WO2018039132A1 - Methods and compositions for the treatment of cancer - Google Patents

Methods and compositions for the treatment of cancer Download PDF

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Publication number
WO2018039132A1
WO2018039132A1 PCT/US2017/047823 US2017047823W WO2018039132A1 WO 2018039132 A1 WO2018039132 A1 WO 2018039132A1 US 2017047823 W US2017047823 W US 2017047823W WO 2018039132 A1 WO2018039132 A1 WO 2018039132A1
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cancer
iqgapl
wrgykqkk
aitciq
agents
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PCT/US2017/047823
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French (fr)
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Richard Allen Anderson
Suyong CHOI
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Wisconsin Alumni Research Foundation
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Publication of WO2018039132A1 publication Critical patent/WO2018039132A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • compositions for the treatment of cancer comprising peptides that inhibit cancer cell proliferation, kits comprising the same, and methods for using the same.
  • IQ motif containing GTPase activating protein 1 (IQGAPl) is also implicated in many cancers (White et al, 2009) and over expression of IQGAPl is correlated to enhanced tumorigenesis (Feigin et al, 2014).
  • PIPKIa has roles in some cancers (Kao et al., 2009; Semenas et al, 2014). Accordingly, agents that modulate the activity of these molecules are advantageous for the treatment of a variety of cancers.
  • Phosphatidylinositol 4,5-bisphosphate is a lipid messenger regulating most aspects of cellular function (Di Paolo and De Camilli, 2006).
  • PI4,5P 2 serves as a substrate for phospholipases and class I phosphoinositide 3 -kinases (PI3Ks) to generate other messengers (Cantley, 2002; Kadamur and Ross, 2013), or PI4,5P 2 directly binds to its effector proteins and regulates their targeting and activities (Choi et al, 2015). To mediate these diverse processes, PI4,5P 2 is present at a total cellular concentration of approximately
  • PI4,5P 2 pool is maintained by phosphorylation at the 5 hydroxyl of the myo-inositol ring of PI4P by type I phosphatidylinositol phosphate kinases (PIPKIs).
  • PIPKIs type I phosphatidylinositol phosphate kinases
  • RTKs receptor tyrosine kinases
  • GPCRs G-protein-coupled receptors
  • integrins leads to activation of PI3Ks, which utilize PI4,5P 2 as a substrate to generate PI3,4,5P 3 (Vanhaesebroeck et al, 2010).
  • PI3Ks catalyze phosphorylation of PI4,5P 2 at the 3 hydroxyl in vitro (Auger et al, 1989) and in vivo (Stephens et al, 1991), the cellular source of PI4,5P 2 used as substrate has not been studied.
  • An assumption has been that sufficient membrane PI4,5P 2 is available for PDKs to synthesize PI3,4,5P 3 , as cellular PI4,5P 2 concentration is orders of magnitude higher than that of PI3,4,5P 3 (Stephens et al, 1993).
  • This assumption is challenged by findings that the majority of PI4,5P 2 is sequestered by binding proteins (McLaughlin and Murray, 2005), suggesting that de novo synthesis of PI4,5P 2 by PIPKIs may be required for efficient
  • PI3,4,5P 3 synthesis by PDKs In support, depletion of a single PIPKI isoform has no significant impact on global PI4,5P 2 levels (Ling et al., 2007; Mao et al., 2009), while Akt phosphorylation, a readout of PI3K activity, is reduced upon inhibition of a PIPKI (Semenas et al, 2014). These suggest that a locally organized specific pool of PI4,5P 2 is responsible for PI3,4,5P 3 synthesis.
  • IQ motif containing GTPase activating protein 1 is a multidomain protein that scaffolds multiple signaling pathways to regulate a plethora of cell functions (Hedman et al, 2015; Smith et al, 2015).
  • the scaffold role of IQGAPl in the mitogen activated protein kinase (MAPK) pathway is best characterized.
  • MAPK pathway components such as Raf, MEK and Erk, directly interact with IQGAPl, which brings these kinases in close proximity to facilitate their sequential phosphorylation (Jameson et al, 2013; Ren et al, 2007).
  • IQGAPl Additional scaffold roles for IQGAPl in GPCR, Wnt and integrin signaling were also identified (Smith et al, 2015). These versatile cellular roles of IQGAPl are in part supported by its abundance. Notably, an unbiased quantitative study reveals that the mRNA and protein copy number of IQGAPl is at least two orders of magnitude higher than that of its interacting proteins (Schwanatorir et al, 2011). This suggests that in a cell IQGAPl might be present in many discrete signaling complexes, and even more IQGAPl -regulated signaling pathways remain to be characterized.
  • IQGAPl is reported to modulate Akt under stress conditions (Sbroggio et al, 201 la; Sbroggio et al, 201 lb), the molecular mechanism by which IQGAPl controls the PI3K-Akt pathway is largely unknown.
  • the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a composition comprising an IQGAPl peptide.
  • the IQGAPl peptide comprises the amino acid sequence
  • the IQGAP1 peptide is selected from the group consisting of KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS , AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
  • the IQGAPl peptide is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe-N-(2-aminoe)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the method of the present disclosure further comprises administering to the subject one or more additional cancer therapeutic agents.
  • the one or more additional cancer therapeutic agents comprise nitric oxide (NO) inducers, statins, negatively charged phospholipids, anti-oxidants, minerals, antiinflammatory agents, anti -angiogenic agents, matrix metalloproteinase inhibitors, carotenoids, vinca alkaloids, microtubule disrupting agents, anti-angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agents, transitional metal complexes, proteasome inhibitors, antimetabolites, alkylating agents, platinum-based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids, or geldanamycin or a derivative thereof.
  • NO nitric oxide
  • the cancer is selected from the group consisting of breast cancer, prostate cancer, head and neck cancer, liver cancer, and colon cancer.
  • the composition further comprises a pharmaceutical carrier. In some embodiments, the composition further comprises a preservative.
  • the present disclosure provides a composition for treating cancer in a subject in need thereof, comprising an IQGAP1 peptide.
  • the peptide comprises the amino acid sequence
  • the peptide is selected from the group consisting of
  • the peptide is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the composition further comprises one or more additional cancer therapeutic agents.
  • the one or more additional cancer therapeutic agents comprise nitric oxide (NO) inducers, statins, negatively charged phospholipids, anti-oxidants, minerals, anti-inflammatory agents, anti -angiogenic agents, matrix metalloproteinase inhibitors, carotenoids, vinca alkaloids, microtubule disrupting agents, anti -angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agents, transitional metal complexes, proteasome inhibitors, antimetabolites, alkylating agents, platinum-based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids, or geldanamycin or a derivative thereof.
  • NO nitric oxide
  • the cancer is selected from the group consisting of breast cancer, prostate cancer, head and neck cancer, liver cancer, and colon cancer.
  • the present disclosure provides a kit for the treatment of cancer in a subject, comprising an IQGAP1 peptide.
  • the peptide comprises the amino acid sequence
  • the peptide is selected from the group consisting of
  • the peptide is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • FIGS la-lf show PIPKIa and IQGAP1 are required for PI3,4,5P 3 generation.
  • FIG. la IQGAP1 associated proteins were analyzed by IP in Hs578T cells. IgG, nonspecific immunoglobulin.
  • FIGS, lb and lc Changes in IQGAP1 (FIG. lb) and PIPKIa (FIG. lc) associated proteins upon EGF stimulation were analyzed by IP. S. free, serum free.
  • FIG. Id Immunoblotting (IB) after RNAi of the indicated proteins in Hs578T cells (top), mean ⁇ standard deviation (SD) of 3 experiments (bottom). Con, non-targeting siRNA.
  • FIG. le Iqgapf 1' mouse embryonic fibroblasts (MEFs) were re-expressed with the indicated IQGAPl proteins (Supplementary FIG. 2a) and IB after EGF treatment (top), mean ⁇ SD of 3 experiments (bottom).
  • FIGS. 2a-2f show PIPKIa and PI3K interact on the WW and IQ domains of
  • FIGS. 2a and 2b PIPKIa and p85 subunit of PI3K binding sites on IQGAPl were mapped with the indicated truncation mutants (Supplementary FIG. 2a) by IP.
  • FIGS. 2c and 2d In vitro binding experiments with the indicated recombinant proteins, representative of at least 3 experiments.
  • FIG. 2e IQGAPl binding sites were mapped in vitro with the indicated bovine p85a fragments.
  • FIG. 2f A model summary of in vitro binding experiments.
  • FIGS. 3a-3f show IQGAPl physically links PIPKIa to PI3K.
  • FIG. 3a The indicated endogenous proteins were IP'ed from Hs578T cell lysates. Associated proteins were analyzed by IB. b-d, Hs578T cells were transfected with non-targeting (siCon),
  • FIG. 3e Hs578T cells were transfected with empty vector (Mock) or Myc-IQGAPl for 48 h. pi 10a was IP'ed and associated PIPKIa was analyzed by IB.
  • FIG. 3f Immunoblots for FIG. 3e were quantified and the graph is shown as mean ⁇ SD of 3 independent experiments. Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
  • FIGS. 4a-4f show PI4,5P 2 produced by PIPKIa is channeled to PI3K for PI3,4,5P 3 synthesis.
  • Liposomes containing 10 mol. % PI4P (for FIG. 4a,4 e) with the indicated amount of PI4,5P 2 (for FIG. 4d) were used as substrates, mean ⁇ SD of 4 independent experiments.
  • FIG. 4a,4 e Liposomes containing 10 mol. % PI4P (for FIG. 4a,4 e) with the indicated amount of PI4,5P 2 (for FIG. 4d) were used as substrates, mean ⁇ SD of 4 independent experiments.
  • FIG. 4b In vitro binding experiments with PIPKIa, PI3K (pi 10 ⁇ / ⁇ 85 ⁇ ) and the WW-IQ fragment (Supplementary FIG. 2a), mean ⁇ SD of 3 independent experiments. The molar ratio of recombinant proteins used is shown.
  • FIG. 4c PI3,4,5P 3 measurement of in vitro kinase assays with PIPKIa, PI3K and WW-IQ in the indicated combinations, mean ⁇ SD of 3 experiments.
  • FIG. 4f IB after replating on type I collagen (COL) for 45 min or 20 ng/ml EGF treatment for 15 min in Hs578T cells expressing the indicated PH domains (top), mean ⁇ SD of 3 experiments (bottom). Sus, suspension. Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
  • FIGS. 5a-5g show membrane receptor signaling activates the IQGAP1 -mediated pathway.
  • FIGS. 5f, 5g, pi 10a subunit of PI3K interactions with IQGAPl and PIPKIa were analyzed by IP in Hs578T cells expressing shRNAs against the indicated proteins (FIG. 5f), mean ⁇ SD of 3 experiments (FIG. 5g). Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
  • FIGS. 6a-6h IGlDPs inhibit cancer cell survival by blocking PI3,4,5P 3 synthesis.
  • FIG. 6a In vitro binding assays of IGlDPs with PIPKIa or PI3K.
  • FIGS. 6b, 6d Effects of IGlDPs on IQGAPl interactions with PIPKIa and PI3K were analyzed by in vitro binding assay (FIG. 6b) or IP in Hs578T cells (FIG. 6d).
  • FIGS. 6c, 6e effects of IGlDPs on
  • FIG. 6f Viability of MDA-MB-231 transfected with a
  • FIGS. 6g, 6h Cell viability was measured after treating with 20 ⁇ IGlDPs in the indicated cells for 48 h, except 30 ⁇ for T47D and Cal51 cells, mean ⁇ SD of 3 independent experiments. Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'. [0029] FIGS. 7a-7e show inhibition of IQGAP1 -mediated PI3,4,5P 3 synthesis is a novel mechanism for targeted cancer therapy. FIG.
  • FIGS. 8a-8d show insulin-stimulated PI3,4,5P 3 synthesis requires IQGAPl .
  • FIG. 8a Insulin-stimulated pS473 Akt was measured in MEFs, mean ⁇ SD of 3 independent experiments.
  • FIG. 8d A model for the IQGAPl -mediated PI3,4,5P 3 generation and downstream signaling.
  • FIGS, la-lh show ⁇ 4 ⁇ , ⁇ and IQGAPl are required for Akt activation, a, RT-PCR analysis of ⁇ mRNA.
  • ⁇ mRNA levels were normalized with GAPDH mRNA.
  • FIGS. 2a-2j show PIPKIa and PI3K directly interact on IQGAP 1 through the IQ3 and WW motifs, a, Schematic representation of IQGAP 1 domains and constructs used in the study, b, 0.1 ⁇ GST-IQGAPl fragments and PIPKIa immobilized on glutathione beads were incubated with 0.5 ⁇ His-PI3K (His-pl 10a/His-p85).
  • PI3K subunits were analyzed by IB with an anti-His antibody.
  • IQGAP 1-N fragment directly binds to PI3K, whereas neither IQGAP 1-C fragment nor PIPKIa binds, c, His-tagged GST alone, GST-WW domain and PIPKIa (0-1 ⁇ ) were incubated with untagged 0.1 ⁇ PI3K.
  • PI3K was immunoprecipitated with an anti-pl 10a antibody and the associated proteins were analyzed by IB with an anti-His antibody, d, The WW domain and IQ motif amino acid sequences.
  • FIGS. 3a-3f show PI3,4,5P3 synthesis requires concerted PI4,5P2 generation by PIPKIa.
  • b Schematic representation of canonical versus IQGAP1 -mediated PI3,4,5P3 synthesis pathways,
  • c The indicated PH domains were stably expressed in Hs578T cells. Cells grown in tissue culture were photographed in bright field and fluorescent channels at 200X magnification. Roughly 70-80% of cells express exogenous proteins.
  • Hs578T cells stably expressing the indicated PH domains were treated with 10 ng/ml EGF for 10 min.
  • PI3,4,5P3 levels were measured by a competitive ELISA and the graph is shown as mean ⁇ SD of three independent experiments (bottom). Paired Student t-tests were used for statistical analysis (*, p ⁇ 0.05; **, p ⁇ 0.01 ; n.s., not significant), e, Hs578T cells were stably expressed with shRNA against IQGAP1.
  • FIGS. 4a-4e show separation of PIPKIa and PI3K binding on IQGAPl attenuates PI3,4,5P3 synthesis
  • a Schematic representation of uncoupling of PI4,5P2 and PI3,4,5P3 synthesis by inserting the 17 aa indicated between the WW and IQ domains
  • b Iqgapl knockout (Iqgapl-/-) mouse embryonic fibroblasts (MEFs) were reconstituted with the indicated GFP-tagged human IQGAPl constructs.
  • Cells were treated with 10 ng/ml EGF for 15 min and cellular PI3,4,5P3 contents were measured by a competitive ELISA.
  • FIGS. 5a-5e show membrane receptor signaling activates the IQGAPl -mediated PI3,4,5P3 synthesis pathway
  • a Hs578T cells stably expressing shRNAs against IQGAPl and PIPKIa were plated on 10 ⁇ g/ml type I collagen for 30 min.
  • FIGS. 6a-6f show the IQGAPl -derived peptides inhibit Akt activation, a, Sequences of cell permeable IGlDPs.
  • b Empty vector (Mock) and HA- tagged IQ domain alone was stably expressed in Hs578T cells.
  • Cell lysates were analyzed by IB with the indicated antibodies, c, Hs578T cells were transfected with empty vector or pi 10a subunit of PI3K for 24 h. Then, cells were treated with the indicated 20 ⁇ of IGlDPs for 24 h.
  • FIGS. 7A-7L' show unprocessed original scans of blots.
  • IQGAPl peptides selectively kill cancer cells, and are useful in the treatment of cancer in subjects in need thereof.
  • the "administration" of an agent, drug, or peptide to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or
  • Administration includes self-administration and the administration by another.
  • amino acid includes naturally-occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally-occurring amino acids.
  • Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally-occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally-occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally- occurring amino acid.
  • Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • the term "effective amount" refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in, the symptoms associated with cancer.
  • the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions can also be administered in combination with one or more additional therapeutic compounds.
  • the IQGAP1 peptides may be administered to a subject having one or more signs or symptoms of cancer.
  • a "therapeutically effective amount" of the IQGAP1 peptides is meant levels in which the physiological effects of cancer are, at a minimum, ameliorated.
  • an "isolated” or “purified” polypeptide or peptide is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the agent is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • an isolated IQGAP1 peptide would be free of materials that would interfere with diagnostic or therapeutic uses of the agent.
  • interfering materials may include enzymes, hormones and other proteinaceous and nonproteinaceous solutes.
  • polypeptide As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins.
  • Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art.
  • the terms “treating” or “treatment” or “alleviation” refers to treatment wherein the object is to prevent, slow down, or lessen the targeted pathologic condition or disorder.
  • a subject is successfully “treated” for cancer if, after receiving a therapeutic amount of one or more IQGAPl peptides according to the methods described herein, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of cancer.
  • the various modes of treatment provided herein include “substantial” treatment, which includes total and less than total treatment and wherein some biologically or medically relevant result is achieved.
  • prevention or “preventing” of a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • prevention refers to the prevention of one or more signs or symptoms of cancer.
  • prevention refers to the prevention of cancer/tumor growth.
  • prevention refers to the prevention of cancer metastasis.
  • IQGAPl peptide refers to a peptide derived from the IQ motif containing GTPase activating protein 1 (IQGAPl).
  • IQGAPl-derived peptides are mimics of IQGAPl sub-domains that mediate PIPKIa and PI3K binding, and are 24 to 28 amino acids in length.
  • IQGAPl-derived peptides completely or partially dissolve in water and become basic when dissolved in water.
  • cancer refers to all types of cancer or neoplasm or malignant or benign tumors found in mammals, including carcinomas and sarcomas.
  • Examples of cancers amendable to methods disclosed herein include, but are not limited to, breast cancer, prostate cancer, head and neck cancer, liver cancer, and colon cancer.
  • Peptides of the present disclosure may be synthesized by any of the methods well known in the art. Suitable methods for chemically synthesizing the protein include, for example, those described by Stuart and Young in Solid Phase Peptide Synthesis, Second Edition, Pierce Chemical Company (1984), and in Methods Enzymol. 289, Academic Press, Inc, New York (1997).
  • the peptide is selected from the group consisting of KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS , AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
  • the peptide comprises
  • RDHINDIIKIQAFIRANKARDDYKTLINAE RDHINDIIKIQAFIRANKARDDYKTLINAE, a functional fragment thereof, or a variant thereof.
  • the peptide is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the peptide comprises a variant of
  • the variant has at least 95%, at least 90%, at least 85%>, at least 80%), at least 75%, at least 70%, at least 65%>, at least 60%>, or at least 50% homology to KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS , AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
  • the variant has more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, or more than 50% homology to KWVKHWVKGGYYYYHNLETQEGGWDEPP,
  • one or more amino acids of the IQGAPl peptide is modified to enhance stability of the peptide or the half-life of the peptide when administered to a subject.
