Identification of Modulators of Autophagy

Methods for identifying compounds that inhibit or stimulate the autophagy pathway are described. Devices for detecting the expression of autophagy-related genes and kits for assaying the expression of autophagy-related genes are also described. Also described are methods for identifying individuals susceptible to or afflicted with a disease state associated with an autophagy pathway defect.

1. A method for identifying compounds that inhibit or stimulate the autophagy pathway for treatment of a disease state associated with an autophagy pathway defect, comprising measuring the effect of one or more test compounds on the inhibition or stimulation of a product of one or more of the genes or gene fragments identified in Table 3 or Table 4.

2. A method for identifying individuals susceptible to or afflicted with a disease state associated with an autophagy pathway defect, comprising testing a biological sample from an individual for a characteristic of one or more polypeptides produced by expression of one or more of the genes or gene fragments identified in Table 3 or Table 4 that is indicative of said disease state, wherein said characteristic is selected from the presence of at least one of said polypeptides, the absence of at least one of said polypeptides, an elevated level of at least one of said polypeptides, a reduced level of at least one of said polypeptides and, for two or more of said polypeptides, combinations thereof.

3. A device for detecting the expression of a plurality of autophagy-related genes associated with an autophagy pathway defect, said device comprising a substrate to which is affixed at known locations a plurality of probes, wherein the probes comprise:

a) a plurality of oligonucleotides or polynucleotides, each of which specifically binds to a different sequence selected from any of the sequences identified in Table 3 or Table 4 or fragments thereof; or

b) a plurality of polypeptide binding agents, each of which specifically binds to a different polypeptide or fragment thereof produced by expression of a gene or gene fragment comprising any of the sequences identified in Table 3 or Table 4 or fragments thereof.

4. A kit for assaying the expression of autophagy-related genes associated with an autophagy pathway defect, comprising at least one container and a collection of two or more probes, wherein the probes comprise:

a) oligonucleotides or polynucleotides that specifically bind to two or more genes or gene fragments comprising any of the sequences identified in Table 3 or Table 4, or fragments thereof; or

b) polypeptide binding agents that specifically bind to polypeptides produced by expression of two or more genes or gene fragments comprising any of the sequences identified in Table 3 or Table 4, or fragments thereof.

The present application is a Continuation of U.S. patent application Ser. No. 13/422,033, filed Mar. 16, 2012, which is a Continuation of U.S. patent application Ser. No. 13/284,923, filed Oct. 30, 2011, which is a Continuation of U.S. patent application Ser. No. 13/046,033, filed Mar. 11, 2011, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 61/313,097, filed Mar. 11, 2010. U.S. patent application Ser. No. 13/046,033 is also a Continuation-in-Part of U.S. patent application Ser. No. 12/622,410, filed Nov. 19, 2009, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 61/116,085, filed Nov. 19, 2008. The disclosures of each of the foregoing applications are hereby incorporated herein by reference in their entireties.

The present application was supported in part by the National Institutes of Health under Grant Nos. R37 CA53370 and RO1 CA130893 and the Department of Defense under DOD W81XWH06-1-0514 and DOD W81XWH05. The U.S. government has certain rights in the invention.

Autophagy is a catabolic, cellular self-digestion process that is activated by starvation and stress whereby double membrane vesicles called autophagosomes form that engulf proteins and organelles. Autophagosomes then fuse with lysosomes where their cargo is degraded. The function of autophagy is to recycle intracellular nutrients to sustain metabolism during nutrient and growth factor deprivation, and to clear damaged proteins and organelles that accumulate during stress. Although elimination of individual proteins occurs by the ubiquitin-mediated proteasome degradation pathway, only the autophagy pathway can eliminate protein aggregates and organelles. Thus, autophagy complements and overlaps with proteasome function to prevent the accumulation of damaged cellular components during starvation and stress. Through these functions, autophagy is an important cellular stress response that functions to maintain protein and organelle quality control, protect the genome from damage, and sustain cell and mammalian viability.

Autophagy is controlled by ATG proteins that were initially identified in yeast for which there are mammalian homologues. ATG proteins are comprised of kinases, proteases, and two ubiquitin-like conjugation systems that likely function in concert with a host of unknown cellular proteins to control autophagosome formation, cargo recognition, engulfment, and trafficking to lysosomes. The ATG6/Beclin1-VPS34-ATG8/LC3 complex regulates autophagosome formation and LC3 cleavage, lipidation, and membrane translocation are frequently utilized to monitor autophagy induction and inhibition of flux through the autophagy pathway.

Targeting of cargo, including proteins and organelles, to autophagosomes for degradation is accomplished by tagging proteins with polyubiquitin. The ubiquitin-binding domain (UBA) on the adaptor protein p62 recognizes and binds these polyubiquitinated proteins. p62 oligomerizes by self-association of its PB1 domain and binds ATG8/LC3 on autophagosome membranes. p62 thereby identifies, collects and delivers cargo to autophagosomes for degradation. p62 itself is an autophagy substrate and is degraded by autophagy along with the cargo. As such, p62 accumulation in aggregates is indicative of autophagy inhibition and clearance of p62 following stress is indicative of functional autophagy. These properties of p62 have been demonstrated in vivo in autophagy-defective mutant mice and are mimicked by expression of EGFP-p62 in cell lines in vitro and in vivo (Mathew, R et al., (2009) Cell 137, 1062-1075).

