Patent Description:
The evolutionarily conserved Hippo pathway plays a key role in tissue homeostasis and organ size control by regulating cell survival, proliferation and differentiation (<NPL>)). This inhibitory pathway is comprised of a core kinase cascade, in which the mammalian sterile <NUM>-like kinases MST1/<NUM> and SAV1 form a complex that phosphorylates and activates the large tumor suppressor kinases LATS1/<NUM>, which in turn phosphorylate and inhibit the activities of the transcriptional co-activators YAP and TAZ (<NPL>); <NPL>)). When liberated from Hippo pathway inhibition, YAP and TAZ accumulate in the nucleus where they drive gene expression by binding to TEAD, the DNA binding transcription factor regulated by the Hippo pathway as well as possibly other transcription factors, to promote cell proliferation and inhibit apoptosis (<NPL>);<NPL>); <NPL>); <NPL>)). In fact, persistent nuclear localization of YAP and/or TAZ due to genetic alterations in the Hippo pathway has been linked mechanistically to oncogenesis (<NPL>); <NPL>)).

The Hippo pathway can be modulated by a variety of stimuli, including G protein-coupled receptor (GPCR) signaling (<NPL>)), actin cytoskeleton changes, cell-cell contact, and cell polarity (<NPL>);<NPL>)). Various tumors have been shown to exhibit loss of function of LATS2 (<NPL>)) or NF2 (<NPL>)), whose functions enforce Hippo negative regulation, or YAP amplification/overexpression (<NPL>); <NPL>); <NPL>)). However, these alterations are relatively infrequent compared to aberrations afflicting oncogenes such as Ras or Raf or tumor suppressors such as p53 (<NPL>); <NPL>); <NPL>); <NPL>)). Nonetheless, increasing interest in Hippo deregulation as an oncogenic driver has led to increased efforts to identify new activating mechanisms, most recently Gq11 activating mutations that up-regulate TEAD/YAP transcription in ocular melanomas (<NPL>); <NPL>)).

In efforts to identify new mechanisms of Hippo deregulation in human tumors, a large panel of human tumor lines was surveyed for activated TEAD/YAP transcription. By searching genomic data bases to identify alterations that might account for the high levels of TEAD activity detected in some, it was noted that a number contained p53 missense mutations, which result in a high level of expression of p53 protein unable to exert normal p53 tumor suppressor functions (<NPL>)). The dominant negative potential of mutant p53 when heterozygous with the wild-type allele has been proposed as an underlying basis for the high preponderance of p53 missense mutations in tumors (<NPL>); <NPL>); <NPL>);<NPL>); <NPL>)). In fact, in Li-Fraumeni patients, germline missense mutations in TP53 consistently show an association with an earlier age of onset when compared with germline deletions. Moreover, mouse genetic models have revealed that some hotspot missense mutations generated as knock-in alleles produce an altered tumor spectrum and/or more metastatic tumors as compared to the loss of one or both wild type p53 alleles.

p53 missense mutants have also been reported to induce various biological effects (<NPL>)). Such phenotypes are generally referred to as gain of function ("GOF"), although it is unclear whether all GOF mutant p53 share the same properties or how many specific GOF mechanisms may exist. Thus, increased understanding of mechanisms by which p53 missense mutations may acquire GOF could be important for prognosis and conceivably for therapy given that p53 missense mutations occur so frequently and in diverse tumor types (<NPL>)). Recently, one research group reported, based on expression array analysis, the upregulation of mevalonate pathway genes by the p53 mutant R273H (<NPL>)). Subsequent evidence indicated that the mutant p53 R280K was able to regulate YAP activity through modulation of the mevalonate pathway in MDA-MB-<NUM> cells, which exhibit NF2 loss of function and constitutive activation of Yap (<NPL>)). To illustrate the lack of clarity in mechanistic understanding of p53 mutant gain of function, these same p53 DNA contact mutants were more recently reported to cooperate with members of the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells (<NPL>)). <NPL>describes the effect of various p53 mutations on the Warburg effect.

The present invention is directed to overcoming deficiencies in the art.

Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy or diagnosis.

The invention is defined in the claims, and provides a Rho-associated protein kinase ("ROCK") inhibitor for use in a method of treating a tumor in a subject, said method comprising: administering to a subject having a tumor comprising a p53 DNA contact mutation a ROCK, wherein the ROCK inhibitor is capable of inhibiting TEAD/YAP dependent transcription and treats the tumor in the subject.

The invention also provides Rho-associated protein kinase ("ROCK") inhibitor for use in a method of treating cancer in a subject, said method comprising:
administering to a subject having a cancer comprising a p53 DNA contact mutation a ROCK inhibitor, wherein the ROCK inhibitor is capable of inhibiting TEAD/YAP dependent transcription and treats the subject for cancer.

Also disclosed but not claimed is a method of identifying a subject as a candidate for treatment. This method involves obtaining a sample from a tumor in a subject and determining the presence of a p53 DNA contact mutation in the sample. The presence of a p53 DNA contact mutation in the sample indicates the tumor is susceptible to targeted treatment with a ROCK inhibitor and the subject is a candidate for treatment.

The present invention establishes that human tumors containing p53 missense mutations affecting amino acids that directly interact with DNA but not those which impair DNA binding through altered conformation of the DNA binding domain or X mutants which encode truncated, unstable p53 proteins show constitutive activation of TEAD/YAP-dependent transcription, which functions as an oncogenic driver. It is shown herein that genetic manipulations, which downregulate either p53 or TEAD/YAP transcription markedly and specifically inhibit proliferation of such tumor cells. Moreover it is demonstrated that inhibitors of ROCK, which act downstream of RhoA to mediate its signaling, phenocopy these effects. The exquisite specificity of these inhibitors for tumor cells bearing p53 DNA contact mutations strongly support the utility of ROCK inhibitors in therapeutically targeting these tumors.

<FIG> illustrate that p53 DNA-contact mutants identify a new class of Hippo deregulated tumors. <FIG> shows a comparison of TEAD4 reporter activity in human tumor lines expressing different p53 mutations, and <FIG> shows mRNA expression of TEAD/YAP target genes (CTGF, CYR61, and ANKRD1) in human tumor lines expressing different p53 mutations. 293T and H2052 cells were used as a negative and positive control, respectively.

<FIG> show p53 knock-down phenocopies TEAD4 inhibition and blocks TEAD/YAP-dependent transcription and proliferation in vitro and in vivo of p53 DNA-contact mutant-containing tumor lines. <FIG> show analysis of TEAD4 reporter activity (<FIG>), CTGF mRNA expression (<FIG>), and proliferation in vitro in the indicated tumor lines upon either overexpression of DN TEAD4 or knock-down of p53 (<FIG> shows the effects of lentiviral transduction of DN TEAD4 or p53 knock-down on the tumor formation in vivo by MDA-MB-<NUM> cells expressing a p53 DNA-contact mutation R280K. <NUM> million cells were inoculated orthotopically into the fat pads of the fifth mammary glands of <NUM>-week-old immunocompromised female NOD/SCID mice. Time-course of tumor growth is shown on the left, tumor size at the time of sacrifice (<NUM> months) is shown on the right.

<FIG> show that activation of TEAD/YAP-dependent transcription is essential for transformed phenotype induced by p53 DNA-contact mutants in MCF10A cells. <FIG> show analysis of TEAD4 reporter activity (<FIG>), mRNA expression of TEAD/YAP target genes (CTGF, CYR61, ANKRD1) (<FIG>), and anchorage-independent growth in soft agar of immortalized MCF10A cells exogenously expressing by lentiviral transduction either YAP WT, p53 R248Q, p53 R273H, p53 R175H, or p53 G245S (<FIG> show analysis of TEAD4 reporter activity (<FIG>), mRNA expression of TEAD/YAP target genes (CTGF, CYR61, ANKRD1) (<FIG>), and anchorage-independent growth in soft agar of immortalized MCF10A cells exogenously expressing by lentiviral transduction YAP WT, p53 R273H, or p53 R175H in the presence or in the absence of concurrent DN TEAD4 lentiviral transduction (<FIG>).