  • one or more covalent bonds of the IQGAPl peptide are modified to enhance stability of the peptide or the half-life of the peptide when administered to a subject.
  • the peptide comprises a protein transduction domain.
  • the protein transduction domain comprises a TAT domain.
  • the protein transduction domain comprises a polyarginine domain.
  • the peptide comprises a protein tag.
  • the protein tag comprises a Myc-tag (e.g., comprising the amino acid sequence
  • Suitable methods include in vitro, ex vivo, or in vivo methods.
  • In vivo methods typically include the administration of an IQGAP1 peptide, such as those described herein, to a mammal, preferably a human.
  • IQGAP1 peptides are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect).
  • the dose and dosage regimen will depend upon the degree of the condition to be treated in the subject, the characteristics of the particular IQGAP1 peptide used, e.g., its therapeutic index, the subject, and the subject's medical history.
  • IQGAP1 peptides are examples of the particular IQGAP1 peptide used, e.g., its therapeutic index, the subject, and the subject's medical history.
  • Effective amounts of IQGAP1 peptides for methods of cancer treatment described herein may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians.
  • An effective amount of a peptide useful in the methods of the present technology, in a pharmaceutical composition, for example, may be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds.
  • the peptide may be administered systemically or locally.
  • compositions for administration, singly or in combination, to a subject for the treatment or prevention of a disorder described herein.
  • Such compositions typically include the active agent and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • Supplementary active compounds can also be incorporated into the compositions.
  • compositions are typically formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the dosing formulation can be provided in a kit containing all necessary equipment (e.g. vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g. 7 days of treatment).
  • compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • Peptide compositions of the present disclosure can include a carrier, which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • a carrier which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like.
  • Glutathione and other antioxidants can be included to prevent oxidation.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the therapeutic compound is formulated into solutions, suspensions, and ointments appropriate for use in the eye.
  • Ophthalmic formulations generally, see Mitra (ed.), Ophthalmic Drug Delivery Systems, Marcel Dekker, Inc., New York, N.Y. (1993) and also Havener, W. H., Ocular Pharmacology, C.V. Mosby Co., St. Louis (1983).
  • Ophthalmic pharmaceutical compositions may be adapted for topical administration to the eye in the form of solutions, suspensions, ointments, creams or as a solid insert, such as for the treatment of eye-related cancers.
  • the ophthalmic preparation may contain non-toxic auxiliary substances such as antibacterial components which are non-injurious in use, for example, thimerosal, benzalkonium chloride, methyl and propyl paraben, benzyldodecinium bromide, benzyl alcohol, or phenyl ethanol; buffering ingredients such as sodium chloride, sodium borate, sodium acetate, sodium citrate, or gluconate buffers; and other conventional ingredients such as sorbitan monolaurate, triethanolamine, polyoxyethylene sorbitan monopalmitylate, ethylenediamine tetraacetic acid, and the like.
  • auxiliary substances such as antibacterial components which are non-injurious in use, for example, thimerosal, benzalkonium chloride, methyl and propyl paraben, benzyldodecinium bromide, benzyl alcohol, or phenyl ethanol
  • buffering ingredients such as sodium chloride, sodium borate, sodium acetate
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • transdermal administration can be accomplished through the use of nasal sprays.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • transdermal administration may be performed my iontophoresis.
  • a therapeutic protein or peptide can be formulated in a carrier system.
  • the carrier can be a colloidal system.
  • the colloidal system can be a liposome, a phospholipid bilayer vehicle.
  • the therapeutic peptide is encapsulated in a liposome while maintaining peptide integrity.
  • there are a variety of methods to prepare liposomes See Lichtenberg et al, Methods Biochem. Anal., 33 :337-462 (1988); Anselem et al, Liposome Technology, CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann. Pharmacother., 34 (7- 8):915-923 (2000)).
  • An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes.
  • Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.
  • the carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix.
  • the therapeutic peptide can be embedded in the polymer matrix, while maintaining protein integrity.
  • the polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly a-hydroxy acids. Examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof.
  • the polymer is poly-lactic acid (PLA) or copoly lactic/glycolic acid (PGLA).
  • PHA poly-lactic acid
  • PGLA copoly lactic/glycolic acid
  • polymer microsphere sustained release formulations are described in PCT publication WO 99/15154 (Tracy et al), U.S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale et al), PCT publication WO 96/40073 (Zale et al), and PCT publication WO 00/38651 (Shah et al).
  • U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.
  • the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylacetic acid.
  • Such formulations can be prepared using known techniques.
  • the materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers.
  • the therapeutic compounds can also be formulated to enhance intracellular delivery.
  • liposomal delivery systems are known in the art, see, e.g., Chonn and Cullis, "Recent Advances in Liposome Drug Delivery Systems," Current Opinion in Biotechnology 6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: Selecting Manufacture and Development Processes,” Immunomethods 4 (3) 201-9 (1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery: Progress and Problems," Trends Biotechnol. 13 (12):527-37 (1995). Mizguchi et al, Cancer Lett. 100:63-69 (1996), describes the use of fusogenic liposomes to deliver a protein to cells both in vivo and in vitro.
  • Dosage, toxicity and therapeutic efficacy of the therapeutic agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • an effective amount of the IQGAPl peptides ranges from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day.
  • the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks.
  • a single dosage of peptide ranges from 0.1-10,000 micrograms per kg body weight.
  • IQGAP1 peptide concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter.
  • An exemplary treatment regime entails administration once per day or once a week. Intervals can also be irregular as indicated by measuring blood levels of glucose or insulin in the subject and adjusting dosage or administration accordingly. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • a therapeutically effective amount of an IQGAP1 peptide comprises a concentration of peptide at the target tissue of 10 "11 to 10 "6 molar, e.g., approximately 10 "7 molar.
  • This concentration may be delivered by systemic doses of 0.01 to 100 mg/kg or equivalent dose by body surface area.
  • the schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, most preferably by single daily or weekly administration, but also including continuous administration (e.g. parenteral infusion or transdermal application).
  • IQGAP1 peptide concentrations range from 10 to 100 micromolar (i.e., 10 "5 molar to 10 "4 molar).
  • treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.
  • the mammal treated in accordance present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits.
  • the mammal is a human.
  • the IQGAPl peptides described herein may be administered in combination with another therapeutic agent.
  • another therapeutic agent such as a pharmaceutically acceptable salt, ester, amide, prodrug, or solvate.
  • an adjuvant i.e., by itself the adjuvant may only have minimal therapeutic benefit, but in combination with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced.
  • the benefit experienced by a patient may be increased by administering one of the compounds described herein with another therapeutic agent (which also includes a therapeutic regimen) that also has therapeutic benefit in the treatment of cancer.
  • another therapeutic agent which also includes a therapeutic regimen
  • the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills).
  • One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses.
  • the timing between the multiple doses may vary from more than zero weeks to less than four weeks.
  • the combination methods, compositions and formulations are not to be limited to the use of only two agents.
  • an IQGAPl peptide may be provided with at least one additional cancer therapeutic and at least one agent for the amelioration or management of side effects associate with the IQGAPl peptide or the additional agent.
  • IQGAPl peptide with nitric oxide (NO) inducers include use of at least one IQGAPl peptide with nitric oxide (NO) inducers, statins, negatively charged phospholipids, anti-oxidants, minerals, anti-inflammatory agents, anti -angiogenic agents, matrix metalloproteinase inhibitors, and carotenoids.
  • suitable combination agents may fall within multiple categories (by way of example only, lutein is an anti-oxidant and a carotenoid).
  • the IQGAPl peptides may also be administered with additional agents that may provide benefit to the patient, including by way of example only cyclosporin A.
  • anti angiogenic or anti-VEGF drugs has also been shown to provide benefit for patients with cancer.
  • suitable anti angiogenic or anti-VEGF drugs that could be used in combination with at least one IQGAPl peptide include Rhufab V2
  • Prinomastat AG3340 ⁇ synthetic matrix metalloproteinase inhibitor, Pfizer
  • fluocinolone acetonide including fluocinolone intraocular implant, Bausch & Lomb/Control Delivery Systems
  • VEGFR inhibitors Sugen
  • VEGF-Trap VEGF-Trap
  • Cancer therapeutic agents suitable for IQGAPl peptide combination therapies include, but are not limited to vinca alkaloids, agents that disrupt microtubule formation (such as colchicines and its derivatives), anti-angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agent (such as tyrosine kinase inhibitors), transitional metal complexes, proteasome inhibitors, antimetabolites (such as nucleoside analogs), alkylating agents, platinum-based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids (such as all-trans retinoic acids or a derivatives thereof); geldanamycin or a derivative thereof (such as 17-AAG), and other cancer therapeutic agents recognized in the art.
  • vinca alkaloids agents that disrupt microtubule formation
  • agents that disrupt microtubule formation such as colchicines and its derivatives
  • anti-angiogenic agents such as therapeutic antibodies
  • therapeutic antibodies such as col
  • cancer therapeutics for use in IQGAPl peptide combination therapies comprise one or more of adriamycin, colchicine, cyclophosphamide, actinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone, fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, interferons, camptothecin and derivatives thereof, phenesterine, taxanes and derivatives thereof (e.g., taxol, paclitaxel and derivatives thereof, taxotere and derivatives thereof, and the like), topetecan, vinblastine, vincristine, tamoxifen, piposulfan, nab-5404, nab-5800, nab- 5801, Irinotecan, HKP, Ortataxe
  • references to a cancer therapeutic agent herein applies to the agent or its derivatives and accordingly the present technology includes either of these embodiments (agent; agent or derivative(s)).
  • “Derivatives” or “analogs” of a cancer therapeutic agent or other chemical moiety include, but are not limited to, compounds that are structurally similar to the cancer therapeutic agent or moiety or are in the same general chemical class as the cancer therapeutic agent or moiety.
  • the derivative or analog of the cancer therapeutic agent or moiety retains similar chemical and/or physical property (including, for example, functionality) of the cancer therapeutic agent or moiety.
  • the cancer therapeutic agent is an antineoplastic agent including, but is not limited to, carboplatin, Navelbine® (vinorelbine), anthracycline
  • kits comprising IQGAPl peptides for the treatment of cancer in a subject.
  • the kit comprises one or more IQGAPl peptides, sterile vessels suitable for handling of the peptides, and instructions for use.
  • the kit comprises one or more IQGAPl peptides together with one or more additional cancer therapeutic agents.
  • the IQGAPl peptide comprises the amino acid sequence KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS ,
  • the IQGAP1 peptide is selected from the group consisting of KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS , AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
  • the IQGAPl peptide is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe-N-(2-aminoe)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • EXAMPLE 1 Agonist Stimulated Phosphatidylinositol 3, 4, 5 -Triphosphate Synthesis by IQGAPl Scaffold Phosphoinositide Kinases
  • peptides disclosed herein including IQGAPl peptides, selectively kill cancer cells in vitro. Accordingly, the peptides are useful in methods and compositions for the treatment of cancer.
  • PI3,4,5P 3 phosphatidylinositol 3,4,5-trisphosphate
  • PDKs class I phosphoinositide 3-kinases
  • PI3K and PIPKIa a PI4,5P 2 generating enzyme
  • IQGAPl a scaffold protein.
  • PI4,5P 2 generated by PIPKIa is channeled to PI3K for PI3,4,5P 3 synthesis. Receptor signaling stimulates this
  • IQGAPl/phosphoinositide kinases complex and is required for PI3,4,5P 3 synthesis.
  • IQGAP 1 interacts with PIPKIa and PI3K bringing the two phosphoinositide kinases in close proximity, which allows for the PI4,5P 2 produced by PIPKIa to be channeled to PI3K for PI3,4,5P 3 synthesis.
  • This IQGAPl -mediated pathway is responsible for PI3,4,5P 3 synthesis stimulated by integrin, RTK or GPCR activation.
  • blockade of the IQGAPl interaction with either PIPKIa or PI3K inhibited PI3,4,5P 3 synthesis and Akt activation, which blocks breast cancer cell proliferation.
  • MDA-MB-231, MDA-MB-468, HEK 293, Hs578T, Cal51, UACC812, SkBr3, HaCaT, Cos7 and MEF cells were purchased from ATCC and maintained in DMEM supplemented with 10% fetal bovine serum (Gibco).
  • MCF-7 Soule et al, 1973
  • HUVEC Beauvais et al, 2009
  • the PIPKIa and IQGAPl constructs used for this work have been described previously (Choi et al, 2013; Loijens et al, 1996).
  • the WW-IQ region was amplified using 5 '-GATGGATCCGGTGAAACTTACC AC AGTGATCTTGCT-3 ' (Forward) and 5 '-GATAAGCTTCT AATCCTC AGC ATTGATGAGAGTCTTGT A-3 ' (Reverse) primers and cloned in pET28a vector (Novagen) within BamHI and Hindlll enzyme sites.
  • Bovine p85a constructs (FIG. 2e) (gifts from D.
  • FIG. 6f constitutively active Aktl (FIG. 6f), Racl and Cdc42 (Supplementary FIG. 6f) constructs used in the study were purchased from Addgene and subcloned in pcDNA3.1 vector (Invitrogen).
  • the GFP-PH domain constructs giftss from T. Balla, National Institutes of Health, USA) (FIG. 4f and Supplementary FIGS. 3c, d) were described previously
  • the 17Insert IQGAPl construct (FIG. le and Supplementary FIG. 4) was generated by a series of site-directed mutagenesis polymerase chain reactions to insert 5'-
  • CATCTCTGGGGTGACTGCCCATATAACCGAGAACAGCTGTGGCTGGTCGAC-3' Constructs were transfected in cells by a lipid-based delivery system from Minis (MDA-MB- 231 cells) or Invitrogen (other cell lines) according to the manufacturer's instructions.
  • cells were infected with retrovirus for 24 h. Then, cells expressing GFP-IQGAPl were first selected for GFP expression, and then further sorted by expression level.
  • cells transfected with lipid were selected by 1.2 mg ml -1 neomycin. Then, GFP-positive cells were further selected in a cell sorter.
  • siRNAs used in the study were obtained from Dharmacon (FIG. Id).
  • Single siRNAs targeting IQGAPl either 5'-GGAAAGCUCUGGCAAUUUAUU- 3' or 5'-GAACGUGGCUUAUGAGUACUU-3'
  • PIPKIa either 5'- AAGTTGGAGC ACTCTTGG-3 ' or 5'-GCACATTATCCCTACCTTA-3 '
  • siRNA targeting the 3 'UTR of PIPKIa (5'-UGACUCCUGGAAGAAUACUCCUGUA-3') was purchased from Thermo Fisher Scientific. Non-targeting siRNA (Dharmacon) was used as a control. siRNAs were delivered to cells by RNAiMAX reagent (Thermo Fisher Scientific) and knockdown efficiency was determined by immunoblotting. Knockdown efficiency greater than 80% was required to observe phenotypic changes in the study. This is consistent with a recent report that at least 80% knockdown of IQGAP1 is required to observe reduced Erk activity
  • Cells were suspended at least for 1 h at 37 °C by rotation before replating on tissue culture plates coated with 10 ⁇ g ml -1 type I collagen (Sigma Aldrich) or fibronectin (Thermo Fisher Scientific). After incubation for 15 to 60 min, cells were lysed and further analysed by immunoblotting or immunoprecipitation.
  • Cell viability assay Cells were plated on 6-well plates at 30-50% confluency at the time of treatment. Cells were changed with fresh media containing class I PI(3)K inhibitors or IQGAP1 -derived peptides at the indicated concentrations for the indicated times (24 to 72 h) (FIGS. 6f-h and 7a-e and Supplementary FIGS. 6c-f). Dead cells floating in the culture medium were removed by washing twice with PBS. Residual PBS was completely removed and cells were lifted by incubating with trypsin-EDTA for 10 m. Cells were stained with 0.4% trypan blue solution (Thermo Fisher Scientific) and counted with a
  • the protein concentration of lysates was measured by the BCA method (Pierce) and equal amounts of protein were used for further analysis. All antibodies were diluted in a 1 : 1,000 ratio for immunoblotting. For immunoprecipitation, 0.5 to 1 mg of proteins were incubated with 1 ⁇ g of antibodies at 4 °C for 8 h and then incubated with a 50% slurry of Protein G Sepharose (GE Life Sciences) for another 2 h. After washing 5 times with lysis buffer, the protein complex was eluted with SDS sample buffer. For immunoblotting, 5 to 20 ⁇ g of proteins were loaded. After developing immunoblots, the film was scanned using a transmitted light scanner (resolution: 600 dpi). Protein bands were quantified using ImageJ, and statistical analysis of the data was performed with Microsoft Excel. The statistical analysis was performed using data from at least three independent experiments.
  • PtdIns(3,4,5)P 3 was performed as previously described (Chagpar et al., 2009; Hammond & Irvine et al., 2009; Sharma et al., 2008) with modifications. Briefly, cells grown in medium were rapidly fixed by adding an equal volume of 8% paraformaldehyde and 0.5%
  • fluorophore- conjugated secondary antibodies were incubated in a solution of buffer A containing 0.1% saponin and 5 vol% of FBS for 1 h at room temperature. Then, cells were washed with buffer A for 45 m at room temperature before post-fixation with 2% paraformaldehyde and 0.125% glutaraldehyde for 10 m at room temperature. Coverslips were washed 5 times with PBS containing 50 mM H 4 CI and once with distilled water. Fluorescence microscopy was performed using a 40 or 60 x plan-fluor objective on a Nikon Eclipse TE2000U equipped with a Photometries Cool SNAP ES CCD (charge-coupled device) camera. Images were captured using MetaMorph v6.3 (Molecular Devices). Images were exported to Photoshop CS2 (Adobe) for final processing and assembly.
  • phosphoinositide measurement were performed using a kit from Echelon Biosciences according to the manufacturer's instructions. Briefly, cells were lysed in ice-cold
  • TCA trichloroacetic acid
  • Pellets were washed in 5% TCA/1 mM EDTA and neutral lipids were extracted with CH 3 OH/CHCI 3 (2: 1) and discarded. Acidic lipids were extracted with CH 3 OH/CHCI 3 /HCI (80:40: 1), recovered by phase-split. Recovered lipids were dried and resuspended for competitive ELISA analysis.
  • PtdIns(3)P, PtdIns(4)P, PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 levels were quantified by competitive ELISA (Echelon Biosciences).
  • PtdIns(4)P and PtdIns(4,5)P 2 cells were harvested from 10 cm plates. For measuring PtdIns(4)P and PtdIns(4,5)P 2 , cells were harvested from 10 cm plates. For measuring PtdIns(4)P and PtdIns(4,5)P 2 , cells were harvested from 10 cm plates.
  • PtdIns(3)P and PtdIns(3,4,5)P 3 cells were harvested from 15 cm plates.
