(12) Patent Application Publication (10) Pub. No.: US 2016/0146783 A1 CHERESH Et Al

US 2016O146783A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0146783 A1 CHERESH et al. (43) Pub. Date: May 26, 2016

(54) COMPOSITIONS AND METHODS FOR Publication Classification TREATING CANCER AND DISEASES AND CONDITIONS RESPONSIVE TO CELL (51) Int. C. GROWTH INHIBITION GOIN33/50 (2006.01) (52) U.S. C. (71) Applicant: THE REGENTS OF THE CPC. G0IN33/5011 (2013.01); G0IN 2333/4724 UNIVERSITY OF CALIFORNLA, (2013.01); G0IN 2333/70557 (2013.01) Oakland, CA (US) (57) ABSTRACT (72) Inventors: David CHERESH, Encinitas, CA (US); In alternative embodiments, the invention provides composi Laetitia SEGUIN, San Diego, CA (US); tions and methods for overcoming or diminishing or prevent Sudarshan ANAND, San Diego, CA ing Growth Factor Inhibitor resistance in a cell, or, a method (US) for increasing the growth-inhibiting effectiveness of a Growth Factor inhibitor on a cell, or, a method for re-sensi (21) Appl. No.: 14/883,398 tizing a cell to a Growth Factor Inhibitor, comprising for example, administration of a combination of a TBK1 inhibi tor and an RTK inhibitor. In alternative embodiments, the cell (22) Filed: Oct. 14, 2015 is a tumor cell, a cancer cell or a dysfunctional cell. In alter native embodiments, the invention provides compositions Related U.S. Application Data and methods for determining: whether an individual or a patient would benefit from or respond to administration of a (63) Continuation of application No. 14/325,288, filed on Growth Factor Inhibitor, or, which individuals or patients Jul. 7, 2014, which is a continuation-in-part of appli would benefit from a combinatorial approach comprising cation No. PCT/US2013/035492, filed on Apr. 5, administration of a combination of at least one growth factor 2013. and at least one compound, composition or formulation used (60) Provisional application No. 61/672.236, filed on Jul. to practice a method of the invention, such as an NFKB 16, 2012, provisional application No. 61/620,725, inhibitor, such as a lenalidomide or a REVLIMIDTM, or IKK filed on Apr. 5, 2012. inhibitor; or an inhibitor of Galectin-3.

Patent Application Publication May 26, 2016 Sheet 2 of 41 US 2016/O146783 A1

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COMPOSITIONS AND METHODS FOR Mutation/amplification in tyrosine kinase receptors or their TREATING CANCER AND DISEASES AND downstream effectors account for the resistance of a broad CONDITIONS RESPONSIVE TO CELL range of tumors. In particular, oncogenic KRAS, the most GROWTH INHIBITION commonly mutated oncogene in human cancer, has been linked to EGFR inhibitor resistance. However, in lung and RELATED APPLICATIONS pancreatic carcinomas, recent studies suggest that oncogenic 0001. This application is a continuation of U.S. patent KRAS is not sufficient to account for EGFR inhibitor resis application Ser. No. 14/325,288, filed Jul. 7, 2014, now pend tance indicating that other factor(s) might control this pro ing, which is a continuation in part (CIP) of Patent Conven CCSS, tion Treaty (PCT) International Application Serial No: PCT/ US2013/035492, filed Apr. 5, 2013, which claims benefit of SUMMARY priority to International Application Serial No: PCT/US2012/ 0006. In alternative embodiments, the invention provides 040390, filed Jun. 2, 2012, and which also claims benefit of methods for: priority to U.S. Provisional Patent Application Ser. No. 0007 overcoming or diminishing or preventing a Growth (“USSN)61/672,236, filed Jul. 16, 2012, and U.S. Ser. No. Factor Inhibitor (GFI) resistance in a cell, or 61/620,725, filed Apr. 5, 2012. This application also claims 0008 increasing the growth-inhibiting effectiveness of a the benefit of priority under 35 U.S.C. S 119(e) of U.S. Pro Growth Factor inhibitor on a cell, or visional Patent Application Ser. No. (“USSN) 61/843,417, 0009 sensitizing, increasing sensitivity to or re-sensitiz filed Jul. 7, 2013. The aforementioned applications are ing a cell to a Growth Factor Inhibitor (GFI), or expressly incorporated herein by reference in their entirety 0010 sensitizing, increasing sensitivity to or re-sensitiz and for all purposes. ing a dysfunctional cell, a tumor or cancer to a drug, 0.011 wherein optionally the drug is a Receptor GOVERNMENT RIGHTS Tyrosine Kinase (RTK) inhibitor, an EGFR1 inhibitor, 0002 This invention was made with government support an EGFR1/EGFR2 inhibitor or an IGF-1R inhibitor, or under grant numbers CA045726, CA050286, CA095262, an erlotinib, a linsitinib, a lapatinib or a lenalidomide, HL057900, and HL103956, awarded by the National Insti 0012 sensitizing, increasing sensitivity to or re-sensitiz tutes of Health (NIH). The government has certain rights in ing a tumor that is resistant to a cancer or anti-tumor drug, or the invention. 0013 reversing a tumor cell, a cancer cell, a cancer stem cellora dysfunctional cell initiation or self-renewal capacity, TECHNICAL FIELD 0.014 wherein optionally the cell is a tumor cell, a can cer cell, a cancer stem cell, or a dysfunctional cell, 0003. This invention generally relates to cell and molecu 00.15 the method comprising: lar biology, diagnostics and oncology. In alternative embodi 0016 (a)(1) providing at least one compound, compo ments, the invention provides compositions and methods for sition or formulation comprising or consisting of overcoming or diminishing or preventing Growth Factor 0017 (i) an inhibitor or depleter of integrin CfB Inhibitor resistance in a cell, or, a method for increasing the (anb3), or growth-inhibiting effectiveness of a Growth Factor inhibitor 0018 an inhibitor of integrin CfB (anb3) protein on a cell, or, a method for re-sensitizing a cell to a Growth activity, or Factor Inhibitor, or, sensitizing a tumor to a drug, wherein 0019 an inhibitor of the formation or activity of an optionally the drug is an erlotinib oralapatinib, or, sensitizing integrin anb3/RalB signaling complex, or a tumor that is resistant to a cancer drug, comprising for 0020 an inhibitor of the formation or signaling activ example, administration of a combination of a TBK1 inhibi ity of an integrin CfB (anb3)/RalB/NFkB signaling tor and an RTK inhibitor. In alternative embodiments, the cell axis, is a tumor cell, a cancer cell, a cancer stem cell or a dysfunc 0021 wherein optionally the inhibitor of integrin tional cell. In alternative embodiments, the invention pro CfB protein activity is an allosteric inhibitor of inte vides compositions and methods for determining: whetheran grin CfB protein activity; individual or a patient would benefit from or respond to 0022 (ii) an inhibitor or depleter of a RalB protein or administration of a Growth Factor Inhibitor, or, which indi an inhibitor of a RalB protein activation, or viduals or patients would benefit from a combinatorial 0023 an inhibitor or depleter of the recruitment of approach comprising administration of a combination of at KRAS/RalB to the plasma membrane or the associa least one growth factor and at least one compound, composi tion of KRAS to RalB, tion or formulation used to practice a method of the invention, 0024 wherein optionally the inhibitor is an allosteric Such as an NFKB (nuclear factor kappa-light-chain-enhancer inhibitor, or of activated B cells, or NF-kB) inhibitor, such as a lenalido (0025 optionally the inhibitor of the RalB protein mide or a REVLIMIDTM, oran IKB kinase (IKK) inhibitor; or activity is an allosteric inhibitor of RalB protein activ an inhibitor of Galectin-3. ity; 0026 (iii) an inhibitor or depleter of a Src or a Tank BACKGROUND Binding Kinase-1 (TBK1) protein or an inhibitor of 0004 Growth factor inhibitors have been used to treat Src or TBK1 protein activation, many cancers including pancreatic, breast, lung and colorec (0027 wherein optionally the inhibitor of the Src or tal cancers. However, resistance to growth factor inhibitors the TBK1 protein activity is: has emerged as a significant clinical problem. 0028 an amlexanox (or 2-amino-7-isopropyl-5-oxo 0005 Tumor resistance to targeted therapies occurs due to 5 H-chromeno2,3-bipyridine-3-carboxylic acid), or a combination of stochastic and instructional mechanisms. an APHTHASOLTM; or US 2016/0146783 A1 May 26, 2016

0029 a Y(1)34.5 protein of herpes simplex viruses 0032 a 6-amino-pyrazolopyrimidine or an analog or (HSV) (see e.g., Ma et al., J. Virol. 2012 February: derivative thereof; or, 86(4):2188-96); or 0030 a BX795 as described in, e.g., Bain et al., Bio 0033 a compound having one of the following for chem J. (2007) December 15:408(3):297-315; Clark mulas, oran analog or derivative thereof (see Hutti, et et al., (2009).J. Biol. Chem. 284:14136-14146; or al., (2012) Development of a High-Throughput Assay 0031 anazabenzimidazole oran analog orderivative for Identifying Inhibitors of TBK1 and IKKe. PLoS thereof, or ONE 7(7):e41494.doi:10.1371/journal.pone):

Molecule IKKe TBK1 IKKB IKKC.

O.77 0.44 -10 >10

>10 OSO >10 >10

u >10 O.64 8.76 -10

OH US 2016/0146783 A1 May 26, 2016

-continued

Molecule IKKe TBK1 /- N >10 O.67 |

N OH N O O O >10 O.87 2 NN4 N N S s NH isk O N \ O O HO

0034 and optionally the inhibitor of the Src or the TARCEVATM, a lapatinib, a TYKERBTM, a cetuxamib, TBK1 protein activity is an allosteric inhibitor of Src an ERBITUXTM, or an insulin growth factor inhibitor; or TBK1 protein activity; 0.045 wherein optionally the combination or the thera 0035 (iv) an inhibitor or depleter of a NFKB or a peutic combination comprises: (i) an inhibitor or depl Interferon regulatory factor 3 (IRF3) protein or an eter of a Src or a Tank Binding Kinase-1 (TBK1) protein inhibitor of RalB protein activation, or an inhibitor of Src or TBK1 protein activation, 0036 wherein optionally the inhibitor of the NFKB wherein optionally the inhibitor of the Src or the TBK1 or the IRF3 protein activity is an allosteric inhibitor of protein activity is an amlexanoX (or 2-amino-7-isopro an NFKB or an Interferon regulatory factor 3 (IRF3) pyl-5-oxo-5H-chromeno2,3-bipyridine-3-carboxylic protein activity; acid) or APHTHASOLTM, and (ii) an RTK inhibitor, 0037 (v) an inhibitor or depleter of NFKB or IKK, or wherein optionally the RTK inhibitor is a Src inhibitor, an inhibitor of NFKB or IKK protein activation, an anti-metabolite inhibitor, a gemcitabine, a 0038 wherein optionally the NFKB inhibitor com GEMZARTM, a mitotic poison, a paclitaxel, a taxol, an prises a lenalidomide or a REVLIMIDTM (Celgene ABRAXANETM, an erlotinib, a TARCEVATM, a lapa Corp., Summit, N.J.) and optionally the IKK inhibitor tinib, a TYKERBTM, a cetuxamib, an ERBITUXTM, or comprises a PS1145 (Millennium Pharmaceuticals, an insulin growth factor inhibitor or a combination Cambridge, Mass.); thereof; 0039 (vi) a lenalidomide or a REVLIMIDTM and 0046 wherein optionally the combination or the thera PS1145; peutic combination comprises an erlotinib with either a 0040 (vii) a lenalidomide or a REVLIMIDTM; a Lenalidomide or a PS-1145, or botha Lenalidomide and PS1145; and, a Receptor Tyrosine Kinase (RTK) a PS-1145; and inhibitor, and optionally the RTK inhibitor comprises 0047 (b) administering a sufficient amount of the at SU14813 (Pfizer, San Diego, Calif.); least one compound, composition or formulation to the 0041 (viii) an inhibitor of Galectin-3; or cell, or the combination of compounds, to: 0042 (ix) any combination of (i) to (viii), or 0048 overcome or diminish or prevent a Growth Factor 0043 (2) one or any combination of the compound, Inhibitor (GFI) resistance in a cell, or composition or formulation, or compounds, composi 0049 increase the growth-inhibiting effectiveness of a tions or formulations, of (1), and at least one growth Growth Factor inhibitor on a cell, or factor inhibitor, 0050 sensitize, increase sensitivity or re-sensitize a cell to 0044 wherein optionally the at least one growth factor a Growth Factor Inhibitor (GFI), or inhibitor comprises a Receptor Tyrosine Kinase (RTK) 0051 sensitize, increase sensitivity or re-sensitize a dys inhibitor, a Src inhibitor, an anti-metabolite inhibitor, a functional cell, a tumor or cancer to a drug, wherein option gemcitabine, a GEMZARTM, a mitotic poison, a pacli ally the drug is a Receptor Tyrosine Kinase (RTK) inhibitor, taxel, a taxol, an ABRAXANETM, an erlotinib, a or an erlotinib, a lapatinib or a lenalidomide, US 2016/0146783 A1 May 26, 2016

0052 sensitize, increase sensitivity or re-sensitize a tumor protease inhibitor that can inhibit an Rel and/or an NFkB, or that is resistant to a cancer or anti-tumor drug, or one or more compositions listed in Table 2, or any combina 0053 reversea tumor cell, a cancer cell, a cancer stem cell tion thereof; or a dysfunctional cell initiation or self-renewal capacity. 0.066 (1) the method of any of (a) to (), wherein the at least 0054 In alternative embodiments of the methods: one compound, composition or formulation, or combination 0055 (a) the at least one compound, composition or for of compounds, comprises an IKBC. (nuclear factor of kappa mulation, or combination of compounds, is formulated as a light polypeptide gene enhancer in B-cells inhibitor, alpha) pharmaceutical composition; phosphorylation and/or degradation inhibitor, or one or more compositions listed in Table 3, or any combination thereof; or 0056 (b) the method of (a), wherein the compound, com 0067 (m) the method of any of (a) to (1), wherein the position or formulation or pharmaceutical composition is method reduces, treats or ameliorates the level of disease in a administered in vitro, ex vivo or in vivo, or is administered to retinal age-related macular degeneration, a diabetic retinopa an individual in need thereof; thy, a cancer or carcinoma, a glioblastoma, a neuroma, a 0057 (c) the method of (a) or (b), wherein the at least one neuroblastoma, a colon carcinoma, a hemangioma, an infec compound, composition or formulation is a pharmaceutical tion and/or a condition with at least one inflammatory com composition is formulated for administration intravenously ponent, and/or any infectious or inflammatory disease. Such (IV), parenterally, nasally, topically, orally, or by liposome or as a rheumatoid arthritis, a psoriasis, a fibrosis, leprosy, mul targeted or vessel-targeted nanoparticle delivery; tiple Sclerosis, inflammatory bowel disease, or ulcerative 0058 (d) the method of any of (a) to (c), wherein the colitis or Crohn's disease. compound or composition comprises or is an inhibitor of 0068. In alternative embodiments, the invention provides transcription, translation or protein expression; kits, blister packages, lidded blisters or blister cards or pack 0059 (e) the method of any of (a) to (d), wherein the ets, clamshells, trays or shrink wraps, comprising: compound or composition is a small molecule, a protein, an 0069 (a) (i) at least one compound, composition or for antibody, a monoclonal antibody, a nucleic acid, a lipid or a mulation used to practice a method of the invention, and (ii); fat, a polysaccharide, an RNA or a DNA; at least one Growth Factor Inhibitor; or 0060 (f) the method of any of (a) to (e), wherein the 0070 (b) the kit of (a), further comprising instructions for compound or composition comprises or is: a VITAXINTM practicing a method of the invention. (Applied Molecular Evolution, San Diego, Calif.) antibody, a 0071. In alternative embodiments, the kit, blister package, humanized version of an LM609 monoclonal antibody, an lidded blister, blister card, packet, clamshell, tray or shrink LM609 monoclonal antibody, or any antibody that function wrap comprises: a combination or a therapeutic combination ally blocks an O?3 integrin or any member of an O?3 inte of drugs comprising: an erlotinib with eithera Lenalidomide grin-comprising complex or an integrin CfB (anb3)/RalB/ or a PS-1145, or both a Lenalidomide and a PS-1145. NFkB signaling axis; 0072. In alternative embodiments, the invention provides 0061 (g) the method of any of (a) to (e), wherein the methods for determining: compound or composition comprises or is a Src inhibitor, a 0073 whether an individual or a patient would benefit dasatinib, a saracatinib; a bosutinib; a NVP-BHG712, or any from or respond to administration of a Growth Factor Inhibi combination thereof; tor, or 0062 (h) the method of any of (a) to (g), wherein Growth 0074 which individuals or patients would benefit from a Factor Inhibitor is or comprises an anti-metabolite inhibitor, a combinatorial approach comprising administration of a com gemcitabine, GEMZARTM, a mitotic poison, a paclitaxel, a bination of at least one growth factor and at least one com taxol, ABRAXANETM, an erlotinib, TARCEVATM, a lapa pound, composition or formulation used to practice a method tinib, TYKERBTM, or an insulin growth factor inhibitor, or of the invention, such as an NfKb inhibitor, any combination thereof; 0075 the method comprising: 0063 (i) the method of any of (a) to (h), wherein the Growth Factor Inhibitor decreases, slows or blocks new blood 0076 detecting the levels or amount of integrin CfB vessel growth, neovascularization or angiogenesis; or, (anb3) and/or active RalB complex in or on a cell, a tissue or wherein administering the Growth Factor Inhibitor treats or a tissue sample, ameliorates conditions that are responsive to blocking or 0077 wherein optionally the detection is by analysis or visualization of a biopsy or a tissue, urine, fluid, serum or slowing cell growth, and/or the development of neovascular blood sample, or a pathology slide taken from the patient or ization or new blood vessels; individual, or by a fluorescence-activated cell sorting (FACS) 0064 () the method of any of (a) to (h), wherein the or flow cytometry analysis or the sample or biopsy, NF-kB inhibitor comprises or consists of one or more of an antioxidant; an O-lipoic acid; an O-tocopherol; a 2-amino-1- 0078 wherein optionally the cellor tissue or tissue sample methyl-6-phenylimidazo 4.5-3pyridine, an allopurinol; an is or is derived from a tumor or a cancer, anetholdithiolthione; a cepharanthine; a beta-carotene; a 0079 wherein optionally the method further comprises dehydroepiandrosterone (DHEA) or a DHEA-sulfate (DH taking a biopsy or a tissue, urine, fluid, serum or blood sample EAS); a dimethyldithiocarbamates (DMDTC); a dimethyl from an individual or a patient, sulfoxide (DMSO); a flavone, a Glutathione; Vitamin C or 0080 wherein a finding of increased levels or amounts of Vitamin B6, or one or more compositions listed in Table 1 or integrin C. B. (anb3) and/or active RalB complexes in or on Table 2, or any combination thereof; the cell, tissue or the tissue sample as compared to normal, 0065 (k) the method of any of (a) to (), wherein the at normalized or wild type cells or tissues, indicates that: least one compound, composition or formulation, or combi I0081 the individual or patient would benefit from a com nation of compounds, comprises a proteasome inhibitor or a binatorial approach comprising administration of a combina US 2016/0146783 A1 May 26, 2016 tion of at least one growth factor and at least one compound, (0091 (ii) an inhibitor or depleter of a RalB protein or composition or formulation used to practice a method of the an inhibitor of a RalB protein activation, or an inhibi invention. tor or depleter of the recruitment of KRAS/RalB to 0082 In alternative embodiments of methods of the inven the plasma membrane or the association of KRAS to tion, the detecting of the levels or amount of integrin CfB RalB, (anb3) and/or active RalB complex in or on the cell, tissue or 0092 wherein optionally the inhibitor is an allosteric the tissue sample is done before or during a drug or a phar inhibitor, or the inhibitor of the RalB protein activity maceutical treatment of an individual using at least one com is an allosteric inhibitor of RalB protein activity; pound, composition or formulation used to practice a method (0093 (iii) an inhibitor or depleter of a Src or a Tank of the invention. Binding Kinase (TBK1) protein oran inhibitor of Src or TBK1 protein activation, 0083. In alternative embodiments, the invention provide (0094 wherein optionally the inhibitor of the Src or uses of a combination of compounds in the manufacture of a the TBK1 protein activity is: an amlexanox (or medicament, 2-amino-7-isopropyl-5-oxo-5H-chromeno2,3-bpy 0084 wherein the combination of compounds comprises: ridine-3-carboxylic acid), or an APHTHASOLTM; or I0085 (1) at least one compound comprising or consist 0.095 a Y(1)34.5 protein of herpes simplex viruses ing of (HSV) (see e.g., Ma et al., J. Virol. 2012 February: I0086 (i) an inhibitor or depleter of integrin C, B, 86(4):2188-96); or, BX795 (as described in, e.g., Bain (anb3), or et al., Biochem J. (2007) December 15; 408(3):297 I0087 an inhibitor of integrin CfB (anb3) protein 315; Clark et al., (2009) J. Biol. Chem. 284:14136 activity, or 14146); or I0088 an inhibitor of the formation or activity of an 0096 anazabenzimidazole oran analog orderivative integrin anb3/RalB signaling complex, or thereof, or a 6-amino-pyrazolopyrimidine or an ana I0089 an inhibitor of the formation or signaling activ log or derivative thereof; or, ity of an integrin CfB (anb3)/RalB/NFkB signaling 0097 a compound having one of the following for axis, mulas, oran analog or derivative thereof (see Hutti, et 0090 wherein optionally the inhibitor of integrin al., (2012) Development of a High-Throughput Assay CfB protein activity is an allosteric inhibitor of inte for Identifying Inhibitors of TBK1 and IKKe. PLoS grin CB protein activity: ONE 7(7):e41494.doi:10.1371/journal.pone):

Molecule IKKe TBK1 IKKB IKKC.

O.77 0.44 >10 >10

>10 OSO >10 >10 US 2016/0146783 A1 May 26, 2016 6

-continued

Molecule IKKe TBK1 IKKB IKKC. u >10 O.64 8.76 -10

rSa N

/- N >10 O.67 O | Nl OH O O O >10 O.87 N21 S. N N N NH sk O N \ O O HO

(0098 and optionally the inhibitor of the Src or the (0103 (vi) a lenalidomide or a REVLIMIDTM and TBK1 protein activity is an allosteric inhibitor of Src PS1145; or TBK1 protein activity; 0104 (vii) a lenalidomide or a REVLIMIDTM; a (0099 (iv) an inhibitor or depleter of a NFKB or a PS1145; and, a Receptor Tyrosine Kinase (RTK) Interferon regulatory factor 3 (IRF3) protein or an inhibitor, and optionally the RTK inhibitor comprises inhibitor of RalB protein activation, SU14813 (Pfizer, San Diego, Calif.); 0100 wherein optionally the inhibitor of the NFKB or the IRF3 protein activity is an allosteric inhibitor of 0105 (viii) an inhibitor of Galectin-3; or an NFKB or an Interferon regulatory factor 3 (IRF3) 0106 (ix) any combination of (i) to (viii), or protein activity; 0.107 (2) one or any combination of the compound, 0101 (v) an inhibitor or depleter of NFKB or IKK, or composition or formulation, or compounds, composi an inhibitor of NFKB or IKK protein activation, tions or formulations, of (1), and at least one growth 0102 wherein optionally the NFKB inhibitor com factor inhibitor, prises a lenalidomide or a REVLIMIDTM (Celgene 0.108 wherein optionally the at least one growth factor Corp., Summit, N.J.) and optionally the IKK inhibitor inhibitor comprises a Receptor Tyrosine Kinase (RTK) comprises a PS1145 (Millennium Pharmaceuticals, inhibitor, a Src inhibitor, an anti-metabolite inhibitor, a Cambridge, Mass.); gemcitabine, a GEMZARTM, a mitotic poison, a pacli US 2016/0146783 A1 May 26, 2016

taxel, a taxol, an ABRAXANETM, an erlotinib, a of a combination of at least two compounds: wherein the at TARCEVATM, a lapatinib, a TYKERBTM, a cetuxamib, least two compounds comprise or consist of an ERBITUXTM, or an insulin growth factor inhibitor; 0109 wherein optionally the combination or the thera 0118 (1) at least one compound comprising or consist peutic combination comprises: (i) an inhibitor or depl ing of eter of a Src or a Tank Binding Kinase-1 (TBK1) protein 0119 (i) an inhibitor or depleter of integrin CfB or an inhibitor of Src or TBK1 protein activation, (anb3), or an inhibitor of integrin O?3 (anb3) protein wherein optionally the inhibitor of the Src or the TBK1 activity, or an inhibitor of the formation or activity of protein activity is an amlexanoX (or 2-amino-7-isopro an integrin anb3/RalB signaling complex, or an pyl-5-oxo-5H-chromeno2,3-bipyridine-3-carboxylic inhibitor of the formation or signaling activity of an acid) or APHTHASOLTM, and (ii) an RTK inhibitor, integrin CfB (anb3)/RalB/NFkB signaling axis, wherein optionally the RTK inhibitor is a Src inhibitor, I0120 wherein optionally the inhibitor of integrin an anti-metabolite inhibitor, a gemcitabine, a CfB protein activity is an allosteric inhibitor of inte GEMZARTM, a mitotic poison, a paclitaxel, a taxol, an grin CfB protein activity; ABRAXANETM, an erlotinib, a TARCEVATM, a lapa I0121 (ii) an inhibitor or depleter of a RalB protein or tinib, a TYKERBTM, a cetuxamib, an ERBITUXTM, or an inhibitor of a RalB protein activation, or an inhibi an insulin growth factor inhibitor or a combination tor or depleter of the recruitment of KRAS/RalB to thereof; the plasma membrane or the association of KRAS to 0110 wherein optionally the combination or the thera peutic combination comprises an erlotinib with either a RalB, Lenalidomide or a PS-1145, or botha Lenalidomide and 0.122 wherein optionally the inhibitor is an allosteric a PS-1145; inhibitor, or the inhibitor of the RalB protein activity 0111 (vii) a lenalidomide or a REVLIMIDTM; a is an allosteric inhibitor of RalB protein activity; PS1145; and, a Receptor Tyrosine Kinase (RTK) inhibi tor, and optionally the RTK inhibitor comprises I0123 (iii) an inhibitor or depleter of a Src or a Tank SU14813 (Pfizer, San Diego, Calif.); Binding Kinase (TBK1) protein oran inhibitor of Src 0112 (viii) an inhibitor of Galectin-3; or or TBK1 protein activation, 0113 (ix) any combination of (i) to (viii); and 0.124 wherein optionally the inhibitor of the Src or 0114 (2) at least one Growth Factor Inhibitor, the TBK1 protein activity is: an amlexanox (or 0115 wherein optionally the Growth Factor Inhibitor is or 2-amino-7-isopropyl-5-oxo-5H-chromeno2,3-bpy comprises an anti-metabolite inhibitor, a gemcitabine, GEMZARTM, a mitotic poison, a paclitaxel, a taxol. ABRAX ridine-3-carboxylic acid), or an APHTHASOLTM; or ANETM, an erlotinib, TARCEVATM, a lapatinib, TYKERBTM, a Y(1)34.5 protein of herpes simplex viruses (HSV) or an insulin growth factor inhibitor, or any combination (see e.g., Ma et al., J Virol. 2012 February: 86(4): thereof; or, the Growth Factor Inhibitor decreases, slows or 2188-96); or, BX795 (as described in, e.g., Bain et al., blocks new blood vessel growth, neovascularization orangio Biochem J. (2007) December 15; 408(3):297-315; genesis; or, wherein administering the Growth Factor Inhibi Clarket al., (2009).J. Biol. Chem. 284:14136-14146); tor treats or ameliorates conditions that are responsive to or an aZabenzimidazole or an analog or derivative blocking or slowing cell growth, and/or the development of thereof, or a 6-amino-pyrazolopyrimidine or an ana neovascularization or new blood vessels, log or derivative thereof; or, a compound having one 0116 wherein optionally the combination or the therapeu of the following formulas, or an analog or derivative tic combination comprises an erlotinib with either a Lenali thereof (see Hutti, et al., (2012) Development of a domide or a PS-1145, or both a Lenalidomide and a PS-1145. High-Throughput Assay for Identifying inhibitors of 0117. In alternative embodiments, the invention provides TBK1 and IKKe. PLoS ONE 7(7):e41494.doi: therapeutic combinations of drugs comprising or consisting 10.1371/journal.pone).

Molecule IKKe TBK1 IKKB IKKC. O.77 0.44 >10 >10 US 2016/0146783 A1 May 26, 2016

-continued

Molecule IKKe TBK1 IKKB IKKC.

>10 OSO >10 >10

>10 O.64 8.76 -10

N N

/- >10 O.67 c-10 >10 O |N

N OH N O O >10 O.87 c-10 >10 O 2NN2 N N N N N NH N ( O \ N O O HO

0.125 and optionally the inhibitor of the Src or the (O127 wherein optionally the inhibitor of the NFKB TBK1 protein activity is an allosteric inhibitor of Src or the IRF3 protein activity is an allosteric inhibitor of or TBK1 protein activity; an NFKB or an Interferon regulatory factor 3 (IRF3) (0.126 (iv) an inhibitor or depleter of a NFKB or a protein activity; Interferon regulatory factor 3 (IRF3) protein or an I0128 (v) an inhibitor or depleter of NFKB or IKK, or inhibitor of RalB protein activation, an inhibitor of NFKB or IKK protein activation, US 2016/0146783 A1 May 26, 2016

I0129 wherein optionally the NFKB inhibitor com 0.143 wherein administering the Growth Factor Inhibitor prises a lenalidomide or a REVLIMIDTM (Celgene treats or ameliorates conditions that are responsive to block Corp., Summit, N.J.) and optionally the IKK inhibitor ing or slowing cell growth, and/or the development of neovas comprises a PS1145 (Millennium Pharmaceuticals, cularization or new blood vessels, Cambridge, Mass.); 0144 wherein optionally the combination or the therapeu I0130 (vi) a lenalidomide or a REVLIMIDTM and tic combination comprises an erlotinib with either a Lenali PS1145; domide or a PS-1145, or both a Lenalidomide and a PS-1145. I0131 (vii) a lenalidomide or a REVLIMIDTM; a 0145. In alternative embodiments, the invention provides PS1145; and, a Receptor Tyrosine Kinase (RTK) combinations, or therapeutic combinations, for overcoming inhibitor, and optionally the RTK inhibitor comprises or diminishing or preventing Growth Factor Inhibitor (GFI) SU14813 (Pfizer, San Diego, Calif.); resistance in a cell, or, a method for increasing the growth (0132 (viii) an inhibitor of Galectin-3; or inhibiting effectiveness of a Growth Factor inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor 0.133 (ix) any combination of (i) to (viii), or Inhibitor (GFI), wherein the combination comprises or con 0.134 (2) one or any combination of the compound, sists of: composition or formulation, or compounds, composi tions or formulations, of (1), and at least one growth 0146 (1) at least one compound comprising or consist factor inhibitor, ing of 0.135 wherein optionally the at least one growth factor I0147 (i) an inhibitor or depleter of integrin C, B, inhibitor comprises a Receptor Tyrosine Kinase (RTK) (anb3), or an inhibitor of integrin CfB (anb3) protein inhibitor, a Src inhibitor, an anti-metabolite inhibitor, a activity, or an inhibitor of the formation or activity of gemcitabine, a GEMZARTM, a mitotic poison, a pacli an integrin anb3/RalB signaling complex, or an taxel, a taxol, an ABRAXANETM, an erlotinib, a inhibitor of the formation or signaling activity of an TARCEVATM, a lapatinib, a TYKERBTM, a cetuxamib, integrin C, B (anb3)/RalB/NFkB signaling axis, an ERBITUXTM, or an insulin growth factor inhibitor; 0148 wherein optionally the inhibitor of integrin 0.136 wherein optionally the combination or the thera O?3 protein activity is an allosteric inhibitor of inte peutic combination comprises: (i) an inhibitor or depl grin CB protein activity; eter of a Src or a Tank Binding Kinase-1 (TBK1) protein 0149 (ii) an inhibitor or depleter of a RalB protein or or an inhibitor of Src or TBK1 protein activation, an inhibitor of a RalB protein activation, or an inhibi wherein optionally the inhibitor of the Src or the TBK1 tor or depleter of the recruitment of KRAS/RalB to protein activity is an amlexanoX (or 2-amino-7-isopro the plasma membrane or the association of KRAS to pyl-5-oxo-5H-chromeno2,3-bipyridine-3-carboxylic RalB, acid) or APHTHASOLTM, and (ii) an RTK inhibitor, wherein optionally the RTK inhibitor is a Src inhibitor, 0150 wherein optionally the inhibitor is an allosteric an anti-metabolite inhibitor, a gemcitabine, a inhibitor, or the inhibitor of the RalB protein activity GEMZARTM, a mitotic poison, a paclitaxel, a taxol, an is an allosteric inhibitor of RalB protein activity; ABRAXANETM, an erlotinib, a TARCEVATM, a lapa 0151 (iii) an inhibitor or depleter of a Src or a Tank tinib, a TYKERBTM, a cetuxamib, an ERBITUXTM, or Binding Kinase (TBK1) protein oran inhibitor of Src an insulin growth factor inhibitor or a combination or TBK1 protein activation, thereof; 0152 wherein optionally the inhibitor of the Src or 0.137 wherein optionally the combination or the thera the TBK1 protein activity is: an amlexanox (or peutic combination comprises an erlotinib with either a 2-amino-7-isopropyl-5-oxo-5H-chromeno2,3-bpy Lenalidomide or a PS-1145, or botha Lenalidomide and ridine-3-carboxylic acid), or an APHTHASOLTM; or a PS-1145; 0153 a Y(1)34.5 protein of herpes simplex viruses I0138 (vii) a lenalidomide or a REVLIMIDTM; a (HSV) (see e.g., Ma et al., J. Virol. 2012 February: PS1145; and, a Receptor Tyrosine Kinase (RTK) inhibi 86(4):2188-96); or, tor, and optionally the RTK inhibitor comprises 0154 a BX795 (as described in, e.g., Bain et al., SU14813 (Pfizer, San Diego, Calif.); Biochem J. (2007) December 15; 408(3):297-315; I0139 (viii) an inhibitor of Galectin-3; or Clarket al., (2009).J. Biol. Chem. 284:14136-14146); 0140 (ix) any combination of (i) to (viii); and O 0155 anazabenzimidazole oran analog orderivative 0141 (2) at least one Growth Factor Inhibitor, thereof, or 0142 wherein optionally the Growth Factor Inhibitor is or comprises an anti-metabolite inhibitor, a gemcitabine, 0156 a 6-amino-pyrazolopyrimidine or an analog or GEMZARTM, a mitotic poison, a paclitaxel, a taxol. ABRAX derivative thereof; or, a compound having one of the ANETM, an erlotinib, TARCEVATM, a lapatinib, TYKERBTM, following formulas, or or an insulin growth factor inhibitor, or any combination 0157 an analog orderivative thereof (see Hutti, et al., thereof; or, the Growth Factor Inhibitor decreases, slows or (2012) Development of a High-Throughput Assay for blocks new blood vessel growth, neovascularization orangio Identifying Inhibitors of TBK1 and IKKe. PLoS ONE genesis; or, 7(7):e41494.doi:10.1371/journal.pone):

US 2016/0146783 A1 May 26, 2016 11

-continued

Molecule IKKe TBK1 IKKB IKKC.

