WO2016149580A2

WO2016149580A2 - Sensitizing agent for cancer chemotherapy and radiation therapy and uses thereof

Info

Publication number: WO2016149580A2
Application number: PCT/US2016/023065
Authority: WO
Inventors: Shawn E. Lupold; Koji Hatano; Binod Kumar; Theodore L. Deweese
Original assignee: The Johns Hopkins University
Priority date: 2015-03-19
Filing date: 2016-03-18
Publication date: 2016-09-22
Also published as: WO2016149580A3

Abstract

The present disclosure provides compositions and methods whereby specific microRNA (miRNA) is utilized as a means to sensitize cancer cells to radiation or chemotherapy.

Description

SENSITIZING AGENT FOR CANCER CHEMOTHERAPY AND RADIATION

THERAPY AND USES THEREOF

RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S.

Provisional Application No: 62/135,429, filed March 19, 2015, which is incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with U.S. Government support under Grant Number R01CA143299 and P50CA058236 from the National Cancer Institute. The Government has certain rights in the invention.

FIELD OF THE DISCLOSURE

[0003] The present invention relates to cancer therapeutics and uses thereof.

BACKGROUND OF THE DISCLOSURE

[0004] MicroRNAs (miRNAs) have been implicated in DNA repair pathways through transcriptional responses to DNA damaging agents and through the predicted regulation of DNA repair pathway genes. Ionizing radiation (IR) is a useful modality to treat multiple types of cancers. The primary cellular injury associated with IR is DNA damage; in particular DNA double strand breaks (DSBs). Activated DNA damage response (DDR) pathways control downstream effectors which can determine cellular fates such as DNA repair, cell cycle arrest or apoptosis. Tumor cells often present radiation protective phenotypes which can lead to IR treatment failure. A number of mechanisms account for this resistance including tumor microenvironment and altered cellular gene expression of DDR pathway components. As such, a better understanding of DDR pathways in cancer is needed for improved treatment design and efficacy.

SUMMARY OF THE DISCLOSURE

[0005] Prior to the invention described herein, methods for sensitizing cancer cells utilized pharmaceutical agents or RNA interference (RNAi) agents such as siRNAs or shRNAs. The present disclosure provides compositions and methods whereby specific microRNA (miRNA) is utilized as a means to sensitize cancer cells to radiation or chemotherapy.

Described herein is the ability of miRNA, including miR-890-5p and miR-744-3p, to enhance the anti-cancer effect of radiation therapy and to inhibit DNA repair processes following radiation therapy.

[0006] The present invention provides a method of improving a cancer therapeutic response to radiation therapy or chemotherapy in a subject, the method comprising administering to a subject an effective amount of miR-890-5p or an agent that enhances the expression of miR- 890-5p or an agent that mimics the effects of miR-890-5p either prior to or in conjunction with a radiation therapy or a chemotherapy. The microRNA miR-890-5p inhibits DNA repair processes by suppressing the expression of several targets (including MAD2L2 (which encodes mitotic spindle assembly checkpoint protein MAD2B) and WEE1). This can be achieved through the administration of miR-890-5p. Alternate methods of activating endogenous miR-890-5p or mimicking miR-890-5p are also considered.

[0007] The present invention provides a method of improving a cancer therapeutic response to radiation therapy or chemotherapy in a subject, the method comprising administering to a subject an effective amount of miR-744-3p or an agent that enhances the expression of miR- 744-3p or an agent that mimics the effects of miR-744-3p either prior to or in conjunction with a radiation therapy or chemotherapy. The present disclosure provides a method to increase the sensitivity of a cancer cell to radiation therapy or chemotherapy through the administration of microRNA miR-744-3p. The microRNA, miR-744-3p, inhibits DNA repair processes by suppressing the expression of several targets (including MAD2L2 and WEE1). This can be achieved through the administration of miR-744-3p. Alternate methods of activating endogenous miR-744-3p or mimicking miR-744-3p are also considered.

[0008] Described herein is a composition for increasing sensitization to ionizing radiation (IR) comprising an miRNA; miRNA mimetic, activator of miRNA, or mixture thereof.

[0009] In certain examples, the miRNA is miR-744. For example, the miRNA is miR-744-3p or miR-744-5p. miR-744-3p comprises SEQ ID NO: 734. In certain examples, the miRNA comprises miR-890. For example, the miRNA comprises miR-890-5p or miR-890-3p. miR- 890-5p comprises SEQ ID NO: 764. The activator of miRNA activates endogenous miR-890- 5p or miR-744-3p when administered to a patient. The miRNA mimetic mimics miR-890-5p or miR-744-3p.

[0010] A composition described herein can further comprise a chemotherapeutic agent. Suitable chemotherapeutic agents are described herein. [0011] The composition is administered 1-5 days prior to treatment with IR. For example, the composition is administered 1 day, 2 days, 3 days, 4 days, or 5 days prior to treatment with IR. Alternatively, the composition is administered 1-5 days after treatment with IR. For example, the composition is administered 1 day, 2 days, 3 days, 4 days, or 5 days after treatment with IR.

[0012] In one aspect, the dose of IR comprises between 0 Gy and 30 Gy in a single fraction of externally applied radiation. Alternatively, the dose of IR comprises between 0 Gy and 20 Gy per fraction in multiple fractions by externally applied radiation. In yet another aspect, the dose of IR comprises low dose rate brachytherapy dosing of 0 - 2 Gy/minute. Optionally, the dose of IR comprises high dose rate brachytherapy dosing of 2 - 20 Gy/minute.

[0013] Also described herein is a method of increasing sensitivity to ionizing radiation (IR) therapy or chemotherapy in a patient in need thereof. The method includes administering to the patient in need thereof a composition comprising an miRNA, miRNA mimetic, activator of miRNA, or mixture thereof. For example, the miRNA is miR-744, e.g., miR-744-3p or miR-744-5p. In one aspect, miR-744-3p comprises SEQ ID NO: 734. Alternatively, the miRNA is miR-890, e.g., miR-890-5p or miR-890-3p. In one aspect, miR-890-5p comprises SEQ ID NO: 764.

[0014] In methods described herein, the composition is administered prior to IR treatment, e.g., 1-5 days prior to treatment with IR. Alternatively, the composition is administered after IR treatment, e.g., 1-5 days after treatment with IR. The dose of IR comprises between 0 Gy and 30 Gy in a single fraction of externally applied radiation. In another aspect, the dose of IR comprises between 0 Gy and 20 Gy per fraction in multiple fractions by externally applied radiation. Alternatively, the dose of IR comprises low dose rate brachytherapy dosing of 0 - 2 Gy/minute. Optionally, the dose of IR comprises high dose rate brachytherapy dosing of 2 - 20 Gy/minute.

[0015] In one aspect, the composition is co-administered with a chemotherapeutic agent. Suitable chemotherapeutic agents are described herein.

[0016] In methods described herein, the patient in need thereof is a patient with cancer or a tumor. Suitable cancers include skin cancer, brain cancer and other central nervous system cancers, head cancer, neck cancer, muscle/sarcoma cancer, bone cancer, lung cancer, esophagus cancer, stomach cancer, pancreas cancer, colon cancer, rectum cancer, uterus cancer, cervix cancer, vagina cancer, vulva cancer, penis cancer, breast cancer, kidney cancer, prostate cancer, bladder cancer, or thyroid cancer. In some cases, the patient is a patient with glioblastoma.

[0017] Also provided are methods of increasing sensitivity to IR therapy or chemotherapy in a tumor cell. The method includes contacting the tumor cell with a composition comprising an miRNA, miRNA mimetic, activator of miRNA, or mixture thereof, thereby increasing sensitivity to IR therapy or chemotherapy.

[0018] As used herein "miRNAs" are small non-coding RNAs which post-transcriptionally regulate gene expression through interaction with the 3' untranslated regions (3'UTRs) of mRNAs. Relatively conserved miRNA sequences with similar functions are referred to as "miRNA families". For example, miR-890 is a member of the miR-743 family (e.g., those found online at mirbase.org/cgi-bin/mirna_summary .pl?fam=MIPF0000386). Furthermore, microRNAs have two functional sequences that are released from either side of a pre-miRNA hairpin structure (the -5p and -3p mature microRNAs). For example, miR-744 forms a hairpin structure of miR-744-5p and miR-744-3p (also referred to as miR-744 and miR-744* respectively). If miR-744 is expressed as a gene, it will produce both miR-744-5p and miR- 744-3p. The -5p and -3p mature microRNAs are different sequences and often have different functions.

[0019] The "seed sequence" of the miRNA comprises nucleotides 2-8 at the 5' end of the miRNA. The miRNA seed sequence is complementary to the target mRNA sequence. The remaining nucleotides of the miRNA is, in some cases, not perfectly complementary to the target mRNA. Thus, nucleotides not present in the seed sequence are not required for binding.

[0020] As used herein, the term "siRNA" refers to a double stranded stretch of RNA or modified RNA monomers, in a typical siRNA compound, the two strands usually have 19 nucleotides complementary to each other, thereby creating a double strand that is 19 nucleotides long and each strand having a 3'-end of two overhanging nucleotides. This is not a strict definition of siRNA, which may be slightly longer or shorter, and with or without overhangs. In siRNA, one strand is guiding and complementary to the target RNA (antisense strand), and the other strand (sense strand) has the same sequence as the target RNA and hence is complementary to the guide antisense strand. As used herein, the term "mRNA" means the presently known mRNA transcript(s) of a targeted gene, and any further transcripts, which may be identified. [0021] As used herein the term "shRNA" means that a single strand RNA of 50 to 100 nucleotides forms a stem-loop structure in a cell, which contains a loop region of 5 to 30 nucleotides, and long complementary RNAs of 15 to 50 nucleotides at both sides of the loop region, which form a double-stranded stem by base pairing between the complementary RNAs; and additional 1 to 500 nucleotides included before and after each complementary strand forming the stem, shRNA is usually transcribed by RNA polymerase in a cell, and subsequently cleaved in the nucleus by Drosha, and the cleaved shRNA is exported from the nucleus to cytosol, and further cleaved in the cytosol by Dicer, Like siRNA, shRNA binds to the target mRNA in a sequence specific manner, thereby cleaving and destroying the target mRNA, and thus suppressing expression of the target mRNA.