  • PtdIns(4)P liposomes that were used as a substrate contain 30% PC, 30% PE, 30% PS and 10% PtdIns(4)P in a molar ratio.
  • Liposomes containing PtdIns(4,5)P 2 were made by decreasing the PC content. For example, liposomes containing 5% PtdIns(4,5)P 2 were generated with 25% PC, 30% PE, 30% PS, 10% PtdIns(4)P and 5% PtdIns(4,5)P 2 . All phospholipids (natural, porcine brain) were purchased from Avanti Polar Lipids. Mixed phospholipids were dried and rehydrated with 50 mM Tris, pH 8.0 for kinase assays.
  • Hydrated lipids were subjected to at least 5 cycles of freeze thawing in liquid nitrogen followed by 1 min bath sonication and extrusion through a 0.1 ⁇ filter.
  • FIG. 3a were from porcine brain (Avanti Polar Lipids) and their purity was extensively tested for the presence of contamination with PtdIns(3)P or PtdIns(3,4,5)P 3 .
  • the identity and purity of brain PtdIns(4)P was tested using mass spectrometry, proton NMR, phosphorus NMR, TLC, and oxidation by UV/VIS.
  • the mass spectrometry also confirms the absence of PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 species as their mlz are not present in the release spectra.
  • phosphorus NMR was used as for the presence or absence of PtdIns(3)P.
  • PtdIns(3)P has two peaks, one at approximately 0.1 and another at approximately -0.75.
  • PtdIns(4)P contains two peaks, one around -0.45 and the second at around 2.5. These shifts can fluctuate slightly, but the pattern is consistent across synthetic and natural PIPs.
  • Brain PtdIns(4,5)P 2 was analysed by mass spectrometry, TLC, proton NMR, phosphorus NMR, and oxidation by UV/VIS.
  • the phosphorus NMR peaks for PtdIns(4,5)P 2 are at approximately 2.4, 1.7 and -0.4.
  • the shifts for PtdIns(3,4,5)P 3 are typically 0.7, 0.0 and 0.8. None of these overlaps with the peaks present in the PtdIns(4,5)P 2 species.
  • mice Male 16-19-week-old wild-type or IqgapF 1' mice (129 background) were fasted for 4 h and received an intraperitoneal injection of saline or insulin (5 U kg -1 ). Mice were euthanized by C0 2 , and 15 min post-injection, tissues (liver and quadriceps muscle) were harvested and immediately frozen in a dry-ice ethanol bath and stored on dry ice until being placed in a -80 °C degree freezer. Lipids were extracted from mouse tissues with the TCA extraction method as above.
  • Lipids extracted from equal weights (approximately 100 mg) of mouse tissues were analysed for PtdIns(3,4,5)P 3 using a competitive ELISA (Echelon Biosciences) and normalized by weight of tissue. Statistical analysis was performed with 6 paired cases.
  • mice were bred in the animal facility at the National Institutes of Health (NTH) and maintained according to NOT guidelines. The studies were performed with approval of the NIH Animal Care and Use Committee.
  • PI3K-Akt pathway requires ⁇ 4 ⁇ . ⁇ and IQGAPl
  • IQGAPl -associated proteins were analyzed by immunoprecipitation (IP). This demonstrated that ⁇ 4 ⁇ , ⁇ and PI3K associate with IQGAPl (FIG. la). IQGAPl also associated with the PI3K downstream effectors of PI3,4,5P 3 phosphoinositide-dependent kinase 1 (PDK1) and Akt. In addition, IQGAP2 and ⁇ associated with IQGAPl confirming known interactors (Schmidt et al., 2008; Choi et al, 2013).
  • FIGS, lb, lc, le Expression of ⁇ did not impact Akt phosphorylation indicating specificity for PIPKI isoforms. Knockdown of PMKIIIa also significantly reduced Akt phosphorylation (Supplementary FIG. lg). These results were replicated in multiple cell lines including MDA-MB-231, Hs578T, MCF7 and HaCaT. These combined data support a model where PMKIIIa, PIPKIa via its association with IQGAPl generates a PI4,5P 2 pool that flows into the PI3K pathway for PI3,4,5P 3 synthesis and Akt activation (Supplementary FIG. le).
  • IQGAPl and PIPKIa loss diminished Akt activation. This was not due to increased expression of the phosphatase and tensin homolog (PTEN), which dephosphorylates
  • PI3,4,5P 3 required for Akt activation (Stephens et al, 1991), PI4P, PI4,5P 2 and PI3,4,5P 3 content was measured. Loss of PMKIIIa, PIPKIa or IQGAPl reduced PI3,4,5P 3 levels -50% and 60%, respectively (FIG. If and Supplementary FIG. lh). Yet, PI3P, PMP and PI4,5P 2 did not change (FIG. If and Supplementary FIG. If) indicating that diminished PI3,4,5P 3 generation or Akt phosphorylation is not due to lack of PMP or PI4,5P 2 . Ectopic expression of IQGAPl or PIPKIa did not increase PI4,5P 2 but dramatically increased PI3,4,5P 3
  • IQGAPl mediates a PIPKIa and PI3K complex
  • IQ domain of IQGAP 1 contains 4 structurally conserved IQ motifs (IQ 1 -IQ4) (Li et al., 2013) .
  • WW and each IQ motif polypeptides were fused to GST (Supplementary FIG. 2d) for binding assay.
  • PIPKIa specifically bound to GST-IQ3, which blocked PIPKIa interaction with IQGAP1-N (Supplementary FIGS. 2e-g).
  • PI3K consisting of pi 10a and p85a bound to GST-WW and -IQ3 and either peptide blocked IQGAPl interaction with PI3K (Supplementary FIGS. 2h-j).
  • IQGAPl significantly increased the PIPKIa co-IP with pi 10a and the generation of PI3,4,5P 3 (FIGS. 3e, 3f and Supplementary FIG. le).
  • PIPKIa and PI3K do not bind to one another (Supplementary FIGS. 2b, 2c), these data indicate that IQGAPl scaffolds PIPKIa and PI3K, bring them in proximity and enhancing generation of PI3,4,5P 3 in vivo. 3.
  • IQGAP1 regulates PI3.4.5P ⁇ synthesis
  • the IQGAPl -tethered PI3K would selectively utilize the PI4,5P 2 generated from PIPKIa.
  • in vitro kinase assays were performed with PIPKIa and PI3K using liposomes containing PI4P or with increasing amounts of PI4,5P 2 (FIG. 4d).
  • PI3,4,5P 3 synthesis increased proportionally with the PI4,5P 2 mole %, suggesting that PI3,4,5P 3 is largely generated from the PI4,5P 2 in the liposomes.
  • PI3P, PI4P and PI4,5P 2 binding proteins were used to sequester free lipid.
  • PI3P-, PI4P- and PI4,5P 2 -specific binding domains (Hammond et al., 2012; Balla et al., 2008; Stenmark et al, 2002) were stably expressed and the impact on collagen- and EGF-stimulated PI3,4,5P 3 synthesis and Akt phosphorylation were quantified.
  • PI4P (Osh2-PH2x) and PI4,5P 2 (PLC51-PH) binding proteins blocked PI3,4,5P 3 synthesis and Akt activation in suspended and serum starved cells but not upon stimulation by collagen or EGF (FIG. 4f and Supplementary FIGS. 3b-d).
  • neomycin which is a PI4,5P 2 sequestration reagent. Neomycin reduced Akt and Akt downstream effectors' activity in unstimulated conditions, whereas neomycin had no impact on insulin-stimulated conditions (Shimaya et al, 2004).
  • PI3,4,5P 3 This indicates at least two distinct pathways of PI3,4,5P 3 synthesis exist in cells, a canonical pathway, where PI3,4,5P 3 is generated from a pool of membrane PI4,5P 2 that is accessible to the binding domains and is inhibited by sequestration of PI4P or PI4,5P 2 (Supplementary FIG. 3b).
  • PI4P and PI4,5P 2 sequestration does not impact PI3,4,5P 3 synthesis as the binding domains would not access the channeling substrates in the IQGAPl -scaffolded complex, consistent with the data (FIG. 4f and Supplementary FIG. 3d).
  • EGF stimulated IQGAP1 interactions with PI3K pathway components (FIGS, lb, lc).
  • IQGAP1 and PIPKIa knockdown cells were treated with agonists for a number of membrane receptor classes. Fibronectin or collagen receptor-mediated Akt activation and PI3,4,5P 3 generation were significantly reduced by IQGAPl or PIPKIa loss (FIGS. 5a, 5b and Supplementary FIGS. 5a-c).
  • FAK Focal adhesion kinase
  • FIG. 5f Summary FIG. 5f.
  • IQGAPl and PIPKIa regulate the PI3K-Akt pathway downstream of receptors
  • IQGAPl complex formation was quantified upon agonist stimulation.
  • the interaction of the PI3K pi 10a subunit with IQGAPl and PIPKIa was enhanced by activation of all three receptor types (FIGS, lb, lc, 5f, 5g).
  • FIGS, lb, lc, 5f, 5g receptor activation stimulates IQGAPl -PIPKIa-PI3K assembly and this ensures efficient PI3,4,5P 3 synthesis and Akt activation.
  • IQGAPl -derived peptides were made membrane-permeable by adding eight arginine residues to the N-terminus (Supplementary FIG. 6a) (Jameson et al.,
  • IQGAPl peptides selectively kill cancer cells
  • IQGAPl modulates Racl and Cdc42 (Li et al., 2013; Mataraza et al., 2003) and Racl and Cdc42 activate pi 10a of PI3K (Fritsch et al., 2013), but constitutively active Racl or Cdc42 did not rescue cell death by IGlDPs (Supplementary FIG. 7f).
  • IQGAPl modulates Ras-dependent Erk pathways via the WW and IQ domains (Ren et al., 2007; Jameson et al., 2013; Sbroggio et al., 2011; Roy, et al., 2005; Matsunaga et al., 2014). Consistently, depletion of IQGAPl reduced Erk phosphorylation and the WW and IQ domains are required for Erk phosphorylation in MEFs (FIG. le). However, knockdown of IQGAPl in MDA-MB-231 and Hs578T cells had no impact on Erk phosphorylation (FIGS. Id, 5c, 5d and Supplementary FIGS. 5a, 5f).
  • Hs578Bst HUVEC (normal), Hs578T and MDA- MB-231 (breast cancer) cells were treated with small molecule PI3K inhibitors that are currently in clinical trials(Engelman, 2009) or the IGlDPs and cell viability was measured (FIG. 7).
  • the PI3K inhibitors used in this study have submicromolar IC 50 , but in many cancer cell lines 1-10 ⁇ concentration is needed to effectively block the PI3K-Akt signaling pathway (Vora et al., 2014; Pei et al., 2011).
  • PI3K inhibitors showed antiproliferative effect without killing.
  • PI3K inhibitors fully blocked the PI3K-Akt pathway (FIG. 7d) resulting in cell death or suppressing proliferation in both normal and cancer cells (FIGS. 7a-c).
  • IGlDPs had selective effect on cancer cells (FIGS. 7a-d).
  • IGlDPs only inhibit a fraction of PI3,4,5P 3 generation in cells (FIG. 7d), consistent with the data indicating that PI3,4,5P 3 generation involves multiple pathways.
  • IQGAPl is required for IGlDPs inhibition of Akt activation as Iqgapl-null MEFs were not impacted by IGlDPs (FIG. 7e), validating the specificity of IGlDPs toward blockade of the IQGAPl - PI3K scaffold.
  • the data indicate that the canonical PI3,4,5P 3 synthesis pathway is responsible for PI3,4,5P 3 synthesis from an accessible pool of PI4P and PI4,5,P 2 , whereas the IQGAPl pathway is for receptor stimulated synthesis.
  • IGlDPs inhibit only the IQGAPl pathway, while PI3K inhibitors inhibit both. This is supported by data showing that IGlDPs partially reduced Akt phosphorylation in Hs578T breast cancer cells, but not in Hs578Bst normal mammary epithelial cells, leading to selective antiproliferative effect on these cancer cells. In contrast, small molecule PI3K inhibitors blocked both the IQGAPl -dependent and - independent pathways, leading to non-specific loss of cells (FIGS.7a-d).
  • IQGAPl is required for insulin signaling in vivo
  • Akt phosphorylation was quantified in WT and Iqgapl-null MEFs stimulated by insulin. Akt activation was significantly reduced in Iqgapl-null MEFs (FIG. 8a).
  • insulin or saline was injected into WT or Iqgapl-null mice.
  • a concerted mechanism for agonist stimulated PI3,4,5P 3 generation and signaling that is key for survival of some cancer cells is described herein.
  • Agonist activated PDKs use
  • PI4,5P 2 as a substrate to synthesize PI3,4,5P 3 and it is shown that IQGAPl spatially organizes PI4,5P 2 and PI3,4,5P 3 producing kinases into a scaffolded complex.
  • PIPKIa and PI3K bind to the WW and IQ domains of IQGAPl .
  • PIPKIa generates
  • PI4,5P 2 that is channeled to PI3K facilitating efficient PI3,4,5P 3 synthesis.
  • the PI3,4,5P 3 in turn activates downstream effectors such as PDK1 and Akt that are also associated (FIG. 8d).
  • FIGS, lg, lh), and is associated with IQGAPl (FIGS. la-c).
  • IQGAPl assembles all phosphoinositide kinases required for sequential phosphorylation of PI to PI3,4,5P 3 and the effectors regulated by PI3,4,5P 3 .
  • Growth factors, extracellular matrix and other agonists stimulate the assembly and signaling of the IQGAPl -PI3K scaffold.
  • This complex summarized in FIG. 8d, has the intrinsic activities to spatially generate PI3,4,5P 3 from PI leading to activation of PDK1 and Akt.
  • As many receptors upon agonist activation are rapidly internalized and continue signaling from endosomal compartments (Sun et al., 2013; van Meer et al., 2008). Even though these endosomal compartments contain little PI4P or
  • PI4,5P 2 (Di Paolo et al, 2006; Sorkin et al, 2009; Balla, 2013), PI is present in all internal membranes (Sorkin et al, 2009; Balla, 2013).
  • the IQGAPl -PI3K scaffold reveals a clear mechanism for efficient PI3,4,5P 3 generation and signaling from these compartments, as the IQGAPl functions as a platform that orchestrates the full PI3K signaling pathway.
  • the results described herein also support an IQGAPl independent pathway(s) for PI3K
  • PI3,4,5P 3 The latter pathways are sensitive to PI4P and PI4,5P 2 binding proteins suggesting that they work on the accessible substrates in membranes.
  • the PI3K pathway(s) that are independent of IQGAPl may explain the survival of Iqgapl-null mice (Li et al., 2000). Whereas, mice lacking IQGAPl are resistant to the development of some tumors (Jameson et al, 2013; Li et a/.,2013; Mataraza et al., 2003) consistent with a role in the PI3K pathway.
  • IQGAPl pathway is present in normal and neoplastic cells, some cancer cells appear to depend on this pathway for survival. These findings provide a potential target for cancer therapies. Unlike conventional PI3K inhibitors by ATP competition (Engelman, 2009), IGlDPs inhibit the IQGAPl -scaffolded kinases assembly and block Akt activation leading to selective cancer cell death. This suggests that some cancer cells become addicted to the IQGAPl -PI3K pathway for their survival (Weinstein et al, 2008).
  • Insulin-stimulated PI3K signaling is implicated in many pathophysiological conditions including diabetes, cardiac function, aging and others (Taniguchi et al., 2006; Lopez-Otin et al, 2013; Alessi et al, 1998; Yao et al, 2014).
  • IQGAPl is required for full insulin stimulation of PI3K and Akt activation that is important for many aspects of insulin function (Yao et al, 2014). This establishes an in vivo role for the IQGAPl -scaffolded phosphoinositide kinases and effectors beyond cancers.
  • IQGAPl binding to the p85 subunit suggests a mechanism for activation of class I PDKs within the scaffold as interaction with the cSH2 domain that could relieve the intramolecular inhibition (Burke et al, 2015).
  • the interactions with both the P-BH-P and cSH2 may link multiple isoforms of class I PDKs explaining the diversity of agonist and receptors that require IQGAPl for the PI3K signaling (Burke et al, 2015).
  • IQGAP1 peptides of the present disclosure selectively kill cancer cells. Accordingly, IQGAP1 peptides described herein are useful in methods and compositions for the treatment of cancer.
  • EXAMPLE 2 Treatment of cancer using IQGAP1 peptides.
  • This example illustrates the use of IQGAP1 peptides described herein in methods for the treatment of cancer in subjects in need thereof.
  • Subjects having or suspected of having cancer are administered one or more IQGAP1 peptides, alone or in conjunction with one or more additional cancer therapeutic agents, or agents directed to the amelioration of side effects of IQGAP1 peptides or cancer therapeutic agents
  • Matched control subjects receive a placebo composition. Timing, dosage, route of administration, and duration of treatment are selected based on criteria described herein and known in the art.
  • subjects are evaluated using criteria known in the art for the evaluation of cancer, such as evaluation of tumor size, tumor number, cancer markers, etc.
  • IQGAP1 peptides [0140] It is predicted that subjects receiving one or more IQGAP1 peptides will show marked improvement in the presence or severity of cancer compared to control subjects not receiving the peptides. These results will show that IQGAP1 peptides disclosed herein are useful in methods and compositions for the treatment of cancer.
  • a plasma membrane pool of phosphatidylinositol 4-phosphate is generated by phosphatidylinositol 4- kinase type-Ill alpha: studies with the PH domains of the oxysterol binding protein and FAPPl . Mol Biol Cell 16, 1282-1295.
  • Syndecan-1 regulates alphavbeta3 and alphavbeta5 integrin activation during angiogenesis and is blocked by synstatin, a novel peptide inhibitor. J Exp Med 206, 691-705.
  • IQGAPl is a novel phosphatidylinositol 4,5 bisphosphate effector in regulation of directional cell migration. EMBO J 32, 2617-2630.
  • PIP kinases define PI4,5P signaling specificity by association with effectors. Biochim Biophys Acta.
  • Membrane ruffling requires coordination between type Ialpha phosphatidylinositol phosphate kinase and Rac signaling. J Biol Chem 278, 23036-23045.
  • G-protein-coupled receptor GPR161 is overexpressed in breast cancer and is a promoter of cell proliferation and invasion. Proc Natl Acad Sci U S A 111, 4191-4196.
  • Fritsch, R. de Krijger, I, Fritsch, K., George, R., Reason, B., Kumar, M.S., Diefenbacher, M., Stamp, G., and Downward, J. (2013).
  • RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms. Cell 153, 1050-1063.
  • PI4P and PI(4,5)P2 are essential but independent lipid determinants of membrane identity. Science 337, 727-730.
  • phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi. J Cell Biol 205, 113-126.
  • IQGAPl scaffold-kinase interaction blockade selectively targets RAS-MAP kinase-driven tumors. Nat Med 19, 626-630.