0158 and optionally the inhibitor of the Src or the ABRAXANETM, an erlotinib, a TARCEVATM, a lapa TBK1 protein activity is an allosteric inhibitor of Src tinib, a TYKERBTM, a cetuxamib, an ERBITUXTM, or or TBK1 protein activity; an insulin growth factor inhibitor or a combination 0159 (iv) an inhibitor or depleter of a NFKB or a thereof; Interferon regulatory factor 3 (IRF3) protein or an 0170 wherein optionally the combination or the therapeu inhibitor of RalB protein activation, tic combination comprises an erlotinib with either a Lenali (0160 wherein optionally the inhibitor of the NFKB domide or a PS-1145, or both a Lenalidomide and a PS-1145. or the IRF3 protein activity is an allosteric inhibitor of (0171 The details of one or more embodiments of the an NFKB or an Interferon regulatory factor 3 (IRF3) invention are set forth in the accompanying drawings and the protein activity; description below. Other features, objects, and advantages of (0161 (v) an inhibitor or depleter of NFKB or IKK, or the invention will be apparent from the description and draw an inhibitor of NFKB or IKK protein activation, ings, and from the claims. All publications, patents, patent 0162 wherein optionally the NFKB inhibitor com applications cited herein are hereby expressly incorporated prises a lenalidomide or a REVLIMIDTM (Celgene by reference for all purposes. Corp., Summit, N.J.) and optionally the IKK inhibitor comprises a PS1145 (Millennium Pharmaceuticals, BRIEF DESCRIPTION OF THE DRAWINGS Cambridge, Mass.); 0172. The drawings set forth herein are illustrative of (0163 (vi) a lenalidomide or a REVLIMIDTM and embodiments of the invention and are not meant to limit the PS1145; Scope of the invention as encompassed by the claims. (0164 (vii) a lenalidomide or a REVLIMIDTM; a (0173 FIGS. 1A-1G illustrate that integrin CVR3 expres PS1145; and, a Receptor Tyrosine Kinase (RTK) sion promotes resistance to EGFR TKI: FIG. 1A illustrates inhibitor, and optionally the RTK inhibitor comprises flow cytometric quantification of cell surface markers after 3 SU14813 (Pfizer, San Diego, Calif.); weeks treatment with erlotinib (pancreatic and colon cancer (0165 (viii) an inhibitor of Galectin-3; or cells) or lapatinib (breast cancer cells); FIG. 1B illustrates 0166 (ix) any combination of (i) to (viii), or flow cytometric analysis of CVB3 expression in FG and Mia 0.167 (2) one or any combination of the compound, paca-2 cells following erlotinib. FIG. 1C illustrates: Top, composition or formulation, or compounds, composi immunofluorescence staining of integrin CVB3 in tissue tions or formulations, of (1), and at least one growth specimens obtained from orthotopic pancreatic tumors factor inhibitor, treated with vehicle or erlotinib. Bottom, Integrin CVB3 0168 wherein optionally the at least one growth factor expression was quantified as ratio of integrin CVB3 pixel area inhibitor comprises a Receptor Tyrosine Kinase (RTK) over nuclei pixel area using METAMORPHTM: FIG. 1D inhibitor, a Src inhibitor, an anti-metabolite inhibitor, a Right, intensity of B3 expression in mouse orthotopic lung gemcitabine, a GEMZARTM, a mitotic poison, a pacli tumors treated with vehicle or erlotinib, Left, immunohis taxel, a taxol, an ABRAXANETM, an erlotinib, a tochemical staining of B3, FIG. 1E illustrates data showing TARCEVATM, a lapatinib, a TYKERBTM, a cetuxamib, that 33 expressing tumor cells were intrinsically more resis an ERBITUXTM, or an insulin growth factor inhibitor; tant to EGFR blockade than B3-negative tumor cell lines, 0169 wherein optionally the combination or the thera where the cells were first screened for CVB3 expression and peutic combination comprises: (i) an inhibitor or depl then analyzed for their sensitivity to EGFR inhibitors erlo eter of a Src or a Tank Binding Kinase-1 (TBK1) protein tinib or lapatinib. FIG.1F illustrates tumor sphere formation or an inhibitor of Src or TBK1 protein activation, assay to establish a dose-response for erlotinib, FIG. 1G wherein optionally the inhibitor of the Src or the TBK1 illustrates orthotopic FG tumors treated for 10 days with protein activity is an amlexanoX (or 2-amino-7-isopro vehicle or erlotinib, results are expressed as % tumor weight pyl-5-oxo-5H-chromeno2,3-bipyridine-3-carboxylic compared to vehicle control, immunoblot analysis for tumor acid) or APHTHASOLTM, and (ii) an RTK inhibitor, lysates after 10 days of erlotinib confirms suppressed EGFR wherein optionally the RTK inhibitor is a Src inhibitor, phosphorylation; as discussed in detail in Example 1, below. an anti-metabolite inhibitor, a gemcitabine, a 0.174 FIGS. 2A-E illustrate that integrin C.VfB3 cooperates GEMZARTM, a mitotic poison, a paclitaxel, a taxol, an with K-RAS to promote resistance to EGFR blockade: FIG. US 2016/0146783 A1 May 26, 2016

2A-B illustrate tumor sphere formation assay of FG tumor erlotinib (0.5uM), OSI-906 (0.1 uM), gemcitabine (0.01 uM) cells expressing (FIG. 2A) or lacking (FIG. 2B) integrin B3 or cisplatin (0.1 uM); FIG. 6C illustrates the effect of dose depleted of KRAS (shKRAS) or not (shCTRL) and treated response of indicated treatments on tumor sphere formation with a dose response of erlotinib. FIG. 2C confocal micros (Error bars represents.d. (n=3 independent experiments); as copy images of PANC-1 and FG-f33 cells grown in suspen discussed in detail in Example 1, below. sion: FIG. 2D illustrates an immunoblot analysis of RAS (0179 FIGS. 7A-B illustrate that integrin C.VfB3 does not activity assay performed in PANC-1 cells using GST-Rafl colocalize with active HRAS, NRAS and RRAS: FIG. 7A RBD immunoprecipitation as described below: FIG. 2E illus illustrates that Ras activity was determined in PANC-1 cells trates an immunoblot analysis of Integrin CVB3 immunopre grown in suspension by using a GST-Rafl-RBD immunopre cipitates from BXPC-3 B3-positive cells grown in suspension cipitation assay as described in Methods, see Example 1 (data and untreated or treated with EGF, and RAS activity was are representative of two independent experiments); FIG. 7B determined using a GST-Rafl-RBD immunoprecipitation illustrates confocal microscopy images of PANC-1 cells assay; as discussed in detail in Example 1, below. grown in suspension and stained for KRAS, RRAS, HRAS, (0175 FIGS. 3A-E illustrate that RalB is a key modulator NRAS (red), integrin Civ|B3 (green) and DNA (TOPRO-3, of integrin CVB3-mediated EGFR TKI resistance: FIG. 3A blue) (Scale bar, 10um. Data are representative of two inde illustrates tumor spheres formation assay of FG-33 treated pendent experiments); as discussed in detail in Example 1, with non-silencing (shCTRL) or RalB-specific shRNA and below. exposed to a dose response of erlotinib. FIG. 3B illustrates 0180 FIGS. 8A-D illustrate that Galectin-3 is required to effects of depletion of RalB on erlotinib sensitivity in promote integrin CVB3/KRAS complex formation: FIGS. B3-positive tumor in a pancreatic orthotopic tumor model; 8A-B illustrate confocal microscopy images of Panc-1 cells FIG.3C illustrates tumor spheres formation assay of FG cells lacking or expressing integrin CVB3 grown in Suspension; ectopically expressing vector control, WT RalB FLAG FIG. 8A illustrates cells stained for KRAS (green), Galec tagged constructs or a constitutively active RalB G23V tin-3 (red), and DNA (TOPRO-3, blue): FIG. 8B illustrates FLAG tagged treated witherlotinib (0.5 uM); FIG. 3D illus cells stained for integrin CVB3 (green), Galectin-3 (red) and trates RalB activity was determined in FG, FG-B3 expressing DNA (TOPRO-3, blue), Scale bar, 10um, data are represen non-silencing or KRAS-specific shRNA, by using a GST tative of three independent experiments: FIG. 8C illustrates RalBP1-RBD immunoprecipitation assay: FIG. 3E illus an immunoblot analysis of Galectin-3 immuno-precipitates trates: Right, overall active Ral immunohistochemical stain from PANC-1 cells expressing non-silencing (sh CTRL) or ing intensity between f33 negative and B3 positive human integrin B3-specific shRNA (sh B3), data are representative of tumors; as discussed in detail in Example 1, below. three independent experiments: FIG.8D illustrates an immu (0176 FIGS. 4A-E illustrate that integrin C.VfB3/RalB com noblot analysis of integrin B3 immunoprecipitates from plex leads to NF-uB activation and resistance to EGFRTKI: PANC-1 cells expressing non-silencing (sh CTRL) or Galec FIG. 4A illustrates an immunoblot analysis of FG, FG-B3 and tin-3-specific shRNA (sh Gal3), data are representative of FG-33 stably expressing non-silencing or RalB-specific three independent experiments; as discussed in detail in ShRNA, grown in suspension and treated with erlotinib (0.5 Example 1, below. uM); FIG. 4B illustrates tumor spheresformation assay of FG 0181 FIGS. 9A-B illustrate that ERK, AKT and RalA are cells ectopically expressing vector control, WT NF-kB not specifically required to promote integrin CVB3-mediated FLAG tagged or constitutively active S276D NF-kB FLAG resistance to EGFRTKI: FIG.9A B3-negative cells, and FIG. tagged constructs treated with erlotinib. FIG. 4C illustrates 9B, B3-positive cells; tumor spheres formation assay of FG tumor spheres formation assay of FG-33 treating with non and FG-B3 expressing non-silencing or ERK 1/2, AKT1 and silencing (shCTRL) or NF-KB-specific shRNA and exposed RalA-specific shRNA and treated with erlotinib (0.5 LM), to erlotinib. FIG. 4D illustrates dose response in FG-B3 cells error bars represent S.d. (n=3 independent experiments); as treated with erlotinib (10 nM to 5uM), lenalidomide (10 nM discussed in detail in Example 1, below. to 5 LM) or a combination of erlotinib (10 nM to 5uM) and 0182 FIGS. 10A-C illustrate that RalB is sufficient to lenalidomide (1 uM); FIG. 4E illustrates Model depicting the promote resistance to EGFR TKI: FIG. 10A illustrates a integrin C.VfB3-mediated EGFRTKI resistance and conquer tumor sphereformation assay of FG expressing non-silencing ing EGFRTKI resistance pathway and its downstream RalB or RalB specific shRNA and treated with a dose response of and NF-kB effectors; as discussed in detail in Example 1, erlotinib. Error bars represents.d. (n=3 independent experi below. ments); FIG. 10B illustrates a tumor spheres formation assay 0177 FIG. 5 illustrates that prolonged exposure to erlo of PANC-1 stably expressing integrin B3-specific shRNA and tinib induces Integrin CVB3 expression in lung tumors; rep ectopically expressing vector control, WT RalB FLAG resentative immunohistochemical staining of integrin B3 in tagged or a constitutively active RalB G23V FLAG tagged mouse tissues obtained from H441 orthotopic lung tumors constructs treated witherlotinib (0.5uM), error bars represent long-term treated with either vehicle or erlotinib (scale bar, S.d. (n=3 independent experiments); FIG. 10C shows that 100 um); as discussed in detail in Example 1, below. integrin CVB3 colocalizes with RalB in cancer cells: illus (0178 FIGS. 6A-C illustrate integrin CVR3, even in its trates confocal microscopy images of Panc-1 cells grown in unligated State, promotes resistance to Growth Factor inhibi Suspension. Cells are stained for integrin CVB3 (green), RalB tors but not to chemotherapies: FIG. 6A illustrates a tumor (red), pFAK (red), and DNA (TOPRO-3, blue), scale bar, 10 sphere formation assay comparing FG lacking B3 (FG), FG um, data are representative of three independent experiments; expressing B3 wild type (FG-B3) or the B3 D119A (FG as discussed in detail in Example 1, below. D119A) ligand binding domain mutant, treated with a dose 0183 FIGS. 11A-B illustrate that integrin CVR3 colocal response of erlotinib (Error bars represents.d. (n=3 indepen izes with RalBinhuman breast and pancreatic tumor biopsies dent experiments); FIG. 6B illustrates tumor sphere forma and interacts with RalB in cancer cells: FIG. 11A illustrates tion assay of FG and FG-B3 cells untreated or treated with confocal microscopy images of integrin CVB3 (green), RalB US 2016/0146783 A1 May 26, 2016

(red) and DNA (TOPRO-3, blue) in tumor biopsies from erlotinib. FIG. 13G illustrates confocal microscopy images breast and pancreatic cancer patients, Scale bar, 20 Jum, FIG. showing immunostaining for integrin B3 (green), KRAS (red) 11B illustrates a Ral activity assay performed in PANC-1 and DNA (TOPRO-3, blue) for PANC-1 cells expressing cells using GST-RalBP1-RBD immunoprecipitation assay, non-target shRNA control or Galectin 3-specific shRNA Immunoblot analysis of RalB and integrin B3, data are rep grown in suspension; FIG. 13H illustrates: Top: immunoblot resentative of three independent experiments; as discussed in analysis of integrin B3 immunoprecipitates from PANC-1 detail in Example 1, below. cells expressing non-target shRNA control (CTRL) or Galec 0184 FIGS. 12A-G illustrate data showing that integrin tin-3-specific shRNA (Gal-3); Bottom: immunoblot analysis B3 is expressed in EGFR inhibitor resistant tumors and is of Galectin-3 immunoprecipitates from PANC-1 cells necessary and sufficient to drive EGFR inhibitor resistance: expressing non-target shRNA control (CTRL) or integrin FIG. 12A schematically illustrates that the identification of B3-specific shRNA (B3); FIG. 13I graphically illustrates erlo the most upregulated tumor progression genes common to tinib dose response of FG-B3 cells expressing a non-target erlotinib resistant carcinomas; FIG.12B in table form shows shRNA control or a Galectin-3-specific shRNA (sh Gal-3); as Erlotinib ICso inapanel of human carcinoma cell lines treated further described in Example 2, below. with erlotinib in 3D culture; FIG. 12C graphically illustrates 0186 FIGS. 14A-I illustrate data showing that RalB is a percentage of integrin B3 positive cells in parental lines vs. central player of integrin B3-mediated EGFR inhibitor resis after 3 or 8 weeks treatment with erlotinib. FIG. 12D graphi tance: FIG. 14A graphically illustrates the effect of RalB cally illustrates quantification of integrin B3 (ITG 33) gene knockdown on erlotinib resistance of B3-positive epithelial expression in human lung cancer biopsies from patients from cancer cell lines, cells were treated with 0.5uM of erlotinib: the BATTLE Study (18) who were previously treated with an FIG. 14B graphically illustrates the effect of RalB knock EGFR inhibitor and progressed (n=27), versus patients who down on erlotinib resistance off33-positive human pancreatic were EGFR inhibitor naive (n=39): FIG. 12E illustrates (FG-B3) orthotopic tumor xenografts, established tumors images of paired human lung cancer biopsies obtained before expressing non-target shRNA, (shCTRL) or a shRNA target and after erlotinib resistance were immunohistochemically ing RalB (sh RalB) were randomized and treated for 10 days stained for integrin B3, scale bar, 50 um: FIG. 12F graphically with vehicle or erlotinib, results are expressed as % of tumor illustrates: Right graph shows effect of integrin B3 knock weight changes after erlotinib treatment compared to vehicle: down on erlotinib resistance of 33-positive cells, and Left FIG. 14C graphically illustrates the effect of expression of a graph shows effect of integrin B3 ectopic expression on erlo constitutively active Ral G23V mutant on erlotinib response tinib resistance in FG and H441 cells; FIG. 12G graphically of B3 negative cells, cells were treated with 0.5 M of erlo illustrates: Right graph shows the effect of integrin B3 knock tinib. FIG. 14D illustrates the effect of expression of integrin down on erlotinib resistance in vivo, A549 shCTRL and A549 B3 on KRAS and RalB membrane localization: FIG. 14E sh integrin B3 (n=8 per treatment group) were treated with illustrates Ral activity that was determined in PANC-1 cells erlotinib (25 mg/kg/day) or vehicle during 16 days, results are grown in suspension by using a GST-RalBP1-RBD immuno expressed as average of tumor volume at day 16. *P<0.05; precipitation assay, immunoblots indicate RalB activity and and Left graph shows orthotopic FG and FG-33 tumors association of active RalB with integrin B3; FIG. 14F illus treated for 30 days with vehicle or erlotinib, results are trates confocal microscopy images of integrin CVB3 (green), expressed as % tumor weight compared to vehicle control; as RalB(red) and DNA (TOPRO-3, blue) intumor biopsies from further described in Example 2, below. pancreatic cancer patients: FIG. 14G illustrates the effect of 0185 FIGS. 13 A-I illustrate data showing that integrin B3 B3 expression and KRAS expression on RalB activity, mea is required to promote KRAS dependency and KRAS-medi sured using a GST-RalBP1-RBD immunoprecipitation assay; ated EGFR inhibitor resistance: FIG. 13A illustrates confocal FIG. 14H illustrates immunoblot analysis of FG and FG-f33 microscopy images showing immunostaining for integrin B3 stably expressing non-target shRNA control or RalB-specific (green), K-, N-, H-, R-Ras (red), and DNA (TOPRO-3, blue) shRNA, grown in suspension and treated with erlotinib (0.5 for BXPc3 cells grown in suspension in media with 10% uM); FIG. 14I graphically illustrates the effect of a Tank serum, arrows indicate clusters where integrin B3 and KRAS Binding Kinase (TBK1) and p65 NFKB on erlotinib resis colocalize (yellow); FIG. 13B-C illustrates confocal micros tance of FG-f33 cells, cells were treated with 0.5 LM of erlo copy images showing immunostaining for integrin B3 tinib; as further described in Example 2, below. (green), KRAS (red) and DNA (Topro-3, blue) for PANC-1 0187 FIGS. 15A-Cillustrate data showing that reversal of (KRAS mutant) and HCC827 (KRAS wild-type) after B3-mediated EGFR inhibitor resistance in oncogenic KRAS acquired resistance to erlotinib (HCC827R) grown in suspen model by pharmacological inhibition: FIG. 15A graphically sion in absence (Vehicle) or in presence of erlotinib (0.5 LM illustrates the effect of NFkB inhibitors on erlotinib response and 0.1 uM respectively), arrows indicate clusters where inte of B3-positive cells (FG-B3, PANC-1 and A549), cells were grin B3 and KRAS colocalize (yellow); FIG.13D graphically treated with vehicle, erlotinib (0.5 LM), lenalidomide (1-2 illustrates the effect of KRAS knockdown on tumorspheres uM), bortezomib (4 nM) alone or in combination; FIG. 15B formation in a panel of lung and pancreatic cancer cells graphically illustrates data from: Left, mice bearing Subcuta expressing or lacking integrin 33: FIG. 13E graphically illus neous B3-positive tumors (FG-B3) were treated with vehicle, trates the effect of KRAS knockdown on tumorsphere forma erlotinib (25 mg/kg/day), lenalidomide (25 mg/kg/day) or the tion in PANC-1 (KRAS mutant) stably expressing non-target combination of erlotinib andlenalidomide, tumor dimensions shRNA control (L3-positive) or specific-integrin B3 shRNA are reported as the fold change relative to size of the same (B3 negative) in FG (KRAS mutant) and BXPc3 (KRAS wild tumor on Day 1, Right, mice bearing Subcutaneous B3-posi type) stably expressing vector control or integrin B3; FIG. tive tumors (FG-R) after acquired resistance to erlotinib were 13F graphically illustrates the effect of KRAS knockdown on treated with vehicle, erlotinib (25 mg/kg/day), bortezomib erlotinib resistance of B3-negative and B3-positive epithelial (0.25 mg/kg), the combination of erlotinib and bortezomib, cancer cell lines, cells were treated with a dose response of tumor dimensions are reported as the fold change relative to US 2016/0146783 A1 May 26, 2016 size of the same tumor on Day 1: FIG. 15C schematically immunoblot analysis showing that Integrin CVB3 immuno illustrates a model depicting an integrin CVB3-mediated precipitates from BXPC-3 cells grown in Suspension in pres KRAS dependency and EGFR inhibitor resistance mecha ence or absence of growth factors; as further described in nism; as further described in Example 2, below. Example 2, below. 0188 FIGS. 16A-C illustrate data showing that illustrates 0193 FIGS. 21A-D illustrate integrin B3 expression pro resistance to EGFR inhibitor is associated with integrin B3 motes KRAS dependency: FIG. 21A illustrates Immunoblots expression in pancreatic and lung human carcinoma cell showing KRAS knockdown efficiency in cells used in FIG. lines: FIG. 16A illustrates immunoblots showing integrin B3 13; FIG. 21B illustrates Representative photographs of crys expression in human cell lines used in FIG. 12; FIG. 16B tal violet-stained tumorspheres of FG and A549 cells express graphically illustrates data showing the effect of erlotinib on ing non-target shRNA control or specific-KRAS shRNA; HCC827 xenograft tumors in immuno-compromised mice FIG. 21C illustrates the effect of an additional KRAS knock relative to vehicle-treated control tumors; FIG. 16C left, down on tumorspheres formation in PANC-1 stably express graphically illustrates data of Integrin CVB3 quantification in ing non-target shRNA control (B3-positive) or specific-inte orthotopic lung (upper panel) and pancreas (lower panel) grin B3 shRNA (B3 negative); FIG. 21D illustrates tumors treated with vehicle or erlotinib until resistance, FIG. immunoblots showing KRAS knockdown efficiency; as fur 16C right, illustrates a representative immunofluorescent ther described in Example 2, below. staining of integrin CVB3 in lung (upper panel)and pancreatic 0194 FIG. 22 illustrates images showing that KRAS and (lower panel) human xenografts treated 4 weeks with vehicle Galectin-3 colocalize in integrin B3-positive cells, in particu or erlotinib; as further described in Example 2, below. lar, confocal microscopy images of FG and FG-B3 cells (0189 FIGS. 17A-B illustrate Integrin B3 expression pre grown in Suspension and stained for KRAS (green), galec dicts intrinsic resistance to EGFR inhibitors in tumors; FIG. tin-3 (red) and DNA (TOPRO-3, blue); as further described in 17A graphically illustrates a plot of progression-free survival Example 2, below. for erlotinib-treated patients with low versus (vs.) high pro tein expression of B3 integrin measured from non-Small cell (0195 FIGS. 23A-Cillustrate Integrin B3-mediated KRAS lung cancer biopsy material (FIG. 17B illustrates: in right dependency and erlotinib resistance is independent of ERK, panel B3 integrin high cells and left panel B3 integrin low AKT and RalA: FIG. 23A graphically illustrates the effect of ERK, AKT, RalA and RalB knockdown on erlotinib response cells) obtained at diagnosis; as further described in Example (erlotinib 0.5uM) of B3-negative FG (left panel) and 33-posi 2, below. tive FG-f33 cells (right panel); FIG. 23B illustrates Immuno (0190 FIGS. 18A-F illustrate Integrin B3 confers Receptor blots showing ERK, AKT RalA and RalB knockdown effi Tyrosine Kinase inhibitor resistance: FIG. 18A illustrates ciency on 33-negative FG (upper panel) and 33-positive immunoblots showing integrin B3 knockdown efficiency in cells used in FIG. 12; FIG. 18B graphically illustrates FG-B3 cells (lower panel); FIG. 23C illustrates Immunoblots response of A549 lung carcinoma cells non-target shRNA showing RalB knockdown efficiency in the B3-positive epi control or shRNA targeting integrin B3 to treatment with thelial cancer cells used in FIG. 14; as further described in either vehicle or erlotinib (25 mg/kg/day) during 16 days: Example 2, below. FIG. 18C illustrates immunoblots showing expression of 0.196 FIGS. 24A-B illustrate constitutive active NFkB is indicated proteins of representative tumors; FIG. 18D illus sufficient to promote erlotinib resistance: FIG.24A illustrates trates representative photographs of crystal violet-stained immunoblots showing a Tank Binding Kinase (TBK1) (upper tumorspheres of B3-negative and B3-positive cells after erlo panel) and NFkB knockdown efficiency (lower panel) used in tinib, OSI-906, gemcitabine and cisplatin treatment; FIG. FIG. 14, FIG.24B graphically illustrates the effect of consti 18Egraphically illustrates the effect of integrin B3 expression tutive active S276D p65NFkBonerlotinib response (erlotinib on lapatinib and OSI-906 (left panel), and cisplatin and gem 0.5uM) of B3-negative cells (FG cells); as further described citabine (right panel); FIG. 18F graphically illustrates data in Example 2, below. from a viability assay of FG and FG-B3 cells grown in sus 0.197 FIGS. 25A-D illustrate NFkB inhibitors in combi pension in media with or without serum; as further described nation witherlotinib increase cell death in Vivo: FIG. 25A and in Example 2, below. FIG. 25B illustrate Immunoblots showing expression of indi (0191 FIGS. 19A-B illustrate integrin B3-mediated EGFR cated proteins of representative tumors from shown in FIG. inhibitor resistance is independent of its ligand binding: FIG. 15B; FIG. 25C illustrates Confocal microscopy images of 19A graphically illustrates the effect of ectopic expression of cleaved caspase 3 (red) and DNA (TOPRO-3, blue) in tumor |B3 wild-type (FG-f3) or the B3 D119A (FG-D119A) ligand biopsies from xenografts tumors used in FIG. 15B treated binding domain mutant on erlotinib response: FIG. 19B illus with vehicle, erlotinib, lenalidomide or lenalidomide and trates an immunoblot showing transfection efficiency of vec erlotinib in combo: FIG. 25D illustrates Confocal micros tor control, integrin B3 wild-type and integrin B3 D119A; as copy images of cleaved caspase 3 (red) and DNA (TOPRO-3, further described in Example 2, below. blue) in tumor biopsies from xenografts tumors used in FIG. (0192 FIGS. 20A-C illustrate integrin B3 colocalizes and 15B treated with vehicle, erlotinib, bortezomib or bortezomib interacts with oncogenic and active wild-type KRAS: FIG. and erlotinib in combo); as further described in Example 2, 20A illustrates confocal microscopy images of FG and FG-33 below. cells grown in suspension in media 10% serum with or with (0198 FIGS. 26, 27, and 28, illustrate supplementary Table out erlotinib (0.5 LM) and stained for KRAS (red), integrin 1 from Example 2, showing that differentially expressed CVB3 (green) and DNA (TOPRO-3, blue); FIG. 20B illus genes in cells resistant to erlotinib (PANC-1, H1650, A459) trates Ras activity was determined in PANC-1 cells grown in compared with the average of two sensitive cells (FG, H441) suspension by using a GST-Rafl-RBD immunoprecipitation and in HCC827 after acquired resistance in vivo (HCC827R) assay, immunoblots indicate KRAS activity and association vs. the HCC827 vehicle-treated control; as further described of active KRAS with integrin B3; FIG. 20O illustrates an in Example 2, below. US 2016/0146783 A1 May 26, 2016

(0199 FIG. 29 illustrates supplementary Table 2, from were treated for 30 days with vehicle or erlotinib. FIG. 33F Example 2, showing KRAS mutational status in pancreatic graphically illustrates Relative mRNA expression of integrin and lung cell lines used in the study of Example 2, below. |B3 (ITGB3) in HCC827 vehicle-treated tumors (n=5) or erlo (0200 FIGS. 30A-B illustrate data showing integrin B3 tinib-treated tumors (n=7) from 33E after acquired resistance: (CD61) is a RTKI (Receptor Tyrosine Kinase (RTK). Inhibi FIG. 33G H&E sections and immunohistochemical analysis tor) drug resistance biomarker on the Surface of circulating of integrin B3 expression in paired human lung cancer biop tumor cells; as discussed in detail in Example 2, below. As sies obtained before and after erlotinib resistance: FIG. 33H schematically illustrated in FIG. 30A, CD61 (B3, or beta3) illustrates images of Limiting dilution in vivo determining the negative human lung cancer cells (HCC827; this lung adeno frequency of tumor-initiating cells for HCC827 vehicle carcinoma has an acquired mutation in the EGFR tyrosine treated (vehicle) and erlotinib-treated tumors from (erlotinib kinase domain (E746-A750 deletion), and they are sensitive resistant non-sorted) (e); FIG. 33I and FIG. 33.J graphically to erlotinib and develop acquired resistance after 6/8 weeks) illustrate the Self-renewal capacity of HCC827 vehicle were injected orthotopically into the lung of mice and treated treated (vehicle), erlotinib-treated (erlotinib resistant non over 3 months with erotinib at 25 mg/kg/day. As graphically Sorted), erlotinib-treated integrin B3-population and erlo illustrated in FIG. 30B, Human lung cancer cells detected in tinib-treated integrin B3+ population; as described in detail in the circulation were positive for CVB3 (or avb3, CD61) Example 3, below. whereas the cells in the untreated group were essentially (0204 FIGS. 34A-I illustrate: Integrin B3/KRAS complex negative for this marker. CD45 negative cells indicates that is critical for integrin B3-mediated stemness: FIG. 34 Confo the detected cells were not leukocytes and pan cytokeratin cal microscopy images show immunostaining for Integrin B3 positive cells indicate tumor cells. CD61 (beta3) positive (green), KRAS (red) and DNA (TOPRO-3, blue) for FG-f3, expression correlated with tumor expression. PANC-1, A549 and HCC827 after acquired resistance to erlo 0201 FIGS. 31 A-C illustrate data showing how targeting tinib (HCC827 ER) grown in suspension, Arrows indicate the NF-kB pathway using compositions and methods of this clusters where integrin B3 and KRAS colocalize (yellow); invention can sensitize resistant tumors to growth factor FIG.34B Ras activity was determined in PANC-1 cells grown inhibitors by showing the effect of NFkB inhibitors on erlo in suspension by using a GST-Rafl-RBD immunoprecipita tinib response of 33-negative (b3-negative) cells (FG) and tion assay, Immunoblots indicate KRAS activity and associa B3-positive cells (FG-B3, MDA-MB231 (intrinsic resistance, tion of active KRAS with integrin B3: FIG. 34C Effect of FIG. 31A) and FG-R (acquired resistance, FIG. 31B), and KRAS knockdown on tumorspheres formation in lung (A549 EGFRTKI (Tyrosine Kinase Inhibitor) sensitive cells, FIG. and H441) and pancreatic (FG and PANC-1) cancer cells 31C. Cells embedded in agar (anchorage independent expressing or lacking integrin B3; FIG. 34D Effect of KRAS growth) were treated with vehicle, erlotinib (0.5uM), Lena knockdown on erlotinib resistance of 33-negative and lidomide (2 uM), PS-1145 (1 uM) alone or in combination for B3-positive epithelial cancer cell lines, Cells were treated 10 to 15 days. Then, the soft agar were stained with crystal with a dose response of erlotinib. FIG. 34E Self-renewal violet and the colonies were counted manually. The results capacity of FG-B3 cells expressing non-target shRNA control show that while B3-positive cells (intrinsic FIG. 31A or (shCTRL) or KRAS-specific shRNA measured by quantify acquired resistant FIG. 31B cells) were resistant to erlotinib ing the number of primary and secondary tumorspheres; FIG. and each NFKB inhibitor alone, the combination of erlotinib 34F Confocal microscopy images show immunostaining for with either Lenalidomide or PS-1145 decreased tumorsphere integrin B3 (green), KRAS (red) and DNA (TOPRO-3, blue) formation. for PANC-1 cells expressing non-target shRNA control or (0202 FIGS. 32A-D illustrate: Integrin B3 expression Galectin 3-specific shRNA grown in suspension; FIG. 34G increase tumor-initiating and self-renewal capacities: FIG. immunoblot analysis of integrin B3 immunoprecipitates from 32A Limiting dilution in vivo determining the frequency of PANC-1 cells expressing non-target shRNA control (CTRL) tumor-initiating cells for A549 cells expressing non-target or Galectin-3-specific shRNA (Gal-3); FIG. 34H Effect of shRNA control or integrin B3-specific shRNA and for FG Galectin-3 knockdown on integrin B3-mediated anchorage cells expressing control vector or integrin B3 (FG-B3); FIG. independent growth and erlotinib resistance: FIG. 34I Self 32(B-C-D) Self-renewal capacity of A549 (FIG. 32B) and renewal capacity of PANC-1 cells expressing non-target PANC-1 (FIG.32C) cells expressing non-target shRNA con shRNA control (shCTRL) or Galectin-3-specific shRNA (sh trol (CTRL) or integrin B3-specific shRNA and of FG Gal-3) measured by quantifying the number of primary and expressing control vector or integrin B3 (FG-B3) (FIG.32D); secondary tumorspheres; as described in detail in Example 3. as described in detail in Example 3, below. below. 0203 FIGS. 33A-J illustrate:Integrin B3 drives resistance (0205 FIGS. 35A-I illustrate: RalB/TBK1 signaling is a to EGFR inhibitors: FIG. 33A graphically illustrates the key modulator of integrin B3-mediated stemness: FIG. 35 Effect of integrin B3 expression (ectopic expression for FG Effect of RalB knockdown on anchorage independence: FIG. and integrin B3-specific knockdown for PANC-1) cells on 35B Self-renewal capacity of FG-B3 cells expressing non drug treatment response: FIG.33B graphically illustrates the target shRNA control (sh CTRL) or RalB-specific shRNA (sh Effect of integrin B3 knockdown on erlotinib response in RalB) measured by quantifying the number of primary and MDA-MB-231 (MDA231), A549 and H1650; FIGS.33C and secondary tumorspheres: FIG. 35C Limiting dilution in vivo 33D graphically illustrate the effect of integrin B3 knock determining the frequency of tumor-initiating cells for FG-f33 down on erlotinib resistance in vivo using A549 shCTRL and cells expressing non-target shRNA control or integrin RalB A549sh B3 treated witherlotinib or vehicle, FIG.33C mea specific shRNA; FIG. 35D. Effect of RalB knockdown on Suring tumorspheres, and 33D measuring tumor Volume in erlotinib resistance of B3-positive epithelial cancer cell lines: A549 shCTRL (integrin B3+), left panel, and A549 (integrin FIG. 35E Effect of RalB knockdown on erlotinib resistance of B3-) (right panel); FIG.33E graphically illustrates Orthotopic B3-positive human pancreatic (FG-f33) orthotopic tumor FG and FG-33 tumors (>1000mm; n=5 per treatment group) Xenografts. Established tumors expressing non-target US 2016/0146783 A1 May 26, 2016

