WO2013152313A1 - Compositions and methods for treating cancer and diseases and conditions responsive to growth factor inhibition - Google Patents

Compositions and methods for treating cancer and diseases and conditions responsive to growth factor inhibition Download PDF

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Publication number
WO2013152313A1
WO2013152313A1 PCT/US2013/035492 US2013035492W WO2013152313A1 WO 2013152313 A1 WO2013152313 A1 WO 2013152313A1 US 2013035492 W US2013035492 W US 2013035492W WO 2013152313 A1 WO2013152313 A1 WO 2013152313A1
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WO
WIPO (PCT)
Prior art keywords
inhibitor
integrin
cell
optionally
growth factor
Prior art date
Application number
PCT/US2013/035492
Other languages
French (fr)
Inventor
David Cheresh
Laetitia SEGUIN
Original Assignee
The Regents Of The University Of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2012/040390 external-priority patent/WO2012167028A2/en
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Publication of WO2013152313A1 publication Critical patent/WO2013152313A1/en
Priority to US14/325,288 priority Critical patent/US9632074B2/en
Priority to US14/883,398 priority patent/US9903855B2/en

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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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Definitions

  • This invention generally relates to cell and molecular biology, diagnostics and oncology.
  • the invention provides compositions and methods for overcoming or diminishing or preventing Growth Factor Inhibitor resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor Inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor, or, sensitizing a tumor to a drug, wherein optionally the drug is an erlotinib or a lapatinib, or, sensitizing a tumor that is resistant to a cancer drug.
  • the cell is a tumor cell, a cancer cell, a cancer stem cell or a dysfunctional cell.
  • the invention provides compositions and methods for determining: whether an individual or a patient would benefit from or respond to administration of a Growth Factor Inhibitor, or, whether the individuals or patients would benefit from a
  • NFKB nuclear factor kappa-light-chain- enhancer of activated B cells, or NF- ⁇
  • VELCADE® or a lenalidomide or a REVLIMIDTM , or an ⁇ kinase (IKK) inhibitor.
  • Growth factor inhibitors have been used to treat many cancers including pancreatic, breast, lung and colorectal cancers. However, resistance to growth factor inhibitors has emerged as a significant clinical problem.
  • Tumor resistance to targeted therapies occurs due to a combination of stochastic and instructional mechanisms. Mutation/amplification in tyrosine kinase receptors or their downstream effectors account for the resistance of a broad range of tumors.
  • oncogenic KRAS the most commonly mutated oncogene in human cancer, has been linked to EGFR inhibitor resistance.
  • oncogenic KRAS is not sufficient to account for EGFR inhibitor resistance indicating that other factor(s) might control this process.
  • the invention provides methods for overcoming or diminishing or preventing Growth Factor Inhibitor (GFI) resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor Inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor (GFI), or, sensitizing or re- sensitizing a dysfunctional cell, a tumor or cancer to a drug, wherein optionally the drug is a Growth Factor Inhibitor drug to which the cell is being sensitized or re- sensitized, e.g., an erlotinib, a lapatinib or a lenalidomide, or, sensitizing a tumor that is resistant to a cancer drug (e.g., a Growth Factor Inhibitor),
  • GFI Growth Factor Inhibitor
  • the cell is a tumor cell, a cancer cell, a cancer stem cell, or a dysfunctional cell,
  • the inhibitor of integrin ⁇ ⁇ ⁇ 3 protein activity is an allosteric inhibitor of integrin ⁇ ⁇ ⁇ 3 protein activity
  • the inhibitor of RalB protein activity is an allosteric inhibitor of RalB protein activity
  • the inhibitor of NFKB or IRF3 protein activity is an allosteric inhibitor of NFKB or IRF3 protein activity
  • the NFKB inhibitor comprises a lenalidomide or a REVLIMIDTM (Celgene Corp., Summit, NJ), or a bortezomib or a
  • the IKK inhibitor comprises a PS 1145
  • a lenalidomide or a REVLIMIDTM a lenalidomide or a REVLIMIDTM ; a PS 1145; and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTK inhibitor comprises SU14813 (Pfizer, San Diego, CA); or
  • the at least one growth factor inhibitor comprises a Receptor Tyrosine Kinase (RTK) inhibitor, a Src inhibitor, an anti-metabolite inhibitor, a gemcitabine, GEMZARTM, a mitotic poison, a paclitaxel, a taxol, ABRAXANETM, an erlotinib, TARCEVATM, a lapatinib, TYKERBTM, or an insulin growth factor inhibitor,
  • RTK Receptor Tyrosine Kinase
  • the combination or the therapeutic combination comprises an erlotinib with either a Lenalidomide or a PS- 1145, or both a Lenalidomide and a PS- 1145; and (b) administering a sufficient amount of the at least one compound, composition or formulation to the cell, or the combination of compounds, to: overcome, diminish or prevent the Growth Factor Inhibitor (GFI) resistance in a cell; increase the growth- inhibiting effectiveness of the Growth Factor inhibitor on the cell; sensitize or re-sensitize the cell to the Growth Factor Inhibitor (GFI); sensitize or re-sensitize the dysfunctional cell, tumor cell or cancer to the drug, or sensitize or re-sensitize the tumor that is resistant to the cancer or anti-tumor drug,
  • GFI Growth Factor Inhibitor
  • the cell is a tumor cell, a cancer cell, a cancer stem cell, or a dysfunctional cell.
  • IV intravenously
  • parenterally parenterally, nasally, topically, orally, or by liposome or targeted or vessel-targeted nanoparticle delivery;
  • the compound or composition is a small molecule, a protein, an antibody, a monoclonal antibody, a nucleic acid, a lipid or a fat, a polysaccharide, an RNA or a DNA;
  • the compound or composition comprises or is: a VITAXINTM (Applied Molecular Evolution, San Diego, CA) antibody, a humanized version of an LM609 monoclonal antibody, an LM609 monoclonal antibody, or any antibody that functionally blocks an ⁇ ⁇ ⁇ 3 integrin or any member of an ⁇ ⁇ ⁇ 3 integrin-comprising complex or an integrin ⁇ ⁇ ⁇ 3 (avb3)/RalB/NFkB signaling axis;
  • VITAXINTM Applied Molecular Evolution, San Diego, CA
  • the NF-kB inhibitor comprises or consists of one or more of: an antioxidant; an a-lipoic acid; an a-tocopherol; a 2-amino-l- methyl-6-phenylimidazo[4,5- ]pyridine; an allopurinol; an anetholdithiolthione; a cepharanthine; a beta-carotene; a dehydroepiandrosterone (DHEA) or a DHEA-sulfate (DHEAS); a dimethyldithiocarbamates (DMDTC); a dimethylsulfoxide (DMSO); a flavone, a Glutathione; Vitamin C or Vitamin B6, or one or more compositions listed in Table 1 or Table 2, or any combination thereof;
  • the NF-kB inhibitor comprises or consists of one or more of: an antioxidant; an a-lipoic acid; an a-tocopherol; a 2-amino-l- methyl-6-pheny
  • the invention provides kits, blister packages, lidded blisters or blister cards or packets, clamshells, trays or shrink wraps, comprising;
  • kit of (a) further comprising instructions for practicing a method of the invention.
  • the kit, blister package, lidded blister, blister card, packet, clamshell, tray or shrink wrap comprises: a combination or a therapeutic combination of drugs comprising: an erlotinib with either a Lenalidomide or a PS-1 145, or both a Lenalidomide and a PS-1 145.
  • the invention provides methods for determining: whether an individual or a patient would benefit from or respond to administration of a Growth Factor Inhibitor, or
  • a combinatorial approach comprising administration of a combination of: at least one growth factor inhibitor and at least one compound, composition or formulation used to practice a method of the invention, such as an NFkB inhibitor,
  • the detection is by analysis or visualization of a biopsy or a tissue, urine, fluid, serum or blood sample, or a pathology slide taken from the patient or individual, or by a fluorescence-activated cell sorting (FACS) or flow cytometry analysis or the sample or biopsy,
  • FACS fluorescence-activated cell sorting
  • the cell or tissue or tissue sample is or is derived from a tumor or a cancer, or the cell is a cancer stem cell,
  • the method further comprises taking a biopsy or a tissue, urine, fluid, serum or blood sample from an individual or a patient,
  • a finding of increased levels or amounts of integrin ⁇ ⁇ ⁇ 3 (anb3) and/or active RalB complexes in or on the cell, tissue or the tissue sample as compared to normal, normalized or wild type cells or tissues indicates that: the individual or patient would benefit from a combinatorial approach comprising administration of a combination of: at least one growth factor inhibitor and at least one compound, composition or formulation used to practice a method of the invention, e.g., an NFKB inhibitor such as a lenalidomide or a REVLIMIDTM , or a bortezomib or a VELCADE®, or an IKK inhibitor such as a PS 1145.
  • an NFKB inhibitor such as a lenalidomide or a REVLIMIDTM
  • a bortezomib or a VELCADE® or an IKK inhibitor such as a PS 1145.
  • the detecting of the levels or amount of integrin ⁇ ⁇ ⁇ 3 (avb3) and/or active RalB complex in or on the cell, tissue or the tissue sample is done before or during a drug or a pharmaceutical treatment of an individual using at least one compound, composition or formulation used to practice a method of the invention.
  • the invention provide uses of a combination of compounds in the manufacture of a medicament
  • the inhibitor of integrin ⁇ ⁇ ⁇ 3 protein activity is an allosteric inhibitor of integrin ⁇ ⁇ ⁇ 3 protein activity
  • the inhibitor of RalB protein activity is an allosteric inhibitor of RalB protein activity
  • the inhibitor of Src or TBK1 protein activity is an allosteric inhibitor of Src or TBK1 protein activity
  • the inhibitor of NFKB or IRF3 protein activity is an allosteric inhibitor of NFKB or IRF3 protein activity; (v) an inhibitor or depleter of NFKB or IKK, or an inhibitor of NFKB or IKK protein activation,
  • the NFKB inhibitor comprises a lenalidomide or a REVLIMIDTM (Celgene Corp., Summit, NJ) or a bortezomib or a
  • the IKK inhibitor comprises a PS 1145
  • a lenalidomide or a REVLIMIDTM a lenalidomide or a REVLIMIDTM ; a PS 1145; and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTK inhibitor comprises
  • the Growth Factor Inhibitor is or comprises an anti-metabolite inhibitor, a gemcitabine, GEMZARTM, a mitotic poison, a paclitaxel, a taxol,
  • ABRAXANETM an erlotinib, TARCEVATM, a lapatinib, TYKERBTM, or an insulin growth factor inhibitor, or any combination thereof; or, the Growth Factor Inhibitor decreases, slows or blocks new blood vessel growth, neovascularization or angiogenesis; or, wherein administering the Growth Factor Inhibitor treats or ameliorates conditions that are responsive to blocking or slowing cell growth, and/or the development of neovascularization or new blood vessels,
  • the combination or the therapeutic combination comprises an erlotinib with either a Lenalidomide or a PS-1 145, or both a Lenalidomide and a PS-1 145.
  • the invention provides therapeutic combinations of drugs comprising or consisting of a combination of at least two compounds: wherein the at least two compounds comprise or consist of:
  • the inhibitor of RalB protein activity is an allosteric inhibitor of RalB protein activity
  • the inhibitor of Src or TBK1 protein activity is an allosteric inhibitor of Src or TBK1 protein activity
  • the inhibitor of NFKB or IRF3 protein activity is an allosteric inhibitor of NFKB or IRF3 protein activity
  • the NFKB inhibitor comprises a lenalidomide or a REVLIMIDTM (Celgene Corp., Summit, NJ) or a bortezomib or a
  • the IKK inhibitor comprises a PS 1145
  • a lenalidomide or a REVLIMIDTM a lenalidomide or a REVLIMIDTM ; a PS 1145; and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTK inhibitor comprises SU14813 (Pfizer, San Diego, CA); or
  • the Growth Factor Inhibitor is or comprises an anti-metabolite inhibitor, a gemcitabine, GEMZARTM, a mitotic poison, a paclitaxel, a taxol,
  • ABRAXANETM an erlotinib, TARCEVATM, a lapatinib, TYKERBTM, or an insulin growth factor inhibitor, or any combination thereof; or, the Growth Factor Inhibitor decreases, slows or blocks new blood vessel growth, neovascularization or angiogenesis; or,
  • administering the Growth Factor Inhibitor treats or ameliorates conditions that are responsive to blocking or slowing cell growth, and/or the development of neovascularization or new blood vessels,
  • the combination or the therapeutic combination comprises an erlotinib with either a Lenalidomide or a PS-1 145, or both a Lenalidomide and a PS-1 145.
  • the invention provides combinations, or therapeutic combinations, for overcoming or diminishing or preventing Growth Factor Inhibitor (GFI) resistance in a cell, or, a method for increasing the growth- inhibiting effectiveness of a Growth Factor Inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor (GFI), wherein the combination comprises or consists of:
  • the inhibitor of integrin ⁇ ⁇ ⁇ 3 protein activity is an allosteric inhibitor of integrin ⁇ ⁇ ⁇ 3 protein activity
  • the inhibitor of RalB protein activity is an allosteric inhibitor of RalB protein activity
  • the inhibitor of Src or TBK1 protein activity is an allosteric inhibitor of Src or TBK1 protein activity
  • the inhibitor of NFKB or IRF3 protein activity is an allosteric inhibitor of NFKB or IRF3 protein activity; (v) an inhibitor or depleter of NFKB or IKK, or an inhibitor of NFKB or IKK protein activation,
  • the NFKB inhibitor comprises a lenalidomide or a REVLIMIDTM (Celgene Corp., Summit, NJ) or a bortezomib or a
  • the IKK inhibitor comprises a PS 1145
  • a lenalidomide or a REVLIMIDTM a lenalidomide or a REVLIMIDTM ; a PS 1145; and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTK inhibitor comprises
  • the Growth Factor Inhibitor is or comprises an anti-metabolite inhibitor, a gemcitabine, GEMZARTM, a mitotic poison, a paclitaxel, a taxol,
  • ABRAXANETM an erlotinib, TARCEVATM, a lapatinib, TYKERBTM, or an insulin growth factor Inhibitor, or any combination thereof; or, the Growth Factor Inhibitor decreases, slows or blocks new blood vessel growth, neovascularization or angiogenesis; or, wherein administering the Growth Factor Inhibitor treats or ameliorates conditions that are responsive to blocking or slowing cell growth, and/or the development of neovascularization or new blood vessels,
  • the cell is a tumor cell, a cancer cell, a cancer stem cell, or a dysfunctional cell,
  • the combination or the therapeutic combination comprises an erlotinib with either a Lenalidomide or a PS-1 145, or both a Lenalidomide and a PS-1 145.
  • Figure 1 illustrates that integrin ⁇ 3 expression promotes resistance to EGFR TKI:
  • Fig. 1(a) illustrates flow cytometric quantification of cell surface markers after 3 weeks treatment with erlotinib (pancreatic and colon cancer cells) or lapatinib (breast cancer cells);
  • Fig. 1(b) illustrates flow cytometric analysis of ⁇ 3 expression in FG and Miapaca-2 cells following erlotinib;
  • Fig. 1(a) illustrates flow cytometric quantification of cell surface markers after 3 weeks treatment with erlotinib (pancreatic and colon cancer cells) or lapatinib (breast cancer cells);
  • Fig. 1(b) illustrates flow cytometric analysis of ⁇ 3 expression in FG and Miapaca-2 cells following erlotinib;
  • Fig. 1(a) illustrates flow cytometric quantification of cell surface markers after 3 weeks treatment with erlotinib (pancreatic and colon cancer cells) or lapati
  • FIG. 1 (c) illustrates: Top, immunofluorescence staining of integrin ⁇ 3 in tissue specimens obtained from orthotopic pancreatic tumors treated with vehicle or erlotinib; Bottom, Integrin ⁇ 3 expression was quantified as ratio of integrin ⁇ 3 pixel area over nuclei pixel area using METAMORPHTM; Fig. 1(d) Right, intensity of ⁇ 3 expression in mouse orthotopic lung tumors treated with vehicle or erlotinib, Left, immunohistochemical staining of ⁇ 3, Fig. 1(f) illustrates tumor sphere formation assay to establish a dose-response for erlotinib, Fig.
  • 1(g) illustrates orthotopic FG tumors treated for 10 days with vehicle or erlotinib
  • results are expressed as % tumor weight compared to vehicle control
  • immunoblot analysis for tumor lysates after 10 days of erlotinib confirms suppressed EGFR phosphorylation; as discussed in detail in Example 1, below.
  • Figure 2 illustrates that integrin ⁇ 3 cooperates with K-RAS to promote resistance to EGFR blockade:
  • Fig. 2(a-b) illustrates tumor sphere formation assay of FG expressing (a) or lacking (b) integrin ⁇ 3 depleted of KRAS (shKRAS) or not (shCTRL) and treated with a dose response of erlotinib;
  • Fig. 2(c) illustrates confocal microscopy images of PANC-1 and FG- ⁇ 3 cells grown in suspension;
  • Fig. 2(d) illustrates RAS activity assay performed in PANC-1 cells using GST-Rafl-RBD immunoprecipitation as described below; as discussed in detail in Example 1, below.
  • FIG. 3 illustrates that RalB is a key modulator of integrin o ⁇ 3-mediated EGFR TKI resistance:
  • Fig. 3(a) illustrates tumor spheres formation assay of FG ⁇ 3 treated with non-silencing (shCTRL) or RalB-specific shRNA and exposed to a dose response of erlotinib;
  • Fig. 3(b) illustrates effects of depletion of RalB on erlotinib sensitivity in ⁇ 3- positive tumor in a pancreatic orthotopic tumor model;
  • Fig. 3(a) illustrates tumor spheres formation assay of FG ⁇ 3 treated with non-silencing (shCTRL) or RalB-specific shRNA and exposed to a dose response of erlotinib
  • Fig. 3(b) illustrates effects of depletion of RalB on erlotinib sensitivity in ⁇ 3- positive tumor in a pancreatic orthotopic tumor model
  • Fig. 3(a) illustrates tumor
  • FIG. 3(c) illustrates tumor spheres formation assay of FG cells ectopically expressing vector control, WT RalB FLAG tagged constructs or a constitutively active RalB G23V FLAG tagged treated with erlotinib (0.5 ⁇ );
  • Fig. 3(d) illustrates RalB activity was determined in FG, FG- 3 expressing non-silencing or KRAS-specific shR A, by using a GST-RalBPl-RBD immunoprecipitation assay;
  • Fig. 3(e) illustrates: Right, overall active Ral
  • Figure 4 illustrates that integrin av 3/RalB complex leads to NF- ⁇ activation and resistance to EGFR TKl:
  • Fig. 4(a) illustrates an immunoblot analysis of FG, FG- 3 and FG- 3 stably expressing non-silencing or RalB-specific ShRNA, grown in suspension and treated with erlotinib (0.5 ⁇ );
  • Fig. 4(b) illustrates tumor spheres formation assay of FG cells ectopically expressing vector control, WT NF- ⁇ FLAG tagged or constitutively active S276D NF- ⁇ FLAG tagged constructs treated with erlotinib;
  • Fig. 4(a) illustrates an immunoblot analysis of FG, FG- 3 and FG- 3 stably expressing non-silencing or RalB-specific ShRNA, grown in suspension and treated with erlotinib (0.5 ⁇ );
  • Fig. 4(b) illustrates tumor spheres formation assay of
  • FIG. 4(c) illustrates tumor spheres formation assay of FG- 3 treating with non- silencing (shCTRL) or NF-KB-specific shRNA and exposed to erlotinib
  • Fig. 4(d) illustrates dose response in FG-P3 cells treated with erlotinib (10 nM to 5 ⁇ ), lenalidomide (10 nM to 5 ⁇ ) or a combination of erlotinib (10 nM to 5 ⁇ ) and lenalidomide (1 ⁇ )
  • FIG. 4(e) illustrates Model depicting the integrin ⁇ 3 -mediated EGFR TKl resistance and conquering EGFR TKl resistance pathway and its downstream RalB and NF- ⁇ effectors; as discussed in detail in Example 1, below.
  • Figure 5 (Supplementary Fig. 1, Example 1) illustrates that prolonged exposure to erlotinib induces Integrin ⁇ 3 expression in lung tumors; representative
  • Figure 6 (Supplementary Fig. 2, Example 1) illustrates integrin ⁇ 3, even in its unligated state, promotes resistance to Growth Factor inhibitors but not to
  • Fig. 6(b) illustrates tumor sphere formation assay of FG and FG- ⁇ 3 cells untreated or treated with erlotinib (0.5 ⁇ ), OSI- 906 (0.1 ⁇ ), gemcitabine (0.01 ⁇ ) or cisplatin (0.1 ⁇ );
  • Figure 7 (Supplementary Fig. 3, Example 1) illustrates that integrin ⁇ 3 does not colocalize with active HRAS, NRAS and RRAS:
  • Fig. 7(a) illustrates that Ras activity was determined in PANC-1 cells grown in suspension by using a GST-Rafl-RBD
  • FIG. 7(b) illustrates confocal microscopy images of PANC-1 cells grown in suspension and stained for KRAS, RRAS, HRAS, NRAS (red), integrin ⁇ 3 (green) and DNA (TOPRO-3, blue) (Scale bar, 10 ⁇ . Data are representative of two independent experiments); as discussed in detail in Example 1, below.
  • Figure 8 (Supplementary Fig. 4, Example 1) illustrates that Galectin-3 is required to promote integrin av 3/KRAS complex formation:
  • Fig. 8(a-b) illustrates confocal microscopy images of Pane- 1 cells lacking or expressing integrin ⁇ 3 grown in suspension;
  • Fig. 8(a) illustrates cells stained for KRAS (green), Galectin-3 (red), and DNA (TOPRO-3, blue);
  • Fig. 8(b) illustrates cells stained for integrin ⁇ 3 (green), Galectin-3 (red) and DNA (TOPRO-3, blue), Scale bar, 10 ⁇ , data are representative of three independent experiments;
  • Fig. 8(a-b) illustrates confocal microscopy images of Pane- 1 cells lacking or expressing integrin ⁇ 3 grown in suspension;
  • Fig. 8(a) illustrates cells stained for KRAS (green), Galectin-3 (red), and DNA (TOPRO-3, blue);
  • FIG. 8(c) illustrates an immunoblot analysis of Galectin-3 immuno-precipitates from PANC- 1 cells expressing non-silencing (sh CTRL) or integrin 3-specific shRNA (sh ⁇ 3), data are representative of three independent experiments;
  • Fig. 8(d) illustrates an immunoblot analysis of integrin ⁇ 3 immunoprecipitates from PANC-1 cells expressing non-silencing (sh CTRL) or Galectin-3 -specific shRNA (sh Gal3), data are representative of three independent experiments; as discussed in detail in Example 1, below.
  • Figure 10 (Supplementary Fig. 6, Example 1) illustrates that RalB is sufficient to promote resistance to EGFR TKI:
  • Fig. 10(c) shows that integrin ⁇ 3 colocalizes with RalB in cancer cells: illustrates confocal microscopy images of Pane- 1 cells grown in suspension. Cells are stained for integrin ⁇ 3 (green), RalB (red), pFAK (red), and DNA (TOPRO-3, blue), scale bar, 10 ⁇ , data are representative of three independent experiments; as discussed in detail in Example 1, below.
  • Figure 1 1 (Supplementary Fig. 8, Example 1) illustrates that integrin ⁇ 3 colocalizes with RalB in human breast and pancreatic tumor biopsies and interacts with RalB in cancer cells:
  • Fig. 1 1(a) illustrates confocal microscopy images of integrin ⁇ 3 (green), RalB (red) and DNA (TOPRO-3, blue) in tumor biopsies from breast and pancreatic cancer patients, Scale bar, 20 ⁇ ;
  • Fig. 1(a) illustrates confocal microscopy images of integrin ⁇ 3 (green), RalB (red) and DNA (TOPRO-3, blue) in tumor biopsies from breast and pancreatic cancer patients, Scale bar, 20 ⁇ ;
  • Fig. 1(a) illustrates confocal microscopy images of integrin ⁇ 3 (green), RalB (red) and DNA (TOPRO-3, blue) in tumor biopsies from breast and pancreatic cancer patients, Scale bar, 20
  • FIG. 11(b) illustrates a Ral activity assay performed in PANC-1 cells using GST-RalBPl-RBD immunoprecipitation assay, Immunoblot analysis of RalB and integrin ⁇ 3, data are representative of three independent experiments; as discussed in detail in Example 1, below.
  • FIG. 12(A) schematically illustrates that the identification of the most upregulated tumor progression genes common to erlotinib resistant carcinomas
  • Fig. 12(B) in table form shows Erlotinib IC5 0 in a panel of human carcinoma cell lines treated with erlotinib in 3D culture
  • Fig. 12(C) graphically illustrates percentage of integrin ⁇ 3 positive cells in parental lines vs. after 3 or 8 weeks treatment with erlotinib
  • Fig. 12(A) schematically illustrates that the identification of the most upregulated tumor progression genes common to erlotinib resistant carcinomas
  • Fig. 12(B) in table form shows Erlotinib IC5 0 in a panel of human carcinoma cell lines treated with erlotinib in 3D culture
  • Fig. 12(C) graphically illustrates percentage of integrin ⁇ 3 positive cells in parental lines vs. after 3 or 8 weeks treatment with erlotinib
  • Fig. 12(A) schematically illustrates
  • Fig. 12(E) illustrates images of paired human lung cancer biopsies obtained before and after erlotinib resistance were immunohistochemically stained for integrin ⁇ 3, scale bar, 50 ⁇ ; Fig.
  • FIG. 12(F) graphically illustrates: Right graph shows effect of integrin ⁇ 3 knockdown on erlotinib resistance of 3-positive cells, and Left graph shows effect of integrin ⁇ 3 ectopic expression on erlotinib resistance in FG and H441 cells;
  • Figure 13 illustrates data showing that integrin ⁇ 3 is required to promote KRAS dependency and KRAS-mediated EGFR inhibitor resistance:
  • Fig. 13(A) illustrates confocal microscopy images showing immunostaining for integrin ⁇ 3 (green), K-, N-, H-, R-Ras (red), and DNA (TOPRO-3, blue) for BxPc3 cells grown in suspension in media with 10% serum, arrows indicate clusters where integrin ⁇ 3 and KRAS colocalize (yellow);
  • Fig. 13(A) illustrates confocal microscopy images showing immunostaining for integrin ⁇ 3 (green), K-, N-, H-, R-Ras (red), and DNA (TOPRO-3, blue) for BxPc3 cells grown in suspension in media with 10% serum, arrows indicate clusters where integrin ⁇ 3 and KRAS colocalize (yellow);
  • Fig. 13(A) illustrates confocal microscopy images
  • FIG. 13(B-C) illustrates confocal microscopy images showing immunostaining for integrin ⁇ 3 (green), KRAs (red) and DNA (Topro-3, blue) for PANC-1 (KRAS mutant) and HCC827 (KRAS wild- type) after acquired resistance to erlotinib (HCC827R) grown in suspension in absence (Vehicle) or in presence of erlotinib (0.5 ⁇ and 0.1 ⁇ respectively), arrows indicate clusters where integrin ⁇ 3 and KRAS colocalize (yellow); Fig.
  • FIG. 13(D) graphically illustrates the effect of KRAS knockdown on tumorspheres formation in a panel of lung and pancreatic cancer cells expressing or lacking integrin ⁇ 3
  • FIG. 13(E) graphically illustrates the effect of KRAS knockdown on tumorsphere formation in PANC-1 (KRAS mutant) stably expressing non-target shRNA control ( ⁇ 3 -positive) or specific-integrin ⁇ 3 shRNA ( ⁇ 3 negative) in FG (KRAS mutant) and BxPc3 (KRAS wild-type) stably expressing vector control or integrin ⁇ 3;
  • FIG. 13(F) graphically illustrates the effect of KRAS knockdown on erlotinib resistance of ⁇ 3-negative and ⁇ 3- ⁇ 8 ⁇ epithelial cancer cell lines, cells were treated with a dose response of erlotinib;
  • Fig. 13(G) illustrates confocal microscopy images showing immunostaining for integrin ⁇ 3 (green), KRAS (red) and DNA (TOPRO-3, blue) for PANC-1 cells expressing non-target shRNA control or Galectin 3 -specific shRNA grown in suspension;
  • Fig. 13(H) illustrates: Top:
  • FIG. 13(1) graphically illustrates erlotinib dose response of FG- 3 cells expressing a non-target shRNA control or a Galectin-3 -specific shRNA (sh Gal-3); as further described in Example 2, below.
  • Figure 14 or Fig. 14/31 ( Figure 3 in Example 2) illustrates data showing that RalB is a central player of integrin 3-mediated EGFR inhibitor resistance:
  • Fig. 14(A) graphically illustrates the effect of RalB knockdown on erlotinib resistance of 3-positive epithelial cancer cell lines, cells were treated with 0.5 ⁇ of erlotinib: Fig.
  • FIG. 14(B) graphically illustrates the effect of RalB knockdown on erlotinib resistance of 3-positive human pancreatic (FG- 3) orthotopic tumor xenografts, established tumors expressing non-target shRNA, (shCTRL) or a shRNA targeting RalB (sh RalB) were randomized and treated for 10 days with vehicle or erlotinib, results are expressed as % of tumor weight changes after erlotinib treatment compared to vehicle;
  • Fig. 14(C) graphically illustrates the effect of expression of a constitutively active Ral G23V mutant on erlotinib response of ⁇ 3 negative cells, cells were treated with 0.5 ⁇ of erlotinib; Fig.
  • FIG. 14(D) illustrates the effect of expression of integrin ⁇ 3 on KRAS and RalB membrane localization
  • Fig. 14(E) illustrates Ral activity that was determined in PANC- 1 cells grown in suspension by using a GST-RalBP l-RBD immunoprecipitation assay, immunoblots indicate RalB activity and association of active RalB with integrin ⁇ 3
  • Fig. 14(F) illustrates confocal microscopy images of integrin ⁇ 3 (green), RalB (red) and DNA (TOPRO-3, blue) in tumor biopsies from pancreatic cancer patients
  • Fig. 14(G) illustrates the effect of ⁇ 3 expression and KRAS expression on RalB activity, measured using a GST-RalBP 1 -RBD
  • FIG. 14(H) illustrates immunoblot analysis of FG and FG ⁇ 3 stably expressing non-target shRNA control or RalB-specific shRNA, grown in suspension and treated with erlotinib (0.5 ⁇ );
  • Fig. 14(1) graphically illustrates the effect of TBK1 and p65 NFKB on erlotinib resistance of FG ⁇ 3 cells, cells were treated with 0.5 ⁇ of erlotinib; as further described in Example 2, below.
  • Figure 15 illustrates data showing that reversal of ⁇ 3 -mediated EGFR inhibitor resistance in oncogenic KRAS model by pharmacological inhibition:
  • Fig. 15(A) graphically illustrates the effect of NFkB inhibitors on erlotinib response of ⁇ 3 -positive cells (FG- 3, PANC-1 and A549), cells were treated with vehicle, erlotinib (0.5 ⁇ ), lenalidomide (1-2 ⁇ ), bortezomib (4 nM) alone or in combination;
  • Fig. 15(A) graphically illustrates the effect of NFkB inhibitors on erlotinib response of ⁇ 3 -positive cells (FG- 3, PANC-1 and A549), cells were treated with vehicle, erlotinib (0.5 ⁇ ), lenalidomide (1-2 ⁇ ), bortezomib (4 nM) alone or in combination;
  • mice bearing subcutaneous ⁇ 3 -positive tumors were treated with vehicle, erlotinib (25 mg/kg/day), lenalidomide (25 mg/kg/day) or the combination of erlotinib and
  • mice bearing subcutaneous 3-positive tumors (FG-R) after acquired resistance to erlotinib were treated with vehicle, erlotinib (25 mg/kg/day), bortezomib (0.25 mg/kg), the combination of erlotinib and bortezomib, tumor dimensions are reported as the fold change relative to size of the same tumor on Day 1;
  • Fig. 15(C) schematically illustrates a model depicting an integrin ⁇ 3 -mediated KRAS dependency and EGFR inhibitor resistance mechanism; as further described in Example 2, below.
  • FIG. 16 illustrates data showing that illustrates resistance to EGFR inhibitor is associated with integrin ⁇ 3 expression in pancreatic and lung human carcinoma cell lines:
  • Fig. 16(A) illustrates immunoblots showing integrin ⁇ 3 expression in human cell lines used in Figure 12;
  • Fig. 16(B) graphically illustrates data showing the effect of erlotinib on HCC827 xenograft tumors in immuno - compromised mice relative to vehicle-treated control tumors;
  • Fig. 16(C) left graphically illustrates data of Integrin ⁇ 3 quantification in orthotopic lung and pancreas tumors treated with vehicle or erlotinib until resistance,
  • Fig. 16(C) right illustrates a representative immunofluorescent staining of integrin ⁇ 3 in pancreatic human xenografts treated 4 weeks with vehicle or erlotinib; as further described in Example 2, below.
  • Figure 17, or Fig. 17/31 illustrates Integrin ⁇ 3 expression predicts intrinsic resistance to EGFR inhibitors in tumors; Fig.
  • FIG. 17A graphically illustrates a plot of progression- free survival for erlotinib-treated patients with low versus (vs.) high protein expression of ⁇ 3 integrin measured from non-small cell lung cancer biopsy material (Fig. 17B illustrates) obtained at diagnosis; as further described in Example 2, below.
  • FIG. 18(A) illustrates immunoblots showing integrin ⁇ 3 knockdown efficiency in cells used in Figure 12
  • Fig. 18(B) graphically illustrates response of A549 lung carcinoma cells non-target shRNA control or shRNA targeting integrin ⁇ 3 to treatment with either vehicle or erlotinib (25 mg/kg/day) during 16 days
  • Fig. 18(C) illustrates immunoblots showing expression of indicated proteins of representative tumors
  • Fig. 18(C) illustrates immunoblots showing expression of indicated proteins of representative tumors.
  • FIG. 18(D) illustrates representative photographs of crystal violet-stained tumorspheres of ⁇ 3 -negative and ⁇ 3 -positive cells after erlotinib, OSI-906, gemcitabine and cisplatin treatment
  • Fig. 18(E) graphically illustrates the effect of integrin ⁇ 3 expression on lapatinib, OSI-906, cisplatin and gemcitabine
  • Fig. 18(F) graphically illustrates data from a viability assay of FG and FG- ⁇ 3 cells grown in suspension in media with or without serum; as further described in Example 2, below.
  • FIG. 19A graphically illustrates the effect of ectopic expression of ⁇ 3 wild-type (FG- ⁇ 3) or the ⁇ 3 Dl 19A (FG-D1 19 A) ligand binding domain mutant on erlotinib response;
  • Fig. 19B illustrates an immunoblot showing transfection efficiency of vector control, integrin ⁇ 3 wild-type and integrin ⁇ 3 D l 19A; as further described in Example 2, below.
  • FIG. 20(A) illustrates confocal microscopy images of FG and FG ⁇ 3 cells grown in suspension in media 10% serum with or without erlotinib (0.5 ⁇ ) and stained for KRAS (red), integrin ⁇ 3 (green) and DNA (TOPRO-3, blue);
  • Fig. 20(B) illustrates Ras activity was determined in PANC-1 cells grown in suspension by using a GST-Rafl-RBD
  • FIG. 20(C) illustrates an immunoblot analysis showing that Integrin ⁇ 3 immunoprecipitates from BxPC-3 cells grown in suspension in presence or absence of growth factors; as further described in Example 2, below.
  • FIG. 21(A) illustrates Immunoblots showing KRAS knockdown efficiency in cells used in Figure 13
  • Fig 21(B) illustrates Representative photographs of crystal violet-stained tumorspheres of FG and A549 cells expressing non-target shRNA control or specific -KRAS shRNA
  • Fig. 21(C) illustrates the effect of an additional KRAS knockdown on tumorspheres formation in PANC-1 stably expressing non-target shRNA control ( ⁇ 3 -positive) or specific-integrin ⁇ 3 shRNA ( ⁇ 3 negative)
  • Fig. 21(D) illustrates immunoblots showing KRAS knockdown efficiency; as further described in Example 2, below.
