WO2009052467A1 - Methods of identifying pi-3-kinase inhibitor resistance - Google Patents

Methods of identifying pi-3-kinase inhibitor resistance Download PDF

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Publication number
WO2009052467A1
WO2009052467A1 PCT/US2008/080429 US2008080429W WO2009052467A1 WO 2009052467 A1 WO2009052467 A1 WO 2009052467A1 US 2008080429 W US2008080429 W US 2008080429W WO 2009052467 A1 WO2009052467 A1 WO 2009052467A1
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ras
px
mutant
method
pi
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PCT/US2008/080429
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French (fr)
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Garth Powis
Nathan Ihle
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Board Of Regents Of The University Of Texas System
Arizona Board Of Regents, Acting On Behalf Of The University Of Arizona
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Priority to US60/981,307 priority
Application filed by Board Of Regents Of The University Of Texas System, Arizona Board Of Regents, Acting On Behalf Of The University Of Arizona filed Critical Board Of Regents Of The University Of Texas System
Publication of WO2009052467A1 publication Critical patent/WO2009052467A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • A61K31/66Phosphorus compounds
    • A61K31/683Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088Compounds having three or more nucleosides or nucleotides
    • A61K31/7105Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links

Abstract

The invention described herein is directed to methods for identifying an individual who may be resistant or susceptible to phosphatidylinositol-3-kinase inhibitors, methods of treating an individual by identifying individuals who are resistant or susceptible to phosphatidylinositol-3-kinase inhibitors, and kits for testing individuals for resistance or susceptibility to phosphatidylinositol-3-kinase inhibitors.

Description

A. Title:

METHODS OF IDENTIFYING PI-3-KINASE INHIBITOR RESISTANCE

B. Cross-Reference to Related Applications:

[0001] This application claims priority to U.S. Provisional Application No. 60/981,307 filed October 19, 2007, herein incorporated by reference in its entirety.

C. Government Interests:

[0002] The United States government may have certain rights to this invention pursuant to Grant Nos. CA52995, CA090821, CAl 7094, CA95060 (GP) and CA99031 (GBM) from the National Institute of Health.

D. Parties to a Joint Research Agreement: Not applicable

E. Incorporation by Reference of Material submitted on a Compact Disc: Not applicable

F. Background

1. Field of Invention: Not applicable

2. Description of Related Art: Not applicable

G. Summary:

[0003] Embodiments of the present invention relate to methods of diagnosing and treating cancer patients. In particular, embodiments of the present invention are directed to methods for determining which cancer patients will benefit from treatment with a phosphatidylinositol-3-kinase (PI-3 kinase) inhibitor.

[0004] Embodiments of the present invention provide diagnostic and prognostic methods for predicting the effectiveness of treatment of a cancer patient with a PI-3 kinase inhibitor. Based upon the surprising results that the sensitivity of tumor cell growth to inhibition by PI-3 kinase inhibitors is dependent on whether such tumor cells express mutant Ras, methods have been devised for determining mutant Ras expression to predict the sensitivity and/or resistance of tumor cells to PI-3 kinase inhibitors.

[0005] One embodiment of the present invention provides a method of predicting the sensitivity of tumor cell growth to inhibition by an PI-3 kinase inhibitor comprising assessing or characterizing the Ras expressed by a tumor cell; and predicting the sensitivity of tumor cell growth to inhibition by a PI-3 kinase inhibitor, wherein expression of wild-type Ras correlates with sensitivity to inhibition by PI-3 kinase inhibitors and wherein expression of mutant Ras correlates with resistance to inhibition by PI-3 kinase inhibitors.

[0006] Embodiments of the present invention also provide for methods for treating cancer patients with PI-3 kinase inhibitors that incorporate the above methodology. Thus, embodiments of the present invention further provide methods for treating tumors or tumor metastases in a patient comprising the steps of diagnosing a patient's likely responsiveness to an PI-3 kinase inhibitor by assessing whether the tumor cells express mutant Ras or wild-type Ras, and administering to said patient a therapeutically effective amount of a PI-3 kinase inhibitor.

[0007] One embodiment of the present invention is a method for determining PI-3 kinase inhibitor resistance comprising identifying the presence of mutant Ras from an individual, wherein the presence of mutant Ras indicates resistance to PI-3 kinase inhibitor therapy.

[0008] A further embodiment of the present invention is a method for determining PI-3 kinase inhibitor susceptibility comprising identifying the presence of wild-type Ras from an individual, wherein the presence of wild-type Ras indicates susceptibility to PI-3 kinase inhibitor therapy. [0009] A further embodiment of the present invention is a method of treating cancer in an individual comprising identifying the absence of mutant Ras in the individual and administering an effective amount of a PI-3 kinase inhibitor. H. Description of Drawings:

[OOIOJ For a better understanding of the disclosure and to show how the same may be carried into effect, reference will now be made to the accompanying drawings. It is stressed that the particulars shown are by way of example only and for purposes of illustrative discussion of the preferred embodiments of the present disclosure only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show experimental details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the accompanying drawings:

[0011] Fig. 1 depicts the PI-3-kinase signaling pathway and PX-866's interaction with this pathway.

[0012] Fig. 2 is a graph depicting the Akt/PKB activity in vivo in sensitive and resistant xenographs following administration of PX-866.

[0013] Fig. 3 is a reverse phase protein array analysis of sensitive and resistant cell lines in 2-D culture.

[0014] Fig. 4 is a graph depicting percent of inhibition of colony formation with PX- 866 in PI-3-kinase pathway specific ras constructs. [0015] Fig. 5 depicts the sensitivity to PX-866 induced cell death in PI-3-K pathway specific ras constructs.

[0016] Fig. 6 depicts a model for the combination of PX-866 with other specific kinase inhibitors.

[0017] Fig. 7 depicts the effects of PX-866 on cell line derived xenografts. Fig. 7A- CeIl line derived xenografts were grown subcutaneously in female SCID mice. Upon reaching 200mm3 the mice were treated with PX-866, 2.5-3.0 mg/kg every other day administered po. At the end of treatment the tumor volume was expressed as a percentage of the increase in the vehicle alone treated tumor volume (T/C%). * p <0.05. Tumor responses were characterized as No response (T/C >70%), Low response (T/C 35-69%), or Antitumor (T/C <35%). The Ras, Raf, PIK3A, LKBl and PTEN mutation status of the tumors is shown: Fig. 7B, phospho-Ser47 - Akt levels measured in representative tumors removed from mice treated with PX-866 2.5-3 mg/kg po at the end of treatment, Images were taken from different fields on the same film.

[0018] Fig. 8 depicts is a protein analysis of sensitive and resistant cell lines. Fig. 8A, Cell lines were analyzed by 52 validated antibodies in a reverse phase protein array (RPPA). Protein levels were quantified and arranged in a heat map, red indicating high expression, black median and green low expression. Fig. 8B, Analysis of expression of components of the PI-3- kinase/Akt pathway and correlation with in vivo antitumor response. The lower panel shows a plot of phospho-Akt levels against in vivo antitumor response. Fig. 8C, Levels of c-Myc and cyclin B proteins showing differences between sensitive and resistant cell lines. * p < 0.05

[0019] Fig. 9 depicts the analysis of RPPA findings and clonogenic potential of HCT- 1 16 H- Ras construct cells. Fig. 9A, Ras constructs used in the study and Western blot analysis of cellular PI-3-kinase activity by phospho-Ser473-Akt, cyclin B and C-Myc. Fig. 9B, Colony formation assay performed on H-Ras construct HCT-1 16 cells treated with 0.5 μM PX-866. * p<0.05 compared to wild type cells.

[0020J Fig. 10 depicts apoptosis and in vivo effects of PX-866 on HCT-1 16 H- Ras construct cells. Fig. 1 OA. Trypan blue and flow cytometry analysis of annexin positive cells treated with 0.5 μM PX-866. * p<0.05 compared to wild type cells. Fig. 1OB, Comparison of final volumes of vehicle (white bars) or PX-866 (black bars) treated tumors, * p<0.05 of treated compared to control.

[0021] Figure 1 1 depicts signaling in resistant and sensitive lines. Diagram showing the interactions of the cell signaling studied in sensitive and resistant lines. Signaling in PX-866 sensitive tumors comes from an increased reliance on the PI-3 -kinase pathway, arising from aberrant activation through growth factors (GF) or mutated components of the pathway itself (*) . Tumors with an activated Ras protein show a minimal response to inhibition of the PI-3 -kinase pathway due to a shared reliance on alternate signaling pathways including the Raf and RaIGDS pathways.

[0022] Figure 12 A, 12B, 12C 12D and 12E depict the structures of certain PI-3 kinase inhibitors in accordance with embodiments of the present invention. I. Detailed Description:

[0023] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular processes, compositions or methodologies described, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0024] It must also be noted that as used herein and in the appended claims, the singular forms "a", "an", and '"the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.

[0025] As used herein, the term "about" means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%- 55%.

[0026] "Administering" when used in conjunction with a therapeutic means to administer a therapeutic systemically or locally, such as directly into or onto a target tissue or to administer a therapeutic to a patient whereby the therapeutic positively impacts the tissue to which it is targeted. Thus, as used herein, the term "administering", when used in conjunction with a phosphatidylinositol-3-kinase (PI-3-kinase) inhibitor, can include, but is not limited to, providing a PI-3-kinase inhibitor into or onto the target tissue; providing a PI-3-kinase inhibitor systemically to a patient by, e.g., intravenous injection whereby the therapeutic reaches the target tissue or cells. "Administering" a composition may be accomplished by injection, topical administration, oral administration or by other methods alone or in combination with other known techniques. [0027J The term "cancer" in an animal refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an animal, or may circulate in the blood stream as independent cells, such as leukemic cells.

[0028] "Abnormal cell growth", as used herein, unless otherwise indicated, refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition). This includes the abnormal growth of: (1) tumor cells (tumors) that proliferate by expressing a mutated tyrosine kinase or overexpression of a receptor tyrosine kinase; (2) benign and malignant cells of other proliferative diseases in which aberrant tyrosine kinase activation occurs; (4) any tumors that proliferate by receptor tyrosine kinases; (5) any tumors that proliferate by aberrant serine/threonine kinase activation; and (6) benign and malignant cells of other proliferative diseases in which aberrant serine/threonine kinase activation occurs.

[0029] The term "individual" as used herein includes, but is not limited to, humans and non-human vertebrates such as wild, domestic and farm animals. The terms "patient" and "subject" are interchangeable and may be taken to mean any living organism which may be treated with compounds of the present disclosure. As such, the terms "patient" and "subject" may include, but are not limited to, any non-human mammal, any primate or a human.

[0030] The term "inhibiting" includes the administration of a compound of the present invention to prevent the onset of the symptoms, alleviating the symptoms or eliminating the disease, condition or disorder. [0031] '"Optional" or "optionally" may be taken to mean that the subsequently described structure, event or circumstance may or may not occur, and that the description includes instances where the events occurs and instances where it does not.

[0032] By "pharmaceutically acceptable", it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0033] The term '"pharmaceutical composition" shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

[0034] As used herein, the term "therapeutic" means an agent utilized to treat, combat, ameliorate, prevent or improve an unwanted condition or disease of a patient. In part, embodiments of the present invention are directed to the treatment of cancer and/or the amelioration of the symptoms of cancer or the decrease in proliferation of cells.

[0035] A "therapeutically effective amount" or "effective amount" of a composition is a predetermined amount calculated to achieve the desired effect, i.e., inhibiting, blocking or reversing the activation, migration or proliferation of cells or to effectively treat cancer or ameliorate the symptoms of cancer. A therapeutically effective amount of a wortmannin analog of this invention is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient to achieve an effective concentration in the plasma or serum or an effective local concentration in a target tissue. Effective amounts of compounds of the present invention can be measured by improvements in tumor size, tumor burden or symptoms experienced by the patient being treated. The activity contemplated by the present methods includes both medical therapeutic and/or prophylactic treatment, as appropriate. The specific dose of a compound administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the route of administration, and the condition being treated.