shRNA, (sh CTRL) or a shRNA targeting RalB (sh RalB); Ral G23V mutant on erlotinib resistance of B3 positive and FIG. 35F Immunoblot analysis of FG and FG-B3 stably negative cells, left panel graphically presenting data and right expressing non-target shRNA control or RalB-specific panel illustrating an immunoblot showing FLAG, RalB and shRNA, grown in 3D and treated with erlotinib (0.5 LM): Hsp90 expression; as described in detail in Example 3, below. FIG. 35G Effect of TBK1 knockdown on PANC-1 self-re 0209 FIGS. 39A-E illustrate: FIG. 39A Immunoblot newal capacity: FIG. 35H Effect of TBK1 knockdown on showing TBK1 knockdown efficiency in PANC-1 cells used erlotinib resistance of PANC-1 cells. Cells were treated with in FIG. 4 (of Example 3); FIG. 39B Effect of the TBK1 0.5 uM of erlotinib. FIG. 35I Mice bearing subcutaneous inhibitor amlexanox on erlotinib response of PANC-1 cells; B3-positive tumors (PANC-1) were treated with vehicle, erlo FIG. 39C Effect of the NFkB inhibitor borthezomib on tinib (25 mg/kg/day), amlexanoX (25 mg/kg/day) or the com B3-positive cells (FG-B3 (left panel), PANC-1 (middle panel) bination of erlotinib and amlexanox; as described in detail in and A549 (right panel)): FIG. 39D Mice bearing subcutane Example 3, below. ous B3-positive tumors (FG-33) were treated with vehicle, 0206 FIGS. 36A-G illustrate: FIG. 36A-B Limiting dilu erlotinib (25 mg/kg/day), bortezomib (0.25 mg/kg), the com tion tables; FIG. 36C Immunoblots showing integrin B3 bination of erlotinib and bortezomib; FIG. 39E Confocal knockdown or ectopic expression efficiency in cells used in microscopy images of cleaved caspase 3 (red) and DNA FIG. 1 (of Example 3); FIG. 36D Viability assay (CelTiter (TOPRO-3, blue) in tumor biopsies from xenografts tumors Glo assay) of FG and FG-f3 cells grown in 3D in media with used in 39D treated with vehicle, erlotinib, bortezomib or or without serum; FIG. 36E Immunohistochemical analysis bortezomib and erlotinib in combo; as described in detail in of integrin B3 expression in paired human lung cancer biop Example 3, below. sies obtained before (upper panel) and after (lower panel) 0210 FIGS. 40A-B graphically illustrate data demonstrat erlotinib resistance: FIG. 36F Limiting dilution table: FIG. ing that depletion of RalB overcomes erlotinib resistance in 36G image of Immunohistochemistry staining of CD166 (up KRAS mutant cells: FIG. 40A graphically illustrates number per panel) and integrin B33 (lower panel) in human lung of tumorspheres as a percent of control for FG, FG-beta3. tumor biopsies after EGFR TKI acquired resistance; as PANC-1, and A539 expressing cells, with or without erlo described in detail in Example 3, below. tinib, in vitro soft agar conditions; and FIG. 40B graphically 0207 FIGS.37A-F illustrate: FIG.37A Effect ofcilenget illustrates tumor weight as a percent of control, in in vivo ide treatment on erlotinib resistance in FG-B3 and PANC-1 orthotopic pancreas Xenograft; as discussed in detail in cells: FIG. 37B Effect of ectopic expression of B3 wild-type Example 2, below. (FG-B3) or the B3 D119A (FG-D119A) ligand binding 0211 FIGS. 41A-C graphically illustrate data demonstrat domain mutant on erlotinib response: FIG. 37C Confocal ing that depletion of TBK1 overcomes erlotinib resistance in microscopy images of FG-33 cells grown in 3D and stained KRAS mutant cells: FIG. 41A illustrates data demonstrating for integrin-B3 (green) and RAS family members (red); FIG. that integrin mediates TBK1 activation through Ralb; FIG. 37D Immunoblots showing KRAS knockdown efficiency in 41B and FIG. 41C graphically illustrate data demonstrating cells used in FIG. 3 (of Example 3); FIG.37E Representative TBK1 depletion (with siRNA) overcomes integrin beta-3- photographs of crystal violet-stained tumorspheres of FG and mediated erlotinib resistance, where FIG. 41A shows the A549 cells expressing non-target shRNA control or specific number of tumorspheres as a percent of non-treated cells with KRAS, FIG. 37F illustrates the Effect of a second KRAS and without siRNA depletion of TBK1, and FIG. 41C shows knockdown (shKRAS 2) on tumorspheres formation in tumor size as a percent of control with erlotinib, amlexanox PANC-1 stably expressing non-target shRNA control and erlotinib+amlexanox; as discussed in detail in Example 2. (3-positive) or specific-integrin-B3 shRNA (3 negative), left below. panel graphically presenting data and right panel illustrating 0212. Like reference symbols in the various drawings an immunoblot showing KRAS expression in sh CTRL, SH indicate like elements. KRAS and sh KRAS 2; as described in detail in Example 3, 0213 Reference will now be made in detail to various below. exemplary embodiments of the invention, examples of which 0208 FIGS. 38A-I illustrate: FIG.38A graphically illus are illustrated in the accompanying drawings. The following trates the Effect of ERK, AKT and RalA knockdown on detailed description is provided to give the reader a better erlotinib response of B3-negative FG and 3-positive FG-3 understanding of certain details of aspects and embodiments cells: FIG.38B Immunoblots showing ERK, AKT and RalA of the invention, and should not be interpreted as a limitation knockdown efficiency in cells used in; FIG. 38C Immunob on the Scope of the invention. lots showing RalB knockdown efficiency in cells used in FIG. 3 (of Example 3); FIG. 38D graphically illustrates the effect DETAILED DESCRIPTION of a second RalB knockdown (shRalB 2) on tumorspheres 0214. In alternative embodiments, the invention provides formation in PANC-1 stably expressing non-target shRNA compositions and methods for overcoming or diminishing or control (B3-positive) or specific-integrin B3 shRNA (3 nega preventing Growth Factor Inhibitor (GFI) resistance in a cell, tive); FIG. 38E Limiting dilution table: FIG.38F Confocal or, a method for increasing the growth-inhibiting effective microscopy images of integrin CVB3 (green), RalB (red) and ness of a Growth Factor inhibitor on a cell, or, a method for DNA (TOPRO-3, blue) in tumor biopsies from pancreatic re-sensitizing a cell to a Growth Factor Inhibitor (GFI). In cancer patients: FIG. 38G Ral activity was determined in alternative embodiments, the cell is a tumor cell, a cancer cell PANC-1 cells grown in suspension by using a GST-RalBP1 or a dysfunctional cell. In alternative embodiments, the inven RBD immunoprecipitation assay. Immunoblots indicate tion provides compositions and methods for determining: RalA and RalB activities: FIG. 38H Effect of B3 expression whether an individual or a patient would benefit from or and KRAS expression on RalB activity, measured using a respond to administration of a Growth Factor Inhibitor, or, GST-RalBP1-RBD immunoprecipitation assay: FIG. 38I which individuals or patients would benefit from a combina illustrates the effect of expression of a constitutively active torial approach comprising administration of a combination US 2016/0146783 A1 May 26, 2016

of at least one growth factor and at least one compound, tion are used to sensitize tumors using NFkB inhibitors, such composition or formulation used to practice a method of the as e.g., lenalidomide or (RS)-3-(4-amino-1-oxo-3H-isoin invention, such as an NfKb inhibitor. dol-2-yl)piperidine-2,6-dione or REVLIMIDTM, or a compo 0215. We found that integrin anb3 is upregulated in cells sition as listed in Table 1. that become resistant to Growth Factor inhibitors. Our find 0219. In alternative embodiments, compositions and ings demonstrate that integrin anb3 promotes de novo and methods of the invention are used to sensitize tumors using an IKK inhibitor, e.g., such as PS1145 (Millennium Pharmaceu acquired resistance to Growth factor inhibitors by interacting ticals, Cambridge, Mass.) (see e.g., Khanbolooki, et al., Mol and activating RalB. RalB activation leads to the activation of Cancer Ther 2006; vol. 5:2251-2260; Published online Sep. Src and TBK1 and the downstream effectors NFKB and 19, 2006; Yemelyanov, et al., Oncogene (2006) vol. 25:387 IRF3. We also found that depletion of RalB or its downstream 398; published online 19 Sep. 2005), or any IKBC. (nuclear signaling (Src/NFKB) in b3-positive cells overcomes resis factor of kappa light polypeptide gene enhancer in B-cells tance to growth factor inhibitors. This invention demonstrates inhibitor, alpha) phosphorylation and/or degradation inhibi that the integrin anb3/RalB signaling complex promotes tor, e.g., one or more compositions listed in Table 3. resistance to growth factor inhibitors; and in alternative 0220. In alternative embodiments, compositions and embodiments, integrin C.B. (anb3) and active RalB are used methods of the invention comprise use of an NFkB inhibitor as biomarkers in patient samples to predict which patients and an IKK inhibitor to treat a drug resistant tumor, e.g., a will respond to growth factor inhibitors and which patients Solid tumor. In alternative embodiments, compositions and might rather benefit from alternative/combinatorial methods of the invention comprise use of an NFkB inhibitor approaches Such as a combination of growth factor inhibitors and an IKK inhibitor to treat a drug resistant tumor in com and Nfkb inhibitors. bination with an anticancer drug, e.g., an NFkB inhibitor and 0216. This invention for the first time identifies integrin an IKK inhibitor are used to sensitize a tumor to drugs such as CVB3 and active RalB as potential biomarker for tumors that erlotinib and lapatinib. In alternative embodiments, the drug are or have become (e.g., de novo and acquired) resistant to combination used to practice the invention comprises lenali growth factors blockade. Accordingly, in alternative embodi domide (such as a REVLIMIDTM) and the IKK inhibitor ments, the invention provides compositions and methods for PS1145 (Millennium Pharmaceuticals, Cambridge, Mass.). the depletion of RalB, Src, NFkB and its downstream signal For example, lenalidomide (such as a REVLIMIDTM) and ing effectors to sensitize CVB3-expressing tumors to growth PS1145 are used to sensitize a tumor that is resistant to a factor blockade. These findings reveal a new role for integrin cancer drug, e.g., an EGFR inhibitor, Such that the tumor is CVB3 in mediating tumor cell resistance to growth factor now responsive to the cancer drug. inhibition and demonstrate that targeting the CVB3/RalB/ 0221. In alternative embodiments, in practicing the inven NfkB/Src signaling pathway will circumvent growth factor tion, an NFkB inhibitor and an IKK inhibitor are used in resistance of a wide range of cancers. combination with a tyrosine kinase receptor (also called 0217. In alternative embodiments, any NF-kB inhibitor Receptor Tyrosine Kinases, or RTKs) inhibitor, e.g., an can be used to practice this invention, e.g., lenalidomide or SU14813 (Pfizer, San Diego, Calif.) or as listed in Table 2 or (RS)-3-(4-amino-1-oxo-3H-isoindol-2-yl)piperidine-2,6-di 3, below, to treat a drug resistant tumor. In alternative embodi one, which can be REVLIMIDTM (Celgene Corp., Summit, ments, compositions and methods of the invention (e.g., N.J.), or thalidomide, or any other derivative of thalidomide, including lenalidomide or PS1145; lenalidomide and or any composition having an equivalent activity. PS1145; or lenalidomide, PS1145 and an RTK inhibitor are 0218. In alternative embodiments, compositions and administered to patients that have become resistant to a can methods of the invention are used to sensitize tumors to drugs, cer drug, e.g., drugs like erotinib or lapatinib, to produce a e.g., Such as erlotinib and lapatinib (which are commonly strong antitumor effect. used to treat a wide range of solid tumors). We have shown 0222. In alternative embodiments, any NF-kB inhibitor that when tumors become resistant to these drugs they can be used to practice this invention, e.g., an antioxidant can become very sensitive to NFkB inhibitors. Thus, in alterna be used to inhibit activation of NF-kB, e.g., including the tive embodiments, compositions and methods of the inven compositions listed in Table 1: TABLE 1.

Antioxidants that have been shown to inhibit activation of NF-kB

Molecule Reference a-Lipoic acid Sen et al., 1998: Suzukiet al., 1992 a-tocopherol Islam et al., 1998 Aged garlic extract (allicin) Ide & Lau, 2001; Langetal, 2004: Hasan et al., 2007 2-Amino-1-methyl-6-phenylimidazo[4,5- Yun et al., 2005 bipyridine (PhIP) N-acetyldopamine dimers (from P. cicadae) Xuetal, 2006 Allopurinol Gomez-Cabrera et al., 2006 Anetholdithiolithione Sen et al., 1996 Apocynin Barbieri et al., 2004 Applejuice extracts Shi & Jiang, 2002; Davisetal, 2006; Jung et al., 2009 Aretensia p7F (5,6,3',5'-tetramethoxy 7,4'- Lee et al., 2004 hydroxyflavone) Astaxanthin Lee et al., 2003 US 2016/0146783 A1 May 26, 2016 18

TABLE 1-continued

Antioxidants that have been shown to inhibit activation of NF-kB

Molecule Reference Autumn olive extracts: olive leaf extracts Wang et al., 2007; Wanget al., 2008 Avenanthramides (from oats) Guo et al., 2007: Suretal, 2008 Bamboo culm extract Lee et al., 2008 Benidipine Matsubara & Hazegawa, 2004 bis-eugenol Murakami et al., 2003 Bruguiera gymnorrhiza compounds Homhual et al., 2006 Butylated hydroxyanisole (BHA) Israel et al., 1992: Schulze-Osthoffet al, 1993 Cepharanthine Okamoto et al., 1994: Tamatani et al., 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 beta-Caroteine Bai et al., 2005; Guruvayoorappan& Kuttan, 2007 Carvedilol Yang et al., 2003 Catecho Derivatives Suzuki & Packer, 1994; Zheng et al., 2008 Centatinea 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 Kwak et al., 2008 phenylamides Cocoa polyphenols Lee et al., 2006 Coffee extract (3-methyl-1,2- Chung et al., 2007 cyclopentanedione) Crataegus pinnatifida polyphenols Kao et al., 2007 Curcumin (Diferulolylmethane); Singh & Aggarwal, 1995: Pae etal, dimethoxycurcumin; EF24 analog 2008; Kasinskiet al., 2008 Dehydroepiandrosterone (DHEA) wasaki et al., 2004: Liuet al., 2005 and DHEA-sulfate (DHEAS) Dibenzylbutyrolactone lignans Cho et al., 2002 Diethyldithiocarbamate (DDC) Schrecket al., 1992 Diferoxamine Sappey et al., 1995; Schreck etal, 1992 Dihydroisoeugenol; isoeugenol; Murakami et al., 1995: Park et al., epoxypseudoisoeugenol-2-methylbutyrate 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 Schrecket al., 1992 Ebselen Schrecket al., 1992 Edaravone Kokura et al., 2005: Ariietal, 2007; Yoshida et al., 2007 EPC-K1 (phosphodiester compound of vitamin Hirano et al., 1998 E and vitamin C) Epigallocatechin-3-gallate (EGCG: green tea Lin & Lin, 1997: Yang et polyphenols) al, 1998: Hou et al., 2007 Ergothioneine Rahman et al., 2003 Ethyl Pyruvate (Glutathione depletion) Song et al., 2004: Tsungetal, 2005; Jimenez-Lopezet al., 2008 Ethylene Glycol Tetraacetic Acid (EGTA) anssen et al., 1999 Eupatilin Lee et al., 2008 Exercise Goto et al., 2007 Fisetin Parket al., 2006; Sunget al., 2007 Flavonoids (Crataeguts; Boerhaavia diffitsa Zhang et al., 2004; Chenetal, root; Xanthohumol; Eupatorium arnottianum; 2004: Pandey et al., 2005; Albini et al., genistein; kaempferol: quercetin, daidzein; 2005; Colgate et al., 2006: Clavin etal, flavone; isorhamnetin; maringenin: 2007: Hamalainen et al., pelargonidin; finestin; Sophora fiavescens; 2008; Zheng et al., 2008; Junget al., Seabuckthorn fruit berry) 2008; Mishra et al., 2008 Folic acid Au-Yeung et al., 2006 Gamma-glutamylcysteine synthetase (gamma Manna et al., 1999 GCS) Ganoderma lucidum polysaccharides Zhang et al., 2003: Ho et al., 2007 Garcinol (from extract of Garcinia indica fruit Liao et al., 2004 rind) Ginkgo biloba extract Chen et al., 2003 Glutathione Cho et al., 1998: Schrecket al., 1992; Wang et al., 2007 US 2016/0146783 A1 May 26, 2016 19

TABLE 1-continued

Antioxidants that have been shown to inhibit activation of NF-kB

Molecule Reference Guaiacol (2-methoxyphenol) Murakami et al., 2007 Hematein Choi et al., 2003 Hinokitiol Byeon et al., 2008 HMCOSherbal extract Kim et al., 2007 Hydroquinone Pyatt et al., 1998; Yanget al., 2006 23-hydroxyursolic acid Shin et al., 2004 RFI O42 (Vitamin E-like compound) Altavilla et al., 2001 ron tetrakis Kang et al., 2001 sostevio Xuetal, 2008 Sovitexin Lin et al., 2005 Soliquiritigenin Kumar et al., 2007; Kimetal, 2008; Kim et al., 2008 Justicia gendarissa root extract Kumar et al., 2011 Kallistatin Shen et al., 2008 Kangen-karyu extract Satoh et al., 2005; Yokozawa et al., 2007 L-cysteine Mihm et al., 1991 Lacidipine Cominacini et al., 1997 Lazaroids Marubayashi et al., 2002 Ligonberries Wang et al., 2005 Lupeol Saleem et al., 2004; Leeet al., 2007 Lutein Kim et al., 2008 Magnolol Chen et al., 2002: Ou et al., 2006; Kim et al., 2007 Maltol Yang et al., 2006 Manganese Superoxide dismutase (Mn-SOD) Manna et al., 1998 Extract of the stem bark of Mangifera indica L. Leiro et al., 2004: Garridoet al., 2005 Melatonin Giladet al., 1998: Mohanetal, 1995; Lietal, 2005 21 (alpha, beta)-methylmelianodiol Zhou et al., 2007 Mulberry anthocyanins Chen et al., 2006 N-acetyl-L-cysteine (NAC) Schrecket al., 1991 Nacyselyn (NAL) Antonicelli et al., 2002 Nordihydroguaiaritic acid (NDGA) Brennan & O'Neill, 1998; Israel et al, 1992: Schulze-Osthoffetal, 1993: Staaletal, 1993 Ochnaflavone Suh et al., 2006 Onion extract (2,3-dihydro-3,5-dihydroxy-6- Ban et al., 2007: Tang et al., 2008 methyl-4H-pyranone) Orthophenanthroline Schrecket al., 1992 N-(3-oxo-dodecanoyl) homoserine lactone Kravchenko et al., 2008 Paricalcitol Tan et al., 2008 Phenolic antioxidants (Hydroquinone and tert Ma et al., 2003 butyl hydroquinone) alkenylphenols from Piper obliquum Valdivia et al., 2008 alpha-phenyl-n-tert-butyl-nitrone (PBN) Kotake et al., 1998: Linet al., 2006 Phenylarsine oxide (PAO, tyrosine phosphatase Arbault et al., 1998 inhibitor) Phyllanthus urinaria Chularojmontrietal, 2005; Shen et al, 2007 Phytosteryl ferulates (rice bran) Islam et al., 2008; Junget al., 2008 Piper longtim Linn. extract Singh et al., 2007 Pitavastatin Tounai et al., 2007: Wang& Kitajima, 2007 Prodelphinidin B2 3,3'-di-O-gallate Hou et al., 2007 Pterostilbene Cichocki et al., 2008: Panet al., 2009 Pyrrolinedithiocarbamate (PDTC) Schrecket al., 1992 Quercetin Musonda & Chipman, 1998; Shih et al, 2004: Garcia-Mediavillaetal, 2006: Ruiz et al., 2007: Min etal, 2007; Kim et al., 2007 Red orange extract Cimini et al., 2008 Red wine Blanco-Colio et al., 2000; Cui & He, 2004 Ref-1 (redox factor 1) Ozaki et al., 2002 Rg(3), a ginseng derivative Keum et al., 2003 Rotenone Schulze-Osthoffetal, 1993 Roxithromycin Ueno et al., 2005; Ou et al., 2008 Rutin Kyung et al., 2008 S-allyl-cysteine (SAC, garlic compound) Geng et al., 1997 Salogaviolide (Centatirea ainetensis) Ghantous et al., 2008 Sauchinone Lee et al., 2003: Hwang et al., 2003 US 2016/0146783 A1 May 26, 2016 20

TABLE 1-continued

Antioxidants that have been shown to inhibit activation of NF-kB

Molecule Reference 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- Kazmi et al., 1995; Ritchieet al., 1995 methoxyphenyl)-N-methyl-1H-pyrazole-3- propanamide) Thioavarol derivatives Amigo et al., 2007; Amigoet al., 2008 Thymoquinone El Gazzar et al., 2007; lSethi etal, 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 Vitamin E and derivatives Suzuki & Packer, 1993: Ekstrand Hammarstrom et al., 2007: Glauert, 2007 a-torphryl Succinate Staal et al., 1993; Suzuki & Packer, 1993 a-torphryl acetate Suzuki & Packer, 1993 PMC (2,2,5,7,8-pentamethyl-6- Suzuki & Packer, 1993 hydroxychromane) Yakuchinone A and B Chun et al., 2002

0223) In alternative embodiments, any proteasome inhibi tor and/or protease inhibitor can be used to practice the inven tion, 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 Proteasone and proteases inhibitors that inhibit Rel/NF-kB Molecule References

Proteasome inhibitors Peptide Aldehydes: Paiombeiia et al., 1994: Grisham etal, 1999; Jobin et al., 1998 ALLnL (N-acetyl-leucinyl-leucynil norleucynal, MG101) LLM (N-acetyl-leucinyl-leucynil methional) Z-LLnV (carbobenzoxyl-leucinyl-leucynil norvalinal, MG115) Z-LLL (carbobenzoxyl-leucinyl-leucynil leucynal, MG132) Lactacystine, beta-lactone Fenteany & Schreiber, 1998; Grisham et al., 1999 Boronic Acid Peptide Grisham et al., 1999; Iqbal et al., 1995 Dithiocarbamate complexes with Cyek & Dvorak, 2007 metals CEP-18770 Piva et al., 2007 Ubiquitin Ligase Inhibitors Yaron et al., 1997 PS-341 (Bortezomib) Adams, 2004 Salinosporamide A (1, NPI-0052) Macheria et al., 2005: Ahn et al., 2007 Cyclosporin A Frantz et al., 1994: Kunz et al., 1995; Marienfeld et al., 1997; McCaffrey et al, 1994; Meyer et al., 1997; Wechsler et al., 1994 US 2016/0146783 A1 May 26, 2016 21

TABLE 2-continued Proteasone and proteases inhibitors that inhibit Rel/NF-kB Molecule References FK506 (Tacrolimus) Okamoto et al., 1994; Venkataraman et al., 1995 Deoxyspergualin Tepper et al., 1995 Disulfiram Lovborg et al., 2005 PT-110 Momose et al., 2007 Protease inhibitors APNE (N-acetyl-DL-phenylalanine-b- Higuchi et al., 1995 naphthylester) BTEE (N-benzoyl L-tyrosine Rossi et al., 1998 ethylester) DCIC (3,4-dichloroisocoumarin) D'Acquisto et al., 1998 DFP (diisopropyl fluorophosphate) TPCK (N-a-tosyl-L-phenylalanine chloromethyl ketone) TLCK (N-a-tosyl-L-lysine chloromethyl ketone)

0224 In alternative embodiments, any IKBC. (nuclear fac tor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) phosphorylation and/or degradation inhibi tor can be used to practice this invention, e.g., including the compositions listed in Table 3: TABLE 3 IkBot phosphorylation and/or degradation inhibitors

Molecule Point of Inhibition References Desloratadine; diphenhydramine Histamine H1 receptor Wu et al., 2004; Scadding, 2005: Rounesian etal, 2008 Bikunin LPS receptor agonists Kobayashi, 2006; Kanayama et al., 2007 Ron Tyrosine kinase receptor Suppresses TNF Lenisch et al., 2007 production TAK-242 TLR4 intracellular Kawamoto et al., 2008 domain Salmeterol, fluticaSone propionate beta2 agonists Baouz et al., 2005 CPUO213 Endothelin receptor He et al., 2006 antagonist Doxazosin alpha1-adrenergic Hui et al., 2007 receptor antagonist Erbin overexpression NOD2 inhibitor McDonald etal, 2005 Protein-bound polysaccharide LPS-CD14 interaction Asai et al., 2005 from basidiomycetes Anti-CD146 antibody AA98 upstream of IKK But et al., 2006 Calagualine (fern derivative) upstream of IKK Manna et al., 2003 (TRAF2-NIK) NS3/4A (HCV protease) upstream of IKK Karayiannis, 2005 golli BG21 (product of myelin upstream of IKK (PKC) Feng et al., 2004 basic protein) NPM-ALK oncoprotein Traf2 inhibition Horie et al., 2004 NS5A (Hepatitis C virus) Traf2 inhibition Parket al., 2002 LY29 and LY30 PI3Kinase inhibitors Choi et al., 2004 Shiga toxin (Enterohemorrhagic E PI3Kinase inhibitor Gobert et al., 2007 coli) Evodiamine (Evodiae Fructus AKT-IKK interaction Takada et al., 2005 component) Rituximab (anti-CD20 antibody) up-regulates Raf-1 Jazirehi et al., 2005 kinase inhibitor Kinase suppressor of ras (KSR2) MEKK3 inhibitor Channavaihaia et al., 2005 Cholecystokinin ocatpeptide p38 kinase Li et al., 2007 (CCK-8) M2L (Vaccinia virus) ERK2 inhibitor Gedey etal, 2006; Hinthong et al., 2008 US 2016/0146783 A1 May 26, 2016 22

TABLE 3-continued IKBC phosphorylation and/or degradation inhibitors Molecule Point of Inhibition References Pefabloc (serine protease inhibitor) upstream of IKK Tando et al., 2002 Rocaglamides (Aglaia derivatives) upstream of IKK Baumann et al., 2002 Ymer Binds to Ub-RIP Bohgaki et al., 2007 Epoxyquinol B TAK1 crosslinker Kamiyama et al., 2008 Betaine NIKIKK Goet al., 2004, 2007 TNAP NIK Hu et al., 2005 Selected peptides NEMO binding to Ub Wvier et al., 2007 Desflurane KK complex formation Li et al., 2008 with TNF-R1 Geldanamycin KK complex formation Chen et al., 2002 Grape seed proanthocyanidins KKa activity Mantena & Katiyar, 2006: Sharma et al., 2007: Cheng etal, 2007; Xu et al., 2008 Laretia acaulis azorellane KKa activity Borquez et al., 2007 diterpenoids MC160 (Molluscum contagiosum KKa activity Nichols & Shisler, 2006 virus) NS5B (Hepatitis C protein) KKa activity Choi et al., 2006 Pomegranate fruit extract KKa activity Afaq et al., 2004; Khan et al, 2006 Tetrandine (plant alkaloid) KKa activity Ho et al., 2004: Xue etal, 2008: Lin et al., 2008 BMS-345541 (4(2'- KKa and IKKb kinase Burke et al., 2002; Yang et Aminoethyl)amino-1.8- activity al, 2006: Beaulieu et al., dimethylimidazo(1,2-a) 2006 quinoxaline) and 4-amino derivatives 1-O-acetylbritannilactone Kb activity Liu et al., 2007 2-amino-3-cyano-4-aryl-6-(2- Kb activity Murata et al., hydroxy-phenyl)pyridine 2003, 2004, 2004 derivatives Acrolein K b activity/p50 DNA Valiacchi et al. d 2005; Lambert et al., 2007 Anandamide Kb activity Sancho et al., 2003 AS6O2868 sKb activity Freiin et al., 2003: Griessinger etal, 2007 Cobrotoxin b activity/p50 DNA Parket al., 2005 l ing Core protein (Hepatitis C) b activity Joo et al., 2005: Shrivastava et al., 1998 1-2-cyano-3,12-dioxooleana KKb activity Yore et al., 2006 1.9(11)-dien-28-oyl imidazole Dihydroxyphenylethanol KKb activity Guichard et al., 2006 Herbimycin A KKb activity Iwasaki etal, 1992; Mahon & O'Neill, 1995; Ogino et al., 2004 Inhibitor 22 b activi Baxter et al., 2004 Isorhapontigenin b activi Li et al., 2005 Manumycin A b activi Bernier et al., 2005; Frassanito et al., 2005 6-methyl-2-propolyimino-6,7- Kim et al., 2008 dihydro-5H benzo.1,3oxathiol-4-One MLB120 (Small molecule) Kb activity Nagashima et al., 2006 Naphthopyrones (6- Kb activity Fulmer et al., 2008 methoxycomaparvin and 6 methooxycomaparvin 5-methyl ether) Novel Inhibitor KKb activity Kanon et al., 2004 vIRF3 (KSHV) KKb activity Seo et al., 2004 Nitric oxide KKb activity/IkB Katsuyama etal, phosphorylation 1998: Matthews etal, 1996; Spieker & Liao, 1999; Reynaert et al., 2004 SC-514 (Small molecule) KKb activity Kishore et al., 2003 Thienopyridine KKb activity Morwicket al., 2006 Acetyl-boswellic acids KK activity Syrovets et al., 2004, 2005 Amino-pyrimidine derivative KK activity Karin et al., 2004 US 2016/0146783 A1 May 26, 2016 23

TABLE 3-continued IKBC phosphorylation and/or degradation inhibitors Molecule Point of Inhibition References Benzoimidazole derivative KK activity Karin et al., 2004 BMS-34S541 KK activity Burke et al., 2003 Butein KKb activity Pandey et al., 2007 Beta-carboline KK activity Yoon et al., 2005 CYL-19s and CYL-26z, two KK activity Huang et al., 2004 synthetic alpha-methylene gamma-butyrolactone derivatives ACHP (2-amino-6-2- KKb activity (ATP Sanda et al., 2006 (cyclopropylmethoxy)-6- analog) hydroxyphenyl-4-piperidin-4-yl nicotinonitrile Berberine KKb activity Hu et al., 2007; Yi etal, 2008: Pandey et al., 2008 Compound A KKb activity (ATP Ziegelbauer et al., 2005 analog) Flavopiridol KK activity and RelA Takada & Aggarwal, phosphor. 2003 Cyclopentones KKb activity Bickley et al., 2004 Dehydroascorbic acid (Vitamin C) KKb activity Carcano et al., 2004 Gossypyin or Gossypium extracts KKb activity Kinnaimakkara etal, 2007:Ji et al., 2008 M protein (SARS-Cornonavirus KKb activity Fang et al., 2007 protein) MD-O3S4 KKb activity Tanaka et al., 2004, 2006; Inayama et al., 2006 esterone dimer KKb activity: DNA Liang et al., 2003, 2006 binding KINK-1 KKb activity Schon et al., 2008 LCY-2-CHO KKb activity Ho et al., 2007 Prolyl hydroxylase-1 KKb activity Cummins et al., 2006 Naphthopyrones (Echinoderm KKb activity Folmer et al., 2007 Comanthus parvicirrus) Neuropeptides CGRP, PACAP and KKb activity Ding et al., 2007 VIP PS-1145 (MLN1145) KKb activity Hideshima et al., 2002 2-(aminocarbonyl)amino-5-(4- KKb activity Bonafotix etal, fluorophenyl)-3- 2005; Podolin et al., 2005 thiophenecarboxamides (TPCA-1) 1'-Acetoxychavicol acetate KK activity Ichikawa et al., (Languas galanga) 2005; Ito et al., 2005 17-Acetoxylolkinolide B KK activity Yan et al., 2008 Acute alcohol exposure KK activity Mandrekar et al., 2007 Anacardic acid (6-nonadecyl- KK activity Sung et al., 2008 Salicylic acid) Apigenin (plant flavinoid) KK activity Shukla & Gupta, 2004: Yoon et al., 2006 Asiatic acid KK activity Yun et al., 2008 Cardamomin KK activity Lee et al., 2005 CDDO-Me (synthetic triterpenoid) KK activity Shishodia et al., 2006 CHS 828 (anticancer drug) KK activity Olsen et al., 2004 CML-1 KK activity Moetal, 2006 Compound 5 (Uredio- KK activity Roshak et al., 2002 thiophenecarboxamide derivative) CT2O126 KK activity/NIK Lee et al., 2008 Diaylpyridine derivative KK activity Murata et al., 2003 3,4-dihydroxybenzalacetone (from KK activity Sung et al., 2008 Chaga) Diosgenin KK activity Shishodia & Aggarwal, 2005; Liagreet al., 2005 E3-14.7K (Adenovirus) KK activity Lietal, 1999 E3-10.4K14.5K (Adenovirus) KK activity Friedman & Horwitz, 2002 E7 (human papillomavirus) KK activity Spitkovsky et al., 2002 Furonaphthoquinone KK activity Shin et al., 2006 3-Formylchromone KKb activity/p65 DNA Yadav et al., 2011 binding Guggulsterone KK activity Ichikawa & Aggarwal, 2006; Deng, 2007; Lyet al, 2008: Lee et al., 2008 HB-EGF (Heparin-binding KK activity Mehta & Besner, 2003 epidermal growth factor-like growth factor) US 2016/0146783 A1 May 26, 2016 24

TABLE 3-continued IKBC phosphorylation and/or degradation inhibitors Molecule Point of Inhibition References Falcarindol KK activity Shiao et al., 2005 Hammerhead ribozyme to IKKalb KK activity Yang et al., 2007 Hepatocyte growth factor KK activity Min et al., 2005; Gong et al, 2006 Honokiol KK activity Tse et al., 2005: Munroe et al, 2007 Humulone KK activity Lee et al., 2007 Hypoestoxide KK activity Ojo-Amaize et al., 2001 Indolecarboxamide derivative KK activity Karin et al., 2004 Labdane diterpenoids KK activity Giron et al., 2008 LF15-0.195 (analog of 15- KK activity Yang et al., 2003 deoxyspergualine) gamma-mangostin (from Garcinia KK activity Nakatani et al., 2004 mangosiana) Garcinone B KK activity Yamakuni et al., 2005 (Amino)imidazolylcarboxaldehyde KK activity Karin et al., 2004 derivative Imidazolylquinoline- KK activity Karin et al., 2004 carboxaldehyde derivative Kahweol KK activity Kim et al., 2004 Kava (Piper methysticum) KK activity Folmer et al., 2006 derivatives Lead KK activity Xu et al., 2006 Marasmius Oreades liquid extract KK activity Petrova et al., 2008 Menatetrenone (vitamin K2 KK activity Ozaki et al., 2007 analogue) Metformin KK activity Huang et al., 2008 Mild hypothermia KK activity Han et al., 2003 ML12OB KK activity Catley et al., 2006 Morin (3,5,7,2',4'- KK activity Manna et al., 2007 Pentahydroxyflavone) Morusin KK activity Lee et al., 2008 MX781 (retinoid antagonist) KK activity Bayon et al., 2003 N-acetylcysteine KK activity Oka et al., 2000 Nitrosylcobalamin (vitamin B12 KK activity Chawia-Sarkar et al., 2003 analog) NSAIDS KK activity Takada et al., 2004 Hepatits C virus NS5B KK activity Choi et al., 2006 PAN1 (aka NALP2 or PYPAF2) KK activity Bruey et al., 2004 Pectin (citrus) KK activity Chen et al., 2006 Pinitol KK activity Sethi et al., 2008 PMX464 KK activity Caister et al., 2008 Pyrazolo4.3-cquinoline KK activity Karin et al., 2004 derivative Pyridooxazinone derivative KK activity Karin et al., 2004 N-(4-hydroxyphenyl) retinamide KK activity Shishodia et al., 2005; Kuefer et al., 2007 Scytonemin KK activity Stevenson et al., 2002 Senecarpus anacardini extract KK activity Singh et al., 2006 SPC-839 KK activity Paianki et al., 2002 Sulforaphane and KK activity Xii etal, phenylisothiocyanate 2005: Murakami etal, 2007: Liu et al., 2008: Hayes et al., 2008 Survanta (Surfactant product) KK activity Raychaudhuri et al., 2003 Torque Teno virus ORF2 KK activity Zheng et al., 2007 Piceatannol KK activity Islam et al., 2004 Plumbagin (5-hydroxy-2-methyl- KK activity Sandur et al., 2006 14-naphthoquinone) IKKb peptide to NEMO binding KK-NEMO interaction May et al., 2000 domain NEMO CC2-LZ peptide NEMO oligomerization Agou et al., 2004 AGRO100 (G-quadraplex NEMO binding Girvan et al., 2006 oligodeoxynucleotide) PTEN (tumor suppressor) Activation of IKK Gustin et al., 2001 Theaflavin (black tea component) Activation of IKK Aneia et al., 2004; Ukill et al, 2006: Kaira et al., 2007 Tilianin Activation of IKK Nam et al., 2005 Withanolides Activation of IKK Ichikawa et al., 2006 Zerumbone Activation of IKK Takada et al., 2005 Silibinin IKKa activity; nuclear Dhanaiakshmi et al., translocation 2002; Singh etal, 2004; Min et al., 2007 US 2016/0146783 A1 May 26, 2016 25