The activation of autophagy by starvation and stress is controlled in part through the PI-3 kinase pathway via the protein kinase mTOR. Growth factor and nutrient availability promote mTOR activation that suppresses autophagy, whereas starvation and mTOR inactivation stimulate autophagy. While there are other mechanisms to regulate autophagy, and those that activate autophagy in response to stress are particularly poorly understood, mTOR provides a link between nutrient and growth factor availability, growth control, autophagy, and metabolism.

Autophagy dysfunction is believed to be a major contributor to human diseases including neurodegeneration, liver disease, and cancer. Many human neurodegenerative diseases are associated with aberrant protein accumulation and excessive neuronal cell death, and neurons of mice with targeted autophagy defects accumulate polyubiquitinated- and p62-containing protein aggregates that result in neurodegeneration. The human liver disease steatohepatitis and a major subset of hepatocellular carcinomas (HCCs) are associated with the formation of p62-containing protein aggregates (Mallory bodies), and livers of mice with autophagy defects have p62-containing protein aggregates, excessive cell death, and HCC.

Evidence from model organism disease models indicates that promoting autophagy with mTOR inhibitors such as rapamycin or CCI-779, and enhancing the clearance of misfolded, damaged or mutated proteins and protein aggregates prevents neurodegeneration, but that there also are mTOR-independent means to increase autophagy. Similarly, genetically eliminating the expression of p62 in hepatocytes and preventing p62 accumulation in autophagy-defective atg7^−/− hepatocytes dramatically suppresses the phenotype of steatohepatitis. In contrast, neurodegeneration due to expression and accumulation of polyglutamate expansion mutant proteins is greatly exacerbated by allelic loss of beclin1 and defective autophagy. Thus, while not intending to be bound by any theory of operation, autophagy is believed to be involved in limiting the buildup of misfolded, mutated proteins in p62-containing protein aggregates, which leads to cellular deterioration and disease.

Analogous to a wound-healing response, chronic tumor cell death in response to stress and induction of inflammation and cytokine production may provide a non-cell-autonomous mechanism by which tumorigenesis is promoted in autophagy-defective cells. Autophagy-defective tumor cells also display an elevated DNA damage response, gene amplification and chromosome instability in response to stress, suggesting that autophagy limits genome damage as a cell-autonomous mechanism of tumor suppression.

Therefore, while not intending to be bound by any theory of operation, stimulating autophagy may be involved in limiting disease progression, particularly neurodegeneration, liver disease, and also cancer, by facilitating the elimination of protein aggregates, damaged organelles, and the toxic consequences of their accumulation.

Autophagy has been identified also as a survival pathway in epithelial tumor cells that enables long-term survival to metabolic stress. Tumor cells with defined defects in autophagy accumulate p62-containing protein aggregates, DNA damage and die in response to stress, whereas those with intact autophagy can survive for weeks utilizing the autophagy survival pathway. Thus, autophagy appears to be required to prevent tumor cell damage and to maintain metabolism. Tumor cells can exploit this survival function to remain dormant only to reemerge under more favorable conditions. Interestingly, roughly half of human cancers may have impaired autophagy, either due to constitutive activation of the PI-3 kinase pathway or allelic loss of the essential autophagy gene beclin1, rendering them particularly susceptible to metabolic stress and autophagy inhibition.

Therefore, identification of the therapeutic means to inhibit the autophagy survival pathway in tumor cells would be advantageous. While not intending to be bound by any theory of operation, this may be of value as many therapeutics currently in use, such as kinase and angiogenesis inhibitors, inflict metabolic stress, which increases the dependency on autophagy for survival. Furthermore, tumor cells with impaired autophagy are particularly vulnerable to metabolic stress and further therapeutic suppression of autophagy may be able to exploit this vulnerability by promoting cell death by metabolic catastrophe or the failure to mitigate cell damage accumulation. Preclinical studies have been conducted using hydroxychloroquine to inhibit lysosome acidification and thereby autophagy in combination therapy. Specific inhibitors of the autophagy survival pathway in tumor cells are may be of great value in combination with agents such as angiogenesis and kinase inhibitors that promote metabolic stress.

Thus, the autophagy pathway represents fertile ground for novel therapeutic target identification for drug discovery for many diseases for both acute treatment and also disease prevention.

Accordingly, a need exists to identify nucleic acid sequences and their encoded proteins which are involved in modulation of the autophagy pathway.

In certain aspects, the present invention relates to methods for identifying compounds that inhibit or stimulate the autophagy pathway.

Further aspects relate to methods for identifying individuals susceptible to or afflicted with a disease state associate with an autophagy pathway defect.