  • Figure 22, or Fig. 22/31 illustrates images showing that KRAS and Galectin-3 colocalize in integrin 3-positive cells, in particular, confocal microscopy images of FG and FG- 3 cells grown in suspension and stained for KRAS (green), galectin-3 (red) and DNA (TOPRO-3, blue); as further described in Example 2, below.
  • FIG. 23 illustrates Integrin 3-mediated KRAS dependency and erlotinib resistance is independent of ERK, AKT and RalA:
  • Fig. 23(A) graphically illustrates the effect of ERK, AKT, RalA and RalB knockdown on erlotinib response (erlotinib 0.5 ⁇ ) of 3-negative FG and ⁇ 3- positive FG- 3 cells;
  • Fig. 23(B) illustrates Immunoblots showing ERK, AKT RalA and RalB knockdown efficiency;
  • Fig. 23(C) illustrates Immunoblots showing RalB knockdown efficiency in cells used in Figure 14; as further described in Example 2, below.
  • FIG. 24 illustrates constitutive active NFkB is sufficient to promote erlotinib resistance:
  • Fig. 24(A) illustrates Immunoblots showing TBK1 and NFkB knockdown efficiency used in Figure 14;
  • Fig. 24(B) graphically illustrates the effect of constitutive active S276D p65NFkB on erlotinib response (erlotinib 0.5 ⁇ ) of 3-negative cells (FG cells); as further described in Example 2, below.
  • Figure 25, or Fig. 25/31 illustrates NFkB inhibitors in combination with erlotinib increase cell death in vivo:
  • Fig. 25(A) and Fig. 25 (B) illustrate Immunoblots showing expression of indicated proteins of representative tumors from shown in Figure 15B;
  • Fig. 25(C) illustrates Confocal microscopy images of cleaved caspase 3 (red) and DNA (TOPRO-3, blue) in tumor biopsies from xenografts tumors used in Fig. 15B treated with vehicle, erlotinib, lenalidomide or lenalidomide and erlotinib in combo;
  • Fig. 25(A) and Fig. 25 (B) illustrate Immunoblots showing expression of indicated proteins of representative tumors from shown in Figure 15B
  • Fig. 25(C) illustrates Confocal microscopy images of cleaved caspase 3 (red) and DNA (TOPRO-3, blue) in tumor biopsies from
  • 25(D) illustrates Confocal microscopy images of cleaved caspase 3 (red) and DNA (TOPRO-3, blue) in tumor biopsies from xenografts tumors used in Figure 15B treated with vehicle, erlotinib, bortezomib or bortezomib and erlotinib in combo); as further described in Example 2, below.
  • Figures 26, 27 and 28, or Fig. 26/31, 27/31 and 28/31 illustrate supplementary Table 1 from Example 2, showing that differentially expressed genes in cells resistant to erlotinib (PANC-1, H I 650, A459) compared with the average of two sensitive cells (FG, H44I) and in HCC827 after acquired resistance in vivo (HCC827R) vs. the HCC827 vehicle- treated control; as further described in Example 2, below.
  • Figure 29, or Fig. 29/31 illustrates supplementary Table 2, from Example 2, showing KRAS mutational status in pancreatic and lung cell lines used in the study of Example 2, below.
  • FIG. 30/31 illustrates data showing integrin ⁇ 3 (CD61) is a RTKI drug resistance biomarker on the surface of circulating tumor cells; as discussed in detail in Example 2, below.
  • CD61 ⁇ 3, or beta3 negative human lung cancer cells (HCC827; this lung adenocarcinoma has an acquired mutation in the EGFR tyrosine kinase domain (E746 - A750 deletion), and they are sensitive to erlotinib and develop acquired resistance after 6/8 weeks) were injected orthotopically into the lung of mice and treated over 3 months with erotinib at 25 mg/kg/day.
  • Human lung cancer cells detected in the circulation were positive for ⁇ 3 (or avb3, CD61) whereas the cells in the untreated group were essentially negative for this marker.
  • CD45 negative cells indicates that the detected cells were not leukocytes and pan cytokeratin positive cells indicate tumor cells.
  • CD61 (beta3) positive expression correlated with tumor expression.
  • Figure 31, or Fig. 31/31 illustrates data showing how targeting the NF-KB
  • compositions and methods of this invention can sensitize resistant tumors to growth factor inhibitors by showing the effect of NFkB inhibitors on erlotinib response of 3-negative (b3-negative) cells (FG) and 3-positive cells (FG- ⁇ 3, MDA-MB231
  • the invention provides compositions and methods for overcoming or diminishing or preventing Growth Factor Inhibitor (GFI) resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor (GFI).
  • the cell is a tumor cell, a cancer cell or a
  • the invention provides compositions and methods for determining: whether an individual or a patient would benefit from or respond to administration of a Growth Factor Inhibitor, or, which individuals or patients would benefit from a combinatorial approach comprising administration of a combination of: at least one growth factor inhibitor and at least one compound, composition or formulation used to practice a method of the invention, such as an NfKb inhibitor.
  • integrin avb3 is upregulated in cells that become resistant to Growth Factor Inhibitors.
  • Our findings demonstrate that integrin avb3 promotes de novo and acquired resistance to Growth Factor Inhibitors by interacting and activating RalB.
  • RalB activation leads to the activation of Src and TBK1 and the downstream effectors NFKB and IRF3.
  • depletion of RalB or its downstream signaling (Src/NFKB) in b3-positive cells overcomes resistance to growth factor inhibitors.
  • integrin avb3/RalB signaling complex promotes resistance to growth factor inhibitors; and in alternative embodiments, integrin ⁇ ⁇ ⁇ 3 (avb3) and active RalB are used as biomarkers in patient samples to predict which patients will respond to growth factor inhibitors and which patients might rather benefit from alternative/combinatorial approaches such as a combination of growth factor inhibitors and NfKb inhibitors.
  • This invention for the first time identifies integrin ⁇ 3 and active RalB as potential biomarker for tumors that are or have become (e.g., de novo and acquired) resistant to growth factors blockade. Accordingly, in alternative embodiments, the invention provides compositions and methods for the depletion of RalB, Src, NFkB and its downstream signaling effectors to sensitize ⁇ 3 -expressing tumors to growth factor blockade. These findings reveal a new role for integrin ⁇ 3 in mediating tumor cell resistance to growth factor inhibition and demonstrate that targeting the ⁇ 3/ RalB/ NfkB/ Src signaling pathway will circumvent growth factor inhibitor resistance of a wide range of cancers.
  • any NF-kB inhibitor can be used to practice this invention, e.g., lenalidomide or (R5)-3-(4-amino-l-oxo-3H-isoindol-2-yl)piperidine-2,6- dione, which can be REVLIMIDTM (Celgene Corp., Summit, NJ), or thalidomide, or any other derivative of thalidomide, or any composition having an equivalent activity.
  • lenalidomide or (R5)-3-(4-amino-l-oxo-3H-isoindol-2-yl)piperidine-2,6- dione which can be REVLIMIDTM (Celgene Corp., Summit, NJ), or thalidomide, or any other derivative of thalidomide, or any composition having an equivalent activity.
  • compositions and methods of the invention are used to sensitize tumors to drugs, e.g., such as erlotinib and lapatinib (which are commonly used to treat a wide range of solid tumors).
  • drugs e.g., such as erlotinib and lapatinib (which are commonly used to treat a wide range of solid tumors).
  • NFkB inhibitors such as e.g., lenalidomide or (R5)-3-(4-amino-l-oxo-3H- isoindol-2-yl)piperidine-2,6-dione or REVLIMIDTM, or a composition as listed in Table 1.
  • compositions and methods of the invention are used to sensitize tumors using an IKK inhibitor, e.g., such as PS 1145 (Millennium
  • phosphorylation and/or degradation inhibitor e.g., one or more compositions listed in Table 3.
  • compositions and methods of the invention comprise use of an NFkB inhibitor and an IKK inhibitor to treat a drug resistant tumor, e.g., a solid tumor.
  • compositions and methods of the invention comprise use of an NFkB inhibitor and an IKK inhibitor to treat a drug resistant tumor in combination with an anticancer drug, e.g., an NFkB inhibitor and an IKK inhibitor are used to sensitize a tumor to drugs such as erlotinib and lapatinib.
  • the drug combination used to practice the invention comprises
  • lenalidomide such as a REVLIMIDTM
  • IKK inhibitor PSl 145 Millennium Pharmaceuticals, Cambridge, MA
  • lenalidomide and PSl 145 are used to sensitize a tumor that is resistant to a cancer drug, e.g., an EGFR inhibitor, such that the tumor is now responsive to the cancer drug.
  • a cancer drug e.g., an EGFR inhibitor
  • an NFkB inhibitor and an IKK inhibitor are used in combination with a tyrosine kinase receptor (also called Receptor Tyrosine Kinases, or RTKs) inhibitor, e.g., an SU14813 (Pfizer, San Diego, CA) or as listed in Table 2 or 3, below, to treat a drug resistant tumor.
  • a tyrosine kinase receptor also called Receptor Tyrosine Kinases, or RTKs
  • SU14813 Pfizer, San Diego, CA
  • Table 2 or 3 listed in Table 2 or 3, below
  • compositions and methods of the invention are administered to patients that have become resistant to a cancer drug, e.g., drugs like erotinib or lapatinib, to produce a strong antitumor effect.
  • a cancer drug e.g., drugs like erotinib or lapatinib
  • any NF-kB inhibitor can be used to practice this invention, e.g., an antioxidant can be used to inhibit activation of NF-kB, e.g., including the compositions listed in Table 1 :
  • Centaurea L extracts Karamenderes et al, 2007
  • Curcumin (Diferulolylmethane); Singh & Aggarwal, 1995;Pae et al, dimethoxycurcumin; EF24 analog 2008; Kasinskiet al, 2008
  • DHEA Dehydroepiandrosterone
  • DHEA-sulfate Iwasaki et al, 2004; Liuet al, 2005
  • Tepoxaline (5 -(4-chlorophenyl)-N-hydroxy-(4- methoxyphenyl) -N-methyl-lH-pyrazole-3- propanamide) Kazmi et al, 1995; Ritchieet al, 1995
  • Vaccinium stamineum (deerberry) extract Wang et al, 2007
  • any proteasome inhibitor and/or protease inhibitor can be used to practice the invention, e.g., any proteasome inhibitor and/or protease inhibitor that can inhibit Rel and/or NF-kB can be used to practice this invention, e.g., including the compositions listed in Table 2:
  • any ⁇ (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) phosphorylation and/or degradation inhibitor can be used to practice this invention, e.g., including the compositions listed in Table 3: Table 3 : ⁇ phosphorylation and/or degradation inhibitors
  • NF-2 Neurofibromatosis-2
  • Phenylarsine oxide (PAO, tyrosine 1998; Singh & Aggarwal, phosphatase inhibitor) Degradation 1995
  • the invention provides pharmaceutical compositions for practicing the methods of the invention, e.g., pharmaceutical compositions for overcoming or diminishing or preventing Growth Factor Inhibitor (GFI) resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor Inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor.
  • GFI Growth Factor Inhibitor
  • compositions used to practice the methods of the invention are formulated with a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions used to practice the methods of the invention can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally.
  • the pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's").
  • compositions administered alone or as a component of a pharmaceutical formulation (composition).
  • the compounds may be formulated for administration in any convenient way for use in human or veterinary medicine.
  • Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • Formulations of the compositions used to practice the methods of the invention include those suitable for oral/ nasal, topical, parenteral, rectal, and/or intravaginal administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • compositions used to practice the methods of the invention can be prepared according to any method known to the art for the manufacture of
  • Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents.
  • a formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture.
  • Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, geltabs, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient.
  • Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores.
  • Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen.
  • Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound (i.e., dosage).
  • Pharmaceutical preparations used to practice the methods of the invention can also be used orally using, e.g., push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol.
  • Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • Aqueous suspensions can contain an active agent (e.g., a composition used to practice the methods of the invention) in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxy ethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono
  • the aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin.
  • preservatives such as ethyl or n-propyl p- hydroxybenzoate
  • coloring agents such as a coloring agent
  • flavoring agents such as aqueous suspension
  • sweetening agents such as sucrose, aspartame or saccharin.
  • Formulations can be adjusted for osmolarity.
  • Oil-based pharmaceuticals are particularly useful for administration hydrophobic active agents used to practice the methods of the invention.
  • Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g., U.S. Patent No. 5,716,928 describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Patent No. 5,858,401).
  • the oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid.
  • an injectable oil vehicle see Minto (1997) J. Pharmacol. Exp. Ther. 281 :93-102.
  • the pharmaceutical formulations of the invention can also be in the form of oil-in-water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these.
  • Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate.
  • the emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent.
  • the pharmaceutical compounds can also be administered by in intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see Rohatagi (1995) J. Clin. Pharmacol. 35: 1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75: 107-11 1).
  • Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug.
  • suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug.
  • Such materials are cocoa butter and polyethylene glycols.
  • the pharmaceutical compounds can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
  • the pharmaceutical compounds can also be delivered as microspheres for slow release in the body.
  • microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.
  • the pharmaceutical compounds can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ.
  • IV intravenous
  • These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier.
  • Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride.
  • sterile fixed oils can be employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter.
  • These formulations may be sterilized by conventional, well known sterilization techniques.
  • the formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs.
  • the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3- butanediol.
  • the administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).
  • the pharmaceutical compounds and formulations used to practice the methods of the invention can be lyophilized.
  • the invention provides a stable lyophilized formulation comprising a composition of the invention, which can be made by lyophilizing a solution comprising a pharmaceutical of the invention and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof.
  • a process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. patent app. no. 20040028670.
  • compositions and formulations used to practice the methods of the invention can be delivered by the use of liposomes (see also discussion, below).
  • liposomes particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Patent Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13 :293-306; Chonn (1995) Curr. Opin.
  • compositions used to practice the methods of the invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a subject already suffering from a condition, infection or disease in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the condition, infection or disease and its complications (a)
  • compositions of the invention are administered in an amount sufficient to treat, prevent and/or ameliorate normal, dysfunction (e.g., abnormally proliferating) cell, e.g., cancer cell, or blood vessel cell, including endothelial and/or capillary cell growth; including neovasculature related to (within, providing a blood supply to) hyperplastic tissue, a granuloma or a tumor.
  • normal, dysfunction e.g., abnormally proliferating
  • cell e.g., cancer cell, or blood vessel cell, including endothelial and/or capillary cell growth
  • neovasculature related to (within, providing a blood supply to) hyperplastic tissue, a granuloma or a tumor.
  • the amount of pharmaceutical composition adequate to accomplish this is defined as a "therapeutically effective dose.”
  • the dosage schedule and amounts effective for this use i.e., the “dosing regimen,” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.
  • the dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:61 1-617; Groning (1996) Pharmazie 51 :337-341 ; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84: 1 144-1 146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24: 103-108; the latest Remington's, supra).
  • pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol.
  • an exemplary pharmaceutical formulation for oral administration of compositions used to practice the methods of the invention can be in a daily amount of between about 0.1 to 0.5 to about 20, 50, 100 or 1000 or more ug per kilogram of body weight per day.
  • dosages are from about 1 mg to about 4 mg per kg of body weight per patient per day are used.
  • Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ.
  • Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation. Actual methods for preparing parenterally or non-parenterally
  • the methods of the invention can further comprise co-administration with other drugs or pharmaceuticals, e.g., compositions for treating cancer, septic shock, infection, fever, pain and related symptoms or conditions.
  • other drugs or pharmaceuticals e.g., compositions for treating cancer, septic shock, infection, fever, pain and related symptoms or conditions.
  • the methods and/or compositions and formulations of the invention can be co-formulated with and/or coadministered with antibiotics (e.g., antibacterial or bacteriostatic peptides or proteins), particularly those effective against gram negative bacteria, fluids, cytokines,
  • immunoregulatory agents such as peptides or proteins comprising collagen-like domains or fibrinogen-like domains (e.g., a ficolin), carbohydrate-binding domains, and the like and combinations thereof.
  • complement activating agents such as peptides or proteins comprising collagen-like domains or fibrinogen-like domains (e.g., a ficolin), carbohydrate-binding domains, and the like and combinations thereof.
  • the invention also provides nanoparticles and liposomal membranes comprising compounds used to practice the methods of the invention.
  • the invention provides nanoparticles and liposomal membranes targeting diseased and/or tumor (cancer) stem cells and dysfunctional stem cells, and angiogenic cells.
  • the invention provides nanoparticles and liposomal membranes comprising (in addition to comprising compounds used to practice the methods of the invention) molecules, e.g., peptides or antibodies, that selectively target abnormally growing, diseased, infected, dysfunctional and/or cancer (tumor) cell receptors.
  • the invention provides nanoparticles and liposomal membranes using IL-11 receptor and/or the GRP78 receptor to targeted receptors on cells, e.g., on tumor cells, e.g., on prostate or ovarian cancer cells. See, e.g., U.S. patent application publication no. 20060239968.
  • compositions used to practice the methods of the invention are specifically targeted for inhibiting, ameliorating and/or preventing endothelial cell migration and for inhibiting angiogenesis, e.g., tumor-associated or disease- or infection- associated neovasculature.
  • angiogenesis e.g., tumor-associated or disease- or infection- associated neovasculature.
  • the invention also provides nanocells to allow the sequential delivery of two different therapeutic agents with different modes of action or different pharmacokinetics, at least one of which comprises a composition used to practice the methods of the invention.
  • a nanocell is formed by encapsulating a nanocore with a first agent inside a lipid vesicle containing a second agent; see, e.g., Sengupta, et al, U.S. Pat. Pub. No. 20050266067.
  • the agent in the outer lipid compartment is released first and may exert its effect before the agent in the nanocore is released.
  • the nanocell delivery system may be formulated in any pharmaceutical composition for delivery to patients suffering from a diseases or condition as described herein, e.g., such as a retinal age-related macular degeneration, a diabetic retinopathy, a cancer or carcinoma, a glioblastoma, a neuroma, a neuroblastoma, a colon carcinoma, a hemangioma, an infection and/or a condition with at least one inflammatory component, and/or any infectious or inflammatory disease, such as a rheumatoid arthritis, a psoriasis, a fibrosis, leprosy, multiple sclerosis, inflammatory bowel disease, or ulcerative colitis or Crohn's disease.
  • a diseases or condition as described herein e.g., such as a retinal age-related macular degeneration, a diabetic retinopathy, a cancer or carcinoma, a glioblastoma, a neuroma, a neuroblasto
  • a traditional antineoplastic agent is contained in the outer lipid vesicle of the nanocell, and an antiangiogenic agent of this invention is loaded into the nanocore. This arrangement allows the antineoplastic agent to be released first and delivered to the tumor before the tumor's blood supply is cut off by the composition of this invention.
  • the invention also provides multilayered liposomes comprising compounds used to practice this invention, e.g., for transdermal absorption, e.g., as described in Park, et al, U.S. Pat. Pub. No. 20070082042.
  • the multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition of this invention.
  • a multilayered liposome used to practice the invention may further include an antiseptic, an antioxidant, a stabilizer, a thickener, and the like to improve stability.
  • Synthetic and natural antiseptics can be used, e.g., in an amount of 0.01% to 20%.
  • Antioxidants can be used, e.g., BHT, erysorbate, tocopherol, astaxanthin, vegetable flavonoid, and derivatives thereof, or a plant-derived antioxidizing substance.
  • a stabilizer can be used to stabilize liposome structure, e.g., polyols and sugars.
  • Exemplary polyols include butylene glycol, polyethylene glycol, propylene glycol, dipropylene glycol and ethyl carbitol; examples of sugars are trehalose, sucrose, mannitol, sorbitol and chitosan, or a monosaccharides or an oligosaccharides, or a high molecular weight starch.
  • a thickener can be used for improving the dispersion stability of constructed liposomes in water, e.g., a natural thickener or an acrylamide, or a synthetic polymeric thickener.
  • exemplary thickeners include natural polymers, such as acacia gum, xanthan gum, gellan gum, locust bean gum and starch, cellulose derivatives, such as hydroxy ethylcellulose, hydroxypropyl cellulose and carboxymethyl cellulose, synthetic polymers, such as polyacrylic acid, poly-acrylamide or polyvinylpyrollidone and polyvinylalcohol, and copolymers thereof or cross-linked materials.
  • Liposomes can be made using any method, e.g., as described in Park, et al, U.S. Pat. Pub. No. 20070042031, including method of producing a liposome by encapsulating a therapeutic product comprising providing an aqueous solution in a first reservoir;
  • an organic lipid solution in a second reservoir wherein one of the aqueous solution and the organic lipid solution includes a therapeutic product; mixing the aqueous solution with said organic lipid solution in a first mixing region to produce a liposome solution, wherein the organic lipid solution mixes with said aqueous solution so as to substantially instantaneously produce a liposome encapsulating the therapeutic product; and immediately thereafter mixing the liposome solution with a buffer solution to produce a diluted liposome solution.
  • the invention also provides nanoparticles comprising compounds used to practice this invention to deliver a composition of the invention as a drug-containing nanoparticles (e.g., a secondary nanoparticle), as described, e.g., in U.S. Pat. Pub. No. 20070077286.
  • the invention provides nanoparticles comprising a fat-soluble drug of this invention or a fat-solubilized water-soluble drug to act with a bivalent or trivalent metal salt.
  • compositions and formulations used to practice the invention can be delivered by the use of liposomes.
  • liposomes particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Patent Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13 :293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46: 1576-1587.
  • compositions and formulations used to practice the invention are delivered by the use of liposomes having rigid lipids having head groups and hydrophobic tails, e.g., as using a polyethyleneglycol-linked lipid having a side chain matching at least a portion the lipid, as described e.g., in US Pat App Pub No. 20080089928.
  • compositions and formulations used to practice the invention are delivered by the use of amphoteric liposomes comprising a mixture of lipids, e.g., a mixture comprising a cationic amphiphile, an anionic amphiphile and/or neutral amphiphiles, as described e.g., in US Pat App Pub No.
  • compositions and formulations used to practice the invention are delivered by the use of liposomes comprising a polyalkylene glycol moiety bonded through a thioether group and an antibody also bonded through a thioether group to the liposome, as described e.g., in US Pat App Pub No. 20080014255.
  • compositions and formulations used to practice the invention are delivered by the use of liposomes comprising glycerides, glycerophospholipides, glycerophosphinolipids, glycerophosphonolipids, sulfolipids, sphingolipids,
  • the invention provides compositions and methods for inhibiting or depleting an integrin ⁇ ⁇ ⁇ 3 (avb3), or inhibiting an integrin ⁇ ⁇ ⁇ 3 (avb3) protein activity, or inhibiting the formation or activity of an integrin avb3/RalB signaling complex, or inhibiting the formation or signaling activity of an integrin ⁇ ⁇ ⁇ 3
  • the invention uses isolated, synthetic or recombinant antibodies that specifically bind to and inhibit an integrin ⁇ ⁇ ⁇ 3 (avb3), or any protein of an integrin ⁇ ⁇ ⁇ 3 (avb3)/RalB/NFkB signaling axis, a RalB protein, a Src or TBK1 protein, or an NFkB protein.
  • the invention provides methods for determining whether an individual or a patient would benefit from or respond to administration of a Growth Factor Inhibitor, or which individuals or patients would benefit from a combinatorial approach comprising administration of a combination of: at least one growth factor inhibitor and at least one compound, composition or formulation used to practice this invention, including for example, an NFkB inhibitor, the method comprising: detecting the levels or amount of integrin ⁇ ⁇ ⁇ 3 (avb3) and/or active RalB complex in or on a cell, a tissue or a tissue sample.
  • avb3 integrin ⁇ ⁇ ⁇ 3
  • the levels or amount of integrin ⁇ ⁇ ⁇ 3 (avb3) and/or active RalB complex in or on a cell, a tissue or a tissue sample is detected or measured using an antibody, e.g., including use of immunofluorescence or FACS analysis.
  • a preferred method for detecting the integrin ⁇ ⁇ ⁇ 3 (avb3) marker is by detecting the heterodimer alpha-v/beta3, and not beta3 alone; the reason for this is that beta3 is also expressed on platelets, so testing for any alpha-v/beta3 heterodimer will exclude platelet expression (if platelets have not already been removed from the sample, or are not excluded from the count).
  • an antibody for practicing the invention can comprise a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. Fundamental Immunology, Third Edition, W.E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97.
  • an antibody for practicing the invention includes antigen-binding portions, i.e., "antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • Single chain antibodies are also included by reference in
  • the invention uses "humanized" antibodies, including forms of non-human (e.g., murine) antibodies that are chimeric antibodies comprising minimal sequence (e.g., the antigen binding fragment) derived from non- human immunoglobulin.
  • humanized antibodies are human immunoglobulins in which residues from a hypervariable region (HVR) of a recipient (e.g., a human antibody sequence) are replaced by residues from a hypervariable region (HVR) of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • HVR hypervariable region
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues to improve antigen binding affinity.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to improve antibody affinity or functional activity.
  • the humanized antibody can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of Ab framework regions are those of a human immunoglobulin sequence.
  • a humanized antibody used to practice this invention can comprise at least a portion of an immunoglobulin constant region (Fc), typically that of or derived from a human immunoglobulin.
  • Fc immunoglobulin constant region
  • completely human antibodies also can be used to practice this invention, including human antibodies comprising amino acid sequence which corresponds to that of an antibody produced by a human.
  • This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues.
  • antibodies used to practice this invention comprise "affinity matured" antibodies, e.g., antibodies comprising with one or more alterations in one or more hypervariable regions which result in an improvement in the affinity of the antibody for antigen; e.g., NFkB, an integrin ⁇ ⁇ ⁇ 3 (avb3), or any protein of an integrin ⁇ ⁇ ⁇ 3 (anb3)/RalB/NFkB signaling axis, a RalB protein, a Src or TBK1 protein, compared to a parent antibody which does not possess those alteration(s).
  • affinity matured antibodies e.g., antibodies comprising with one or more alterations in one or more hypervariable regions which result in an improvement in the affinity of the antibody for antigen; e.g., NFkB, an integrin ⁇ ⁇ ⁇ 3 (avb3), or any protein of an integrin ⁇ ⁇ ⁇ 3 (anb3)/RalB/NFkB signaling axis
  • antibodies used to practice this invention are matured antibodies having nanomolar or even picomolar affinities for the target antigen, e.g., NFkB, an integrin ⁇ ⁇ ⁇ 3 (avb3), or any protein of an integrin ⁇ ⁇ ⁇ 3 (avb3)/RalB/NFkB signaling axis, a RalB protein, a Src or TBK1 protein.
  • Affinity matured antibodies can be produced by procedures known in the art.
  • the invention provides compositions and methods for inhibiting or depleting an integrin ⁇ ⁇ ⁇ 3 (avb3), or inhibiting an integrin ⁇ ⁇ ⁇ 3 (avb3) protein activity, or inhibiting the formation or activity of an integrin anb3/RalB signaling complex, or inhibiting the formation or signaling activity of an integrin ⁇ ⁇ ⁇ 3
  • inhibitory nucleic acids e.g., siRNA, antisense nucleic acids, and/or inhibitory microRNAs.
  • compositions used to practice the invention are formulated with a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions used to practice the invention can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally.
  • the pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's").
  • miRNAs are short (20-24 nt) non-coding RNAs that are involved in post- transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. miRNAs are transcribed by RNA polymerase II as part of capped and polyadenylated primary transcripts (pri-miRNAs) that can be either protein-coding or non-coding.
  • the primary transcript is cleaved by the Drosha ribonuclease III enzyme to produce an approximately 70-nt stem-loop precursor miRNA (pre-miRNA), which is further cleaved by the cytoplasmic Dicer ribonuclease to generate the mature miRNA and antisense miRNA star (miRNA*) products.
  • pre-miRNA stem-loop precursor miRNA
  • miRNA* miRNA and antisense miRNA star
  • the mature miRNA is incorporated into a RNA-induced silencing complex (RISC), which recognizes target mRNAs through imperfect base pairing with the miRNA and most commonly results in translational inhibition or destabilization of the target mRNA.
  • RISC RNA-induced silencing complex
  • compositions used to practice the invention are administered in the form of a dosage unit, e.g., a tablet, capsule, bolus, spray.
  • pharmaceutical compositions comprise a compound, e.g., an antisense nucleic acid, e.g., an siRNA or a microRNA, in a dose: e.g., 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg : 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg , 145 mg , 150 mg , 155 mg , 160 mg , 165 mg , 170 mg , 175 mg , 180 mg , 185 mg , 190 mg , 195 mg ,200 mg ,205 mg ,210 mg ,215 mg ,220 mg ,225 mg ,230 mg ,
  • an siRNA or a microRNA used to practice the invention is administered as a pharmaceutical agent, e.g., a sterile formulation, e.g., a lyophilized siRNA or microRNA that is reconstituted with a suitable diluent, e.g., sterile water for injection or sterile saline for injection.
  • a suitable diluent e.g., sterile water for injection or sterile saline for injection.
  • the reconstituted product is administered as a subcutaneous injection or as an intravenous infusion after dilution into saline.
  • the lyophilized drug product comprises siRNA or microRNA prepared in water for injection, or in saline for injection, adjusted to pH 7.0-9.0 with acid or base during preparation, and then lyophilized.
  • a lyophilized siRNA or microRNA of the invention is between about 25 to 800 or more mg, or about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mg of a siRNA or microRNA of the invention.
  • the lyophilized siRNA or microRNA of the invention can be packaged in a 2 mL Type I, clear glass vial (e.g., ammonium sulfate-treated), e.g., stoppered with a bromobutyl rubber closure and sealed with an aluminum overseal.
  • Type I, clear glass vial e.g., ammonium sulfate-treated
  • stoppered with a bromobutyl rubber closure e.g., stoppered with a bromobutyl rubber closure and sealed with an aluminum overseal.
  • the invention provides compositions and methods comprising in vivo delivery of antisense nucleic acids, e.g., siRNA or microRNAs.
  • the antisense nucleic acids, siRNAs, or microRNAs can be modified, e.g., in alternative embodiments , at least one nucleotide of antisense nucleic acid, e.g., siRNA or microRNA, construct is modified, e.g., to improve its resistance to nucleases, serum stability, target specificity, blood system circulation, tissue distribution, tissue penetration, cellular uptake, potency, and/or cell-permeability of the
  • the antisense nucleic acid, siRNA or microRNA construct is unmodified. In other embodiments, at least one nucleotide in the antisense nucleic acid, siRNA or microRNA construct is modified.
  • guide strand modifications are made to increase nuclease stability, and/or lower interferon induction, without significantly decreasing antisense nucleic acid, siRNA or microRNA activity (or no decrease in antisense nucleic acid, siRNA or microRNA activity at all).
  • the modified antisense nucleic acid, siRNA or microRNA constructs have improved stability in serum and/or cerebral spinal fluid compared to an unmodified structure having the same sequence.
  • a modification includes a 2'-H or 2'-modified ribose sugar at the second nucleotide from the 5'-end of the guide sequence.
  • the guide strand e.g., at least one of the two single-stranded
  • polynucleotides comprises a 2'-0-alkyl or 2'-halo group, such as a 2'-0-methyl modified nucleotide, at the second nucleotide on the 5'-end of the guide strand, or, no other modified nucleotides.
  • polynucleotide constructs having such modification may have enhanced target specificity or reduced off-target silencing compared to a similar construct without the 2'-0-methyl modification at the position.
  • a second nucleotide is a second nucleotide from the
  • a "2'-modified ribose sugar” comprises ribose sugars that do not have a 2'-OH group.
  • a "2'-modified ribose sugar” does not include 2'-deoxyribose (found in unmodified canonical DNA nucleotides), although one or more DNA nucleotides may be included in the subject constructs (e.g., a single deoxyribonucleotide, or more than one deoxyribonucleotide in a stretch or scattered in several parts of the subject constructs).
  • the 2'-modified ribose sugar may be 2'-0-alkyl nucleotides, 2'-deoxy-2'- fluoro nucleotides, 2'-deoxy nucleotides, or combination thereof.
  • an antisense nucleic acid, siRNA or microRNA construct used to practice the invention comprises one or more 5'-end modifications, e.g., as described above, and can exhibit a significantly (e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more) less "off-target" gene silencing when compared to similar constructs without the specified 5 '-end modification, thus greatly improving the overall specificity of the antisense nucleic acid, siRNA or microRNA construct of the invention.
  • an antisense nucleic acid, siRNA or microRNA construct to practice the invention comprises a guide strand modification that further increase stability to nucleases, and/or lowers interferon induction, without significantly decreasing activity (or no decrease in microRNA activity at all).
  • the 5'-stem sequence comprises a 2'-modified ribose sugar, such as 2'-0- methyl modified nucleotide, at the second nucleotide on the 5'-end of the polynucleotide, or, no other modified nucleotides.
  • the hairpin structure having such modification has enhanced target specificity or reduced off-target silencing compared to a similar construct without the 2'-0-methyl modification at same position.
  • the 2'-modified nucleotides are some or all of the pyrimidine nucleotides (e.g., C/U).
  • Examples of 2'-0-alkyl nucleotides include a 2'-0- methyl nucleotide, or a 2'-0-allyl nucleotide.
  • the modification comprises a 2'-0-methyl modification at alternative nucleotides, starting from either the first or the second nucleotide from the 5'-end.
  • the modification comprises a 2'-0-methyl modification of one or more randomly selected pyrimidine nucleotides (C or U).
  • the modification comprises a 2'-0-methyl modification of one or more nucleotides within the loop.
  • the modified nucleotides are modified on the sugar moiety, the base, and/or the phosphodiester linkage.
  • the modification comprise a phosphate analog, or a phosphorothioate linkage; and the phosphorothioate linkage can be limited to one or more nucleotides within the loop, a 5'- overhang, and/or a 3 '-overhang.
  • the phosphorothioate linkage may be limited to one or more nucleotides within the loop, and 1, 2, 3, 4, 5, or 6 more nucleotide(s) of the guide sequence within the double-stranded stem region just 5' to the loop.
  • the total number of nucleotides having the phosphorothioate linkage may be about 12-14.
  • all nucleotides having the phosphorothioate linkage are not contiguous.
  • the modification comprises a 2'- O-methyl modification, or, no more than 4 consecutive nucleotides are modified.
  • all nucleotides in the 3 '-end stem region are modified.
  • all nucleotides 3' to the loop are modified.
  • the 5'- or 3'-stem sequence comprises one or more universal base-pairing nucleotides.
  • universal base-pairing nucleotides include extendable nucleotides that can be incorporated into a polynucleotide strand (either by chemical synthesis or by a polymerase), and pair with more than one pairing type of specific canonical nucleotide.
  • the universal nucleotides pair with any specific nucleotide.
  • the universal nucleotides pair with four pairings types of specific nucleotides or analogs thereof.
  • the universal nucleotides pair with three pairings types of specific nucleotides or analogs thereof.
  • the universal nucleotides pair with two pairings types of specific nucleotides or analogs thereof.
  • an antisense nucleic acid, siRNA or microR A used to practice the invention comprises a modified nucleoside, e.g., a sugar-modified nucleoside.
  • the sugar-modified nucleosides can further comprise a natural or modified heterocyclic base moiety and/or a natural or modified internucleoside linkage; or can comprise modifications independent from the sugar modification.
  • a sugar modified nucleoside is a 2'-modified nucleoside, wherein the sugar ring is modified at the 2' carbon from natural ribose or 2'- deoxy-ribose.
  • a 2'-modified nucleoside has a bicyclic sugar moiety.
  • the bicyclic sugar moiety is a D sugar in the alpha configuration.
  • the bicyclic sugar moiety is a D sugar in the beta configuration.
  • the bicyclic sugar moiety is an L sugar in the alpha configuration.
  • the bicyclic sugar moiety is an L sugar in the beta configuration.
