WO2016134246A2 - Compositions and methods for treating protein conformational diseases - Google Patents

Abstract

The present invention relates to the field of protein conformational diseases. More specifically, the present invention provides compositions and methods for treating protein conformational diseases including amyotrophic lateral sclerosis (ALS). In one embodiment, a method comprises the step of administering to a patient an effective amount of a Ube4B inhibitor and a LSD1 inhibitor. In another embodiment, the method further comprises the step of administering a p53 agonist.

Description

COMPOSITIONS AND METHODS FOR TREATING PROTEIN

CONFORMATIONAL DISEASES

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62/118,503, filed February 20, 2015, which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant no. 5R00NS062089 and grant no. NS074324, awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of protein conformational diseases. More specifically, the present invention provides compositions and methods for treating protein conformational diseases including amyotrophic lateral sclerosis (ALS).

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

This application contains a sequence listing. It has been submitted electronically via EFS-Web as an ASCII text file entitled "P11672-02_ST25.txt." The sequence listing is 9,470 bytes in size, and was created on February 19, 2016. It is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Living organisms endure environmental stress and metabolic errors that inflict damage to macromolecules such as DNA and protein, which are either repaired or removed by quality control programs in the cell. Toxicities from protein misfolding and aggregation, or proteotoxicity, are thought to underlie many neurodegenerative diseases including Creutzfeldt- Jakob disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, and amyotrophic lateral sclerosis (ALS) (Balch et al, 2008;

Prusiner, 2012). The cell coordinates several major quality control systems to guard against proteotoxicity including molecular chaperones, ubiquitin-proteasome system (UPS) and autophagy (Bukau and Horwich, 1998; Ciechanover and Brundin, 2003; Hartl et al, 2011; Mizushima et al, 2008). The regulation of protein quality control occurs at different scales from individual proteins to whole organisms (Wolff et al, 2014). The protein quality control systems might be harnessed to defend against proteotoxicity associated with neurodegenerative diseases. However, how the cell might reprogram its protein quality control systems is not fully understood.

Mutant Cu/Zn superoxide dismutase (SOD1), linked to -20% of familial ALS, represents a simple molecular model for protein misfolding and aggregation. The wild-type (WT) SOD1 protein has a stable β-barrel structure with a two-state folding process (Parge et al., 1992), whereas mutant SOD1 proteins gain heightened propensity to aggregate in vitro and in vivo (Bruijn et al, 1997; Lindberg et al, 2005; Wang et al, 2003a). There is increasing evidence that misfolding and aggregation are a common feature of

neurodegeneration-associated proteins including ALS-linked TDP-43 and FUS (Kwiatkowski et al, 2009; Vance et al., 2009). Identifying mechanisms that suppress the toxicity of protein misfolding and aggregation may help understand the pathogenesis of neurodegenerative diseases and also provide potential targets for corrections.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery that Lysine-Specific Demethylase 1 (LSD1) and Ubiquitination Factor E4B (Ube4B), can be targeted to suppress proteotoxicity and treat protein conformational diseases.

Protein conformational diseases refer to all the pathological conditions that are associated with protein misfolding, including major forms of neurodegenerative diseases. Neurodegenerative diseases in humans present formidable medical and economic challenge. With a doubling of the average human lifespan over the last century, neurodegenerative diseases have become a major aging-related public health challenge in the US and many other countries. Unfortunately, no curative treatments exist for these debilitating conditions.

Neurodegenerative processes are often accompanied by pathological protein aggregation. There is a strong correlation between the accumulation of misfolded and aggregated proteins and the onset of number of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The present inventors' work and that of others have contributed to the recognition that the misfolding and aggregation of Cu/Zn superoxide dismutase (SOD1), and other proteins, play an important role in the pathogenesis of ALS. A recent study by Brown and colleagues showed that misfolded SOD1 is a common pathology in a significant fraction of sporadic ALS patients. This enormous medical problem calls for innovative approaches to alleviate the burden of accumulated toxic proteins in nervous system. To solve this problem, the present inventors identified novel molecular targets of neurodegeneration, the Lysine- Specific Demethylase 1 (LSD1) and Ubiquitination Factor E4B (Ube4B). As described herein, the present inventors showed in a mammalian cell-culture model system that reduction of levels of these molecular targets lead to reduction in protein aggregation, improved proteasome function and activation of pathways beneficial to cells under stress. Mouse models of neurodegeneration are being used to test the efficacy of RNAi and drug inhibitors in reducing Ube4b/LSD1 function and reducing neurotoxic burden of aggregated proteins. The outcome of such efficacy studies in is instructive towards collaborative human clinical studies.