Additional aspects relate to devices for detecting the expression of autophagy-related genes.

Further aspects relate to kits for assaying expression of autophagy-related genes.

Other aspects are readily apparent from the following description.

The following definitions are provided to facilitate an understanding of the present invention:

A “polynucleotide,” “polynucleotide molecule” or “polynucleotide sequence” refers to a chain of nucleotides. It may refer to a DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In some instances, the sequences will be fully complementary (no mismatches) when aligned. In other instances, there may be up to about a 30% mismatch in the sequences.

The term “oligonucleotide,” as used herein refers to sequences, primers and probes, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.

The term “probe” as used herein refers to either a probe for a nucleic acid or a probe for a protein. When used in connection with nucleic acids, a “probe” refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single stranded or double stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and method of use. The probes herein are selected to be “substantially” complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to “specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non complementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically. When used in connection with a polypeptide, a “probe” is a protein- or polypeptide-binding substance or agent, capable of specifically binding a particular protein or protein fragment to the substantial exclusion of other proteins or protein fragments. Such binding agents may be any molecule to which the protein or peptide specifically binds, including DNA (for DNA binding proteins), antibodies (as described in greater detail herein), cell membrane receptors, peptides, cofactors, lectins, sugars, polysaccharides, cells, cell membranes, organelles and organellar membranes.

“Array” refers to an ordered arrangement of at least two probes on a substrate. At least one of the probes represents a control or standard, and the other, a probe of diagnostic or screening interest.

“Specific binding” refers to a special and precise interaction between two molecules which is dependent upon their structure, particularly their molecular side groups; for example, the intercalation of a regulatory protein into the major groove of a DNA molecule, the hydrogen bonding along the backbone between two single stranded nucleic acids, or the binding between an epitope of a protein and an agonist, antagonist, or antibody.

The term “specifically hybridize” refers to the association between two single stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under predetermined conditions generally used in the art (sometimes termed “substantially complementary”). For example, the term may refer to hybridization of a nucleic acid probe with a substantially complementary sequence contained within a single stranded DNA or RNA molecule according to an aspect of the invention, to the substantial exclusion of hybridization of the nucleic acid probe with single stranded nucleic acids of non-complementary sequence. When used in connection with the association between single stranded nucleic acid molecules, the term “specifically bind” may be used to indicate that the molecules “specifically hybridize” as described herein.

An “antibody” or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. The term includes polyclonal, monoclonal, chimeric, and bispecific antibodies. As used herein, antibody or antibody molecule contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule such as those portions known in the art as Fab, Fab′, F(ab′)2 and F(v).

“Sample” is used in its broadest sense as containing nucleic acids, proteins, antibodies, and the like. A sample may comprise, for example, a bodily fluid; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue or a tissue biopsy; a tissue print; a fingerprint, buccal cells, skin, or hair; and the like. Bodily fluids include, without limitation, whole blood, blood plasma, blood serum, sputum, urine, sweat, and lymph.

As used herein, the term “subject” or “patient” refers to both humans and animals, unless specified that the “subject” or “patient” is an animal or a human. An “individual” also refers to both humans and animals, unless specified that the “individual” is an animal or a human. Animal subjects are preferably vertebrates, and more preferably, mammals.

“Autophagy-associated” or “autophagy-related” as used herein with respect to a disease, condition or disorder refers to that which results from an increase or decrease in normal autophagy function and/or that which may be treated and/or prevented by modulation of the autophagy pathway. As used herein with respect to a biological molecule, such as, for example, a polynucleotide or polypeptide, “autophagy-associated” or “autophagy-related” refers to a molecule for which alteration of the expression, abundance and/or activity thereof leads to modulation of the autophagy pathway.

In certain embodiments, the present invention relates to the identification of genes whose expression modulates autophagy. These genes and their gene products may represent targets for therapeutic intervention in the autophagy pathway.

In accordance with aspects of the present invention, a number of polynucleotides comprising at least a fragment of a gene have been identified as representing molecules whose knockdown of expression modulates the function of the autophagy pathway. In certain aspects, knockdown of gene expression stimulates autophagy. In other aspects, knockdown of gene expression inhibits autophagy.

Embodiments of the invention include validation of the candidate genes and gene fragments described herein using known techniques for in vitro and in vivo analysis.

In accordance with various aspects of the present invention, combinations, compositions, devices and kits are provided that may be used in the practice of methods provided according to certain embodiments of the invention.

Certain embodiments relate to methods of detection of alterations in the autophagy pathway. Certain of these methods may be used to detect conditions in which autophagy is reduced. Certain of these methods may be used to detect conditions in which autophagy is increased.

In accordance with certain aspects of the invention, a combination is provided comprising a plurality of polynucleotide molecules wherein the polynucleotide molecules encode gene products associated with modulation of the autophagy pathway. In certain embodiments, the combination comprises a plurality of polynucleotides whose knockdown stimulates autophagy. In certain embodiments, the plurality of polynucleotide molecules comprise two or more molecules identified in Table 3 or fragments thereof.