TABLE 3-continued IKBC phosphorylation and/or degradation inhibitors Molecule Point of Inhibition References Sulfasalazine KKa and IKKb kinase Wahl etal, activity 1998: Weber et al., 2000 SulfaSalazine analogs KK kinase activity Habens et al., 2005 Quercetin KK activity Peet & Li, 1999 RoSmarinic acid KK activity Lee et al., 2006 Staurosporine KK activity Peet & Li, 1999 gamma-Tocotrienol KK activity Shah & Sylvester, 2005: Ahn et al., 2006 Wedelolactone KK activity Kobori et al., 2003 Betulinic acid KKa activity and pé5 Takada & Aggarwal, phosphorylation 2003; Rabi et al., 2008 Ursolic acid KKa activity and pé5 Shishodia et al., phosphorylation 2003: Manu & Kuttan, 2008 Thalidomide (and thalidomide KK activity Keifer et al., 2001: Geet analogs) al, 2006: Carcache de Bianco et al., 2007 Salubrinal KK Huang et al., 2011 activity degradation Fas-associated factor-1 KK assembly Parket al., 2007 Interleukin-10 Reduced IKKa and Tabary et al., 2003 KKb expression MC160 (molluscum contagiosum Reduced IKKa. Nichols & Shisler, 2006 virus) expression Monochloramine and glycine Oxidizes IkB Kim et al., chloramine (NH2Cl) 2005: Midwinter et al., 2006 GS143 Blocks IkB Nakajima et al., ubiquitylation 2008: Hirose et al., 2008 Salmonella Secreted Factor L Blocks IkB Le Negrate et al., 2008 ubiquitylation Anethole Phosphorylation Chainy et al., 2000 Anti-thrombin III Phosphorylation Oelschlager et al., 2002 Artemisia vestita Phosphorylation Sun et al., 2006 Aspirin, sodium salicylate Phosphorylation, Frantz & O'Neill, KKbeta 1995; Kopp & Ghosh, 1994; Yin et al., 1998 Azidothymidine (AZT) Phosphorylation Ghosh etal, 2003: Kurokawa et al., 2005 Baoganning Phosphorylation Tan et al., 2005 BAY-11-7082 Phosphorylation Pierce etal, 1997 (E3((4-methylphenyl)-sulfonyl)-2- propenenitrile) BAY-117083 Phosphorylation Pierce etal, 1997 (E3((4-t-butylphenyl)-sulfonyl)-2- propenenitrile) Benzyl isothiocyanate Phosphorylation Srivastava & Singh, 2004 Black raspberry extracts (cyanidin Phosphorylation Huang etal, 3-O-glucoside, cyanidin 3-O- 2002; Hecht et al., 2006 (2(G)-xylosylrutinoside), cyanidin 3-O-rutinoside) Buddlejasaponin IV Phosphorylation Won et al., 2006 Cacospongionolide B Phosphorylation Posadas et al., 2003 Calagualine Phosphorylation Manna et al., 2003 Carbon monoxide Phosphorylation Sarady et al., 2002 Carboplatin Phosphorylation Singh & Bhat, 2004 Cardamonin Phosphorylation Israfetal, 2006 Chorionic gonadotropin Phosphorylation Manna et al., 2000 Cordycepin Phosphorylation Kim et al., 2006; Huang et al., 2007 Crassocephalum rabens Phosphorylation Hou et al., 2007 galactolipid Cycloepoxydon: 1-hydroxy-2- Phosphorylation Gehrt etal, 1998 hydroxymethyl-3-pent-1- enylbenzene Cytomegalovirus Phosphorylation Jarvis et al., 2006 Decursin Phosphorylation Kim et al., 2006 Delphinidin Phosphorylation Syed et al., 2008 Dexanabinol Phosphorylation Juttier et al., 2004 Digitoxin Phosphorylation Srivastava etal, 2004; Jagielska et al., 2009 US 2016/0146783 A1 May 26, 2016 26

TABLE 3-continued IKBC phosphorylation and/or degradation inhibitors Molecule Point of Inhibition References Dihydrotestosterone Phosphorylation Xu et al., 2011 Diterpenes (synthetic) Phosphorylation Chao et al., 2005 Docosahexaenoic acid Phosphorylation Chen et al., 2005; Zandet al, 2008 Entamoeba histolytica Phosphorylation Kannanadiminii & Chadee, 2006 Extensively oxidized low density Phosphorylation Brand et al., 1997; Page et lipoprotein (OX-LDL), 4- al, 1999 Hydroxynonenal (HNE) FBD Phosphorylation Lin et al., 2008 FHIT (Fragile histidine triad Phosphorylation Nakagawa & Akao, 2006 protein) Frticius Ligustrain lucidi Phosphorylation An et al., 2007 Gabexate mesilate Phosphorylation Uchiba et al., 2003 6-gingerol; casparol Phosphorylation Kim et al., 2005: Akitan et al, 2006: Ishiguro etal, 2007 Gieditsia sinensis thorns extract Phosphorylation Ha et al., 2008 Gleevec (Imatanib) Phosphorylation Wolfetal, 2005 Glossogyne tentifolia Phosphorylation Wii et al., 2004: Haetal, 2006 Guggulsterone Phosphorylation Shishodia & Aggarwal, 2004 4-hydroxy-3,6,7,8,3',4'- Phosphorylation Lai et al., 2007 hexamethoxyflavone Hydroquinone Phosphorylation Kerzic et al., 2003 Ibuprofen Phosphorylation Palayoor et al., 1998 Indirubin-3-oxime Phosphorylation Mak et al., 2004 Inonotus obliquit is ethanol extract Phosphorylation Kim et al., 2007 Interferon-alpha Phosphorylation Manna et al., 2000 Inhaled isobutyl nitrite Phosphorylation Ponnappan et al., 2004 Kaempferol Phosphorylation Garcia-Media viiia et al., 2006; Kim et al., 2007 Kushen flavonoids and kurairinone Phosphorylation Han et al., 2006 Licorce extracts Phosphorylation Kim et al., 2006: Kwon et al, 2007 Melatonin Phosphorylation Alonso etal, 2006; Tamura et al., 2009 Marine natural products (several) KKb proteasome Folmer et al., 2009 Methotrexate Phosphorylation Majumdar & Aggarwal, 2001: Yozai et al., 2005 Monochloramine Phosphorylation Omori et al., 2002 Nafamostat mesilate Phosphorylation Noguchi et al., 2003 Obovatol Phosphorylation Lee et al., 2008 Oleandrin Phosphorylation Manna et al., 2000: Sreeivasan et al., 2003 Oleanolic acid (Aralia eiata) Phosphorylation Suh et al., 2007 Omega 3 fatty acids Phosphorylation Novak et al., 2003 Panduratin A (from Kaempferia Phosphorylation Yun et al., 2003 pandiirata, Zingiberaceae) Petrosaspongiolide M Phosphorylation Posadas et al., 2003 Pinosylvin Phosphorylation Lee et al., 2006 Plagius fioscillost is extract Phosphorylation Calzado et al., 2005 polyacetylene spiroketal Phytic acid (inositol Phosphorylation Ferry et al., 2002 hexakisphosphate) Pomegranate fruit extract Phosphorylation Ahmed et al., 2005 Prostaglandin A1 Phosphorylation/IKK Rossi etal, 1997, 2000 Protocatechuic Aldehyde Phosphorylation Xu et al., 2011 20(S)-Protopanaxatriol Phosphorylation Oh et al., 2004; Lee etal, (ginsenoside metabolite) 2005 Rengyolone Phosphorylation Kim et al., 2006 Rottlerin Phosphorylation Kim et al., 2005: Torricelli et al., 2008 Saikosaponin-d Phosphorylation; Leung et al., 2005; Dang et increased IkB al, 2007 Saline (low Na+ istonic) Phosphorylation Tabary et al., 2003 Saivia militiorrhizae water-soluble Phosphorylation Kim et al., 2005 extract US 2016/0146783 A1 May 26, 2016 27

TABLE 3-continued IKBC phosphorylation and/or degradation inhibitors Molecule Point of Inhibition References Sanguinarine Phosphorylation Chaturvedi et al., 1997 (pseudochelerythrine, 13-methyl 1,3-benzodioxolo-5,6-c-1,3- dioxolo-4.5 phenanthridinium) Scoparone Phosphorylation Jong et al., 2005 Sesaminol glucosides Phosphorylation Lee et al., 2006 Shikonins Phosphorylation Nam et al., 2008 Sillymarin Phosphorylation Manna et al., 1999; Saiou et al., 1998 Snake venom toxin (Vipera Phosphorylation Son et al., 2007 lebetina turanica) SOCS1 Phosphorylation Kinivo etal, 2002; Nakagawa etal, 2002 Spilanthol Phosphorylation Wu et al., 2008 Statins (several) Phosphorylation Hilgendorfetal, 2003; Han et al., 2004: Piana via et al., 2005 Sulindac KK/Phosphorylation Yamamato et al., 1999 THI 52 (1-naphthylethyl-6,7- Phosphorylation Kang et al., 2003 dihydroxy-1,2,3,4- tetrahydroisoquinoline) 1,2,4-thiadiazolidine derivatives Phosphorylation Manna et al., 2004 Tomatidine Phosphorylation Chiu & Lin, 2008 Vesnarinone Phosphorylation Manna & Aggarwal, 2000; Harada et al., 2005 Xanthoangelol D Phosphorylation Sugii et al., 2005 YC-1 Phosphorylation Huang et al., 2005 Yop (encoded by Yersinia Deubiquintinase for Schesser etal, pseudotuberculosis) (kBa: Acetylation of 1998: Zhou et al., KKbeta 2005; Mittal etal, 2006; Mukherjee & Orth, 2008 Osmotic stress kB ubiquitination Huangfu et al., 2007 Acetaminophen Degradation Mancini et al., 2003 Activated Protein C (APC) Degradation Yiiksei et al., 2002 Alachlor Degradation Shimomura-Shimizu et al, 2005 Allylpyrocatechol Degradation Sarkar et al., 2008 a-melanocyte-stimulating hormone Degradation Manna & Aggarwal, 1998 (a-MSH) Amentoflavone Degradation Banerjee etal, 2002; Guruvayoorappan & Kuttan, 2007 Angelica dahirica radix extract Degradation Kang et al., 2006 Apple extracts Degradation/proteasome Yoon & Liu, 2007 Artemisia capillaris Thunb extract Degradation Hong et al., 2004: Kim et (capillarisin) al, 2007; Lee et al., 2007 Artemisia iwavonogi extract Degradation Kim et al., 2005 L-ascorbic acid Degradation Han et al., 2004 Antrodia Camphorata Degradation Hseu et al., 2005 Aucubin Degradation Jeong et al., 2002 Baicalein Degradation Ma et al., 2004 N-(quinolin-8- Degradation Xie et al., 2007 yl)benzenesulfonamindes beta-lapachone Degradation Manna et al., 1999 Blackberry extract Degradation Pergola et al., 2006 1-Bromopropane Degradation Yoshida et al., 2006 Buchang-tang Degradation Shin et al., 2005 Capsaicin (8-methyl-N-vanillyl-6- Degradation Singh et al., 1996; Mori et nonenamide) al, 2006; Kang et al., 2007 Catalposide Degradation Kim et al., 2004 Cierodendron trichotomum Degradation Park & Kim, 2007 Tunberg Leaves Clomipraminefimipramine Degradation Hwang et al., 2008 Coptidis rhizoma extract Degradation Kim et al., 2007 Cyclolinteinone (sponge Degradation D'Acquisto et al., 2000 sesterterpene) DA-9601 (Artemisia asiatica Degradation Choi et al., 2006 extract) US 2016/0146783 A1 May 26, 2016 28

TABLE 3-continued IKBC phosphorylation and/or degradation inhibitors Molecule Point of Inhibition References Diamide (tyrosine phosphatase Degradation Toledano & Leonard, inhibitor) 1991; Singh & Aggarwal, 1995 Dihydroarteanniun Degradation Li et al., 2006 Dobutamine Degradation Loop et al., 2004 Docosahexaenoic acid Degradation Weldon et al., 2006 E-73 (cycloheximide analog) Degradation Sugimoto et al., 2000 Ecabet sodium Degradation Kim et al., 2003 Electrical stimulation of vagus Degradation Guarini et al., 2003 le:We Emodin (3-methyl-1,6,8- Degradation Kumar etal, trihydroxyanthraquinone) 1998; Huang et al., 2004 Ephedrae herba (Mao) Degradation Aoki et al., 2005 Equol Degradation Kang et al., 2005 Erbstatin (tyrosine kinase Degradation Nataraian et al., 1998 inhibitor) Estrogen (E2) Degradation and various Sun etal, other steps 1998; Kalaitzidis & Gilmore, 2005; Stefan et al, 2006 Ethacrynic acid Degradation and DNA Han et al., 2004 binding) Fludarabine Degradation Nishioka et al., 2007 Fosfomycin Degradation Yoneshima et al., 2003 Fungal gliotoxin Degradation Pahl et al., 1999 Gabexate mesilate Degradation Yuksel et al., 2003 Gamisanghyulyunbueum Degradation Shin et al., 2005 Genistein (tyrosine kinase Degradation; caspase Nataraian et al., inhibitor) cleavage of IkBa. 1998: Baixa & Yoshimura, 2003 Genipin Degradation Koo et al., 2004 Glabridin Degradation Kang et al., 2004 Ginsenoside Re Degradation Zhang et al., 2007 Glimepiride Degradation Schiekofer et al., 2003 Glucosamine (sulfate or Degradation Largo et al., 2003; Rafi et carboxybutyrylated) al., 2007: Rajapakse etal, 2008 gamma-glutamylcysteine Degradation Manna et al., 1999 synthetase Glutamine Degradation Singleton etal, 2005: Fillmann etal, 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 Heat shock protein-70 Degradation Chan et al., 2004; Shi etal, 2006 Herbal mixture (Cinnamomiramulus, Degradation Jeong et al., 2008 Anemarrheriae rhizoma, Officinari rhizoma) Hypochlorite Degradation Mohri et al., 2002 Ibudilast Degradation Kiebala cc Maggirwar, 1998 IL-13 Degradation Manna & Aggarwal, 1998 Incensole acetate Degradation Moussaieffet al., 2007 Intravenous immunoglobulin Degradation Ichiyama et al., 2004 Isomalotochromanol and Degradation Ishii et al., 2003 isomalotochromene K1L (Vaccinia virus protein) Degradation Shisier & Jin, 2004 Kochia scoparia fruit (methanol Degradation Shin et al., 2004 extract) Kummerowia striata (Thunb.) Degradation Tao et al., 2008 Schindl (ethanol extract) Leflunomide metabolite (A77 Degradation Manna & Aggarwal, 1999 1726) Lidocaine Degradation Feng et al., 2007: Lahat et al, 2008 Lipoxin A4 Degradation Zhang et al., 2007 Losartan Degradation/NF-kB Chen et al., 2002; Zhu et expression al, 2007 US 2016/0146783 A1 May 26, 2016 29

TABLE 3-continued IKBC phosphorylation and/or degradation inhibitors Molecule Point of Inhibition References Low level laser therapy Degradation Rizzi et al., 2006 LY294.002 (PI3-kinase Degradation Parket al., 2002 inhibitor) 2-(4-morpholinyl)-8- phenylchromone MC159 (Molluscum contagiosum Degradation of IkBb Murao & Shisler, 2005 virus) 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 Mosia dianihera extract Degradation Lee et al., 2006 Mume fructus extract Degradation Choi et al., 2007 Murr1 gene product Degradation Ganesh et al., 2003 Neurofibromatosis-2 (NF-2: Degradation Kim et al., 2002 merlin) protein Opiiniia fictis indica va saboten Degradation Lee et al., 2006 extract Ozone (aqueous) Degradation Huth et al., 2007 Paeony total glucosides Degradation Chen et al., 2007 Pectenotoxin-2 Degradation Kim et al., 2008 Penetratin Degradation Letoya et al., 2006 Pervanadiate (tyrosine phosphatase Degradation Singh & Aggarwal, inhibitor) 1995: Singh et al., 1996 Phenylarsine oxide (PAO, tyrosine Degradation Mahboubi etal, phosphatase inhibitor) 1998; Singh & Aggarwal, 1995 beta-Phenylethyl (PEITC) and 8- Degradation Rose et al., 2005 methylsulphinyloctyl isothiocyanates (MSO) (watercress) Phenytoin Degradation Kato et al., 2005 c-phycocyanin Degradation Chering et al., 2007 Platycodin Saponins Degradation Ahn et al., 2005: Lee etal, 2008 Polymeric formula Degradation de Jong et al., 2007 Polymyxin B Degradation Jiang et al., 2006 Poncirus trifoliata fruit extract Degradation; Shin et al., 2006: Kim et phosphorylation of IkBa al., 2007 Probiotics Degradation Petrofetal, 2004 Pituitary adenylate cyclase- Degradation Delgado & Ganea, 2001 activating polypeptide (PACAP) Prostaglandin 15-deoxy- Degradation Cuzzo crea et al., Delta(12,14)-PGJ(2) 2003; Chatterjee et al., 2004 Prodigiosin (Haheila cheiuensis) 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 bouliardii Degradation Sougioultzis et al., 2006 anti-inflammatory factor) Sanguis Draconis Degradation Choy et al., 2007 San-Huang-Xie-Xin-Tang Degradation Shih et al., 2007 Schisandra fructus extract Degradation Kang et al., 2006; Guo et al, 2008 Scutellarin Degradation Tan et al., 2007 Sesquiterpene lactones Degradation Hehner et al., 1998: Whan (parthenolide; ergolide; Han et al., 2001; Schorr et guaianolides; alpha-humulene; al, 2002: Medeiros etal, trans-caryophyllene) 2007 Sevoflurane isoflurane Degradation Boost et al., 2009 Siegeskaurolic acid (from Degradation Parket al., 2007 Siegesbeckia pubescens root) ST2 (IL-1-like receptor secreted Degradation Takezako et al., 2006 form) Synadenium carinatum latex lectin Degradation Rogerio et al., 2007 Taiwanofinguis Camphoratiis Degradation Liu et al., 2007 Taurene bromamine Degradation Tokunaga et al., 2007 Thiopental Degradation Loop et al., 2002 Tipifarnib Degradation Xue et al., 2005 US 2016/0146783 A1 May 26, 2016 30

TABLE 3-continued IKBC phosphorylation and/or degradation inhibitors Molecule Point of Inhibition References Titanium Degradation Yang et al., 2003 TNP-470 (angiogenesis inhibitor) Degradation Mauriz et al., 2003 Stinging nettle (Urtica dioica) Degradation Riehemann et al., 1999 plant extracts Trichomomas vaginalis infection Degradation Chang et al., 2004 Triglyceride-rich lipoproteins Degradation Kunwenda et al., 2002 TuSSilagone (Farfarae fios) Degradation Lim et al., 2008 UO126 (MEK inhibitor) Degradation Takaya et al., 2003 Ursodeoxycholic acid Degradation Joo et al., 2004 Xanthium strumarium L. Degradation Kim et al., 2005: Yoon et (methanol extract) al, 2008 Yulda-Hanso-Tang Degradation Jeong et al., 2007 Zinc Degradation Uzzo et al., 2006: Bao et al, 2006 Molluscum contagiosum virus (kBbeta degradation Murao di Shisier; 2005 MC159 protein Vasoactive intestinal peptide Degradation (and CBP- Delgado & Ganea, RelA. interaction) 2001; Delgado, 2002 HIV-1 Vpu protein TrCP ubiquitin ligase Bour et al., 2001 inhibitor Epoxyquinone A monomer KKb/DNA binding Liang et al., 2006 Ro106-9920 (small molecule) kBa ubiqutination Swinney et al., 2002 inhibitor Furonaphthoquinone KK activity Shin et al., 2006

Pharmaceutical Compositions nasal, topical, parenteral, rectal, and/or intravaginal adminis tration. The formulations may conveniently be presented in 0225. In alternative embodiments, the invention provides unit dosage form and may be prepared by any methods well pharmaceutical compositions for practicing the methods of known in the art of pharmacy. The amount of active ingredient the invention, e.g., pharmaceutical compositions for over which can be combined with a carrier material to produce a coming or diminishing or preventing Growth Factor Inhibitor single dosage form will vary depending upon the host being (GFI) resistance in a cell, or, a method for increasing the treated, the particular mode of administration. The amount of growth-inhibiting effectiveness of a Growth Factor inhibitor active ingredient which can be combined with a carrier mate on a cell, or, a method for re-sensitizing a cell to a Growth rial to produce a single dosage form will generally be that Factor Inhibitor. amount of the compound which produces atherapeutic effect. 0226. In alternative embodiments, compositions used to 0229. Pharmaceutical formulations used to practice the practice the methods of the invention are formulated with a methods of the invention can be prepared according to any pharmaceutically acceptable carrier. In alternative embodi method known to the art for the manufacture of pharmaceu ments, the pharmaceutical compositions used to practice the ticals. Such drugs can contain Sweetening agents, flavoring methods of the invention can be administered parenterally, agents, coloring agents and preserving agents. A formulation topically, orally or by local administration, Such as by aerosol can be admixtured with nontoxic pharmaceutically accept or transdermally. The pharmaceutical compositions can be able excipients which are suitable for manufacture. Formula formulated in any way and can be administered in a variety of tions may comprise one or more diluents, emulsifiers, preser unit dosage forms depending upon the condition or disease Vatives, buffers, excipients, etc. and may be provided in Such and the degree of illness, the general medical condition of forms as liquids, powders, emulsions, lyophilized powders, each patient, the resulting preferred method of administration sprays, creams, lotions, controlled release formulations, tab and the like. Details on techniques for formulation and lets, pills, gels, on patches, in implants, etc. administration are well described in the scientific and patent 0230 Pharmaceutical formulations for oral administra literature, see, e.g., the latest edition of Remington’s Pharma tion can be formulated using pharmaceutically acceptable ceutical Sciences, Maack Publishing Co., Easton Pa. (“Rem carriers well known in the art in appropriate and Suitable ington's'). dosages. Such carriers enable the pharmaceuticals to be for 0227. Therapeutic agents used to practice the methods of mulated in unit dosage forms as tablets, geltabs, pills, powder, the invention can be administered alone or as a component of dragees, capsules, liquids, lozenges, gels, syrups, slurries, a pharmaceutical formulation (composition). The com Suspensions, etc., Suitable for ingestion by the patient. Phar pounds may be formulated for administration in any conve maceutical preparations for oral use can be formulated as a nient way for use in human or veterinary medicine. Wetting Solid excipient, optionally grinding a resulting mixture, and agents, emulsifiers and lubricants, such as Sodium lauryl Sul processing the mixture of granules, after adding Suitable addi fate and magnesium Stearate, as well as coloring agents, tional compounds, if desired, to obtaintablets or dragee cores. release agents, coating agents, Sweetening, flavoring and per Suitable solid excipients are carbohydrate or protein fillers fuming agents, preservatives and antioxidants can also be include, e.g., Sugars, including lactose. Sucrose, mannitol, or present in the compositions. Sorbitol; starch from corn, wheat, rice, potato, or other plants; 0228. Formulations of the compositions used to practice cellulose such as methyl cellulose, hydroxypropylmethyl the methods of the invention include those suitable for oral/ cellulose, or sodium carboxy-methylcellulose; and gums US 2016/0146783 A1 May 26, 2016 including arabic and tragacanth; and proteins, e.g., gelatin agents include naturally-occurring gums, such as gum acacia and collagen. Disintegrating or solubilizing agents may be and gum tragacanth, naturally occurring phosphatides. Such added, such as the cross-linked polyvinyl pyrrolidone, agar, as soybean lecithin, esters or partial esters derived from fatty alginic acid, or a salt thereof. Such as sodium alginate. acids and hexitol anhydrides, such as Sorbitan mono-oleate, 0231 Dragee cores are provided with suitable coatings and condensation products of these partial esters with ethyl Such as concentrated Sugar Solutions, which may also contain ene oxide. Such as polyoxyethylene Sorbitan mono-oleate. gum arabic, talc, polyvinylpyrrolidone, carbopol gel, poly The emulsion can also contain Sweetening agents and flavor ethylene glycol, and/or titanium dioxide, lacquer Solutions, ing agents, as in the formulation of syrups and elixirs. Such and Suitable organic solvents or solvent mixtures. Dyestuffs formulations can also contain a demulcent, a preservative, or or pigments may be added to the tablets or dragee coatings for a coloring agent. product identification or to characterize the quantity of active 0234. In practicing this invention, the pharmaceutical compound (i.e., dosage). Pharmaceutical preparations used to compounds can also be administered by in intranasal, practice the methods of the invention can also be used orally intraocular and intravaginal routes including Suppositories, using, e.g., push-fit capsules made of gelatin, as well as Soft, insufflation, powders and aerosol formulations (for examples sealed capsules made of gelatin and a coating Such as glycerol of steroid inhalants, see Rohatagi (1995) J. Clin. Pharmacol. or Sorbitol. Push-fit capsules can contain active agents mixed 35:1187-1193: Tiwa (1995) Ann. Allergy Asthma Immunol. with a filler or binders such as lactose or starches, lubricants 75:107-111). Suppositories formulations can be prepared by Such as talc or magnesium Stearate, and, optionally, stabiliz mixing the drug with a suitable non-irritating excipient which ers. In soft capsules, the active agents can be dissolved or is solid at ordinary temperatures but liquid at body tempera Suspended in Suitable liquids, such as fatty oils, liquid paraf tures and will therefore melt in the body to release the drug. fin, or liquid polyethylene glycol with or without stabilizers. Such materials are cocoa butter and polyethylene glycols. 0232 Aqueous Suspensions can contain an active agent 0235. In practicing this invention, the pharmaceutical (e.g., a composition used to practice the methods of the inven compounds can be delivered by transdermally, by a topical tion) in admixture with excipients suitable for the manufac route, formulated as applicator Sticks, Solutions, Suspensions, ture of aqueous Suspensions. Such excipients include a sus emulsions, gels, creams, ointments, pastes, jellies, paints, pending agent, such as Sodium carboxymethylcellulose, powders, and aerosols. methylcellulose, hydroxypropylmethylcellulose, sodium 0236. In practicing this invention, the pharmaceutical alginate, polyvinylpyrrolidone, gum tragacanthandgum aca compounds can also be delivered as microspheres for slow cia, and dispersing or wetting agents such as a naturally release in the body. For example, microspheres can be admin occurring phosphatide (e.g., lecithin), a condensation product istered via intradermal injection of drug which slowly release of an alkylene oxide with a fatty acid (e.g., polyoxyethylene subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. Stearate), a condensation product of ethylene oxide with a 7:623-645; as biodegradable and injectable gel formulations, long chain aliphatic alcohol (e.g., heptadecaethylene oxycet see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as anol), a condensation product of ethylene oxide with a partial microspheres for oral administration, see, e.g., Eyles (1997) ester derived from a fatty acid and a hexitol (e.g., polyoxy J. Pharm. Pharmacol. 49:669-674. ethylene Sorbitol mono-oleate), or a condensation product of 0237. In practicing this invention, the pharmaceutical ethylene oxide with a partial ester derived from fatty acid and compounds can be parenterally administered, such as by a hexitol anhydride (e.g., polyoxyethylene Sorbitan mono intravenous (IV) administration or administration into a body oleate). The aqueous Suspension can also contain one or more cavity or lumenofan organ. These formulations can comprise preservatives Such as ethyl or n-propyl p-hydroxybenzoate, a solution of active agent dissolved in a pharmaceutically one or more coloring agents, one or more flavoring agents and acceptable carrier. Acceptable vehicles and solvents that can one or more Sweetening agents, such as Sucrose, aspartame or be employed are water and Ringer's solution, an isotonic saccharin. Formulations can be adjusted for osmolarity. sodium chloride. In addition, sterile fixed oils can be 0233. Oil-based pharmaceuticals are particularly useful employed as a solvent or Suspending medium. For this pur for administration hydrophobic active agents used to practice pose any bland fixed oil can be employed including synthetic the methods of the invention. Oil-based suspensions can be mono- or diglycerides. In addition, fatty acids such as oleic formulated by Suspending an active agent in a vegetable oil, acid can likewise be used in the preparation of injectables. Such as arachis oil, olive oil, sesame oil or coconut oil, or in These solutions are sterile and generally free of undesirable a mineral oil such as liquid paraffin, or a mixture of these. See matter. These formulations may be sterilized by conventional, e.g., U.S. Pat. No. 5,716,928 describing using essential oils or well known sterilization techniques. The formulations may essential oil components for increasing bioavailability and contain pharmaceutically acceptable auxiliary Substances as reducing inter- and intra-individual variability of orally required to approximate physiological conditions such as pH administered hydrophobic pharmaceutical compounds (see adjusting and buffering agents, toxicity adjusting agents, e.g., also U.S. Pat. No. 5,858.401). The oil suspensions can con Sodium acetate, sodium chloride, potassium chloride, cal tain a thickening agent, such as beeswax, hard paraffin or cium chloride, sodium lactate and the like. The concentration cetyl alcohol. Sweetening agents can be added to provide a ofactive agent in these formulations can vary widely, and will palatable oral preparation, Such as glycerol, Sorbitol or be selected primarily based on fluid volumes, viscosities, sucrose. These formulations can be preserved by the addition body weight, and the like, in accordance with the particular of an antioxidant such as ascorbic acid. As an example of an mode of administration selected and the patient’s needs. For injectable oil vehicle, see Minto (1997) J. Pharmacol. Exp. IV administration, the formulation can be a sterile injectable Ther. 281:93-102. The pharmaceutical formulations of the preparation, Such as a sterile injectable aqueous or oleaginous invention can also be in the form of oil-in-water emulsions. Suspension. This suspension can be formulated using those The oily phase can be a vegetable oil or a mineral oil, Suitable dispersing or wetting agents and Suspending agents. described above, or a mixture of these. Suitable emulsifying The sterile injectable preparation can also be a Suspension in US 2016/0146783 A1 May 26, 2016 32 a nontoxic parenterally-acceptable diluent or solvent, such as guidance to determine the dosage regiment, i.e., dose sched a solution of 1,3-butanediol. The administration can be by ule and dosage levels, administered practicing the methods of bolus or continuous infusion (e.g., Substantially uninter the invention are correct and appropriate. rupted introduction into a blood vessel for a specified period 0242 Single or multiple administrations of formulations of time). can be given depending on the dosage and frequency as 0238. The pharmaceutical compounds and formulations required and tolerated by the patient. The formulations should used to practice the methods of the invention can be lyo provide a sufficient quantity of active agent to effectively philized. The invention provides a stable lyophilized formu treat, prevent or ameliorate a conditions, diseases or symp lation comprising a composition of the invention, which can toms as described herein. For example, an exemplary phar be made by lyophilizing a solution comprising a pharmaceu maceutical formulation for oral administration of composi tical of the invention and a bulking agent, e.g., mannitol, tions used to practice the methods of the invention can be in a trehalose, raffinose, and Sucrose or mixtures thereof. A pro daily amount of between about 0.1 to 0.5 to about 20, 50, 100 cess for preparing a stable lyophilized formulation can or 1000 or more ugper kilogram of body weight per day. In an include lyophilizing a solution about 2.5 mg/mL protein, alternative embodiment, dosages are from about 1 mg to about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a about 4 mg per kg of body weight per patient per day are used. sodium citrate buffer having a pH greater than 5.5 but less Lower dosages can be used, in contrast to administration than 6.5. See, e.g., U.S. patent app. no. 2004.0028670. orally, into the blood stream, into a body cavity or into a 0239. The compositions and formulations used to practice lumen of an organ. Substantially higher dosages can be used the methods of the invention can be delivered by the use of in topical or oral administration or administering by powders, liposomes (see also discussion, below). By using liposomes, spray or inhalation. Actual methods for preparing parenter particularly where the liposome surface carries ligands spe ally or non-parenterally administrable formulations will be cific for target cells, or are otherwise preferentially directed to known or apparent to those skilled in the art and are described a specific organ, one can focus the delivery of the active agent in more detail in Such publications as Remington's, Supra. into target cells in vivo. See, e.g., U.S. Pat. Nos. 6,063,400; 0243 The methods of the invention can further comprise 6,007,839; Al-Muhammed (1996).J. Microencapsul. 13:293 co-administration with other drugs or pharmaceuticals, e.g., 306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro compositions for treating cancer, septic shock, infection, (1989) Am. J. Hosp. Pharm. 46:1576-1587. fever, pain and related symptoms or conditions. For example, the methods and/or compositions and formulations of the 0240. The formulations used to practice the methods of the invention can be co-formulated with and/or co-administered invention can be administered for prophylactic and/or thera with antibiotics (e.g., antibacterial or bacteriostatic peptides peutic treatments. In therapeutic applications, compositions or proteins), particularly those effective against gram nega are administered to a Subject already suffering from a condi tive bacteria, fluids, cytokines, immunoregulatory agents, tion, infection or disease in an amount Sufficient to cure, anti-inflammatory agents, complement activating agents, alleviate or partially arrest the clinical manifestations of the condition, infection or disease and its complications (a Such as peptides or proteins comprising collagen-like “therapeutically effective amount'). For example, in alterna domains or fibrinogen-like domains (e.g., a ficolin), carbohy tive embodiments, pharmaceutical compositions of the inven drate-binding domains, and the like and combinations tion are administered in an amount Sufficient to treat, prevent thereof. and/or ameliorate normal, dysfunction (e.g., abnormally pro Nanoparticles and Liposomes liferating) cell, e.g., cancer cell, or blood vessel cell, includ ing endothelial and/or capillary cell growth; including 0244. The invention also provides nanoparticles and lipo neovasculature related to (within, providing a blood Supply Somal membranes comprising compounds used to practice to) hyperplastic tissue, a granuloma or a tumor. The amount of the methods of the invention. In alternative embodiments, the pharmaceutical composition adequate to accomplish this is invention provides nanoparticles and liposomal membranes defined as a “therapeutically effective dose.” The dosage targeting diseased and/or tumor (cancer) stem cells and dys schedule and amounts effective for this use, i.e., the "dosing functional stem cells, and angiogenic cells. regimen.” will depend upon a variety of factors, including the 0245. In alternative embodiments, the invention provides stage of the disease or condition, the severity of the disease or nanoparticles and liposomal membranes comprising (in addi condition, the general state of the patient’s health, the tion to comprising compounds used to practice the methods patient’s physical status, age and the like. In calculating the of the invention) molecules, e.g., peptides or antibodies, that dosage regimen for a patient, the mode of administration also selectively target abnormally growing, diseased, infected, is taken into consideration. dysfunctional and/or cancer (tumor) cell receptors. In alter 0241 The dosage regimen also takes into consideration native embodiments, the invention provides nanoparticles pharmacokinetics parameters well known in the art, i.e., the and liposomal membranes using IL-11 receptor and/or the active agents’ rate of absorption, bioavailability, metabolism, GRP78 receptor to targeted receptors on cells, e.g., on tumor clearance, and the like (see, e.g., Hidalgo-Aragones (1996).J. cells, e.g., on prostate or ovarian cancer cells. See, e.g., U.S. Steroid Biochem. Mol. Biol. 58:611-617: Groning (1996) patent application publication no. 200602399.68. Pharmazie 51:337-341; Fotherby (1996) Contraception 0246. In one aspect, the compositions used to practice the 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; methods of the invention are specifically targeted for inhibit Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. ing, ameliorating and/or preventing endothelial cell migra J. Clin. Pharmacol. 24:103-108; the latest Remington's, tion and for inhibiting angiogenesis, e.g., tumor-associated or supra). The state of the art allows the clinician to determine disease- or infection-associated neovasculature. the dosage regimen for each individual patient, active agent 0247 The invention also provides nanocells to allow the and disease or condition treated. Guidelines provided for sequential delivery of two different therapeutic agents with similar compositions used as pharmaceuticals can be used as different modes of action or different pharmacokinetics, at US 2016/0146783 A1 May 26, 2016 least one of which comprises a composition used to practice as to Substantially instantaneously produce a liposome encap the methods of the invention. A nanocell is formed by encap Sulating the therapeutic product; and immediately thereafter Sulating a nanocore with a first agent inside a lipid vesicle mixing the liposome solution with a buffer solution to pro containing a secondagent; see, e.g., Sengupta, et al., U.S. Pat. duce a diluted liposome solution. Pub. No. 20050266067. The agent in the outer lipid compart 0252. The invention also provides nanoparticles compris ment is released first and may exert its effect before the agent ing compounds used to practice this invention to deliver a in the nanocore is released. The nanocell delivery system may composition of the invention as a drug-containing nanopar be formulated in any pharmaceutical composition for deliv ticles (e.g., a secondary nanoparticle), as described, e.g., in ery to patients suffering from a diseases or condition as U.S. Pat. Pub. No. 20070077286. In one embodiment, the described herein, e.g., Such as a retinal age-related macular invention provides nanoparticles comprising a fat-soluble degeneration, a diabetic retinopathy, a cancer or carcinoma, a drug of this invention or a fat-solubilized water-soluble drug glioblastoma, a neuroma, a neuroblastoma, a colon carci to act with a bivalent or trivalent metal salt. noma, a hemangioma, an infection and/or a condition with at Liposomes least one inflammatory component, and/or any infectious or 0253 inflammatory disease, Such as a rheumatoid arthritis, a pso 0254 The compositions and formulations used to practice riasis, a fibrosis, leprosy, multiple Sclerosis, inflammatory the invention can be delivered by the use of liposomes. By bowel disease, or ulcerative colitis or Crohn's disease. using liposomes, particularly where the liposome Surface car 0248. In treating cancer, a traditional antineoplastic agent ries ligands specific for target cells, or are otherwise prefer is contained in the outer lipid vesicle of the nanocell, and an entially directed to a specific organ, one can focus the delivery antiangiogenic agent of this invention is loaded into the nano of the active agent into target cells in vivo. See, e.g., U.S. Pat. core. This arrangement allows the antineoplastic agent to be Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. released first and delivered to the tumor before the tumors Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Bio technol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. blood supply is cut off by the composition of this invention. 46:1576-1587. For example, in one embodiment, composi 0249. The invention also provides multilayered liposomes tions and formulations used to practice the invention are comprising compounds used to practice this invention, e.g., delivered by the use of liposomes having rigid lipids having for transdermal absorption, e.g., as described in Park, et al., head groups and hydrophobic tails, e.g., as using a polyeth U.S. Pat. Pub. No. 20070082042. The multilayered liposomes yleneglycol-linked lipid having a side chain matching at least can be prepared using a mixture of oil-phase components a portion the lipid, as described e.g., in US Pat App Pub No. comprising squalane, Sterols, ceramides, neutral lipids or oils, 2008.0089928. In another embodiment, compositions and for fatty acids and lecithins, to about 200 to 5000 nm in particle mulations used to practice the invention are delivered by the size, to entrap a composition of this invention. use of amphoteric liposomes comprising a mixture of lipids, 0250) A multilayered liposome used to practice the inven e.g., a mixture comprising a cationic amphiphile, an anionic tion may further include an antiseptic, an antioxidant, a sta amphiphile and/or neutral amphiphiles, as described e.g., in bilizer, a thickener, and the like to improve stability. Synthetic US Pat App Pub No. 20080088046, or 20080031937. In and natural antiseptics can be used, e.g., in an amount of another embodiment, compositions and formulations used to 0.01% to 20%. Antioxidants can be used, e.g., BHT, erysor practice the invention are delivered by the use of liposomes bate, tocopherol, astaxanthin, vegetable flavonoid, and comprising a polyalkylene glycol moiety bonded through a derivatives thereof, or a plant-derived antioxidizing sub thioether group and an antibody also bonded through a thio stance. A stabilizer can be used to stabilize liposome struc ether group to the liposome, as described e.g., in US Pat App ture, e.g., polyols and Sugars. Exemplary polyols include Pub No. 20080014255. In another embodiment, composi butylene glycol, polyethylene glycol, propylene glycol, tions and formulations used to practice the invention are dipropylene glycol and ethyl carbitol; examples of Sugars are delivered by the use of liposomes comprising glycerides, trehalose. Sucrose, mannitol, Sorbitol and chitosan, or a glycerophospholipides, glycerophosphinolipids, glycero monosaccharides oran oligosaccharides, or a high molecular phosphonolipids, Sulfolipids, sphingolipids, phospholipids, weight starch. A thickener can be used for improving the isoprenolides, steroids, Stearines, sterols and/or carbohydrate dispersion stability of constructed liposomes in water, e.g., a containing lipids, as described e.g., in US Pat App Pub No. natural thickener or an acrylamide, or a synthetic polymeric 2007O14822O. thickener. Exemplary thickeners include natural polymers, Such as acacia gum, Xanthan gum, gellan gum, locust bean gum and starch, cellulose derivatives, such as hydroxyethyl Antibodies as Pharmaceutical Compositions cellulose, hydroxypropyl cellulose and carboxymethyl cellu 0255. In alternative embodiments, the invention provides lose, synthetic polymers, such as polyacrylic acid, poly-acry compositions and methods for inhibiting or depleting an inte lamide or polyvinylpyrollidone and polyvinylalcohol, and grin O?3 (anb3), or inhibiting an integrin CfB (anb3) protein copolymers thereof or cross-linked materials. activity, or inhibiting the formation or activity of an integrin 0251 Liposomes can be made using any method, e.g., as anb3/RalB signaling complex, or inhibiting the formation or described in Park, et al., U.S. Pat. Pub. No. 20070042031, signaling activity of an integrin CfB (anb3)/RalB/NFkB sig including method of producing a liposome by encapsulating naling axis; or inhibiting or depleting a RalB protein or an a therapeutic product comprising providing an aqueous solu inhibitor of RalB protein activation; or inhibiting or depleting tion in a first reservoir, providing an organic lipid solution in a Src or TBK1 protein or an inhibitor of Src or TBK1 protein a second reservoir, wherein one of the aqueous Solution and activation. In alternative embodiments, this is achieved by the organic lipid solution includes atherapeutic product; mix administration of inhibitory antibodies. For example, in alter ing the aqueous solution with said organic lipid solution in a native embodiments, the invention uses isolated, synthetic or first mixing region to produce a liposome solution, wherein recombinant antibodies that specifically bind to and inhibit an the organic lipid solution mixes with said aqueous Solution so integrin CfB (anb3), or any protein of an integrin CVB3 US 2016/0146783 A1 May 26, 2016 34

