WO2009155659A1 - Combination therapy - Google Patents

Combination therapy Download PDF

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Publication number
WO2009155659A1
WO2009155659A1 PCT/AU2009/000821 AU2009000821W WO2009155659A1 WO 2009155659 A1 WO2009155659 A1 WO 2009155659A1 AU 2009000821 W AU2009000821 W AU 2009000821W WO 2009155659 A1 WO2009155659 A1 WO 2009155659A1
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Prior art keywords
pi3k
hdaci
akt pathway
modulator
cancer
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PCT/AU2009/000821
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French (fr)
Inventor
Nicholas A. Saunders
Rafael Behring Erlich
Alenxander Guminski
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The University Of Queensland
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Priority to AU2008903284A priority Critical patent/AU2008903284A0/en
Priority to AU2008903284 priority
Application filed by The University Of Queensland filed Critical The University Of Queensland
Publication of WO2009155659A1 publication Critical patent/WO2009155659A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365Lactones
    • A61K31/366Lactones having six-membered rings, e.g. delta-lactones
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/16Amides, e.g. hydroxamic acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/16Amides, e.g. hydroxamic acids
    • A61K31/165Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/185Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic, hydroximic acids
    • A61K31/19Carboxylic acids, e.g. valproic acid
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K31/535Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5355Non-condensed oxazines and containing further heterocyclic rings
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/04Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/15Depsipeptides; Derivatives thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Abstract

The present invention relates to the use of histone deacetylase inhibitors (HDACIs) in combination with modulators of the PI3K/Akt pathway to treat or prevent cancer. In particular embodiments, the present invention relates to compositions, methods and kits involving HDACIs in combination with inhibitors of the PI3K/Akt pathway to treat or prevent squamous cell carcinomas, and in particular, head and neck squamous cell carcinomas (HNSCCs).

Description

Combination Therapy

Field of the invention

The present invention relates to the use of histone deacetylase inhibitors (HDACIs) in combination with modulators of the PI3K/Akt pathway to treat or prevent cancer. In particular embodiments, the present invention relates to compositions, methods and kits involving HDACIs in combination with inhibitors of the PI3K/Akt pathway to treat or prevent squamous cell carcinomas.

Background of the invention

Head and neck squamous cell carcinoma (HNSCC) is currently the sixth most common cancer in the developed world. The disease has an annual incidence of more than 500,000 cases worldwide, and represents 3.2% of all newly diagnosed cancers in the United States alone. Tobacco use and alcohol intake are well established as the major risk factors for the development of HNSCC, but the molecular mechanisms by which these carcinogens induce transformation and malignant progression are not fully understood. Irrespective of the etiologic agent, HNSCCs are associated with multiple genetic defects, which in turn lead to deregulation of basic biological processes such as proliferation, differentiation and apoptosis. The burden of such locally recurrent and metastatic disease is high in terms of morbidity, pain and distress, and survival is relatively short.

HNSCCs1 like all cancers, share a relatively restricted set of characteristics crucial to their phenotype: proliferation in the absence of external growth stimuli, avoidance of apoptosis and no limits to replication, escape from both external growth suppressive forces and the immune response, formation of new blood vessels and the ability to invade normal tissues. Deregulation of signal transduction, which accounts for aberrant responses to distinct soluble factors, is also a common feature of HNSCCs, and modulation of signalling pathways has become an option for targeted therapies.

Advanced HNSCC is usually treated with a combination of surgery, radiotherapy and chemotherapy. However, recent advances have been made in traditional modalities such as improvements in radiotherapy techniques. In addition, biological agents such as antibodies to epidermal growth factor (EGF) receptor have been trialled, as EGFR has been reported to be over- expressed in a number of cancers. For example, Cetuximab, an antibody that binds to the EGF receptor, has been indicated for use in combination with radiotherapy for the treatment of HNSCC. Although important advances in the treatment of HNSCC have therefore occurred in the last few decades, HNSCCs are still associated with severe disease- and treatment-related morbidity. Indeed, patients with HNSCC have a 5-year survival rate of approximately 50%. Further improvements in curative rates in HNSCC will therefore require significant advances in the development of chemotherapeuthic drugs and strategies. There is accordingly a clear need for more effective and efficient therapies for treatment of HNSCC.

Histone deacetylase inhibitors (HDACIs) are an emerging class of drugs that have shown promise as anticancer agents when used alone or in combination with some specific conventional therapies. Whilst the precise mechanism of action of HDACIs remains unknown, there is a growing body of evidence that the effects of HDACIs on both histone and non-histone proteins are important for their anti-proliferative and pro-apoptotic activities. HDACIs are thought to modify the acetylation status of nucleosomal proteins and therefore regulate transcriptional activity. Increased histone acetylation attenuates the electrostatic interaction with negatively charged bases, thereby decreasing the interaction of histones with DNA and allowing the access of transcription factors to target genes.

One particular HDACI is suberoylanilide hydroxamic acid (SAHA) which inhibits all histone deacetylases (HDACs) of class I and Il at a concentration of about 50 nM. SAHA, also known as Vorinostat, arrests cell growth of several cancer cell lines at low micromolar concentrations. Similarly to other HDACIs, the mechanisms of the anticancer effects of SAHA are not completely understood. Previous studies have suggested that SAHA can modulate 1) the activity of transcription factor complexes; 2) the acetylation, and consequently the activity, of proteins involved in the control of proliferation, protein stability, apoptosis, cell motility and angiogenesis; and 3) the expression of proteins related to the control of oxidative metabolism.

Many tumor suppressor genes inactivated in cancer cells are re-expressed following treatment with HDACI resulting in favorable changes such as apoptosis, cell-cycle arrest and differentiation. Important proteins such as p53, Hsp90 and tubulin are targets for HDACI and some of their anti-cancer effects may occur though these mechanisms. However, the exact mechanisms by which HDACIs induce these effects is not fully understood.

In addition, HDACIs have been used in combination with other anti-tumour drugs in cancer therapy with highly varying degrees of success. When used with chemotherapeutic agents, for example, combined effects have been observed to range from synergistic to antagonistic, or even increasing toxicity.

Significantly, it has been observed that the effects of HDACIs are extremely cell-type specific. Accordingly, when dealing with a molecule with a completely different mode of action to any of the known therapeutics, it has not been possible to predict how a combination therapy involving HDACIs will operate. Hence, there is a clear need to characterize suitable combination therapies involving HDACIs for use in cancer treatment.

The present inventors have now surprisingly found that HDACI-induced cell death is enhanced when combined with modulators of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway. In particular, the present inventors have demonstrated enhancement of HDACI-induced cell death in combination with a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway in squamous cell carcinoma- derived cell lines, wherein the molecular mechanism underlying these observations appears distinct from that reported in relation to other disease states.

Summary of the invention

According to a first aspect of the invention, there is provided a composition for preventing or treating a squamous cell carcinoma in a subject, wherein the composition comprises a histone deacetylase inhibitor (HDACI) and a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway.

In one preferred embodiment the modulator of the (PI3K)/AKT pathway is an inhibitor of the PI3K/AKT pathway. Any HDACI is contemplated to be useful and effective in combination with any (PI3K)/AKT pathway inhibitor. Any HDACI is contemplated to be within the scope of the invention regardless of any structural similarities or dissimilarities of compounds or compositions that are an HDAC inhibitor. Similarly, any inhibitor of the (PI3K)/AKT pathway is contemplated to be within the scope of the invention regardless of any structural similarities or dissimilarities of compounds or compositions that are inhibitors of the (PI3K)/AKT pathway.

In particular embodiments, the HDACI may be selected from the group comprising hydroxamic acids, cyclic peptides, benzamides and aliphatic acids.

In further embodiments, the HDACI may be selected from the group comprising suberoylanilide hydroxamic acid (SAHA), valproic acid, depsipeptide, LAQ824/LBH589, CI994, MS275 and MGCD0103.

The modulator of the PI3K/AKT pathway may be selected from the group comprising LY294002, Wortmannin analogues, PEG Wortmannin, PX-866, SF1124, SF1126, BEZ235, BGT226, BKM120,TGX115 and TGX126.

The squamous cell carcinoma may be any squamous cell carcinoma (SCC). The SCC may be of the skin, head and neck, esophagus, lung, penis, prostate, vagina and cervix. In particular aspects, the carcinoma is head and neck squamous cell carcinoma (HNSCC). The HNSCC may be an upper aerodigestive tract cancer. The upper aerodigestive tract cancer may be selected from the group comprising nasal cancer, paranasal cancer, mouth cancer, oropharyngeal cancer, laryngeal cancer, oesophageal cancer and cutaneous SCC arising on the head and neck region or lip.

The subject may be selected from the group consisting of human, non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline or canine. In preferred embodiments, the subject may be human.

The composition may further comprise a pharmaceutically acceptable carrier, excipient or diluent.

The composition may sensitize the squamous cell carcinoma to the HDACI.

The composition may be synergistic.

According to a second aspect of the invention, there is provided a method for preventing or treating a squamous cell carcinoma in a subject, wherein the method comprises administering to the subject the composition according to the first aspect, wherein the administering prevents or treats the squamous cell carcinoma in the subject.

The squamous cell carcinoma may be head and neck squamous cell carcinoma (HNSCC). The HNSCC may be an upper aerodigestive tract cancer. The upper aerodigestive tract cancer may be selected from the group comprising nasal cancer, paranasal cancer, mouth cancer, oropharyngeal cancer, pharyngeal, nasopharyngeal, llaryngeal cancer, oesophageal cancer and cutaneous SCC arising on the head and neck region or lip.

The subject may be selected from the group consisting of human, non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline or canine. In preferred embodiments, the subject may be human.

According to a third aspect of the invention, there is provided a method for preventing or treating a squamous cell carcinoma in a subject, wherein the method comprises administering to the subject a histone deacetylase inhibitor (HDACI) and a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway, wherein the administering prevents or treats the squamous cell carcinoma in the subject.

In one preferred embodiment the modulator of the (PI3K)/AKT pathway is an inhibitor of the PI3K/AKT pathway. Any HDACI is contemplated to be useful and effective in combination with any (PI3K)/AKT pathway inhibitor. Any HDACI is contemplated to be within the scope of the invention regardless of any structural similarities or dissimilarities of compounds or compositions that are an HDAC inhibitor. Similarly, any inhibitor of the (PI3K)/AKT pathway is contemplated to be within the scope of the invention regardless of any structural similarities or dissimilarities of compounds or compositions that are inhibitors of the (PI3K)/AKT pathway.

The HDACI may be selected from the group comprising hydroxamic acids, cyclic peptides, benzamides and aliphatic acids. The HDACI may be selected from the group comprising suberoylanilide hydroxamic acid (SAHA), valproic acid, depsipeptide, LAQ824/LBH589, CI994, MS275 and MGCD0103.