<FIG> show that the ROCK inhibitor Y-<NUM> phenocopies p53 knockdown or DNTEAD4 overexpression in specifically antagonizing the TEAD/YAP transformed phenotype. The graphs in <FIG> show analysis of TEAD4 reporter activity in representative tumor lines harboring a p53 DNA-contact mutation. <FIG> shows the effects of treatment with Verteporfin, Simvastatin, or Y-<NUM> on proliferation of in vitro tumor lines with different p53 mutations. Of note, Y-<NUM> specifically inhibited proliferation of Hippo deregulated tumor cells with p53 DNA-contact mutations, whereas Verteporfin and Simvastatin exerted nonspecific effects by inhibiting proliferation also in tumor lines expressing p53 conformational mutations. <FIG> shows the effects treatment with the ROCK inhibitor Y-<NUM> on the colony formation in soft agar of a representative p53 DNA-contact mutant (R273H) or a different p53 mutant (R175H) or HRAS G12V in MCF10A cells. Note the lack of effects of Y-<NUM> treatment on the transformed phenotype induced by either p53 R175H or HRAS G12V.

<FIG> show that ROCK inhibitors are a new class of inhibitors of Hippo pathway deregulated tumors. <FIG> shows analysis of TEAD4 reporter activity in MDA-MB-<NUM>, a representative tumor line harboring a p53 DNA-contact mutation treated with either increasing doses of Y-<NUM>, <NUM> Glycyl-H-<NUM>, or <NUM> Fasudil. Note the lack of effect of <NUM> Fasudil on the TEAD reporter activity. <FIG> shows the effects of treatment with either increasing doses of Y-<NUM>, <NUM> Glycyl-H-<NUM>, or <NUM> Fasudil on proliferation of in vitro tumor lines with different p53 mutations. Note the specific inhibition of proliferation of tumor lines with p53 DNA-contact mutations at a concentration of <NUM>-<NUM>. Of note, Glycyl-H-<NUM> was able to specifically inhibit the proliferation of the same cells at concentrations <NUM>-fold lower than Y-<NUM>, whereas <NUM> Fasudil had no effect on proliferation of either MDA-MB-<NUM> or SK-BR-<NUM> cells, which are representative tumor lines harboring a p53 DNA-contact or conformational mutation, respectively.

<FIG> shows that XAV939 and Y-<NUM> cooperate to specifically target tumor cells harboring p53 DNA-contact mutations. In particular, <FIG> shows that suboptimal concentrations of the tankyrase inhibitor XAV939 and the ROCK inhibitor Y-<NUM> cooperate in specifically inhibiting the proliferation of tumor cells with p53 DNA contact mutations.

<FIG> provides a comparison of the abilities of XAV939 and Y-<NUM> to inhibit proliferation of tumor cells with different lesions that deregulate the Hippo inhibitor pathway and upregulate TEAD dependent transcription. <FIG> shows that neither XAV939 nor Y-<NUM> inhibits proliferation of non-tumorigenic cells or p53 conformational mutant tumor cells without lesions in Hippo pathway core components. Further, ROCK inhibitors generally do not inhibit proliferation of tumor cells with lesions in Hippo pathway core components in contrast to XAV939, which inhibits proliferation of such tumor cells with p53 DNA contact mutants.

Disclosed herein are methods of treating a tumor and cancer in a subject by administering a Rho-associated protein kinase (ROCK) inhibitor to a subject with a tumor comprising a p53 DNA contact mutation, and identifying a subject as a candidate for such treatment.

A first aspect of the present invention relates to a Rho-associated protein kinase ("ROCK") inhibitor for use in a method of treating a tumor in a subject, said method comprising:
administering to a subject having a tumor comprising a p53 DNA contact mutation a ROCK, wherein the ROCK inhibitor is capable of inhibiting TEAD/YAP dependent transcription and treats the tumor in the subject.

Another aspect of the present invention relates to a Rho-associated protein kinase ("ROCK") inhibitor for use in a method of treating cancer in a subject, said method comprising:
administering to a subject having a cancer comprising a p53 DNA contact mutation a ROCK inhibitor, wherein the ROCK inhibitor is capable of inhibiting TEAD/YAP dependent transcription and treats the subject for cancer.

In accordance with all aspects of the present invention, a "subject" encompasses any animal, but preferably a mammal, e.g., human, non-human primate, a dog, a cat, a horse, a cow, or a rodent. More preferably, the subject is a human.

As used herein, a "tumor" is any kind of new growth, benign or malignant. The term "cancer", as used herein, refers to a form of a tumor, namely malignant.

Cancers and tumors to be treated according to the methods of the present invention include, without limitation, carcinoma of the bladder, breast, colon, kidney, liver, lung, head and neck, gall-bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, or skin; a hematopoietic tumor of lymphoid lineage (i.e., leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma); a hematopoietic tumor of myeloid lineage (i.e., acute myelogenous leukemia, chronic myelogenous leukemia, multiple myelogenous leukemia, myelodysplastic syndrome, and promyelocytic leukemia); a tumor of mesenchymal origin (i.e., fibrosarcoma and rhabdomyosarcoma); a tumor of the central or peripheral nervous system (i.e., astrocytoma, neuroblastoma, glioma, and schwannomas); melanoma; seminoma; teratocarcinoma; osteosarcoma; thyroid follicular cancer; Kaposi's sarcoma; hepatoma; and mesothelioma.

As used herein, a "p53 DNA contact mutation" is a p53 mutation that affects amino acids that directly interact with DNA, but that does not impair DNA binding through altered conformation of the DNA binding domain or encode a truncated, unstable p53 protein. Mutations may include an insertion, a truncation, a deletion, a nonsense mutation, a frameshift mutation, a splice-site mutation, or a missense mutation.

In one embodiment of this and all other aspects of the present invention, the mutation comprises a non-synonymous single nucleotide base substitution. Such mutations can occur in the coding region of a p53 nucleic acid sequence, more particularly in any of the identified domains involved in contact with DNA. However, the present invention also encompasses mutations in p53 other than those specifically identified below. These mutations may be in coding or non-coding regions of p53.

P53 comprises the nucleotide sequence of SEQ ID NO: <NUM> as follows:
<IMG>
<IMG>.

The amino acid sequence of p53 (SEQ ID NO:<NUM>) is as follows:
<IMG>.