[0036] An important cell survival mechanism for many cancers is mediated by the phosphatidylinositol-3 -kinase (PI-3-kinase) /Akt (protein kinase B) signaling pathway. Class I PI-3 -kinases phosphorylate membrane phosphatidylinositols to give PI(3,4,5)P3, which then binds and recruits the serine/threonine kinase Akt through its N-terminal pleckstrin homology (PH) domain, in a process reversed by PTEN phosphatase . The membrane associated Akt is activated by Thr308 phosphorylation by membrane associated phosphoinositide dependent kinase- 1 (PDKl) and Ser473 phosphorylation most likely through the TORC2 complex. Activated Akt detaches from the plasma membrane and moves to the cytoplasm and the nucleus where it phosphorylates a battery of targets leading to changes in cellular functions.

[0037] Aberrant activation of phosphatidylinositol-3-kinase (PI-3-kinase) has been shown to contribute to abnormal cell proliferation and tumorigenesis by increasing cellular levels of phosphoinositide polyphosphate (PI(3,4,5)P or PIP3) and inducing the formation of molecular complexes that act in signal transduction pathways to suppress apoptosis and promote cell survival. Activation of the PI-3-kinase/ Akt pathway can occur due to upstream inputs including deregulated growth factor signaling, activating mutations in the proto-oncogene Ras, point mutations or overexpression of the PI-3-kinase alpha catalytic subunit (PIK3CA), mutation or loss of PTEN, and activating mutations in the PH domain of Akt. Alterations in the PI-3-kinase regulation pathways and irregular activation of PI-3-kinase have been shown to be associated with a variety of cancer types including, but not limited to, breast, glioma, prostrate, non small cell lung, ovarian, head and neck, urinary tract, cervical, ovarian, pancreatic and colon cancers.

[0038] PI-3-kinase consists of a heterodimer of p85 and pi 10 subunits. Four distinct Class I PI-3-kinases, designated Pl-3-kinase alpha, beta, delta, and gamma, have been identified to date and each consists of a distinct 1 10 kDa catalytic subunit and a regulatory subunit. Three of the catalytic subunits, pl lOα, pl lOβ and pl lOδ, interact with the same regulatory subunit, p85, while pi lOγ interacts with a different regulatory subunit, plOl . PI-3-kinase alpha, beta and delta localize to the plasma membrane by the interaction of an SH2 domain with phosphorylated tyrosine residues of target proteins. A wealth of information has been accumulated in the recent past on the cellular functions of PI-3-kinases. The roles played by the individual isoforms of PI- 3-kinases have yet to be clearly defined.

[0039] Small GTPase proteins called Ras proteins are known to bind PI-3-kinase and upregulate PI-3-kinase activity. For example, Ras has been shown to increase PI-3-kinase activity in Fischer rat liver epithelial cells, increased expression of Ras increases PI-3-kinase activity in COS-I cells, and a dominant negative Asn-17 Ras mutant has been shown to inhibit formation of PI-3-kinase phosphorylated lipids in PC- 12 rat pheochromocytoma cells. Moreover, Ras has since been shown to bind to all four Class I PI-3-kinase catalytic subunits, pi 10a, pi lOβ pi lOδ and pi 1Oy, in a GTP-dependent manner, and pi 10a and pi lOγ have been shown to be activated by Ras GTP in vitro.

[0040] Three isoforms of Ras have been identified to date, K-Ras (SEQ ID No. 1), H- Ras (SEQ ID No. 2) and N-Ras (SEQ ID No. 3), and these isoforms share a significant sequence homology. In fact, the only region that exhibits significant sequence divergence is the final 24 amino acid residues, which make up a hypervariable domain. Although, H-Ras, K-Ras and N- Ras are nearly identical in sequence, the Ras isoforms appear to have distinct cellular functions. For example, K-Ras has been shown to be important for normal mouse development, a K-Ras knockout is embryonic lethal at 12-14 days of gestation, while H-Ras and N-Ras knockout mice exhibit no distinct phenotypes. The distinct biological functions of Ras isoforms are also exerted through the selective activation of downstream effectors, such as the serine/threonine kinases Raf, PI-3-kinase, and RaI-GDS, the exchange factor for RaI GTPase. Additionally, each Ras isoform can differentially activate PI-3-kinase and Raf. For example, H-Ras is a more potent activator of PI-3-kinase than K-Ras, whereas K-Ras is a more potent activator of the Rac pathway and recruits Raf-1 to the cell membrane more efficiently than H-Ras.

[0041] The differential ability of H-Ras and K-Ras to increase PI-3-kinase signaling in cells appears to effect radiation-induced apoptosis. Specifically, the overexpression of an active isoform of H-Ras (12V-H-Ras) in Rat2 cells has been shown to increase PI-3-kinase signaling as measured by phosphorylated-Akt which appears to cause resistance in these cells to the ionizing radiation. PI-3-K inhibitor, LY294002, or a dominant-negative Akt has been shown to attenuate H-Ras induced radiation resistance. In contrast, Rat2 cells overexpressing activated K-Ras showed no activation of PI-3-kinase signaling measured by phosphorylated-Akt and decreased radiation resistance.

[0042] PI-3-kinase inhibitors have recently been introduced into clinical testing as antitumor agents and are generally used in combination with other agent and/or radiation to enhance antitumor activity. However, it has been observed that some tumor cells are sensitive to the antitumor effects of PI-3-kinase inhibition while others are not. There is an active search for biomarkers in the tumor cells resistant to PI-3-kinase inhibitors that will predict which patients are most likely to respond to the therapeutic effects of these inhibitors. Such a marker could lead to a diagnostic test to predict which patients will respond to PI-3-kinase inhibitor therapy and may benefit patients who would otherwise be subjected to ineffective therapy.

[0043] Embodiments of the present invention relate to methods of diagnosing and treating cancer patients. In particular, embodiments of the present invention are directed to methods for determining which cancer patients will benefit from treatment with a phosphatidylinositol-3-kinase (Pl-3 kinase) inhibitor.

[0044] Embodiments of the present invention provide diagnostic and prognostic methods for predicting the effectiveness of treatment of a cancer patient with a PI-3 kinase inhibitor. Based upon the surprising results that the sensitivity of tumor cell growth to inhibition by PI-3 kinase inhibitors is dependent on whether such tumor cells express mutant Ras, methods have been devised for determining mutant Ras expression to predict the sensitivity and/or resistance of tumor cells to PI-3 kinase inhibitors. In certain embodiments, mutant Ras is selected from mutant K-Ras, mutant N-Ras and mutant H-Ras.

[0045] One embodiment of the present invention provides a method of predicting the sensitivity of tumor cell growth to inhibition by a PI-3 kinase inhibitor comprising assessing or characterizing the Ras expressed by a tumor cell; and predicting the sensitivity of tumor cell growth to inhibition by a PI-3 kinase inhibitor, wherein expression of wild-type Ras correlates with sensitivity to inhibition by PI-3 kinase inhibitors and wherein expression of mutant Ras correlates with resistance to inhibition by PI-3 kinase inhibitors. In certain embodiments, mutant Ras is selected from mutant K-Ras, mutant N-Ras and mutant H-Ras. In certain embodiments, the mutant Ras is mutant K-Ras. [0046] In another aspect of the invention there is provided a method for identifying a tumor in a human subject that is susceptible to treatment with a PI-3 kinase inhibitor comprising (i) determining the presence of a wild-type Ras protein or gene in a sample of said tumor whereby the presence of a wild-type RAS protein or gene indicates that the tumor is susceptible to treatment with a PI-3 kinase inhibitor or (ii) determining the presence of a mutated Ras protein or gene in a sample of said tumor whereby the absence of a mutated Ras protein or gene indicates that the tumor is susceptible to treatment with a PI-3 kinase inhibitor. I n certain embodiments, mutant Ras is selected from mutant K-Ras, mutant N-Ras and mutant H-Ras. In certain embodiments, the mutant Ras is mutant K-Ras.

[0047] In another aspect of the invention there is provided a method for identifying a tumor in a human subject that is susceptible to treatment with a PI-3 kinase inhibitor comprising (i) determining the presence of a wild-type K-Ras protein or gene in a sample of said tumor whereby the presence of a wild-type K-Ras protein or gene indicates that the tumor is susceptible to treatment with a PI-3 kinase inhibitor or (ii) determining the presence of a mutated K-Ras protein or gene in a sample of said tumor whereby the absence of a mutated K-Ras protein or gene indicates that the tumor is susceptible to treatment with a PI-3 kinase inhibitor.

[0048] In embodiments of the present invention, the tumor samples are analyzed for mutations in Ras. Ras may be selected from K-Ras, H-Ras, N-Ras and combinations thereof. In certain embodiments, mutations in Ras are considered any non-wild-type Ras. For example, wild-type K-Ras has the sequence of SEQ ID No. 1 , wild-type H-Ras has the sequence of SEQ ID NO. 2, and wild-type N-Ras has the sequence of SEQ ID No. 3. In certain embodiments, the Ras mutations occur in exon 1. Exemplary K-Ras mutations include, but are not limited to, G12D, G12V, G12S, G12A, G12C, G13A, G13D. G12R, G13C, G13D, E37G, T35S, Y40C, 12V 35S, 12V 37G, 12V 4OC and combinations thereof. In certain embodiments, the K-Ras mutation is selected from .G12D, G 12V, G12S. G12A, G12C, Gl 3 A. Gl 3D, G12R, Gl 3C, Gl 3D and combinations thereof. Exemplary H-Ras mutations include, but are not limited to, G12V, E37G, T35S, G15A, T35S, Y40C, 17N, 12V 35S, 12V 37G, 12V 4OC and combinations thereof. Exemplary N-Ras mutations include, but are not limited to, G12D, G12V, E37G, T35S, Y40C, 12V 35S, 12V 37G, 12V 4OC and combinations thereof.

[0049] Ras mutations are more fully described in White et al., Multiple Ras functions can contribute to mammalian cell transformation, 80 Cell 533-41 (1995); Khosravi-Far R et al. Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation, 16 Molecular and cellular biology 3923-33 (1996); Hamad NM, et al. Distinct requirements for Ras oncogenesis in human versus mouse cells, 16 Genes & development 2045-57 (2002); Lim KH, Reduction in the requirement of oncogenic Ras signaling to activation of P 13 KJ AKT pathway during tumor maintenance 8 Cancer cell :381-92 (2002); and Shirasawa S et al, Altered Growth of Human Colon Cancer Cell Lines Disrupted at Activated Ki-r as, 260 Science 85-88 (1993), each of which are herein incorporated by reference in their entireties.

[0050] Accordingly, embodiments of the present invention provide for a method of identifying patients not responsive to therapy of a PI-3 kinase inhibitor alone or in combination with chemotherapy comprising determining the presence or absence of a Ras mutation whereby the presence of said mutation indicates a patient will not respond to said therapy. Alternatively, there is provided a method for identifying a tumor in a human subject that is susceptible to treatment with PI-3 kinase inhibitor comprising (i) determining the presence of a wild-type Ras protein or gene in a sample of said tumor whereby the presence of a wild-type Ras protein or gene indicates that the tumor is susceptible to treatment with a PI-3 kinase inhibitor or (ii) determining the presence of a mutated Ras protein or gene in a sample of said tumor whereby the absence of a mutated Ras protein or gene indicates that the tumor is susceptible to treatment with a PI-3 kinase inhibitor. In certain embodiments, wild-type Ras is selected from wild-type K-Ras, wild-type H-Ras, wild-type N-Ras and a combination thereof. Wild-type K-Ras may be SEQ ID No. 1, wild-type H-Ras may be SEQ ID No. 2, and wild-type N-Ras may be SEQ ID No. 3. In certain embodiments a Ras mutation may be selected from a mutation in a Ras gene, a Ras protein or a combination thereof. In certain embodiments, the Ras mutation may be a mutation in K-Ras, H-Ras, N-Ras or a combination thereof. Exemplary K-Ras mutations include, but are not limited to, G12D, G12V, G12S, G12A, G12C, G13A, G13D, G12R, G13C, G13D, E37G, T35S, Y40C, 12V 35S, 12V 37G, 12V 4OC and combinations thereof. Exemplary H-Ras mutations include, but are not limited to, G12V, E37G, T35S, G15A, T35S, Y40C, 17N, 12V 35S, 12V 37G, 12V 4OC and combinations thereof. Exemplary N-Ras mutations include, but are not limited to, G12D, G12V, E37G, T35S, Y40C, 12V 35S, 12V 37G, 12V 4OC and combinations thereof.

