of Flag-Actin-R62D. Cells were maintained in 1 mg/ml BSA for 36 hours, stimulated with 15% serum for one hour, then fixed and immunostained with rabbit-anti Flag and mouse 12CA5 antibodies.

Figure 15. MAL is phosphorylated upon signal-induction. (A) MAL is phosphorylated at serine/threonine after serum stimulation. NIH3T3 cells were transfected with a plasmid expressing MAL-HA, maintained in 0.5% serum for 18 hours. 15%FCS serum was added to the cells for one hour before immunoprecipitation and λphosphatase treatment performed as described in Materials and Methods. Blots were probed with anti-HA-HRP conjugated antibodies and anti-phosphotyrosine monoclonal antibodies (B) MAL is phosphorylated at its C-terminus. NIH3T3 cells were transfected with 200ng of plasmids expressing various Flag-MAL deletion mutants. Cells were maintained in 0.5% serum for 18 hours before stimulation with 15%FCS serum for an hour. Whole cell extracts were obtained by direct lysis of the cells in lxSDS/PAGE sample buffer. Proteins were run on a 7% gel. NS indicates non-specific bands. (C) MAL phosphorylation requires alterations in actin dynamics. Cells were co-transfected with plasmids expressing Mal-HA and various Dial derivatives in the presence or absence of Flag-Actin-R62D. Analysis of protein mobility shift was performed as in (B). (D) MAL is phosphorylated in response to all signals that activate SRF. NIH3T3 cells were cotransfected with 50ng of plasmid expressing MAL-HA and expression plasmids for proteins that regulate actin dynamics as indicated. Cells were maintained in 0.5% serum for 18 hours. Whole cell lysates were run on a 7% gel and blots were probed with anti-HA-HRP conjugated antibodies.

Figure 16. Inhibition of SRF-mediated gene activation by MAL dominant- negative mutants. (A) MAL 1-471 inhibits signalling to SRF. NIH3T3 cells were transfected with SRF reporter and increasing amounts (100, 500 and lOOOng) of plasmid expressing MAL 1-471. When indicated, lOOng of plasmids expressing proteins that control actin dynamics where co-transfected. Cells were maintained in 0.5%) serum for 24 hours. Serum stimulation was for 7 hours before reporter analysis (B) MAL22 (1-471) forms a ternary complex with SRF. Whole cell extracts were made from cells rrasfected with MLV.plink vector or MAL 1-471 -expression plasmid. Binding reactions included recombinant SRF (133-265) and probe SRE.L. Complexes marked MAL refer to complex between MAL and the SRF DNA binding domain fragment (133-265).

Figure 17. Signalling to SRF is inhibited by cytoplasmic forms of MAL containing the LZL domain. (A) NIH3T3 cells were transfected with SRF reporter and increasing amounts (5 and 50ng) of plasmids expressing the indicated cytoplasmic Mai derivatives. Cells were maintained in 0.5% serum for 36 hours before serum stimulation, which was for 7 hours before reporter analysis. (B) The LZL domain of MAL is required to retain wild-type MAL in the cytoplasm. NIH3T3 cells were transfected with 250ng of plasmids expressing cytoplasmic Flag-Mai derivatives (ΔB1ΔB2 and ΔB1ΔB2ΔLZL) together with 50 ng of a plasmid expressing wild-type HA-tagged MAL. Cells were maintained in 1 mg/ml BSA for 24 hours, and then serum-stimulated for 1 hour before fixing and subsequent immunostaining with rabbit-anti Flag and mouse 12CA5 antibodies.

Figure 18. MAL contacts the hydrophobic pocket of the SRF DNA Binding Domain. Gelshift assays were performed using MALΔN whole cell extracts, reticulocyte lysates expressing different SRF point mutant derivatives of the SRF DNA binding domain (the SRF derivatives extended beyond the DNA binding domain and included residues 120-265) and DNA probe from the c- fos SRE.L mutant.

Figure 19. The MAL Bl box peptide competes for MAL-SRF complex formation. Binding reactions included whole cell extracts prepared from cells expressing MALΔN, recombinant SRF( 133-265), SRE.WT DNA probe and increasing amounts of Bl box MAL peptides (0.8, 4.0 or 20.0 pmol; for sequences see Figure 20a).

Figure 20. The B 1 box of MAL is necessary and sufficient to mediate the MAL-SRF interaction. (A) Sequences of the MAL peptides. (B) Complex formation between MAL B1Q and Bl peptides and SRF (133-265). Mutations in the MAL Bl box abolish complex formation. Gelshift assays were performed using MAL peptides, recombinant SRF (133-265) and probe from the c-fos SRE. Amounts used were: Bl and B1Y238A, 0.8 or 4.0 pmol; B1Q and B1QY238A, 0.36, 1.8 and 9 pmol. Peptides Bl, B1Y238A, B1Q and B1QY238A are SEQ ID Nos: 15, 16, 17 and 18, respectively. (C) The MAL Bl box peptide does not interact with SRF-M2. Assays were performed using reticulocyte lysates expressing SRF(120-265) and SRF.M2 (120-265) with 0.8 and 4.0 pmol wildtype or mutant Bl box peptide.

Example I: Mai is an SRF co-activator linked to the Rho pathway

Figure 1 shows the MAL DNA and protein sequences (mouse mMAL and human hMAL). Figure 2 is a map of mouse and human MAL with the position and residue numbers of the different domains indicated.

