WO2018085275A1 - Targeting lats1/2 and the hippo intracellular signaling pathway for cancer immunotherapy - Google Patents

Targeting lats1/2 and the hippo intracellular signaling pathway for cancer immunotherapy Download PDF

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WO2018085275A1
WO2018085275A1 PCT/US2017/059310 US2017059310W WO2018085275A1 WO 2018085275 A1 WO2018085275 A1 WO 2018085275A1 US 2017059310 W US2017059310 W US 2017059310W WO 2018085275 A1 WO2018085275 A1 WO 2018085275A1
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tumor
cells
kinase
map4k
cancer
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Kun-Liang Guan
Toshiro MOROISHI
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The Regents Of The University Of California
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5152—Tumor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response

Definitions

  • the one or more guide RNAs guide CRISPR/Cas9 to specifically cleave one or more nucleic acid sequences encoding one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7), and optionally STK3, and partially, substantially, or completely deletes, silences, inactivates, or down-regulates one or more of LATSl, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g.,
  • the inhibitor is a polypeptide, peptide or small organic compound.
  • the methods comprise administering to the subject an inhibitor of LATS 1 and/or LATS2 selected from the group consisting of A443654, Lestaurtinib, GSK-690693, lysophosphatidic acid (LP A), sphingosine-1 -phosphate (S IP) and thrombin.
  • the methods comprise administering to the subject an inhibitor of STK4 and/or STK3 selected from 9E1 and XMU-MP-1.
  • the inhibitor is an antibody or fragment thereof.
  • XM_011529018.2 XP_011527320.1; and XM_005260532.3 ⁇ XP_005260589.1; (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of a STK4 polypeptide; or an amino acid sequence encoded by a STK4 nucleic acid, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a STK4 protein, and conservatively modified variants thereof; and/or (4) have a nucleic acid sequence that has greater than about 90%, preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the full-length, to a
  • NM_001195045.1 ⁇ NP_001181974.1 yorkie homolog isoform 4 (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of a YAP polypeptide; or an amino acid sequence encoded by a YAP nucleic acid, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a YAP protein, and conservatively modified variants thereof; and/or (4) have a nucleic acid sequence that has greater than about 90%, preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the full-length, to a YAP nucleic acid (e.g., described above).
  • TAZ transcriptional coactivator with PDZ binding motif
  • TAZ refers to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 90% amino acid sequence identity, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 400, or more amino acids, or over the full- length, to an amino acid sequence encoded by a TAZ nucleic acid (see, e.g., GenBank Accession Nos. M_001168278.1 ⁇ P_001161750.1; 2. M_001168280.1 ⁇
  • TAZ is a transcriptional coactivator that binds to Sd in its active, unphosphorylated form to activate expression of transcriptional targets that promote cell growth, cell proliferation, and prevent apoptosis.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., share at least about 80% identity, for example, at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a specified region to a reference sequence, e.g., LATS1/2, MST1, STK3, MAP4K, YAP or TAZ polynucleotide or polypeptide sequence as described herein, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • a reference sequence e.g., LATS1/2, MST1, STK3, MAP4K, YAP or TAZ polynucleotide or polypeptide sequence as described herein
  • a polypeptide is typically
  • systemic administration and “systemically administered” refer to a method of administering the agent that inhibits one or more proteins within or associated with the HIPPO intracellular signaling pathway to a mammal so that the agent is delivered to sites in the body, including the targeted site of pharmaceutical action, via the circulatory system.
  • Systemic administration includes, but is not limited to, oral, intranasal, rectal and parenteral (i.e., other than through the alimentary tract, such as intramuscular, intravenous, intra-arterial, transdermal and subcutaneous) administration.
  • the phrase "cause to be administered” refers to the actions taken by a medical professional (e.g., a physician), or a person controlling medical care of a subject, that control and/or permit the administration of the agent(s)/compound(s) at issue to the subject.
  • Causing to be administered can involve diagnosis and/or determination of an appropriate therapeutic or prophylactic regimen, and/or prescribing particular
  • patient refers to a mammal, for example, a human or a non-human mammal, including primates (e.g., macaque, pan troglodyte, pongo), a domesticated mammal (e.g., felines, canines), an agricultural mammal (e.g., bovine, ovine, porcine, equine) and a laboratory mammal or rodent (e.g., rattus, murine, lagomorpha, hamster).
  • primates e.g., macaque, pan troglodyte, pongo
  • domesticated mammal e.g., felines, canines
  • an agricultural mammal e.g., bovine, ovine, porcine, equine
  • rodent e.g., rattus, murine, lagomorpha, hamster
  • the terms "inhibiting,” “reducing,” “decreasing” with respect to tumor or cancer growth or progression refers to inhibiting the growth, spread, metastasis of a tumor or cancer in a subject.
  • the growth, progression or spread of a tumor or cancer is inhibited, reduced or decreased if the tumor burden is at least about 10%, 20%, 30%, 50%, 80%, or 100%) reduced, e.g., in comparison to the tumor burden prior to inhibition of one or more protein within or associated with the HIPPO intracellular signaling pathway selected from large tumor suppressor kinase 1 (LATS 1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7)) (and optionally serine/threonine kinase 3 (STK3)), optionally in combination with another active agent (e.g., an immune checkpoint inhibitor and
  • antibody is used in the broadest sense and includes fully assembled antibodies, tetrameric antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind an antigen (e.g., Fab', F(ab)2, Fv, single chain antibodies, diabodies), and recombinant peptides comprising the forgoing as long as they exhibit the desired biological activity.
  • An "immunoglobulin” or “tetrameric antibody” is a tetrameric glycoprotein that consists of two heavy chains and two light chains, each comprising a variable region and a constant region.
  • Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antibody fragments or antigen-binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibody (dAb), complementarity determining region (CDR) fragments, CDR-grafted antibodies, single-chain antibodies (scFv), single chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibody, linear antibody; chelating recombinant antibody, a tribody or bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), an antigen- binding-domain immunoglobulin fusion protein, a camelized antibody, a VHH containing antibody, or a variant or a derivative thereof, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the
  • Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • target-binding polypeptides, or fragments, variants, or derivatives thereof will bind with a greater affinity to human target as compared to its binding affinity to target of other, i.e., non-human, species, but binding polypeptides that recognize and bind orthologs of the target are within the scope provided.
  • Wild-type (WT) and LATSl/2 double knockout (dKO) 4T1 breast cancer cells were subjected to immunoblot (IB) and phos-tag analysis.
  • F Loss of LATSl/2 promotes YAP/TAZ nuclear localization.
  • LatB-treated or non-treated (control) 4T1 cells were subjected to immunostaining analysis.
  • YAP/TAZ subcellular localization was determined by immunofluorescence staining for endogenous YAP/TAZ (green) along with DAPI for DNA (blue). Representative images are presented in the left panel.
  • Light panel Cells in five randomly selected views ( ⁇ 100 cells) were selected for the quantification of YAP/TAZ localization.
  • N nuclear
  • C cytoplasmic.
  • LATS1/2 dKO SCC7 cells confer cancer immunity.
  • C3H/HeOu mice were first injected with non-irradiated LATS1/2 dKO SCC7 cells. 60 days after the initial injection, mice designated tumor-free were re-challenged with WT SCC7 cells and tumor growth was monitored [LATS1/2 dKO(#l) ⁇ WT].
  • the tumor growth curves shown in Figure 4B are presented in a lighter color for reference, p value was determined using two-way ANOVA test, comparing WT EV- immunized group [WT + WT EVs ⁇ WT] to LATS 1/2 dKO EV-immunized group [WT + LATSl/2 dKO EVs ⁇ WT]. ***p ⁇ 0.001.
  • LATSl/2-null tumor cells secrete more EV proteins. EVs were isolated from culture supernatants of equal numbers of WT or LATSl/2 dKO B16-OVA cells and protein concentrations were determined. Data are means ⁇ SEM of 6 independent experiments. ***p ⁇ 0.001, unpaired t-test.
  • F EVs from LATSl/2-deficient or YAP(5SA)-overexpressing tumor cells contain higher amounts of RNA than EVs from WT tumor cells.
  • FIG. 12A-D illustrate that EVs from LATS 1/2-depleted tumor cells stimulate anti-tumor immunity via the Toll-like receptors (TLRs)-type I interferon (IFN) pathway.
  • TLRs Toll-like receptors
  • IFN I interferon
  • FIGS 13A-I illustrate that LATSl/2-depleted tumor EVs stimulate antitumor immunity via the Toll-like receptors (TLRs)-type I interferon (IFN) pathway. See also, Figure 12.
  • TLRs Toll-like receptors
  • IFN I interferon
  • the tumor cell is rendered replication incompetent, e.g., via irradiation.
  • transduction involves introducing a nucleic acid molecule via viral methods into a cancerous cell.
  • a nucleic acid molecule is introduced into a cancerous cell via viral vectors.
  • the viral vectors used to introduce a nucleic acid molecule are adenoviral, retroviral, lentiviral, or adeno-associated viral vectors.
  • the viral vectors are introduced into a cancerous cell via the chemical based, non-chemical based, and particle based transfection methods described above.
  • RNA as well as altered RNA or analogs of naturally occurring RNA.
  • TALENs Transcription Activator-Like Effector Nucleases
  • activity or expression of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase is partially, substantially, or completely deleted, silenced, inactivated, down-regulated, reduced or inhibited by introducing a nucleic acid, preferably an expression vector, containing a nucleic acid encoding transcription activator-like effector nucleases (TALEN).
  • a nucleic acid preferably an expression vector, containing a nucleic acid encoding transcription activator-like effector nucleases (TALEN).
  • TALEN are restriction enzymes that are designed to specifically cleave nucleic acid sequences encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3).
  • LATS 1, LATS2, STK4 and/or a MAP4K family kinase e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3
  • LAGLIDADG is the amino acid sequence that is generally conserved in all proteins of this family.
  • the DNA binding recognition site and cleavage function of the meganucleases are intertwined in a single domain. Meganucleases bind adjacent DNA target sites and induce double-strand breaks between the target sequences.
  • megaTAL are restriction enzymes that are designed to specifically cleave nucleic acid sequences encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1 , LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3).
  • a cancerous cell is transfected with a vector containing nucleic acid encoding megaTAL, wherein the megaTAL specifically cleave one or more nucleic acid sequences encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) and partially, substantially, or completely deletes, silences, inactivates, or down-regulates one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3).
  • the megaTAL specifically cleave one or more nucleic acid sequences encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS
  • the cancerous cell in which one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) is partially, substantially, or completely deleted, silenced, inactivated, or down-regulated is transformed with a nucleic acid, preferably an expression vector, containing a Cas9 molecule and a nucleic acid encoding a crRNA and tracrRNA.
  • the CRISPR/Cas system is originally an RNA-mediated bacterial immune system that provides a form of acquired immunity against viruses and plasmids; it comprises three components: a Cas9 (CRISPR associated protein 9)
  • endonuclease is programmed by a crRNA and tracrRNA hybrid to cleave a LATS l, LATS2, or LATS 1/2 sequence.
  • the gRNA sequence is at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary, or any range derivable therein, to a portion of one or more genes encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3).
  • the CRISPR/Cas9 system is delivered by using a plasmid.
  • the CRISPR/Cas9 system is delivered by using a ribonucleoprotein complex.
  • the ribonucleoprotein complex comprises a Cas9 protein and a nucleic acid sequence encoding crRNA and tracrRNA.
  • Exemplary endocrine and glandular tissue cancers that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include without limitation pancreatic carcinoma, medullary thyroid carcinoma, follicular thyroid carcinoma, anaplastic thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, adrenal tumors and adenocarcinoma.
  • MAP4K family kinase e.g., MAP4K 1/2/3/4/5/6/7
  • an immune checkpoint inhibitor and/or chemotherapeutic agent include without limitation pancreatic carcinoma, medullary thyroid carcinoma, follicular thyroid carcinoma, anaplastic thyroid carcinoma, papillary
  • Additional cancers that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include without limitation teratomas. 4.
  • the cellular material e.g., tumor cells, extracellular vesicles (EVs) and cell lysates thereof
  • the cellular material deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS 1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4), a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7), and optionally serine/threonine kinase 3 (STK3) are employed as the immunogen.
  • LATS 1 large tumor suppressor kinase 1
  • LATS2 large tumor suppressor kinase 2
  • STK4 serine/threonine kinase 4
  • STK3 a MAP4K family kinase
  • STK3 optionally serine/threonine kinase 3
  • Illustrative TAAs that find use as an immunogen to be co-administered with cellular material deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) include without limitation melanoma associated antigens (MAGE-1, MAGE-3, TRP-2, melanosomal membrane glycoprotein gplOO, gp75 and MUC-1 (mucin- 1) associated with melanoma); CEA (carcinoembryonic antigen) which can be associated, e.g., with ovarian, melanoma or colon cancers; folate receptor alpha, WAP four-disulfide core domain 2 (HE4) or mesothelin expressed by ovarian carcinoma; free human chorionic gonadotropin beta (hCGP) subunit expressed by many different tumors
  • MAP4K 1/2/3/4/5/6/7) (and optionally STK3) (e.g., tumor cells, EVs and cell lysates thereof) is obtained from and autologous to the subject.
  • the methods may further comprise prior to administration of the cellular material the steps of: a) isolating a population of tumor cells from the subject; and b) reducing or eliminating expression of one or more proteins within or associated with the HIPPO intracellular signaling selected from L ATS 1 , LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) in the isolated tumor cells.
  • the tumor cell immunogen and/or the cellular material deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and a MAP4K family kinase selected from LATS 1, LATS2, STK4 and a MAP4K family kinase (e.g.,
  • one or more inhibitors of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of LATS1, LATS2, STK4 and a MAP4K family kinase (e.g.,
  • MAP4K1/2/3/4/5/6/7) (and optionally STK3) is an inhibitory nucleic acid.
  • the one or more inhibitory nucleic acids that inhibit the expression and/or activity levels of one or more proteins selected from the group consisting of LATSl, LATS2, heat shock protein 90 (HSP90), STK4 and a MAP4K family kinase e.g.,
  • Polymer reagents for delivery of an inhibitory and expression vectors may incorporate compounds that increase their utility. These groups can be incorporated into monomers prior to polymer formation or attached to polymers after their formation.
  • An expression vector transfer enhancing moiety is typically a molecule that modifies a nucleic acid complex and can direct it to a cell location (such as tissue cells) or location in a cell (such as the nucleus) either in culture or in a whole organism. By modifying the cellular or tissue location of the complex, the desired localization and activity of the inhibitory nucleic acid or expression vector can be enhanced.
  • the transfer enhancing moiety can be, for example, a protein, peptide, lipid, steroid, sugar, carbohydrate, nucleic acid, cell receptor ligand, or synthetic compound.
  • the transfer enhancing moieties can enhance cellular binding to receptors, cytoplasmic transport to the nucleus and nuclear entry or release from endosomes or other intracellular vesicles.
  • the carrier system is a protein-based carrier system such as a cationic peptide-nucleic acid complex.
  • one or more inhibitors of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of LATS1, LATS2, STK4 and a MAP4K family kinase is a peptide, a polypeptide or a small organic compound.
  • Small organic inhibitors of one or more proteins in the HIPPO intracellular signaling pathway selected from the group consisting of LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) are known in the art and find use.
  • LATS1 and/or LATS2 include without limitation A443654 (chemical name: (2S)-l-(lH-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin- 3-yl]oxypropan-2-amine; CAS Registry Number: 552325-16-3), Lestaurtinib (chemical name: (5S,6S,8R)-6-Hydroxy-6-(hydroxymethyl)-5-methyl-7,8, 14,15-tetrahydro-5H-16- oxa-4b,8a,14-triaza-5,8-methanodibenzo[b,h]cycloocta[jkl]cyclopenta[e]-as-indacen-
  • A443654 chemical name: (2S)-l-(lH-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin- 3-yl]oxyprop
  • HSP90 Numerous inhibitors of HSP90 are known in the art and can find use in the present methods. Illustrative inhibitors of HSP90 are described, e.g., in U.S. Patent Nos.
  • MAP4K1/2/3/4/5/6/7) an analog thereof can be formulated for administration orally, parenterally, (intravenously (IV), intramuscularly (IM), depo-IM, subcutaneously (SQ), and depo-SQ), intratumorally, sublingually, intranasally (inhalation), intrathecally,
  • the dosage form is selected to facilitate delivery to an intracellular target (e-g-, using nanoparticles, liposomes, viral vectors).
  • an intracellular target e-g-, using nanoparticles, liposomes, viral vectors.
  • the compounds described herein are readily delivered to the brain. Dosage forms known to those of skill in the art are suitable for delivery of the compound.
  • the one or more inhibitors of one or more HIPPO pathway proteins selected from LATS1, LATS2, STK4 and a MAP4K family kinase can be administered in the "native" form or, if desired, in the form of salts, esters, amides, prodrugs, derivatives, and the like, provided the salt, ester, amide, prodrug or derivative is suitable pharmacologically effective, e.g., effective in the present method(s).
  • physiologically acceptable salt or ester is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutical practice.
  • a physiologically acceptable vehicle carrier, excipient, binder, preservative, stabilizer, flavor, etc.
  • the amount of active substance in those compositions or preparations is such that a suitable dosage in the range indicated is obtained.
  • compositions are preferably formulated in a unit dosage form, each dosage containing from about 1-1000 mg, 2-800 mg, 5-500 mg, 10-400 mg, 50-200 mg, e.g., about 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg or 1000 mg of the active ingredient.
  • unit dosage from refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • compositions the compound is mixed with a suitable solvent
  • the compounds and/or analogs thereof can be enclosed in multiple or single dose containers.
  • the enclosed compounds and compositions can be provided in kits, for example, including component parts that can be assembled for use.
  • a compound inhibitor in lyophilized form and a suitable diluent may be provided as separated components for combination prior to use.
  • a kit may include a compound inhibitor and a second therapeutic agent for co-administration. The inhibitor and second therapeutic agent may be provided as separate component parts.
  • a kit may include a plurality of containers, each container holding one or more unit dose of the compounds.
  • composition will depend on absorption, inactivation, and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors.
  • the oral dosage forms can be administered to the patient 1, 2, 3, or 4 times daily. It is preferred that the compound be administered either three or fewer times, more preferably once or twice daily. Hence, it is preferred that the compound be administered in oral dosage form. It is preferred that whatever oral dosage form is used, that it be designed so as to protect the compound from the acidic environment of the stomach. Enteric coated tablets are well known to those skilled in the art. In addition, capsules filled with small spheres each coated to protect from the acidic stomach, are also well known to those skilled in the art.
  • an administered amount therapeutically effective to prevent, delay inhibit or reverse tumor growth is from about 0.1 mg/day to about 200 mg/day, for example, from about 1 mg/day to about 100 mg/day, for example, from about 5 mg/day to about 50 mg/day.
  • the subject is administered the compound at a dose of about 0.05 to about 0.50 mg/kg, for example, about 0.05 mg/kg, 0.10 mg/kg, 0.20 mg/kg, 0.33 mg/kg, 0.50 mg/kg. It is understood that while a patient may be started at one dose, that dose may be varied (increased or decreased, as appropriate) over time as the patient's condition changes. Depending on outcome evaluations, higher doses may be used.
  • the compounds and/or analogs thereof can be administered intratum orally.
  • compound and/or analogs thereof can be administered intrathecally.
  • the appropriate dosage form can be a parenteral dosage form as is known to those skilled in the art.
  • the dosage of compound and/or analog thereof for intrathecal administration is the amount described above for IM administration.
  • erythrohydroxynonyladenine fludarabine, cladribine
  • plant alkaloid(s) and/or terpenoid(s) e.g., vincristine, vinblastine, vinorelbine, and vindesine
  • vinca alkaloid(s) e.g., vincristine, vinblastine, vinorelbine, and vindesine
  • podophyllotoxin(s) e.g., etoposide and teniposide
  • camptothecin(s) e.g., irinotecan and topotecan
  • anthracycline e.g., aromatase inhibitor(s)
  • taxane(s) e.g., paclitaxel, taxol and docetaxel
  • topoisomerase inhibitor(s) e.g., (Type I inhibitors: camptothecins, including irinotecan and topotecan; Type II Inhibitors: amsacrine, etoposide, etoposide phosphate, and teniposide), antibiotic(s) (e.g., dactinomycin, daunorubicin, doxorubicin, idarubicin, epirubicin, bleomycins, mitomycin), hormone(s), differentiating agent(s), kinase inhibitor(s
  • a control value of tumor burden (e.g., a mean and standard deviation) is determined from a control population of individuals who have undergone treatment with tumor cells tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells, optionally in combination with a chemotherapeutic agent.
  • LATS1 large tumor suppressor kinase 1
  • LATS2 large tumor suppressor kinase 2
  • STK4 serine/threonine kinase 4
  • Measured values of tumor burden in a patient are compared with the control value. If the measured level in a patient is not significantly different (e.g., more than one standard deviation) from the control value, treatment can be discontinued. If the tumor burden level in a patient is significantly above the control value, continued administration of agent is warranted.
  • Immune response signals can include but are not limited to for example assessing the enhancement of the lymphoma-specific cytotoxic effect of human peripheral blood mononuclear cells (PBMCs). If the value for the immune response signal does not change significantly, or decreases, a negative treatment outcome is indicated.
  • PBMCs peripheral blood mononuclear cells
  • DNA was then diluted and used for quantification by real-time PCR, which was performed using KAPA SYBR FAST qPCR Kit (Kapa Biosy stems) and the 7300 real-time PCR system (Applied Biosystems).
  • the sequences of the PCR primers (forward and reverse, respectively) are 5'-GCCTGGAGAAACCTGCCAAGTATG-3' and
  • C57BL/6 mice were immunized intradermally at the base of the tail with irradiated B16-OVA cells (100 Gy, 1 ⁇ 10 6 ) 12 days prior to challenge with B16-OVA cells (one time vaccination, without any adjuvant).
  • B16-OVA cells 100 Gy, 1 ⁇ 10 6
  • EVs freshly isolated from culture supernatants of B16-OVA cells (6 ⁇ 10 6 ) were injected every 3 days (days 0, 3, 6, and 9) into the same place until challenged with B16-OVA cells at day 12.
  • EL4 cells were mixed at 1 : 1 ratio, and then co-cultured with CD8+ T cells isolated from splenocytes of C57BL/6 mice challenged (or not) with WT or LATSl/2 dKO B16-OVA cells at 8: 1 effector to target cell ratio (E:T).
  • CD8+ T cells were isolated using EasySepTM Mouse CD8a Positive Selection Kit (STEMCELL, #18753) from pooled splenocytes of 3-4 mice per group for each experiment.
  • the frequencies of CFSElow and CSFEhigh EL4 cells in CFSE positive fraction were determined by flow cytometric analysis 18 h after incubation and the percent of specific killing was calculated.
  • BMDCs Bone marrow derived dendritic cells
  • Cytokine enzyme-linked immunosorbent assay ELISA. IFN- ⁇ or IL-12 levels in culture supernatants were determined by ELISA.
  • IFN- ⁇ or IL-12 levels in culture supernatants were determined by ELISA.
  • draining lymph nodes (inguinal lymph nodes) were isolated from C57BL/6 mice challenged (or not) with B16-OVA cells and cultured with OVA protein (100 ⁇ g/ml) for 3 days.
  • B16-OVA cells were seeded in 150 mm culture plate and incubated in DMEM supplemented with 10% FBS for 24 h at 37°C to allow cell attachment. The cells were then washed with PBS twice, and culture medium was switched to 35 ml of DMEM without serum. After incubation for 48 h, conditioned medium was collected and centrifuged at 2,000 g for 10 min at 4°C to thoroughly remove cell debris.
  • the resulting supernatant was then filtered through a 0.22 ⁇ PVDF filter (Millipore, #SLGV033RB) to remove cell debris and microvesicles (for the detergent treatment experiment, the resulting flow-through was treated with 1% Triton X-100 for 10 min at 4°C prior to the ultracentrifugation).
  • the flow-through was transferred into ultracentrifuge tubes (BECKMAN COULTER, #344058) and then ultracentrifuged in a Beckman SW32Ti rotor at 30,000 rpm for 90 min at 4°C.
  • the resulting pellets were washed with 35 ml of PBS and then ultracentrifuged again at 30,000 rpm for 90 min at 4°C.
  • LATS 1/2 -deficiency enhances tumor vaccine efficacy via adaptive immunity.
  • TLRs endosomal Tolllike receptors
  • cytosolic non-TLR sensors Figure 12 A
  • LATS1/2 mRNA expression levels and patient outcome in different types of human cancer (Table S2).
  • 26 studies show significant (p ⁇ 0.05) correlation between LATS2 mRNA levels and patient outcome, which includes 17 studies showing better patient survival with low LATS2 expression.
  • 12 studies show significant correlation between LATSl mRNA levels and patient outcome, which includes 5 studies showing better patient survival with low LATSl expression.
  • low YAP expression predicted worse patient survival in human colorectal cancer (Barry et al., 2013, (2013). Nature 493, 106-110) and multiple myeloma (Cottini et al., (2014) Nat. Med. 20, 599-606).
  • LATS1/2 inhibition to improve immunogenicity of tumor cells enhances immune checkpoint inhibitor efficacy.
  • a combination of LATS1/2 inhibitors and immune checkpoint inhibitors is a novel and exciting therapeutic approach for poorly immunogenic cancers, especially in cases where malignancy is driven by oncogenic alterations that leave the Hippo signaling pathway intact. It is noteworthy that germline or somatic mutations affecting the core components of the Hippo pathway are uncommon in human cancers (Harvey et al., 2013, supra; Moroishi et al., (2015) Nat. Rev. Cancer 15, 73-79). Therefore, inhibition of LATS 1/2 enhances tumor immunity in most cancer types.
  • a physician obtains a population of cancerous cells from an individual via a biopsy.
  • a vaccine comprising allogenic cancerous cells with one or more genes selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) having ablated or reduced molecule activity and extracellular vesicles (EVs) as an adjuvant is generated.
  • the allogenic cancerous cells isolated from the individual are irradiated.
  • the crRNA and tracrRNA activates Cas9 and guides it to the portion of the one or more genes selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) to be cleaved.
  • Cas9 cleaves a portion of the one or more genes selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), rendering them inactive.
  • the cancerous cells are expanded in vitro and an allogenic cancerous cell line deficient for one or more genes selected from LATS l, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) is established.
  • the EVs produced by the LATS l/2-deficient allogenic cancerous cells are isolated.
  • the one or more small molecule drug are inhibitors of enzymatic activity of one or more HIPPO pathway proteins selected from LATS l, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), e.g., by competitive, non-competitive, or uncompetitive mechanisms that binds to one or more HIPPO pathway proteins selected from LATS l, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) and inhibits their function.
  • Decrease of the molecular activity of one or more HIPPO pathway proteins selected from LATS l, LATS2, STK4 and a MAP4K family kinase e.g.,
  • MAP4K1/2/3/4/5/6/7) increases the immune response to the tumor and thereby leads to inhibition of tumor growth.

Abstract

Provided are tumor cells having reduced or eliminated expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), as well as extracellular vesicles and cell lysates from such tumor cells. The tumor cells may also have reduced or eliminated expression and/or activity of serine/threonine kinase 3 (STK3) and/or overexpress one or both of YAP and/or TAZ, or hyperactive mutants thereof. Further provided are methods of inducing, promoting and/or enhancing an immune response against a tumor in a subject by administering tumor cells having reduced or eliminated expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), or extracellular vesicles or cell lysates of such tumor cells. Further provided are methods of inducing, promoting and/or enhancing an immune response against a tumor in a subject by administering inhibitors of expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally further inhibiting the expression and/or activity of serine/threonine kinase 3 (STK3).

Description

TARGETING LATS1/2 AND THE HIPPO INTRACELLULAR SIGNALING PATHWAY FOR CANCER IMMUNOTHERAPY
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. § 1 19(e) of U.S.
Provisional Appl. No. 62/416,414, filed on November 2, 2016, which is hereby incorporated herein by reference in its entirety for all purposes.
STATEMENT OF GOVERNMENTAL SUPPORT
[0002] This work was supported in part by Grant No. R35CA196878 from the
National Institutes of Health. The Government has certain rights in this invention.
SUMMARY
[0003] In one aspect, provided is a tumor cell or a population of tumor cells having reduced or eliminated expression or function of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATS 1), large tumor suppressor kinase 2 (LATS2),
serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7). In some embodiments, the function or activity of serine/threonine kinase 3 (STK3) is further reduced or eliminated in the tumor cell. In some embodiments, the function or activity of the one or more proteins within or associated with the HIPPO intracellular signaling pathway has been reduced and/or eliminated. In some embodiments, the expression of the one or more proteins within or associated with the HIPPO intracellular signaling pathway has been reduced and/or eliminated. In some embodiments, genes encoding the one or more proteins within or associated with the HIPPO intracellular signaling pathway have been knocked down or knocked out. In some embodiments, the genes encoding both of LATS 1 and LATS2 have been knocked down or knocked out. In some embodiments, the tumor cell has elevated expression and/or activity levels of YAP and/or TAZ. In some embodiments, the tumor cell comprises one or more recombinant polynucleotides encoding (hyper)active mutants of YAP and/or TAZ. In some embodiments, one or more recombinant
polynucleotides encoding YAP and/or TAZ, or (hyper)active mutants thereof, are incorporated into the genome. In some embodiments, the tumor cell expresses or overexpresses a tumor-associated antigen. In some embodiments, the tumor cell is irradiated. In some embodiments, the tumor cell has been irradiated with a sufficient dose of radiation and for a sufficient time such that it is viable but unable to proliferate. In some embodiments, the tumor cell has retained function of YAP and/or TAZ. In some embodiments, the tumor cell is selected from the group consisting of a melanoma, a glioma, a colon cancer, a squamous cell carcinoma and a breast carcinoma. In some embodiments, the tumor cell is from a cancer selected from the group consisting of sarcoma, lymphoma, hematological cancer, skin cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, colon cancer, rectal cancer, urogenital cancer, hepatic cancer, thyroid cancer, esophageal cancer, bladder cancer, renal cancer, brain cancer (e.g., glioma) and head and neck cancer. In a related aspect, provided is an extracellular vesicle (EV) or a population of EVs from a tumor cell or a population of tumor cell, as described above and herein. In a related aspect, provided is cell lysate from a tumor cell or a population of tumor cell, as described above and herein. In a further aspect, provided is an immunogenic composition comprising a tumor cell or a population of tumor cells, an extracellular vesicle (EV) or population of EVs, and/or a cell lysate of claim 18 and a pharmaceutically acceptable excipient. In some embodiments, the immunogenic composition further comprises an adjuvant.
