POLYPEPTIDES FOR IMPROVED RESPONSE TO ANTI-CANCER THERAPY
20190255146 ยท 2019-08-22
Assignee
Inventors
- Besim OGRETMEN (Mount Pleasant, SC, US)
- Raquela J. THOMAS (Charleston, SC, US)
- Natalia OLEINIK (Charleston, SC, US)
Cpc classification
C12N2710/16632
CHEMISTRY; METALLURGY
A61K9/0019
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K9/0073
HUMAN NECESSITIES
A61K31/164
HUMAN NECESSITIES
A61K48/00
HUMAN NECESSITIES
C12N2710/16641
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61K9/00
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
A61K31/164
HUMAN NECESSITIES
Abstract
The present disclosure provides E2F5 mimetic polypeptides. Further provided are methods for the treatment of cancer, such as head and neck cancer, comprising administering the E2F5 mimetic polypeptides alone or in combination with an additional anti-cancer therapy, such as a chemotherapeutic.
Claims
1. An isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 2, wherein the polypeptide comprises no more than 100 contiguous amino acids of SEQ ID NO: 13.
2. The polypeptide of claim 1, wherein the polypeptide comprises no more than 50 contiguous amino acids of SEQ ID NO: 13.
3. The polypeptide of claim 1, wherein the polypeptide is less than 100 amino acids in length.
4. The polypeptide of claim 1, wherein the polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 1.
5. The polypeptide of claim 4, wherein the polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:l.
6. The polypeptide of claim 1, wherein the polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 2.
7. The polypeptide of claim 1, wherein the polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 2.
8. The polypeptide of claim 7, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO: 2.
9. The polypeptide of claim 1, further comprising a cell penetration sequence.
10. The polypeptide of claim 9, wherein the cell penetration sequence is a polyarginine sequence.
11. A pharmaceutical composition comprising the isolated polypeptide of any one of claims 1-10 and a pharmaceutical carrier.
12. The composition of claim 11, wherein the pharmaceutical composition is formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection.
13. An isolated nucleic acid encoding the polypeptide of any one of claims 1-7.
14. The nucleic acid of claim 13, wherein the nucleic acid is comprised in a vector.
15. The nucleic acid of claim 14, wherein the vector comprises a mammalian expression vector.
16. A host cell comprising the nucleic acid of claim 13.
17. A method for treating cancer in a subject comprising administering an effective amount of the polypeptide of any one of claims 1-10 or a nucleic acid of claim 15 to the subject.
18. The method of claim 17, wherein the cancer is head and neck cancer.
19. The method of claim 17, wherein the subject is diagnosed as Human papillomavirus (HPV) negative.
20. The method of claim 17, further comprising administering at least a second anti-cancer therapy.
21. The method of claim 20, wherein the second anti-cancer therapy is selected from the group consisting of a chemotherapy, a radiotherapy, an immunotherapy, or a surgery.
22. The method of claim 21, wherein the chemotherapy is cisplatin.
23. The method of claim 20, wherein the at least a second anti-cancer therapy is a ceramide analogue drug.
24. The method of claim 23, wherein the ceramide analogue drug is C.sub.18-pyridinium-ceramide (C.sub.18-pyr-cer).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
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DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0032] Human papillomavirus (HPV) infection is linked to improved survival in response to chemo-radiotherapy for patients with oropharynx head and neck squamous cell carcinoma (HNSCC). However, mechanisms involved in increased HNSCC cell death by HPV signaling in response to therapy are largely unknown. Studies in the present disclosure used molecular, pharmacologic and genetic tools to show that HPV early protein 7 (E7) enhances ceramide-mediated lethal mitophagy in response to chemotherapy-induced cellular stress in HPV-positive HNSCC cells by selectively targeting retinoblastoma protein (RB). Inhibition of RB by HPV-E7 relieves E2F5, which then associates with DRP1, providing a scaffolding platform for Drp1 activation and mitochondrial translocation, leading to mitochondrial fission and increased lethal mitophagy. Ectopic expression of a constitutively active mutant RB, which is not inhibited by HPV-E7, attenuated ceramide-dependent mitophagy and cell death in HPV+HNSCC cells. Moreover, mutation of E2F5 to prevent Drp1 activation inhibited mitophagy in HPV+cells. Activation of Drp1 with E2F5-mimetic peptide for inducing Drp1 mitochondrial localization, enhanced ceramide-mediated mitophagy, and led to tumor suppression in HPV-negative HNSCCderived xenograft tumors in response to cisplatin in SCID mice.
[0033] Thus, the present disclosure provides an E2F5-mimetic polypeptide and its use thereof for the treatment of cancers, particularly head and neck cancer. In particular, the E2F5 polypeptide provided herein is administered in combination with a chemotherapeutic such as cisplatin to provide an improved response to therapy in subjects with head and neck cancer, without pathological HPV infection.
I. DEFINITIONS
[0034] As used herein, essentially free, in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
[0035] As used herein in the specification and claims, a or an may mean one or more. As used herein in the specification and claims, when used in conjunction with the word comprising, the words a or an may mean one or more than one. As used herein, in the specification and claim, another or a further may mean at least a second or more.
[0036] As used herein in the specification and claims, the term about is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
[0037] Treatment and treating refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, a treatment may include administration of a T cell therapy.
[0038] Subject and patient refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.
[0039] The term therapeutic benefit or therapeutically effective as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.
[0040] An anti-cancer agent is capable of negatively affecting a cancer cell/tumor in a subject, for example, by promoting killing of cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.
[0041] The phrases pharmaceutical or pharmacologically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. The preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.
[0042] As used herein, pharmaceutically acceptable carrier includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.
[0043] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of sequence identity or homology to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60, expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR.