[0022] Ionizing radiation may be quantified and expressed in terms of a gray (Gy). One gray is the absorption of one joule of energy, in the form of ionizing radiation, by one kilogram of matter. For X-rays and gamma rays, these are the same units as the sievert (Sv). To avoid any risk of confusion between the absorbed dose and the equivalent dose, one must use the corresponding special units, namely the gray instead of the joule per kilogram for absorbed dose and the sievert instead of the joule per kilogram for the dose equivalent. The gray measures the deposited energy of radiation. The biological effects vary by the type and energy of the radiation and the organism and tissues involved. The sievert attempts to account for these variations.

[0023] As used herein "cancer" may refer to any one of a cancerous cell proliferative disorder including, for example, cancers or tumors of the skin, brain and other central nervous system sites, head, neck, muscle/sarcoma, bone, lung, esophagus, stomach, pancreas, colon, rectum, uterus, cervix, vagina, vulva, penis, breast, kidney, prostate, bladder, and thyroid.

[0024] As used herein, "treating" or "treat" describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a composition of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder.

[0025] A composition of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof, can also be used to prevent a disease, condition or disorder. As used herein, "preventing" or "prevent" describes reducing or eliminating the onset of the symptoms or complications of the disease, condition or disorder. [0026] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation.

[0027] A "purified" or "biologically pure" nucleic acid is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the nucleic acid or cause other adverse consequences. That is, a nucleic acid of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0028] Similarly, by "substantially pure" is meant a nucleotide molecule that has been separated from the components that naturally accompany it. Typically, the nucleotides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated.

[0029] By "isolated nucleic acid" is meant a nucleic acid that is free of the genes which flank it in the naturally-occurring genome of the organism from which the nucleic acid is derived. The term covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule, but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner, such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a synthetic cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleic acid molecules according to the present invention further include molecules produced synthetically, as well as any nucleic acids that have been altered chemically and/or that have modified backbones. For example, the isolated nucleic acid is a purified cDNA or RNA polynucleotide. Isolated nucleic acid molecules also include messenger ribonucleic acid (mRNA) molecules.

[0030] By "reduces" is meant a negative alteration of at least 5%, 10%, 25%, 50%, 75%, or 100%.

[0031] By "substantially identical" is meant a nucleic acid molecule exhibiting at least 50% identity to a reference nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the nucleic acid to the sequence used for comparison.

[0032] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.

[0033] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0034] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0035] Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%,

3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0036] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. [0037] The transitional term "comprising," which is synonymous with "including,"

"containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase

"consisting of excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel

characteristic(s)" of the claimed invention.

[0038] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figs. 1A-D show high-throughput functional screening for miRNAs which modulate prostate cancer viability and radiosensitivity. Fig. 1A is a schematic representation of the high-throughput miRNA functional screening. Fig. IB shows the average viable cell density (represented by MLuc activity) of irradiated (red) and non-irradiated (blue) LNCaP-MLuc cells on day 11. The data is organized as a waterfall plot for each miRNA ranked by sensitivity to IR. Average cell response to control miRNAs, with and without IR, are represented by dashed lines. RLU; Relative Light Units. Fig. 1C shows the average LNCaP-MLuc viable cell density (represented by MLuc activity) after treatment with 810 different miRNAs, in the absence of IR. Results are a waterfall plot ranked by viable cell density. Average signal with a control miRNA is represented as a dashed line. Fig. ID shows four different categories of miRNA responses based on cell viability and radiation sensitivity modulated by 810 miRNAs. The MLuc activity for each miRNA was normalized to control miRNAs and quantified as relative cell viability with or without IR.

[0040] Figs.2A -B demonstrate candidate radiation sensitizing miRNA validation by MLuc assay and clonogenic survival assay. Fig. 2A shows LNCaP-MLuc or PC3-MLuc cell radiosensitivity with candidate miRNAs, negative control miRNA (cel-miR-239b) or positive control DNAPK siRNA (siDNAPK). The % cell viability (mean + S.E., n=3) following IR (4Gy) is presented by the ratio of MLuc activity for irradiated cells to non- irradiated cells for each miRNA on day 11. Control miRNA is noted by the dash line. *, P < 0.05. Fig. 2B plots a clonogenic survival assay of DU145 cells transfected with candidate radiation sensitizing miRNAs or control miRNA. The surviving fraction is reported (mean + S.D., n=3) following the indicated doses of IR. *, P < 0.05. DMF. Dose Modifying Factor.

[0041] Figs. 3A-D demonstrate DSB repair delay by radiation sensitizing miRNAs. Fig. 3A is a series of images showing immunofluorescent staining of γ-Η2ΑΧ (green) in DU145 cells transfected with control, miR-890-5p or miR-744-3p miRNAs in untreated (0 Gy) or 1 and 8 h after IR (4 Gy) treatment. Nuclei were stained with DAPI (blue). Fig. 3B is a graphic quantification of γ-Η2ΑΧ foci. The percentage of cells containing >10 γ-Η2ΑΧ foci (mean + S.E., n≥3) is reported. *, P < 0.05. Fig. 3C is an image of a comet assay in DU145 cells transfected with control, miR-890-5p or miR-744-3p miRNAs and untreated (0 Gy) or 4 h after IR (4 Gy) treatment. Fig. 3D is a graphic quantification of the average tail moment (mean + S.E., n≥50) is reported. *, P < 0.05.

[0042] Figs. 4A-C demonstrate that miR-890-5p and miR-744-3p directly target MAD2L2 and RAD23B. Fig. 4A is an image of western blot assays of four prostate cancer cell lines for MAD2L2 (left) and RAD23B (right) 48 h after miRNA mimetic transfection. Fig. 4B is a graph representation of the MAD2L2 and RAD23B 3'UTR miRNA binding sites. Mutated (Mut) MAD2L2 and RAD23B seed sequences are indicated (red). Fig. 4C is a graphic representation of Lucif erase activities following transfection with indicated 3'UTR reporters and miRNA mimetics. Relative luciferase activity (mean + S.E., n=4) is normalized to control miRNA for each reporter. *, P < 0.05 relative to control miRNAs.

[0043] Figs. 5A-D show that multiple DDR pathway genes targeted by radiation sensitizing miRNAs. Figs. 5A-B are images of western blot analyses of (Fig. 5A) MAD2L2, WEE1, XPC, and KU80 in and (Fig. 5B) RAD23B, XLF and MCL1 48 h after LNCaP transfection with indicated miRNA mimetics. Asterisk represents a nonspecific band. Figs. 5C-D are graphs of quantified percent protein knock-down (mean + S.E., n=3) by (Fig. 5C) miR-890- 5p and (Fig. 5D) miR-744-3p, relative to control (from three separate experiments).

[0044] Figs. 6A-C show that miR-890-5p targets multiple proteins to enhance IR therapeutic effect. Fig. 6 A is an image of a western blot 48 h after transfection showing MAD2L2 knockdown by siRNA (siMAD2L2), miR-890, or combined siRNA and miR-890-5p in LNCaP cells (10 nM). Fig. 6B is a graphic representation of IR sensitization potency of siMAD2L2 and/or miR-890-5p (0.08-10 nM) in LNCaP-MLuc cells. Relative cell viability (mean + S.E., n=12) is presented as the MLuc activity after IR (4Gy), as normalized by control miRNA. *, P < 0.05. Fig. 6C is a graph of the calculated icso value of each treatment group, based on relative cell viability after IR.

[0045] Figs. 7A-B show that miR-890-5p mimetic injection enhances IR therapy of established prostate tumors. Fig. 7A is a graph of tumor volume for established subcutaneous DU145 tumors (n=3-5 per group) that were directly injected with PBS or liposomal-loaded miRNA mimetics. Two day after injection (day 0) tumors were irradiated with 6 Gy or no IR. miR-890-5p significantly reduced tumor volume in irradiated groups when compared to controls. ***P < 0.001, **P < 0.01, *P < 0.05; 2-way ANOVA. Mean + SEM. Fig. 7B is a graph showing the extension of tumor quadrupling for DU145 tumor model. Events (animals whose tumor volume was not yet 4-fold the size at injection) were plotted by Kaplan-Meier curve. miR-890-5p significantly extended time to tumor quadrupling in irradiated groups when compared to controls. P < 0.01; log-rank (Mantel-Cox) test.

[0046] Figs. 8A-B demonstrate the radiation sensitization efficacy of DNAPK siRNA in LNCaP-MLuc cells. DNAPK siRNA was used as a positive control for each 96 well LNCaP- MLuc plate of the high throughput miRNA radiosensitivity screening. Two days before IR, each plate contained wells which were transfected with either DNAPK siRNA or control. On day 0, plates were irradiated at 4 Gy or remained untreated. On day 11, the cell viability and radiation sensitivity were determined by the MLuc viability assay (RLU; Relative Light Units). Fig. 8A is a graph of the MLuc activity of each group (mean + S.E., n=132). Fig. 8B is a graph of the relative cell viability after IR (mean + S.E., n=132) is presented as the MLuc activity of irradiated cells normalized by that of untreated cells. *, P < 0.05.

[0047] Figs. 9A-B demonstrate the reproducibility of the high-throughput functional miRNA screening. The high-throughput miRNA screening for cell growth and IR response was performed with duplicate wells for each miRNA and treatment condition, forming Group 1 and Group 2. The correlation of viable cell number for each miRNA, as measured by relative MLuc activity normalized by control in Group 1 and Group 2, is plotted for the (Fig. 9A) non-irradiated and (Fig. 9B) IR samples. The linear correlation coefficient (R²) between the two groups is noted.