In certain embodiments, the combination comprises a plurality of polynucleotides whose knockdown inhibits autophagy. In certain embodiments, the plurality of polynucleotide molecules comprise two or more molecules identified in Table 4 or fragments thereof.

An embodiment of the invention provides a method for identifying compounds that modulate autophagy-associated gene expression comprising: a) measuring standard expression by measuring transcription or translation products of one or more of the genes or gene fragments identified in Table 3 and/or 4, or fragments thereof, in a standard sample in the absence of a test compound; b) measuring test expression by measuring the transcription or translation products of one or more of the genes or gene fragments identified in Table 3 and/or 4, or fragments thereof, in a test sample in the presence of the test compound; and c) comparing the standard expression to the test expression, wherein a change in the test expression compared to the standard expression is indicative of an effect of the test compound on the expression of genes whose expression modulates the autophagy pathway. In certain embodiments, a plurality of two or more of the genes or gene fragments identified in Table 3 and/or 4, or fragments thereof are used.

One embodiment of the invention provides a method for identifying compounds that inhibit or stimulate the autophagy pathway for treatment of a disease state associated with an autophagy pathway defect, comprising measuring the effect of one or more test compounds on the inhibition or stimulation of a product of one or more of the genes or gene fragments identified in Table 3 or Table 4.

An embodiment provides a method for the detection of differential expression of autophagy-associated polypeptides in a sample, comprising the steps of: a) reacting protein binding molecules with polypeptides of the sample, thereby allowing specific binding to occur, wherein the polypeptides bound by the protein-binding molecules comprise one or more polypeptides encoded by the genes or gene fragments identified in Table 3 and/or Table 4 or fragments thereof; b) detecting specific binding; and c) comparing the specific binding in the sample with that of a standard, wherein differences between the standard and sample specific binding indicate differential expression of polypeptides in the sample. In certain embodiments, the protein-binding molecules are directed to polypeptides comprising a plurality of two or more polypeptides encoded by the genes or gene fragments identified in Table 3 and/or Table 4 or fragments thereof.

Another embodiment provides a method for the detection of differential expression of autophagy-associated nucleic acids in a sample, comprising the steps of: a) hybridizing polynucleotides comprising one or more molecules identified in Table 3 and/or Table 4 or fragments thereof with nucleic acids of the sample, thereby forming one or more hybridization complexes; b) detecting the hybridization complexes; and c) comparing the hybridization complexes with those of a standard, wherein differences between the standard and sample hybridization complexes indicate differential expression of nucleic acids in the sample. In certain embodiments, the polynucleotides comprise a plurality of two or more molecules identified in Table 3 and/or Table 4 or fragments thereof.

Another embodiment comprises a composition of matter comprising one or more probes for detecting expression of autophagy-associated genes, wherein the probes comprise one or more of: a) nucleic acid molecules that specifically hybridize to one or more of the genes or gene fragments identified in Table 3 and/or Table 4, or fragments thereof; or b) polypeptide binding agents that specifically bind to polypeptides produced by expression of one or more nucleic acid molecules comprising sequences selected from one or more of genes or gene fragments identified in Table 3 and/or Table 4, or fragments thereof. In certain embodiments, the composition of matter comprises a collection of two or more probes.

Another embodiment provides a device for detecting expression of a plurality of autophagy-related genes, comprising a substrate to which is affixed, at known locations, a plurality of probes, wherein the probes comprise: a) a plurality of oligonucleotides or polynucleotides, each of which specifically hybridizes to a different sequence selected from any of the sequences identified in Table 3 and/or Table 4 or fragments thereof; or b) a plurality of polypeptide binding agents, each of which specifically binds to a different polypeptide or fragment thereof produced by expression of a nucleic acid molecule comprising a sequence selected from the genes or gene fragments comprising any of the sequences identified in Table 3 and/or Table 4 or fragments thereof.

In certain embodiments, a device is provided for detecting the expression of a plurality of autophagy-related genes associated with an autophagy pathway defect, said device comprising a substrate to which is affixed at known locations a plurality of probes, wherein the probes comprise:

• • a) a plurality of oligonucleotides or polynucleotides, each of which specifically binds to a different sequence selected from any of the sequences identified in Table 3 or Table 4 or fragments thereof; or
  • b) a plurality of polypeptide binding agents, each of which specifically binds to a different polypeptide or fragment thereof produced by expression of a gene or gene fragment comprising any of the sequences identified in Table 3 or Table 4 or fragments thereof.

Another embodiment provides a method for measuring the effect of a test compound on expression of an autophagy-associated gene, wherein the gene is selected from the group consisting of the genes or gene fragments identified in Table 3 and/or 4, the method comprising measuring production of transcription or translation products produced by expression of the gene or gene fragment in the presence or absence of the test compound, wherein a change in the production of transcription or translation products in the presence of the test compound is indicative of an effect of the test compound on expression of the gene or gene fragment.