[0004] In another aspect, provided is a method of inducing, promoting and/or enhancing an immune response against a tumor in a subject in need thereof. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of cellular material (e.g., a population of tumor cells, a population of extracellular vesicles (EVs) or a cell lysate) having reduced or eliminated expression or function of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATS l), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally reduced or eliminated expression and/or activity of STK3), thereby inducing, promoting and/or enhancing the immune response against the tumor in the subject. In some embodiments, the methods comprise coadministering to the subject a tumor associated antigen and a therapeutically effective amount of cellular material (e.g., a population of tumor cells, a population of extracellular vesicles (EVs) or a cell lysate) having reduced or eliminated expression or function of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATS l), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally reduced or eliminated expression and/or activity of STK3), thereby inducing, promoting and/or enhancing the immune response against the tumor in the subject. In some embodiments, the tumor associated antigen is a synthetic or recombinant peptide or polypeptide. In some embodiments, the tumor associated antigen is in a tumor cell or tumor cell lysate obtained from the subject. In some embodiments, the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is injected intradermally, epicutaneously or subcutaneously. In some
embodiments, the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate are administered multiple times. In some embodiments, the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is co-administered with an adjuvant. In some embodiments, the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is autologous to the subject. In some embodiments, the methods further the steps prior to administration of: a) isolating a population of tumor cells from the subject; and b) reducing or eliminating expression and/or activity in the isolated tumor cells of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1
(LATS l), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7). In some embodiments, the function or activity of serine/threonine kinase 3 (STK3) is further reduced or eliminated. In some embodiments, the methods further comprise knocking-out or knocking down in the population of tumor cells genes encoding the one or more proteins within or associated with the HIPPO intracellular signaling. In some embodiments, the methods further comprise contacting the population of tumor cells with one or more inhibitor compounds that inhibit the one or more proteins within or associated with the HIPPO intracellular signaling. In varying embodiments, the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is syngeneic, allogeneic or xenogeneic to the subject.
[0005] In a related aspect, provided are methods of inducing, promoting and/or enhancing an immune response against a tumor in a subject in need thereof. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of a population of dendritic cells with the major histocompatibility complex (MHC) proteins loaded with antigens from cellular material (e.g., a population of tumor cells, a population of extracellular vesicles (EVs) or a cell lysate) having reduced or eliminated expression or function of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATSl), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally reduced or eliminated expression and/or activity of STK3), thereby inducing, promoting and/or enhancing the immune response against the tumor in the subject. In some embodiments, the dendritic cells are autologous, syngeneic, allogeneic or xenogeneic to the subject. In some embodiments, the population of dendritic cells is injected intradermally, epicutaneously or subcutaneously. In some embodiments, the population of dendritic cells is administered multiple times. In some embodiments, the population of dendritic cells is co-administered with an adjuvant.
[0006] In a related aspect, provided are methods of inducing, promoting and/or enhancing an immune response against a tumor in a subject in need thereof. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of an inhibitor of the expression or function of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7). In some embodiments, the methods further comprise inhibiting serine/threonine kinase 3 (STK3). In some embodiments, the inhibitor comprises one or more inhibitory nucleic acids. In some embodiments, the one or more inhibitory nucleic acids specifically hybridize to one or more of LATS 1, LATS2, heat shock protein 90 (HSP90), STK4 a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) and optionally STK3. In some embodiments, the methods comprise administering to the subject a one or more inhibitory nucleic acids that partially, substantially, or completely deletes, silences, inactivates, down-regulates, reduces, or inhibits, activity or expression of one or more of LATS 1, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) and optionally STK3. In some embodiments, the one or more inhibitory nucleic acids comprise a small interfering RNA (siRNA). In some embodiments, the one or more inhibitory nucleic acids encode one or more transcription activator-like effector nucleases (TALEN). In some embodiments, the one or more TALENs specifically cleave one or more nucleic acid sequences encoding one or more of LATS 1, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7), and optionally STK3, and partially, substantially, or completely deletes, silences, inactivates, or down-regulates one or more of LATS 1, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3. In some embodiments, the one or more inhibitory nucleic acids encode one or more meganucleases. In some embodiments, the one or more meganucleases specifically cleave one or more nucleic acid sequences encoding one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3, and partially, substantially, or completely deletes, silences, inactivates, or down- regulates one or more of L ATS 1 , LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3. In some embodiments, the one or more inhibitory nucleic acids encode one or more transcription activator-like effector meganucleases (megaTALs). In some embodiments, the one or more megaTALs specifically cleave one or more nucleic acid sequences encoding one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7), and optionally STK3, and partially, substantially, or completely deletes, silences, inactivates, or down-regulates one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3. In some embodiments, the one or more inhibitory nucleic acids encode one or more zinc finger nucleases (ZFNs). In some embodiments, the one or more ZFNs specifically cleave one or more nucleic acid sequences encoding one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7), and optionally STK3, and partially, substantially, or completely deletes, silences, inactivates, or down-regulates one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3. In some embodiments, the one or more inhibitory nucleic acids encode one or more guide RNAs (sgRNA). In some embodiments, the one or more guide RNAs guide CRISPR/Cas9 to specifically cleave one or more nucleic acid sequences encoding one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7), and optionally STK3, and partially, substantially, or completely deletes, silences, inactivates, or down-regulates one or more of LATSl, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7), and optionally STK3. In some embodiments, the one or more inhibitory nucleic acids encode one or more crRNAs, one or more tracrRNAs, and one or more Cas9 endonucleases, wherein the crRNA, tracrRNA and Cas9 endonuclease operatively coordinate. In some embodiments, the one or more Cas9 endonucleases is programmed by a coordinating crRNA and a coordinating tracrRNA hybrid to cleave one or more nucleic acid sequences encoding one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3, thereby partially, substantially, or completely deleting, silencing, inactivating, or down-regulating one or more of LATS 1, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3. In some embodiments, the inhibitor is a polypeptide, peptide or small organic compound. In some embodiments, the methods comprise administering to the subject an inhibitor of LATS 1 and/or LATS2 selected from the group consisting of A443654, Lestaurtinib, GSK-690693, lysophosphatidic acid (LP A), sphingosine-1 -phosphate (S IP) and thrombin. In some embodiments, the methods comprise administering to the subject an inhibitor of STK4 and/or STK3 selected from 9E1 and XMU-MP-1. In some embodiments, the inhibitor is an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof partially, substantially, or completely deletes, silences, inactivates, down-regulates, reduces, or inhibits, activity or expression of one or more of LATS 1, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3. In some embodiments, the inhibitor of the expression or function of one or more proteins are administered by a route selected from the group consisting of orally, intravenously, intramuscularly, subcutaneously, intradermally, intralesionally and intratumorally. In some embodiments, the methods further comprise administering to the individual cellular material from a population of cancerous cells in which expression or activity of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) is partially, substantially, or completely deleted, silenced, inactivated, down-regulated, reduced or inhibited. In some embodiments, the cellular material comprises a population of replication-deficient tumor cells, a population of extracellular vesicles (EVs) and/or cell lysate. In some embodiments, the tumor is from a cancer that has retained function of YAP and/or TAZ. In some embodiments, the tumor is from a cancer selected from the group consisting of a melanoma, a glioma, a colon cancer, a squamous cell carcinoma and a breast carcinoma. In some embodiments, the tumor is from a cancer selected from the group consisting of sarcoma, lymphoma, hematological cancer, skin cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, colon cancer, rectal cancer, urogenital cancer, hepatic cancer, thyroid cancer, esophageal cancer, bladder cancer, renal cancer, brain cancer (e.g., glioma) and head and neck cancer. In some embodiments, the subject is a human. In some embodiments, the methods further comprise coadministering an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of cytotoxic T-lymphocyte associated protein 4
(CTLA4), programmed cell death 1 (PCD1), CD274 molecule (PD-L1), phosphoinositide 3- kinase γ (ΡΒΚγ) or indoleamine 2,3-dioxygenase (IDO). In some embodiments, the immune checkpoint inhibitor is an antibody or fragment or variant thereof. In varying embodiments, the checkpoint inhibitor is administered before, after or concurrently with the inhibitor of the expression or function of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATS l), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7). [0007] In a further aspect, provided is a vaccine composition comprising: (a) cellular material from a population of cancerous cells in which expression or activity of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATS l), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) is partially, substantially, or completely deleted, silenced, inactivated, down-regulated, reduced or inhibited, and (b) a pharmaceutically- acceptable excipient. In varying embodiments, the cellular material comprises a population of replication-deficient tumor cells, a population of extracellular vesicles (EVs) and/or cell lysate. In varying embodiments, the vaccine composition is formulated for intradermal, epicutaneous or subcutaneous administration. In varying embodiments, the cellular material is autologous, syngeneic, allogeneic or xenogeneic to an individual to be treated. In some embodiments, the replication-deficient tumor cells have been irradiated. In some embodiments, the replication-deficient tumor cells have been irradiated with a sufficient dose of radiation and for a sufficient time such that they are viable but unable to proliferate. DEFINITIONS
[0008] The terms "large tumor suppressor kinase 1" or "LATS l" interchangeably refer to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 90% amino acid sequence identity, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99% or 100% amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 400, or more amino acids, or over the full-length, to an amino acid sequence encoded by a LATS l nucleic acid (see, e.g., GenBank Accession No.
NM_004690.3→NP_004681.1 (serine/threonine-protein kinase LATS l isoform 1); M_001270519.1→ P_001257448.1 (serine/threonine-protein kinase LATSl isoform 2); see also, XM_017011474.1→ XP_016866963.1; XM_017011478.1→ XP_016866967.1; XM_017011476.1→ XP_016866965.1; XM_017011480.1→ XP_016866969.1 ;
XM_017011475.1→ XP_016866964.1; XM_017011481.1→ XP_016866970.1 ;
XM_017011479.1→ XP_016866968.1; XM_017011477.1→ XP O 16866966.1 ;
XM_011536252.2→XP_011534554.1; XM_006715603.3→ XP_006715666.1 and XM_017011482.1→ XP O 16866971.1); (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of a LATSl polypeptide; or an amino acid sequence encoded by a LATSl nucleic acid, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a LATSl protein, and conservatively modified variants thereof; and/or (4) have a nucleic acid sequence that has greater than about 90%, preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%o, 99%o or 100%) nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the full-length, to a LATSl nucleic acid {e.g., described above). Based on the knowledge of LATSl homologs, those of skill can readily determine residue positions that are more tolerant to substitution. For example, amino acid residues conserved amongst species are less tolerant of
substitution or deletion. Similarly, amino acid residues that are not conserved amongst species are more tolerant of substitution or deletion, while retaining the function of the LATSl protein. LATSl is a protein of the HIPPO signaling pathway. Functionally, LATSl is a Nuclear DBF-2-related kinase that phosphorylates and inactivates Yki.
[0009] The terms "large tumor suppressor kinase 2" or "LATS2" interchangeably refer to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 90% amino acid sequence identity, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o, 99%o or 100%) amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 400, or more amino acids, or over the full-length, to an amino acid sequence encoded by a LATS2 nucleic acid {see, e.g., GenBank Accession No.
NM_014572.2→NP_055387.2; see also, XM_017020542.1→ XP_016876031.1;
XM_005266342.1→ XP_005266399.1; XM_017020541.1→ XP_016876030.1 ; and XM_011535042.2→ XP_011533344.1); (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of a LATS2 polypeptide; or an amino acid sequence encoded by a LATS2 nucleic acid, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a LATS2 protein, and conservatively modified variants thereof; and/or (4) have a nucleic acid sequence that has greater than about 90%, preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%), 98%), 99% or 100% nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the full-length, to a LATS2 nucleic acid (e.g., described above). Based on the knowledge of LATS2 homologs, those of skill can readily determine residue positions that are more tolerant to substitution. For example, amino acid residues conserved amongst species are less tolerant of substitution or deletion. Similarly, amino acid residues that are not conserved amongst species are more tolerant of substitution or deletion, while retaining the function of the LATS2 protein. LATS2 is a protein of the HIPPO signaling pathway. Functionally, LATS2 is a Nuclear DBF-2-related kinase that phosphorylates and inactivates Yki.
[0010] As used herein, LATS1/2 means a combination of LATS1 and LATS2. [0011] The terms "serine/threonine kinase 4" or "STK4" or "mammalian STE20- like protein kinase 1" or "MST1" interchangeably refer to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 90% amino acid sequence identity, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 400, or more amino acids, or over the full-length, to an amino acid sequence encoded by a STK4 nucleic acid (see, e.g., NM_006282.3→ NP 006273.1 ; see also, XM 005260533.2→ XP 005260590.1;
XM_017028033.1 XP_016883522.1; XM_017028030.1→ XP_016883519.1 ;
XM_017028029.1 XP_016883518.1; XM_017028032.1→ XP_016883521.1 ;
XM 005260531.3 XP_005260588.1; XM_005260530.2→ XP_005260587.1 ;
XM_011529020.2 XP_011527322.1; XM_017028031.1→ XP_016883520.1 ;
XM_011529018.2 XP_011527320.1; and XM_005260532.3→ XP_005260589.1); (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of a STK4 polypeptide; or an amino acid sequence encoded by a STK4 nucleic acid, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a STK4 protein, and conservatively modified variants thereof; and/or (4) have a nucleic acid sequence that has greater than about 90%, preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the full-length, to a STK4 nucleic acid (e.g., described above). Based on the knowledge of STK4 homologs, those of skill can readily determine residue positions that are more tolerant to substitution. For example, amino acid residues conserved amongst species are less tolerant of substitution or deletion. Similarly, amino acid residues that are not conserved amongst species are more tolerant of substitution or deletion, while retaining the function of the STK4 protein. STK4 is a protein of the HIPPO signaling pathway. Functionally, STK4 is a Sterile-20-type kinase that phosphorylates and activates Wts. [0012] The terms "serine/threonine kinase 3" or "STK3" or "mammalian STE20- like protein kinase 2" or "MST2" interchangeably refer to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 90% amino acid sequence identity, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 400, or more amino acids, or over the full-length, to an amino acid sequence encoded by a STK3 nucleic acid (see, e.g., NM 006281.3→ P_006272.2 (isoform 1); NM_001256312.1→NP_001243241.1 (isoform 2); M_001256313.1→ P_001243242.1 (isoform 3)); (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of a STK3 polypeptide; or an amino acid sequence encoded by a STK3 nucleic acid, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a STK3 protein, and conservatively modified variants thereof; and/or (4) have a nucleic acid sequence that has greater than about 90%, preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the full-length, to a STK3 nucleic acid (e.g., described above). Based on the knowledge of STK3 homologs, those of skill can readily determine residue positions that are more tolerant to substitution. For example, amino acid residues conserved amongst species are less tolerant of substitution or deletion. Similarly, amino acid residues that are not conserved amongst species are more tolerant of substitution or deletion, while retaining the function of the STK3 protein. STK3 is a protein of the HIPPO signaling pathway. Functionally, STK3 is a Sterile-20-type kinase that phosphorylates and activates Wts. [0013] The term "MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7)" refers collectively to Misshapen (Msn, MAP4K4/6/7 in mammals) and Happyhour (Hppy, MAP4K1/2/3/5 in mammals) homologs, in particular to "mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1)," "mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2)," "mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3)," "mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4)," "mitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5)," and "misshapen like kinase 1 (MINK1, also known as MAP4K6)" and interchangeably refers to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 90% amino acid sequence identity, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 400, or more amino acids, or over the full-length, to an amino acid sequence encoded by a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) nucleic acid (see, e.g., M_001042600.2→ P_001036065.1 MAP4K1 isoform 1); NM 007181.5→ P_009112.1 (MAP4K1 isoform 2);
M_004579.4→ P_004570.2 (MAP4K2 isoform 1); M_001307990.1→
P_001294919.1 (MAP4K2 isoform 2); M_003618.3→ P_003609.2 (MAP4K3 isoform 1); M_001270425.1→ P_001257354.1 (MAP4K3 isoform 2); M_004834.4 → P_004825.3 (MAP4K4 isoform 1); M_145686.3→ P_663719.2 (MAP4K4 isoform 2); M_145687.3→ P_663720.1 (MAP4K4 isoform 3); M_001242559.1→ P_001229488.1 (MAP4K4 isoform 4); M_001242560.1→ P_001229489.1 (MAP4K4 isoform 5); M_006575.4→ P_006566.2 (MAP4K5 variant 1); M_198794.2→
P_942089.1 (MAP4K5 variant 2); M_015716.4→ P_056531.1 (MINK1 isoform 1); M_170663.4→ P_733763.1 (MINK1 isoform 2); NM_153827.4→ P_722549.2 (MINK1 isoform 3); NM_001024937.3→ P_001020108.1 (MINK1 isoform 4);
M_001321236.1→ P_001308165.1 (MINK1 isoform 5)); (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) polypeptide; or an amino acid sequence encoded by a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) nucleic acid, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) protein, and conservatively modified variants thereof; and/or (4) have a nucleic acid sequence that has greater than about 90%, preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the full-length, to a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) nucleic acid (e.g., described above). Based on the knowledge of MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) homologs, those of skill can readily determine residue positions that are more tolerant to substitution. For example, amino acid residues conserved amongst species are less tolerant of substitution or deletion. Similarly, amino acid residues that are not conserved amongst species are more tolerant of substitution or deletion, while retaining the function of the MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) protein. MAP4K family kinases (e.g.,
MAP4K 1/2/3/4/5/6/7) are proteins associated with the HIPPO intracellular signaling pathway, and can be considered a part of the HIPPO intracellular signaling pathway.
Misshapen (Msn, MAP4K4/6/7 in mammals) and Happyhour (Hppy, MAP4K 1/2/3/5 in mammals) act in parallel to STK3/4 (a.k.a, MST1/2) to activate LTS1/2. See, Meng, et al, Nat Commun. (2015) 6:8357; Zheng, et al, Developmental Cell. (2015) 34 (6): 642-655; and Li, et al, Developmental Cell. (2014) 31 (3): 291-304.
[0014] The "Yes-associated protein (YAP) transcriptional co-activator" refers to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 90% amino acid sequence identity, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 400, or more amino acids, or over the full-length, to an amino acid sequence encoded by a YAP nucleic acid (see, e.g., GenBank Accession No. NM_001130145.2→
NP_001123617.1 yorkie homolog isoform 1; NM_006106.4→ NP_006097.2 yorkie homolog isoform 2; NM_001195044.1→ NP_001181973.1 yorkie homolog isoform 3; 3. NM_001195045.1→ NP_001181974.1 yorkie homolog isoform 4); (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of a YAP polypeptide; or an amino acid sequence encoded by a YAP nucleic acid, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a YAP protein, and conservatively modified variants thereof; and/or (4) have a nucleic acid sequence that has greater than about 90%, preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the full-length, to a YAP nucleic acid (e.g., described above). Based on the knowledge of YAP homologs, those of skill can readily determine residue positions that are more tolerant to substitution. For example, amino acid residues conserved amongst species are less tolerant of substitution or deletion. Similarly, amino acid residues that are not conserved amongst species are more tolerant of substitution or deletion, while retaining the function of the YAP protein. YAP is the human ortholog of chicken YAP protein which binds to the SH3 domain of the Yes proto-oncogene product. This protein contains a WW domain that is found in various structural, regulatory and signaling molecules in yeast, nematode, and mammals, and may be involved in protein-protein interaction; a TEAD interaction domain, and a transactivation domain. Functionally, YAP is a transcriptional co- activator and a major downstream effector of the HIPPO intracellular signaling (Dong et al.,
(2007) Cell 130, 1120-1133). LATS1/2 inhibit YAP by direct phosphorylation, which results in YAP binding to 14-3-3σ and cytoplasmic sequestration (Dong et al., 2007; Hao et al., 2008; Zhao et al., (2007) Genes Dev. 21, 2747-2761). The unphosphorylated YAP localizes in the nucleus and acts mainly through TEAD family transcription factors to stimulate expression of genes that promote proliferation and inhibit apoptosis (Zhao et al.,
(2008) Genes Dev. 22, 1962-1971). Phosphorylation of YAP by Lats 1/2 kinases can also promote its ubiquitination-dependent degradation (Zhao et al., (2010) Genes Dev. 24(1):72- 85). YAP is a transcriptional coactivator that binds to Sd in its active, unphosphorylated form to activate expression of transcriptional targets that promote cell growth, cell proliferation, and prevent apoptosis.
[0015] The terms "WW domain containing transcription regulator 1 (WWTR1)" and
"transcriptional coactivator with PDZ binding motif (TAZ)" refers to a YAP paralog in mammals and is also regulated by the HIPPO intracellular signaling through both cytoplasmic retention and proteasome degradation (Lei et al., 2008). TAZ is a WW domain containing a transcriptional coactivator that modulates mesenchymal differentiation and development of multiple organs. TAZ refers to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 90% amino acid sequence identity, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 400, or more amino acids, or over the full- length, to an amino acid sequence encoded by a TAZ nucleic acid (see, e.g., GenBank Accession Nos. M_001168278.1→ P_001161750.1; 2. M_001168280.1→
P_001161752.1; M_015472.4→ P_056287.1; see also, Kanai, et al, The EMBO Journal (2000) 19(24):6778-6791); (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of a TAZ polypeptide; or an amino acid sequence encoded by a TAZ nucleic acid, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding a TAZ protein, and conservatively modified variants thereof; (4) have a nucleic acid sequence that has greater than about 90%, preferably greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, 2000 or more nucleotides, or over the full-length, to a TAZ nucleic acid (e.g., described above). Based on the knowledge of TAZ homologs, those of skill can readily determine residue positions that are more tolerant to substitution. For example, amino acid residues conserved amongst species are less tolerant of substitution or deletion. Similarly, amino acid residues that are not conserved amongst species are more tolerant of substitution or deletion, while retaining the function of the TAZ protein. TAZ is a transcriptional coactivator that binds to Sd in its active, unphosphorylated form to activate expression of transcriptional targets that promote cell growth, cell proliferation, and prevent apoptosis.
[0016] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., share at least about 80% identity, for example, at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a specified region to a reference sequence, e.g., LATS1/2, MST1, STK3, MAP4K, YAP or TAZ polynucleotide or polypeptide sequence as described herein, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the compliment of a test sequence. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, for example, over a region that is 50, 100, 200, 300, 400 amino acids or nucleotides in length, or over the full-length of a reference sequence. [0017] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins to LATS1/2, STK4, STK3, MAP4K, YAP or TAZ nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and the default parameters are used.
[0018] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is
immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically
substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
[0019] The terms "systemic administration" and "systemically administered" refer to a method of administering the agent that inhibits one or more proteins within or associated with the HIPPO intracellular signaling pathway to a mammal so that the agent is delivered to sites in the body, including the targeted site of pharmaceutical action, via the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal and parenteral (i.e., other than through the alimentary tract, such as intramuscular, intravenous, intra-arterial, transdermal and subcutaneous) administration.
[0020] The term "co-administration" or "concurrent administration", when used, for example with respect to the compounds (e.g., inhibitors of one or more proteins in the
HIPPO intracellular signaling pathway selected from LATSl, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3)) and/or analogs thereof and another active agent (e.g., a cognition enhancer), refers to administration of the compound and/or analogs and the active agent such that both can simultaneously achieve a physiological effect. The two agents, however, need not be administered together. In certain embodiments, administration of one agent can precede administration of the other. Simultaneous physiological effect need not necessarily require presence of both agents in the circulation at the same time. However, in certain embodiments, co-administering typically results in both agents being simultaneously present in the body (e.g,. in blood, plasma, serum) at a significant fraction (e.g., 20% or greater, preferably 30% or 40% or greater, more preferably 50% or 60% or greater, most preferably 70% or 80% or 90% or greater) of their maximum serum concentration for any given dose. [0021] The term "effective amount" or "pharmaceutically effective amount" refer to the amount and/or dosage, and/or dosage regime of one or more compounds necessary to bring about the desired result e.g., an amount sufficient to mitigating in a mammal one or more symptoms associated with cancer, or an amount sufficient to lessen the severity or delay the progression of cancer in a mammal (e.g., therapeutically effective amounts), an amount sufficient to reduce the risk or delaying the onset, and/or reduce the ultimate severity of a cancer in a mammal (e.g., prophylactically effective amounts).
[0022] The phrase "cause to be administered" refers to the actions taken by a medical professional (e.g., a physician), or a person controlling medical care of a subject, that control and/or permit the administration of the agent(s)/compound(s) at issue to the subject. Causing to be administered can involve diagnosis and/or determination of an appropriate therapeutic or prophylactic regimen, and/or prescribing particular
agent(s)/compounds for a subject. Such prescribing can include, for example, drafting a prescription form, annotating a medical record, and the like.
[0023] The terms "patient," "subject" or "individual" interchangeably refers to a mammal, for example, a human or a non-human mammal, including primates (e.g., macaque, pan troglodyte, pongo), a domesticated mammal (e.g., felines, canines), an agricultural mammal (e.g., bovine, ovine, porcine, equine) and a laboratory mammal or rodent (e.g., rattus, murine, lagomorpha, hamster).
[0024] The terms "inhibiting," "reducing," "decreasing" with respect to tumor or cancer growth or progression refers to inhibiting the growth, spread, metastasis of a tumor or cancer in a subject. The growth, progression or spread of a tumor or cancer is inhibited, reduced or decreased if the tumor burden is at least about 10%, 20%, 30%, 50%, 80%, or 100%) reduced, e.g., in comparison to the tumor burden prior to inhibition of one or more protein within or associated with the HIPPO intracellular signaling pathway selected from large tumor suppressor kinase 1 (LATS 1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7)) (and optionally serine/threonine kinase 3 (STK3)), optionally in combination with another active agent (e.g., an immune checkpoint inhibitor and/or a chemotherapeutic agent). In some embodiments, the growth, progression or spread of a tumor or cancer is inhibited, reduced or decreased by at least about 1-fold, 2-fold, 3-fold, 4-fold, or more in comparison to the tumor burden prior to inhibition of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with another active agent (e.g., an immune checkpoint inhibitor and/or a chemotherapeutic agent).
[0025] The term "antibody" is used in the broadest sense and includes fully assembled antibodies, tetrameric antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind an antigen (e.g., Fab', F(ab)2, Fv, single chain antibodies, diabodies), and recombinant peptides comprising the forgoing as long as they exhibit the desired biological activity. An "immunoglobulin" or "tetrameric antibody" is a tetrameric glycoprotein that consists of two heavy chains and two light chains, each comprising a variable region and a constant region. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antibody fragments or antigen-binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibody (dAb), complementarity determining region (CDR) fragments, CDR-grafted antibodies, single-chain antibodies (scFv), single chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibody, linear antibody; chelating recombinant antibody, a tribody or bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), an antigen- binding-domain immunoglobulin fusion protein, a camelized antibody, a VHH containing antibody, or a variant or a derivative thereof, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the
polypeptide, such as one, two, three, four, five or six CDR sequences, as long as the antibody retains the desired biological activity.
[0026] Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
[0027] Antibody variant" as used herein refers to an antibody polypeptide sequence that contains at least one amino acid substitution, deletion, or insertion in the variable region of the reference antibody variable region domains. Variants may be substantially homologous or substantially identical to the unmodified antibody.
[0028] A "neutralizing antibody" is an antibody molecule which is able to eliminate or significantly reduce a biological function of a target antigen to which it binds.
Accordingly, a "neutralizing" anti-target antibody is capable of eliminating or significantly reducing a biological function, such as enzyme activity, ligand binding, or intracellular signaling.
[0029] An "isolated" antibody is one that has been identified and separated and recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non- proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N- terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
[0030] As used herein, an antibody that "specifically binds" is "target specific", is
"specific for" target or is "immunoreactive" with the target antigen refers to an antibody or antibody substance that binds the target antigen with greater affinity than with similar antigens. In one aspect of the disclosure, the target-binding polypeptides, or fragments, variants, or derivatives thereof, will bind with a greater affinity to human target as compared to its binding affinity to target of other, i.e., non-human, species, but binding polypeptides that recognize and bind orthologs of the target are within the scope provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figures 1 A-E illustrate that LATS1/2 deletion enhances anchorage- independent tumor cell growth in vitro. See also, Figure 2. (A) Deletion of LATS1/2 abolishes YAP phosphorylation in response to serum starvation and actin polymerization inhibitor, Latrunculin B (LatB) treatment. Wild-type (WT) and two independent clones of LATSl/2 double knockout (dKO) B16-OVA melanoma cells were subjected to immunoblot (IB) analysis with antibodies to the indicated proteins. (B) Loss of LATSl/2 causes constitutive YAP/TAZ nuclear localization. LatB-treated or non-treated (control) B 16- OVA cells were subjected to immunostaining analysis. YAP/TAZ subcellular localization was determined by immunofluorescence staining for endogenous YAP/TAZ (green) along with DAPI for DNA (blue). Representative images are presented in the left panel. (Right panel) Cells in five randomly selected views (~ 100 cells) were selected for the
quantification of YAP/TAZ localization. N, nuclear; C, cytoplasmic. (C) LATSl/2 deletion promotes anchorage-independent growth of B 16-OVA cells in vitro. Soft-agar colony-formation assay was performed and the colonies were stained with crystal violet for quantification. (D) LATSl/2 deletion promotes anchorage-independent growth of SCC7 squamous cell carcinoma cells in vitro. Soft-agar colony-formation assay was performed and the colonies were quantified. (E) LATSl/2 deletion promotes anchorage-independent growth of 4T1 breast cancer cells in vitro. Soft-agar colony-formation assay was performed and the colonies were quantified. Data are means ± SD from 3 independent experiments (C-E). p values were determined using one-way ANOVA test followed by Tukey's multiple comparison test (C and D) or unpaired t-test (E). **p < 0.01; ***p < 0.001.
[0032] Figures 2A-G illustrate that LATSl/2 deletion enhances anchorage- independent tumor cell growth in vitro. Related to Figure 1. (A) LATSl/2 dKO B16-OVA cells grow similarly to WT on regular cell culture plates. Wild-type (WT) and two independent clones of LATSl/2 double knockout (dKO) B16-OVA melanoma cells (1 χ 105) were plated in 6-well culture dishes and cell number was determined with a
hemocytometer after the indicated times. Data are means ± SD of triplicate cultures from a representative experiment, ns, not significant (p > 0.05, two-way ANOVA test). (B) Deletion of LATSl/2 in SCC7 cells abolishes YAP phosphorylation in response to actin polymerization inhibitor Latrunculin B (LatB) treatment and glycolysis inhibitor 2-deoxy- D-glucose (2-DG) treatment. Wild-type (WT) and two independent clones of LATSl/2 double knockout (dKO) SCC7 squamous cell carcinoma cells were subjected to immunoblot (IB) analysis with antibodies to the indicated proteins. The mouse YAP SI 12 is equivalent to human YAP SI 27, which is the major regulatory site responsible for YAP cytoplasmic localization. Where indicated, gels containing phos-tag were employed for assessment of YAP phosphorylation status. YAP proteins can be separated into multiple bands in the presence of phos-tag depending on differential phosphorylation levels, with phosphorylated proteins migrating more slowly. (C) Loss of LATSl/2 promotes YAP/TAZ nuclear localization. LatB-treated or non-treated (control) SCC7 cells were subjected to
immunostaining analysis. YAP/TAZ subcellular localization was determined by immunofluorescence staining for endogenous YAP/TAZ (green) along with DAPI for DNA (blue). Representative images are presented in the left panel. (Right panel) Cells in five randomly selected views (~100 cells) were selected for the quantification of YAP/TAZ localization. N, nuclear; C, cytoplasmic. (D) LATSl/2 deletion promotes anchorage- independent growth of SCC7 cells in vitro. Representative images of the soft-agar colony- formation assay in Figure ID are shown. (E) Deletion of LATSl/2 in 4T1 cells abolishes YAP phosphorylation in response to serum starvation, LatB treatment, and 2-DG treatment. Wild-type (WT) and LATSl/2 double knockout (dKO) 4T1 breast cancer cells were subjected to immunoblot (IB) and phos-tag analysis. (F) Loss of LATSl/2 promotes YAP/TAZ nuclear localization. LatB-treated or non-treated (control) 4T1 cells were subjected to immunostaining analysis. YAP/TAZ subcellular localization was determined by immunofluorescence staining for endogenous YAP/TAZ (green) along with DAPI for DNA (blue). Representative images are presented in the left panel. (Right panel) Cells in five randomly selected views (~ 100 cells) were selected for the quantification of YAP/TAZ localization. N, nuclear; C, cytoplasmic.