II. E2F5 POLYPEPTIDES
[0044] Embodiments of the present disclosure concern E2F5 mimetic polypeptides. In particular embodiments, the E2F5 mimetic polypeptide comprises a biologically active fragment of a E2F5 polypeptide. The amino acid sequence of a wild type E2F5 polypeptide (GenBank Accession No. CAB01634.1) is provided below:
TABLE-US-00001 (SEQIDNO:13) 1 maaaepassgqqapagqgqgqrpppqppqaqapqpppppqlggagggssrhekslglltt 61 kfvsllqeakdgvldlkaaadtlavrqkrriyditnvlegidliekksknsiqwkgvgag 121 cntkevidrlrylkaeiedlelkereldqqklllqqsiknvmddsinnrfsyvthedicn 181 cfngdtllaiqapsgtqlevpipemgqngqkkyqinlkshsgpihvllinkesssskpvv 241 fpvpppddltqpssqsltpvtpqkssmatqnlpeqhvsersqalqqtsatdissagsisg 301 diidelmssdvfpllrlsptpaddynfnlddnegvcdlfdvqilny
[0045] In certain preferred aspects, a E2F5 fragment of the embodiments comprises a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent sequence identity with the dimerization domain of E2F5 provided as SEQ ID NO: 1 (e.g., amino acids 84-177 of GenBank Accession No. CAB01634.1) or with SEQ ID NO: 2. In certain aspects, polypeptide comprises no more that 100, 95, 85, 80, 75, 70, 65, 60, 55, 50, 45, 49, 48, 47, 46, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 contiguous amino acids of SEQ ID NO: 13. In particular aspects, the polypeptide comprises, consists of or consists essentially of the amino acid sequence ELDQQKLWLQQSIKNVMDDSINNRFSYVTHED (SEQ ID NO: 2).
[0046] An E2F1 polypeptide may be a recombinant polypeptide, synthetic polypeptide, purified polypeptide, immobilized polypeptide, detectably labeled polypeptide, encapsulated polypeptide, or a vector-expressed polypeptide (e.g., a polypeptide encoded by a nucleic acid in a vector comprising a heterologous promoter operably linked to the nucleic acid). In some embodiments, an E2F5 polypeptide may be administered to a subject, such as a human patient, for the treatment of cancer, such as head and neck cancer.
[0047] The E2F5 polypeptides that can be used in various embodiments include the amino acid sequences described herein, as well as analogues and derivatives thereof. The analogues and derivatives can include, but are not limited to, additions or substitutions of amino acid residues within the amino acid sequences encoded by a nucleotide sequence, but that result in a silent change, thus producing a functionally equivalent gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example: nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
[0048] Amino acid substitutions may alternatively be made on the basis of the hydropathic index of amino acids. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (0.4); threonine (0.7); serine (0.8); tryptophan (0.9); tyrosine (1.3); proline (1.6); histidine (3.2); glutamate (3.5); glutamine (3.5); aspartate (3.5); asparagine (3.5); lysine (3.9); and arginine (4.5). The use of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (Kyte and Doolittle, J. Mol. Biol. 157:105-132, 1982). It is known that in certain instances, certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments the substitution of amino acids whose hydropathic indices are within 2 is included, while in other embodiments amino acid substitutions that are within 1 are included, and in yet other embodiments amino acid substitutions within 0.5 are included.
[0049] Amino acid substitutions may alternatively be made on the basis of hydrophilicity, particularly where the biologically functional protein or polypeptide thereby created is intended for use in immunological embodiments. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 1); glutamate (+3.0 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (0.4); proline (0.5 1); alanine (0.5); histidine (0.5); cysteine (1.0); methionine (1.3); valine (1.5); leucine (1.8); isoleucine (1.8); tyrosine (2.3); phenylalanine (2.5) and tryptophan (3.4). In making changes based upon similar hydrophilicity values, in certain embodiments the substitution of amino acids whose hydrophilicity values are within 2 is included, in certain embodiments those that are within 1 are included, and in certain embodiments those within 0.5 are included. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity.
[0050] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.
[0051] A. Cell Penetration Sequence
[0052] The E2F5 polypeptide of the present disclosure may comprise or be coupled to a cell importation peptide or a cellular internalization transporter (e.g., via a peptide bond, linker, or cleavable linker). As used herein the term cell penetrating peptide refers to segments of polypeptide sequence that allow or promote a polypeptide to cross the cell membrane, such as the plasma membrane of a eukaryotic cell. Examples of cell importation signals include, but are not limited to, polyarginine sequences (e.g., RRRRRRRR (SEQ ID NO: 7), segments derived from HIV Tat (e.g., GRKKRRQRRRPPQ, SEQ ID NO:8; or RKKRRQRRR, SEQ ID NO: 9), herpes virus VP22, the Drosophila Antennapedia homeobox gene product (RQPKIWFPNRRKPWKK; SEQ ID NO:10), protegrin I, Penetratin (RQIKIWFQNRRMKWKK; SEQ ID NO:11), Antp-3A (Antp mutant), Buforin II Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-I, SynBl, Pep-7, HN-1, KALA, R11, K11, or melittin (GIGAVLKVLTTGLPALISWIKRKRQQ; SEQ ID NO:12). Poly-R sequences may vary in length, e.g., from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 R amino acids in length.
[0053] Cell importation signals for use herein may be covalently conjugated (e.g., chemically fused or attached, expressed as a fusion construct, etc.) with an E2F5 polypeptide to promote transport of the E2F5 polypeptide across a cell membrane. Cell importation signals that may be used include, e.g., peptides (e.g., cell penetration peptides), polypeptides, hormones, growth factors, cytokines, aptamers or avimers. Furthermore, a cell importation signal may mediate non-specific cell internalization or may be a cell targeting moiety that is internalized in a subpopulation of targeted cells.
[0054] B. Cell-Targeting Moiety
[0055] In some embodiments, an E2F5 polypeptide may be expressed as a fusion protein or chemically attached to a cell targeting moiety to selectively target the construct containing the E2F5 polypeptide to a particular subset of cells such as, e.g., cancerous cells, tumor cells, endothelial cells. For example, in some embodiments, the cell targeting moiety is an antibody. In general the term antibody includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies, humanized antibodies, minibodies, dibodies, tribodies as well as antibody fragments, such as Fab, Fab, F(ab)2, single domain antibodies and any mixture thereof. In some cases it is preferred that the cell targeting moiety is a single chain antibody (scFv). In a related embodiment, the cell targeting domain may be an avimer polypeptide. Therefore, in certain cases the cell targeting constructs of the present disclosure are fusion proteins comprising an E2F5 polypeptide and a scFv or an avimer.
[0056] In certain aspects of the present disclosure, a cell targeting moiety may be a growth factor. For example, transforming growth factor, epidermal growth factor, insulin-like growth factor, fibroblast growth factor, B lymphocyte stimulator (BLyS), heregulin, platelet-derived growth factor, vascular endothelial growth factor (VEGF), or hypoxia inducible factor may be used as a cell targeting moiety according to the invention. These growth factors enable the targeting of constructs to cells that express the cognate growth factor receptors.