In an embodiment, the gene expression is measured by providing a DNA construct comprising a reporter gene coding sequence operably linked to transcription regulatory sequences of the autophagy-associated gene, and measuring formation of a reporter gene product in the presence or absence of the test compound.

Another embodiment provides a kit for assaying the expression of autophagy-related genes, comprising at least one container comprising a collection of two or more probes, wherein the probes comprise: a) oligonucleotides or polynucleotides that specifically hybridize to two or more genes or gene fragments comprising any of the sequences identified in Table 3 and/or Table 4, or fragments thereof; or b) polypeptide binding agents that specifically bind to polypeptides produced by expression of two or more genes or gene fragments comprising any of the sequences identified in Table 3 and/or Table 4, or fragments thereof. The kit preferably comprises instructions for performing an assay of gene expression.

In certain embodiments, the invention provides a kit for assaying the expression of autophagy-related genes associated with an autophagy pathway defect, comprising at least one container and a collection of two or more probes, wherein the probes comprise:

• • a) oligonucleotides or polynucleotides that specifically bind to two or more genes or gene fragments comprising any of the sequences identified in Table 3 or Table 4, or fragments thereof; or
  • b) polypeptide binding agents that specifically bind to polypeptides produced by expression of two or more genes or gene fragments comprising any of the sequences identified in Table 3 or Table 4, or fragments thereof. The kit preferably comprises instructions for performing an assay of gene expression.

The invention provides, in certain embodiments, methods for identifying compounds that are useful in modulating the autophagy pathway. Preferably, the methods include contacting at least one polypeptide encoded by the genes and/or gene fragments identified in Table 3 and/or Table 4 with a test substance and determining whether the test substance binds to the polypeptide. Further, in certain embodiments of the invention, a test substance may be determined to stimulate or inhibit the biological activity of the relevant gene product comprising at least one polypeptide encoded by the genes and/or gene fragments identified in Table 3 and/or Table 4 and thereby be identified as a compound useful for the modulation of the autophagy pathway. Such assays may, in certain embodiments, be performed in vitro and may, in certain embodiments, be performed in a cell-based assay. In some embodiments, substances identified as modulating expression or biological activity in vitro may be further tested in vivo to confirm relevant and effective activity.

Test substances or compounds contemplated by aspects of the invention include compounds from chemical libraries, including natural products and/or synthetic products from combinatorial chemical synthesis. Such substances may include, without limitation, polypeptides, oligonucleotides, polynucleotides, or organic molecules.

In a further embodiment is provided a method of modulating autophagy-associated gene expression in a cell by administering an effective amount of a composition under appropriate conditions to affect the expression of at least one gene associated with autophagy having a sequence selected from the sequences identified in Table 3 and/or Table 4, or fragments thereof.

In preferred embodiments, the composition comprises an inhibitor of gene expression. The inhibitor of gene expression may be selected from molecules including, but not limited to, an antisense RNA, a morpholino polynucleotide, and an interfering RNA (RNAi).

According to a still further aspect of the invention, there is provided a genetically-modified non-human animal that has been transformed to express higher, lower or absent levels of a protein according to any one of the aspects of the invention described herein. Preferably, said genetically-modified animal is a transgenic or knockout animal. Preferably, the genetically-modified animal is a rodent, most preferably a mouse.

An embodiment of the invention also provides a method for screening for a substance effective to treat an autophagy-associated disease condition, by contacting a non-human genetically-modified animal as described above with a candidate substance and determining the effect of the substance on the physiological state of the animal.

Certain embodiments of the invention provide methods and kits for diagnosis of, determining susceptibility to and/or developing a prognosis for an autophagy-associated disease state in a subject. In certain aspects, these may involve tests on subject samples. In certain embodiments, these may be nucleic acid based tests or polypeptide-based tests. In some embodiments, the method or kit may include probes that bind to at least one polynucleotide encoding an autophagy-associated polypeptode. In some embodiments, the a plurality of two or more probes may be used. In some embodiments, the method or kit may include polypeptide binding agents that bind to at least one autophagy-associated polypeptide. In some embodiments, a plurality of two or more polypeptide binding agents may be used. In certain embodiments, the polypeptide-binding agent comprises antibodies and/or antigen-binding portions of an antibody that specifically binds to one or more autophagy-associated polypeptides. Preferably, the autophagy-associated polypeptides are encoded by the gene or gene fragments identified in Table 3 and/or Table 4.

One embodiment provides a method for identifying individuals susceptible to or afflicted with a disease state associated with an autophagy pathway defect, comprising testing a biological sample from an individual for a characteristic of one or more polypeptides produced by expression of one or more of the genes or gene fragments identified in Table 3 or Table 4 that is indicative of said disease state, wherein said characteristic is selected from the presence of at least one of said polypeptides, the absence of at least one of said polypeptides, an elevated level of at least one of said polypeptides, a reduced level of at least one of said polypeptides and, for two or more of said polypeptides, combinations thereof.