  • the bicyclic sugar moiety comprises a bridge group between the 2' and the 4'-carbon atoms. In alternative embodiments, the bridge group comprises from 1 to 8 linked biradical groups. In alternative embodiments, the bicyclic sugar moiety comprises from 1 to 4 linked biradical groups. In alternative embodiments, the bicyclic sugar moiety comprises 2 or 3 linked biradical groups.
  • the bicyclic sugar moiety comprises 2 linked biradical groups.
  • the bicyclic sugar moiety is bridged between the 2' and 4' carbon atoms with a biradical group selected from— 0 ⁇ (CH 2 )x ⁇ ,— O— CH 2 ⁇ ,— O— CH2CH2-, -O-CH(alkyl)-, -NH-(CH2)P-, -N(alkyl)-(CH 2 )x-, -O-CH(alkyl)-, - (CH(alkyl))-(CH2)x-, -NH-0-(CH2)x-, -N(alkyl)-0-(CH 2 )x-, or -O-N(alkyl)- (CH 2 )x ⁇ , wherein x is 1, 2, 3, 4 or 5 and each alkyl group can be further substituted. In certain embodiments, x is 1, 2 or 3.
  • These 2'- substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (N0 2 ), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
  • a 2'-modified nucleoside comprises a 2'-substituent group selected from F, O-CH 3 , and OCH 2 CH20CH 3 .
  • a sugar-modified nucleoside is a 4'-thio modified nucleoside.
  • a sugar-modified nucleoside is a 4'-thio-2'- modified nucleoside.
  • a 4'-thio modified nucleoside has a .beta.-D-ribonucleoside where the 4'-0 replaced with 4'-S.
  • a 4'-thio-2'-modified nucleoside is a 4'-thio modified nucleoside having the 2'-OH replaced with a 2'- substituent group.
  • 2'-substituent groups include 2'-OCH3, 2'- 0-(CH2) 2 -OCH 3 , and 2'-F.
  • a modified oligonucleotide of the present invention comprises one or more internucleoside modifications.
  • each internucleoside linkage of a modified oligonucleotide is a modified internucleoside linkage.
  • a modified internucleoside linkage comprises a phosphorus atom.
  • a modified antisense nucleic acid, siRNA or microRNA comprises at least one phosphorothioate internucleoside linkage.
  • each internucleoside linkage of a modified oligonucleotide is a phosphorothioate internucleoside linkage.
  • a modified internucleoside linkage does not comprise a phosphorus atom.
  • an internucleoside linkage is formed by a short chain alkyl internucleoside linkage.
  • an internucleoside linkage is formed by a short chain alkyl internucleoside linkage.
  • internucleoside linkage is formed by a cycloalkyl internucleoside linkages. In alternative embodiments, an internucleoside linkage is formed by a mixed heteroatom and alkyl internucleoside linkage. In alternative embodiments, an internucleoside linkage is formed by a mixed heteroatom and cycloalkyl internucleoside linkages. In alternative embodiments, an internucleoside linkage is formed by one or more short chain heteroatomic internucleoside linkages. In alternative embodiments, an internucleoside linkage is formed by one or more heterocyclic internucleoside linkages. In alternative embodiments, an internucleoside linkage has an amide backbone, or an internucleoside linkage has mixed N, O, S and CH2 component parts.
  • a modified oligonucleotide comprises one or more modified nucleobases.
  • a modified oligonucleotide comprises one or more 5-methylcytosines, or each cytosine of a modified oligonucleotide comprises a 5-methylcytosine.
  • a modified nucleobase comprises a 5 -hydroxy methyl cytosine, 7-deazaguanine or 7-deazaadenine, or a modified nucleobase comprises a 7- deaza-adenine, 7-deazaguanosine, 2-aminopyridine or a 2-pyridone, or a modified nucleobase comprises a 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O- 6 substituted purines, or a 2 aminopropyladenine, 5-propynyluracil or a 5- propynylcytosine.
  • a modified nucleobase comprises a polycyclic heterocycle, or a tricyclic heterocycle; or, a modified nucleobase comprises a
  • phenoxazine derivative or a phenoxazine further modified to form a nucleobase or G- clamp.
  • compounds, compositions, pharmaceutical compositions and formulations used to practice the invention can be administered for prophylactic and/or therapeutic treatments; for example, the invention provides compositions and methods for overcoming or diminishing or preventing Growth Factor Inhibitor (GFI) resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor Inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor.
  • GFI Growth Factor Inhibitor
  • the invention provides compositions and methods for treating, preventing or ameliorating: a disease or condition associated with dysfunctional stem cells or cancer stem cells, a retinal age-related macular degeneration, a diabetic retinopathy, a cancer or carcinoma, a glioblastoma, a neuroma, a neuroblastoma, a colon carcinoma, a hemangioma, an infection and/or a condition with at least one inflammatory component, and/or any infectious or inflammatory disease, such as a rheumatoid arthritis, a psoriasis, a fibrosis, leprosy, multiple sclerosis, inflammatory bowel disease, or ulcerative colitis or Crohn's disease.
  • a disease or condition associated with dysfunctional stem cells or cancer stem cells a retinal age-related macular degeneration, a diabetic retinopathy, a cancer or carcinoma, a glioblastoma, a neuroma, a neuroblastoma, a colon carcinoma
  • compositions are administered to a subject already suffering from a condition, infection or disease in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the condition, infection or disease (e.g., disease or condition associated with dysfunctional stem cells or cancer stem cells) and its complications (a "therapeutically effective amount").
  • a pharmaceutical composition is administered in an amount sufficient to treat (e.g., ameliorate) or prevent a disease or condition associated with dysfunctional stem cells or cancer stem cells.
  • the amount of pharmaceutical composition adequate to accomplish this is defined as a "therapeutically effective dose.”
  • the dosage schedule and amounts effective for this use i.e., the “dosing regimen,” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.
  • kits comprising compositions for practicing the methods of the invention, including instructions for use thereof.
  • the invention provides kits, blister packages, lidded blisters or blister cards or packets, clamshells, trays or shrink wraps comprising a combination of compounds.
  • the combination of compounds comprises:
  • At least one compound comprising or consisting of: (i) an inhibitor or depleter of integrin ⁇ ⁇ ⁇ 3 (avb3), or an inhibitor of integrin ⁇ ⁇ ⁇ 3 (avb3) protein activity, or an inhibitor of the formation or activity of an integrin avb3/RalB signaling complex, or an inhibitor of the formation or signaling activity of an integrin ⁇ ⁇ ⁇ 3 (anb3)/RalB/NFkB signaling axis,
  • the inhibitor of RalB protein activity is an allosteric inhibitor of RalB protein activity
  • the inhibitor of Src or TBK1 protein activity is an allosteric inhibitor of Src or TBK1 protein activity
  • the inhibitor of NFKB or IRF3 protein activity is an allosteric inhibitor of NFKB or IRF3 protein activity
  • kits, blister packages, lidded blisters or blister cards or packets, clamshells, trays or shrink wraps further comprise instructions for practicing a method of the invention.
  • EXAMPLE 1 Methods of the invention are effective for sensitizing and re-sensitizing cancer cells to growth factor inhibitors
  • the data presented herein demonstrates the effectiveness of the compositions and methods of the invention in sensitizing and re-sensitizing cancer cells, and cancer stem cells, to growth factor inhibitors, and validates this invention's therapeutic approach to overcome growth factor inhibitor, e.g., EGFR inhibitor, resistance for a wide range of cancers.
  • growth factor inhibitor e.g., EGFR inhibitor
  • the data presented herein demonstrates that genetic and pharmacological inhibition of RalB or NF- ⁇ was able to re-sensitize ⁇ 3 -expressing tumors to EGFR inhibitors.
  • EGFR epidermal growth factor receptor
  • TKIs EGFR Tyrosine Kinase inhibitors
  • a number of resistance mechanisms have been identified, including KRAS and EGFR mutations, resulting in constitutive activation of the ERK pathway 5"7 .
  • KRAS-mediated ERK signaling is associated with resistance to EGFR inhibition, KRAS also induces PI3K and Ral activation leading to tumor cell survival and proliferation 8 ' 9 .
  • pancreatic (FG, Miapaca-2), breast (BT474, SKBR3 and MDAMB468) and colon (SW480) human tumor cell lines to increasing concentrations of erlotinib or lapatinib for three weeks, to select cell subpopulations that were at least 10-fold more resistant to these targeted therapies than their parental counterparts.
  • Parent or resistant cells were then evaluated for a panel of stem/progenitor cell markers previously identified to be upregulated in the most aggressive metastatic tumor cells 11"13 .
  • ⁇ 3 is a marker of the most malignant tumor cells in a wide range of cancers 16 ' 17 .
  • various breast, lung and pancreatic tumor cells were first screened for ⁇ 3 expression and then analyzed for their sensitivity to EGFR inhibitors (Supplementary Table 1).
  • ⁇ 3 expressing tumor cells were intrinsically more resistant to EGFR blockade than 3-negative tumor cell lines (Fig. le).
  • ⁇ 3 was required for resistance to EGFR inhibitors, since knockdown of ⁇ 3 in PANC-1 cells resulted in a 10-fold increase in tumor cell sensitivity to erlotinib (Fig. If).
  • integrin ⁇ 3 was sufficient to induce erlotinib resistance since ectopic expression of ⁇ 3 in FG cells lacking this integrin dramatically increased erlotinib resistance both, in vitro and in orthotopic pancreatic tumors after systemic treatment in vivo (Fig. If and g).
  • Integrin ⁇ 3 not only promotes adhesion-dependent signaling via activation of focal adhesion kinase FAK 16 but it can also activate a FAK-independent signaling cascade in the absence of integrin ligation that is associated with increased survival and tumor metastasis 17 .
  • FG cells transfected with either WT ⁇ 3 or a ligation deficient mutant of the integrin (Dl 19A) 17 were treated with erlotinib.
  • the same degree of erlotinib resistance was observed in cells expressing either the ligation competent or incompetent form of integrin ⁇ 3, see Figure 6a (Supplementary Fig.
  • KRAS promotes multiple effector pathways including those regulated by RAF, phosphatidylinositol-3-OH kinases (PBKs) and RalGEFs leading to a variety of cellular functions 24 .
  • PBKs phosphatidylinositol-3-OH kinases
  • RalGEFs leading to a variety of cellular functions 24 .
  • RalB an effector of RAS has been shown to induce TBKl/NF- ⁇ activation leading to enhanced tumor cell survival 25 ' 26 .
  • NF-KB signaling is essential for KRAS-driven tumor growth and resistance to EGFR blockade 27" 29 . This prompted us to ask whether ⁇ 3 could regulate NF- ⁇ activity through RalB activation and thereby promote tumor cell resistance to EGFR targeted therapy.
  • tumor cells expressing or lacking integrin ⁇ 3 and/or RalB were grown in the presence or absence of erlotinib and lysates of these cells were analyzed for activated downstream effectors of RalB.
  • integrins can promote adhesion dependent cell survival and induce tumor progression 16
  • integrin ⁇ 3 even in the unligated state, can drive tumor cell survival and resistance to EGFR blockade by interaction with KRAS.
  • This action leads to the recruitment and activation of RalB and its downstream signaling effector NF- ⁇ .
  • NF- ⁇ inhibition re-sensitizes ⁇ 3- bearing tumors to EGFR blockade.
  • Integrin ⁇ 3 cooperates with KRAS to promote resistance to EGFR blockade.
  • RalB is a key modulator of integrin ⁇ 3 -mediated EGFR TKI resistance.
  • pTBKl refers to phospho-S172 TBK1
  • p-p65 NF- ⁇ refers to phospho-p65 NF- ⁇ S276
  • pFAK refers to phospho-FAK Tyr 861.
  • Data are representative of three independent experiments, (b) Tumor spheres formation assay of FG cells ectopically expressing vector control, WT NF- ⁇ FLAG tagged or constitutively active S276D NF- ⁇ FLAG tagged constructs treated with erlotinib (0.5 ⁇ ).
  • FG Human pancreatic (FG, PANC-1, Miapaca-2 (MP2), CFPAC-1, XPA-1, CAPAN- 1 , BxPc3 ), breast (MDAMB231 , MDAMB468 (MDA468), BT20, SKBR3 , BT474), colon (SW480) and lung (A549, H441) cancer cell lines were grown in ATCC recommended media supplemented with 10% fetal bovine serum, glutamine and nonessential amino acids.
  • FG- 3, FG-D119A mutant and PANC-sh 3 cells as previously described 17 .
  • Erlotinib, OSI-906, Gemcitabine and Lapatinib were purchased from Chemietek.
  • Cisplatin was generated from Sigma-Aldrich. Lenalidomide was purchased from LC Laboratories. We established acquired EGFR TKI resistant cells by adding an increasing concentration of erlotinib (50 nM to 15 ⁇ ) or lapatinib (10 nM to 15 ⁇ ), daily in 3D culture in 0.8% methylcellulose.
  • Tumor spheres formation assays were performed essentially as described previously 11 . Briefly, cells were seeded at 1000 to 2000 cells per well and grown for 12 days to 3 weeks. Cells were treated with vehicle (DMSO), erlotinib (10 nM to 5 ⁇ ), lapatinib (10 nM to 5 ⁇ ), gemcitabine (0.001 nM to 5 ⁇ ), OSI-906 (10 nM to 5 ⁇ ), lenalidomide (10 nM to 5 ⁇ ), or cisplatin (10 nM to 5 ⁇ ), diluted in DMSO. The media was replaced with fresh inhibitor every day for erlotinib, lapatinib, lenalidomide and 3 times a week for cisplatin and gemcitabine. Colonies were stained with crystal violet and scored with an Olympus SZH10 microscope. Survival curves were generated at least with five concentration points.
  • DMSO vehicle
  • erlotinib 10 nM to 5 ⁇
  • lapatinib 10
  • Tissue sections were washed and then incubated with biotinylated secondary antibody (1 :500, Jackson ImmunoResearch) in blocking solution for lh. Sections were washed and incubated with Vectastain ABC (Vector Labs) for 30 min. Staining was developed using a Nickel- enhanced diamino-benzidine reaction (Vector Labs) and sections were counter-stained with hematoxylin. Sections stained with integrin ⁇ 3 and active Ral were scored by a H- score according to the staining intensity (SI) on a scale 0 to 3 within the whole tissue section.
  • SI staining intensity
  • Cells were lysed in either RIPA lysis buffer (50 mM Tris pH 7.4, 100 mM NaCL, 2 mM EDTA, 10% DOC, 10% Triton, 0.1% SDS) or Triton lysis buffer (50 mM Tris pH 7.5, 150 mN NaCl, 1 mM EDTA, 5 mM MgC12, 10% Glycerol, 1% Triton) supplemented with complete protease and phosphatase inhibitor mixtures (Roche) and centrifuged at 13,000 g for 10 min at 4°C. Protein concentration was determined by BCA assay.
  • RIPA lysis buffer 50 mM Tris pH 7.4, 100 mM NaCL, 2 mM EDTA, 10% DOC, 10% Triton, 0.1% SDS
  • Triton lysis buffer 50 mM Tris pH 7.5, 150 mN NaCl, 1 mM EDTA, 5 mM MgC12, 10% Glycerol, 1% Trit
  • RAS and Ral activation assays were performed in accordance with the manufacturer's (Upstate) instruction. Briefly, cells were cultured in suspension for 3h, lysed and protein concentration was determined. 10 ⁇ g of Ral Assay Reagent (Ral BP 1, agarose) or RAS assay reagent (Raf-1 RBD, agarose) was added to 500 mg to 1 mg of total cell protein in MLB buffer (Millipore). After 30 min of rocking at 4°C, the activated (GTP) forms of RAS/Ral bound to the agarose beads were collected by centrifugation, washed, boiled in Laemmli buffer, and loaded on a 15% SDS-PAGE gel.
  • Ral Assay Reagent Ral BP 1, agarose
  • RAS assay reagent Raf-1 RBD, agarose
  • Frozen sections from tumors from the orthotopic xenograft pancreas cancer mouse model or from patients diagnosed with pancreas or breast cancers (as approved by the institutional Review Board at University of California, San Diego) or tumor cell lines were fixed in cold acetone or 4% paraformaldehyde for 15 min, permeabilized in PBS containing 0.1% Triton for 2 min and blocked for lh at room temperature with 2% BSA in PBS.
  • Orthotopic pancreas cancer xenograft model Orthotopic pancreas cancer xenograft model.
  • Tumors were generated by injection of FG human pancreatic carcinoma cells (10 6 tumor cells in 30 ⁇ ⁇ of sterile PBS) into the tail of the pancreas of 6-8 week old male immune compromised nu/nu mice. Tumors were established for 2-3 weeks (tumor sizes were monitored by ultrasound) before beginning dosing. Mice were dosed by oral gavage with vehicle (6% Captisol) or 100 mg/kg/day erlotinib for 10 to 30 days prior to harvest.
  • vehicle 6% Captisol
  • Orthotopic lung cancer xenograft model Orthotopic lung cancer xenograft model.
  • Tumors were generated by injection of H441 human lung adenocarcinoma cells (10 6 tumor cells per mouse in 50 ⁇ ⁇ of HBSS containing 50 mg growth factor-reduced Matrigel (BD Bioscience) into the left thorax at the lateral dorsal axillary line and into the left lung, as previously described 14 of 8 week old male immune-compromised nu/nu mice. 3 weeks after tumor cell injection, the mice were treated with vehicle or erlotinib (100 mg/kg/day) by oral gavage until moribund (approximately 50 and 58 days, respectively).
  • vehicle or erlotinib 100 mg/kg/day
  • Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation. Nature Cell Biology 9, 201-209 (2007).
  • Schmitt, C.A., et al. A senescence program controlled by p53 and pl6INK4a contributes to the outcome of cancer therapy. Cell 109, 335-346 (2002).
  • Galectin-3 is an important mediator of VEGF- and bFGF-mediated angiogenic response. The Journal of Experimental Medicine 207, 1981-1993 (2010).
  • EXAMPLE 2 Methods of the invention are effective for sensitizing and re-sensitizing cancer cells to growth factor inhibitors
  • compositions and 40 methods of the invention demonstrate the effectiveness of the compositions and 40 methods of the invention in sensitizing and re-sensitizing cancer cells, and cancer stem cells, to growth factor inhibitors, and validates this invention's therapeutic approach to overcome growth factor inhibitor resistance for a wide range of cancers.
  • the data presented in this Example demonstrates that ⁇ 3 integrin induces erlotinib resistance in cancer cells by switching tumor dependency from EGFR to KRAS.
  • the compositions and methods of the invention overcome tumor drug resistance that limits the long-term success of therapies targeting EGFR.
  • integrin ⁇ 3 as a biomarker of intrinsic and acquired resistance to erlotinib in human pancreatic and lung carcinomas irrespective of their KRAS mutational status.
  • ⁇ 3 is necessary and sufficient for this resistance where it acts in the unligated state as a scaffold to recruit active KRAS into membrane clusters switching tumor dependency from EGFR to KRAS.
  • the KRAS effector RalB is recruited to this complex, where it mediates erlotinib resistance via a TBK-1/NF-KB pathway. Disrupting assembly of this complex or inhibition of its downstream effectors fully restores tumor sensitivity to EGFR blockade.
  • Our findings uncouple KRAS mutations from erlotinib resistance, revealing an unexpected requirement for integrin ⁇ 3 in this process.
  • integrin ⁇ 3 was both necessary and sufficient to account for erlotinib resistance in vitro and during systemic treatment of lung and orthotopic pancreatic tumors in vivo (Fig. 12F, G and Fig. 18A-C).
  • integrin ⁇ 3 expression did not impact resistance to chemotherapeutic agents such as gemcitabine and cisplatin while conferring resistance to inhibitors targeting EGFR1/EGFR2 or IGFR (Fig. 18C-E), suggesting this integrin plays a specific role in tumor cell resistance to RTK inhibitors.
  • integrin ⁇ 3 is functions as an adhesion receptor
  • ligand binding inhibitors could represent a therapeutic strategy to sensitize tumors to EGFR inhibitors.
  • ⁇ 3 expression induced drug resistance in cells growing in suspension.
  • neither function blocking antibodies nor cyclic peptide inhibitors sensitized integrin ⁇ 3- expressing tumors to EGFR inhibitors (not shown), and tumor cells expressing wild-type integrin ⁇ 3 or the ligati on-deficient mutant ⁇ 3 D 119A ( /_/) showed equivalent drug resistance (Fig. 19). Since the contribution of integrin ⁇ 3 to erlotinib resistance appears to involve a non-canonical, ligation-independent mechanism that is not sensitive to traditional integrin antagonists, understanding the molecular mechanisms driving this pathway could provide therapeutic opportunities.
  • Integrins function in the context of RAS family members. Interestingly, we found that ⁇ 3 associated with KRAS but not N- , H- or R-RAS (Fig. 13 A). While oncogenic KRAS has been linked to erlotinib resistance, there are many notable exceptions (6 ⁇ 9). In fact, we observed a number of tumor cell lines with oncogenic KRAS to be sensitive to erlotinib (FG, H441, and CAPAN1), whereas H1650 cells were erlotinib resistant despite their expression of wildtype KRAS and mutant EGFR (Fig. 17). In fact, ⁇ 3 expression consistently correlated with erlotinib resistance for all cell lines tested (Pearson's correlation coefficient R 2 0.87) making a better predictor of erlotinib resistance.
  • ⁇ 3 integrin expression switches tumor cell dependency from EGFR to KRAS, and that the localization of ⁇ 3 with KRAS at the plasma membrane appears to be a critical determinant of tumor cell resistance to erlotinib. Also, our results reveal that tumors expressing oncogenic KRAS without ⁇ 3 remain sensitive to EGFR blockade.
  • galectin-3 can interact with either KRAS (12) or ⁇ 3 (73) so we asked whether this protein might serve as an adaptor to promote KRAS ⁇ 3 complex formation.
  • integrin ⁇ 3, KRAS, and Galectin-3 were co-localized in membrane clusters (Fig. 13G and Fig. 22), and knockdown of either integrin ⁇ 3 or Galectin-3 prevented complex formation, KRAS membrane localization, and importantly sensitized ⁇ 3 expressing tumors to erlotinib (Fig. 13G-I).
  • Gemcitabine Bortezomib and Lapatinib were purchased from Chemietek. Cisplatin was generated from Sigma-Aldrich. Lenalidomide was purchased from LC Laboratories. Gene expression analysis.
  • the Tumor Metastasis PCR Array (Applied Biosystem), consisting of 92 genes known to be involved in tumor progression and metastasis, was used to profile the common genes upregulated in erlotinib-resistant cells compared to erlotinib-sensitive cells according to the manufacturer's instructions. Briefly, total RNA was extracted and reverse transcribed into cDNA using the RNeasy kit (Qiagen).
  • the cDNA was combined with a SYBR Green qPCR Master Mix (Qiagen), and then added to each well of the same PCR Array plate that contained the predispensed gene-specific primer sets.
  • Tumor digestion and Flow Cytometry Fresh tumor tissue from lung cancer cell lines was mechanically dissociated and then enzymatically digested in trypsin. The tissue was further filtered through a cell strainer to obtain a suspension of single tumor cells. Then, cells were washed were washed with PBS and incubated for 20 minutes with the Live/Dead reagent (Invitrogen) according to the manufacturer's instruction, then, cells were fixed with 4% paraformaldehyde for 15 min and blocked for 30 min with 2% BSA in PBS. Cells were stained with fluorescent-conjugated antibodies to integrin ⁇ 3 (LM609, Cheresh Lab), After washing several times with PBS, cells were analyzed by FACS.
  • LM609 fluorescent-conjugated antibodies to integr
  • Tumorsphere assay was performed as previously described (10).
  • DMSO vehicle
  • erlotinib 10 nM to 5 ⁇
  • lapatinib 10 nM to 5 ⁇
  • gemcitabine 0.001 nM to 5 ⁇
  • OSI-906 10 nM to 5 ⁇
  • lenalidomide ⁇
  • cisplatin 10 nM to 5 ⁇
  • bortezomib 4 nM
  • FG pancreatic carcinoma cells (1 x 106 tumor cells in 30 ⁇ of PBS) were injected into the pancreas of 6-to 8-week-old male nude mice as previously described (10). Tumors were established for 2-3 weeks (tumor sizes were monitored by ultrasound) before beginning dosing. Mice were dosed by oral gavage with vehicle (6% Captisol) or 10, 25 and 50 mg/kg/day erlotinib for 10 to 30 days prior to harvest. H441 lung adenocarcinoma cells were generated as previously described (21).
  • mice were treated with vehicle or erlotinib (100 mg/kg/day) by oral Mouse cancer models. All research was conducted under protocol S05018 and approved by the University of California-San Diego Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • FG pancreatic carcinoma cells (1 x 106 tumor cells in 30 ⁇ of PBS) were injected into the pancreas of 6-to 8-week-old male nude mice as previously described (10). Tumors were established for 2-3 weeks (tumor sizes were monitored by ultrasound) before beginning dosing. Mice were dosed by oral gavage with vehicle (6% Captisol) or 10, 25 and 50 mg/kg/day erlotinib for 10 to 30 days prior to harvest.
  • H441 lung adenocarcinoma cells were generated as previously described (21). 3 weeks after tumor cell injection, the mice were treated with vehicle or erlotinib (100 mg/kg/day) by oral gavage until moribund (approximately 50 and 58 days, respectively).
  • FG- 3, FG-R (after erlotinib resistance) and HCC-827 human carcinoma cells (5 x 106 tumor cells in 200 ⁇ of PBS) were injected subcutaneous ly to the left or right flank of 6-8-week-old female nude mice. Tumors were measured every 2-3 days with calipers until they were harvested at day 10, 16 or after acquired resistance.
  • NSCLC specimens from the BATTLE trial were randomized phase II, single-center, open-label study in patients with advanced NSCLC refractory to prior chemotherapy and included patients with and without prior EGFR inhibitor treatment (12). Patients underwent a tumor new biopsy prior to initiating study treatment.
  • the microarray analysis of mRNA expression on frozen tumor core biopsies was conducted using the Affymetrix Human Gene l .STTM platform as previously described (22).
  • Cells were transfected with vector control, WT, G23V RalB-FLAG, WT and S276D NF-KB-FLAG using a lentiviral system.
  • cells were transfected with a pool of RalA, RalB, AKT1, ERK1/2 siRNA (Qiagen) using the lipofectamine reagent (Invitrogen) following manufacturer's protocol or transfected with shRNA (integrin ⁇ 3, KRAS, Galectin-3, RalB, TBK1 and p65NF-kB) (Open Biosystems) using a lentiviral system.
  • shRNA integrated ⁇ 3, KRAS, Galectin-3, RalB, TBK1 and p65NF-kB
  • Immunohistochemical analysis was performed according to the manufacturer's recommendations (Vector Labs) on 5 ⁇ sections of paraffin-embedded tumors from tumor biopsies from lung cancer patients. Tumor sections were processed as previously described (23) using integrin ⁇ 3 (Abeam clone EP2417Y). Sections stained with integrin ⁇ 3 were scored by a H-score according to the staining intensity (SI) on a scale 0 to 3 within the whole tissue section.
  • SI staining intensity
  • Lysates from cell lines and xenograft tumors were generated using standard methods and RIPA or Triton buffers.
  • Immunoprecipitation experiments were performed as previously described (23) with anti-integrin ⁇ 3 (LM609) or Galectin-3.
  • 25 ⁇ g of protein was boiled in Laemmli buffer and resolved on 8% to 15% gel.
  • the following antibodies were used: anti-integrin ⁇ 3, KRAS, NRAS, RRAS, HRAS, Hsp60 and Hsp90 from Santa Cruz, phospho-S 172 NAK/TBK1 from Epitomics, TBK1, phospho-p65NF-KB S276, p65NF-KB, RalB, phospho-EGFR, EGFR, from Cell Signaling Technology, and Galectin 3 from BioLegend.
  • Membrane extract Membrane fraction from FG and FG ⁇ 3 grown in suspension in media complemented with 0.1% BSA were isolated using the MEM-PER membrane extraction kit (Fisher) according to the manufacturer's instructions. Affinity pull-down assays for Ras and Ral. RAS and Ral activation assays were performed in accordance with the manufacturer's (Upstate) instruction. Briefly, cells were cultured in suspension for 3h. 10 ⁇ g of Ral Assay Reagent (Ral BP1, agarose) or RAS assay reagent (Raf-1 RBD, agarose) was added to 500 mg to 1 mg of total cell protein in MLB buffer
  • Figure 1 illustrates data showing that integrin ⁇ 3 is expressed in EGFR inhibitor resistant tumors and is necessary and sufficient to drive EGFR inhibitor resistance.
  • A Identification of the most upregulated tumor progression genes common to erlotinib resistant carcinomas.
  • C Percentage of integrin ⁇ 3 positive cells in parental lines vs. after 3 or 8 weeks treatment with erlotinib.
  • *P 0.04 using a Student's / test).
  • Figure 2 (Fig. 13/31) illustrates data showing that integrin ⁇ 3 is required to promote KRAS dependency and KRAS-mediated EGFR inhibitor resistance.
  • n 3 mean ⁇ SEM. *P ⁇ 0.05, **P ⁇ 0.01.
  • E Effect of KRAS knockdown on tumorsphere formation in PANC-1 (KRAS mutant) stably expressing non-target shRNA control ( ⁇ 3 -positive) or specific-integrin ⁇ 3 shRNA ( ⁇ 3 negative) in FG (KRAS mutant) and BxPc3 (KRAS wild-type) stably expressing vector control or integrin ⁇ 3.
  • *n 3; mean + SEM. *P ⁇ 0.05. **P ⁇ 0.01.
  • F Effect of KRAS knockdown on erlotinib resistance of ⁇ 3 -negative and ⁇ 3 -positive epithelial cancer cell lines.
  • Figure 3 illustrates data showing that RalB is a central player of integrin ⁇ 3 -mediated EGFR inhibitor resistance.
  • (A) Effect of RalB knockdown on erlotinib resistance of ⁇ 3- ⁇ 8 ⁇ epithelial cancer cell lines. Cells were treated with 0.5 ⁇ of erlotinib. n 3; mean ⁇ SEM, *P ⁇ 0.05, **P ⁇ 0.01.
  • (B) Effect of RalB knockdown on erlotinib resistance of 3-positive human pancreatic (FG- 3) orthotopic tumor xenografts. Established tumors expressing non-target shRNA, (shCTRL) or a shRNA targeting RalB (sh RalB) (>1000 mm 3 ; n 13 per treatment group) were randomized and treated for 10 days with vehicle or erlotinib.
  • shCTRL non-target shRNA
  • sh RalB shRNA targeting RalB
  • Results are expressed as % of tumor weight changes after erlotinib treatment compared to vehicle. **P ⁇ 0.01.
  • D Effect of expression of integrin ⁇ 3 on KRAS and RalB membrane localization. Data are representative of two independent experiments.
  • E Ral activity was determined in PANC-1 cells grown in suspension by using a GST-RalBP l- RBD immunoprecipitation assay.
  • Immunoblots indicate RalB activity and association of active RalB with integrin ⁇ 3. Data are representative of three independent experiments.
  • F Confocal microscopy images of integrin ⁇ 3 (green), RalB (red) and DNA (TOPRO- 3, blue) in tumor biopsies from pancreatic cancer patients. Scale bar, 20 ⁇ .
  • G Effect of ⁇ 3 expression and KRAS expression on RalB activity, measured using a GST-RalBPl- RBD immunoprecipitation assay. Data are representative of three independent experiments.
  • H Immunoblot analysis of FG and FG ⁇ 3 stably expressing non-target shRNA control or RalB-specific shRNA, grown in suspension and treated with erlotinib (0.5 ⁇ ).
  • Figure 4 (Fig. 15/31) illustrates data showing that reversal of ⁇ 3 -mediated EGFR inhibitor resistance in oncogenic KRAS model by pharmacological inhibition.
  • mice bearing subcutaneous ⁇ 3- ⁇ 8 ⁇ tumors were treated with vehicle, erlotinib (25 mg/kg/day), lenalidomide (25 mg/kg/day) or the combination of erlotinib and lenalidomide.
  • FIG. 16/31) illustrates resistance to EGFR inhibitor is associated with integrin ⁇ 3 expression in pancreatic and lung human carcinoma cell lines.
  • A Immunoblots showing integrin ⁇ 3 expression in human cell lines used in Figure 1A and Figure IB.
  • C Integrin ⁇ 3 quantification in orthotopic lung and pancreas tumors treated with vehicle or erlotinib until resistance.
  • integrin ⁇ 3 expression was scored (scale 0 to 3) and representative images are shown.
  • FIG. 1 A Immunoblots showing integrin ⁇ 3 knockdown efficiency in cells used in Figure 1.
  • C Immunoblots showing expression of indicated proteins of representative tumors.
  • D Representative photographs of crystal violet-stained tumorspheres of ⁇ 3 -negative and ⁇ 3 -positive cells after erlotinib, OSI-906, gemcitabine and cisplatin treatment.
  • Fig. S4 (Fig. 19/31) illustrates Integrin 3-mediated EGFR inhibitor resistance is independent of its ligand binding.
  • Fig. S5 (Fig. 20/31) illustrates Integrin ⁇ 3 colocalizes and interacts with oncogenic and active wild-type KRAS.
  • Fig. S6 (Fig. 21/31) illustrates Integrin ⁇ 3 expression promotes KRAS dependency.
  • FIG. 1 Immunoblots showing KRAS knockdown efficiency in cells used in Figure 2.
  • FIG. 2 Representative photographs of crystal violet-stained tumorspheres of FG and A549 cells expressing non-target shRNA control or specific -KRAS shRNA.
  • Fig. 22/31) illustrates KRAS and Galectin-3 colocalize in integrin ⁇ 3 -positive cells.
  • Fig. S8 (Fig. 23/31) illustrates Integrin 3-mediated KRAS dependency and erlotinib resistance is independent of ERK, AKT and RalA.
  • (A) Effect of ERK, AKT, RalA and RalB knockdown on erlotinib response (erlotinib 0.5 ⁇ ) of 3-negative FG and 3-positive FG- 3 cells. n triplicate.
  • Fig. 24/31) illustrates Constitutive active NFkB is sufficient to promote erlotinib resistance.
  • Fig. S10 (Fig. 25/31) illustrates NFkB inhibitors in combination with erlotinib increase cell death in vivo.
  • FIG. 4B Immunoblots showing expression of indicated proteins of representative tumors from shown in Figure 4B
  • C Confocal microscopy images of cleaved caspase 3 (red) and DNA (TOPRO-3, blue) in tumor biopsies from xenografts tumors used in Fig. 4B treated with vehicle, erlotinib, lenalidomide or lenalidomide and erlotinib in combo. Scale bar, 20 ⁇ .
  • Fig 26/31 shows differentially expressed genes in cells resistant to erlotinib (PANC-1, H1650, A459) compared with the average of two sensitive cells (FG, H441) and in HCC827 after acquired resistance in vivo (HCC827R) vs. the
  • HCC827 vehicle-treated control.
  • the genes upregulated more than 2.5 fold are in red.
  • TBK1 couples innate immune signaling to tumor cell survival.
  • Cell 127, 157 (Oct)

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Abstract

In alternative embodiments, the invention provides compositions and methods for overcoming or diminishing or preventing Growth Factor Inhibitor resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor. In alternative embodiments, the cell is a tumor cell, a cancer cell or a dysfunctional cell. In alternative embodiments, the invention provides compositions and methods for determining: whether an individual or a patient would benefit from or respond to administration of a Growth Factor Inhibitor, or, which individuals or patients would benefit from a combinatorial approach comprising administration of a combination of: at least one growth factor inhibitor and at least one compound, composition or formulation used to practice a method of the invention, such as an NFKB inhibitor, such as a lenalidomide or a REVLIMIDTM, or a bortezomib or a VELCADE®, or IKK inhibitor.