[0048] Fig. 10 is a graph showing radiosensitization by miRNAs from individual miRNA families. The radiation sensitization of LNCaP-MLuc cells from high-throughput miRNA screening for the individual miRNA families miR-15/16, miR-1/133, and miR-106b. Each relative cell viability following IR (mean + S.E., n≥4) is presented as the MLuc activity of irradiated cells relative to those miRNA transfected non-irradiated cells. The dashed line indicates control cell viability following IR.

[0049] Fig. 11 is a graph showing miRNA mediated radiation sensitization in PC3-MLuc cells. PC3-MLuc Clonogenic Assay. PC3-MLuc cells were transfected with 20 nM of miR- 890-5p or control miRNA (cel-miR-239b) and grown for 2 days, after which cells were irradiated at the indicated doses. The cells were grown for 14 days and colonies with greater than 30 cells were scored, and surviving fraction was calculated (mean + S.D., n=3). *, P < 0.05 relative to control. DMF; Dose Modifying Factor.

[0050] Fig. 12 is an image of western blot analyses demonstrating the knock-down efficacy of target genes by miR-890-5p and MAD2L2 siRNA. LNCaP cells were transfected with 20 nM of control, miR-890-5p mimics or serial dilutions of MAD2L2 siRNA and were incubated for 48 h. Western blot analyses were performed to detect MAD2L2 and WEE1. ACTB was used as a control for protein loading.

[0051] Figs. 13A-E are graphs of individual DU145 tumor growth curves. Subcutaneous DU145 tumors were directly injected with PBS, liposomal control miRNA mimetic, or liposomal miR-890-5p mimetic on day -2. On day 0, animals were divided into groups that received either 6 Gy IR or non-irradiated. Tumors were measured every other day and individual tumor growth curves are reported. (Fig. 13A) PBS (n=3); (Fig. 13B) Control miRNA (n=4); (Fig. 13C) miR-890-5p (n=5); (Fig. 13D) Control miRNA + IR (n=4); (Fig. 13E) miR-890-5p + IR (n=4).

DETAILED DESCRIPTION OF THE DISCLOSURE

[0052] siRNA library screening has been utilized to determine the influence of DDR genes on prostate cancer sensitivity to IR (Ni X, et al. (2011) J Clin Invest 121(6):2383-2390.). Potent target genes DNAPK, MAD2L2 and BRCA2 were identified, all of which are components of DNA DSB repair. In addition to these genes, RAD23B, a component of nucleotide excision repair, significantly sensitized cells to IR. These studies demonstrate the utility of high-throughput radiation sensitivity screens to identify vital genes in DNA repair and radiation response. Described herein is a design that examined the role of non-coding microRNA (miRNA) genes in cellular DNA repair and sensitivity to IR.

[0053] miRNAs are small non-coding RNAs which post-transcriptionally regulate gene expression through interaction with the 3' untranslated regions (3'UTRs) of mRNAs. While miRNAs mediate a variety of normal developmental and physiological processes, their expression is commonly deregulated in cancer (Calin GA, Croce CM (2006) Nat Rev Cancer 6(l l):857-866.). Notably, individual miRNAs have been shown to possess tumor suppressor and oncogenic properties (Kent OA, Mendell JT (2006) Oncogene 25(46):6188-6196.). Growing evidence also supports that miRNA genes are responsive to DNA damage and that they can regulate DDR pathways (Zhao L, Bode AM, Cao Y, Dong Z (2012) Carcinogenesis 33(l l):2220-2227.; Czochor JR, Glazer PM (2014) Antioxid Redox Signal 21(2):293-312.). However, prior to the invention described herein, the role of miRNAs in DNA repair was not fully elucidated. The present disclosure details a high-throughput functional screen of miRNA mimetics to identify candidate miRNAs which altered DNA repair and cellular sensitivity to IR. The resulting miRNAs and pathways are useful in the future radiologic management of cancer patients.

[0054] miRNAs target complementary mRNA through the binding of a small 'seed' recognition sequence, often comprising 6-8 nucleotides (Friedman RC, Farh KK, Burge CB, Bartel DP (2009) Genome Res 19(1):92-105.). From this, it is calculable that a single miRNA can regulate hundreds of different of genes (Lewis BP, Burge CB, Bartel DP (2005) Cell 120(1): 15-20.). On the other hand, it is generally accepted that most miRNAs regulate a few key genes or pathways to achieve a given phenotype. Prior to the invention described herein, there were few studies to characterize the polyvalent nature of a single miRNA within a specific pathway or phenotype. Studies described herein have uncovered two miRNAs, miR- 890-5p and miR-744-3p, which regulate multiple components of DNA damage repair to contribute to cellular IR sensitivity. Pre-treatment of established tumors with miR-890-5p mimetics significantly enhanced IR efficacy, demonstrating its potential as an IR-sensitizing agent.

[0055] The present disclosure describes a systematic screen of 810 different miRNA mimetics for the ability to alter DNA damage response caused by ionizing radiation. A comprehensive analysis of miRNA-induced changes in prostate cancer cell growth, survival and radiosensitivity is described herein. Two potent radiosensitizing miRNAs, miR-890-5p and miR-744-3p, were identified and shown to inhibit DNA repair. Each miRNA targets multiple components of the DNA damage response. As described in detail below, miR-890- 5p demonstrated superior radiosensitization when compared to an siRNA against a primary target, MAD2L2. Moreover, MAD2L2 knock-down was not sufficient to ablate miR-890-5p radiosensitization, indicating that additional targets beyond MAD2L2 contribute to the radiosensitization phenotype. These results validate the polyvalent nature of miRNA- regulated pathways and support the use of miR-890-5p as a radiosensitizing agent. DDR pathways influence cellular sensitivity to IR. Growing evidence supports that miRNAs are involved in DDR and repair (Zhao L, Bode AM, Cao Y, Dong Z (2012) Carcinogenesis 33(l l):2220-2227; Czochor JR, Glazer PM (2014) Antioxid Redox Signal 21(2):293-312). The inventors hypothesized that additional DNA repair modifying miRNAs could be identified through a high-throughput screen of miRNA mimetics for the ability to modify cell response to IR. Disclosed herein are data demonstrating that two potent radiation sensitizing miRNAs, miR-890-5p and miR-744-3p, specifically target multiple components of DSB repair, nucleotide excision repair and cell cycle progression checkpoints. These miRNAs were among 324 candidate radiation sensitizing miRNAs and 75 candidate radiation protective miRNAs identified in the screen. As described in detail below, the data is evaluated for previously reported DDR miRNAs. In the same way, the data from non- irradiated cells is analyzed for miRNAs reported in cell growth and cell survival pathways. Multiple miRNAs have been reported to regulate DDR. A number of these miRNAs were found through transcriptional responses to IR treatment or through differential expression in IR-resistant cancer cells (Czochor JR, Glazer PM (2014) Antioxid Redox Signal 21(2):293- 312; Weidhaas JB, et al. (2007) Cancer Res 67(23): 11111-11116.; Josson S, Sung SY, Lao K, Chung LW, Johnstone PA (2008) Prostate 68(15): 1599-1606.; Chen G, et al. (2010) Oncol Rep 23(4):997-1003.; Wagner-Ecker M, Schwager C, Wirkner U, Abdollahi A, Huber PE (2010) Radiat Oncol 5:25.; Wang XC, et al. (2011) Lung Cancer 72(l):92-99.; Li B, et al. (2011) Prostate 71(6):567-574.; Qu C, et al. (2012) Cell Cycle l l(4):785-796.; Mueller AC, Sun D, Dutta A (2013) Oncogene 32(9): 1164-1172.). For example, the miR-34 gene family is induced by IR through direct transcriptional activation by p53. Elevated miR- 34 expression results in decreased cellular proliferation and increased apoptosis (He L, et al. (2007) Nature 447(7148): 1130-1134.; Chang TC, et al. (2007) Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell 26(5):745-752.; Ji Q, et al. (2009) PLoS One 4(8):e6816.). In the screen of the present disclosure, miR-34c was identified as the most toxic of the 810 miRNAs transfected in the absence of irradiation. Candidate DDR regulating miRNAs have also been found through in silico analyses of known DNA repair pathway genes. For example, miR-421 and miR-101 were identified as ATM and DNAPK targeting miRNAs through 3 'UTR analyses (Hu H, Du L, Nagabayashi G, Seeger RC, Gatti RA (2010) Proc Natl Acad Sci USA 107(4): 1506-1511.; Yan D, et al.

(2010) PLoS One 5(7):el l397.). Both of these reported IR- sensitizing miRNAs were identified as strong radiation sensitizing miRNAs in screens described herein (Table 1).

Table 1.

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Table 1.

[0056] Several miRNAs have also been reported to protect cells from IR. As such, inhibition of these miRNAs is an attractive treatment strategy to help overcome radiation resistance.

Similarly, these miRNAs serve as biomarkers to predict tumor susceptibility to IR.

miR-21 is an oncogenic miRNA that is up-regulated in multiple cancers (Volinia S, et al.

(2006) Proc Natl Acad Sci USA 103(7):2257-2261.) Previous studies have reported miR-21 to be radiation protective through the regulation of PI3K/AKT signaling and autophagy (Gwak HS, et al. (2012) PLoS One 7(10):e47449.). In another example, the cancer associated miR-106b family (Volinia S, et al. (2006) Proc Natl Acad Sci USA 103(7):2257-2261.; Hudson RS, et al. (2013) Oncogene 32(35):4139-4147.) provides a radiation protective phenotype through the regulation of cell cycle progression (Li B, et al. (2011) Prostate 71(6):567-574.). Both miR-21 and miR- 106b were confirmed as radiation protective miRNAs in screens described herein. Several additional radiation modifying candidate miRNAs (Li B, et al. (2011) Prostate 71(6):567-574.; Czochor JR, Glazer PM (2014) Antioxid Redox Signal 21(2):293-312.; Weidhaas JB, et al.