The p53 DNA contact mutation can encode an amino acid substitution at one or more amino acid residues corresponding to amino acid positions <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and/or <NUM> of SEQ ID NO: <NUM>. Exemplary mutations in the nucleotide sequence encoding these amino acid substitutions include, without limitation, those that result in a lysine (K) to glutamic acid (E) substitution at an amino acid position corresponding to position <NUM> (K120E) of SEQ ID NO: <NUM>; a lysine (K) to asparagine (N) substitution at an amino acid position corresponding to position <NUM> (K120N) of SEQ ID NO: <NUM>; a serine (S) to phenylalanine (F) substitution at an amino acid position corresponding to position <NUM> (S241F) of SEQ ID NO: <NUM>; a serine (S) to cysteine (C) substitution at an amino acid position corresponding to position <NUM> (S241C) of SEQ ID NO: <NUM>; a serine (S) to tyrosine (Y) substitution at an amino acid position corresponding to position <NUM> (S241Y) of SEQ ID NO: <NUM>; a serine (S) to proline (P) substitution at an amino acid position corresponding to position <NUM> (S241P) of SEQ ID NO: <NUM>; an arginine (R) to glutamine (Q) substitution at an amino acid position corresponding to position <NUM> (R248Q) of SEQ ID NO: <NUM>; an arginine (R) to tryptophan (W) substitution at an amino acid position corresponding to position <NUM> (R248W) of SEQ ID NO: <NUM>; an arginine (R) to leucine (L) substitution at an amino acid position corresponding to position <NUM> (R248L) of SEQ ID NO: <NUM>; an arginine (R) to proline (P) substitution at an amino acid position corresponding to position <NUM> (R248P) of SEQ ID NO: <NUM>; an arginine (R) to glycine (G) substitution at an amino acid position corresponding to position <NUM> (R248G) of SEQ ID NO: <NUM>; an arginine (R) to cysteine (C) substitution at an amino acid position corresponding to position <NUM> (R273C) of SEQ ID NO: <NUM>; an arginine (R) to histidine (H) substitution at an amino acid position corresponding to position <NUM> (R273H) of SEQ ID NO: <NUM>; an arginine (R) to leucine (L) substitution at an amino acid position corresponding to position <NUM> (R273L) of SEQ ID NO: <NUM>; an arginine (R) to proline (P) substitution at an amino acid position corresponding to position <NUM> (R273P) of SEQ ID NO: <NUM>; an arginine (R) to serine (S) substitution at an amino acid position corresponding to position <NUM> (R273S) of SEQ ID NO: <NUM>; an arginine (R) to tyrosine (Y) substitution at an amino acid position corresponding to position <NUM> (R273Y) of SEQ ID NO: <NUM>; an alanine (A) to proline (P) substitution at an amino acid position corresponding to position <NUM> (A276P) of SEQ ID NO: <NUM>; an alanine (A) to aspartic acid (D) substitution at an amino acid position corresponding to position <NUM> (A276D) of SEQ ID NO: <NUM>; an alanine (A) to glycine (G) substitution at an amino acid position corresponding to position <NUM> (A276G) of SEQ ID NO: <NUM>; an alanine (A) to valine (V) substitution at an amino acid position corresponding to position <NUM> (A276V) of SEQ ID NO: <NUM>; a cysteine (C) to phenylalanine (F) substitution at an amino acid position corresponding to position <NUM> (C277F) of SEQ ID NO: <NUM>; an arginine (R) to threonine (T) substitution at an amino acid position corresponding to position <NUM> (R280T) of SEQ ID NO: <NUM>; an arginine (R) to lysine (K) substitution at an amino acid position corresponding to position <NUM> (R280K) of SEQ ID NO: <NUM>; an arginine (R) to glycine (G) substitution at an amino acid position corresponding to position <NUM> (R280G) of SEQ ID NO: <NUM>; an arginine (R) to isoleucine (I) substitution at an amino acid position corresponding to position <NUM> (R280I) of SEQ ID NO: <NUM>; an arginine (R) to serine (S) substitution at an amino acid position corresponding to position <NUM> (R280S) of SEQ ID NO: <NUM>; and an arginine (R) proline (P) substitution at an amino acid position corresponding to position <NUM> (R283P) of SEQ ID NO.

In some embodiments of the present invention, the p53 DNA contact mutation is selected from the group consisting of R280K, R273H, and R248Q.

Rho family of small GTPases is a class of small G-proteins which play a critical role in signaling pathways and control organelle development, cytoskeletal dynamics, cell growth and division, cell movement, and other cellular functions. Rho must be located at the interior of the plasma membrane and is translocated by attachment of the C-<NUM> geranyl group to a C-terminal. The GTP bound form of Rho is "switched on" and interacts with a variety of downstream effectors, including the Rho-associated protein kinases (ROCKs) (<NPL>); <NPL>)).

Two ROCK isoforms have been identified in the art and include ROCK1 and ROCK2. Both ROCK1 and ROCK2 are serine/threonine protein kinases that are activated by the GTP-bound form of RhoA. Activation of ROCKs results in phosphorylation of substrates involved in cell signaling. ROCK signaling pathways are implicated in cell morphology, motility, smooth muscle contraction, formation of stress fiber, focal adhesion, cell transformation, and cytokinesis. Based on the broad involvement of the ROCK signaling pathway in a variety of cellular functions, ROCK inhibitors have been under investigation for treating many diseases, including diabetes, neurodegenerative diseases such as Parkinson's disease, cardiovascular diseases such as pulmonary hypertension, inflammation, and glaucoma. In addition, recent findings have shown that ROCK inhibitors can be used to establish 3D-organoid cultures derived from patients with tumors and to grow stem cells in culture (<NPL>);<NPL>); <NPL>)).

ROCK1 comprises the nucleotide sequence (NCBI Reference Sequence: NM_005406) of SEQ ID NO:<NUM> as follows:
<IMG>
<IMG>
<IMG>
<IMG>.

ROCK2 comprises the nucleotide sequence (NCBI Reference Sequence: NM_004850) of SEQ ID NO:<NUM> as follows:
<IMG>
<IMG>
<IMG>
<IMG>.

In another embodiment of the methods of the present invention, a tankyrase inhibitor is also administered. Thus, according to various embodiments of the methods of the present invention, both a ROCK inhibitor that is capable of inhibiting TEAD/YAP dependent transcription and a tankyrase inhibitor are administered to a subject to treat a tumor in a subject or to treat cancer in a subject.

In one embodiment, both the ROCK inhibitor capable of inhibiting TEAD/YAP dependent transcription and the tankyrase inhibitor are combined in a single pharmaceutical formulation. In an alterntive embodiment, the ROCK inhibitor and the tankyrase inhibitor are separately formulated into two different pharmaceutical formulations as described herein and administered in two separate dosages. Thus, according to various embodiments, the ROCK inhibitor and the tankyrase inhibitor may be administered together, separately, or as cotreatments.

According to one embodiment, the ROCK inhibitor capable of inhibiting TEAD/YAP dependent transcription and the tankyrase inhibitor are each administered at a dose sufficient in their combination to treat a tumor in a subject or to treat a subject for cancer, but in a dosage not sufficient (e.g., too low of an amount) for either the ROCK inhibitor or the tankyrase inhibitor alone to treat a tumor in a subject or to treat a subject for cancer. Thus, according this embodiment, the ROCK inhibitor and the tankyrase inhibitor are administered at dosages such that they cause a synergistic treatment effect.

There are two human tankyrases-tankyrase <NUM> and tankyrase <NUM>. Human tankyrase <NUM> has a published nucleotide sequence as set forth in Accession No. NM_003747 (SEQ ID NO:<NUM>), as follows:
<IMG>
<IMG>
<IMG>
<IMG>
<IMG>.

The human tankyrase <NUM> protein encoded by this nucleotide sequence is as follows (SEQ ID NO:<NUM>):
<IMG>.

Human tankyrase <NUM> has a published nucleotide sequence as set forth in Accession No. NM_025235 (SEQ ID NO:<NUM>), as follows:
<IMG>
<IMG>
<IMG>
<IMG>.

The human tankyrase <NUM> protein encoded by this nucleotide sequence is as follows (SEQ ID NO:<NUM>):
<IMG>
<IMG>.

The ROCK inhibitor capable of inhibiting TEAD/YAP dependent transcription and/or the tankyrase inhibitor may include any of the following: nucleic acid inhibitory molecules, inhibitory peptides, antibodies, and small molecules, each of which is described in more detail below. Inhibitors of both ROCK1 and ROCK2 and tankyrase are encompassed in the methods of the present invention.