Description

COMPOSITIONS AND METHODS FOR TREATING CANCER AND DISEASES AND CONDITIONS RESPONSIVE TO GROWTH FACTOR INHIBITION GOVERNMENT RIGHTS
This invention was made with government support under grant numbers
CA045726, CA050286, CA095262, HL057900, and HL103956, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.
TECHNICAL FIELD
This invention generally relates to cell and molecular biology, diagnostics and oncology. In alternative embodiments, the invention provides compositions and methods for overcoming or diminishing or preventing Growth Factor Inhibitor resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor Inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor, or, sensitizing a tumor to a drug, wherein optionally the drug is an erlotinib or a lapatinib, or, sensitizing a tumor that is resistant to a cancer drug. In alternative embodiments, the cell is a tumor cell, a cancer cell, a cancer stem cell or a dysfunctional cell. In alternative embodiments, the invention provides compositions and methods for determining: whether an individual or a patient would benefit from or respond to administration of a Growth Factor Inhibitor, or, whether the individuals or patients would benefit from a
combinatorial approach comprising administration of a combination of: at least one growth factor inhibitor and at least one compound, composition or formulation used to practice a method of the invention, such as an NFKB (nuclear factor kappa-light-chain- enhancer of activated B cells, or NF-κΒ) inhibitor, such as a bortezomib or a
VELCADE®, or a lenalidomide or a REVLIMID™ , or an ΙκΒ kinase (IKK) inhibitor.
BACKGROUND
Growth factor inhibitors have been used to treat many cancers including pancreatic, breast, lung and colorectal cancers. However, resistance to growth factor inhibitors has emerged as a significant clinical problem.
Tumor resistance to targeted therapies occurs due to a combination of stochastic and instructional mechanisms. Mutation/amplification in tyrosine kinase receptors or their downstream effectors account for the resistance of a broad range of tumors. In particular, oncogenic KRAS, the most commonly mutated oncogene in human cancer, has been linked to EGFR inhibitor resistance. However, in lung and pancreatic carcinomas, recent studies suggest that oncogenic KRAS is not sufficient to account for EGFR inhibitor resistance indicating that other factor(s) might control this process. SUMMARY
In alternative embodiments, the invention provides methods for overcoming or diminishing or preventing Growth Factor Inhibitor (GFI) resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor Inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor (GFI), or, sensitizing or re- sensitizing a dysfunctional cell, a tumor or cancer to a drug, wherein optionally the drug is a Growth Factor Inhibitor drug to which the cell is being sensitized or re- sensitized, e.g., an erlotinib, a lapatinib or a lenalidomide, or, sensitizing a tumor that is resistant to a cancer drug (e.g., a Growth Factor Inhibitor),
wherein optionally the cell is a tumor cell, a cancer cell, a cancer stem cell, or a dysfunctional cell,
the method comprising:
(a) (1) providing at least one compound, composition or formulation comprising or consisting of:
(i) an inhibitor or depleter of integrin ανβ3 (avb3), or an inhibitor of integrin ανβ3 (avb3) protein activity, or an inhibitor of the formation or activity of an integrin avb3/RalB signaling complex, or an inhibitor of the formation or signaling activity of an integrin ανβ3 (avb3)/RalB/NFkB signaling axis,
wherein optionally the inhibitor of integrin ανβ3 protein activity is an allosteric inhibitor of integrin ανβ3 protein activity;
(ii) an inhibitor or depleter of RalB protein or an inhibitor of RalB protein activation,
wherein optionally the inhibitor of RalB protein activity is an allosteric inhibitor of RalB protein activity;
(iii) an inhibitor or depleter of a Src kinase or TANK-binding kinase 1 (TBK1) protein or an inhibitor of a Src or a TBK1 protein activation, wherein optionally the inhibitor of the Src or the TBK1 protein activity is an allosteric inhibitor of Src or TAN -binding kinase 1 (TBK 1 ) protein activity;
(iv) an inhibitor or depleter of a NFKB or an Interferon regulatory factor 3 (IRF3) protein or an inhibitor of RalB protein activation,
wherein optionally the inhibitor of NFKB or IRF3 protein activity is an allosteric inhibitor of NFKB or IRF3 protein activity;
(v) an inhibitor or depleter of NFKB or IKK, or an inhibitor of NFKB or IKK protein activation,
wherein optionally the NFKB inhibitor comprises a lenalidomide or a REVLIMID™ (Celgene Corp., Summit, NJ), or a bortezomib or a
VELCADE®, and optionally the IKK inhibitor comprises a PS 1145
(Millennium Pharmaceuticals, Cambridge, MA);
(vi) a lenalidomide or a REVLIMID™ and PS 1145;
(vii) an erlotinib or a TARCEVA® and a bortezomib or a VELCADE®;
(viii) a lenalidomide or a REVLIMID™ ; a PS 1145; and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTK inhibitor comprises SU14813 (Pfizer, San Diego, CA); or
(ix) any combination of (i) to (viii); or
(2) one or any combination of the compound, composition or formulation, or compounds, compositions or formulations, of (1), and at least one growth factor inhibitor,
wherein optionally the at least one growth factor inhibitor comprises a Receptor Tyrosine Kinase (RTK) inhibitor, a Src inhibitor, an anti-metabolite inhibitor, a gemcitabine, GEMZAR™, a mitotic poison, a paclitaxel, a taxol, ABRAXANE™, an erlotinib, TARCEVA™, a lapatinib, TYKERB™, or an insulin growth factor inhibitor,
wherein optionally the combination or the therapeutic combination comprises an erlotinib with either a Lenalidomide or a PS- 1145, or both a Lenalidomide and a PS- 1145; and (b) administering a sufficient amount of the at least one compound, composition or formulation to the cell, or the combination of compounds, to: overcome, diminish or prevent the Growth Factor Inhibitor (GFI) resistance in a cell; increase the growth- inhibiting effectiveness of the Growth Factor inhibitor on the cell; sensitize or re-sensitize the cell to the Growth Factor Inhibitor (GFI); sensitize or re-sensitize the dysfunctional cell, tumor cell or cancer to the drug, or sensitize or re-sensitize the tumor that is resistant to the cancer or anti-tumor drug,
wherein optionally the cell is a tumor cell, a cancer cell, a cancer stem cell, or a dysfunctional cell.
In alternative embodiments of the methods:
(a) the at least one compound, composition or formulation, or combination of compounds, is formulated as a pharmaceutical composition;
(b) the method of (a), wherein the compound, composition or formulation or pharmaceutical composition is administered in vitro, ex vivo or in vivo, or is administered to an individual in need thereof;
(c) the method of (a) or (b), wherein the at least one compound, composition or formulation is a pharmaceutical composition is formulated for administration
intravenously (IV), parenterally, nasally, topically, orally, or by liposome or targeted or vessel-targeted nanoparticle delivery;
(d) the method of any of (a) to (c), wherein the compound or composition comprises or is an inhibitor of transcription, translation or protein expression;
(e) the method of any of (a) to (d), wherein the compound or composition is a small molecule, a protein, an antibody, a monoclonal antibody, a nucleic acid, a lipid or a fat, a polysaccharide, an RNA or a DNA;
(f) the method of any of (a) to (e), wherein the compound or composition comprises or is: a VITAXIN™ (Applied Molecular Evolution, San Diego, CA) antibody, a humanized version of an LM609 monoclonal antibody, an LM609 monoclonal antibody, or any antibody that functionally blocks an ανβ3 integrin or any member of an ανβ3 integrin-comprising complex or an integrin ανβ3 (avb3)/RalB/NFkB signaling axis;
(g) the method of any of (a) to (e), wherein the compound or composition comprises or is a Src inhibitor, a dasatinib, a saracatinib; a bosutinib; a VP-BHG712, or any combination thereof; (h) the method of any of (a) to (g), wherein Growth Factor Inhibitor is or comprises an anti-metabolite inhibitor, a gemcitabine, GEMZAR™, a mitotic poison, a paclitaxel, a taxol, ABRAXANE™, an erlotinib, TARCEVA™, a lapatinib, TYKERB™, or an insulin growth factor inhibitor, or any combination thereof;
(i) the method of any of (a) to (h), wherein the Growth Factor Inhibitor decreases, slows or blocks new blood vessel growth, neovascularization or angiogenesis; or, wherein administering the Growth Factor Inhibitor treats or ameliorates conditions that are responsive to blocking or slowing cell growth, and/or the development of
neovascularization or new blood vessels;
(j) the method of any of (a) to (h), wherein the NF-kB inhibitor comprises or consists of one or more of: an antioxidant; an a-lipoic acid; an a-tocopherol; a 2-amino-l- methyl-6-phenylimidazo[4,5- ]pyridine; an allopurinol; an anetholdithiolthione; a cepharanthine; a beta-carotene; a dehydroepiandrosterone (DHEA) or a DHEA-sulfate (DHEAS); a dimethyldithiocarbamates (DMDTC); a dimethylsulfoxide (DMSO); a flavone, a Glutathione; Vitamin C or Vitamin B6, or one or more compositions listed in Table 1 or Table 2, or any combination thereof;
(k) the method of any of (a) to (j), wherein the at least one compound, composition or formulation, or combination of compounds, comprises a proteasome inhibitor or a protease inhibitor that can inhibit an Rei and/or an NFkB, or one or more compositions listed in Table 2, or any combination thereof;
(1) the method of any of (a) to (j), wherein the at least one compound, composition or formulation, or combination of compounds, comprises an ΙκΒα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) phosphorylation and/or degradation inhibitor, or one or more compositions listed in Table 3, or any combination thereof; or
(m) the method of any of (a) to (1), wherein the method reduces, treats or ameliorates the level of disease in a retinal age-related macular degeneration, a diabetic retinopathy, a cancer or carcinoma, a glioblastoma, a neuroma, a neuroblastoma, a colon carcinoma, a hemangioma, an infection and/or a condition with at least one inflammatory component, and/or any infectious or inflammatory disease, such as a rheumatoid arthritis, a psoriasis, a fibrosis, leprosy, multiple sclerosis, inflammatory bowel disease, or ulcerative colitis or Crohn's disease. In alternative embodiments, the invention provides kits, blister packages, lidded blisters or blister cards or packets, clamshells, trays or shrink wraps, comprising;
(a) (i) at least one compound, composition or formulation used to practice a method of the invention, and (ii); at least one Growth Factor Inhibitor; or
(b) the kit of (a), further comprising instructions for practicing a method of the invention.
In alternative embodiments, the kit, blister package, lidded blister, blister card, packet, clamshell, tray or shrink wrap comprises: a combination or a therapeutic combination of drugs comprising: an erlotinib with either a Lenalidomide or a PS-1 145, or both a Lenalidomide and a PS-1 145.
In alternative embodiments, the invention provides methods for determining: whether an individual or a patient would benefit from or respond to administration of a Growth Factor Inhibitor, or
whether the individuals or patients would benefit from a combinatorial approach comprising administration of a combination of: at least one growth factor inhibitor and at least one compound, composition or formulation used to practice a method of the invention, such as an NFkB inhibitor,
the method comprising:
detecting the levels or amount of integrin ανβ3 (avb3) and/or active RalB complex in or on a cell, a tissue or a tissue sample,
wherein optionally the detection is by analysis or visualization of a biopsy or a tissue, urine, fluid, serum or blood sample, or a pathology slide taken from the patient or individual, or by a fluorescence-activated cell sorting (FACS) or flow cytometry analysis or the sample or biopsy,
wherein optionally the cell or tissue or tissue sample is or is derived from a tumor or a cancer, or the cell is a cancer stem cell,
wherein optionally the method further comprises taking a biopsy or a tissue, urine, fluid, serum or blood sample from an individual or a patient,
wherein a finding of increased levels or amounts of integrin ανβ3 (anb3) and/or active RalB complexes in or on the cell, tissue or the tissue sample as compared to normal, normalized or wild type cells or tissues, indicates that: the individual or patient would benefit from a combinatorial approach comprising administration of a combination of: at least one growth factor inhibitor and at least one compound, composition or formulation used to practice a method of the invention, e.g., an NFKB inhibitor such as a lenalidomide or a REVLIMID™ , or a bortezomib or a VELCADE®, or an IKK inhibitor such as a PS 1145.
In alternative embodiments of methods of the invention, the detecting of the levels or amount of integrin ανβ3 (avb3) and/or active RalB complex in or on the cell, tissue or the tissue sample is done before or during a drug or a pharmaceutical treatment of an individual using at least one compound, composition or formulation used to practice a method of the invention.
In alternative embodiments, the invention provide uses of a combination of compounds in the manufacture of a medicament,
wherein the combination of compounds comprises:
(1) at least one compound comprising or consisting of:
(i) an inhibitor or depleter of integrin ανβ3 (avb3), or an inhibitor of integrin ανβ3 (avb3) protein activity, or an inhibitor of the formation or activity of an integrin avb3/RalB signaling complex, or an inhibitor of the formation or signaling activity of an integrin ανβ3 (avb3)/RalB/NFkB signaling axis,
wherein optionally the inhibitor of integrin ανβ3 protein activity is an allosteric inhibitor of integrin ανβ3 protein activity;
(ii) an inhibitor or depleter of RalB protein or an inhibitor of RalB protein activation,
wherein optionally the inhibitor of RalB protein activity is an allosteric inhibitor of RalB protein activity;
(iii) an inhibitor or depleter of Src or TBK1 protein or an inhibitor of Src or TBK1 protein activation,
wherein optionally the inhibitor of Src or TBK1 protein activity is an allosteric inhibitor of Src or TBK1 protein activity;
(iv) an inhibitor or depleter of NFKB or IRF3 protein or an inhibitor of RalB protein activation,
wherein optionally the inhibitor of NFKB or IRF3 protein activity is an allosteric inhibitor of NFKB or IRF3 protein activity; (v) an inhibitor or depleter of NFKB or IKK, or an inhibitor of NFKB or IKK protein activation,
wherein optionally the NFKB inhibitor comprises a lenalidomide or a REVLIMID™ (Celgene Corp., Summit, NJ) or a bortezomib or a
VELCADE®, and optionally the IKK inhibitor comprises a PS 1145
(Millennium Pharmaceuticals, Cambridge, MA);
(vi) a lenalidomide or a REVLIMID™ and PS 1145;
(vii) an erlotinib or a TARCEVA® and a bortezomib or a VELCADE®;
(viii) a lenalidomide or a REVLIMID™ ; a PS 1145; and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTK inhibitor comprises
SU14813 (Pfizer, San Diego, CA); or
(ix) any combination of (i) to (viii); and/or
(2) at least one Growth Factor Inhibitor,
wherein optionally the Growth Factor Inhibitor is or comprises an anti-metabolite inhibitor, a gemcitabine, GEMZAR™, a mitotic poison, a paclitaxel, a taxol,
ABRAXANE™, an erlotinib, TARCEVA™, a lapatinib, TYKERB™, or an insulin growth factor inhibitor, or any combination thereof; or, the Growth Factor Inhibitor decreases, slows or blocks new blood vessel growth, neovascularization or angiogenesis; or, wherein administering the Growth Factor Inhibitor treats or ameliorates conditions that are responsive to blocking or slowing cell growth, and/or the development of neovascularization or new blood vessels,
wherein optionally the combination or the therapeutic combination comprises an erlotinib with either a Lenalidomide or a PS-1 145, or both a Lenalidomide and a PS-1 145.
In alternative embodiments, the invention provides therapeutic combinations of drugs comprising or consisting of a combination of at least two compounds: wherein the at least two compounds comprise or consist of:
(1) at least one compound comprising or consisting of:
(i) an inhibitor or depleter of integrin ανβ3 (avb3), or an inhibitor of integrin ανβ3 (avb3) protein activity, or an inhibitor of the formation or activity of an integrin avb3/RalB signaling complex, or an inhibitor of the formation or signaling activity of an integrin ανβ3 (avb3)/RalB/NFkB signaling axis, wherein optionally the inhibitor of integrin ανβ3 protein activity is an allosteric inhibitor of integrin ανβ3 protein activity;
(ii) an inhibitor or depleter of RalB protein or an inhibitor of RalB protein activation,
wherein optionally the inhibitor of RalB protein activity is an allosteric inhibitor of RalB protein activity;
(iii) an inhibitor or depleter of Src or TBK1 protein or an inhibitor of Src or TBK1 protein activation,
wherein optionally the inhibitor of Src or TBK1 protein activity is an allosteric inhibitor of Src or TBK1 protein activity;
(iv) an inhibitor or depleter of NFKB or IRF3 protein or an inhibitor of RalB protein activation,
wherein optionally the inhibitor of NFKB or IRF3 protein activity is an allosteric inhibitor of NFKB or IRF3 protein activity;
(v) an inhibitor or depleter of NFKB or IKK, or an inhibitor of NFKB or IKK protein activation,
wherein optionally the NFKB inhibitor comprises a lenalidomide or a REVLIMID™ (Celgene Corp., Summit, NJ) or a bortezomib or a
VELCADE®, and optionally the IKK inhibitor comprises a PS 1145
(Millennium Pharmaceuticals, Cambridge, MA);
(vi) a lenalidomide or a REVLIMID™ and PS 1145;
(vii) an erlotinib or a TARCEVA® and a bortezomib or a VELCADE®;
(viii) a lenalidomide or a REVLIMID™ ; a PS 1145; and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTK inhibitor comprises SU14813 (Pfizer, San Diego, CA); or
(ix) any combination of (i) to (viii); and/or
(2) at least one Growth Factor Inhibitor,
wherein optionally the Growth Factor Inhibitor is or comprises an anti-metabolite inhibitor, a gemcitabine, GEMZAR™, a mitotic poison, a paclitaxel, a taxol,
ABRAXANE™, an erlotinib, TARCEVA™, a lapatinib, TYKERB™, or an insulin growth factor inhibitor, or any combination thereof; or, the Growth Factor Inhibitor decreases, slows or blocks new blood vessel growth, neovascularization or angiogenesis; or,
wherein administering the Growth Factor Inhibitor treats or ameliorates conditions that are responsive to blocking or slowing cell growth, and/or the development of neovascularization or new blood vessels,
wherein optionally the combination or the therapeutic combination comprises an erlotinib with either a Lenalidomide or a PS-1 145, or both a Lenalidomide and a PS-1 145.
In alternative embodiments, the invention provides combinations, or therapeutic combinations, for overcoming or diminishing or preventing Growth Factor Inhibitor (GFI) resistance in a cell, or, a method for increasing the growth- inhibiting effectiveness of a Growth Factor Inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor (GFI), wherein the combination comprises or consists of:
(1) at least one compound comprising or consisting of:
(i) an inhibitor or depleter of integrin ανβ3 (avb3), or an inhibitor of integrin ανβ3 (avb3) protein activity, or an inhibitor of the formation or activity of an integrin avb3/RalB signaling complex, or an inhibitor of the formation or signaling activity of an integrin ανβ3 (avb3)/RalB/NFkB signaling axis,
wherein optionally the inhibitor of integrin ανβ3 protein activity is an allosteric inhibitor of integrin ανβ3 protein activity;
(ii) an inhibitor or depleter of RalB protein or an inhibitor of RalB protein activation,
wherein optionally the inhibitor of RalB protein activity is an allosteric inhibitor of RalB protein activity;
(iii) an inhibitor or depleter of Src or TBK1 protein or an inhibitor of Src or TBK1 protein activation,
wherein optionally the inhibitor of Src or TBK1 protein activity is an allosteric inhibitor of Src or TBK1 protein activity;
(iv) an inhibitor or depleter of NFKB or IRF3 protein or an inhibitor of RalB protein activation,
wherein optionally the inhibitor of NFKB or IRF3 protein activity is an allosteric inhibitor of NFKB or IRF3 protein activity; (v) an inhibitor or depleter of NFKB or IKK, or an inhibitor of NFKB or IKK protein activation,
wherein optionally the NFKB inhibitor comprises a lenalidomide or a REVLIMID™ (Celgene Corp., Summit, NJ) or a bortezomib or a
VELCADE®, and optionally the IKK inhibitor comprises a PS 1145
(Millennium Pharmaceuticals, Cambridge, MA);
(vi) a lenalidomide or a REVLIMID™ and PS 1145;
(vii) an erlotinib or a TARCEVA® and a bortezomib or a VELCADE®;
(viii) a lenalidomide or a REVLIMID™ ; a PS 1145; and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTK inhibitor comprises
SU14813 (Pfizer, San Diego, CA); or
(ix) any combination of (i) to (viii); and/or
(2) at least one Growth Factor Inhibitor,
wherein optionally the Growth Factor Inhibitor is or comprises an anti-metabolite inhibitor, a gemcitabine, GEMZAR™, a mitotic poison, a paclitaxel, a taxol,
ABRAXANE™, an erlotinib, TARCEVA™, a lapatinib, TYKERB™, or an insulin growth factor Inhibitor, or any combination thereof; or, the Growth Factor Inhibitor decreases, slows or blocks new blood vessel growth, neovascularization or angiogenesis; or, wherein administering the Growth Factor Inhibitor treats or ameliorates conditions that are responsive to blocking or slowing cell growth, and/or the development of neovascularization or new blood vessels,
wherein optionally the cell is a tumor cell, a cancer cell, a cancer stem cell, or a dysfunctional cell,
wherein optionally the combination or the therapeutic combination comprises an erlotinib with either a Lenalidomide or a PS-1 145, or both a Lenalidomide and a PS-1 145.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS
The drawings set forth herein are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Figure 1 illustrates that integrin ανβ3 expression promotes resistance to EGFR TKI: Fig. 1(a) illustrates flow cytometric quantification of cell surface markers after 3 weeks treatment with erlotinib (pancreatic and colon cancer cells) or lapatinib (breast cancer cells); Fig. 1(b) illustrates flow cytometric analysis of ανβ3 expression in FG and Miapaca-2 cells following erlotinib; Fig. 1 (c) illustrates: Top, immunofluorescence staining of integrin ανβ3 in tissue specimens obtained from orthotopic pancreatic tumors treated with vehicle or erlotinib; Bottom, Integrin ανβ3 expression was quantified as ratio of integrin ανβ3 pixel area over nuclei pixel area using METAMORPH™; Fig. 1(d) Right, intensity of β3 expression in mouse orthotopic lung tumors treated with vehicle or erlotinib, Left, immunohistochemical staining of β3, Fig. 1(f) illustrates tumor sphere formation assay to establish a dose-response for erlotinib, Fig. 1(g) illustrates orthotopic FG tumors treated for 10 days with vehicle or erlotinib, results are expressed as % tumor weight compared to vehicle control, immunoblot analysis for tumor lysates after 10 days of erlotinib confirms suppressed EGFR phosphorylation; as discussed in detail in Example 1, below.
Figure 2 illustrates that integrin ανβ3 cooperates with K-RAS to promote resistance to EGFR blockade: Fig. 2(a-b) illustrates tumor sphere formation assay of FG expressing (a) or lacking (b) integrin β3 depleted of KRAS (shKRAS) or not (shCTRL) and treated with a dose response of erlotinib; Fig. 2(c) illustrates confocal microscopy images of PANC-1 and FG- β3 cells grown in suspension; Fig. 2(d) illustrates RAS activity assay performed in PANC-1 cells using GST-Rafl-RBD immunoprecipitation as described below; as discussed in detail in Example 1, below.
Figure 3 illustrates that RalB is a key modulator of integrin o^3-mediated EGFR TKI resistance: Fig. 3(a) illustrates tumor spheres formation assay of FG^3 treated with non-silencing (shCTRL) or RalB-specific shRNA and exposed to a dose response of erlotinib; Fig. 3(b) illustrates effects of depletion of RalB on erlotinib sensitivity in β3- positive tumor in a pancreatic orthotopic tumor model; Fig. 3(c) illustrates tumor spheres formation assay of FG cells ectopically expressing vector control, WT RalB FLAG tagged constructs or a constitutively active RalB G23V FLAG tagged treated with erlotinib (0.5 μΜ); Fig. 3(d) illustrates RalB activity was determined in FG, FG- 3 expressing non-silencing or KRAS-specific shR A, by using a GST-RalBPl-RBD immunoprecipitation assay; Fig. 3(e) illustrates: Right, overall active Ral
immunohistochemical staining intensity between β3 negative and β3 positive human tumors; as discussed in detail in Example 1, below.
Figure 4 illustrates that integrin av 3/RalB complex leads to NF-μΒ activation and resistance to EGFR TKl: Fig. 4(a) illustrates an immunoblot analysis of FG, FG- 3 and FG- 3 stably expressing non-silencing or RalB-specific ShRNA, grown in suspension and treated with erlotinib (0.5 μΜ); Fig. 4(b) illustrates tumor spheres formation assay of FG cells ectopically expressing vector control, WT NF-κΒ FLAG tagged or constitutively active S276D NF-κΒ FLAG tagged constructs treated with erlotinib; Fig. 4(c) illustrates tumor spheres formation assay of FG- 3 treating with non- silencing (shCTRL) or NF-KB-specific shRNA and exposed to erlotinib; Fig. 4(d) illustrates dose response in FG-P3 cells treated with erlotinib (10 nM to 5 μΜ), lenalidomide (10 nM to 5 μΜ) or a combination of erlotinib (10 nM to 5 μΜ) and lenalidomide (1 μΜ) ; Fig. 4(e) illustrates Model depicting the integrin ανβ3 -mediated EGFR TKl resistance and conquering EGFR TKl resistance pathway and its downstream RalB and NF-κΒ effectors; as discussed in detail in Example 1, below.
Figure 5 (Supplementary Fig. 1, Example 1) illustrates that prolonged exposure to erlotinib induces Integrin ανβ3 expression in lung tumors; representative
immunohistochemical staining of integrin β3 in mouse tissues obtained from H441 orthotopic lung tumors long-term treated with either vehicle or erlotinib (scale bar, 100 μιη); as discussed in detail in Example 1, below.
Figure 6 (Supplementary Fig. 2, Example 1) illustrates integrin ανβ3, even in its unligated state, promotes resistance to Growth Factor inhibitors but not to
chemotherapies: Fig. 6(a) illustrates a tumor sphere formation assay comparing FG lacking β3 (FG), FG expressing β3 wild type (FG^3) or the β3 Dl 19A (FG-D119A) ligand binding domain mutant, treated with a dose response of erlotinib (Error bars represent s.d. (n = 3 independent experiments); Fig. 6(b) illustrates tumor sphere formation assay of FG and FG- β3 cells untreated or treated with erlotinib (0.5 μΜ), OSI- 906 (0.1 μΜ), gemcitabine (0.01 μΜ) or cisplatin (0.1 μΜ); Fig. 6(c) illustrates the effect of dose response of indicated treatments on tumor sphere formation (Error bars represent s.d. (n = 3 independent experiments); as discussed in detail in Example 1, below.
Figure 7 (Supplementary Fig. 3, Example 1) illustrates that integrin ανβ3 does not colocalize with active HRAS, NRAS and RRAS: Fig. 7(a) illustrates that Ras activity was determined in PANC-1 cells grown in suspension by using a GST-Rafl-RBD
immunoprecipitation assay as described in Methods, see Example 1 (data are
representative of two independent experiments); Fig. 7(b) illustrates confocal microscopy images of PANC-1 cells grown in suspension and stained for KRAS, RRAS, HRAS, NRAS (red), integrin ανβ3 (green) and DNA (TOPRO-3, blue) (Scale bar, 10 μιη. Data are representative of two independent experiments); as discussed in detail in Example 1, below.
Figure 8 (Supplementary Fig. 4, Example 1) illustrates that Galectin-3 is required to promote integrin av 3/KRAS complex formation: Fig. 8(a-b) illustrates confocal microscopy images of Pane- 1 cells lacking or expressing integrin ανβ3 grown in suspension; Fig. 8(a) illustrates cells stained for KRAS (green), Galectin-3 (red), and DNA (TOPRO-3, blue); Fig. 8(b) illustrates cells stained for integrin ανβ3 (green), Galectin-3 (red) and DNA (TOPRO-3, blue), Scale bar, 10 μιη, data are representative of three independent experiments; Fig. 8(c) illustrates an immunoblot analysis of Galectin-3 immuno-precipitates from PANC- 1 cells expressing non-silencing (sh CTRL) or integrin 3-specific shRNA (sh β3), data are representative of three independent experiments; Fig. 8(d) illustrates an immunoblot analysis of integrin β3 immunoprecipitates from PANC-1 cells expressing non-silencing (sh CTRL) or Galectin-3 -specific shRNA (sh Gal3), data are representative of three independent experiments; as discussed in detail in Example 1, below.
Figure 9 (Supplementary Fig. 5, Example 1) illustrates that ERK, AKT and RalA are not specifically required to promote integrin o^3-mediated resistance to EGFR TKI; tumor spheres formation assay of FG and FG^3 expressing non-silencing or ERK1/2, AKTl and RalA-specific shRNA and treated with erlotinib (0.5 μΜ), error bars represent s.d. (n = 3 independent experiments); as discussed in detail in Example 1, below.
Figure 10 (Supplementary Fig. 6, Example 1) illustrates that RalB is sufficient to promote resistance to EGFR TKI: Fig. 10(a) (supplementary Figure 6, Example 1) illustrates a tumor sphere formation assay of FG expressing non-silencing or RalB specific shRNA and treated with a dose response of erlotinib. Error bars represent s.d. (n = 3 independent experiments); Fig. 10(b) (supplementary Figure 6) illustrates a tumor spheres formation assay of PANC-1 stably expressing integrin 3-specific shRNA and ectopically expressing vector control, WT RalB FLAG tagged or a constitutively active RalB G23V FLAG tagged constructs treated with erlotinib (0.5 μΜ), error bars represent s.d. (n = 3 independent experiments); Fig. 10(c) (Supplementary Figure 7, Example 1) shows that integrin ανβ3 colocalizes with RalB in cancer cells: illustrates confocal microscopy images of Pane- 1 cells grown in suspension. Cells are stained for integrin ανβ3 (green), RalB (red), pFAK (red), and DNA (TOPRO-3, blue), scale bar, 10 μιη, data are representative of three independent experiments; as discussed in detail in Example 1, below.
Figure 1 1 (Supplementary Fig. 8, Example 1) illustrates that integrin ανβ3 colocalizes with RalB in human breast and pancreatic tumor biopsies and interacts with RalB in cancer cells: Fig. 1 1(a) illustrates confocal microscopy images of integrin ανβ3 (green), RalB (red) and DNA (TOPRO-3, blue) in tumor biopsies from breast and pancreatic cancer patients, Scale bar, 20 μιη; Fig. 11(b) illustrates a Ral activity assay performed in PANC-1 cells using GST-RalBPl-RBD immunoprecipitation assay, Immunoblot analysis of RalB and integrin β3, data are representative of three independent experiments; as discussed in detail in Example 1, below.
Figure 12, or Fig. 12/31 (Figure 1 in Example 2) illustrates data showing that integrin β3 is expressed in EGFR inhibitor resistant tumors and is necessary and sufficient to drive EGFR inhibitor resistance: Fig. 12(A) schematically illustrates that the identification of the most upregulated tumor progression genes common to erlotinib resistant carcinomas; Fig. 12(B) in table form shows Erlotinib IC50 in a panel of human carcinoma cell lines treated with erlotinib in 3D culture; Fig. 12(C) graphically illustrates percentage of integrin β3 positive cells in parental lines vs. after 3 or 8 weeks treatment with erlotinib; Fig. 12(D) graphically illustrates quantification of integrin β3 (ΠΌβ3) gene expression in human lung cancer biopsies from patients from the BATTLE Study (18) who were previously treated with an EGFR inhibitor and progressed (n = 27), versus patients who were EGFR inhibitor naive (n = 39); Fig. 12(E) illustrates images of paired human lung cancer biopsies obtained before and after erlotinib resistance were immunohistochemically stained for integrin β3, scale bar, 50 μιη; Fig. 12(F) graphically illustrates: Right graph shows effect of integrin β3 knockdown on erlotinib resistance of 3-positive cells, and Left graph shows effect of integrin β3 ectopic expression on erlotinib resistance in FG and H441 cells; Fig. 12(G) graphically illustrates: Right graph shows the effect of integrin β3 knockdown on erlotinib resistance in vivo, A549 shCTRL and A549 sh integrin β3 (n=8 per treatment group) were treated with erlotinib (25 mg/kg/day) or vehicle during 16 days, results are expressed as average of tumor volume at day 16. *P < 0.05; and Left graph shows orthotopic FG and FG- 3 tumors treated for 30 days with vehicle or erlotinib, results are expressed as % tumor weight compared to vehicle control; as further described in Example 2, below.
Figure 13, or Fig. 13/31 (Figure 2 in Example 2) illustrates data showing that integrin β3 is required to promote KRAS dependency and KRAS-mediated EGFR inhibitor resistance: Fig. 13(A) illustrates confocal microscopy images showing immunostaining for integrin β3 (green), K-, N-, H-, R-Ras (red), and DNA (TOPRO-3, blue) for BxPc3 cells grown in suspension in media with 10% serum, arrows indicate clusters where integrin β3 and KRAS colocalize (yellow); Fig. 13(B-C) illustrates confocal microscopy images showing immunostaining for integrin β 3 (green), KRAs (red) and DNA (Topro-3, blue) for PANC-1 (KRAS mutant) and HCC827 (KRAS wild- type) after acquired resistance to erlotinib (HCC827R) grown in suspension in absence (Vehicle) or in presence of erlotinib (0.5 μΜ and 0.1 μΜ respectively), arrows indicate clusters where integrin β3 and KRAS colocalize (yellow); Fig. 13(D) graphically illustrates the effect of KRAS knockdown on tumorspheres formation in a panel of lung and pancreatic cancer cells expressing or lacking integrin β3; Fig. 13(E) graphically illustrates the effect of KRAS knockdown on tumorsphere formation in PANC-1 (KRAS mutant) stably expressing non-target shRNA control (μ3 -positive) or specific-integrin β3 shRNA (β3 negative) in FG (KRAS mutant) and BxPc3 (KRAS wild-type) stably expressing vector control or integrin β3; Fig. 13(F) graphically illustrates the effect of KRAS knockdown on erlotinib resistance of β3-negative and β3-ρο8Ϊίϊνε epithelial cancer cell lines, cells were treated with a dose response of erlotinib; Fig. 13(G) illustrates confocal microscopy images showing immunostaining for integrin β3 (green), KRAS (red) and DNA (TOPRO-3, blue) for PANC-1 cells expressing non-target shRNA control or Galectin 3 -specific shRNA grown in suspension; Fig. 13(H) illustrates: Top:
immunoblot analysis of integrin β3 immunoprecipitates from PANC-1 cells expressing non-target shRNA control (CTRL) or Galectin-3 -specific shRNA (Gal-3); Bottom:
immunoblot analysis of Galectin-3 immunoprecipitates from PANC-1 cells expressing non-target shRNA control (CTRL) or integrin 3-specific shRNA (β3); Fig. 13(1) graphically illustrates erlotinib dose response of FG- 3 cells expressing a non-target shRNA control or a Galectin-3 -specific shRNA (sh Gal-3); as further described in Example 2, below.
Figure 14, or Fig. 14/31 (Figure 3 in Example 2) illustrates data showing that RalB is a central player of integrin 3-mediated EGFR inhibitor resistance: Fig. 14(A) graphically illustrates the effect of RalB knockdown on erlotinib resistance of 3-positive epithelial cancer cell lines, cells were treated with 0.5 μΜ of erlotinib: Fig. 14(B) graphically illustrates the effect of RalB knockdown on erlotinib resistance of 3-positive human pancreatic (FG- 3) orthotopic tumor xenografts, established tumors expressing non-target shRNA, (shCTRL) or a shRNA targeting RalB (sh RalB) were randomized and treated for 10 days with vehicle or erlotinib, results are expressed as % of tumor weight changes after erlotinib treatment compared to vehicle; Fig. 14(C) graphically illustrates the effect of expression of a constitutively active Ral G23V mutant on erlotinib response of β3 negative cells, cells were treated with 0.5 μΜ of erlotinib; Fig. 14(D) illustrates the effect of expression of integrin β3 on KRAS and RalB membrane localization; Fig. 14(E) illustrates Ral activity that was determined in PANC- 1 cells grown in suspension by using a GST-RalBP l-RBD immunoprecipitation assay, immunoblots indicate RalB activity and association of active RalB with integrin β3; Fig. 14(F) illustrates confocal microscopy images of integrin ανβ3 (green), RalB (red) and DNA (TOPRO-3, blue) in tumor biopsies from pancreatic cancer patients; Fig. 14(G) illustrates the effect of β3 expression and KRAS expression on RalB activity, measured using a GST-RalBP 1 -RBD
immunoprecipitation assay; Fig. 14(H) illustrates immunoblot analysis of FG and FG^3 stably expressing non-target shRNA control or RalB-specific shRNA, grown in suspension and treated with erlotinib (0.5 μΜ); Fig. 14(1) graphically illustrates the effect of TBK1 and p65 NFKB on erlotinib resistance of FG^3 cells, cells were treated with 0.5 μΜ of erlotinib; as further described in Example 2, below.