[0051] Embodiments of the present invention also provide for methods for treating cancer patients or tumors and/or tumor metastases with PI-3 kinase inhibitors that incorporate the above methodology are also provided. Thus, embodiments of the present invention further provide methods for treating tumors or tumor metastases in a patient comprising the steps of diagnosing a patient's likely responsiveness to an PI-3 kinase inhibitor by assessing whether the tumor cells express mutant Ras or wild-type Ras, and administering to said patient a therapeutically effective amount of an PI-3 kinase inhibitor. In certain embodiments, wild-type Ras is selected from wild-type K-Ras, wild-type H-Ras, wild-type N-Ras and a combination thereof. Wild-type K-Ras may be SEQ ID No. 1 , wild-type H-Ras may be SEQ ID No. 2, and wild-type N-Ras may be SEQ ID No. 3. In certain embodiments a Ras mutation may be selected from a mutation in a Ras gene, a Ras protein or a combination thereof. In certain embodiments, the Ras mutation may be a mutation in K-Ras, H-Ras, N-Ras or a combination thereof. Exemplary K-Ras mutations include, but are not limited to, G12D, G 12V, G12S, G12A, G12C, G13A, G13D, G12R, G13C, G13D, E37G, T35S, Y40C, 12V 35S, 12V 37G, 12V 4OC and combinations thereof. Exemplary H-Ras mutations include, but are not limited to, G 12V, E37G, T35S, Gl 5A, T35S, Y40C, 17N, 12V 35S, 12V 37G, 12V 4OC and combinations thereof. Exemplary N-Ras mutations include, but are not limited to, G12D, G12V, E37G, T35S, Y40C, 12V 35S, 12V 37G, 12V 4OC and combinations thereof.

[0052] One embodiment of the present invention is a method for determining PI-3 kinase inhibitor resistance comprising identifying the presence of mutant Ras from an individual, wherein the presence of mutant Ras indicates resistance to PI-3 kinase inhibitor therapy. In certain embodiments a Ras mutation may be selected from a mutation in a Ras gene, a Ras protein or a combination thereof. In certain embodiments, the Ras mutation may be a mutation in K-Ras, H-Ras, N-Ras or a combination thereof. Exemplary K-Ras mutations include, but are not limited to, G12D, G12V, G12S, G12A, G12C, G13A, G13D, G12R, G13C, G13D, E37G, T35S, Y40C, 12V 35S, 12V 37G, 12V 4OC and combinations thereof. Exemplary H-Ras mutations include, but are not limited to, G12V, E37G, T35S, G15A, T35S, Y40C, 17N, 12V 35S, 12V 37G, 12V 4OC and combinations thereof. Exemplary N-Ras mutations include, but are not limited to, G12D, G12V, E37G, T35S, Y40C, 12V 35S, 12V 37G, 12V 4OC and combinations thereof. [0053] A further embodiment of the present invention is a method for determining PI-3 kinase inhibitor susceptibility comprising identifying the presence of wild-type Ras from an individual, wherein the presence of wild-type Ras indicates susceptibility to PI-3 kinase inhibitor therapy. In certain embodiments, wild-type Ras is selected from wild-type K-Ras, wild-type H- Ras, wild-type N-Ras and a combination thereof. Wild-type K-Ras may be SEQ ID No. 1 , wild- type H-Ras may be SEQ ID No. 2, and wild-type N-Ras may be SEQ ID No. 3.

[0054] A further embodiment of the present invention is a method of treating cancer in an individual comprising identifying the absence of mutant Ras in the individual and administering an effective amount of a PI-3 kinase inhibitor. In certain embodiments a Ras mutation may be selected from a mutation in a Ras gene, a Ras protein or a combination thereof. In certain embodiments, the Ras mutation may be a mutation in K-Ras, H-Ras, N-Ras or a combination thereof. Exemplary K-Ras mutations include, but are not limited to, G12D, Gl 2 V, G12S, G12A, G12C, G13A, G13D, G12R, G13C, G13D, E37G, T35S, Y40C, 12V 35S, 12V 37G, 12V 4OC and combinations thereof. Exemplary H-Ras mutations include, but are not limited to, G12V, E37G, T35S, G15A, T35S, Y40C, 17N, 12V 35S, 12V 37G, 12V 4OC and combinations thereof. Exemplary N-Ras mutations include, but are not limited to, G12D, G 12V, E37G, T35S, Y40C, 12V 35S, 12V 37G, 12V 4OC and combinations thereof.

[0055] Embodiments of the invention described herein is generally directed to a method for identifying a marker for resistance to a PI-3-kinase inhibitor, a method for treating cancer in a patient by identifying a marker for resistance to a PI-3-kinase inhibitor and a kit including materials for identifying a marker for resistance to a PI-3-kinase. As used herein, PI-3-kinase inhibitors include, but are not limited to, wortmannin, and analogs thereof, including, for example, PX-866, PX-867, DJM2-181 , DJM2-170, DJM2-171 , DJM2-177, DJM- 190, PX-868, PX-870, PX-871, PX-880, PX-881, PX-882, PX-889 and any metabolites, such as 17-hydroxy or 1 1, 17-dihydroxy metabolites, including, for example, PX-866-1, PX-866-2 and PX-867-1 or analogs thereof, and any salts thereof. Such PI-3-kinase inhibitors are more fully described in, for example, U.S. Patent No. 7,081,475, U.S. Application No. 1 1/187,553 (Publication No. 2006/0063824) and U.S. Application No. 1 1/618,036 (Publication No. 2007/0191466), each incorporated by reference in their entireties. For example, a wortmannin analog may have the following structure:

Figure imgf000020_0001
wherein Y is selected from a heteroatom, such as oxygen, nitrogen and sulfur, Rl is selected from an unsaturated alkyl, non-linear alkyl, and a substituted alkyl and R2 is selected from an unsaturated alkyl, non-linear alkyl, and a substituted alkyl.

[0056] Examples of PI-3 kinase inhibitors that fall within this general formula are set forth in Figure 12. In certain embodiments of the present invention, the methods include PX-866 and PX-867. PX-866 and PX-867 have the following structures, respectively:

Figure imgf000021_0001

[0057] Other PI-3 -kinase inhibitors include, but are not limited to, LY294002 and analogs thereof, BEZ235 (Novartis), GSK615 (Glaxo Smith Kline), GSK 690693 (Glaxo Smith Kline) XL 418 (Exelexis), XL 147 (Exelexis), XL 665 (Exelixis), SF 1 126 (Semafore), CAL 101 (Calistoga), compound 1 of Aeterna Zentaris, perifosine, and archexin LY29002 (Eli Lilly), ZSTK474 (Zenyaku Kogyo), GDC-0941 (Genentech/Piramed) BGT-226 (Novartis), AS041 164 (Merck Serono), XL-765 (Exelixis), PL- 103 (Piramed), CHR-4432 (Chroma Therapeutics) and AS-604850.

[0058] Other PI-3 kinase inhibitors include, but are not limited to, those PI-3 kinase delta inhibitors that are set forth in U.S. Application No. 11/110,204 (Publication No. 2005/0261317) and triciribine and related compounds as set forth in U.S. Application No. 1 1/733,001 (Publication No. 2007/0238745), each incorporated by reference in their entireties, and other inhibitors targeting the ATP binding site and other portions of the Akt enzyme. [0059] In one embodiment, a method for identifying an individual that is not resistant to PI-3-kinase inhibitors is provided. The method may comprise identifying the absence of a mutant K-Ras protein or a mutant K-Ras gene and administering a PI-3-kinase inhibitor to a patient in need thereof.

[0060] In the methods described herein, the step of identifying the presence or absence or mutant Ras or otherwise characterizing the Ras expression in an individual can be readily assessed by one of skill in the art, for example by using any of the standard bioassay procedures known in the art for determination of the level of expression of a gene, including for example ELISA, RIA, immunoprecipitation, immunoblotting, immunofluorescence microscopy, immunohistochemistry (IHC), RT-PCR, in situ hybridization, cDNA microarray, complete sequencing, pyrosequencing or the like, preferably RT-PCR, in situ hybridization, complete sequence and pyrosequencing.

[0061] In the methods of this invention, the expression Ras or mutant Ras is preferably assessed by assaying a tumor biopsy. However, in an alternative embodiment, expression level of Ras or mutant Ras can be assessed in bodily fluids or excretions containing detectable levels of Ras or mutant Ras originating from the tumor or tumor cells. Bodily fluids or excretions useful in the present invention include blood, urine, saliva, stool, pleural fluid, lymphatic fluid, sputum, ascites, prostatic fluid, cerebrospinal fluid (CSF), or any other bodily secretion or derivative thereof. By blood it is meant to include whole blood, plasma, serum or any derivative of blood. Assessment of tumor epithelial or mesenchymal biomarkers in such bodily fluids or excretions can sometimes be preferred in circumstances where an invasive sampling method is inappropriate or inconvenient. [0062] For assessment of tumor cell Ras expression, patient samples containing tumor cells, or proteins or nucleic acids produced by these tumor cells, may be used in the methods of the present invention. In these embodiments, the expression of Ras or mutant Ras can be assessed by assessing the amount (e.g. absolute amount or concentration) of the marker in a tumor cell sample, e.g., a tumor biopsy obtained from a patient, or other patient sample containing material derived from the tumor (e.g. blood, serum, urine, or other bodily fluids or excretions as described herein above). The cell sample can, of course, be subjected to a variety of well-known post-collection preparative and storage techniques (e.g., nucleic acid and/or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.) prior to assessing the amount of the marker in the sample. Likewise, tumor biopsies may also be subjected to post-collection preparative and storage techniques, e.g., fixation.

[0063] In the methods of the invention, one can detect expression of Ras or mutant Ras proteins having at least one portion which is displayed on the surface of tumor cells which express it. It is a simple matter for the skilled artisan to determine whether a marker protein, or a portion thereof, is exposed on the cell surface. For example, immunological methods may be used to detect such proteins on whole cells, or well known computer-based sequence analysis methods may be used to predict the presence of at least one extracellular domain (i.e. including both secreted proteins and proteins having at least one cell-surface domain). Expression of a marker protein (i.e. mutant Ras) having at least one portion which is displayed on the surface of a cell which expresses it may be detected without necessarily lysing the tumor cell (e.g. using a labeled antibody which binds specifically with a cell-surface domain of the protein). [0064] Expression of Ras or mutant Ras may be assessed by any of a wide variety of well known methods for detecting expression of a transcribed nucleic acid or protein. Non- limiting examples of such methods include immunological methods for detection of secreted, cell-surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.

[0065] In one embodiment, expression of Ras or mutant Ras may be assessed using an antibody (e.g. a radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g. an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair {e.g. biotin-streptavidin}), or an antibody fragment (e.g. a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically with Ras or mutant Ras protein or fragment thereof, including a protein which has undergone either all or a portion of post-translational modifications to which it is normally subjected in the tumor cell (e.g. glycosylation, phosphorylation, methylation etc.).