(2007) Cancer Res 67(23): 11111-11116.; Chen G, et al. (2010) Oncol Rep 23(4):997-1003.) were supported in studies described herein, indicating the reliability of the screen and analysis. However, some contradictory results were also observed. For example, IR sensitivity was not potently affected by miR-155 in the screens described herein (Gasparini P, et al. (2014) Proc Natl Acad Sci USA 111(12):4536-4541.). These results may reflect differences in radiation sensitivity between cancer cell types or anomalies within the library screen. These differences underscore the need to validate each candidate miRNAs for its role in radiation response and DNA repair.

[0057] The p53 status of cancer cells can influence radiation response due to its role in DDR and repair (Mirzayans R, Andrais B, Scott A, Wang YW, Murray D (2013) Int J Mol Sci 14(l l):22409-22435.; Simone CB 2nd, et al. (2013) Transl Oncol 6(5):573-585.). Over half of human cancers have mutated or deleted p53, and p53 is often lost in more advanced disease (Harris CC (1996) J Natl Cancer Inst 88(20): 1442- 1455.). Here, multiple cell lines of differential p53 gene status were applied. Specifically, LNCaP cells are p53 wild-type, where PC3 and DU145 cells are p53 null and mutant, respectively (Simone CB 2nd, et al. (2013) Transl Oncol 6(5):573-585.). While the functional screen was completed with WT p53 LNCaP cells, the candidate miRNAs showed common radiation sensitizing effect in p53 wild type and ablated cell types. Therefore, many of these miRNAs are strong agents to use in combination with IR regardless of the p53 status.

[0058] Examples herein describe two radiation sensitizing miRNAs, miR-890-5p and miR-744- 3p. miRNAs may also be administered with a chemotherapeutic agent to sensitize cancer cells to the chemotherapeutic agent. Mechanistic studies confirmed that these miRNAs delayed γ-Η2ΑΧ resolution and DNA repair. Further experiments demonstrated the polyvalent nature of these miRNAs with their ability to target multiple genes within DDR pathways. miR-890-5p transfection reduced the protein levels of four DDR genes, MAD2L2, WEE1, XPC, and KU80. Suppression of MAD2L2, a mitotic spindle assembly checkpoint protein, is known to cause hypersensitivity to IR and increased γ-Η2ΑΧ foci formation (Cheung HW, et al. (2006) Cancer Res 66(8):4357-4367.; Zhao J, et al. (2011) J Clin Neurosci 18(6):827-833.). WEE1, a mitotic checkpoint protein and tyrosine kinase, is also a validated target for enhancing cellular sensitivity to DNA damaging agents (Wang Y, Decker SJ, Sebolt-Leopold J (2004) Cancer Biol Ther 3(3):305-313.; Bridges KA, et al. (2011) Clin Cancer Res 17(17):5638-5648.). XPC is mutated in xeroderma pigmentosum and functions in nucleotide excision repair (Chavanne F, et al. (2000) Cancer Res 60(7): 1974- 1982.). XPC knock-down also reduces DSB repair and increases cellular sensitivity to DNA damaging agents (Despras E, et al. (2007) Cancer Res 67(6):2526-2534.). KU80, encoded by XRCC5, is also a well characterized DDR and repair protein involved in non-homologous end joining and radiation sensitivity (Taccioli GE, et al. (1994) Science 265(5177): 1442-1445.; Liang F, Romanienko PJ, Weaver DT, Jeggo PA, Jasin M (1996) Proc Natl Acad Sci USA 93(17):8929-8933.).

[0059] Thus, miR-890-5p enhances cellular sensitivity to DNA damaging agents through a variety of pathway targets. While it has been hypothesized that individual miRNAs may target multiple genes within the same pathway to produce a given phenotype (Petrocca F, et al.

(2008) Cancer Cell 13(3):272-286.), this concept has not been significantly challenged. To investigate the multifunctional nature of miR-890-5p to mediate radiation sensitization, siRNA was used to knock-down a primary miR-890-5p target, MAD2L2, to determine if additional radiation sensitivity could be detected through alternate targets. The results demonstrate that miR-890-5p enhances IR sensitivity even after significant MAD2L2 knockdown, supporting the concept that miRNAs can function by targeting multiple genes in related pathways. The knockdown of MAD2L2 did not appear to influence the efficiency of miR-890-5p to suppress a secondary target, WEE1, suggesting that these mRNAs do not significantly compete for miR- 890-5p binding.

[0060] The primary transcript for miR-890-5p is not well characterized, but it is presumed to include miR-888, miR-892a, and miR-892b due to their proximity. The mature miRNAs from this cluster were radiation sensitizing in screens described herein, indicating that this gene region plays a specialized role in DNA repair. Interestingly, miRNAs from this gene region are uniquely expressed at high levels in human epididymis, suggesting a potential role for these miRNAs in sperm maturation or fertility (Landgraf P, et al. (2007) Cell 129(7): 1401- 1414.; Belleannee C, et al. (2012) PLoS One 7(4):e34996.). miR-890-5p expression is low or absent in other human tissues, including the prostate (Landgraf P, et al. (2007) Cell 129(7): 1401-1414.). Notably, the incidence of epididymal tumors is very rare and represents at most 0.03% of all male cancers, in sharp contrast to almost 20% for prostate cancer in Western countries (Yeung CH, Wang K, Cooper TG (2012) Asian J Androl 14(3):465-475.). As described herein, the miR- 890-5p gene cluster contributes to this differential cancer susceptibility due to increased cellular sensitivity to DNA damage.

[0061] The results presented herein support the potential use of miR-890-5p and miR-744-3p as a radiation sensitizing agent. miRNA mimetics and inhibitors have shown promise as therapeutic agents in pre-clinical models and a few are being translated for human clinical trials. For example a miR-122 inhibitor, Miravirsen, is currently in clinical trials for the treatment of Hepatitis C (Janssen HL, et al. (2013) N Engl J Med 368(18): 1685-1694.). Further, a liposome-based miR-34 mimic (MRX34) entered Phase I clinical trials in patients with advanced hepatocellular carcinoma in 2013 (Ling H, Fabbri M, Calin GA (2013) Nat Rev Drug Discov 12(11):847-865.). Demonstrated herein, a liposomally-delivered miR-890-5p mimetic enhanced the therapeutic effect of IR in established prostate tumors. IR is a commonly applied treatment for multiple cancer types, including prostate cancer. It can be administered through targeted external beam radiation therapy, localized radioactive seed placement, or systemically targeted

radiotherapeutics. Thus the selective delivery miR-890-5p to tumors, or activation of endogenous miR-890-5p in cancer cells, may significantly enhance the therapeutic index of IR or other DNA damaging therapies by preventing DNA repair.

[0062] In summary, a series of high-throughput functional screens have identified several miRNAs capable of regulating cancer cell radiation sensitivity and DNA repair. miR-890-5p sensitizes cancer cells to IR through multiple gene targets, including MAD2L2 supporting the concept that a single miRNA can simultaneously regulate multiple genes within a single pathway. These results suggest that miRNAs may have therapeutic potential in the treatment of cancers with IR or with an additional chemotherapeutic agent.

[0063] A composition of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof, may be administered in combination with a

chemotherapeutic agent. The chemotherapeutic agent (also referred to as an anti-neoplastic agent or anti-proliferative agent) can be an alkylating agent; an antibiotic; an anti-metabolite; a detoxifying agent; an interferon; a polyclonal or monoclonal antibody; an EGFR inhibitor; a HER2 inhibitor; a histone deacetylase inhibitor; a hormone; a mitotic inhibitor; an MTOR inhibitor; a multi-kinase inhibitor; a serine/threonine kinase inhibitor; a tyrosine kinase inhibitors; a VEGF/VEGFR inhibitor; a taxane or taxane derivative, an aromatase inhibitor, an anthracycline, a microtubule targeting drug, a topoisomerase poison drug, an inhibitor of a molecular target or enzyme (e.g., a kinase inhibitor), a cytidine analogue drug or any

chemotherapeutic, anti-neoplastic or anti-proliferative agent listed in

www .cancer. org/docroot/cdg/cdg_0. asp .

[0064] Exemplary alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan (Alkeran); carmustine (BiCNU); busulfan

(Busulfex); lomustine (CeeNU); dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel); ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran); carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide (Temodar); thiotepa (Thioplex);

bendamustine (Treanda); or streptozocin (Zanosar).

[0065] Exemplary antibiotics include, but are not limited to, doxorubicin (Adriamycin);

doxorubicin liposomal (Doxil); mitoxantrone (Novantrone); bleomycin (Blenoxane);

daunorubicin (Cerubidine); daunorubicin liposomal (DaunoXome); dactinomycin (Cosmegen); epirubicin (Ellence); idarubicin (Idamycin); plicamycin (Mithracin); mitomycin (Mutamycin); pentostatin (Nipent); or valrubicin (Valstar).

[0066] Exemplary anti-metabolites include, but are not limited to, fluorouracil (Adrucil);

capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine (Purinethol); pemetrexed

(Alimta); fludarabine (Fludara); nelarabine (Arranon); cladribine (Cladribine Novaplus);

clofarabine (Clolar); cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposomal (DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine (FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine (Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall); thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS).

[0067] Exemplary detoxifying agents include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

[0068] Exemplary interferons include, but are not limited to, interferon alfa-2b (Intron A) or interferon alfa-2a (Roferon-A).

[0069] Exemplary polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab (Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab (Vectibix); tositumomab/iodine 131 tositumomab (Bexxar); alemtuzumab (Campath); ibritumomab (Zevalin; In-I l l; Y-90 Zevalin); gemtuzumab

(Mylotarg); eculizumab (Soliris) ordenosumab.

[0070] Exemplary EGFR inhibitors include, but are not limited to, gefitinib (Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva); panitumumab (Vectibix); PKI-166;

canertinib (CI- 1033); matuzumab (Emd7200) or EKB-569.

[0071] Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin); lapatinib (Tykerb) or AC-480.

[0072] Histone Deacetylase Inhibitors include, but are not limited to, vorinostat (Zolinza).