According to one embodiment, the ROCK inhibitor is a small molecule. Exemplary small molecule ROCK inhibitors include, but are not limited to, Y-<NUM> , Glycyl-H-<NUM>, Thiazovivin, GSK429286, CAY10622, AS1892802, and SR <NUM>. Other small molecule ROCK inhibitors are described in<NPL>).

According to one embodiment, the tankyrase inhibitor is a small molecule. Exemplary small molecule tankyrase inhibitors include, without limitation, XAV939, MN-<NUM>, IWRI, a pyrimidinone nicotinamide mimetic (e.g., AZ-<NUM>) (see<NPL>), and combinations thereof.

According to another embodiment, the ROCK inhibitor and/or the tankyrase inhibitor is an inhibitory molecule (e.g., a nucleic acid inhibitor). Exemplary nucleic acid ROCK inhibitors and tankyrase inhibitors include antisense RNAs or RNAi, such as short interfering RNAs (siRNA), short hairpin RNAs (shRNA), and microRNAs.

The use of antisense methods to inhibit the in vivo translation of genes and subsequent protein expression is well known in the art (see e.g., <CIT>; <CIT>; <CIT>; <CIT>). Antisense nucleic acids are nucleic acid molecules (e.g., molecules containing DNA nucleotides, RNA nucleotides, or modifications (e.g., modification that increase the stability of the molecule, such as <NUM>'-O-alkyl (e.g., methyl) substituted nucleotides) or combinations thereof) that are complementary to, or that hybridize to, at least a portion of a specific nucleic acid molecule, such as an mRNA molecule (see e.g., <NPL>)). The antisense nucleic acid molecule hybridizes to its corresponding target nucleic acid molecule, such as ROCK1, ROCK2, or tankyrase mRNA, to form a double-stranded molecule, which interferes with translation of the mRNA, as the cell will not translate a double-stranded mRNA. Antisense nucleic acids used in the methods of the present invention are typically at least <NUM>-<NUM> nucleotides in length, for example, at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> nucleotides in length. The antisense nucleic acid can also be as long as the target nucleic acid with which it is intended to form an inhibitory duplex. Antisense nucleic acids can be introduced into cells as antisense oligonucleotides, or can be produced in a cell in which a nucleic acid encoding the antisense nucleic acid has been introduced, for example, using gene therapy methods.

siRNAs are double stranded synthetic RNA molecules approximately <NUM>-<NUM> nucleotides in length with short <NUM>-<NUM> nucleotide <NUM>' overhangs on both ends. The double stranded siRNA molecule represents the sense and anti-sense strand of a portion of the target mRNA molecule, in this case a portion of the ROCK1, ROCK2, or tankyrase nucleotide sequence (the nucleotide sequences of ROCK1, ROCK2, and tankyrase are provided supra). siRNA molecules are typically designed to target a region of the mRNA target approximately <NUM>-<NUM> nucleotides downstream from the start codon. Upon introduction into a cell, the siRNA complex triggers the endogenous RNA interference (RNAi) pathway, resulting in the cleavage and degradation of the target mRNA molecule. Various improvements of siRNA compositions, such as the incorporation of modified nucleosides or motifs into one or both strands of the siRNA molecule to enhance stability, specificity, and efficacy, have been described and are suitable for use in accordance with this aspect of the invention (see e.g., <CIT>, <CIT>, <CIT> and <CIT>. , and <CIT>).

Short or small hairpin RNA molecules are similar to siRNA molecules in function, but comprise longer RNA sequences that make a tight hairpin turn. shRNA is cleaved by cellular machinery into siRNA and gene expression is silenced via the cellular RNA interference pathway.

Nucleic acid aptamers that specifically bind to ROCK1, ROCK2, or tankyrase are also useful in the methods of the present invention. Nucleic acid aptamers are single-stranded, partially single-stranded, partially double-stranded, or double-stranded nucleotide sequences, advantageously a replicatable nucleotide sequence, capable of specifically recognizing a selected non-oligonucleotide molecule or group of molecules by a mechanism other than Watson-Crick base pairing or triplex formation. Aptamers include, without limitation, defined sequence segments and sequences comprising nucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified nucleotides, and nucleotides comprising backbone modifications, branchpoints, and non-nucleotide residues, groups, or bridges. Nucleic acid aptamers include partially and fully single-stranded and double-stranded nucleotide molecules and sequences; synthetic RNA, DNA, and chimeric nucleotides; hybrids; duplexes; heteroduplexes; and any ribonucleotide, deoxyribonucleotide, or chimeric counterpart thereof and/or corresponding complementary sequence, promoter, or primer-annealing sequence needed to amplify, transcribe, or replicate all or part of the aptamer molecule or sequence.

ROCK inhibitors and tankyrase inhibitors suitable for use in the methods of the present invention may also include inhibitory peptides. Suitable inhibitory peptides include, without limitation, modified ROCK1, ROCK2, or tankyrase peptides that bind, preferably, specifically to the ROCK1, ROCK2, or tankyrase protein but prevent normal ROCK or tankyrase function. Such inhibitory peptides may be chemically synthesized using known peptide synthesis methodology or may be prepared and purified using recombinant technology. Such peptides are usually at least about <NUM> amino acids in length, but can be anywhere from <NUM> to <NUM> amino acids in length. Such peptides may be identified without undue experimentation using well known techniques. Techniques for screening peptide libraries for peptides that are capable of specifically binding to a polypeptide target, in this case ROCK1, ROCK2, and/or tankyrase are well known in the art (see e.g., <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; and <CIT> and <CIT>).

In one embodiment, a subject with a tumor comprising a p53 DNA contact mutation is identified prior to administering a ROCK inhibitor (and, optionally, a tankyrase inhibitor).

In another embodiment, identifying a subject with a tumor comprising a p53 DNA contact mutation involves obtaining a tissue sample from the tumor and testing the sample for a p53 DNA contact mutation.

"Obtaining a tissue sample" as used herein, refers to obtaining possession of a sample by "directly acquiring" or "indirectly acquiring" the sample. "Directly acquiring a sample" means performing a process (e.g., performing a physical method such as a surgery, biopsy, or extraction) to obtain the sample. "Indirectly acquiring a sample" refers to receiving the sample from another party or source (e.g., a third party laboratory that directly acquired the sample). Methods described herein can include obtaining a tissue sample from a tumor.

The source of the tissue sample can be solid tissue as from a fresh, frozen, and/or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any time in gestation or development of the subject. Preferably, the tissue sample is from a tumor. The tissue sample can contain compounds that are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like. The sample may be preserved as a frozen sample or as formaldehyde- or paraformaldehyde-fixed paraffin-embedded ("FFPE") tissue preparation. For example, the sample can be embedded in a matrix, e.g., an FFPE block or a frozen sample. Typically, the sample is a tumor sample, e.g., includes one or more premalignant or malignant cells. In certain, embodiments, the sample, e.g., the tumor sample, is acquired from a solid tumor, a soft tissue tumor, or a metastatic lesion. In other embodiments, the sample, e.g., the tumor sample, includes tissue or cells from a surgical margin. In an embodiment, the sample, e.g., tumor sample, includes one or more circulating tumor cells ("CTC") (e.g., a CTC acquired from a blood sample). In certain, embodiments, the sample, e.g., the tumor sample, is acquired from a solid tumor, a soft tissue tumor or a metastatic lesion.