(G) LATSl/2 deletion promotes anchorage-independent growth of 4T1 cells in vitro.
Representative images of the soft-agar colony-formation assay in Figure IE are shown. [0033] Figures 3A-G illustrate that Loss of LATSl/2 in tumors inhibits tumor growth in vivo. See also, Figure 4. (A) Deletion of LATSl/2 in B16-OVA melanoma inhibits tumor growth in vivo. Equal numbers of WT or LATSl/2 dKO B 16-OVA cells were transplanted into C57BL/6 mice and tumor growth was monitored after the indicated times. Data are represented as mean ± SEM; n = 8 tumors for each group. ***p < 0.001, two-way ANOVA test. (B) C57BL/6 mice were inj ected with WT or LATS 1/2 dKO B 16- OVA melanoma and tumor weight was determined 20 days after transplantation. Data are represented as mean ± SEM; n = 6 tumors for WT, n = 8 tumors for LATSl/2 dKO (note that two of the tumors were completely rejected). **p < 0.01, Mann-Whitney test. (C) LATSl/2 deletion in B16-OVA melanoma protects mice from tumor challenge. Kaplan- Meier tumor-free survival curves for mice injected with WT or LATSl/2 dKO B 16-OVA cells are shown (n = 14 mice for each group). ***p < 0.001, log-rank test. (D) Deletion of LATSl/2 in SCC7 squamous cell carcinoma inhibits tumor growth in vivo. WT or two independent clones of LATSl/2 dKO SCC7 cells were transplanted into C3H/HeOu mice and tumor growth was monitored after the indicated times. Data are represented as mean ± SEM; n = 8 tumors for each group, p values were determined using two-way ANOVA test, comparing each group to WT group. ***p < 0.001. (E) LATSl/2 deletion in SCC7 squamous cell carcinoma protects mice from tumor challenge. Kaplan-Meier tumor-free survival curves for mice injected with WT or LATSl/2 dKO SCC7 cells are shown (n = 4 mice for each group), p values were determined using log-rank test, comparing each group to WT group. **p < 0.01. (F) Deletion of LATSl/2 in 4T1 breast cancer inhibits tumor growth in vivo. BALB/c mice were injected with WT or LATSl/2 dKO 4T1 cells and primary tumor weight was determined 28 days after transplantation. Data are represented as mean ± SEM; n = 16 tumors for each group. ***p < 0.001, Mann-Whitney test. (G)
Deletion of LATSl/2 abolishes metastasis of 4T1 breast cancer cells. WT or LATSl/2 dKO 4T1 cells were transplanted into the mammary fat pad of BALB/c mice and lung metastasis of the primary tumor was determined 28 days after transplantation. Normal lung tissue was stained with black India ink, whereas tumor nodules remain white. The gross appearance of the lungs (left panel) and tumor nodules on lungs (right panel) were examined. Data are represented as mean ± SEM; n = 8 mice for each group. ***p < 0.001, Mann-Whitney test.
[0034] Figures 4A-C illustrate that loss of LATSl/2 in tumor cells inhibits tumor growth in vivo. Related to Figure 3. (A) Deletion of LATSl/2 in B 16-OVA melanoma inhibits tumor growth in vivo. Wild-type (WT) or clone #2 of LATSl/2 double knockout (dKO) B16-OVA cells were injected into C57BL/6 mice and tumor weight was determined 16 days after transplantation. Data are represented as mean ± SEM; n = 6 tumors for each genotype. **p < 0.01, Mann-Whitney test. (B) Deletion of LATSl/2 in SCC7 squamous cell carcinoma inhibits tumor growth in vivo. The gross appearance of C3H/HeOu mice challenged with WT or two independent clones of LATSl/2 dKO SCC7 cells was examined 18 days after transplantation. (C) Deletion of LATSl/2 in 4T1 breast cancer inhibits tumor growth in vivo. The gross appearance of the primary tumors of WT or LATSl/2 dKO 4T1 cells injected into BALB/c mice was examined 28 days after transplantation.
[0035] Figures 5A-I illustrate that LATSl/2-deficiency in tumor cells induces host anti -tumor immunity. See also, Figure 6. (A) LATSl/2 deletion induces immune responses. WT or LATSl/2 dKO B 16-OVA melanoma cells were injected into C57BL/6 mice. Tumors were paraffin-embedded and stained with hematoxylin and eosin (H&E) 12 days after transplantation. Arrowheads indicate infiltration of inflammatory cells. (B) CD45+ leukocytes infiltrate into LATSl/2-null tumors. Frozen sections from WT or LATSl/2 dKO B16-OVA melanomas were subjected to immunostaining analysis of CD45 (red) along with DAPI for DNA (blue). (C) LATSl/2 deletion does not affect ovalbumin (OVA) expression. WT and two independent clones of LATSl/2 dKO B16-OVA melanoma cells were subjected to immunoblot (IB) analysis with antibodies to the indicated proteins. (D) Loss of LATSl/2 in tumors enhances OVA-specific antibody production.
C57BL/6 mice were injected (or not) with WT or LATSl/2 dKO B 16-0 VA melanoma cells and serum anti-OVA IgG concentrations were determined by enzyme-linked
immunosorbent assay (ELISA) 12 days after transplantation. (E) LATSl/2-null tumors activate CD8+ T cells. Splenocytes from C57BL/6 mice injected as in (D) were re- stimulated ex vivo with SIINFEKL peptide and then subjected to flow cytometric analysis. SIINFEKL is an OVA-derived peptide being presented through the class I major histocompatibility complex (MHC class I) molecule, H-2Kb. Frequency of CD8+ T cells expressing activation markers, Granzyme B or interferon γ (IFN-γ), was determined. (F) LATSl/2 deletion in tumors increases tumor-specific CD8+ T cells. Splenocytes from C57BL/6 mice injected as in (D) were subjected to flow cytometric analysis. OVA-specific CD8+ T cells were quantified using Kb-SIINFEKL-tetramers and plotted as a percentage of total CD8+ T cells. Data are represented as mean ± SEM; n = 4 mice for no injected group, n = 10 mice for WT-injected group, n = 10 mice for LATSl/2 dKO-injected group (D-F). ***p < 0.001, one-way ANOVA test followed by Tukey's multiple comparison test (D-F). (G) Lymph node cells from LATSl/2 dKO tumor-injected mice show increased OVA- specific T cell response. C57BL/6 mice were injected as in (D) and the inguinal lymph nodes were cultured ex vivo with OVA protein (100 μg/ml) for 3 days. IFN-γ levels in the culture supernatants were determined by ELISA. Data are means ± SEM of triplicate cultures of pooled lymph node cells from 4 mice per group. ***p < 0.001, one-way ANOVA test followed by Tukey's multiple comparison test. (H) CD8+ T cells from
LATSl/2 dKO tumor-injected mice show increased OVA-specific cytotoxicity. C57BL/6 mice were injected as in (D) and CD8+ T cells were isolated from splenocytes. T cell cytotoxicity assay was performed with CFSE-labeled EL4 cells ex vivo. The frequency of CSFEhigh (irrelevant peptide control) and CFSElow (SIINFEKL loaded target) EL4 cells was determined by flow cytometric analysis 18 h after incubation and the percent of specific killing was plotted. Data are means ± SEM of 5 independent experiments with pooled CD8+ T cells from 3-4 mice per group. ***p < 0.001, one-way ANOVA test followed by Tukey's multiple comparison test. (I) CD8+ T cells infiltrate into LATSl/2 dKO tumors. WT or LATSl/2 dKO B16-OVA melanoma cells were injected into C57BL/6 mice and tumors were subjected to flow cytometric analysis 12 days after transplantation. Data are represented as mean ± SEM of the percentage of CD8+ T cells infiltrating into tumors among total CD45+ cells; n = 4 tumors for each group. ***p < 0.001, unpaired t-test.
[0036] Figures 6A-F illustrate that LATSl/2-deficiency in tumor cells stimulates host anti -tumor immunity. Related to Figure 5. (A) LATS1/2 deletion induces immune responses. Wild-type (WT) or LATS1/2 double knockout (dKO) 4T1 breast cancer cells were injected into BALB/c mice. Tumors were paraffin-embedded and stained with hematoxylin and eosin (H&E) 28 days after transplantation. Arrowheads indicate infiltration of inflammatory cells. (B) CD45+ leukocytes infiltrate into LATSl/2-null tumors. Frozen sections from WT or LATS1/2 dKO 4T1 breast cancers were subjected to immunostaining analysis of CD45 (red) along with DAPI for DNA (blue). (C) LATS 1/2- null tumors activate CD8+ T cells. Representative scatter plots of the gated CD8+ T cells in Figure 5E are shown. Gating of CD8+ T cells was performed after background assessment. Numbers indicate the percentage of Granzyme B or interferon γ (IFN-γ) positive cells in the gated CD8+ population. FSC, forward scatter. (D) LATS 1/2 deletion in tumors increases tumor-specific CD8+ T cells. Representative scatter plots of the gated CD8+ T cells in Figure 5F are shown. Gating of CD8+ T cells was performed after background assessment. Numbers indicate the percentage of tetramer positive cells in the gated CD8+ population. (E) CD8+ T cells from LATS 1/2 dKO tumor-challenged mice show increased OVA- specific cytotoxicity. Representative histograms of the gated CSFE+ EL4 cells in Figure 5H are shown in the upper panel. Gating of CSFE+ EL4 cells was performed after background assessment. Numbers indicate the percentage of the gated cells in CSFE+ population. Schematic representation of ex vivo cytotoxicity assay using CFSE-labeled EL4 cells is shown in the lower panel. CFSE, carboxyfluorescein succinimidyl ester. (F) CD8+ T cells infiltrate into LATS 1/2 dKO tumors. Representative scatter plots of the gated CD45+ cells in Figure 51 are shown. Gating of CD45+ T cells was performed after background assessment. Numbers indicate the percentage of CD8 positive cells in the gated CD45+ population.
[0037] Figures 7A-G illustrate that LATS 1/2 deletion in tumors stimulates host adaptive immunity and enhances tumor vaccine efficacy. (A) Co-injection of LATSl/2-null tumors suppresses tumor growth of the corresponding LATS 1/2 wild-type tumors in vivo. WT or LATS 1/2 dKO B16-OVA melanoma cells were injected into C57BL/6 mice and tumor growth was monitored after the indicated times (left panel). For co-injection experiments, WT and LATS1/2 dKO cells were injected into opposite flanks in the same mouse (right panel). "WT [with LATS1/2 dKO(#l)]" (blue line) indicates WT tumor growth, and "LATS1/2 dKO(#l) [with WT]" (yellow line) indicates LATS1/2 dKO tumor growth, in the co-injected mice. Data are represented as mean ± SEM; n = 8 tumors for WT or LATS1/2 dKO group, n = 6 tumors for each group co-injected, p value was determined using two-way ANOVA test, comparing WT [with LATS1/2 dKO(#l)] group to WT group. ***p < 0.001. (B) Deletion of LATS1/2 in tumors enhances tumor vaccine efficacy.
C57BL/6 mice were immunized with irradiated WT or LATS1/2 dKO B 16-0 VA cells (or PBS control). 12 days after immunization, mice were challenged with WT B16-OVA melanoma and tumor growth was monitored after the indicated times. Data are represented as mean ± SEM; n = 8 tumors for each group. PBS, phosphate buffered saline. (C) Kaplan- Meier tumor-free survival curves for mice immunized and challenged as in (B) are shown (n = 12 mice for each group). The survival curve of C57BL/6 mice challenged with WT B16- OVA melanoma without vaccination in Figure 2C is also shown in light grey color for reference. Schematic representation of vaccination experiment with irradiated-tumor cells is shown in the lower panel. Briefly, C57BL/6 mice were immunized intradermally at the base of the tail with equal numbers of irradiated WT or LATS1/2 dKO B16-OVA cells (one time vaccination, without any adjuvant), and then challenged with WT B16-OVA
melanoma 12 days after the initial immunization, p value was determined using two-way ANOVA test (B) or log-rank test (C), comparing WT-immunized group [WT→ WT] to LATS1/2 dKO-immunized group [LATS1/2 dKO(#l)→ WT]. ***p < 0.001. (D)
LATS1/2 dKO SCC7 cells confer cancer immunity. C3H/HeOu mice were first injected with non-irradiated LATS1/2 dKO SCC7 cells. 60 days after the initial injection, mice designated tumor-free were re-challenged with WT SCC7 cells and tumor growth was monitored [LATS1/2 dKO(#l)→ WT]. The tumor growth curve of WT SCC7 injected into naive C3H/HeOu mice in Figure 2D is also shown in light grey for reference [WT]. Data are represented as mean ± SEM; n = 8 tumors for each group. ***p < 0.001, two-way ANOVA test. (E) LATS1/2 dKO tumors grow similarly to WT in Rag-1 knockout (KO) mice that lack mature B and T lymphocytes. WT or LATS1/2 dKO B 16-0 VA cells were transplanted into Rag-1 KO mice and tumor growth was monitored after the indicated times. Data are represented as mean ± SEM; n = 8 tumors for each group, ns, not significant (p > 0.05, two-way ANOVA test). (F) Kaplan-Meier tumor-free survival curves for mice transplanted as in (E) are shown (n = 10 mice for each group), ns, not significant (p > 0.05, log-rank test). (G) Co-injection of LATSl/2-null tumors does not suppress WT tumor growth in Rag-1 KO mice. WT and LATS1/2 dKO B16-OVA melanoma cells were injected into opposite flanks in the same Rag-1 KO mouse. Data are represented as mean ± SEM; n = 6 tumors for each group. The tumor growth curves shown in (E) are shown in a lighter color for reference, p value was determined using two-way ANOVA test, comparing "WT [with LATS1/2 dKO(#l)]" group to "WT" group, ns, not significant (p > 0.05).
[0038] Figures 8A-H illustrate that overexpression of YAP or TAZ in tumor cells partially suppresses tumor growth in vivo. Related to Figure 9. (A) YAP/TAZ activation in LATSl/2-null tumors. Wild-type (WT) or LATS1/2 double knockout (dKO) B 16-0 VA melanoma cells were injected into C57BL/6 mice. 20 days after transplantation, tumor samples were harvested and then subjected to immunoblot (IB) analysis with antibodies to the indicated proteins. YAP dephosphorylation and TAZ accumulation were evident in tumors deficient for LATS1/2. n = 3 tumors for each group. (B) Increased YAP/TAZ transcriptional activity in LATSl/2-null tumors. The tumor samples, same as in (A), were subjected to RT and real-time PCR analysis of the indicated YAP/TAZ target gene mRNA. Normalized data are expressed relative to the corresponding value for WT tumors and are mean ± SEM; n = 3 tumors for each group. *p < 0.05, unpaired t-test. (C) Expression levels of YAP(5SA) or TAZ(4SA) in B16-OVA melanoma cells. YAP(5SA) and
TAZ(4SA) are active mutants of YAP/TAZ with all five/four LATS phosphorylation sites mutated to alanine, thereby unresponsive to inhibition by the LATS 1/2 kinase. B16-OVA cells stably expressing YAP(5SA), TAZ(4SA), or control vector were subjected to immunoblot (IB) analysis with antibodies to the indicated proteins. (D) YAP(5SA) or TAZ(4SA) overexpression promotes anchorage-independent growth of B 16-OVA cells in vitro. Soft-agar colony-formation assay was performed and the colonies were stained with crystal violet for quantification. Data are means ± SD from 3 independent experiments. **p < 0.01; ***p < 0.001, one-way ANOVA test followed by Tukey's multiple comparison test. (E) YAP(5SA) or TAZ(4SA) overexpression in B16-OVA melanoma inhibits tumor growth in vivo. B 16-OVA cells stably expressing YAP(5SA), TAZ(4SA), or control vector were injected into C57BL/6 mice and tumor growth was monitored after the indicated times. Data are represented as mean ± SEM; n = 8 tumors for each group, p values were determined using two-way ANOVA test, comparing each group to control group. ***p < 0.001. (F) Expression levels of YAP(5SA) or YAP(5SA/S94A) in B16-OVA melanoma cells. TEADl-4 are the major YAP-associated transcription factors, and their binding to YAP requires the Ser94 residue in YAP. YAP(5SA/S94A) is unable to bind TEADl-4 and thus fails to promote TEAD-dependent transcription. (G) YAP(5SA), but not YAP(5SA/S94A), promotes downstream target gene transcription in B 16-OVA cells. Total RNA extracted from B16-OVA cells stably expressing the indicated constructs was subjected to RT and real-time PCR analysis of the indicated YAP/TAZ target genes. Data are means ± SD of triplicates from a representative experiment. (H) Tumor growth suppression by YAP in vivo requires TEAD-binding of YAP. B16-OVA cells stably expressing YAP(5SA/S94A) were injected into C57BL/6 mice and tumor growth was monitored after the indicated times. The tumor growth curves shown in (H) are presented in a lighter color for reference. Data are represented as mean ± SEM; n = 8 tumors for each group, p value was determined using two-way ANOVA test, comparing YAP(5SA) group to YAP(5SA/S94A) group. ***p < 0.001.
[0039] Figures 9A-F illustrate that extracellular vesicles (EVs) released from
LATSl/2-null tumor cells stimulate host immune responses. See also Figures 8-13. (A) Conditioned medium from LATSl/2-deficient tumor cells stimulates cross-presentation in vitro as indicated by the increased OT-I CD8+ T cell proliferation. Bone marrow-derived dendritic cells (BMDCs) were pretreated for 18 h with conditioned medium from WT or LATSl/2 dKO B 16-0 VA melanoma cells (or control medium) and pulsed with OVA protein (10 μg/ml) for the last 4 h. BMDCs were then subjected to an in vitro cross- presentation assay using CFSE-labeled CD8+ T cells isolated from OVA-specific T cell receptor transgenic OT-I mice. OT-I CD8+ T cells proliferate when they were stimulated with OVA antigen via cross-presentation by BMDCs, resulting in dilution of CFSE content. Representative histograms of the gated CD8+ T cells are shown in the left panel. Gating of CD8+ T cells was performed after background assessment. The division index was calculated and data are presented as means ± SEM of 3 independent experiments in the right panel. ***p < 0.001, one-way ANOVA test followed by Tukey's multiple comparison test. (B) EVs from LATSl/2-deficient tumor cells are more potent to stimulate IL-12 production in BMDCs. BMDCs were stimulated for 18 h with conditioned medium or EVs from WT or LATSl/2 dKO B 16-OVA cell culture supematants and then IL-12 levels in the culture supematants were determined by ELISA. Data are represented as mean ± SEM; n = 3 independent experiments for conditioned medium stimulation, n = 4 independent experiments for EVs stimulation. *p < 0.05; ***p < 0.001, one-way ANOVA test followed by Tukey's multiple comparison test. (C) EVs from LATSl/2-deficient tumor cells increase immunogenicity of LATSl/2 wild-type tumor cells. C57BL/6 mice were inoculated with irradiated WT B 16-OVA cells at the base of the tail and EVs freshly isolated from culture supematants of equal numbers of WT or LATSl/2 dKO B16-OVA cells were injected every 3 days (day 0, 3, 6, and 9). At day 12, mice were challenged with WT B16-0VA melanoma and tumor growth was monitored. Data are represented as mean ± SEM; n = 8 tumors for each group. The tumor growth curves shown in Figure 4B are presented in a lighter color for reference, p value was determined using two-way ANOVA test, comparing WT EV- immunized group [WT + WT EVs→ WT] to LATS 1/2 dKO EV-immunized group [WT + LATSl/2 dKO EVs→ WT]. ***p < 0.001. (D) LATSl/2-null tumor cells secrete more EV particles. EVs isolated from culture supernatants of equal numbers of WT or LATSl/2 dKO B16-OVA cells were subjected to nanoparticle tracking analysis (NanoSight) to quantify the number and size distribution. The numbers of particles are presented as means ± SEM of 3 independent experiments. **p < 0.01, unpaired t-test. (E) LATSl/2-null tumor cells secrete more EV proteins. EVs were isolated from culture supernatants of equal numbers of WT or LATSl/2 dKO B16-OVA cells and protein concentrations were determined. Data are means ± SEM of 6 independent experiments. ***p < 0.001, unpaired t-test. (F) EVs from LATSl/2-deficient or YAP(5SA)-overexpressing tumor cells contain higher amounts of RNA than EVs from WT tumor cells. EVs were isolated from culture supernatants of equal numbers of WT, LATSl/2 dKO, or YAP(5SA)-overexpressing B16- OVA cells and RNA concentrations were determined by Agilent TapeStation. Data are means ± SEM of 3 independent experiments. ***p < 0.001, one-way ANOVA test followed by Tukey's multiple comparison test. [0040] Figures 10A-B illustrate that extracellular vesicles (EVs) released from
LATSl/2-null tumor cells stimulate host immune responses. Related to Figure 9. (A) Enrichment of the EV protein markers, such as CD81, ALIX, and FLOT1 in the EV preparation. Whole cell lysate (WCL) of Wild-type (WT) or LATSl/2 double knockout (dKO) B16-OVA melanoma cells as well as EVs isolated from their culture supernatants were subjected to immunoblot (IB) analysis with antibodies to the indicated proteins. Equal amounts of protein samples (2.5 μg) were resolved in SDS-PAGE in non-reducing conditions. (B) Detergent treatment abolishes the activity of EV preparations in stimulating bone marrow-derived dendritic cells (BMDCs). Culture supernatants of WT or LATSl/2 dKO B16-OVA melanoma cells were either treated or untreated with detergent (1% Triton X-100) and then ultracentrifuged to isolate EVs. The resulting EV pellets were re- suspended in PBS and used to stimulate BMDCs. 18 h after incubation, IL-12 levels in the culture supernatants of BMDC were determined by ELISA. Data are represented as mean ± SEM of 3 independent experiments, p value was determined using one-way ANOVA test followed by Tukey's multiple comparison test. ***p < 0.001; ns, not significant (p > 0.05). [0041] Figures 11 A-F illustrate that extracellular vesicles (EVs) released from
LATSl/2-null tumors contain more nucleic-acid-binding proteins in comparison to wild- type EVs. Related to Figure 9. (A) LATSl/2-null tumor cells secrete more EVs. EVs isolated from culture supematants of WT or LATSl/2 dKO B16-OVA melanoma cells were subjected to nanoparticle tracking analysis (NanoSight) to quantify the number and size distribution. Representative histograms in Figure 9D are shown. (B) Proteomic profiling of total proteins identified in EVs show enrichment of previously reported exosomal and microvesicle cargo proteins. EVs were isolated from culture supematants of WT or LATSl/2 dKO B16-OVA cells and subjected to mass spectrometry analysis. Enrichment analysis of the Gene Ontology (GO) cellular component of total EV proteins identified
(1,772 proteins) was done using the PANTHER program. The enrichment p-value of each term was transformed to a -logio(p-value). The top 3 most significantly enriched cellular components are indicated. (C) Heatmap of the total EV proteins identified. Absolute protein abundances were estimated using the iBAQ algorithm. The iBAQ-scaled protein expression (Ex) was transformed to a log2(Ex) and the scale indicates relative expression. Data represent two independent biological replicates. (D) RNA binding and nucleic-acid- binding proteins are enriched in EVs from LATSl/2-null tumor cells. Enrichment analysis of the GO molecular function of the top 100 most significantly increased proteins in LATSl/2 dKO EVs is shown. The enrichment p-value of each term was transformed to a - logio(p-value). (E) EVs from LATSl/2-deficient tumor cells or YAP(5SA)-overexpressing tumor cells contain higher amounts of RNA than EVs from WT tumor cells. EVs were isolated from culture supematants of equal numbers of WT, LATSl/2 dKO, or YAP(5SA)- overexpressing B16-OVA cells and RNA concentrations were determined by Agilent TapeStation. Representative histograms in Figure 9F are shown. (F) EVs from B16-OVA cells contain single stranded RNA. EVs were isolated from culture supematants of equal numbers of WT or LATSl/2 dKO B16-OVA cells. RNA was then purified from EV samples and either treated or untreated with single-strand-specific ribonuclease (RNase A, the reaction was performed under high salt concentrations to achieve single-strand specificity), followed by agarose gel electrophoresis in non-denaturing conditions. [0042] Figures 12A-D illustrate that EVs from LATS 1/2-depleted tumor cells stimulate anti-tumor immunity via the Toll-like receptors (TLRs)-type I interferon (IFN) pathway. Related to Figure 13. (A) Schematic representation of the endogenous nucleic- acid-sensing pathways and TLR signaling. TLR, toll-like receptor; LPS,
lipopolysaccharide; dsRNA, double-stranded RNA; ssRNA, single-stranded RNA; rRNA, ribosomal RNA; TRIF, TIR-domain-containing adapter-inducing interferon-γ; MYD88, myeloid differentiation primary response 88; STING, stimulator of interferon genes; IL, interleukin; IFN, interferon. (B) WT or LATS l/2 dKO B 16-OVA cells were injected into Myd88 knockout (KO) mice and tumor growth was monitored after the indicated times. Data are represented as mean ± SEM; n = 8 tumors for each group. (C) WT or LATSl/2 dKO B 16-OVA cells were injected into Ticaml (also known as TRIF) KO mice and tumor growth was monitored after the indicated times. Data are represented as mean ± SEM; n = 10 tumors for each group. (D) LATS l/2 deletion has little effect on the mRNA abundance of type I IFN in tumor cells. WT or LATS l/2 dKO B 16-OVA cells were stimulated (or not) with 2 μ^ν ΐ poly(LC) complexed with 4 μg/ml Poly Jet™ for 24 h. Total RNA was extracted from the cells and then subjected to RT and real-time PCR analysis of the indicated mRNA. Data are means ± SD of triplicates from a representative experiment.