[0073] Exemplary hormones include, but are not limited to, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); megestrol (Megace); leuprolide (Lupron; Lupron Depot; Eligard; Viadur) ; fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar LA; Trelstar Depot) ; exemestane (Aromasin) ; goserelin (Zoladex) ; bicalutamide (Casodex); anastrozole (Arimidex);

fluoxymesterone (Androxy; Halotestin); medroxyprogesterone (Provera; Depo-Provera);

estramustine (Emcyt); flutamide (Eulexin); toremifene (Fareston); degarelix (Firmagon);

nilutamide (Nilandron); abarelix (Plenaxis); or testolactone (Teslac).

[0074] Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin; Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos; VePesid); teniposide (Vumon); ixabepilone (Ixempra);

nocodazole; epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan (Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D). [0075] Exemplary MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torisel); rapamune, ridaforolimus; or AP23573.

[0076] Exemplary multi-kinase inhibitors include, but are not limited to, sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474; PKC-412; motesanib; or AP24534.

[0077] Exemplary serine/threonine kinase inhibitors include, but are not limited to,

ruboxistaurin; eril/easudil hydrochloride; flavopiridol; seliciclib (CYC202; Roscovitrine); SNS- 032 (BMS-387032); Pkc412; bryostatin; KAI-9803;SF1126; VX-680; Azdl l52; Arry-142886 (AZD-6244); SCIO-469; GW681323; CC-401; CEP-1347 or PD 332991.

[0078] Exemplary tyrosine kinase inhibitors include, but are not limited to, erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib (Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab (Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux); panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath);

gemtuzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient); dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584); CEP-701; SU5614; MLN518; XL999; VX- 322; Azd0530; BMS-354825; SKI-606 CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG888.

[0079] Exemplary VEGF/VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent); ranibizumab; pegaptanib; or vandetinib.

[0080] Exemplary microtubule targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristin, vinblastin, nocodazole, epothilones and navelbine.

[0081] Exemplary topoisomerase poison drugs include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin and idarubicin.

[0082] Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.

[0083] Exemplary general chemo therapeutic, anti-neoplastic, anti-proliferative agents include, but are not limited to, altretamine (Hexalen); isotretinoin (Accutane; Amnesteem; Clara vis; Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade) asparaginase (Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine (Matulane); pegaspargase

(Oncaspar); denileukin diftitox (Ontak); porfimer (Photofrin); aldesleukin (Proleukin);

lenalidomide (Revlimid); bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel); arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosine (Leucenol); (1M tegafur - 0.4 M 5-chloro-2,4-dihydroxypyrimidine - 1 M potassium oxonate) or lovastatin.

[0084] In another aspect, the second chemotherapeutic agent can be a cytokine such as G-CSF (granulocyte colony stimulating factor). In another aspect, a composition the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof, may be administered in combination with radiation therapy. Radiation therapy can also be administered in combination with a composition of the present invention and another chemotherapeutic agent described herein as part of a multiple agent therapy. In yet another aspect, a composition of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, mimetic, analog or derivative thereof, may be administered in combination with standard chemotherapy

combinations such as, but not restricted to, CMF (cyclophosphamide, methotrexate and 5- fluorouracil), CAF (cyclophosphamide, adriamycin and 5-fluorouracil), AC (adriamycin and cyclophosphamide), FEC (5-fluorouracil, epirubicin, and cyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide, and paclitaxel), rituximab, Xeloda (capecitabine), Cisplatin (CDDP), Carboplatin, TS-1 (tegafur, gimestat and otastat potassium at a molar ratio of 1:0.4: 1), Camptothecin-11 (CPT-11, Irinotecan or Camptosar™) or CMFP (cyclophosphamide, methotrexate, 5-fluorouracil and prednisone).

[0085] In preferred embodiments, a composition of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof, may be administered with an inhibitor of an enzyme, such as a receptor or non-receptor kinase. Receptor and non-receptor kinases of the invention are, for example, tyrosine kinases or serine/threonine kinases. Kinase inhibitors of the invention are small molecules, polynucleic acids, polypeptides, or antibodies.

[0086] Exemplary kinase inhibitors include, but are not limited to, Bevacizumab (targets VEGF), BIBW 2992 (targets EGFR and Erb2), Cetuximab/Erbitux (targets Erbl), Imatinib/Gleevic (targets Bcr-Abl), Trastuzumab (targets Erb2), Gefitinib/Iressa (targets EGFR), Ranibizumab (targets VEGF), Pegaptanib (targets VEGF), Erlotinib/Tarceva (targets Erbl), Nilotinib (targets Bcr-Abl), Lapatinib (targets Erbl and Erb2/Her2), GW-572016/lapatinib ditosylate (targets HER2/Erb2), Panitumumab/Vectibix (targets EGFR), Vandetinib (targets RET/VEGFR), E7080 (multiple targets including RET and VEGFR), Herceptin (targets HER2/Erb2), PKI-166 (targets EGFR), Canertinib/CI-1033 (targets EGFR), Sunitinib/SU-11464/Sutent (targets EGFR and FLT3), Matuzumab/Emd7200 (targets EGFR), EKB-569 (targets EGFR), Zd6474 (targets EGFR and VEGFR), PKC-412 (targets VEGR and FLT3), Vatalanib/Ptk787/ZK222584 (targets VEGR), CEP-701 (targets FLT3), SU5614 (targets FLT3), MLN518 (targets FLT3), XL999 (targets FLT3), VX-322 (targets FLT3), Azd0530 (targets SRC), BMS-354825 (targets SRC), SKI-606 (targets SRC), CP-690 (targets JAK), AG-490 (targets JAK), WHI-P154 (targets JAK), WHI-P131 (targets JAK), sorafenib/Nexavar (targets RAF kinase, VEGFR- 1, VEGFR-2, VEGFR-3, PDGFR- β, KIT, FLT-3, and RET), Dasatinib/Sprycel (BCR/ABL and Src), AC-220 (targets Flt3), AC-480 (targets all HER proteins, "panHER"), Motesanib diphosphate (targets VEGF1-3, PDGFR, and c-kit), Denosumab (targets RANKL, inhibits SRC), AMG888 (targets HER3), and AP24534 (multiple targets including Flt3).

[0087] Exemplary serine/threonine kinase inhibitors include, but are not limited to, Rapamune (targets mTOR/FRAPl), Deforolimus (targets mTOR), Certican/Everolimus (targets

mTOR/FRAPl), AP23573 (targets mTOR/FRAPl), Eril/Fasudil hydrochloride (targets RHO), Flavopiridol (targets CDK), Seliciclib/CYC202/Roscovitrine (targets CDK), SNS-032/BMS- 387032 (targets CDK), Ruboxistaurin (targets PKC), Pkc412 (targets PKC), Bryostatin (targets PKC), KAI-9803 (targets PKC), SF1126 (targets PI3K), VX-680 (targets Aurora kinase), Azdl l52 (targets Aurora kinase), Arry-142886/AZD-6244 (targets MAP/MEK), SCIO-469 (targets MAP/MEK), GW681323 (targets MAP/MEK), CC-401 (targets JNK), CEP-1347 (targets JNK), and PD 332991 (targets CDK).

[0088] The present invention also provides pharmaceutical compositions comprising a composition of each of the miRNA described herein in combination with at least one

pharmaceutically acceptable excipient or carrier.

[0089] A "pharmaceutical composition" is a formulation containing the compositions of the present invention in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler or a vial. The quantity of active ingredient (e.g. , a formulation of the disclosed composition or salt, hydrate, solvate or isomer thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like. Dosage forms for the topical or transdermal administration of a composition of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. In one embodiment, the active composition is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers or propellants that are required.

[0090] As used herein, the phrase "pharmaceutically acceptable" refers to those compositions, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0091] "Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. A "pharmaceutically acceptable excipient" as used in the

specification and claims includes both one and more than one such excipient.

[0092] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g. , inhalation), transdermal (topical), and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens;

antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as

ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0093] A composition or pharmaceutical composition of the invention can be administered to a subject in many of the well-known methods currently used for chemotherapeutic treatment. For example, for treatment of cancers, a composition of the invention may be injected directly into tumors, injected into the blood stream or body cavities or taken orally or applied through the skin with patches. The dose chosen should be sufficient to constitute effective treatment but not as high as to cause unacceptable side effects. The state of the disease condition (e.g. , cancer, precancer, and the like) and the health of the patient should preferably be closely monitored during and for a reasonable period after treatment.

[0094] The term "therapeutically effective amount", as used herein, refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician. In a preferred aspect, the disease or condition to be treated is cancer. In another aspect, the disease or condition to be treated is a cell proliferative disorder.

[0095] For any composition, the therapeutically effective amount can be estimated initially either in cell culture assays, e.g. , of neoplastic cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic/prophylactic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage may vary within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[0096] Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and

tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

[0097] The pharmaceutical compositions containing active compositions of the present invention may be manufactured in a manner that is generally known, e.g. , by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and/or auxiliaries that facilitate processing of the active compositions into

preparations that can be used pharmaceutically. Of course, the appropriate formulation is dependent upon the route of administration chosen.

[0098] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of

microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0099] Sterile injectable solutions can be prepared by incorporating the active composition in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active composition into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0100] Oral compositions generally include an inert diluent or an edible pharmaceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active composition can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the composition in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compositions of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0101] For administration by inhalation, the compositions are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g. , a gas such as carbon dioxide, or a nebulizer.

[0102] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or

suppositories. For transdermal administration, the active compositions are formulated into ointments, salves, gels, or creams as generally known in the art.

[0103] The active compositions can be prepared with pharmaceutically acceptable carriers that will protect the composition against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0104] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active composition and the particular therapeutic effect to be achieved.