[0057] In further aspects of the present disclosure, a cell targeting moiety may be a hormone. Some examples of hormones for use in the invention include, but are not limited to, human chorionic gonadotropin, gonadotropin releasing hormone, an androgen, an estrogen, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, prolactin, growth hormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin, thyrotropin-releasing hormone, growth hormone releasing hormone, corticotropin-releasing hormone, somatostatin, dopamine, melatonin, thyroxine, calcitonin, parathyroid hormone, glucocorticoids, mineralocorticoids, adrenaline, noradrenaline, progesterone, insulin, glucagon, amylin, erythropoitin, calcitriol, calciferol, atrial-natriuretic peptide, gastrin, secretin, cholecystokinin, neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor-1, leptin, thrombopoietin, angiotensinogen, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, or IL-36. Targeting constructs that comprise a hormone may enable methods of targeting cell populations that comprise extracelluar receptors for the indicated hormone.
[0058] In certain aspects, a cell targeting moiety of the present disclosure may be a cancer cell-targeting moiety. It is well known that certain types of cancer cells aberrantly express surface molecules that are unique as compared to surrounding tissue. Thus, cell targeting moieties that bind to these surface molecules may enable the targeted delivery of E2F5 peptides specifically to the cancers cells. For example, a cell targeting moiety may bind to and be internalized by a lung, breast, brain, prostate, spleen, pancreatic, cervical, ovarian, head and neck, esophageal, liver, skin, kidney, leukemia, bone, testicular, colon, or bladder cancer cell. The skilled artisan will understand that the effectiveness of a cancer cell-targeted E2F5 polypeptide may, in some cases, be contingent upon the expression or expression level of a particular cancer marker on the cancer cell. Thus, in certain aspects, there are provided methods for treating a cancer with a targeted E2F5 polypeptide comprising determining whether (or to what extent) the cancer cell expresses a particular cell surface marker and administering targeted E2F5 polypeptide therapy (or another anticancer therapy) to the cancer cells depending on the expression level of a marker gene or polypeptide.
[0059] C. Linkers/Coupling Agents
[0060] In some embodiments, an E2F5 polypeptide of the present disclosure may be chemically attached to another group such as, e.g., a cell targeting moiety. If desired, the compound of interest may be joined via a biologically-releasable bond, such as a selectively-cleavable linker or amino acid sequence. For example, peptide linkers that include a cleavage site for an enzyme preferentially located or active within a tumor environment are contemplated. Exemplary forms of such peptide linkers are those that are cleaved by urokinase, plasmin, thrombin, Factor IXa, Factor Xa, or a metallaproteinase, such as collagenase, gelatinase, or stromelysin.
[0061] Additionally, while numerous types of disulfide-bond containing linkers are known which can successfully be employed to conjugate moieties, certain linkers will generally be preferred over other linkers, based on differing pharmacologic characteristics and capabilities. For example, linkers that contain a disulfide bond that is sterically hindered may be preferred, due to their greater stability in vivo, thus preventing release of the moiety prior to binding at the site of action.
[0062] Additionally, any other linking/coupling agents and/or mechanisms known to those of skill in the art can be attached to a peptide of the present invention, such as, for example, amide linkages, ester linkages, thioester linkages, ether linkages, thioether linkages, phosphoester linkages, phosphoramide linkages, anhydride linkages, disulfide linkages, ionic and hydrophobic interactions, or combinations thereof.
[0063] Cross-linking reagents are used to form molecular bridges that tie together functional groups of two different molecules, e.g., a stablizing and coagulating agent. However, it is contemplated that dimers or multimers of the same analog can be made or that heteromeric complexes comprised of different analogs can be created. To link two different compounds in a step-wise manner, hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.
III. METHODS OF TREATMENT
[0064] Provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount an E2F5 polypeptide provided herein. Examples of cancers contemplated for treatment include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphomas, pre-neoplastic lesions in the lung, colon cancer, melanoma, and bladder cancer. In particular aspects, the cancer is head and neck cancer.
[0065] The E2F5 polypeptide may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage of the E2F5 polypeptide may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.
[0066] Intratumoral injection, or injection into the tumor vasculature is specifically contemplated for discrete, solid, accessible tumors. Local, regional or systemic administration also may be appropriate. For tumors of >4 cm, the volume to be administered will be about 4-10 ml (in particular 10 ml), while for tumors of <4 cm, a volume of about 1-3 ml will be used (in particular 3 ml). Multiple injections delivered as single dose comprise about 0.1 to about 0.5 ml volumes.
[0067] A. Pharmaceutical Compositions
[0068] Also provided herein are pharmaceutical compositions and formulations comprising the E2F5 polypeptide and a pharmaceutically acceptable carrier.
[0069] Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (such as an antibody or a polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22.sup.nd edition, 2012), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn- protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
[0070] B. Combination Therapies
[0071] In certain embodiments, the compositions and methods of the present embodiments involve an E2F5 polypeptide in combination with at least one additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In particular aspects, the at least one additional therapy is a chemotherapy (e.g., cisplatin) or a ceramide analogue drug.
[0072] In some embodiments, the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side- effect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is therapy targeting PBK/AKT/mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or chemopreventative agent. The additional therapy may be one or more of the chemotherapeutic agents known in the art.
[0073] An E2F5 polypeptide composition may be administered before, during, after, or in various combinations relative to an additional cancer therapy. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the E2F5 polypeptide composition is provided to a patient separately from an additional therapeutic agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the antibody therapy and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.
[0074] Various combinations may be employed. For the example below an E2F5 polypeptide therapy is A and an anti-cancer therapy is B:
TABLE-US-00002 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0075] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy.
[0076] 1. Chemotherapy
[0077] A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. The term chemotherapy refers to the use of drugs to treat cancer. A chemotherapeutic agent is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
[0078] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegaIl); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2,2-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine,plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above
[0079] 2. Radiotherapy
[0080] Other factors that cause DNA damage and have been used extensively include what are commonly known as -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
[0081] 3. Immunotherapy
[0082] The skilled artisan will understand that additional immunotherapies may be used in combination or in conjunction with methods of the embodiments. In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells
[0083] Antibody-drug conjugates have emerged as a breakthrough approach to the development of cancer therapeutics. Cancer is one of the leading causes of deaths in the world. Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) that are covalently linked to cell-killing drugs. This approach combines the high specificity of MAbs against their antigen targets with highly potent cytotoxic drugs, resulting in armed MAbs that deliver the payload (drug) to tumor cells with enriched levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in decreased toxicity and improved therapeutic index. The approval of two ADC drugs, ADCETRIS (brentuximab vedotin) in 2011 and KADCYLA (trastuzumab emtansine or T-DM1) in 2013 by FDA validated the approach. There are currently more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization are becoming more and more mature, the discovery and development of new ADCs are increasingly dependent on the identification and validation of new targets that are suitable to this approach and the generation of targeting MAbs. Two criteria for ADC targets are upregulated/high levels of expression in tumor cells and robust internalization.