We tested whether MAL would transactivate an SRF reporter gene. Expression of increasing amounts of MAL potentiates SRF reporter activity in serum-deprived cells and at low doses potentiates the response to serum stimulation (Figure 3A). At low MAL inputs activity is dependent on functional Rho (Figure 3B). Deletion of the N-terminus of MAL (residues 1- 80, including the MAL domain) renders the protein constitutively active and insensitive to inactivation of Rho, suggesting that the N-terminus sensitises the protein to Rho signalling. MAL expression does not potentiate activity of the altered binding specificity SRF derivative SRF-M2, which does not respond to the Rho signalling pathway (Figure 3C) (Hill et al, 1993). Biochemical studies indicate that extracts of cells expressing MAL form a ternary complex with recombinant SRF DNA binding domain in vitro (Figure 4A) Removal of the MAL domain increases DNA binding affinity, while removal of the B 1 box (residues 224-249) completely abolishes binding, and removal of the Q box (residues 264-285) reduces binding slightly (Figure 4A). MAL does not make a ternary complex with the altered specificity SRF derivative SRF-M2 on its cognate site SRE.M (Figure 4B). Point mutagenesis of the MAL Bl box shows that conserved hydrophobic residues essential for complex formation with SRF are located at positions analogous to similar residues involved in the TCF-SRF interaction, suggesting that the two complexes may be structurally related (Figure 4C).

In addition to the sequence alterations present in SRF-M2, sequences within a hydrophobic pocket on the SRF DNA binding domain are required for interaction with MAL. Point mutations within this pocket which decrease its depth (I206F or 1215F) or introduce charge (V194E or T196E) inhibit binding of MAL to SRF (Figure 18).

The B 1 box is sufficient for interaction with SRF, since addition of a B 1 box peptide (see Figure 20a) to binding reactions inhibits complex formation between MAL and the SRF DNA binding domain (Figure 19).

MAL and SRF interact directly. Purified peptides comprising the Bl box alone or the Bl and Q boxes (Figure 20a), effectively bind purified recombinant SRF DNA binding domain, and these interactions are blocked by mutations which block interactions of the intact proteins (Figure 20b). The Bl peptide, like intact MAL, cannot bind the SRF derivative SRF-M2 (Figure 20c). Deletion of the MAL LZL motif causes the reduction of binding affinity and increase in mobility of the MAL-SRF complex, indicating that MAL acts as a dimer and that its formation is mediated through the LZL motif (Figure 4C).

Targeting of the TCF Elk-1 derivative GAL4-ElkΔ33NA, which can bind DNA autonomously but cannot be activated by ERK signalling, to a DNA binding site neighbouring SRF inhibits serum-stimulation, provided the TCF B-box is intact (Murai and Treisman, 2002). In a similar experiment, targeting of GAL4-ElkΔ33NA inhibited transactivation of the SRF reporter by MAL. In contrast a derivative of GAL4-ElkΔ33NA containing a mutation that prevents interaction with SRF (Ling et al, 1997) was unable to inhibit SRF coactivation by MAL (Figure 5). These data indicate that MAL has the SRF- binding properties predicted of the presumptive co-activator which mediates Rho-actin signalling (Hill et al, 1993; Hill et al, 1994; Murai and Treisman, 2002).

Taken together these data support the claim that MAL binds directly to SRF; that it has the DNA binding properties expected of the putative Rho-actin co- activator; that it acts as an SRF co-activator; and that the N-terminal MAL domain sensitises the activity of the protein to Rho signalling.

Example II: Cellular localisation of MAL is regulated by extracellular signalling

We raised an antiserum directed against the MAL N-terminus, which does not cross react with MC, and used it to investigate MAL cellular localisation. In cycling and serum-deprived cells, endogenous MAL exhibited predominantly cytoplasmic staining with strong exclusion from the cell nuclei. Upon serum stimulation MAL accumulated in the cell nucleus (Figure 6). Similar results were obtained when HA- or Flag- tagged MAL was transiently expressed in NIH3T3 cells, with significant nuclear accumulation observed by 10 minutes following stimulation. MAL translocation was also induced by Lysophosphatidic acid and by the phorbol ester TPA (data not shown). In unstimulated cells, cytoplasmic MAL did not colocalise with the F-actin fibre network.

Example III: Inhibitors of Rho signalling block signal-induced accumulation of nuclear MAL

To investigate the signalling to MAL we tested the ability of known inhibitors of Rho-actin signalling and other pathways to inhibited nuclear accumulation of transfected- Flag-MAL following serum stimulation. Results are shown in Figure 7. Serum-induced nuclear shuttling of MAL was inhibited upon inactivation of Rho with C3 transferase or Toxin B, or by a blockade of actin polymerisation with Latrunculin B, which sequesters actin monomer. Both of these treatments inhibit the Rho-dependent signal pathway to SRF (Gineitis and Treisman, 2001 ; Sotiropoulos et al, 1999). Upon expression in NIH3T3 cells, BSAC also migrated to the nucleus upon serum stimulation, and this was also inhibited by C3 -transferase co-expression (not shown). No inhibition of translocation was observed upon treatment of cells with the ERK inhibitor U0126, upon inactivation of Gai and ERK signalling by pertussis toxin, upon inhibition of the Rho effector kinase ROCK with Y27632, or upon inhibition of PI-3 kinase signalling with LY294002 (data not shown). Nuclear Flag- MAL accumulation was also inhibited in cells expressing deleted forms of mDia (F1F2Δ1) and VASP (VASPΔB) which specifically interfere with operation of the Rho-dependent signalling pathway to SRF (Copeland and Treisman, 2002; Grosse et al, 2003).