The modulator of the PI3K/AKT pathway may be selected from the group comprising LY294002, Wortmannin analogues, PEG Wortmannin, PX-866, SF1124, SF1126, BEZ235, BGT226, BKM120, TGX115 and TGX126.The squamous cell carcinoma may be head and neck squamous cell carcinoma (HNSCC). The HNSCC may be an upper aerodigestive tract cancer. The upper aerodigestive tract cancer may be selected from the group comprising nasal cancer, paranasal cancer, mouth cancer, oropharyngeal cancer, laryngeal cancer, oesophageal cancer and cutaneous SCC arising on the head and neck region or lip.

The subject may be selected from the group consisting of human, non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline or canine. In preferred embodiments, the subject may be human.

The HDACI may be administered simultaneously with, sequentially with or separately to the modulator of the PI3K/AKT pathway.

The HDACI and the modulator of the PI3K/AKT pathway may act synergistically.

According to a fourth aspect of the present invention, there is provided a method for sensitizing a squamous cell carcinoma in a subject to treatment with a histone deacetylase inhibitor (HDACI), wherein the method comprises administering a modulator of the PI3K/AKT pathway.

The HDACI may be selected from the group comprising hydroxamic acids, cyclic peptides, benzamides and aliphatic acids.

The HDACI may be selected from the group comprising suberoylanilide hydroxamic acid (SAHA), valproic acid, depsipeptide, LAQ824/LBH589, CI994, MS275 and MGCD0103.

In one preferred embodiment the modulator of the (PI3K)/AKT pathway is an inhibitor of the PI3K/AKT pathway. Any HDACI is contemplated to be useful and effective in combination with any (PI3K)/AKT pathway inhibitor. Any HDACI is contemplated to be within the scope of the invention regardless of any structural similarities or dissimilarities of compounds or compositions that are an HDAC inhibitor. Similarly, any inhibitor of the (PI3K)/AKT pathway is contemplated to be within the scope of the invention regardless of any structural similarities or dissimilarities of compounds or compositions that are inhibitors of the (PI3K)/AKT pathway.

The modulator of the PI3K/AKT pathway may be selected from the group comprising LY294002, Wortmannin analogues, PEG Wortmannin, PX-866, SF1124, SF1126, BEZ235, BGT226, BKM 120, TGX115 and TGX126.The squamous cell carcinoma may be head and neck squamous cell carcinoma (HNSCC). The HNSCC may be an upper aerodigestive tract cancer. The upper aerodigestive tract cancer may be selected from the group comprising nasal cancer, paranasal cancer, mouth cancer, oropharyngeal cancer, pharyngeal, nasopharyngeal, laryngeal cancer and oesophageal cancer.

The subject may be selected from the group consisting of human, non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline or canine. In preferred embodiments, the subject may be human.

In some embodiments, a HDACI may be administered simultaneously with, sequentially with or separately to the modulator of the PI3K/AKT pathway.

In some embodiments, a HDACI and the modulator of the PI3K/AKT pathway may act synergistically.

According to a fifth aspect of the invention there is provided use of a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway and a histone deacetylase inhibitor (HDACI) for preventing or treating a squamous cell carcinoma in a subject, wherein the use comprises simultaneously, sequentially or separately administering the modulator of the PI3K/AKT pathway and the HDACI.

According to a sixth aspect of the invention, there is provided use of a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway and a histone deacetylase inhibitor (HDACI) in the preparation of a medicament for preventing or treating a squamous cell carcinoma in a subject.

According to a seventh aspect of the invention, there is provided a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway for use in sensitizing a squamous cell carcinoma in a subject to treatment with a histone deacetylase inhibitor (HDACI).

According to an eighth aspect of the present invention, there is provided a kit for:

(a) preventing or treating a squamous cell carcinoma in a subject; or

(b) sensitizing a squamous cell carcinoma in a subject to treatment with a histone deacetylase inhibitor (HDACI) or to treatment with a modulator of the PI3K/AKT pathway wherein the kit comprises:

(c) the composition according to the first aspect; or

(d) a histone deacetylase inhibitor (HDACI) and a modulator of the phosphatidylinositol-3- kinase (PI3K)/AKT pathway.

According to a ninth aspect of the present invention, there is provided a method for screening for a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway that sensitizes a squamous cell carcinoma in a subject to treatment with a histone deacetylase inhibitor (HDACI), wherein the method comprises culturing a tumour cell line with the HDACI in the presence and in the absence of the modulator of the PI3K/AKT pathway, wherein increased cell death in the tumour cell line cultured with both the HDACI and the modulator of the PI3K/AKT pathway compared with the tumour cell line cultured with the HDACI without the modulator of the PI3K/AKT pathway is indicative that the modulator of the PI3K/AKT pathway sensitizes the squamous cell carcinoma in the subject to treatment with the HDACI.

According to a tenth aspect of the present invention, there is provided a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway that sensitizes a squamous cell carcinoma in a subject to treatment with a histone deacetylase inhibitor (HDACI) screened by the method of the ninth aspect.

Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well. For instance, any embodiment discussed in the context of one modulator of the (PI3K)/AKT pathway may be applied to any other modulator of the (PI3K)/AKT pathway. Similarly, any embodiment discussed in the context of one HDAC inhibitor may be applied to any other HDAC inhibitor and any embodiment discussed in the context of one combination of (PI3K)/AKT pathway modulator and HDACI may be applied to any other combination. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined. For example, any HDACI is contemplated to be useful and effective in combination with any (PI3K)/AKT pathway inhibitor. Any HDACI is contemplated to be within the scope of the invention regardless of any structural similarities or dissimilarities of compounds or compositions that are an HDAC inhibitor. Similarly, any inhibitor of the (PI3K)/AKT pathway is contemplated to be within the scope of the invention regardless of any structural similarities or dissimilarities of compounds or compositions that are inhibitors of the (PI3K)/AKT pathway. HDACIs and inhibitors of the (PI3K)/AKT pathway are further described herein.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in cell biology, chemistry, molecular biology and cell culture). Standard techniques used for molecular and biochemical methods can be found in Sambrook et a/., Molecular Cloning: A Laboratory Manual, 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. and Ausubel et a/., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc. - and the full version entitled Current Protocols in Molecular Biology).

Throughout this specification and the claims, reference to numerical values, unless stated otherwise, is taken to indicate that a value includes the inherent variation of error for the device and the method being employed to determine the value, or the variation that exists among the study subjects.

Throughout this specification and the claims, reference to numerical values, unless stated otherwise, is to be taken as meaning "about" that numerical value. The term "about" is used to indicate that a value includes the inherent variation of error for the device and the method being employed to determine the value, or the variation that exists among the study subjects.

Throughout this specification and the claims, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Following long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in the claims or specification, denotes one or more, unless specifically noted. The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

Brief description of the figures

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1 shows SCC cells and normal human keratinocytes (HKs) treated with distinct SAHA concentrations for 24 hrs. Cytotoxicity was determined by quantitative measurement of LDH release to the culture media, and the results were normalized with respect to the rate of LDH release in untreated cells.

Figure 2 shows A. SCC25 cells treated with SAHA (5 uM) alone or in combination with LY294002 or U0126 for 24 hrs. Cytotoxicity was determined by quantitative measurement of LDH release to the culture media, and the results were normalized with respect to the rate of LDH release in untreated cells; B. SCC25 cells subjected to distinct treatments for 24hrs, followed by lysis and analysis by western blot for phospho-Akt levels. Total Akt was used as a loading control. Figure 3 shows A. Cal27 cells treated with SAHA (5 uM) alone or in combination with LY294002 or U0126 for 24 hrs. SCC25 cells were treated with Depsipeptide (Dep) (5nM); B. or valproic acid (VA) (3mM); C. alone or in combination with LY294002 for 24 hrs. Cytotoxicity was determined by quantitative measurement of LDH release to the culture media, and the results were normalized with respect to the rate of LDH release in untreated cells.

Figure 4 shows A. HK cells treated with SAHA (5 uM) alone or in combination with LY294002 or U0126 for 24 hrs. Cytotoxicity was determined by quantitative measurement of LDH release to the culture media, and the results were normalized with respect to the rate of LDH release in untreated cells; B. HK and SCC25 cells were left untreated or treated with SAHA for 24hrs, followed by lysis and analysis by western blot for phospho-Akt levels. Total Akt was used as a loading control.

Figure 5 shows SCC cell lines (SCC25, Cal27, SCC9) and HKs subjected to SAHA treatment at distinct concentrations for 24 h. (A) Proliferation and (B) cytotoxicity were determined as described in the text. (B) reproduces the data of Figure 1. Values are means ± standard error of two independent experiments performed in triplicate. (C, D) SCC25 cells were treated with SAHA (5 μM) for 24 h and the actual percentage of cell death was accessed through LDH release assays (C) or Pl staining followed by FACS analysis (D). *** indicates p<0.001 versus CTR. Values are means ± standard error of at least three independent experiments performed in triplicate.

Figure 6 shows PI3K-Akt inhibitors potentiate caspase-dependent cell death induced by HDACI. (A) LDH viability assay for SCC25 cells subjected to SAHA treatment (5 uM) alone or in combination with LY (10 uM) or U0126 (10 uM) for 24 h. (B, C) Western blots show lysates from SCC25 cells subjected to SAHA treatment (5 uM) alone or in combination with LY294002 (10 uM) or U0126 (10 uM) for 10 minutes (B) or 24 h (C) probed against distinct antibodies. (D) SCC25 cells were subjected to distinct treatments for the indicated times. (E) SCC25 cells were assayed for viability following combination treatment with LY (10 uM) or Wortmannin (1 uM). (F) Similar assay following combination treatments with LY (10 uM) or Akt VIII (10 uM). (G) Western blots show effect upon Akt phosphorylation following distinct treatments for 24h. (H) SCC25 cells were subjected to distinct treatments in the presence or absence of ZVAD-FMK (100 uM) and assayed for viability. *** indicates p≤0.001 versus CTR. Values are means ± standard error of at least three independent experiments performed in triplicate. Western blot figures are representative of three independent experiments with similar results. Ct, control; SA, SAHA; LY, LY294002; UO, U0126; s+l, SAHA+LY294002; Akti, Akt VIII; wo, Wortmannin.

Figure 7 shows ectopic expression of constitutively active Akt attenuates increase in cytotoxicity mediated by SAHA / LY294002 co-treatment. (A) SCC25 cells transfected with constitutively active Akt (myr-Akt) or its correspondent empty vector were subjected to distinct treatments for 24 h and assayed for viability. * indicates p<0.05. Values are means ± standard error of three independent experiments performed in triplicate. (B) Western blots show lysates from SCC25 cells left untreated or subjected to SAHA treatment for 24 h, probed against distinct antibodies. Western blot figures are representative of two independent experiments with similar results.

Figure 8 shows enhanced cell death induced by SAHA/LY combination in SCC cells correlates with ROS accumulation. (A, B) SCC25 cells were subjected to distinct treatments for 24 h, labelled with oxidative-sensitive dye CM-H2DCFDA and analyzed by FACS for increase in FITC fluorescence. Values are means of three independent experiments. VE 0.1, vitamin E (0.1 mM); VE 1, vitamin E (1 mM). (C) Viability assay of SCC25 cells subjected to distinct treatments for 24 h with or without vitamin E pre-treatment. * indicates p<0.05 and ** indicates p≤0.01 versus respective controls. Values are means ± standard error of two independent experiments performed in triplicate.