[0066] In another embodiment, expression of Ras or mutant Ras is assessed by preparing mRNA/cDNA (i.e. a transcribed polynucleotide) from cells in a patient sample, and by hybridizing the mRNA/cDNA with a reference polynucleotide which is a complement of a Ras/mutant Ras nucleic acid, or a fragment thereof. cDNA can, optionally, be amplified using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide. Expression can likewise be detected using quantitative PCR to assess the level of expression of the biomarker(s). Alternatively, any of the many known methods of detecting mutations or variants (e.g. single nucleotide polymorphisms, deletions, etc.) of a biomarker of the invention may be used to detect occurrence of a biomarker in a patient. [0067] In a related embodiment, a mixture of transcribed polynucleotides obtained from the sample is contacted with a substrate having fixed thereto a polynucleotide complementary to or homologous with at least a portion (e.g. at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or more nucleotide residues) of a Ras or mutant Ras nucleic acid. If polynucleotides complementary to or homologous with are differentially detectable on the substrate (e.g. detectable using different chromophores or fluorophores, or fixed to different selected positions), then the levels of expression of a plurality of biomarkers can be assessed simultaneously using a single substrate (e.g. a "gene chip" microarray of polynucleotides fixed at selected positions). When a method of assessing expression is used which involves hybridization of one nucleic acid with another, it is preferred that the hybridization be performed under stringent hybridization conditions.

[0068] An exemplary method for detecting the presence or absence of a wild-type or mutant Ras protein or nucleic acid in a biological sample involves obtaining a biological sample (e.g. a tumor-associated body fluid) from a test subject and contacting the biological sample with a compound or an agent capable of detecting the polypeptide or nucleic acid (e.g., mRNA, genomic DNA, or cDNA). The detection methods of the invention can thus be used to detect mRNA, protein, cDNA, or genomic DNA, for example, in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of a biomarker protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of genomic DNA include Southern hybridizations. In vivo techniques for detection of mRNA include polymerase chain reaction (PCR), Northern hybridizations and in situ hybridizations. Furthermore, in vivo techniques for detection of a biomarker protein include introducing into a subject a labeled antibody directed against the protein or fragment thereof. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0069] A general principle of such diagnostic and prognostic assays involves preparing a sample or reaction mixture that may contain a biomarker, and a probe, under appropriate conditions and for a time sufficient to allow the biomarker and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways.

[0070] For example, one method to conduct such an assay would involve anchoring the biomarker or probe onto a solid phase support, also referred to as a substrate, and detecting target biomarker/probe complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, a sample from a subject, which is to be assayed for presence and/or concentration of biomarker, can be anchored onto a carrier or solid phase support. In another embodiment, the reverse situation is possible, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored component of the assay.

[0071] There are many established methods for anchoring assay components to a solid phase. These include, without limitation, biomarker or probe molecules which are immobilized through conjugation of biotin and streptavidin. Such biotinylated assay components can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin- coated 96 well plates (Pierce Chemical). In certain embodiments, the surfaces with immobilized assay components can be prepared in advance and stored. [0072] Other suitable carriers or solid phase supports for such assays include any material capable of binding the class of molecule to which the biomarker or probe belongs. Well- known supports or carriers include, but are not limited to, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.

[0073] In order to conduct assays with the above mentioned approaches, the non- immobilized component is added to the solid phase upon which the second component is anchored. After the reaction is complete, uncomplexed components may be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the solid phase. The detection of biomarker/probe complexes anchored to the solid phase can be accomplished in a number of methods outlined herein.

[0074] In one embodiment, the probe, when it is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are well-known to one skilled in the art.

[0075] It is also possible to directly detect biomarker/probe complex formation without further manipulation or labeling of either component (biomarker or probe), for example by utilizing the technique of fluorescence energy transfer (i.e. FET, see for example, Lakowicz et al., U.S. Pat. No. 5,631 ,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, "donor" molecule is selected such that, upon excitation with incident light of appropriate wavelength, its emitted fluorescent energy will be absorbed by a fluorescent label on a second "acceptor" molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the "donor" protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the acceptor' molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

[0076] In another embodiment, determination of the ability of a probe to recognize a biomarker can be accomplished without labeling either assay component (probe or biomarker) by utilizing a technology such as real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C, 1991, Anal. Chem. 63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705). As used herein, "BIA" or "surface plasmon resonance" is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

[0077] Alternatively, in another embodiment, analogous diagnostic and prognostic assays can be conducted with biomarker and probe as solutes in a liquid phase. In such an assay, the complexed biomarker and probe are separated from uncomplexed components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. In differential centrifugation, biomarker/probe complexes may be separated from uncomplexed assay components through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A. P., 1993, Trends Biochem Sci. 18(8):284-7). Standard chromatographic techniques may also be utilized to separate complexed molecules from uncomplexed ones. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components. Similarly, the relatively different charge properties of the biomarker/probe complex as compared to the uncomplexed components may be exploited to differentiate the complex from uncomplexed components, for example through the utilization of ion-exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard, N. H., 1998, J. MoI. Recognit. Winter 1 1(1-6): 141-8; Hage, D. S., and Tweed, S. A. J. Chromatogr B Biomed Sci Appl 1997 Oct. 10; 699(1 -2):499-525). Gel electrophoresis may also be employed to separate complexed assay components from unbound components (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987-1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, non-denaturing gel matrix materials and conditions in the absence of reducing agent are typically preferred. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art.

[0078] In a particular embodiment, the expression of wild-type or mutant Ras mRNA can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. The term "biological sample" is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from tumor cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Pat. No. 4,843,155).

[0079] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a biomarker of the present invention. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that the biomarker in question is being expressed.

[0080] In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. Λ skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the biomarkers of the present invention.

[008 IJ An alternative method for determining the level of mRNA biomarker in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987. U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6: 1 197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[0082] For in situ methods, mRNA does not need to be isolated from the tumor cells prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the biomarker. [0083] As an alternative to making determinations based on the absolute expression level of the biomarker, determinations may be based on the normalized expression level of the biomarker. Expression levels are normalized by correcting the absolute expression level of a biomarker by comparing its expression to the expression of a gene that is not a biomarker, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-tumor sample, or between samples from different sources.

[0084] Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a biomarker (e.g. a mesenchymal biomarker), the level of expression of the biomarker is determined for 10 or more samples of normal versus cancer cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the biomarker. The expression level of the biomarker determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that biomarker. This provides a relative expression level.

[0085] In another embodiment of the present invention, a biomarker protein is detected. A preferred agent for detecting biomarker protein of the invention is an antibody capable of binding to such a protein or a fragment thereof, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment or derivative thereof (e.g., Fab or F(ab').sub.2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

[0086] Proteins from tumor cells can be isolated using techniques that are well known to those of skill in the art. The protein isolation methods employed can, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.).

[0087] A variety of formats can be employed to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunoabsorbant assay (ELISA). A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether tumor cells express a biomarker of the present invention.

[0088] In one format, antibodies, or antibody fragments or derivatives, can be used in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins. In such uses, it is generally preferable to immobilize either the antibody or proteins on a solid support. Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. [0089] One skilled in the art will know many other suitable carriers for binding antibody or antigen, and will be able to adapt such support for use with the present invention. For example, protein isolated from tumor cells can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose. The support can then be washed with suitable buffers followed by treatment with the detectably labeled antibody. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on the solid support can then be detected by conventional means.

[0090] For ELISA assays, specific binding pairs can be of the immune or non-immune type. Immune specific binding pairs are exemplified by antigen-antibody systems or hapten/anti- hapten systems. There can be mentioned fluorescein/anti-fluorescein, dinitrophenyl/anti- dinitrophenyl, biotin/anti-biotin, peptide/anti-peptide and the like. The antibody member of the specific binding pair can be produced by customary methods familiar to those skilled in the art. Such methods involve immunizing an animal with the antigen member of the specific binding pair. If the antigen member of the specific binding pair is not immunogenic, e.g., a hapten, it can be covalently coupled to a carrier protein to render it immunogenic. Non-immune binding pairs include systems wherein the two components share a natural affinity for each other but are not antibodies. Exemplary non-immune pairs are biotin-streptavidin, intrinsic factor-vitamin B 12, folic acid-folate binding protein and the like.

[0091] A variety of methods are available to covalently label antibodies with members of specific binding pairs. Methods are selected based upon the nature of the member of the specific binding pair, the type of linkage desired, and the tolerance of the antibody to various conjugation chemistries. Biotin can be covalently coupled to antibodies by utilizing commercially available active derivatives. Some of these are biotin-N-hydroxy-succinimide which binds to amine groups on proteins; biotin hydrazide which binds to carbohydrate moieties, aldehydes and carboxyl groups via a carbodiimide coupling; and biotin maleimide and iodoacetyl biotin which bind to sulfhydryl groups. Fluorescein can be coupled to protein amine groups using fluorescein isothiocyanate. Dinitrophenyl groups can be coupled to protein amine groups using 2,4-dinitrobenzene sulfate or 2,4-dinitrofluorobenzene. Other standard methods of conjugation can be employed to couple monoclonal antibodies to a member of a specific binding pair including dialdehyde, carbodiimide coupling, homofunctional crosslinking, and heterobifunctional crosslinking. Carbodiimide coupling is an effective method of coupling carboxyl groups on one substance to amine groups on another. Carbodiimide coupling is facilitated by using the commercially available reagent l-ethyl-3-(dimethyl-aminopropyl)- carbodiimide (EDAC).

[0092] Homobifunctional crosslinkers, including the bifunctional imidoesters and bifunctional N-hydroxysuccinimide esters, are commercially available and are employed for coupling amine groups on one substance to amine groups on another. Heterobifunctional crosslinkers are reagents which possess different functional groups. The most common commercially available heterobifunctional crosslinkers have an amine reactive N- hydroxysuccinimide ester as one functional group, and a sulfhydryl reactive group as the second functional group. The most common sulfhydryl reactive groups are maleimides, pyridyl disulfides and active halogens. One of the functional groups can be a photoactive aryl nitrene, which upon irradiation reacts with a variety of groups.

[0093J The detectably-labeled antibody or detectably-labeled member of the specific binding pair is prepared by coupling to a reporter, which can be a radioactive isotope, enzyme, fluorogenic, chemiluminescent or electrochemical materials. Two commonly used radioactive isotopes are 1251 and 3H. Standard radioactive isotopic labeling procedures include the chloramine T, lactoperoxidase and Bolton-Hunter methods for 1251 and reductive methylation for 3H. The term "detectably-labeled" refers to a molecule labeled in such a way that it can be readily detected by the intrinsic enzymic activity of the label or by the binding to the label of another component, which can itself be readily detected.

[0094] Enzymes suitable for use include, but are not limited to, horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase, glucose oxidase, luciferases, including firefly and renilla, .beta.-lactamase, urease, green fluorescent protein (GFP) and lysozyme. Enzyme labeling is facilitated by using dialdehyde, carbodiimide coupling, homobifunctional crosslinkers and heterobifunctional crosslinkers as described above for coupling an antibody with a member of a specific binding pair.

[0095] The labeling method chosen depends on the functional groups available on the enzyme and the material to be labeled, and the tolerance of both to the conjugation conditions. The labeling method used in the present invention can be one of, but not limited to, any conventional methods currently employed including those described by Engvall and Pearlmann, Immunochemistry 8, 871 (1971), Avrameas and Ternynck, Immunochemistry 8, 1 175 (1975), Ishikawa et al., J. Immunoassay 4(3):209-327 (1983) and Jablonski, Anal. Biochem. 148:199 (1985).

[0096] Labeling can be accomplished by indirect methods such as using spacers or other members of specific binding pairs. An example of this is the detection of a biotinylated antibody with unlabeled streptavidin and biotinylated enzyme, with streptavidin and biotinylated enzyme being added either sequentially or simultaneously. Thus, according to the present invention, the antibody used to detect can be detectably-labeled directly with a reporter or indirectly with a first member of a specific binding pair. When the antibody is coupled to a first member of a specific binding pair, then detection is effected by reacting the antibody-first member of a specific binding complex with the second member of the binding pair that is labeled or unlabeled as mentioned above.