Identifying a p53 DNA contact mutation in a tumor can be carried out using methods that are well known in the art. In one embodiment, detecting or identifying a p53 DNA contact mutation comprises sequencing at least a portion of the nucleotide sequence of p53 comprising the mutation. This can be performed by direct sequencing of the gene, such as gene regions comprising the mutation, from a tissue sample obtained from the tumor of a subject. Direct sequencing assays typically involve isolating a DNA sample from the subject using any suitable method known in the art, and cloning the region of interest to be sequenced into a suitable vector for amplification by growth in a host cell (e.g., bacteria) or direct amplification by PCR or other amplification assay. Following amplification, the DNA can be sequenced using any suitable method. One sequencing method involves high-throughput next generation sequencing ("NGS") to identify genetic variation. Various NGS sequencing chemistries are available and suitable for use in carrying out the claimed invention, including pyrosequencing (Roche® <NUM>), sequencing by reversible dye terminators (Illumina® HiSeq, Genome Analyzer and MiSeq systems), sequencing by sequential ligation of oligonucleotide probes (Life Technologies® SOLiD), and hydrogen ion semiconductor sequencing (Life Technologies®, Ion Torrent™). Alternatively, classic sequencing methods, such as the Sanger chain termination method or Maxam-Gilbert sequencing, which are well known to those of ordinary skill in the art, can be used to carry out the methods of the present invention (i.e., to identify or detect a p53 DNA contact mutation).

In another embodiment, the DNA contact mutation in p53 is identified or detected in a hybridization assay utilizing one or more oligonucleotide probes comprising a nucleotide sequence that is complementary to a nucleic acid molecule comprising p53. In a hybridization assay, the presence or absence of a gene mutation is determined based on the hybridization of one or more oligonucleotide probes to one or more nucleic acid molecules in a sample from the subject. The oligonucleotide probe or probes comprise a nucleotide sequence that is complementary to at least the region of the gene that contains the identified mutation. The oligonucleotide probes are designed to be complementary to the wild type, non-mutant nucleotide sequence and/or the mutant nucleotide sequence of the one or more genes to effectuate the detection of the presence or the absence of the mutation in the sample from the subject upon contacting the sample with the oligonucleotide probe(s).

A variety of hybridization assays that are known in the art are suitable for use in the methods of the present invention. These methods include, without limitation, direct hybridization assays, such as northern blot or Southern blot (see e.g., <NPL>)). Alternatively, direct hybridization can be carried out using an array based method where oligonucleotide probe(s) designed to be complementary to a particular non-mutant or mutant gene region are affixed to a solid support. A labeled DNA or cDNA sample from the subject is contacted with the array containing the oligonucleotide probe(s), and hybridization of nucleic acid molecules from the sample to their complementary oligonucleotide probes on the array surface is detected. Examples of direct hybridization array platforms include, without limitation, the Affymetrix GeneChip or SNP arrays and Illumina's Bead Array.

In another embodiment, identifying is carried out with an amplification-based assay which amplifies a nucleic acid molecule comprising p53 or a portion thereof. Amplification based assays include assays such as molecular beacon assays, nucleic acid arrays, and allele-specific PCR. Other common genotyping methods include, but are not limited to, restriction fragment length polymorphism assays; primer extension assays, such as allele-specific primer extension (e.g., Illumina® Infinium® assay), arrayed primer extension (see<NPL>)), homogeneous primer extension assays, primer extension with detection by mass spectrometry (e.g., Sequenom® iPLEX SNP genotyping assay) (see <NPL>)), multiplex primer extension sorted on genetic arrays; flap endonuclease assays (e.g., the Invader® assay) (see <NPL>)); <NUM>' nuclease assays, such as the TaqMan® assay (see <CIT> and <CIT>); and oligonucleotide ligation assays, such as ligation with rolling circle amplification, homogeneous ligation, OLA (see <CIT>), multiplex ligation reactions followed by PCR, wherein zipcodes are incorporated into ligation reaction probes, and amplified PCR products are determined by electrophoretic or universal zipcode array readout (see <CIT> and <CIT>). Such methods may be used in combination with detection mechanisms such as, for example, luminescence or chemiluminescence detection, fluorescence detection, time-resolved fluorescence detection, fluorescence resonance energy transfer, fluorescence polarization, mass spectrometry, and electrical detection.

According to one embodiment, once the p53 DNA contact mutation is identified, a ROCK inhibitor capable of inhibiting TEAD/YAP dependent transcription may be administered to the subject.

Pharmaceutical compositions containing a ROCK inhibitor suitable for use in the methods of the present invention can include a pharmaceutically acceptable carrier as described infra, one or more active agents (i.e., the ROCK inhibitor), and a suitable delivery vehicle. Suitable delivery vehicles include, but are not limited to, viruses, bacteria, biodegradable microspheres, microparticles, nanoparticles, liposomes, collagen minipellets, and cochleates.

In one embodiment, the pharmaceutical composition or formulation containing an inhibitory nucleic acid molecule (e.g., siRNA molecule) is encapsulated in a lipid formulation to form a nucleic acid-lipid particle as described in <NPL>), <CIT>, <CIT>, and <CIT>.

In another embodiment, the delivery vehicle is a nanoparticle. A variety of nanoparticle delivery vehicles are known in the art and are suitable for delivery of a ROCK inhibitor (see e.g.,<NPL>)). Suitable nanoparticles include, without limitation, poly(beta-amino esters) (<NPL>)), polyethylenimine-alt-poly(ethylene glycol) copolymers (<NPL>) and <NPL>)), and liposome-entrapped siRNA nanoparticles (<NPL>)). Other nanoparticle delivery vehicles suitable for use in the present invention include microcapsule nanotube devices disclosed in <CIT>.

In another embodiment, the pharmaceutical composition is contained in a liposome delivery vehicle. The term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

Several advantages of liposomes include: their biocompatibility and biodegradability, incorporation of a wide range of water and lipid soluble drugs; and they afford protection to encapsulated drugs from metabolism and degradation. Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Methods for preparing liposomes for use in the present invention include those disclosed in <NPL>); <CIT>; <CIT>; <CIT>; <CIT>, and <CIT>.

A liposome containing a ROCK inhibitor can be contacted with the target primary cancer (or tumor) cells under conditions effective for delivery of the inhibitory agent into the cancer (or tumor) cell. For administration to a primary tumor site, the liposomal vesicles need not be targeted to the cancer (or tumor) cells per se.

A liposome and nanoparticle delivery system can be made to accumulate at a target organ, tissue, or cell via active targeting (e.g., by incorporating an antibody or other ligand on the surface of the delivery vehicle). For example, when the target cell is a cancer (or tumor) cell as in the present invention, delivery vehicle may be conjugated to an anti-C3B(I) antibody as disclosed by <CIT> Alternatively, the delivery vehicle may be conjugated to an alphafeto protein receptor as disclosed by <CIT>, or to a monoclonal GAH antibody as disclosed by <CIT>.

In another embodiment, the delivery vehicle is a viral vector. Viral vectors are particularly suitable for the delivery of inhibitory nucleic acid molecules, such as siRNA or shRNA molecules, but can also be used to deliver molecules encoding an anti-ROCK antibody. Suitable gene therapy vectors include, without limitation, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and herpes viral vectors.

Adenoviral viral vector delivery vehicles can be readily prepared and utilized as described in<NPL>); <NPL>); <CIT>; <CIT>; and <CIT> Adeno-associated viral delivery vehicles can be constructed and used to deliver an inhibitory nucleic acid molecule of the present invention to cells as described in <NPL>); <NPL>);<NPL>);<NPL>); and<NPL>). In vivo use of these vehicles is described in<NPL>) and<NPL>). Additional types of adenovirus vectors are described in <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; and <CIT>.