  • Akt a pleckstrin homology domain containing kinase
  • Type I gamma phosphatidylinositol phosphate kinase modulates adherens junction and E-cadherin trafficking via a direct interaction with mu IB adaptin. J Cell Biol 77(5, 343- 353.
  • Type I phosphatidylinositol-4-phosphate 5- kinases are distinct members of this novel lipid kinase family. J Biol Chem 277, 32937- 32943.
  • IQGAPl modulates activation of B-Raf. Proc Natl Acad Sci U S A 104, 10465-10469.
  • IQGAPl is a scaffold for mitogen-activated protein kinase signaling. Mol Cell Biol 25, 7940-7952.
  • ERKl/2 activation in heart is controlled by melusin, focal adhesion kinase and the scaffold protein IQGAPl . J Cell Sci 124, 3515- 3524.
  • IQGAPl regulates ERKl/2 and AKT signalling in the heart and sustains functional remodelling upon pressure overload.
  • IQGAPs choreograph cellular signaling from the membrane to the nucleus.
  • LAPTM4B is a PtdIns(4,5)P2 effector that regulates EGFR signaling, lysosomal sorting, and degradation.
  • Phosphoinositide signaling regulates the exocyst complex and polarized integrin trafficking in directionally migrating cells. Dev Cell 22, 116-130.
  • IQGAPs in cancer a family of scaffold proteins underlying tumorigenesis. FEBS Lett 583, 1817-1824.

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Abstract

The present disclosure provides compositions for the treatment of cancer, comprising peptides that inhibit cancer cell proliferation, kits comprising the same, and methods for using the same.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF CANCER
FIELD OF TECHNOLOGY
[0001] The present disclosure provides compositions for the treatment of cancer, comprising peptides that inhibit cancer cell proliferation, kits comprising the same, and methods for using the same.
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under GM057549 and
CA104708 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
[0003] Alteration of the PI3K-Akt pathway is linked to many human diseases including cancer (Engelman, 2009; Liu et al, 2009; Luo et al, 2003). IQ motif containing GTPase activating protein 1 (IQGAPl) is also implicated in many cancers (White et al, 2009) and over expression of IQGAPl is correlated to enhanced tumorigenesis (Feigin et al, 2014). PIPKIa has roles in some cancers (Kao et al., 2009; Semenas et al, 2014). Accordingly, agents that modulate the activity of these molecules are advantageous for the treatment of a variety of cancers.
[0004] Phosphatidylinositol 4,5-bisphosphate (PI4,5P2) is a lipid messenger regulating most aspects of cellular function (Di Paolo and De Camilli, 2006). PI4,5P2 serves as a substrate for phospholipases and class I phosphoinositide 3 -kinases (PI3Ks) to generate other messengers (Cantley, 2002; Kadamur and Ross, 2013), or PI4,5P2 directly binds to its effector proteins and regulates their targeting and activities (Choi et al, 2015). To mediate these diverse processes, PI4,5P2 is present at a total cellular concentration of approximately
50 μΜ (Stephens et al, 1993). The majority PI4,5P2 pool is maintained by phosphorylation at the 5 hydroxyl of the myo-inositol ring of PI4P by type I phosphatidylinositol phosphate kinases (PIPKIs). In humans, there are three PIPKI isoforms (α, β and γ) with multiple splice variants (van den Bout and Divecha, 2009). Stimulation of receptor tyrosine kinases (RTKs),
G-protein-coupled receptors (GPCRs) and integrins leads to activation of PI3Ks, which utilize PI4,5P2 as a substrate to generate PI3,4,5P3 (Vanhaesebroeck et al, 2010). Although
PI3Ks catalyze phosphorylation of PI4,5P2 at the 3 hydroxyl in vitro (Auger et al, 1989) and in vivo (Stephens et al, 1991), the cellular source of PI4,5P2 used as substrate has not been studied. An assumption has been that sufficient membrane PI4,5P2 is available for PDKs to synthesize PI3,4,5P3, as cellular PI4,5P2 concentration is orders of magnitude higher than that of PI3,4,5P3 (Stephens et al, 1993). This assumption is challenged by findings that the majority of PI4,5P2 is sequestered by binding proteins (McLaughlin and Murray, 2005), suggesting that de novo synthesis of PI4,5P2 by PIPKIs may be required for efficient
PI3,4,5P3 synthesis by PDKs. In support, depletion of a single PIPKI isoform has no significant impact on global PI4,5P2 levels (Ling et al., 2007; Mao et al., 2009), while Akt phosphorylation, a readout of PI3K activity, is reduced upon inhibition of a PIPKI (Semenas et al, 2014). These suggest that a locally organized specific pool of PI4,5P2 is responsible for PI3,4,5P3 synthesis.
[0005] IQ motif containing GTPase activating protein 1 (IQGAPl) is a multidomain protein that scaffolds multiple signaling pathways to regulate a plethora of cell functions (Hedman et al, 2015; Smith et al, 2015). The scaffold role of IQGAPl in the mitogen activated protein kinase (MAPK) pathway is best characterized. MAPK pathway components, such as Raf, MEK and Erk, directly interact with IQGAPl, which brings these kinases in close proximity to facilitate their sequential phosphorylation (Jameson et al, 2013; Ren et al, 2007).
Additional scaffold roles for IQGAPl in GPCR, Wnt and integrin signaling were also identified (Smith et al, 2015). These versatile cellular roles of IQGAPl are in part supported by its abundance. Notably, an unbiased quantitative study reveals that the mRNA and protein copy number of IQGAPl is at least two orders of magnitude higher than that of its interacting proteins (Schwanhausser et al, 2011). This suggests that in a cell IQGAPl might be present in many discrete signaling complexes, and even more IQGAPl -regulated signaling pathways remain to be characterized. Although IQGAPl is reported to modulate Akt under stress conditions (Sbroggio et al, 201 la; Sbroggio et al, 201 lb), the molecular mechanism by which IQGAPl controls the PI3K-Akt pathway is largely unknown.
SUMMARY
[0006] In some aspects, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a composition comprising an IQGAPl peptide.
[0007] In some embodiments, the IQGAPl peptide comprises the amino acid sequence
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS ,
AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD, DIIKIQAFIRANKARDDYKT, LA EGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRANKARDDYKTLINAE.
[0008] In some embodiments, the IQGAP1 peptide is selected from the group consisting of KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS , AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, and
RDHINDIIKIQAFIRANKARDDYKTLINAE.
[0009] In some embodiments, the IQGAPl peptide is
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS, AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRANKARDDYKTLINAE.
[0010] In some embodiments, the method of the present disclosure further comprises administering to the subject one or more additional cancer therapeutic agents. In some embodiments, the one or more additional cancer therapeutic agents comprise nitric oxide (NO) inducers, statins, negatively charged phospholipids, anti-oxidants, minerals, antiinflammatory agents, anti -angiogenic agents, matrix metalloproteinase inhibitors, carotenoids, vinca alkaloids, microtubule disrupting agents, anti-angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agents, transitional metal complexes, proteasome inhibitors, antimetabolites, alkylating agents, platinum-based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids, or geldanamycin or a derivative thereof.
[0011] In some embodiments, the cancer is selected from the group consisting of breast cancer, prostate cancer, head and neck cancer, liver cancer, and colon cancer. [0012] In some embodiments, the composition further comprises a pharmaceutical carrier. In some embodiments, the composition further comprises a preservative.
[0013] In some aspects, the present disclosure provides a composition for treating cancer in a subject in need thereof, comprising an IQGAP1 peptide.
[0014] In some embodiments, the peptide comprises the amino acid sequence
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS ,
AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LA EGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRA KARDDYKTLINAE.
[0015] In some embodiments, the peptide is selected from the group consisting of
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS,
AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, and
RDHINDIIKIQAFIRANKARDDYKTLINAE.
[0016] In some embodiments, the peptide is
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS,
AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRANKARDDYKTLINAE.
[0017] In some embodiments, the composition further comprises one or more additional cancer therapeutic agents. In some embodiments, the one or more additional cancer therapeutic agents comprise nitric oxide (NO) inducers, statins, negatively charged phospholipids, anti-oxidants, minerals, anti-inflammatory agents, anti -angiogenic agents, matrix metalloproteinase inhibitors, carotenoids, vinca alkaloids, microtubule disrupting agents, anti -angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agents, transitional metal complexes, proteasome inhibitors, antimetabolites, alkylating agents, platinum-based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids, or geldanamycin or a derivative thereof.
[0018] In some embodiments, the cancer is selected from the group consisting of breast cancer, prostate cancer, head and neck cancer, liver cancer, and colon cancer.
[0019] In some aspects, the present disclosure provides a kit for the treatment of cancer in a subject, comprising an IQGAP1 peptide.
[0020] In some embodiments, the peptide comprises the amino acid sequence
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQARCRGYLVRQEFRS,
AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRANKARDDYKTLINAE.
[0021] In some embodiments, the peptide is selected from the group consisting of
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQARCRGYLVRQEFRS,
AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, and
RDHINDIIKIQAFIRANKARDDYKTLINAE.
[0022] In some embodiments, the peptide is
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQARCRGYLVRQEFRS,
AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRANKARDDYKTLINAE. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS la-lf show PIPKIa and IQGAP1 are required for PI3,4,5P3 generation. FIG. la, IQGAP1 associated proteins were analyzed by IP in Hs578T cells. IgG, nonspecific immunoglobulin. FIGS, lb and lc, Changes in IQGAP1 (FIG. lb) and PIPKIa (FIG. lc) associated proteins upon EGF stimulation were analyzed by IP. S. free, serum free. FIG. Id, Immunoblotting (IB) after RNAi of the indicated proteins in Hs578T cells (top), mean ± standard deviation (SD) of 3 experiments (bottom). Con, non-targeting siRNA. *, ><0.05; **, ><0.01; n.s., not significant (Student's t-test). FIG. le, Iqgapf1' mouse embryonic fibroblasts (MEFs) were re-expressed with the indicated IQGAPl proteins (Supplementary FIG. 2a) and IB after EGF treatment (top), mean ± SD of 3 experiments (bottom). FIG. If, PI4,5P2 and PI3,4,5P3 contents after knockdown of the indicated proteins in Hs578T, mean ± SD of 6 experiments. Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
[0024] FIGS. 2a-2f show PIPKIa and PI3K interact on the WW and IQ domains of
IQGAPl . FIGS. 2a and 2b, PIPKIa and p85 subunit of PI3K binding sites on IQGAPl were mapped with the indicated truncation mutants (Supplementary FIG. 2a) by IP. FIGS. 2c and 2d, In vitro binding experiments with the indicated recombinant proteins, representative of at least 3 experiments. FIG. 2e, IQGAPl binding sites were mapped in vitro with the indicated bovine p85a fragments. FIG. 2f, A model summary of in vitro binding experiments.
Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
[0025] FIGS. 3a-3f show IQGAPl physically links PIPKIa to PI3K. FIG. 3a, The indicated endogenous proteins were IP'ed from Hs578T cell lysates. Associated proteins were analyzed by IB. b-d, Hs578T cells were transfected with non-targeting (siCon),
IQGAPl, pi 10a or PIPKIa siRNA for 48 h. The indicated proteins were IP'ed and associated molecules were analyzed by IB. FIG. 3e, Hs578T cells were transfected with empty vector (Mock) or Myc-IQGAPl for 48 h. pi 10a was IP'ed and associated PIPKIa was analyzed by IB. FIG. 3f, Immunoblots for FIG. 3e were quantified and the graph is shown as mean ± SD of 3 independent experiments. Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
[0026] FIGS. 4a-4f show PI4,5P2 produced by PIPKIa is channeled to PI3K for PI3,4,5P3 synthesis. FIGS. 4a,4 d, 4e, PI4,5P2 and/or PI3,4,5P3 measurement of in vitro kinase assays with ΡΙΡΚΙα, PI3K, IQGAP1-N or -C in the indicated combinations. Liposomes containing 10 mol. % PI4P (for FIG. 4a,4 e) with the indicated amount of PI4,5P2 (for FIG. 4d) were used as substrates, mean ± SD of 4 independent experiments. FIG. 4b, In vitro binding experiments with PIPKIa, PI3K (pi 10α/ρ85α) and the WW-IQ fragment (Supplementary FIG. 2a), mean ± SD of 3 independent experiments. The molar ratio of recombinant proteins used is shown. FIG. 4c, PI3,4,5P3 measurement of in vitro kinase assays with PIPKIa, PI3K and WW-IQ in the indicated combinations, mean ± SD of 3 experiments. FIG. 4f, IB after replating on type I collagen (COL) for 45 min or 20 ng/ml EGF treatment for 15 min in Hs578T cells expressing the indicated PH domains (top), mean ± SD of 3 experiments (bottom). Sus, suspension. Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
[0027] FIGS. 5a-5g show membrane receptor signaling activates the IQGAP1 -mediated pathway. FIGS. 5a,5b, IB after replating on fibronectin (FN) or COL for 30 min in MDA- MB-231 cells transfected with siRNAs against the indicated proteins (FIG. 5a), mean ± SD of 3 experiments (FIG. 5b). FIGS 5c-5e, IB after treating with the indicated agonists (10 ng/ml EGF, 30 ng/ml PDGF, 20 ng/ml IGF-1, 15 μΜ LP A, or 30 ng/ml SDF-Ια) for 15 min in MDA-MB-231 cells transfected with siRNAs against control, IQGAPl or PIPKIa (FIG. 5c, 5d), mean ± SD of 3 experiments (FIG. 5e). FIGS. 5f, 5g, pi 10a subunit of PI3K interactions with IQGAPl and PIPKIa were analyzed by IP in Hs578T cells expressing shRNAs against the indicated proteins (FIG. 5f), mean ± SD of 3 experiments (FIG. 5g). Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
[0028] FIGS. 6a-6h IGlDPs inhibit cancer cell survival by blocking PI3,4,5P3 synthesis. FIG. 6a, In vitro binding assays of IGlDPs with PIPKIa or PI3K. FIGS. 6b, 6d, Effects of IGlDPs on IQGAPl interactions with PIPKIa and PI3K were analyzed by in vitro binding assay (FIG. 6b) or IP in Hs578T cells (FIG. 6d). FIGS. 6c, 6e, effects of IGlDPs on
PI3,4,5P3 synthesis were analyzed in vitro (FIG. 6c) or in Hs578T cells (FIG. 6e) of 3 independent experiments. FIG. 6f, Viability of MDA-MB-231 transfected with a
constitutively active Aktl after 30 μΜ IG1DP treatment, mean ± SD of 3 independent experiments. FIGS. 6g, 6h, Cell viability was measured after treating with 20 μΜ IGlDPs in the indicated cells for 48 h, except 30 μΜ for T47D and Cal51 cells, mean ± SD of 3 independent experiments. Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'. [0029] FIGS. 7a-7e show inhibition of IQGAP1 -mediated PI3,4,5P3 synthesis is a novel mechanism for targeted cancer therapy. FIG. 7a, Viability after IG1DP or PI3K inhibitor treatment in Hs578Bst, HUVEC, Hs578T (20 μΜ) and MDA-MB-231 cells (30 μΜ), mean ± SD of 3 independent experiments. FIGS. 7b, 7c, Viability of Hs578T and HUVEC cells after 1-50 μΜ IG1DP or PI3K inhibitor treatment, mean ± SD of 3 independent experiments. FIG. 7d, IB after treating with DMSO (5 μΜ), BKM120 (1 μΜ), BYL719 (5 μΜ), ZSTK474 (5 μΜ) or IGlDPs (20 μΜ) in Hs578T and Hs578Bst cells, representative of 3 independent experiments. FIG. 7e, WT or IqgapT 'MEFs treated with 40 μΜ of the indicated peptides were stimulated with 20 ng/ml EGF for 15 min. Cell lysates were analyzed by IB with the indicated antibodies (top). pS473Akt immunoblots were quantified and the graph is shown as mean ± SD of 3 independent experiments (bottom). Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
[0030] FIGS. 8a-8d show insulin-stimulated PI3,4,5P3 synthesis requires IQGAPl . FIG. 8a, Insulin-stimulated pS473Akt was measured in MEFs, mean ± SD of 3 independent experiments. FIG. 8b, WT or Iqgapf1' mice were injected with insulin and the indicated tissues were analyzed for PI3,4,5P3. p value of paired /-test result is shown (n=6). FIG. 8c, pS473Akt immunoblots with lysates from tissues were quantified (n=3). FIG. 8d, A model for the IQGAPl -mediated PI3,4,5P3 generation and downstream signaling.
[0031] Supplementary FIGS, la-lh show ΡΙ4ΚΙΙΙα, ΡΙΡΚΙα and IQGAPl are required for Akt activation, a, RT-PCR analysis of ΡΙΡΚΙβ mRNA. ΡΙΡΚΙβ mRNA levels were normalized with GAPDH mRNA. The graph is shown as mean ± SD of n=3 independent experiments. Paired Student t-tests were used for statistical analysis (*, p<0.05; **, p<0.01; n.s., not significant), b, c, Indicated proteins were knocked down and/or overexpressed in MDA-MB-231 cells and cell lysates were analyzed by IB. d, Indicated proteins were overexpressed in MDA-MB-231 cells and PI4,5P2 and PI3,4,5P3 contents were analyzed by a competitive ELISA. Paired Student t-tests were used for statistical analysis (*, p<0.05; **, p<0.01; n.s., not significant), e, Wild type or Iqgapl-/- MEFs were overexpressed with PIPKIa and cells were treated with 10 ng/ml EGF for 10 min. Cell lysates were analyzed by IB and the graph is shown as mean ± SD of n=3 independent experiments. Paired Student t- tests were used for statistical analysis (*, p<0.05; **, p<0.01; n.s., not significant), f, Stable Hs578T cells growing in normal culture conditions were harvested PI3P and PI4P were measured by a competitive ELISA (Echelon Biosciences). The graph is shown as mean ± SD of n=4 independent experiments, g, h, MDA-MB-231 cells were transfected with the indicated siRNAs. Akt phosphorylation (i) and cellular PI3,4,5P3 content (j) were measured. The graphs are shown as mean ± SD of n=3 independent experiments. Paired Student t-tests were used for statistical analysis (*, p<0.05; **, p<0.01; n.s., not significant). Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
[0032] Supplementary FIGS. 2a-2j show PIPKIa and PI3K directly interact on IQGAP 1 through the IQ3 and WW motifs, a, Schematic representation of IQGAP 1 domains and constructs used in the study, b, 0.1 μΜ GST-IQGAPl fragments and PIPKIa immobilized on glutathione beads were incubated with 0.5 μΜ His-PI3K (His-pl 10a/His-p85).