[0084] In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.
[0085] Examples of immunotherapies currently under investigation or in use are immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, e.g., interferons , , and , IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the antibody therapies described herein.
[0086] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either turn up a signal (e.g., co-stimulatory molecules) or turn down a signal. Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA). In particular, the immune checkpoint inhibitors target the PD-1 axis and/or CTLA-4.
[0087] The immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligand or receptors, or, in particular, are antibodies, such as human antibodies (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both incorporated herein by reference). Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used. As the skilled person will know, alternative and/or equivalent names may be in use for certain antibodies mentioned in the present disclosure. Such alternative and/or equivalent names are interchangeable in the context of the present invention. For example it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.
[0088] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and/or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Pat. Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art such as described in U.S. Patent Application No. US20140294898, US2014022021, and US20110008369, all incorporated herein by reference.
[0089] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP- 224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO, is an anti-PD-1 antibody described in WO2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA, and SCH-900475, is an anti-PD-1 antibody described in WO2009/114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342.
[0090] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an off switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.
[0091] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
[0092] Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in: U.S. Pat. No. 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17): 10067-10071; Camacho et al. (2004) J Clin Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. WO2001014424, WO2000037504, and U.S. Pat. No. 8,017,114; all incorporated herein by reference.
[0093] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy) or antigen binding fragments and variants thereof (see, e.g., WOO 1/14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on CTLA-4 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).
[0094] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as described in U.S. Pat. Nos. 5,844,905, 5,885,796 and International Patent Application Nos. WO1995001994 and WO1998042752; all incorporated herein by reference, and immunoadhesins such as described in U.S. Pat. No. 8,329,867, incorporated herein by reference.
[0095] 4. Surgery
[0096] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs' surgery).
[0097] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
[0098] 5. Other Agents
[0099] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.
IV. ARTICLES OF MANUFACTURE OR KITS
[0100] An article of manufacture or a kit is provided comprising E2F5 polypeptides is also provided herein. The article of manufacture or kit can further comprise a package insert comprising instructions for using the E2F5 polypeptides to treat or delay progression of cancer in an individual or to enhance immune function of an individual having cancer. Any of the E2F5 peptides described herein may be included in the article of manufacture or kits. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation and the label on, or associated with, the container may indicate directions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more of another agent (e.g., a chemotherapeutic agent, and anti-neoplastic agent). Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.
V. EXAMPLES
[0101] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
[0102] Example 1Head and Neck Squamous Cell Carcinoma (HNSCC) Studies
[0103] To determine whether HPV(+) compared to HPV() HNSCC cells are more sensitive to stress-mediated growth inhibition in response to chemotherapeutic drugs, it was first established HPV(+) UM-SCC-47- and HPV() UM-SCC-22A-derived xenograft tumors in SCID mice. After the xenograft-derived tumors were 75-100 mm.sup.3, the mice were treated with cisplatin (3.5 mg/kg) for two weeks, which is below its maximum tolerated doses (van Moorsel et al, 1999). Then, tumor volumes were measured at days 0 and 14 of treatment. The data suggest that the growth of HPV(+) UM-SCC-47 derived HNSCC xenografts was inhibited in response to cisplatin (75% inhibition), whereas there was no detectable tumor suppression when HPV() UM-SCC-22A-derived xenografts were treated with this drug in SCID mice (
[0104] HPV-E7 signaling induces cell death by CerS1/ceramide-dependent mitophagy. Endogenous ceramide (C.sub.18-ceramide), which induces lethal mitophagy, is generated by ceramide synthase 1 (CerS1) (Pewzner-Jung et al, 2006; Koybasi et al, 2004; Venkataraman et al, 2002). This is first confirmed by treatment of mouse embryonic fibroblasts (MEFs) isolated from wild type (WT) and CerS1-toppler mutant mice (CerS1.sup.top/top), which encodes for inactive enzyme for defective C.sub.18-ceramide generation (Spassieva et al, 2016; Zhao et al, 2011) by sodium selenite, a known inducer of mitophagy (Sentelle et al, 2012). Sodium selenite markedly induced mitophagy in WT-MEFs, but not in CerS1.sup.top/top-MEFs, as detected by the co-localization of lysosomes (lysotracker green, LTG) and mitochondria (mitotracker red, MTR) using live cell imaging/confocal microscopy or lipidation of LC3B using Western blotting (
[0105] To determine the roles of HPV16-E6 and E7 proteins in HNSCC growth inhibition in response to cisplatin, the effects of siRNA-mediated knockdown of these proteins on the growth of UM-SCC-47 cells were measured in response to cisplatin compared to scrambled (Scr)-siRNA-transfected controls. Knockdown of E6 and E7 proteins, confirmed by Western blotting resulted in 8-fold resistance to cisplatin-mediated growth inhibition in UM-SCC-47 cells compared to controls (