We previously showed that overexpression of either wild type β-actin or its nonpolymerisable mutant derivatives β-actin G13R and R62D is sufficient to inhibit activation of SRF via the Rho pathway (Posern et al, 2002; Sotiropoulos et al, 1999). Expression of these actins significantly inhibited nuclear accumulation of transfected Flag-MAL (Figure 8 A).

We also tested the inhibition of endogenous MAL translocation. Expression of interfering mDial, wild type actin or actin G13R resulted in the inhibition of endogenous MAL translocation of (Figure 8B).

Example IV: Activation of Rho-actin signalling induces nuclear MAL accumulation

Many proteins involved in actin dynamics controlled by Rho GTPases can induce activation of SRF either when overexpressed as wild type forms or as mutant activated derivatives. These include RhoA, Cdc42 and Rac (Hill et al, 1995); LIM kinase (Geneste et al, 2002; Sotiropoulos et al, 1999); profilin (Sotiropoulos et al, 1999); mDial and mDia2 (Copeland and Treisman, 2002 Sotiropoulos et al, 1999; Tominaga et al, 2000); VASP (Grosse et al, 2003 Sotiropoulos et al, 1999); and WASP and N-WASP (Geneste et al, 2002 Sotiropoulos et al, 1999). Each of these proteins activates nuclear translocation of MAL in the absence of extracellular signals (Figure 9).

In addition to signalling molecules, SRF can also be activated by direct interference with the actin treadmilling cycle. Drugs such as cytochalasin D, swinholide A or mycalolide, which can bind G-actin but do not promote actin polymerisation, activate SRF (Sotiropoulos et al, 1999). Treatment of cells with these drugs also induced nuclear MAL accumulation (Figure 7B). Nuclear MAL accumulation was also induced upon treatment of cells with jasplakinolide, which stabilises F-actin filaments and activates SRF (Sotiropoulos et al, 1999); Figure 10).

Previous work by others has shown that the yeast actin mutation V159N promotes increased stability of the mutant actin filament (Belmont and Drubin, 1998; Belmont et al, 1999). The same mutation in human β-actin has properties consistent with a similar effect, and strongly activates SRF in the absence of signal, and we have identified two further actin mutants with similar properties, S14C and G15S (Posern et al, 2002). Expression of each of these activating actin mutants promoted nuclear translocation of MAL.

Example V: The MAL N- and C-terminal sequences are required for regulated shuttling

To investigate the mechanism of relocalisation of MAL we generated mutant derivatives of the protein and tested their ability to relocalise in response to extracellular signals upon expression in NIH3T3 cells. Data are summarised in Figure 11.

Removal of the N-terminal MAL domain (residues 1-80) generated a form of the protein, which was constitutively nuclear-localised. Within this region, point mutations disrupting the conserved RPEL motifs (P34A, P78A, R33D, R77D, and combined double mutations) caused constitutive nuclear localisation, but deletion of the short basic B2 box had no effect. We next examined C-terminal deletions of flag-MAL. Removal of residues C-terminal to 631 generated a derivative, which apparently constitutively localised to the intermediate filament network. The N-terminal half of MAL (1-471) localised exclusively to the nucleus. Fragments of MAL comprising residues 80-306 or 1-256 were also constitutively nuclear, demonstrating that these sequences are sufficient for nuclear localisation. In contrast, the C-terminal region of the protein (residues 471-929) was localised throughout the cell.

The above data suggest that the MAL N- and C-termini are both involved in its regulated redistribution in response to signals. To test this, we examined internal deletion derivatives of MAL. Deletion of residues 170-581 generated a form of the protein, which was regulated normally. A deletion lacking residues 170-712 was distributed throughout nucleus and cytoplasm in the absence of signal, but was still induced to accumulate in the nucleus in response to signal. We conclude that regulation of MAL localisation involves the N- and C-termini of the protein.

Having identified regions sufficient involved in regulated relocalisation of MAL we investigated those sequences which mediate required for relocalisation. We first focussed on the conserved basic boxes since similar sequences are frequently associated with nuclear localisation signals, and because small MAL derivatives containing them are localised exclusively in the nucleus. Deletion of box Bl appeared to decrease the efficiency with which MAL redistributed to the nucleus; in contrast, deletion of box B2 had no effect. Since the presence of box Bl is not required for regulated redistribution of MAL, we tested whether other conserved sequences in the region between residues 170 and 471 affect MAL localisation. Deletion of the SAP domain, LZL or other conserved elements in the C-terminus of the protein had no effect on regulation. Strikingly, deletion of the Q box resulted in nuclear accumulation of the protein but a deletion of both the Bl and Q boxes restored regulation. These data are consistent with a model in which the Bl and Q boxes are respectively involved in nuclear import and export of MAL, but their function dependent on the presence of the N- and C-terminal regions.