Accordingly, in one aspect, the present invention provides compositions and methods for treating a protein conformational disease. In one embodiment, a method comprises the step of administering to a patient an effective amount of a Ube4B inhibitor and a LSD1 inhibitor. In another embodiment, the method further comprises the step of administering a p53 agonist. The present invention also provides methods for treating a protein

conformational disease comprising the step of administering to a patient an effective amount of a p53 agonist, a Ube4B inhibitor and a LSD1 inhibitor. In yet another embodiment, a method for treating a protein conformational disease comprises the step of administering to a patient an effective amount of a p53 agonist. In specific embodiments, the method further comprises administering an effective amount of a Ube4B inhibitor and/or a LSD1 inhibitor.

In certain embodiments, the protein conformational disease comprises a

neurodegenerative disease. In specific embodiments, the neurodegenerative disease is Creutzfeldt- Jakob disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, or amyotrophic lateral sclerosis (ALS).

In particular embodiments, the inhibitor is a small molecule, an antibody or an inhibitory nucleic acid molecule. Specifically, the inhibitory nucleic acid molecule can be an siRNA, shRNA, antisense RNA or a ribozyme. In particular embodiments, the inhibitory nucleic acid molecule is an siRNA. Examples of LSD 1 and Ube4B siRNA molecules are shown in SEQ ID NOS: 14-19 and SEQ ID NOS:20-49, respectively. In other embodiments, the inhibitory nucleic acid molecules is an shRNA. Examples of LSD1 and Ube4B shRNA molecules are shown in SEQ ID NOS:4-8 and SEQ ID NOS:9-13, respectively.

Thus, in certain embodiments, the present invention provides pharmaceutical compositions comprising the siRNA and/or shRNA described here. In certain embodiments, a pharmaceutical composition comprises one or more siRNA encoded by SEQ ID NOS: 15-19 and a pharmaceutical carrier. In other embodiments, a pharmaceutical composition comprises one or more siRNA encoded by SEQ ID NOS: 20-49 and a pharmaceutical carrier. A pharmaceutical composition can also comprise one or more shRNA encoded by SEQ ID NOS:4-8 and a pharmaceutical carrier. In further embodiments, a pharmaceutical composition comprises one or more shRNA encoded by SEQ ID NOS:9-13 and a pharmaceutical carrier. The composition can comprise a vector encoding a siRNA and/or shRNA.

p53 agonists/activating drugs include, but are not limited to, RG7112 (Ro5045337 (Roche), RG7112 with cytarabine (Roche), RG7112 with doxorubicin (Roche), RO5503781 (Roche), RO5503781 with cytarabine (Roche), MI-773 (SAR405838) (Sanofi), DS-3032b (Daiichi Sankyo), and PRIMA-l^¹ (APR246) (Aprea). p53 activating drugs can include the molecules listed above whose mechanism of action is antagonizing MDM2. Other p53 agonists include XI-011 (NSC146109), CGM097 (Novartis), MK-8242 (SCH900242) (Merck), Tenovin-1, Tenovin-6, and CP31398.