Figure 15, or Fig. 15/31 (Figure 4 in Example 2) illustrates data showing that reversal of β3 -mediated EGFR inhibitor resistance in oncogenic KRAS model by pharmacological inhibition: Fig. 15(A) graphically illustrates the effect of NFkB inhibitors on erlotinib response of β3 -positive cells (FG- 3, PANC-1 and A549), cells were treated with vehicle, erlotinib (0.5 μΜ), lenalidomide (1-2 μΜ), bortezomib (4 nM) alone or in combination; Fig. 15(B) graphically illustrates data from: Left, mice bearing subcutaneous β3 -positive tumors (FG- 3) were treated with vehicle, erlotinib (25 mg/kg/day), lenalidomide (25 mg/kg/day) or the combination of erlotinib and
lenalidomide, tumor dimensions are reported as the fold change relative to size of the same tumor on Day 1 ; Right, mice bearing subcutaneous 3-positive tumors (FG-R) after acquired resistance to erlotinib were treated with vehicle, erlotinib (25 mg/kg/day), bortezomib (0.25 mg/kg), the combination of erlotinib and bortezomib, tumor dimensions are reported as the fold change relative to size of the same tumor on Day 1; Fig. 15(C) schematically illustrates a model depicting an integrin ανβ3 -mediated KRAS dependency and EGFR inhibitor resistance mechanism; as further described in Example 2, below.
Figure 16, or Fig. 16/31 (supplementary Figure S I, in Example 2) illustrates data showing that illustrates resistance to EGFR inhibitor is associated with integrin β3 expression in pancreatic and lung human carcinoma cell lines: Fig. 16(A) illustrates immunoblots showing integrin β3 expression in human cell lines used in Figure 12; Fig. 16(B) graphically illustrates data showing the effect of erlotinib on HCC827 xenograft tumors in immuno - compromised mice relative to vehicle-treated control tumors; Fig. 16(C) left, graphically illustrates data of Integrin ανβ3 quantification in orthotopic lung and pancreas tumors treated with vehicle or erlotinib until resistance, Fig. 16(C) right, illustrates a representative immunofluorescent staining of integrin ανβ3 in pancreatic human xenografts treated 4 weeks with vehicle or erlotinib; as further described in Example 2, below.
Figure 17, or Fig. 17/31 (supplementary Figure S2, in Example 2) illustrates Integrin β3 expression predicts intrinsic resistance to EGFR inhibitors in tumors; Fig.
17A graphically illustrates a plot of progression- free survival for erlotinib-treated patients with low versus (vs.) high protein expression of β3 integrin measured from non-small cell lung cancer biopsy material (Fig. 17B illustrates) obtained at diagnosis; as further described in Example 2, below.
Figure 18, or Fig. 18/31 (supplementary Figure S3, in Example 2) illustrates
Integrin β3 confers Receptor Tyrosine Kinase inhibitor resistance: Fig. 18(A) illustrates immunoblots showing integrin β3 knockdown efficiency in cells used in Figure 12; Fig. 18(B) graphically illustrates response of A549 lung carcinoma cells non-target shRNA control or shRNA targeting integrin β3 to treatment with either vehicle or erlotinib (25 mg/kg/day) during 16 days; Fig. 18(C) illustrates immunoblots showing expression of indicated proteins of representative tumors; Fig. 18(D) illustrates representative photographs of crystal violet-stained tumorspheres of β3 -negative and β3 -positive cells after erlotinib, OSI-906, gemcitabine and cisplatin treatment; Fig. 18(E) graphically illustrates the effect of integrin β3 expression on lapatinib, OSI-906, cisplatin and gemcitabine; Fig. 18(F) graphically illustrates data from a viability assay of FG and FG- β3 cells grown in suspension in media with or without serum; as further described in Example 2, below.
Figure 19, or Fig. 19/31 (supplementary Figure S4, in Example 2) illustrates integrin β3-mediated EGFR inhibitor resistance is independent of its ligand binding: Fig. 19A graphically illustrates the effect of ectopic expression of β3 wild-type (FG- β3) or the β3 Dl 19A (FG-D1 19 A) ligand binding domain mutant on erlotinib response; Fig. 19B illustrates an immunoblot showing transfection efficiency of vector control, integrin β3 wild-type and integrin β3 D l 19A; as further described in Example 2, below.
Figure 20, or Fig. 20/31 (supplementary Figure S5, in Example 2) illustrates integrin β3 colocalizes and interacts with oncogenic and active wild-type KRAS: Fig. 20(A) illustrates confocal microscopy images of FG and FG^3 cells grown in suspension in media 10% serum with or without erlotinib (0.5 μΜ) and stained for KRAS (red), integrin ανβ3 (green) and DNA (TOPRO-3, blue); Fig. 20(B) illustrates Ras activity was determined in PANC-1 cells grown in suspension by using a GST-Rafl-RBD
immunoprecipitation assay, immunoblots indicate KRAS activity and association of active KRAS with integrin β3; Fig. 20(C) illustrates an immunoblot analysis showing that Integrin ανβ3 immunoprecipitates from BxPC-3 cells grown in suspension in presence or absence of growth factors; as further described in Example 2, below.
Figure 21, or Fig. 21/31 (supplementary Figure S6, in Example 2) illustrates integrin β3 expression promotes KRAS dependency: Fig. 21(A) illustrates Immunoblots showing KRAS knockdown efficiency in cells used in Figure 13; Fig 21(B) illustrates Representative photographs of crystal violet-stained tumorspheres of FG and A549 cells expressing non-target shRNA control or specific -KRAS shRNA; Fig. 21(C) illustrates the effect of an additional KRAS knockdown on tumorspheres formation in PANC-1 stably expressing non-target shRNA control (β3 -positive) or specific-integrin β3 shRNA (β3 negative); Fig. 21(D) illustrates immunoblots showing KRAS knockdown efficiency; as further described in Example 2, below.
Figure 22, or Fig. 22/31 (supplementary Figure S7, in Example 2) illustrates images showing that KRAS and Galectin-3 colocalize in integrin 3-positive cells, in particular, confocal microscopy images of FG and FG- 3 cells grown in suspension and stained for KRAS (green), galectin-3 (red) and DNA (TOPRO-3, blue); as further described in Example 2, below.
Figure 23, or Fig. 23/31 (supplementary Figure S8, in Example 2) illustrates Integrin 3-mediated KRAS dependency and erlotinib resistance is independent of ERK, AKT and RalA: Fig. 23(A) graphically illustrates the effect of ERK, AKT, RalA and RalB knockdown on erlotinib response (erlotinib 0.5 μΜ) of 3-negative FG and β3- positive FG- 3 cells; Fig. 23(B) illustrates Immunoblots showing ERK, AKT RalA and RalB knockdown efficiency; Fig. 23(C) illustrates Immunoblots showing RalB knockdown efficiency in cells used in Figure 14; as further described in Example 2, below.
Figure 24, or Fig. 24/31 (supplementary Figure S9, in Example 2) illustrates constitutive active NFkB is sufficient to promote erlotinib resistance: Fig. 24(A) illustrates Immunoblots showing TBK1 and NFkB knockdown efficiency used in Figure 14; Fig. 24(B) graphically illustrates the effect of constitutive active S276D p65NFkB on erlotinib response (erlotinib 0.5 μΜ) of 3-negative cells (FG cells); as further described in Example 2, below.
Figure 25, or Fig. 25/31 (supplementary Figure S10, in Example 2) illustrates NFkB inhibitors in combination with erlotinib increase cell death in vivo: Fig. 25(A) and Fig. 25 (B) illustrate Immunoblots showing expression of indicated proteins of representative tumors from shown in Figure 15B; Fig. 25(C) illustrates Confocal microscopy images of cleaved caspase 3 (red) and DNA (TOPRO-3, blue) in tumor biopsies from xenografts tumors used in Fig. 15B treated with vehicle, erlotinib, lenalidomide or lenalidomide and erlotinib in combo; Fig. 25(D) illustrates Confocal microscopy images of cleaved caspase 3 (red) and DNA (TOPRO-3, blue) in tumor biopsies from xenografts tumors used in Figure 15B treated with vehicle, erlotinib, bortezomib or bortezomib and erlotinib in combo); as further described in Example 2, below.
Figures 26, 27 and 28, or Fig. 26/31, 27/31 and 28/31 illustrate supplementary Table 1 from Example 2, showing that differentially expressed genes in cells resistant to erlotinib (PANC-1, H I 650, A459) compared with the average of two sensitive cells (FG, H44I) and in HCC827 after acquired resistance in vivo (HCC827R) vs. the HCC827 vehicle- treated control; as further described in Example 2, below.
Figure 29, or Fig. 29/31 illustrates supplementary Table 2, from Example 2, showing KRAS mutational status in pancreatic and lung cell lines used in the study of Example 2, below.
Figure 30, or Fig. 30/31 illustrates data showing integrin β3 (CD61) is a RTKI drug resistance biomarker on the surface of circulating tumor cells; as discussed in detail in Example 2, below. CD61 (β3, or beta3) negative human lung cancer cells (HCC827; this lung adenocarcinoma has an acquired mutation in the EGFR tyrosine kinase domain (E746 - A750 deletion), and they are sensitive to erlotinib and develop acquired resistance after 6/8 weeks) were injected orthotopically into the lung of mice and treated over 3 months with erotinib at 25 mg/kg/day. Human lung cancer cells detected in the circulation were positive for ανβ3 (or avb3, CD61) whereas the cells in the untreated group were essentially negative for this marker. CD45 negative cells indicates that the detected cells were not leukocytes and pan cytokeratin positive cells indicate tumor cells. CD61 (beta3) positive expression correlated with tumor expression.
Figure 31, or Fig. 31/31 illustrates data showing how targeting the NF-KB
pathway using compositions and methods of this invention can sensitize resistant tumors to growth factor inhibitors by showing the effect of NFkB inhibitors on erlotinib response of 3-negative (b3-negative) cells (FG) and 3-positive cells (FG- β3, MDA-MB231
(intrinsic resistance) and FG-R (acquired resistance). Cells embedded in agar (anchorage independent growth) were treated with vehicle, erlotinib (0.5 μΜ), Lenalidomide (2 μΜ), PS- 1145 (1 μΜ) alone or in combination for 10 to 15 days. Then, the soft agar were stained with crystal violet and the colonies were counted manually. The results show that while 3-positive cells (intrinsic or acquired resistant cells) were resistant to erlotinib and each NFKB inhibitor alone, the combination of erlotinib with either Lenalidomide or PS-1 145 decreased tumorsphere formation. Like reference symbols in the various drawings indicate like elements.
Reference will now be made in detail to various exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. The following detailed description is provided to give the reader a better understanding of certain details of aspects and embodiments of the invention, and should not be interpreted as a limitation on the scope of the invention.
DETAILED DESCRIPTION
In alternative embodiments, the invention provides compositions and methods for overcoming or diminishing or preventing Growth Factor Inhibitor (GFI) resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor (GFI). In alternative embodiments, the cell is a tumor cell, a cancer cell or a
dysfunctional cell. In alternative embodiments, the invention provides compositions and methods for determining: whether an individual or a patient would benefit from or respond to administration of a Growth Factor Inhibitor, or, which individuals or patients would benefit from a combinatorial approach comprising administration of a combination of: at least one growth factor inhibitor and at least one compound, composition or formulation used to practice a method of the invention, such as an NfKb inhibitor.
We found that integrin avb3 is upregulated in cells that become resistant to Growth Factor Inhibitors. Our findings demonstrate that integrin avb3 promotes de novo and acquired resistance to Growth Factor Inhibitors by interacting and activating RalB. RalB activation leads to the activation of Src and TBK1 and the downstream effectors NFKB and IRF3. We also found that depletion of RalB or its downstream signaling (Src/NFKB) in b3-positive cells overcomes resistance to growth factor inhibitors. This invention demonstrates that the integrin avb3/RalB signaling complex promotes resistance to growth factor inhibitors; and in alternative embodiments, integrin ανβ3 (avb3) and active RalB are used as biomarkers in patient samples to predict which patients will respond to growth factor inhibitors and which patients might rather benefit from alternative/combinatorial approaches such as a combination of growth factor inhibitors and NfKb inhibitors.
This invention for the first time identifies integrin ανβ3 and active RalB as potential biomarker for tumors that are or have become (e.g., de novo and acquired) resistant to growth factors blockade. Accordingly, in alternative embodiments, the invention provides compositions and methods for the depletion of RalB, Src, NFkB and its downstream signaling effectors to sensitize ανβ3 -expressing tumors to growth factor blockade. These findings reveal a new role for integrin ανβ3 in mediating tumor cell resistance to growth factor inhibition and demonstrate that targeting the ανβ3/ RalB/ NfkB/ Src signaling pathway will circumvent growth factor inhibitor resistance of a wide range of cancers.
In alternative embodiments, any NF-kB inhibitor can be used to practice this invention, e.g., lenalidomide or (R5)-3-(4-amino-l-oxo-3H-isoindol-2-yl)piperidine-2,6- dione, which can be REVLIMID™ (Celgene Corp., Summit, NJ), or thalidomide, or any other derivative of thalidomide, or any composition having an equivalent activity.
In alternative embodiments, compositions and methods of the invention are used to sensitize tumors to drugs, e.g., such as erlotinib and lapatinib (which are commonly used to treat a wide range of solid tumors). We have shown that when tumors become resistant to these drugs they become very sensitive to NFkB inhibitors. Thus, in alternative embodiments, compositions and methods of the invention are used to sensitize tumors using NFkB inhibitors, such as e.g., lenalidomide or (R5)-3-(4-amino-l-oxo-3H- isoindol-2-yl)piperidine-2,6-dione or REVLIMID™, or a composition as listed in Table 1.
In alternative embodiments, compositions and methods of the invention are used to sensitize tumors using an IKK inhibitor, e.g., such as PS 1145 (Millennium
Pharmaceuticals, Cambridge, MA) (see e.g., Khanbolooki, et al, Mol Cancer Ther 2006; vol. 5:2251-2260; Published online September 19, 2006; Yemelyanov, et al, Oncogene (2006) vol. 25:387-398; published online 19 September 2005), or any ΙκΒα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha)
phosphorylation and/or degradation inhibitor, e.g., one or more compositions listed in Table 3.
In alternative embodiments, compositions and methods of the invention comprise use of an NFkB inhibitor and an IKK inhibitor to treat a drug resistant tumor, e.g., a solid tumor. In alternative embodiments, compositions and methods of the invention comprise use of an NFkB inhibitor and an IKK inhibitor to treat a drug resistant tumor in combination with an anticancer drug, e.g., an NFkB inhibitor and an IKK inhibitor are used to sensitize a tumor to drugs such as erlotinib and lapatinib. In alternative embodiments, the drug combination used to practice the invention comprises
lenalidomide (such as a REVLIMID™) and the IKK inhibitor PSl 145 (Millennium Pharmaceuticals, Cambridge, MA). For example, lenalidomide (such as a REVLIMID™) and PSl 145 are used to sensitize a tumor that is resistant to a cancer drug, e.g., an EGFR inhibitor, such that the tumor is now responsive to the cancer drug.
In alternative embodiments, in practicing the invention, an NFkB inhibitor and an IKK inhibitor are used in combination with a tyrosine kinase receptor (also called Receptor Tyrosine Kinases, or RTKs) inhibitor, e.g., an SU14813 (Pfizer, San Diego, CA) or as listed in Table 2 or 3, below, to treat a drug resistant tumor. In alternative embodiments, compositions and methods of the invention (e.g., including lenalidomide or PSl 145; lenalidomide and PSl 145; or lenalidomide, PSl 145 and an RTK inhibitor are administered to patients that have become resistant to a cancer drug, e.g., drugs like erotinib or lapatinib, to produce a strong antitumor effect.
In alternative embodiments, any NF-kB inhibitor can be used to practice this invention, e.g., an antioxidant can be used to inhibit activation of NF-kB, e.g., including the compositions listed in Table 1 :
Table 1 : Antioxidants that have been shown to inhibit activation of NF-kB
Figure imgf000026_0001
Bruguiera gymnorrhiza compounds Homhual et al, 2006
Israel et al, 1992; Schulze-Osthoffet
Butylated hydroxyanisole (BHA) al, 1993
Okamoto et al, 1994;Tamatani et al,
Cepharanthine 2007
Caffeic Acid Phenethyl Ester (3,4- Natarajan et al, 1996;Nagasaka et al, dihydroxycinnamic acid, CAPE) 2007
Carnosol Lo et al, 2002; Huang et al, 2005
Bai et al, 2005;Guruvayoorappan& beta-Carotene Kuttan, 2007
Carvedilol Yang et al, 2003
Suzuki & Packer, 1994;Zheng et al,
Catechol Derivatives 2008
Centaurea L (Asteraceae) extracts Karamenderes et al, 2007
Chalcone Liu et al, 2007
Chlorogenic acid Feng et al, 2005
5 -chloroacetyl-2-amnio- 1 ,3 -selenazoles Nam et al, 2008
Cholestin Lin et al, 2007
Chroman-2-carboxylic acid N-substituted
phenylamides Kwak et al, 2008
Cocoa polyphenols Lee et al, 2006
Coffee extract (3-methyl-l,2- cyclopentanedione) Chung et al, 2007
Crataegus pinnatifida polyphenols Kao et al, 2007
Curcumin (Diferulolylmethane); Singh & Aggarwal, 1995;Pae et al, dimethoxycurcumin; EF24 analog 2008; Kasinskiet al, 2008
Dehydroepiandrosterone (DHEA)
and DHEA-sulfate (DHEAS) Iwasaki et al, 2004; Liuet al, 2005
Dibenzylbutyrolactone lignans Cho et al, 2002
Diethyldithiocarbamate (DDC) Schreck et al, 1992
Sappey et al, 1995;Schreck et al,
Diferoxamine 1992
Dihydroisoeugenol; isoeugenol; Murakami et al, 1995;Park et al, epoxypseudoisoeugenol-2-methyl butyrate 2007; Ma et al, 2008
Dihydrolipoic Acid Suzuki et al, 1992, 1995
Dilazep + fenofibric acid Sonoki et al, 2003; Yanget al, 2005
Dimethyldithiocarbamates (DMDTC) Pyatt et al, 1998
Dimethylsulfoxide (DMSO) Kelly et al, 1994
Disulfiram Schreck et al, 1992
Ebselen Schreck et al, 1992
Kokura et al, 2005; Ariiet al,
Edaravone 2007; Yoshida et al, 2007
EPC-Kl (phosphodiester compound of vitamin
E and vitamin C) Hirano et al, 1998
Epigallocatechin-3-gallate (EGCG; green tea Lin & Lin, 1997; Yang et polyphenols) al,1998; Hou et al, 2007
Figure imgf000028_0001
Magnolol 2006; Kim et al, 2007
Figure imgf000029_0001
-a y-cystene , gar c compoun engeta, 7 Salogaviolide (Centaurea ainetensis) Ghantous et al, 2008
Lee et al, 2003; Hwang et al,
Sauchinone 2003
Schisandrin B Giridharan et all, 2011
Silybin Gazak et al, 2007
Spironolactone Han et al, 2006
Strawberry extracts Wang et al, 2005
Taxifolin Wang et al, 2005
Tempol Cuzzocrea et al, 2004
Tepoxaline (5 -(4-chlorophenyl)-N-hydroxy-(4- methoxyphenyl) -N-methyl-lH-pyrazole-3- propanamide) Kazmi et al, 1995; Ritchieet al, 1995
Thio avarol derivatives Amigo et al, 2007; Amigoet al, 2008
El Gazzar et al, 2007;lSethi et al,
Thymoquinone 2008
Tocotrienol (palm oil) Wu et al, 2008
Tomato peel polysaccharide De Stefano et al, 2007
UDN glycoprotein (Ulmus davidiana Nakai) Lee & Lim, 2007
Vaccinium stamineum (deerberry) extract Wang et al, 2007
Vanillin (2-hydroxy-3 -methoxybenzaldehyde) Murakami et al, 2007
Vitamin C Staal et al, 1993; Son et al, 2004
Vitamin B6 Yanaka et al, 2005
Suzuki & Packer, 1993;Ekstrand- Hammarstrom et al, 2007; Glauert,
Vitamin E and derivatives 2007
Staal et al, 1993; Suzuki & Packer, a-torphryl succinate 1993
a-torphryl acetate Suzuki & Packer, 1993
PMC (2,2,5,7,8-pentamethyl-6- hydroxychromane) Suzuki & Packer, 1993
Yakuchinone A and B Chun et al, 2002
In alternative embodiments, any proteasome inhibitor and/or protease inhibitor can be used to practice the invention, e.g., any proteasome inhibitor and/or protease inhibitor that can inhibit Rel and/or NF-kB can be used to practice this invention, e.g., including the compositions listed in Table 2:
Table 2: Proteasome and proteases inhibitors that inhibit Rei/NF-kB
Figure imgf000030_0001
Figure imgf000031_0001
chloromethyl ketone) In alternative embodiments, any ΙκΒα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) phosphorylation and/or degradation inhibitor can be used to practice this invention, e.g., including the compositions listed in Table 3: Table 3 : ΙκΒα phosphorylation and/or degradation inhibitors
Figure imgf000032_0001
Figure imgf000033_0001
Cobrotoxin binding Park et al. 2005
Figure imgf000034_0001
nicotinonitrile analog) Sanda et al. 2006
Figure imgf000035_0001
Figure imgf000036_0001
Marasmius oreades liquid extract IKK activity Petrova et al, 2008
Figure imgf000037_0001
Figure imgf000038_0001
Aspirin, sodium salicylate [KKbeta 1995; Kopp & Ghosh.
Figure imgf000039_0001
Figure imgf000040_0001
mega atty ac s P osp oryaton Nova et a.
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
gamma-glutamylcysteine Degradation Manna et al, 1999 synthetase
Singleton et al,
2005; Fillmann et al,
Glutamine Degradation 2007; Chen et al. 2008
Glycochenodeoxycholate Degradation Bucher et al. 2006
Guave leaf extract Degradation Choi et al. 2008
Gumiganghwaltang Degradation Kim et al. 2005
Gum mastic Degradation He et al, 2007
Chan et al, 2004;Shi et al,
Heat shock protein-70 Degradation 2006
Herbal mixture (Cinnamomi
ramulus, Anemarrheriae rhizoma,
Officinari rhizoma) Degradation Jeong et al. 2008
Hypochlorite Degradation Mohri et al. 2002
Kiebala & Maggirwar,
Ibudilast Degradation 1998
IL-13 Degradation Manna & Aggarwal. 1998
Incensole acetate Degradation Moussaieff et al, 2007
Intravenous immunoglobulin Degradation Ichivama et al, 2004
Isomallotochromanol and
is omallotochromene Degradation Ishii et al. 2003
K1L (Vaccinia virus protein) Degradation Shisler & Jin. 2004
Kochia scoparia fruit (methanol
extract) Degradation Shin et al. 2004
Kummerowia striata (Thunb.)
Schindl (ethanol extract) Degradation Tao et al. 2008
Leflunomide metabolite (A77
1726) Degradation Manna & Aggarwal, 1999
Feng et al, 2007;Lahat et
Lidocaine Degradation al, 2008
Lipoxin A4 Degradation Zhang et al. 2007
Degradation/NF-kB Chen et al. 2002;Zhu et
Losartan expression al, 2007
Low level laser therapy Degradation izzi et al. 2006
LY294002 (PI3-kinase
inhibitor) [2-(4-morpholinyl)-8- phenylchromone] Degradation Park et al. 2002
MCI 59 (Molluscum contagiosum
virus) Degradation of IkBb Murao & Shisler. 2005
Melatonin Degradation Zhang et al. 2004
Meloxicam Degradation Liu et al, 2007
5'-methylthioadenosine Degradation Hevia et al. 2004
Midazolam Degradation Kim et al. 2006 Momordin I Degradation Hwang et al. 2005
Morinda officinalis extract Degradation Kim et al. 2005
Mosla dianthera extract Degradation Lee et al. 2006
Mume fructus extract Degradation Choi et al. 2007
Murrl gene product Degradation Ganesh et al. 2003
Neurofibromatosis-2 (NF-2;
merlin) protein Degradation Kim et al. 2002
Opuntia ficus indica va saboten
extract Degradation Lee et al, 2006
Ozone (aqueous) Degradation Huth et al. 2007
Paeony total glucosides Degradation Chen et al. 2007
Pectenotoxin-2 Degradation Kim et al. 2008
Penetratin Degradation Letova et al. 2006
Pervanadate (tyrosine phosphatase Singh & Aggarwal, inhibitor) Degradation 1995; Singh et al. 1996
Mahboubi et al.
Phenylarsine oxide (PAO, tyrosine 1998; Singh & Aggarwal, phosphatase inhibitor) Degradation 1995
beta-Phenylethyl (PEITC) and 8- methylsulphinyloctyl
isothiocyanates (MSO)
(watercress) Degradation Rose et al. 2005
Phenytoin Degradation Kato et al, 2005 c-phycocyanin Degradation Cherng et al. 2007
Ahn et al, 2005; Leeet al,
Platycodin saponins Degradation 2008
Polymeric formula Degradation de Jong et al, 2007
Polymyxin B Degradation Jiang et al, 2006
Degradation; Shin et al. 2006;Kim et
Poncirus trifoliata fruit extract phosphorylation of IkBa al, 2007
Probiotics Degradation Petrof et al. 2004
Pituitary adenylate cyclase- activating polypeptide (PA CAP) Degradation Delgado & Ganea, 2001
Cuzzocrea et al,
Prostaglandin 15-deoxy- 2003; Chatteriee et al. Delta(12, 14)-PGJ(2) Degradation 2004
Prodigiosin (Hahella chejuensis) Degradation Huh et al. 2007
PS-341 Degradation/proteasome Hideshima et al, 2002
Radix asari extract Degradation Song et al. 2007
Radix clematidis extract Degradation Lee et al, 2009
Resiniferatoxin Degradation Singh et al. 1996
Sabaeksan Degradation Choi et al. 2005
SAIF (Saccharomyces boulardii Degradation Sougioultzis et al. 2006
Figure imgf000046_0001
Ro 106-9920 (small molecule) inhibitor Swinnev et al. 2002 jFuronaphthoquinone |IKK activity Shin et al, 2006
Pharmaceutical compositions
In alternative embodiments, the invention provides pharmaceutical compositions for practicing the methods of the invention, e.g., pharmaceutical compositions for overcoming or diminishing or preventing Growth Factor Inhibitor (GFI) resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor Inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor.
In alternative embodiments, compositions used to practice the methods of the invention are formulated with a pharmaceutically acceptable carrier. In alternative embodiments, the pharmaceutical compositions used to practice the methods of the invention can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's").
Therapeutic agents used to practice the methods of the invention
administered alone or as a component of a pharmaceutical formulation (composition). The compounds may be formulated for administration in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Formulations of the compositions used to practice the methods of the invention include those suitable for oral/ nasal, topical, parenteral, rectal, and/or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
Pharmaceutical formulations used to practice the methods of the invention can be prepared according to any method known to the art for the manufacture of
pharmaceuticals. Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, geltabs, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores. Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen. Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound (i.e., dosage). Pharmaceutical preparations used to practice the methods of the invention can also be used orally using, e.g., push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
Aqueous suspensions can contain an active agent (e.g., a composition used to practice the methods of the invention) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxy ethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity.
Oil-based pharmaceuticals are particularly useful for administration hydrophobic active agents used to practice the methods of the invention. Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g., U.S. Patent No. 5,716,928 describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Patent No. 5,858,401). The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281 :93-102. The pharmaceutical formulations of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent.
In practicing this invention, the pharmaceutical compounds can also be administered by in intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see Rohatagi (1995) J. Clin. Pharmacol. 35: 1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75: 107-11 1). Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug. Such materials are cocoa butter and polyethylene glycols.
In practicing this invention, the pharmaceutical compounds can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
In practicing this invention, the pharmaceutical compounds can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.
In practicing this invention, the pharmaceutical compounds can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ. These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3- butanediol. The administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).
The pharmaceutical compounds and formulations used to practice the methods of the invention can be lyophilized. The invention provides a stable lyophilized formulation comprising a composition of the invention, which can be made by lyophilizing a solution comprising a pharmaceutical of the invention and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof. A process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. patent app. no. 20040028670.
The compositions and formulations used to practice the methods of the invention can be delivered by the use of liposomes (see also discussion, below). By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Patent Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13 :293-306; Chonn (1995) Curr. Opin.
Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46: 1576-1587.
The formulations used to practice the methods of the invention can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a subject already suffering from a condition, infection or disease in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the condition, infection or disease and its complications (a
"therapeutically effective amount"). For example, in alternative embodiments, pharmaceutical compositions of the invention are administered in an amount sufficient to treat, prevent and/or ameliorate normal, dysfunction (e.g., abnormally proliferating) cell, e.g., cancer cell, or blood vessel cell, including endothelial and/or capillary cell growth; including neovasculature related to (within, providing a blood supply to) hyperplastic tissue, a granuloma or a tumor. The amount of pharmaceutical composition adequate to accomplish this is defined as a "therapeutically effective dose." The dosage schedule and amounts effective for this use, i.e., the "dosing regimen," will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.
The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:61 1-617; Groning (1996) Pharmazie 51 :337-341 ; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84: 1 144-1 146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24: 103-108; the latest Remington's, supra). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods of the invention are correct and appropriate.
Single or multiple administrations of formulations can be given depending on the dosage and frequency as required and tolerated by the patient. The formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate a conditions, diseases or symptoms as described herein. For example, an exemplary pharmaceutical formulation for oral administration of compositions used to practice the methods of the invention can be in a daily amount of between about 0.1 to 0.5 to about 20, 50, 100 or 1000 or more ug per kilogram of body weight per day. In an alternative embodiment, dosages are from about 1 mg to about 4 mg per kg of body weight per patient per day are used. Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ. Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation. Actual methods for preparing parenterally or non-parenterally
administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's, supra.
The methods of the invention can further comprise co-administration with other drugs or pharmaceuticals, e.g., compositions for treating cancer, septic shock, infection, fever, pain and related symptoms or conditions. For example, the methods and/or compositions and formulations of the invention can be co-formulated with and/or coadministered with antibiotics (e.g., antibacterial or bacteriostatic peptides or proteins), particularly those effective against gram negative bacteria, fluids, cytokines,
immunoregulatory agents, anti-inflammatory agents, complement activating agents, such as peptides or proteins comprising collagen-like domains or fibrinogen-like domains (e.g., a ficolin), carbohydrate-binding domains, and the like and combinations thereof.
Nanoparticles and Liposomes
The invention also provides nanoparticles and liposomal membranes comprising compounds used to practice the methods of the invention. In alternative embodiments, the invention provides nanoparticles and liposomal membranes targeting diseased and/or tumor (cancer) stem cells and dysfunctional stem cells, and angiogenic cells.
In alternative embodiments, the invention provides nanoparticles and liposomal membranes comprising (in addition to comprising compounds used to practice the methods of the invention) molecules, e.g., peptides or antibodies, that selectively target abnormally growing, diseased, infected, dysfunctional and/or cancer (tumor) cell receptors. In alternative embodiments, the invention provides nanoparticles and liposomal membranes using IL-11 receptor and/or the GRP78 receptor to targeted receptors on cells, e.g., on tumor cells, e.g., on prostate or ovarian cancer cells. See, e.g., U.S. patent application publication no. 20060239968.
In one aspect, the compositions used to practice the methods of the invention are specifically targeted for inhibiting, ameliorating and/or preventing endothelial cell migration and for inhibiting angiogenesis, e.g., tumor-associated or disease- or infection- associated neovasculature.
The invention also provides nanocells to allow the sequential delivery of two different therapeutic agents with different modes of action or different pharmacokinetics, at least one of which comprises a composition used to practice the methods of the invention. A nanocell is formed by encapsulating a nanocore with a first agent inside a lipid vesicle containing a second agent; see, e.g., Sengupta, et al, U.S. Pat. Pub. No. 20050266067. The agent in the outer lipid compartment is released first and may exert its effect before the agent in the nanocore is released. The nanocell delivery system may be formulated in any pharmaceutical composition for delivery to patients suffering from a diseases or condition as described herein, e.g., such as a retinal age-related macular degeneration, a diabetic retinopathy, a cancer or carcinoma, a glioblastoma, a neuroma, a neuroblastoma, a colon carcinoma, a hemangioma, an infection and/or a condition with at least one inflammatory component, and/or any infectious or inflammatory disease, such as a rheumatoid arthritis, a psoriasis, a fibrosis, leprosy, multiple sclerosis, inflammatory bowel disease, or ulcerative colitis or Crohn's disease.
In treating cancer, a traditional antineoplastic agent is contained in the outer lipid vesicle of the nanocell, and an antiangiogenic agent of this invention is loaded into the nanocore. This arrangement allows the antineoplastic agent to be released first and delivered to the tumor before the tumor's blood supply is cut off by the composition of this invention.
The invention also provides multilayered liposomes comprising compounds used to practice this invention, e.g., for transdermal absorption, e.g., as described in Park, et al, U.S. Pat. Pub. No. 20070082042. The multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition of this invention. A multilayered liposome used to practice the invention may further include an antiseptic, an antioxidant, a stabilizer, a thickener, and the like to improve stability.
Synthetic and natural antiseptics can be used, e.g., in an amount of 0.01% to 20%.
Antioxidants can be used, e.g., BHT, erysorbate, tocopherol, astaxanthin, vegetable flavonoid, and derivatives thereof, or a plant-derived antioxidizing substance. A stabilizer can be used to stabilize liposome structure, e.g., polyols and sugars. Exemplary polyols include butylene glycol, polyethylene glycol, propylene glycol, dipropylene glycol and ethyl carbitol; examples of sugars are trehalose, sucrose, mannitol, sorbitol and chitosan, or a monosaccharides or an oligosaccharides, or a high molecular weight starch. A thickener can be used for improving the dispersion stability of constructed liposomes in water, e.g., a natural thickener or an acrylamide, or a synthetic polymeric thickener. Exemplary thickeners include natural polymers, such as acacia gum, xanthan gum, gellan gum, locust bean gum and starch, cellulose derivatives, such as hydroxy ethylcellulose, hydroxypropyl cellulose and carboxymethyl cellulose, synthetic polymers, such as polyacrylic acid, poly-acrylamide or polyvinylpyrollidone and polyvinylalcohol, and copolymers thereof or cross-linked materials.
Liposomes can be made using any method, e.g., as described in Park, et al, U.S. Pat. Pub. No. 20070042031, including method of producing a liposome by encapsulating a therapeutic product comprising providing an aqueous solution in a first reservoir;
providing an organic lipid solution in a second reservoir, wherein one of the aqueous solution and the organic lipid solution includes a therapeutic product; mixing the aqueous solution with said organic lipid solution in a first mixing region to produce a liposome solution, wherein the organic lipid solution mixes with said aqueous solution so as to substantially instantaneously produce a liposome encapsulating the therapeutic product; and immediately thereafter mixing the liposome solution with a buffer solution to produce a diluted liposome solution.