Removal of the basic region within the MAL domain, the B2 box, did not affect regulation of MAL. However, deletion of both the Bl and B2 boxes generated a derivative which exhibited exclusively cytoplasmic localisation, in contrast to the Bl box deletion mutant. A mutant lacking Bl, B2 and Q was also exclusively cytoplasmic.

Taken together the above data are consistent with the view that the B 1 and B2 boxes represent nuclear import signals, while the Q box contains a nuclear export signal. These signals are subject to regulation by the Rho-actin pathway via a mechanism involving the N- and C-terminal sequences of MAL, for which the integrity of the RPEL motifs is required. We addressed this model in two ways. We first tested whether deletion of the MAL domain (including box B2 and the RPEL motifs) can induce signal-independent nuclear localisation of a MAL, derivative lacking the Bl and Q boxes. Consistent with the model, this mutant remained cytoplasmically localised upon serum stimulation. Secondly, we asked whether the nuclear localisation of any MAL mutants required basal Rho signalling by co-expressing them with C3 transferase or inactivate Rho. This experiment showed that ΔQ nuclear localisation required functional Rho, whereas ΔN nuclear localisation was independent of Rho. These results are consistent with the notion that the ΔQ mutation affects a constitutive nuclear import / function and not regulation itself.

We tested whether MC could exhibit similar relocalisation in response to external signals. Upon expression in NIH3T3 cells, MC was exclusively nuclear localised, and in the reporter assay it exhibited efficient Rho- independent activation of the SRF reporter even at low doses. Since the MC MAL domain contains point changes within the RPEL motif required for MAL cytoplasmic retention, we tested the effect of exchanging the two MAL domains. Strikingly, substitution of the MAL domain of MAL with that of MC generated a derivative which was constitutively nuclear-localised, while substitution of the MC MAL domain with that of MC generated a derivative which exhibited signal-dependent regulation. Conesponding effects were seen in the SRF reporter assay. These results demonstrate that the structure of the MAL domain is crucial for regulation, and that the C-terminal domains of both MC and MAL can mediate regulation.

Example VI: MAL associates with unpolymerised actin We tested whether MAL is an actin-binding protein using co- immunoprecipitation. Epitope-tagged MAL or derivatives were co-expressed with either wild type β-actin or its different mutants and actin immunoprecipitates analysed for the presence of coprecipitated MAL proteins. In this assay, MAL was specifically recovered in immunoprecipitates of extracts expressing wild type actin but only at background levels in cells transfected with vector alone. MAL was recovered in extracts from cells expressing the nonpolymerisable actins G13R and R62D, and from the stabilising mutant G15S, but was not recovered in immunoprecipitates of the stabilising actin mutants V159N and S14C (Figure 12).

These data indicate that MAL interacts specifically with actin. It can interact with unpolymerised actin, consistent with the immunofluorescence data, and its interaction is affected by mutations which alter the polymerisation properties of actin. To study which MAL sequences are required for these interactions, we examined the behaviour of MAL mutants. Deletion of the MAL domain, or point mutations in either RPEL motif, specifically abolished interaction of MAL within wild type actin, actin R62D, and actin G15S (Figure 12).

To conoborate these data we tested the ability of actin to interact with MAL in vivo by targeting actin to different locations within the cell. A membrane targeting sequence derived from GAP-43 protein (MLCCMRRTKQV; SEQ ID No: 19) was added to the unpolymerisable R62D mutant. In immunofluorescence assays many cells expressing the membrane-tagged R62D mutant exhibited strong perinuclear staining presumably at the Golgi; these cells also showed perinuclear concentration of MAL at the Golgi, consistent with physical interaction between the proteins (Figure 13). In a second approach, we added an NLS signal to actin, which promotes its nuclear accumulation but still inhibits SRF activation (Posern et al, 2002). Cells expressing this protein showed strong accumulation of nuclear actin, and also displayed nuclear MAL (Figure 13).

To gain further insight into the interaction between MAL and actin, we tested the ability of the nonpolymerisable actin mutant R62D to inhibit signal- induced nuclear translocation of MAL mutants. R62D expression was unable to retain either MALΔN in the cytoplasm; in contrast MALΔQ was relocalised to the cytoplasm in the presence of R62D (Figure 14). These data are again consistent with the model that the MAL domain mediates both regulation by Rho and interaction with actin.

Taken together these data show that MAL interacts with actin in vivo; that unpolymerisable actin can interact with MAL; and that at least one activating actin mutant retains the ability to bind MAL. We propose that interaction of actin with MAL somehow either masks nuclear localisation signals in the protein or anchors the protein in the cytoplasm, and suggest that the activating G15S mutant, while able to bind actin, does so in a manner incompatible with NLS masking or cytoplasmic anchoring.

Example VII: Signal-induced phosphorylation of MAL

Upon serum stimulation, both endogenous and transfected MAL proteins undergo a change in mobility on SDS-PAGE analysis; phosphatase treatment indicates that this modification is due to serine / threonine phosphorylation (Figure 15 A). Phosphorylation takes place in the C-terminal sequences of the protein required for regulation (Figure 15B). Phosphorylation can be induced by expression of activated forms of actin remodelling proteins such as mDial, and analysis of mDial mutants indicates that only those mutants competent to induce actin assembly induce phosphorylation (Figure 15C) mDia-induced phosphorylation is inhibited by actin R62D, suggesting that it requires alterations in actin dynamics (Figure 15D). MAL is phosphorylated in response to all signals which induce changes in actin dynamics, suggesting that phosphorylation may be essential for activation in response to signals.