LSD1 inhibitors include, but are not limited to, poly amine analogs (see Huang et al, 104 PROC. NATL. ACAD. SCI. U.S.A. 8023-28 (2007), CBB-1007 (see Wang et al, 71 CANCER RES. 7238-49 (2011), namoline (see Willmann et al., 131 INT. J. CANCER 2704-09 (2012)), amidoximes (see Hazeldine et al, 55 J. MED. CHEM. 7378-91 (2012)), phenyl oxazoles (see Dulla et al, 11 ORG. BlOMOL. CHEM. 3103-07 (2013)), GSK-354 (see Hitchin et al, 4 MED. CHEM. COMM. 1513-22 (2014)), aminothiazole (see Hitchin et al, 4 MED. CHEM. COMM. 1513-22 (2014)), huntsman (see Soma et al, 58 J. MED. CHEM. 9496-9508 (2013), and dithiocarbamates (see Duan et al, 62 EUR. J. MED. CHEM. 11-19 (2013) and Nobel et al, 270 J. BIOL. CHEM. 26202-08 (1995)).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 A-1H. Identification and characterization of a robust suppressor that ameliorates the locomotion defects in the C. elegans model of SOD 1 -associated ALS. (A) Work flow of the suppressor screen identifying mutant C. elegans (red) with saliently improved movement. (B) Locomotor behavior, measured by thrashing rates in liquid medium, in the C. elegans strains with neuronal expression of human WT SOD1 or ALS- linked mutant SOD1G85R, in the presence (Ml/Ml) or absence (+/+) of the suppressor mutation (n=16). (C) Northern (top panel) and western (middle, bottom panels) blot analyses of total RNA and protein from SOD1G85R strains, with (Ml/Ml) or without (+/+) suppressor mutations, demonstrating that the levels of SOD1G85R mRNA and protein are not changed by the suppressor mutation (Ml/Ml). (D) Sequence analysis of the Ml strain, revealing that independent mutations in two genes, a lysine-specific demethylase, spr-5 (R646Q), and a ubiquitin ligase, ufd-2 (W824X), are required for the full suppressor phenotype. (E) C. elegans SPR-5 and UFD-2 and their mammalian homologs LSD1 and UBE4B share all major protein domains. These include the Swi3-Rsc8-Moira (SWIRM), amine oxidase-like (AOL), and TOWER domains in LSD1/SPR-5; and the Ufd-2 Core and U-box domains in UBE4B/UFD-2. Positions of the missense, nonsense, and deletion mutations in mutant C. elegans are indicated. (F) Locomotor behaviors, measured by thrashing rates, of the C. elegans carrying the SOD1G85R transgene on the normal background (WT), with the null mutation of either ufd-2(tml 380) or spr-5(byl34), the double mutation ufd-2(tml380);spr-5(byl34), or the Ml suppressor ufd-2(W824X);spr-5(R646Q) (n=16). (G) Western blotting of the total proteins from C. elegans indicates that the ufd-2 and spr-5 mutations do not alter the SOD1G85R expression levels. (H) The overexpression (OE) of wild-type UFD-2 or SPR-5 in the C. elegans nervous system blocks the protection conferred by the double mutation ufd-2 (tml 380); spr- 5(byl34) (n=16). Data represent means ± SEM.

FIG. 2A-2E. Neuron-specific suppression of aggregation of diverse proteins is correlated with improved locomotion in ufd-2;spr-5 mutant animals. (A) Schematic drawing at the top depicts pan-neuronal expression of YFP in head and ventral neurons in the context of the C. elegans body plan. Micrographs show the SOD1G85R-YFP (top panels) and TDP- C25-YFP (bottom panels) proteins expressed in the WT or the double mutant spr- 5(byl34);ufd-2(tml380) background. The double-mutant worms show a marked decrease in protein aggregation in neurons. (B) Schematic drawing of muscle-directed YFP expression. Micrographs show the polyQYFP proteins expressed in the C. elegans neurons (top panels) and body wall muscles (bottom panels). Only the neuronal protein aggregates are significantly decreased by spr-5(byl34);ufd- 2(tml380). (C) A decrease in aggregated SOD1G85R-YFP or TDP-c25-YFP protein in the presence of spr-5(byl34);ufd-2(tml380), as shown by western blot analyses of the supernatant fractions (S) and the pellet fractions (P). (D) Quantification of locomotion in the spr-5(byl34);ufd- 2(tml380) and the WT C. elegans with neuronal (Neurons) and muscular polyQ-YFP aggregates (Muscles). n=12 for both "Neurons" and "Muscles". Data represent means ± SEM. (E) Neurodegenerative rough-eye phenotype of adult flies is alleviated by the knockdown of the homologs of ufd-2/UBE4B and spr-5/LSO\, CG9934 and Su(Var)3-3, respectively. Eye-specific expression of TDP- 43M337V, FUSR521C and RNAi was driven by GMR-Gal4. Arrowhead: nerve rings; lines: ventral cords.