[0043] Figures 13A-I illustrate that LATSl/2-depleted tumor EVs stimulate antitumor immunity via the Toll-like receptors (TLRs)-type I interferon (IFN) pathway. See also, Figure 12. (A-G) Tumor protection by LATS l/2 deletion requires the host TLR-
MYD88/TRIF pathway. WT or LATSl/2 dKO B 16-OVA cells were transplanted into mice deficient in Myd88 (A; n = 12 mice for WT group, n = 13 mice for LATS l/2 dKO group), Ticaml (also known as TRIF) (B; n = 6 mice for each group), Tmeml73 (also known as STING) (C; n = 4 mice for each group), Caspl (also known as Caspase-1) (D; n = 4 mice for WT group, n = 5 mice for LATS l/2 dKO group), Tlr4 (E; n = 9 mice for each group), Tlr7 (F; n = 8 mice for each group), or Tlr9 (G; n = 7 mice for each group) and Kaplan- Meier tumor-free survival curves are shown. The survival curves of wild-type C57BL/6 mice injected with WT or LATS l/2 dKO B 16-OVA in Figure 2C are shown in a lighter color for reference, p value was determined using log-rank test, comparing each KO mice injected with LATS l/2 dKO B 16-0 VA cells (orange) to corresponding wild-type C57BL/6 mice injected with LATS l/2 dKO B 16-OVA cells (light red), ns, not significant (p > 0.05); *p < 0.05; **p < 0.01 ; ***p < 0.001. (H) Functional type I IFN signaling is required for tumor protection by LATSl/2-deficiency. WT or LATS l/2 dKO B 16-OVA cells were injected into Ifnarl KO mice that lack functional type I IFN receptor and tumor growth was monitored after the indicated times. The tumor growth curves of WT or LATS l/2 dKO B 16-OVA cells injected into wild-type C57BL/6 mice in Figure 2A are shown in a lighter color for reference. Data are represented as mean ± SEM; n = 8 tumors for each group. (I) Kaplan-Meier tumor-free survival curves for mice injected as in (H) are shown (n = 8 mice for each group). The survival curves of wild-type C57BL/6 mice injected with WT or LATSl/2 dKO B16-OVA in Figure 2C are shown in a lighter color for reference, p value was determined using log-rank test, comparing Ifnarl KO mice injected with LATSl/2 dKO B16-OVA cells (orange) to corresponding wild-type C57BL/6 mice injected with LATSl/2 dKO B16-OVA cells (light red). ***p < 0.001. [0044] Figure 14 illustates a model for the regulation of anti -tumor immunity by the
HIPPO intracellular signaling in tumors. Poorly immunogenic tumor cells evade host immune defenses despite expressing antigenic neoepitopes. LATSl/2 deletion in tumor cells stimulates nucleic-acid-rich EV secretion, which induces a type I IFN response via the TLRs-MYD88/TRTF pathway. Type I IFN stimulates multiple components of host immune responses, including cross-presentation of tumor-derived antigens by antigen-presenting cells and T cell activation. Activated T cells, in turn, facilitate tumor-specific responses of cytotoxic T cells and antibody production by B cells, promoting tumor destruction. Loss of LATSl/2 in tumors thus leads to rejection of both LATSl/2-deficient and LATS Inadequate tumor cells by enhancing host anti-tumor immune responses. [0045] Figures 15 A-D. (A) Deletion of LATSl/2 in 168FARN breast cancer inhibits tumor growth in vivo. 168FARN cells were transfected with control or LATS1/2- targeting CRISPR plasmids and then selected with puromycin for 3 days. After expansion, equal numbers of control (blue line) or LATSl/2-targetting (red line) CRISPR-transfected 168FARN cells were transplanted into BALB/c mice and tumor growth was monitored after the indicated times. Data are represented as mean ± SEM; n = 8 tumors for each group. (B) Identification of candidate LATSl/2 inhibitors. A total of 17,666 previously characterized small compounds were screened for LATSl/2 inhibitors based on their kinome profiling in the public databases (DSigDB: http://tanlab.ucdenver.edu/DSigDB/DSigDBvLO/, LINCS: http://lincs.hms.harvard.edu). 22 compounds were selected with the threshold of Kd < 1 μΜ or POC < 15% for either LATS1 or LATS2. Among them, 3 compounds shown in this figure were validated as direct LATS inhibitors. (C) A443654, Lestaurtinib, and GSK- 690693 directly inhibit LATS1 kinase activity in vitro. Endogenous LATS1 was immunoprecipitated from HEK293 A cells and the resulting immunoprecipitates were subjected to in vitro kinase assay in the presence of the indicated small compounds. For performing the in vitro kinase assay, the immunoprecipitated LATS1 were washed two times with lysis buffer, followed by once with wash buffer (40 mM HEPES pH 7.5, 200 mM NaCl) and once with kinase assay buffer (30 mM HEPES pH 7.5, 50 mM potassium acetate, 5 mM MgCl2). The immunoprecipitates were then subjected to a kinase assay in 30 μΐ of kinase assay buffer supplemented with 500 μΜ ATP, and 1 μ of GST- YAP expressed and purified from Escherichia coli as substrates. The reaction mixtures were incubated for 30 min at 30°C, terminated with SDS sample buffer, and subjected to immunoblot analysis. (D) A443654, Lestaurtinib, and GSK-690693 inhibit LATS1/2 kinase activity and induce YAP dephosphorylation in SCC7 squamous cell carcinoma cells. SCC7 cells were treated with the indicated small compounds for 2 h and then subjected to phos-tag analysis. YAP proteins can be separated into multiple bands in the presence of phos-tag depending on differential phosphorylation levels, with phosphorylated proteins migrating more slowly. [0046] Figures 16A-G illustrate (A) Deletion of LATS1/2 in 67 R breast cancer inhibits tumor growth in vivo. 67NR cells were transfected with control or LATS1/2- targetting CRISPR plasmids and then selected with puromycin for 3 days. After expansion, equal numbers of control (black line) or LATSl/2-targetting (red line) CRISPR-transfected 67 R cells were transplanted into BALB/c mice and tumor growth was monitored after the indicated times. Data are represented as mean ± SEM; n = 8 tumors for each group. ***p < 0.001, two-way ANOVA test. (B) Deletion of LATS1/2 in GL261 glioma inhibits tumor growth in vivo. Equal numbers of WT (black line) or LATS1/2 dKO (red line) GL261 cells were transplanted into C57BL/6 mice and tumor growth was monitored after the indicated times. LATSl/2-deficient GL261 cells were created through the CRISPR system. GL261 cells were transiently transfected with LATSl/2-targetting CRISPR plasmids and selected with puromycin for 3 days. Cells were then single-cell sorted by FACS and knockout clones were selected by immunoblot analysis for the lack of LATS1/2 proteins and YAP phosphorylation. Data are represented as mean ± SEM; n = 8 tumors for each group. ***p < 0.001, two-way ANOVA test. (C) Deletion of LATS1/2 in MB49 bladder cancer inhibits tumor growth in vivo. Equal numbers of WT (black line) or LATS1/2 dKO (red line) MB49 cells were transplanted into C57BL/6 mice and tumor growth was monitored after the indicated times. LATSl/2-deficient MB49 cells were created through the CRISPR system. MB49 cells were transiently transfected with LATSl/2-targetting CRISPR plasmids and selected with puromycin for 3 days. Cells were then single-cell sorted by FACS and knockout clones were selected by immunoblot analysis for the lack of LATS1/2 proteins and YAP phosphorylation. Data are represented as mean ± SEM; n = 8 tumors for each group. ***p < 0.001, two-way ANOVA test. (D) Deletion of LATS1/2 in MC38 colon cancer inhibits tumor growth in vivo. Equal numbers of WT (black line) or LATS1/2 dKO (red line) MC38 cells were transplanted into C57BL/6 mice and tumor growth was monitored after the indicated times. LATSl/2-deficient MC38 cells were created through the CRISPR system. MC38 cells were transiently transfected with LATSl/2-targetting CRISPR plasmids and selected with puromycin for 3 days. Cells were then single-cell sorted by FACS and knockout clones were selected by immunoblot analysis for the lack of LATS1/2 proteins and YAP phosphorylation. Data are represented as mean ± SEM; n = 8 tumors for each group. ***p < 0.001, two-way ANOVA test. (E) Schematic representation of LATS1/2 inhibitor experiment with SCC7 squamous cell carcinoma is shown. Briefly, C3H/HeOu mice were injected with WT SCC7. Five days after injection, mice were then treated (or not treated) with intratumoral injection of DMSO control or A443654 (50 nmol) every day for 6 days (day 5, 6, 7, 8, 9, and 10). Tumor growth was monitored until mice were sacrificed at day 21. (F) Intratumoral injection of the LATS1/2 inhibitor A443654 inhibits tumor growth in vivo. SCC7 cells were transplanted into C3H/HeOu mice and LATS1/2 inhibitor experiment was performed as described in (E). Data are represented as mean ± SEM; n = 4 tumors for each group, p value was determined using two-way
ANOVA test, comparing DMSO group to A443654 group. *p < 0.05. (G) Intratumoral injection of the LATS1/2 inhibitor A443654 activates CD8+ T cells. Tumor-infiltrating leukocytes from C3H/HeOu mice injected and treated as in (F) were subjected to flow cytometric analysis at day 21. Frequency of CD8+ T cells expressing activation markers, Granzyme B or interferon γ (IFN-γ), was determined. Data are means of pooled leukocytes from 4 tumors per group.
DETAILED DESCRIPTION
[0047] Disclosed herein, in certain embodiments, are methods of treating or preventing a cancer in an individual in need thereof, comprising reducing or inhibiting the activity in a cancerous cell of one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATSl), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase {e.g., MAP4K1/2/3/4/5/6/7) (and optionally serine/threonine kinase 3 (STK3). Further disclosed herein, in certain embodiments, are tumor cells and
populations of tumor cells having reduced or eliminated expression of one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATSl), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase {e.g.,
MAP4K1/2/3/4/5/6/7) (and optionally serine/threonine kinase 3 (STK3), and extracellular vesicles (EVs) and populations of EVs and cell lysates of such tumor cells. Additionally, disclosed herein, in certain embodiments, are vaccine compositions (e.g., capable of inducing an therapeutically effective immune response) comprising: (a) a cancerous cell having reduced activity or expression of one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally serine/threonine kinase 3 (STK3), and (b) a pharmaceutically-acceptable excipient.
1. Introduction [0048] Cellular transformation, tumor growth, and metastasis comprise a multistep process that requires continuous rewiring of signaling pathways and alterations of the reciprocal interaction between cancer cells and the tumor microenvironment, thereby allowing cells to acquire features to become fully neoplastic and eventually malignant (Hanahan and Weinberg, (2011) Cell 144, 646-674). The Hippo pathway has gained great interest in recent years as being strongly involved in several of these key hallmarks of cancer progression (Harvey et al., (2013) Nat. Rev. Cancer 13, 246-257; Moroishi et al., (2015) Nat. Rev. Cancer 15, 73-79), and in general, serves important regulatory functions in organ development, regeneration, and stem cell biology (Johnson and Haider, (2014) Nat. Rev. DrugDiscov. 13, 63-79; Wackerhage et al., (2014) Sci. Signal. 7, re4; Yu et al., (2015) Cell 163, 811-828). The heart of the mammalian Hippo pathway is a kinase cascade involving mammalian STE20-like protein kinase 1 (MST1; also known as STK4) and MST2 (also known as STK3) (homologs of Drosophila Hippo), as well as two groups of MAP4Ks (mitogen-activated protein kinase kinase kinase kinase); MAP4K 1/2/3/5
(homologs of Drosophila Happyhour) and MAP4K4/6/7 (homologs of Drosophila
Misshapen), and in addition, the large tumour suppressor 1 (LATS1) and LATS2 (homologs of Drosophila Warts) (Meng et al., (2016) Genes Dev. 30, 1-17; Sun and Irvine, (2016) Trends Cell Biol. 26(9):694-704. When the Hippo pathway is activated, MST1/2 or MAP4Ks phosphorylate and activate the LATS1/2 kinases, which in turn directly phosphorylate and inactivate Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ; also known as WWTR1), the two major downstream effectors that mediate transcriptional output of the Hippo pathway (Hansen et al., (2015) Trends Cell Biol. 25, 499-513). Activation of LATS1/2 kinases (and inactivation of YAP/TAZ) represents the major functional output of the Hippo pathway. [0049] Previous studies have convincingly established the Hippo pathway as a suppressor signal for cellular transformation and tumorigenesis, though other studies revealed its oncogenic functions in certain contexts (Moroishi et al., (2015) Nat. Rev.
Cancer 15, 73-79; Wang et al., (2014) Cancer Metastasis Rev. 33, 173-181). Deletion of MST1/2 in mouse liver results in tissue overgrowth and tumor development, demonstrating the tumor suppressor function of these kinases (Zhou et al., (2009) Cancer Cell 16, 425- 438). Complementary, overexpression of YAP in mouse liver also promotes tissue overgrowth and tumorigenesis (Camargo et al., (2007) Curr. Biol. 17, 2054-2060; Dong et al., (2007) Cell 130, 1120-1133). These studies have demonstrated an inhibitory role of the Hippo pathway in tumor initiation. However, effects of the Hippo pathway in tumor growth, especially in the context of reciprocal interactions between tumor cells and host anti-tumor immune responses, remain largely unknown. In the current study, we investigate the role of the LATS1/2 kinases in the growth of established tumors in the context of antitumor immunity. Surprisingly, inactivation of the "tumor suppressor" LATS1/2 in tumor cells strongly suppresses tumor growth in immune competent, but not immune
compromised, mice due to the induction of host anti -tumor immune responses. Our data indicate a new paradigm for how tumor immunogenicity is regulated through the Hippo signaling pathway in tumor cells and also have implications for targeting LATS1/2 in cancer immunotherapy. [0050] The present compositions and methods are based, in part, on the discovery of an unexpected role of the HIPPO intracellular signaling in suppressing anti-tumor immunity. We demonstrate that, in three different murine syngeneic tumor models (B16, SCC7, and 4T1), loss of the HIPPO intracellular signaling kinases LATS1/2 in tumor cells inhibits tumor growth. Tumor regression by deletion of one or more proteins within or associated with the HIPPO intracellular signaling selected from large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally serine/threonine kinase 3 (STK3) involves adaptive immune responses, and deficiency of one or more proteins within or associated with the HIPPO intracellular signaling selected from large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase {e.g., MAP4K1/2/3/4/5/6/7) (and optionally serine/threonine kinase 3 (STK3) enhances tumor vaccine efficacy.
Deletion in tumors of one or more proteins within or associated with the HIPPO
intracellular signaling selected from large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally serine/threonine kinase 3 (STK3) improves tumor immunogenicity, leading to tumor destruction by enhancing anti-tumor immune responses. 2. HIPPO Pathway Protein-Deficient Cellular Material a. Compositions
[0051] Provided are tumor cells and populations of tumor cells having reduced or eliminated expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATSl), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4
(STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and extracellular vesicles (EVs) and populations of EVs and cell lysates of such tumor cells. In certain embodiments, the expression and/or activity of serine/threonine kinase 3 (STK3) is also reduced or eliminated. [0052] In varying embodiments, the expression and/or activity levels of LATSl,
LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) is reduced or knocked-down. Reduction in LATSl, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) expression levels can be achieved using any method known in the art, e.g., using inhibitory nucleic acids that specifically hybridize to LATSl and/or LATS2. For purposes of reducing the activity of sEH, siRNAs to the gene encoding sEH can be specifically designed using computer programs. Numerous algorithms for designing siRNAs are available on the internet, including those listed, e.g., at rnaiweb.com; by ThermoFisher at
rnaidesigner.thermofisher.com/rnaiexpress/; and by IDT at
biotools.idtdna.com/site/order/designtool/index/DSIRNA_CUSTOM. LATSl and/or
LATS2 expression levels are considered to be reduced, e.g., if the detected levels of LATSl and/or LATS2 mRNA and/or protein in comparison to wild-type levels is reduced by at least 10%, e.g., by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
[0053] In varying embodiments, the genes encoding one or both of LATSl and LATS2 have been knocked out, eliminated or rendered non-functional. In varying embodiments, the genes encoding both of LATSl and LATS2 have been knocked out, eliminated or rendered non-functional. Genes encoding one or both of LATSl and LATS2 can be knocked out or rendered non-functional using any method known in the art. In varying embodiments one or both genes encoding of LATS1 and/or LATS2 are knocked out or rendered non-functional using CRISPR or homologous recombination.
[0054] Additionally, in varying embodiments, the tumor cell has elevated expression levels of YAP and/or TAZ, e.g., by recombinant expression of YAP and/or TAZ, or active mutants of YAP and/or TAZ. The active mutants of YAP and TAZ are defined that they at higher nuclear localization, or more stable, or higher transcription stimulating activity. In varying embodiments, one or more recombinant polynucleotides encoding YAP and/or TAZ are incorporated into the genome of the tumor cell. In varying embodiments, one or more recombinant polynucleotides encoding YAP and/or TAZ have been introduced episomally into the tumor cell. Introduction of one or more recombinant polynucleotides encoding YAP and/or TAZ into the tumor cell can be accomplished using any method known in the art, e.g., by infection with a virus (e.g., an adenovirus, an adeno-associated virus, a lentivirus, a retrovirus), or by introduction of a plasmid (e.g., via electroporation, a liposome, a nanoparticle). In varying embodiments, the recombinant polynucleotides encoding YAP and/or TAZ can be in a recombinant expression cassette, under the control of and operably linked to a constitutive or inducible promoter. YAP and/or TAZ expression levels are considered to be elevated or increased, e.g., if the detected levels of YAP and/or TAZ mPvNA and/or protein in comparison to wild-type levels is elevated or increased by at least 10%, e.g., by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, or more.
[0055] In varying embodiments, the tumor cell expresses or overexpresses a tumor- associated antigen (TAA). Examples of known TAAs include without limitation, melanoma associated antigens (MAGE-1, MAGE-3, TRP-2, melanosomal membrane glycoprotein gplOO, gp75 and MUC-1 (mucin- 1) associated with melanoma); CEA (carcinoembryonic antigen) which can be associated, e.g., with ovarian, melanoma or colon cancers; folate receptor alpha, WAP four-disulfide core domain 2 (HE4) or mesothelin expressed by ovarian carcinoma; free human chorionic gonadotropin beta (hCGP) subunit expressed by many different tumors, including but not limited to myeloma; HER-2/neu associated with breast cancer; encephalomyelitis antigen HuD associated with small-cell lung cancer;
tyrosine hydroxylase associated with neuroblastoma; prostate-specific antigen (PSA) associated with prostate cancer; CA125 associated with ovarian cancer; and the idiotypic determinants of a B cell lymphoma can generate tumor-specific immunity (attributed to idiotype-specific humoral immune response). Moreover, antigens of human T cell leukemia virus type 1 have been shown to induce specific CTL responses and antitumor immunity against the virus-induced human adult T cell leukemia (ATL). See, e.g., Haupt, et al., Experimental Biology and Medicine (2002) 227:227-237; Ohashi, et al., Journal of
Virology (2000) 74(20):9610-9616. Other TAAs are known and find use for co- administration with tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS 1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells, optionally in combination with a chemotherapeutic agent.
[0056] In varying embodiments, the tumor cell is rendered replication incompetent, e.g., via irradiation.
[0057] In varying embodiments, the tumor cell is obtained from a patient to be treated by reinjection of the tumor cell after expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7) (and optionally serine/threonine kinase 3 (STK3) has been reduced or eliminated. In varying embodiments, the tumor cell is selected from the group consisting of a melanoma, a glioma, a colon cancer, a squamous cell carcinoma and a breast carcinoma. In varying embodiments, the tumor cell is from a cancer selected from the group consisting of sarcoma, lymphoma, hematological cancer, skin cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, colon cancer, rectal cancer, urogenital cancer, hepatic cancer, thyroid cancer, esophageal cancer, bladder cancer, renal cancer, brain cancer (e.g., glioma) and head and neck cancer. b. Methods Of Making Cellular Material Deficient For One Or More
HIPPO Pathway Proteins
[0058] In some embodiments, the nucleic acid molecule that partially, substantially, or completely deletes, silences, inactivates, down-regulates, reduces, or inhibits, activity or expression of one or more genes within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7) (and optionally STK3) is introduced into a cancerous cell via an expression vector, under the appropriate conditions, to induce or cause partial, substantial, or complete deletion, silencing, inactivation, down-regulation, reduction, or inhibition of one or more genes encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3).
[0059] In some embodiments, activity or expression of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) is partially, substantially, or completely deleted, silenced, inactivated, down-regulated, reduced or inhibited by introducing into the cancerous cell nucleic acid molecules by transfection or transduction. In some embodiments, transfection involves introducing a nucleic acid molecule via non-viral methods into a cancerous cell. In some embodiments, the non-viral methods used when transfecting cancerous cells are chemical based, non- chemical based, and particle based. In some embodiments, the chemical based methods to transfect cancerous cells are calcium phosphate precipitation, dendrimer binding, lipofection comprising cationic liposomes, or cationic polymer complex uptake. In some embodiments, non-chemical based methods to transfect cancerous cells are electroporation, cell squeezing, sonoporation, optical transfection, impalefection, or hydrodynamic delivery. In some embodiments, particle based methods to transfect cancerous cells are gene gun delivery, magnetofection, impalefection, or particle bombardment. In some embodiments, other methods to transfect cancerous cells utilize cell-penetrating peptides; virosomes, which are a hybrid of liposomes and an inactivated virus; inorganic nanoparticles such as gold, silica, or iron oxide; or polymersomes.
[0060] In some embodiments, transduction involves introducing a nucleic acid molecule via viral methods into a cancerous cell. In some embodiments, a nucleic acid molecule is introduced into a cancerous cell via viral vectors. In some embodiments, the viral vectors used to introduce a nucleic acid molecule are adenoviral, retroviral, lentiviral, or adeno-associated viral vectors. In some embodiments, the viral vectors are introduced into a cancerous cell via the chemical based, non-chemical based, and particle based transfection methods described above.
[0061] In some embodiments, the nucleic acid vector is a plasmid. In some embodiments, nucleic acid vectors introduced into the cancerous cell are small interfering RNA (siRNA) also known as short interfering RNA or silencing RNA. In some embodiments, nucleic acid vectors introduced into the cancerous cells are messenger RNA or mRNA.
[0062] In some embodiments, the terms "small interfering," "short interfering nucleic acid," "siNA" or "SINA" molecules, "short interfering RNA," "siRNA," "short interfering nucleic acid molecule," "short interfering oligonucleotide molecule," as used herein, refer to any nucleic acid molecule capable of inhibiting or down-regulating gene expression by an RNA interference mechanism. The term "RNA" as used herein means a molecule comprising at least one ribonucleotide residue and includes double stranded RNA, single stranded RNA, isolated RNA, partially purified, pure or synthetic RNA,
recombinantly produced RNA, as well as altered RNA or analogs of naturally occurring RNA.
Transcription Activator-Like Effector Nucleases (TALENs)
[0063] In some embodiments, activity or expression of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) is partially, substantially, or completely deleted, silenced, inactivated, down-regulated, reduced or inhibited by introducing a nucleic acid, preferably an expression vector, containing a nucleic acid encoding transcription activator-like effector nucleases (TALEN).
[0064] In some embodiments, TALEN are restriction enzymes that are designed to specifically cleave nucleic acid sequences encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3).
TALEN are produced by the fusion of a transcription activator-like (TAL) effector DNA- binding domain, which is derived from TALE proteins, to a nuclease or Fokl DNA cleavage domain. Fokl is a type IIS restriction endonuclease that is naturally found in the gram-negative bacteria Flavobacterium okeanokoites. TALE proteins originate from the bacteria genus Xanthomonas and contain DNA-binding domains, 33-35-amino-acid repeat regions, which are able to recognize a single base pair. This amino acid repeat region in the TAL effectors is readily customizable and determines binding specificity. TALEN bind adjacent DNA target sites and induce double-strand breaks between the target sequences. In some embodiments, a cancerous cell is transfected with a vector containing nucleic acid encoding TALEN, wherein the TALEN specifically cleave one or more nucleic acid sequences encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) and partially, substantially, or completely deletes, silences, inactivates, or down-regulates one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3).
Meganucleases
[0065] In some embodiments, activity or expression of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) is partially, substantially, or completely deleted, silenced, inactivated, down-regulated, reduced or inhibited by introducing a nucleic acid, preferably an expression vector, containing a nucleic acid encoding meganucleases. In some embodiments, meganucleases are restriction enzymes that are designed to specifically cleave nucleic acid sequences encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1 , LATS2, STK4 and/or a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7) (and optionally STK3). Meganucleases are double stranded endodeoxyribonucleases comprising a large recognition site of 12 to 40 base pairs. In some embodiments, a meganuclease from the LAGLIDADG family of homing endonucleases is utilized to specifically cleave nucleic acid sequences encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1 , LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3).
LAGLIDADG is the amino acid sequence that is generally conserved in all proteins of this family. The DNA binding recognition site and cleavage function of the meganucleases are intertwined in a single domain. Meganucleases bind adjacent DNA target sites and induce double-strand breaks between the target sequences. In some embodiments, a cancerous cell is transfected with a vector containing nucleic acid encoding a meganuclease, wherein the meganuclease specifically cleave one or more nucleic acid sequences encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3), and partially, substantially, or completely deletes, silences, inactivates, or down- regulates LATS 1, LATS2, or LATS 1/2. Transcription Activator-Like Meganucleases (MegaTAL)
[0066] In some embodiments, activity or expression of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) is partially, substantially, or completely deleted, silenced, inactivated, down-regulated, reduced or inhibited by introducing a nucleic acid, preferably an expression vector, containing a nucleic acid encoding transcription activator-like effector meganucleases (megaTAL). In some embodiments, megaTAL are restriction enzymes that are designed to specifically cleave nucleic acid sequences encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1 , LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3).
MegaTAL are produced by the fusion of a transcription activator-like (TAL) effector DNA- binding domain, which is derived from TALE proteins, to a meganuclease. MegaTAL bind adjacent DNA target sites and induce double-strand breaks between the target sequences. In some embodiments, a cancerous cell is transfected with a vector containing nucleic acid encoding megaTAL, wherein the megaTAL specifically cleave one or more nucleic acid sequences encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) and partially, substantially, or completely deletes, silences, inactivates, or down-regulates one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3).
Zinc Finger Nucleases (ZFN)
[0067] In some embodiments, activity or expression of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1 , LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) is partially, substantially, or completely deleted, silenced, inactivated, down-regulated, reduced or inhibited by introducing a nucleic acid, preferably an expression vector, containing a nucleic acid encoding zinc finger nucleases (ZFN). In some embodiments, ZFN are restriction enzymes that are designed to specifically cleave one or more nucleic acid sequences encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3). ZFN are produced by the fusion of a Cys2-His2 zinc finger DNA-binding domain to a DNA-cleavage domain. The DNA- cleavage domain is a Fokl type IIS restriction endonuclease. The Cys2-His2 zinc finger DNA-binding domain is one of the most common DNA-binding motifs found in eukaryotes. An individual zinc finger comprises 30 amino acids and is able to contact three base pairs in the major groove of DNA. Zinc finger DNA-binding domains contain between 3 and 6 zinc finger repeats and can be customized to recognize 9 to 18 target base pairs. In some embodiments, zinc finger DNA-binding domains are generated via a modular assembly process, wherein 3 individual zinc fingers are used to generate a 3 -finger array that can recognize 9 target base pairs. In some embodiments, zinc finger DNA-binding domains are generated via a modular assembly process, wherein 2-finger modules are used. In some embodiments, zinc finger DNA-binding domains are generated via a modular assembly process, wherein 1 -finger modules are used. ZFN dimers bind adjacent DNA target sites and induce double-strand breaks between the target sequences. In some embodiments, a cancerous cell is transfected with a vector containing nucleic acid encoding ZFN, wherein the ZFN specifically cleave one or more nucleic acid sequences encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3), and partially, substantially, or completely deletes, silences, inactivates, or down- regulates one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1 , LATS2, STK4 and/or a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7) (and optionally STK3).
CRISPR/Cas System
[0068] In some embodiments, activity or expression of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) is partially, substantially, or completely deleted, silenced, inactivated, down-regulated, reduced or inhibited by introducing a nucleic acid, preferably an expression vector, containing a nucleic acid encoding a crRNA, tracrRNA, and a Cas9 molecule. In some embodiments, the cancerous cell in which one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) is partially, substantially, or completely deleted, silenced, inactivated, or down-regulated is transformed with a nucleic acid, preferably an expression vector, containing a Cas9 molecule and a nucleic acid encoding a crRNA and tracrRNA. The CRISPR/Cas system is originally an RNA-mediated bacterial immune system that provides a form of acquired immunity against viruses and plasmids; it comprises three components: a Cas9 (CRISPR associated protein 9)
endonuclease, a crRNA (CRISPR RNA), and a tracrRNA (transactivating crRNA).
Clustered regularly interspaced short palindromic repeats (CRISPR) are short repetitions of bacterial DNA followed by short repetitions of spacer DNA from viruses or plasmids. The Cas9 endonuclease contains two nuclease domains and is programmed by a crRNA and tracrRNA hybrid to cleave the target sequence. In some embodiments, the Cas9
endonuclease is programmed by a crRNA and tracrRNA hybrid to cleave a LATS l, LATS2, or LATS 1/2 sequence.
[0069] In some embodiments, the crRNA sequence is substantially homologous to a portion of the gene encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3). In some embodiments the gRNA sequence is substantially homologous to a portion of one or more genes encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3). In some embodiments, the gRNA sequence is at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary, or any range derivable therein, to a portion of one or more genes encoding one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3). In some embodiments, the CRISPR/Cas9 system is delivered by using a plasmid. In some embodiments, the CRISPR/Cas9 system is delivered by using a ribonucleoprotein complex. In some embodiments, the ribonucleoprotein complex comprises a Cas9 protein and a nucleic acid sequence encoding crRNA and tracrRNA.
3. Subjects and Conditions Amenable to Treatment
[0070] Reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) finds use in the treatment and/or prevention of cancer in subjects who have a competent immune system sufficient to mount an immune response. Further, reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, can be co-administered to a patient to effect the inhibition, reduction, retraction or prevention of proliferation or growth of a tumor or a cancer cell. In the context of effecting treatment, the patient has a cancer or a tumor burden, and reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, can reverse, delay or inhibit progression of the disease. In the context of effecting prevention, the patient may be in remission, or may have undergone the removal of a primary tumor, and reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, can reduce, inhibit or eliminate growth of metastasis. The subject may or may not already be undergoing a regime of an immune checkpoint inhibitor and/or chemotherapeutic agent. [0071] Exemplary cancers that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include cancers from any tissue type but which have maintained regulation of YAP or which have retained YAP/TAZ function. Cancers subject to treatment include without limitation skin cancers (including melanoma), lymphoma, lung cancer, breast cancer, ovarian cancer, gastric and intestinal cancers (including colon cancer and rectal cancer), hepatic cancer, esophageal cancer, bladder cancer, renal cancer, head and neck cancers. In some embodiments, the cancer produces solid tumors. In some embodiments, the cancer is an epithelial cancer or a carcinoma, a sarcoma, or a hematological cancer. [0072] Exemplary hematologic malignancies that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include without limitation lymphomas (such as but not limited to, non-Hodgkin's lymphoma, including Burkitt' s lymphoma, and Hodgkin' s lymphoma, as well as all subtypes associated with each), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and adult T-cell leukemia lymphoma.
[0073] Exemplary lung cancers that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include without limitation adenocarcinoma, squamous carcinoma, bronchial carcinoma, broncoalveloar carcinoma, large cell carcinoma, small-cell carcinoma, non- small cell lung carcinoma and metastatic lung cancer refractory to conventional chemotherapy. [0074] Exemplary hematological cancers that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include without limitation leukemia, multiple myeloma and plasmocytoma.
[0075] Exemplary sarcomas that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include without limitation rhabdomyosarcoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma and Ewing' s sarcoma. [0076] Exemplary gastric, digestive and intestinal cancers that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include without limitation intestinal carcinoma, rectal carcinoma, colon carcinoma, familial adenomatous polyposis carcinoma, hereditary non-polyposis colorectal cancer, gastric carcinoma, craniopharyngioma, gall bladder carcinoma, esophageal carcinoma, pancreatic carcinoma and adenocarcinoma (including
adenocarcinomas of the esophagus and stomach).
[0077] Exemplary cancers of the head and neck that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include without limitation larynx carcinoma, hypopharynx carcinoma, tongue carcinoma and salivary gland carcinoma.
[0078] Exemplary urogenital cancers that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1 , LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include without limitation labial carcinoma, ovarian carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, prostate carcinoma, testis carcinoma, seminoma, urinary carcinoma, kidney carcinoma, renal carcinoma, and adenocarcinoma (including adenocarcinomas of the vagina, cervix, prostate, and urachus).
[0079] Exemplary nervous and sensory system cancers that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include without limitation neuroblastoma, brain tumors, meningioma, ependymoma, medulloblastoma, peripheral neuroectodermal tumors, glioblastoma, astrocytoma, oligodendroglioma and retinoblastoma.
[0080] Exemplary endocrine and glandular tissue cancers that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include without limitation pancreatic carcinoma, medullary thyroid carcinoma, follicular thyroid carcinoma, anaplastic thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, adrenal tumors and adenocarcinoma.
[0081] Exemplary hepatic cancers that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include without limitation hepatocellular carcinoma.
[0082] Exemplary skin cancers that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include without limitation melanoma, basal cell carcinoma, squamous cell carcinoma and choroids melanoma.
[0083] Additional cancers that can be treated or prevented by reducing or inhibiting in tumor cells the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and/or a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3), optionally in combination with an immune checkpoint inhibitor and/or chemotherapeutic agent, include without limitation teratomas. 4. Methods of Tumor Immunotherapy
[0084] Disclosed herein, in certain embodiments, are methods of treating or preventing a cancer in an individual in need thereof, comprising administering to the individual a cancerous cell in which the expression and/or activity of one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7), and optionally serine/threonine kinase 3 (STK3 or MST2) has been partially, substantially, or completely deleted, silenced, inactivated, down-regulated, reduced or inhibited.
[0085] Cellular material deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS 1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (e.g., tumor cells, EVs and cell lysates thereof) find use in tumor immunotherapy as immunogen, as adjuvant and as antigen loaded into MHC proteins and presented on the surface of dendritic cells. a. HIPPO Pathway Protein Deficient Cellular Material as Immunogen [0086] In varying embodiments, the cellular material (e.g., tumor cells, extracellular vesicles (EVs) and cell lysates thereof) deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS 1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4), a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7), and optionally serine/threonine kinase 3 (STK3) are employed as the immunogen.