[0105] In therapeutic applications, the dosages of the pharmaceutical compositions used in accordance with the invention vary depending on the agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage. Generally, the dose should be sufficient to result in slowing, and preferably regressing, the growth of the tumors and also preferably causing complete regression of the cancer. An effective amount of a

pharmaceutical agent is that which provides an objectively identifiable improvement as noted by the clinician or other qualified observer. For example, regression of a tumor in a patient may be measured with reference to the diameter of a tumor. Decrease in the diameter of a tumor indicates regression. Regression is also indicated by failure of tumors to reoccur after treatment has stopped. As used herein, the term "dosage effective manner" refers to amount of an active composition to produce the desired biological effect in a subject or cell.

[0106] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. EXAMPLES

[0107] Example 1. Materials and Methods

[0108] The human prostate cancer cell lines LNCaP, C4-2, PC3 and DU145 were purchased from the American Type Culture Collection (Manassas, VA). The cells were maintained in RPMI 1640 medium (Cellgro, Manassas, VA) containing 10% fetal bovine serum (FBS), 5μg/ml ciprofloxacin (USBio, Swampscott, MA) and 5μg/ml Gentamicin (Quality Biological, Gaithersburg, MD). LNCaP-MLuc and PC3-MLuc cells were previously developed (Lupoid SE, Johnson T, Chowdhury WH, Rodriguez R (2012) PLoS One 7(5):e36535.) and maintained in RPMI 1640 containing 10% FBS, 5μg/ml ciprofloxacin and 5μg/ml Gentamicin and 5 μg/mL Blasticidin (Invitrogen, Grand Island, NY). The cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% C02. Athymic nude mice (nu/nu) aged 6 weeks were purchased from Harlan Laboratories Inc. (Indianapolis, IN) and maintained in a temperature-controlled, pathogen-free room. All animals were handled according to the approved protocols and guidelines of Animal Care and Use Committee at Johns Hopkins University.

[0109] MLuc viability assay

[0110] MLuc cell viability assays were completed as previously described (Lupoid SE, Johnson T, Chowdhury WH, Rodriguez R (2012) PLoS One 7(5):e36535.). Two days before IR, LNCaP- MLuc and PC3-MLuc cells stably transfected with the pDonor-hp-Actin-hMLuc vector were transfected with miRNA mimetics and/or siRNAs. On day 0, the cells were irradiated (4Gy) or untreated. On day 11, the cell culture media was then assayed for MLuc activity. The prostate cancer MLuc cell viability assays were completed as previously described (Lupoid SE, Johnson T, Chowdhury WH, Rodriguez R (2012) PLoS One 7(5):e36535.). Two days before IR, LNCaP-MLuc and PC3-MLuc stably transfected with the pDonor-hp-Actin- hMLuc vector were plated at low density and transfected with miRNA mimetics and/or siRNAs using Lipofectamine 2000 or Lipofectamine RNAiMax (Invitrogen). On day 0, the cells were irradiated (4 Gy in a Gammacell 40 [Nordion] 137Cs radiator at approximately 0.5 Gy/min) or untreated. On day 11, 50 μΐ cell culture media was then assayed for MLuc activity to quantify the relative cell density using a Renilla buffer, consisting of PBS with 1.43 μΜ coelenterazine. The MLuc activities were read in a Perkin Elmer Micro Beta luminometer. [0111] High-throughput functional screening using miRNA library

[0112] Two days before IR, LNCaP-MLuc cells were transfected with 20 nM of 810 different miRNA mimics. Each miRNA was transfected in quadruplicate. Control miRNA and DNAPK siRNA were included in each 96 well plate (sequences in Table 2).

Table 2. The sequence of siRNAs used in this study.

Half of the plates were irradiated (4Gy) 48 h after transfection, while the other half remained untreated. Eleven days after IR, MLuc activity was quantified and normalized to the control miRNA. Two days before IR, 2xl0³ LNCaP-MLuc cells were transfected with 20 nM of 810 different miRNA mimics using Lipofectamine 2000 (Invitrogen) in individual wells of 96 well plates. Each miRNA was transfected in quadruplicate with control miRNA and DNAPK siRNA in each 96 well plate. Half of the plates were irradiated 48 h after transfection (4 Gy), while the other half remained untreated. Seven days after irradiation, 50 μΐ of media was removed and replaced with 50 μΐ of fresh media. Eleven days after irradiation, MLuc activity was examined to quantify viable cell density, normalizing to the control miRNA. Toxic miRNAs, which inhibited cell growth by over 50% in the absence of IR on day 11, were excluded from the classification for radiation sensitization. Radiosensitizing or radioprotective miRNAs were defined as those which increased cell death by over 50% or increased cell survival by over 2 fold, respectively.

[0113] Clonogenic assay

[0114] DU145 cells were transfected with 20 nM control or candidate miRNAs and grown for 48 h, after which cell dilutions were plated and irradiated immediately at different doses. The cells were grown for 14 days. Colonies with greater than 30 cells were scored and surviving fraction was calculated as previously described (Lupoid SE, Johnson T, Chowdhury WH, Rodriguez R (2012) PLoS One 7(5):e36535.). Clonogenic survival assays were performed with 8 x 10⁵ DU145 cells transfected with 20 nM control or candidate miRNAs and grown for 48 h, after which cell dilutions were plated into 25 cm culture flasks and irradiated immediately. Radiation exposures were carried out as described above at 0, 2.5, 5, 7.5 and 10 Gy. The cells were grown for 14 days, fixed and stained with crystal violet; colonies with greater than 30 cells were scored, and surviving fraction was calculated as previously described (Ni X, et al. (2011) Clin Invest 121(6):2383-2390). Survival curves were fitted by GraphPad Prism software

(GraphPad Software, La Jolla, CA) by nonlinear regression analysis and a user defined equation for cell survival analysis due to radiation injury.

[0115] Y-H2AX foci formation

[0116] DU145 cells were transfected with 20 nM of control or candidate miRNAs and were seeded on glass slides. The cells were incubated for 48 h and then irradiated (4 Gy) or untreated. The γ-Η2ΑΧ foci formation was evaluated at 1, 4, 8, 12 and 24 h after IR by immunofluore scent microscopy. The percentage of cells containing >10 fluorescent foci was calculated as previously reported (Cheung HW, et al. (2006) Cancer Res 66(8):4357-4367.). DU145 cells were transfected with 20 nM of control or candidate miRNAs and were seeded at 8 x 10⁴ cells/well on 4 chamber polystyrene vessel tissue culture treated glass slides. The cells were incubated for 48 h and then irradiated (4 Gy) or untreated. The γ-Η2ΑΧ foci formation was evaluated at 1, 4, 8, 12 and 24 h after IR. The cells were washed with PBS, and fixed with 4% formaldehyde for 15 min, followed by treatment with 0.2% Triton X-100 for 10 min. The cells were blocked with 1% BSA in PBS for 1 h and were incubated with γ-Η2ΑΧ antibody (1: 1000) for 30 min. The cells were washed and labeled with a 1: 1000 dilution of Alexa Fluor antibody (Invitrogen) for 30 min. Cellular DNA was counterstained with Prolong Gold with DAPI (Invitrogen). Fluorescent signals were visualized with a Nikon eclipse TE 2000E microscope and analyzed by NIS- Elements advanced Research version 3.2 software (Nikon, Tokyo, Japan). Images were photographed at the same exposure time under a x20 objective. Over 300 cells were counted from more than three random fields under a x20 objective for each experiment, and the percentage of cells containing >10 fluorescent foci was calculated as previously reported (Cheung HW, et al. (2006) Cancer Res 66(8):4357-4367.).

[0117] Comet assay

[0118] The comet assay was carried out under neutral pH conditions using CometAssay® from Trevigen (Gaithersburg, MD). Comets were imaged by fluorescent microscopy and analyzed using CometScore. The comet assay was carried out under neutral pH conditions using the CometAssay® from Trevigen (Gaithersburg, MD). Briefly, cells were mixed with low melting point agarose and plated on microscope slides and allowed to gel. Cells were then lysed under neutral buffer followed by rinse in TBE buffer (10.8 % [w/v] tris base, 5.5% [w/v] boric acid, 0.93% [w/v] EDTA). After electrophoresis in TBE buffer for 40 min at 1 V/cm, slides were washed in water and dehydrated with ethanol, air dried overnight and then treated with SYBR Green for DNA staining. Comets were imaged Zeiss Imager.Zl fluorescent microscopy (Carl Zeiss AG, Oberkochen, Germany) and analyzed using the software CometScore

(Autocomet.com). The tail moment was calculated as the average of at least 50 comets.

[0119] 3'UTR constructs

[0120] The 3'UTR regions of the targets were amplified from 293T genomic DNA (human embryonic kidney cells) by PCR. Primer sequences were designed to incorporate Spel and Hindlll restriction enzyme sites (Table 3). PCR products were inserted into the Spel and Hindlll cloning site of the pMIR-REPORT luciferase expression reporter vector (Ambion, Grand Island, NY) to generate pMIR-3'UTR. The mutant pMIR-3'UTR reporter vector was generated using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Columbia, MD) according to the manufacturer's instructions (Table 3).

Table 3. The sequence of primers used in this study.

[0121] 3'UTR Dual Lucif erase Assay

[0122] LNCaP cells were transfected with 20 nM control or miRNA mimics, 80 ng pMIR- 3'UTR reporter vector and 20 ng of reference pRL-CMV Renilla reporter vector. After 48 h incubation, the reporter activity was assessed using a Dual Luciferase Enzyme Assay. Firefly luciferase was normalized against the internal control Renilla luciferase. LNCaP cells were transfected by Lipofectamine 2000 with 20 nM control or miRNA mimics, 80 ng pMIR-3'UTR reporter vector and 20 ng of reference pRL-CMV Renilla reporter vector. After 48 h incubation, the reporter activity was assessed using a Dual Luciferase Enzyme Assay. The firefly luciferase assay buffer consisted of 25 mM glycylglycine, 15 mM K_XPO4 (pH 7.8), 4 mM EGTA, 2 mM ATP, 1 mM DTT, 15 mM MgS04, 60 μΜ CoA, 75 μΜ luciferin, with the final pH adjusted to 8.0. The Renilla assay buffer was made as follows: 1.1 M NaCl, 2.2 mM Na2EDTA, 0.22 M K_XPO4 (pH 5.1), 0.44 mg/mL BSA, 1.3 mM NaN3, 1.43 μΜ coelenterazine, with the final pH adjusted to 5.0. Light emission was measured in a Wallac 1450 Microbeta Jet Luminescence Reader. Firefly luciferase was first read and signal normalized against the internal control Renilla luciferase.