Example VIII: Inhibition of SRF-mediated gene activation through MAL mutants

Evaluation of inactive MAL mutants for their ability to inhibit signalling to SRF showed that Mai 1-471 could abrogate induction of SRF reporter activity in response to serum and signalling molecules involved in the control of actin dynamics (for example RhoA, mDial, LiMK, VASP) (Figure 16A). Mai 1- 471 could also reduce SRF reporter activity by drugs that interfere with the actin treadmilling cycle (cytochalasin D and jasplakinolide) and by actin mutants that promote increased stability of the actin filaments.

Band-shift assays indicated that Mai 1-471 can strongly bind to SRF (Figure 16B), indicating that this Mai mutant most likely exerts its effects by competing with endogenous Mai binding to SRF.

In addition, we have shown that the cytoplasmically restricted MAL mutant ΔB1ΔB2 acts as a dominant interfering mutant inhibiting SRF-mediated gene activation (Figure 17A). Mutants which lack the Bl box do not form a complex with SRF (Figure 4) and ΔB1ΔB2 does not enter the nucleus upon stimulation (Figure 11). Instead MAL ΔB1ΔB2 acts by complexing the wild- type MAL and preventing it from nuclear entry in a manner dependent on the integrity of the LZL motif (Figure 17B). As expected, the deletion of the LZL motif from ΔB1ΔB2 generated a protein (MAL ΔB1ΔB2ΔLZL) which is not able to dimerise with the wild-type MAL and therefore had no effect on translocation of wild-type MAL. As a result, MAL ΔB1ΔB2ΔLZL did not interfere with signal-induced SRF reporter activation (Figure 17). The generation of these dominant-negative mutants demonstrates that interference of MAL activity through the disruption of SRF-MAL interaction, MAL shuttling or MAL dimerisation results in the modulation of SRF- mediated gene activation.

Materials and Methods for Examples I to VIII

Plasmids

Mouse mMAL was PCR-amplified from a NIH 3T3 cDNA library (Clontech) and cloned into derivatives of EFplink canying N-terminal Flag, myc or HA epitope tags (Sotiropoulos et al, 1999). Deletion and point mutants of MAL were generated by standard procedures. Mouse myocardin was amplified from reverse-transcribed mouse heart RNA. Expression plasmids encoding either wild type or mutant forms of mDial, VASP, profilin, WASP, N-WASP, B-actin, C3 -transferase, RhoV14 and Cdc42Hs have been described previously (Copeland, 2002, Stotiropoulos, 1999, Grosse, 2003, Posern, 2002, Hill et α/.,1994 and Hill et α/.,1993). The following plasmids are as described MLV- LacZ (Sotiropoulos et al, 1999); 3D.Aluc(Geneste, 2002), MLV.SRF, MLV.SRF-M2, MLV.SRF (1-265), MLV.SRF-M2 (1-265), MLV.SRF .VP16, MLV.SRF-M2.VP16 (Hill et α/.,1994 and Hill et al.,\993), Gal-ElkΔ33, Gal- ElkY59A and ASIF/ATCF (Murai, 2002). The c-fos promoter mutants SRE.L and SRE.LM are derivatives of pF711 and were described in Hill et al, 1994 and Hill et α/.,1993. The reporter plasmids SRE.L2.1uc and SRE.LM2.luc were derived from SRE.L2-TKCAT and SRE.LM2-TKCAT (Hill et al, 1994; Hill et al, 1993).

Transfections .

NIH3T3 cell were transfected using Lipofectamine (Invitrogen). For luciferase assays, cells were transfected with 40 ng 3DA.Luc, 150 ng reference plasmid MLV-LacZ, and expression plasmids as in the Figure Legends, and empty EFplink plasmid to make up a total of 1 μg DNA per 6 well dish. When indicated, 50ng of SRE.L2.1uc or SRE.LM2.luc reporter genes were used (Hill et al, 1994; Hill et α .,1993). For activation experiments the transfected cells were maintained in 0.5% FCS and harvested 24hr later for standard luciferase assay (Promega), with transfection efficiency standardised by β-galactosidase assay. Data were expressed relative to reporter activation by the constitutively active SRF derivative SRFVP16 (50 ng), included in every set of transfections. For interference assays stimulation was 40hr following transfection. For immunofluorescence experiments 50ng of Flag- tagged and HA-tagged-MAL expression vectors were transfected. alone or in combination with expression vectors (100 to 200 ng for activating molecules and 200 to 1000 ng for interfering mutants). When indicated, cells were treated with cytochalasin D (Calbiochem), jasplakinolide (Molecular Probes) and swinholide A (Alexis) for 1 hour; or pretreated with latrunculin B (Calbiochem) or toxin B (Gineitis, 2002) for 1 hour before serum stimulation.

Antibodies

To generate antibodies against mouse MAL, the N-terminal residues 1-170 were fused to glutathione S-transferase and expressed in Bal21 bacterial strain. Recombinant protein was purified using glutathione Sepharose beads (Pharmacia) according to the manufacturer's instructions and was used for rabbit immunisation. Antibodies from rabbit antisera were affinity purified on protein A (Cancer Research UK) and, subsequently, anti-GST reactivity was removed by incubation with recombinant GST protein.