FIG. 3A-3E. UBE4B and LSD1 double-knockdown accelerates SOD1G85R protein degradation. (A) Western blots of cell lysates derived from mock (CTRL), single UBE4B or LSD1, or double UBE4B and LSD1 knockdowns. Supernatant (S) and pellet (P) fractions were probed with indicated antibodies. While the LSDl or UBE4B single-knockdown reduces the SOD1G85R aggregates in both supernatant and pellet fractions, the combined knockdown produces the strongest reduction in the aggregates. (B) Quantification of SOD1G85R protein levels by western blotting (A). n=3 (supernatant); n=8 (pellet). (C) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half-lives in the double UBE4B and LSDl knockdown cells versus controls. (D) Quantification of SOD1G85R clearance, as analyzed by western blotting in (C). The graph indicates the relative band intensity of SOD1G85R at each chase time point. n=5; Overall p=0.02 (paired r-test, CTRL vs. UBE4B and LSDl double-knockdown). Individual p=0.03 (3h), p=0.003 (6h), p=0.06 (9h), and p=0.004 (12-21h). (E) The half-life of SOD1G85R is reduced from 8.5 h to 5 h upon knockdown of UBE4B and LSDl. See also FIG. 8.

FIG. 4A-4H. UBE4B and LSDl knockdown activates transcription mediated by p53 and FOXOs. (A) Venn diagram of upstream activators (z-score >2) that are differentially activated in single, UBE4B or LSDl, and double UBE4B and LSDl knockdowns, compared to the control. Activation state of an upstream regulator is predicted from differential mRNA levels of its downstream target genes. (B) The volcano scatter plot indicates fold changes in the levels of gene transcripts affected differentially by the UBE4B and LSDl double- knockdown versus control shRNA. Gray spots represents 22,148 annotated transcripts. Red spots are predicted p53- activated targets, and blue spots are predicted p53-inhibited targets. The enrichment of red spots in the up-regulated genes (right upper quadrant, >1.2-fold change, p<0.04) and blue spots in the down-regulated genes (left upper quadrant) indicates that the p53-mediated transcription is activated by the UBE4B and LSDl double-knockdown. (C) RT-qPCR validation of the expression levels of representative p53 target genes in the UBE4B and LSDl double-knockdown samples. n=(2 to 6), P<0.05. (D) The p53 protein level is significantly increased by UBE4B and LSDl double-knockdown. Left:

Representative western blots showing p53 levels, the knockdown of UBE4B and LSDl, and the actin control in HEK293 cells. Right: Quantification of the p53 protein levels normalized against actin (n=3). (E) The p53-mediated transcriptional activity is measured by a luciferase reporter under the control of a p53-response element promoter, which was transfected into HEK293T cells 72-96 h after the initiation of the UBE4B and LSD 1 single- or double- knockdowns (n=6). (F) Increased interaction of p53 with its activating partner 53BP1 in response to UBE4B and LSDl knockdown. (G) RT-qPCR validation of the expression levels of FOX03a, FOX04, and PSMD11 (n=6). (H) The FOX03a-mediated transcriptional activity was measured with a luciferase reporter under the control of a FOXO-response element promoter. HEK293T cells were transfected with shRNAs targeting mock, LSD1, UBE4B, or both LSD1 and UBE4B, followed 72-96 h later by transfection of the luciferase reporter together with a constitutively active form of FOX03a (TM) for measurement of its specific activity (n=6). Data represent means ± SEM. See also FIGS. 9 and 10.

FIG. 5A-5D. UBE4B and LSD1 double-knockdown activates both proteasomes and autophagy. (A) Increased protein levels of proteasome subunits upon the UBE4B and LSD1 double knockdown in HEK293T cells. (B) The UBE4B and LSD1 single- or double- knockdowns increase the proteasomal degradation of a reporter substrate, Suc-LLVY- Luciferin, whose degradation is measured in a luciferase release assay. The double- knockdown shows synergistic proteasomal activation when compared with the individual knockdowns. n=6. (C) The autophagy activity is significantly increased in cells with the UBE4B and LSD1 double-knockdown. The in vivo activity of the ATG4B protease, which cleaves LC3 precursors, was quantified via a Gaussia luciferase release assay (see FIGS. 1 IB and 11C). The cells were analyzed 48 h after the initiation of the knockdown. (D)

Quantification of LC3-I and LC3-II levels in HCT116 cells with the UBE4B and LSD1 double-knockdown or the mock control. The cells were treated with or without 10 mM 3- methyladenine (3-MA) to block the recycling and degradation of LC3-II. Western blots indicate an increase in LC3 levels (top panels) in the double UBE4B and LSD 1 -knockdown cells (middle panels), while the actin control is unchanged (bottom panels). The graph shows the quantification of LC3-II levels as normalized to actin levels (n=3). See also FIG. 11.