The invention also provides nanoparticles comprising compounds used to practice this invention to deliver a composition of the invention as a drug-containing nanoparticles (e.g., a secondary nanoparticle), as described, e.g., in U.S. Pat. Pub. No. 20070077286. In one embodiment, the invention provides nanoparticles comprising a fat-soluble drug of this invention or a fat-solubilized water-soluble drug to act with a bivalent or trivalent metal salt. Liposomes
The compositions and formulations used to practice the invention can be delivered by the use of liposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Patent Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13 :293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46: 1576-1587. For example, in one embodiment, compositions and formulations used to practice the invention are delivered by the use of liposomes having rigid lipids having head groups and hydrophobic tails, e.g., as using a polyethyleneglycol-linked lipid having a side chain matching at least a portion the lipid, as described e.g., in US Pat App Pub No. 20080089928. In another embodiment, compositions and formulations used to practice the invention are delivered by the use of amphoteric liposomes comprising a mixture of lipids, e.g., a mixture comprising a cationic amphiphile, an anionic amphiphile and/or neutral amphiphiles, as described e.g., in US Pat App Pub No. 20080088046, or 20080031937. In another embodiment, compositions and formulations used to practice the invention are delivered by the use of liposomes comprising a polyalkylene glycol moiety bonded through a thioether group and an antibody also bonded through a thioether group to the liposome, as described e.g., in US Pat App Pub No. 20080014255. In another embodiment, compositions and formulations used to practice the invention are delivered by the use of liposomes comprising glycerides, glycerophospholipides, glycerophosphinolipids, glycerophosphonolipids, sulfolipids, sphingolipids,
phospholipids, isoprenolides, steroids, stearines, sterols and/or carbohydrate containing lipids, as described e.g., in US Pat App Pub No. 20070148220. Antibodies as Pharmaceutical compositions
In alternative embodiments, the invention provides compositions and methods for inhibiting or depleting an integrin ανβ3 (avb3), or inhibiting an integrin ανβ3 (avb3) protein activity, or inhibiting the formation or activity of an integrin avb3/RalB signaling complex, or inhibiting the formation or signaling activity of an integrin ανβ3
(avb3)/RalB/NFkB signaling axis; or inhibiting or depleting a RalB protein or an inhibitor of RalB protein activation; or inhibiting or depleting a Src or TBK1 protein or an inhibitor of Src or TBK1 protein activation. In alternative embodiments, this is achieved by administration of inhibitory antibodies. For example, in alternative embodiments, the invention uses isolated, synthetic or recombinant antibodies that specifically bind to and inhibit an integrin ανβ3 (avb3), or any protein of an integrin ανβ3 (avb3)/RalB/NFkB signaling axis, a RalB protein, a Src or TBK1 protein, or an NFkB protein.
In alternative embodiments, the invention provides methods for determining whether an individual or a patient would benefit from or respond to administration of a Growth Factor Inhibitor, or which individuals or patients would benefit from a combinatorial approach comprising administration of a combination of: at least one growth factor inhibitor and at least one compound, composition or formulation used to practice this invention, including for example, an NFkB inhibitor, the method comprising: detecting the levels or amount of integrin ανβ3 (avb3) and/or active RalB complex in or on a cell, a tissue or a tissue sample. In alternative embodiments, the levels or amount of integrin ανβ3 (avb3) and/or active RalB complex in or on a cell, a tissue or a tissue sample is detected or measured using an antibody, e.g., including use of immunofluorescence or FACS analysis. In alternative embodiments, a preferred method for detecting the integrin ανβ3 (avb3) marker is by detecting the heterodimer alpha-v/beta3, and not beta3 alone; the reason for this is that beta3 is also expressed on platelets, so testing for any alpha-v/beta3 heterodimer will exclude platelet expression (if platelets have not already been removed from the sample, or are not excluded from the count).
In alternative aspects, an antibody for practicing the invention can comprise a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. Fundamental Immunology, Third Edition, W.E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. In alternative aspects, an antibody for practicing the invention includes antigen-binding portions, i.e., "antigen binding sites," (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term "antibody."
In alternative embodiments, the invention uses "humanized" antibodies, including forms of non-human (e.g., murine) antibodies that are chimeric antibodies comprising minimal sequence (e.g., the antigen binding fragment) derived from non- human immunoglobulin. In alternative embodiments, humanized antibodies are human immunoglobulins in which residues from a hypervariable region (HVR) of a recipient (e.g., a human antibody sequence) are replaced by residues from a hypervariable region (HVR) of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In alternative embodiments, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues to improve antigen binding affinity.
In alternative embodiments, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to improve antibody affinity or functional activity. In alternative embodiments, the humanized antibody can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of Ab framework regions are those of a human immunoglobulin sequence.
In alternative embodiments, a humanized antibody used to practice this invention can comprise at least a portion of an immunoglobulin constant region (Fc), typically that of or derived from a human immunoglobulin.
However, in alternative embodiments, completely human antibodies also can be used to practice this invention, including human antibodies comprising amino acid sequence which corresponds to that of an antibody produced by a human. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues.
In alternative embodiments, antibodies used to practice this invention comprise "affinity matured" antibodies, e.g., antibodies comprising with one or more alterations in one or more hypervariable regions which result in an improvement in the affinity of the antibody for antigen; e.g., NFkB, an integrin ανβ3 (avb3), or any protein of an integrin ανβ3 (anb3)/RalB/NFkB signaling axis, a RalB protein, a Src or TBK1 protein, compared to a parent antibody which does not possess those alteration(s). In alternative
embodiments, antibodies used to practice this invention are matured antibodies having nanomolar or even picomolar affinities for the target antigen, e.g., NFkB, an integrin ανβ3 (avb3), or any protein of an integrin ανβ3 (avb3)/RalB/NFkB signaling axis, a RalB protein, a Src or TBK1 protein. Affinity matured antibodies can be produced by procedures known in the art.
Antisense, siRNAs and microRNAs as Pharmaceutical compositions
In alternative embodiments, the invention provides compositions and methods for inhibiting or depleting an integrin ανβ3 (avb3), or inhibiting an integrin ανβ3 (avb3) protein activity, or inhibiting the formation or activity of an integrin anb3/RalB signaling complex, or inhibiting the formation or signaling activity of an integrin ανβ3
(avb3)/RalB/NFkB signaling axis; or inhibiting or depleting a RalB protein or an inhibitor of RalB protein activation; or inhibiting or depleting a Src or TBK1 protein or an inhibitor of Src or TBK1 protein activation. In alternative embodiments, this is achieved by administration of inhibitory nucleic acids, e.g., siRNA, antisense nucleic acids, and/or inhibitory microRNAs.
In alternative embodiments, compositions used to practice the invention are formulated with a pharmaceutically acceptable carrier. In alternative embodiments, the pharmaceutical compositions used to practice the invention can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's").
While the invention is not limited by any particular mechanism of action:
microRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that are involved in post- transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. miRNAs are transcribed by RNA polymerase II as part of capped and polyadenylated primary transcripts (pri-miRNAs) that can be either protein-coding or non-coding. The primary transcript is cleaved by the Drosha ribonuclease III enzyme to produce an approximately 70-nt stem-loop precursor miRNA (pre-miRNA), which is further cleaved by the cytoplasmic Dicer ribonuclease to generate the mature miRNA and antisense miRNA star (miRNA*) products. The mature miRNA is incorporated into a RNA-induced silencing complex (RISC), which recognizes target mRNAs through imperfect base pairing with the miRNA and most commonly results in translational inhibition or destabilization of the target mRNA.
In alternative embodiments pharmaceutical compositions used to practice the invention are administered in the form of a dosage unit, e.g., a tablet, capsule, bolus, spray. In alternative embodiments, pharmaceutical compositions comprise a compound, e.g., an antisense nucleic acid, e.g., an siRNA or a microRNA, in a dose: e.g., 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg: 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg , 145 mg , 150 mg , 155 mg , 160 mg , 165 mg , 170 mg , 175 mg , 180 mg , 185 mg , 190 mg , 195 mg ,200 mg ,205 mg ,210 mg ,215 mg ,220 mg ,225 mg ,230 mg ,235 mg ,240 mg ,245 mg ,250 mg ,255 mg ,260 mg ,265 mg ,270 mg ,270 mg ,280 mg ,285 mg ,290 mg ,295 mg , 300 mg ,305 mg ,310 mg ,315 mg , 320 mg ,325 mg ,330 mg ,335 mg ,340 mg ,345 mg , 350 mg ,355 mg ,360 mg ,365 mg , 370 mg ,375 mg ,380 mg ,385 mg ,390 mg ,395 mg ,400 mg ,405 mg ,410 mg ,415 mg ,420 mg ,425 mg ,430 mg ,435 mg ,440 mg ,445 mg ,450 mg ,455 mg ,460 mg ,465 mg ,470 mg ,475 mg ,480 mg ,485 mg ,490 mg ,495 mg , 500 mg ,505 mg ,510 mg ,515 mg , 520 mg ,525 mg ,530 mg ,535 mg ,540 mg ,545 mg , 550 mg ,555 mg ,560 mg ,565 mg , 570 mg ,575 mg ,580 mg ,585 mg ,590 mg ,595 mg , 600 mg ,605 mg ,610 mg ,615 mg , 620 mg ,625 mg ,630 mg ,635 mg ,640 mg ,645 mg , 650 mg ,655 mg ,660 mg ,665 mg , 670 mg ,675 mg ,680 mg ,685 mg ,690 mg ,695 mg , 700 mg ,705 mg ,710 mg ,715 mg , 720 mg ,725 mg ,730 mg ,735 mg ,740 mg ,745 mg , 750 mg ,755 mg ,760 mg ,765 mg , 770 mg ,775 mg ,780 mg ,785 mg ,790 mg ,795 mg , or 800 mg or more.
In alternative embodiments, an siRNA or a microRNA used to practice the invention is administered as a pharmaceutical agent, e.g., a sterile formulation, e.g., a lyophilized siRNA or microRNA that is reconstituted with a suitable diluent, e.g., sterile water for injection or sterile saline for injection. In alternative embodiments the reconstituted product is administered as a subcutaneous injection or as an intravenous infusion after dilution into saline. In alternative embodiments the lyophilized drug product comprises siRNA or microRNA prepared in water for injection, or in saline for injection, adjusted to pH 7.0-9.0 with acid or base during preparation, and then lyophilized. In alternative embodiments a lyophilized siRNA or microRNA of the invention is between about 25 to 800 or more mg, or about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mg of a siRNA or microRNA of the invention. The lyophilized siRNA or microRNA of the invention can be packaged in a 2 mL Type I, clear glass vial (e.g., ammonium sulfate-treated), e.g., stoppered with a bromobutyl rubber closure and sealed with an aluminum overseal.
In alternative embodiments, the invention provides compositions and methods comprising in vivo delivery of antisense nucleic acids, e.g., siRNA or microRNAs. In practicing the invention, the antisense nucleic acids, siRNAs, or microRNAs can be modified, e.g., in alternative embodiments , at least one nucleotide of antisense nucleic acid, e.g., siRNA or microRNA, construct is modified, e.g., to improve its resistance to nucleases, serum stability, target specificity, blood system circulation, tissue distribution, tissue penetration, cellular uptake, potency, and/or cell-permeability of the
polynucleotide. In alternative embodiments, the antisense nucleic acid, siRNA or microRNA construct is unmodified. In other embodiments, at least one nucleotide in the antisense nucleic acid, siRNA or microRNA construct is modified.
In alternative embodiments, guide strand modifications are made to increase nuclease stability, and/or lower interferon induction, without significantly decreasing antisense nucleic acid, siRNA or microRNA activity (or no decrease in antisense nucleic acid, siRNA or microRNA activity at all). In certain embodiments, the modified antisense nucleic acid, siRNA or microRNA constructs have improved stability in serum and/or cerebral spinal fluid compared to an unmodified structure having the same sequence.
In alternative embodiments, a modification includes a 2'-H or 2'-modified ribose sugar at the second nucleotide from the 5'-end of the guide sequence. In alternative embodiments, the guide strand (e.g., at least one of the two single-stranded
polynucleotides) comprises a 2'-0-alkyl or 2'-halo group, such as a 2'-0-methyl modified nucleotide, at the second nucleotide on the 5'-end of the guide strand, or, no other modified nucleotides. In alternative embodiments, polynucleotide constructs having such modification may have enhanced target specificity or reduced off-target silencing compared to a similar construct without the 2'-0-methyl modification at the position.
In alternative embodiments, a second nucleotide is a second nucleotide from the
5'-end of the single-stranded polynucleotide. In alternative embodiments, a "2'-modified ribose sugar" comprises ribose sugars that do not have a 2'-OH group. In alternative embodiments, a "2'-modified ribose sugar" does not include 2'-deoxyribose (found in unmodified canonical DNA nucleotides), although one or more DNA nucleotides may be included in the subject constructs (e.g., a single deoxyribonucleotide, or more than one deoxyribonucleotide in a stretch or scattered in several parts of the subject constructs). For example, the 2'-modified ribose sugar may be 2'-0-alkyl nucleotides, 2'-deoxy-2'- fluoro nucleotides, 2'-deoxy nucleotides, or combination thereof.
In alternative embodiments, an antisense nucleic acid, siRNA or microRNA construct used to practice the invention comprises one or more 5'-end modifications, e.g., as described above, and can exhibit a significantly (e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more) less "off-target" gene silencing when compared to similar constructs without the specified 5 '-end modification, thus greatly improving the overall specificity of the antisense nucleic acid, siRNA or microRNA construct of the invention.
In alternative embodiments, an antisense nucleic acid, siRNA or microRNA construct to practice the invention comprises a guide strand modification that further increase stability to nucleases, and/or lowers interferon induction, without significantly decreasing activity (or no decrease in microRNA activity at all). In alternative embodiments, the 5'-stem sequence comprises a 2'-modified ribose sugar, such as 2'-0- methyl modified nucleotide, at the second nucleotide on the 5'-end of the polynucleotide, or, no other modified nucleotides. In alternative embodiments the hairpin structure having such modification has enhanced target specificity or reduced off-target silencing compared to a similar construct without the 2'-0-methyl modification at same position.
In alternative embodiments, the 2'-modified nucleotides are some or all of the pyrimidine nucleotides (e.g., C/U). Examples of 2'-0-alkyl nucleotides include a 2'-0- methyl nucleotide, or a 2'-0-allyl nucleotide. In alternative embodiments, the modification comprises a 2'-0-methyl modification at alternative nucleotides, starting from either the first or the second nucleotide from the 5'-end. In alternative embodiments, the modification comprises a 2'-0-methyl modification of one or more randomly selected pyrimidine nucleotides (C or U). In alternative embodiments, the modification comprises a 2'-0-methyl modification of one or more nucleotides within the loop.
In alternative embodiments, the modified nucleotides are modified on the sugar moiety, the base, and/or the phosphodiester linkage. In alternative embodiments the modification comprise a phosphate analog, or a phosphorothioate linkage; and the phosphorothioate linkage can be limited to one or more nucleotides within the loop, a 5'- overhang, and/or a 3 '-overhang.
In alternative embodiments, the phosphorothioate linkage may be limited to one or more nucleotides within the loop, and 1, 2, 3, 4, 5, or 6 more nucleotide(s) of the guide sequence within the double-stranded stem region just 5' to the loop. In alternative embodiments, the total number of nucleotides having the phosphorothioate linkage may be about 12-14. In alternative embodiments, all nucleotides having the phosphorothioate linkage are not contiguous. In alternative embodiments, the modification comprises a 2'- O-methyl modification, or, no more than 4 consecutive nucleotides are modified. In alternative embodiments, all nucleotides in the 3 '-end stem region are modified. In alternative embodiments, all nucleotides 3' to the loop are modified.
In alternative embodiments, the 5'- or 3'-stem sequence comprises one or more universal base-pairing nucleotides. In alternative embodiments universal base-pairing nucleotides include extendable nucleotides that can be incorporated into a polynucleotide strand (either by chemical synthesis or by a polymerase), and pair with more than one pairing type of specific canonical nucleotide. In alternative embodiments, the universal nucleotides pair with any specific nucleotide. In alternative embodiments, the universal nucleotides pair with four pairings types of specific nucleotides or analogs thereof. In alternative embodiments, the universal nucleotides pair with three pairings types of specific nucleotides or analogs thereof. In alternative embodiments, the universal nucleotides pair with two pairings types of specific nucleotides or analogs thereof.
In alternative embodiments, an antisense nucleic acid, siRNA or microR A used to practice the invention comprises a modified nucleoside, e.g., a sugar-modified nucleoside. In alternative embodiments, the sugar-modified nucleosides can further comprise a natural or modified heterocyclic base moiety and/or a natural or modified internucleoside linkage; or can comprise modifications independent from the sugar modification. In alternative embodiments, a sugar modified nucleoside is a 2'-modified nucleoside, wherein the sugar ring is modified at the 2' carbon from natural ribose or 2'- deoxy-ribose.
In alternative embodiments, a 2'-modified nucleoside has a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety is a D sugar in the alpha configuration. In certain such embodiments, the bicyclic sugar moiety is a D sugar in the beta configuration. In certain such embodiments, the bicyclic sugar moiety is an L sugar in the alpha configuration. In alternative embodiments, the bicyclic sugar moiety is an L sugar in the beta configuration.
In alternative embodiments, the bicyclic sugar moiety comprises a bridge group between the 2' and the 4'-carbon atoms. In alternative embodiments, the bridge group comprises from 1 to 8 linked biradical groups. In alternative embodiments, the bicyclic sugar moiety comprises from 1 to 4 linked biradical groups. In alternative embodiments, the bicyclic sugar moiety comprises 2 or 3 linked biradical groups.
In alternative embodiments, the bicyclic sugar moiety comprises 2 linked biradical groups. In alternative embodiments, a linked biradical group is selected from -0—,— S— , -N(R1)-, -C(R1)(R2)-, -C(R1)=C(R1)-, -C(R1)=N-, -C(=NR1)-, -Si(Rl)(R2)-, - S(=0)2-, ~S(=0)~, ~C(=0)- and -C(=S)-; where each Rl and R2 is, independently, H, hydroxyl, CI to Ci2 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-Ci2 alkenyl, C2-Ci2 alkynyl, substituted C2-C12 alkynyl, C2-C20 aryl, substituted C2-C20 aryl, a heterocycle radical, a substituted heterocycle radical, heteroaryl, substituted heteroaryl, C2-C7 alicyclic radical, substituted C2-C7 alicyclic radical, halogen, substituted oxy (—0—), amino, substituted amino, azido, carboxyl, substituted carboxyl, acyl, substituted acyl, CN, thiol, substituted thiol, sulfonyl (S(=0) 2— H), substituted sulfonyl, sulfoxyl (S(=0)-H) or substituted sulfoxyl; and each substituent group is, independently, halogen, Cl-Ci2 alkyl, substituted Cl-C12 alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-Ci2 alkynyl, substituted C2-Ci2 alkynyl, amino, substituted amino, acyl, substituted acyl, Cl-Ci2 aminoalkyl, Cl-Ci2 aminoalkoxy, substituted Cl-Ci2 aminoalkyl, substituted Cl-Ci2 aminoalkoxy or a protecting group. In alternative embodiments, the bicyclic sugar moiety is bridged between the 2' and 4' carbon atoms with a biradical group selected from— 0~(CH2)x~,— O— CH2~,— O— CH2CH2-, -O-CH(alkyl)-, -NH-(CH2)P-, -N(alkyl)-(CH2)x-, -O-CH(alkyl)-, - (CH(alkyl))-(CH2)x-, -NH-0-(CH2)x-, -N(alkyl)-0-(CH2)x-, or -O-N(alkyl)- (CH2)x~, wherein x is 1, 2, 3, 4 or 5 and each alkyl group can be further substituted. In certain embodiments, x is 1, 2 or 3.
In alternative embodiments, a 2'-modified nucleoside comprises a 2'-substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, 0-, S— , or N(Rm)-alkyl; 0-, S-, or N(Rm)-alkenyl; 0-, S- or N(Rm)-alkynyl; O-alkylenyl-0- alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, 0(CH2) 2SCH3, 0-(CH2) 2-0- N(Rm)(Rn) or 0-CH2-C(=0)--N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl. These 2'- substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (N02), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
In alternative embodiments, a 2'-modified nucleoside comprises a 2'-substituent group selected from F, O-CH3, and OCH2CH20CH3.
In alternative embodiments, a sugar-modified nucleoside is a 4'-thio modified nucleoside. In alternative embodiments, a sugar-modified nucleoside is a 4'-thio-2'- modified nucleoside. In alternative embodiments a 4'-thio modified nucleoside has a .beta.-D-ribonucleoside where the 4'-0 replaced with 4'-S. A 4'-thio-2'-modified nucleoside is a 4'-thio modified nucleoside having the 2'-OH replaced with a 2'- substituent group. In alternative embodiments 2'-substituent groups include 2'-OCH3, 2'- 0-(CH2)2-OCH3, and 2'-F.
In alternative embodiments, a modified oligonucleotide of the present invention comprises one or more internucleoside modifications. In alternative embodiments, each internucleoside linkage of a modified oligonucleotide is a modified internucleoside linkage. In alternative embodiments, a modified internucleoside linkage comprises a phosphorus atom.
In alternative embodiments, a modified antisense nucleic acid, siRNA or microRNA comprises at least one phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage of a modified oligonucleotide is a phosphorothioate internucleoside linkage.
In alternative embodiments, a modified internucleoside linkage does not comprise a phosphorus atom. In alternative embodiments, an internucleoside linkage is formed by a short chain alkyl internucleoside linkage. In alternative embodiments, an
internucleoside linkage is formed by a cycloalkyl internucleoside linkages. In alternative embodiments, an internucleoside linkage is formed by a mixed heteroatom and alkyl internucleoside linkage. In alternative embodiments, an internucleoside linkage is formed by a mixed heteroatom and cycloalkyl internucleoside linkages. In alternative embodiments, an internucleoside linkage is formed by one or more short chain heteroatomic internucleoside linkages. In alternative embodiments, an internucleoside linkage is formed by one or more heterocyclic internucleoside linkages. In alternative embodiments, an internucleoside linkage has an amide backbone, or an internucleoside linkage has mixed N, O, S and CH2 component parts.
In alternative embodiments, a modified oligonucleotide comprises one or more modified nucleobases. In certain embodiments, a modified oligonucleotide comprises one or more 5-methylcytosines, or each cytosine of a modified oligonucleotide comprises a 5-methylcytosine.
In alternative embodiments, a modified nucleobase comprises a 5 -hydroxy methyl cytosine, 7-deazaguanine or 7-deazaadenine, or a modified nucleobase comprises a 7- deaza-adenine, 7-deazaguanosine, 2-aminopyridine or a 2-pyridone, or a modified nucleobase comprises a 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O- 6 substituted purines, or a 2 aminopropyladenine, 5-propynyluracil or a 5- propynylcytosine.
In alternative embodiments, a modified nucleobase comprises a polycyclic heterocycle, or a tricyclic heterocycle; or, a modified nucleobase comprises a
phenoxazine derivative, or a phenoxazine further modified to form a nucleobase or G- clamp.
Therapeutically effective amount and doses
In alternative embodiment, compounds, compositions, pharmaceutical compositions and formulations used to practice the invention can be administered for prophylactic and/or therapeutic treatments; for example, the invention provides compositions and methods for overcoming or diminishing or preventing Growth Factor Inhibitor (GFI) resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor Inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor. In alternative embodiments, the invention provides compositions and methods for treating, preventing or ameliorating: a disease or condition associated with dysfunctional stem cells or cancer stem cells, a retinal age-related macular degeneration, a diabetic retinopathy, a cancer or carcinoma, a glioblastoma, a neuroma, a neuroblastoma, a colon carcinoma, a hemangioma, an infection and/or a condition with at least one inflammatory component, and/or any infectious or inflammatory disease, such as a rheumatoid arthritis, a psoriasis, a fibrosis, leprosy, multiple sclerosis, inflammatory bowel disease, or ulcerative colitis or Crohn's disease. In therapeutic applications, compositions are administered to a subject already suffering from a condition, infection or disease in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the condition, infection or disease (e.g., disease or condition associated with dysfunctional stem cells or cancer stem cells) and its complications (a "therapeutically effective amount"). In the methods of the invention, a pharmaceutical composition is administered in an amount sufficient to treat (e.g., ameliorate) or prevent a disease or condition associated with dysfunctional stem cells or cancer stem cells. The amount of pharmaceutical composition adequate to accomplish this is defined as a "therapeutically effective dose." The dosage schedule and amounts effective for this use, i.e., the "dosing regimen," will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.
Kits and Instructions
The invention provides kits comprising compositions for practicing the methods of the invention, including instructions for use thereof. In alternative embodiments, the invention provides kits, blister packages, lidded blisters or blister cards or packets, clamshells, trays or shrink wraps comprising a combination of compounds. In alternative embodiments, the combination of compounds comprises:
(1) at least one compound comprising or consisting of: (i) an inhibitor or depleter of integrin ανβ3 (avb3), or an inhibitor of integrin ανβ3 (avb3) protein activity, or an inhibitor of the formation or activity of an integrin avb3/RalB signaling complex, or an inhibitor of the formation or signaling activity of an integrin ανβ3 (anb3)/RalB/NFkB signaling axis,
(ii) an inhibitor or depleter of RalB protein or an inhibitor of RalB protein activation,
wherein optionally the inhibitor of RalB protein activity is an allosteric inhibitor of RalB protein activity;
(iii) an inhibitor or depleter of Src or TBK1 protein or an inhibitor of Src or TBK1 protein activation,
wherein optionally the inhibitor of Src or TBK1 protein activity is an allosteric inhibitor of Src or TBK1 protein activity;
(iv) an inhibitor or depleter of NFKB or IRF3 protein or an inhibitor of RalB protein activation,
wherein optionally the inhibitor of NFKB or IRF3 protein activity is an allosteric inhibitor of NFKB or IRF3 protein activity; or
(v) any combination of (i) to (iv); and
(2) at least one Growth Factor Inhibitor.
In alternative embodiments, the kits, blister packages, lidded blisters or blister cards or packets, clamshells, trays or shrink wraps further comprise instructions for practicing a method of the invention.
The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples. EXAMPLES
EXAMPLE 1 : Methods of the invention are effective for sensitizing and re-sensitizing cancer cells to growth factor inhibitors
The data presented herein demonstrates the effectiveness of the compositions and methods of the invention in sensitizing and re-sensitizing cancer cells, and cancer stem cells, to growth factor inhibitors, and validates this invention's therapeutic approach to overcome growth factor inhibitor, e.g., EGFR inhibitor, resistance for a wide range of cancers. The data presented herein demonstrates that genetic and pharmacological inhibition of RalB or NF-κΒ was able to re-sensitize ανβ3 -expressing tumors to EGFR inhibitors.
Resistance to epidermal growth factor receptor (EGFR) inhibitors has emerged as a significant clinical problem in oncology owing to various resistance mechanisms1'2. Since cancer stem cells have been associated with drug resistance3, we examined the expression of stem/progenitor cell markers for breast, pancreas and colon tumor cells with acquired resistance to EGFR inhibitors. We found that CD61 (β3 integrin) was the one marker consistently upregulated on EGFR inhibitor resistant tumor cells. Moreover, integrin ανβ3 expression was markedly enhanced in murine orthotopic lung and pancreas tumors following their acquired resistance to systemically delivered EGFR inhibitors. In fact, ανβ3 was both necessary and sufficient to account for the tumor cell resistance to EGFR inhibitors and other growth factor receptor inhibitors but not cytotoxic drugs.
Mechanistically, in drug resistant tumors ανβ3 forms a complex with KRAS via the adaptor Galectin-3 resulting in recruitment of RalB and activation of its effector TBKl/NF-κΒ, revealing a previously undescribed integrin-mediated pathway.
Accordingly, genetic or pharmacological inhibition of RalB or NF-κΒ was able to re- sensitize ανβ3 -expressing tumors to EGFR inhibitors, demonstrating the effectiveness of the compositions and methods of the invention and validating this invention's therapeutic approach to overcome EGFR inhibitor resistance for a wide range of cancers.
Despite some level of clinical success achieved with EGFR Tyrosine Kinase inhibitors (TKIs), intrinsic and acquired cellular resistance mechanisms limit their efficacy1'2'4. A number of resistance mechanisms have been identified, including KRAS and EGFR mutations, resulting in constitutive activation of the ERK pathway5"7. While KRAS-mediated ERK signaling is associated with resistance to EGFR inhibition, KRAS also induces PI3K and Ral activation leading to tumor cell survival and proliferation8'9.
Nevertheless, it is clear that treatment of tumors with EGFR inhibitors appears to select for a cell population that remains insensitive to EGFR blockade1'2. Prolonged administration of tumors with EGFR TKIs also selects for cells characterized by a distinct array of membrane proteins, including cancer stem/progenitor cell markers known to be associated with increased cell survival and metastasis10. While a number of EGFR- inhibitor resistance mechanisms have been defined, it is not clear whether a single unifying mechanism might drive the resistance of a broad range of cancers. To investigate this, we exposed pancreatic (FG, Miapaca-2), breast (BT474, SKBR3 and MDAMB468) and colon (SW480) human tumor cell lines to increasing concentrations of erlotinib or lapatinib for three weeks, to select cell subpopulations that were at least 10-fold more resistant to these targeted therapies than their parental counterparts. Parent or resistant cells were then evaluated for a panel of stem/progenitor cell markers previously identified to be upregulated in the most aggressive metastatic tumor cells11"13.
As expected, the expression of some of these markers was significantly increased in one or more of these resistant cell populations. Surprisingly, we observed that CD61 (integrin β3) was the one marker upregulated in all resistant cell lines tested, Figure la. The longer cells were exposed to erlotinib the greater the expression level of ανβ3 was observed, Figure lb. These findings were extended in vivo as mice bearing orthotopic FG pancreatic tumors with minimal integrin ανβ3 evaluated following four weeks of erlotinib treatment showed a 10-fold increase in ανβ3 expression, Figure lc. Moreover, H441 human lung adenocarcinoma orthotopic tumors14 exposed to systemic erlotinib treatment in vivo for 7-8 weeks developed resistance and a qualitative increase in integrin ανβ3 expression compared with vehicle-treated tumors, see Fig. Id and Figure 5
(Supplementary Fig. 1). Thus, exposure of histologically distinct tumor cells in vitro or in vivo to EGFR inhibitors selects for a tumor cell population expressing high levels of ανβ3.
In addition to being expressed on a subpopulation of stem/progenitor cells during mammary development15, ανβ3 is a marker of the most malignant tumor cells in a wide range of cancers16'17. To determine whether endogenous expression of integrin ανβ3 might predict tumor cell resistance to EGFR blockade, various breast, lung and pancreatic tumor cells were first screened for ανβ3 expression and then analyzed for their sensitivity to EGFR inhibitors (Supplementary Table 1).
Seguin et a!.. Supplementary Table 1 Table 1. KRAS mutation, integrin ανβδ expression and EGFR TKi sensitivity of cancer cell lines
Figure imgf000071_0001
In all cases, β3 expressing tumor cells were intrinsically more resistant to EGFR blockade than 3-negative tumor cell lines (Fig. le). In fact, ανβ3 was required for resistance to EGFR inhibitors, since knockdown of ανβ3 in PANC-1 cells resulted in a 10-fold increase in tumor cell sensitivity to erlotinib (Fig. If). Moreover, integrin ανβ3 was sufficient to induce erlotinib resistance since ectopic expression of ανβ3 in FG cells lacking this integrin dramatically increased erlotinib resistance both, in vitro and in orthotopic pancreatic tumors after systemic treatment in vivo (Fig. If and g).
Integrin ανβ3 not only promotes adhesion-dependent signaling via activation of focal adhesion kinase FAK16 but it can also activate a FAK-independent signaling cascade in the absence of integrin ligation that is associated with increased survival and tumor metastasis17. To determine whether ανβ3 ligation was required for its causative role in erlotinib resistance, FG cells transfected with either WT β3 or a ligation deficient mutant of the integrin (Dl 19A)17 were treated with erlotinib. The same degree of erlotinib resistance was observed in cells expressing either the ligation competent or incompetent form of integrin ανβ3, see Figure 6a (Supplementary Fig. 2a) indicating that expression of ανβ3, even in the unligated state, was sufficient to induce tumor cell resistance to erlotinib. Tumor cells with acquired resistance to one drug can often display resistance to a wide range of drugs18'19. Therefore, we examined whether ανβ3 expression also promotes resistance to other growth factor inhibitors and/or cytotoxic agents. Interestingly, while ανβ3 expression accounted for EGFR inhibitor resistance, it also induced resistance to the IGFR inhibitor OSI-906, yet failed to protect cells from the antimetabolite agent gemcitabine and the chemotherapeutic agent cisplatin, see Figure 6b and Figure 6c (Supplementary Fig. 2b and c). These results demonstrate that integrin ανβ3 accounts for tumor cell resistance to drugs that target growth factor receptor mediated pathways but does not promote for a more general resistant phenotype to all drugs, particularly those that induce cell cytotoxicity.
In some cases oncogenic KRAS has been associated with EGFR TKIs
resistance20, however, it remains unclear whether oncogenic KRAS is a prerequisite for EGFR resistance21. Thus, we examined the KRAS mutational status in various tumor cell lines and found that KRAS oncogenic status did not account for resistance to EGFR inhibitors (Supplementary Table 1). Nevertheless, knockdown of KRAS in ανβ3 expressing cells rendered them sensitive to erlotinib while KRAS knockdown in cells lacking ανβ3 had no such effect, see Figure 6a and Figure 6b, indicating that ανβ3 and KRAS function cooperatively to promote tumor cell resistance to erlotinib. Interestingly, even in non-adherent cells, ανβ3 colocalized with oncogenic KRAS in the plasma membrane (Figure 2c) and could be co-precipitated in a complex with KRAS , see Figure 6d. This interaction was specific for KRAS, as ανβ3 was not found to associate with N-, R- or H- RAS isoforms in these cells, see Figure 6d and Figure 7a and Figure 7b
(Supplementary Fig. 3 a and b). Furthermore, in BXPC3 human pancreatic tumor cells expressing wildtype KRAS, ανβ3 showed increased association with KRAS only after these cells were stimulated with EGF, see Figure 6e. Previous studies have indicated that the KRAS interacting protein Galectin-3 can also couple to integrins22'23. Therefore, we considered whether Galectin-3 might serve as an adaptor facilitating an interaction between ανβ3 and KRAS in epithelial tumor cells. In PANC-1 cells with endogenous β3 expression, ανβ3, KRAS, and Galectin-3 co-localized to membrane clusters, see Figure 8a and Figure 8b (Supplementary Fig.4a-b). Furthermore, knockdown of either β3 or Galectin-3 prevented the localization of KRAS to these membrane clusters or their co- immunoprecipitation, see Figure 8 (Supplementary Fig 4). KRAS promotes multiple effector pathways including those regulated by RAF, phosphatidylinositol-3-OH kinases (PBKs) and RalGEFs leading to a variety of cellular functions24. To investigate whether one or more KRAS effector pathway(s) may contribute to integrin 3/KRAS-mediated tumor cell resistance to EGFR inhibitors, we individually knocked-down or inhibited each downstream RAS effector in cells expressing or lacking integrin ανβ3. While suppression of AKT, ERK and RalA sensitized tumor cells to erlotinib, regardless of the ανβ3 expression status, see Figure 9 (Supplementary Fig.5), knockdown of RalB selectively sensitized ανβ3 expressing tumor cells to erlotinib, see Figure 7a and Figure 10a (Supplementary Fig. 6a). This was relevant to pancreatic tumor growth in vivo since, knockdown of RalB re-sensitized ανβ3- expressing pancreatic orthotopic tumors to erlotinib in mice, see Figure 7b. In fact, expression of a constitutively active RalB (G23V) mutant in β3 -negative cells was sufficient to confer resistance to EGFR inhibition, see Figure 7c and Figure 10b
(Supplementary Fig. 6b). Furthermore, ectopic expression of ανβ3 enhanced RalB activity in tumor cells in a KRAS-dependent manner, see Figure 7d). Accordingly, integrin ανβ3 and RalB were co-localized in tumor cells, see Figure 10c (Supplementary Fig. 7) and in human breast and pancreatic cancer biopsies, see Figure 11 (Supplementary Fig. 8) and a strong correlation was found between ανβ3 expression and Ral GTPase activity in patients biopsies suggesting the av33/RalB signaling module is clinically relevant, see Figure 7e. Together, these findings indicate that integrin ανβ3 promotes erlotinib resistance of cancer cells by complexing with KRAS and RalB resulting in RalB activation.