[0123] Western blot analysis

[0124] Cells were transfected with miRNA mimics or siRNAs and incubated for 48 h. MAD2L2, RAD23B, WEE1, XPC, KU80, XLF and MCL1 antibodies were applied to western blotting. ACTB was used as a control for protein loading. Prostate cancer cells were transfected with miRNA mimics or siRNAs and were incubated for 48 h. Cells were harvested and lysed in RIPA lysis buffer. Protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred onto polyvinylidene fluoride

membranes, and the membranes were then blocked with odyssey blocking buffer and incubated overnight at 4°C with anti-MAD2L2 (1: 1,000), anti-RAD23B (1:500), anti-WEEl (1: 1000), anti- XPC (1: 1000), anti-KU80 (1: 1000), anti-XLF (1: 1000), anti-MCLl (1: 1000) or anti-ACTB (1: 10,000) antibodies. The membranes were washed and labelled with a 1:20,000 dilution of IRDye 680LT anti-mouse or a 1: 15,000 dilution of IRDye 800CW anti-rabbit secondary antibody (LI-COR, Lincoln, NE) at room temperature for approximately 1 h. Images were captured and analyzed with an Odyssey infrared imaging system (LI-COR).

[0125] In vivo tumor models [0126] The animal studies were performed according to the protocols approved by the Animal Care and Use Committee at Johns Hopkins University. Athymic nude mice (nu/nu) aged 6 weeks were inoculated with 5 x 10⁶ DU145 cells subcutaneously. Cohorts of similar tumor volume were randomized and directly injected with PBS, 12.5 μg of liposomal control miRNA mimetic, or liposomal miR-890-5p mimetic. Two days after injection (day 0) radiation groups received 6 Gy local IR. The tumor volume was calculated according to the formula: tumor volume (cm ) = 1 x w x h x 0.52. Tumor response was determined as reaching 4 times as its initial size as previously reported (Lupoid SE, Johnson T, Chowdhury WH, Rodriguez R (2012) PLoS One 7(5):e36535.). Athymic nude mice (nu/nu) aged 6 weeks were inoculated with 5 x 10⁶ DU 145 cells subcutaneously in left thigh. When tumor size reached 0.1 - 0.3 cm in diameter, the treatment was started. Tumors were randomized into 5 groups, including PBS, control miRNA, miR-890, control miRNA plus IR and miR-890-5p plus IR. Liposomal control miRNA mimetic, or liposomal miR-890-5p mimetic were prepared using the MaxSuppressorTM In Vivo RNA- LANCEr II kit (Bioo Scientific, Austin, TX) according to the manufacturer's instructions. Mice were anesthetized with isoflurane, and then, tumors were directly injected with total 50 μΐ of reagents; PBS, 12.5 μg of liposomal control miRNA mimetic, or liposomal miR-890-5p mimetic. Two days after injection (day 0), radiation groups received 6 Gy local IR (7.14 Gy/min) using a J.L. Shepherd Mark 137Cs irradiator with the body shielded from the source. Tumors were measured every 2 days and the tumor volume was calculated according to the formula; tumor volume (cm ) = length x width x height x 0.52. Tumor response was determined as reaching 4 times its volume as its initial size as previously reported (Ni X, et al. (2011) Clin Invest 121(6):2383-2390). Two animals were eliminated from the study due to illness and failure of progressive tumor growth. The animal studies were performed according to the protocols approved by the Animal Care and Use Committee at Johns Hopkins University.

[0127] Statistical analyses

[0128] Assays were analyzed by 2-tailed, unpaired Student's t-test or 2-way ANOVA. Kaplan- Meier analysis was performed by log-rank (Mantel-Cox). P < 0.05 was considered statistically significant. The results are reported as the mean + standard error (S.E.) or standard deviation (S.D.). The differences between groups were evaluated by 2-tailed, unpaired Student's t-test. The IC₅₀ value was calculated using GraphPad Prism 5 software. Tumor volume was evaluated by 2-way ANOVA. For the extension of tumor quadrupling experiments, events (animals whose tumor volume was not yet 4-fold the size at injection) were plotted on a Kaplan-Meier curve and analyzed by log-rank (Mantel-Cox) test as previously reported (Ni X, et al. (2011) Clin Invest 121(6):2383-2390). P < 0.05 was considered statistically significant.

[0129] Example 2. High-throughput screening identifies miRNAs which modulate prostate cancer growth, survival and radiosensitivity.

[0130] A previously developed bioluminescent Metridia Luciferase (MLuc) viability assay (Lupoid SE, Johnson T, Chowdhury WH, Rodriguez R (2012) PLoS One 7(5):e36535.), was used to measure cellular sensitivity to IR following the transfection of miRNA mimetics. With this assay the expression of a secreted humanized MLuc reporter is controlled by the human β- actin promoter and enhancer, and bioluminescent activity corresponds linearly with viable cell number (Lupoid SE, Johnson T, Chowdhury WH, Rodriguez R (2012) PLoS One

7(5):e36535.9). One prostate cancer cell line, LNCaP-MLuc, was applied to screen a library of 810 human miRNA mimetics. A schematic representation of the experimental design is shown in Fig. 1A. Briefly, LNCaP-MLuc cells were separately transfected with individual miRNA mimetics, or control miRNAs, in replicates in multiwell plate format. After 48 h, one group of cells was irradiated (4 Gy), while the other group remained untreated. The secreted MLuc activity was then quantified on the eleventh day to measure relative cell viability and therapeutic effect. A positive control siRNA targeting DNAPK was utilized for radiation sensitization (Figs. 8A and 8B). Analysis of duplicate samples indicates assay reproducibility (Figs. 9A and 9B).

[0131] MLuc activity, representing viable cell density over eleven days, is shown in Fig. IB for both irradiated cells (red) and non-irradiated cells (blue). The results are organized as a waterfall plot based on radiosensitivity, with the most radiation sensitizing miRNAs on the left, leading to the most radiation protective miRNAs on the right. The results indicate that over half of the miRNAs studied enhanced cellular sensitivity to IR, when compared to a control miRNA, while a much smaller percentage of miRNAs was radiation protective. Many miRNAs belonging to the same families produced matching radiosensitization phenotypes. For example, miRNAs from the miR-15/16 and miR-1/133 families were each observed to be radiation sensitizing, whereas miRNAs from the miR-106b family were found to be radioprotective (Fig. 10). The results for all miRNAs are available in Table 1. [0132] The experimental design also provided data on cellular responses to miRNA mimetics in the absence of irradiation (Fig. 1C). It is notable that the majority of miRNAs reduced viable cell density, when compared to the control miRNA. These results are consistent with the common global miRNA down-regulation observed in human cancers (Lu J, et al. (2005) Nature 435(7043):834-838.). For the purpose of characterizing cellular responses, the miRNAs were grouped into four categories (Fig. ID, Table 1). miRNAs which reduced growth or viability by over 50% in the absence of IR were termed as toxic miRNAs (purple). Non-toxic miRNAs which enhanced radiation induced cell death by over 50% were considered radiation sensitizing miRNAs (red), and miRNAs which increased cell survival by over 2-fold after irradiation were considered radioprotective (blue). Among 810 miRNAs, 75 (9.3%) were identified as radioprotective, 324 (40.0%) were identified as radiosensitizing, and 127 (15.7%) were considered toxic (Fig. ID, Table 1).

[0133] Validation of candidate radiation sensitizing miRNAs

[0134] The top 15 candidate radiation sensitizing miRNAs were selected for repeat assays using two reporter cell lines, LNCaP-MLuc and PC3-MLuc (Fig. 2). All 15 candidates were validated as radiation sensitizing in LNCaP-MLuc cells, and 7 miRNAs were confirmed to also sensitize PC3-MLuc cells (Fig. 2A). Four miRNAs, miR-890, miR-744-3p, miR-32-3p and miR-130b-5p demonstrated equal or greater radiosensitization efficiency, when compared to the DNAPK siRNA positive control, in both cells. The radiation sensitizing potential of these miRNAs was further quantified by clonogenic survival assays (Fig. 2B, and Fig. 11). The dose-modifying factor (DMF) for each miRNA was calculated as the ratio of IR dose required to cause 90% cell death by control miRNA versus the dose required to cause 90% cell death by the candidate miRNAs. The two most potent miRNAs, miR-890-5p and miR-744-3p, achieved DMFO.I of 1.52 and 1.46, respectively (Fig. 2B). It was anticipated that these miRNAs function through targeted regulation of DNA repair processes.

[0135] miR-890-5p and miR-744-3p radiosensitize cells through inhibition of DNA repair

[0136] DNA repair pathways play an important role in cellular response to IR. A hallmark of DNA DSB detection and repair is phosphorylation of H2AX. Following DNA repair, these γ- H2AX foci are resolved by phosphatase activity (Firsanov DV, Solovjeva LV, Svetlova MP (2011) Clin Epigenetics 2(2):283-297.). The influence of miR-890-5p and miR-744-3p on the formation and resolution of γ-Η2ΑΧ foci was examined over 24 h using irradiated (4 Gy) DU145 cells. Within 1 h of IR, γ-Η2ΑΧ foci formation was visible in all treatment groups (Fig. 3A). Within 8 h, the control treated cells had resolved most of the γ-Η2ΑΧ foci. However, the radiation sensitizing miRNAs, miR-890-5p and miR-744-3p, significantly delayed γ-Η2ΑΧ resolution over the 24 h time period (Fig. 3A and 3B).