(anb3)/RalB/NFkB signaling axis, a RalB protein, a Src or one or more hyperVariable regions which result in an TBK1 protein, or an NFkB protein. improvement in the affinity of the antibody for antigen; e.g., 0256 In alternative aspects, an antibody for practicing the NFkB, an integrin C. B. (anb3), or any protein of an integrin invention can comprise a peptide or polypeptide derived CfB (anb3)/RalB/NFkB signaling axis, a RalB protein, a Src from, modeled after or Substantially encoded by an immuno or TBK1 protein, compared to a parent antibody which does globulin gene or immunoglobulin genes, or fragments not possess those alteration(s). In alternative embodiments, thereof, capable of specifically binding an antigen or epitope, antibodies used to practice this invention are matured anti see, e.g. Fundamental Immunology. Third Edition, W. E. bodies having nanomolar or even picomolar affinities for the Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immu target antigen, e.g., NFkB, an integrin CfB (anb3), or any nol. Methods 175:267-273:Yarmush (1992).J. Biochem. Bio protein of an integrin CfB (anb3)/RalB/NFkB signaling axis, phys. Methods 25:85-97. In alternative aspects, an antibody a RalB protein, a Src or TBK1 protein. Affinity matured for practicing the invention includes antigen-binding por antibodies can be produced by procedures known in the art. tions, i.e., “antigen binding sites.” (e.g., fragments, Subse Antisense, siRNAs and microRNAs as Pharmaceutical Com quences, complementarity determining regions (CDRS)) that positions retain capacity to bind antigen, including (i) a Fab fragment, 0262. In alternative embodiments, the invention provides a monovalent fragment consisting of the VL, VH, CL and compositions and methods for inhibiting or depleting an inte CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment grin C. B. (anb3), or inhibiting an integrin CfB (anb3) protein comprising two Fab fragments linked by a disulfide bridge at activity, or inhibiting the formation or activity of an integrin the hinge region; (iii) a Fd fragment consisting of the VHand anb3/RalB signaling complex, or inhibiting the formation or CH1 domains; (iv) a Fv fragment consisting of the VL and VH signaling activity of an integrin CfB (anb3)/RalB/NFkB sig domains of a single arm of an antibody, (v) a dAb fragment naling axis; or inhibiting or depleting a RalB protein or an (Wardet al., (1989) Nature 341:544-546), which consists of a inhibitor of RalB protein activation; or inhibiting or depleting VH domain; and (vi) an isolated complementarity determin a Src or TBK1 protein or an inhibitor of Src or TBK1 protein ing region (CDR). Single chain antibodies are also included activation. In alternative embodiments, this is achieved by by reference in the term “antibody.” administration of inhibitory nucleic acids, e.g., siRNA, anti 0257. In alternative embodiments, the invention uses sense nucleic acids, and/or inhibitory microRNAs. "humanized' antibodies, including forms of non-human 0263. In alternative embodiments, compositions used to (e.g., murine) antibodies that are chimeric antibodies com practice the invention are formulated with a pharmaceutically prising minimal sequence (e.g., the antigen binding frag acceptable carrier. In alternative embodiments, the pharma ment) derived from non-human immunoglobulin. In alterna ceutical compositions used to practice the invention can be tive embodiments, humanized antibodies are human administered parenterally, topically, orally or by local admin immunoglobulins in which residues from a hyperVariable istration, Such as by aerosol or transdermally. The pharma region (HVR) of a recipient (e.g., a human antibody ceutical compositions can be formulated in any way and can sequence) are replaced by residues from a hyperVariable be administered in a variety of unit dosage forms depending region (HVR) of a non-human species (donorantibody) Such upon the condition or disease and the degree of illness, the as mouse, rat, rabbit or nonhuman primate having the desired general medical condition of each patient, the resulting pre specificity, affinity, and capacity. In alternative embodiments, ferred method of administration and the like. Details on tech framework region (FR) residues of the human immunoglo niques for formulation and administration are well described bulin are replaced by corresponding non-human residues to in the Scientific and patent literature, see, e.g., the latest edi improve antigen binding affinity. tion of Remington’s Pharmaceutical Sciences, Maack Pub 0258. In alternative embodiments, humanized antibodies lishing Co., Easton Pa. (“Remington's'). may comprise residues that are not found in the recipient 0264. While the invention is not limited by any particular antibody or the donor antibody. These modifications may be mechanism of action: microRNAs (miRNAs) are short (20 made to improve antibody affinity or functional activity. In 24 nt) non-coding RNAS that are involved in post-transcrip alternative embodiments, the humanized antibody can com tional regulation of gene expression in multicellular organ prise Substantially all of at least one, and typically two, Vari isms by affecting both the stability and translation of mRNAs. able domains, in which all or substantially all of the hyper miRNAs are transcribed by RNA polymerase II as part of variable regions correspond to those of a non-human capped and polyadenylated primary transcripts (pri-miR immunoglobulin and all or substantially all of Ab framework NAS) that can be either protein-coding or non-coding. The regions are those of a human immunoglobulin sequence. primary transcript is cleaved by the Drosha ribonuclease III 0259. In alternative embodiments, a humanized antibody enzyme to produce an approximately 70-nt stem-loop precur used to practice this invention can comprise at least a portion sor miRNA (pre-miRNA), which is further cleaved by the of an immunoglobulin constant region (Fc), typically that of cytoplasmic Dicer ribonuclease to generate the mature or derived from a human immunoglobulin. miRNA and antisense miRNA star (miRNA) products. The 0260. However, in alternative embodiments, completely mature miRNA is incorporated into a RNA-induced silencing human antibodies also can be used to practice this invention, complex (RISC), which recognizes target mRNAs through including human antibodies comprising amino acid sequence imperfect base pairing with the miRNA and most commonly which corresponds to that of an antibody produced by a results in translational inhibition or destabilization of the human. This definition of a human antibody specifically target mRNA. excludes a humanized antibody comprising non-human anti 0265. In alternative embodiments pharmaceutical compo gen binding residues. sitions used to practice the invention are administered in the 0261. In alternative embodiments, antibodies used to prac form of a dosage unit, e.g., a tablet, capsule, bolus, spray. In tice this invention comprise “affinity matured antibodies, alternative embodiments, pharmaceutical compositions com e.g., antibodies comprising with one or more alterations in prise a compound, e.g., an antisense nucleic acid, e.g., an US 2016/0146783 A1 May 26, 2016

siRNA or a microRNA, in a dose: e.g., 25 mg, 30 mg, 35 mg. activity at all). In certain embodiments, the modified anti 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 sense nucleic acid, siRNA or microRNA constructs have mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg. improved stability in serum and/or cerebral spinal fluid com 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg. pared to an unmodified structure having the same sequence. 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 0269. In alternative embodiments, a modification includes 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg. a 2-H or 2'-modified ribose sugar at the second nucleotide 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg. from the 5'-end of the guide sequence. In alternative embodi 260 mg, 265 mg, 270 mg, 270 mg, 280 mg, 285 mg, 290 mg. ments, the guide Strand (e.g., at least one of the two single 295 mg, 300 mg. 305 mg. 310 mg, 315 mg, 320 mg. 325 mg. stranded polynucleotides) comprises a 2'-O-alkyl or 2'-halo 330 mg, 335 mg, 340 mg, 345 mg, 350 mg, 355 mg, 360 mg. group, such as a 2'-O-methyl modified nucleotide, at the 365 mg, 370 mg, 375 mg, 380 mg. 385 mg,390 mg. 395 mg. second nucleotide on the 5'-end of the guide Strand, or, no 400 mg. 405 mg, 410 mg, 415 mg, 420 mg, 425 mg. 430 mg. other modified nucleotides. In alternative embodiments, 435 mg. 440 mg. 445 mg, 450 mg. 455 mg, 460 mg. 465 mg. polynucleotide constructs having Such modification may 470 mg. 475 mg, 480 mg, 485 mg. 490 mg. 495 mg, 500 mg. have enhanced target specificity or reduced off-target silenc 505 mg, 510 mg, 515 mg, 520 mg, 525 mg, 530 mg, 535 mg. ing compared to a similar construct without the 2'-O-methyl 540 mg, 545 mg, 550 mg, 555 mg, 560 mg, 565 mg, 570 mg. modification at the position. 575 mg, 580 mg, 585 mg, 590 mg, 595 mg, 600 mg. 605 mg. 0270. In alternative embodiments, a second nucleotide is a 610 mg. 615 mg. 620 mg. 625 mg, 630 mg, 635 mg, 640 mg. second nucleotide from the 5'-end of the single-stranded 645 mg, 650 mg. 655 mg. 660 mg. 665 mg, 670 mg. 675 mg. polynucleotide. In alternative embodiments, a '2'-modified 680 mg, 685 mg. 690 mg. 695 mg, 700 mg, 705 mg, 710 mg. ribose Sugar comprises ribose Sugars that do not have a 715 mg, 720 mg, 725 mg, 730 mg, 735 mg, 740 mg, 745 mg. 2'-OH group. In alternative embodiments, a '2'-modified 750 mg, 755 mg, 760 mg, 765 mg, 770 mg, 775 mg, 780 mg. ribose sugar does not include 2'-deoxyribose (found in 785 mg, 790 mg, 795 mg. or 800 mg or more. unmodified canonical DNA nucleotides), although one or 0266. In alternative embodiments, an siRNA or a more DNA nucleotides may be included in the subject con microRNA used to practice the invention is administered as a structs (e.g., a single deoxyribonucleotide, or more than one pharmaceutical agent, e.g., a sterile formulation, e.g., a lyo deoxyribonucleotide in a stretch or scattered in several parts philized siRNA or microRNA that is reconstituted with a of the subject constructs). For example, the 2'-modified ribose Suitable diluent, e.g., Sterile water for injection or sterile sugar may be 2'-O-alkyl nucleotides. 2'-deoxy-2'-fluoro saline for injection. In alternative embodiments the reconsti nucleotides. 2'-deoxynucleotides, or combination thereof. tuted product is administered as a Subcutaneous injection or 0271. In alternative embodiments, an antisense nucleic as an intravenous infusion after dilution into Saline. In alter acid, siRNA or microRNA construct used to practice the native embodiments the lyophilized drug product comprises invention comprises one or more 5'-end modifications, e.g., as siRNA or microRNA prepared in water for injection, or in described above, and can exhibit a significantly (e.g., at least saline for injection, adjusted to pH 7.0-9.0 with acid or base about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%. 65%, during preparation, and then lyophilized. In alternative 70%, 75%, 80%, 85%, 90% or more) less “off-target' gene embodiments a lyophilized siRNA or microRNA of the silencing when compared to similar constructs without the invention is between about 25 to 800 or more mg, or about 25, specified 5'-end modification, thus greatly improving the 50, 75, 100, 125, 150, 175, 200,225, 250,275,300,325,350, overall specificity of the antisense nucleic acid, siRNA or 375, 425,450, 475,500, 525,550, 575, 600,625, 650, 675, microRNA construct of the invention. 700, 725,750, 775, and 800 mg of a siRNA or microRNA of 0272. In alternative embodiments, an antisense nucleic the invention. The lyophilized siRNA or microRNA of the acid, siRNA or microRNA construct to practice the invention invention can be packaged in a 2 mL. Type I, clear glass vial comprises a guide Strand modification that further increase (e.g., ammonium sulfate-treated), e.g., stoppered with a bro stability to nucleases, and/or lowers interferon induction, mobutyl rubber closure and sealed with an aluminum over without significantly decreasing activity (or no decrease in seal. microRNA activity at all). In alternative embodiments, the 0267 In alternative embodiments, the invention provides 5'-stem sequence comprises a 2'-modified ribose Sugar, Such compositions and methods comprising in vivo delivery of as 2'-O-methyl modified nucleotide, at the second nucleotide antisense nucleic acids, e.g., siRNA or microRNAS. In prac on the 5'-end of the polynucleotide, or, no other modified ticing the invention, the antisense nucleic acids, siRNAS, or nucleotides. In alternative embodiments the hairpin structure microRNAs can be modified, e.g., in alternative embodi having Such modification has enhanced target specificity or ments, at least one nucleotide of antisense nucleic acid, e.g., reduced off-target silencing compared to a similar construct siRNA or microRNA, construct is modified, e.g., to improve without the 2'-O-methyl modification at same position. its resistance to nucleases, serum stability, target specificity, 0273. In alternative embodiments, the 2'-modified nucle blood system circulation, tissue distribution, tissue penetra otides are some or all of the pyrimidine nucleotides (e.g., tion, cellular uptake, potency, and/or cell-permeability of the C/U). Examples of 2'-O-alkyl nucleotides include a 2'-O- polynucleotide. In alternative embodiments, the antisense methyl nucleotide, or a 2'-O-allyl nucleotide. In alternative nucleic acid, siRNA or microRNA construct is unmodified. In embodiments, the modification comprises a 2'-O-methyl other embodiments, at least one nucleotide in the antisense modification at alternative nucleotides, starting from either nucleic acid, siRNA or microRNA construct is modified. the first or the second nucleotide from the 5'-end. In alterna 0268. In alternative embodiments, guide strand modifica tive embodiments, the modification comprises a 2'-O-methyl tions are made to increase nuclease stability, and/or lower modification of one or more randomly selected pyrimidine interferon induction, without significantly decreasing anti nucleotides (C or U). In alternative embodiments, the modi sense nucleic acid, siRNA or microRNA activity (or no fication comprises a 2'-O-methyl modification of one or more decrease in antisense nucleic acid, siRNA or microRNA nucleotides within the loop. US 2016/0146783 A1 May 26, 2016 36

0274. In alternative embodiments, the modified nucle embodiments, a linked biradical group is selected from otides are modified on the Sugar moiety, the base, and/or the phosphodiester linkage. In alternative embodiments the modification comprise a phosphate analog, or a phospho rothioate linkage; and the phosphorothioate linkage can be where each R1 and R is, independently, H., hydroxyl, C1 to limited to one or more nucleotides within the loop, a 5'-over Calkyl, substituted C1-C12 alkyl, C-C12 alkenyl, substi hang, and/or a 3'-overhang. tuted C-C alkenyl, C-C alkynyl. Substituted C-C 12 0275. In alternative embodiments, the phosphorothioate alkynyl, C-C20 aryl, substituted C-C20 aryl, a heterocycle linkage may be limited to one or more nucleotides within the radical, a Substituted heterocycle radical, heteroaryl, Substi loop, and 1, 2, 3, 4, 5, or 6 more nucleotide(s) of the guide tuted heteroaryl, C-C, alicyclic radical, Substituted C-C, sequence within the double-stranded stem region just 5' to the alicyclic radical, halogen, Substituted oxy ( O—), amino, loop. In alternative embodiments, the total number of nucle Substituted amino, azido, carboxyl. Substituted carboxyl, otides having the phosphorothioate linkage may be about acyl, substituted acyl, CN, thiol, substituted thiol, sulfonyl 12-14. In alternative embodiments, all nucleotides having the (S(=O). H), substituted sulfonyl, sulfoxyl (S(=O)—H) phosphorothioate linkage are not contiguous. In alternative or Substituted Sulfoxyl; and each Substituent group is, inde embodiments, the modification comprises a 2'-O-methyl pendently, halogen, C1-C alkyl, substituted C1-C alkyl, modification, or, no more than 4 consecutive nucleotides are C-C alkenyl, Substituted C-C alkenyl, C-C alkynyl, modified. In alternative embodiments, all nucleotides in the Substituted C-C alkynyl, amino, Substituted amino, acyl, 3'-end stem region are modified. In alternative embodiments, Substituted acyl, C1-C aminoalkyl, C1-C aminoalkoxy, all nucleotides 3' to the loop are modified. Substituted C1-C aminoalkyl, Substituted C1-C ami 0276. In alternative embodiments, the 5'- or 3'-stem noalkoxy or a protecting group. sequence comprises one or more universal base-pairing 0281. In alternative embodiments, the bicyclic sugar moi nucleotides. In alternative embodiments universal base-pair ety is bridged between the 2' and 4' carbon atoms with a ing nucleotides include extendable nucleotides that can be biradical group selected from —O—(CH)X-, —O—CH2—, incorporated into a polynucleotide Strand (either by chemical —O—CH2CH2—, —O CH(alkyl)-, - NH-(CH2)P . synthesis or by a polymerase), and pair with more than one —N(alkyl)-(CH)x-, - O -CH(alkyl)-, —(CH(alkyl)- pairing type of specific canonical nucleotide. In alternative (CH2)x-, - NH-O-(CH2)x-, - N(alkyl)-O-(CH2)x-, or embodiments, the universal nucleotidespair with any specific —O N(alkyl)-(CH2)x-, wherein X is 1, 2, 3, 4 or 5 and each nucleotide. In alternative embodiments, the universal nucle alkyl group can be further substituted. In certain embodi otides pair with four pairings types of specific nucleotides or ments, X is 1, 2 or 3. analogs thereof. In alternative embodiments, the universal 0282. In alternative embodiments, a 2'-modified nucleo nucleotides pair with three pairings types of specific nucle side comprises a 2-substituent group selected from halo, otides or analogs thereof. In alternative embodiments, the allyl, amino, azido, SH, CN, OCN, CF. OCF. O. , S: , or universal nucleotides pair with two pairings types of specific N(Rm)-alkyl; O – S , or N(Rm)-alkenyl; O – S or nucleotides or analogs thereof. N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, 0277. In alternative embodiments, an antisense nucleic aralkyl, O-alkaryl, O-aralkyl, O(CH2)SCH, O—(CH)— acid, siRNA or microRNA used to practice the invention O N(Rm)(Rn) or O CH2-C(=O) N(Rm)(Rn), where comprises a modified nucleoside, e.g., a Sugar-modified each Rm and Rn is, independently, H, an amino protecting nucleoside. In alternative embodiments, the Sugar-modified group or substituted or unsubstituted C1-C10 alkyl. These nucleosides can further comprise a natural or modified het 2-substituent groups can be further substituted with one or erocyclic base moiety and/or a natural or modified inter more substituent groups independently selected from nucleoside linkage; or can comprise modifications indepen hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro dent from the Sugar modification. In alternative (NO), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alk embodiments, a Sugar modified nucleoside is a 2'-modified enyl and alkynyl. nucleoside, wherein the Sugar ring is modified at the 2 carbon from natural ribose or 2'-deoxy-ribose. 0283. In alternative embodiments, a 2'-modified nucleo 0278. In alternative embodiments, a 2'-modified nucleo side comprises a 2-substituent group selected from F, side has a bicyclic Sugar moiety. In certain such embodi O CH, and OCHCH2OCH. ments, the bicyclic Sugar moiety is a D Sugar in the alpha 0284. In alternative embodiments, a sugar-modified configuration. In certain Such embodiments, the bicyclic nucleoside is a 4'-thio modified nucleoside. In alternative Sugar moiety is a D Sugar in the beta configuration. In certain embodiments, a Sugar-modified nucleoside is a 4'-thio-2'- Such embodiments, the bicyclic Sugar moiety is an L Sugar in modified nucleoside. In alternative embodiments a 4'-thio the alpha configuration. In alternative embodiments, the bicy modified nucleoside has a beta.-D-ribonucleoside where the clic Sugar moiety is an L Sugar in the beta configuration. 4'-O replaced with 4-S. A 4'-thio-2'-modified nucleoside is a 0279. In alternative embodiments, the bicyclic sugar moi 4'-thio modified nucleoside having the 2'-OH replaced with a ety comprises a bridge group between the 2' and the 4-carbon 2-substituent group. In alternative embodiments 2'-substitu atoms. In alternative embodiments, the bridge group com ent groups include 2'-OCH, 2'-O-(CH2)-OCH, and 2'-F. prises from 1 to 8 linked biradical groups. In alternative 0285. In alternative embodiments, a modified oligonucle embodiments, the bicyclic Sugar moiety comprises from 1 to otide of the present invention comprises one or more inter 4 linked biradical groups. In alternative embodiments, the nucleoside modifications. In alternative embodiments, each bicyclic Sugar moiety comprises 2 or 3 linked biradical internucleoside linkage of a modified oligonucleotide is a groups. modified internucleoside linkage. In alternative embodi 0280. In alternative embodiments, the bicyclic sugar moi ments, a modified internucleoside linkage comprises a phos ety comprises 2 linked biradical groups. In alternative phorus atom. US 2016/0146783 A1 May 26, 2016 37

0286. In alternative embodiments, a modified antisense amount sufficient to cure, alleviate or partially arrest the nucleic acid, siRNA or microRNA comprises at least one clinical manifestations of the condition, infection or disease phosphorothioate internucleoside linkage. In certain embodi (e.g., disease or condition associated with dysfunctional stem ments, each internucleoside linkage of a modified oligonucle cells or cancer stem cells) and its complications (a “therapeu otide is a phosphorothioate internucleoside linkage. tically effective amount'). In the methods of the invention, a 0287. In alternative embodiments, a modified internucleo pharmaceutical composition is administered in an amount side linkage does not comprise a phosphorus atom. In alter Sufficient to treat (e.g., ameliorate) or prevent a disease or native embodiments, an internucleoside linkage is formed by condition associated with dysfunctional stem cells or cancer a short chain alkyl internucleoside linkage. In alternative stem cells. The amount of pharmaceutical composition embodiments, an internucleoside linkage is formed by a adequate to accomplish this is defined as a “therapeutically cycloalkyl internucleoside linkages. In alternative embodi effective dose.” The dosage schedule and amounts effective ments, an internucleoside linkage is formed by a mixed het for this use, i.e., the “dosing regimen will depend upon a eroatom and alkyl internucleoside linkage. In alternative variety of factors, including the stage of the disease or con embodiments, an internucleoside linkage is formed by a dition, the severity of the disease or condition, the general mixed heteroatom and cycloalkyl internucleoside linkages. In state of the patient's health, the patient’s physical status, age alternative embodiments, an internucleoside linkage is and the like. In calculating the dosage regimen for a patient, formed by one or more short chain heteroatomic internucleo the mode of administration also is taken into consideration. side linkages. In alternative embodiments, an internucleoside linkage is formed by one or more heterocyclic internucleo Kits and Instructions side linkages. In alternative embodiments, an internucleoside linkage has an amide backbone, oran internucleoside linkage 0292. The invention provides kits comprising composi has mixed N, O, S and CH2 component parts. tions for practicing the methods of the invention, including 0288. In alternative embodiments, a modified oligonucle instructions for use thereof. In alternative embodiments, the otide comprises one or more modified nucleobases. In certain invention provides kits, blister packages, lidded blisters or embodiments, a modified oligonucleotide comprises one or blister cards or packets, clamshells, trays or shrink wraps more 5-methylcytosines, or each cytosine of a modified oli comprising a combination of compounds. In alternative gonucleotide comprises a 5-methylcytosine. embodiments, the combination of compounds comprises: 0289. In alternative embodiments, a modified nucleobase 0293 (1) at least one compound comprising or consisting comprises a 5-hydroxymethyl cytosine, 7-deazaguanine or of: 7-deaZaadenine, or a modified nucleobase comprises a 0294 (i) an inhibitor or depleter of integrin C, B (anb3), 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine or a oran inhibitor of integrin O?3 (anb3) proteinactivity, or 2-pyridone, or a modified nucleobase comprises a 5-substi an inhibitor of the formation or activity of an integrin tuted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 anb3/RalB signaling complex, or an inhibitor of the Substituted purines, or a 2 aminopropyladenine, 5-propyny formation or signaling activity of an integrin CfB luracil or a 5-propynylcytosine. (anb3)/RalB/NFkB signaling axis, 0290. In alternative embodiments, a modified nucleobase 0295 wherein optionally the inhibitor of integrin CfB comprises a polycyclic heterocycle, or atricyclic heterocycle; protein activity is an allosteric inhibitor of integrin CfB or, a modified nucleobase comprises a phenoxazine deriva protein activity; tive, or a phenoxazine further modified to form a nucleobase 0296 (ii) an inhibitor or depleter of RalB protein or an or G-clamp. inhibitor of RalB protein activation, 0297 wherein optionally the inhibitor of RalB protein Therapeutically Effective Amount and Doses activity is an allosteric inhibitor of RalB protein activity; 0298 (iii) an inhibitor or depleter of Src or a Tank 0291. In alternative embodiment, compounds, composi Binding Kinase (TBK1) protein or an inhibitor of Src or tions, pharmaceutical compositions and formulations used to TBK1 protein activation, practice the invention can be administered for prophylactic 0299 wherein optionally the inhibitor of the Src or the and/or therapeutic treatments; for example, the invention pro TBK1 protein activity is an amlexanox (or 2-amino-7- vides compositions and methods for overcoming or dimin isopropyl-5-oxo-5H-chromeno2,3-bipyridine-3-car ishing or preventing Growth Factor Inhibitor (GFI) resistance boxylic acid) or APHTHASOLTM, in a cell, or, a method for increasing the growth-inhibiting 0300 and optionally the inhibitor of the Src or the effectiveness of a Growth Factor inhibitor on a cell, or, a TBK1 protein activity is an allosteric inhibitor of Src or method for re-sensitizing a cell to a Growth Factor Inhibitor. TBK1 protein activity; In alternative embodiments, the invention provides composi tions and methods for treating, preventing or ameliorating: a 0301 (iv) an inhibitor or depleter of NFKB or IRF3 disease or condition associated with dysfunctional stem cells protein or an inhibitor of RalB protein activation, or cancer stem cells, a retinal age-related macular degenera 0302 wherein optionally the inhibitor of NFKB or tion, a diabetic retinopathy, a cancer or carcinoma, a glioblas IRF3 protein activity is an allosteric inhibitor of NFKB toma, a neuroma, a neuroblastoma, a colon carcinoma, a or IRF3 protein activity; or hemangioma, an infection and/or a condition with at least one 0303 (v) any combination of (i) to (iv); and inflammatory component, and/or any infectious or inflamma 0304 (2) at least one Growth Factor Inhibitor. tory disease, Such as a rheumatoid arthritis, a psoriasis, a 0305. In alternative embodiments, the kit further com fibrosis, leprosy, multiple Sclerosis, inflammatory bowel dis prises instructions for practicing a method of the invention. ease, or ulcerative colitis or Crohn's disease. In therapeutic 0306 The invention will be further described with refer applications, compositions are administered to a subject ence to the following examples; however, it is to be under already Suffering from a condition, infection or disease in an stood that the invention is not limited to such examples. US 2016/0146783 A1 May 26, 2016 38