RalB, an effector of RAS has been shown to induce TBKl/NF-κΒ activation leading to enhanced tumor cell survival25'26. In addition, it has been shown that NF-KB signaling is essential for KRAS-driven tumor growth and resistance to EGFR blockade27" 29. This prompted us to ask whether ανβ3 could regulate NF-κΒ activity through RalB activation and thereby promote tumor cell resistance to EGFR targeted therapy. To test this, tumor cells expressing or lacking integrin ανβ3 and/or RalB were grown in the presence or absence of erlotinib and lysates of these cells were analyzed for activated downstream effectors of RalB. We found that erlotinib treatment of ανβ3 negative cells reduced levels of phosphorylated TBK1 and NF-κΒ, whereas in β3-ρο8Ϊίίνε cells these effectors remained activated unless RalB was depleted, see Figure 4a. NF-κΒ activity was sufficient to account for EGFR inhibitor resistance since ectopically expressed a constitutively active NF-κΒ (S276D) in β3 -negative FG pancreatic tumor cells30 conferred resistance to EGFR inhibition, see Figure 4b). Accordingly, genetic or pharmacological inhibition of NF-κΒ in 3-positive cells completely restored erlotinib sensitivity31, see Figure 4c and d). These findings demonstrate that RalB, the effector of the av 3/KRAS complex, promotes tumor cell resistance to EGFR targeted therapy via TBKl/NF-κΒ activation. Together, our studies describe a role for ανβ3 mediating resistance to EGFR inhibition via RalB activation and its downstream effector NF-KB, opening new avenues to target tumors that are resistant to EGFR targeted therapy, see Figure 4e.
Recent studies have shown that, upon prolonged treatment with EGFR inhibitors, tumor cells develop alternative or compensatory pathways to sustain cell survival, leading to drug resistance1'32. Here we show that integrin ανβ3 is specifically upregulated in histologically distinct tumors where it accounts for resistance to EGFR inhibition. At present, it is not clear whether exposure to EGFR inhibitors may promote increased ανβ3 expression or whether these drugs simply eliminate cells lacking ανβ3 allowing the expansion of ανβ3 -expressing tumor cells. Given that integrin ανβ3 is a marker of mammary stem cells15, it is possible that acquired resistance to EGFR inhibitors selects for a tumor stem-like cell population3'33. While integrins can promote adhesion dependent cell survival and induce tumor progression16, here, we show that integrin ανβ3, even in the unligated state, can drive tumor cell survival and resistance to EGFR blockade by interaction with KRAS. This action leads to the recruitment and activation of RalB and its downstream signaling effector NF-κΒ. In fact, NF-κΒ inhibition re-sensitizes ανβ3- bearing tumors to EGFR blockade. Taken together, our findings not only identify ανβ3 as a tumor cell marker of drug resistance but reveal that inhibitors of EGFR and NF-KB should provide synergistic activity against a broad range of cancers.
Figure legends
Figure 1. Integrin ανβ3 expression promotes resistance to EGFR TKI.
(a) Flow cytometric quantification of cell surface markers after 3 weeks treatment with erlotinib (pancreatic and colon cancer cells) or lapatinib (breast cancer cells), (b) Flow cytometric analysis of ανβ3 expression in FG and Miapaca-2 cells following erlotinib. Error bars represent s.d. (n = 3 independent experiments), (c) Top, immunofluorescence staining of integrin ανβ3 in tissue specimens obtained from orthotopic pancreatic tumors treated with vehicle (n = 3) or erlotinib (n = 4). Scale bar, 50 μιη. Bottom, Integrin ανβ3 expression was quantified as ratio of integrin ανβ3 pixel area over nuclei pixel area using Metamorph (*P = 0.049 using Mann-Whitney U test), (d) Right, intensity (scale 0 to 3) of β3 expression in mouse orthotopic lung tumors treated with vehicle (n = 8) or erlotinib (n = 7). Left, immunohistochemical staining of β3. Scale bar, 100 μιη. (**P = 0.0012 using Mann- Whitney U test) (e) IC50 for cells treated with erlotinib or lapatinib. (f) Tumor sphere formation assay to establish a dose-response for erlotinib. Error bars represent s.d. (n = 3 independent experiments), (g) Orthotopic FG tumors (>1000 mm3; n = 10 per treatment group) were treated for 10 days with vehicle or erlotinib. Results are expressed as % tumor weight compared to vehicle control. *P < 0.05. Immunoblot analysis for tumor lysates after 10 days of erlotinib confirms suppressed EGFR phosphorylation.
Figure 2. Integrin ανβ3 cooperates with KRAS to promote resistance to EGFR blockade.
(a-b) Tumor sphere formation assay of FG expressing (a) or lacking (b) integrin β3 depleted of KRAS (shKRAS) or not (shCTRL) and treated with a dose response of erlotinib. Error bars represent s.d. (n = 3 independent experiments), (c) Confocal microscopy images of PANC-1 and FG- β3 cells grown in suspension. Cells are stained for integrin ανβ3 (green), KRAS (red), and DNA (TOPRO-3, blue). Scale bar, 10 m. Data are representative of three independent experiments, (d) RAS activity assay performed in PANC-1 cells using GST-Rafl-RBD immunoprecipitation as described in Methods. Immunoblot analysis of KRAS, NRAS, HRAS, RRAS, integrin βΐ and integrin β3. Data are representative of three independent experiments, (e) Immunoblot analysis of Integrin ανβ3 immunoprecipitates from BxPC-3 β3 -positive cells grown in suspension and untreated or treated with EGF 50 ng / ml for 5 minutes. RAS activity was determined using a GST-Rafl-RBD immunoprecipitation assay. Data are representative of three independent experiments.
Figure 3. RalB is a key modulator of integrin ανβ3 -mediated EGFR TKI resistance.
(a) Tumor spheres formation assay of FG^3 treated with non-silencing (shCTRL) or RalB-specific shRNA and exposed to a dose response of erlotinib. Error bars represent s.d. (n = 3 independent experiments). Immunoblot analysis showing RalB knockdown, (b) Effects of depletion of RalB on erlotinib sensitivity in β3 -positive tumor in a pancreatic orthotopic tumor model. Established 3-positive tumors expressing non-silencing (shCTRL) or RalB-specific shRNA (>1000 mm3; n = 13 per treatment group) were randomized and treated for 10 days with erlotinib. Results are expressed as % of tumor weight changes after erlotinib treatment compared to control. *P < 0.05, **P < 0.01. Tumor images, average weights +/- s.e are shown, (c) Tumor spheres formation assay of FG cells ectopically expressing vector control, WT RalB FLAG tagged constructs or a constitutively active RalB G23V FLAG tagged treated with erlotinib (0.5 μΜ). Error bars represent s.d. (n = 3 independent experiments). *P < 0.05, NS = not significant.
Immunoblot analysis showing RalB WT and RalB G23 FLAG tagged constructs transfection efficiency, (d) RalB activity was determined in FG, FG- 3 expressing non- silencing or KRAS-specific shRNA, by using a GST-RalBP l-RBD immunoprecipitation assay as described in Methods. Data are representative of three independent experiments. (e) Right, overall active Ral immunohistochemical staining intensity between β3 negative (n = 15) and β3 positive (n = 70) human tumors. Active Ral staining was compared between each group by Fisher's exact test (*P < 0.05, P = 0.036, two-sided). Left, representative immunohistochemistry images of human tumor tissues stained with an integrin β3 -specific antibody and an active Ral antibody. Scale bar, 50 μιη.
Figure 4. Integrin av 3/RalB complex leads to NF-μΒ activation and resistance to
EGFR TKI.
Immunoblot analysis of FG, FG- 3 and FG- 3 stably expressing non-silencing or RalB-specific ShRNA, grown in suspension and treated with erlotinib (0.5 μΜ). pTBKl refers to phospho-S172 TBK1, p-p65 NF-κΒ refers to phospho-p65 NF-κΒ S276, pFAK refers to phospho-FAK Tyr 861. Data are representative of three independent experiments, (b) Tumor spheres formation assay of FG cells ectopically expressing vector control, WT NF-κΒ FLAG tagged or constitutively active S276D NF-κΒ FLAG tagged constructs treated with erlotinib (0.5 μΜ). Error bars represent s.d. (n = 3 independent experiments). *P < 0.05, **P < 0.001, NS = not significant. Immunoblot analysis showing NF-κΒ WT and S276D NF-κΒ FLAG transfection efficiency, (c) Tumor spheres formation assay of FG- 3 treating with non-silencing (shCTRL) or NF-KB- specific shRNA and exposed to erlotinib (0.5 μΜ). Error bars represent s.d. (n = 3 independent experiments). *P < 0.05, NS = not significant, (d) Dose response in FG-P3 cells treated with erlotinib (10 nM to 5 μΜ), lenalidomide (10 nM to 5 μΜ) or a combination of erlotinib (10 nM to 5 μΜ) and lenalidomide (1 μΜ). Error bars represent s.d. (n = 3 independent experiments). *P < 0.05, NS = not significant, (e) Model depicting the integrin ανβ3 -mediated EGFR TKI resistance and conquering EGFR TKI resistance pathway and its downstream RalB and NF-κΒ effectors.
METHODS
Compounds and cell culture.
Human pancreatic (FG, PANC-1, Miapaca-2 (MP2), CFPAC-1, XPA-1, CAPAN- 1 , BxPc3 ), breast (MDAMB231 , MDAMB468 (MDA468), BT20, SKBR3 , BT474), colon (SW480) and lung (A549, H441) cancer cell lines were grown in ATCC recommended media supplemented with 10% fetal bovine serum, glutamine and nonessential amino acids. We obtained FG- 3, FG-D119A mutant and PANC-sh 3 cells as previously described17. Erlotinib, OSI-906, Gemcitabine and Lapatinib were purchased from Chemietek. Cisplatin was generated from Sigma-Aldrich. Lenalidomide was purchased from LC Laboratories. We established acquired EGFR TKI resistant cells by adding an increasing concentration of erlotinib (50 nM to 15 μΜ) or lapatinib (10 nM to 15 μΜ), daily in 3D culture in 0.8% methylcellulose.
Lentiviral studies and Transfection.
Cells were transfected with vector control, WT, G23V RalB-FLAG, WT and
S276D NF-KB-FLAG using a lentiviral system. For knock-down experiments, cells were transfected with KRAS, RalA, RalB, AKTl, ERKl/2, p65 NF-κΒ siRNA (Qiagen) using the lipofectamine reagent (Invitrogen) following manufacturer's protocol or transfected with shRNA (Open Biosystems) using a lentiviral system. Gene silencing was confirmed by immunoblots analysis.
Tumor sphere formation.
Tumor spheres formation assays were performed essentially as described previously 11. Briefly, cells were seeded at 1000 to 2000 cells per well and grown for 12 days to 3 weeks. Cells were treated with vehicle (DMSO), erlotinib (10 nM to 5 μΜ), lapatinib (10 nM to 5 μΜ), gemcitabine (0.001 nM to 5 μΜ), OSI-906 (10 nM to 5 μΜ), lenalidomide (10 nM to 5 μΜ), or cisplatin (10 nM to 5 μΜ), diluted in DMSO. The media was replaced with fresh inhibitor every day for erlotinib, lapatinib, lenalidomide and 3 times a week for cisplatin and gemcitabine. Colonies were stained with crystal violet and scored with an Olympus SZH10 microscope. Survival curves were generated at least with five concentration points.
Flow Cytometry.
200, 000 cells, after drug or vehicle treatment, were washed with PBS and incubated for 20 minutes with the Live/Dead reagent (Invitrogen) according to the manufacturer's instruction, then, cells were fixed with 4% paraformaldehyde for 15 min and blocked for 30 min with 2% BSA in PBS. Cells were stained with fluorescent- conjugated antibodies to CD61 (LM609), CD44 (eBioscience), CD24 (eBioscience), CD34 (eBioscience), CD133 (Santa Cruz), CD56 (eBioscience), CD29 (P4C10) and CD49f (eBioscience). All antibodies were used at 1 : 100 dilutions, 30 minutes at 4°C. After washing several times with PBS, cells were analyzed by FACS.
Immunohistochemical analysis.
Immunostaining was performed according to the manufacturer's
recommendations (Vector Labs) on 5 μΜ sections of paraffin-embedded tumors from the orthotopic xenograft pancreas and lung cancer mouse models14 or from a metastasis tissue array purchased from US Biomax (MET961). Antigen retrieval was performed in citrate buffer pH 6.0 at 95°C for 20 min. Sections were treated with 0.3% H2O2 for 30 min, blocked in normal goat serum, PBS-T for 30 min followed by Avidin-D and then incubated overnight at 4°C with primary antibodies against integrin β3 (Abeam) and active Ral (NewEast) diluted 1 : 100 and 1 :200 in blocking solution. Tissue sections were washed and then incubated with biotinylated secondary antibody (1 :500, Jackson ImmunoResearch) in blocking solution for lh. Sections were washed and incubated with Vectastain ABC (Vector Labs) for 30 min. Staining was developed using a Nickel- enhanced diamino-benzidine reaction (Vector Labs) and sections were counter-stained with hematoxylin. Sections stained with integrin β3 and active Ral were scored by a H- score according to the staining intensity (SI) on a scale 0 to 3 within the whole tissue section.
Immunoprecipitation and Immunoblot analysis.
Cells were lysed in either RIPA lysis buffer (50 mM Tris pH 7.4, 100 mM NaCL, 2 mM EDTA, 10% DOC, 10% Triton, 0.1% SDS) or Triton lysis buffer (50 mM Tris pH 7.5, 150 mN NaCl, 1 mM EDTA, 5 mM MgC12, 10% Glycerol, 1% Triton) supplemented with complete protease and phosphatase inhibitor mixtures (Roche) and centrifuged at 13,000 g for 10 min at 4°C. Protein concentration was determined by BCA assay. 500 μg to 1 mg of protein were immunoprecipitated with 3 μg of anti-integrin ανβ-3 (LM609) overnight at 4°C following by capture with 25 μΐ of protein A/G (Pierce). Beads were washed five times, eluted in Laemmli buffer, resolved on NuPAGE 4-12% Bis-Tris Gel (Invitrogen) and immunoblotting was performed with anti-integrin β3 (Santa Cruz), anti- RalB (Cell Signaling Technology), anti KRAS (Santa Cruz). For immunoblot analysis, 25 μg of protein was boiled in Laemmli buffer and resolved on 8% to 15% gel. The following antibodies were used: KRAS (Santa Cruz), NRAS (Santa Cruz), RRAS (Santa Cruz), HRAS (Santa Cruz), phospho-S 172 NAK/TBK1 (Epitomics), TBK1 (Cell Signaling Technology), phospho-p65NF-KB S276 (Cell Signaling Technology), p65NF- KB (Cell Signaling Technology), RalB (Cell Signaling Technology), phospho-EGFR (Cell Signaling Technology), EGFR (Cell Signaling Technology), FLAG (Sigma), phospho-FAK Tyr 861 (Cell Signaling Technology), FAK (Santa Cruz), Galectin 3 (BioLegend) and Hsp90 (Santa Cruz).
Affinity pull-down assays for Ras and Ral.
RAS and Ral activation assays were performed in accordance with the manufacturer's (Upstate) instruction. Briefly, cells were cultured in suspension for 3h, lysed and protein concentration was determined. 10 μg of Ral Assay Reagent (Ral BP 1, agarose) or RAS assay reagent (Raf-1 RBD, agarose) was added to 500 mg to 1 mg of total cell protein in MLB buffer (Millipore). After 30 min of rocking at 4°C, the activated (GTP) forms of RAS/Ral bound to the agarose beads were collected by centrifugation, washed, boiled in Laemmli buffer, and loaded on a 15% SDS-PAGE gel.
Immunofluorescence Microscopy.
Frozen sections from tumors from the orthotopic xenograft pancreas cancer mouse model or from patients diagnosed with pancreas or breast cancers (as approved by the institutional Review Board at University of California, San Diego) or tumor cell lines were fixed in cold acetone or 4% paraformaldehyde for 15 min, permeabilized in PBS containing 0.1% Triton for 2 min and blocked for lh at room temperature with 2% BSA in PBS. Cells were stained with antibodies to integrin ανβ3 (LM609), RalB (Cell Signaling Technology), Galectin 3 (BioLegend), pFAK (Cell Signaling Technology), NRAS (Santa Cruz), RRAS (Santa Cruz), HRAS (Santa Cruz) and KRAS (Abgent). All primary antibodies were used at 1 : 100 dilutions, overnight at 4°C. Where mouse antibodies were used on mouse tissues, we used the MOM kit (Vector Laboratory). After washing several times with PBS, cells were stained for two hours at 4°C with secondary antibodies specific for mouse or rabbit (Invitrogen), as appropriate, diluted 1 :200 and co- incubated with the DNA dye TOPRO-3 (1 :500) (Invitrogen). Samples were mounted in VECTASHIELD hard-set media (Vector Laboratories) and imaged on a Nikon Eclipse CI confocal microscope with 1.4 NA 60x oil-immersion lens, using minimum pinhole (30 m). Images were captured using 3.50 imaging software. Colocalization between Integrin ανβ3 and KRAS was studied using the Zenon Antibody Labeling Kits
(Invitrogen).
Orthotopic pancreas cancer xenograft model.
All mouse experiments were carried out in accordance with approved protocols from the UCSD animal subjects committee and with the guidelines set forth in the NIH Guide for the Care and Use of Laboratory Animals. Tumors were generated by injection of FG human pancreatic carcinoma cells (106 tumor cells in 30 μΐ^ of sterile PBS) into the tail of the pancreas of 6-8 week old male immune compromised nu/nu mice. Tumors were established for 2-3 weeks (tumor sizes were monitored by ultrasound) before beginning dosing. Mice were dosed by oral gavage with vehicle (6% Captisol) or 100 mg/kg/day erlotinib for 10 to 30 days prior to harvest.
Orthotopic lung cancer xenograft model.
Tumors were generated by injection of H441 human lung adenocarcinoma cells (106 tumor cells per mouse in 50 μΐ^ of HBSS containing 50 mg growth factor-reduced Matrigel (BD Bioscience) into the left thorax at the lateral dorsal axillary line and into the left lung, as previously described14 of 8 week old male immune-compromised nu/nu mice. 3 weeks after tumor cell injection, the mice were treated with vehicle or erlotinib (100 mg/kg/day) by oral gavage until moribund (approximately 50 and 58 days, respectively).
Statistical Analyses.
All statistical analyses were performed using Prism software (GraphPad). Two- tailed Mann Whitney U tests, Fisher's exact tests, or t-tests were used to calculate statistical significance. A P value < 0.05 was considered to be significant.
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35
EXAMPLE 2: Methods of the invention are effective for sensitizing and re-sensitizing cancer cells to growth factor inhibitors
The data presented herein demonstrates the effectiveness of the compositions and 40 methods of the invention in sensitizing and re-sensitizing cancer cells, and cancer stem cells, to growth factor inhibitors, and validates this invention's therapeutic approach to overcome growth factor inhibitor resistance for a wide range of cancers. In particular, the data presented in this Example demonstrates that β3 integrin induces erlotinib resistance in cancer cells by switching tumor dependency from EGFR to KRAS. In alternative embodiments, the compositions and methods of the invention overcome tumor drug resistance that limits the long-term success of therapies targeting EGFR. Here, we identify integrin ανβ3 as a biomarker of intrinsic and acquired resistance to erlotinib in human pancreatic and lung carcinomas irrespective of their KRAS mutational status. Functionally, ανβ3 is necessary and sufficient for this resistance where it acts in the unligated state as a scaffold to recruit active KRAS into membrane clusters switching tumor dependency from EGFR to KRAS. The KRAS effector RalB is recruited to this complex, where it mediates erlotinib resistance via a TBK-1/NF-KB pathway. Disrupting assembly of this complex or inhibition of its downstream effectors fully restores tumor sensitivity to EGFR blockade. Our findings uncouple KRAS mutations from erlotinib resistance, revealing an unexpected requirement for integrin ανβ3 in this process.
We hypothesized that upregulation of specific genes common to multiple tumor types exposed to erlotinib drives a conserved pathway that governs both intrinsic and acquired resistance. To identify genes associated with erlotinib (N-(3-ethynylphenyl)-6,7- bis(2-methoxyethoxy) quinazolin-4-amine) resistance, we analyzed the expression of a tumor progression gene array for human cell lines with intrinsic resistance or murine xenografts following the acquisition of resistance in vivo. The most upregulated gene common to all drug resistant carcinomas tested was the cell surface ITGB3, integrin β3 (Fig. 12, and Fig. 26) associated with the integrin ανβ3 whose expression has been linked to tumor progression . ανβ3 expression completely predicted erlotinib resistance for a panel of histologically distinct tumor cell lines (Fig. 12B and Fig. 16). Moreover, chronic treatment of the erlotinib sensitive lines resulted in the induction of β3 expression concomitantly with drug resistance (Fig.12 and Fig. 16B, C). We also detected increased β3 expression in lung carcinoma patients who had progressed on erlotinib therapy (Fig. 17). In addition, we examined both treatment naive and erlotinib resistant NSCLC patients from the BATTLE Study (JO) of non-small cell lung cancer (NSCLC) and found β3 gene expression was significantly higher in patients who progressed on erlotinib (Fig. 12D). Finally, we examined serial primary lung tumors biopsies from patients before treatment or after erlotinib resistance and found a qualitative increase in integrin β3 expression concurrent with the loss of erlotinib sensitivity (Fig. 12E). Taken together, our findings show that integrin β3 is a marker of acquired and intrinsic erlotinib resistance for pancreas and lung cancer.
To assess the functional role of ανβ3 in erlotinib resistance we used a gain and loss-of-function approach and found that integrin β3 was both necessary and sufficient to account for erlotinib resistance in vitro and during systemic treatment of lung and orthotopic pancreatic tumors in vivo (Fig. 12F, G and Fig. 18A-C). Interestingly, integrin β3 expression did not impact resistance to chemotherapeutic agents such as gemcitabine and cisplatin while conferring resistance to inhibitors targeting EGFR1/EGFR2 or IGFR (Fig. 18C-E), suggesting this integrin plays a specific role in tumor cell resistance to RTK inhibitors.
As integrin ανβ3 is functions as an adhesion receptor, ligand binding inhibitors could represent a therapeutic strategy to sensitize tumors to EGFR inhibitors. However, ανβ3 expression induced drug resistance in cells growing in suspension. Also, neither function blocking antibodies nor cyclic peptide inhibitors sensitized integrin ανβ3- expressing tumors to EGFR inhibitors (not shown), and tumor cells expressing wild-type integrin β3 or the ligati on-deficient mutant β3 D 119A ( /_/) showed equivalent drug resistance (Fig. 19). Since the contribution of integrin ανβ3 to erlotinib resistance appears to involve a non-canonical, ligation-independent mechanism that is not sensitive to traditional integrin antagonists, understanding the molecular mechanisms driving this pathway could provide therapeutic opportunities.
Integrins function in the context of RAS family members. Interestingly, we found that ανβ3 associated with KRAS but not N- , H- or R-RAS (Fig. 13 A). While oncogenic KRAS has been linked to erlotinib resistance, there are many notable exceptions (6^9). In fact, we observed a number of tumor cell lines with oncogenic KRAS to be sensitive to erlotinib (FG, H441, and CAPAN1), whereas H1650 cells were erlotinib resistant despite their expression of wildtype KRAS and mutant EGFR (Fig. 17). In fact, ανβ3 expression consistently correlated with erlotinib resistance for all cell lines tested (Pearson's correlation coefficient R2=0.87) making a better predictor of erlotinib resistance.
Interestingly, we observed active KRAS to be distributed within the cytoplasm in β3- negative cells (Fig. 20A) whereas in cells expressing β3 endogenously or ectopically, KRAS was localized to β3-containing membrane clusters, even in the presence of erlotinib (Fig. 13B,C and Fig.20A) a relationship that was not observed for βΐ integrin (Fig. 20B and C). Furthermore, knockdown of KRAS impaired tumorsphere formation and restored erlotinib sensitivity in 3-positive cells (Fig. 13D-F and Fig. 21A-C). In contrast, KRAS was dispensable for tumorsphere formation and erlotinib response the in cells lacking β3 expression (Fig. 13D-F). Thus, β3 integrin expression switches tumor cell dependency from EGFR to KRAS, and that the localization of β3 with KRAS at the plasma membrane appears to be a critical determinant of tumor cell resistance to erlotinib. Also, our results reveal that tumors expressing oncogenic KRAS without β3 remain sensitive to EGFR blockade.
Independent studies have shown that galectin-3 can interact with either KRAS (12) or β3 (73) so we asked whether this protein might serve as an adaptor to promote KRAS^3 complex formation. Under anchorage-independent growth conditions, integrin β3, KRAS, and Galectin-3 were co-localized in membrane clusters (Fig. 13G and Fig. 22), and knockdown of either integrin β3 or Galectin-3 prevented complex formation, KRAS membrane localization, and importantly sensitized ανβ3 expressing tumors to erlotinib (Fig. 13G-I).
We next evaluated the signaling pathways driven by the integrin β3/ΚΡΑ8 complex. Erlotinib resistance of β3-ρο8Ϊίίνε cells was not affected by depletion of known KRAS effectors, including AKT, ERK, or RalA (Fig. 23A,B). However, knockdown of RalB sensitized β3 -expressing cells to erlotinib in vitro (Fig. 14A and Fig. 23A-C) and in pancreatic orthotopic tumors in vivo (Fig. 14B). Accordingly, expression of
constitutively active RalB in β3-negative cells conferred erlotinib resistance (Fig. 14C). Mechanistically, RalB was recruited to the β3/ΚΡΑ8 membrane clusters (Fig. 14D-F) where it became activated in a KRAS-dependent manner (Fig. 14G). Recent studies have reported that TBK1 and NF-κΒ are RalB effectors linked to KRAS dependency (] ) and erlotinib resistance (75). We found that erlotinib decreased the activation of these effectors only in the absence of integrin β3 (Fig. 14H). In fact, loss of RalB in β3- expressing cells restored erlotinib-mediated inhibition of TBK1 and NF-κΒ (Fig. 14H). Accordingly, depletion of either TBK1 or NF-κΒ sensitized β3-ρο8Ϊίϊνε cells to erlotinib (Fig. 141 and Fig. 24A), while ectopic expression of activated NF-κΒ was sufficient to promote drug resistance in β3-negative cells (Fig. 24B). To evaluate the therapeutic potential of targeting this pathway, we examined whether erlotinib resistance of β3- expressing tumors could be reversed with approved drugs known to suppress NF-KB activation, lenalidomide/ REVLIMID® (16) and bortezomib/VELCADE® (I T). While monotherapy with these drugs failed to impact tumor growth, either drug used combination with erlotinib decreased tumorsphere formation in vitro (Fig. 4A) and completely suppressed tumor growth in vivo (Fig. 15B, C and Fig. 25). These findings support the model depicted in Fig. 15D where inhibition of NF-κΒ restores erlotinib sensitivity in β3 expressing tumors. These findings support the model depicted in Fig. 15D that ανβ3 expression in lung and pancreatic tumors recruits oncogenic KRAS facilitating NFKB activity leading to erlotinib resistance which can be overcome by a combination of currently approved inhibitors of NF-κΒ and EGFR.
Our observations demonstrate that the ability of β3 integrin to recruit KRAS into a membrane complex along with Galectin-3 and RalB functions to switch tumor cell dependency from EGFR to KRAS. In fact, oncogenic KRAS requires this non-canonical β3 -mediated pathway to drive erlotinib resistance. We show that currently available approved inhibitors of this pathway can be used to practice the methods of this invention to treat patients with solid tumors, rendering them sensitive to EGFR inhibitors such as erlotinib.
Material and Methods
Compounds and cell culture. Human pancreatic (FG, PANC-1, CFPAC-1, XPA-1, HPAFII, CAPAN-1, BxPC3) and lung (A549, H441, HCC827 and HI 650) cancer cell lines were grown in ATCC recommended media supplemented with 10% fetal bovine serum, glutamine and non-essential amino acids. We obtained FG^3, FG-D1 19A mutant and PA C-sl^3 cells as previously described (10). Erlotinib, OSI-906,
Gemcitabine, Bortezomib and Lapatinib were purchased from Chemietek. Cisplatin was generated from Sigma-Aldrich. Lenalidomide was purchased from LC Laboratories. Gene expression analysis. The Tumor Metastasis PCR Array (Applied Biosystem), consisting of 92 genes known to be involved in tumor progression and metastasis, was used to profile the common genes upregulated in erlotinib-resistant cells compared to erlotinib-sensitive cells according to the manufacturer's instructions. Briefly, total RNA was extracted and reverse transcribed into cDNA using the RNeasy kit (Qiagen). The cDNA was combined with a SYBR Green qPCR Master Mix (Qiagen), and then added to each well of the same PCR Array plate that contained the predispensed gene-specific primer sets. Tumor digestion and Flow Cytometry. Fresh tumor tissue from lung cancer cell lines was mechanically dissociated and then enzymatically digested in trypsin. The tissue was further filtered through a cell strainer to obtain a suspension of single tumor cells. Then, cells were washed were washed with PBS and incubated for 20 minutes with the Live/Dead reagent (Invitrogen) according to the manufacturer's instruction, then, cells were fixed with 4% paraformaldehyde for 15 min and blocked for 30 min with 2% BSA in PBS. Cells were stained with fluorescent-conjugated antibodies to integrin ανβ3 (LM609, Cheresh Lab), After washing several times with PBS, cells were analyzed by FACS.
Tumorsphere assay. Tumorsphere assay was performed as previously described (10).
Cells were treated with vehicle (DMSO), erlotinib (10 nM to 5 μΜ), lapatinib (10 nM to 5 μΜ), gemcitabine (0.001 nM to 5 μΜ), OSI-906 (10 nM to 5 μΜ), lenalidomide (ΙμΜ), cisplatin (10 nM to 5 μΜ), or bortezomib (4 nM) diluted in DMSO. The media was replaced with fresh inhibitor 2/6 times a week. Survival curves were generated at least with five concentration points.
Mouse cancer models. All research was conducted under protocol S05018 and approved by the University of California-San Diego Institutional Animal Care and Use Committee (IACUC). FG pancreatic carcinoma cells (1 x 106 tumor cells in 30 μΐ of PBS) were injected into the pancreas of 6-to 8-week-old male nude mice as previously described (10). Tumors were established for 2-3 weeks (tumor sizes were monitored by ultrasound) before beginning dosing. Mice were dosed by oral gavage with vehicle (6% Captisol) or 10, 25 and 50 mg/kg/day erlotinib for 10 to 30 days prior to harvest. H441 lung adenocarcinoma cells were generated as previously described (21). 3 weeks after tumor cell injection, the mice were treated with vehicle or erlotinib (100 mg/kg/day) by oral Mouse cancer models. All research was conducted under protocol S05018 and approved by the University of California-San Diego Institutional Animal Care and Use Committee (IACUC). FG pancreatic carcinoma cells (1 x 106 tumor cells in 30 μΐ of PBS) were injected into the pancreas of 6-to 8-week-old male nude mice as previously described (10). Tumors were established for 2-3 weeks (tumor sizes were monitored by ultrasound) before beginning dosing. Mice were dosed by oral gavage with vehicle (6% Captisol) or 10, 25 and 50 mg/kg/day erlotinib for 10 to 30 days prior to harvest. H441 lung adenocarcinoma cells were generated as previously described (21). 3 weeks after tumor cell injection, the mice were treated with vehicle or erlotinib (100 mg/kg/day) by oral gavage until moribund (approximately 50 and 58 days, respectively). To generate subcutaneous tumors, FG- 3, FG-R (after erlotinib resistance) and HCC-827 human carcinoma cells (5 x 106 tumor cells in 200 μΐ of PBS) were injected subcutaneous ly to the left or right flank of 6-8-week-old female nude mice. Tumors were measured every 2-3 days with calipers until they were harvested at day 10, 16 or after acquired resistance.
NSCLC specimens from the BATTLE trial. The BATTLE (Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination) trial was a randomized phase II, single-center, open-label study in patients with advanced NSCLC refractory to prior chemotherapy and included patients with and without prior EGFR inhibitor treatment (12). Patients underwent a tumor new biopsy prior to initiating study treatment. The microarray analysis of mRNA expression on frozen tumor core biopsies was conducted using the Affymetrix Human Gene l .ST™ platform as previously described (22).
Serial biopsies from NSCLC patients. Tumor biopsies from University of California, San Diego (UCSD) Medical Center stage IV non-small cell lung cancer patients were obtained before erlotinib treatment and 3 patients before and after erlotinib resistance. All biopsies are from lung or pleural effusion. Patients 1 had a core biopsy from the primary lung tumor, and Patient 2 and 3 had a fine needle biopsy from a pleural effusion. All patients had an initial partial response, followed by disease progression after 920, 92, and 120 days of erlotinib therapy, respectively. This work was approved by the UCSD Institutional Review Board (IRB).
Immunofluorescence microscopy. Frozen sections from tumors from orthotopic pancreatic tumors, from patients diagnosed with pancreas cancers (as approved by the institutional Review Board at University of California, San Diego) or tumor cell lines were processed as previously described (23). Cells were stained with indicated primary, followed by secondary antibodies specific for mouse or rabbit (Invitrogen), as appropriate. Samples imaged on a Nikon ECLIPSE CI™ confocal microscope with 1.4 NA 60x oil-immersion lens, using minimum pinhole (30 μιη). The following antibodies were used: anti-integrin β3 (LM609), KRAS (Pierce and Abgent M01), Galectin-3, NRAS, RRAS, Genetic knockdown and expression of mutant constructs. Cells were transfected with vector control, WT, G23V RalB-FLAG, WT and S276D NF-KB-FLAG using a lentiviral system. For knock-down experiments, cells were transfected with a pool of RalA, RalB, AKT1, ERK1/2 siRNA (Qiagen) using the lipofectamine reagent (Invitrogen) following manufacturer's protocol or transfected with shRNA (integrin β3, KRAS, Galectin-3, RalB, TBK1 and p65NF-kB) (Open Biosystems) using a lentiviral system. Gene silencing was confirmed by immunoblots analysis.
Immunohistochemical analysis. Immunostaining was performed according to the manufacturer's recommendations (Vector Labs) on 5 μΜ sections of paraffin-embedded tumors from tumor biopsies from lung cancer patients. Tumor sections were processed as previously described (23) using integrin β3 (Abeam clone EP2417Y). Sections stained with integrin β3 were scored by a H-score according to the staining intensity (SI) on a scale 0 to 3 within the whole tissue section.
Immunoprecipitation and immunoblots. Lysates from cell lines and xenograft tumors were generated using standard methods and RIPA or Triton buffers.
Immunoprecipitation experiments were performed as previously described (23) with anti-integrin ανβ3 (LM609) or Galectin-3. For immunoblot analysis, 25 μg of protein was boiled in Laemmli buffer and resolved on 8% to 15% gel. The following antibodies were used: anti-integrin β3, KRAS, NRAS, RRAS, HRAS, Hsp60 and Hsp90 from Santa Cruz, phospho-S 172 NAK/TBK1 from Epitomics, TBK1, phospho-p65NF-KB S276, p65NF-KB, RalB, phospho-EGFR, EGFR, from Cell Signaling Technology, and Galectin 3 from BioLegend.