Retroviral vectors which have been modified to form infective transformation systems can also be used to deliver a nucleic acid molecule to a target cell or tissue. One such type of retroviral vector is disclosed in <CIT> Other suitable nucleic acid delivery vehicles include those disclosed in <CIT>.

Regardless of the type of infective transformation system employed, it should be targeted for delivery of the nucleic acid to the desired cell type. For example, for delivery into a cluster of cells (e.g., cancer or tumor cells) a high titer of the infective transformation system can be injected directly within the site of those cells so as to enhance the likelihood of cell infection. The infected cells will then express the inhibitory nucleic acid molecule targeting the inhibition of integrin expression. The expression system can further contain a promoter to control or regulate the strength and specificity of expression of the nucleic acid molecule in the target tissue or cell.

In one embodiment, the administering step is carried out to treat a tumor in a subject. Such administration can be carried out systemically or via direct or local administration to the tumor or tumor site. By way of example, suitable modes of systemic administration include, without limitation orally, topically, transdermally, parenterally, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, or by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterialy, intralesionally, or by application to mucous membranes. Suitable modes of local administration include, without limitation, catheterization, implantation, direct injection, dermal/transdermal application, or portal vein administration to relevant tissues, or by any other local administration technique, method, or procedure generally known in the art. The mode of affecting delivery of an agent will vary depending on the type of therapeutic agent (e.g., an antibody, an inhibitory nucleic acid molecule, or a small molecule) and the tumor or cancer to be treated.

A ROCK inhibitor of the present invention may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or it may be enclosed in hard or soft shell capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. ROCK inhibitors may also be administered in a time release manner incorporated within such devices as time-release capsules or nanotubes. Such devices afford flexibility relative to time and dosage. For oral therapeutic administration, ROCK inhibitors may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and preparations should contain at least <NUM>% of the inhibitor, although lower concentrations may be effective and indeed optimal. The percentage of the inhibitor in these compositions may, of course, be varied and may be between about <NUM>% to about <NUM>% of the weight of the unit. The amount of an inhibitor of the present invention in such therapeutically useful compositions is such that a suitable dosage will be obtained.

When the ROCK inhibitor of the present invention is administered parenterally, solutions or suspensions of the agent can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, may be preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

Pharmaceutical formulations suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

When it is desirable to deliver the inhibitors of the present invention systemically, they may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.

Intraperitoneal or intrathecal administration of ROCK inhibitors can also be achieved using infusion pump devices. Such devices allow continuous infusion of desired compounds avoiding multiple injections and multiple manipulations.

In addition to the formulations described previously, the inhibitors may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.

Effective doses of the compositions containing an inhibitor may vary depending upon many different factors, including type and stage of the tumor or cancer, means of administration, target site, physiological state of the subject, other medications or therapies administered, and physical state of the subject relative to other medical complications. Treatment dosages may need to be titrated to optimize safety and efficacy.

For the treatment of tumors, the inhibitors can be administered to a subject in need of treatment alone, or in combination with other antitumor or anticancer substances and/or with radiation therapy and/or with surgical treatment to remove a tumor or cancerous tissue. These other substances or radiation treatments may be given at the same or different times as administering the inhibitor. For example, administration of an inhibitor can be used in combination with mitotic inhibitors, such as taxol or vinblastine; alkylating agents, such as cisplatin, cyclophosamide, or ifosfamide; antimetabolites, such as <NUM>-fluorouracil or hydroxyurea; DNA intercalators, such as adriamycin or bleomycin; topoisomerase inhibitors, such as etoposide or camptothecin; antiangiogenic agents, such as angiostatin; antiestrogens, such as tamoxifen; and/or other drugs or antibodies that inhibit cancer or tumor cells, such as, for example, GLEEVEC (Novartis) and HERCEPTIN (Genetech).

The term "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, where the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of a tumor or cancer. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and/or remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Subjects in need of treatment include those already with the condition or disorder (i.e., a tumor or cancer) as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. The term "treat" or "treatment" with respect to a tumor or tumor cells refers to stopping the progression of said cells, slowing down growth, inducing regression, or amelioration of symptoms associated with the presence of said cells.

Disclosed herein but not claimed is a method of identifying a subject as a candidate for treatment. This method involves obtaining a sample from a tumor in a subject and determining the presence of a p53 DNA contact mutation in the sample. The presence of a p53 DNA contact mutation in the sample indicates the tumor is susceptible to targeted treatment with a ROCK inhibitor and the subject is a candidate for treatment.

p53 DNA contact mutations and ROCK inhibitors are described supra.

In some scenarios, a course of treatment is assigned to the subject based on determining the presence of a p53 DNA contact mutation in the sample. Determining the presence of a DNA p53 mutation in a sample, or identifying the presence of a p53 DNA contact mutation in a sample can be carried out as described supra. For example, and without limitation, determining the presence of a p53 DNA contact mutation may be carried out using a hybridization assay or an amplification assay. Assigning a suitable treatment can involve assigning a treatment as described supra. For example, and according to one embodiment, the assigned course of treatment comprises administering a ROCK inhibitor as described supra.

Also disclosed herein but not claimed is a method of treating a tumor in a subject. This method involves administering to a subject having a tumor comprising increased YAP-dependent transcription a Rho-associated protein kinase (ROCK) inhibitor, where the ROCK inhibitor treats the tumor in the subject.

As discussed supra, YAP is a potent transcriptional co-activator that functions as a nuclear effector of the Hippo signaling pathway. In particular, YAP interacts with a variety of DNA-binding transcription factors in the nucleus to activate target gene expression (<NPL>); <NPL>)).

YAP activity is linked to its cellular abundance and nuclear localization. Amplification of the YAP gene has been observed in several cancer types, including breast (<NPL>)), medulloblastoma (<NPL>)), hepatocellular (HCC) (<NPL>)), and squamous cell carcinomas (<NPL>)). Increased YAP abundance is also seen in liver (<NPL>); <NPL>)), breast (<NPL>)), prostate (<NPL>)) and colorectal (<NPL>)) cancers, squamous cell (<NPL>)), lung and colon adenocarcinomas, and ovarian carcinomas (<NPL>)).

As described herein, tumors containing p53 DNA contact mutations show constitutive activation of TEAD/YAP transcription.

The details described supra regarding other aspects of the present invention also apply to carrying the method of this aspect of the present invention.

The following examples are provided to illustrate embodiments of the present invention but they are by no means intended to limit its scope.

HEK293T, MDA-MB-<NUM>, MDA-MB-<NUM>, U373MB, U251MG, SK-LMS-<NUM>, U138MG, LN229, M059J, M059K, and BT-<NUM> cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with <NUM>% Fetal Bovine Serum (FBS) (Sigma-Aldrich, St. Louis, MO), <NUM> units/ml of penicillin/streptomycin. HCC193, SF295, SK-BR-<NUM>, HCC1395, HCC1954, H1299, HCC1937, and HCC1806 cells were grown in RPMI-<NUM> medium supplemented with <NUM>% FBS and <NUM> units/ml of penicillin. SK-MEL-<NUM> cells were grown in Eagle's Minimum Essential Medium (MEM) supplemented with <NUM>% non-essential amino acids (NEAA), <NUM>% Fetal Bovine Serum (FBS) (Sigma-Aldrich, St. Louis, MO), <NUM> units/ml of penicillin/streptomycin. MCF10A cells were grown in DMEM/F12 medium supplemented with, <NUM>% horse serum, <NUM> ug/ml insulin, <NUM> ng/ml cholera toxin, <NUM>/ml hydrocortisone, <NUM> ng/ml EGF, and <NUM> units/ml of penicillin/streptomycin. All cells were cultured at <NUM> and <NUM>% humidity in a <NUM>% CO<NUM> incubator. All inhibitors were dissolved in DMSO and treatments were as indicated. DMSO was used as a control in all experiments. ROCK inhibitors were as follows: Y-<NUM> (Tocris Bioscience, <NUM>), Glycyl-H-<NUM> (Cayman Chemical, <NUM>), Fasudil (Abcam, Ab120306); Verteporfin (Sigma-Aldrich, SML0534), and Simvastatin (Sigma-Aldrich, S6196).

pQCXIH-Myc-YAP was a gift from Kunliang Guan (Addgene plasmid # <NUM>) <NUM>. A pQCXIH vector control was made by removing YAP. pBABE vector control and H-RAS (V12) were as previously described (<NPL>)). DN TEAD4 was cloned from the pSPORT6 Vector (Dharmacon) with primers containing the restriction sites compatible with the NSPI-CMV-MCS lentiviral vector (<NPL>)). The DN mutation, Y429H (TAC-->CAC), was introduced by PCR amplification with the mutated <NUM>' primer. Primers were as follows:.