[0106] Lethal mitophagy is mediated by mitochondria-targeted C.sub.18-ceramide via HPV-E7 signaling. To determine whether ceramide accumulation in mitochondria is sufficient to replicate the cellular response to cisplatin, HPV(+) UM-SCC-47 cells were treated with C.sub.18-pyr-cer. A pyridinium ring conjugated to the sphingosine backbone targets this ceramide analogue to mitochondria (Senkal et al, 2006). C.sub.18-pyr-cer increased mitophagy detected by decreased oxygen consumption rate, measured using the Sea Horse compared to vehicle-treated controls (
[0107] Inhibition of Rb by HPV-E7 targets HNSCC mitochondria for ceramide-dependent mitophagy. To examine a possible role for HPV-E6/E7 oncoproteins in the enhanced response of HPV-positive HNSCC, HPV-E6/E7 in HPV-positive cells were knocked down and the response to treatment with C.sub.18-pyr-cer was examined. It was found that knockdown of E6/E7 resulted in resistance to C.sub.18-pyr-cer-induced cell death compared to Scr-siRNA-transfected controls (Fig. EV3, A). Moreover, siRNA-mediated knockdown of E6/E7 attenuated C.sub.18-pyr-cer-induced mitophagy, measured by change in oxygen consumption rate (OCR), and co-localization of LTG and MTR (
[0108] To further confirm that HPV-E7, but not HPV-E6, plays an important role in sensitizing HNSCC to C.sub.18-pyr-cer, the E6 target p53 or the E7 target RB were knocked down in HPV-negative cells and their effects on cell death were measured. The data demonstrated that shRNA-mediated knockdown of RB, but not p53, increased C.sub.18-pyr-cer-induced cell death (
[0109] Activation of E2F5 via inhibition of RB by HPV-E7 enhances ceramide-induced mitophagy. To identify the downstream mediators of HPV-E7/RB signaling in enhancing ceramide-mediated lethal mitophagy, the roles of E2F proteins, the canonical downstream targets of RB, were investigated. Because E2F1-5 proteins have been associated with autophagy, cell death, or inhibition of cell growth previously (Morales et al, 2014; Jiang et al, 2010; Polager et al, 2008), the effects of shRNA-mediated knockdown of E2F1, E2F4, or E2F5 on ceramide-induced mitophagy were assessed. The data showed that knockdown of E2F5, but not E2F1 or E2F4, attenuated cell death in response to C.sub.18-pyr-cer in HPV(+) HNSCC cells (
[0110] HPV-E7/ceramide-mediated mitophagy is induced by E2F5-Drp1 complex. To further define the mechanism by which E2F5 enhances HPV-E7/ceramide-mediated mitophagy, the involvement of dynamin related protein 1 (Drp1) in this process was investigated, due to the increased mitochondrial fission observed in TEM micrographs containing images of C.sub.18-pyr-cer-treated UM-SCC-47 cells (
[0111] Interestingly, the data also demonstrated that alterations of E2F5 abundance by shRNA transfections had no effect on Drp1, LC3 or ATGS mRNAs compared to controls (
[0112] To determine the molecular details of Drp1-E2F5 complex, a specific domain of E2F5 was identified involved in Drp1 interaction by molecular docking using the ZDOCK server. These data suggested that the association between Drp1 and E2F5 might involve the dimerization domain of E2F5, where E2F5 is known to bind the activating dimerization partner (DP) protein, between residues 84 and 177 (
[0113] It is known that Drp1 engages with its mitochondrial receptor MFF to induce mitochondrial fission and mitophagy (Koirala et al, 2013). Thus, to determine the mechanism by which E2F5-Drp1 association enhances HPV-E7/ceramide-induced lethal mitophagy, the mitochondrial localization of Drp1 and its association with mitochondrial receptor MFF were examined. The data showed that C.sub.18-pyr-cer induced Drp1-MFF association, which was attenuated by stable knockdown of E2F5 in UM-SCC47 cells compared to controls (
[0114] Reconstitution of E2F5-Drp1 association by E2F5-peptide mimetic enhances mitophagy in HPV() cells. To determine whether the putative Drp1-binding domain of E2F5 alone was sufficient to reconstitute E2F5 activity for mitophagy induction in HPV-negative cells, a peptide based on the minimal stretch of amino acids was generated, corresponding to Drp1 binding E2F5 residues (146-175, biotin-RRRRRRRR-ELDQQKLWLQQSIKNVMDDSINNRFSYVTHED (SEQ ID NO. 2)) and scrambled control peptide, which contains the same amino acids as E2F5-pept in randomized/scrambled order (biotin-RRRRRRRR-LILFVIKLHQDVNDMRNSNQDQTQSE-DRESKWY (SEQ ID NO. 3)), as predicted by molecular docking studies. Eight arginine residues (R8) were included on the N-terminus to enhance cell penetration of the E2F5 peptide (Rancher and Ryu, 2015). Importantly, treatment of UM-SCC-22A cells with the E2F5-pept largely increased cisplatin or C.sub.18-pyr-cer-induced cell death compared to scr-pept (
[0115] Example 2Materials and Methods
[0116] Reagents. C.sub.18-pyridium-ceramide was synthesized at the synthetic Lipidomics Core at the Medical University of South Carolina (MUSC). Cisplatin was purchased from Sigma. Treatments were performed using 40 M cisplatin in DMSO, 20 M C.sub.18-pyr-cer in EtOH for 1-4 h for mitophagy detection, or corresponding amount of vehicle control. Peptides were synthesized by LifeTein, Inc. Peptides contained C-terminal amidation. E2F5-pept: Biotin-RRRRRRRR-ELDQQKLWLQQSIKNVMDDSINNRFSYVTHED (SEQ ID NO. 2). Scr-pep: Biotin-RRRRRRRR-LILFVIKLHQDVNDMRNSNQDQTQSEDRESKWY (SEQ ID NO. 3).
[0117] Antibodies used were as follows: TOM20(F-10) Santa Cruz (sc)-17764; Actin-Sigma Rb A2066; HPV16E6-sc-1584 (N-17); HPV16E7sc-65711 (NM2); pRBBDBiosciences 554136 (G3-254); p53BDBiosciences 554294 clone DO-7; CerS1 (Lass1)(C-14) sc-65096; Ceramide(MID 15B4) ALX-804-196-T050; E2F5sc-999, Drp1BD Biosciences.
[0118] Cell lines and culture conditions. HPV(+) cell lines were provided by Drs. Tom Carey, University of Michigan (UM-SCC-47) and Susan Gollin, University of Pittsburgh Cancer Institute (UPI-SCC-90). UM-SCC-22A, UM-SCC-1A, and UM-SCC-47 were grown in DMEM (Corning) with 10% FBS (Atlanta Biologicals) and 1% Penicillin and streptomycin (Cellgro) at 37 C. with 5% CO.sub.2. UPI-SCC-90 were grown in DMEM with 10% FBS (Atlanta Biologicals), 2mM L-glutamine, 1X non-essential amino acids solution, and 500 ug/ml gentamicin (Gibco).
[0119] Stable shRNA-mediated knockdown of E2F5. pLK0.1 plasmids expressing shRNA to E2F5 or scrambled control (MUSC shRNA Shared Technology Resource) were co-transfected with pCMV-psPAX2 and pMD2 plasmids in Plat A cells using the viral transduction protocol as described by the RNA interference (RNAi) Consortium. Viral supernatants were added to UM-SCC47 cells, and selection was performed using puromycin (1 g/ml) for 14 days.