Figure 9 shows SAHA/LY combination treatment does not induce cell death and Akt inhibition in normal human keratinocytes. (A) Western blots show lysates from HKs and SCC25 cells subjected to SAHA treatment for 24 h, probed against p21 antibody. Figure are representative of three independent experiments with similar results. (B) LDH viability assay for HKs subjected to SAHA treatment (5 uM) alone or in combination with LY294002 (10 uM) or U0126 (10 uM) for 24 h. Values are means ± standard error of three independent experiments performed in triplicate. (C) Western blots show lysates from HKs subjected to distinct treatments. Ct, control; SA, SAHA; LY, LY294002; s+l, SAHA+LY294002. Figures are representative of two independent experiments with similar results.

Figure 10 shows PI3K-Akt inhibition potentiates cell death induced by distinct HDACIs and is not cell type-specific. (A) SCC25 cells were subjected to valproic acid (3 mM) or depsipeptide (5 nM) treatment alone or in combination with LY (10 uM) for 24 h and assayed for viability. (B) Viability assays of Cal27 cells subjected to SAHA treatment (5 uM) alone or in combination with LY294002 (10 uM) or U0126 (10 uM) for 24 h. *** indicates p<0.001 versus CTR or indicated groups. Values are means ± standard error of at least three independent experiments performed in triplicate.

Detailed description of the invention

The inventors have surprisingly found that HDACI-induced cell death is enhanced when combined with a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway. In particular, the inventors have demonstrated enhancement of HDACI-induced cell death in combination with a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway in squamous cell carcinoma- derived cell lines, wherein the molecular mechanism underlying these observations appears distinct from that reported in relation to other disease states. The PI3K/AKT signalling pathway is involved in the regulation of apoptosis and is activated in cancer. Binding of external growth factors with the EGF receptor activates the PI3K/AKT cascade. AKT which is downstream of PI3K has multiple targets. AKT signalling inactivates several pro-apoptotic factors such as BAD, pro-caspase-9 and Forkhead (FKHR) transcription factors and the tumour suppression protein p53 through activation of mdm2. AKT mediated activation of Mammalian Target of Rapamycin (mTOR) is also associated with stimulating cell proliferation.

For example, the inventors have shown that SAHA is an effective cytotoxic agent against SCC cell lines but that it does not induce cell death in normal human keratinocytes (HK). The inventors have yet determined that specific inhibition of the PI3K/Akt signaling pathway causes a marked increase in SAHA-induced cytotoxicity in SCC cell lines. Importantly, normal keratinocytes are insensitive to this combination regimen, thereby providing a therapeutic window that can be explored in a clinical setting. These data provide a rational basis for the specific targeting of the PI3K/Akt pathway in order to enhance the anticancer effects of HDACIs.

Compositions

The present invention provides compositions for preventing or treating a squamous cell carcinoma in a subject, wherein the composition comprises a histone deacetylase inhibitor (HDACI) and a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway.

The HDACI may include any molecule having the ability to inhibit a histone deacetylase. The term "inhibit" is defined in the context of the biological role of the HDACI and may include any molecule having the ability to inhibit, prevent, abrogate, retard or down-regulate any biological function, signal or signal transduction cascade in any form within, related to or otherwise associated with a histone deacetylase The HDACI may therefore include without limitation any peptide, non-peptide small molecule, antibodies including polyclonal, monoclonal, chimeric, humanised or single chain antibodies, Fab fragments and Fab expression libraries, antibody fragments, antisense molecules including antisense mRNA and oligonucleotide decoys including interfering RNA such as siRNA or RNAi that is capable of inhibiting a histone deacetylase. In some embodiments, the HDACI may be selected from the group comprising hydroxamic acids, cyclic peptides, benzamides and aliphatic acids. In other embodiments, the HDACI may be selected from the group comprising suberoylanilide hydroxamic acid (SAHA), valproic acid, depsipeptide, LAQ824/LBH589, CI994, MS275 and MGCD0103. In preferred embodiments, the HDACI may be selected from the group comprising SAHA, valproic acid and depsipeptide. In particularly preferred embodiments, the HDACI may be SAHA.

The HDACI may include short-chain fatty acids (butyrate and valproic acid), hydroxamates (SAHA, trichostatin A, ITF2357, LBH589, oxamflatin, PCI-24781, and PXD101), benzamides (MS-275, CI-994, and MGCD-0103), cyclic tetrapeptides (depsipeptide, trapoxin A, and apicidin), electrophilic ketones (trifluoromethylketone) , depudecin, SNDX-275, and isothiocyanates. The HDACI may include CI-994 (N-acetyldinaline, [4-(acetylamino)-N-(2-amino-phenyl) benzamide), FK228 (FR901228, depsipeptide, romidepsin), ITF2357, LBH589 (panobinostat), MGCD0103, MS-275 (MS-27-275; Λ/-(2- aminophenyl)-4-[Λ/-(pyridin-3-yl-methoxycarbonyl) aminomethyl] benzamide), PCI-24781 (CRA- 024781), Phenylbutyrate, PXD101 (belinostat), NVP-LAQ824 ((2E)-N-hydroxy-3-[4-[[(2-hydroxyethyl)[2- (1 H-indol-3-yl)ethyl]amino]methyl]phenyl]-2-propenamide), and SNDX-275.

Modulators of the PI3K pathway may include any molecule having the ability to modulate a biological function of a component of the PI3K/AKT pathway. The term "modulator" is defined in the context of the biological role of the components of the PI3K pathway and without limitation, includes any peptide, non-peptide small molecule, antibodies including polyclonal, monoclonal, chimeric, humanised or single chain antibodies, Fab fragments and Fab expression libraries, antibody fragments, antisense molecules including antisense mRNA and oligonucleotide decoys including interfering RNA such as siRNA or RNAi. In some embodiments, the modulator of the PI3K/AKT pathway may be selected from the group comprising LY294002, Wortmannin analogues, PEG Wortmannin, PX-866, SF1124, SF1126, BEZ235, BGT226, BKM120, TGX115 and TGX126. In preferred embodiments, the modulator of the PI3K/AKT pathway may be LY294002.

The modulator of the PI3K/AKT pathway may be an inhibitor of the PI3K/AKT pathway. The term "inhibitor" is defined in the context of the biological role of the components of the PI3K/AKT pathway and without limitation, includes any peptide, non-peptide small molecule, antibodies including polyclonal, monoclonal, chimeric, humanised or single chain antibodies, Fab fragments and Fab expression libraries, antibody fragments, antisense molecules including antisense mRNA and oligonucleotide decoys including interfering RNA such as siRNA or RNAi that is capable of inhibiting any biological function, signal or signal transduction cascade in any form within, related to or otherwise associated with the PI3K/AKT pathway.

The modulator of the PI3K/AKT pathway may include SF1126 (Semafore Pharmaceuticals, Inc.), BEZ235 (Novartis), XL765, BGT226 (Novartis), BKM120 (Novartis), CAL-101 (Calistoga), ZSTK474, SF1126, PX-866 (a small-molecule wortmannin analogue inhibitor of the alpha, gamma, and delta isoforms of PI3K), SF1126 (a pro-drug that produces LY294002), XL147 (a PI3K inhibitor from Exelixis), ONC-201 (Oncalis), AS 252424 (a PI3K p110γ inhibitor), GDC-0941 (PI3K inhibitor), Pl 103 (PI3K inhibitor), PIK 75 (PI3K p110α inhibitor), PIK 90 (PI3K p110α inhibitor), and TGX 221 (PI3K p110β inhibitor). Additional modulators of the PI3K/AKT pathway may include those described in each of WO 2006/065601, WO 2007/090913, WO 2008/0318947, WO 2008/144463, WO 2009/071888, WO 2009/032651, WO 2009/032652, WO 2009/032653, each of which is incorporated herein by reference in their entirety. Modulators of the PI3K/AKT pathway may include inhibitors or modulators of AKT such as those described in US Patents: 7,544,677; 7,524,850; 7,511,041; and US published applications 2009/0098135; 2009/0029997; and 2009/0029998, each of which is incorporated herein by reference in their entirety.

In view of the data presented herein and the discovery of the inventors, any HDACI is contemplated to be within the scope of the invention regardless of any structural similarities or dissimilarities of compounds or compositions that are an HDAC inhibitor. Similarly, any modulator of the (PI3K)/AKT pathway is contemplated to be within the scope of the invention regardless of any structural similarities or dissimilarities of compounds or compositions that are modulators of the (PI3K)/AKT pathway. Chemically or structurally unrelated HDACIs and chemically or structurally unrelated modulators of the (PI3K)/AKT pathway have been shown herein to be effective in the invention.

The squamous cell carcinoma may be head and neck squamous cell carcinoma (HNSCC). The HNSCC may be an upper aerodigestive tract cancer. The upper aerodigestive tract cancer may be selected from the group comprising nasal cancer, paranasal cancer, mouth cancer, oropharyngeal cancer, pharyngeal, nasopharyngeal, laryngeal cancer, oesophageal cancer and cutaneous SCC arising on the head and neck region or lip.

The subject may be selected from the group consisting of human, non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline or canine. In preferred embodiments, the subject may be human.

The composition may sensitize the squamous cell carcinoma to the HDACI or the composition may sensitize the squamous cell carcinoma to the modulator of the PI3K/AKT pathway. The composition may be synergistic.

Compositions of the present invention may be administered either therapeutically or preventatively. In a therapeutic application, compositions are administered to a subject already suffering from a condition, in an amount sufficient to cure or at least partially arrest the condition and any complications. The quantity of the composition should be sufficient to effectively treat the patient. Compositions may be prepared according to methods which are known to those of ordinary skill in the art and accordingly may include a pharmaceutically acceptable carrier, excipient or diluent. Methods for preparing administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., hereby incorporated herein by reference. Compositions of the present invention may further comprise other therapeutic agents. The other therapeutic agents may be selected from the group comprising chemotherapeutic agents, radioisotopes, differentiation agents and epigenetic agents.

The compositions may incorporate any suitable surfactant such as an anionic, cationic or non- ionic surfactant such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

The compositions may also be administered in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances, and are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The compositions in liposome form may contain stabilisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, and in relation to this, specific reference is made to: Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 ef seq., the contents of which are incorporated herein by reference.

Carriers, excipients and diluents

Compositions of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent. Carriers, excipients and diluents must be "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Such carriers, excipients and diluents may be used for enhancing the integrity and half-life of the compositions of the present invention. These may also be used to enhance or protect the biological activities of the compositions of the present invention.

Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils, arachis oil or coconut oil; silicone oils, including polysiloxanes; such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrolidone; agar; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

The compositions of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection.

For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.

Some examples of suitable carriers, excipients and diluents for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin. In addition these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which delay disintegration.

Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents. Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof. Suspensions for oral administration may further comprise dispersing agents and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or di- oleate, -stearate or -laurate and the like.

The emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.