Gel Mobility Shift Assays

Cells were transfected as described above using lμg of mMAL or SRF expression plasmid and l μg MLV.plink to make up to a total of 2μg DNA/6cm dish. Cells were kept in 0.5% serum for 24 hours and whole cell extracts were prepared as described in Murai and Treisman, 2002, after 30min. of serum stimulation. Probes were generated by PCR as described in Murai and Treisman, 2002. Assays were performed as described in Murai and Treisman, 2002, using lμg of cell extract and lng/μl c-fos promoter mutant probe. Peptides were added to binding reactions as specified in the figure legends.

Immunofluorescence

NIH3T3 cells were transfected as above, fixed in 4% formaldehyde/PBS, and permeabilised in 0.2% Triton-X-100/PBS. Immunofluorescence staining was as described (Sotiropoulos et al, 1999; Tran Quang et al, 2000). Primary antibodies were rabbit anti-Flag (Sigma), and anti-12CA5 (Cancer Research

UK and Roche), at 1/100 to 1/300 dilution. Affinity purified rabbit anti-Mai serum was used at 1/100. Secondary FITC- and TRITC-anti-mouse and anti- rabbit antibodies (DAKO, Sigma T2659, Molecular Probes) were used according to the manufacturer's recommendations. FITC- or TRITC-labelled phalloidin (Molecular Probes) was used at 33-66nM. Images were generated using an Zeiss Axioplan II microscope with Plan-Neofluar 63x objective and appropriate filters, with a Quantix CCD camera (Photometries) and SmartCapture 2 software (Applied Imaging).

Immunoprecipitation and Western Blotting.

For protein mobility shift assays, cells were transfected as described above. Following 18h incubation in 0.5%FBS, serum was added for one hour. Cells were lysed directly onto the plates with lx SDS/PAGE sample buffer, sonicated and run on a 7% Laemli gel. For immunoprecipitation and dephosphorylation assays, NIH3T3 cells (3.0 x 10⁵ cells per 60 mm dish) were transfected and maintained for 24 h in DMEM / 0.5% FCS until lysed for 10 min in 1 ml of ice-cold lysis buffer per dish (20 mM Tris-HCl, pH 7.5, 160 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM NaF, 1 μM Na₃V0₄, 1% Triton X-100, 10% glycerol, lOmM B- glycerolphosphate, 1 mM phenylmethylsulfonyl fluoride, 1 μM leupeptin, and 0.1 μM aprotinin). Lysates were sonicated briefly and precleared by centrifugation for 10 min at 13000 rpm and subsequently incubated with anti- HA 12CA5 and protein A beads at 4°C for 120min. Precipitates were collected by pulse centrifugation for lOsec, washed four times in 1 ml cold lysis buffer. Following the last wash, the beads were split into two equal parts and incubated with λphosphatase buffer with or without enzyme (New England Biolabs) for 3 hours at 30°C. After three washes in immunoprecipitation buffer, the beads were resuspended in SDS/PAGE sample buffer. Immunoprecipitates were first probed with and anti- phosphotyrosine monoclonal antibody (Transduction laboratories) and subsequently reprobed with 12CA5 antibody.

Coimmunoprecipitation of Actin-MAL complexes was performed as above, except that transfection was performed in 10cm dishes, the cells were lysed in RIPA buffer and immunoprecipitation was carried out using anti-M2 agarose- conjugated beads. The beads were resuspended in lxSDS/PAGE buffer and run on a 10% gel.

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Claims

1. An agent that modulates a MAL activity.

2. An agent according to Claim 1 that modulates an interaction of MAL with SRF.

3. An agent according to Claim 2 that inhibits an interaction of MAL with SRF.

4. An agent according to Claim 3 comprising an anti-MAL antibody that inhibits SRF binding to MAL; an anti-SRF antibody that inhibits MAL binding to SRF; a MAL fragment or mutant that binds to SRF; or an SRF fragment or mutant that binds to MAL.

5. An agent according to any of Claims 1 to 4 that selectively binds to the Bl domain and/or the Q domain of MAL.

6. An agent according to any of Claims 1 to 4 that selectively binds to the DNA binding domain of SRF, preferably to the hydrophobic pocket in the DNA binding domain .

7. A MAL mutant according to Claim 4 comprising MAL 1 -471.

8. An SRF fragment according to Claim 4 or 5 consisting of or comprising the DNA binding domain of SRF.

9. A MAL fragment according to Claim 4 or 6 consisting of or comprising the Bl domain of MAL, the Q domain of MAL, or both the Bl and Q domains of MAL.

10. An agent according to Claim 1 that modulates translocation of MAL to and/or from the nucleus.

11. An agent according to Claim 10 that inhibits translocation of MAL to the nucleus.

12. An agent according to Claim 11 that selectively binds to the Bl domain or to both the Bl and the B2 domains of MAL.

13. An agent according to Claim 11 that selectively binds to the LZL domain of MAL.

14. An agent according to Claim 11 comprising MAL ΔB1ΔB2.

15. An agent according to Claim 10 that inhibits translocation of MAL from the nucleus.

16. An agent according to Claim 15 that selectively binds to the Q domain of MAL.

17. An agent according to Claim 1 that modulates C-terminal phosphorylation of MAL.

18. An agent according to Claim 17 that inhibits C-terminal phosphorylation of MAL.

19. An agent according to Claim 18 that binds to at least one dephosphorylated C-terminal serine or threonine MAL residue, thus inhibiting phosphorylation of the at least one dephosphorylated C-terminal serine or threonine MAL residue.