EXAMPLES colon (SW480) human tumor cell lines to increasing concen trations of erlotinib or lapatinib for three weeks, to select cell Example 1 subpopulations that were at least 10-fold more resistant to these targeted therapies than their parental counterparts. Par Methods of the Invention are Effective for ent or resistant cells were then evaluated for a panel of stem/ Sensitizing and Re-Sensitizing Cancer Cells to progenitor cell markers previously identified to be upregu Growth Factor Inhibitors lated in the most aggressive metastatic tumor cells''. 0307 The data presented herein demonstrates the effec tiveness of the compositions and methods of the invention in 0313 As expected, the expression of some of these mark sensitizing and re-sensitizing cancer cells, and cancer stem ers was significantly increased in one or more of these resis cells, to growth factor inhibitors, and validates this inven tant cell populations. Surprisingly, we observed that CD61 tions therapeutic approach to overcome growth factor inhibi (integrin B3) was the one marker upregulated in all resistant tor, e.g., EGFR inhibitor, resistance for a wide range of can cell lines tested, FIG. 1A. The longer cells were exposed to cers. The data presented herein demonstrates that genetic and erlotinib the greater the expression level of C.VfB3 was pharmacological inhibition of RalB or NF-kB was able to observed, FIG. 1B. These findings were extended in vivo as re-sensitize CVB3-expressing tumors to EGFR inhibitors. mice bearing orthotopic FG pancreatic tumors with minimal 0308 Resistance to epidermal growth factor receptor integrin CVB3 evaluated following four weeks of erlotinib (EGFR) inhibitors has emerged as a significant clinical prob treatment showed a 10-fold increase in CVB3 expression, FIG. lem in oncology owing to various resistance mechanisms". 1C. Moreover, H441 human lung adenocarcinoma orthotopic Since cancer stem cells have been associated with drug resis tumors' exposed to systemic erlotinib treatment in vivo for tance, we examined the expression of stem/progenitor cell 7-8 weeks developed resistance and a qualitative increase in markers for breast, pancreas and colon tumor cells with integrin CVB3 expression compared with vehicle-treated acquired resistance to EGFR inhibitors. We found that CD61 tumors, see FIG. 1D and FIG. 5 (Supplementary FIG. 1). (B3 integrin) was the one marker consistently upregulated on Thus, exposure of histologically distinct tumor cells in vitro EGFR inhibitor resistant tumor cells. Moreover, integrin or in vivo to EGFR inhibitors selects for a tumor cell popu CVB3 expression was markedly enhanced in murine orthoto lation expressing high levels of CVB3. pic lung and pancreas tumors following their acquired resis tance to systemically delivered EGFR inhibitors. In fact, 0314. In addition to being expressed on a subpopulation of CVB3 was both necessary and sufficient to account for the stem/progenitor cells during mammary development', C.VfB3 tumor cell resistance to EGFR inhibitors and other growth is a marker of the most malignant tumor cells in a wide range factor receptor inhibitors but not cytotoxic drugs. of cancers'''. To determine whetherendogenous expression 0309 Mechanistically, in drug resistant tumors CVB3 of integrin C.VfB3 might predict tumor cell resistance to EGFR forms a complex with KRAS via the adaptor Galectin-3 blockade, various breast, lung and pancreatic tumor cells resulting in recruitment of RalB and activation of its effector were first screened for CVB3 expression and then analyzed for TBK1/NF-kB, revealing a previously undescribed integrin their sensitivity to EGFR inhibitors (Supplementary Table 1). mediated pathway. Accordingly, genetic or pharmacological inhibition of Galectin-3, RalB or NF-kB was able to re TABLE 1 sensitize CVB3-expressing tumors to EGFR inhibitors, dem KRAS mutation, integrin CVB3 expression and onstrating the effectiveness of the compositions and methods EGFRTKI sensitivity of cancer cell lines of the invention and validating this inventions therapeutic approach to overcome EGFR inhibitor resistance for a wide Mutated integrin Civ3 EGFRTKI range of cancers. Cell line Origin KRAS expression sensitive 0310. Despite some level of clinical success achieved with PANC-1 pancreas yes yes O FG pancreas yes O yes EGFR Tyrosine Kinase inhibitors (TKIs), intrinsic and Mapaca-2 (MP2) pancreas yes O yes acquired cellular resistance mechanisms limit their efficacy' CAPAN-1 pancreas yes O yes 2.4. A number of resistance mechanisms have been identified, XPA-1 pancreas O O yes including KRAS and EGFR mutations, resulting in constitu CFPAC-1 pancreas yes yes O A549 lung yes yes O tive activation of the ERK pathway 7. While KRAS-medi SKBR3 breast O O yes ated ERK signaling is associated with resistance to EGFR MDAMB231 breast yes yes O inhibition, KRAS also induces PI3K and Ral activation lead MDAMB468 breast O O yes ing to tumor cell survival and proliferation. (MDA468) BT474 breast O O yes 0311. Nevertheless, it is clear that treatment of tumors BT2O breast O yes O with EGFR inhibitors appears to select for a cell population T47D breast yes O yes that remains insensitive to EGFR blockade'. Prolonged SW48O colon yes O yes 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 0315. In all cases, B3 expressing tumor cells were intrin associated with increased cell survival and metastasis'. sically more resistant to EGFR blockade than 33-negative While a number of EGFR-inhibitor resistance mechanisms tumor cell lines (FIG. 1E). In fact, CVB3 was required for have been defined, it is not clear whether a single unifying resistance to EGFR inhibitors, since knockdown of CVB3 in mechanism might drive the resistance of a broad range of PANC-1 cells resulted in a 10-fold increase in tumor cell CaCCS. sensitivity to erlotinib (FIG. 1F). Moreover, integrin CVB3 0312 To investigate this, we exposed pancreatic (FG, was sufficient to induce erlotinib resistance since ectopic Miapaca-2), breast (BT474, SKBR3 and MDAMB468) and expression of CVB3 in FG cells lacking this integrin dramati US 2016/0146783 A1 May 26, 2016 39 cally increased erlotinib resistance both, in vitro and in ortho 0319. KRAS promotes multiple effector pathways includ topic pancreatic tumors after systemic treatment in vivo ing those regulated by RAF, phosphatidylinositol-3-OH (FIGS. 1F and G). kinases (PI3Ks) and RalGEFs leading to a variety of cellular functions. To investigate whether one or more KRAS effec 0316 Integrin CVB3 not only promotes adhesion-depen tor pathway(s) may contribute to integrin B3/KRAS-medi dent signaling via activation of focal adhesion kinase FAK' ated tumor cell resistance to EGFR inhibitors, we individu but it can also activate a FAK-independent signaling cascade ally knocked-down or inhibited each downstream RAS in the absence of integrin ligation that is associated with effector in cells expressing or lacking integrin C.VfB3. While increased survival and tumor metastasis''. To determine suppression of AKT, ERK and RalA sensitized tumor cells to whether CVB3 ligation was required for its causative role in erlotinib, regardless of the CVB3 expression status, see FIG.9 erlotinib resistance, FG cells transfected with either WTB3 or (Supplementary FIG. 5), knockdown of RalB selectively sen a ligation deficient mutant of the integrin (D119A)'7 were sitized CVB3 expressing tumor cells to erlotinib, see FIG. 7A treated witherlotinib. The same degree of erlotinib resistance and FIG. 10A (Supplementary FIG. 6A). This was relevant to was observed in cells expressing either the ligation competent pancreatic tumor growth in Vivo since, knockdown of RalB or incompetent form of integrin O.VfB3, see FIG. 6A) (Supple re-sensitized CLV33-expressing pancreatic orthotopic tumors mentary FIG. 2a) indicating that expression of CVB3, even in to erlotinib in mice, see FIG. 7B. In fact, expression of a the unligated State, was sufficient to induce tumor cell resis constitutively active RalB (G23V) mutant in B3-negative tance to erlotinib. cells was sufficient to confer resistance to EGFR inhibition, 0317 Tumor cells with acquired resistance to one drug can see FIG.7C and FIG. 10B (Supplementary FIG. 6b). Further often display resistance to a wide range of drugs''. There more, ectopic expression of CVB3 enhanced RalB activity in fore, we examined whether CVB3 expression also promotes tumor cells in a KRAS-dependent manner, see FIG. 7D). resistance to other growth factor inhibitors and/or cytotoxic Accordingly, integrin CVB3 and RalB were co-localized in agents. Interestingly, while CVB3 expression accounted for tumor cells, see FIG. 10C (Supplementary FIG. 7) and in EGFR inhibitor resistance, it also induced resistance to the human breast and pancreatic cancer biopsies, see FIG. 11 IGFR inhibitor OSI-906, yet failed to protect cells from the (Supplementary FIG. 8) and a strong correlation was found antimetabolite agent gemcitabine and the chemotherapeutic between C.VfB3 expression and Ral GTPase activity in patients agent cisplatin, see FIG. 6B and FIG. 6C (Supplementary biopsies suggesting the CVB3/RalB signaling module is clini FIGS.2b and c). These results demonstrate that integrin CVB3 cally relevant, see FIG. 7E. Together, these findings indicate accounts for tumor cell resistance to drugs that target growth that integrin CVB3 promotes erlotinib resistance of cancer factor receptor mediated pathways but does not promote for a cells by complexing with KRAS and RalB resulting in RalB more general resistant phenotype to all drugs, particularly activation. those that induce cell cytotoxicity. 0320 RalB, an effector of RAS has been shown to induce 0318. In some cases oncogenic KRAS has been associated TBK1/NF-kB activation leading to enhanced tumor cell sur with EGFR TKIs resistance', however, it remains unclear vival. In addition, it has been shown that NF-kB signal whether oncogenic KRAS is a prerequisite for EGFR resis ing is essential for KRAS-driven tumor growth and resistance tance'. Thus, we examined the KRAS mutational status in to EGFR blockade’’’. This prompted us to ask whether various tumor cell lines and found that KRAS oncogenic CVB3 could regulate NF-KB activity through RalB activation status did not account for resistance to EGFR inhibitors and thereby promote tumor cell resistance to EGFR targeted (Supplementary Table 1). Nevertheless, knockdown of therapy. To test this, tumor cells expressing or lacking integrin KRAS in CVB3 expressing cells rendered them sensitive to CVB3 and/or RalB were grown in the presence or absence of erlotinib while KRAS knockdown in cells lacking CVB3 had erlotinib and lysates of these cells were analyzed for activated no such effect, see FIG. 6A and FIG. 6B, indicating that CVB3 downstream effectors of RalB. We found that erlotinib treat and KRAS function cooperatively to promote tumor cell ment of CVB3 negative cells reduced levels of phosphorylated resistance to erlotinib. Interestingly, even in non-adherent TBK1 and NF-KB, whereas in B3-positive cells these effec cells, CVB3 colocalized with oncogenic KRAS in the plasma tors remained activated unless RalB was depleted, see FIG. membrane (FIG. 2C) and could be co-precipitated in a com 4A. NF-kB activity was sufficient to account for EGFR plex with KRAS, see FIG. 6D. This interaction was specific inhibitor resistance since ectopically expressed a constitu for KRAS, as C.VfB3 was not found to associate with N-, R- or tively active NF-kB (S276D) in B3-negative FG pancreatic HRAS isoforms in these cells, see FIG. 6D and FIG. 7A and tumor cells' conferred resistance to EGFR inhibition, see FIG. 7B (Supplementary FIGS. 3a and b). Furthermore, in FIG. 4B). Accordingly, genetic orpharmacological inhibition BXPC3 human pancreatic tumor cells expressing wildtype of NF-kB in B3-positive cells completely restored erlotinib KRAS, CVB3 showed increased association with KRAS only sensitivity, see FIGS. 4C and D). These findings demon after these cells were stimulated with EGF, see FIG. 6E. strate that RalB, the effector of the CVB3/KRAS complex, Previous studies have indicated that the KRAS interacting promotes tumor cell resistance to EGFR targeted therapy via protein Galectin-3 can also couple to integrins’’. There TBK1/NF-kB activation. Together, our studies describe a role fore, we considered whether Galectin-3 might serve as an for C.VfB3 mediating resistance to EGFR inhibition via RalB adaptor facilitating an interaction between CVB3 and KRAS activation and its downstream effector NF-KB, opening new in epithelial tumor cells. In PANC-1 cells with endogenous B3 avenues to target tumors that are resistant to EGFR targeted expression, CVB3, KRAS, and Galectin-3 co-localized to therapy, see FIG. 4E. membrane clusters, see FIG. 8A and FIG. 8B (Supplementary 0321 Recent studies have shown that, upon prolonged FIG. 4a–b). Furthermore, knockdown of either B3 or Galec treatment with EGFR inhibitors, tumor cells develop alterna tin-3 prevented the localization of KRAS to these membrane tive or compensatory pathways to Sustain cell Survival, lead clusters or their co-immunoprecipitation, see FIG. 8 (Supple ing to drug resistance'''. Here we show that integrin C.VfB3 is mentary FIG. 4). specifically upregulated in histologically distinct tumors US 2016/0146783 A1 May 26, 2016 40 where it accounts for resistance to EGFR inhibition. At or treated with EGF 50 ng/ml for 5 minutes. RAS activity was present, it is not clear whether exposure to EGFR inhibitors determined using a GST-Rafl-RBD immunoprecipitation may promote increased CVB3 expression or whether these assay. Data are representative of three independent experi drugs simply eliminate cells lacking CVB3 allowing the mentS. expansion of CVB3-expressing tumor cells. Given that inte 0326 FIG. 3. RalB is a key modulator of integrin C.VfB3 grin CIVfB3 is a marker of mammary stem cells', it is possible mediated EGFRTKI resistance. that acquired resistance to EGFR inhibitors selects for a 0327 (a) Tumor spheres formation assay of FG-B3 treated tumor stem-like cell population. While integrins can pro with non-silencing (shCTRL) or RalB-specific shRNA and mote adhesion dependent cell Survival and induce tumor pro exposed to a dose response of erlotinib. Error bars represent gression', here, we show that integrin C.VfB3, even in the S.d. (n-3 independent experiments). Immunoblot analysis unligated State, can drive tumor cell Survival and resistance to showing RalB knockdown. (b) Effects of depletion of RalB EGFR blockade by interaction with KRAS. This action leads on erlotinib sensitivity in B3-positive tumor in a pancreatic to the recruitment and activation of RalB and its downstream orthotopic tumor model. Established B3-positive tumors signaling effector NF-KB. In fact, NF-kB inhibition re-sensi expressing non-silencing (shCTRL) or RalB-specific shRNA tizes CVB3-bearing tumors to EGFR blockade. Taken (>1000 mm; n=13 per treatment group) were randomized together, our findings not only identify CVB3 as a tumor cell and treated for 10 days witherlotinib. Results are expressed as marker of drug resistance but reveal that inhibitors of EGFR % of tumor weight changes after erlotinib treatment com and NF-KB should provide synergistic activity against a pared to control. *P<0.05, **P<0.01. Tumor images, average broad range of cancers. weights+/-S.e are shown. (c) Tumor spheres formation assay of FG cells ectopically expressing vector control, WT RalB FIGURE LEGENDS FLAG tagged constructs or a constitutively active RalB 0322 FIG. 1. Integrin O.VfB3 expression promotes resis G23V FLAG tagged treated with erlotinib (0.5 M). Error tance to EGFRTKI. bars represents.d. (n=3 independent experiments). *P-0.05, 0323 (a) Flow cytometric quantification of cell surface NS=not significant. Immunoblot analysis showing RalBWT markers after 3 weeks treatment with erlotinib (pancreatic and RalB G23 FLAG tagged constructs transfection effi and colon cancer cells) or lapatinib (breast cancer cells). (b) ciency. (d) RalB activity was determined in FG, FG-f33 Flow cytometric analysis of CVB3 expression in FG and Mia expressing non-silencing or KRAS-specific shRNA, by using paca-2 cells followingerlotinib. Error bars represents.d. (n=3 a GST-RalBP1-RBD immunoprecipitation assay as independent experiments). (c) Top, immunofluorescence described in Methods. Data are representative of three inde staining of integrin CVB3 in tissue specimens obtained from pendent experiments. (e) Right, overall active Ral immuno orthotopic pancreatic tumors treated with vehicle (n-3) or histochemical staining intensity between 33 negative (n=15) erlotinib (n=4). Scale bar, 50 Lum. Bottom, Integrin CVB3 and B3 positive (n=70) human tumors. Active Ral staining expression was quantified as ratio of integrin CVB3 pixel area was compared between each group by Fisher's exact test over nuclei pixel area using Metamorph (P=0.049 using (*P-0.05, P=0.036, two-sided). Left, representative immu Mann-Whitney Utest). (d) Right, intensity (scale 0 to 3) of B3 nohistochemistry images of human tumor tissues stained with expression in mouse orthotopic lung tumors treated with an integrin B3-specific antibody and an active Ral antibody. vehicle (n=8) or erlotinib (n=7). Left, immunohistochemical Scale bar, 50 lum. staining of B3. Scale bar, 100 p.m. (*P=0.0012 using Mann 0328 FIG. 4. Integrin C.VfB3/RalB complex leads to NF Whitney U test) (e) ICs for cells treated with erlotinib or uB activation and resistance to EGFRTKI. lapatinib. (O Tumor sphere formation assay to establish a 0329 Immunoblot analysis of FG, FG-B3 and FG-B3 sta dose-response for erlotinib. Error bars represent S.d. (n=3 bly expressing non-silencing or RalB-specific ShRNA, independent experiments). (g) Orthotopic FG tumors (>1000 grown in suspension and treated with erlotinib (0.5 LM). mm. n=10 per treatment group) were treated for 10 days with pTBK1 refers to phospho-S172TBK1, p-p65 NF-kB refers to vehicle or erlotinib. Results are expressed as % tumor weight phospho-p65 NF-KBS276, pFAKrefers to phospho-FAKTyr compared to vehicle control. *P<0.05. Immunoblot analysis 861. Data are representative of three independent experi for tumor lysates after 10 days of erlotinib confirms sup ments. (b) Tumor spheres formation assay of FG cells ectopi pressed EGFR phosphorylation. cally expressing vector control, WTNF-kB FLAG tagged or 0324 FIG. 2. Integrin CVB3 cooperates with KRAS to constitutively active S276D NF-KB FLAG tagged constructs promote resistance to EGFR blockade. treated with erlotinib (0.5uM). Error bars represents.d. (n=3 0325 (a-b) Tumor sphere formation assay of FG express independent experiments).*P<0.05, **P<0.001, NS=not sig ing (a) or lacking (b) integrin B3 depleted of KRAS (sh nificant. Immunoblot analysis showing NF-kB WT and KRAS) or not (shCTRL) and treated with a dose response of S276D NF-kB FLAG transfection efficiency. (c) Tumor erlotinib. Error bars represents.d. (n=3 independent experi spheresformation assay of FG-33 treating with non-silencing ments). (c) Confocal microscopy images of PANC-1 and (shCTRL) or NF-kB-specific shRNA and exposed to erlo FG-B3 cells grown in suspension. Cells are stained for inte tinib (0.5 LM). Error bars represent S.d. (n=3 independent grin C.VfB3 (green), KRAS (red), and DNA (TOPRO-3, blue). experiments). *P-0.05, NS=not significant. (d) Dose Scale bar, 10 . Data are representative of three indepen response in FG-B3 cells treated with erlotinib (10 nM to 5 dent experiments. (d) RAS activity assay performed in uM), lenalidomide (10 nM to 5 LM) or a combination of PANC-1 cells using GST-Raf1-RBD immunoprecipitation as erlotinib (10 nM to 5 uM) and lenalidomide (1 uM). Error described in Methods. Immunoblot analysis of KRAS, bars represents.d. (n=3 independent experiments). *P-0.05, NRAS, HRAS, RRAS, integrin B1 and integrin B3. Data are NS=not significant. (e) Model depicting the integrin C.VfB3 representative of three independent experiments. (e) Immu mediated EGFR TKI resistance and conquering EGFRTKI noblot analysis of Integrin CVB3 immunoprecipitates from resistance pathway and its downstream RalB and NF-KB BxPC-3 B3-positive cells grown in suspension and untreated effectors. US 2016/0146783 A1 May 26, 2016

Methods active Ral (NewEast) diluted 1:100 and 1:200 in blocking 0330 Compounds and Cell Culture. Solution. Tissue sections were washed and then incubated 0331 Human pancreatic (FG, PANC-1, Miapaca-2 with biotinylated secondary antibody (1:500, Jackson Immu (MP2), CFPAC-1, XPA-1, CAPAN-1, BxBc3), breast noResearch) in blocking solution for 1 h. Sections were (MDAMB231, MDAMB468 (MDA468), BT20, SKBR3, washed and incubated with Vectastain ABC (Vector Labs) for BT474), colon (SW480) and lung (A549, H441) cancer cell 30 min. Staining was developed using a Nickel-enhanced lines were grown in ATCC recommended media Supple diamino-benzidine reaction (Vector Labs) and sections were mented with 10% fetal bovine serum, glutamine and non counter-stained with hematoxylin. Sections stained with inte essential amino acids. We obtained FG-33, FG-D119A grin B3 and active Ral were scored by a H-score according to mutant and PANC-shf3 cells as previously described'. Erlo the staining intensity (SI) on a scale 0 to 3 within the whole tinib, OSI-906, Gemcitabine and Lapatinib were purchased tissue section. from Chemietek. Cisplatin was generated from Sigma-Ald 0340 Immunoprecipitation and Immunoblot Analysis. rich. Lenalidomide was purchased from LC Laboratories. We (0341 Cells were lysed in either RIPA lysis buffer (50 mM established acquired EGFRTKI resistant cells by adding an Tris pH 7.4, 100 mM NaCL, 2 mM EDTA, 10% DOC, 10% increasing concentration of erlotinib (50 nM to 15 uM) or Triton, 0.1% SDS) or Triton lysis buffer (50 mM Tris pH 7.5, lapatinib (10 nM to 15 uM), daily in 3D culture in 0.8% 150 mN NaCl, 1 mM EDTA, 5 mM MgCl2, 10% Glycerol, methylcellulose. 1% Triton) supplemented with complete protease and phos 0332 Lentiviral Studies and Transfection. phatase inhibitor mixtures (Roche) and centrifuged at 13,000 0333 Cells were transfected with vector control, WT. gfor 10 min at 4°C. Protein concentration was determined by G23V RalB-FLAG, WT and S276D NF-kB-FLAG using a BCA assay. 500 ug to 1 mg of protein were immunoprecipi lentiviral system. For knock-down experiments, cells were tated with 3 ug of anti-integrin CVB-3 (LM609) overnight at transfected with KRAS, RalA, RalB, AKT1, ERK1/2, p.65 4°C. following by capture with 25ul of protein A/G (Pierce). NF-kB siRNA (Qiagen) using the lipofectamine reagent (In Beads were washed five times, eluted in Laemmli buffer, vitrogen) following manufacturer's protocol or transfected resolved on NuPAGE 4-12% Bis-Tris Gel (Invitrogen) and with shRNA (Open Biosystems) using a lentiviral system. immunoblotting was performed with anti-integrin B3 (Santa Gene silencing was confirmed by immunoblots analysis. Cruz), anti-RalB (Cell Signaling Technology), anti KRAS 0334] Tumor Sphere Formation. (Santa Cruz). For immunoblot analysis, 25 ug of protein was 0335 Tumor spheres formation assays were performed boiled in Laemmli buffer and resolved on 8% to 15% gel. The essentially as described previously '7. Briefly, cells were following antibodies were used: KRAS (Santa Cruz), NRAS seeded at 1000 to 2000 cells per well and grown for 12 days (Santa Cruz), RRAS (Santa Cruz), HRAS (Santa Cruz), phos to 3 weeks. Cells were treated with vehicle (DMSO), erlotinib pho-S172 NAK/TBK1 (Epitomics), TBK1 (Cell Signaling (10 nM to 5 uM), lapatinib (10 nM to 5 LM), gemcitabine Technology), phospho-p65NF-KB S276 (Cell Signaling (0.001 nM to 5uM), OSI-906 (10 nM to 5uM), lenalidomide Technology), p.65NF-kB (Cell Signaling Technology), RalB (10 nM to 5 uM), or cisplatin (10 nM to 5 uM), diluted in (Cell Signaling Technology), phospho-EGFR (Cell Signaling DMSO. The media was replaced with fresh inhibitor every Technology), EGFR (Cell Signaling Technology), FLAG day for erlotinib, lapatinib, lenalidomide and 3 times a week (Sigma), phospho-FAKTyr861 (Cell Signaling Technology), for cisplatin and gemcitabine. Colonies were stained with FAK (Santa Cruz), Galectin 3 (BioLegend) and Hsp90 (Santa crystal violet and scored with an Olympus SZH10 micro Cruz). scope. Survival curves were generated at least with five con 0342 Affinity Pull-Down Assays for Ras and Ral. centration points. 0343 RAS and Ral activation assays were performed in 0336 Flow Cytometry. accordance with the manufacturers (Upstate) instruction. 0337 200,000 cells, after drug or vehicle treatment, were Briefly, cells were cultured in suspension for 3 h, lysed and washed with PBS and incubated for 20 minutes with the protein concentration was determined. 10 ug of Ral Assay Live/Dead reagent (Invitrogen) according to the manufactur Reagent (Ral BP1, agarose) or RAS assay reagent (Raf-1 er's instruction, then, cells were fixed with 4% paraformal RBD, agarose) was added to 500 mg to 1 mg of total cell dehyde for 15 min and blocked for 30 min with 2% BSA in protein in MLB buffer (Millipore). After 30 min of rocking at PBS. Cells were stained with fluorescent-conjugated antibod 4°C., the activated (GTP) forms of RAS/Ral bound to the ies to CD61 (LM609), CD44 (eBioscience), CD24 (eBio agarose beads were collected by centrifugation, washed, science), CD34 (eBioscience), CD133 (Santa Cruz), CD56 boiled in Laemmli buffer, and loaded on a 15% SDS-PAGE (eBioscience), CD29 (P4C10) and CD49f (eBioscience). All gel. antibodies were used at 1:100 dilutions, 30 minutes at 4°C. 0344 Immunofluorescence Microscopy. Afterwashing several times with PBS, cells were analyzed by 0345 Frozen sections from tumors from the orthotopic FACS. Xenograft pancreas cancer mouse model or from patients 0338 Immunohistochemical Analysis. diagnosed with pancreas or breast cancers (as approved by the 0339 Immunostaining was performed according to the institutional Review Board at University of California, San manufacturer's recommendations (Vector Labs) on 5uM sec Diego) or tumor cell lines were fixed in cold acetone or 4% tions of paraffin-embedded tumors from the orthotopic paraformaldehyde for 15 min, permeabilized in PBS contain Xenograft pancreas and lung cancer mouse models or from a ing 0.1% Triton for 2 min and blocked for 1 h at room metastasis tissue array purchased from US Biomax temperature with 2% BSA in PBS. Cells were stained with (MET961). Antigen retrieval was performed in citrate buffer antibodies to integrin CVB3 (LM609), RalB (Cell Signaling pH 6.0 at 95°C. for 20 min. Sections were treated with 0.3% Technology), Galectin 3 (BioLegend), pFAK (Cell Signaling HO for 30 min, blocked in normal goat serum, PBS-T for 30 Technology), NRAS (Santa Cruz), RRAS (Santa Cruz), min followed by Avidin-D and then incubated overnight at 4 HRAS (Santa Cruz) and KRAS (Abgent). All primary anti C. with primary antibodies against integrin B3 (Abcam) and bodies were used at 1:100 dilutions, overnight at 4°C. Where US 2016/0146783 A1 May 26, 2016 42 mouse antibodies were used on mouse tissues, we used the 0358 7. Zoppoli, G., et al. Ras-induced resistance to lapa MOM kit (Vector Laboratory). After washing several times tinib is overcome by MEK inhibition. Current Cancer Drug with PBS, cells were stained for two hours at 4° C. with Targets 10, 168-175 (2010). secondary antibodies specific for mouse or rabbit (Invitro 0359 8. Gupta, S., et al. Binding of ras to phosphoi gen), as appropriate, diluted 1:200 and co-incubated with the nositide 3-kinase p110alpha is required for ras-driven tum DNA dye TOPRO-3 (1:500) (Invitrogen). Samples were origenesis in mice. Cell 129,957-968 (2007). mounted in VECTASHIELD hard-set media (Vector Labora 0360 9. Lim, K. H., et al. Activation of RalA is critical for tories) and imaged on a Nikon Eclipse C1 confocal micro Ras-induced tumorigenesis of human cells. Cancer Cell 7. Scope with 1.4 NA 60x oil-immersion lens, using minimum 533-545 (2005). pinhole (30 ). Images were captured using 3.50 imaging 0361 10. Sharma, S. V., et al. A chromatin-mediated software. Colocalization between Integrin CVB3 and KRAS reversible drug-tolerant state in cancer cell Subpopulations. was studied using the Zenon Antibody Labeling Kits (Invit Cell 141, 69-80 (2010). rogen). 0362 11. Liu, C., et al. The microRNA miR-34a inhibits 0346 Orthotopic Pancreas Cancer Xenograft Model. prostate cancer stem cells and metastasis by directly 0347 All mouse experiments were carried out in accor repressing CD44. Nature Medicine 17, 211-215 (2011). dance with approved protocols from the UCSD animal sub 0363 12. Vaillant, F., et al. The mammary progenitor jects committee and with the guidelines set forth in the NIH marker CD61/beta3 integrin identifies cancer stem cells in Guide for the Care and Use of Laboratory Animals. Tumors mouse models of mammary tumorigenesis. Cancer were generated by injection of FG human pancreatic carci Research 68,7711-7717 (2008). noma cells (10° tumor cells in 30 uL of sterile PBS) into the 0364 13. Adhikari, A. S. Agarwal, N. & Iwakuma, T. tail of the pancreas of 6-8 week old male immune compro Metastatic potential of tumor-initiating cells in Solid mised nu/nu mice. Tumors were established for 2-3 weeks tumors. Front Biosci 16, 1927-1938 (2011). (tumor sizes were monitored by ultrasound) before beginning 0365. 14. Cascone, T., et al. Unregulated stromal EGFR dosing. Mice were dosed by oral gavage with vehicle (6% and vascular remodeling in mouse Xenograft models of Captisol) or 100 mg/kg/day erlotinib for 10 to 30 days prior to angiogenesis inhibitor-resistant human lung adenocarci harvest. noma. J Clin Invest 121, 1313-1328 (2011). 0348 Orthotopic Lung Cancer Xenograft Model. 0366 15. Asselin-Labat, M.L., et al. Gata-3 is an essential 0349 Tumors were generated by injection of H441 human regulator of mammary-gland morphogenesis and luminal lung adenocarcinoma cells (10° tumor cells per mouse in 50 cell differentiation. Nature Cell Biology 9, 201-209 uL of HBSS containing 50 mg growth factor-reduced Matri (2007). gel (BD Bioscience) into the left thorax at the lateral dorsal 0367. 16. Desgrosellier, J. S. & Cheresh, D. A. Integrins in axillary line and into the left lung, as previously described' cancer: biological implications and therapeutic opportuni of 8 week old male immune-compromised nu/nu mice. 3 ties. Nature Reviews 10, 9-22 (2010). weeks after tumor cell injection, the mice were treated with 0368 17. Desgrosellier, J. S., et al. An integrin alpha(v) vehicle or erlotinib (100 mg/kg/day) by oral gavage until beta(3)-c-Src oncogenic unit promotes anchorage-inde moribund (approximately 50 and 58 days, respectively). pendence and tumor progression. Nature Medicine 15, 0350 Statistical Analyses. 1163-1169 (2009). 0351 All statistical analyses were performed using Prism 0369. 18. Borst, P., Jonkers, J. & Rottenberg, S. What software (GraphPad). Two-tailed Mann Whitney U tests, makes tumors multidrug resistant? Cell Cycle 6, 2782 Fisher's exact tests, ort-tests were used to calculate statistical 2787 (2007). significance. AP values 0.05 was considered to be significant. 0370. 19. Schmitt, C. A., et al. A senescence program controlled by p53 and p16INK4a contributes to the out REFERENCES come of cancer therapy. Cell 109, 335-346 (2002). 0371. 20. Baselga, J. & Rosen, N. Determinants of RASis Example 1 tance to anti-epidermal growth factor receptor agents. J 0352 1. Wheeler, D. L., Dunn, E. F. & Harari, P. M. Clin Oncol 26, 1582-1584 (2008). Understanding resistance to EGFR inhibitors-impact on 0372. 21. Moore, M. J., et al. Erlotinib plus gemcitabine future treatment strategies. Nat Rev Clin Oncol 7,493-507 compared with gemcitabine alone in patients with (2010). advanced pancreatic cancer: a phase III trial of the National 0353 2. Dorans, K. Outpacing cancer. Nature Medicine Cancer Institute of Canada Clinical Trials Group. J. Clin 15,718–722 (2009). Oncol 25, 1960-1966 (2007). 0354 3. Dean, M., Fojo, T. & Bates, S. Tumour stem cells 0373). 22. Levy, R. Grafi-Cohen, M., Kraiem, Z. & Kloog, and drug resistance. Nature Reviews 5, 275-284 (2005). Y. Galectin-3 promotes chronic activation of K-Ras and 0355 4. Engelman, J. A. & Janne, P. A. Mechanisms of differentiation block in malignant thyroid carcinomas. acquired resistance to epidermal growth factor receptor Molecular Cancer Therapeutics 9, 2208-2219 (2010). tyrosine kinase inhibitors in non-small cell lung cancer. 0374) 23. Markowska, A. I., Liu, F. T. & Panjwani, N. Clin Cancer Res 14, 2895-2899 (2008). Galectin-3 is an important mediator of VEGF- and bFGF 0356 5. Montagut, C., et al. Identification of a mutation in mediated angiogenic response. The Journal of Experimen the extracellular domain of the Epidermal Growth Factor tal Medicine 207, 1981-1993 (2010). Receptor conferring cetuximab resistance in colorectal 0375. 24. Buday, L. & Downward, J. Many faces of Ras cancer. Nature Medicine 18, 221-223 (2012). activation. Biochim Biophys Acta 1786, 178-187 (2008). 0357 6. Sharma, S.V., Bell, D.W., Settleman, J. & Haber, 0376) 25. Barbie, D. A., et al. Systematic RNA interfer D. A. Epidermal growth factor receptor mutations in lung ence reveals that oncogenic KRAS-driven cancers require cancer. Nature Reviews 7, 169-181 (2007). TBK1. Nature 462, 108-112 (2009). US 2016/0146783 A1 May 26, 2016 43