Dosages

The therapeutically effective dose level for any particular patient will depend upon a variety of factors including the condition being treated and the severity of the condition, the activity of the compound or agent employed, the composition employed, the age, body weight, general health, sex and diet of the patient, the time of administration, the route of administration, the rate of sequestration, the duration of the treatment, and any drugs used in combination or coincidental with the treatment, together with other related factors well known in the art. One skilled in the art would therefore be able, by routine experimentation, to determine an effective, non-toxic amount of the compositions which would be required to treat applicable conditions.

Generally, an effective dosage is expected to be in the range of about 0.0001 mg to about IOOOmg per kg body weight per 24 hours; typically, about 0.001 mg to about 750mg per kg body weight per 24 hours; about 0.01 mg to about 500mg per kg body weight per 24 hours; about 0.1 mg to about 500mg per kg body weight per 24 hours; about 0.1 mg to about 250mg per kg body weight per 24 hours; about 1.Omg to about 250mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range about 1.Omg to about 200mg per kg body weight per 24 hours; about 1.0mg to about 100mg per kg body weight per 24 hours; about lOmg to about 50mg per kg body weight per 24 hours; about 1.Omg to about 25mg per kg body weight per 24 hours; about 5.0mg to about 50mg per kg body weight per 24 hours; about 5.0mg to about 20mg per kg body weight per 24 hours; about 5.0mg to about 15mg per kg body weight per 24 hours. Alternatively, an effective dosage may be up to about 500mg/m2. Generally, an effective dosage is expected to be in the range of about 25 to about 500mg/m2, preferably about 25 to about 350mg/m2, more preferably about 25 to about 300mg/m2, still more preferably about 25 to about 250mg/m2, even more preferably about 50 to about 250mg/m2, and still even more preferably about 75 to about 150mg/m2. Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages of the composition will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the nature of the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques.

It will also be apparent to one of ordinary skill in the art that the optimal course of treatment, such as the number of doses of the composition given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

Routes of Administration

The compositions of the present invention can be administered by standard routes. In general, the compositions may be administered by the parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular), oral or topical route.

Methods for preventing or treating squamous cell carcinoma

The present invention provides methods for preventing or treating a squamous cell carcinoma in a subject, wherein the methods comprise administering to the subject any one or more of the compositions herein described, wherein the administering prevents or treats the squamous cell carcinoma in the subject.

The method may sensitize the squamous cell carcinoma to a HDACI or the composition may sensitize the carcinoma to a modulators of the PI3K/AKT pathway. The method may result in the compositions employed acting synergistically.

The present invention also provides methods for preventing or treating a squamous cell carcinoma in a subject, wherein the methods comprise administering to the subject a histone deacetylase inhibitor (HDACI) and a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway, wherein the administering prevents or treats the squamous cell carcinoma in the subject. '

For the methods described herein involving use of a HDACI, the HDACI may include any molecule having the ability to inhibit a histone deacetylase. The term "inhibit" is defined in the context of the biological role of the HDACI and may include any molecule having the ability to inhibit, prevent, abrogate, retard or down-regulate any biological function, signal or signal transduction cascade in any form within, related to or otherwise associated with a histone deacetylase The HDACI may therefore include without limitation any HDACI inhibitor described or referenced herein, including a peptide, non- peptide small molecule, antibodies including polyclonal, monoclonal, chimeric, humanised or single chain antibodies, Fab fragments and Fab expression libraries, antibody fragments, antisense molecules including antisense mRNA and oligonucleotide decoys including interfering RNA such as siRNA or RNAi that is capable of inhibiting a histone deacetylase. In some embodiments, the HDACI may be selected from the group comprising hydroxamic acids, cyclic peptides, benzamides and aliphatic acids. In other embodiments, the HDACI may be selected from the group comprising suberoylanilide hydroxamic acid (SAHA), valproic acid, depsipeptide, LAQ824/LBH589, CI994, MS275 and MGCD0103. In preferred embodiments, the HDACI may be selected from the group comprising SAHA, valproic acid and depsipeptide. In particularly preferred embodiments, the HDACI may be SAHA.

For the methods described herein involving use of a modulator of the PI3K/AKT pathway, the modulator of the PI3K/AKT pathway may be an inhibitor of the PI3K/AKT pathway. The term "inhibitor" is defined in the context of the biological role of the components of the PI3K/AKT pathway and without limitation, includes any peptide, non-peptide small molecule, antibodies including polyclonal, monoclonal, chimeric, humanised or single chain antibodies, Fab fragments and Fab expression libraries, antibody fragments, antisense molecules including antisense mRNA and oligonucleotide decoys including interfering RNA such as siRNA or RNAi that is capable of inhibiting any biological function, signal or signal transduction cascade in any form within, related to or otherwise associated with the PI3K/AKT pathway.

Modulators of the PI3K pathway may include any molecule having the ability to modulate a biological function of a component of the PI3K/AKT pathway. The term "modulator" is defined in the context of the biological role of the components of the PI3K pathway and without limitation, includes any modulator of the PI3K/AKT pathway described or referenced herein, including any peptide, non- peptide small molecule, antibodies including polyclonal, monoclonal, chimeric, humanised or single chain antibodies, Fab fragments and Fab expression libraries, antibody fragments, antisense molecules including antisense mRNA and oligonucleotide decoys including interfering RNA such as siRNA or RNAi. In some embodiments, the modulator of the PI3K/AKT pathway may be selected from the group comprising LY294002, Wortmannin analogues, PEG Wortmannin, PX-866, SF1124, SF1126, BEZ235, BGT226, BKM120, TGX115 and TGX126. In preferred embodiments, the modulator of the PI3K/AKT pathway may be LY294002.

The HDACI may be administered simultaneously with, sequentially with or separately to the modulator of the PI3K/AKT pathway. The HDACI and the modulator of the PI3K/AKT pathway may be administered so that they act synergistically.

The method may further comprise administering other therapeutic agents. The other therapeutic agents may be selected from the group comprising chemotherapeutic agents, radioisotopes, differentiation agents and epigenetic agents. The method may further comprise administering other treatments. The other treatments may be selected from the group comprising radiotherapy, chemotherapy, surgical therapy such as neck dissection, photosensitizers and interstitial laser therapy, immune therapy and gene therapy.

The radiotherapy may involve radiation dosage in excess of 6000 cGy with a boost to areas of high risk. As known to the skilled artisan, indications for radiotherapy may include a bulky tumour with significant risk of recurrence, histologically positive margins and perineural or perivascular invasion by the tumour. For the neck, indications for radiotherapy may include elective treatment of the neck not treated surgically where risk of micrometastasis is high, gross residual tumour in the neck following neck dissection, multiple positive lymph nodes, and extranodal extension of tumour.

The chemotherapy may involve administration of bleomycin with or without electroporation. The chemotherapy may involve administration of cisplatin or taxol. A combination of cisplatin, taxol, or other chemotherapeutic agents with interstitial laser therapy may be used, wherein hyperthermia produced by the laser augments cytotoxic effects of both radiation therapy and chemotherapeutic agents.

The surgical therapy may involve surgical resection. Because patients with cancers of the head and neck often have had previous radiation therapy, flaps must have an adequate blood supply.

Neck dissection may be used to preserve the spinal accessory nerve, the great auricular, and the sternocleidomastoid muscle. The jugular vein and submandibular gland may also be preserved using neck dissection. In addition, successful results may be achieved through less than complete lymph node removal, selectively removing only those lymph nodes likely to be invaded by metastases. Modified radical neck dissection usually involves removal of all five lymph node levels, preserving one or all of the spinal accessory nerves, jugular vein, and sternocleidomastoid muscle.

Selective neck dissection may be used to remove HNSCC via either supraomohyoid neck dissection, anterior neck dissection, or anterolateral neck dissection. Selective neck dissection is usually limited to patients without pathologically involved lymph nodes on the side of the dissection.

Classic radical neck dissection may also be used to remove the sternocleidomastoid muscle, submandibular gland, jugular vein, and spinal accessory nerve. This operation remains the best procedure for definitive control of neck disease. Radical neck dissection can be combined with resection of the primary cancer and postoperative radiation therapy. Radical neck dissection has significant morbidity because of the resection of the spinal accessory nerve and, in bilateral dissection, the sacrifice of the internal jugular veins. Severing the spinal accessory nerve results in paralysis of the trapezius muscle in approximately 70% of subjects. In most subject, the shoulder subsequently loses support, rotates forward, and droops, and the subject has pain and difficulty lifting his or her arm.

Photosensitizing drugs that concentrate in cancer cells may also be used as another treatment in the form of photodynamic therapy. Activation of the drug with light results in cancer cell death. Laser photothermal ablation may be an alternative to surgery for the palliative treatment of head and neck cancer because of its tissue-sparing access, the possibility of repeated treatment, and the lower recurrence at tumour margins compared with surgery. The combination of interstitial laser therapy with regional chemotherapy agents that are activated by light or heat may also used as a combined therapeutic regimen.

Recruitment of immune cells and administration of stimulatory immune factors to augment treatment of cancer through the host immune response may also be used.

Gene therapy may also be used. Therapeutic genes may encode a polypeptide that induces a biologic response, such as activation of the immune system with transferred interleukin sequences. Head and neck cancers are known to have high levels of p53 mutations. Normal functions of p53 are cell growth regulation. Head and neck cancers are accessible to injection therapy and are therefore good candidates for p53 gene therapy. Another form of therapeutic gene delivery is the adenovirus vector, which uses a genetically engineered virus that is replication incompetent. Prodrug gene therapy, also known as suicide gene therapy, is designed to induce negative selection of cancer cells. By transducing cancer cells with a gene encoding an enzyme that metabolizes a nontoxic prodrug into its toxic form, cancer cells can be selectively killed.

Herpes simplex virus is a common human virus that produces a unique thymidine kinase. This viral enzyme preferentially phosphorylates the prodrug ganciclovir, a guanine nucleoside analogue, to produce a metabolite that, after cellular phosphorylation, is incorporated into replicating DNA, thereby inhibiting DNA polymerase and ultimately killing the cancer cell. This therapy is most effective in treating cancer cells growing in tissues where normal cells are not proliferating.

The squamous cell carcinoma to be treated or prevented by the methods of the present invention may include head and neck squamous cell carcinoma (HNSCC). The HNSCC may be an upper aerodigestive tract cancer. The upper aerodigestive tract cancer may be selected from the group comprising nasal cancer, paranasal cancer, mouth cancer, oropharyngeal cancer, pharyngeal, nasopharyngeal, laryngeal cancer, oesophageal cancer and cutaneous SCC arising on the head and neck region or lip.

As would be apparent to the person skilled in the art, squamous cell carcinomas usually begin as surface lesions with erythema and a slight physical elevation. They are termed erythroplasia and usually are subjected to biopsy. Such early red lesions are often asymptomatic and may represent either carcinoma in situ or invasive carcinoma. One third of lesions are pure white and are known as leukoplakia, but only 10% of white lesions are carcinoma in situ or invasive carcinoma. The most common sites for squamous cell carcinoma are the floor of the mouth, the tongue, soft palate, anterior tonsillar pillar, and the retromolar trigone. Tender, painful lesions are usually suggestive of perineural invasions. When lesions become palpable masses, symptoms such as a vague persistent sore throat or ear infection may occur. In more advanced cases, dissemination to ipsilateral submandibular and jugulodigastric nodes is common, and subjects may present with a mass in the neck. When lymph node or remote bone and organ metastases are associated with an early oral primary lesion, often a second, more advanced primary upper aerodigestive or lung cancer is responsible for the metastases.