Associated PI3K subunits were analyzed by IB with an anti-His antibody. IQGAP 1-N fragment directly binds to PI3K, whereas neither IQGAP 1-C fragment nor PIPKIa binds, c, His-tagged GST alone, GST-WW domain and PIPKIa (0-1 μΜ) were incubated with untagged 0.1 μΜ PI3K. PI3K was immunoprecipitated with an anti-pl 10a antibody and the associated proteins were analyzed by IB with an anti-His antibody, d, The WW domain and IQ motif amino acid sequences. 28 aa from the WW domain and 20 aa (in black) from each IQ motif (IQ1-IQ4) were used in the study, e, 0.1 μΜ His-PIPKIa was incubated with 0.05 μΜ GST-WW domain or -IQ motifs immobilized on beads. GST- polypeptides were pulled down and associated PIPKIa was analyzed by immunoblotting. f, g, 0.02 μΜ PIPKIa and 0.02 μΜ IQGAP1-N were incubated with 0.1 μΜ GST-tagged polypeptides. PIPKIa was pulled down and the associated proteins were analyzed by immunoblotting. For g, 0, 0.05 and 0.1 μΜ GST-IQ3 were used, h, 0.02 μΜ PI3K (pi 10a/p85) was incubated with 0.05 μΜ GST-WW domain or -IQ motifs immobilized on beads. GST-polypeptides were pulled down and associated PI3K subunits were analyzed by immunoblotting. i, j, 0.02 μΜ PI3K and 0.02 μΜ IQGAP1-N were incubated with 0.1 μΜ GST-tagged polypeptides. PI3K was pulled down with and the associated molecules were analyzed by immunoblotting. For j, 0, 0.05 and 0.1 μΜ GST- WW or IQ3 were used. The experiments described above were performed independently at least four times. Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
[0033] Supplementary FIGS. 3a-3f show PI3,4,5P3 synthesis requires concerted PI4,5P2 generation by PIPKIa. a, PI3,4,5P3 generated by PI3K and IQGAP 1 fragments from 25 μΜ liposomes containing 10 molar % of PI4,5P2 was measured. The graph is shown as mean ± SD of n=3 independent experiments, b, Schematic representation of canonical versus IQGAP1 -mediated PI3,4,5P3 synthesis pathways, c, The indicated PH domains were stably expressed in Hs578T cells. Cells grown in tissue culture were photographed in bright field and fluorescent channels at 200X magnification. Roughly 70-80% of cells express exogenous proteins. Scale bar, 100 μπι. d, Hs578T cells stably expressing the indicated PH domains were treated with 10 ng/ml EGF for 10 min. Cell lysates were analyzed by IB (top) and pS473 Akt immunoblots of n=4 independent experiments were quantified (middle). PI3,4,5P3 levels were measured by a competitive ELISA and the graph is shown as mean ± SD of three independent experiments (bottom). Paired Student t-tests were used for statistical analysis (*, p<0.05; **, p<0.01 ; n.s., not significant), e, Hs578T cells were stably expressed with shRNA against IQGAP1. Cells expressing non-targeting shRNA were used as a control. Cells were grown to confluence, wounded and fixed 3 h later, followed by immunostaining for PIPKIa and PI3,4,5P3. Cells were photographed at 400X magnification. Scale bar, 100 μπι. f, Immunostaining images of e were analyzed and percent of cells (over 100 cells counted for each condition) that are positive for both PIPKIa and PI3,4,5P3 signals at the leading edges were shown in the graph (n= 120 for shCon and 110 for shIQGAPl, mean ± SD of three independent experiments). Unpaired Student t-tests were used for statistical analysis (*, p<0.05; **, p<0.01; n.s., not significant). The experiments described above were performed independently at least n=3 times. Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
[0034] Supplementary FIGS. 4a-4e show separation of PIPKIa and PI3K binding on IQGAPl attenuates PI3,4,5P3 synthesis, a, Schematic representation of uncoupling of PI4,5P2 and PI3,4,5P3 synthesis by inserting the 17 aa indicated between the WW and IQ domains, b, Iqgapl knockout (Iqgapl-/-) mouse embryonic fibroblasts (MEFs) were reconstituted with the indicated GFP-tagged human IQGAPl constructs. Cells were treated with 10 ng/ml EGF for 15 min and cellular PI3,4,5P3 contents were measured by a competitive ELISA. The graph is shown as mean ± SD of n=3 independent
experiments. Unpaired Student t-tests were used for statistical analysis (*, p<0.05; **, p<0.01 ; n.s., not significant), c, Using cell lysates from the reconstituted MEFs, IQGAPl proteins were IP'ed with an anti-GFP antibody and associated proteins were analyzed by IB. d, e, The reconstituted MEFs were transfected with constitutively active Aktl or PDK1 and Aktl or PDK1 was IP'ed and the associated IQGAPl proteins were analyzed by IB. The experiments described above were performed independently at least three times. Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
[0035] Supplementary FIGS. 5a-5e show membrane receptor signaling activates the IQGAPl -mediated PI3,4,5P3 synthesis pathway, a, Hs578T cells stably expressing shRNAs against IQGAPl and PIPKIa were plated on 10 μg/ml type I collagen for 30 min. Cell lysates were analyzed by IB with the indicated antibodies, b, pS473 Akt immunoblots were quantified and the graph is shown as mean ± SD of n=3 independent experiments. Paired Student t-tests were used for statistical analysis (*, p<0.05; **, p<0.01; n.s., not significant), c, MDA-MB-231 cells were transfected with the indicated siRNAs for 48 h. Serum starved cells were plated on collagen I-coated dish or treated with 20 ng/ml EGF or 15 μΜ LP A for 15 min. Lipids were extracted from equal number of cells and analyzed for PI3,4,5P3 content using kits from Echelon Biosciences. The graph is shown as mean ± SD of n=3 independent experiments. Paired Student t-tests were used for statistical analysis (*, p<0.05; **, p<0.01; n.s., not significant), d, Hs578T cells stably expressing indicated shRNAs were plated on 10 μg/ml type I collagen (COL) for the indicated times. Cell lysates analyzed by IB and pS473 Akt and pY397FAK immunoblots were quantified and the graph is shown as mean ± SD of n=3 independent experiments. Paired Student t-tests were used for statistical analysis (*, p<0.05; **, p<0.01; n.s., not significant), e, pY397FAK immunoblots in FIG. 4a were quantified and the graph is shown as mean ± SD of n=3 independent experiments. Paired Student t-tests were used for statistical analysis (*, p<0.05; **, p<0.01; n.s., not significant), f, Hs578T cells were transfected with the indicated siRNAs for 24 h. Cells were serum starved for 18 h before treating with 0-100 ng/ml EGF for 15 min. Cell lysates were analyzed by IB for the indicated molecules. pS473 Akt and pEGFR immunoblots were quantified and the graph is shown as mean ± SD of three independent experiments. The experiments described above were performed independently at least three times. Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
[0036] Supplementary FIGS. 6a-6f show the IQGAPl -derived peptides inhibit Akt activation, a, Sequences of cell permeable IGlDPs. b, Empty vector (Mock) and HA- tagged IQ domain alone was stably expressed in Hs578T cells. Cell lysates were analyzed by IB with the indicated antibodies, c, Hs578T cells were transfected with empty vector or pi 10a subunit of PI3K for 24 h. Then, cells were treated with the indicated 20 μΜ of IGlDPs for 24 h. Cell lysates were analyzed by IB (top) and pS473 Akt immunoblots were quantified and the graph is shown as mean ± SD of three independent experiments (bottom), d, Cells containing PIK3CA mutations were treated with 30 μΜ IGlDPs for 48 h. Cell lysates were analyzed by IB with the indicated antibodies, e, pS473 Akt blots of FIG. 7d were quantified and the graph is shown as mean ± SD of n=3 independent experiments. Paired Student t-tests were used for statistical analysis (*, p<0.05; **, p<0.01; n.s., not significant), f, Hs578T cells were transfected with a constitutively active Racl or Cdc42 for 24. Then, cells were treated with 20 μΜ of the indicated IGlDPs for 48 h. Cell viability and protein expression were measured and the graph is shown as mean ± SD of n=3 independent experiments. Paired Student t-tests were used for statistical analysis (*, p<0.05; **, p<0.01; n.s., not significant). Unprocessed original scans of blots are shown in Supplementary FIGS. 7A-7L'.
[0037] Supplementary FIGS. 7A-7L' show unprocessed original scans of blots.
DETAILED DESCRIPTION
[0038] The present disclosure provides methods, compositions, and kits for the treatment of cancer, comprising IQGAPl peptides. As described herein, IQGAPl peptides selectively kill cancer cells, and are useful in the treatment of cancer in subjects in need thereof.
[0039] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.
[0040] As used herein, the "administration" of an agent, drug, or peptide to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or
subcutaneously), or topically. Administration includes self-administration and the administration by another.
[0041] As used herein, the term "amino acid" includes naturally-occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally-occurring amino acids. Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally-occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally-occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally- occurring amino acid. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
[0042] As used herein, the term "effective amount" refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in, the symptoms associated with cancer. The amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the IQGAP1 peptides may be administered to a subject having one or more signs or symptoms of cancer. For example, a "therapeutically effective amount" of the IQGAP1 peptides is meant levels in which the physiological effects of cancer are, at a minimum, ameliorated.
[0043] An "isolated" or "purified" polypeptide or peptide is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the agent is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. For example, an isolated IQGAP1 peptide would be free of materials that would interfere with diagnostic or therapeutic uses of the agent. Such interfering materials may include enzymes, hormones and other proteinaceous and nonproteinaceous solutes.
[0044] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art.
[0045] As used herein, the terms "treating" or "treatment" or "alleviation" refers to treatment wherein the object is to prevent, slow down, or lessen the targeted pathologic condition or disorder. A subject is successfully "treated" for cancer if, after receiving a therapeutic amount of one or more IQGAPl peptides according to the methods described herein, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of cancer. It is also to be appreciated that the various modes of treatment provided herein include "substantial" treatment, which includes total and less than total treatment and wherein some biologically or medically relevant result is achieved.
[0046] As used herein, "prevention" or "preventing" of a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample. In some embodiments, prevention refers to the prevention of one or more signs or symptoms of cancer. In some embodiments, prevention refers to the prevention of cancer/tumor growth. In some embodiments, prevention refers to the prevention of cancer metastasis.
[0047] As used herein, "IQGAPl peptide" refers to a peptide derived from the IQ motif containing GTPase activating protein 1 (IQGAPl). IQGAPl-derived peptides are mimics of IQGAPl sub-domains that mediate PIPKIa and PI3K binding, and are 24 to 28 amino acids in length. IQGAPl-derived peptides completely or partially dissolve in water and become basic when dissolved in water.
[0048] As used herein, "cancer" refers to all types of cancer or neoplasm or malignant or benign tumors found in mammals, including carcinomas and sarcomas. Examples of cancers amendable to methods disclosed herein include, but are not limited to, breast cancer, prostate cancer, head and neck cancer, liver cancer, and colon cancer. Peptides
[0049] Peptides of the present disclosure may be synthesized by any of the methods well known in the art. Suitable methods for chemically synthesizing the protein include, for example, those described by Stuart and Young in Solid Phase Peptide Synthesis, Second Edition, Pierce Chemical Company (1984), and in Methods Enzymol. 289, Academic Press, Inc, New York (1997).
[0050] In some embodiments, the peptide is selected from the group consisting of KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS , AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF,
RDHINDIIKIQAFIRANKARDDYKTLINAE, functional fragments thereof, and variants thereof.
[0051] In some embodiments, the peptide comprises
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS, AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF,
RDHINDIIKIQAFIRANKARDDYKTLINAE, a functional fragment thereof, or a variant thereof.
[0052] In some embodiments, the peptide is
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS, AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF,
RDHINDIIKIQAFIRANKARDDYKTLINAE, a functional thereof, or a variant thereof.
[0053] In some embodiments, the peptide comprises a variant of
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS, AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LA EGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHrNDIIKIQAFIRA KARDDYKTLINAE having one or more conservative amino acid substitutions.
[0054] In some embodiments, the variant has at least 95%, at least 90%, at least 85%>, at least 80%), at least 75%, at least 70%, at least 65%>, at least 60%>, or at least 50% homology to KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS , AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRANKARDDYKTLINAE.
[0055] In some embodiments, the variant has more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, or more than 50% homology to KWVKHWVKGGYYYYHNLETQEGGWDEPP,
LITRLQARCRGYL VRQEFRS, AITCIQSQWRGYKQKKAYQD,
E VVKIQ SL ARMHQ ARKRYRD, DIIKIQAFIRANKARDDYKT,
LANEGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRANKARDDYKTLINAE.
[0056] In some embodiments, one or more amino acids of the IQGAPl peptide is modified to enhance stability of the peptide or the half-life of the peptide when administered to a subject. In some embodiments, one or more covalent bonds of the IQGAPl peptide are modified to enhance stability of the peptide or the half-life of the peptide when administered to a subject.
[0057] In some embodiments, the peptide comprises a protein transduction domain. In some embodiments, the protein transduction domain comprises a TAT domain. In some embodiments, the protein transduction domain comprises a polyarginine domain. [0058] In some embodiments, the peptide comprises a protein tag. In some embodiments, the protein tag comprises a Myc-tag (e.g., comprising the amino acid sequence
EQKLISEEDL) for the efficient detection of the peptides.
Compositions/Modes of Administration/Effective Dosages
[0059] Any method known to those in the art for contacting a cell, organ or tissue with a peptide or peptide composition may be employed. Suitable methods include in vitro, ex vivo, or in vivo methods. In vivo methods typically include the administration of an IQGAP1 peptide, such as those described herein, to a mammal, preferably a human. When used in vivo for therapy, IQGAP1 peptides are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect). The dose and dosage regimen will depend upon the degree of the condition to be treated in the subject, the characteristics of the particular IQGAP1 peptide used, e.g., its therapeutic index, the subject, and the subject's medical history. When used in vitro or ex vivo for therapy, IQGAP1 peptides are
administered to cells, tissues, or organs removed from the subject. Following peptide administration, the cells, tissues or organs are returned/replaced.
[0060] Effective amounts of IQGAP1 peptides for methods of cancer treatment described herein may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount of a peptide useful in the methods of the present technology, in a pharmaceutical composition, for example, may be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. The peptide may be administered systemically or locally.
[0061] The IQGAP1 peptides described herein can be incorporated into pharmaceutical compositions for administration, singly or in combination, to a subject for the treatment or prevention of a disorder described herein. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
[0062] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For convenience of the patient or treating physician, the dosing formulation can be provided in a kit containing all necessary equipment (e.g. vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g. 7 days of treatment).
[0063] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
[0064] Peptide compositions of the present disclosure can include a carrier, which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants can be included to prevent oxidation. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
[0065] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0066] For ophthalmic applications, the therapeutic compound is formulated into solutions, suspensions, and ointments appropriate for use in the eye. For ophthalmic formulations generally, see Mitra (ed.), Ophthalmic Drug Delivery Systems, Marcel Dekker, Inc., New York, N.Y. (1993) and also Havener, W. H., Ocular Pharmacology, C.V. Mosby Co., St. Louis (1983). Ophthalmic pharmaceutical compositions may be adapted for topical administration to the eye in the form of solutions, suspensions, ointments, creams or as a solid insert, such as for the treatment of eye-related cancers.
[0067] The ophthalmic preparation may contain non-toxic auxiliary substances such as antibacterial components which are non-injurious in use, for example, thimerosal, benzalkonium chloride, methyl and propyl paraben, benzyldodecinium bromide, benzyl alcohol, or phenyl ethanol; buffering ingredients such as sodium chloride, sodium borate, sodium acetate, sodium citrate, or gluconate buffers; and other conventional ingredients such as sorbitan monolaurate, triethanolamine, polyoxyethylene sorbitan monopalmitylate, ethylenediamine tetraacetic acid, and the like.
[0068] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
[0069] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
[0070] Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one embodiment, transdermal administration may be performed my iontophoresis.
[0071] A therapeutic protein or peptide can be formulated in a carrier system. The carrier can be a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one embodiment, the therapeutic peptide is encapsulated in a liposome while maintaining peptide integrity. As one skilled in the art would appreciate, there are a variety of methods to prepare liposomes. (See Lichtenberg et al, Methods Biochem. Anal., 33 :337-462 (1988); Anselem et al, Liposome Technology, CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann. Pharmacother., 34 (7- 8):915-923 (2000)). An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.
[0072] The carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix. In one embodiment, the therapeutic peptide can be embedded in the polymer matrix, while maintaining protein integrity. The polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly a-hydroxy acids. Examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is poly-lactic acid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and
nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Ann. Pharmacother., 34 (7-8): 915-923 (2000)). A polymer formulation for human growth hormone (hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).
[0073] Examples of polymer microsphere sustained release formulations are described in PCT publication WO 99/15154 (Tracy et al), U.S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale et al), PCT publication WO 96/40073 (Zale et al), and PCT publication WO 00/38651 (Shah et al). U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.
[0074] In some embodiments, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylacetic acid. Such formulations can be prepared using known techniques. The materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. [0075] The therapeutic compounds can also be formulated to enhance intracellular delivery. For example, liposomal delivery systems are known in the art, see, e.g., Chonn and Cullis, "Recent Advances in Liposome Drug Delivery Systems," Current Opinion in Biotechnology 6:698-708 (1995); Weiner, "Liposomes for Protein Delivery: Selecting Manufacture and Development Processes," Immunomethods 4 (3) 201-9 (1994); and Gregoriadis, "Engineering Liposomes for Drug Delivery: Progress and Problems," Trends Biotechnol. 13 (12):527-37 (1995). Mizguchi et al, Cancer Lett. 100:63-69 (1996), describes the use of fusogenic liposomes to deliver a protein to cells both in vivo and in vitro.
[0076] Dosage, toxicity and therapeutic efficacy of the therapeutic agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
[0077] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
[0078] Typically, an effective amount of the IQGAPl peptides, sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Preferably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of peptide ranges from 0.1-10,000 micrograms per kg body weight. In one embodiment, IQGAP1 peptide concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per day or once a week. Intervals can also be irregular as indicated by measuring blood levels of glucose or insulin in the subject and adjusting dosage or administration accordingly. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
[0079] In some embodiments, a therapeutically effective amount of an IQGAP1 peptide comprises a concentration of peptide at the target tissue of 10"11 to 10"6 molar, e.g., approximately 10"7 molar. This concentration may be delivered by systemic doses of 0.01 to 100 mg/kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, most preferably by single daily or weekly administration, but also including continuous administration (e.g. parenteral infusion or transdermal application). In some embodiments, depending on the cancer cell type, IQGAP1 peptide concentrations range from 10 to 100 micromolar (i.e., 10"5 molar to 10"4 molar).
[0080] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.