FIG. 6A-6C. p53 promotes the clearance of misfolded SODl mutant proteins. (A) p53 small molecule activators Tenovin-1 and CP-31398 reduce the levels of misfolded SODl proteins, as determined by the SOD1G85R aggregation assay in HEK293 cells. Increasing concentrations of the p53 activators significantly decrease the levels of SOD1G85R but not the endogenous WT SODl proteins in western blots of both supernatant and pellet fractions. (B) A decrease in p53 as the result of shRNA knockdown increases the levels of SOD1G85R but not WT SODl proteins in the SOD1G85R aggregation assay, as shown by western blots of both supernatant (S) (n=2) and pellet (P) (n=3) fractions. (C) A complete absence of p53 increases the accumulation of SOD1G85R mutant proteins in p53-/- HCT116 cells when compared to controls. Representative western blots (left panels) and quantification of

SOD1G85R levels in the supernatant ly sates, where it is detectable, are shown. The middle graph indicates the ratio of G85R to WT SODl proteins in the presence or absence of p53 with varying amounts of transfected mutant SODl . The right graph panel shows the same data as shown in the middle panel, but normalized to the average SOD1G85R level for each amount of the transfected plasmid. See also FIG. 12.

FIG. 7. Suppression of protein aggregates by UBE4B and LSDl knockdown depends on p53. (A) p53 knockdown reverses the suppression of SOD1G85R protein aggregates in the UBE4B and LSDl double-knockdown. Quantification of SOD1G85R in pellet fractions from HEK293T cells transfected with mock, double (UBE4B/LSD1), or triple

(UBE4B/LSDl/p53) shRNAs (n=2). (B) The degenerative TDP-43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 (p53-RNAi), or by the overexpression of a dominant negative p53 mutant (p53.R155H). Expression of p53-RNAi, p53.R155H, and TDP-43M337V are driven by GMR-Gal4. (C) The p53 activator drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SODlG85R-induced proteotoxicity. Toxicity assay on mixed spinal cord cultures treated with vehicle (DMSO, VEH) or Tenovin-1 (TEN1). Rat spinal cord cultures were grown as described herein and treated with 0.8 μΜ TEN1 and vehicle (DMSO, VEH), followed by infection of HSV- SOD1G85R or HSV-LacZ after 24 h. At 5 days post-infection, cells were fixed and stained with a motor neuron-specific anti-NF-H antibody. Left: Representative images of motor neurons in each condition. Scale bar=5C^m. Right: Quantification of motor neuron survival (ANOVA, F(3,12)=27.96, pO.0001). (D) Schematic of the SUNS pathway. A reduction in UBE4B and LSDl function synergistically activates p53- and FOXO-mediated

transcriptional programs, suppressing protein aggregates via increased proteasomal and autophagic clearance. See also FIG. 13.

FIG. 8A-8C. Knockdown of UBE4B and LSDl reduces TDP-43<2 ^1K protein aggregation in mammalian cells (related to FIG. 3). (A) The flow chart of the mammalian cell-based protein aggregation assay as described herein. (B) The western blots of TDP- 43<2^331K protein aggregation assay. HEK293 cells were transfected with a TDP-43^{Q 1K} expression plasmid, together with a control shRNA (CTRL) or the mixed UBE4B and LSDl shRNA plasmids. Following cell lysis and fractionation, supernatant (S) and pellet (P) fractions were run on 15% SDS-PAGE gels. (C) Quantification of TDP-43<2 ^1K in the pelleted (P) fraction containing large insoluble aggregates indicates a significant reduction caused by the UBE4B and LSDl knockdown (n=3).

FIG. 9A-9D. The transcriptional profiling and network analysis (related to FIG. 4). (A) The heat map of microarray signals of differentially regulated genes (p<0.05) upon the knockdown of LSDl alone, UBE4B alone, or both, in triplicates. Hierarchical clustering of the samples indicates that the single UBE4B knockdown induces similar transcriptional changes as the double-knockdown, consistent with the pattern of anti-proteotoxic activities shown in FIGS. 3A and 3B. (B) The heat map of p53 transcriptional targets. The hierarchical clustering of the samples demonstrates the same partem as shown above for all differentially regulated genes. (C) The p53 network is activated in the UBE4B and LSD1 double-knockdown cells (p53: z-score=2.0; p-value of overlap=2.49 x 10^"2). The transcriptional targets with changes consistent with p53 activation are shown, with up- regulated genes in red and downregulated genes in green. (D) RT-qPCR validation of the expression levels of FOX03a, FOX04, and PSMD11 (n=6).