[0137] To more directly study DNA damage and repair, irradiated cells were examined by the single cell gel electrophoresis comet assay. As above, DU145 cells were transfected with control or radiation sensitizing miRNAs and then treated with 4 Gy of IR 48 h post transfection. Within 4 h of IR, DNA comet tails moments were significantly extended in cells transfected with miR-890-5p and miR-744-3p, when compared to cells transfected with control miRNA (Fig. 3C and 3D). Collectively these data support that miR-890-5p and miR-744-3p inhibited DNA repair processes, which likely contributed to the radiation sensitization phenotype.

[0138] miR-890-5p and miR-744-3p regulate multiple components of DDR pathways

[0139] Several DDR genes were previously characterized as potent radiation sensitizing targets, including DNAPK, MAD2L2, BRCA2, NBN, RAD23B and RAD54L (Ni X, et al. (2011) J Clin

Invest 121(6):2383-2390.). These genes were examined to determine whether they may be direct targets of the identified radiation sensitizing miRNAs by in silico analysis (Table 4).

Table 4. Radiation sensitizing miRNAs predicted to target radiation sensitizing DDR pathway genes using in silico analysis, microRNA.org.

The bolded values corresponding to the miRNAs with a mirSVR score < -0.5.

miR-890-5p and miR-744-3p were predicted to target two of these genes, MAD2L2 and

RAD23B, respectively, with mirSVR Scores < -0.5, indicating a high probability of target gene down-regulation (Betel D, Koppal A, Agius P, Sander C, Leslie C (2010) Genome Biol

11(8):R90.). Transfection of miR-890-5p resulted in reduced MAD2L2 protein levels in multiple prostate cancer cell lines, as detected by western blot (Fig. 4A). As also predicted, miR-744-3p transfection resulted in reduced RAD23B protein levels (Fig. 4A). The 3'UTR regions of the respective genes were then subcloned downstream of firefly luciferase to generate miRNA specific reporters (Fig. 4B). [0140] The respective 3'UTR lucif erase activities of MAD2L2 and RAD23B were significantly down-regulated by miR-890-5p and miR-744-3p transfection (Fig. 4C). Mutation of the corresponding miRNA seed binding sites ablated miRNA mediated suppression (Fig. 4C), indicating direct miRNA regulation of these genes.

[0141] Because a single miRNA has the potential to regulate the expression of multiple genes (Lewis BP, Burge CB, Bartel DP (2005) Cell 120(1): 15-20.), it is anticipated that miR-890-5p and miR-744-3p may regulate additional DDR pathway components. In silico analysis using microRNA.org predicted several additional DDR pathway targets for miR-890-5p or miR-744- 3p, each with high mirSVR scores (Table 5).

Table 5. Potential DDR pathway genes predicted to be targeted by miR-890-5p or miR-744-3p using in silico analysis, microRNA.org, and corresponding mirSVR scores.

DDR pathway miRNA mirSVR score

genes

WEE1 miR-890 -1.1805

XPC miR-890 -0.9251

MAD2L2 miR-890 -0.5108

KU80 miR-890 -0.4843

XLF miR-744* -0.8632

RAD23B miR-744* -0.7604

MCL1 miR-744* -0.6699

Western blot analyses confirmed that miR-890-5p transfection also reduced the target genes WEE1, XPC, and KU80 (Fig. 5A and C). In addition, miR-744-3p transfection reduced the levels of XLF and MCL1 (Fig. 5B and D).

[0142] Analysis of miR-890-5p and its multiple targets on cellular sensitivity to IR

[0143] The multifunctional nature of the identified miRNAs suggests that they may be more potent radiation sensitizing agents than rationally designed siRNAs engineered to target a single DDR gene. When cells were treated with an equal dose of MAD2L2-targeting siRNA or miR- 890-5p miRNA (10 nM), the expression of MAD2L2 was most strongly down-regulated by the siRNA (Fig. 6A). However, miR-890-5p transfection resulted in greater radiation sensitizing efficacy, when compared to MAD2L2 siRNA, at multiple dose levels (Fig. 6B). To further study whether miR-890-5p radiation sensitization was dependent upon MAD2L2 silencing, cells were co-transfected with the miR-890-5p mimetic and MAD2L2 siRNA (Fig. 6B). The addition of miR-890-5p to MAD2L2 siRNA resulted in greater IR sensitization at all doses studied (Fig. 6B), including at higher doses where no additional MAD2L2 knock-down was detected (Fig. 6A). This supports that miR-890-5p functions as a radiation sensitizer by targeting additional genes beyond MAD2L2. The miR-890-5p target WEEl, was not observed to be further reduced by the knock-down of MAD2L2 mRNA (Fig. 12), indicating that miR-890-5p regulates these genes independently. The calculated icso of MAD2L2 siRNA, miR-890-5p and combined MAD2L2 siRNA and miR-890-5p were 0.49 nM, 0.36 nM and 0.15 nM, respectively (Fig. 6C).

[0144] miR-890-5p mimetic sensitizes established prostate cancer tumors to IR

[0145] In view of the potent and multifunctional nature of miR-890-5p radiation sensitization in vitro, it was determined whether a miR-890-5p mimetic could enhance the therapeutic effect of IR in vivo. Established subcutaneous DU145 prostate tumors were directly treated with miR-890- 5p or control miRNA mimetics, in liposomal formulation, with a single intratumoral

administration. Two days after injection, one cohort of tumors was irradiated with a single dose of 6 Gy, while the other cohort was not irradiated. A final control cohort was treated with PBS only. Tumor volume was evaluated over 35 days and the time to tumor volume quadrupling was calculated.

[0146] In the absence of irradiation, the tumor grew comparably with miR-890, control miRNA, or PBS treatment (Fig. 7A and Figs. 13A-E). On the other hand, after irradiation, the tumor volume of miR-890-5p treated mice was significantly reduced when compared to the miRNA control. The miR-890-5p mimetic significantly delayed the time to tumor quadrupling of irradiated mice by 14 days, when compared to the control miRNA (Fig. 7B). These results support the use of miR-890-5p as a potent radiation sensitizing agent.

EQUIVALENTS

[0147] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0148] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0149] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

CLAIMS What is claimed is:

1. A composition for increasing sensitization to ionizing radiation (IR) comprising an

miRNA; miRNA mimetic, activator of miRNA, or mixture thereof.

2. The composition of claim 1, wherein the miRNA comprises miR-744.

3. The composition of claim 2, wherein the miRNA comprises miR-744-3p or miR-744-5p.

4. The composition of claim 3, wherein the miR-744-3p comprises SEQ ID NO: 734.

5. The composition of claim 1, wherein the miRNA comprises miR-890.

6. The composition of claim 5, wherein the miRNA comprises miR-890-5p or miR-890-3p.

7. The composition of claim 6, wherein miR-890-5p comprises SEQ ID NO: 764.

8. The composition of claim 1, wherein the activator of miRNA activates endogenous miR- 890-5p or miR-744-3p when administered to a patient.

9. The composition of claim 1, wherein the miRNA mimetic, mimics miR-890-5p or miR- 744-3p.

10. The composition of claim 1, wherein the composition further comprises a

chemotherapeutic agent.

11. The composition of claim 1, wherein the composition is administered 1-5 days prior to treatment with IR.

12. The composition of claim 11, wherein the dose of IR comprises between 0 Gy and 30 Gy in a single fraction of externally applied radiation.

13. The composition of claim 11, wherein the dose of IR comprises between 0 Gy and 20 Gy per fraction in multiple fractions by externally applied radiation.

14. The composition of claim 11, wherein the dose of IR comprises low dose rate

brachy therapy dosing of 0 - 2 Gy /minute.

15. The composition of claim 11, wherein the dose of IR comprises high dose rate

brachy therapy dosing of 2 - 20 Gy /minute.

16. A method of increasing sensitivity to ionizing radiation (IR) therapy or chemotherapy in a patient in need thereof, the method comprising:

administering to the patient in need thereof a composition comprising an miRNA, miRNA mimetic, activator of miRNA, or mixture thereof.

17. The method of claim 16, wherein the miRNA comprises miR-744.

18. The method of claim 17, wherein the miRNA comprises miR-744-3p or miR-744-5p.

19. The method of claim 18, wherein the miR-744-3p comprises SEQ ID NO: 734.

20. The method of claim 16, wherein the miRNA comprises miR-890.

21. The method of claim 20, wherein the miRNA comprises miR-890-5p or miR-890-3p.

22. The method of claim 21, wherein miR-890-5p comprises SEQ ID NO: 764.

23. The method of claim 16, wherein the composition is administered prior to IR treatment.

24. The method of claim 23, wherein the composition is administered 1-5 days prior to

treatment with IR.

25. The method of claim 23, wherein the dose of IR comprises between 0 Gy and 30 Gy in a single fraction of externally applied radiation.

26. The method of claim 23, wherein the dose of IR comprises between 0 Gy and 20 Gy per fraction in multiple fractions by externally applied radiation.

27. The method of claim 23, wherein the dose of IR comprises low dose rate brachytherapy dosing of 0 - 2 Gy/minute.

28. The method of claim 23, wherein the dose of IR comprises high dose rate brachytherapy dosing of 2 - 20 Gy/minute.

29. The method of claim 16, wherein the composition is co-administered with a

chemotherapeutic agent.

30. The method of claim 16, wherein the patient in need thereof is a patient with cancer or tumors of the skin, brain and other central nervous system sites, head, neck,

muscle/sarcoma, bone, lung, esophagus, stomach, pancreas, colon, rectum, uterus, cervix, vagina, vulva, penis, breast, kidney, prostate, bladder, or thyroid.

31. The method of claim 16, wherein the patient in need thereof is a patient with prostate cancer.

32. A method of increasing sensitivity to IR therapy or chemotherapy in a tumor cell, the method comprising:

contacting the tumor cell with a composition comprising an miRNA, miRNA mimetic, activator of miRNA, or mixture thereof,

thereby increasing sensitivity to IR therapy or chemotherapy.