The mutant p53 constructs containing the substitution of a single amino acid, were obtained by PCR site-directed mutagenesis using the QuickChange® Lightning Site-Directed Mutagenesis Kit (Agilent Technology, Agilent Technology, Milano, Italy) and the WT-p53 cDNA as template as previously described. Primers were as follow:.

All constructs were verified by DNA sequencing.

To create retroviral stocks, HEK293T cells were co-transfected with the appropriate retroviral expression vector and pCL-ampho packaging plasmid. To create lentiviral stocks, HEK293T cells were co-transfected with the appropriate lentiviral expression vector, pCMVΔR8. <NUM> packaging vector and pMD2 VSVG envelope vector. Titers for each virus stock were determined by colony formation following marker selection in the same assay cell, HT1080, making it possible to compare results using similar amounts of virus in different experiments. Retroviral and lentiviral infections were carried out on all cell lines in the presence of <NUM>µg/ml polybrene (Sigma). Cells were subsequently selected for antibiotic resistance (<NUM>µg/ml puromycin or <NUM>µg/ml hygromycin) and expanded as mass populations. In all cases, similar MOIs were used.

Cells were plated at <NUM> × <NUM><NUM> cells/well in <NUM> well plates, unless otherwise stated, <NUM> hours before collection and treated or genetically manipulated as described. Firefly and renilla luciferase activities were assayed with the dual luciferase assay system (Promega, Madison WI, USA), as directed, and firefly luciferase activity was normalized to renilla luciferase activity. Firefly and renilla luminescence were measured with the TD-20e Luminometer (Turner).

Growth in soft agar was determined by seeding <NUM> × <NUM><NUM> MCF10A cells in <NUM> of growth media containing <NUM>% agar (BD <NUM>) on top of <NUM> of <NUM>% agar in <NUM>-mm dishes. Cells were fed every <NUM> days for <NUM> weeks with <NUM> of growth medium. Colonies were stained with <NUM>% crystal violet in ethanol and photographed. Colony density was measured using Image J. Assays were conducted in triplicate.

Total RNA was extracted from cells using QIAshredder (Qiagen, Valencia, CA, USA) and RNeasy Mini kit (Qiagen) following the manufacturer's instructions. First-strand cDNA synthesis was performed using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Quantitative PCR was carried out using the ViiA™ <NUM> Real-Time PCR System (Life Technologies) using the FastStart SYBR Green Master mix (Roche, <NUM>). Primers were as follows:.

PCR was performed in <NUM> well plates using <NUM>µl volumes under the following conditions: <NUM> for <NUM>, followed by <NUM> cycles of <NUM> for <NUM> sec, <NUM> for <NUM> sec, and <NUM> for <NUM> sec. Specificity was verified by a dissociation curve. Results were analyzed with ViiA7 RUO software (Life Technologies).

For clonogenic assay, cells were plated in triplicate at <NUM> × <NUM><NUM> cells in <NUM>-well plates. Cells were treated or genetically manipulated as described. After <NUM> to <NUM> days, colonies were stained with <NUM>% crystal violet in ethanol and photographed.

<NUM> × <NUM><NUM> MDA-MB-<NUM> cells either overexpressing DN TEAD4 or silenced for p53 were inoculated orthotopically into the fat pads of the fifth mammary glands of <NUM>-week-old immunocompromised female SCID mice. The tumor volume was measured with a caliper every <NUM> weeks, using the formula volume = length × width<NUM>/<NUM>. At the end of the <NUM> months observation period, the mice bearing xenograft tumors were sacrificed and the tumor tissues were removed for formalin fixation and preparation of paraffin-embedded sections.

The paraffin-embedded tissue sections were used for examination of TEAD4 and p53 expression, and HE staining. For immunohistochemistry study, sections were incubated with primary antibodies (<NUM>:<NUM> dilutions) overnight at <NUM>, followed by biotin-labeled secondary antibody (<NUM>:<NUM> dilutions) for <NUM> at room temperature. Sections were then incubated with ABC-peroxidase and DAB (diaminobenzedine), counterstained with hematoxylin, and visualized using light microscope.

Missense mutations reside within the DNA binding domain (DBD), some of which inhibit DNA-contact (e.g., R248, R273), which affect amino acids that directly interact with DNA, while p53 conformational mutations (e.g., R175, G245, R282) profoundly alter the 3D conformation of the DBD (<NPL>)). Thus, the level of TEAD/YAP transcriptional activity was systematically compared in human tumor lines harboring different types of p53 missense mutations, including the four most frequent hotspot mutations found in human cancers as well as p53 null tumor cells (Table <NUM>).

A very high TEAD luciferase reporter activity was detected in all tumor lines analyzed containing p53 DNA-contact mutations but lack of this activity in any with a p53 conformational mutation or null genotype (<FIG>). Moreover, p53 DNA-contact mutant-expressing cells exhibited much higher levels of expression of TEAD specific target genes, CTGF, CYR61, and ANKRD1 than p53 conformational mutant-expressing cells (<FIG>). These findings suggested that p53 DNA-contact mutants comprised a new class of genetic alterations that upregulate TEAD/YAP transcription in human tumors.

To test whether p53 DNA contact mutations were responsible for TEAD/YAP transcriptional upregulation in tumor cells, the abilities of DN TEAD4, which lacks the TEAD DNA binding domain and functions as a dominant negative for TEAD/YAP transcription (<NPL>)) and shp53 to impact TEAD reporter activity were compared. <FIG> shows that each of these genetic manipulations markedly inhibited the elevated TEAD reporter activity specifically observed in each p53 DNA contact mutant tumor line analyzed. This same pattern of inhibition was observed for inhibition of expression of the elevated levels of TEAD dependent target genes such as CTGF observed in these same p53 DNA contact mutant tumor lines (<FIG>).

The biological effects of these genetic manipulations on tumor cell proliferation in serum containing medium were next compared. It was observed that colony formation by all p53 mutant tumor cells was strikingly inhibited in response to shp53, which had no effect on colony formation by H1299, a p53 null mutant colon tumor line as a specificity control. These results were consistent with evidence that p53 missense mutations exhibit gain of function (GOF), which appear to be addictive for tumor cells possessing them. Of note, DNTEAD4 was markedly inhibitory to colony formation, specifically by p53 DNA contact mutant containing tumor cells (<FIG>). These results correlated DN TEAD4 inhibition of deregulated TEAD/YAP transcription in these p53 mutant tumor cells with specific inhibition of their proliferation. Finally, the effects of DNTEAD4 or shp53 on growth in vivo of MDA-MB-<NUM> breast carcinoma cells, which contain a p53 DNA-contact mutant, R280K, were tested. <FIG> demonstrates that either manipulation caused a profound inhibition in the in vivo growth of orthotopically inoculated tumor cells, establishing the importance of p53 DNA contact mutants as oncogenic drivers through upregulation of TEAD dependent transcription.