The subject may be selected from the group consisting of human, non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline or canine. In preferred embodiments, the subject may be human.

Methods for sensitizing a squamous cell carcinoma to treatment with a HDACI

The present invention provides methods for sensitizing a squamous cell carcinoma in a subject to treatment with a histone deacetylase inhibitor (HDACI), wherein the methods comprise administering a modulator of the PI3K/AKT pathway.

In some embodiments, a HDACI may be administered simultaneously with, sequentially with or separately to the modulator of the PI3K/AKT pathway.

In some embodiments, a HDACI and the modulator of the PI3K/AKT pathway may act synergistically.

For such methods, the identity of the modulator of the PI3K pathway is described elsewhere in the present disclosure. In circumstance where a HDACI inhibitor is administered, the identity of the HDACI is described elsewhere in the present disclosure.

The subject may be selected from the group consisting of human, non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline or canine. In preferred embodiments, the subject may be human.

Methods for sensitizing a squamous cell carcinoma to treatment with a modulator of the PI3K/AKT pathway

The present invention provides methods for sensitizing a squamous cell carcinoma in a subject to treatment with a modulator of the PI3K/AKT pathway, wherein the methods comprise administering a HDACI.

In some embodiments, a a modulator of the PI3K/AKT pathway may be administered simultaneously with, sequentially with or separately to the HDACI.

In some embodiments, a HDACI and the modulator of the PI3K/AKT pathway may act synergistically. For such methods, the identity of the modulator of the PI3K pathway is described elsewhere in the present disclosure. In circumstance where a HDACI inhibitor is administered, the identity of the HDACI is described elsewhere in the present disclosure.

The subject may be selected from the group consisting of human, non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline or canine. In preferred embodiments, the subject may be human.

Uses of modulators of the PI3K/AKT pathway and HDACIs

The present invention provides uses of a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway and a histone deacetylase inhibitor (HDACI) for preventing or treating a squamous cell carcinoma in a subject, wherein the uses comprise simultaneously, sequentially or separately administering the modulator of the PI3K/AKT pathway and the HDACI.

The present invention also provides uses of a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway and a histone deacetylase inhibitor (HDACI) in the preparation of a medicament for preventing or treating a squamous cell carcinoma in a subject.

The invention moreover provides modulators of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway for use in sensitizing a squamous cell carcinoma in a subject to treatment with a histone deacetylase inhibitor (HDACI).

Kits

The present invention also provides kits for preventing or treating a squamous cell carcinoma in a subject, or for sensitizing a squamous cell carcinoma in a subject to treatment with a histone deacetylase inhibitor (HDACI), wherein the kits comprise any one or more of the compositions described herein, or a histone deacetylase inhibitor (HDACI) and a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway.

Kits of the present invention facilitate the employment of the methods of the present invention. Typically, kits for carrying out a method of the invention contain all the necessary reagents to carry out the method. For example, in one embodiment the kit may comprise a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway as described herein, and/or or a histone deacetylase inhibitor (HDACI) as described herein, and/or any one or more of the compositions described herein.

Typically, the kits described herein will also comprise one or more containers. In the context of the present invention, a compartmentalised kit includes any kit in which compounds or compositions are contained in separate containers, and may include small glass containers, plastic containers or strips of plastic or paper. Such containers may allow the efficient transfer of compounds or compositions from one compartment to another compartment whilst avoiding cross-contamination of samples, and the addition of agents or solutions of each container from one compartment to another in a quantitative fashion.

Typically, a kit of the present invention will also include instructions for using the kit components to conduct the appropriate methods.

Methods and kits of the present invention are equally applicable to any animal, including humans and other animals, for example including non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline and canine species. Accordingly, for application to different species, a single kit of the invention may be applicable, or alternatively different kits, for example containing compounds or compositions specific for each individual species, may be required.

Methods and kits of the present invention find application in any circumstance in which it is desirable to prevent or treat cancer in a subject.

Methods for screening for modulators and for inhibiting the PI3K/AKT pathway

The present invention provides methods for screening for a modulator of the phosphatidylinositol- 3-kinase (PI3K)/AKT pathway that sensitizes a squamous cell carcinoma in a subject to treatment with a histone deacetylase inhibitor (HDACI), wherein the methods comprise culturing a tumour cell line with the HDACI in the presence and in the absence of the modulator of the PI3K/AKT pathway, wherein increased cell death in the tumour cell line cultured with both the HDACI and the modulator of the PI3K/AKT pathway compared with the tumour cell line cultured with the HDACI without the modulator of the PI3K/AKT pathway is indicative that the modulator of the PI3K/AKT pathway sensitizes the squamous cell carcinoma in the subject to treatment with the HDACI.

It will be apparent to persons of skill in the art that candidate modulators of the PI3K/AKT pathway that sensitize a squamous cell carcinoma in a subject to treatment with a HDACI may be identified using different techniques. Such modulators may exert a modulatory effect by activating, stimulating, increasing, inhibiting or preventing expression or activity of polypeptides and/or polynucleotides involved in the PI3K/AKT pathway. Modulators may exert their effect by virtue of either a direct (for example binding) or indirect interaction.

Interaction and/or binding of a candidate modulator to a molecule involved in the PI3K/AKT pathway may be determined using standard competitive binding assays or two-hybrid assay systems.

For example, the two-hybrid assay is a yeast-based genetic assay system typically used for detecting protein-protein interactions. This assay takes advantage of the multi-domain nature of transcriptional activators. For example, the DNA-binding domain of a known transcriptional activator may be fused to a known polypeptide, and the activation domain of the transcriptional activator fused to a candidate modulator. Interaction between the candidate modulator and the known polypeptide will bring the DNA-binding and activation domains of the transcriptional activator into close proximity. Interaction can thus be detected by virtue of transcription of a specific reporter gene activated by the transcriptional activator.

Alternatively, affinity chromatography may be used to identify binding partners. For example, a polypeptide may be immobilised on a support (such as sepharose) and cell lysates passed over the column. Proteins binding to the immobilised polypeptide can then be eluted from the column and identified. Initially such proteins may be identified by N-terminal amino acid sequencing for example.

Alternatively, in a modification of the above technique, a fusion protein may be generated by fusing a polypeptide to a detectable tag, such as alkaline phosphatase, and using a modified form of immunoprecipitation as described by Flanagan and Leder (1990).

Methods for detecting candidate modulators of the PI3K/AKT pathway may also involve combining in an assay the candidate modulator with a suitable labelled substrate indicative of PI3K/AKT activity and monitoring the effect of the candidate modulator by changes in the substrate (which may be determined as a function of time). Suitable labelled substrates include those labelled for colourimetric, radiometric, fluorimetric or fluorescent resonance energy transfer (FRET) based methods.

The present invention also provides methods for screening for candidate modulators which may exert their modulatory effect on the PI3K/AKT pathway by altering expression of a polypeptide. In this case, such candidate modulators may be identified by comparing the level of expression of the polypeptide in the presence of the candidate modulator with the level of expression in the absence of the candidate modulator.

It will be appreciated that the above described methods are merely examples of the types of methods which may be employed to identify candidate modulators that are capable of interacting with, or modulating the activity of the PI3K/AKT pathway. Other suitable methods will be known to persons skilled in the art and are within the scope of the present invention.

Candidate modulators, for screening by the above methods, may be generated by a number of techniques known to those skilled in the art. For example, various forms of combinatorial chemistry may be used to generate putative non-peptide modulators. Additionally, techniques such as nuclear magnetic resonance (NMR) and X-ray crystallography may be used to model the structure of polypeptides and computer predictions used to generate possible modulators that will fit the shape of the substrate binding cleft of polypeptides involved in the PI3K/AKT pathway.

Modulators, including antagonists or agonists, of the PI3K/AKT pathway may include antibodies. Suitable antibodies include, but are not limited to polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanised antibodies, single chain antibodies and Fab fragments. Antibodies may be prepared from discrete regions or fragments of the polypeptide of interest. An antigenic polypeptide contains at least about 5, and preferably at least about 10, amino acids. Methods for the generation of suitable antibodies will be readily appreciated by those skilled in the art. For example, a suitable monoclonal antibody, typically containing Fab portions, may be prepared using the hybridoma technology described in Antibodies-A Laboratory Manual, (Harlow and Lane, eds.) Cold Spring Harbor Laboratory, N.Y. (1988), the disclosure of which is incorporated herein by reference.

Similarly, there are various procedures known in the art which may be used for the production of polyclonal antibodies. For the production of polyclonal antibodies, various host animals, including but not limited to rabbits, mice, rats, sheep, goats can be immunized by injection with a polypeptide. Further, the polypeptide can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Also, various adjuvants may be used to increase the immunological response, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.

Screening for the desired antibody can also be accomplished by a variety of techniques known in the art. Assays for immunospecific binding of antibodies may include, but are not limited to, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays, Western blots, precipitation reactions, agglutination assays, complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays as described in, for example, Ausubel et a/., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). Antibody binding may be detected by virtue of a detectable label on the primary antibody. Alternatively, a primary antibody may be detected by virtue of its binding with a secondary antibody or reagent which is appropriately labelled. A variety of methods is known in the art for detecting binding in an immunoassay and such methods are within the scope of the present invention.

Embodiments of the invention may also utilise antisense technology to inhibit the expression of a polynucleotide by blocking translation of the encoded polypeptide, wherein the polypeptide is involved in the PI3K/AKT pathway. Antisense technology takes advantage of the fact that nucleic acids pair with complementary sequences. Suitable antisense molecules can be manufactured by chemical synthesis or, in the case of antisense RNA, by transcription in vitro or in vivo when linked to a promoter, by methods known to those skilled in the art. For example, antisense oligonucleotides, typically of 18-30 nucleotides in length, may be generated which are at least substantially complementary across their length to a region of the nucleotide sequence of the polynucleotide of interest. Binding of the antisense oligonucleotide to their complementary cellular nucleotide sequences may interfere with transcription, RNA processing, transport, translation and/or mRNA stability. Suitable antisense oligonucleotides may be prepared by methods well known to those of skill in the art and may be designed to target and bind to regulatory regions of the nucleotide sequence or to coding (exon) or non-coding (intron) sequences. Typically antisense oligonucleotides will be synthesized on automated synthesizers. Suitable antisense oligonucleotides may include modifications designed to improve their delivery into cells, their stability once inside a cell, and/or their binding to the appropriate target. For example, the antisense oligonucleotide may be modified by the addition of one or more phosphorothioate linkages, or the inclusion of one or morpholine rings into the backbone (so-called 'morpholino' oligonucleotides).