20. An agent according to Claim 17 that inhibits C-terminal dephosphorylation of MAL.

21. An agent according to Claim 20 that binds to at least one phosphorylated C-terminal serine or threonine MAL residue, thus inhibiting dephosphorylation of the at least one phosphorylated C-terminal serine or threonine MAL residue.

22. An agent according to Claim 1 that modulates an interaction of actin with MAL.

23. An agent according to Claim 22 that inhibits an interaction of actin with MAL.

24. An agent according to Claim 23 wherein the agent comprises an anti- MAL antibody that inhibits actin binding to MAL; an anti-actin antibody that inhibits MAL binding to actin; a MAL fragment that binds to actin; or an actin fragment that binds to MAL.

25. An agent according to Claim 23 or 24 that selectively binds to one or both of the RPEL domains of MAL.

26. A MAL fragment according to Claim 23 or 24 comprising or consisting of one or both of the RPEL domains of MAL.

27. An agent according to Claim 22 that stimulates an interaction of actin with MAL.

28. An agent according to any of Claims 5, 6, 12, 13, 16, 19, 21 and 25 wherein the agent is an antibody.

29. An agent according to Claim 1 that modulates MAL gene expression.

30. An agent according to Claim 29 that stimulates MAL gene expression.

31. An agent according to Claim 29 that inhibits MAL gene expression.

32. An agent according to Claim 31 wherein the agent comprises a antisense RNA, a small interfering RNA, or an engineered transcription repressor that inhibits MAL gene transcription.

33. A polynucleotide encoding an agent according to any one of Claims 4 to 9, 12 to 14, 16, 19, 21, 24 to 26, 28 or 32.

34. A genetic construct comprising the RNA of Claim 32 or the polynucleotide according to Claim 33.

35. A host cell comprising the genetic construct of Claim 34.

36. A pharmaceutical preparation comprising an agent which modulates a MAL activity and a pharmaceutically acceptable carrier.

37. A pharmaceutical preparation according to Claim 36 wherein the agent is as defined in any one of Claims 2 to 32.

38. A pharmaceutical preparation comprising a polynucleotide encoding an agent which modulates a MAL activity and a pharmaceutically acceptable carrier.

39. A pharmaceutical preparation according to Claim 38 wherein the polynucleotide is as defined in Claim 33.

40. A method of combating a disorder in an individual, the method comprising modulating a MAL activity in the individual.

41. A method according to Claim 40 wherein the disorder is selected from cancer, such as leukaemia, particularly the childhood leukaemia AML-M7; wounds; myopathies such as muscle hypertrophy; and any disorder that would benefit from enhanced angiogenesis.

42. A method according to Claim 40 or 41 wherein the disorder is cancer, and modulating a MAL activity combats tumour cell growth, adhesion, cellular mobility, invasion or metastasis.

43. A method according to any one of Claims 40 to 42 comprising inhibiting a MAL activity in the individual.

44. A method according to any one of Claims 40 to 42 comprising stimulating a MAL activity in the individual.

45. A method according to any one of Claims 40 to 44 comprising administering an agent that modulates a MAL activity to the individual.

46. A method according to Claim 45 wherein the agent is as defined in any one of Claims 2 to 32.

47. A method according to any one of Claims 40 to 44 comprising administering a polynucleotide that encodes an agent that modulates a MAL activity to the individual.

48. A method according to Claim 47 wherein the polynucleotide is as defined in Claim 33.

49. An agent that modulates a MAL activity for use in medicine.

50. An agent according to Claim 49, wherein the agent is as defined in any one of Claims 2 to 32.

51. A polynucleotide that encodes an agent that modulates a MAL activity for use in medicine.

52. A polynucleotide according to Claim 51, wherein the polynucleotide is as defined in Claim 33.

53. Use of an agent which modulates a MAL activity in the manufacture of a medicament for combating a disorder that would benefit from a modulation of MAL activity.

54. Use according to Claim 53 wherein the agent is as defined in any one of Claims 2 to 32.

55. Use of a polynucleotide that encodes an agent which modulates a MAL activity in the manufacture of a medicament for combating a disorder that would benefit from a modulation of MAL activity.

56. Use according to Claim 55 wherein the polynucleotide is as defined in Claim 33.

57. Use according to any one of Claims 53 to 56 wherein the disorder is selected from cancer, such as leukaemia, particularly the childhood leukaemia AML-M7; wounds; myopathies such as muscle hypertrophy; and any disorder that would benefit from enhanced angiogenesis.

58. Use according to Claim 57 wherein the medicament for combating cancer combats tumour cell growth, adhesion, cellular mobility, invasion or metastasis.

59. A method of screening for an agent that modulates the MAL/SRF interaction, comprising (a) providing a composition comprising SRF and MAL;

(b) providing a test agent; and

(c) assessing the interaction of MAL with SRF in the composition, wherein a change in the interaction of MAL with SRF in the presence of the test agent indicates that the test agent may modulate the MAL/SRF interaction.

60. A method according to Claim 59 wherein the MAL comprises a fragment, variant, derivative or fusion of MAL which binds to SRF.

61. A method according to Claim 59 wherein the SRF comprises a fragment, variant, derivative or fusion of SRF which binds to MAL.