[0081] The mammal treated in accordance present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In a preferred embodiment, the mammal is a human. Combination Therapies
[0082] In certain instances, it may be appropriate to administer at least one of the IQGAPl peptides described herein (or a pharmaceutically acceptable salt, ester, amide, prodrug, or solvate) in combination with another therapeutic agent. By way of example only, if one of the side effects experienced by a patient upon receiving one of the IQGAPl peptides herein is inflammation, then it may be appropriate to administer an anti-inflammatory agent in combination with the initial therapeutic agent. Or, by way of example only, the therapeutic effectiveness of one of the compounds described herein may be enhanced by administration of an adjuvant (i.e., by itself the adjuvant may only have minimal therapeutic benefit, but in combination with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced). Or, by way of example only, the benefit experienced by a patient may be increased by administering one of the compounds described herein with another therapeutic agent (which also includes a therapeutic regimen) that also has therapeutic benefit in the treatment of cancer. By way of example only, in a treatment for cancer involving
administration of one of the IQGAPl peptides described herein, increased therapeutic benefit may result by also providing the patient with other therapeutic agents or therapies for cancer. The overall benefit experienced by the patient may simply be additive of the two therapeutic agents or the patient may experience a synergistic benefit.
[0083] Multiple therapeutic agents may be administered in any order or even
simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between the multiple doses may vary from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents. By way of example only, an IQGAPl peptide may be provided with at least one additional cancer therapeutic and at least one agent for the amelioration or management of side effects associate with the IQGAPl peptide or the additional agent.
[0084] Specific, non-limiting examples of possible combination therapies include use of at least one IQGAPl peptide with nitric oxide (NO) inducers, statins, negatively charged phospholipids, anti-oxidants, minerals, anti-inflammatory agents, anti -angiogenic agents, matrix metalloproteinase inhibitors, and carotenoids. In several instances, suitable combination agents may fall within multiple categories (by way of example only, lutein is an anti-oxidant and a carotenoid). Further, the IQGAPl peptides may also be administered with additional agents that may provide benefit to the patient, including by way of example only cyclosporin A.
[0085] The use of anti angiogenic or anti-VEGF drugs has also been shown to provide benefit for patients with cancer. Examples of suitable anti angiogenic or anti-VEGF drugs that could be used in combination with at least one IQGAPl peptide include Rhufab V2
(Lucentis™), Tryptophanyl-tRNA synthetase (TrpRS), EyeOOl (Anti-VEGF Pegylated Aptamer), squalamine, Retaane™ 15 mg (anecortave acetate for depot suspension; Alcon, Inc.), Combretastatin A4 Prodrug (CA4P), Macugen™, Mifeprex™ (mifepristone~ru486), subtenon triamcinolone acetonide, intravitreal crystalline triamcinolone acetonide,
Prinomastat (AG3340~synthetic matrix metalloproteinase inhibitor, Pfizer), fluocinolone acetonide (including fluocinolone intraocular implant, Bausch & Lomb/Control Delivery Systems), VEGFR inhibitors (Sugen), and VEGF-Trap (Regeneron/Aventis).
[0086] Cancer therapeutic agents suitable for IQGAPl peptide combination therapies include, but are not limited to vinca alkaloids, agents that disrupt microtubule formation (such as colchicines and its derivatives), anti-angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agent (such as tyrosine kinase inhibitors), transitional metal complexes, proteasome inhibitors, antimetabolites (such as nucleoside analogs), alkylating agents, platinum-based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids (such as all-trans retinoic acids or a derivatives thereof); geldanamycin or a derivative thereof (such as 17-AAG), and other cancer therapeutic agents recognized in the art.
[0087] In some embodiments, cancer therapeutics for use in IQGAPl peptide combination therapies comprise one or more of adriamycin, colchicine, cyclophosphamide, actinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone, fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, interferons, camptothecin and derivatives thereof, phenesterine, taxanes and derivatives thereof (e.g., taxol, paclitaxel and derivatives thereof, taxotere and derivatives thereof, and the like), topetecan, vinblastine, vincristine, tamoxifen, piposulfan, nab-5404, nab-5800, nab- 5801, Irinotecan, HKP, Ortataxel, gemcitabine, Oxaliplatin, Herceptin®, vinorelbine, Doxil®, capecitabine, Alimta®, Avastin®, Velcade®, Tarceva®, Neulasta®, lapatinib, sorafenib, erlotinib, erbitux, derivatives thereof, cancer therapeutic agents known in the art, and the like. In some embodiments, the cancer therapeutic agent is a composition comprising nanoparticles comprising a thiocolchicine derivative and a carrier protein (such as albumin).
[0088] Reference to a cancer therapeutic agent herein applies to the agent or its derivatives and accordingly the present technology includes either of these embodiments (agent; agent or derivative(s)). "Derivatives" or "analogs" of a cancer therapeutic agent or other chemical moiety include, but are not limited to, compounds that are structurally similar to the cancer therapeutic agent or moiety or are in the same general chemical class as the cancer therapeutic agent or moiety. In some embodiments, the derivative or analog of the cancer therapeutic agent or moiety retains similar chemical and/or physical property (including, for example, functionality) of the cancer therapeutic agent or moiety.
[0089] In some embodiments, the cancer therapeutic agent is an antineoplastic agent including, but is not limited to, carboplatin, Navelbine® (vinorelbine), anthracycline
(Doxil®), lapatinib (GW57016), Herceptin®, gemcitabine (Gemzar®), capecitabine
(Xeloda®), Alimta®, cisplatin, 5-fluorouracil (5-Fu), epirubicin, cyclophosphamide, Avastin®, Velcade®, etc.
Kits
[0090] In one aspect, the present disclosure provides kits comprising IQGAPl peptides for the treatment of cancer in a subject. In some aspects, the kit comprises one or more IQGAPl peptides, sterile vessels suitable for handling of the peptides, and instructions for use. In some embodiments, the kit comprises one or more IQGAPl peptides together with one or more additional cancer therapeutic agents.
[0091] In some embodiments, the IQGAPl peptide comprises the amino acid sequence KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS ,
AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
Din IQAFIRA KARDDYKT, LA EGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRA KARDDYKTLINAE. [0092] In some embodiments, the IQGAP1 peptide is selected from the group consisting of KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS , AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LA EGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, and
RDHINDIIKIQAFIRA KARDDYKTLINAE.
[0093] In some embodiments, the IQGAPl peptide is
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS, AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRANKARDDYKTLINAE.
[0094] The following examples are presented in order to more fully illustrate the preferred embodiments of the present technology. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims.
EXAMPLES
EXAMPLE 1 : Agonist Stimulated Phosphatidylinositol 3, 4, 5 -Triphosphate Synthesis by IQGAPl Scaffold Phosphoinositide Kinases
[0095] This example shows that peptides disclosed herein, including IQGAPl peptides, selectively kill cancer cells in vitro. Accordingly, the peptides are useful in methods and compositions for the treatment of cancer.
Introduction
[0096] Generation of phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P3) by the class I phosphoinositide 3-kinases (PDKs) requires PI4,5P2 as a substrate. The PI3K signaling pathway has been intensively studied, but a direct role for PI4,5P2 synthesis in the pathway has been largely neglected. A prevailing assumption is that PI3K utilizes free PI4,5P2 in the plasma membrane. Yet, the majority of PI4,5P2 appears to be sequestered suggesting that PI3K might require de novo PI4,5P2 synthesis for efficient PI3,4,5P3 production. Here, it is shown that PI3K and PIPKIa, a PI4,5P2 generating enzyme, are organized by IQGAPl, a scaffold protein. In the IQGAPl -scafolded complex PI4,5P2 generated by PIPKIa is channeled to PI3K for PI3,4,5P3 synthesis. Receptor signaling stimulates this
IQGAPl/phosphoinositide kinases complex and is required for PI3,4,5P3 synthesis.
Blockade of the IQGAPl interactions with the phosphoinositide kinases inhibits PI3,4,5P3 synthesis and signaling. Collectively, the data demonstrate a concerted mechanism for PI3,4,5P3 synthesis and approaches to therapeutically block PI3K signaling in cancer cells.
[0097] Here, it is shown that IQGAP 1 interacts with PIPKIa and PI3K bringing the two phosphoinositide kinases in close proximity, which allows for the PI4,5P2 produced by PIPKIa to be channeled to PI3K for PI3,4,5P3 synthesis. This IQGAPl -mediated pathway is responsible for PI3,4,5P3 synthesis stimulated by integrin, RTK or GPCR activation. Further, blockade of the IQGAPl interaction with either PIPKIa or PI3K inhibited PI3,4,5P3 synthesis and Akt activation, which blocks breast cancer cell proliferation. Collectively, these results establish that agonist stimulated PI3,4,5P3 synthesis requires spatiotemporal organization of PIPKIa and PI3K by IQGAPl .
Materials and Methods
[0098] Cell culture and constructs. MDA-MB-231, MDA-MB-468, HEK 293, Hs578T, Cal51, UACC812, SkBr3, HaCaT, Cos7 and MEF cells were purchased from ATCC and maintained in DMEM supplemented with 10% fetal bovine serum (Gibco). MCF-7 (Soule et al, 1973), HUVEC (Beauvais et al, 2009) (a gift from A. Rapraeger, University of
Wisconsin-Madison, USA), MCF10A (Soule et al, 1990) and Hs578Bst (Hackett et al, 1977) cells were maintained as described. All the cell lines used in this study are routinely tested for mycoplasma contamination and mycoplasma-negative cells were used. No cell lines used in this study were found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI Biosample. The cell lines were not authenticated. For cell viability assays, cells were switched to DMEM supplemented with 10% fetal bovine serum at least 24 h before assay. The PIPKIa and IQGAPl constructs used for this work have been described previously (Choi et al, 2013; Loijens et al, 1996). The WW-IQ region was amplified using 5 '-GATGGATCCGGTGAAACTTACC AC AGTGATCTTGCT-3 ' (Forward) and 5 '-GATAAGCTTCT AATCCTC AGC ATTGATGAGAGTCTTGT A-3 ' (Reverse) primers and cloned in pET28a vector (Novagen) within BamHI and Hindlll enzyme sites. Bovine p85a constructs (FIG. 2e) (gifts from D. Anderson, University of Saskatchewan, Canada) were described previously (Chagpar et al., 2009). p85 (FIG. 2b), plOOa, constitutively active Aktl (FIG. 6f), Racl and Cdc42 (Supplementary FIG. 6f) constructs used in the study were purchased from Addgene and subcloned in pcDNA3.1 vector (Invitrogen). The GFP-PH domain constructs (gifts from T. Balla, National Institutes of Health, USA) (FIG. 4f and Supplementary FIGS. 3c, d) were described previously
(Hammond et al, 2012; Kim et al., 2011; Balla et al., 2005; Varnai & Balla, 1998). The 17Insert IQGAPl construct (FIG. le and Supplementary FIG. 4) was generated by a series of site-directed mutagenesis polymerase chain reactions to insert 5'-
CATCTCTGGGGTGACTGCCCATATAACCGAGAACAGCTGTGGCTGGTCGAC-3'. Constructs were transfected in cells by a lipid-based delivery system from Minis (MDA-MB- 231 cells) or Invitrogen (other cell lines) according to the manufacturer's instructions.
Typically, 3-9 μg of DNA and 6-12 μΐ of lipid were used for transfecting in 6-well plates. In all overexpression experiments, GFP construct was transfected in parallel to monitor the transfection efficiency. Cells having at least 70% transfection efficiency were used for further analysis.
[0099] Stable cell line generation. To generate stable MDA-MB-231 and Hs578T cell lines expressing shRNAs against human IQGAPl, PIPKIa or vector control, a pLL3.7 vector- based lentiviral delivery system was used. pLL3.7 lentiviral vector was cloned to express shRNAs against IQGAPl (Sakurai-Yageta et al., 2008) and PIPKIa (Mellman et al., 2008). After infection, cells were passaged at least 5 times before sorting GFP-positive cells in a cell sorter to select cells expressing shRNAs as pLL3.7 vector also expresses GFP bicistronically (Thapa et al., 2012). For generation of stable MEFs, cells were infected with retrovirus for 24 h. Then, cells expressing GFP-IQGAPl were first selected for GFP expression, and then further sorted by expression level. For generation of stable Hs578T cells expressing GFP- tagged PH domains, cells transfected with lipid were selected by 1.2 mg ml-1 neomycin. Then, GFP-positive cells were further selected in a cell sorter.
[0100] Antibodies and siRNAs. Polyclonal and monoclonal antibodies against total and PIPKIa and ΡΙΡΚΙγ were produced as described previously (Schill & Anderson, 2009;
Doughman et al., 2003). Pooled siRNAs used in the study were obtained from Dharmacon (FIG. Id). Single siRNAs targeting IQGAPl (either 5'-GGAAAGCUCUGGCAAUUUAUU- 3' or 5'-GAACGUGGCUUAUGAGUACUU-3') and PIPKIa (either 5'- AAGTTGGAGC ACTCTTGG-3 ' or 5'-GCACATTATCCCTACCTTA-3 ') were used elsewhere (Sakurai-Yageta et al, 2008; Mellman et al, 2008). siRNA targeting the 3 'UTR of PIPKIa (5'-UGACUCCUGGAAGAAUACUCCUGUA-3') was purchased from Thermo Fisher Scientific. Non-targeting siRNA (Dharmacon) was used as a control. siRNAs were delivered to cells by RNAiMAX reagent (Thermo Fisher Scientific) and knockdown efficiency was determined by immunoblotting. Knockdown efficiency greater than 80% was required to observe phenotypic changes in the study. This is consistent with a recent report that at least 80% knockdown of IQGAP1 is required to observe reduced Erk activity
(Monteleon et al., 2015).
[0101] Replating assay on extracellular matrix. To measure Akt and FAK phosphorylation in response to integrin activation on type I collagen and fibronectin (FIGS. 4f and 5a, b, f, g and Supplementary FIGS. 3d and 5a-e) replating assays were performed. Briefly, cells were serum starved for at least 4 h and then lifted by treating with trypsin-EDTA for a short time (less than 1 min). Cells were treated with serum-free DMEM containing 0.2% bovine serum albumin and pelleted to remove trypsin-EDTA. For the remaining steps, cells were maintained in serum-free DMEM containing 0.2% bovine serum albumin. Cells were suspended at least for 1 h at 37 °C by rotation before replating on tissue culture plates coated with 10 μg ml-1 type I collagen (Sigma Aldrich) or fibronectin (Thermo Fisher Scientific). After incubation for 15 to 60 min, cells were lysed and further analysed by immunoblotting or immunoprecipitation.
[0102] Cell viability assay. Cells were plated on 6-well plates at 30-50% confluency at the time of treatment. Cells were changed with fresh media containing class I PI(3)K inhibitors or IQGAP1 -derived peptides at the indicated concentrations for the indicated times (24 to 72 h) (FIGS. 6f-h and 7a-e and Supplementary FIGS. 6c-f). Dead cells floating in the culture medium were removed by washing twice with PBS. Residual PBS was completely removed and cells were lifted by incubating with trypsin-EDTA for 10 m. Cells were stained with 0.4% trypan blue solution (Thermo Fisher Scientific) and counted with a
haemocytometer. The number of trypan blue-negative cells was counted and used for the statistical analysis.
[0103] Immunoprecipitation and immunoblotting. Cells were lysed in a buffer containing 1% Brij58, 150 mM NaCl, 20 mM HEPES, pH 7.4, 2 mM MgCl2, 2 mM CaCl2, 1 mM
Na3V04, 1 mM Na2Mo04 and protease inhibitors. The protein concentration of lysates was measured by the BCA method (Pierce) and equal amounts of protein were used for further analysis. All antibodies were diluted in a 1 : 1,000 ratio for immunoblotting. For immunoprecipitation, 0.5 to 1 mg of proteins were incubated with 1 μg of antibodies at 4 °C for 8 h and then incubated with a 50% slurry of Protein G Sepharose (GE Life Sciences) for another 2 h. After washing 5 times with lysis buffer, the protein complex was eluted with SDS sample buffer. For immunoblotting, 5 to 20 μg of proteins were loaded. After developing immunoblots, the film was scanned using a transmitted light scanner (resolution: 600 dpi). Protein bands were quantified using ImageJ, and statistical analysis of the data was performed with Microsoft Excel. The statistical analysis was performed using data from at least three independent experiments.
[0104] In vitro binding assay. Recombinant proteins were expressed in the BL21 E. coli strain. GST-tagged proteins were then purified with GST Sepharose 4B (GE Life Sciences) and His-tagged proteins were purified with His-Bind Resin (Novagen). Recombinant PI(3)K (pi 10a and p85a) proteins were purchased from Echelon Biosciences. GST-tagged proteins were incubated with glutathione beads before binding assays. The binding assay was performed in the lysis buffer used for immunoprecipitation by adding 10 nM to 5 μΜ of His- tagged proteins and 20 μΐ of GST-tagged protein-bound glutathione beads. After incubation for 1 h at 25 °C, unbound proteins were washed out and the protein complex was analysed by immunoblotting. For the triple binding assay with recombinant GST-IQGAPl, His-PIPKIa and PI(3)K (His-pl 10a and untagged p85a), PIPKIa was pulled down with an anti-PIPKIa antibody pre-bound on Protein G Sepharose beads. For binding assays with p85a, wild-type or deletion mutants of bovine p85a (Chagpar et al., 2009) were expressed in HEK293 cells and cell lysates were used for binding experiments with recombinant IQGAPl proteins.
[0105] Immunofluorescence microscopy. Glass coverslips were coated with 10 μg ml-1 collagen, fibronectin or 10% serum before seeding cells. Cells were grown on coverslips placed inside 6-well plates until experimental manipulation. Immunostaining of
PtdIns(3,4,5)P3 was performed as previously described (Chagpar et al., 2009; Hammond & Irvine et al., 2009; Sharma et al., 2008) with modifications. Briefly, cells grown in medium were rapidly fixed by adding an equal volume of 8% paraformaldehyde and 0.5%
glutaraldehyde to medium for 15 min at room temperature. After a 30 min wash with PBS containing 50 mM H4CI, cells were permeabilized and blocked with a solution of buffer A (20 mM PIPES, pH 6.8, 135 mM NaCl, 5 mM KC1) containing 0.5% saponin and 5 vol% FBS for 45 min at room temperature. Primary antibodies were incubated in a solution of buffer A containing 0.1% saponin and 5 vol% of FBS for 12 h at 4 °C. Concentrations of 2- 4 μg ml-1 primary antibodies were used. After a 30 min wash with buffer A, fluorophore- conjugated secondary antibodies were incubated in a solution of buffer A containing 0.1% saponin and 5 vol% of FBS for 1 h at room temperature. Then, cells were washed with buffer A for 45 m at room temperature before post-fixation with 2% paraformaldehyde and 0.125% glutaraldehyde for 10 m at room temperature. Coverslips were washed 5 times with PBS containing 50 mM H4CI and once with distilled water. Fluorescence microscopy was performed using a 40 or 60 x plan-fluor objective on a Nikon Eclipse TE2000U equipped with a Photometries Cool SNAP ES CCD (charge-coupled device) camera. Images were captured using MetaMorph v6.3 (Molecular Devices). Images were exported to Photoshop CS2 (Adobe) for final processing and assembly.