An embodiment provides a method to diagnose or develop a prognosis for an autophagy-related disease in a subject, the method comprising: a) obtaining a sample from the subject; b) measuring in the sample the production of transcription or translation products produced by the expression of one or more autophagy-associated genes or gene fragments comprising any of the sequences identified in Table 3 and/or Table 4, or fragments thereof; c) comparing the transcription or translation products of the sample with that of a standard, wherein a difference in the expression of any of the autophagy-associated genes or gene fragments is indicative of autophagy-related disease.

In one embodiment is provided a kit for the diagnosis of an autophagy-associated disease in a subject comprising polynucleotide probes that specifically bind to one or more autophagy-associated polynucleotides or a fragment thereof. Preferably the autophagy-associated polynucleotides or fragments thereof are selected from the polynucleotide sequences identified in Table 3 and/or Table 4 or fragments thereof. In certain embodiments, the kit comprises a plurality of two or more polynucleotide probes that specifically bind to polynucleotide sequences identified in Table 3 and/or Table 4 or fragments thereof. Preferably, the kit comprises also instructions for use.

The invention also provides kits for diagnosis of autophagy-associated conditions from patient samples that may be nucleic acid based tests or polypeptide-based tests. In some embodiments, the kit contains at least one polynucleotide that binds to a polynucleotide encoding an autophagy-related gene product. In some embodiments, the kit contains, preferably in separate containers, a plurality of probes to detect two or more polynucleotides encoding one or more autophagy-associated gene products. In preferred embodiments, the gene products are encoded by one or more of the genes or gene fragments identified in Table 3 and/or Table 4. In other embodiments, the kit contains at least one polypeptide binding agent that specifically binds to at least one autophagy-associated polypeptide. In some embodiments, the kit contains, preferably in separate containers, a plurality of polypeptide binding agents (or mixtures thereof) to detect one or more autophagy-associated polypeptide. In certain embodiments, the polypeptide binding agent may be an antibody or antigen-binding portion of an antibody. In certain embodiments, the autophagy-associated polypeptides include at least one polypeptide encoded by the genes or gene fragments identified in Table 3 and/or Table 4. In certain embodiments, the autophagy-associated polypeptides identified by the kit include a plurality of two or more polypeptides encoded by the genes or gene fragments identified in Table 3 and/or Table 4. In certain embodiments, the kits may also include instructions for use.

In certain embodiments, methods according to the invention may be used for high-throughput screening assays.

In certain embodiments, methods and kits useful in the methods of the invention may utilize nucleic acid, antibody and/or polypeptide arrays.

Using a cell-based loss-of function screen, the present inventors have identified candidate genes whose expression is involved in the autophagy pathway. In particular, the screen has been used to identify genes whose knockdown stimulates autophagy. Results from this screen are shown in Table 1. The screen has also been used to identify genes whose knockdown inhibits autophagy. Results from this screen are shown in Table 2.

A high-efficiency delivery method that enables stable long-term gene suppression in a broad range of cell types is virus-mediated integration of an RNAi expression cassette. After integration, the cassette produces a short dsRNA molecule, usually in the form of a hairpin structure, a short or small hairpin RNA (shRNA), which is processed into active small interfering RNA (siRNA). Although many types of viruses are suitable for this purpose, lentiviral vectors generate viruses of both high titer and broad tropism, permitting the infection of both dividing and nondividing cells. Lentiviral shRNA libraries for mouse gene clones were utilized that allow gene silencing in most dividing and nondividing cell types.

An image based, arrayed shRNA screen was employed. Lentiviral shRNA libraries developed by the RNA Consortium (TRC) at the Broad Institute were used in a cell-based screen. The screens utilized the publicly available kinase and vesicle trafficking lentiviral library subsets at the Broad Institute, as well as a custom library containing shRNAs targeting mouse GTPases. Lentiviruses are high-titer, individual clones with representation of at least five independent hairpins for each target gene supplied in a high-throughput format (Root, D. E., et al. (2006), Nature Methods 3, 715-719.) Fluorescence image analysis was used to capture the data. Gene were identified that were shown to promote or suppress autophagy (bimodal analysis).

The high content arrayed shRNA screen used to identify autophagy modulators utilized autophagy defective beclin1^+/− iBMK cells stably expressing the autophagy substrate EGFP-p62 (Mathew, R., Karantza-Wadsworth, V., and White, E. (2009) Methods Enzymol 453, 53-81; Mathew, R., et al. (2009) Cell 137, 1062-1075; and Mathew, R., et al. (2007) Genes Dev 21, 1367-1381). p62 accumulates and aggregates in response to metabolic stress and requires autophagy for degradation. p62 also accumulates in degenerative neuronal and liver diseases and in autophagy-defective mouse tissues, beclin1^+/− and atg5^−/− iBMK cells, and tumors. Genes were identified whose inactivation compensates for defective autophagy and restores p62 protein turnover. Since the image analysis captured every hairpin's p62 aggregation score (EGFP-p62 intensity divided by the nuclei in the field) it was also possible to identify genes whose inactivation lead to further accumulation of p62 aggregates, predicted to be autophagy inhibitors (FIG. 2). This was possible because the cell line employed in this screen is autophagy impaired rather than fully autophagy defective. Therefore, the disposition of p62 in the test cells serves as readout for both autophagy promotion (p62 degradation, low p62) and inhibition (autophagy inhibition, high p62) and is the basis for the identification of autophagy modulators in cell-based screens.