0377 26. Chien, Y., et al. RalB GTPase-mediated activa resistance. To identify genes associated with erlotinib (N-(3- tion of the IkappaB family kinase TBK1 couples innate ethynylphenyl)-6,7-bis(2-methoxyethoxy) quinazolin-4- immune signaling to tumor cell survival. Cell 127, 157-170 amine) resistance, we analyzed the expression of a tumor (2006). progression gene array for human cell lines with intrinsic 0378. 27. Ling, J., et al. Kras(G12D)-Induced IKK2/beta/ resistance or murine Xenografts following the acquisition of NF-kappaB Activation by IL-1 alpha and p62 Feedforward resistance in vivo. The most upregulated gene common to all Loops Is Required for Development of Pancreatic Ductal drug resistant carcinomas tested was the cell surface ITGB3, Adenocarcinoma. Cancer Cell 21, 105-120 (2012). integrin (FIG. 1A, and table S1) associated with the integrin 0379 28. Bivona, T.G., et al. FAS and NF-kappaB signal CVB3 whose expression has been linked to tumor progression. ling modulate dependence of lung cancers on mutant CVB3 expression completely predicted erlotinib resistance for EGFR. Nature 471, 523-526 (2011). a panel of histologically distinct tumor cell lines (FIG. 1 Band 0380 29. Min, J., et al. An oncogene-tumor suppressor FIG.S1B). Moreover, chronic treatment of the erlotinib sen cascade drives metastatic prostate cancer by coordinately sitive lines resulted in the induction of B3 expression con activating Ras and nuclear factor-kappaB. Nature Medi comitantly with drug resistance (FIG. 1C and FIG.S1B, C). cine 16, 286-294 (2010). We also detected increased B3 expression in lung carcinoma 0381 30. Dong, J., Jimi, E. Zeiss, C., Hayden, M. S. & patients who had progressed on erlotinib therapy (FIG.S2). In Ghosh, S. Constitutively active NF-kappaB triggers sys addition, we examined both treatment naive and erlotinib temic TNFalpha-dependent inflammation and localized resistant NSCLC patients from the BATTLE Study (10) of TNFalpha-independent inflammatory disease. Genes & non-small cell lung cancer (NSCLC) and found B3 gene Development 24, 1709-1717 (2011). expression was significantly higher in patients who pro 0382. 31. Braun, T., et al. Targeting NF-kappaB in hema gressed on erlotinib (FIG. 1D). Finally, we examined serial tologic malignancies. Cell Death Differ 13, 748-758 primary lung tumors biopsies from patients before treatment (2006). or after erlotinib resistance and found a qualitative increase in 0383 32. Workman, P. & Clarke, P. A. Resisting targeted integrin expression concurrent with the loss of erlotinib sen therapy: fifty ways to leave your EGFR. Cancer Cell 19, sitivity (FIG. 1E). Taken together, our findings show that 437-440 (2011). integrin B3 is a marker of acquired and intrinsic erlotinib 0384 33. Lewis, M. T. & Wicha, M. S. Tumor-initiating resistance for pancreas and lung cancer. cells and treatment resistance: how goes the war? Journal 0388 To assess the functional role of C.VfB3 in erlotinib of Mammary Gland Biology and Neoplasia 14, 1-2 (2009). resistance we used again and loss-of-function approach and found that integrin B3 was both necessary and sufficient to Example 2 account for erlotinib resistance in vitro and during systemic treatment of lung and orthotopic pancreatic tumors in vivo Methods of the Invention are Effective for (FIG. 1F, G and FIG. S3A-C). Interestingly, integrin B3 Sensitizing and Re-Sensitizing Cancer Cells to expression did not impact resistance to chemotherapeutic Growth Factor Inhibitors agents such as gemcitabine and cisplatin while conferring 0385. The data presented herein demonstrates the effec resistance to inhibitors targeting EGFR1/EGFR2 or IGFR tiveness of the compositions and methods of the invention in (FIG.S3C-E), Suggesting this integrin plays a specific role in sensitizing and re-sensitizing cancer cells, and cancer stem tumor cell resistance to RTK inhibitors. cells, to growth factor inhibitors, and validates this inven 0389. As integrin CVB3 is functions as an adhesion recep tions therapeutic approach to overcome growth factor inhibi tor, ligand binding inhibitors could represent a therapeutic tor resistance for a wide range of cancers. In particular, the strategy to sensitize tumors to EGFR inhibitors. However, data presented in this Example demonstrates that B3 integrin CVB3 expression induced drug resistance in cells growing in induces erlotinib resistance in cancer cells by Switching Suspension. Also, neither function blocking antibodies nor tumor dependency from EGFR to KRAS. cyclic peptide inhibitors sensitized integrin CVB3-expressing 0386. In alternative embodiments, the compositions and tumors to EGFR inhibitors (not shown), and tumor cells methods of the invention overcome tumor drug resistance that expressing wild-type integrin B3 or the ligation-deficient limits the long-term success of therapies targeting EGFR. mutant f83 D119A (11) showed equivalent drug resistance Here, we identify integrin C.VfB3 as a biomarker of intrinsic (FIG.S4). Since the contribution of integrin CVB3 to erlotinib and acquired resistance to erlotinib in human pancreatic and resistance appears to involve a non-canonical, ligation-inde lung carcinomas irrespective of their KRAS mutational sta pendent mechanism that is not sensitive to traditional integrin tus. Functionally, CVB3 is necessary and sufficient for this antagonists, understanding the molecular mechanisms driv resistance where it acts in the unligated State as a scaffold to ing this pathway could provide therapeutic opportunities. recruit active KRAS into membrane clusters switching tumor 0390 Integrins function in the context of RAS family dependency from EGFR to KRAS. The KRAS effector RalB members. Interestingly, we found that CVB3 associated with is recruited to this complex, where it mediates erlotinib resis KRAS but not N-, H- or R-RAS (FIG. 2A). While oncogenic tance via a TBK-1/NF-kB pathway. Disrupting assembly of KRAS has been linked to erlotinib resistance, there are many this complex or inhibition of its downstream effectors fully notable exceptions (6-9). In fact, we observed a number of restores tumor sensitivity to EGFR blockade. Our findings tumor cell lines with oncogenic KRAS to be sensitive to uncouple KRAS mutations from erlotinib resistance, reveal erlotinib (FG, H441, and CAPAN1), whereas H1650 cells ing an unexpected requirement for integrin CVB3 in this pro were erlotinib resistant despite their expression of wildtype CCSS, KRAS and mutant EGFR (table S2). In fact, CVB3 expression 0387 We hypothesized that upregulation of specific genes consistently correlated with erlotinib resistance for all cell common to multiple tumor types exposed to erlotinib drives a lines tested (Pearson's correlation coefficient R-0.87) mak conserved pathway that governs both intrinsic and acquired ing a better predictor of erlotinib resistance. Interestingly, we US 2016/0146783 A1 May 26, 2016 44 observed active KRAS to be distributed within the cytoplasm activity leading to erlotinib resistance which can be overcome in B3-negative cells (FIG. S.5A) whereas in cells expressing by a combination of currently approved inhibitors of NF-KB B3 endogenously or ectopically, KRAS was localized to and EGFR. B3-containing membrane clusters, even in the presence of 0393. See also FIG. 40 and FIG. 41, graphically illustrat erlotinib (FIG. 2B,C and FIG. S.5A) a relationship that was ing data demonstrating that depletion of RalB overcomes not observed for B1 integrin (FIGS. S5B and C). Furthermore, erlotinib resistance in KRAS mutant cells, and depletion of knockdown of KRAS impaired tumorsphere formation and TBK1 overcomes erlotinib resistance in KRAS mutant cells, restored erlotinib sensitivity in B3-positive cells (FIG. 2D-F respectively. In FIG. 41: Integrin b3 mediates TBK1 activa and FIG. S.6A-C). In contrast, KRAS was dispensable for tion through RalB and TBK1 depletion overcomes integrin tumorsphere formation and erlotinib response the in cells b3-mediated erlotinib resistance. lacking B3 expression (FIG. 2D-F). Thus, B3 integrin expres 0394 Our observations demonstrate that the ability of B3 integrin to recruit KRAS into a membrane complex along sion switches tumor cell dependency from EGFR to KRAS, with Galectin-3 and RalB functions to switch tumor cell and that the localization of B3 with KRAS at the plasma dependency from EGFR to KRAS. In fact, oncogenic KRAS membrane appears to be a critical determinant of tumor cell requires this non-canonical B3-mediated pathway to drive resistance to erlotinib. Also, our results reveal that tumors erlotinib resistance. We show that currently available expressing oncogenic KRAS without 33 remain sensitive to approved inhibitors of this pathway can be used to practice EGFR blockade. the methods of this invention to treat patients with solid 0391 Independent studies have shown that galectin-3 can tumors, rendering them sensitive to EGFR inhibitors such as interact with either KRAS (12) or B3 (13) so we asked erlotinib. whether this protein might serve as an adaptor to promote KRAS/B3 complex formation. Under anchorage-indepen Material and Methods dent growth conditions, integrin B3, KRAS, and Galectin-3 Compounds and Cell Culture. were co-localized in membrane clusters (FIG. 2G and FIG. S7), and knockdown of either integrin B3 or Galectin-3 pre 0395 Human pancreatic (FG, PANC-1, CFPAC-1, XPA vented complex formation, KRAS membrane localization, 1, HPAFII, CAPAN-1, BXPC3) and lung (A549, H441, and importantly sensitized CVB3 expressing tumors to erlo HCC827 and H1650) cancer cell lines were grown in ATCC tinib (FIG. 2G-I). recommended media supplemented with 10% fetal bovine serum, glutamine and non-essential amino acids. We obtained 0392 We next evaluated the signaling pathways driven by FG-B3, FG-D119A mutant and PANC-shf3 cells as previ the integrin B3/KRAS complex. Erlotinib resistance of ously described (10). Erlotinib, OSI-906, Gemcitabine, Bort B3-positive cells was not affected by depletion of known eZomib and Lapatinib were purchased from Chemietek. Cis KRAS effectors, including AKT, ERK, or RalA (FIG. S8A, platin was generated from Sigma-Aldrich. Lenalidomide was B). However, knockdown of RalB sensitized f3-expressing purchased from LC Laboratories. Gene expression analysis. cells to erlotinib in vitro (FIG. 3A and FIG. S8A-C) and in The Tumor Metastasis PCR Array (Applied Biosystem), con pancreatic orthotopic tumors in vivo (FIG.3B). Accordingly, sisting of 92 genes known to be involved in tumor progression expression of constitutively active RalB in B3-negative cells and metastasis, was used to profile the common genes conferred erlotinib resistance (FIG. 3C). Mechanistically, upregulated in erlotinib-resistant cells compared to erlotinib RalB was recruited to the B3/KRAS membrane clusters (FIG. sensitive cells according to the manufacturers instructions. 3D-F) where it became activated in a KRAS-dependent man Briefly, total RNA was extracted and reverse transcribed into ner (FIG. 3G). Recent studies have reported that TBK1 and cDNA using the RNeasy kit (Qiagen). The cDNA was com NF-kB are RalB effectors linked to KRAS dependency (14) bined with a SYBR Green qPCR Master Mix (Qiagen), and and erlotinib resistance (15). We found that erlotinib then added to each well of the same PCR Array plate that decreased the activation of these effectors only in the absence contained the predispensed gene-specific primer sets. of integrin B3 (FIG.3H). In fact, loss of RalB in B3-express ing cells restored erlotinib-mediated inhibition of TBK1 and Tumor Digestion and Flow Cytometry. NF-kB (FIG.3H). Accordingly, depletion of either TBK1 or 0396 Fresh tumor tissue from lung cancer cell lines was NF-kB sensitized B3-positive cells to erlotinib (FIG. 3I and mechanically dissociated and then enzymatically digested in FIG.S9A), while ectopic expression of activated NF-kB was trypsin. The tissue was further filtered through a cell strainer Sufficient to promote drug resistance in B3-negative cells to obtain a suspension of single tumor cells. Then, cells were (FIG.S9B). To evaluate the therapeutic potential of targeting washed were washed with PBS and incubated for 20 minutes this pathway, we examined whether erlotinib resistance of B3-expressing tumors could be reversed with approved drugs with the Live/Dead reagent (Invitrogen) according to the known to suppress NF-kB activation, lenalidomide/REV manufacturers instruction, then, cells were fixed with 4% LIMIDR (16) and bortezomib/VELCADE(R) (17). While paraformaldehyde for 15 min and blocked for 30 min with 2% monotherapy with these drugs failed to impact tumor growth, BSA in PBS. Cells were stained with fluorescent-conjugated either drug used combination with erlotinib decreased tumor antibodies to integrin CVB3 (LM609, Cheresh Lab). After sphere formation in vitro (FIG. 4A) and completely sup washing several times with PBS, cells were analyzed by pressed tumor growth in vivo (FIG. 4B, C and FIG. S10). FACS These findings support the model depicted in FIG. 4D where Tumorsphere Assay. inhibition of NF-kB restores erlotinib sensitivity in B3 expressing tumors. These findings Support the model 0397 Tumorsphere assay was performed as previously depicted in FIG. 4D that CVB3 expression in lung and pan described (10). Cells were treated with vehicle (DMSO), creatic tumors recruits oncogenic KRAS facilitating NFKB erlotinib (10 nM to 5 LM), lapatinib (10 nM to 5 LM), gem US 2016/0146783 A1 May 26, 2016

citabine (0.001 nM to 5 uM), OSI-906 (10 nM to 5 uM), Immunofluorescence Microscopy. lenalidomide (1 uM), cisplatin (10 nM to 5 uM), or bort 04.01. Frozen sections from tumors from orthotopic pan eZomib (4 nM) diluted in DMSO. The media was replaced creatic tumors, from patients diagnosed with pancreas can with fresh inhibitor 2/6 times a week. Survival curves were cers (as approved by the institutional Review Board at Uni generated at least with five concentration points. versity of California, San Diego) or tumor cell lines were Mouse Cancer Models. processed as previously described (23). Cells were stained with indicated primary, followed by secondary antibodies 0398 All research was conducted under protocol S05018 specific for mouse or rabbit (Invitrogen), as appropriate. and approved by the University of California—San Diego Samples imaged on a Nikon ECLIPSEC1TM confocal micro Institutional Animal Care and Use Committee (IACUC). FG Scope with 1.4 NA 60x oil-immersion lens, using minimum pancreatic carcinoma cells (1x106 tumor cells in 30 ul of pinhole (30 m). The following antibodies were used: anti PBS) were injected into the pancreas of 6-to 8-week-old male integrin B3 (LM609), KRAS (Pierce and Abgent M01), nude mice as previously described (10). Tumors were estab Galectin-3, NRAS, RRAS, lished for 2-3 weeks (tumor sizes were monitored by ultra Sound) before beginning dosing. Mice were dosed by oral Genetic Knockdown and Expression of Mutant Constructs. gavage with vehicle (6% Captisol) or 10, 25 and 50 mg/kg/ day erlotinib for 10 to 30 days prior to harvest. H441 lung 0402 Cells were transfected with vector control, WT. adenocarcinoma cells were generated as previously described G23V RalB-FLAG, WT and S276D NF-kB-FLAG using a (21). 3 weeks after tumor cell injection, the mice were treated lentiviral system. For knock-down experiments, cells were with vehicle or erlotinib (100 mg/kg/day) by oral Mouse transfected with a pool of RalA, RalB, AKT1, ERK1/2 cancer models. All research was conducted under protocol siRNA (Qiagen) using the lipofectamine reagent (Invitrogen) S05018 and approved by the University of California San following manufacturer's protocol or transfected with Diego Institutional Animal Care and Use Committee shRNA (integrin B3, KRAS, Galectin-3, RalB, TBK1 and (IACUC). FG pancreatic carcinoma cells (1x106 tumor cells p65NF-kB) (Open Biosystems) using a lentiviral system. in 30 ul of PBS) were injected into the pancreas of 6-to Gene silencing was confirmed by immunoblots analysis. 8-week-old male nude mice as previously described (10). Tumors were established for 2-3 weeks (tumor sizes were Immunohistochemical Analysis. monitored by ultrasound) before beginning dosing. Mice 0403. Immunostaining was performed according to the were dosed by oral gavage with vehicle (6% Captisol) or 10, manufacturer's recommendations (Vector Labs) on 5 LM sec 25 and 50 mg/kg/day erlotinib for 10 to 30 days prior to tions of paraffin-embedded tumors from tumor biopsies from harvest. H441 lung adenocarcinoma cells were generated as lung cancer patients. Tumor sections were processed as pre previously described (21). 3 weeks after tumor cell injection, viously described (23) using integrin B3 (Abcam clone the mice were treated with vehicle or erlotinib (100 mg/kg/ EP2417Y). Sections stained with integrin B3 were scored by day) by oral gavage until moribund (approximately 50 and 58 a H-score according to the staining intensity (SI) on a scale 0 days, respectively). To generate Subcutaneous tumors, FG to 3 within the whole tissue section. B3, FG-R (after erlotinib resistance) and HCC-827 human carcinoma cells (5x106 tumor cells in 200 ul of PBS) were Immunoprecipitation and Immunoblots. injected subcutaneously to the left or right flank of 6-8-week old female nude mice. Tumors were measured every 2-3 days 0404 Lysates from cell lines and xenograft tumors were with calipers until they were harvested at day 10, 16 or after generated using standard methods and RIPA or Triton buffers. acquired resistance. Immunoprecipitation experiments were performed as previ NSCLC Specimens from the BATTLE Trial. ously described (23) with anti-integrin CVB3 (LM609) or 0399. The BATTLE (Biomarker-integrated Approaches of Galectin-3. For immunoblot analysis, 25 ug of protein was Targeted Therapy for Lung Cancer Elimination) trial was a boiled in Laemmli buffer and resolved on 8% to 15% gel. The randomized phase II, single-center, open-label study in following antibodies were used: anti-integrin B3, KRAS, patients with advanced NSCLC refractory to prior chemo NRAS, RRAS, HRAS, Hsp60 and Hsp90 from Santa Cruz, therapy and included patients with and without prior EGFR phospho-S172 NAK/TBK1 from Epitomics, TBK1, phos inhibitor treatment (12). Patients underwent a tumor new pho-p65NF-kB S276, p.65NF-kB, RalB, phospho-EGFR, biopsy prior to initiating study treatment. The microarray EGFR, from Cell Signaling Technology, and Galectin 3 from analysis of mRNA expression on frozen tumor core biopsies BioLegend. was conducted using the Affymetrix Human Gene 1. STTM platform as previously described (22). Membrane Extract. Serial Biopsies from NSCLC Patients. 04.05 Membrane fraction from FG and FG-B3 grown in 0400 Tumor biopsies from University of California, San suspension in media complemented with 0.1% BSA were Diego (UCSD) Medical Center stage IV non-small cell lung isolated using the MEM-PER membrane extraction kit cancer patients were obtained before erlotinib treatment and (Fisher) according to the manufacturers instructions. Affin 3 patients before and after erlotinib resistance. All biopsies ity pull-down assays for Ras and Ral. RAS and Ral activation are from lung or pleural effusion. Patients 1 had a core biopsy assays were performed in accordance with the manufactur from the primary lung tumor, and Patient 2 and 3 had a fine er's (Upstate) instruction. Briefly, cells were cultured in sus needle biopsy from a pleural effusion. All patients had an pension for 3 h. 10 ug of Ral Assay Reagent (Ral BP1, initial partial response, followed by disease progression after agarose) or RAS assay reagent (Raf-1 RBD, agarose) was 920, 92, and 120 days of erlotinib therapy, respectively. This added to 500 mg to 1 mg of total cell protein in MLB buffer work was approved by the UCSD Institutional Review Board (Millipore). After 30 min of rocking at 40 C, the activated (IRB). (GTP) forms of RAS/Ral bound to the agarose beads were US 2016/0146783 A1 May 26, 2016 46 collected by centrifugation, washed, boiled in Laemmli calize (yellow). Scale bar, 10 Lim. Data are representative of buffer, and loaded on a 15% SDS-PAGE gel. three independent experiments. (D) Effect of KRAS knock down on tumorspheres formation in a panel of lung and Statistical Analyses. pancreatic cancer cells expressing or lacking integrin B3. n=3 0406 All statistical analyses were performed using Prism meanti-SEM. *P<0.05, **P<0.01. (E) Effect of KRAS knock software (GRAPHPADTM). Two-tailed Mann Whitney U down on tumorsphere formation in PANC-1 (KRAS mutant) tests, Chi-squared tests, one way ANOVA tests ort-tests were stably expressing non-target shRNA control (L3-positive) or used to calculate statistical significance. A P values 0.05 was specific-integrin B3 shRNA (B3 negative) in FG (KRAS considered to be significant. mutant) and BXPc3 (KRAS wild-type) stably expressing vec tor control or integrin B3. *n=3; mean+SEM. *P<0.05. FIGURE LEGENDS **P<0.01. (F) Effect of KRAS knockdown on erlotinib resis tance of B3-negative and B3-positive epithelial cancer cell 04.07 FIG. 1 (FIG. 12/31) illustrates data showing that integrin B3 is expressed in EGFR inhibitor resistant tumors lines. Cells were treated with a dose response of erlotinib. and is necessary and sufficient to drive EGFR inhibitor resis n=3; meanti-SEM, *P-0.05, **P<0.01. (G) Confocal micros tance. copy images show immunostaining for integrin B3 (green), 0408 (A) Identification of the most upregulated tumor KRAS (red) and DNA (TOPRO-3, blue) for PANC-1 cells progression genes common to erlotinib resistant carcinomas. expressing non-target shRNA control or Galectin 3-specific (B) Erlotinib ICso in a panel of human carcinoma cell lines shRNA grown in suspension. Scale bar=10 p.m. Data are treated with erlotinib in 3D culture. n=3 independent experi representative of three independent experiments. (H) Top: ments. (C) Percentage of integrin B3 positive cells in parental immunoblot analysis of integrin B3 immunoprecipitates from lines vs. after 3 or 8 weeks treatment with erlotinib. (D) PANC-1 cells expressing non-target shRNA control (CTRL) Quantification of integrin B3 (ITG 33) gene expression in or Galectin-3-specific shRNA (Gal-3). Bottom: immunoblot human lung cancer biopsies from patients from the BATTLE analysis of Galectin-3 immunoprecipitates from PANC-1 Study (18) who were previously treated with an EGFR inhibi cells expressing non-target shRNA control (CTRL) or inte tor and progressed (n=27), versus patients who were EGFR grin B3-specific shRNA (B3). Data are representative of three inhibitor naive (n=39). (*P=0.04 using a Student's t test). (E) independent experiments. (I) Erlotinib dose response of FG Paired human lung cancer biopsies obtained before and after B3 cells expressing a non-target shRNA controlora Galectin erlotinib resistance were immunohistochemically stained for 3-specific shRNA (sh Gal-3). n=3; meantSEM. integrin B3. Scale bar, 50 um. (F) Right, effect of integrin B3 knockdown on erlotinib resistance of 3-positive cells. Cells 0411 FIG. 3 (FIG. 14/31) illustrates data showing that were treated with 0.5uM of erlotinib. Results are normalized RalB is a central player of integrin B3-mediated EGFR inhibi using non-treated cells as controls. n=3; meant SEM. *P-0. tor resistance. 05, **P<0.001. Left, effect of integrin B3 ectopic expression 0412 (A) Effect of RalB knockdown on erlotinib resis on erlotinib resistance in FG and H441 cells. Cells were tance of B3-positive epithelial cancer cell lines. Cells were treated with 0.5 M of erlotinib. n=3; meantSEM. *P<0.05, treated with 0.5uM of erlotinib. n=3; meantSEM, *P<0.05, **P<0.001. (G) Right, effect of integrin B3 knockdown on **P<0.01. (B) Effect of RalB knockdown on erlotinib resis erlotinib resistance in vivo, A549 shCTRL and A549 sh inte tance of B3-positive human pancreatic (FG-B3) orthotopic grin B3 (n=8 per treatment group) were treated with erlotinib tumor Xenografts. Established tumors expressing non-target (25 mg/kg/day) or vehicle during 16 days. Results are shRNA, (shCTRL) or a shRNA targeting RalB (sh RalB) expressed as average of tumor volume at day 16. *P<0.05. (>1000 mm. n=13 per treatment group) were randomized Left, orthotopic FG and FG-?33 tumors (>1000mm: n=5 per and treated for 10 days with vehicle or erlotinib. Results are treatment group) were treated for 30 days with vehicle or expressed as % of tumor weight changes after erlotinib treat erlotinib. Results are expressed as % tumor weight compared ment compared to vehicle. **P<0.01. (C) Effect of expres to vehicle control. P<0.05. sion of a constitutively active Ral G23V mutant on erlotinib 04.09 FIG. 2 (FIG. 13/31) illustrates data showing that response of B3 negative cells. Cells were treated with 0.5uM integrin B3 is required to promote KRAS dependency and oferlotinib. n=3; meaniSEM. *P<0.05. (D) Effect of expres KRAS-mediated EGFR inhibitor resistance. sion of integrin B3 on KRAS and RalB membrane localiza 0410 (A) Confocal microscopy images show immun tion. Data are representative of two independent experiments. ostaining for integrin B3 (green), K-, N-, H-, R-Ras (red), and (E) Ral activity was determined in PANC-1 cells grown in DNA (TOPRO-3, blue) for BXPc3 cells grown in suspension suspension by using a GST-RalBP1-RBD immunoprecipita in media with 10% serum. Arrows indicate clusters where tion assay. Immunoblots indicate RalB activity and associa integrin B3 and KRAS colocalize (yellow). Scale bar, 10um. tion of active RalB with integrin B3. Data are representative of Data are representative of three independent experiments. three independent experiments. (F) Confocal microscopy Erlotinib ICso in a panel of human carcinoma cell lines images of integrin CVB3 (green), RalB (red) and DNA (TO expressing non-target shRNA control or KRAS-specific PRO-3, blue) in tumor biopsies from pancreatic cancer shRNA and treated with erlotinib. n=3 meaniSEM.*P<0.05, patients. Scale bar, 20 p.m. (G) Effect of B3 expression and **P<0.01. (B-C) Confocal microscopy images show immu KRAS expression on RalB activity, measured using a GST nostaining for integrin B3 (green), KRAS (red) and DNA RalBP1-RBD immunoprecipitation assay. Data are represen (Topro-3, blue) for PANC-1 (KRAS mutant) and HCC827 tative of three independent experiments. (H) Immunoblot (KRAS wild-type) after acquired resistance to erlotinib analysis of FG and FG-33 stably expressing non-target (HCC827R) grown in suspension in absence (Vehicle) or in shRNA control or RalB-specific shRNA, grown in suspen presence of erlotinib (0.5 LM and 0.1 uM respectively). sion and treated with erlotinib (0.5uM). Data are representa Arrows indicate clusters where integrin B3 and KRAS colo tive of three independent experiments. (I) Effect of TBK1 and US 2016/0146783 A1 May 26, 2016 47 p65 NFkB on erlotinib resistance of FG-B3 cells. Cells were spheres of 33-negative and 33-positive cells after erlotinib, treated with 0.5 M of erlotinib. n=3; meantSEM. *P<0.05, OSI-906, gemcitabine and cisplatin treatment. (E) Effect of ** P&O.O1. integrin B3 expression on lapatinib, OSI-906, cisplatin and 0413 FIG. 4 (FIG. 15/31) illustrates data showing that gemcitabine n=3; meant-SEM. (F) Viability assay (CelTiter reversal of B3-mediated EGFR inhibitor resistance in onco Glo assay) of FG and FG-B3 cells grown in suspension in genic KRAS model by pharmacological inhibition. media with or without serum. n=2; mean+SEM. *P<0.05. 0414 (A) Effect of NFkB inhibitors on erlotinib response **P&O.O1. of B3-positive cells (FG-B3, PANC-1 and A549). Cells were 0418 Supplementary FIG. S4 (FIG. 19/31) illustrates treated with vehicle, erlotinib (0.5 M), lenalidomide (1-2 Integrin B3-mediated EGFR inhibitor resistance is indepen uM), bortezomib (4 nM) alone or in combination. n=3; dent of its ligand binding. meaniSEM. *P<0.05, *P<0.01. (B) Left, mice bearing sub Effect of ectopic expression of B3 wild-type (FG-B3) or the cutaneous 3-positive tumors (FG-33) were treated with B3 D119A (FG-D119A) ligand binding domain mutant on vehicle, erlotinib (25 mg/kg/day), lenalidomide (25 mg/kg/ erlotinib response. n=3; meaniSEM. Immunoblot showing day) or the combination of erlotinib and lenalidomide. Tumor transfection efficiency of vector control, integrin B3 wild dimensions are reported as the fold change relative to size of type and integrin B3 D119A. the same tumor on Day 1. Mean-SEM, (A)*P=0.042 using a 0419 Supplementary FIG. S5 (FIG. 20/31) illustrates one way ANOVA test. n=6 mice per group. Right, mice bear Integrin 133 colocalizes and interacts with oncogenic and ing Subcutaneous B3-positive tumors (FG-R) after acquired active wild-type KRAS. resistance to erlotinib were treated with vehicle, erlotinib (25 (A) Confocal microscopy images of FG and FG-B3 cells mg/kg/day), bortezomib (0.25 mg/kg), the combination of grown in Suspension in media 10% serum with or without erlotinib and bortezomib. Tumor dimensions are reported as erlotinib (0.5uM) and stained for KRAS (red), integrin CVB3 the fold change relative to size of the same tumor on Day 1. (green) and DNA (TOPRO-3, blue). Scale bar, 10um. Data *P=0.0134 using a one way ANOVA test. n=8 mice per group. are representative of three independent experiments. (B) Ras (C) Model depicting the proposed integrin C.VfB3-mediated activity was determined in PANC-1 cells grown in suspension KRAS dependency and EGFR inhibitor resistance mecha by using a GST-Rafl-RBD immunoprecipitation assay. 1S. Immunoblots indicate KRAS activity and association of 0415 Supplementary FIG. S1 (FIG. 16/31) illustrates active KRAS with integrin B3. Data are representative of resistance to EGFR inhibitor is associated with integrin B3 three independent experiments. (C) Immunoblot analysis of expression in pancreatic and lung human carcinoma cell Integrin CVB3 immunoprecipitates from BXPC-3 cells grown lines. (A) Immunoblots showing integrin B3 expression in in Suspension in presence or absence of growth factors. human cell lines used in FIG. 1A and FIG. 1B. (B) Effect of 0420 Supplementary FIG. S6 (FIG. 21/31) illustrates erlotinib on HCC827 xenograft tumors in immuno-compro Integrin B3 expression promotes KRAS dependency. mised mice (n=5 mice per treatment group) relative to (A) Immunoblots showing KRAS knockdown efficiency in vehicle-treated control tumors. Representative Integrin B3 cells used in FIG. 2. (B) Representative photographs of crys cell surface quantification in HCC827 treated with vehicle or tal violet-stained tumorspheres of FG and A549 cells express erlotinib during 64 days. (C) Integrin CVB3 quantification in ing non-target shRNA control or specific-KRAS shRNA. (C) orthotopic lung and pancreas tumors treated with vehicle or Effect of an additional KRAS knockdown on tumorspheres erlotinib until resistance. For lung cancer, integrin 33 expres formation in PANC-1 stably expressing non-target shRNA sion was scored (scale 0 to 3) and representative images are control (B3-positive) or specific-integrin B3 shRNA (B3 nega shown. For pancreatic cancer, integrin B3 expression was tive). n=3; mean+SEM. *P-0.05. Immunoblots showing quantified as ratio of integrin CVB3 pixel area over nuclei KRAS knockdown efficiency. pixel area using METAMORPHTM (**P=0.0012, *P=0.049 using Mann-Whitney U test). Representative immunofluo 0421 Supplementary FIG. S7 (FIG. 22/31) illustrates rescent staining of integrin CVB3 in pancreatic human KRAS and Galectin-3 colocalize in integrin B3-positive cells. xenografts treated 4 weeks with vehicle or erlotinib. Confocal microscopy images of FG and FG-f33 cells grown in 0416) Supplementary FIG. S2 (FIG. 17/31) illustrates Suspension and stained for KRAS (green), galectin-3 (red) Integrin B3 expression predicts intrinsic resistance to EGFR and DNA (TOPRO-3, blue). Scale bar, 10 p.m. Data are inhibitors in tumors. Plot of progression-free survival for representative of three independent experiments. erlotinib-treated patients with low vs. high protein expression 0422 Supplementary FIG. S8 (FIG. 23/31) illustrates of B3 integrin measured from non-Small cell lung cancer Integrin B3-mediated KRAS dependency and erlotinib resis biopsy material obtained at diagnosis (P=0.0122, using tance is independent of ERK, AKT and RalA. Mann-Whitney U test). Representative images showing (A) Effect of ERK, AKT, RalA and RalB knockdown on immunohistochemical staining for B3 integrin (brown) are erlotinib response (erlotinib 0.5 LM) of 33-negative FG and shown. B3-positive FG-B3 cells. n=triplicate. (B) Immunoblots 0417 Supplementary FIG. S3 (FIG. 18/31) illustrates showing ERK, AKT RalA and RalB knockdown efficiency. Integrin B3 confers Receptor Tyrosine Kinase inhibitor resis (C) Immunoblots showing RalB knockdown efficiency in tance. (A) Immunoblots showing integrin B3 knockdown effi cells used in FIG. 3. ciency in cells used in FIG. 1. (B) Response of A549 lung 0423 Supplementary FIG. S9 (FIG. 24/31) illustrates carcinoma cells non-target shRNA control or shRNA target Constitutive active NFkB is sufficient to promote erlotinib ing integrin B3 to treatment with either vehicle or erlotinib (25 resistance. mg/kg/day) during 16 days. Tumor Volumes are expressed as (A) Immunoblots showing TBK1 and NFkB knockdown effi meant-SEM. n=8 mice per group. (C) Immunoblots showing ciency used in FIG.3. (B) Effect of constitutive active S276D expression of indicated proteins of representative tumors. (D) p65NFkB on erlotinib response (erlotinib 0.5 M) of Representative photographs of crystal violet-stained tumor |B3-negative cells (FG cells). n=3; mean+SEM. *P<0.05. US 2016/0146783 A1 May 26, 2016 48

0424 Supplementary FIG. S10 (FIG. 25/31) illustrates 0437 11. J. S. Desgrosellier et al., An integrin alpha(v) NFkB inhibitors in combination with erlotinib increase cell beta(3)-c-Src oncogenic unit promotes anchorage-inde death in vivo. pendence and tumor progression. Nature medicine 15, (A-B) Immunoblots showing expression of indicated proteins 1163 (October, 2009). of representative tumors from shown in FIG. 4B (C) Confocal 0438 12. A.U. Newlaczyl, L. G. Yu, Galectin-3 a jack microscopy images of cleaved caspase 3 (red) and DNA of-all-trades in cancer. Cancer letters 313, 123 (Dec. 27. (TOPRO-3, blue) in tumor biopsies from xenografts tumors 2011). used in FIG. 4B treated with vehicle, erlotinib, lenalidomide 0439 13. A. I. Markowska, F.T. Liu, N. Panjwani, Galec or lenalidomide and erlotinib in combo. Scale bar, 20 Lum. (D) tin-3 is an important mediator of VEGF- and bFGF-medi Confocal microscopy images of cleaved caspase 3 (red) and ated angiogenic response. The Journal of experimental DNA (TOPRO-3, blue) in tumor biopsies from xenografts medicine 207, 1981 (Aug. 30, 2010). tumors used in FIG. 4B treated with vehicle, erlotinib, bort 0440 14. D. A. Barbie et al., Systematic RNA interference eZomib or bortezomib and erlotinib in combo. reveals that oncogenic KRAS-driven cancers require 0425 Supplementary Table 1: shows differentially TBK1. Nature 462, 108 (Nov. 5, 2009). expressed genes in cells resistant to erlotinib (PANC-1, 0441 15. Y. Chien et al., RalB GTPase-mediated activa H1650, A459) compared with the average of two sensitive tion of the IkappaB family kinase TBK1 couples innate cells (FG, H441) and in HCC827 after acquired resistance in immune signaling to tumor cell survival. Cell 127, 157 vivo (HCC827R) vs. the HCC827 vehicle-treated control. (Oct. 6, 2006). The genes upregulated more than 2.5 fold are in red. 0442. 16.Y.Yang et al., Exploiting Synthetic Lethality for 0426 Supplementary Table 2: shows KRAS mutational the Therapy of ABC Diffuse Large B Cell Lymphoma. status of the pancreatic and lung cancer cell lines used in this Cancer Cell 21, 723 (Jun. 12, 2012). study. 0443) 17. M. S. Kumar et al., The GATA2 transcriptional network is requisite for RAS oncogene-driven non-Small REFERENCES cell lung cancer. Cell 149, 642 (Apr. 27, 2012). 0444, 18. E. S. Kim et al., The BATTLE Trial: Personal Example 2 izing Therapy for Lung Cancer. Cancer discovery, (Apr. 3, 2011, 2011). 0427 1. R. J. Gillies, D. Verduzco, R. A. Gatenby, Evolu tionary dynamics of carcinogenesis and why targeted Example 3 therapy does not work. Nature reviews. Cancer 12, 487 (July, 2012). A B3 Integrin/KRAS Complex Shift Tumor 0428 2. S. Zhang et al., Combating trastuzumab resis Phenotype Toward Stemness tance by targeting SRC, a common node downstream of multiple resistance pathways. Nature medicine 17, 461 0445. The data presented herein demonstrates the effec (April, 2011). tiveness of the compositions and methods of the invention in reversing tumor initiation and self-renewal, and resensitizing 0429. 3. J. S. Duncan et al., Dynamic reprogramming of tumors to Receptor Tyrosine Kinase (RTK) inhibition. the kinome in response to targeted MEK inhibition in 0446 Integrin C.VfB3 expression is a marker of tumor pro triple-negative breast cancer. Cell 149,307 (Apr. 13, 2012). gression for a wide range of histologically distinct cancers', 0430 4. D. L. Wheeler, E. F. Dunn, P. M. Harari, Under yet the molecular mechanism by which CVB3 influences the standing resistance to EGFR inhibitors-impact on future growth and malignancy of cancer is poorly understood. Here, treatment strategies. Nature reviews 7, 493 (September, we reveal that integrin CVB3, in the unligated state, is both 2010). necessary and Sufficient to promote tumor initiation and self 0431 5. F. Ciardiello, G. Tortora, EGFR antagonists in renewal through its recruitment of KRAS/RalB to the plasma cancer treatment. The New England journal of medicine membrane leading to the activation of TBK-1/NFkB. Accord 358, 1160 (Mar. 13, 2008). ingly, this pathway also drives KRAS-mediated resistance to 0432 6. C. M. Ardito et al., EGF receptor is required for receptor tyrosine kinases inhibitors such as erlotinib. Inhibi KRAS-induced pancreatic tumorigenesis. Cancer Cell 22, tion of RalB or its effectors not only reverses tumor initiation 304 (Sep. 11, 2012). and self renewal but resensitizes tumors to Receptor Tyrosine 0433 7. C. Navas et al., EGF receptor signaling is essen Kinase (RTK) inhibition. These findings provide a molecular tial for k-ras oncogene-driven pancreatic ductal adenocar basis to explain how CVB3 drives tumor progression and cinoma. Cancer Cell 22, 318 (Sep. 11, 2012). reveals a therapeutic strategy to target and destroy these cells. 0447 Tumor-initiating cells (also known as cancer stem 0434 8. C. Ferte et al. Durable responses to Erlotinib cells). EMT, and drug resistance have recently been linked despite KRAS mutations in two patients with metastatic together as a challenge for cancer therapy. Here, we propose lung adenocarcinoma. Ann Oncol 21, 1385 (June, 2010). integrin CVB3 as a potential lynchpin capable of influencing 0435 9. M. J. Moore et al., Erlotinib plus gemcitabine and integrating these three critical determinants of cancer compared with gemcitabine alone in patients with progression. Indeed, expression off33 integrin has long been advanced pancreatic cancer: a phase III trial of the National associated with poor outcome and higher incidence of Cancer Institute of Canada Clinical Trials Group. J Clin metastasis for a variety of epithelial cancers', its expression Oncol 25, 1960 (May 20, 2007). has been reported on a subpopulation of breast and myeloid 0436 10. E. S. Kim et al., The BATTLE Trial: Personal leukemia cancer stem cells, and 33 has been implicated in the izing Therapy for Lung Cancer. Cancer discovery 1, 44 process of epithelial-to-mesenchymal transition, especially (June, 2012). in the context of TGF-?3. US 2016/0146783 A1 May 26, 2016 49