[0106] Cellular phosphoinositide measurement. Lipid extraction and cellular
phosphoinositide measurement were performed using a kit from Echelon Biosciences according to the manufacturer's instructions. Briefly, cells were lysed in ice-cold
trichloroacetic acid (TCA, 0.5 M). Pellets were washed in 5% TCA/1 mM EDTA and neutral lipids were extracted with CH3OH/CHCI3 (2: 1) and discarded. Acidic lipids were extracted with CH3OH/CHCI3/HCI (80:40: 1), recovered by phase-split. Recovered lipids were dried and resuspended for competitive ELISA analysis. PtdIns(3)P, PtdIns(4)P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels were quantified by competitive ELISA (Echelon Biosciences). For measuring PtdIns(4)P and PtdIns(4,5)P2, cells were harvested from 10 cm plates. For
PtdIns(3)P and PtdIns(3,4,5)P3, cells were harvested from 15 cm plates.
[0107] In vitro phosphoinositide kinase assay. In vitro phosphoinositide kinase assays were performed as described previously (Schill & Anderson, 2009; Bazenet & Anderson, 1992) with modification. Briefly, 0.02-0.1 μΜ of recombinant IQGAPl, PIPKIa and PI(3)K were incubated with 250 μΜ phosphoinositide in liposomes in a kinase buffer (50 mM Tris HC1, pH 8.0, 10 mM MgCl2, 0.5 mM EDTA). The liposomes containing 10 mol% of
phosphoinositides were generated as described previously (Choi et al, 2013). Kinase reaction was initiated by adding 20 μΜ ATP and incubated for 30 m at 25 °C. A 50 μΐ reaction was terminated by adding 100 μΐ 1 M HC1 and 200 μΐ CHC13/CH30H (1 : 1). Extracted lipids were dried and PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels were quantified by competitive ELISA (Echelon Biosciences) as above. [0108] PtdIns(4)P liposomes that were used as a substrate contain 30% PC, 30% PE, 30% PS and 10% PtdIns(4)P in a molar ratio. Liposomes containing PtdIns(4,5)P2 were made by decreasing the PC content. For example, liposomes containing 5% PtdIns(4,5)P2 were generated with 25% PC, 30% PE, 30% PS, 10% PtdIns(4)P and 5% PtdIns(4,5)P2. All phospholipids (natural, porcine brain) were purchased from Avanti Polar Lipids. Mixed phospholipids were dried and rehydrated with 50 mM Tris, pH 8.0 for kinase assays.
Hydrated lipids were subjected to at least 5 cycles of freeze thawing in liquid nitrogen followed by 1 min bath sonication and extrusion through a 0.1 μιη filter.
[0109] PtdIns(4)P and PtdIns(4,5)P2 lipids used in kinase assays (FIG. 4 and
Supplementary FIG. 3a) were from porcine brain (Avanti Polar Lipids) and their purity was extensively tested for the presence of contamination with PtdIns(3)P or PtdIns(3,4,5)P3. The identity and purity of brain PtdIns(4)P was tested using mass spectrometry, proton NMR, phosphorus NMR, TLC, and oxidation by UV/VIS. In addition to confirming the identity of brain PtdIns(4)P, the mass spectrometry also confirms the absence of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 species as their mlz are not present in the release spectra. As for the presence or absence of PtdIns(3)P, phosphorus NMR was used. There are distinctly different shifts for the phosphorus peaks in PtdIns(3)P and PtdIns(4)P. PtdIns(3)P has two peaks, one at approximately 0.1 and another at approximately -0.75. PtdIns(4)P contains two peaks, one around -0.45 and the second at around 2.5. These shifts can fluctuate slightly, but the pattern is consistent across synthetic and natural PIPs. Brain PtdIns(4,5)P2 was analysed by mass spectrometry, TLC, proton NMR, phosphorus NMR, and oxidation by UV/VIS. The phosphorus NMR peaks for PtdIns(4,5)P2 are at approximately 2.4, 1.7 and -0.4. The shifts for PtdIns(3,4,5)P3 are typically 0.7, 0.0 and 0.8. None of these overlaps with the peaks present in the PtdIns(4,5)P2 species.
[0110] Ptdlns(3 A5 P^ measurement in mouse tissues. Male 16-19-week-old wild-type or IqgapF1' mice (129 background) were fasted for 4 h and received an intraperitoneal injection of saline or insulin (5 U kg-1). Mice were euthanized by C02, and 15 min post-injection, tissues (liver and quadriceps muscle) were harvested and immediately frozen in a dry-ice ethanol bath and stored on dry ice until being placed in a -80 °C degree freezer. Lipids were extracted from mouse tissues with the TCA extraction method as above. Lipids extracted from equal weights (approximately 100 mg) of mouse tissues were analysed for PtdIns(3,4,5)P3 using a competitive ELISA (Echelon Biosciences) and normalized by weight of tissue. Statistical analysis was performed with 6 paired cases.
[0111] Mice were bred in the animal facility at the National Institutes of Health (NTH) and maintained according to NOT guidelines. The studies were performed with approval of the NIH Animal Care and Use Committee.
[0112] Peptide production and treatment in cells. Octo-arginine-conjugated WW domain and IQ motif peptides were synthesized by Genscript. Peptides were dissolved in water to a 2.5-5 mM working solution. Small-molecule class I PI(3)K inhibitors were purchased from Selleckchem and dissolved in dimethylsulfoxide to a 5 mM working solution. Peptide and PI(3)K inhibitors were added directly into the tissue culture medium at the concentrations indicated. Dimethylsulfoxide was used as a vehicle control.
[0113] Statistics and reproducibility. ImageJ software was used to quantify the intensity of immunoblotting images. Non-saturating exposure of immunoblot films was used for quantification with the appropriate loading controls as standards. Two-tailed unpaired t-tests were used for pairwise significance unless otherwise indicated. For consistency in these comparisons, the following denotes significance in figures: *, P < 0.05; **, P < 0.01. No power calculations were used. Sample sizes were determined on the basis of previously published experiments where differences were observed. Each experiment was repeated independently at least three times and sample sizes and number of repeats are defined in each figure legend. 3-7 cases were used for statistical analysis. No samples were excluded. Investigators were blinded to allocation during experiments and outcome assessment.
Results
1. PI3K-Akt pathway requires ΡΙ4ΚΙΙΙα. ΡΙΡΚΙα and IQGAPl
[0114] To define roles with phosphoinositide kinases, IQGAPl -associated proteins were analyzed by immunoprecipitation (IP). This demonstrated that ΡΙ4ΚΙΙΙα, ΡΙΡΚΙα and PI3K associate with IQGAPl (FIG. la). IQGAPl also associated with the PI3K downstream effectors of PI3,4,5P3 phosphoinositide-dependent kinase 1 (PDK1) and Akt. In addition, IQGAP2 and ΡΙΡΚΙγ associated with IQGAPl confirming known interactors (Schmidt et al., 2008; Choi et al, 2013). MEK1 and Erk associate with IQGAPl in MEFs (Roy et al, 2005) and are regulated (FIG. le) but these interactions were not detected in the cancer cells examined here, suggesting that these interactions are cell type specific. IQGAPl associations with PMKIIIa, PIPKIa, PI3K, PDK1 and Akt were increased by EGF stimulation (FIGS, lb, lc), indicating that these interactions are enhanced by agonist stimulation. Increased Ras association was also detected, consistent with its interaction with and activation of PI3K (FIG. lc).
[0115] The associations of all components of the PI3K pathway required for PI3,4,5P3 generation and Akt activation suggest that PI3K signaling may be regulated by an IQGAPl scaffold. To explore if IQGAPl modulates PI3K signaling, protein expression was depleted by RNAi and PI3K activation was measured by Akt phosphorylation, which mirrors
PI3,4,5P3 generation (Cantley, 2002) (FIG. Id and Supplementary FIGS, la, lg). Loss of IQGAPl, but not IQGAP2, significantly reduced Akt phosphorylation. PIPKIa was the only PIPKI isoform whose knockdown decreased Akt phosphorylation (FIG. Id). These results are not due to off-target effects of siRNAs as Iqgapl-null MEF also have reduced EGF- stimulated Akt phosphorylation that is rescued by re-expression of wild type (WT) IQGAPl (FIG. le). Significantly, PIPKIa kinase activity was required as ectopic expression of WT PIPKIa, but not the kinase dead (KD) mutant, increased Akt phosphorylation
(Supplementary FIGS, lb, lc, le). Expression of ΡΙΡΚΙγ did not impact Akt phosphorylation indicating specificity for PIPKI isoforms. Knockdown of PMKIIIa also significantly reduced Akt phosphorylation (Supplementary FIG. lg). These results were replicated in multiple cell lines including MDA-MB-231, Hs578T, MCF7 and HaCaT. These combined data support a model where PMKIIIa, PIPKIa via its association with IQGAPl generates a PI4,5P2 pool that flows into the PI3K pathway for PI3,4,5P3 synthesis and Akt activation (Supplementary FIG. le).
[0116] IQGAPl and PIPKIa loss diminished Akt activation. This was not due to increased expression of the phosphatase and tensin homolog (PTEN), which dephosphorylates
PI3,4,5P3 to PI4,5P2 (Cully et al, 2006) (FIG. Id). As PMKIIIa and PIPKIa loss could reduce cellular PI4,5P2 levels, diminishing its availability for PI3K generation of the
PI3,4,5P3 required for Akt activation (Stephens et al, 1991), PI4P, PI4,5P2 and PI3,4,5P3 content was measured. Loss of PMKIIIa, PIPKIa or IQGAPl reduced PI3,4,5P3 levels -50% and 60%, respectively (FIG. If and Supplementary FIG. lh). Yet, PI3P, PMP and PI4,5P2 did not change (FIG. If and Supplementary FIG. If) indicating that diminished PI3,4,5P3 generation or Akt phosphorylation is not due to lack of PMP or PI4,5P2. Ectopic expression of IQGAPl or PIPKIa did not increase PI4,5P2 but dramatically increased PI3,4,5P3
(Supplementary FIG. Id). Collectively these data suggest that PI4,5P2 generation by PIPKIa leads to PI3A5P^ synthesis and Akt activation that is regulated by IQGAPl .
2. IQGAPl mediates a PIPKIa and PI3K complex
[0117] The PIPKIa and PI3K binding sites on IQGAPl were mapped (Supplementary FIG. 2a) (Choi et al, 2013; Li et al, 2013). Deletion of the IQ domain abrogated PIPKIa co-IP and binding, indicating the IQ domain is required, while interaction with PI3K required both the WW and IQ domains (FIGS. 2a-d). To define IQGAPl binding sites on PI3K, p85a domain constructs were used (Chagpar et al., 2009). IQGAPl -N bound to the P-BH-P and cSH2 domains of p85a (FIG. 2e).
[0118] The IQ domain of IQGAP 1 contains 4 structurally conserved IQ motifs (IQ 1 -IQ4) (Li et al., 2013) . WW and each IQ motif polypeptides were fused to GST (Supplementary FIG. 2d) for binding assay. PIPKIa specifically bound to GST-IQ3, which blocked PIPKIa interaction with IQGAP1-N (Supplementary FIGS. 2e-g). PI3K consisting of pi 10a and p85a bound to GST-WW and -IQ3 and either peptide blocked IQGAPl interaction with PI3K (Supplementary FIGS. 2h-j). In summary, PIPKIa and PI3K could bind distinct regions on IQGAPl (FIG. 2f). Consistently, ectopic expression of the IQ domain that binds PIPKIa and PI3K reduced Akt phosphorylation (Supplementary FIG. 6b). Also, the WW and IQ domains were required for EGF-stimulated Akt activation (FIG. le).
[0119] Endogenous IQGAPl, PIPKIa and PI3K form a complex in vivo (FIG. 3a) and loss of IQGAPl eliminated the PI3K interaction with PIPKIa (FIG. 3b) indicating that IQGAPl is necessary for the phosphoinositide kinases interaction. Loss of PIPKIa or PI3K (pi 10a) had no impact on the interaction of the other kinase with IQGAPl (FIGS. 3c, 3d).
Overexpression of IQGAPl significantly increased the PIPKIa co-IP with pi 10a and the generation of PI3,4,5P3 (FIGS. 3e, 3f and Supplementary FIG. le). As PIPKIa and PI3K do not bind to one another (Supplementary FIGS. 2b, 2c), these data indicate that IQGAPl scaffolds PIPKIa and PI3K, bring them in proximity and enhancing generation of PI3,4,5P3 in vivo. 3. IQGAP1 regulates PI3.4.5P^ synthesis
[0120] The scaffolding of PIPKIa and PI3K by IQGAP1 (FIG. 3) positions these kinases to coordinate enzymatic processivity. To test this, in vitro kinase assays were performed using PI4P-containing liposomes. PIPKIa phosphorylates PI4P to generate PI4,5P2, which is then phosphorylated by PI3K to PI3,4,5P3. PIPKIa alone or with IQGAP1 did not generate PI3,4,5P3. PIPKIa in combination with PI3K generated PI3,4,5P3 and this was significantly enhanced (-4.5 -fold) by adding IQGAPl-N (FIG. 4a). These data indicate that IQGAPl-N, which scaffolds PIPKIa and PI3K, enhances PI3,4,5P3 synthesis. Importantly, PI4,5P2 accumulation was reduced by added IQGAPl-N, suggesting that PI4,5P2 generated by PIPKIa is utilized by PI3K for PI3,4,5P3 synthesis. As IQGAPl-N does not enhance enzyme activity of PIPKIa or PI3K (FIG. 3 a and Supplementary FIG. 3 a), the data indicate that PI4,5P2 produced by PIPKIa is selectively passed to PI3K for PI3,4,5P3 synthesis in the scaffold.
[0121] To further examine if IQGAP1 mediates concerted PI3,4,5P3 synthesis by scaffolding these kinases in vitro, the WW-IQ fragment was used as a minimal scaffold (Supplementary FIG. 2a). PIPKIa, PI3K and WW-IQ formed a ternary complex that peaks at a 1 : 1 : 1 ratio, but the complex was diminished with increasing WW-IQ (FIG. 4b).
Consistently, addition of WW-IQ enhanced PI3,4,5P3 synthesis from PI4P and this peaked when the ratio was 1 : 1 : 1, but diminished at a higher ratio of WW-IQ (FIG. 4c). Collectively, these data signify that each IQGAPl binds a single PIPKIa and PI3K, and PIPKIa and PI3K require scaffolding by IQGAPl for efficient PI3, 4, 5P3 synthesis.
[0122] In a concerted mechanism, the IQGAPl -tethered PI3K would selectively utilize the PI4,5P2 generated from PIPKIa. To test this, in vitro kinase assays were performed with PIPKIa and PI3K using liposomes containing PI4P or with increasing amounts of PI4,5P2 (FIG. 4d). Without IQGAPl, PI3,4,5P3 synthesis increased proportionally with the PI4,5P2 mole %, suggesting that PI3,4,5P3 is largely generated from the PI4,5P2 in the liposomes. Addition of IQGAPl with PIPKIa and PI3K increased synthesis of PI3,4,5P3 ~8-fold (compare green bars), with only a modest increase when PI4,5P2 was added. This indicates that the PI3,4,5P3 is preferentially generated from the PI4,5P2 synthesized by PIPKIa. Most significant, the PIPKIa-PDK-IQGAPl complex synthesized PI3,4,5P3 ~20-fold more rapidly than PIPKIa or PI3K alone (FIG. 4e, green bars), confirming that IQGAPl -bound PI3K preferentially utilizes the PI4,5P2 generated by PIPKIa.
[0123] To explore a concerted mechanism in vivo, PI3P, PI4P and PI4,5P2 binding proteins were used to sequester free lipid. PI3P-, PI4P- and PI4,5P2-specific binding domains (Hammond et al., 2012; Balla et al., 2008; Stenmark et al, 2002) were stably expressed and the impact on collagen- and EGF-stimulated PI3,4,5P3 synthesis and Akt phosphorylation were quantified. The PI4P (Osh2-PH2x) and PI4,5P2 (PLC51-PH) binding proteins, but not the PI3P binding protein (Hrs-FYVE2x), blocked PI3,4,5P3 synthesis and Akt activation in suspended and serum starved cells but not upon stimulation by collagen or EGF (FIG. 4f and Supplementary FIGS. 3b-d). These results are consistent with a previous report using neomycin which is a PI4,5P2 sequestration reagent. Neomycin reduced Akt and Akt downstream effectors' activity in unstimulated conditions, whereas neomycin had no impact on insulin-stimulated conditions (Shimaya et al, 2004). This indicates at least two distinct pathways of PI3,4,5P3 synthesis exist in cells, a canonical pathway, where PI3,4,5P3 is generated from a pool of membrane PI4,5P2 that is accessible to the binding domains and is inhibited by sequestration of PI4P or PI4,5P2 (Supplementary FIG. 3b). In the IQGAPl - mediated pathway, PI4P and PI4,5P2 sequestration does not impact PI3,4,5P3 synthesis as the binding domains would not access the channeling substrates in the IQGAPl -scaffolded complex, consistent with the data (FIG. 4f and Supplementary FIG. 3d).
[0124] If PIPKIa and PI3K are functionally linked by IQGAPl then separating the PIPKIa and PI3K binding sites on IQGAPl could uncouple PI4,5P2 and PI3,4,5P3 synthesis and this was assessed by inserting 17 aa between the WW domain and IQ3 motif (Supplementary FIG. 4a). Consistent with this hypothesis, a 17 residue insert mutant reduced EGF-stimulated PI3,4,5P3 synthesis and Akt phosphorylation (FIG. le and Supplementary FIG.5b). Yet, the quantity of PIPKIa, PI3K, PDKl and Aktl that co-IP' ed with the insert IQGAPl mutant did not change (Supplementary FIGS. 5b-e). This supports a model where IQGAPl scaffolds PIPKIa and PI3K and the close proximity is required for the sequential phosphorylation of PI4P to PI3,4,5P3.
[0125] The above data indicate that the IQGAPl -PI3K scaffold functional assembly on a membrane compartment would be linked to a biological role. During cell migration PI3K activation and PI3,4,5P3 synthesis are localized to the leading edge of migrating cells
(Devreotes et al, 2003; Huang et al, 2003; Iijima et al, 2002) and IQGAPl is required for migration (Choi et al., 2013). To examine a physiological role of the IQGAPl -mediated ΡΓ3,4,5Ρ3 synthesis, cells migrating into scratch wounds were immunostained (Supplementary FIGS. 3e, 3f). PIPKIa co-localized with PI3,4,5P3 at the leading edges in control cells, whereas PIPKIa and PI3,4,5P3 at the leading edges was lost by IQGAP1 knockdown. These data demonstrate that the IQGAP1-PI3K scaffold synthesizes PI3,4,5P3 at the leading edge of migrating cells.