[0120] Cell transfections. Plasmids containing target gene, shRNA, or empty vector were transfected into cells using effectene transfection reagent (QIAGEN) following the manufacturer's instructions, followed by PBS rinse six hours after transfection. CerS6, Drp1, p53, Rb, and E2F1, 4, and 5 shRNAs were from the MUSC shRNA Shared Technology Resource. siRNA transfections were performed using DharmaFECT (ThermoScientific, Dharmacon). SiRNAs used: E6/E7 (ThermoScientific, custom sequence AGGAGGAUGAAAUAGAUGGUU (SEQ ID NO. 4)); CerS1 , ATG5, LC3B (Thermo Scientific, Dharmacon); or non-targeting scrambled siRNA (Qiagen).
[0121] Trypan blue exclusion assay. Cells were seeded in 6-well plates, and allowed to adhere for 20 h. After treatments (for 24 h), medium containing dead cells was pelleted with trypsinized cells, then re-suspended in 1 PBS then counted in a hemocytometer after addition of trypan blue dye (Sigma-Aldrich) at a 1:10 dilution.
[0122] IC.sub.50 determination by MTT assay. Cells were plated in 96-well plates and allowed to adhere for 20 h, then treated with the indicated concentrations of drug. After 48 h treatment, the MTT assay (ATCC) was performed as described previously (Sentelle et al, 2012).
[0123] Immunoblotting. Cells were lysed in RIPA buffer plus protease inhibitor cocktail on ice for 15 minutes then centrifuged. 30ug of protein from the supernatant were run on Criterion TGX Precast Gels (Bio-Rad).
[0124] Quantitative RT-PCR. RNA was extracted from cell pellets using RNeasy kit (Qiagen) per the manufacturer's instructions. cDNA was generated using equal amounts of RNA from each sample and iScript cDNA synthesis kit (Bio-Rad) per manufacturer's instructions. Reactions were carried out using SsoFast probes mix (Bio-Rad) and TaqMan primer probes (ThermoFisher Scientific) in a StepOne Plus qPCR cycler as described by the manufacturer.
[0125] Site-directed mutagenesis. E2F5.sup.84-177 mutant was generated using Q5 site-directed mutagenesis kit (New England Biolabs) per manufacturer's instructions. Primers were designed using NEBaseChanger (New England Biolabs). Forward: GAG GTG GAG GTC TAG ATC ACC AAT GTC TTA GAG GG (SEQ ID NO. 5), Reverse: CAG TGT GGT GGA ATT CTA TGT ATC ACC ATG AAA GC (SEQ ID NO. 6).
[0126] Molecular modeling of protein-protein interactions. Phyre2 was used to predict secondary structure of E2F5 based on the sequence in GenBank (NP_001942.2). The generated PDB file was input along with the PDB for Drp1(4BEJ) from RSCB (www.rscb.org) into ZDOCK Server (http://zdock.umassmed.edu/). The top model was then used to predict the sites of association between E2F5 and Drp1.
[0127] Measurement of cellular respiration using Seahorse XF analyzer. Cells were plated in a Seahorse Biosciences 96-well plate and allowed to adhere for 20 hours. They were then treated with 20 uM C.sub.18-pyr-cer or equivalent amount of vehicle (EtOH) for two hours, then oxygen consumption rate was measured using a Seahorse XF96 (Seahorse Biosciences) as described by the manufacturer (Sentelle et al, 2012).
[0128] Proximity ligation assay. Proximity ligation assays were performed using Duolink in situ red kit (Sigma) per manufacturer's instructions, then analyzed as described (Panneer-Selvam et al, 2015).
[0129] Immunofluorescence. Cells were plated on glass coverslips in 6-well plates and allowed to adhere for 20 hours. Treatment was performed with 40 uM cisplatin or equivalent amount of DMSO for 8 hr, or 5 M peptide for 2 hours. Fixation was in 4% paraformaldehyde, followed by permeabilization with 0.1% Triton-X100, and blocking in 1% BSA in PBS. Samples were incubated at 4 C. overnight with primary antibodies in blocking solution. TOM20 (SCBT) 1:200, Lass1/CerS1 (SCBT) 1:50, ceramide (Enzo Life Sciences) 1:100, and biotin (SCBT) 1:200. Immunofluorescent-conjugated secondary antibodies (AlexaFluor 488 or 594, Jackson Immuno) were added at 1:500 for one hour. Coverslips were then mounted onto glass slides with ProLong Gold Antifade Mountant (Molecular Probes).
[0130] Laser scanning confocal microscopy. For live cell imaging, cultured cells were incubated with 500 nM of mitotracker far red and 500 nM lysotracker green in DMSO for 30 min at 37 C. Cells were treated with 20 uM C.sub.18-pyr-cer or 40 uM cisplatin and kept in an incubator with 5% CO.sub.2 at 37 C. during imaging. An Olympus FV10i confocal microscope was used for imaging. 543- and 488-nm channels were used for visualizing red or green fluorescence, respectively, with pinholes set to 1.0 Airy units. At least three random fields were imaged for each sample (Sentelle et al, 2012).
[0131] Ultra-structure analysis using transmission electron microscopy. Cells were washed with 1 PBS then fixed in 2% glutaraldehyde (w/v) in 0.1 M cacodylate. After post-fixation in 2% (v/v) osmium tetroxide, specimens were embedded in Epon 812, and sections were cut orthogonally to the cell monolayer with a diamond knife. Thin sections were visualized in a JEOL 1010 transmission electron microscope (Saddoughi et al, 2013).
[0132] Cell fractionation. Cells were treated with 40 uM cisplatin or vehicle for eight hours, 20 uM C.sub.18-pyr-cer or vehicle and/or 5 uM scr-pept or E2F5-pept for 1.5 hours. Mitochondria isolation kit (ThermoFisher Scientific) was used as described by the manufacturer.
[0133] Co-immunoprecipitation. Cells were lysed in 500 ul Pierce IP lysis/wash buffer (ThermoFisher) with protease inhibitor cocktail (Sigma) on ice for 15 minutes. 350 g of protein were used with SureBeads Protein A Magnetic Beads (BioRad) per manufacturer's instructions, using 10 g antibody or corresponding normal IgG control (SCBT).
[0134] Image Quantification. Images were quantified by ImageJ. Co-localization in micrographs was measured using FIJI. Duolink ImageTool software was used for quantification of PLA signals (Panneer-Selvam et al, 2015).