[0097] Moreover, the unlabeled detector antibody can be detected by reacting the unlabeled antibody with a labeled antibody specific for the unlabeled antibody. In this instance "detectably-labeled" as used above is taken to mean containing an epitope by which an antibody specific for the unlabeled antibody can bind. Such an anti-antibody can be labeled directly or indirectly using any of the approaches discussed above. For example, the anti-antibody can be coupled to biotin which is detected by reacting with the streptavidin-horseradish peroxidase system discussed above.

[0098] In one immunoassay format for practicing this invention, a forward sandwich assay is used in which the capture reagent has been immobilized, using conventional techniques, on the surface of a support. Suitable supports used in assays include synthetic polymer supports, such as polypropylene, polystyrene, substituted polystyrene, e.g. aminated or carboxylated polystyrene, polyacrylamides, polyamides, polyvinylchloride, glass beads, agarose, or nitrocellulose.

[0099] The invention also encompasses kits for detecting the presence of a wild-type or mutant Ras protein or nucleic acid in a biological sample. Such kits can be used to determine if a subject is suffering from or is at increased risk of developing a tumor that is less susceptible to inhibition by PI-3 kinase inhibitors. For example, the kit can comprise a labeled compound or agent capable of detecting a biomarker protein or nucleic acid in a biological sample and means for determining the amount of the protein or mRNA in the sample (e.g., an antibody which binds the protein or a fragment thereof, or an oligonucleotide probe which binds to DNA or mRNA encoding the protein). Kits can also include instructions for interpreting the results obtained using the kit.

[00100] For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a biomarker protein; and, optionally, (2) a second, different antibody which binds to either the protein or the first antibody and is conjugated to a detectable label.

[00101] For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a biomarker protein or (2) a pair of primers useful for amplifying a biomarker nucleic acid molecule. The kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

[00102] In the various methods of the present invention, tumors include, but are not limited to, neuroblastoma, intestine carcinoma such as rectum carcinoma, colon carcinoma, familiary adenomatous polyposis carcinoma and hereditary non-polyposis colorectal cancer, esophageal carcinoma, labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tong carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, medullary thyroidea carcinoma, papillary thyroidea carcinoma, renal carcinoma, kidney parenchym carcinoma, ovarian carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, pancreatic carcinoma, prostate carcinoma, testis carcinoma, breast carcinoma, urinary carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, Hodgkin lymphoma. non-Hodgkin lymphoma, Burkitt lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), adult T-cell leukemia lymphoma, hepatocellular carcinoma, gall bladder carcinoma, bronchial carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyo sarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma and plasmacytoma. Particular tumors include those of the brain, liver, kidney, bladder, breast, gastric, ovarian, colorectal, prostate, pancreatic, breast, lung, vulval, thyroid, colorectal, oesophageal, hepatic carcinomas, sarcomas, glioblastomas, head and neck, leukemias and lymphoid malignancies. In particular, methods, the cancer and/or tumor may include, but are not limited to, breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, glioma, liver cancer, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, renal cancer, prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma, skin cancer, lymphoma and blood cancer or a tumor cell from any of a variety of other cancers as described herein below. In certain embodiments, the cancer and/or tumor may include lung (e.g. non-small cell lung cancer (NSCLC) or small-cell lung cancer)), pancreatic, colon, prostate, ovarian, breast, head and neck, gastric, myeloma. In certain embodiments, the cancer and/or tumor may include prostate, pancreatic, color, ovarian and breast cancer.

[00103] In another embodiment, the method may comprise identifying an individual or patient that is not resistant to PI-3 -kinase inhibitors comprising identifying the absence of a mutant K-Ras protein or a mutant K-Ras gene and administering a PI-3-kinase inhibitor and a chemotherapeutic agent to a patient in need thereof. Preferably, the patient has cancer. The PI- 3-kinase inhibitor may be administered prior to, during or following administration of a chemotherapeutic agent. Chemotherapeutic agents include both cytotoxic agents and anti-tumor targeting agents. Exemplary cytotoxic agents include, but are not limited to, gemcitabine (Gemzar®), paclitaxel (Taxol®), and cisplatin (Platinol®). Exemplary anti-tumor targeting agents include, but are not limited to, gefitinib (Iressa®), erlotinib (Tarceva®), trastuzumab (Herceptin®), cetuximab (Erbitux®), bevacizumab (Avastin®) sorafenib (Nexavar®) and BAY 43-9006. In certain embodiments the PI-3-kinase inhibitor and the chemotherapeutic agent may be in a combination formulation. In other embodiments, the PI-3-kinase inhibitor and the chemotherapeutic agent may be administered separately, either prior to, substantially simultaneously or after administration of the other agent. Such exemplary combinatorial methods are further described in co-pending U.S. Application No. 1 1/178,553 (2006/0063824), and is herein incorporated by reference in its entirety.

[00104) Further embodiments of the present invention related to methods of administering to a subject in need thereof a PI-3-kinase inhibitor in combination with other specific kinase inhibitors. Such specific kinase inhibitors preferably include inhibitors or RAF and/or RAL, including for example chemotherapeutic agents, including both cytotoxic agents and anti-tumor targeting agents. [00105] In various embodiments of the invention, the marker identified may be a mutant K-Ras protein or a mutant K-Ras gene. Without wishing to be bound by theory, mutations in K- Ras may be utilized to identify an individual who may be resistant to PI-3-kinase inhibitors, and identification of such individuals may provide the basis for treatment of certain individuals having a malady which symptomatically includes uncontrolled cellular proliferation, such as, but not limited to, cancer, with PI-3-kinase inhibitors. For example, individuals having a mutation in K-Ras may be resistant to treatment with PI-3-kinase inhibitors while such treatment may be effective for individuals having wild type K-Ras.

[00106] The material through which K-Ras mutants are identified may vary in embodiments. For example, a K-Ras mutant may be identified from one or more genetic material, such as, DNA or RNA, or protein, and in some embodiments, a K-Ras mutant may be identified from both one or more genetic material and protein. Such genetic material or protein may be derived from any number of sources and the embodiments, described herein are not limited to any particular source. For example, the genetic material or protein may be derived from a cell, a group of cells or tissue, and the cell, group of cells or tissue may be from an individual or a cultured cell line. Moreover, the individual or cell line may exhibit a normal or diseased phenotype. For example, in one embodiment, the genetic material or protein may be derived from tumorigenic tissue of an individual, such as, a cancer patient. In another embodiment, the genetic material or protein may be derived from normal, non-tumorigenic tissue of an individual having cancer or an individual who does not have cancer or may be predisposed to cancer. In still another embodiment, the genetic material or protein may be derived from an immortalized tumor cell line, a cultured tissue or an individual used in research, such as, a rat, mouse, dog or monkey. [00107] The genetic material and/or protein may be collected by any method known in the art, and the skilled artisan may choose from among a diverse number of techniques depending upon the type of material being collected and the individual, cells or tissues from which the material is collected. For example, in some embodiments, the method may include the step of collecting genetic material from an individual, isolating genetic material encoding a K- Ras gene or a K-Ras transcript, and testing the isolated genetic material for a K-Ras mutant. Other embodiments may include the steps of collecting proteins from an individual, isolating K- Ras proteins and identifying mutant K-Ras. In still other embodiments, the method may include the steps of collecting genetic material and collecting proteins from an individual and isolating K-Ras genes or mRNA and K-Ras proteins and identifying mutant K-Ras genes and mutant K- Ras proteins.

[00108] The step of collecting genetic material or proteins may be carried out using any technique known in the art, and the various embodiments described herein are not limited by the technique used. For example, in one embodiment, genetic material and/or proteins may be collected from tissue harvested from a biopsy, and in another, the genetic material and/or proteins may be collected from sputum or other excreted material. In still another embodiment, the genetic material and/or protein may be collected from cultured tissue from a xenograft, and in yet another embodiment, the genetic material may be collected from cultured tumorigenic cells. As described above, the tissue or cells from which the genetic material and/or protein is collected may be normal or diseased.

[00109] In some embodiments, genetic material or proteins may be collected and these materials may be directly screened for mutant K-Ras genes or mutant K-Ras proteins. In other embodiments, the K-Ras genetic material and/or K-Ras proteins may be isolated from the collected material, and the isolated genetic material and/or proteins may be screened to identify mutant K-Ras. Isolation of K-Ras genetic material and/or K-Ras proteins may be carried out by any method known in the art, such as, for example, column chromatography, ion exchange chromatography, affinity chromatography, immuno-affinity chromatography, precipitation, electrophoresis, and the like and any combination thereof.

[00110] The step of identifying mutant K-Ras genetic material or mutant K-Ras proteins may be carried up using any procedure known in the art. For example, in some embodiments, collected and/or isolated genetic material may be sequenced directly using techniques and processors known in the art, such as, but not limited to, an automated sequencer. In other embodiments, collected and/or isolated genetic material may be cloned and amplified prior to sequencing. In still other embodiments, collected and/or isolated proteins may be sequenced using, for example, Edmond degradation which may or may not be carried out using an automated sequencer. In further embodiments, K-Ras mutants may be identified from genetic material and/or protein using, for example, fluorescently or radio-labeled antibodies, and in certain embodiments, K-Ras mutants may be identified using, for example, electrophoresis isoelectric focusing, native gel electrophoresis and the like.

[00111] In various embodiments, following the identification of a mutant K-Ras or K- Ras gene, the individual may be administered a PI-3-kinase inhibitor alone or in combination with a chemotherapeutic agent. Embodiments described herein may utilize any PI-3-kinase inhibitor known in the art at this time or developed in the future, and such PI-3-kinase inhibitors may be administered by any method known in the art and useful for whatever malady is being treated. Without wishing to be bound by theory, evidence provided by the methods embodied hereinabove that a individual may not possess a mutant K-Ras may allow that individual to be effectively treated with a PI-3-kinase inhibitor. For example, an individual having lung cancer may be tested for mutant K-Ras by assessing the presence of mutant K-Ras from a biopsy of tumorigenic tissue from a lung using any of the methods embodied hereinabove. If a negative result is obtained indicating that the patient's K-Ras is not mutated, the patient may be effectively treated with a PI-3-kinase inhibitor. If a positive result is obtained indicating that the patient has a mutant K-Ras, a different course of treatment other than PI-3-kinase treatment may be pursued. In certain embodiments, treatment with a PI-3-kinase inhibitor may be carried out concurrently with another separate type of therapy. For example, an individual may be treated with a PI-3-kinase inhibitor and an additional form of chemo-therapy, radiation therapy, and/or surgery.

[00112] Embodiments described herein also provide for a kit for testing for the presence of a K-Ras mutant. In various embodiments, such a kit may contain reagents and/or materials necessary to collect genetic material and/or protein from an individual and identify mutant K-Ras from the collected genetic material and/or protein. In some embodiments, a kit may further contain materials useful for isolating K-Ras genes, K-Ras mRNA or K-Ras proteins from the collected genetic material and/or protein. The type of materials and/or reagents supplied in a kit may vary depending on the techniques utilized to collect, identify, and in some cases isolate, and it is well within the purview of the skilled artisan to determine which materials and/or reagents are necessary in developing such a kit.

[00113] This invention and embodiments illustrating the method and materials used may be further understood by reference to the following non-limiting examples. The data discussed herein relates to other mammalian species, particularly a mouse. It is well understood in the art that the data presented herein will correlate to humans. EXAMPLE 1

[00114] The antitumor effect of PI-3-kinase inhibitor, PX-866, has been tested in a number of cell lines derived human tumor xenograft models in immunodeficient scid mice. Briefly, human cancer cell lines were grown as xenografts in scid mice and their antitumor response to PX-866 was monitored. Cell lines that showed a response to PX-866 are classified as responders or moderate responders depending on the extent to which they respond. Cell lines that do not respond to PX-866 are classified as non-responders. The genotype of the cell lines is shown in Table 1.