62. A method according to Claim 60 wherein the SRF comprises a fragment, variant, derivative or fusion of SRF which binds to MAL, and wherein the respective SRF and MAL fragments, variants, derivatives or fusions bind together.

63. A method of screening for an agent that modulates the MAL/actin interaction, comprising

(a) providing a composition comprising MAL and actin;

(b) providing a test agent; and

(c) assessing the interaction of MAL with actin in the composition, wherein a change in the interaction of MAL with actin in the presence of the test agent indicates that the test agent may modulate the MAL/actin interaction.

64. A method of screening for an agent that modulates MAL dimerisation, comprising

(a) providing a composition comprising MAL; (b) providing a test agent; and

(c) assessing the dimerisation of MAL in the composition, wherein a change in the level, extent or rate of dimerisation MAL in the presence of the test agent indicates that the test agent may modulate dimerisation of MAL.

65. A method of screening for an agent that modulates the translocation of MAL to and/or from the nucleus, comprising

(a) providing a cell culture comprising MAL; (b) providing a test agent; and

(c) assessing the translocation of MAL to and/or from the nucleus in the cells, wherein a change in the cellular localisation of MAL in the presence of the test agent indicates that the test agent may modulate MAL nuclear translocation.

66. A method of screening for an agent that modulates the phosphorylation of specific C-terminal residue(s) of residues of MAL, comprising (a) providing a composition comprising MAL;

(b) providing a protein kinase;

(c) providing a source of phosphate;

(d) providing a test agent; and

(e) assessing the phosphorylation of specific MAL residue(s), wherein a change in phosphorylation status of the specific MAL residue(s) in the presence of the test agent indicates that the test agent may modulate MAL phosphorylation.

67. A method of screening for an agent that modulates the expression of the MAL gene, comprising (a) providing a cell culture in which at least some cells in the culture comprise a polynucleotide having a MAL regulatory sequence fused to a polynucleotide encoding a detectable product;

(b) providing a test agent; and

(c) assessing the expression of the detectable product in the cells, wherein a change in the expression of the detectable product in the presence of the test agent indicates that the test agent may modulate MAL gene expression.

68. A method according to any one of Claims 59 to 67 wherein an agent identified as a result of the screen is modified and rescreened.

69. A method according to any one of Claims 59 to 68 wherein an agent having or expected to have similar properties to a an agent identified as a result of the screen is screened.

70. A method according to any one of Claims 59 to 69 wherein an agent identified as a result of the screening is tested for efficacy in a cell model of a disorder selected from cancer, such as leukaemia, particularly the childhood leukaemia AML-M7; wounds; myopathies such as muscle hypertrophy; and any disorder that would benefit from enhanced angiogenesis.

71. A method according to any one of Claims 59 to 70 wherein an agent identified as a result of the screening and/or testing is further tested for efficacy in an animal model of a disorder selected from cancer, such as leukaemia, particularly the childhood leukaemia AML-M7; wounds; myopathies such as muscle hypertrophy; and any disorder that would benefit from enhanced angiogenesis.

72. A method according to any one of Claims 59 to 71 wherein an agent identified as a result of the screening and/or testing is further tested for efficacy and safety in a clinical trial for a disorder selected from cancer, such as leukaemia, particularly the childhood leukaemia AML-M7; wounds; myopathies such as muscle hypertrophy; and any disorder that would benefit from enhanced angiogenesis.

73. A method according to any one of Claims 59 to 72 wherein an agent identified as a result of the screening and/or testing is packaged and presented for use in medicine.

74. MAL ΔB1ΔB2.

75. MAL 1-471.

76. An agent that selectively binds to a RPEL motif of MAL.

77. An agent according to Claim 76 that selectively binds to the amino acid sequence DYLKRKIRSRPERSELVRMHILEETS (SEQ ID No: 3) or DDLNEKIAQRPGPMELVEKNILPVES (SEQ ID No: 5).

78. An agent that selectively binds to a Bl box domain of MAL.

79. An agent according to Claim 78 that selectively binds to the amino acid sequence KKAKELKPKVKKLKYHQYIPPDQKQDR (SEQ ID No: 20).

80. An agent that selectively binds to a B2 box domain of MAL.

81. An agent according to Claim 80 that selectively binds to the amino acid sequence KQLKLKRAR (SEQ ID No: 21).

82. An agent that selectively binds to a Q box domain of MAL.

83. An agent according to Claim 82 that selectively binds to the amino acid sequence QQQQLFLQLQILNQQQQQ (SEQ ID No: 22).

84. An agent that selectively binds to a SAP domain of MAL.

85. An agent according to Claim 84 that selectively binds to the amino acid sequence LDDMKVAELKQELKLRSLPVSGTKTELIERLRAYQ (SEQ ID No: 23).

86. An agent that binds to a selectively LZL domain of MAL.

87. An agent according to Claim 86 that selectively binds to the amino acid sequence LEGRDKDQMLQEKDKQIEALTRMLRQKQQLVERLKLQLEQE (SEQ ID

No: 24).

88 . An agent according to any one of Claim 76 to 87 wherein the agent is an antibody.

89. Use of an agent according to any of Claims 2 to 32, or a polynucleotide according to Claim 33, or a genetic construct according to Claim 34, for modulating an activity of MAL in vitro.