[0135] In vivo studies. Severe combined immunodeficient (SCID) mice were purchased from Jackson Laboratories. Age- and sex-matched mice were used. All animal protocols were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina. UM-SCC22A or UM-SCC47 cells (75,000) were implanted into the flanks of SCID mice (n=5-8 mice). When the tumors were palpable, the mice were treated every three days with 3.5 mg/kg cisplatin, 20 mg/kg C.sub.18-pyr-cer, or corresponding amount of vehicle control and/or 3.76 g E2F5-peptide or scrambled control peptide. Tumor volume was measured using calipers. At the end of the 14-day treatment, the mice were euthanized and tumor tissues were collected (Sentelle et al, 2007; Saddoughi et al, 2013).
[0136] Statistical analysis. Data were reported as meanstandard error. Mean values were compared using the student t test and p<0.05 was considered statistically significant (Saddoughi et al, 2013).
[0137] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
[0138] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
[0139] Ambrus A M, Islam A B, Holmes K B, Moon N S, Lopez-Bigas N, Benevolenskaya E V, Frolov M V (2013) Loss of dE2F compromises mitochondrial function. Dev Cell 27: 438-451
[0140] Ang K K, Harris J, Wheeler R, Weber R, Rosenthal D I, Nguyen-Tan P F, Westra W H, Chung C H, Jordan R C, Lu C et al (2010) Human papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med 363: 24-35
[0141] Apostolova M D, Ivanova I A, Dagnino C, D'Souza S J, Dagnino L (2002) Active nuclear import and export pathways regulate E2F-5 subcellular localization. J Biol Chem 277: 34471-34479
[0142] Bol V, Gregoire V (2014) Biological basis for increased sensitivity to radiation therapy in HPV-positive head and neck cancers. Biomed Res Int 2014: 1-6
[0143] Chang W Y, Bryce D M, D'Souza SJA, Dagnino L (2004) The DP-1 transcription factor is required for keratinocyte growth and epidermal stratification. J Biol Chem 279: 51343-51353
[0144] Chen H Z, Tsai S Y, Leone G (2009) Emerging roles of E2Fs in cancer: an exit from cell cycle control. Nat Rev Cancer 9: 785-797
[0145] Cosway B, Lovat P (2016) The role of autophagy in squamous cell carcinoma of the head and neck. Oral Oncol 54: 1-6
[0146] Dany M, Ogretmen B (2015) Ceramide induced mitophagy and tumor suppression. Biochim Biophys Acta-Mol Cell Res 1853: 2834-2845
[0147] Dick F A, Sailhamer E, Dyson N J (2000) Mutagenesis of the pRB pocket reveals that cell cycle arrest functions are separable from binding to viral oncoproteins. Mol Cell Biol 20: 3715-3727
[0148] D'Souza G, Kreimer A R, Viscidi R, Pawlita M, Fakhry C, Koch W M, Westra W H, Gillison M L (2007) Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med 356: 1944-1956
[0149] Dyson N, Howley P M, Munger K, Harlow E (1989) The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243: 934-937
[0150] Fakhry C, Westra W H, Li S, Cmelak A, Ridge J A, Pinto H, Forastiere A, Gillison M L (2008) Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial. J Natl Cancer Inst 100: 261-269
[0151] Garcia-Zepeda S P, Garcia-Villa E, Diaz-Chavez J, Hernandez-Pando R, Gariglio P (2013) Resveratrol induces cell death in cervical cancer cells through apoptosis and autophagy. Eur J Cancer Prey 22: 577-584
[0152] Gemin A, Sweet S, Preston T J, Singh G (2005) Regulation of the cell cycle in response to inhibition of mitochondrial generated energy. Biochem Biophys Res Commun 332: 1122-1132
[0153] Halbert C L, Demers G W, Galloway D A (1992) The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells. J Virol 66: 2125-2134
[0154] Hanada K, Kumagai K, Yasuda S, Miura Y, Kawano M, Fukasawa M, Nishijima M (2003) Molecular machinery for non-vesicular trafficking of ceramide. Nature 426: 803-809
[0155] Helt A M, Funk J O, Galloway D A (2002), Inactivation of both the retinoblastoma tumor suppressor and p21 by the human papillomavirus type 16 E7 oncoprotein is necessary to inhibit cell cycle arrest in human epithelial cells. J Virol 76: 10559-10568
[0156] Hijmans E M, Voorhoeve P M, Beijersbergen R L, van 't Veer U, Bernards R (1995) E2F-5, a new E2F family member that interacts with p130 in vivo. Mol Cell Biol 15: 3082-3089
[0157] Itoh A, Levinson S F, Morita T, Kourembanas S, Brody J S, Mitsialis S A (1995) Structural characterization and specificity of expression of E2F-5: a new member of the E2F family of transcription factors. Cell Mol Biol Res 41: 147-154
[0158] Jiang H, Martin V, Gomez-Manzano C, Johnson D G, Alonso M, White E, Xu J, McDonnell T J, Shinojima N, Fueyo J (2010) The RB-E2F1 pathway regulates autophagy. Cancer Res 70,7882-7893
[0159] Killock D (2015) Therapeutic HPV vaccine holds promise. Nat Rev Clin Oncol 12: 686
[0160] Koirala S, Guo Q, Kalia R, Bui H T, Eckert D M, Frost A, Shaw J M (2013) Interchangeable adaptors regulate mitochondrial dynamin assembly for membrane scission. Proc Natl Acad Sci USA 110: E1342-E1351
[0161] Koybasi S, Senkal C E, Sundararaj K, Spassieva S, Bielawski J, Osta W, Day T A, Jiang J C, Jazwinski S M, Hannun Y A (2004) Defects in cell growth regulation by C18:0-ceramide and longevity assurance gene 1 in human head and neck squamous cell carcinomas. J Biol Chem 279:44311-44319
[0162] Lee B K, Bhinge A A, Iyer V R (2011) Wide-ranging functions of E2F4 in transcriptional activation and repression revealed by genome-wide analysis. Nucleic Acids Res 39: 3558-3573
[0163] Leemans C R, Braakhuis B J, Brakenhoff R H (2011) The molecular biology of head and neck cancer. Nat Rev Cancer 11: 9-22