[0087] Accordingly, provided are methods of inducing, promoting and/or enhancing an immune response against a tumor in a subject in need thereof. In some embodiments, the methods comprise administering to the subj ect a therapeutically effective amount of a population of tumor cells, a population of extracellular vesicles (EVs), or a cell lysate deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7) (and optionally STK3), as described above and herein, thereby inducing, promoting and/or enhancing the immune response against the tumor in the subject. [0088] In certain embodiments, provided are methods of treating or preventing a cancer in an individual in need thereof, comprising administering to the individual a vaccine composition comprising (a) cancerous cell material (whole cells, lysates, extracellular vesicles) in which the activity or expression of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATSl, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) is partially, substantially, or completely deleted, silenced, inactivated, down-regulated, reduced or inhibited; and (b) a pharmaceutically-acceptable excipient. In some embodiments, the cancer is a melanoma, a glioma, a colon cancer, a squamous cell carcinoma and a breast carcinoma.
[0089] In varying embodiments, the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is injected intradermally, epicutaneously or subcutaneously. In varying embodiments, a therapeutically effective dose involves administering cellular material deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1 , LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) (e.g., tumor cells, EVs and cell lysates thereof) corresponding to or equivalent to at least about 5xl05 tumor cells, e.g., at least about lxlO6, 2xl06, 3xl06, 4xl06, 5xl06, 6xl06, 7xl06, 8xl06, 9xl06, or lxlO7 tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1 , LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3), or an equivalent amount of EVs or tumor cell lysate.
[0090] In varying embodiments, the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate are administered multiple times. For example, in varying embodiments, the subject is administered a regime of a primary immunization with cellular material deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from LATSl, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) (e.g., tumor cells, EVs and cell lysates thereof), followed by subsequent one or more booster immunizations. As appropriate, the subsequent one or more booster immunizations can be administered at 1 month, 2 month, 3 month, 6 month or 12 month intervals after the initial immunization, e.g., depending on the health of the subject and the robustness of the immune response.
[0091] In varying embodiments, the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is co-administered with an adjuvant. Adjuvants suitable for administration to a human subject include without limitation, e.g., aluminum gels or aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate), interleukins (e.g., IL-12, IL-2, IL-15), as well as natural and synthetic variants of saponin adjuvant QS-21. See, e.g., Ragupathi, et al., Expert Rev Vaccines. 2011 Apr; 10(4):463-70; Fernandez-Tejada, et al., Acc Chem Res. 2016 Sep 20;49(9): 1741-56; and Fernandez-Tejada, et al., Nat Chem. 2014 Jul; 6(7): 635 -43 Additional adjuvants useful in human vaccines are described, e.g., in Alving, et al, Curr Opin Immunol. (2012) 24(3):310-5. [0092] In varying embodiments, the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is obtained from and autologous to the subject. When using autologous cellular material deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) as immunogen, the methods may further comprise prior to administration of the cellular material the steps of: a) isolating a population of tumor cells from the subject; and b) reducing or eliminating expression of one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7) (and optionally STK3) in the isolated tumor cells. [0093] In varying embodiments, the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is syngeneic, allogeneic or xenogeneic to the subject. In varying embodiments, the subject is a human, canine or feline. b. HIPPO Pathway Protein-Deficient Cellular Material as Adjuvant
[0094] In varying embodiments, the cellular material of tumor cells (e.g., tumor cells, extracellular vesicles (EVs) and cell lysates thereof) deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7), and optionally serine/threonine kinase 3 (STK3) are co-administered as an adjuvant to induce, enhance and/or promote the response to an immunogen, e.g., a tumor associated antigen.
[0095] Accordingly, provided are methods of inducing, promoting and/or enhancing an immune response against a tumor in a subject in need thereof. In some embodiments, the methods comprise co-administering to the subject a tumor associated antigen and a therapeutically effective amount of a population of tumor cells, a population of extracellular vesicles (EVs), or cell lysate deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), as described above and herein, thereby inducing, promoting and/or enhancing the immune response against the tumor in the subject.
[0096] In varying embodiments, the tumor associated antigen is in a tumor cell or tumor cell lysate obtained from the subject. In varying embodiments, the tumor associated antigen (TAA) is a synthetic or recombinant peptide or polypeptide. Illustrative TAAs that find use as an immunogen to be co-administered with cellular material deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) include without limitation melanoma associated antigens (MAGE-1, MAGE-3, TRP-2, melanosomal membrane glycoprotein gplOO, gp75 and MUC-1 (mucin- 1) associated with melanoma); CEA (carcinoembryonic antigen) which can be associated, e.g., with ovarian, melanoma or colon cancers; folate receptor alpha, WAP four-disulfide core domain 2 (HE4) or mesothelin expressed by ovarian carcinoma; free human chorionic gonadotropin beta (hCGP) subunit expressed by many different tumors, including but not limited to myeloma; HER-2/neu associated with breast cancer; encephalomyelitis antigen HuD associated with small-cell lung cancer; tyrosine hydroxylase associated with neuroblastoma; prostate-specific antigen (PSA) associated with prostate cancer; CA125 associated with ovarian cancer; and others, as discussed above.
[0097] In varying embodiments, the immunogen and population of tumor cells, population of extracellular vesicles (EVs) or cell lysate are formulated in a pharmaceutically acceptable carrier (e.g., phosphate-buffered saline) and co-injected intradermally, epicutaneously or subcutaneously. The immunogen and the cellular material deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from LATSl, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) can be co-injected via the same or different routes of administration. The immunogen and the cellular material can be concurrently co-administered or administered sequentially. In varying embodiments, a therapeutically effective dose of cellular material (e.g., tumor cells, EVs and cell lysates thereof) as adjuvant corresponds to or is the equivalent of at least about lxlO5 tumor cells, e.g., at least about 2xl05, 3xl05, 4xl05, 5xl05, 6xl05, 7xl05, 8xl05, 9xl05, lxlO6, 2xl06, 3xl06, 4xl06, 5xl06, 6xl06, 7xl06, 8xl06, 9xl06, or lxlO7 tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS l, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), or an equivalent amount of EVs or tumor cell lysate.
[0098] In varying embodiments, the immunogen and population of tumor cells, population of extracellular vesicles (EVs) or cell lysate are administered multiple times. For example, in varying embodiments, the subject is co-administered a regime of a primary immunization with immunogen in combination with cellular material (e.g., tumor cells, EVs and cell lysates thereof), followed by subsequent one or more booster immunizations of immunogen in combination with cellular material. As appropriate, the subsequent one or more booster immunizations can be administered at 1 month, 2 month, 3 month, 6 month or 12 month intervals after the initial immunization, e.g., depending on the health of the subject and the robustness of the immune response.
[0099] In varying embodiments, the immunogen and population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is co-administered with an additional adjuvant, e.g., aluminum gels or aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate), interleukins (e.g., IL-12, IL-2, IL- 15), as well as natural and synthetic variants of saponin adjuvant QS-21, as discussed above.
[0100] In varying embodiments, the tumor cell immunogen and/or the cellular material deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7) (and optionally STK3) (e.g., tumor cells, EVs and cell lysates thereof) is obtained from and autologous to the subject. When using autologous tumor cell immunogen and/or cellular material as adjuvant, the methods may further comprise prior to administration of the cellular material the steps of: a) isolating a population of tumor cells from the subject; and b) reducing or eliminating expression of one or more proteins within or associated with the HIPPO intracellular signaling selected from L ATS 1 , LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) in the isolated tumor cells.
[0101] In varying embodiments, the tumor cell immunogen and/or the cellular material deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS 1, LATS2, STK4 and a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7) (and optionally STK3) (e.g., tumor cells, EVs and cell lysates thereof) is syngeneic, allogeneic or xenogeneic to the subject. In varying embodiments, the subject is a human, canine or feline. c. Dendritic Cells Loaded with HIPPO Pathway Protein-Deficient Cellular Material
[0102] In varying embodiments, the cellular material (e.g., tumor cells, EVs, apoptotic tumor cell preparations and cell lysates thereof) deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7), and optionally serine/threonine kinase 3 (STK3) are loaded into the MHC molecules on the surface of a population of dendritic cells. The loaded dendritic cells or exosomes derived from the loaded dendritic cells are administered to the subject.
[0103] Accordingly, provided are methods inducing, promoting and/or enhancing an immune response against a tumor in a subject in need thereof. In varying embodiments, the methods comprise administering to the subject a therapeutically effective amount of a population of dendritic cells with the major histocompatibility complex (MHC) proteins (e-g-, MHC class I and/or MHC class II) loaded with antigens from cellular material deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7) (and optionally STK3) (e.g., tumor cells, EVs, apoptotic tumor cell preparations and cell lysates thereof), as described above and herein, or exosomes derived from the dendritic cells, thereby inducing, promoting and/or enhancing the immune response against the tumor in the subject.
[0104] In varying embodiments, the MHC class I and/or MHC class II proteins of the dendritic cells are at least partially loaded. Methods for loading MHC class I and/or MHC class II proteins of the dendritic cells with tumor cell antigens, including from whole tumor cell preparations, are known in the art and described in, e.g., Strome, et al, Cancer Res. 2002 Mar 15;62(6): 1884-9; Thumann, et al, Journal of Immunological Methods 277 (2003) 1-16; Neidhardt-Berard, et al, Breast Cancer Research (2004) 6:R322; Inzkirweli, et al, ANTICANCER RESEARCH 27: 2121-2130 (2007); Van Nuffel, et al, Intl. Society of Blood Transfusion (ISBT) Science Series, Volume 8, Issue 1, June 2013, Pages 161-164; Sabado, et al., J. Vis. Exp. (78), e50085, doi: 10.3791/50085 (2013); Rizzo, et al., in Cancer Vaccines, Volume 1139 of the series Methods in Molecular Biology pp 41-44 (2014) (Print ISBN 978-1-4939-0344-3). Dendritic cells can be generated by culturing bone marrow derived dendritic cells (BMDCs) in culture media comprising a sufficient amount (e.g., 20 ng/ml) of granulocyte-macrophage colony stimulating factor (GM-CSF) and optionally IL-4. Additional protocols for antigen loading of MHC molecules on the surface of dendritic cells can be found, e.g., in Coligan, et al. Editor, Current Protocols in
Immunology, USA, 1987-2016. Apoptotic tumor cell preparations for loading into MHC proteins on the surface of dendritic cells can be prepared according to the methods of Inzkirweli, et al, ANTICANCER RESEARCH 21 : 2121-2130 (2007).
[0105] In varying embodiments, the dendritic cells are autologous, syngeneic, allogeneic, or xenogeneic to the subject. In varying embodiments, the population of dendritic cells is administered by injection intradermally, epicutaneously or subcutaneously. In varying embodiments, a regime of the population of dendritic cells is administered multiple times. In varying embodiments, the population of dendritic cells is coadministered with an adjuvant (e.g., aluminum gels or aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate), interleukins (e.g., IL-12, IL-2, IL-15), as well as natural and synthetic variants of saponin adjuvant QS-21). In varying embodiments, the subject is a human, canine or feline.
5. Methods Entailing Inhibiting Expression and/or Activity of One or More
Proteins Within or Associated with The HIPPO Intracellular Signaling
Pathway [0106] Disclosed herein, in certain embodiments, are methods of treating or preventing a cancer in an individual in need thereof, comprising partially, substantially, or completely deleting, silencing, inactivating, down-regulating, reducing or inhibiting the activity or expression in a cancerous cell in the individual one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally serine/threonine kinase 3 (STK3 or MST2).
[0107] In varying embodiments, an inhibitor of one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) is administered to the subject. Optionally, an inhibitor of serine/threonine kinase 3 (STK3) or MST2 is additionally co-administered. a. Inhibitor Compounds
[0108] The inhibitor of the one or more proteins within or associated with the
HIPPO intracellular signaling pathway selected from LATSl, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) can be an inhibitory nucleic acid, a polypeptide, a peptide, a small organic compound or an antibody or fragment thereof. Inhibitors of one or more proteins within or associated with the HIPPO
intracellular signaling selected from the group consisting of LATSl, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) are known in the
Figure imgf000057_0001
[0109] In varying embodiments, the inhibitor of one or more proteins within or associated with the HIPPO intracellular signaling pathway is formulated for intracellular delivery. Strategies for intracellular drug delivery are known in the art and find use in the present methods. Illustrative strategies for intracellular drug delivery include nanoparticles (Xu, et al., ACS Appl Mater Interfaces. 2016 Oct 19; PMID 27759366; An, et al., Mater Sci Eng C Mater Biol Appl. 2016 Dec l;69:292-300; Shen, et al., ACS Appl Mater Interfaces. 2016 Sep 21;8(37):24502-8; Yang, et al, Langmuir. 2016 Aug 8; PMID 27467698; Wang, et al., Biomacromolecules. 2016 Sep 12; 17(9):2920-9; El-Boubbou, Bioconjug Chem. 2016 Jun 15;27(6): 1471-83; Yang, et al, ACS Appl Mater Interfaces . 2016 Jun 1 ;8(21): 13251-61 ; Huang, et al., Acta Biomater. 2016 Aug;40:263-72; Zhang, et al ., ACS Appl Mater
Interfaces. 2015 Dec 9;7(48):26666-73), liposomal nanocarriers (Kang, et al., Mol Pharm. 2015 Dec 7; 12(12):4200-13), liposomal nanohybrid cerosomes (Zhou, et al, Colloids Surf B Biointerfaces. 2016 Sep 23; 148:518-525), nanogels (Zhang, et al, ACS ApplMater Interfaces. 2016 May 4;8(17): 10673-82), heparosan-based nanocarriers (Chen, et al., J Control Release . 2015 Sep 10;213 :e54-5), polycaprolactone/maltodextrin nanocarriers (Korang-Yeboah, Int J Nanomedicine . 2015 Jul 29; 10:4763-81), diatomite-based
nanovectors (Terracciano, Nanoscale. 2015 Dec 21;7(47):20063-74), mesoporous silica particles (Shokry, et al., Nanoscale. 2015 Sep 14;7(34): 14434-43; Zheng, et al., Anal Chem. 2015 Dec 1;87(23): 11739-45; Yang, et al., ACS ApplMater Interfaces. 2015 Aug
12;7(31): 17399-407), amphipathic polymers (Mercado, et al, Mater Sci Eng C Mater Biol Appl. 2016 Dec l;69: 1051-7; Zhao, et al., ACS Appl Mater Interfaces. 2016 Aug
31;8(34):22127-34), liposomes (Chen, et al., ACS Appl Mater Interfaces. 2016 Aug
31;8(34):22457-67), micelles (Nesporova, Int J Pharm. 2016 Sep 10;51 l(l):638-47; Chen, et al., J Control Release. 2015 Sep 10;213 :e55; Wang, et al ., J Control Release . 2015 Sep 10;213 :el34-5; Fang, et al, Int JPharm. 2016 Apr 1 l;502(l-2):28-37; Wang, et al., Colloids Surf B Biointerfaces . 2015 Nov l; 135:283-90) and viral vectors {e.g., retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors).
Accordingly, in certain embodiments, one or more inhibitors of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of LATS1, LATS2, STK4 and a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7) (and optionally STK3) is conjugated to, encapsulated within or administered in conjunction with a modality that facilitates intracellular delivery, e.g., nanoparticle, a nanocarrier, a nanovector, a mesoporous silica particle, a cerosome, a lipsome, a micelle, a nanogel, an amphipathic polymer, or any other modality known in the art to facilitate intracellular delivery of the inhibitor. Viral vectors additionally find use for delivery of nucleic acid inhibitors.
[0110] In varying embodiments, one or more inhibitors of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of LATS1, LATS2, STK4 and a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7) (and optionally STK3) can be conjugated to or is encapsulated within a delivery vehicle conjugated to a tumor targeting moiety. Tumor targeting strategies are known in the art and find use. The selection of tumor targeting moiety will depend on the tumor and/or cancerous tissue being targeted. In varying embodiments, the tumor target is a tumor associated antigen, expressed on the surface of the tumor to be targeted. Illustrative known tumor associated antigens that can serve as a tumor target to facilitate delivery of the inhibitor to desired target cells while reducing off-target toxicities are described herein. The tumor targeting moiety can be any ligand binding partner that specifically binds to a desired tumor associated or tissue associated antigen, including an antibody or fragment thereof, an antibody mimic (e.g., based on fibronectin, lipocalin, calixarene scaffolds), a peptide ligand, an aptamer. In varying embodiments, the tumor targeting moiety can be conjugated to the surface of a modality that facilitates intracellular drug delivery, which in turn is conjugated to and/or encapsulates one or more inhibitors of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), as described above. i. Inhibitory Nucleic Acids
[0111] In varying embodiments, one or more inhibitors of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of LATSl, LATS2, STK4 and a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7) (and optionally STK3) is an inhibitory nucleic acid. For example, in varying embodiments, the one or more inhibitory nucleic acids that inhibit the expression and/or activity levels of one or more proteins selected from the group consisting of LATSl, LATS2, heat shock protein 90 (HSP90), STK4 and a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7) (and optionally STK3) can be a small interfering RNA (siRNA), a microRNA (miRNA), a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), and/or an antisense oligonucleotide. In varying embodiments, the one or more inhibitory nucleic acid encode one or more transcription activator-like effector nucleases (TALENs), meganucleases, transcription activator-like effector meganucleases (megaTALs), or zinc finger nucleases (ZFNs) that specifically cleave one or more nucleic acid sequences encoding one or more of LATSl, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3, and partially,
substantially, or completely deletes, silences, inactivates, or down-regulates one or more of LATSl, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3. In varying embodiments, the one or more inhibitory nucleic acid encode one or more tracrRNAs, and one or more Cas9
endonucleases, wherein the crRNA, tracrRNA and Cas9 endonuclease operatively coordinate to specifically cleave one or more nucleic acid sequences encoding one or more of LATSl, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7), and optionally STK3, and partially, substantially, or completely deletes, silences, inactivates, or down-regulates one or more of LATSl, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3. Heat Shock Protein 90 Inhibition Depletes LATS 1 and LATS2.
Huntoon, et al, Cancer Research (2010) 70(21): 8642-50.
[0112] Delivery of oligonucleotides and/or expression vectors to a target cell can be achieved in a variety of ways. In some embodiments, a transfection agent is used. A transfection agent, or transfection reagent or delivery vehicle, is a compound or compounds that bind(s) to or complex(es) with oligonucleotides and polynucleotides, and enhances their entry into cells. Examples of transfection reagents include, but are not limited to, cationic liposomes and lipids, polyamines, calcium phosphate precipitates, polycations, histone proteins, polyethylenimine, polylysine, and polyampholyte complexes. Transfection reagents are well known in the art. One transfection reagent suitable for delivery of an inhibitory nucleic acid is siPORT™, NeoFX™ transfection agent (Ambion), which can be used to transfect a variety of cell types with inhibitory nucleic acids. Inhibitory nucleic acids can be readily electroporated into primary cells without inducing significant cell death. In addition, the inhibitory nucleic acid can be transfected at different concentrations. The transfection efficiency of synthetic miRNAs has been shown to be very good, and around 100% for certain cell types (Ambion™ miRNA Research Guide, page 12. See also, www.ambion.com/miRNA).
[0113] Reagents for delivery of an inhibitory nucleic acid, and expression vectors can include, but are not limited to protein and polymer complexes (polyplexes), lipids and liposomes (lipoplexes), combinations of polymers and lipids (lipopolyplexes), and multilayered and recharged particles. Transfection agents may also condense nucleic acids. Transfection agents may also be used to associate functional groups with a polynucleotide. Functional groups can include cell targeting moieties, cell receptor ligands, nuclear localization signals, compounds that enhance release of contents from endosomes or other intracellular vesicles (such as membrane active compounds), and other compounds that alter the behavior or interactions of the compound or complex to which they are attached
(interaction modifiers). For delivery in vivo, complexes made with sub-neutralizing amounts of cationic transfection agent may be preferred. Additional illustrative lipid formulations for delivery are described, e.g., in U.S. Patent Publ. No. 2011/0256175. [0114] In some embodiments, polycations are mixed with polynucleotides for delivery to a cell. Polycations are a very convenient linker for attaching specific receptors to DNA and as result, DNA/polycation complexes can be targeted to specific cell types. Here, targeting is preferably to cells involved in innate immunity. An endocytic step in the intracellular uptake of DNA/polycation complexes is suggested by results in which functional DNA delivery is increased by incorporating endosome disruptive capability into the polycation transfection vehicle. Polycations also cause DNA condensation. The volume which one DNA molecule occupies in complex with polycations is drastically lower than the volume of a free DNA molecule. The size of DNA/polymer complex may be important for gene delivery in vivo. In some embodiments, the one or more inhibitory nucleic acids and a transfection reagent are delivered systematically such as by injection. In other embodiments, they may be injected into particular areas comprising target cells, such as target tumor cells and cancerous tissues. [0115] Polymer reagents for delivery of an inhibitory and expression vectors may incorporate compounds that increase their utility. These groups can be incorporated into monomers prior to polymer formation or attached to polymers after their formation. An expression vector transfer enhancing moiety is typically a molecule that modifies a nucleic acid complex and can direct it to a cell location (such as tissue cells) or location in a cell (such as the nucleus) either in culture or in a whole organism. By modifying the cellular or tissue location of the complex, the desired localization and activity of the inhibitory nucleic acid or expression vector can be enhanced. The transfer enhancing moiety can be, for example, a protein, peptide, lipid, steroid, sugar, carbohydrate, nucleic acid, cell receptor ligand, or synthetic compound. The transfer enhancing moieties can enhance cellular binding to receptors, cytoplasmic transport to the nucleus and nuclear entry or release from endosomes or other intracellular vesicles.
[0116] In alternative embodiments, the one or more inhibitory nucleic acids may be effectively delivered to target cancer cells, by a variety of methods known to those skilled in the art. Such methods include but are not limited to liposomal encapsulation/delivery, vector-based gene transfer, fusion to peptide or immunoglobulin sequences (peptides described, e.g., in Intl. Publ. No. 2011/038142) for enhanced cell targeting and other techniques. Suitable viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, etc. In alternative embodiments, the one or more inhibitory nucleic acids may also be formulated in pharmaceutical compositions well known to those in the field. These include liposomal formulations and combinations with other agents or vehicles/excipients such as cyclodextrins which may enhance delivery of the inhibitory nucleic acid. In alternative embodiments, suitable carriers include lipid-based carriers such as a stabilized nucleic acid-lipid particle (e.g., SNALP or SPLP), cationic lipid or liposome nucleic acid complexes (i.e., lipoplexes), a liposome, a micelle, a virosome, or a mixture thereof. In other embodiments, the carrier system is a polymer-based carrier system such as a cationic polymer-nucleic acid complex (i.e., polyplex). In alternative
embodiments, the carrier system is a cyclodextrin-based carrier system such as a
cyclodextrin polymer-nucleic acid complex. In further embodiments, the carrier system is a protein-based carrier system such as a cationic peptide-nucleic acid complex. ii. Peptides and Small Organic Compounds
[0117] In varying embodiments, one or more inhibitors of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) is a peptide, a polypeptide or a small organic compound. Small organic inhibitors of one or more proteins in the HIPPO intracellular signaling pathway selected from the group consisting of LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) are known in the art and find use. Illustrative inhibitors of LATS1 and/or LATS2 include without limitation A443654 (chemical name: (2S)-l-(lH-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin- 3-yl]oxypropan-2-amine; CAS Registry Number: 552325-16-3), Lestaurtinib (chemical name: (5S,6S,8R)-6-Hydroxy-6-(hydroxymethyl)-5-methyl-7,8, 14,15-tetrahydro-5H-16- oxa-4b,8a,14-triaza-5,8-methanodibenzo[b,h]cycloocta[jkl]cyclopenta[e]-as-indacen-
13(6H)-one; CAS Registry Number: 111358-88-4), GSK-690693 (chemical name: 4-[2-(4- Amino-l,2,5-oxadiazol-3-yl)-l -ethyl -7-[(3S)-3-piperidinylmethoxy)-lH-imidazo[4,5- c]pyridin-4-yl]-2-methyl-3-butyn-2-ol; CAS Registry No. 937174-76-0), lysophosphatidic acid (LP A), sphingosine-1 -phosphate (SIP) and thrombin. See, e.g., Figure 15B; Johnson and Haider, Nat Rev Drug Discov. (2014) 13(1): 63-79. Illustrative inhibitors of STK4 and/or STK3/MST2 include without limitation the enantiopure organoruthenium inhibitor, 9E1 (described in Anand, et al, J Med Chem. 2009 Mar 26;52(6): 1602-11), XMU-MP-1 (chemical name: 4-((5,10-dimethyl-6-oxo-6, 10-dihydro-5H-pyrimido[5,4-b]thieno[3,2- e][l,4]diazepin-2-yl)amino)benzenesulfonamide; described in Fan, et al., Sci Transl Med. (2016) 8(352):352ral08) and a truncated peptide of STK4, e.g., in particular, the C-terminus portion, e.g., the C-terminal 63 amino acid residues of STK4.
Figure imgf000062_0001
Structure of STK4 inhibitor 9E1 from Anand, et al., J Med Chem. 2009 Mar 26;52(6): 1602- 11. [0118] Numerous inhibitors of STK3/MST2 are known in the art and can find use in the present methods. Illustrative inhibitors of STK3/MST2 include without limitation staurosporine, CEP-1 1981, lestaurtinib, sunitinib, neratinib, SU-14813, JNJ-28312141, midostaurin, NVP-TAE684, bosutinib, dovitinib, staurosporine, Cdkl/2 inhibitor, midostaurin, JAK3 inhibitor VI, K-252a, Go 6976, SB 218078, PKR inhibitor, indirubin derivative E804, JAK inhibitor I {see,
guidetopharmacology.org/GRAC/Obj ectDisplayForward?obj ectld=2219) and those described in WO 2008/045627.
[0119] Numerous inhibitors of HSP90 are known in the art and can find use in the present methods. Illustrative inhibitors of HSP90 are described, e.g., in U.S. Patent Nos.
9,457,045; 9,439,899; 9,402,831; 9,249,159; 9,205,086; 9,056,104; 8,816,070; 8,628,752; 8,530,469; 8,518,897; 8,426,396; 8,399,464; 8,357,676; 8, 158,638; 7,947,670; 7,825,094; 7,691,838; 7,538,241; 7,405,208; 7,211,562; 6,887,853; 6,872,715 and in Bobrov, et al, Oncotarget. 2016 Oct 13; PMID 27779106; Kim, et al, Bioorg Med Chem. 2016 Nov 15;24(22):6082-6093; Teracciano, et al, Chem Commun (Camb). 2016 Oct
25;52(87): 12857-12860; and Courtin, et al, Br J Cancer. 2016 Oct 25; 115(9): 1069-1077.
iii. Antibodies and Fragments thereof
[0120] In varying embodiments, one or more inhibitors of one or more proteins selected from LATSl, LATS2, STK4, HSP90 and a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7) (and optionally STK3) is an antibody or fragment thereof, or an antibody mimic, as described herein. Antibodies against one or more proteins selected from LATSl, LATS2, STK4, HSP90 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) are known in the art, are commercially available and can find use. Illustrative antibodies that specifically bind to one or more proteins selected from LATSl, LATS2, STK4, HSP90 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) can be purchased from Abeam (abcam.com), Santa Cruz Biotechnology (scbt.com), Cell Signaling Technologies (cellsignal.com), Origene (acris-antibodies.com), Novus Biologicals (novusbio.com), Bethyl Laboratories (bethyl.com), Thermo Fisher (thermofisher.com), Sigma Aldrich (sigmaaldrich.com), Atlas Antibodies (atlas
antibodies.com), EMD Millipore (emdmillipore.com), R&D Systems (rndsystems.com), In varying embodiments, the antibodies are monoclonal or polyclonal. In varying
embodiments, the antibodies or fragments thereof are humanized for therapeutic administration to a human. In varying embodiments, the antibody fragments or antigen- binding portions that specifically bind to one or more proteins selected from LATS 1, LATS2, STK4, HSP90 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) can include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibody (dAb), complementarity determining region (CDR) fragments, CDR-grafted antibodies, single- chain antibodies (scFv), single chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibody, linear antibody; chelating recombinant antibody, a tribody or bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), an antigen-binding-domain immunoglobulin fusion protein, a camelized antibody, a VHH containing antibody, or a variant or a derivative thereof, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide, such as one, two, three, four, five or six CDR sequences, as long as the antibody retains the desired biological activity. b. Formulation and Administration i. Formulation
[0121] The inhibitors (particularly the small organic compounds, polypeptides, peptides and antibodies and fragments thereof) of one or more HIPPO pathway proteins selected from large tumor suppressor kinase 1 (LATS 1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7) an analog thereof can be formulated for administration orally, parenterally, (intravenously (IV), intramuscularly (IM), depo-IM, subcutaneously (SQ), and depo-SQ), intratumorally, sublingually, intranasally (inhalation), intrathecally,
transdermally (e.g., via transdermal patch), topically, ionophoretically or rectally. In some embodiments, the dosage form is selected to facilitate delivery to an intracellular target (e-g-, using nanoparticles, liposomes, viral vectors). In this context it is noted that the compounds described herein are readily delivered to the brain. Dosage forms known to those of skill in the art are suitable for delivery of the compound.
[0122] Compositions are provided that contain therapeutically effective amounts of the compound. The compounds are preferably formulated into suitable pharmaceutical preparations such as tablets, capsules, or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration. Typically the compounds described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art. [0123] The one or more inhibitors of one or more HIPPO pathway proteins selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) can be administered in the "native" form or, if desired, in the form of salts, esters, amides, prodrugs, derivatives, and the like, provided the salt, ester, amide, prodrug or derivative is suitable pharmacologically effective, e.g., effective in the present method(s). Salts, esters, amides, prodrugs and other derivatives of the active agents can be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed. N. Y. Wiley-Interscience. [0124] Methods of formulating such derivatives are known to those of skill in the art. For example, the disulfide salts of a number of delivery agents are described in PCT Publication WO 2000/059863 which is incorporated herein by reference. Similarly, acid salts of therapeutic peptides, peptoids, or other mimetics, and can be prepared from the free base using conventional methodology that typically involves reaction with a suitable acid. Generally, the base form of the drug is dissolved in a polar organic solvent such as methanol or ethanol and the acid is added thereto. The resulting salt either precipitates or can be brought out of solution by addition of a less polar solvent. Suitable acids for preparing acid addition salts include, but are not limited to both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, orotic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. An acid addition salt can be reconverted to the free base by treatment with a suitable base. Certain particularly preferred acid addition salts of the active agents herein include halide salts, such as may be prepared using hydrochloric or hydrobromic acids. Conversely, preparation of basic salts of the active agents of this invention are prepared in a similar manner using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or the like. In certain embodiments basic salts include alkali metal salts, e.g., the sodium salt, and copper salts.