To directly demonstrate the ability of p53 DNA-contact mutants to specifically activate TEAD/YAP transcription, two p53 DNA-contact mutants (R248Q and R273H) and two p53 conformational mutants were exogenously expressed by lentiviral mediated transduction of immortalized MCF10A cells. <FIG> show that like overexpression of YAP, which served as a positive control, both DNA contact mutants induced high levels of constitutive TEAD/YAP reporter activity and increased levels of TEAD/YAP endogenous target genes, not observed with either p53 conformational mutant tested. Both DNA contact and conformational p53 mutants as well as YAP promoted anchorage-independent colony formation in soft agar by MCF10a cells as previously reported for YAP (<NPL>)). A p53 mutant (G245S) that lacked the ability to upregulate TEAD dependent transcription, failed to induce colonies in soft agar, suggesting that this mutant does not exert gain of function (<FIG>).

To further establish that the ability of p53 DNA contact mutations to function as oncogenic drivers was mediated by their activation of TEAD/YAP dependent transcription, the effects of DN TEAD4 on both TEAD transcription and the transformed phenotype were tested. <FIG> shows that this genetic manipulation markedly inhibited both transcription of the TEAD reporter and the over expression of endogenous Hippo target genes in response to YAP overexpression or p53R273H, a representative DNA contact mutant (<FIG>). Moreover, DNTEAD4 specifically blocked colony formation in agar in response to YAP and p53R273H, with no effect on transformation induced by p53R175H, a representative conformational mutant (<FIG>). All of these findings provide strong evidence that p53 DNA contact mutations acquire transforming GOF by a mechanism, which causes Hippo pathway deregulation and constitutive TEAD/YAP transcriptional activation.

There are few if any agents yet available that specifically target Hippo pathway mutant tumors. The above findings identifying a major new class of these tumors led to seeking to identify potential inhibitors, which would inhibit both TEAD/YAP transcription and transformation by p53 DNA contact mutants with a high degree of specificity. Verteporfin has been reported to inhibit Hippo deregulated transcription at the level of TEAD/YAP protein/protein interactions (<NPL>)). Freed-Pastor and colleagues performed expression array analysis of MDA-MB-<NUM> p53 mutated cells and identified mutant p53 dependent upregulation of several genes involved in the cholesterol synthesis pathway (<NPL>)). Moreover, the mevalonate pathway has been proposed as upstream regulator of YAP activity (<NPL>)). Thus, the activity of Simvastatin, a potent inhibitor of the mevalonate pathway, which might inhibit Hippo pathway deregulation by decreasing RhoA posttranslational lipidation, blocking its accumulation at the plasma membrane, was tested. Inhibitors that antagonize the functions of ROCKs that act downstream of RhoA, are known to have diverse biological effects, including enhancing IPS generation (<NPL>)) and the propagation of normal and tumor cells in organoid culture (<NPL>);<NPL>)). Despite these apparent growth positive effects, a prototype ROCK inhibitor, Y-<NUM>, was also tested on growth of the same battery of p53 mutated human tumor cells.

At a concentration level sufficiently high to inhibit proliferation of representative human tumor lines with p53 DNA contact mutations, both Verteporfin and Simvastatin also inhibited the proliferation of representative tumor lines expressing p53 conformational mutants. Simvastatin also inhibited proliferation of SK-LMS-<NUM>, expressing a p53 conformational mutation, and p53-null H1299 cells, neither of which like the other p53 conformational mutant tumor cells showed up-regulated TEAD/YAP transcription or was detectably inhibited by DN TEAD4 (see <FIG>). Thus, neither of these inhibitors showed a high degree of specificity. In striking contrast, Y-<NUM> phenocopied the effects of DNTEAD4 in specifically inhibiting the proliferation of p53 DNA contact mutant expressing tumor cells without any obvious growth inhibitory effects on any of the other tumor cells analyzed (<FIG>). Similarly, the ROCK inhibitor antagonized both TEAD reporter and TEAD endogenous target gene expression in a manner similar to that of DN TEAD4 (<FIG>). As shown in <FIG>, Y-<NUM> also specifically and markedly impaired the transforming ability of MCF10a cells overexpressing YAP and p53 R273H (DNA contact) but not p53 R175H (conformational) or HRAS (V12G) mutants tested under the same conditions. These results confirmed its exquisite specificity as well as potent inhibitory ability for cells transformed by mechanisms involving TEAD/YAP dependent transcription.

Several ROCK inhibitors with varying potencies in inhibiting in vitro kinase activities of ROCK1 and ROCK2 have been developed (<NPL>)). <FIG> shows a titration of Y-<NUM> in which marked and specific inhibition of proliferation of tumor cells containing different p53 DNA contact mutants. There was almost complete inhibition of colony formation at concentrations ranging from <NUM>-<NUM> of <NUM> different p53 contact mutant tumor cells with no detectable inhibition of colony formation even at <NUM> for tumor cells with p53 DNA conformational mutants (<FIG>). Glycyl-H -<NUM> is a more potent ROCK inhibitor with a reported ICD<NUM> for inhibition of ROCK1 and ROCK2 of <NUM> and <NUM>, respectively (<NPL>); <NPL>); <NPL>)). At <NUM>, Glycyl-H-<NUM> treatment strikingly and specifically inhibited proliferation of tumor lines expressing p53 contact mutants, correlated with marked inhibition of TEAD/YAP dependent transcription (<FIG>). Fasudil, which is in the clinic for treatment of pulmonary hypertension and other cardiovascular disorders because of its ability to act as a potent vasodilator, is a far less potent ROCK inhibitor with reported ICD<NUM> of <NUM> for ROCK2 (<NPL>); <NPL>)). At <NUM>, Fasudil caused little if any inhibition of growth of representative tumor lines containing either a p53 DNA contact or conformational mutant, and caused little if any detectable inhibition of TEAD/YAP dependent transcription. All of these findings argue strongly that potent ROCK inhibitors have the ability to specifically inhibit the proliferation of tumors containing the newly identified class of p53 mutants with lesions that directly impair DNA binding.

Y-<NUM>, a prototype inhibitor of Rho kinases (ROCK1 and ROCK2), is able to specifically inhibit the proliferation of p53 DNA-contact mutant tumor lines (<FIG>), but was not shown to affect the growth of tumor lines harboring mutations in Hippo pathway core components. XAV939 was tested on a newly identified class of Hippo deregulated tumors that harbor p53 DNA-contact mutations. Strikingly, XAV939 was able to inhibit the proliferation of p53-DNA contact tumor lines, MDA-MB-<NUM> and HCC-<NUM>, but had no effect on the proliferation of p53 conformational mutant lines, HCC-<NUM> and SK-LMS-<NUM>, which do not show a deregulation in the Hippo pathway (<FIG>). These findings indicate that tankyrase inhibitors may be more broadly effective than ROCK inhibitors in treating Hippo pathway deregulated tumor cells (<FIG>).

Claim 1:
A Rho-associated protein kinase ("ROCK") inhibitor for use in a method of treating a tumor in a subject, said method comprising:
administering to a subject having a tumor comprising a p53 DNA contact mutation a ROCK inhibitor,
wherein the tumor shows constitutive activation of TEAD/YAP transcription; and
wherein the ROCK inhibitor is capable of inhibiting TEAD/YAP dependent transcription and treats the tumor in the subject.