4. Stimulated PI3 Α5Ρ^ synthesis needs IQGAP1
[0126] EGF stimulated IQGAP1 interactions with PI3K pathway components (FIGS, lb, lc). To further investigate receptor regulation of the IQGAP1 -mediated PI3,4,5P3 synthesis pathway, control, IQGAP1 and PIPKIa knockdown cells were treated with agonists for a number of membrane receptor classes. Fibronectin or collagen receptor-mediated Akt activation and PI3,4,5P3 generation were significantly reduced by IQGAPl or PIPKIa loss (FIGS. 5a, 5b and Supplementary FIGS. 5a-c). Focal adhesion kinase (FAK)
phosphorylation is reported to regulate integrin-mediated PI3K activation (Chen et al., 1996), but FAK phosphorylation remained unchanged upon knockdown of IQGAPl and PIPKIa consistent with the complex functioning downstream of FAK (FIG. 5a and Supplementary FIGS. 5d, 5e). IQGAPl and PIPKIa loss reduced Akt phosphorylation and PI3,4,5P3 generation by ligands for multiple RTKs and GPCRs (FIGS. 5c-e and Supplementary FIG. 5c). This is not due to a loss of EGF receptor signaling upon IQGAPl knockdown
(Supplementary FIG. 5f). To investigate how IQGAPl and PIPKIa regulate the PI3K-Akt pathway downstream of receptors, IQGAPl complex formation was quantified upon agonist stimulation. The interaction of the PI3K pi 10a subunit with IQGAPl and PIPKIa was enhanced by activation of all three receptor types (FIGS, lb, lc, 5f, 5g). These data support a model where receptor activation stimulates IQGAPl -PIPKIa-PI3K assembly and this ensures efficient PI3,4,5P3 synthesis and Akt activation.
5. IQGAPl derived peptides block PI3 A5P3 synthesis
[0127] In vitro the WW domain and IQ3 motif mediate the interaction of IQGAPl with
PIPKIa and PI3K (Supplementary FIG. 2). To expand on this, peptides corresponding to the
WW domain and IQ motifs were synthesized and their impact on the PI3K-Akt pathway examined. These IQGAPl -derived peptides (IGlDPs) were made membrane-permeable by adding eight arginine residues to the N-terminus (Supplementary FIG. 6a) (Jameson et al.,
2013). Consistent with the GST-fusion peptide data (Supplementary FIG. 2), IG1DPIQ3 bound to PIPKIa, and both IG1DPW W and IGlDPiyj bound to PI3K in vitro and blocked interactions with IQGAPl in vitro and in vivo (FIGS. 6a, 6b, 6d). The IQGAPl -mediated PI3,4,5P3 synthesis was blocked by IGIDP** and IG1DPIQ3 in vitro and in cells grown in normal culture (FIGS. 6c, 6e) and activation of Akt was also diminished (Supplementary FIGS. 6c-e). Overexpression of PI3K and PIPKIa increased PI3,4,5P3 synthesis and this also required IQGAPl (Supplementary FIGS. Id, le, 6c). Treatment with both IG1DPWW and IG1DPIQ3 did not further inhibit Akt activation, consistent with blockade of the same pathway.
6. IQGAPl peptides selectively kill cancer cells
[0128] The PI3K-Akt pathway regulates survival of many cell types (Cantley, 2002;
Vanhaesebroeck et al, 2010). To examine the roles of IGlDPs in cell survival, a variety of breast cancer cells and normal cells were treated with IGlDPs and cell viability was quantified. IGIDP** and IG1DPIQ3 reduced the number of viable cells in breast cancer cell lines (FIGS. 6f, 6h) with little or no effect on survival of normal cells (FIG. 6g). Expression of a constitutively active Aktl (Kohn et al, 1996) partially rescued cell death by IG1DPIQ3 and IG1DPWW (FIG. 6f), suggesting that cell death induced by IGlDPs is not solely attributable to blockade of Akt. Akt-independent signaling downstream of PI3K, such as the PDKl-mediated pathway, is also reported to contribute to survival (Tan et al., 2013).
IQGAPl modulates Racl and Cdc42 (Li et al., 2013; Mataraza et al., 2003) and Racl and Cdc42 activate pi 10a of PI3K (Fritsch et al., 2013), but constitutively active Racl or Cdc42 did not rescue cell death by IGlDPs (Supplementary FIG. 7f).
[0129] IQGAPl modulates Ras-dependent Erk pathways via the WW and IQ domains (Ren et al., 2007; Jameson et al., 2013; Sbroggio et al., 2011; Roy, et al., 2005; Matsunaga et al., 2014). Consistently, depletion of IQGAPl reduced Erk phosphorylation and the WW and IQ domains are required for Erk phosphorylation in MEFs (FIG. le). However, knockdown of IQGAPl in MDA-MB-231 and Hs578T cells had no impact on Erk phosphorylation (FIGS. Id, 5c, 5d and Supplementary FIGS. 5a, 5f). Also, overexpression of the IQ domain or treating with IGlDPs (WW or IQ3 peptides) had no impact on Erk phosphorylation in MDA- MB-231 and Hs578T cells (FIG. 7d and Supplementary FIG. 6b). Further, it was not possible to co-IP Erk and MEKl with IQGAPl in those cells (FIG. la). These data indicate that IQGAPl scaffolds the MAP kinase pathway only in certain cells (MEFs, MCF-7 and Hek293 cells (Roy et al., 2005; McNulty, et al, 2011; Roy, et al., 2004)) but not in the cancer cells that were tested in this study. Suggesting, that these cancer cells become addicted to the IQGAP1-PI3K pathway for survival and the MAPK pathway is not modulated by IQGAP1 in these cells. In support, a recent study indicates that WW peptide in a skin cancer model had no impact on Erk activity (Monteleon et al, 2015).
[0130] To investigate the combined roles of PI3K in PI3,4,5P3 generation compared to IQGAP1 selective PI3,4,5P3 generation, Hs578Bst, HUVEC (normal), Hs578T and MDA- MB-231 (breast cancer) cells were treated with small molecule PI3K inhibitors that are currently in clinical trials(Engelman, 2009) or the IGlDPs and cell viability was measured (FIG. 7). The PI3K inhibitors used in this study have submicromolar IC50, but in many cancer cell lines 1-10 μΜ concentration is needed to effectively block the PI3K-Akt signaling pathway (Vora et al., 2014; Pei et al., 2011). PI3K inhibitors at concentrations of 1-5 μΜ led to cell killing. However, in some cell lines (including MDA-MB-231, MDA-MB-468 and Cal51), PI3K inhibitors showed antiproliferative effect without killing. Within these conditions, PI3K inhibitors fully blocked the PI3K-Akt pathway (FIG. 7d) resulting in cell death or suppressing proliferation in both normal and cancer cells (FIGS. 7a-c). In contrast, IGlDPs had selective effect on cancer cells (FIGS. 7a-d).
[0131] IGlDPs only inhibit a fraction of PI3,4,5P3 generation in cells (FIG. 7d), consistent with the data indicating that PI3,4,5P3 generation involves multiple pathways. IQGAPl is required for IGlDPs inhibition of Akt activation as Iqgapl-null MEFs were not impacted by IGlDPs (FIG. 7e), validating the specificity of IGlDPs toward blockade of the IQGAPl - PI3K scaffold. The data indicate that the canonical PI3,4,5P3 synthesis pathway is responsible for PI3,4,5P3 synthesis from an accessible pool of PI4P and PI4,5,P2, whereas the IQGAPl pathway is for receptor stimulated synthesis. IGlDPs inhibit only the IQGAPl pathway, while PI3K inhibitors inhibit both. This is supported by data showing that IGlDPs partially reduced Akt phosphorylation in Hs578T breast cancer cells, but not in Hs578Bst normal mammary epithelial cells, leading to selective antiproliferative effect on these cancer cells. In contrast, small molecule PI3K inhibitors blocked both the IQGAPl -dependent and - independent pathways, leading to non-specific loss of cells (FIGS.7a-d).
7. IQGAPl is required for insulin signaling in vivo
[0132] The PI3K-Akt pathway plays a key role in insulin signaling (Taniguchi et al, 2006). To assess the role of IQGAPl in insulin stimulation, Akt phosphorylation was quantified in WT and Iqgapl-null MEFs stimulated by insulin. Akt activation was significantly reduced in Iqgapl-null MEFs (FIG. 8a). To establish the in vivo role, insulin or saline was injected into WT or Iqgapl-null mice. This demonstrated that insulin-stimulated PI3,4,5P3 synthesis and Akt activation were significantly reduced in classic insulin stimulated tissues, namely skeletal muscle and liver in the Iqgapl-null compared to WT mice (FIGS. 8b, 8c). These data demonstrate that IQGAPl is required for maximal activation of the PI3K-Akt pathway downstream of the insulin receptor in cells and mice.
8. Discussion
[0133] A concerted mechanism for agonist stimulated PI3,4,5P3 generation and signaling that is key for survival of some cancer cells is described herein. Agonist activated PDKs use
PI4,5P2 as a substrate to synthesize PI3,4,5P3 and it is shown that IQGAPl spatially organizes PI4,5P2 and PI3,4,5P3 producing kinases into a scaffolded complex. In this complex PIPKIa and PI3K bind to the WW and IQ domains of IQGAPl . PIPKIa generates
PI4,5P2 that is channeled to PI3K facilitating efficient PI3,4,5P3 synthesis. The PI3,4,5P3 in turn activates downstream effectors such as PDK1 and Akt that are also associated (FIG. 8d).
The type ΙΙΙα PI4K, which generates PI4P is also required for Akt activation (Supplementary
FIGS, lg, lh), and is associated with IQGAPl (FIGS. la-c). IQGAPl assembles all phosphoinositide kinases required for sequential phosphorylation of PI to PI3,4,5P3 and the effectors regulated by PI3,4,5P3. Growth factors, extracellular matrix and other agonists stimulate the assembly and signaling of the IQGAPl -PI3K scaffold. This complex, summarized in FIG. 8d, has the intrinsic activities to spatially generate PI3,4,5P3 from PI leading to activation of PDK1 and Akt. As many receptors upon agonist activation are rapidly internalized and continue signaling from endosomal compartments (Sun et al., 2013; van Meer et al., 2008). Even though these endosomal compartments contain little PI4P or
PI4,5P2 (Di Paolo et al, 2006; Sorkin et al, 2009; Balla, 2013), PI is present in all internal membranes (Sorkin et al, 2009; Balla, 2013). Thus, the IQGAPl -PI3K scaffold reveals a clear mechanism for efficient PI3,4,5P3 generation and signaling from these compartments, as the IQGAPl functions as a platform that orchestrates the full PI3K signaling pathway. The results described herein also support an IQGAPl independent pathway(s) for PI3K
generation of PI3,4,5P3. The latter pathways are sensitive to PI4P and PI4,5P2 binding proteins suggesting that they work on the accessible substrates in membranes. The PI3K pathway(s) that are independent of IQGAPl may explain the survival of Iqgapl-null mice (Li et al., 2000). Whereas, mice lacking IQGAPl are resistant to the development of some tumors (Jameson et al, 2013; Li et a/.,2013; Mataraza et al., 2003) consistent with a role in the PI3K pathway.
[0134] Alteration of the PI3K-Akt pathway is linked to many human diseases including cancers (Engelman, 2009; Luo et al, 2003; Liu et al., 2009). IQGAPl is overexpressed in many cancers (White et al., 2009) and correlated with tumorigenesis (Feigin et al., 2014; Jadeski et al, 2008). Recently, inhibition of PIPKIa was shown to significantly reduce Akt activation in prostate cancers (Semeans et al., 2014) and selectively kills cancer cells. This study supports our conclusion that PIPKIa directly integrates with the IQGAPl -PI3K pathway to regulate cell survival. Although the IQGAPl pathway is present in normal and neoplastic cells, some cancer cells appear to depend on this pathway for survival. These findings provide a potential target for cancer therapies. Unlike conventional PI3K inhibitors by ATP competition (Engelman, 2009), IGlDPs inhibit the IQGAPl -scaffolded kinases assembly and block Akt activation leading to selective cancer cell death. This suggests that some cancer cells become addicted to the IQGAPl -PI3K pathway for their survival (Weinstein et al, 2008).
[0135] Insulin-stimulated PI3K signaling is implicated in many pathophysiological conditions including diabetes, cardiac function, aging and others (Taniguchi et al., 2006; Lopez-Otin et al, 2013; Alessi et al, 1998; Yao et al, 2014). IQGAPl is required for full insulin stimulation of PI3K and Akt activation that is important for many aspects of insulin function (Yao et al, 2014). This establishes an in vivo role for the IQGAPl -scaffolded phosphoinositide kinases and effectors beyond cancers. IQGAPl binding to the p85 subunit suggests a mechanism for activation of class I PDKs within the scaffold as interaction with the cSH2 domain that could relieve the intramolecular inhibition (Burke et al, 2015). The interactions with both the P-BH-P and cSH2 may link multiple isoforms of class I PDKs explaining the diversity of agonist and receptors that require IQGAPl for the PI3K signaling (Burke et al, 2015).
[0136] In summary, here is shown a previously unrecognized mechanism for PI3,4,5P3 generation and signaling. This is achieved by the scaffold IQGAPl, where PI4KIIIa, PIPKIa and PI3K are linked for sequential phosphorylation of PI for robust and efficient PI3,4,5P3 synthesis that leads to the activation of associated effectors. [0137] This example shows that IQGAP1 peptides of the present disclosure selectively kill cancer cells. Accordingly, IQGAP1 peptides described herein are useful in methods and compositions for the treatment of cancer.
EXAMPLE 2: Treatment of cancer using IQGAP1 peptides.
[0138] This example illustrates the use of IQGAP1 peptides described herein in methods for the treatment of cancer in subjects in need thereof.
[0139] Subjects having or suspected of having cancer are administered one or more IQGAP1 peptides, alone or in conjunction with one or more additional cancer therapeutic agents, or agents directed to the amelioration of side effects of IQGAP1 peptides or cancer therapeutic agents Matched control subjects receive a placebo composition. Timing, dosage, route of administration, and duration of treatment are selected based on criteria described herein and known in the art. At the conclusion of treatment, subjects are evaluated using criteria known in the art for the evaluation of cancer, such as evaluation of tumor size, tumor number, cancer markers, etc.
[0140] It is predicted that subjects receiving one or more IQGAP1 peptides will show marked improvement in the presence or severity of cancer compared to control subjects not receiving the peptides. These results will show that IQGAP1 peptides disclosed herein are useful in methods and compositions for the treatment of cancer.
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Claims

CLAIMS What is claimed is:
1. A method of treating cancer in a subject in need thereof, comprising administering to the subject a composition comprising an IQGAP1 peptide.
2. The method of claim 1, wherein the peptide comprises the amino acid sequence KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQARCRGYLVRQEFRS, AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL, KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRANKARDDYKTLINAE.
3. The method of claim 1, wherein the peptide is selected from the group consisting of KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQARCRGYLVRQEFRS, AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL, KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, and
RDHINDIIKIQAFIRANKARDDYKTLINAE.
4. The method of claim 1, wherein the peptide is
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQARCRGYLVRQEFRS, AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL, KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRANKARDDYKTLINAE.
5. The method of claim 1, further comprising administering to the subject one or more additional cancer therapeutic agents.
6. The method of claim 5, wherein the one or more additional cancer therapeutic agents comprise nitric oxide (NO) inducers, statins, negatively charged phospholipids, antioxidants, minerals, anti-inflammatory agents, anti-angiogenic agents, matrix metalloproteinase inhibitors, carotenoids, vinca alkaloids, microtubule disrupting agents, anti -angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agents, transitional metal complexes, proteasome inhibitors, antimetabolites, alkylating agents, platinum-based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids, or geldanamycin or a derivative thereof.
7. The method of claim 1, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, head and neck cancer, liver cancer, and colon cancer.
8. The method of claim 1, wherein the composition further comprises a pharmaceutical carrier.
9. The method of claim 1, wherein the composition further comprises a preservative.
10. A composition for treating cancer in a subject in need thereof, comprising an IQGAP1 peptide.
11. The composition of claim 10, wherein the peptide comprises the amino acid sequence KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS , AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LA EGLITRLQARCRGYLVRQEFRSRMNFL, KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRA KARDDYKTLINAE.
12. The composition of claim 10, wherein the peptide is selected from the group
consisting of KWVKHWVKGGYYYYHNLETQEGGWDEPP,
LITRLQARCRGYL VRQEFRS, AITCIQSQWRGYKQKKAYQD,
E VVKIQ SL ARMHQ ARKRYRD, DIIKIQAFIRANKARDDYKT,
LANEGLITRLQARCRGYLVRQEFRSRMNFL,
KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, and
RDHINDIIKIQAFIRANKARDDYKTLINAE.
13. The composition of claim 10, wherein the peptide is
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS , AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL, KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRANKARDDYKTLINAE.
14. The composition of claim 10, further comprising one or more additional cancer
therapeutic agents.
15. The composition of claim 14, wherein the one or more additional cancer therapeutic agents comprise nitric oxide (NO) inducers, statins, negatively charged phospholipids, anti-oxidants, minerals, anti-inflammatory agents, anti-angiogenic agents, matrix metalloproteinase inhibitors, carotenoids, vinca alkaloids, microtubule disrupting agents, anti-angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agents, transitional metal complexes, proteasome inhibitors, antimetabolites, alkylating agents, platinum-based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids, or geldanamycin or a derivative thereof.
16. The composition of claim 10, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, head and neck cancer, liver cancer, and colon cancer.
17. A kit for the treatment of cancer in a subject, comprising an IQGAP1 peptide.
18. The kit of claim 17, wherein the peptide comprises the amino acid sequence
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQ ARCRGYL VRQEFRS, AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL, KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRANKARDDYKTLINAE.
19. The kit of claim 17, wherein the peptide is selected from the group consisting of KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQARCRGYLVRQEFRS, AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL, KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, and
RDHINDIIKIQAFIRANKARDDYKTLINAE.
20. The kit of claim 17, wherein the peptide is
KWVKHWVKGGYYYYHNLETQEGGWDEPP, LITRLQARCRGYLVRQEFRS, AITCIQ S Q WRGYKQKK A YQD , E VVKIQ SL ARMHQ ARKRYRD,
DIIKIQAFIRANKARDDYKT, LANEGLITRLQARCRGYLVRQEFRSRMNFL, KKQIP AITCIQ S Q WRGYKQKK A YQDRL A YL,
RSHKDEVVKIQSLARMHQARKRYRDRLQYF, or
RDHINDIIKIQAFIRANKARDDYKTLINAE.
PCT/US2017/047823 2016-08-24 2017-08-21 Methods and compositions for the treatment of cancer WO2018039132A1 (en)

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