[0125] For the preparation of salt forms of basic drugs, the pKa of the counterion is preferably at least about 2 pH lower than the pKa of the drug. Similarly, for the preparation of salt forms of acidic drugs, the pKa of the counterion is preferably at least about 2 pH higher than the pKa of the drug. This permits the counterion to bring the solution's pH to a level lower than the pHmax to reach the salt plateau, at which the solubility of salt prevails over the solubility of free acid or base. The generalized rule of difference in pKa units of the ionizable group in the active pharmaceutical ingredient (API) and in the acid or base is meant to make the proton transfer energetically favorable. When the pKa of the API and counterion are not significantly different, a solid complex may form but may rapidly disproportionate (e.g., break down into the individual entities of drug and counterion) in an aqueous environment.
[0126] Preferably, the counterion is a pharmaceutically acceptable counterion. Suitable anionic salt forms include, but are not limited to acetate, benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate (embonate), phosphate and diphosphate, salicylate and disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide, valerate, and the like, while suitable cationic salt forms include, but are not limited to aluminum, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, zinc, and the like.
[0127] In various embodiments preparation of esters typically involves
functionalization of hydroxyl and/or carboxyl groups that are present within the molecular structure of the active agent. In certain embodiments, the esters are typically acyl- substituted derivatives of free alcohol groups, e.g., moieties that are derived from carboxylic acids of the formula RCOOH where R is alky, and preferably is lower alkyl. Esters can be reconverted to the free acids, if desired, by using conventional hydrogenolysis or hydrolysis procedures.
[0128] Amides can also be prepared using techniques known to those skilled in the art or described in the pertinent literature. For example, amides may be prepared from esters, using suitable amine reactants, or they may be prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine. [0129] About 1 to 1000 mg of a compound or mixture of the one or more inhibitors of one or more HIPPO pathway proteins selected from LATS 1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) or a
physiologically acceptable salt or ester is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutical practice. The amount of active substance in those compositions or preparations is such that a suitable dosage in the range indicated is obtained. The compositions are preferably formulated in a unit dosage form, each dosage containing from about 1-1000 mg, 2-800 mg, 5-500 mg, 10-400 mg, 50-200 mg, e.g., about 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg or 1000 mg of the active ingredient. The term "unit dosage from" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
[0130] To prepare compositions, the compound is mixed with a suitable
pharmaceutically acceptable carrier. Upon mixing or addition of the compound(s), the resulting mixture may be a solution, suspension, emulsion, or the like. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for lessening or ameliorating at least one symptom of the disease, disorder, or condition treated and may be empirically determined.
[0131] Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. In addition, the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action. The compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
[0132] Where the compounds exhibit insufficient solubility, methods for solubilizing may be used. Such methods are known and include, but are not limited to, using cosolvents such as dimethylsulfoxide (DMSO), using surfactants such as Tween™, and dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as salts or prodrugs may also be used in formulating effective pharmaceutical compositions. [0133] The concentration of the compound is effective for delivery of an amount upon administration that lessens or ameliorates at least one symptom of the disorder for which the compound is administered and/or that is effective in a prophylactic context. Typically, the compositions are formulated for single dosage (e.g., daily) administration. [0134] The compounds may be prepared with carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems. The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated disorder. A therapeutically or prophylactically effective dose can be determined by first administering a low dose, and then incrementally increasing until a dose is reached that achieves the desired effect with minimal or no undesired side effects. [0135] In various embodiments, the compounds and/or analogs thereof can be enclosed in multiple or single dose containers. The enclosed compounds and compositions can be provided in kits, for example, including component parts that can be assembled for use. For example, a compound inhibitor in lyophilized form and a suitable diluent may be provided as separated components for combination prior to use. A kit may include a compound inhibitor and a second therapeutic agent for co-administration. The inhibitor and second therapeutic agent may be provided as separate component parts. A kit may include a plurality of containers, each container holding one or more unit dose of the compounds. The containers are preferably adapted for the desired mode of administration, including, but not limited to tablets, gel capsules, sustained-release capsules, and the like for oral administration; depot products, pre-filled syringes, ampules, vials, and the like for parenteral administration; and patches, medipads, creams, and the like for topical administration.
[0136] The concentration and/or amount of active compound in the drug
composition will depend on absorption, inactivation, and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
[0137] The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed
compositions. [0138] If oral administration is desired, the compound can be provided in a formulation that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient. [0139] Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules, or troches. Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the
composition.
[0140] In various embodiments, the tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, gum tragacanth, acacia, corn starch, or gelatin; an excipient such as microcrystalline cellulose, starch, or lactose; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a gildant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.
[0141] When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials, which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be
administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors.
[0142] The active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action. [0143] Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, and the like, or a synthetic fatty vehicle such as ethyl oleate, and the like, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens;
antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, and phosphates; and agents for the adjustment of tonicity such as sodium chloride and dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required.
[0144] Where administered intravenously, suitable carriers include physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and
solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, and mixtures thereof. Liposomal suspensions including tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known for example, as described in U.S. Pat. No. 4,522,811.
[0145] The active compounds may be prepared with carriers that protect the compound against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and the like. Methods for preparation of such formulations are known to those skilled in the art. ii. Routes of Administration and Dosing
[0146] In various embodiments, the compounds and/or analogs thereof can be administered orally, parenterally (IV, IM, depo-IM, SQ, and depo-SQ), sublingually, intranasally (inhalation), intratumorally, intrathecally, transdermally (e.g., via transdermal patch), topically, or rectally. Dosage forms known to those skilled in the art are suitable for delivery of the compounds and/or analogs thereof.
[0147] In various embodiments, the compounds and/or analogs thereof may be administered enterally or parenterally. When administered orally, the compounds can be administered in usual dosage forms for oral administration as is well known to those skilled in the art. These dosage forms include the usual solid unit dosage forms of tablets and capsules as well as liquid dosage forms such as solutions, suspensions, and elixirs. When the solid dosage forms are used, it is preferred that they be of the sustained release type so that the compound needs to be administered only once or twice daily.
[0148] The oral dosage forms can be administered to the patient 1, 2, 3, or 4 times daily. It is preferred that the compound be administered either three or fewer times, more preferably once or twice daily. Hence, it is preferred that the compound be administered in oral dosage form. It is preferred that whatever oral dosage form is used, that it be designed so as to protect the compound from the acidic environment of the stomach. Enteric coated tablets are well known to those skilled in the art. In addition, capsules filled with small spheres each coated to protect from the acidic stomach, are also well known to those skilled in the art.
[0149] When administered orally, an administered amount therapeutically effective to prevent, delay inhibit or reverse tumor growth is from about 0.1 mg/day to about 200 mg/day, for example, from about 1 mg/day to about 100 mg/day, for example, from about 5 mg/day to about 50 mg/day. In some embodiments, the subject is administered the compound at a dose of about 0.05 to about 0.50 mg/kg, for example, about 0.05 mg/kg, 0.10 mg/kg, 0.20 mg/kg, 0.33 mg/kg, 0.50 mg/kg. It is understood that while a patient may be started at one dose, that dose may be varied (increased or decreased, as appropriate) over time as the patient's condition changes. Depending on outcome evaluations, higher doses may be used. For example, in certain embodiments, up to as much as 1000 mg/day can be administered, e.g., 5 mg/day, 10 mg/day, 25 mg/day, 50 mg/day, 100 mg/day, 200 mg/day, 300 mg/day, 400 mg/day, 500 mg/day, 600 mg/day, 700 mg/day, 800 mg/day, 900 mg/day or 1000 mg/day. [0150] The compounds and/or analogs thereof may also be advantageously delivered in a nano crystal dispersion formulation. Preparation of such formulations is described, for example, in U.S. Pat. No. 5, 145,684. Nano crystalline dispersions of HIV protease inhibitors and their method of use are described in U.S. Pat. No. 6,045,829. The nano crystalline formulations typically afford greater bioavailability of drug compounds.
[0151] In various embodiments, the compounds and/or analogs thereof can be administered parenterally, for example, by IV, IM, depo-IM, SC, or depo-SC. When administered parenterally, a therapeutically effective amount of about 0.5 to about
100 mg/day, preferably from about 5 to about 50 mg daily should be delivered. When a depot formulation is used for injection once a month or once every two weeks, the dose should be about 0.5 mg/day to about 50 mg/day, or a monthly dose of from about 15 mg to about 1,500 mg. In various embodiments, the compounds and/or analogs thereof can be administered intratum orally.
[0152] In various embodiments, the compounds and/or analogs thereof can be administered sublingually. When given sublingually, the compounds and/or analogs thereof can be given one to four times daily in the amounts described above for IM administration.
[0153] In various embodiments, the compounds and/or analogs thereof can be administered intranasally. When given by this route, the appropriate dosage forms are a nasal spray or dry powder, as is known to those skilled in the art. The dosage of compound and/or analog thereof for intranasal administration is the amount described above for IM administration.
[0154] In various embodiments, compound and/or analogs thereof can be administered intrathecally. When given by this route the appropriate dosage form can be a parenteral dosage form as is known to those skilled in the art. The dosage of compound and/or analog thereof for intrathecal administration is the amount described above for IM administration.
[0155] In certain embodiments, the compound and/or analog thereof can be administered topically. When given by this route, the appropriate dosage form is a cream, ointment, or patch. When administered topically, the dosage is from about 1.0 mg/day to about 200 mg/day. Because the amount that can be delivered by a patch is limited, two or more patches may be used. The number and size of the patch is not important, what is important is that a therapeutically effective amount of compound be delivered as is known to those skilled in the art. The compound can be administered rectally by suppository as is known to those skilled in the art. When administered by suppository, the therapeutically effective amount is from about 1.0 mg to about 500 mg.
[0156] In various embodiments, the compound and/or analog thereof can be administered by implants as is known to those skilled in the art. When administering the compound by implant, the therapeutically effective amount is the amount described above for depot administration.
[0157] It should be apparent to one skilled in the art that the exact dosage and frequency of administration will depend on the particular condition being treated, the severity of the condition being treated, the age, weight, general physical condition of the particular patient, and other medication the individual may be taking as is well known to administering physicians who are skilled in this art.
6. Combination Therapies
[0158] In various embodiments, tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells, are co-administered with one or more chemotherapeutic agents and/or one or more immune checkpoint inhibitors. a. Immune Checkpoint Inhibitors
[0159] In some embodiments, the method further comprising co-administering to the individual a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a synthetic oligonucleotide. In some embodiments, the checkpoint inhibitor is a molecule that specifically interacts with the immune checkpoint protein, to inhibit immune system downregulation mediated by the immune checkpoint protein. In some embodiments, the . iimmune checkpoint inhibitor is an antibody (e.g., commercially available antibodies) or fragment thereof, or small molecule drug.
[0160] In some embodiments, the checkpoint inhibitor is administered before inhibition the activity and/or expression of one or more proteins selected from LATS 1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3). In some embodiments, the checkpoint inhibitor is administered after inhibition of LATSl, LATS2, or LATS1/2. In some embodiments, the checkpoint inhibitor is administered concurrently with inhibition of LATSl, LATS2, or LATS1/2.
[0161] Examples of immune checkpoint inhibitors that can be co-administered with tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATSl), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells are known in the art and include without limitation inhibitors of cytotoxic T-lymphocyte associated protein 4 (CTLA4), programmed cell death 1 (PCD1 or PD-1), CD274 molecule (PD-L1), phosphoinositide 3-kinase γ (ΡΒΚγ) or indoleamine 2,3- dioxygenase (IDO). In varying embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof. Illustrative CTLA4 inhibitors known in the art and of use include, e.g., Ipilimumab and Tremelimumab. Illustrative PCD1 inhibitors known in the art and of use include, e.g., Nivolumab and Pembrolizumab. Illustrative PD-L1 inhibitors known in the art and of use include, e.g., BMS-936559, MPDL3280A and MEDI4736. Illustrative ΡΙ3Κγ inhibitors are known in the art and include, e.g., AS252424 (Jin, et al., Inflammation. 2014 Aug;37(4): 1254-60); AS605240 (Saito, et al, Pulm Pharmacol Ther. 2014 Apr;27(2): 164-9); CZC24832 (Bergamini, et al, Nat Chem Biol. 2012 Apr
29;8(6):576-82); and U.S. Patent Publication Nos. 2016/0303134 and 2016/0303123.
[0162] As appropriate, the immune checkpoint inhibitor can be co-administered via the same or different route of administration as the cellular material or the HIPPO intracellular pathway protein inhibitor. As appropriate, the immune checkpoint inhibitor can be co-administered concurrently with or sequentially to the cellular material or the HIPPO intracellular pathway protein inhibitor. b. Chemotherapeutic Agents
[0163] Examples of chemotherapeutic agents that can be co-administered with tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATSl), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase {e.g., MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells are known in the art and include without limitation alkylating agent(s) (e.g., nitrogen mustards, nitrogen ureas, ethylenimines, methylmelamines, alkyl sulfonates, carmustine, triazenes), platinum-coordination complexes (e.g., cisplatin, carboplatin, and oxaliplatin), anti-metabolite(s) (e.g., folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., capecitabine, 5-fluorouracil, 5-fluorodeoxyuridine, 5- fluorodeoxyuridine monophosphate, cytosine arabinoside, 5-azacytidine, gemcitabine), purine analogs (e.g., mercaptopurine, thioguanine, azathioprine, pentostatin,
erythrohydroxynonyladenine, fludarabine, cladribine)), plant alkaloid(s) and/or terpenoid(s), vinca alkaloid(s) (e.g., vincristine, vinblastine, vinorelbine, and vindesine),
podophyllotoxin(s) (e.g., etoposide and teniposide), camptothecin(s) (e.g., irinotecan and topotecan), anthracycline(s), aromatase inhibitor(s), taxane(s) (e.g., paclitaxel, taxol and docetaxel), topoisomerase inhibitor(s) (e.g., (Type I inhibitors: camptothecins, including irinotecan and topotecan; Type II Inhibitors: amsacrine, etoposide, etoposide phosphate, and teniposide), antibiotic(s) (e.g., dactinomycin, daunorubicin, doxorubicin, idarubicin, epirubicin, bleomycins, mitomycin), hormone(s), differentiating agent(s), kinase inhibitor(s) (e.g., Bevacizumab, BIBW 2992, Cetuximab, Imatinib, Trastuzumab, Gefitinib,
Ranibizumab, Pegaptanib, Sorafenib, Dasatinib, Sunitinib, Erlotinib, Nilotinib, Lapatinib, Panitumumab, Vandetanib, E7080, Pazopanib, Mubritinib and Fostamatinib) and antineoplastic agent(s) (e.g., (dactinomycin, doxorubicin, epirubicin, fludarabine and bleomycin). Any chemotherapeutic agent being used to treat the cancer of interest can be co-administered in a combination therapy regime with tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells. Chemotherapeutic agents of use are known in the art and described in reference texts, e.g., Physicians' Desk Reference, by PDR Network; 70th 2016 edition, or Brunton, et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, 12th edition, 201 1, McGraw-Hill Education). 7. Methods of Monitoring
[0164] A variety of methods can be employed in determining efficacy of therapeutic and prophylactic treatment with tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells, optionally in combination with a chemotherapeutic agent. Generally, efficacy is the capacity to produce an effect without significant toxicity. Efficacy indicates that the therapy provides therapeutic or prophylactic effects for a given intervention (examples of interventions can include by are not limited to administration of a pharmaceutical formulation, employment of a medical device, or employment of a surgical procedure). Efficacy can be measured by comparing treated to untreated individuals or by comparing the same individual before and after treatment. Efficacy of a treatment can be determined using a variety of methods, including pharmacological studies, diagnostic studies, predictive studies and prognostic studies. Examples of indicators of efficacy include but are not limited to inhibition of tumor cell growth and promotion of tumor cell death. [0165] The efficacy of an anti-cancer treatment can be assessed by a variety of methods known in the art. Tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells, optionally in combination with a chemotherapeutic agent, can be screened for prophylactic or therapeutic efficacy in animal models in comparison with untreated or placebo controls. Tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells, optionally in combination with a chemotherapeutic agent, identified by such screens can be then analyzed for the capacity to induce tumor cell death or enhanced immune system activation. For example, multiple dilutions of sera can be tested on tumor cell lines in culture and standard methods for examining cell death or inhibition of cellular growth can be employed. See, e.g., Green and Sambrook, et al, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Lab., New York, 2012; Ausubel, et al. Editor, Current Protocols in Molecular Biology, USA, 1984-2016; and Coligan, et al. Editor, Current Protocols in Immunology, USA, 1987-2016; Bonifacino, et al., Editor, Current Protocols in Cell Biology, USA, 2010-2016; all of which are incorporated herein by reference in their entirety. [0166] The methods of the present invention provide for detecting inhibition disease in patient suffering from or susceptible to various cancers. A variety of methods can be used to monitor both therapeutic treatment for symptomatic patients and prophylactic treatment for asymptomatic patients.
[0167] Monitoring methods entail determining a baseline value of a tumor burden in a patient before administering a dosage of tumor cells tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATSl), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells, optionally in combination with a chemotherapeutic agent, and comparing this with a value for the tumor burden after treatment, respectively.
[0168] With respect to therapies using tumor cells tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATSl), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells, optionally in combination with a chemotherapeutic agent, a significant decrease (i.e., greater than the typical margin of experimental error in repeat measurements of the same sample, expressed as one standard deviation from the mean of such measurements) in value of the tumor burden signals a positive treatment outcome (i.e., that administration of tumor cells tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATSl), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells, optionally in combination with a chemotherapeutic agent has blocked or inhibited, or reduced progression of tumor growth and/or metastasis).
[0169] In other methods, a control value of tumor burden (e.g., a mean and standard deviation) is determined from a control population of individuals who have undergone treatment with tumor cells tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells, optionally in combination with a chemotherapeutic agent. Measured values of tumor burden in a patient are compared with the control value. If the measured level in a patient is not significantly different (e.g., more than one standard deviation) from the control value, treatment can be discontinued. If the tumor burden level in a patient is significantly above the control value, continued administration of agent is warranted.
[0170] In other methods, a patient who is not presently receiving treatment but has undergone a previous course of treatment is monitored for tumor burden to determine whether a resumption of treatment is required. The measured value of tumor burden in the patient can be compared with a value of tumor burden previously achieved in the patient after a previous course of treatment. A significant decrease in tumor burden relative to the previous measurement (i.e., greater than a typical margin of error in repeat measurements of the same sample) is an indication that treatment can be resumed. Alternatively, the value measured in a patient can be compared with a control value (mean plus standard deviation) determined in a population of patients after undergoing a course of treatment. Alternatively, the measured value in a patient can be compared with a control value in populations of prophylactically treated patients who remain free of symptoms of disease, or populations of therapeutically treated patients who show amelioration of disease characteristics. In all of these cases, a significant increase in tumor burden relative to the control level (i.e., more than a standard deviation) is an indicator that treatment should be resumed in a patient. [0171] The tissue sample for analysis is typically blood, plasma, serum, mucous, tissue biopsy, tumor, ascites or cerebrospinal fluid from the patient. The sample can be analyzed for indication of neoplasia. Neoplasia or tumor burden can be detected using any method known in the art, e.g., visual observation of a biopsy by a qualified pathologist, or other visualization techniques, e.g., radiography, ultrasound, magnetic resonance imaging (MRI).
[0172] Further, the level of immune system activity in conjunction with tumor burden in a patient before administering a dosage of tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells, optionally in combination with a chemotherapeutic agent can be compared this with a value for the immune system activity in conjunction with tumor burden after treatment, again respectively.
[0173] With respect to therapies involving enhanced immune system activity, a significant increase (i.e., greater than the typical margin of experimental error in repeat measurements of the same sample, expressed as one standard deviation from the mean of such measurements) in value of immune response signals a positive treatment outcome (i.e., that administration of tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and EVs and lysates thereof, optionally in combination with an adjuvant or loaded into the MHC class I and/or MHC class II molecules of dendritic cells, optionally in combination with a chemotherapeutic agent has achieved or augmented an immune response). Immune response signals can include but are not limited to for example assessing the enhancement of the lymphoma-specific cytotoxic effect of human peripheral blood mononuclear cells (PBMCs). If the value for the immune response signal does not change significantly, or decreases, a negative treatment outcome is indicated. In general, patients undergoing an initial course of treatment with an immunogenic agent are expected to show an increase in immune response activity with successive dosages, which eventually reaches a plateau. Administration of an agent is often continued while the immune response is increasing.
Once a plateau is obtained, that is an indicator if the treatment is solely for the immune the administration of the treatment can be discontinued or reduced in dosage or frequency. 8. Kits
[0174] Further provided are kits. In some embodiments, the kits comprise tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally serine/threonine kinase 3 (STK3 or MST2), and/or EVs and lysates thereof. As appropriate, the tumor cells may be irradiated, frozen and/or lyophilized. In varying embodiments, kits comprise dendritic cells having at least partially loaded MHC class I and/or MHC class II molecules with cellular material from tumor cells deficient for one or more proteins within or associated with the HIPPO intracellular signaling selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7). In varying embodiments, the kits may further comprise an adjuvant. In varying embodiments, the kits may further comprise one or more chemotherapeutic agents. In varying embodiments, the kits may further comprise one or more immune checkpoint inhibitors (e.g., antibodies or fragments against cytotoxic T-lymphocyte associated protein 4 (CTLA4), programmed cell death 1 (PCD1), CD274 molecule (PD-L1), phosphoinositide 3-kinase γ (ΡΙ3Κγ). indoleamine 2,3- dioxygenase (IDO)), as described above and herein. The cellular material, dendritic cells and optionally adjuvant and chemotherapeutic agents can be provided in unitary dosage vials or containers (e.g., ampules).
EXAMPLES
[0175] The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1
The HIPPO intracellular signaling Kinases LATS1/2 Suppress Cancer Immunity
EXPERIMENTAL MODEL AND SUBJECT DETAILS
[0176] Animals. C57BL/6, C3H/HeOu, or BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Myd88, Tlr4, Tlr7, and Tlr9 KO mice were kind gifts from Dr. Shizuo Akira (Osaka University, Osaka, Japan). Ticaml (also known as TRIF) KO mice were kindly provided by Dr. Bruce Beutler (University of Texas Southwestern Medical Center, Dallas, TX, USA). Caspl (also known as Caspase-1) KO mice were kindly provided by Dr. Richard A. Flavell (Yale University School of
Medicine, New Haven, CT, USA). Ragl KO mice, Tmeml73 (also known as STING) KO mice, and OT-I transgenic mice were purchased from The Jackson Laboratory. Ifnarl KO mice were purchased from B&K Universal (East Yorkshire, United Kingdom). These mouse strains were backcrossed for 10 generations onto the C57BL/6 background at the University of California, San Diego. Mutant mice were bled by the University of
California, San Diego Animal Care Program. 7-12 weeks old mice were used and all animal experiments were approved by the University of California, San Diego, Institutional Animal Care and Use Committee.
MATERIALS AND METHODS
[0177] Cell culture and gene deletion by CRISPR/Cas9 system. All cell lines were cultured under an atmosphere of 5% C02 at 37°C. B16-OVA cells (B 16F10 cells expressing ovalbumin) were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/ml), and streptomycin (100 mg/ml). SCC7, 4T1, EL4, bone marrow-derived dendritic cells (BMDCs), mouse primary lymph node cells and CD8+ T cells were cultured in RPMI 1640 (Gibco) supplemented with 10% FBS (Gibco), penicillin (100 U/ml), and streptomycin (100 mg/ml).
[0178] LATS 1/2-deficient cells were created through the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system (Ran et al., (2013) Nat.
Protoc. 8, 2281-2308). We use a transient CRISPR strategy for the deletion of LATSl/2 to avoid any potential unspecific effects mediated by stable Cas9/sgRNA genome integration.
Cells were transiently transfected with a Cas9 and single-guide RNA (sgRNA) expression plasmid encoding puromycin resistance (PX459; Addgene plasmid #48139). The CRISPR- transfected cells will thus acquire transient resistance to puromycin. The guide sequences were designed using the Optimized CRISPR Design at http://crispr.mit.edu. The guide sequences used are 5'-AGACGTTCTGCTCCGAAATC-3' or 5'-
ACGTTTCC ATTGGCGAATGA-3 ' for mouse Latsl; 5'-GAGTGTCCAGCTTACAAGCG- 3' or 5'-GCTGGGTGGTGCAAACTACG-3 ' for mouse Lats2. Following transfection and transient selection with puromycin for 3 days, cells were single-cell sorted by fluorescence- activated cell sorting (FACS) into 96-well plate without puromycin. Knockout clones were selected by immunoblot analysis for the lack of LATSl/2 proteins and YAP
phosphorylation. We confirmed that LATSl/2 dKO cells were sensitive to puromycin after expansion, indicating a transient expression of CRISPR/Cas9 system in those cells. Two independent clones were analyzed as indicated and the parental LATS1/2 WT cells (not transfected with PX459) were used as control.
[0179] Retroviral infection. B16-OVA cells stably expressing empty vector, YAP(5SA), YAP(5SA/S94A), or TAZ(4SA) were generated by retroviral infection. 293 Phoenix retrovirus packaging cells were transfected with pBABE empty vector, pBABE YAP(5SA), pBABE YAP(5SA/S94A), or pBABE TAZ(4SA) constructs. Forty-eight hours after transfection, retroviral supernatant was supplemented with 5 μg/ml polybrene, filtered through a 0.45 μπι filter, and used for infection. Forty-eight hours after infection, cells were selected with 4 μg/ml puromycin in culture medium.
[0180] Immunoblot analysis. Equal amount of protein samples were resolved in
SDS-PAGE in reducing conditions unless otherwise mentioned in the Figure Legends. Antibodies to YAP (#14074), pYAP (SI 27 in humans and SI 12 in mice; #4911), YAP/TAZ (#8418), LATS1 (#3477), CD81 (#10037), and ALIX (#2171) were obtained from Cell Signaling; those to actin (#ab3280) and ovalbumin (OVA, #abl221) were from Abeam; those to LATS2 (#A300-479A, also weakly recognize LATS1) were from Bethyl
Laboratories; those to FLOT1 (#610821) and HSP90 (#610418) were from BD Biosciences. The phos-tag electrophoresis was performed as described previously (Moroishi et al., (2015) Genes Dev. 29, 1271-1284). YAP proteins can be separated into multiple bands in the presence of phos-tag depending on differential phosphorylation levels, with phosphorylated proteins migrating more slowly. Where indicated, cells were treated with serum starvation (DMEM or RPMI 1640 without other supplements), 1 μg/ml Latrunculin B (LatB), or 25 mM 2-deoxy-D-glucose (2-DG) for 1 h before harvest.
[0181] Immunostaining of cells. Cells were treated with or without 1 μg/ml Latrunculin B (LatB) for 1 h before harvest. Cells were then fixed for 10 min at room temperature with 4% paraformaldehyde in phosphate-buffered saline (PBS) and were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Cells were then incubated consecutively with primary antibodies to YAP/TAZ (Santa Cruz, #sc- 101199) (overnight at 4°C) and Alexa Fluor 488-labeled goat secondary antibodies (for 90 min at room temperature) in PBS containing 1% bovine serum albumin (BSA). Cells were covered with a drop of ProLong Gold antifade reagent with DAPI (Invitrogen) for observation. Cells in five randomly selected views (~ 100 cells) were selected for the quantification of YAP/TAZ localization. Reverse transcription (RT) and real-time PCR analysis. Total RNA (500 ng) isolated from cells with the use of RNeasy Plus Mini Kit (Qiagen) was reverse-transcribed to
complementary DNA using iScript™ cDNA Synthesis Kit (Bio-Rad). Complementary
DNA was then diluted and used for quantification by real-time PCR, which was performed using KAPA SYBR FAST qPCR Kit (Kapa Biosy stems) and the 7300 real-time PCR system (Applied Biosystems). The sequences of the PCR primers (forward and reverse, respectively) are 5'-GCCTGGAGAAACCTGCCAAGTATG-3' and
5'-GAGTGGGAGTTGCTGTTGAAGTCG-3' for mouse Gapdh;
5'-AGCTGACCTGGAGGAAAACA-3' and 5'-GACAGGCTTGGCGATTTTAG-3' for mouse Ctgf; 5'-GCTCAGTCAGAAGGCAGACC-3' and
5'-GTTCTTGGGGACACAGAGGA-3' for mouse Cyr61;
5'-AGGAGAAGAGTTGCCCACCTATGAG-3' and
5'-TCGAAGAGCTTCATCCTGTCGC-3' for mouse Amotl2;
5'-CCTGAGAAAGAAGAAAC ACAGCCTC-3 ' and
5'-GCAAGTTGGTTGAGGAAGAGAGGG-3' for mouse Ifna4;
5'-GAAGAGTTACACTGCCTTTGCCATC-3' and
5'-AAACACTGTCTGCTGGTGGAGTTC-3' for mouse Ifnbl . Reactions for Gapdh mRNA were performed concurrently on the same plate as those for the test mRNAs, and results were normalized by the corresponding amount of Gapdh mRNA. Soft agar colony formation assay. Each 6-well plate was coated with 1.5 ml of bottom agar (DMEM or RPMI 1640 containing 10% FBS and 0.5% Difco agar noble). Cells (5 χ 103 cells for B16-OVA and SCC7, 2.5 χ 103 cells for 4T1) were suspended in 1.5 ml of top agar (DMEM or RPMI 1640 containing 10% FBS and 0.35% Difco agar noble) into each well. Cells were cultured for approximately two weeks and replaced with fresh medium every three days. Colonies were stained using 0.005% crystal violet in 5% methanol and quantified using ImageJ software.
[0182] Tumor transplantation and immunization. B 16-OVA cells (2 x 105) were subcutaneously transplanted into both back flanks of C57BL/6 mice. Tumor height and width were measured with a caliper every 2-3 days to calculate tumor volume (= width2 χ height x 0.523). Mice were sacrificed when tumors reached maximum allowed size (15 mm in diameter) or when signs of ulceration were evident. Likewise, 1 χ 105 of SCC7 cells were subcutaneously transplanted into both back flanks of C3H/HeOu mice and 2.5 χ 105 of 4T1 cells were transplanted into both mammary fat pads of BALB/c mice. For 4T1 lung metastasis assay, lungs were tracheally injected with India ink 28 days after transplantation, and then destained in Fekete's solution to count tumor nodules.