The shRNA libraries were screened using autophagy-impaired test cells expressing a marker of protein aggregation, subjecting the test cell to metabolic stress, and performing analysis on the test cell to determine the level of the marker. The marker of protein aggregation is a p62 protein linked to enhanced green fluorescent protein (EGFP) label. Image analysis is performed to determine the level of p62 aggregates in cells. The level of the marker found in p62 aggregates in the test cell is compared with that of a control cell. A lower level of p62 aggregates comprising the marker in the test cell compared to that demonstrated by the control cell demonstrates the rescue of the impairment in p62 clearance, indicating the lowered expression of a gene whose knockdown stimulates autophagy. A greater level of p62 aggregates in a test cell compared to that of a control cell demonstrates suppression of p62 clearance, indicating the knockdown of a gene whose lowered level of expression leads to inhibition of autophagy.

The cell-based screen utilized autophagy-deficient beclin1^+/− immortalized baby mouse kidney (iBMK) cells stably expressing EGFP-p62. p62 accumulates and aggregates in response to metabolic stress and requires autophagy for degradation. FIG. 1 illustrates a cell-based shRNA screen for rescue of autophagy deficiency and p62 protein aggregate accumulation in metabolic stress and therefore represents a screen for autophagy stimulators. The autophagy-deficient beclin1^+/− iBMK cell line stably expressing EGFP-p62 accumulates p62-containing protein aggregates under stress, which fail to be cleared following recovery. Those shRNAs that facilitate p62 aggregate clearance, compensating for defective autophagy, are identified. The autophagy wild type beclin1^+/+ iBMK cell line stably expressing EGFP-p62 that effectively clears p62 aggregates following stress is used as a positive control.

FIG. 2 illustrates representative images of cells contacted with shRNAs that modulate p62 aggregate elimination. The screen is designed to identify genes whose loss results in restoration of autophagy (autophagy stimulators), manifested by successful clearance of p62 aggregates following a time course of stress and recovery. mTOR, a master negative regulator of autophagy, is shown here as an example of a gene whose loss restores autophagy and clearance of p62. Alternatively, loss of some genes is predicted to further inhibit autophagy (autophagy inhibitors). Loss of Ikbkb, a known autophagy promoter (Criollo, A, et al. EMBO J 29, 619-631), results in marked accumulation of p62. This accumulation is greater than observed in cells infected with an shRNA targeting luciferase.

The shRNAs shown to promote p62 elimination (autophagy stimulators) identify potential targets for drug discovery efforts for development of modulators of autophagy, including autophagy inhibitors. While not intending to be bound by any theory of operation, autophagy inhibitors are potentially useful as anti-cancer therapeutics by promoting cancer cell death.

The shRNAs shown to enhance p62 accumulation (autophagy inhibitors) identify potential targets for drug discovery efforts for development of modulators of autophagy, including autophagy stimulators. While not intending to be bound by any theory of operation, autophagy stimulators are potentially useful in preventing or delaying disease manifestation in the setting of cancer, neurodegenerative conditions, Crohn's disease, liver disease, aging and inflammatory diseases and in combating infections.

The following examples serve to more fully describe the manner of using the above-described invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes.

Murine kidney epithelial cells were isolated from beclin1^+/− mice and immortalized with dominant negative p53 and EIA as described previously (Degenhardt, K., and White, E. (2006). Clin Cancer Res 12, 5298-5304; Mathew, R., Degenhardt, K., Haramaty, L., Karp, C. M., and White, E. (2008) Methods Enzymol 446, 77-106.). The cells were subsequently engineered to overexpress Bcl-2 and eGFP-p62. Thus, these cells, known as 3BC2 EGFP-P62, contain an autophagy defect, are apoptotically impaired, and stably express EGFP-P62. (Mathew, R., et al. (2009). Cell 137, 1062-1075.).

Screening Protocol for Beclin^+/− eGFP-P62

Cells were plated into black barcoded 384 well plates (Corning 8793BC) at a density of 700 cells/well by the Biotek microfill and allowed to attach overnight. Infection and media changes for plates were achieved by use of two robotic liquid handlers at the Broad Institute, the Perkin Elmer Janus and EP3. Each viral plate was used to infect four target plates. Each virus plate contained 20 control hairpins (targeting either RFP, luciferase, or EGFP) in addition to wells containing no virus. Two hairpins targeting p62 were spiked into each plate at the time of infection to ensure that positive and negative controls were present on all plates. Immediately prior to infection, media was changed with the Janus robot (Perkin Elmer) to DMEM containing 8 ug/mL polybrene. The Perkin Elmer EP3 robot was used to add 6 ul of virus to each well. Cells were spin infected (2250 rpm 30 mins, 30° C.) in the presence of 6 ul of virus and Bug/ml of polybrene before returning to the 37 C incubator. Virus and polybrene containing media was removed 4 hours post infection and cells were incubated in normal growth media overnight (DMEM high glucose, 10% FBS, 1% penicillin streptomycin (PS)). Twenty four hours post infection, media was changed with the Janus to DMEM containing 3 ug/mL puromycin (for the three puro plus plates) or DMEM alone (for the puro minus replicate). Selection was allowed to continue for 72 hrs.