[00115] Analysis of the antitumor response of these and other human tumor xenograft models reveal a clear relationship between the presence of mutant K-Ras in the cell line and resistance of the tumor to PX-866 (See, Table 1). All resistant tumors have a mutant K-Ras while responding tumors have wild type Ras. A-549 is a PX-866 sensitive tumor which has been reported to possess an atypical 12S mutation. However, another report suggests that K-Ras is wild type. Significantly, no other mutation marker, including N-Ras, PI-3-kinase pi 10a, PTEN, p53, LKB or B-Raf is capable of predicting sensitivity or resistance to PI-3-kinase inhibitors.

Table 1 :

Figure imgf000046_0001

[00116] Increased expression of wild-type Ras or the presence of a constitutively activated mutant Ras appears to increase the activity of PI-3 -kinase signaling pathways, which may be a factor in the resistance of cancer cells to chemotherapy therapy. These data suggest that increased PI-3-kinase signaling may make cells more sensitive to an inhibitor of PI-3-kinase and that H-Ras would be more active than K-Ras in this regard. It was, therefore, unexpected that the antitumor activity of PI-3-kinase inhibitor PX-866 in cell line-derived human tumor xenografts in immuno-deficient mice was uniformly inhibited by the presence of a K-Ras mutation. Thus, K-Ras may be a marker for decreased or non-responsiveness to a PI-3-kinase inhibitor. [00117] It is important also to point out that our findings are made in tumors not in cells in vitro. The concentrations of the PI-3-kinase inhibitor PX-866 required to kill cancer cells in culture (0.5 to 5 μM) would appear to be an order of magnitude or higher than required to inhibit PI-3-kinase in cells (0.025 to 0.05 μM). Cancer cell lines in culture are not stressed and most likely do not require PI-3-K for survival. Cell killing by PX-866 is most likely due to another target it inhibits. Cancer cells in a tumor are much more stressed because of poor blood perfusion, hypoxia and nutrient and growth factor deprivation so that it is likely that the effects of a PI-3-kinase inhibitor will be only apparent in a tumor.

EXAMPLE 2

[00118] Approximately 107 cells in log cell growth were injected s.c. in 0.1 mL saline into the flanks of severe combined immunodeficient (SCID) mice. When the tumors reached 200 mm", the mice were stratified into groups of 8-10 animals having approximately equal mean tumor volumes and oral administration of 2.5 to 5 mg/kg of PX-866 was begun. The tumor was treated 1 -3 weeks depending of the tumor burden of the mice. Test/ Control was determined by dividing the volume of the PX-866 treated by the volume of the control tumors at the same time point, subtracted from the tumor volume at time first treatment.

[00119] As shown in Table 2 below, mutations in PIK3CA/PTEN were associated with an increased likelihood of response to PX-866, but are not required for a cytostatic response to PX-866, and Ras mutations are associated with increased reisistance to PX-866 regardless of the presence or absence of PIK3CA mutations. Table 2. Cell Line Derived Xenografts Treated with PX-866.

Sensitive (Cytostatic) p110 alpha PTEN LKB BRAF Kras Nras Test/Control Sig.

Figure imgf000048_0001

[00120] Xenografts were grown in groups of four by the protocol above. After treatment xenografts were removed and processed in RPPA lysis buffer. These samples were spotted onto nitrocellulose-coated slides Each sample interpolated from a supercurve constructed for each protein in a script written in R. The samples were then normalized, the normalized values for the phospho proteins were divided by the normalized values for the total proteins to determine protein activity. PC-3 cells treated with 9mg/kg PX-866 IV, Panc-1 cells treated with 2.5 mg/kg PX-866 PO, doses previously determined to inhibit akt activation in vivo. As shown in Figure 2, PX-866 inhibits Akt/PKB activity in sensitive and resistant cell lines in vivo.

[00121] Cell lines were grown in ATCC recommended media with 10% FBS were harvested in RPPA lysis buffer during log phase growth. These samples were spotted onto nitrocellulose-coated slides and treated with the 54 validated antibodies listed above. Each sample interpolated from a supercurve constructed for each protein in a script written in R. The samples were then normalized and these values were then median centered. Normalized values were evaluated using a Student's t test to detectsignificant differences between the sensitive and resistant cell lines. As shown in Figure 3, no correlation was found between Akt activation as measured at the (1) Ser 473, (2) Thr 308 phosphorylation sites or expression levels of the (3) pi 10 alpha subunit of PI-3K protein. E cadherin, β cadherin, p-Mek and Mek were found to have significantly lower expression (*p<0.05) in cell lines resistant to PX-866, Cyclin B was found to be very significantly (p<0.01)higher expression in resistant cell lines.

[00122] Cells were assayed for the effect of PX-866 on cell survival. Cells were plated (250-2,000 cells per 60 mm dish) and 12h after plating treated with vehicle (DMSO) or (PX866, 500 nM) for 4h. The media was changed 4h after drug treatment. The cells were then permitted to continue growing for 10-14 days. Colonies were fixed and stained with crystal violet. To generate the survival data, individual assays were performed at multiple dilutions with a total of six plates per data point. * indicates significance p<0.05 vs Kras Gl 3D. As shown in Fig. 4, pathway specific Ras constructs show the ability of oncogenic Ras to utilize multiple signaling pathways for growth.

[00123] Cells were treated with PX866 (500 nM, 4h) and harvested 48h after exposure by trypsinization. As some apoptotic cells detached from the culture substratum into the medium, these cells were also collected by centrifugation of the medium at 1,500 rpm for 5 min. the Annexin V/propidium iodide assay was carried to determine cell viability out as per the manufacturer's instructions using a FACScan flow cytometer. * indicates significance p<0.05 vs Kras Gl 3D. As shown in Fig. 5, pathway specific Ras constructs show the ability of Oncogenic Ras to utilize multiple signaling pathways for survival.

[00124] Based upon the foregoing, a model for the combination of PX-866 with other specific kinase inhibitors has been developed, as set forth in Fig. 6. [00125] The phosphatidylinositol-3-kinase (PI-3-K)/Akt signaling pathway plays a major role in cell growth, survival and the resistance of cancer cells to therapy. Several inhibitors of PI-3-K signaling are rapidly moving towards or have reached early clinical trials. As the patient population begins receiving these agents, defining measurable patient parameters to predict a subset who will receive a maximal survival benefit will prove increasingly important. Loss of the PTEN phosphatase and mutation of the PIK3CA catalytic subunit of PI-3-K have previously been reported to be correlated with sensitivity to PI-3-K inhibitors. PX-866, a semisynthetic viridin inhibitor of PI-3-K, exhibits antitumor activity in a number of in vivo tumor models and has been shown to have a toxicity profile suitable for entry into clinical trials. We have screened PX-866 in vivo against multiple tissue tumor cell line-derived xenografts for its ability to inhibit tumor growth. PX-866, when administered at a schedule sufficient for the prolonged inhibition of AKT activity, showed pronounced single agent cytostatic effects in several different tumor types. Additionally, a subset of tumor types was identified which displayed little or no growth arrest after prolonged treatment with PX-866 at pharmacologically relevant doses. The magnitude of the responses to PX-866 in vivo were not reflected in standard cell killing assays under monolayer culture conditions. An analysis of the mutations in the cell line xenografts screened revealed that the five showing moderate to complete resistance to PX-866 all harbored an oncogenic, activating mutation in the K or N Ras signaling protein at codons 12, 13, or 61. The activated Ras protein has been shown to have the ability to simultaneously activate a number of signaling pathways involved in oncogenic processes, including the RaI, Raf and PI-3-K pathways. It was also notable that oncogenic Ras conferred resistance in the presence of an activating PI-3-K mutation. This is relevant PIK3CA and Ras mutations have been found to coexist in several tumor types and these tumors may not respond to PI-3-K inhibitors. Identification of active Ras and/or markers associated with its activation as an indicator of lack of response may serve as important tools in the selection of individuals best suited for treatment with inhibitors of the PI-3-K signaling pathway for cancer therapy. Additionally, identification of signaling pathways driving this resistance may serve as a guide for the rational combination of these inhibitors, including PX-866, with other current and emerging targeted therapies.

EXAMPLE 3

[00126] Materials and Methods. Cells. A-549, H460 and H1299 human non small cell (NSC) lung cancer, HT-29, HCT-15 and HCT-1 16 human colon cancer, MDA-MB-361 human breast cancer, Panc-1, BxPC-3 and MiaPaCa-2 pancreatic cancer, PC-3 prostate cancer, Skov-3 ovarian cancer, and RPMA-8226 multiple myeloma cancer cells were obtained from the American Tissue Type Collection (ATTC, Rockville, MD). Th e cell lines were grown in humidified 95% air, 5% CO2 at 370C in their ATCC recommended media with 10% fetal bovine serum (FBS). All cell lines were tested to be mycoplasma free using a PCR ELlSA assay (Roche Diagnostics Inc., Indianapolis). HCTl 16 K-Ras-deleted cells generated by homologous deletion of the mutant K-Ras allele [20, 21] were transfected by electroporation at 600 V for 60 milliseconds using a Multiporator Eppendorf (Hamburg, Germany) with G418 selectable plasmids expressing mutant active H-Ras (H-Ras V 12), and the selective effectors H-Ras V12S35, H-Ras V12G37, or H-Ras V12C40, which preferentially activate Raf, RaIGDS, and PI- 3-kinase enzymes, respectively, and individual colonies isolated. The plasmids were generously provided (Cold Spring Harbor, NY). These Ras mutations have been previously characterized in a number of models.

[00127] Antitumor studies Approximately 107 cells in log cell growth were injected subcutaneously in 0.1 ml sterile 0.9% NaCl into the flanks of severe combined immunodeficient (SCID) mice. Cell lines with H-Ras constructs were removed from G418 one passage before injection. When the tumors reached 200 mm2, the mice were stratified into groups of 8-10 animals having approximately equal mean tumor volumes and oral administration of 2.5 to 3 mg/kg of PX-866 begun every other day for 1 to 3 weeks. For oral (po) administration to mice PX-866 (45',4ai?,5/?,6aSr,9a/?,£)- 1 -((diallylamino)methylene)- 1 1 -hydroxy-4-(methoxymethyl)- 4a,6a-dimethyl-2,7,10-trioxo-l , 2,4 ,4a,5,6,6a,7, 8,9,9a, 10-dodecahydroindeno[4,5-Λ]isochromen- 5-yl acetate) was dissolved at 0.3 to 0.5 mg/ml in 5% ethanol in water and dosed by oral gavage At the end of the study antitumor activity was expressed as percent test/control (T/C%) determined by dividing the increase in volume of the PX-866 treated tumors by the increase in volume of the control tumors, from the start of treatment. Information on mutations in the cell lines was obtained from the Sanger Institute data base (http://www.sanger.ac.uk) and confirmed by matrix-assisted laser desorption/ionization time-of- flight mass spectrometric sequencing.

[00128] Tumor PI-3-kinase activity. Mice were killed 24 hr after the last PX-866 treatment, the tumors excised and immediately frozen in liquid N2. For assay, the tumors were homogenized in lysis buffer containing 50 mM HEPES buffer, pH 7.5, 50 mM NaCl, 0.2 mM NaF, 0.2 mM sodium orthovanadate, 1 mM, phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 1% NP-40, and 0.25% sodium deoxycholate. Protein concentration was determined by BCA assay and 50 μg of cell lysate protein was boiled for 5 minutes with denaturing buffer containing 0.25 M Tris, pH 6.8, 35% glycerol, 8% sodium dodecyl sulfate, and 10% 2-mercaptoethanol, loaded on a 10% acrylamide/bisacrylamide gel, and separated by electrophoresis at 150 V for 40 minutes. Proteins were electrophoretically transferred to a polyvinylidene fluoride membrane, preincubated with a blocking buffer of 137 mM NaCl, 2.7 mM KCl, 897 mM CaCl2, 491 mM MgCl2, 3.4 mM Na2HPO4, 593 mM KH2PO4, and 5% bovine serum albumin, and incubated overnight with anti-ρhospho-Ser473-Akt, anti-Akt, anti-phospho- Ser -Raf, or anti-Raf polyclonal antibodies (Cell Signaling, Beverly, MA). Detection used donkey anti-rabbit IgG peroxidase-coupled secondary antibody (GE Healthcare, Buckinghamshire, UK). Band density was measured using the Renaissance chemiluminescence system on Kodak X-0mat Blue ML films (Eastman Kodak, New Haven, CT).