0448. Although the primary influence of integrins is con erlotinib but invariably develop resistance through multiple sidered to be their regulation of cell-matrix adhesion events mechanisms including acquired or selected mutations, gene leading to clustering of focal adhesions to drive intracellular amplification and alternate routes of kinase pathway activa signaling cascades, we have recently made the Surprising tion. Recent studies indicate that multiple resistance mecha observation that CVB3 integrin is capable of forming clusters nisms may operate within an individual tumor to promote on the Surface of non-adherent cells to recruit signaling com acquired resistance to EGFRTKIs in persons with NSCLC plexes that can drive cell survival in the absence of ligand and accumulating evidence Supports the concept that the binding". This property is not shared by other integrins, tumor-initiating cells contribute to EGFR TKI resistance in including 31, Suggesting that CVB3 expression may provide a lung. critical Survival signal for cells invading hostile environ 0454. To assess the clinical relevance of our findings, mice ments. Indeed, exposing quiescent endothelial cells to angio with established HCC827 (human NSCLC cells with deletion genic growth factors results in the upregulation of CVB3 of exon 19 of EGFR) have been treated with erlotinib until expression that is required for their conversion to the angio development of acquired resistance (FIG. 2F, or FIG. 33F). genic/invasive state'. We propose that expression of CVB3 Integrin B3 expression was significantly higher in erlotinib offers tumor cells an equivalent Survival advantage, and that resistant tumors compared to vehicle-treated tumors (FIG.2G targeting this pathway could undercut a tumors ability to or FIG.33G). metastasize and resist therapy. 0455 To validate these findings, we examined biopsies 0449 Since we previously reported that integrin CVB3 from lung cancer patients harboring an EGFR mutation expression was associated with increased anchorage-inde before erlotinib treatment and after acquired resistance and pendent growth", we postulated that (B3 expression may play we found that integrin B3 expression was qualitatively higher a role in tumor progression by shifting epithelial tumor cells after acquired resistance to erlotinib (FIG. 2H, or FIG.33H; toward a stem-like phenotype. To evaluate a possible effect of FIG.S1e, or, or FIG. 36E). To investigate the role of integrin B3 expression on tumor stemness in Vivo, we knocked down B3 in this context, we sorted erlotinib-resistant HCC827 integrin B3 in various human carcinoma cells expressing this tumors into integrin B3" and Integrin B3 populations and receptor, or ectopically expressed B3 in tumor cells lacking tested them for tumor initiating cell abilities. As expected, the this integrin. Compared with their respective B3-negative integrin B3' population showed enhanced tumor initiating counterparts, 33-positive cells showed a 50-fold increased and self-renewal capacities compared to the integrin B3 tumor-initiating capacity, measured as a higher frequency of population (FIG. 2I-J, or FIG.33I-J; FIG. S.1?, or FIG. 36F) tumor initiating cells in a limiting dilution assay (see FIG. 1A suggesting that integrin B3 contribute to the stem-like pheno and FIG. S1a-c (of Example 3), which are FIG. 32A and type of the drug resistance tumor. In addition integrin B3 has FIGS. 36A, 36B and 36C, respectfully). been found in a subpopulation of the CD166+ cells in human 0450. In vitro, tumor stemness is also associated with an adenocarcinoma after acquired resistance to erlotinib (FIG. increased capacity to form tumorspheres and undergo self S1g, or FIG. 36g). Together these findings reveal that B3 renewal. Consequently, we measured the capacity of 33 expression is both necessary and Sufficient to account for expressing tumor cells to form primary and secondary tumor tumor stem-like properties in vitro and in vivo. spheres. Notably, the ratio of secondary tumorspheres to pri 0456. Our results suggest that targeting integrin B3 func mary tumorspheres was 2-4 fold higher for cells expressing tion may represent a viable approach to reverse stem-like integrin B3 (see FIG. 1B-Db-d and FIG.S1c (of Example 3): properties and sensitize tumors to RTK inhibitors. However, which are FIG. 32B-Db-d and FIG. 36C, respectively). integrin antagonists that compete for ligand binding sites and Together, these findings indicate that B3 expression enhances disrupt cell adhesion are not likely to have an impact on the the stem-like behavior of these tumors. stemness and drug resistance properties that are represented 0451. Tumor-initiating cells are known to be particularly by 3D growth of tumor cells under anchorage-independent resistant to cellular stresses, such as nutrient deprivation or conditions. Accordingly, neither expression of a mutant inte exposure to anti-cancer drugs. Indeed, B3-positive cells Sur grin B3 (D119A) incapable of binding ligand nor treating Vived to a greater degree when stressed by removal of serum cells with cyclic peptides that compete with CVB3 for ligand from their growth media compared with cells lacking this binding impacted the B3-mediated enhancement of 3D integrin (FIG. S1d (of Example 3), or FIG. 36D). However, colony formation in the presence of erlotinib (FIG.S2a-b, or B3 expression did not impact the response to the chemothera FIG. 37A-B). Thus, the contribution of B3 integrin to stem peutic agent cisplatin or the anti-metabolite agent gemcitab ness and drug resistance appears to involve a non-canonical ine for cells growing in 3D (FIG. 2A, or FIG. 33A). Under function for this integrin, independent from its traditional role these same conditions, B3 expression did strongly correlate as a mediator of cell adhesion to specific B3 ligands. If this is with reduced sensitivity to Receptor Tyrosine Kinase (RTK) the case, then blocking this pathway will require understand inhibitors, including the EGFR1 inhibitor erlotinib, the ing the downstream molecular mechanism(s) that become EGFR1/EGFR2 inhibitor lapatinib, and the IGF-1R inhibitor engaged in the presence of 33. linsitinib (OSI906) (FIG. 2B-C, or FIG.33 B-C). 0457 To study how B3 integrin influences tumor stem 0452. This link between B3 expression and RTK inhibitor ness, we considered that integrins frequently transmit signals resistance was also observed in Vivo, as knockdown of inte in the context of RAS family members'. To examine a pos grin B3 overcame erlotinib resistance for subcutaneous A549 sible link between 33 expression and RAS, tumor cells grow xenografts (FIG. 2D, or FIG.33D), while ectopic expression ing in 3D were stained for B3 and various RAS family mem of integrin B3 conferred erlotinib resistance to FG tumors bers. Interestingly, in cells growing in Suspension, B3 growing orthotopically in the pancreas (FIG. 2E, or FIG. co-localized in clusters at the plasma membrane with KRAS, 33E). but not with NRAS, RRAS, or HRAS (FIG. 3A, or FIG.3A, 0453. In clinic, human non-small cell lung cancer harbor FIG.S2c, or FIG. 37C). In fact, KRAS could be specifically ing activating mutations in EGFR often initially respond to co-immunoprecipitated with B3 but not B1 integrin (FIG.3B, US 2016/0146783 A1 May 26, 2016 50 or FIG.34B), indicating a specific interaction between B3 and 0461) Despite numerous advances in our knowledge of KRAS in cells undergoing anchorage-independent growth. cancer, most advanced cancers remain incurable. At present, Finally, we observed that KRAS knockdown abolished the conventional therapies can control tumor growth initially but B3-induced anchorage independence, self-renewal, and erlo most patients ultimately relapse, highlighting the urgent need tinib resistance (FIG. 3C-E, or FIG. 34 C-E), indicating that for new approaches to treat cancerous tumors. One Such B3 and KRAS cooperate to drive B3-mediated stem-like phe approach may be to target the tumor-initiating cells. An notype. emerging picture is that tumor-initiating cells do not consti tute a homogenous population of cells explaining the lack of 0458 Since there are no known KRAS binding sites on the reliability of cancer stem markers. We discovered an integrin B3 cytoplasmic tail, it is likely that this KRAS/B3 interaction B3+ subpopulation of tumor-initiating cells that are specifi occurs through an intermediary. Galectin-3 is a carbohydrate cally resistant to RTKIs. Several studies have shown that binding lectin linked to tumor progression'' that is known to integrin-mediated cellular adhesion to extracellular matrix separately interact with KRAS' and integrin C.VfB3". There components is an important determinant of therapeutic fore, we considered whether Galectin-3 might serve as an response. In fact, integrin B3 increases adhesion-mediated adaptor facilitating the B3/KRAS interaction in anchorage cell Survival, drug resistance and Suppresses antitumor immu independent tumor cells. Indeed, we observed co-localization nity' suggesting that blocking integrin (33 could offerathera of B3, KRAS, and Galectin-3 within membrane clusters for peutic strategy. We and other previously established that cells grown under anchorage-independent conditions (FIG. besides the adhesion-dependent functions, integrins can also 3F, or FIG. 34F). Knockdown of Galectin-3 not only pre be involved in different cellular mechanisms. In fact, we vented formation of the KRAS/B3 complex (FIG. 3F-G, or recently showed the ability of B3 to drive anchorage-indepen FIG. 34F-G), but also reversed the advantage of B3 expres dent growth in 3D without providing any growth or survival sion for anchorage independence erlotinib resistance and advantage in 2D. Since there is also evidence that 3D cul self-renewal (FIG. 3H-I, or FIG. 34H). These findings pro tures mimic drug sensitivity in Vivo more accurately than 2D vide evidence that Galectin-3 facilitates an interaction cultures'', we focused on the role of B3 in promoting stem between B3 and KRAS that is required for the promotion of ness and drug resistance using 3D culture models in vitro and StemneSS. tumor growth in vivo. 0459. The activation of KRAS elicits changes in cellular 0462 Although KRAS mutations, present in 95% of pan function by signaling through a number of downstream effec creatic tumors and 25% of lung cancers, have been linked to tors, most prominently AKT/PI3K, RAF/MEK/ERK, and Ral RTK inhibitor resistance, recent studies have demonstrated GTPases''. Depletion of Akt, Erk, or RalA inhibited the 3D that expression of oncogenic KRAS is an incomplete predic growth off.3" versus B3 tumor cells equally (FIG.S3a-b, or tor of erlotinib resistance in pancreatic and lung cancer, since FIG.38A-B), suggesting these effectors were not selectively a number of individual patients presenting with KRAS muta involved in the ability of B3 to enhance stemness. In contrast, tion unexpectedly respond to therapy. In fact, for 3D growth knockdown of RalB not only selectively impaired colony in Soft agar and in vivo experiments, we found that erlotinib formation for B3k cells (FIG. 4A, or FIG.35A; FIG. S3c-d), resistance could be predicted by evaluating integrin B3 but it also negated the effect of B3 expression and stem-like expression in KRAS mutant cancers suggesting that onco phenotype (FIG. 4B-C; FIG. S3e, or FIG.38E) and erlotinib genic KRAS is not sufficient to drive erlotinib resistance. It resistance (FIG. 4D-E, or FIG. 35D-E). Mechanistically, the has been demonstrated that its localization to the plasma association between KRAS and integrin B3 at the plasma membrane is a critical component to its function and inhibit membrane was able to recruit and activate RalB (Supplemen ing its membrane localization could represent a therapeutic tary Information, FIG. S3f.h, or FIG. 38F-H). In fact, the strategy. Here, we revealed an unexpected role for integrin b3 activation of RalB alone is sufficient to drive this pathway, that can maintain KRAS in membrane clusters through its since expression of a constitutively active RalBG23V mutant interaction with Galectin-3 representing a potential therapeu in B3-negative tumor cells conferred erlotinib resistance tic opportunity. KRAS dependency had previously been (FIG.S3i, or FIG.38I). linked to erlotinib sensitivity for tumor cells growing in 2D. 0460 Consistent with recent studies that have linked the These results emphasize the contribution of B3 integrin to RalB effectors TBK1 and RelA to RTKI resistance and stem tumor cell behavior for cells grown in 3D, and Suggest that ness', f3" tumor cells showed activation of these effectors alternative or even opposing pathways may dominate when even in the presence of erlotinib (FIG.4F or FIG. 35F). Loss cells are grown in 2D under adherent conditions. of RalB restored erlotinib-mediated inhibition of TBK1 and 0463. The invention thus provides methods for determin RelA for B3" tumor cells (FIG. 4F, or FIG. 35F), suggesting ing or predicting the course of cancer therapy in terms of these as therapeutic targets relevant for this pathway. Since personalized medicine. Our results demonstrate that biopsies targeting integrin ligation events cannot perturb this pathway, taken at diagnosis can be screened for B3 expression to pre and RAS inhibitors have underperformed expectations in the dict a poor response to RTK-targeted therapies. If a biopsy is clinic, interrupting signaling downstream of RalB could positive, we would predict that co-administering an inhibitor reverse the stemness potential of B3" tumor cells. Indeed, of RalB/TBK1/RelA could improve the response. Since B3" genetic or pharmacological inhibition of TBK1 or RelA over tumor cells are particularly sensitive to KRAS knockdown, came self-renewal and B3-mediated erlotinib resistance (FIG. Such tumors represent a population of particularly good can 4G-I, or FIG. 35G-I; FIG. S4a-e, or FIG. 39A-E). Taken didates for KRAS-directed therapies which have shown only together, our observations indicate that integrin B3 expression poor responses thus far. promotes a cancer stem-like program by cooperating with 0464 Our work demonstrates that a tumor could be sen KRAS to regulate the activity of RalB, and that elements of sitized to therapy by reversing the advantages of B3 expres this pathway can be disrupted to provide therapeutic benefit in sion. We demonstrate this can beachieved by inhibiting RalB mouse models of lung and pancreatic cancer. mediated signaling using genetic knockdown or by treating US 2016/0146783 A1 May 26, 2016 with a number of FDA-approved drugs. We focused our 0477 Cell Viability Assay. efforts on the role of B3 expression on lung and pancreatic 0478 Cell viability assays were performed as described'. cancers in the context of erlotinib therapy, since it is approved Briefly cells were seeded in low adherent plates 7 days in for these patients. However, we were able to correlate KRAS DMEM containing 10% or 0% serum, 0.1% BSA. dependency and B3 expression for a diverse panel of epithe 0479 Genetic Knockdown and Expression of Mutant lial cancer cells. Constructs. 0480 Cells were transfected with vector control, WT. Methods G23V RalB-FLAG, using a lentiviral system. For knock 0465 Compounds and Cell Culture. down experiments, cells were transfected with KRAS, RalA, 0466 Human pancreatic (FG, PANC-1), breast RalB, AKT1, ERK1/2, TBK1, siRNA (Qiagen) using the (MDAMB231 (MDA231) and lung (A549 and H1650) can lipofectamine reagent (Invitrogen) following manufacturers cer cell lines were grown in ATCC recommended media protocol or transfected with shRNA (Open Biosystems) using supplemented with 10% fetal bovine serum, glutamine and a lentiviral system. Gene silencing was confirmed by immu non-essential amino acids. We obtained FG-B3, FG-D119A noblots analysis. mutant and PANC-shf3 cells as previously described. Erlo 0481 Immunohistochemical Analysis. tinib, linsitinib, Gemcitabine, Bortezomib and Lapatinib 0482 Immunostaining was performed according to the were purchased from Chemietek. Cisplatin was generated manufacturer's recommendations (Vector Labs) on 5 DM from Sigma-Aldrich. sections of paraffin-embedded tumors from tumor biopsies 0467 Self Renewal Tumorsphere Assay and Soft Agar from lung cancer patients. Tumor sections were processed as Assay. previously described using integrin B3 (Abcam)+stem 0468 Tumorsphere assay was performed as previously markers, diluted 1:200. Sections stained with integrin B3 described. Soft agar formation assays were performed essen were scored by a H-score according to the staining intensity tially as described previously. Cells were treated with vehicle (SI) on a scale 0 to 3 within the whole tissue section. (DMSO), erlotinib (10 nM to 5 uM), lapatinib (10 nM to 5 0483 RNA Extraction PCR uM), gemcitabine (0.001 nM to 5uM), linsitinib (10 nM to 5 0484 Immunoprecipitation and Immunoblots. uM), cisplatin (10 nM to 5 LM), or bortezomib (4 nM) diluted 0485 Lysates from cell lines and xenograft tumors were in DMSO. The media was replaced with fresh inhibitor 2/5 generated using standard methods and RIPA or Triton buffers. times a week. Survival curves were generated at least with Immunoprecipitation experiments were performed as previ five concentration points. ously described with anti-integrin-3 (LM609) or Galectin 0469 Limiting Dilution. 3. For immunoblot analysis, 25 ug of protein was boiled in 0470 All mouse experiments were carried out in accor Laemmli buffer and resolved on 8% to 15% gel. The follow dance with approved protocols from the UCSD animal sub ing antibodies were used: anti-integrin B3 (), KRAS, NRAS, jects committee and with the guidelines set forth in the NIH RRAS, HRAS, FAK and Hsp90 from Santa Cruz, phospho Guide for the Care and Use of Laboratory Animals. 10, 10, S172 NAK/TBK1 from Epitomics, TBK1, phospho 10, 10 and 10° of A549 NS, A549 shB3, FG, FG-B3 and p65NFkB S276, p.65NFkB, RalB, phospho-EGFR, EGFR, FG-33 sh RalB cells were suspended in a mixture of Base phospho-FAK Tyr 861 from Cell Signaling Technology, and ment Membrane Matrix Phenol Red-free (BD Biosciences) Galectin 3 from BioLegend. and PBS 1:1 and injected in the flanks of 6/8 weeks old female 0486 Affinity Pull-Down Assays for Ras and Ral. immune compromised nu/nu mice. After 30/40 days, pal 0487 RAS and Ral activation assays were performed in pable tumors were counted and the tumor-initiating cells accordance with the manufacturers (Upstate) instruction. frequency was calculated using the ELDA Software. Briefly, cells were cultured in suspension for 3 h. 10 ug of Ral 0471 Orthotopic Pancreas Cancer Xenograft Model. Assay Reagent (Ral BP1, agarose) or RAS assay reagent 0472 Tumors were generated as previously described (Raf-1 RBD, agarose) was added to 500 mg to 1 mg of total (JAY). Tumors were established for 2-3 weeks (tumor sizes cell protein in MLB buffer (Millipore). After 30 min of rock were monitored by ultrasound) before beginning dosing. ing at 4°C., the activated (GTP) forms of RAS/Ral bound to Mice were dosed by oral gavage with vehicle (6% captisol) or the agarose beads were collected by centrifugation, washed, 10, 25 and 50 mg/kg/day erlotinib for 10 to 30 days prior to boiled in Laemmli buffer, and loaded on a 15% SDS-PAGE harvest. gel. 0473 Immunofluorescence Microscopy. 0488 Statistical Analyses. 0474 Frozen sections from tumors from patients diag 0489 All statistical analyses were performed using Prism nosed with pancreas or tumor cell lines were processed as software (GraphPad). Two-tailed Mann Whitney Utests, Chi previously described (Mielgo). Cells were stained with indi squared tests, Fisher's exact tests, one way ANOVA tests or cated primary, followed by secondary antibodies specific for t-tests were used to calculate statistical significance. A P mouse or rabbit (Invitrogen), as appropriate. Samples imaged values 0.05 was considered to be significant. on a Nikon Eclipse C1 confocal microscope with 1.4NA 60x oil-immersion lens, using minimum pinhole (30 um). Colo FIGURE LEGENDS calization between Integrin B3 and KRAS was studied using the Zenon Antibody Labeling Kits (Invitrogen) and the Example 3 KRAS rabbit antibody. 0490 FIG. 1: Integrin B3 expression increase tumor-initi 0475 Biopsies from NSCLC Patients. ating and self-renewal capacities: 0476 Tumor biopsies from University of California, San 0491 (a) Limiting dilution in vivo determining the fre Diego (UCSD) Medical Center breast, pancreas and non quency of tumor-initiating cells for A549 cells expressing Small cell lung cancer patients were obtained. This work was non-target shRNA control or integrin B3-specific shRNA and approved by the UCSD Institutional Review Board (IRB). for FG cells expressing control vector or integrin B3 (FG-B3). US 2016/0146783 A1 May 26, 2016 52

The frequency of tumor-initiating cells per 10,000 cells was integrin B3. n=3 mean+SEM. *P<0.05, **P<0.01. (d) Effect calculated using the ELDA extreme limiting dilution soft of KRAS knockdown on erlotinib resistance of 33-negative ware. (b-c-d) Self-renewal capacity of A549 and PANC-1 and 33-positive epithelial cancer cell lines. Cells were treated cells expressing non-target shRNA control (CTRL) or inte with a dose response of erlotinib. n=3; meaniSEM, *P<0.05, grin B3-specific shRNA and of FG expressing control vector **P<0.01. (e) Self-renewal capacity of FG-B3 cells express or integrin B3 (FG-B3), measured by quantifying the number ing non-target shRNA control (shCTRL) or KRAS-specific of primary and secondary tumorspheres. Representative shRNA measured by quantifying the number of primary and images of tumorspheres are shown. n=3; meant SEM. *P<0. secondary tumorspheres. n=3; meaniSEM. *P<0.05, *P<0. 05, **P<0.01. 01. (f) Confocal microscopy images show immunostaining 0492 FIG. 2: Integrin B3 drives resistance to EGFR for integrin B3 (green), KRAS (red) and DNA (TOPRO-3, inhibitors: blue) for PANC-1 cells expressing non-target shRNA control 0493 (a) Effect of integrin B3 expression (ectopic expres or Galectin 3-specific shRNA grown in suspension. Scale sion for FG and integrin B3-specific knockdown for PANC-1) bar=10 Lum. Data are representative of three independent cells on drug treatment response. Cells were treated with a experiments. (g) immunoblot analysis of integrin B3 immu dose response of gemcitabine, cisplatin, erlotinib, lapatinib noprecipitates from PANC-1 cells expressing non-target and linsitinib. Results are normalized using non-treated cells shRNA control (CTRL) or Galectin-3-specific shRNA (Gal as controls. n=3; mean+SEM. *P<0.05, **P<0.001. (b) 3). Data are representative of three independent experiments. Effect of integrin B3 knockdown on erlotinib response in (h) Effect of Galectin-3 knockdown on integrin B3-mediated MDA-MB-231 (MDA231), A549 and H1650. n=3; anchorage independent growth and erlotinib resistance. meanti-SEM. *P<0.05, **P<0.001. (c) Effect of integrin B3 PANC-1 cells expressing a non-target shRNA control or a knockdown on erlotinib resistance in vivo, A549 shCTRL and Galectin-3-specific shRNA (sh Gal-3) were treated with A549 sh B3 (n=8 per treatment group) were treated with vehicle or erlotinib (0.5 M). n=3; mean+SEM. (i) Self-re erlotinib (25 mg/kg/day) or vehicle during 16 days. Tumor newal capacity of PANC-1 cells expressing non-target volumes are expressed as meant SEM. *P-0.05. (d) Orthoto shRNA control (shCTRL) or Galectin-3-specific shRNA (sh pic FG and FG-f3 tumors (>1000 mm; n=5 per treatment Gal-3) measured by quantifying the number of primary and group) were treated for 30 days with vehicle or erlotinib. secondary tumorspheres. n=3; meaniSEM. *P<0.05, *P<0. Results are expressed as % tumor weight compared to vehicle O1. control. *P<0.05. (e) Effectoferlotinib treatmenton HCC827 Xenograft tumors (n=8 tumors per treatment group). HCC827 0496 FIG. 4. RalB/TBK1 signaling is a key modulator of cells were treated with vehicle control or erlotinib (12.5 integrin B3-mediated stemness: mg/kg/day) until acquired resistance. (f) Relative mRNA 0497 (a) Effect of RalB knockdown on anchorage inde expression of integrin B3 (ITGB3) in HCC827 vehicle pendence. n=3; meanti-SEM, *P<0.05, **P<0.01. (b) Self treated tumors (n=5) or erlotinib-treated tumors (n=7) from renewal capacity of FG-B3 cells expressing non-target (e) after acquired resistance. Data are meaniSE: **P<0.001. shRNA control (sh CTRL) or RalB-specific shRNA (sh RalB) (g) H&E sections and immunohistochemical analysis of inte measured by quantifying the number of primary and second grin B3 expression in paired human lung cancer biopsies ary tumorspheres. n=3; mean+SEM. *P<0.05, **P<0.01. (c) obtained before and after erlotinib resistance. Scale bar, 50 Limiting dilution in vivo determining the frequency of tumor um. (h) Limiting dilution in vivo determining the frequency initiating cells for FG-B3 cells expressing non-target shRNA of tumor-initiating cells for HCC827 vehicle-treated (ve control or integrin RalB-specific shRNA. (d) Effect of RalB hicle) and erlotinib-treated tumors from (erlotinib resistant knockdown on erlotinib resistance of B3-positive epithelial non-sorted) (e). The HCC827 erlotinib-treated tumors have cancer cell lines. Cells were treated with 0.5uM of erlotinib. been digested and sorted in two groups: the integrin B3- and n=3; meanti-SEM, *P<0.05, **P<0.01. (e) Effect of RalB the integrin B3+ population. (i) and () Self-renewal capacity knockdown on erlotinib resistance off33-positive human pan of HCC827 vehicle-treated (vehicle), erlotinib-treated (erlo creatic (FG-f33) orthotopic tumor xenografts. Established tinib resistant non-Sorted), erlotinib-treated integrin tumors expressing non-target shRNA, (sh CTRL) or a shRNA B3-population and erlotinib-treated integrin B3+ population, targeting RalB (sh RalB) (>1000 mm; n=13 per treatment measured by quantifying the number of primary and second group) were randomized and treated for 10 days with vehicle ary tumorspheres. n=3; meaniSEM. *P<0.05, **P<0.01. or erlotinib. Results are expressed as % of tumor weight 0494 FIG. 3: Integrin B3/KRAS complex is critical for changes after erlotinib treatment compared to vehicle. *P<0. integrin B3-mediated stemness: 05. (f) Immunoblot analysis of FG and FG-f33 stably express 0495 (a) Confocal microscopy images show immun ing non-target shRNA control or RalB-specific shRNA, ostaining for Integrin B3 (green), KRAS (red) and DNA (TO grown in 3D and treated with erlotinib (0.5 LM). Data are PRO-3, blue) for FG-B3, PANC-1, A549 and HCC827 after representative of three independent experiments. (g) Effect of acquired resistance to erlotinib (HCC827 ER) grown in sus TBK1 knockdown on PANC-1 self-renewal capacity. n=3; pension. Arrows indicate clusters where integrin B3 and meanti-SEM. *P<0.05, **P<0.01. (h) Effect of TBK1 knock KRAS colocalize (yellow). Scale bar-10 um. Data are repre down on erlotinib resistance of PANC-1 cells. Cells were sentative of three independent experiments. (b) Ras activity treated with 0.5uM of erlotinib. n=3; meantSEM. *P<0.05, was determined in PANC-1 cells grown in suspension by **P<0.01. (i) Mice bearing subcutaneous 3-positive tumors using a GST-Rafl-RBD immunoprecipitation assay. Immu (PANC-1) were treated with vehicle, erlotinib (25 mg/kg/ noblots indicate KRAS activity and association of active day), amlexanoX (25 mg/kg/day) or the combination of erlo KRAS with integrin B3. Data are representative of three inde tinib and amlexanox. Tumor dimensions are reported as the pendent experiments. (c) Effect of KRAS knockdown on fold change relative to size of the same tumor on Day 1. tumorspheres formation in lung (A549 and H441) and pan Mean+SEM, (A) *P=0.042 using a one way ANOVA test. creatic (FG and PANC-1) cancer cells expressing or lacking n=8 mice per group. US 2016/0146783 A1 May 26, 2016

Figure S1 Figure S4 Example 3 Example 3 0498 (a-b) Limiting dilution tables. (c) Immunoblots 0501 (a) Immunoblot showing TBK1 knockdown effi showing integrin B3 knockdown or ectopic expression effi ciency in PANC-1 cells used in FIG. 4. (b) Effect of the TBK1 ciency in cells used in FIG. 1. (d) Viability assay (CelTiter inhibitor amlexanox on erlotinib response of PANC-1 cells. Glo assay) of FG and FG-f3 cells grown in 3D in media with Cells were treated with vehicle, erlotinib (0.5 M), amlex or without serum. n=3; mean+SEM. *P<0.05. **P<0.01. (e) anox alone or in combination. (c) Effect of the NFkB inhibitor Immunohistochemical analysis of integrin 33 expression in borthezomib on B3-positive cells (FG-B3, PANC-1 and paired human lung cancer biopsies obtained before and after A549). Cells were treated with vehicle, erlotinib (0.5 uM), erlotinib resistance. Scale bar, 50 lum. (f) Limiting dilution bortezomib (4 nM) alone or in combination. n=3; table. (g) Immunohistochemistry staining of CD166 and inte meaniSEM. *P<0.05, *P<0.01. (d) Mice bearing subcuta grin B3 in human lung tumor biopsies after EGFR TKI neous B3-positive tumors (FG-B3) were treated with vehicle, acquired resistance. erlotinib (25 mg/kg/day), bortezomib (0.25 mg/kg), the com bination of erlotinib and bortezomib. Tumor dimensions are Figure S2 reported as the fold change relative to size of the same tumor on Day 1. *P-X using a one way ANOVA test. n=8 mice per Example 3 group. (e) Confocal microscopy images of cleaved caspase 3 (red) and DNA (TOPRO-3, blue) in tumor biopsies from 0499 (a) Effect of cilengetide treatment on erlotinib resis xenografts tumors used in (d) treated with vehicle, erlotinib, tance in FG-B3 and PANC-1 cells. n=3; mean+SEM. (b) bortezomib or bortezomib and erlotinib in combo. Scale bar, Effect of ectopic expression of B3 wild-type (FG-B3) or the 20 Lum. B3 D119A (FG-D119A) ligand binding domain mutant on erlotinib response. n=3; meaniSEM. Immunoblot showing REFERENCES transfection efficiency of vector control, integrin B3 wild type and integrin 3 D119A. (c) Confocal microscopy images Example 3 of FG-B3 cells grown in 3D and stained for integrin-B3 0502. 1. Desgrosellier, J. S. & Cheresh, D. A. Integrins in (green) and RAS family members (red). Scale bar, 10 um. cancer: biological implications and therapeutic opportuni Data are representative of three independent experiments. (d) ties. Nat Rev Cancer 10, 9-22 (2010). Immunoblots showing KRAS knockdown efficiency in cells 0503 2. Singh, A. & Settleman, J. EMT, cancer stem cells used in FIG. 3. (e) Representative photographs of crystal and drug resistance: an emerging axis of evil in the war on violet-stained tumorspheres of FG and A549 cells expressing cancer. Oncogene 29, 4741-4751 (2010). non-target shRNA control or specific-KRAS. (f) Effect of a (0504 3. Lo, P. K., et al. CD49f and CD61 identify Her2/ second KRAS knockdown (shKRAS 2) on tumorspheres for neu-induced mammary tumor-initiating cells that are mation in PANC-1 stably expressing non-target shRNA con potentially derived from luminal progenitors and main trol (3-positive) or specific-integrin-B3 shRNA (3 negative). tained by the integrin-TGFbeta signaling. Oncogene n=3; mean+SEM. *P<0.05. (2011). 0505. 4. Vaillant, F., et al. The mammary progenitor Figure S3 marker CD61/beta3 integrin identifies cancer stem cells in mouse models of mammary tumorigenesis. Cancer Res 68, Example 3 7711-7717 (2008). (0500 (a) Effect of ERK, AKT and RalA knockdown on (0506 5. Galliher, A.J. & Schiemann, W. P. Beta3 integrin erlotinib response of B3-negative FG and 3-positive FG-3 and Src facilitate transforming growth factor-beta medi cells. (b) Immunoblots showing ERK, AKT and RalA knock ated induction of epithelial-mesenchymal transition in down efficiency in cells used in (a). (c) Immunoblots showing mammary epithelial cells. Breast cancer research. BCR 8, RalB knockdown efficiency in cells used in FIG. 3. (d) Effect R42 (2006). of a second RalB knockdown (shRalB 2) on tumorspheres 0507 6. Mamuya, F.A. & Duncan, M.K. aV integrins and formation in PANC-1 stably expressing non-target shRNA TGF-beta-induced EMT: a circle of regulation. Journal of control (B3-positive) or specific-integrin B3 shRNA (B3 nega cellular and molecular medicine 16, 445-455 (2012). tive). n=3; mean+SEM. *P<0.05. (e) Limiting dilution table. 0508 7. Desgrosellier, J.S., et al. An integrin alpha(v)beta (f) Confocal microscopy images of integrin CVB3 (green), (3)-c-Src oncogenic unit promotes anchorage-indepen RalB (red) and DNA (TOPRO-3, blue) intumor biopsies from dence and tumor progression. Nat Med 15, 1163-1169 pancreatic cancer patients. Scale bar, 20 p.m. (g) Ral activity (2009). was determined in PANC-1 cells grown in suspension by 0509 8. Boudreau, N., et al. Induction of the angiogenic using a GST-RalBP1-RBD immunoprecipitation assay. phenotype by Hox D3.J Cell Biol 139, 257-264 (1997). Immunoblots indicate RalA and RalB activities. Data are 0510) 9. Dean, M., Fojo, T. & Bates, S. Tumour stem cells representative of three independent experiments. (h) Effect of and drug resistance. Nature Reviews Cancer 5, 275-284 B3 expression and KRAS expression on RalB activity, mea (2005). sured using a GST-RalBP1-RBD immunoprecipitation assay. 0511 10. Martin, K. H., et al. Integrin Connections Map: Data are representative of three independent experiments. (i) To Infinity and Beyond. Science 296, 1652-1653 (2002). Effect of expression of a constitutively active Ral G23V 0512 11. Newlaczyl, A. U. & Yu, L. G. Galectin-3-a mutant on erlotinib resistance of B3 positive and negative jack-of-all-trades in cancer. Cancer letters 313, 123-128 cells. n=3; mean-SEM. *P<0.05. (2011). US 2016/0146783 A1 May 26, 2016 54

0513 12. Shalom-Feuerstein, R., et al. K-ras nanocluster (c) mixing or combining the testagent or molecule with the ing is subverted by overexpression of the scaffold protein Galactin-3 and the integrin CVB3 protein under condi galectin-3. Cancer research 68, 6608-6616 (2008). tions wherein the Galactin-3 and the integrin CVB3 pro 0514 13. Markowska, A. I., Liu, F.T. & Panjwani, N. teins bind to each other; and Galectin-3 is an important mediator of VEGF- and bFGF (d) determining or measuring if the test agent or molecule mediated angiogenic response.J Exp Med207, 1981-1993 inhibits or blocks the binding of the Galactin-3 to the (2010). integrin CVB3 protein, 0515 14. Pylayeva-Gupta, Y., Grabocka, E. & Bar-Sagi, wherein a test agent or molecule that inhibits or blocks the D. RAS oncogenes: weaving a tumorigenic web. Nat Rev binding of the Galactin-3 to the integrin CVB3 protein is Cancer 11, 761-774 (2011). identified as an inhibitor of the Galactin-3 function. 0516) 15. Delhase, M., et al. TANK-binding kinase 1 12: The method or assay of claim 9, wherein the method or (TBK1) controls cell survival through PAI-2/serpinB2 and assay further comprises use of a positive and a negative con transglutaminase 2. Proceedings of the National Academy trol, of Sciences of the United States of America 109, E177-186 and optionally the amount of inhibiting or blocking of the (2012). Galactin-3 to the integrin C.VfB3 protein in the presence 0517. 16. Jinushi, M., et al. ATM-mediated DNA damage of the test agent or molecule is compared to the amount signals mediate immune escape through integrin-alphav of inhibiting or blocking of the Galactin-3 to the integrin beta3-dependent mechanisms. Cancer Res 72, 56-65 CVB3 protein in the absence of the test agent or mol (2012). ecule, and/or in the presence of known inhibitor or 0518 17. Schmeichel, K. L. & Bissell, M. J. Modeling blocker of the binding of the Galactin-3 to the integrin tissue-specific signaling and organ function in three CVB3 protein, optionally the positive and/or negative dimensions. Journal of cell science 116, 2377-2388 control assays are done under conditions identical or (2003). Substantially similar to the test assay with the test agent 0519. 18. Singh, A., et al. A gene expression signature or molecule. associated with "K-Ras addiction” reveals regulators of 13: The method or assay of claim 9, wherein the Galactin-3 EMT and tumor cell survival. Cancer Cell 15, 489-500 and/or the integrin CVB3 protein is a synthetic protein, a (2009). recombinant protein and/or a Substantially isolated protein. 0520. A number of embodiments of the invention have 14: The method or assay of claim 9, wherein the Galactin-3 been described. Nevertheless, it will be understood that vari or the integrin CVB3 protein is immobilized on a surface, ous modifications may be made without departing from the wherein optionally the surface is a gel, a glass, a membrane, spirit and scope of the invention. Accordingly, other embodi a glass or a plastic, and optionally the Galactin-3 or the ments are within the scope of the following claims. integrin CLV33 protein is immobilized on a plate surface, 1-8. (canceled) wherein optionally the immobilized protein is in a well. 9: A method or assay for Screening for and/or identifying an 15: The method or assay of claim 9, wherein the amount of agent or molecule that can block or inhibit a Galactin-3 func Galectin-3 bound to the immobilized CVB3 is measured or tion, comprising: quantified using a fluorescent anti-Galectin-3 antibody or Screening for an agent or molecule that can block or inhibit Galectin-3 binding fragment thereof, and optionally the integrin C.VfB3/Galactin-3 complex formation; or, the amount of Galectin-3 bound to the immobilized CVB3 is binding of Galactin-3 to integrin CVB3. measured or quantified as fluorescent anti-Galectin-3 immu 10: The method or assay of claim 9, wherein the screening noreactivity. is in vitro or in vivo. 16: The method or assay of claim 9, wherein the test agent 11: The method or assay of claim 9, comprising: or the test molecule comprises a small molecule, a protein, a (a) providing a test agent or a test molecule; peptide, an antibody, a nucleic acid, a Sugar or a polysaccha (b) providing a Galactin-3 protein and an integrin CVB3 ride, a fatty acid or a fat. protein; k k k k k