[0164] Lehtinen M, Dillner J (2013) Clinical trials of human papillomavirus vaccines and beyond. Nat Rev Clin Oncol 10: 400-410
[0165] Mirghani H, Amen F, Tao Y, Deutsch E, Levy A (2015) Increased radiosensitivity of HPV-positive head and neck cancers: Molecular basis and therapeutic perspectives. Cancer Treat Rev 41: 844-852
[0166] Moody C A, Fradet-Turcotte A, Archambault J, Laimins L A (2007) Human papillomaviruses activate caspases upon epithelial differentiation to induce viral genome amplification. Proc Natl Acad Sci U S A 104: 19541-19546
[0167] Morales L D, Casillas Pavan E A, Shin J W, Garcia A, Capetillo M, Kim D J, Lieman J H, Bhattacharya S (2014) Protein tyrosine phosphatases PTP-1B, SHP-2, and PTEN facilitate Rb/E2F-associated apoptotic signaling. PLoS One 9: e97104
[0168] Panneer Selvam S, De Palma R M, Oaks J J, Oleinik N, Peterson Y K, Stahelin R V, Skordalakes E, Ponnusamy S, Garrett-Mayer E, Smith C D et al (2015) Binding of the sphingolipid SIP to hTERT stabilizes telomerase at the nuclear periphery by allosterically mimicking protein phosphorylation. Sci Signal 8: ra58
[0169] Pewzner-Jung Y, Ben-Dor S, Futerman A H (2006) When do Lasses (longevity assurance genes) become CerS (ceramide synthases)?: Insights into the regulation of ceramide synthesis. J Biol Chem 281: 25001-25005
[0170] Polager S, Ofir M, Ginsberg D (2008) E2F1 regulates autophagy and the transcription of autophagy genes. Oncogene 27: 4860-4864
[0171] Sadasivam S, DeCaprio J A (2013) The DREAM complex: master coordinator of cell cycle-dependent gene expression. Nat Rev Cancer 13: 585-595
[0172] Saddoughi S A, Gencer S, Peterson Y K, Ward K E, Mukhopadhyay A, Oaks J, Bielawski J, Szulc Z M, Thomas R J, Panneer Selvam S et al (2013) Sphingosine analogue drug FTY720 targets I2PP2A/SET and mediates lung tumour suppression via activation of PP2A-RIPK1-dependent necroptosis. EMBO Mol Med 5: 105-121
[0173] Scheffner M, Werness B A, Huibregtse J M, Levine A J, Howley P M (1990) The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63: 1129-1136
[0174] Senkal C E, Ponnusamy S, Rossi M J, Sundararaj K, Szulc Z, Bielawski J, Bielawska A, Meyer M, Cobanoglu B, Koybasi S (2006) Potent antitumor activity of a novel cationic pyridinium-ceramide alone or in combination with gemcitabine against human head and neck squamous cell carcinomas in vitro and in vivo. J Pharmacol Exp Ther 317: 1188-1199
[0175] Sentelle R D, Senkal C E, Jiang W, Ponnusamy S, Gencer S, Panneer Selvam S, Ramshesh V K, Peterson Y K, Lemasters J J, Szulc Z M (2012) Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy. Nat Chem Biol 8: 831-838
[0176] Smirnova E, Griparic L, Shurland D L, van der Bliek A M (2001) Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells. Mol Biol Cell 12: 2245-2256
[0177] Spassieva S D, Ji X, Liu Y, Gable K, Bielawski J, Dunn T M, Bieberich E, Zhao L (2016) Ectopic expression of ceramide synthase 2 in neurons suppresses neurodegeneration induced by ceramide synthase 1 deficiency. Proc Natl Acad Sci USA 113: 5928-5933
[0178] Strack S, Cribbs J T (2012) Allosteric modulation of Drp1 mechanoenzyme assembly and mitochondrial fission by the variable domain. J Biol Chem 287: 10990-11001 Raucher D, Ryu J S (2015) Cell-penetrating peptides: strategies for anticancer treatment. Trends Mol Med 21: 560-570
[0179] Rieckmann T, Tribius S, Grob T J, Meyer F, Busch C J, Petersen C, Dikomey E, Kriegs M (2013) HNSCC cell lines positive for HPV and p16 possess higher cellular radiosensitivity due to an impaired DSB repair capacity. Radiother Oncol 107: 242-246
[0180] Tao Y, Kassatly R F, Cress W D (1997) Subunit composition determines E2F DNA-binding site specificity. Mol Cell Biol 17: 6994-7007
[0181] van Moorsel C J, Pinedo H M, Veerman G, Vermorken J B, Postmus P E, Peters G J (1999). Scheduling of gemcitabine and cisplatin in Lewis lung tumour bearing mice. Eur J Cancer 35: 808-814
[0182] Venkataraman K, Riebeling C, Bodennec J, Riezman H, Allegood J C, Sullards M C, Merrill Jr A H, Futerman A H (2002) Upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene 1 (LAG1), regulates N-stearoyl-sphinganine (C.sub.18-(dihydro)ceramide) synthesis in a fumonisin Bl-independent manner in mammalian cells. J Biol Chem 277: 35642-35649
[0183] Weinberg S E, Chandel N S (2015) Targeting mitochondria metabolism for cancer therapy. Nat Chem Biol 11: 9-15
[0184] White E A, Sowa M E, Tan M J, Jeudy S, Hayes S D, Santha S, Munger K, Harper J W, Howley P M (2012) Systematic identification of interactions between host cell proteins and E7 oncoproteins from diverse human papillomaviruses. Proc Natl Acad Sci U S A 109: E260-E267
[0185] Wu W, Lin C, Wu K, Jiang L, Wang X, Li W, Zhuang H, Zhang X, Chen H, Li S et al (2016) FUNDC1 regulates mitochondrial dynamics at the ER-mitochondrial contact site under hypoxic conditions. EMBO J: e201593102
[0186] Zhang C S, Lin SC (2016) AMPK promotes autophagy by facilitating mitochondrial fission. Cell Metab 23: 399-401
[0187] Zhao L, Spassieva S D, Jucius T J, Shultz L D, Shick H E, Macklin W B, Hannun Y A, Obeid L M, Ackerman S L (2011) A deficiency of ceramide biosynthesis causes cerebellar purkinje cell neurodegeneration and lipofuscin accumulation. PLoS Genet 7: e1002063
[0188] Ziemann F, Arenz A, Preising S, Wittekindt C, Klussmann J P, Engenhart-Cabillic R, Wittig A (2015) Increased sensitivity of HPV-positive head and neck cancer cell lines to x-irradiation and cisplatin due to decreased expression of E6 and E7 oncoproteins and enhanced apoptosis. Am J Cancer Res 5: 1017-1031