An alternative antisense technology, known as RNA interference (RNAi), may also be used according to known methods in the art, as exemplified in, for example, WO 99/49029 and WO 01/70949 (the disclosures of which are incorporated herein by reference), to inhibit the expression of a polynucleotide. RNAi refers to a means of selective post-transcriptional gene silencing by destruction of specific mRNA by small interfering RNA molecules (siRNA). The siRNA is generated by cleavage of double stranded RNA, where one strand is identical to the message to be inactivated. Double-stranded RNA molecules may be synthesised in which one strand is identical to a specific region of an mRNA transcript and introduced directly. Alternatively corresponding dsDNA can be employed, which, once presented intracellular^ is converted into dsRNA. Methods for the synthesis of suitable molecule for use in RNAi and for achieving post-transcriptional gene silencing are known to those of skill in the art.

A further means of inhibiting nucleic acid and/or polypeptide expression may be achieved by introducing catalytic antisense nucleic acid constructs, such as ribozymes, which are capable of cleaving mRNA transcripts and thereby preventing the production of wildtype protein. Ribozymes are targeted to and anneal with a particular sequence by virtue of two regions of sequence complementary to the target flanking the ribozyme catalytic site. After binding the ribozyme cleaves the target in a site- specific manner. The design and testing of ribozymes which specifically recognise and cleave sequences of interest can be achieved by techniques well known to those in the art as disclosed in, for example, Lieber and Strauss, 1995, Molecular and Cellular Biology, 15:540-551, the disclosure of which is incorporated herein by reference.

Combination Therapies

Those skilled in the art will also appreciate that the compositions as herein disclosed may be administered as part of a combination therapy approach, employing one or more of the compositions as herein disclosed in conjunction with other therapeutic approaches to the methods disclosed herein. For such combination therapies, each component of the combination may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired therapeutic effect. When administered separately, it may be preferred for the components to be administered by the same route of administration, although it is not necessary for this to be so. Alternatively, the components may be formulated together in a single dosage unit as a combination product. Suitable agents which may be used in combination with the compositions of the present invention will be known to those of ordinary skill in the art.

In preferred embodiments, the methods of the present invention may further comprise administering an additional therapeutic agent or treatment. Exemplary additional therapeutic agents and treatments are disclosed herein.

Timing of Therapies

Those skilled in the art will appreciate that the compositions as herein disclosed may be administered as a single agent or as part of a combination therapy approach to the methods disclosed herein, either at diagnosis or subsequently thereafter, for example, as follow-up treatment or consolidation therapy as a compliment to currently available therapies for such treatments. The compositions as herein disclosed may also be used as preventative therapies for subjects who are genetically or environmentally predisposed to developing such diseases.

Although the present invention is exemplified herein with particular reference to the treatment of head and neck squamous cell carcinoma (HNSCC), it will be appreciated by persons of skill in the art that the compositions, methods, uses and kits described herein may also be suitable for the treatment of other types of squamous cell carcinoma, including but not limited to skin, prostate and cervix cancer.

The present invention will now be further described in greater detail by reference to the following specific examples, which should not be construed as in any way limiting the scope of the invention.

Examples

Example 1. Materials and methods

1.1 Chemicals

SAHA was obtained from Merck (NJ1USA). Valproic acid and α-tocopherol were purchased from Sigma (USA). Depsipeptide was obtained from Gloucester Pharmaceutocals (USA). U0126, LY294002, Wortmannin and Akt VIII were purchased from Cell Signaling (USA). ZVAD-fmk was purchased from Alexis Biochemicals (USA). Some of the drug preparations were made in cell culture-grade dimethylsulfoxide (DMSO) (Sigma, USA). The final concentrations of DMSO in the culture medium in all experiments were a maximum of 0.2% (v/v). Polyclonal antibodies recognizing total Akt, phosphorylated Akt (Ser 473), phosphorylated p44/p42 MAPK, phosphorylated GSK3βand α/βtubulin were obtained from Cell Signaling (USA). Polyclonal antibody recognizing Erk2 was purchased from Santa Cruz (USA). Polyclonal antibody recognizing Myc was purchased from Upstate (USA). Peroxidase- conjugated anti-rabbit IgG secondary antibody was purchased from GE Healthcare (UK). Chemicals and reagents were analytical grade or better.

1.2 Treatments

In co-treatments, LY294002, U0126, wortmannin and Akt VIII were added 10 minutes prior to SAHA. Vitamin E and ZVAD-fmk were added 30 minutes prior to other treatments.

1.3 Maintenance of cells

Normal human keratinocytes (HKs) were isolated and cultured from neonatal foreskins following circumcision as previously described (Jones SJ, Dicker AJ, Dahler AL, Saunders NA. E2F as a regulator of keratinocyte proliferation: implications for skin tumor development. J Invest Dermatol 1997 Aug;109(2): 187-93). The SCC9, SCC25 and Cal27 tumour cell lines were grown and maintained in Dulbecco's modified Eagle medium-F12 (Gibco), supplemented with Penicillin/Streptomycin/Glutamine solution (10ml/L, Gibco), hydrocortisone (0.4ug/ml), gentamycin (10ug/ml) and fetal bovine serum 5% (v/v) (Gibco). Culture flasks were maintained at 37° C in 5% CO2. Exponentially growing cells were detached from the culture flasks with 0.25% trypsin/ethylene diammine tetraacetic acid (EDTA) and seeded at different densities depending on the assay.

1.4 Proliferation assays

HKs and tumor cells were seeded at 104 cells per well and maintained in their respective culture media in 96-well plates overnight. Cells were exposed to different SAHA concentrations for 24 hours. A BrDu pulse (6h) was added (1OuM final concentration) after 18h of treatment. BrDU incorporation measurements were performed with the Cell Proliferation assay kit (Roche, US, #11647229001) according to the manufacturer's protocol.

1.5 LDH Cytotoxicity assays HKs and tumour cells were seeded at 104 cells per well and maintained in their respective culture media in 96-well plates overnight. Cells were exposed to different drug concentrations for 24 hours. Measurement of LDH lactate dehydrogenase release after treatments was performed with the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, US, #G1780) according to the manufacturer's protocol. Actual percentages of cell death for each experimental group were obtained according to the manufacturer's protocol. Briefly, complete lysis of all cells for each experimental group following distinct treatments was obtained by adding Triton X100 (final concentration 0.8%) to respective wells 45 minutes prior to harvesting supernatants for absorbance readings. Percentages were determined by the ratio between "experimental LDH release (OD490)" and "maximum LDH release (OD490)".

1.6 Transfections

SCC25 cells were cultured overnight in 6-well plates and transfected with the PUSE empty vector or the myrystoilated-AKT construct (Upstate, USA) using FuGENE 6 (Roche, USA) according to manufacturer's protocol. Twenty-four hours later, cells were subjected to Geneticin (G418, Gibco- Invitrogen, USA) selection at 200 ug/ml during 15 days. Stable transfectants were then subjected to distinct treatments and assays as described in the text.

1.7 Reactive oxygen species measurement

CM-H2DCFDA (C6827, Molecular Probes, USA) is a stable, nonfluorescent, and nonpolar compound that can diffuse through cell membranes. Once inside the cell, the acetyl groups are cleaved by cytosolic enzymes to form the polar non-fluorescent dichlorofluorescein (DCFH), which is rapidly oxidized to highly fluorescent dichlorofluorescein (DCF) in the presence of ROS. Cells were plated in 6- well plates at 2.5 x105 cells / well. After distinct treatments, cells were harvested, washed twice- with PBS, suspended in PBS with CM-H2DCFDA to a final concentration of 10 M, and incubated at 37 0C for 20 min. ROS accumulat ion was measured by fluorescence intensity (FL-1, 530 nm) of 10,000 cells using a FACS Calibur flow cytometer (Becton Dickinson). Mean fluorescence intensity was obtained by histogram statistics using the CellQuest software.

1.8 Propidium Iodide (Pl) viability assay

Cells were plated in 6-well plates at 2.5 x105 cells / well. After SAHA treatment, cells were harvested, washed and suspended in PBS and stained with 2 μg/ml of Pl for 10 minutes. Cell viability was assayed by flow cytometry (FACS Calibur -Becton Dickinson) and the FacsDiva software. 1.9 Statistical analysis

The number of experimental replicates is given in figure legends. Data were analyzed by Student's t-test when two groups are compared or ANOVA followed by post-hoc comparisons (Tukey's test) when multiple groups are compared.

1.10 Western blotting

After treatments cells were washed twice with cold phosphate-buffered saline (PBS) and lysed in 1% NP40, 1% Tryton X-100, 1% Sodium Deoxycholate, 1OmM Tris-HCI pH 7.5, 10OmM NaCI, 0.1% SDS, 5mM EDTA, supplemented with Complete protease and phosphatase inhibitory cocktails (Roche, USA). Samples (30-60 μg) were resolved by SDS-PAGE and transferred to lmmobilon P membranes (Millipore). The membranes were blocked with 5% nonfat milk in Tris-buffered saline, 0.1% Tween-20 (TBS-T) for one hour, incubated with primary antibodies (phospho-Erk 1:1000, phospho-Akt (S473) 1:1000, phospho-GSK3β1:1000, Erk2 1:8000, Akt 1:5000, myc 1:2000, α/βtubulin 1:1000, actin 1:8000) overnight at 4° C, washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000) for one hour. The reactions were developed using enhanced chemiluminescence (Pierce, USA).

Example 2. SAHA induces cell death in SCC cell lines but not in normal human keratinocytes

Proliferation was determined by quantitative measurement of BrDU (10M final concentration, 6 hours pulse) incorporation and the results were normalized with respect to the rate of proliferation in untreated cells (Fig 5A). After a 24h treatment period, SAHA (1 - 10 mM) caused a dose-dependent inhibition of proliferation in HNSCC cells and HKs. Previous studies have shown that HDACIs often exhibit tumour cell-specific cytotoxicity. To confirm these observations, the cytotoxic effects of SAHA upon HNSCC cell lines and HKs were tested. Cytotoxicity was determined by quantitative measurement of lactate dehydrogenase (LDH) release to the culture media and the results were normalized with respect to the rate of LDH release in untreated cells. SAHA induced cell death in a dose-and cell type-dependent manner in all tested cancer cell lines but in contrast to the effects upon proliferation, did not induce cell death in HKs (Fig. 1; Fig. 5B). The actual percentage of cell death induced by 24h SAHA (5 μM) treatment in SCC25 cells was measured through LDH release and Pl staining assays. Both methods yielded similar results (Figs. 5C, 5D). These data are consistent with earlier reports that HDACIs are cancer cell specific-cytotoxic agents.

Example 3. Inhibition of the PI3K-Akt pathway increases SAHA-induced cytotoxicity in cancer cells

To investigate the involvement of the PI3K and MAPK signalling pathways in SAHA-induced cell death, SCC25 cells were treated with SAHA in the presence of LY294002, an inhibitor of PI3K or U0126, or an inhibitor of MEK. Inhibition of PI3K caused a marked increase in SAHA-induced cell death as compared to cells treated with SAHA alone. On the other hand, inhibition of MEK did not affect cytotoxicity induced by SAHA (Fig 2A). Importantly, treatment with LY294002 or U0126 alone did not induce cell death.