[00129] Reverse phase protein array (RPPA). Cells were lysed with buffer containing 150 mM NaCl, 50 niM HEPES, pH 7.4, 1.5 mM MgCl2, 1 mM EGTA, 100 niM NaF, 10 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-IOO supplemented with Complete Protease Inhibitor Cocktail Tablets (Roche Applied Science, Indianapolis, IN) and cleared by centrifugation at 15,000 rpm for 10 min at 4°C. Samples were denatured by the addition of 1 part denaturing buffer to 3 parts cell lysate and boiling for 5 min. Sample concentrations were adjusted to lmg/ml with dilution buffer (1 part denaturing buffer and 3 parts cell lysis buffer) and printed as serial dilutions on glass slides, and specific proteins quantified using 52 validated antibodies.

[00130] Apoptosis measurement. Cells were treated with 0.5 μM PX866 for 48 hr and harvested by 10 min exposure to trypsin/EDTA at 37 0C. Apoptotic cells that detached from the culture surface were collected by centrifugation of the medium at 1,500 rpm for 5 min. The pooled cell pellets were resuspended and mixed with trypan blue dye. Dye incorporation into non-viable cells was measured by counting 500 cells from randomly chosen fields with a light microscope and a hemocytometer, and expressed as a percentage of the total number of cells counted. For confirmatory purposes the extent of apoptosis was evaluated by assessing Hoechst and TUNEL stained cytospin slides under fluorescent light microscopy and scoring the number of cells exhibiting the "classic" morphological features of apoptosis and necrosis. For each condition, 10 randomly selected fields per slide were evaluated, encompassing at least 1500 cells. Alternatively, the Annexin V/propidium iodide assay was carried to determine cell viability out as per the manufacturer's instructions (BD PharMingen) using a Becton Dickinson FACScan flow cytometer.

[00131] Colony formation. For studies of the effects of PX-866 on cell survival 250 to 2,000 cells were plated in a 60 mm dish and 12 hr later treated with 0.5 μM PX-866 for 4 hr. The media was changed and the cells grown for 10-14 days. After fixation and staining with crystal violet colonies of more than 50 cells were counted using a colony counter (Oxford Optronics, Oxford, England). Individual assays were performed at multiple dilutions with a total of six plates per data point.

[00132] Data analysis. Comparison of the effects of treatments and comparison of protein levels in vitro used a two-tailed t test. Differences with a p < 0.05 were considered statistically significant. A two tailed t test and a Pearson correlation was performed between normalized data obtained from the RPPA analysis. For studies of the in vivo antitumor activity of PX-866, a Whitney Mann U test was performed using SPSS software. (SPSS Inc. Chicago, II).

[00133] Results. In vivo activity of PX-866. The antitumor activity of PX-866 was measured in 13 human tumor cell line-derived xenografts in SCID mice. These tumors were then classified into three groups: resistant tumors that showed minimal or no response (No Response, T/C >70%); tumors that displayed a slowed but continued growth through treatment (Low Response, T/C 35-69%) and sensitive tumors that displayed a antitumor response to PX- 866 (Antitumor, T/C <35%), which included two that showed a cytostatic response and one that showed a regression. The mutation status of the cell lines was obtained from the Sanger Institute database and confirmed by mass spectral sequencing (Figure 7A). [00134] PC-3 prostate, BxPC3 pancreatic, HT-29 colon, Skov-3 ovarian and MDA-MB- 361 breast cancer were all sensitive to PX-866. PC-3 prostate cancer is PTEN null while HT-29 colon, Skov-3 ovarian and MDA-MB-361 breast cancer all have activating mutations in PIK3CA. HT-29 colon cancer has a coexisting activating mutation in B-Raf, but this was insufficient to cause resistance to PX-866 antitumor activity. Of the sensitive tumors only BxPC3 has no reported mutation in the Pl-3-kinase/Akt pathway.

[00135] All tumors that have an activating mutation in Ras displayed moderate to marked resistance to the antitumor activity of PX-866. This includes HCT-116, HCT-15, and H460 colon cancer which have an activating PIK3CA mutation as well as an activating Ras mutation. The resistant lines A549 and H460 NSC lung cancer also have a mutation resulting in a dysfunctional LKBl concurrent with an activating mutation in Ras. LKBl is a known tumor suppressor that down-regulates mTor mediated protein translation in the presence on low energy conditions in the cell. How LKBl contributes to the sensitivity of the tumors to PI-3-kinase inhibition has not been determined, but in the two xenografts studied the intermediate response to PX-866 observed was similar to tumors with a PIK3CA mutation together with oncogenic Ras. PX-866 was tested for its ability to inhibit tumor PI-3-kinase activity measured by the phosphorylation of Akt at Ser47 by Western blotting, which was found to be equally inhibited in tumors that showed varying degrees of sensitivity to PX-866 treatment (Figure 7B). Thus RAS mutation appears to be an indicator of resistance to PX-866, dominant over the sensitizing effects of PI-3-kinase pathway mutations.

[00136] PX-866 resistant cells display characteristics of Ras transformed cells. Next RPPA technology was used to address whether response to PX-886 was dependent on the level of expression or activation of proteins of the PI-3-kinase/Akt pathway (Figure 8A). Neither PI- 3-kinase protein levels, nor AKT activation measured by Thr308 or Ser47j phosphorylation, nor the phosphorylation of the downstream Λkt target GSK-3 were significantly altered in sensitive compared to resistant lines, or when correlated with in vivo antitumor response (Figure 8B). Two proteins on the array did show a significant difference between sensitive and resistant lines, with levels of c-Myc and cyclin B being significantly higher in lines resistant to PX-866 in vivo. (Figure 8C). It is noteworthy that an increase in both of these proteins has been reported as a result of ras induced transformation.

[00137] H-Ras or H-Ras mutants preferentially activating Raf or RaIGDS, but not RAS mutants linked to PI-3-kinase, are resistant to PX-866 in vitro and in vivo.

[00138] Wild type HCT-1 16 K-Ras positive cells, HCT-1 16 K-Ras-null cells and K-Ras- null cells constitutively expressing an active H-Ras, an H-Ras modified to preferentially activate Raf (H-Ras V12S35), RaIGDS (H-Ras V12G37), or Pl-3-kinase (H-Ras V12C40) were used as a model for the simultaneous and individual activation of proteins effected by Ras signaling. (Figure 9A). This model was used to determine p-AKT activation, cyclin B, and c-Myc in the context of individual downstream Ras targets (Figure 9B). HCT-1 16 cells with activated H-Ras and PI-3-Kinase activating H-Ras showed a robust activation of PI-3-kinase signaling measured by phospho-Ser473-Akt. Surprisingly, FI-Ras activating RaIGDS also retained the ability to activate Akt to a similar level, which may reflect cell line specific signaling, although notably these proteins have been linked. Wild type HCT-1 16 showed moderate levels of phospho-Ser473- Akt and the H-Ras cells specific for Raf had the lowest expression. Both wild type HCT-116 and the K- Ras null FlCT-1 16 transfected with activated H-Ras showed a robust expression of cyclin B, as did the H-Ras activating Raf, while the H-Ras linked to RaIGDS and PI-3-kinase showed basal levels of activation. Wild type HCT-1 16 and the K-Ras null HCT-1 16 transfected with activated H-Ras showed high levels of c-Myc protein, while the three conditional Ras lines all showed lower levels. The lines were also studied for their sensitivity to PX-866 measured by colony formation (Figure 9B). K-Ras null cells, H-Ras, Raf, and RaIGDS activated cell lines behaved similar to the wild type HCT-116 (mutant K-Ras, mutant PIK3CA) line when treated with PX-866. In contrast, an H-Ras mutant that preferentially activates PI-3 -kinase without activating RaIGDS or Raf showed significant inhibition of colony formation by PX-866.

[00139] Apoptosis was measured in the cell lines both by trypan blue assay and flow cytometry, Cells with active Raf and RaIGDS lines showed levels of apoptosis similar to wild type HCT-116 cells while H-Ras cells showed a moderate but significant increase in apoptosis. In contrast H-Ras cells with active PI-3-kinase, but not Raf or RaIGDS activation, showed a large and significant increase in apoptosis. (Figure 10A).

[00140] Tumors lacking the mutant K-Ras allele have previously been shown to be non- tumorigenic indicating that ras is a dominant tumorigenic factor in this cell line. Tumors derived from implanted ras driven cell lines treated with vehicle or 2.5 mg/kg PX-866 were measured at the final day of treatment (Figure 10B). The T/C percentage for the wild type HCT-1 16 cells was 49%, for K-Ras null, H-Ras tumors 54% and for Raf driven tumors 53% (p = 0.05) RaIGDS driven tumors showed a significant decrease in T/C to 37% (p - 0.01 ) and PI-3-kinase driven tumors showed the greatest sensitivity with a T/C value of 31% (p = 0.02).

[00141] Discussion. The PI-3-kinase /Akt signaling pathway is critical for cancer cell growth and survival and a number of inhibitors of PI-3-kinase or Akt have been, or will soon be introduced into clinical trial as antitumor agents. Determining which patients respond to these drugs will play a role in how, and at what pace they move through clinical development. The in vivo antitumor studies in a panel of 13 molecularly characterized human tumor cell line-derived xenografts described herein found that tumors with mutant PIK3CA or PTEN null, but without mutant Ras were sensitive to PX-866. The three most sensitive lines, which displayed a cytostatic or regression response had activating mutations in Pl-3-kinase. It is noteworthy that an activating mutation in Raf in the IJT-29 derived xenograft together with a PIK3CA activating mutation was insufficient to reverse the cytostatic effects of PX-866. The BxPC3 pancreatic cancer cell line with no reported mutations in the PI-3 -kinase/ Akt pathway also showed a cytostatic response to PX-866. This cell line has been characterized as having an inactivating mutation in Smad 4, which has recently been shown to allow these cells to down-regulate PTEN and thus activate PI-3-kinase signaling. This illustrates that while individual mutations or deletions in the PI-3 -kinase/ Akt/ PTEN signaling pathway may be sufficient to predict response to PX-866, they are not be necessary for response as multiple inputs can influence this pathway. Importantly it was found that mutant oncogenic Ras is a negative predictor of response to the PI- 3-kinase inhibitor PX-866 in xenografts, even those with concurrent activating mutations in PI-3- kinase, thus PI-3 -Kinase mutation cannot be used as individual marker for sensitivity.

[00142J RPPA technology was used to measure protein levels and activation in the cell lines in vivo . Several proteins known to be directly involved in PI-3-kinase/ Akt signaling were studied, including PIK3CA, Akt, and GSK. Neither total nor phosphoprotein levels were significantly different between sensitive and resistant lines nor was there a significant association with the in vivo antitumor response. Thus, the expression level and activation of these PI-3- kinase/Akt pathway proteins in vitro under basal conditions does not translate into in vivo tumor sensitivity. Although this finding excludes the level of Akt Thr308 or Ser473 phosphorylation as a predictive biomarker, AKT phosphorylation remains a surrogate endpoint for measuring the efficacy of target inhibition as phosphorylation of Akt was inhibited by PX-866 in vivo, independent of the sensitivity or resistance of the tumor to PX-866 in terms of its growth. Cyclin B and c-Myc were shown to be significantly overexpressed in cell lines forming PX-866 resistant xenografts compared to cell lines showing sensitivity to PX-866. Oncogenic Ras has been shown to have the ability to upregulate total c-Myc levels both through an increase in niRNA levels and increases in protein stability. Cyclin B was increased in cells resistant to PX-866 treatment and showed a significant negative association with antitumor response. Cyclin B has been shown to be upregulated during Ras induced transformation and is associated with an increased mitotic rate. Whether cyclin B and c-Myc act are factors contributing to the resistance of mutant Ras tumors to PX-866, or only serve as markers of mutant Ras is not known.