[0183] For tumor vaccination experiments, C57BL/6 mice were immunized intradermally at the base of the tail with irradiated B16-OVA cells (100 Gy, 1 χ 106) 12 days prior to challenge with B16-OVA cells (one time vaccination, without any adjuvant). For immunization with EVs, C57BL/6 mice were inoculated with irradiated B 16-0 VA cells (100 Gy, 1 x 106) at the base of the tail and EVs freshly isolated from culture supernatants of B16-OVA cells (6 χ 106) were injected every 3 days (days 0, 3, 6, and 9) into the same place until challenged with B16-OVA cells at day 12. [0184] Histopathology and immunostaining of tumors. Tumors were fixed with 4% paraformaldehyde in PBS, embedded in paraffin, sectioned with a microtome, and stained with hematoxylin-eosin according to standard procedures. Immunostaining of tumors was performed with frozen cryostat sections with PE-conjugated antibodies to CD45
(eBioscience, #12-0451-82). [0185] Measurement of OVA specific antibodies. Serum anti -OVA IgG
concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Briefly, half area 96-well plates (Corning) were coated with 5 μg/ml OVA protein (Worthington Biochemical, #LS003056) in PBS overnight at 4°C. Plates were washed and then blocked for 3 h at room temperature with blocking buffer [1% BSA (bovine serum albumin) in PBS], followed by wash and incubation with serum samples tested at a 1 : 100 to 1 : 125,600 dilutions in blocking buffer overnight at 4°C. Plates were then washed and incubated with HRP-conjugated detecting antibody in blocking buffer at room temperature for 2 h. Plates were washed and incubated with TMB substrate (KPL, #95059-286), and then read at 450 nm and 650 nm after stopping the development with 1 M phosphoric acid. Each ELISA plate contained a titration of a previously quantified serum to generate a standard curve. Anti-OVA IgG concentrations were determined from the lowest dilution of serum samples within a standard curve and reported as U/ml.
[0186] Flow cytometry. Flow cytometry was performed using a BD LSRFortessa and results were analyzed using FlowJo software (Treestar). Cell suspensions were incubated in mouse Fc block (anti CD16/CD32; BD Biosciences, #553142) prior to staining. Fluorochrome conjugated anti-mouse CD45 (clone 30F-11), CD3e (clone 145- 2C11), CD8a (clone 53-6.7), Granzyme B (clone GB 11), and IFN-γ (clone XMG1.2) antibodies were used following the manufacturers protocol. Kb-SIINFEKL-tetramer was used for identifying OVA-specific CD8+ T cells. Propidium iodide (PI) was used to stain dead cells.
[0187] To analyze intracellular cytokine expression, cells were re-stimulated ex vivo with 10 μ^ηύ SIINFEKL peptide (AnaSpec, #AS-60193-1) for 5 hours in the presence of protein transport inhibitor (BD biosciences, #555029) for the last 4 hours. Intracellular cytokine staining was then performed using Fixation/Permeabilization Solution Kit (BD Biosciences, #554714).
[0188] Ex vivo cytotoxicity assay. EL4 cells were pulsed with 8 μg/ml SIINFEKL peptide or irrelevant peptides for 2 h at 37°C, and then labeled with 0.25 μΜ or 2.5 μΜ CFSE (carboxyfluorescein succinimidyl ester; Thermo Fisher Scientific, #C34554) for 10 min at 37°C, respectively. CFSElow (SIINFEKL loaded target) and CSFEhigh
(irrelevant peptide control) EL4 cells were mixed at 1 : 1 ratio, and then co-cultured with CD8+ T cells isolated from splenocytes of C57BL/6 mice challenged (or not) with WT or LATSl/2 dKO B16-OVA cells at 8: 1 effector to target cell ratio (E:T). CD8+ T cells were isolated using EasySepTM Mouse CD8a Positive Selection Kit (STEMCELL, #18753) from pooled splenocytes of 3-4 mice per group for each experiment. The frequencies of CFSElow and CSFEhigh EL4 cells in CFSE positive fraction were determined by flow cytometric analysis 18 h after incubation and the percent of specific killing was calculated. Specific killing (%) = [1-" Sample ratio'V'Negative control ratio"] χ 100; "Sample ratio" = [CFSElow(target)/CSFEhigh(irrelevant)] value of each samples co-cultured with CD8+ T cells; "Negative control ratio" = [CFSElow(target)/CSFEhigh(irrelevant)] value of EL4 cells not cultured with CD8+ T cells.
[0189] In vitro cross-presentation assay. For conditioned medium preparation, B16-
OVA cells were seeded on culture plates and incubated in DMEM supplemented with 10% FBS for 24 h at 37°C to allow cell attachment. The cells were then washed with PBS, and culture medium was switched to DMEM without serum. After incubation for 48 h, conditioned medium was collected and centrifuged at 2,000 g for 10 min at 4°C to remove cell debris. The resulting supernatant was used for the experiment.
[0190] Naive CD8+ T cells were isolated from OVA-specific T cell receptor transgenic OT-I mice using EasySepTM Mouse CD8a Positive Selection Kit (STEMCELL) and labeled with 2 μΜ CFSE. Bone marrow derived dendritic cells (BMDCs) were generated by 6 days of GM-CSF (20 ng/ml; Applied Biosystems, #14-8331-80)
differentiation, and then incubated (or not) for 18 h with conditioned medium (10% of the total volume) from WT or LATS1/2 dKO B 16-OVA melanoma cells and pulsed with OVA protein (10 μ§/ιη1) for the last 4 h. The cells were washed and cultured with CFSE-labeled OT-I CD8+ T cells at 1 : 1 ratio for 3 days. OT-1 T cell proliferation was monitored by CFSE dilution using a flow cytometer and a division index was determined using FlowJo software (Treestar).
[0191] Cytokine enzyme-linked immunosorbent assay (ELISA). IFN-γ or IL-12 levels in culture supernatants were determined by ELISA. For ex vivo IFN-γ production from lymph node cells, draining lymph nodes (inguinal lymph nodes) were isolated from C57BL/6 mice challenged (or not) with B16-OVA cells and cultured with OVA protein (100 μg/ml) for 3 days. For IL-12 production from BMDCs, BMDCs were generated by 6 days of GM-CSF (20 ng/ml) differentiation and stimulated (or not) for 18 h with conditioned medium (10% of the total volume) or EVs isolated from culture supernatants of equal numbers of WT or LATS1/2 dKO B16-OVA cells (EVs from 2 105 cells were used to stimulate 1 χ 106 BMDCs). Both cultures were done in RPMI 1640 supplemented with 10%) FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml) under an atmosphere of 5%> C02 at 37°C, and then aliquots of cell culture supernatants were used for cytokine ELISA. For cell conditioned medium preparation, B16-OVA cells were seeded on culture plates and incubated in DMEM supplemented with 10% FBS for 24 h at 37°C to allow cell attachment. The cells were then washed with PBS, and culture medium was switched to DMEM without serum. After incubation for 48 h, conditioned medium was collected and centrifuged at
2,000 g for 10 min at 4°C to remove cell debris. The resulting supernatant was used for EV isolation, which is described in the "EV isolation and analysis" section.
[0192] IFN-γ concentrations were determined using Mouse IFN-γ DuoSet ELISA
(R&D Systems, #DY485-05) according to a manufacturer's protocol. For IL-12 ELISA, half area 96-well plates were coated with capture antibody (Purified Rat Anti -Mouse IL-12 p40/p70; BD Biosciences, #551219) in PBS overnight at 4°C. Plates were washed and then blocked for 3 h at room temperature with blocking buffer (1%> BSA in PBS), followed by wash and incubation with culture supernatants overnight at 4°C. Plates were then washed and incubated with biotinylated detection antibody (Biotin Rat Anti -Mouse IL-12 (p40/p70; BD Biosciences, #554476) in blocking buffer at room temperature for 1 h, followed by wash and incubation with streptavidin-URP conjugate for 20 min. Plates were washed and incubated with TMB substrate (KPL, #95059-286), and then read at 450 nm and 650 nm after stopping the development with 1 M phosphoric acid. Concentrations were determined by comparison to a standard curve.
[0193] EV isolation and analysis. B16-OVA cells were seeded in 150 mm culture plate and incubated in DMEM supplemented with 10% FBS for 24 h at 37°C to allow cell attachment. The cells were then washed with PBS twice, and culture medium was switched to 35 ml of DMEM without serum. After incubation for 48 h, conditioned medium was collected and centrifuged at 2,000 g for 10 min at 4°C to thoroughly remove cell debris. The resulting supernatant was then filtered through a 0.22 μπι PVDF filter (Millipore, #SLGV033RB) to remove cell debris and microvesicles (for the detergent treatment experiment, the resulting flow-through was treated with 1% Triton X-100 for 10 min at 4°C prior to the ultracentrifugation). The flow-through was transferred into ultracentrifuge tubes (BECKMAN COULTER, #344058) and then ultracentrifuged in a Beckman SW32Ti rotor at 30,000 rpm for 90 min at 4°C. The resulting pellets were washed with 35 ml of PBS and then ultracentrifuged again at 30,000 rpm for 90 min at 4°C. The resulting EV pellets were re-suspended in PBS for experimental use. Protein concentrations of EVs were determined using Micro BCA Protein Assay Kit (Thermo, #23235). RNA in EVs was isolated using TRIzol reagents (Thermo, #15596026) according to the manufacturer's protocol and concentrations were determined using Agilent 2200 TapeStation (Agilent Technologies). For ribonuclease treatment, total RNA isolated from EVs was digested for 30 min at 37°C with 100 RNase A (Thermo, #EN0531) in a buffer comprising 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 300 mM NaCl. RNA was then resolved in agarose gels in non- denaturing conditions. Nanoparticle tracking analysis was performed using NanoSight NS300 system (Malvern Instruments Inc., Ranch Cucamonga, CA, USA) on isolated EVs diluted 5,000-fold with PBS for analysis. [0194] LC-MS/MS. EV samples were resolved in SDS-PAGE and the gels were cut into three regions, and then digested with trypsin. Extracted peptides were analyzed using a CI 8 column and an EASY-nLC-1000 (Thermo Scientific) coupled to a hybrid quadrupole- orbitrap Q-Exactive mass spectrometer (Thermo Scientific). A data-dependent, top 50 method was utilized for analysis. The resulting .RAW files were analyzed with Proteome Discoverer 1.4 and MASCOT. Results were filtered with 1% FDR at the protein level and exported to our in-house FileMakerPro database iSPEC and analyzed with Align!, which calculated intensity based absolute quantification (iBAQ) values (Schwanhausser et al., (2011) Nature 473, 337-342) that were used for subsequent analysis. The ratio of the iBAQ values for WT and LATS1/2 dKO EVs (DKO/WT ratio) in the individual experiments was calculated and scored according to the following criteria: score 2, >5-fold; score 1, 5- to 2- fold; score 0, 2- to 0.5-fold; score -1, 0.5- to 0.2-fold; and score -2, <0.2-fold. We then added each scores from the individual experiments and set the threshold as a score of >3 for the top 100 most significantly increased proteins in LATS1/2 dKO EVs. Gene Ontology (GO) analysis was done using the PANTHER program (Mi et al., (2013) Nat. Protoc. 8, 1551-1566). Heatmaps were generated using NetWalker (Komurov et al., (2012) BMC Genomics 13, 282).
QUANTIFICATION AND STATISTICAL ANALYSIS [0195] Statistical Analysis. Statistical analyses were performed using GraphPad
Prism 5 software (GraphPad Software, Inc, La Jolla, CA, USA). Statistical parameters and methods are reported in the Figures and the Figure Legends. A value of p < 0.05 was considered statistically significant. Epidemiological data are obtained using the PrognoScan database (Mizuno et al., (2009) BMC Med. Genomics 2, 18). Association of gene expression with the survival of patients was evaluated using log-rank test and a value of p < 0.05 was considered statistically significant.
RESULTS
[0196] LATS1/2 deletion enhances anchorage -independent growth in vitro. To elucidate the role of the Hippo pathway in anti-tumor immunity, we took advantage of murine syngeneic tumor models of three different cancer types in three different host genetic backgrounds; B16-OVA melanoma [B16F10 melanoma expressing ovalbumin (OVA)] in C57BL/6 mice, SCC7 head and neck squamous cell carcinoma in C3H/HeOu mice, and 4T1 breast cancer in BALB/c mice. These syngeneic allograft models have been well characterized and extensively used to study reciprocal interactions between tumor cells and host anti -turn or immune responses (Dranoff, (2012) Nat. Rev. Immunol. 12, 61-66;
Lei et al., (2016) OncoTargets Ther. 9, 545-555). We have recently shown that deletion of LATS1/2 almost completely abolished YAP/TAZ regulation by the Hippo pathway while deletion of other components had only a partial or minor effect on YAP/TAZ activity (Meng et al., (2015) Nat. Commun. 6, 8357). We therefore deleted LATS 1/2 in B16-OVA melanoma cells using CRISPR/Cas9 genome-editing technology (Ran et al., (2013) Nat. Protoc. 8, 2281-2308). We obtained multiple independent LATS1/2 double knockout (dKO) clones verified by the lack of protein expression of both LATS1 and LATS2 (Figure 1 A). Two different clones generated by two independent CRISPR guide sequences were used for this study. Because YAP is a direct substrate of LATSl/2, of which phosphorylation can be readily detected with a phospho-YAP antibody or by mobility shift on a phos-tag gel, we use YAP phosphorylation status as an indicator of LATSl/2 activity. We found that YAP phosphorylation levels were regulated in response to LATS1/2- activating signals in wild-type (WT) B16-OVA cells, however, loss of LATSl/2 abolished YAP phosphorylation (Figure 1A). Phosphorylation of YAP/TAZ by LATSl/2 is known to promote YAP/TAZ cytoplasmic localization and inactivation (Zhao et al., (2007) Genes Dev. 21, 2747-2761). Indeed, YAP/TAZ localized in the cytoplasm in response to filamentous actin disruption (which activates LATSl/2) in WT B16-OVA cells, yet YAP/TAZ remain localized in the nucleus in LATSl/2 dKO cells under the same condition (Figure IB). LATSl/2 inactivation or YAP/TAZ hyperactivation is known to promote cell growth (Zhao et al., (2008) Genes Dev. 22, 1962-1971). Although LATSl/2 dKO B 16- OVA cells showed identical growth on regular cell culture plates compared with WT cells (Figure 2A), LATSl/2 dKO B16-OVA cells showed a significant increase in anchorage- independent growth in comparison to WT cells, both in terms of colony number and colony size (Figure 1C). These observations indicate that the Hippo pathway is still operational in B16-OVA melanoma cells, and in addition, LATSl/2-deficiency activates YAP/TAZ and can further potentiate anchorage-independent growth of B 16-OVA cells in vitro.
[0197] We also deleted LATSl/2 in SCC7 squamous cell carcinoma cells and found that LATSl/2-deficiency almost completely blocked YAP phosphorylation (Figure 2B) as well as YAP/TAZ cytoplasmic localization (Figure 2C) in response to LATSl/2-activating signals. Notably, WT SCC7 cells showed high YAP phosphorylation and cytoplasmic localization of YAP/TAZ even in the absence of LATSl/2-activating signals, suggesting high basal LATSl/2 activity in these cancer cells. Loss of LATSl/2 again increased anchorage-independent growth of SCC7 cells (Figures ID and 2D). LATSl/2-dependent regulation of YAP phosphorylation (Figure 2E), YAP/TAZ subcellular localization (Figure 2F), and anchorage-independent cell growth (Figures IE and 2G) were similarly observed in 4T1 breast cancer cells. Together, our data demonstrate that deletion of LATSl/2 in tumor cells promotes anchorage-independent tumor cell growth in vitro. [0198] Loss of LATSl/2 inhibits tumor growth in vivo. To investigate the role of the
Hippo pathway in tumor growth in vivo, we subcutaneously transplanted equal numbers of WT or LATSl/2 dKO B 16-0 VA cells into the back flanks of C57BL/6 mice and monitored their growth. Unexpectedly, deletion of LATSl/2 in B 16-OVA cells strongly inhibited tumor growth in vivo (Figures 3 A and 3B). All mice died before day 22 in the WT B 16- OVA injected group, whereas injection with LATSl/2 dKO B 16-0 VA cells resulted in tumor-free survival in more than half of the mice (Figure 3C). We confirmed the growth suppressive effect of LATSl/2 deletion with an independent clone of LATSl/2 dKO B16- OVA cells (Figure 4A). We next examined tumor growth of LATS 1/2 dKO and WT SCC7 squamous cell carcinoma cells in syngeneic C3H/HeOu mice. All mice injected with the parental SCC7 cells showed aggressive tumor growth (Figures 3D and 4B) and 100% died before day 21 (Figure 3E). In contrast, none of the mice injected with LATSl/2 dKO SCC7 cells developed tumors and all survived tumor free (Figures 3D, 3E, and 4B). The differences in tumor growth potentials between WT and LATSl/2 dKO SCC7 cells are astonishing. In a 4T1 orthotopic allograft model, 4T1 breast cancer cells grow into solid tumors and can readily metastasize to the lung, liver, and brain when transplanted into the mammary fat pads of syngeneic BALB/c mice. Consistently, the parental 4T1 cells developed tumors and metastasized to the lung in BALB/c mice (Figures 3F, 3G, and 4C). On the other hand, LATSl/2 dKO 4T1 cells developed little tumors and had no metastasis when allografted in BALB/c mice (Figure 3F, 3G, and 4C). Thus, collectively, our observations indicate that loss of LATSl/2 in tumor cells dramatically inhibits tumor growth in vivo in multiple types of cancer in different host backgrounds. Based on the current dogma, these results are totally unexpected as LATSl/2 supposedly function as tumor suppressors.
[0199] LATSl/2 deletion enhances immunogenicity of tumor cells. Since LATSl/2 deletion exerts completely opposite effects on tumor cell growth in vitro and in vivo (Figures 1 and 3), we investigated whether host factors may contribute to the apparent discrepancy between in vitro and in vivo phenotypes of LATSl/2 dKO tumor cells.
Therefore, we examined histopathology of tumors from allografted mice. We found massive infiltration of inflammatory cells in LATSl/2 dKO B16-OVA melanomas (Figure 5 A) as well as in LATSl/2 dKO 4T1 breast cancers (Figure 6A), which were confirmed by staining with the pan-leukocyte marker CD45 (Figures 5B and 6B). These observations prompted us to investigate whether immune cells infiltrate and thereby eliminate LATSl/2 dKO tumor cells. Both innate and adaptive immune responses work together to constitute host anti-tumor immunity, but the adaptive immune system plays a pivotal role in mediating robust and highly specific immune responses against tumors (Gajewski et al., (2013) Nat. Immunol. 14, 1014-1022). Therefore, we examined host adaptive immune responses against tumor cells. We chose a B 16-OVA melanoma model to explore this because B16- OVA melanoma cells express a non-secreted form of chicken OVA as a surrogate tumor antigen that can be conveniently used to follow immune responses directed against the OVA antigen. In addition, B16-OVA has been extensively used to study cancer immunity and many genetically altered syngeneic mouse C57BL/6 strains are available. [0200] Although WT and LATS 1/2 dKO B l 6-0 VA cells showed identical expression of OVA (Figure 5C), we detected significantly higher levels of serum anti-OVA antibody in mice injected with LATS 1/2 dKO B16-OVA cells (Figure 5D), suggesting an enhanced tumor-specific humoral immune response in the LATS 1/2 dKO B16-OVA injected mice. We next examined cellular immune responses. CD8+ T cells isolated from the spleens of LATS 1/2 dKO B 16-0 VA-injected mice produced multiple effector cytokines (Figures 5E and 6C), indicative of T cell activation. We observed significantly higher CD8+ T cell cross-priming when mice were injected with LATS 1/2 dKO B 16-OVA cells (Figures 5F and 6D), and in addition, lymph node cells isolated from draining lymph nodes of LATS1/2 dKO B 16-0 VA-injected mice showed a remarkably higher OVA-specific T cell response than lymph node cells isolated from the parental B 16-0 VA-injected mice as measured by interferon γ (IFN-γ) production (Figure 5G). These observations suggest that tumor-specific cellular immune responses, particularly CD8+ T cell responses, are induced in mice injected with LATS 1/2 dKO B16-OVA cells. Indeed, CD8+ T cells in LATS 1/2 dKO B 16-0 VA-injected mice possessed OVA-specific cytotoxic activity ex vivo (Figures 5H and 6E), and infiltrated into tumors in vivo (Figures 51 and 6F). Together, the above data demonstrate that LATS 1/2 deletion in tumor cells stimulate tumor-specific humoral and cellular immune responses, leading to the establishment of robust anti-tumor immunity.
[0201] LATS 1/2 -deficiency enhances tumor vaccine efficacy via adaptive immunity.
Given that LATS 1/2 deletion in tumor cells enhances host anti -tumor immune responses, we investigated whether LATSl/2-null tumor cells, by stimulating anti -tumor immunity, may protect the host from challenge with the corresponding LATS 1/2 WT tumor cells. To test this, we performed two sets of experiments; co-injection of LATS 1/2 dKO and WT tumor cells into each side of the same mouse (Figure 7A), or immunization of mice with LATS 1/2 dKO tumor cells prior to LATS 1/2 WT tumor cell injection (Figures 7B and 7C). Strikingly, co-injection of LATS1/2 dKO B 16-OVA cells significantly suppressed tumor growth of the co-injected WT B 16-OVA cells (Figure 7A). Moreover, immunization of mice with irradiated LATS 1/2 dKO B16-OVA cells, which were viable but unable to proliferate, strongly inhibited the corresponding LATS 1/2 WT tumor growth whereas immunization with irradiated parental B16-OVA cells produced a much weaker effect (Figure 7B). These data indicate that LATSl/2 dKO B 16-0 VA cells are much more potent than LATSl/2 WT cells in inducing anti -tumor immunity. Notably, in our experimental setting, a single dose of tumor vaccination with irradiated WT B16-OVA cells was not sufficient to extend survival (Figure 7C). In contrast, a single dose of tumor vaccination with irradiated LATSl/2 dKO B 16-OVA cells showed a significant delay in tumor growth and prolonged survival (Figure 7C). Approximately 25% of the mice immunized with irradiated LATSl/2 dKO B16-OVA cells were tumor free when challenged with WT B16- OVA cells. The above observations suggest that LATSl/2-deficiency renders B16-OVA cells highly immunogenic and improves tumor vaccine efficacy. We further confirmed enhanced anti-tumor immunity by LATSl/2 deletion with a different syngeneic model. Mice having rejected LATSl/2 dKO SCC7 cells were resistant to rechallenge with the parental SCC7 cells (Figure 7D), indicating that these animals have established
immunological memory against the given tumor cells. [0202] Next, we tested whether adaptive immunity is required for tumor suppression by LATSl/2 deletion. We subcutaneously transplanted WT or LATSl/2 dKO B16-OVA cells into RAG-1 (recombination activating gene 1) knockout (KO) mice that are immune compromised due to the lack of mature T and B cells. LATSl/2 dKO B 16-OVA tumor cells grew similarly to WT cells (Figure 7E) and showed comparable mortality (Figure 7F) in the absence of an adaptive immune system. Consistently, co-injection of LATSl/2 dKO B16-OVA cells failed to inhibit the corresponding LATSl/2 WT tumor growth in RAG-1 KO mice (Figure 7G). Based on the above data, we conclude that LATSl/2 deletion in tumor cells enhances immunogenicity and provokes an adaptive immune response to eliminate tumor cells. [0203] YAP or TAZ over expression in tumor cells suppresses tumor growth in vivo.
YAP and TAZ are the most characterized downstream effectors of the Hippo pathway. LATSl/2 directly phosphorylate YAP/TAZ on multiple serine residues, leading to cytoplasmic retention, degradation, and thereby inactivation of YAP/TAZ. Because we observed high YAP/TAZ activation in LATSl/2 dKO B 16-0 VA tumors in vivo (Figures 8 A and 8B), we examined whether YAP/TAZ hyperactivation phenocopies the effect of LATSl/2 deletion in tumor growth. To this end, we generated B16-OVA cells stably overexpressing YAP(5SA) (an active mutant of YAP with all five LATSl/2
phosphorylation sites mutated to alanine, and thus unresponsive to inhibition by the LATS1/2 kinases) or TAZ(4SA) (an active mutant of TAZ with all four LATS1/2 phosphorylation sites replaced by alanine). Notably, we observed a mutual inhibition between YAP and TAZ protein abundance in YAP(5SA)- or TAZ(4SA)-overexpressing B16-OVA cells (Figure 8C), consistent with the previously described negative feedback response (Moroishi et al., (2015) Genes Dev. 29, 1271-1284). YAP(5SA)- or TAZ(4SA)- overexpressing B16-OVA cells showed increased anchorage-independent growth potential in comparison to the control cells in vitro (Figure 8D), while their tumor growth in vivo was significantly delayed (Figure 8E). We next investigated whether the effect of YAP(5SA) requires its transcriptional activity. YAP mainly binds to the TEAD family of transcription factors (TEAD 1-4) to induce gene expression and Ser94 in YAP is required for TEAD binding (Zhao et al., (2008) Genes Dev. 22, 1962-1971). As expected, mutating Ser94 abolished the ability of YAP(5SA) to induce target gene transcription (Figures 8F and 8G). Importantly, the TEAD binding defective YAP(5SA/S94A) was unable to suppress B16- OVA tumor growth (Figure 8H), suggesting that tumor suppression by YAP requires TEAD-dependent transcription. Together, these observations indicate that hyperactivation of YAP and TAZ significantly, though may not entirely, contributes to the in vivo tumor growth suppression by LATS1/2 deletion through a mechanism requiring TEAD-mediated transcription.
[0204] Extracellular vesicles released from LATS 1/2 -null tumor cells stimulate immune responses. We next explored how LATSl/2-deficiency in tumors stimulates host anti-tumor immune responses. Because we observed a preeminent CD8+ T cell cross- priming in mice injected with LATS 1/2 dKO B16-OVA cells (Figure 5F), we investigated whether LATS 1/2 dKO B16-OVA cells stimulate cross-presentation by antigen-presenting cells. To test this, we examined the effects of LATS 1/2 dKO B16-OVA cells on MHC (major histocompatibility complex) class I-restricted cross-presentation using bone marrow- derived dendritic cells (BMDCs) as antigen-presenting cells. We found that pre-treatment of BMDCs with conditioned medium from LATS1/2 dKO B 16-0 VA cells significantly augmented antigen cross-presentation in comparison to WT conditioned medium (Figure 9A), and consistent with this, LATS 1/2 dKO conditioned medium enhanced BMDCs activation as assessed by interleukin-12 (IL-12) production (Figure 9B). These results imply that factors released from LATS 1/2 dKO B 16-0 VA cells activate BMDCs and thereby enhance antigen cross-presentation. We sought to identify the secreted factor responsible for the increased immune stimulation. Recent studies have revealed the emerging roles of extracellular vesicles (EVs) in immune regulation, both in an immunosuppressive and immunostimulatory manner (Robbins and Morelli, (2014) Nat. Rev. Immunol. 14, 195-208). We therefore investigated whether EVs secreted from LATS1/2 dKO B16-OVA cells are capable of stimulating immune responses. We isolated EVs from culture supernatants of WT or LATS1/2 dKO B16-OVA melanoma cells by ultracentrifugation (Figure 1 OA) and found that EVs from LATS 1/2-deficient B 16-OVA cells were more potent than EVs from WT B 16-OVA cells in activating BMDCs as assessed by IL-12 production in vitro (Figure 9B). To discriminate EVs from extracellular non-membranous particles that may be enriched by ultracentrifugation, we treated the culture supernatants with detergent (Triton X-100) prior to EV purification. The detergent- treated EV pellets failed to activate BMDCs (Figure 1 OB). More importantly, LATS 1/2 dKO EVs improved tumor vaccine efficacy of irradiated WT B 16-OVA cells and conferred a strong immunity against tumor challenge in vivo (Figure 9C). Thus, our results show that EVs released from LATSl/2-deficient tumor cells induce immune responses and are sufficient to render LATSl/2-adequate tumor cells highly immunogenic. [0205] LATS 1/2 -deficient tumor cells secrete nucleic-acid-rich extracellular vesicles. To elucidate the mechanistic basis for immunostimulatory effects of LATS 1/2 dKO EVs, we characterized the nature of EVs released from WT or LATS 1/2 dKO B16- OVA cells. We found that LATS 1/2 dKO Bl 6-OVA cells produced slightly more EVs compared with WT cells, as assessed by nanoparticle tracking analysis (Figures 9D and 10A) as well as by protein quantification (Figure 9E). We then analyzed the proteome of EVs using quantitative mass spectrometry. We identified a total of 1,772 proteins in EVs, which showed enrichment of previously reported exosomal and microvesicle cargo proteins (Figure 10B), supporting the quality of our EV purification. Most of the protein expression was almost identical between WT and LATS 1/2 dKO EVs, but a subset of proteins were highly elevated in LATS 1/2 dKO EVs (Figure 11C). Among the top increased proteins in LATS 1/2 dKO EVs were those involved in RNA and nucleic acid binding (Figure 1 ID). These observations prompted us to investigate whether LATSl/2-null tumor EVs contain higher amounts of nucleic acids, which are previously reported contents of EVs (Yanez-Mo et al., (2015) . J. Extracell. Vesicles 4, 27066) and are also well-known
immunostimulators (Junt and Barchet, (2015) Nat. Rev. Immunol. 15, 529-544). Because RNA is the most abundant nucleic acid in EVs (Robbins and Morelli, (2014) Nat. Rev. Immunol. 14, 195-208), we characterized total RNA isolated from EVs. We found that RNA contents in LATS 1/2 dKO or YAP(5SA)-overexpressing EVs were dramatically increased in comparison to WT EVs (Figures 9F and 1 IE). The RNAs in EVs were single stranded RNA as they were sensitive to single-strand-specific ribonuclease treatment (Figures 10F). Taken together, our observations are consistent with a model that LATS1/2- deficient tumor cells secrete higher amounts of nucleic-acid-rich EVs that may contribute to the potent immunostimulatory effects. [0206] Extracellular vesicles from LATS1/2 dKO tumor cells stimulate the Toll-like receptors-type I interferon pathway. To investigate whether nucleic-acid-rich EVs released from LATSl/2-null tumors stimulate host anti -tumor immunity, we examined whether alterations in the host nucleic-acid-sensing pathways impair the tumor protective effects of LATS1/2 deletion in vivo. Both microbial (non-self) and self nucleic acids can be recognized by distinct families of pattern recognition receptors, including endosomal Tolllike receptors (TLRs) and cytosolic non-TLR sensors (Figure 12 A). Activation of these pathways results in the production of inflammatory cytokines as well as type I IFN, which stimulates innate and adaptive immunity (Junt and Barchet, (2015) Nat. Rev. Immunol. 15, 529-544). We subcutaneously transplanted WT or LATS1/2 dKO B16-OVA cells into C57BL/6 mice deficient in the following key molecules in the endogenous nucleic-acid- sensing pathways; MYD88 (myeloid differentiation primary response 88) or TRIF (TIR- domain-containing adapter-inducing interferon-γ, also known as TICAMl), two adaptor proteins required for TLR signaling; STING (stimulator of interferon genes, also known as TMEM173), an adaptor protein required for the cGAS (cyclic GMP-AMP synthase, also known as MB21D1) cytoplasmic DNA-sensing pathway; and Caspase-1 (also known as CASP1), an effector protein involved in IL-Ιβ maturation under the AEVI2 (absent in melanoma 2) cytoplasmic DNA-sensing pathway. We found that deletion of MYD88 largely (Figures 13A and 12B), and TRIF -deficiency considerably (Figures 13B and 12C), attenuated the tumor suppressive effects of LATS1/2 deletion as assessed by tumor mortality. In contrast, deletion of STING (Figure 13C) or Caspase-1 (Figure 13D) in recipient mice had no effect on tumor protection by LATSl/2-deficiency, suggesting that the TLR-MYD88/TRIF nucleic-acid-sensing pathway, not the cytoplasmic DNA-sensing pathway, is required for immunostimulatory effects of LATS1/2 deletion.