The inventors used the phosphorylation level of Akt at Ser-473, which is required for maximal activation of this kinase, as a marker for the activity status of the PI3K-Akt pathway. Western blots of lysates from SCC25 cells showed that 24hrs of SAHA treatment caused a partial inhibition of Akt phosphorylation. A similar level of inhibition was observed in cells treated with LY294002 alone. In contrast, phosphorylation of Akt was completely abrogated in cells treated simultaneously with both drugs (Fig 2B). Taken together these data indicate a specific involvement of the PI3K-Akt pathway in SAHA-induced cytotoxicity and show that pharmacological inhibition of this pathway synergizes with SAHA, resulting in a marked increase in cell death.

To further investigate if disruption of these pathways was able to modulate SAHA-induced cell death, SCC25 cells were treated for 24 h with SAHA alone or in combination with LY or U0126, respectively a PI3K and a MEK inhibitor (Fig. 6A). Co-treatment with LY induced a marked increase in SAHA cytotoxicity. On the other hand, U0126 co-treatment did not affect SAHA cytotoxic effects. Treatment with LY or U0126 alone did not induce cell death as compared to untreated cells. The inhibitory effect of LY and U0126 upon Akt and ERK respectively was confirmed by western blots of total lysates from SCC25 cells treated for 10 minutes with the inhibitors alone or in combination with SAHA and probed against phospho-Akt (S473) and phospho-p42/44 antibodies (Fig. 6B). Next, the activation status of Akt and ERK following 24 h of the distinct treatments was measured, similarly to the cell death assays (Fig. 6C). While treatment with SAHA or the inhibitors alone caused a small inhibition of Akt and ERK activities, SAHA treatment in combination with LY or U0126 induced a pronounced inhibition of Akt and ERK respectively. Since the combination treatment SAHA/LY related to potentiation of SAHA-induced cell death and robust inhibition of Akt , the dynamics of Akt phosphorylation status was investigated in more detail by western blot assays at distinct time points (Fig. 6D). Akt inhibition induced by SAHA treatment in the presence of LY was much more intense and durable (lanes 3, 6, 9, 12, 15) as compared to cells treated with SAHA alone (lanes 2, 5, 8, 11, 14). On the other hand, LY treatment alone caused an intense but short-lived Akt inhibition (lanes 4, 7, 10, 13, 16). These data indicate that potentiation of SAHA cytotoxic effects mediated by LY correlates with a long and robust Akt inhibition, which is not achieved by isolated drug treatments.

To further confirm the observation that PI3K-Akt pathway inhibition enhances HDACI cytotoxicity, the effects of SAHA in combination with Wortmannin, a PI3K inhibitor chemically unrelated to LY, or with an isoform-specific Akt 1/2 inhibitor (Akt VIII), were examined. Both combination treatments caused a significant increase in cell death when compared to the effects of SAHA alone. Similar to LY, treatment with Wortmannin or Akt VIII alone did not induce cell death in SCC25 cells (Figs. 6E, 6F). Moreover, SAHA/Wortmannin or SAHA/Akt VIII combinations also induced a more intense inhibition of Akt phosphorylation as compared to cells treated with Wortmannin or Akt VIII alone (Fig. 6G).

Previous studies have shown that SAHA can induce cell death through caspase-dependent and independent pathways in transformed cells. In this context, the effect of pre-treatment with a pan- caspase inhibitor, ZVAD-FMK, upon SCC25 cells subjected to SAHA or SAHA/LY treatments was tested. Irrespective of the treatment, pan-caspase inhibition completely abrogated cell death, an indication that both treatments induce a caspase-dependent cell death (Fig.6H).

Example 4. PI3K inhibition synergizes with distinct histone deacetylase inhibitors in distinct cell lines.

Although HNSCC are classified in different groups based on histological features, it is known that these tumours are constituted by a heterogeneous set of cell populations, which renders each tumour a unique pathologic process and, to date, has prevented the development of broadly effective therapeutic regimens. In addition, different HDACIs present specific structural characteristics and may inhibit distinct HDACs with specific selectivity.

To test if the observed synergy between PI3K inhibitors and SAHA was restricted to the SCC25 cell line, the effect of this combination upon Cal27 cells was tested. Figure 3A shows that Cal27 cells responded to the treatments similarly to SCC25 cells. In addition, SCC25 cells were tested with distinct HDACIs (depsipeptide or valproic acid) alone or in combination with LY294002 to investigate if these drugs would also synergize. Figures 3B and 3C show that the cytotoxicity of both depsipeptide and valproic acid were markedly enhanced by the addition of LY294002. Similar to SCC25 cells, CaI 27 cells were sensitive to SAHA-induced cell death and this effect was significantly increased by co-treatment with LY, but not U0126 (Fig. 6B). In addition, SCC25 cells were tested with two structurally dissimilar HDACIs chemically unrelated to SAHA, valproic acid or depsipeptide, alone or in combination with LY. Similar to the effect observed with SAHA co-treatments, cytotoxicity mediated by valproic acid and depsipeptide was markedly increased by LY (Fig. 6A). These data indicate that LY enhances cytotoxicity mediated by a range of structurally unrelated HDACIs and that these effects are conserved between distinct HNSCC cell lines. Taken together, these results suggest that synergy between a PI3K inhibitor and HDACIs is a more general phenomenon that could be used to enhance the potency of these drugs in distinct contexts.

Example 5. Combination treatments do not induce cell death in normal human keratinocytes

Normal human keratinocytes (HK) obtained from neonatal foreskins were treated with SAHA alone or in combination with LY294002 or U0126. In contrast to cancer cell lines, SAHA or TSA treatment had no effect upon viability of HK even when combined with LY294002 (Figs 4A, B). To test whether the activation status of the PI3K-Akt pathway related to HKs resistance to SAHA treatment, the level of Akt (Ser 473) phosphorylation following SAHA treatment was measured by western blots in SCC25 cells and HKs. In contrast to cancer cells, SAHA treatment was unable to inhibit Akt activity in HKs (Fig 4C). These results indicate that combination treatments present significant selectivity towards SCC cells and further implicate the PI3K-Akt pathway as an important mediator of SAHA-induced cell death.

The relative resistance of normal cells to cytotoxicity induced by SAHA and other HDACIs when compared to cancer cells is one of the most important characteristics of this class of drugs in the clinical context. Indeed, the data of Fig. 1 and Fig. 5B show that this cancer selectivity is also present in the experimental model. The cyclin-/CIP1> dependent kinase inhibitor CDKN1A (encoding p21WAF1) is upregulated by SAHA and other HDACIs, an effect that correlates with arrest of cells in the G1 cell- cycle phase and was suggested to induce protection from cytotoxic effects mediated by HDACIs. Consistent with the ability of SAHA to induce inhibition of proliferation in HKs and SCC25 cells (Fig. 5A), western blots showed that p21 expression was induced to a similar extent in both normal and cancer cells (Fig. 9A). Most importantly, HKs were still resistant to SAHA treatment in combination with PI3K or MEK inhibitors. Similar to SAHA treatment alone, cytotoxicity assays showed that none of the combination treatments were able to induce cell death in HKs (Fig. 9B). Moreover, in contrast to cancer cells, SAHA/LY combination treatment did not induce inhibition of Akt in HKs (Fig. 9C). These results indicate that the cancer selectivity of SAHA is maintained in the presence of LY and that SAHA/LY treatment induces a cancer cell-selective inhibition of the Akt pathway. Taken together, these data reinforce the therapeutic benefits of combining HDACIs with PI3-Akt inhibitors.

Example 6. PI3K-Akt inhibitors potentiate caspase-dependent cell death induced by SAHA

PI3K-Akt and MAPK signalling pathways commonly relate to cell proliferation and survival and are often deregulated in HNSCC. To investigate if disruption of these pathways was able to modulate SAHA-induced cell death, SCC25 cells were treated for 24 h with SAHA alone or in combination with LY or U0126, respectively a PI3K and a MEK inhibitor (Fig. 6A). Co-treatment with LY induced a marked increase in SAHA cytotoxicity. On the other hand, U0126 co-treatment did not affect SAHA cytotoxic effects. Treatment with LY or U0126 alone did not induce cell death as compared to untreated cells. The inhibitory effect of LY and U0126 upon Akt and ERK respectively was confirmed by western blots of total lysates from SCC25 cells treated for 10 minutes with the inhibitors alone or in combination with SAHA and probed against phospho-Akt (S473) and phospho-p42/44 antibodies (Fig. 6B). The activation status of Akt and ERK following 24h of the distinct treatments was measured similarly to the cell death assays (Fig. 6C). While treatment with SAHA or the inhibitors alone caused a small inhibition of Akt and ERK activities, SAHA treatment in combination with LY or U0126 induced a pronounced inhibition of Akt and ERK, respectively. Since the combination treatment SAHA/LY related to potentiation of SAHA-induced cell death and robust inhibition of Akt , the dynamics of Akt phosphorylation status was investigated in more detail by western blot assays at distinct time points (Fig. 6D). Akt inhibition induced by SAHA treatment in the presence of LY was much more intense and durable (lanes 3, 6, 9, 12, 15) as compared to cells treated with SAHA alone (lanes 2, 5, 8, 11 , 14). On the other hand, LY treatment alone caused an intense but short-lived Akt inhibition (lanes 4, 7, 10, 13, 16). These data indicate that potentiation of SAHA cytotoxic effects mediated by LY correlates with a long and robust Akt inhibition, which is not achieved by isolated drug treatments.

To further confirm the observation that PI3K-Akt pathway inhibition enhances SAHA cytotoxicity, we examined the effects of SAHA in combination with Wortmannin, a PI3K inhibitor chemically unrelated to LY, or with an isoform-specific Akt 1/2 inhibitor (Akt VIII). Both combination treatments caused a significant increase in cell death when compared to the effects of SAHA alone. Similar to LY, treatment with Wortmannin or Akt VIII alone did not induce cell death in SCC25 cells (Figs. 6E, F). Moreover, SAHA/Wortmannin or SAHA/Akt VIII combinations also induced a more intense inhibition of Akt phosphorylation as compared to cells treated with Wortmannin or Akt VIII alone (Fig. 6G). Previous studies have shown that SAHA can induce cell death through caspase-dependent and independent pathways in transformed cells. Pre-treatment with a pan-caspase inhibitor, ZVAD-FMK, upon SCC25 cells subjected to SAHA or SAHA/LY treatments was tested. Irrespective of the treatment, pan-caspase inhibition completely abrogated cell death, an indication that both treatments induce a caspase-dependent cell death (Fig. 6H).

Example 7. Ectopic expression of constitutively active Akt attenuates increase in cytotoxicity mediated by SAHA/LY co-treatment

To investigate the functional importance of Akt activity inhibition in the potentiation of SAHA- induced cell death mediated by co-treatment with LY, SCC25 cells were stably transfected with a constitutively active myc-tagged myristoylated Akt (myr-Akt), or with the correspondent "empty vector", and treated with SAHA alone or in combination with LY. Expression of myr-Akt caused a significant attenuation of cytotoxicity induced by the combination treatment (Fig. 7A). Western blot assays of total lysates confirmed expression of myr-Akt and hyperactivation of the Akt pathway in transfected SCC25 cells as measured by phosphorylation levels of GSK3β, a well established Akt target in untreated and SAHA treated cells (Fig. 7B). Taken together with the results presented in Figs. 6A-6D, these. data indicate that inhibition of Akt activity directly relates to the marked increase in SAHA-induced cell death mediated by SAHA/LY combined treatment.