The assay employed in this screen is predicated on the ability of autophagy competent cells to successfully eliminate p62 aggregates that accumulate during metabolic stress during a recovery phase during which time oxygen and glucose are restored. Optimization experiments were conducted comparing the ability of autophagy competent beclin1^+/+-EGFP-p62 cells (WB3-EGFP-p62) and autophagy deficient beclin1^+/−-EGFP-p62 cells (3Bc2-EGFP-p62) to eliminate p62 aggregates during various time courses of metabolic stress (1% oxygen, glucose deprivation) and recovery within the setting of 384 well plates post infection and selection with puromycin. 7.5 hours of metabolic stress followed by 18 hours of recovery in high glucose DMEM 10% FBS was optimal, and these conditions were chosen for the large-scale screen.

Following puromycin selection, media containing DMEM high glucose was removed, and cells were washed twice in ischemia media (DMEM containing no glucose, 10% FBS, 1% PS) to remove residual glucose in wells prior to transfer into a hypoxia incubator set to 1% oxygen for 7.5 hours. They were then transferred to an incubator which could lower ambient oxygen levels to 1% by virtue of its attachment to a nitrogen tank. Cells stayed in this 1% oxygen, no glucose conditions, referred to as metabolic stress, for 7.5 h.

At the conclusion of the metabolic stress, normal growth media (DMEM high glucose 10% FBS, 1% PS) was added to the plates and the cells were allowed to recover overnight at 37° C. 18 hours post recovery, media was removed from plates, and cells were fixed by addition of 4% paraformaldehyde/PBS for 10 mins at RT.

Nuclei were visualized by inclusion of Hoechst 33342 at a dilution of 1:10,000 in the fixation solution. Plates were washed 3× with the ELx405 automated plate washer (Biotek). 80 ul of filtered PBS was left in each well at the end of washing to allow for evaporation during imaging.

Plates were imaged on the Arrayscan VTI (Thermo Scientific) housed within the Genome technology Core of the Whitehead Institute using a modified version of the Cellomics compartmental analysis bioapplication. Nuclei were visualized in channel 1. EGFP-p62 aggregates were visualized in channel 2. Nine images per channel were captured for each field, with an autofocus field interval of 3. MEAN_valid object count channel 1 represents the mean nuclear count within the field. MEAN_ring spot average integrated intensity channel 2 represents the mean intensity of the p62 aggregates in the field. To properly identify the p62 aggregates the following settings were employed: Spot kernel radius: 10, ring distance from nucleus: 0, ring width: 10 pixels. Data was exported to Excel for further analysis. Data quality (batch-to-batch variation, similarity of replicates) was examined with Spotfire decision software and RNAeyes, in house software developed by The RNAi Consortium (TRC) of the Broad Institute.

A p62 aggregate score equal to Mean Ring Spot total intensity/Mean nuclei was calculated for each well. Viral infections were done in quadruplicate, with three plates receiving puromycin, one not. A comparison of the nuclei counts from the puro+/puro− plates allowed calculation of the infection efficiency of each hairpin. Hairpins with less than 1500 nuclei per well or those that had an infection efficiency less than 25% were omitted from subsequent analysis. A robust Z-score, a standard metric for high throughput assays (Birmingham A., et al. (2009) Nat Methods 6, 569-575), was calculated for each well. The three puromycin selected replicates were averaged, and this value was used for further analysis.

The in-house Gene-E software ranked genes at both a hairpin and a gene level. Attached to this application are candidate results (‘hits’) from either end of our analysis: those that resulted in profound elimination of p62, predicted to be autophagy inducers (Table 1), and those that resulted in profound accumulation of p62, predicted to be autophagy inhibitors (Table 2). These tables represent the weighted sum ranking of the data. In this metric, 75% of the score is based on the robust z-score of the second best hairpin for a given gene, while the other 25% of the score is based on the rank of the robust z-score of the best hairpin. Similar data was obtained when three other analysis measures were employed: cut-off based on a given standard deviation from controls, second best ranking, or RNA Interference Gene Enrichment Rank (RIGER) analysis based on the KS statistic as described previously (Luo B., et al. (2008) Proc Natl Acad Sci USA 105, 20380-20385).

A subset of viral plates were re-screened to ensure reproducibility of the assay and analyses.

Table 1 shows results of a screen that led to elimination of p62.

Table 2 shows the results of a screen that led to accumulation of p62. The results are presented in a positive to negative ranking according to p62.