[00143] In colony formation assays the HCT-1 16 H-Ras line with specific activation of PI-3-Kinase was found to be the only line sensitive to the effects of PI-3-kinase inhibition, indicating this line had been made more dependent on PI-3-Kinase signaling. Cells with the wild type H-Ras showed greater amounts of apoptosis than cells with mutant K-Ras, which may reflect differences between the two Ras isoforms in their utilization of downstream signaling, or the ability of parental K-Ras to utilize less characterized pathways downstream of Ras. This also suggests the slowing of growth seen in mutant Ras tumors in vivo following PX-866 treatment may be a result increased apoptosis. The highest apoptosis was seen in the H-Ras cell line, as it lacked the resistance provided by parallel signaling pathways. Of interest, HCT-1 16 K-Ras null cells which retain a mutant Pl-3 -Kinase were not sensitized to PX-866 suggesting that in cell lines arising from a Ras mutation, in contrast to tumors with exclusive Pl-3 -Kinase mutations, the input from Ras may be essential for PI-3-Kinase signaling to be utilized in tumorigenic processes. This observation agrees with previous studies that found that despite this PI-3-kinase heterozygous mutation, to sensitize HCT-1 16 to the effects of PI-3-Kinase inhibitors in colony formation and growth inhibition assays, a homozygous PIK3CA variant need to be created and the assays performed in low serum conditions, additionally HCT-1 16 cells retaining mutant K- Ras but with only a homozygous wild type PI-3-kinase were able to form tumors. In contrast, K-Ras null HCT-1 16 cells have been shown to lack the ability to form tumors.

{00144] When injected into mice, the oncogenic H-Ras and selective H-Ras cells all retained the ability to form tumors. Despite both having increased Akt activity compared with the parental HCT-116 line, the cells with an active H-Ras responded similarly to PX-866 as the parental HCT-116, while PI-3-kinase activated H-Ras cells showed a 23% greater response to PX-866 than control cells. H-Ras cells displaying low levels of Akt activity but higher levels of cyclin B than the other constructs showed activity similar to the parental HCT-1 16 or HCT-1 16 H-Ras lines. The H-Ras line specific for RaIGDS signaling showed an intermediate response, with a 17% increase in activity of PX-866 against the tumor. Together these results show that the magnitude of Akt activation does not determine response to PI-3-kinase inhibition but that activation of pathways downstream of oncogenic Ras, parallel or compensatory for active PI-3- kinase, can rescue tumors from inhibition.

[00145] Mutant active Ras has recently been described to predict resistance to small molecule inhibitors and antibodies against the EGF receptor presumably due to the down-stream location of Ras in EGF receptor signaling. Additionally, Ras-driven cell lines exhibit a modest response to MEK inhibitors, showing a growth delay when grown as xenografts, while B-Raf driven xenografts display a cytostatic response. This is similar to the current observation with PX-866 in the context of PI-3-kinase signaling and derive from the ability of Raf and PI-3-kinase to converge on redundant downstream mediators, "funnel factors", of translation, such as eiF4e, survival factors such as Bad, and cyclin D for cell cycle progression. Ultimately, treatment of Ras-driven tumors may lie in the direct inhibition of the active Ras protein itself, or the combination of PI-3-kinase inhibitors, including PX-866, with other agents targeting endpoints of the Ras pathway.

[00146] The relationships of the signaling pathways we have studied are shown in Figure 5. Oncogenic Ras effectively negates the effects of other potential predictors such as mutant PIK3CA which has been proposed to be a positive marker of response to inhibitors of PI-3- kinase/Akt signaling and decreases the reliance of the tumor on Pl-3-kinase signaling, in favor of other Ras dependent signaling pathways. Thus, it may be necessary to know the mutational status of K-Ras, PIK3CA and PTEN as markers for predicting response to PI-3-kinase inhibition. This information may have considerable significance in tumor types, such as ovarian, endometrial, and colon where mutations in PIK3CA or PTEN and Ras have been found to coexist at relevant rates.

[00147] In summary, we have studied the activity of the PI-3-kinase inhibitor PX-866 in a panel of tumor cell line-derived xenografts and shown mutant oncogenic Ras to be a negative predictor of response to PX-866, both independently and in the presence of positive prognostic indictors such as PI-31CA and loss of PTEN activity. Known effects of Ras-induced transformation such as increased cyclin B and c-Myc were also negatively associated with antitumor response to PX-866 The level of activation of PI-3-kinase signaling measured with phospho-Akt was not sufficient to predict in vivo antitumor response to PX-866. Ras constructs modified to activate specific components of the Ras signaling pathway showed that multiple pathways are utilized for growth and survival both in vitro and in vivo in Ras dependent signaling. (00148J It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

J. CLAIMSWhat is claimed is:
1. A method for determining phosphatidylinositol-3-kinase inhibitor resistance of tumor cells comprising: characterizing the Ras expression of said tumor cells; and determining the phosphatidylinositol-3-kinase inhibitor resistance of said tumor cells, wherein the presence of mutant Ras indicates resistance to a phosphatidylinositol-3-kinase inhibitor.
2. The method of claim 1, wherein the Ras is selected from K-Ras, H-Ras, N-Ras and a combination thereof.
3. The method of claim 1, wherein the Ras is K-Ras.
4. The method of claim 3, wherein the mutant Ras is at least one of G12D, G 12V, G12S, G12A, G12C, G13A, G13D, G12R, G13C, G13D, E37G, T35S, Y40C, 12V 35S, 12V 37G, 12V 4OC and combinations thereof.
5. The method of claim 1, wherein the phosphatidylinositol-3-kinase inhibitor is selected from PX-866, PX-867, DJM2-181, DJM2-170, DJM2-171, DJM2-177, DJM- 190, PX- 868, PX-870, PX-871, PX-880, PX-881 , PX-882, PX-889 PX-866-1, PX-866-2, PX-867-1, LY294002, LY29002, BEZ235, GSK615, GSK 690693, XL 418, XL 147, XL 665, XL-765, SF
1 126, CAL 101, compound 1 , perifosine, archexin, ZSTK474, GDC-0941 , BGT-226, AS041 164, PL- 103, CHR-4432, AS-604850, triciribine and salts thereof.
6. A method of predicting whether a cancer patient is afflicted with a tumor that will respond effectively to treatment with a phosphatidylinositol-3-kinase inhibitor comprising: assessing the presence of wild-type Ras in said cancer patient; and predicting if the tumor will respond effectively to treatment with a phosphatidylinositoi- 3-kinase inhibitor, wherein the presence of wild-type Ras indicates that the tumor will respond effectively to treatment with a phosphatidylinositol-3-kinase inhibitor.
7. The method of claim 6, wherein the wild-type Ras is selected from wild-type K- Ras, wild-type H-Ras, wild-type N-Ras and a combination thereof.
8. The method of claim 6, wherein the wild-type Ras is wild-type K-Ras.
9. The method of claim 6, wherein the phosphatidylinositol-3-kinase inhibitor is selected from PX-866, PX-867, DJM2-181, DJM2-170, DJM2-171, DJM2-177, DJM- 190, PX- 868, PX-870, PX-871, PX-880, PX-881, PX-882, PX-889 PX-866-1, PX-866-2, PX-867-1, LY294002, LY29002, BEZ235, GSK615, GSK 690693, XL 418, XL 147, XL 665, XL-765, SF
1 126, CAL 101, compound 1, perifosine, archexin, ZSTK474, GDC-0941, BGT-226, AS041 164, PL-103, CHR-4432, AS-604850, triciribine and salts thereof.
10. A method of identifying a patient nonresponsive to treatment with a phosphatidylinositol-3-kinase inhibitor comprising determining the presence or absence of a Ras mutation in a tumor of said patient, whereby the presence of a Ras mutation indicates a patient will not respond to said phosphatidylinositol-3-kinase inhibitor treatment.
11. The method of claim 10, wherein the Ras mutation is selected from mutant K- Ras, mutant H-Ras, mutant N-Ras and a combination thereof.
12. The method of claim 11 , wherein the Ras mutation is mutant K-Ras.
13. The method of claim 12, wherein the mutant K-Ras selected from G12D, G 12V, G12S, G12A, G12C, G13A, G13D, G12R, G13C, G13D, E37G, T35S, Y40C, 12V 35S, 12V 37G, 12V 4OC and combinations thereof.
14. The method of claim 10, wherein the phosphatidylinositol-3-kinase inhibitor is selected from PX-866, PX-867, DJM2-181, DJM2-170, DJM2-171 , DJM2-177, DJM- 190, PX- 868, PX-870, PX-871 , PX-880, PX-88I , PX-882, PX-889 PX-866- 1, PX-866-2, PX-867- 1, LY294002, LY29002, BEZ235, GSK615, GSK 690693, XL 418, XL 147, XL 665, XL-765, SF
1 126, CAL 101, compound 1 , perifosine, archexin, ZSTK474, GDC-0941, BGT-226, AS041 164, PL- 103, CHR-4432, AS-604850, triciribine and salts thereof.
15. A method of identifying a patient nonresponsive to treatment with a phosphatidylinositol-3-kinase inhibitor in combination with a chemotherapeutic agent comprising determining the presence or absence of a Ras mutation in a tumor of said patient, whereby the presence of a Ras mutation in said tumor indicates a patient will not respond to said phosphatidylinositol-3-kinase inhibitor treatment in combination with chemotherapy.
16. The method of claim 15, wherein the Ras mutation is selected from mutant K- Ras, mutant H-Ras, mutant N-Ras and a combination thereof.
17. The method of claim 16, wherein the Ras mutation is mutant K-Ras.
18. The method of claim 17, wherein the mutant K-Ras selected from G12D, G12V, G12S, G12A, G12C, G13A, G13D, G12R, G13C, G13D, E37G, T35S, Y40C, 12V 35S, 12V 37G, 12V 4OC and combinations thereof.
19. The method of claim 15, wherein the phosphatidylinositol-3-kinase inhibitor is selected from PX-866, PX-867, DJM2-181 , DJM2-170, DJM2-171, DJM2-177, DJM- 190, PX- 868, PX-870, PX-871, PX-880, PX-881 , PX-882, PX-889 PX-866-1, PX-866-2, PX-867-1 , LY294002, LY29002, BEZ235, GSK615, GSK 690693, XL 418, XL 147, XL 665, XL-765, SF
1 126, CAL 101, compound 1, perifosine, archexin, ZSTK474, GDC-0941, BGT-226, AS041 164, PL-103, CHR-4432, AS-604850, triciribine and salts thereof.
20. A method for treating cancer in a patient comprising: determining the presence of wild-type Ras in said patient; and administering an effective amount of a phosphatidylinositol-3-kinase inhibitor.
21. The method of claim 20, wherein the wild-type Ras is selected from wild-type K- Ras, wild-type H-Ras, wild-type N-Ras and a combination thereof.
22. The method of claim 20, wherein the wild-type Ras is wild-type K-Ras.
23. The method of claim 20, wherein the phosphatidylinositol-3-kinase inhibitor is selected from PX-866, PX-867, DJM2-181, DJM2-170, DJM2-171, DJM2-177, DJM-190, PX- 868, PX-870, PX-871 , PX-880, PX-881, PX-882, PX-889 PX-866-1, PX-866-2, PX-867-1, LY294002, LY29002, BEZ235, GSK615, GSK 690693, XL 418, XL 147, XL 665, XL-765, SF
1 126, CAL 101, compound 1 , perifosine, archexin, ZSTK474, GDC-0941, BGT-226, AS041164, PL- 103, CHR-4432, AS-604850, triciribine and salts thereof.
24. The method of claim 20 further comprising determining the absence of mutant Ras in said patient.
25. The method of claim 24, wherein the mutant Ras is selected from mutant K-Ras, mutant H-Ras, mutant N-Ras and a combination thereof.
26. The method of claim 20, wherein the mutant Ras is mutant K-Ras.
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