Membrane extract. Membrane fraction from FG and FG^3 grown in suspension in media complemented with 0.1% BSA were isolated using the MEM-PER membrane extraction kit (Fisher) according to the manufacturer's instructions. Affinity pull-down assays for Ras and Ral. RAS and Ral activation assays were performed in accordance with the manufacturer's (Upstate) instruction. Briefly, cells were cultured in suspension for 3h. 10 μg of Ral Assay Reagent (Ral BP1, agarose) or RAS assay reagent (Raf-1 RBD, agarose) was added to 500 mg to 1 mg of total cell protein in MLB buffer
(Millipore). After 30 min of rocking at 40C, the activated (GTP) forms of RAS/Ral bound to the agarose beads were collected by centrifugation, washed, boiled in Laemmli buffer, and loaded on a 15% SDS-PAGE gel. Statistical Analyses. All statistical analyses were performed using Prism software (GRAPHPAD™). Two-tailed Mann Whitney U tests, Chi-squared tests, one way ANOVA tests or t-tests were used to calculate statistical significance. A P value < 0.05 was considered to be significant.
Figure legends
Figure 1 (Fig. 12/31) illustrates data showing that integrin β3 is expressed in EGFR inhibitor resistant tumors and is necessary and sufficient to drive EGFR inhibitor resistance.
(A) Identification of the most upregulated tumor progression genes common to erlotinib resistant carcinomas. (B) Erlotinib IC50 in a panel of human carcinoma cell lines treated with erlotinib in 3D culture, n = 3 independent experiments. (C) Percentage of integrin β3 positive cells in parental lines vs. after 3 or 8 weeks treatment with erlotinib. (D) Quantification of integrin β3 (ΠΌβ3) gene expression in human lung cancer biopsies from patients from the BATTLE Study (18) who were previously treated with an EGFR inhibitor and progressed (n = 27), versus patients who were EGFR inhibitor naive (n = 39). (*P = 0.04 using a Student's / test). (E) Paired human lung cancer biopsies obtained before and after erlotinib resistance were immunohistochemically stained for integrin β3. Scale bar, 50 μιη. (F) Right, effect of integrin β3 knockdown on erlotinib resistance of β3-ρο8Ϊίίνε cells. Cells were treated with 0.5 μΜ of erlotinib. Results are normalized using non-treated cells as controls, n = 3; mean ± SEM. *P < 0.05, **P < 0.001. Left, effect of integrin β3 ectopic expression on erlotinib resistance in FG and H441 cells. Cells were treated with 0.5 μΜ of erlotinib. n = 3; mean ± SEM. *P < 0.05, **P < 0.001. (G) Right, effect of integrin β3 knockdown on erlotinib resistance in vivo, A549 shCTRL and A549 sh integrin β3 (n=8 per treatment group) were treated with erlotinib (25 mg/kg/day) or vehicle during 16 days. Results are expressed as average of tumor volume at day 16. *P < 0.05. Left, orthotopic FG and FG^3 tumors (>1000 mm3; n = 5 per treatment group) were treated for 30 days with vehicle or erlotinib. Results are expressed as % tumor weight compared to vehicle control. *P < 0.05.
Figure 2 (Fig. 13/31) illustrates data showing that integrin β3 is required to promote KRAS dependency and KRAS-mediated EGFR inhibitor resistance.
(A) Confocal microscopy images show immunostaining for integrin β3 (green), K-, N-, H-, R-Ras (red), and DNA (TOPRO-3, blue) for BxPc3 cells grown in suspension in media with 10% serum. Arrows indicate clusters where integrin β3 and KRAS colocalize (yellow). Scale bar, 10 μιη. Data are representative of three independent experiments. Erlotinib IC50 in a panel of human carcinoma cell lines expressing non- target shRNA control or KRAS-specific shRNA and treated with erlotinib. n = 3 mean ± SEM. *P < 0.05, **P < 0.01. (B-C) Confocal microscopy images show immunostaining for integrin β 3 (green), KRAs (red) and DNA (Topro-3, blue) for PANC-1 (KRAS mutant) and HCC827 (KRAS wild-type) after acquired resistance to erlotinib (HCC827R) grown in suspension in absence (Vehicle) or in presence of erlotinib (0.5 μΜ and 0.1 μΜ respectively). Arrows indicate clusters where integrin β3 and KRAS colocalize (yellow). Scale bar, 10 μιη. Data are representative of three independent experiments. (D) Effect of KRAS knockdown on tumorspheres formation in a panel of lung and pancreatic cancer cells expressing or lacking integrin β3. n = 3 mean ± SEM. *P < 0.05, **P < 0.01. (E) Effect of KRAS knockdown on tumorsphere formation in PANC-1 (KRAS mutant) stably expressing non-target shRNA control (μ3 -positive) or specific-integrin β3 shRNA (β3 negative) in FG (KRAS mutant) and BxPc3 (KRAS wild-type) stably expressing vector control or integrin β3. *n = 3; mean + SEM. *P < 0.05. **P < 0.01. (F) Effect of KRAS knockdown on erlotinib resistance of β3 -negative and β3 -positive epithelial cancer cell lines. Cells were treated with a dose response of erlotinib. n = 3; mean ± SEM, *P < 0.05, **P < 0.01. (G) Confocal microscopy images show immunostaining for integrin β3 (green), KRAS (red) and DNA (TOPRO-3, blue) for PANC-1 cells expressing non-target shRNA control or Galectin 3 -specific shRNA grown in suspension. Scale bar = 10 μιη. Data are representative of three independent experiments. (H) Top: immunoblot analysis of integrin β3 immunoprecipitates from PANC-1 cells expressing non-target shRNA control (CTRL) or Galectin-3 -specific shRNA (Gal-3). Bottom: immunoblot analysis of Galectin-3 immunoprecipitates from PANC-1 cells expressing non-target shRNA control (CTRL) or integrin p3-specific shRNA (β3). Data are representative of three independent experiments. (I) Erlotinib dose response of FG^3 cells expressing a non-target shRNA control or a Galectin-3 -specific shRNA (sh Gal-3). n = 3; mean ± SEM.
Figure 3 (Fig. 14/31) illustrates data showing that RalB is a central player of integrin β3 -mediated EGFR inhibitor resistance.
(A) Effect of RalB knockdown on erlotinib resistance of β3-ρο8Ϊίϊνε epithelial cancer cell lines. Cells were treated with 0.5 μΜ of erlotinib. n = 3; mean ± SEM, *P < 0.05, **P < 0.01. (B) Effect of RalB knockdown on erlotinib resistance of 3-positive human pancreatic (FG- 3) orthotopic tumor xenografts. Established tumors expressing non-target shRNA, (shCTRL) or a shRNA targeting RalB (sh RalB) (>1000 mm3; n = 13 per treatment group) were randomized and treated for 10 days with vehicle or erlotinib. Results are expressed as % of tumor weight changes after erlotinib treatment compared to vehicle. **P < 0.01. (C) Effect of expression of a constitutively active Ral G23V mutant on erlotinib response of β3 negative cells. Cells were treated with 0.5 μΜ of erlotinib. n = 3; mean ± SEM. *P < 0.05. (D) Effect of expression of integrin β3 on KRAS and RalB membrane localization. Data are representative of two independent experiments. (E) Ral activity was determined in PANC-1 cells grown in suspension by using a GST-RalBP l- RBD immunoprecipitation assay. Immunoblots indicate RalB activity and association of active RalB with integrin β3. Data are representative of three independent experiments. (F) Confocal microscopy images of integrin ανβ3 (green), RalB (red) and DNA (TOPRO- 3, blue) in tumor biopsies from pancreatic cancer patients. Scale bar, 20 μιη. (G) Effect of β3 expression and KRAS expression on RalB activity, measured using a GST-RalBPl- RBD immunoprecipitation assay. Data are representative of three independent experiments. (H) Immunoblot analysis of FG and FG^3 stably expressing non-target shRNA control or RalB-specific shRNA, grown in suspension and treated with erlotinib (0.5 μΜ). Data are representative of three independent experiments. (I) Effect of TBK1 and p65 NFKB on erlotinib resistance of FG^3 cells. Cells were treated with 0.5 μΜ of erlotinib. n = 3; mean ± SEM. * P < 0.05, **P < 0.01.
Figure 4 (Fig. 15/31) illustrates data showing that reversal of β3 -mediated EGFR inhibitor resistance in oncogenic KRAS model by pharmacological inhibition.
(A) Effect of NFkB inhibitors on erlotinib response of β3-ρο8Ϊίίνε cells (FG^3, PANC-1 and A549). Cells were treated with vehicle, erlotinib (0.5 μΜ), lenalidomide (1- 2 μΜ), bortezomib (4 nM) alone or in combination, n = 3; mean ± SEM. *P < 0.05, **P < 0.01. (B) Left, mice bearing subcutaneous β3-ρο8Ϊίίνε tumors (FG^3) were treated with vehicle, erlotinib (25 mg/kg/day), lenalidomide (25 mg/kg/day) or the combination of erlotinib and lenalidomide. Tumor dimensions are reported as the fold change relative to size of the same tumor on Day 1. Mean ± SEM, (A) *P =0.042 using a one way ANOVA test, n = 6 mice per group. Right, mice bearing subcutaneous β3 -positive tumors (FG-R) after acquired resistance to erlotinib were treated with vehicle, erlotinib (25 mg/kg/day), bortezomib (0.25 mg/kg), the combination of erlotinib and bortezomib. Tumor dimensions are reported as the fold change relative to size of the same tumor on Day 1. *P =0.0134 using a one way ANOVA test, n = 8 mice per group. (C) Model depicting the proposed integrin ανβ3 -mediated KRAS dependency and EGFR inhibitor resistance mechanism.
Supplementary Fig. SI (Fig. 16/31) illustrates resistance to EGFR inhibitor is associated with integrin β3 expression in pancreatic and lung human carcinoma cell lines. (A) Immunoblots showing integrin β3 expression in human cell lines used in Figure 1A and Figure IB. (B) Effect of erlotinib on HCC827 xenograft tumors in immuno - compromised mice (n = 5 mice per treatment group) relative to vehicle-treated control tumors. Representative Integrin β3 cell surface quantification in HCC827 treated with vehicle or erlotinib during 64 days. (C) Integrin ανβ3 quantification in orthotopic lung and pancreas tumors treated with vehicle or erlotinib until resistance. For lung cancer, integrin β3 expression was scored (scale 0 to 3) and representative images are shown. For pancreatic cancer, integrin β3 expression was quantified as ratio of integrin ανβ3 pixel area over nuclei pixel area using METAMORPH™ (**P = 0.0012, *P = 0.049 using Mann- Whitney U test). Representative immunofluorescent staining of integrin ανβ3 in pancreatic human xenografts treated 4 weeks with vehicle or erlotinib.
Supplementary Fig. S2 (Fig. 17/31) illustrates Integrin β3 expression predicts intrinsic resistance to EGFR inhibitors in tumors. Plot of progression-free survival for erlotinib-treated patients with low vs. high protein expression of β3 integrin measured from non-small cell lung cancer biopsy material obtained at diagnosis (*P=0.0122, using Mann- Whitney U test). Representative images showing immunohistochemical staining for β3 integrin (brown) are shown.
Supplementary Fig. S3 (Fig. 18/31) illustrates Integrin β3 confers Receptor
Tyrosine Kinase inhibitor resistance.
(A) Immunoblots showing integrin β3 knockdown efficiency in cells used in Figure 1. (B) Response of A549 lung carcinoma cells non-target shRNA control or shRNA targeting integrin β3 to treatment with either vehicle or erlotinib (25 mg/kg/day) during 16 days. Tumor volumes are expressed as mean ± SEM. n = 8 mice per group. (C) Immunoblots showing expression of indicated proteins of representative tumors. (D) Representative photographs of crystal violet-stained tumorspheres of β3 -negative and β3 -positive cells after erlotinib, OSI-906, gemcitabine and cisplatin treatment. (E) Effect of integrin β3 expression on lapatinib, OSI-906, cisplatin and gemcitabine n = 3; mean ± SEM. (F) Viability assay (CellTiter-Glo assay) of FG and FG- 3 cells grown in suspension in media with or without serum, n = 2; mean + SEM. *P < 0.05. **P < 0.01.
Supplementary Fig. S4 (Fig. 19/31) illustrates Integrin 3-mediated EGFR inhibitor resistance is independent of its ligand binding.
Effect of ectopic expression of β3 wild-type (FG- β3) or the β3 Dl 19A (FG-D1 19A) ligand binding domain mutant on erlotinib response, n = 3; mean ± SEM. Immunoblot showing transfection efficiency of vector control, integrin β3 wild-type and integrin β3 D119A.
Supplementary Fig. S5 (Fig. 20/31) illustrates Integrin β3 colocalizes and interacts with oncogenic and active wild-type KRAS.
(A) Confocal microscopy images of FG and FG^3 cells grown in suspension in media 10% serum with or without erlotinib (0.5 μΜ) and stained for KRAS (red), integrin ανβ3 (green) and DNA (TOPRO-3, blue). Scale bar, 10 μιη. Data are representative of three independent experiments. (B) Ras activity was determined in PANC-1 cells grown in suspension by using a GST-Rafl-RBD immunoprecipitation assay. Immunoblots indicate KRAS activity and association of active KRAS with integrin β3. Data are representative of three independent experiments. (C) Immunoblot analysis of Integrin ανβ3
immunoprecipitates from BxPC-3 cells grown in suspension in presence or absence of growth factors.
Supplementary Fig. S6 (Fig. 21/31) illustrates Integrin β3 expression promotes KRAS dependency.
(A) Immunoblots showing KRAS knockdown efficiency in cells used in Figure 2. (B) Representative photographs of crystal violet-stained tumorspheres of FG and A549 cells expressing non-target shRNA control or specific -KRAS shRNA. (C) Effect of an additional KRAS knockdown on tumorspheres formation in PANC- 1 stably expressing non-target shRNA control (β3 -positive) or specific-integrin β3 shRNA (β3 negative), n = 3; mean +SEM. *P < 0.05. Immunoblots showing KRAS knockdown efficiency.
Supplementary Fig. S7 (Fig. 22/31) illustrates KRAS and Galectin-3 colocalize in integrin β3 -positive cells.
Confocal microscopy images of FG and FG^3 cells grown in suspension and stained for KRAS (green), galectin-3 (red) and DNA (TOPRO-3, blue). Scale bar, 10 μηι. Data are representative of three independent experiments.
Supplementary Fig. S8 (Fig. 23/31) illustrates Integrin 3-mediated KRAS dependency and erlotinib resistance is independent of ERK, AKT and RalA.
(A) Effect of ERK, AKT, RalA and RalB knockdown on erlotinib response (erlotinib 0.5 μΜ) of 3-negative FG and 3-positive FG- 3 cells. n= triplicate. (B) Immunoblots showing ERK, AKT RalA and RalB knockdown efficiency. (C) Immunoblots showing RalB knockdown efficiency in cells used in Figure 3.
Supplementary Fig. S9 (Fig. 24/31) illustrates Constitutive active NFkB is sufficient to promote erlotinib resistance.
(A) Immunoblots showing TBK1 and NFkB knockdown efficiency used in Figure 3. (B) Effect of constitutive active S276D p65NFkB on erlotinib response (erlotinib 0.5 μΜ) of 3-negative cells (FG cells), n = 3; mean ± SEM. *P < 0.05.
Supplementary Fig. S10 (Fig. 25/31) illustrates NFkB inhibitors in combination with erlotinib increase cell death in vivo.
(A-B) Immunoblots showing expression of indicated proteins of representative tumors from shown in Figure 4B (C) Confocal microscopy images of cleaved caspase 3 (red) and DNA (TOPRO-3, blue) in tumor biopsies from xenografts tumors used in Fig. 4B treated with vehicle, erlotinib, lenalidomide or lenalidomide and erlotinib in combo. Scale bar, 20 μιη. (D) Confocal microscopy images of cleaved caspase 3 (red) and DNA (TOPRO-3, blue) in tumor biopsies from xenografts tumors used in Figure 4B treated with vehicle, erlotinib, bortezomib or bortezomib and erlotinib in combo.
Supplementary Table 1 (Fig 26/31): shows differentially expressed genes in cells resistant to erlotinib (PANC-1, H1650, A459) compared with the average of two sensitive cells (FG, H441) and in HCC827 after acquired resistance in vivo (HCC827R) vs. the
HCC827 vehicle-treated control. The genes upregulated more than 2.5 fold are in red.
Supplementary Table 2 (Fig 29/31): shows KRAS mutational status of the pancreatic and lung cancer cell lines used in this study.
References - Example 2
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A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A method for:
overcoming or diminishing or preventing a Growth Factor Inhibitor (GFI) resistance in a cell, or
increasing the growth-inhibiting effectiveness of a Growth Factor inhibitor on a cell, or
sensitizing or re-sensitizing a cell to a Growth Factor Inhibitor (GFI), or sensitizing or re-sensitizing a dysfunctional cell a tumor or cancer to a drug, wherein optionally the drug is an erlotinib, a lapatinib, a bortezomib, or a lenalidomide, or sensitizing or re-sensitizing a tumor that is resistant to a cancer or anti-tumor drug, wherein optionally the cell is a tumor cell, a cancer cell, a cancer stem cell, or a dysfunctional cell,
the method comprising:
(a) (1) providing at least one compound, composition or formulation comprising or consisting of:
(i) an inhibitor or depleter of integrin ανβ3 (avb3), or an inhibitor of integrin ανβ3 (avb3) protein activity, or an inhibitor of the formation or activity of an integrin avb3/RalB signaling complex, or an inhibitor of the formation or signaling activity of an integrin ανβ3 (avb3)/RalB/NFkB signaling axis,
wherein optionally the inhibitor of integrin ανβ3 protein activity is an allosteric inhibitor of integrin ανβ3 protein activity;
(ii) an inhibitor or depleter of a RalB protein or an inhibitor of a RalB protein activation,
wherein optionally the inhibitor of the RalB protein activity is an allosteric inhibitor of RalB protein activity;
(iii) an inhibitor or depleter of a Src or a TBK1 protein or an inhibitor of Src or TBK1 protein activation,
wherein optionally the inhibitor of the Src or the TBK1 protein activity is an allosteric inhibitor of Src or TBK1 protein activity;
(iv) an inhibitor or depleter of a NFKB or a Interferon regulatory factor 3
(IRF3) protein or an inhibitor of RalB protein activation, wherein optionally the inhibitor of the NFKB or the IRF3 protein activity is an allosteric inhibitor of an NFKB or an Interferon regulatory factor 3 (IRF3) protein activity;
(v) an inhibitor or depleter of NFKB or IKK, or an inhibitor of NFKB or IKK protein activation,
wherein optionally the NFKB inhibitor comprises a lenalidomide or a REVLIMID™ (Celgene Corp., Summit, NJ), or a bortezomib or a
VELCADE® , and optionally the IKK inhibitor comprises a PS 1145
(Millennium Pharmaceuticals, Cambridge, MA);
(vi) a lenalidomide or a REVLIMID™ and PS 1145;
(vii) an erlotinib or a TARCEVA® and a bortezomib or a VELCADE®;
(viii) a lenalidomide or a REVLIMID™ ; a PS 1145; and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTK inhibitor comprises SU14813 (Pfizer, San Diego, CA); or
(ix) any combination of (i) to (viii); or
(2) one or any combination of the compound, composition or formulation, or compounds, compositions or formulations, of (1), and at least one growth factor inhibitor,
wherein optionally the at least one growth factor inhibitor comprises a Receptor Tyrosine Kinase (RTK) inhibitor, a Src inhibitor, an anti-metabolite inhibitor, a gemcitabine, GEMZAR™, a mitotic poison, a paclitaxel, a taxol, ABRAXANE™, an erlotinib, TARCEVA™, a lapatinib, TYKERB™, or an insulin growth factor inhibitor;
wherein optionally the combination or the therapeutic combination comprises an erlotinib with either a Lenalidomide or a PS- 1145, or both a Lenalidomide and a PS- 1145; and
(b) administering a sufficient amount of the at least one compound, composition or formulation to the cell, or the combination of compounds, to: overcome, diminish or prevent the Growth Factor Inhibitor (GFI) resistance in a cell; increase the growth- inhibiting effectiveness of the Growth Factor Inhibitor on the cell; sensitize or re-sensitize the cell to the Growth Factor Inhibitor (GFI); sensitize or re-sensitize the dysfunctional cell, tumor cell or cancer to the drug, or sensitize or re-sensitize the tumor that is resistant to the cancer or anti-tumor drug,
wherein optionally the cell is a tumor cell, a cancer cell, a cancer stem cell, or a dysfunctional cell.
2. The method of claim 1, wherein:
(a) the at least one compound, composition or formulation, or combination of compounds, is formulated as a pharmaceutical composition;
(b) the method of (a), wherein the compound, composition or formulation or pharmaceutical composition is administered in vitro, ex vivo or in vivo, or is administered to an individual in need thereof;
(c) the method of (a) or (b), wherein the at least one compound, composition or formulation is a pharmaceutical composition is formulated for administration
intravenously (IV), parenterally, nasally, topically, orally, or by liposome or targeted or vessel-targeted nanoparticle delivery;
(d) the method of any of (a) to (c), wherein the compound or composition comprises or is an inhibitor of transcription, translation or protein expression;
(e) the method of any of (a) to (d), wherein the compound or composition is a small molecule, a protein, an antibody, a monoclonal antibody, a nucleic acid, a lipid or a fat, a polysaccharide, an R A or a DNA;
(f) the method of any of (a) to (e), wherein the compound or composition comprises or is: a VITAXIN™ (Applied Molecular Evolution, San Diego, CA) antibody, a humanized version of an LM609 monoclonal antibody, an LM609 monoclonal antibody, or any antibody that functionally blocks an ανβ3 integrin or any member of an av 3 integrin-comprising complex or an integrin ανβ3 (anb3)/RalB/NFkB signaling axis;
(g) the method of any of (a) to (e), wherein the compound or composition comprises or is a Src inhibitor, a dasatinib, a saracatinib; a bosutinib; a VP-BHG712, or any combination thereof;
(h) the method of any of (a) to (g), wherein Growth Factor Inhibitor is or comprises an anti-metabolite inhibitor, a gemcitabine, GEMZAR™, a mitotic poison, a paclitaxel, a taxol, ABRAXANE™, an erlotinib, TARCEVA™, a lapatinib, TYKERB™, or an insulin growth factor inhibitor, or any combination thereof;
(i) the method of any of (a) to (h), wherein the Growth Factor Inhibitor decreases, slows or blocks new blood vessel growth, neovascularization or angiogenesis; or, wherein administering the Growth Factor Inhibitor treats or ameliorates conditions that are responsive to blocking or slowing cell growth, and/or the development of
neovascularization or new blood vessels;
(j) the method of any of (a) to (h), wherein the NF-kB inhibitor comprises or consists of one or more of: an antioxidant; an a-lipoic acid; an a-tocopherol; a 2-amino-l- methyl-6-phenylimidazo[4,5- ]pyridine; an allopurinol; an anetholdithiolthione; a cepharanthine; a beta-carotene; a dehydroepiandrosterone (DHEA) or a DHEA-sulfate
(DHEAS); a dimethyldithiocarbamates (DMDTC); a dimethylsulfoxide (DMSO); a flavone, a Glutathione; Vitamin C or Vitamin B6, or one or more compositions listed in
Table 1 or Table 2, or any combination thereof;
(k) the method of any of (a) to (j), wherein the at least one compound, composition or formulation, or combination of compounds, comprises a proteasome inhibitor or a protease inhibitor that can inhibit an Rei and/or an NFkB, or one or more compositions listed in Table 2, or any combination thereof;
(1) the method of any of (a) to (j), wherein the at least one compound, composition or formulation, or combination of compounds, comprises an ΙκΒα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) phosphorylation and/or degradation inhibitor, or one or more compositions listed in Table 3, or any combination thereof; or
(m) the method of any of (a) to (1), wherein the method reduces, treats or ameliorates the level of disease in a retinal age-related macular degeneration, a diabetic retinopathy, a cancer or carcinoma, a glioblastoma, a neuroma, a neuroblastoma, a colon carcinoma, a hemangioma, an infection and/or a condition with at least one inflammatory component, and/or any infectious or inflammatory disease, such as a rheumatoid arthritis, a psoriasis, a fibrosis, leprosy, multiple sclerosis, inflammatory bowel disease, or ulcerative colitis or Crohn's disease.
3. A kit, a blister package, a lidded blister or a blister card or packet, a clamshell, a tray or a shrink wrap, comprising;
(a) (i) at least one compound, composition or formulation used to practice the method of claim 1, and (ii); at least one Growth Factor Inhibitor,
wherein optionally the Growth Factor Inhibitor is or comprises an anti-metabolite inhibitor, a gemcitabine, GEMZAR™, a mitotic poison, a paclitaxel, a taxol,
ABRAXANE™, an erlotinib, TARCEVA™, a lapatinib, TYKERB™, or an insulin growth factor inhibitor, or any combination thereof; or, the Growth Factor Inhibitor decreases, slows or blocks new blood vessel growth, neovascularization or angiogenesis; or, wherein administering the Growth Factor Inhibitor treats or ameliorates conditions that are responsive to blocking or slowing cell growth, and/or the development of neovascularization or new blood vessels; or
(b) the kit of (a), further comprising instructions for practicing the method of claim 1 or claim 2,
wherein optionally the kit, blister package, lidded blister, blister card, packet, clamshell, tray or shrink wrap comprises: an erlotinib with either lenalidomide or PS- 1 145, or lenalidomide and PS-1 145.
4. A method for determining:
whether an individual or a patient would benefit from or respond to administration of a Growth Factor Inhibitor, or
whether an individual or a patient would benefit from a combinatorial approach comprising administration of a combination of: at least one growth factor inhibitor and at least one compound, composition or formulation used to practice the method of claim 1, wherein optionally the least one compound comprises an NFkB inhibitor or an
IKK inhibitor, and optionally the NFKB inhibitor comprises a lenalidomide or a
REVLIMID™ (Celgene Corp., Summit, NJ) , or a bortezomib or a VELCADE® , and optionally the IKK inhibitor comprises a PS 1 145 (Millennium Pharmaceuticals, Cambridge, MA),
the method comprising:
detecting the levels or amount of integrin ανβ3 (avb3) and/or active RalB complex in or on a cell, a tissue or a tissue sample, wherein optionally the detection is by analysis or visualization of a biopsy or a tissue, urine, fluid, serum or blood sample, or a pathology slide taken from the patient or individual, or by a fluorescence-activated cell sorting (FACS) or flow cytometry analysis or the sample or biopsy,
wherein optionally the cell or tissue or tissue sample is or is derived from a tumor or a cancer, or the cell is a cancer stem cell,
wherein optionally the method further comprises taking a biopsy or a tissue, urine, fluid, serum or blood sample from an individual or a patient,
wherein a finding of increased levels or amounts of integrin ανβ3 (avb3) and/or active RalB complexes in or on the cell, tissue or the tissue sample as compared to normal, normalized or wild type cells or tissues, indicates that:
the individual or patient would benefit from a combinatorial approach comprising administration of a combination of: at least one growth factor and at least one compound, composition or formulation used to practice the method of claim 1.
5. The method of claim 4, wherein the detecting of the levels or amount of integrin ανβ3 (avb3) and/or active RalB complex in or on the cell, tissue or the tissue sample is done before or during a drug or a pharmaceutical treatment of an individual using at least one compound, composition or formulation used to practice the method of claim 1.
6. Use of a combination of compounds in the manufacture of a medicament, wherein the combination of compounds comprises:
(1) at least one compound comprising or consisting of:
(i) an inhibitor or depleter of integrin ανβ3 (avb3), or an inhibitor of integrin ανβ3 (avb3) protein activity, or an inhibitor of the formation or activity of an integrin avb3/RalB signaling complex, or an inhibitor of the formation or signaling activity of an integrin ανβ3 (avb3)/RalB/NFkB signaling axis,
wherein optionally the inhibitor of integrin ανβ3 protein activity is an allosteric inhibitor of integrin ανβ3 protein activity;
(ii) an inhibitor or depleter of RalB protein or an inhibitor of RalB protein activation, wherein optionally the inhibitor of RalB protein activity is an allosteric inhibitor of RalB protein activity;
(iii) an inhibitor or depleter of Src or TBK1 protein or an inhibitor of Src or TBK1 protein activation,
wherein optionally the inhibitor of Src or TBK1 protein activity is an allosteric inhibitor of Src or TBK1 protein activity;
(iv) an inhibitor or depleter of NFKB or IRF3 protein or an inhibitor of RalB protein activation,
wherein optionally the inhibitor of NFKB or IRF3 protein activity is an allosteric inhibitor of NFKB or IRF3 protein activity;
(v) an inhibitor or depleter of NFKB or IKK, or an inhibitor of NFKB or IKK protein activation,
wherein optionally the NFKB inhibitor comprises a lenalidomide or a REVLIMID™ (Celgene Corp., Summit, NJ) , or a bortezomib or a
VELCADE® , and optionally the IKK inhibitor comprises a PS 1145
(Millennium Pharmaceuticals, Cambridge, MA);
(vi) a lenalidomide or a REVLIMID™ and PS 1145;
(vii) an erlotinib or a TARCEVA® and a bortezomib or a VELCADE®;
(viii) a lenalidomide or a REVLIMID™ ; a PS 1145; and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTK inhibitor comprises
SU14813 (Pfizer, San Diego, CA); or
(ix) any combination of (i) to (viii); and/or
(2) at least one Growth Factor Inhibitor,
wherein optionally the Growth Factor Inhibitor is or comprises an anti-metabolite inhibitor, a gemcitabine, GEMZAR™, a mitotic poison, a paclitaxel, a taxol,
ABRAXANE™, an erlotinib, TARCEVA™, a lapatinib, TYKERB™, or an insulin growth factor inhibitor, or any combination thereof; or, the Growth Factor Inhibitor decreases, slows or blocks new blood vessel growth, neovascularization or angiogenesis; or, wherein administering the Growth Factor Inhibitor treats or ameliorates conditions that are responsive to blocking or slowing cell growth, and/or the development of neovascularization or new blood vessels, wherein optionally the combination or the therapeutic combination comprises an erlotinib with either Lenalidomide or PS-1145, or Lenalidomide and PS-1 145.
7. A therapeutic combination of drugs comprising or consisting of a combination of at least two compounds: wherein the at least two compounds comprise or consist of:
(1) at least one compound comprising or consisting of:
(i) an inhibitor or depleter of integrin ανβ3 (avb3), or an inhibitor of integrin ανβ3 (avb3) protein activity, or an inhibitor of the formation or activity of an integrin anb3/RalB signaling complex, or an inhibitor of the formation or signaling activity of an integrin ανβ3 (avb3)/RalB/NFkB signaling axis,
wherein optionally the inhibitor of integrin ανβ3 protein activity is an allosteric inhibitor of integrin ανβ3 protein activity;
(ii) an inhibitor or depleter of RalB protein or an inhibitor of RalB protein activation,
wherein optionally the inhibitor of RalB protein activity is an allosteric inhibitor of RalB protein activity;
(iii) an inhibitor or depleter of Src or TBK1 protein or an inhibitor of Src or TBK1 protein activation,
wherein optionally the inhibitor of Src or TBK1 protein activity is an allosteric inhibitor of Src or TBK1 protein activity;
(iv) an inhibitor or depleter of NFKB or IRF3 protein or an inhibitor of RalB protein activation,
wherein optionally the inhibitor of NFKB or IRF3 protein activity is an allosteric inhibitor of NFKB or IRF3 protein activity;
(v) an inhibitor or depleter of NFKB or IKK, or an inhibitor of NFKB or IKK protein activation,
wherein optionally the NFKB inhibitor comprises a lenalidomide or a REVLIMID™ (Celgene Corp., Summit, NJ) , or a bortezomib or a
VELCADE® , and optionally the IKK inhibitor comprises a PS 1145
(Millennium Pharmaceuticals, Cambridge, MA);
(vi) a lenalidomide or a REVLIMID™ and PS 1145; (vii) an erlotinib or a TARCEVA® and a bortezomib or a VELCADE®;
(viii) a lenalidomide or a REVLIMID™ ; a PS 1145; and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTK inhibitor comprises SU14813 (Pfizer, San Diego, CA); or
(ix) any combination of (i) to (viii); and/or
(2) at least one Growth Factor Inhibitor,
wherein optionally the Growth Factor Inhibitor is or comprises an anti-metabolite inhibitor, a gemcitabine, GEMZAR™, a mitotic poison, a paclitaxel, a taxol,
ABRAXANE™, an erlotinib, TARCEVA™, a lapatinib, TYKERB™, or an insulin growth factor inhibitor, or any combination thereof; or, the Growth Factor Inhibitor decreases, slows or blocks new blood vessel growth, neovascularization or angiogenesis; or, wherein administering the Growth Factor Inhibitor treats or ameliorates conditions that are responsive to blocking or slowing cell growth, and/or the development of neovascularization or new blood vessels,
wherein optionally the combination or the therapeutic combination comprises an erlotinib with either Lenalidomide or PS-1145, or Lenalidomide and PS-1 145.
8. A combination, or a therapeutic combination, for overcoming or diminishing or preventing Growth Factor Inhibitor (GFI) resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor (GFI), wherein the combination comprises or consists of:
(1) at least one compound comprising or consisting of:
(i) an inhibitor or depleter of integrin ανβ3 (avb3), or an inhibitor of integrin ανβ3 (avb3) protein activity, or an inhibitor of the formation or activity of an integrin avb3/RalB signaling complex, or an inhibitor of the formation or signaling activity of an integrin ανβ3 (avb3)/RalB/NFkB signaling axis,
wherein optionally the inhibitor of integrin ανβ3 protein activity is an allosteric inhibitor of integrin ανβ3 protein activity;
(ii) an inhibitor or depleter of RalB protein or an inhibitor of RalB protein activation, wherein optionally the inhibitor of RalB protein activity is an allosteric inhibitor of RalB protein activity;
(iii) an inhibitor or depleter of Src or TBK1 protein or an inhibitor of Src or TBK1 protein activation,
wherein optionally the inhibitor of Src or TBK1 protein activity is an allosteric inhibitor of Src or TBK1 protein activity;
(iv) an inhibitor or depleter of NFKB or IRF3 protein or an inhibitor of RalB protein activation,
wherein optionally the inhibitor of NFKB or IRF3 protein activity is an allosteric inhibitor of NFKB or IRF3 protein activity;
(v) an inhibitor or depleter of NFKB or IKK, or an inhibitor of NFKB or IKK protein activation,
wherein optionally the NFKB inhibitor comprises a lenalidomide or a REVLIMID™ (Celgene Corp., Summit, NJ) , or a bortezomib or a
VELCADE® , and optionally the IKK inhibitor comprises a PS 1145
(Millennium Pharmaceuticals, Cambridge, MA);
(vi) a lenalidomide or a REVLIMID™ and PS 1145;
(vii) an erlotinib or a TARCEVA® and a bortezomib or a VELCADE®;
(viii) a lenalidomide or a REVLIMID™ ; a PS 1145; and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTK inhibitor comprises
SU14813 (Pfizer, San Diego, CA); or
(ix) any combination of (i) to (viii); and/or
(2) at least one Growth Factor Inhibitor,
wherein optionally the Growth Factor Inhibitor is or comprises an anti-metabolite inhibitor, a gemcitabine, GEMZAR™, a mitotic poison, a paclitaxel, a taxol,
ABRAXANE™, an erlotinib, TARCEVA™, a lapatinib, TYKERB™, or an insulin growth factor inhibitor, or any combination thereof; or, the Growth Factor Inhibitor decreases, slows or blocks new blood vessel growth, neovascularization or angiogenesis; or, wherein administering the Growth Factor Inhibitor treats or ameliorates conditions that are responsive to blocking or slowing cell growth, and/or the development of neovascularization or new blood vessels; wherein optionally the cell is a tumor cell, a cancer cell, a cancer stem cell, or a dysfunctional cell;
wherein optionally the combination or the therapeutic combination comprises an erlotinib with either Lenalidomide or PS-1145, or Lenalidomide and PS-1 145.
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