Example 8. Enhanced cell death induced by SAHA/LY combination in SCC cells correlates with ROS accumulation

Previous studies indicated that SAHA and other HDACIs induce ROS accumulation in several cell types, and that this effect is relevant to SAHA-induced cell death. In addition, PI3K inhibitors were shown to potentiate peroxide accumulation induced by chemotherapy. Therefore, the effect of LY treatment upon SAHA-induced ROS accumulation was investigated. SAHA treatment induced accumulation of ROS in SCC25 cells and this effect was enhanced by co-treatment with LY. In contrast, LY treatment alone induced only a small increase in ROS levels as compared to untreated cells (Fig 8A). Pre-treatment with the anti-oxidant α-tocopherol (vitamin E) caused a small decrease in levels of ROS accumulation induced by SAHA and SAHA/LY treatments and partially protected against cytotoxic effects in a dose-dependent manner (Figs. 8B, 8C). These data indicate that ROS accumulation induced by SAHA is potentiated by LY and suggest that ROS accumulation is involved in both SAHA- and SAHA/LY-induced cytotoxicity. Example 9. LY294002 potentiates cell death induced by distinct HDACIs and is not cell type-specific

To address the generality of cell death increase caused by PI3K-Akt inhibition, Cal27 cells, a distinct HNSCC cell line, were treated with SAHA alone or in combination with LY or U0126. Similar to SCC25 cells, CaI 27 cells were sensitive to SAHA-induced cell death and this effect was significantly increased by co-treatment with LY, but not U0126 (Fig. 1OA). In addition, SCC25 cells were treated with two structurally dissimilar HDACIs chemically unrelated to SAHA, valproic acid or depsipeptide, alone or in combination with LY. Similar to the effect observed with SAHA co-treatments, cytotoxicity mediated by valproic acid and depsipeptide was markedly increased by LY (Fig. 10B). These data show that LY enhances cytotoxicity mediated by a range of structurally unrelated HDACIs and that these effects are conserved between distinct HNSCC cell lines.

Claims

WE CLAIM:
1. A composition for preventing or treating a squamous cell carcinoma in a subject, wherein the composition comprises a histone deacetylase inhibitor (HDACI) and a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway.
2. . The composition according to claim 1, wherein the HDACI is selected from the group comprising hydroxamic acids, cyclic peptides, benzamides and aliphatic acids.
3. The composition according to claim 1, wherein the HDACI is selected from the group comprising suberoylanilide hydroxamic acid (SAHA), valproic acid, depsipeptide, LAQ824/LBH589, CI994, MS275 and MGCD0103.
4. The composition according to any one of claims 1 to 3, wherein the HDACI is SAHA.
5. The composition according to any one of claims 1 to 4, wherein the modulator of the PI3K/AKT pathway is an inhibitor of the PI3K/AKT pathway.
6. The composition according to any one of claims 1 to 4, wherein the modulator of the PI3K/AKT pathway is selected from the group comprising LY294002, Wortmannin analogues, PEG
Wortmannin, PX-866, SF1124, SF1126, BEZ235, BGT226, BKM120, TGX115 and TGX126.
7. The composition according to any one of claims 1 to 6, wherein the modulator of the PI3K/AKT pathway is LY294002.
8. The composition according to any one of claims 1 to 7, wherein the squamous cell carcinoma is head and neck squamous cell carcinoma (HNSCC).
9. The composition according to claim 8, wherein the HNSCC is an upper aerodigestive tract cancer.
10. The composition according to claim 9, wherein the upper aerodigestive tract cancer is selected from the group comprising nasal cancer, paranasal cancer, pharyngeal, nasopharyngeal, mouth cancer, oropharyngeal cancer, laryngeal cancer, oesophageal cancer and cutaneous SCC arising on the head and neck region or lip.
11. The composition according to any one of claims 1 to 10, wherein the subject is human.
12. The composition according to any one of claims 1 to 11, further comprising a pharmaceutically acceptable carrier, excipient or diluent.
13. The composition according to any one of claims 1 to 12, wherein the composition sensitizes the squamous cell carcinoma to the HDACI.
14. The composition according to any one of claims 1 to 12, wherein the composition is synergistic.
15. A method for preventing or treating a squamous cell carcinoma in a subject, wherein the method comprises administering to the subject the composition according to any one of claims 1 to 14, wherein the administering prevents or treats the squamous cell carcinoma in the subject.
16. The method according to claim 15, wherein the squamous cell carcinoma is head and neck squamous cell carcinoma (HNSCC).
17. The method according to claim 16, wherein the HNSCC is an upper aerodigestive tract cancer.
18. The method according to claim 17, wherein the upper aerodigestive tract cancer is selected from the group comprising nasal cancer, pharyngeal, nasopharyngeal, paranasal cancer, mouth cancer, oropharyngeal cancer, laryngeal cancer, oesophageal cancer and cutaneous SCC arising on the head and neck region or lip.
19. The method according to any one of claim 15 to 18, wherein the subject is human.
20. A method for preventing or treating a squamous cell carcinoma in a subject, wherein the method comprises administering to the subject a histone deacetylase inhibitor (HDACI) and a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway, wherein the administering prevents or treats the squamous cell carcinoma in the subject.
21. A method for sensitizing a squamous cell carcinoma in a subject to treatment with a histone deacetylase inhibitor (HDACI), wherein the method comprises administering a modulator of the PI3K/AKT pathway.
22. The method according to claim 20 or claim 21, wherein the HDACI is selected from the group comprising hydroxamic acids, cyclic peptides, benzamides and aliphatic acids.
23. The method according to claim 20 or claim 21, wherein the HDACI is selected from the group comprising suberoylanilide hydroxamic acid (SAHA), valproic acid, depsipeptide, LAQ824/LBH589, CI994, MS275 and MGCD0103.
24. The method according to any one of claims 20 to 23, wherein the HDACI is SAHA.
25. The method according to any one of claims 20 to 24, wherein the modulator of the PI3K/AKT pathway is an inhibitor of the PI3K/AKT pathway.
26. The method according to any one of claims 20 to 24, wherein the modulator of the PI3K/AKT pathway is selected from the group comprising LY294002, Wortmannin analogues, PEG Wortmannin, PX-866, SF1124, SF1126, BEZ235, BGT226, BKM120, TGX115 and TGX126.
27. The method according to any one of claims 20 to 26, wherein the modulator of the PI3K/AKT pathway is LY294002.
28. The method according to any one of claims 20 to 27, wherein the squamous cell carcinoma is head and neck squamous cell carcinoma (HNSCC).
29. The method according to claim 28, wherein the HNSCC is an upper aerodigestive tract cancer.
30. The method according to claim 29, wherein the upper aerodigestive tract cancer is selected from the group comprising nasal cancer, paranasal cancer, pharyngeal, nasopharyngeal, mouth cancer, oropharyngeal cancer, laryngeal cancer, oesophageal cancer and cutaneous SCC arising on the head and neck region or lip.
31. The method according to any one of claims 20 to 30, wherein the subject is human.
32. The method according to any one of claims 20 or 22 to 30, wherein the HDACI is administered simultaneously with, sequentially with or separately to the modulator of the PI3K/AKT pathway.
33. The method according to any one of claims 20 or 22 to 31, wherein the HDACI and the modulator of the PI3K/AKT pathway act synergistically.
34. The method according to any one of claims 22 to 33, further comprising administering to the subject a HDACI.
35. Use of a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway and a histone deacetylase inhibitor (HDACI) for preventing or treating a squamous cell carcinoma in a subject, wherein the use comprises simultaneously, sequentially or separately administering the modulator of the PI3K/AKT pathway and the HDACI.
36. Use of a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway and a histone deacetylase inhibitor (HDACI) in the preparation of a medicament for preventing or treating a squamous cell carcinoma in a subject.
37. A modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway for use in sensitizing a squamous cell carcinoma in a subject to treatment with a histone deacetylase inhibitor
(HDACI).
38. A kit for:
(a) preventing or treating a squamous cell carcinoma in a subject; or
(b) sensitizing a squamous cell carcinoma in a subject to treatment with a histone deacetylase inhibitor (HDACI) wherein the kit comprises:
(c) the composition according to any one of claims 1 to 14; or (d) a histone deacetylase inhibitor (HDACI) and a modulator of the phosphatidylinositol-3- kinase (PI3K)/AKT pathway.
39. A method for screening for a modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway that sensitizes a squamous cell carcinoma in a subject to treatment with a histone deacetylase inhibitor (HDACI)1 wherein the method comprises culturing a tumour cell line with the HDACI in the presence and in the absence of the modulator of the PI3K/AKT pathway, wherein increased cell death in the tumour cell line cultured with both the HDACI and the modulator of the PI3K/AKT pathway compared with the tumour cell line cultured with the HDACI without the modulator of the PI3K/AKT pathway is indicative that the modulator of the PI3K/AKT pathway sensitizes the squamous cell carcinoma in the subject to treatment with the HDACI.
40. A modulator of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway that sensitizes a squamous cell carcinoma in a subject to treatment with a histone deacetylase inhibitor (HDACI) screened by the method of claim 39.
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US8367663B2 (en) 2009-01-08 2013-02-05 Curis, Inc. Phosphoinositide 3-kinase inhibitors with a zinc binding moiety
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US8906909B2 (en) 2009-01-08 2014-12-09 Curis, Inc. Phosphoinositide 3-kinase inhibitors with a zinc binding moiety
WO2010130779A3 (en) * 2009-05-15 2013-03-28 Novartis Ag Combination of (a) a phosphoinositide 3-kinase inhibitor and (b) an antidiabetic compound for use in the treatment of proliferative diseases
EP2557923A4 (en) * 2010-04-16 2013-10-23 Curis Inc Treatment of cancers having k-ras mutations
EP2557923A1 (en) * 2010-04-16 2013-02-20 Curis, Inc. Treatment of cancers having k-ras mutations
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GB2493982A (en) * 2011-08-26 2013-02-27 Univ Leicester Disease treatments involving histone deacetylases
WO2013049300A1 (en) * 2011-09-30 2013-04-04 Dana-Farber Cancer Institute, Inc. Method of treating mucoepidermoid carcinoma
WO2014150996A1 (en) * 2013-03-15 2014-09-25 Cba Pharma, Inc. Cancer treatment
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WO2014181252A1 (en) * 2013-05-07 2014-11-13 Novartis Ag Combination of a pi3 kinase inhibitor with paclitaxel for use in the treatment or prevention of a cancer of the head and neck
AU2014264318B2 (en) * 2013-05-07 2017-03-16 Novartis Ag Combination of a PI3 kinase inhibitor with paclitaxel for use in the treatment or prevention of a cancer of the head and neck
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