[0207] Distinct types of TLRs utilize MYD88 or TRIF as adaptor proteins and specifically respond to a wide range of ligands on the cell surface as well as in the endosome (Figure 12A). The endosomal TLRs are intrinsically capable of detecting nucleic acids. We further investigated which TLR is required for tumor suppression by LATS1/2 loss. Whereas LATS1/2 deletion in tumors still protected mice from tumor challenge in TLR4 (which senses bacterial lipopoly saccharides) KO mice (Figure 13E), deletion of TLR7 (which senses single stranded RNA) (Figure 13F) or TLR9 (which senses double stranded DNA) (Figure 13G) in recipient mice partially but significantly impaired tumor protection by LATS1/2 loss. Thus, our data are consistent with the conclusion that multiple TLRs, and probably not a single TLR, cooperatively sense the nucleic-acid-rich EVs secreted from LATSl/2-null tumors and trigger immune responses through the
MYD88/TRIF signaling pathway.
[0208] Activation of TLRs-MYD88/TRIF signaling results in pro-inflammatory cytokine production as well as type I IFN (in particular IFN-γ and IFN-γ) production, which stimulates anti-tumor immune responses (Figure 12A). Particularly, type I IFN plays a central role in anti-tumor immunity by promoting dendritic cell maturation, antigen cross- presentation, and CD8+ T cell clonal expansion (Fuertes et al., (2013) Trends Immunol . 34, 67-73). We therefore examined whether host type I IFN signaling is required for establishing host anti -tumor immunity induced by LATS1/2 dKO tumor cells. To test this, we subcutaneously transplanted WT or LATS 1/2 dKO B 16-OVA cells into IFNARl
(interferon a and β receptor subunit 1) KO mice that are deficient in a functional type I IFN receptor. We found that loss of host type I IFN signaling largely obliterated the protective role of LATS 1/2 deletion in tumor growth (Figure 13H) as well as tumor mortality (Figure 131). Thus, collectively, our data provide in vivo evidence supporting a model that EVs secreted from LATS 1/2-deficient tumor cells stimulate the host TLRs-M YD 88/TRIF nucleic-acid-sensing pathways to incite type I IFN signaling and establish robust anti-tumor immunity.
DISCUSSION
[0209] In this study, we demonstrate that LATS 1/2 deletion unmasks a malignant cell's immunogenic potential and restrains tumor growth due to induction of anti -tumor immune responses. The effects of LATS 1/2 deletion on tumor growth are striking insofar as LATS 1/2 dKO completely abolishes the tumor growth potential of SCC7 and
dramatically reduces tumor growth and metastasis of B16 and 4T1 cells. LATSl/2-null B16 melanomas secrete nucleic-acid-rich EVs that stimulate the host TLRs-MYD88/TRIF- IFN pathways to induce anti-tumor immunity and eventual elimination of tumor cells
(Figure 14). LATS 1/2 deletion similarly stimulates host immune responses in both SCC7 and 4T1 syngeneic models (Figures 7D, 6A, and 6B), though the involvement of EVs has only been examined in the B 16 model in this study. [0210] Dual functions of LATS1/2 in cancer. It is generally accepted that the Hippo pathway is a tumor suppressor that inhibits cell proliferation and survival of normal cells, preventing tumorigenesis (Harvey et al., (2013) Nat. Rev. Cancer 13, 246-257; Moroishi et al., (2015) Nat. Rev. Cancer 15, 73-79; Wang et al., (2014) Cancer Metastasis Rev. 33, 173-181), yet a few studies did suggest an oncogenic role of the Hippo pathway in certain contexts (Barry et al., (2013). Nature 493, 106-110; Cottini et al., (2014) Nat. Med. 20, 599-606). We have analyzed human epidemiological data using the PrognoScan database (Mizuno et al., (2009) BMC Med. Genomics 2, 18) to find any correlation between
LATS1/2 mRNA expression levels and patient outcome in different types of human cancer (Table S2). Among 107 epidemiological data sets available, 26 studies show significant (p < 0.05) correlation between LATS2 mRNA levels and patient outcome, which includes 17 studies showing better patient survival with low LATS2 expression. Moreover, 12 studies show significant correlation between LATSl mRNA levels and patient outcome, which includes 5 studies showing better patient survival with low LATSl expression. In addition, low YAP expression predicted worse patient survival in human colorectal cancer (Barry et al., 2013, (2013). Nature 493, 106-110) and multiple myeloma (Cottini et al., (2014) Nat. Med. 20, 599-606). Therefore, although YAP/TAZ hyperactivation is frequently observed in human cancers (Harvey et al., 2013, supra; Moroishi et al., (2015) Nat. Rev. Cancer 15, 73-79), the precise role of the Hippo pathway in human cancer might be context dependent. In this study, however, we show that deletion of LATS1/2 in tumor cells strongly suppresses tumor growth in vivo. On the surface, our data cannot be easily reconciled with the tumor suppressor model of LATS1/2 in the Hippo field.
[0211] The data are consistent with the following model: LATS1/2 suppress tumor initiation as well as inhibit immunogenicity. These two activities are important for the physiological role of LATS1/2 in maintaining tissue and organ homeostasis. LATS1/2 normally provide growth inhibitory signals to the cells, and therefore they function cell- autonomously to limit tissue overgrowth. We also propose that LATS1/2 suppress immunogenicity, serving as a built-in mechanism to prevent overgrowth of undesirable cells at wrong places in the organism. For example, inactivation of LATS1/2 is needed to promote cell proliferation during wound healing and tissue regeneration. However, cells with impaired LATS1/2 activity may over-proliferate and migrate to wrong place. Such undesirable cells should be eliminated to maintain tissue homeostasis and integrity. This can be achieved because inactivation of LATS1/2 in these cells can induce a strong immune response. Therefore, the immunosuppressive function of LATSl/2 is consistent with its physiological roles in tissue and organ homeostasis.
[0212] In the established tumor cell lines of B16, SCC7, and 4T1, YAP/TAZ are not constitutively active. In fact, YAP/TAZ are readily regulated (in B16 and 4T1 cells) or even largely inactive (in SCC7 cells). Therefore, the tumorigenicity of these cancer cell lines is independent of the Hippo pathway. Nevertheless, deletion of LATSl/2 causes a moderate increase of anchorage-independent growth of these tumor cells in vitro, consistent with the growth inhibitory effect of LATSl/2. However, the enhanced immunogenicity unmasked by the LATSl/2 deletion in these cells can induce strong immune responses and overwhelm any growth advantage that might be gained due to LATSl/2 deletion, leading to strong inhibition of tumor growth in the immune competent mice. Consistent with this model, tumor growth of LATSl/2 dKO cells is not inhibited in the immune compromised RAG-1 mutant mouse. Moreover, immunization of mice with LATSl/2 dKO cells or EVs from LATSl/2 dKO cells also inhibits tumor growth of the parental cells. The dual functions of LATSl/2 in suppressing cell growth and immunogenicity can explain previous observations along with our current data. LATSl/2 inhibition in normal cells contributes to tumor initiation while LATSl/2 inactivation in tumor cells induces host anti -tumor immunity. The dual nature of the Hippo pathway in cancer is not totally eccentric. Many signaling pathways, such as TGFP, INK, and NRF2 (Ikushima and Miyazono, (2010) Nat. Rev. Cancer 10, 415-424; Menegon et al., (2016) Trends Mol. Med. 22, 578-593; Wagner and Nebreda, (2009) Nat. Rev. Cancer 9, 537-549), have been shown to both promote and inhibit tumors in a context dependent manner. The unique nature of the LATSl/2 in tumor suppression is its dependency on the host immune system.
[0213] Hippo pathway in inflammation and tumor immunogenicity. Our results indicate that inactivation of the Hippo pathway in tumor cells induces host inflammatory responses. Interestingly, recent studies revealed that the Hippo pathway can respond to (Nowell et al., (2016) Nat. Cell Biol. 18, 168-180; Taniguchi et al., (2015) Nature 519, 57- 62) and mediate (Liu et al., (2016) Cell 164, 406-419) inflammatory signals. Our study together with these findings suggests a reciprocal interaction between the Hippo pathway and inflammatory responses. EVs secreted from LATSl/2-null tumor cells are important for inducing inflammation, though the molecular mechanisms by which LATS1/2- deficiency alters the quality and quantity of EVs are currently not well understood.
LATSl/2-deficient tumor derived EVs contain higher amounts of nucleic acids, which stimulate the host TLRs-MYD88/TRIF nucleic-acid-sensing pathways, provoking a type I IFN response to establish robust anti-tumor immunity. Recent studies indicate that tumor cells themselves can produce type I IFN in response to chemotherapy, thus enhancing antitumor immune responses (Chiappinelli et al., (2015) Cell 162, 974-986; Sistigu et al., (2014) Nat. Med. 20, 1301-1309). Because WT and LATS 1/2 dKO B 16-OVA cells showed similar expression levels of type I IFN genes, such as Ifna4 and Ifnbl (Figure 12D), it is unlikely that type I IFN secreted from LATSl/2-null tumor cells is the main mechanism conferring the anti -tumor immunity evoked by LATS 1/2 deletion. Given that nucleic-acid- rich EVs from LATSl/2-deficient tumors can stimulate dendritic cells in vitro (Figure 9B), and that the host TLRs-MYD88/TRIF nucleic-acid-sensing pathways are required for immunostimulatory effects of LATS 1/2 deletion in vivo (Figure 13), host immune cells in the tumor microenvironment may be the major source of type I IFN (Corrales et al., (2015) Cell Rep. 11, 1018-1030; Fuertes et al., (2013) Trends Immunol. 34, 67-73).
[0214] A series of unbiased Hippo pathway interactome studies have linked endosomal compartments to the Hippo pathway (Moya and Haider, (2014) Cell Res. 24, 137-138). It is possible that the Hippo pathway may regulate endocytic trafficking and therefore regulate EV biogenesis. Little is known about the signaling mechanisms involved in EV biogenesis and incorporation of proteins or nucleic acids into EVs. Given the known effect of YAP on global miRNA biogenesis (Mori et al., (2014) Cell 156, 893-906) and the functional importance of miRNA in EVs (Yanez-Mo et al., (2015) . J. Extracell. Vesicles 4, 27066), the effect of YAP/TAZ on miRNA biogenesis may increase immunogenicity of LATSl/2-null cells. However, we show that TEAD-mediated transcription is required for tumor suppression by YAP (Figure 8H) whereas TEAD dependent transcription is dispensable for YAP-influenced miRNA biogenesis (Mori et al., (2014) Cell 156, 893-906). Moreover, LATS 1/2 dKO does not increase miRNA contents in EVs (Figure 1 IE).
Therefore, YAP/TAZ hyperactivation suppresses tumor growth in vivo via a transcription dependent, but miRNA biogenesis independent, mechanism. The tumor growth suppression by YAP/TAZ overexpression (Figure 8E) is not as strong as that of LATS 1/2 deletion (Figure 3 A), suggesting that LATS 1/2 may have additional targets to suppress immune responses. Recent studies revealed new LATS1/2 substrates in spindle orientation (Dewey et al., (2015) Curr. Biol. 25, 2751-2762; Keder et al., (2015) Curr. Biol. 25, 2739-2750). Because aneuploidy plays a role in tumor immunogenicity (Senovilla et al., (2012) Science 337, 1678-1684), these new LATS1/2 substrates in spindle regulation could contribute to immunosuppression. Further investigations delineating the mechanistic link between the Hippo pathway and EV biogenesis will have important implications in understanding both the basic biology of EVs and inflammation provoked by alteration of the Hippo pathway.
[0215] Targeting the Hippo pathway for cancer immunotherapy. Recent advances in cancer immunotherapy have provided new therapeutic approaches for cancer, and several immune checkpoint inhibitors indeed show impressive effects in the clinic (Sharma and Allison, (2015) Cell 161, 205-214). However, individual immune response to cancer immunotherapy often relies on tumor immunogenicity that varies extensively between different cancer types and different individuals, and therefore immune checkpoint inhibitors may not work in cases where tumor immunogenicity is intrinsically limited (Pico de Coana et al., (2015) Trends Mol. Med. 21, 482-491). Our study revealed that inactivation of LATS1/2 in tumor cells increases tumor immunogenicity and enhances tumor vaccine efficacy. Therefore, inhibiting LATS1/2 enhances anti -tumor immune response, and therefore, is an attractive approach to treat cancer. Furthermore, LATS1/2 inhibition to improve immunogenicity of tumor cells enhances immune checkpoint inhibitor efficacy. Thus, a combination of LATS1/2 inhibitors and immune checkpoint inhibitors is a novel and exciting therapeutic approach for poorly immunogenic cancers, especially in cases where malignancy is driven by oncogenic alterations that leave the Hippo signaling pathway intact. It is noteworthy that germline or somatic mutations affecting the core components of the Hippo pathway are uncommon in human cancers (Harvey et al., 2013, supra; Moroishi et al., (2015) Nat. Rev. Cancer 15, 73-79). Therefore, inhibition of LATS 1/2 enhances tumor immunity in most cancer types.
Example 2
Autologous Treatment Of A Tumor Using Cells Deficient For One Or More
HIPPO Intracellular Signaling Pathway Proteins [0216] A physician obtains a population of cancerous cells from an individual via a biopsy. An autologous cancerous cell line with ablated or reduced LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) molecule activity is generated. The cancerous cells isolated from the individual are irradiated. One or more genes selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) in the cancerous cells isolated from the individual is knocked down using a CRISPR/Cas9 system. The cancerous cells isolated from an individual are transfected with a plasmid containing nucleic acid sequences encoding a crRNA, a tracrRNA, and a Cas9 molecule directed to a portion of the one or more genes selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3). The crRNA and tracrRNA activates Cas9 and guides it to the portion of one or more genes selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) to be cleaved. One or more Cas9 proteins cleaves a portion of one or more genes LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) rendering them inactive. The cancerous cells are expanded in vitro and a cancerous cell line deficient for one or more genes in the HIPPO intracellular signaling pathway selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) is established. The physician injects intravenously the cells deficient for one or more genes selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7) (and optionally STK3) back into the same individual from which the cancerous cells were isolated.
Example 3
Treatment Of A Tumor Using An Allogenic Vaccine Deficient For One Or
More HIPPO Intracellular Signaling Pathway Proteins
[0217] A physician obtains a population of cancerous cells from an individual via a biopsy. A vaccine comprising allogenic cancerous cells with one or more genes selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3) having ablated or reduced molecule activity and extracellular vesicles (EVs) as an adjuvant is generated. The allogenic cancerous cells isolated from the individual are irradiated. One or more genes selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) in the cancerous cells isolated from the individual are knocked down using a CRISPR/Cas9 system. The cancerous cells isolated from an individual are transfected with a plasmid containing nucleic acid sequences encoding a crRNA, a tracrRNA, and one or more Cas9 molecules directed to a portion of one or more genes selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3). The crRNA and tracrRNA activates Cas9 and guides it to the portion of the one or more genes selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) to be cleaved. Cas9 cleaves a portion of the one or more genes selected from LATS1, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), rendering them inactive. The cancerous cells are expanded in vitro and an allogenic cancerous cell line deficient for one or more genes selected from LATS l, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) is established. The EVs produced by the LATS l/2-deficient allogenic cancerous cells are isolated. The physician injects intravenously a vaccine formulation comprising the allogenic cells deficient for one or more genes selected from LATS l, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) in addition to the EVs, as an adjuvant, back into the a different individual from which the cancerous cells were isolated.
Example 4
Treatment Of A Tumor Using A Small Molecule Drug Targeting One Or More
HIPPO Intracellular Signaling Pathway Proteins
[0218] A physician administers one or more small molecule drugs that inhibit or reduce the molecule activity of one or more HIPPO pathway proteins selected from LATS l, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) to an individual suffering from a cancer. The one or more small molecule drug are inhibitors of enzymatic activity of one or more HIPPO pathway proteins selected from LATS l, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7) (and optionally STK3), e.g., by competitive, non-competitive, or uncompetitive mechanisms that binds to one or more HIPPO pathway proteins selected from LATS l, LATS2, STK4 and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) (and optionally STK3) and inhibits their function. Decrease of the molecular activity of one or more HIPPO pathway proteins selected from LATS l, LATS2, STK4 and a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7) (and optionally STK3) increases the immune response to the tumor and thereby leads to inhibition of tumor growth.
[0219] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMS What is claimed is:
1. A tumor cell having reduced or eliminated expression or function of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATS l), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7).
2. The tumor cell of claim 1, further wherein function or activity of serine/threonine kinase 3 (STK3) is reduced or eliminated.
3. The tumor cell of any one of claims 1 to 2, wherein function or activity of the one or more proteins within or associated with the HIPPO intracellular signaling pathway has been reduced and/or eliminated.
4. The tumor cell of any one of claims 1 to 2, wherein the expression of the one or more proteins within or associated with the HIPPO intracellular signaling pathway has been reduced and/or eliminated.
5. The tumor cell of claim 4, wherein genes encoding the one or more proteins within or associated with the HIPPO intracellular signaling pathway have been knocked down.
6. The tumor cell of claim 4, wherein the genes encoding the one or more proteins within or associated with the HIPPO intracellular signaling pathway have been knocked out.
7. The tumor cell of claim 4, wherein the genes encoding both of LATS l and LATS2 have been knocked down or knocked out.
8. The tumor cell of any one of claims 1 to 7, wherein the tumor cell has elevated expression and/or activity levels of YAP and/or TAZ.
9. The tumor cell of claim 8, wherein the tumor cell comprises one or more recombinant polynucleotides encoding (hyper)active mutants of YAP and/or TAZ.
10. The tumor cell of any one of claims 8 to 9, wherein one or more recombinant polynucleotides encoding YAP and/or TAZ, or mutants thereof, are incorporated into the genome.
1 1. The tumor cell of any one of claims 1 to 10, wherein the tumor cell expresses or overexpresses a tumor-associated antigen.
12. The tumor cell of any one of claims 1 to 1 1, wherein the tumor cell is irradiated.
13. The tumor cell of claim 12, wherein the tumor cell has been irradiated with a sufficient dose of radiation and for a sufficient time such that it is viable but unable to proliferate.
14. The tumor cell of any one of claims 1 to 13, wherein the tumor cell has retained function of YAP and/or TAZ.
15. The tumor cell of any one of claims 1 to 14, wherein the tumor cell is selected from the group consisting of a melanoma, a glioma, a colon cancer, a squamous cell carcinoma and a breast carcinoma.
16. The tumor cell of any one of claims 1 to 14, wherein the tumor cell is from a cancer selected from the group consisting of sarcoma, lymphoma, hematological cancer, skin cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, colon cancer, rectal cancer, urogenital cancer, hepatic cancer, thyroid cancer, esophageal cancer, bladder cancer, renal cancer, brain cancer (e.g., glioma) and head and neck cancer.
17. An extracellular vesicle (EV) from a tumor cell or a population of tumor cells of any one of claims 1 to 16.
18. A cell lysate from a tumor cell or a population of tumor cells of any one of claims 1 to 16.
19. An immunogenic composition comprising a tumor cell or a population of tumor cells of any one of claims 1 to 16, an extracellular vesicle (EV) of claim 17 and/or a cell lysate of claim 18 and a pharmaceutically acceptable excipient.
20. The immunogenic composition of claim 19, further comprising an adjuvant.
21. A method of inducing, promoting and/or enhancing an immune response against a tumor in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a population of tumor cells of any one of claims 1 to 16, a population of extracellular vesicles (EVs) of claim 17, or a cell lysate of claim 18, thereby inducing, promoting and/or enhancing the immune response against the tumor in the subject.
22. A method of inducing, promoting and/or enhancing an immune response against a tumor in a subject in need thereof, comprising co-administering to the subject a tumor associated antigen and a therapeutically effective amount of a population of tumor cells of any one of claims 1 to 16, a population of extracellular vesicles (EVs) of claim 17, or a cell lysate of claim 18, thereby inducing, promoting and/or enhancing the immune response against the tumor in the subject.
23. The method of claim 22, wherein the tumor associated antigen is a synthetic or recombinant peptide or polypeptide.
24. The method of claim 22, wherein the tumor associated antigen is in a tumor cell or tumor cell lysate obtained from the subject.
25. The method of any one of claims 21 to 24, wherein the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is injected
intradermally, epicutaneously or subcutaneously.
26. The method of any one of claims 21 to 25, wherein the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate are administered multiple times.
27. The method of any one of claims 21 to 26, wherein the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is co-administered with an adjuvant.
28. The method of any one of claims 21 to 27, wherein the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is autologous to the subject.
29. The method of any one of claims 21 to 28, further comprising the steps prior to administration of:
a) isolating a population of tumor cells from the subject; and
b) reducing or eliminating expression and/or activity in the isolated tumor cells of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATS l), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7).
30. The method of claim 29, further wherein the function or activity of serine/threonine kinase 3 (STK3) is reduced or eliminated.
31. The method of any one of claims 29 to 30, comprising knocking-out or knocking down in the population of tumor cells genes encoding the one or more proteins within or associated with the HIPPO intracellular signaling.
32. The method of any one of claims 29 to 30, comprising contacting the population of tumor cells with one or more inhibitor compounds that inhibit the one or more proteins within or associated with the HIPPO intracellular signaling.
33. The method of any one of claims 21 to 32, wherein the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is syngeneic to the subject.
34. The method of any one of claims 21 to 32, wherein the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is allogeneic to the subject.
35. The method of any one of claims 21 to 32, wherein the population of tumor cells, population of extracellular vesicles (EVs) or cell lysate is xenogeneic to the subject.
36. A method of inducing, promoting and/or enhancing an immune response against a tumor in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a population of dendritic cells with the major histocompatibility complex (MHC) proteins loaded with antigens from a population of tumor cells of any one of claims 1 to 16, a population of extracellular vesicles (EVs) of claim 17, an apoptotic tumor cell preparation or a cell lysate of claim 18 or exosomes derived from the dendritic cells, thereby inducing, promoting and/or enhancing the immune response against the tumor in the subject.
37. The method of claim 36, wherein the dendritic cells are autologous to the subject.
38. The method of claim 36, wherein the dendritic cells are syngeneic to the subject.
39. The method of claim 36, wherein the dendritic cells are allogeneic to the subject.
40. The method of claim 36, wherein the dendritic cells are xenogeneic to the subject.
41. The method of any one of claims 36 to 40, wherein the population of dendritic cells is injected intradermally, epicutaneously or subcutaneously.
42. The method of any one of claims 36 to 41, wherein the population of dendritic cells is administered multiple times.
43. The method of any one of claims 36 to 42, wherein the population of dendritic cells is co-administered with an adjuvant.
44. A method of inducing, promoting and/or enhancing an immune response against a tumor in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an inhibitor of the expression or function of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATS l), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7).
45. The method of claim 44, further comprising inhibiting serine/threonine kinase 3 (STK3).
46. The method of any one of claims 44 to 45, wherein the inhibitor comprises one or more inhibitory nucleic acids.
47. The method of claim 46, wherein the one or more inhibitory nucleic acids specifically hybridize to one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4 a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) and optionally STK3.
48. The method of claim 46, comprising administering to the subject a one or more inhibitory nucleic acids that partially, substantially, or completely deletes, silences, inactivates, down-regulates, reduces, or inhibits, activity or expression of one or more of LATSl, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) and optionally STK3.
49. The method of any one of claims 46 to 48, wherein the one or more inhibitory nucleic acids comprise a small interfering RNA (siRNA).
50. The method of any one of claims 46 to 48, wherein the one or more inhibitory nucleic acids encode one or more transcription activator-like effector nucleases (TALEN).
51. The method of claim 50, wherein the one or more TALENs specifically cleave one or more nucleic acid sequences encoding one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7), and optionally STK3, and partially, substantially, or completely deletes, silences, inactivates, or down-regulates one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3.
52. The method of any one of claims 46 to 48, wherein the one or more inhibitory nucleic acids encode one or more meganucleases.
53. The method of claim 52, wherein the one or more meganucleases specifically cleave one or more nucleic acid sequences encoding one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7), and optionally STK3, and partially, substantially, or completely deletes, silences, inactivates, or down-regulates one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3.
54. The method of any one of claims 46 to 48, wherein the one or more inhibitory nucleic acids encode one or more transcription activator-like effector
meganucleases (megaTALs).
55. The method of claim 54, wherein the one or more megaTALs specifically cleave one or more nucleic acid sequences encoding one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7), and optionally STK3, and partially, substantially, or completely deletes, silences, inactivates, or down-regulates one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3.
56. The method of any one of claims 46 to 48, wherein the one or more inhibitory nucleic acids encode one or more zinc finger nucleases (ZFNs).
57. The method of claim 56, wherein the one or more ZFNs specifically cleave one or more nucleic acid sequences encoding one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7), and optionally STK3, and partially, substantially, or completely deletes, silences, inactivates, or down-regulates one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3.
58. The method of any one of claims 46 to 48, wherein the one or more inhibitory nucleic acids encode one or more crRNAs, one or more tracrRNAs, and one or more Cas9 endonucleases, wherein the crRNA, tracrRNA and Cas9 endonuclease operatively coordinate.
59. The method of claim 58, wherein the one or more Cas9 endonucleases is programmed by a coordinating crRNA and a coordinating tracrRNA hybrid to cleave one or more nucleic acid sequences encoding one or more of LATS l, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g.,
MAP4K 1/2/3/4/5/6/7), and optionally STK3, thereby partially, substantially, or completely deleting, silencing, inactivating, or down-regulating one or more of LATS 1, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7), and optionally STK3.
60. The method of any one of claims 44 to 45, wherein the inhibitor is a polypeptide, peptide or small organic compound.
61. The method of claim 60, comprising administering to the subject an inhibitor of LATS 1 and/or LATS2 selected from the group consisting of A443654,
Lestaurtinib, GSK-690693, lysophosphatidic acid (LP A), sphingosine-1 -phosphate (S IP) and thrombin.
62. The method of any one of claims 60 to 61, comprising administering an inhibitor of STK4 and/or STK3 selected from 9E1 and XMU-MP-1.
63. The method of any one of claims 44 to 45, wherein the inhibitor is an antibody or fragment thereof.
64. The method of claim 63, wherein the antibody or fragment thereof partially, substantially, or completely deletes, silences, inactivates, down-regulates, reduces, or inhibits, activity or expression of one or more of LATS 1, LATS2, heat shock protein 90 (HSP90), STK4, a MAP4K family kinase (e.g., MAP4K1/2/3/4/5/6/7), and optionally STK3.
65. The method of any one of claims 44 to 64, wherein the inhibitor of the expression or function of one or more proteins are administered by a route selected from the group consisting of orally, intravenously, intramuscularly, subcutaneously,
intradermally, intralesionally and intratumorally.
66. The method of any one of claims 44 to 65, further comprising administering to the individual cellular material from a population of cancerous cells in which expression or activity of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATS1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7) is partially, substantially, or completely deleted, silenced, inactivated, down-regulated, reduced or inhibited.
67. The method of claim 66, wherein the cellular material comprises a population of replication-deficient tumor cells, a population of extracellular vesicles (EVs) and/or cell lysate.
68. The method of any one of claims 21 to 65, wherein the tumor is from a cancer that has retained function of YAP and/or TAZ.
69. The method of any one of claims 21 to 68, wherein the tumor is from a cancer selected from the group consisting of a melanoma, a glioma, a colon cancer, a squamous cell carcinoma and a breast carcinoma.
70. The method of any one of claims 21 to 65, wherein the tumor is from a cancer selected from the group consisting of sarcoma, lymphoma, hematological cancer, skin cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, colon cancer, rectal cancer, urogenital cancer, hepatic cancer, thyroid cancer, esophageal cancer, bladder cancer, renal cancer, brain cancer (e.g., glioma) and head and neck cancer.
71. The method of any one of claims 21 to 70, wherein the subject is a human.
72. The method of any one of claims 21 to 71, further comprising co-administering an immune checkpoint inhibitor.
73. The method of claim 72, wherein the immune checkpoint inhibitor is an inhibitor of cytotoxic T-lymphocyte associated protein 4 (CTLA4), programmed cell death 1 (PCD1), CD274 molecule (PD-L1) , phosphoinositide 3 -kinase γ (ΡΙ3Κγ) or indoleamine 2,3 -di oxygenase (IDO).
74. The method of any one of claims 72 to 73, wherein the immune checkpoint inhibitor is an antibody or fragment or variant thereof.
75. The method of any one of claims 72 to 74, wherein the checkpoint inhibitor is administered before the inhibitor of the expression or function of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATS 1), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7).
76. The method of any one of claims 72 to 74, wherein the checkpoint inhibitor is administered after the inhibitor of the expression or function of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATS l), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7).
77. The method of any one of claims 72 to 74, wherein the checkpoint inhibitor is administered concurrently with the inhibitor of the expression or function of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATS l), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g., MAP4K 1/2/3/4/5/6/7).
78. A vaccine composition comprising: (a) cellular material from a population of cancerous cells in which expression or activity of one or more proteins within or associated with the HIPPO intracellular signaling pathway selected from the group consisting of large tumor suppressor kinase 1 (LATSl), large tumor suppressor kinase 2 (LATS2), serine/threonine kinase 4 (STK4) and a MAP4K family kinase (e.g.,
MAP4K1/2/3/4/5/6/7) is partially, substantially, or completely deleted, silenced, inactivated, down-regulated, reduced or inhibited, and (b) a pharmaceutically-acceptable excipient.
79. The vaccine composition of claim 78, wherein the cellular material comprises a population of replication-deficient tumor cells, a population of extracellular vesicles (EVs) and/or cell lysate.
80. The vaccine composition of any one of claims 78 to 79, wherein the vaccine composition is formulated for intradermal, epicutaneous or subcutaneous administration.
81. The vaccine composition of any one of claims 78 to 80, wherein the cellular material is autologous to an individual to be treated.
82. The vaccine composition of any one of claims 78 to 80, wherein the cellular material is syngeneic to an individual to be treated.
83. The vaccine composition of any one of claims 78 to 80, wherein the cellular material is allogeneic to an individual to be treated.
84. The vaccine composition of any one of claims 78 to 80, wherein the cellular material is xenogeneic to an individual to be treated.
85. The vaccine composition of any one of claims 79 to 84, wherein the replication-deficient tumor cells have been irradiated.
86. The vaccine composition of claim 85, wherein replication-deficient tumor cells have been irradiated with a sufficient dose of radiation and for a sufficient time such that they are viable but unable to proliferate.
Ill
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