FIG. 10. The regulation of p53 transcriptional activity by UBE4B and LSD1 is specific (related to FIG. 4). Elevation of MDM2, the p53-targeting ubiquitin ligase, significantly reduced the p53 response element-mediated activity that was induced by the UBE4B and LSD1 knockdown (n=5). MDM2 is co-transfected with the p53 reporter upon knockdown of UBE4B and/or LSD1.

FIG. 1 lA-11C. The proteasomal and autophagic activity assays (related to FIG. 5). (A) A schematic of a luciferase-based proteasomal activity assay. Isolated cytosol is mixed with a peptide substrate, Suc-LLVY-luciferin. The chymotrypsin-like activity of proteasomes cleaves off the Suc-LLVY peptide, releasing the amino-luciferin, which produces strong chemiluminescence in the presence of luciferase and ATP. The detected chemiluminescence is used to quantify the proteasomal activity. (B) The flow chart of an autophagic activity assay to measure LC3 cleavage based on a luciferase (GLuc) reporter. Cells were transfected with a set of plasmids to knockdown LSD1 and UBE4B (or non- targeting shRNA, CTRL) and to express the GLuc reporters and SEAP (Secreted Embryonic Alkaline Phosphatase). SEAP is constitutively secreted and serves as a transfection normalization control. S0D1^G85R is expressed concurrently to match the condition with the increased burden of misfolded proteins, as described earlier (FIG. 3). (C) A schematic of the LC3 cleavage and Glue release assay. A cleavable fusion protein, Actin(Act)-LC3-GLuc, or its uncleavable negative control, Act-GLuc, is anchored to the actin cytoskeleton inside the cell. When Act-LC3-GLuc is cleaved by the autophagy-associated protease ATG4B, the GLuc fragment is released from its actin anchor and rapidly secreted out of the cell. The activity of GLuc in the cell medium is assayed over a period of several days using the Dual Luminescence Assay kit.

FIG. 12. p53-activating drugs enhance protein clearance of misfolded mutant SOD1 (related to FIG. 6). (A) The p53 small molecule activator, Tenovin-1 (TEN1, 2μΜ), significantly decreased the levels of misfolded S0D1^G85R but not WT SOD1 proteins in both supernatant and pellet fractions as compared to vehicle-treated controls (VEH) (n=4). The S0D1^G85R solubility assay in HEK293 cells is described herein. (B) Another p53 small molecule activator, CP-31398 ^g/ml), also significantly decreased the levels of misfolded S0D1^G85R SOD1 proteins in both supernatant and pellet fractions in similar quantification of western blots (n=3). (C) Knockdown of p53 blocked the improved clearance of misfolded SOD 1^G85R proteins by Tenovin-1 or CP-31398. HEK393T cells were transfected with S0D1^G85R and treated with p53-activating drugs Tenovin-1 or CP31398. Protein aggregation assays were performed to evaluate he levels of S0D1^G85R proteins in the supernatant (S) and pellet (P) fractions. The reduction of S0D1^G85R aggregation in cells treated with Tenovin-1 or CP-31398 is dependent on p53, as the knockdown of p53 abolishes the ability of the drugs to remove aggregates. (D) p53-activating drugs activate autophagy as indicated by LC3 protein levels. HEK293T cells treated with Tenovin-1 or CP-31398 for 24 h were lysed in 1% SDS buffer, and LC3-I and LC-II levels were analyzed by western blots. For both Tenovin-1 and CP-31398, the LC-II levels are augmented with increasing drug

concentrations. Both panels were from the same gel and western blot with identical imaging settings.

FIG. 13A-13B. p53 mediates improved protein clearance induced by the knockdown of UBE4B and LSDl (related to FIG. 7). (A) Stable knockdown of p53 partially blocks the improved clearance of SOD 1^G85R proteins conferred by the knockdown of UBE4B and LSDl. A stable cell line with inducible knockdown of p53 via an integrated shRNA (in a pR4R3- TET-PURO vector) is used to conditionally remove p53 upon the induction of Doxycycline (DOX). The protein aggregation assay was used to analyze the S0D1^G85R protein levels in S and P fractions. The knockdown of UBE4B and LSDl substantially enhances the clearance of misfolded S0D1^G85R proteins, but this effect is partially reversed upon the conditional knockdown of p53 (+DOX). The western blots for the S and P fractions are from the same gels, respectively. (B) Transient knockdown of p53 similarly blocks the improved clearance of S0D1^G85R proteins conferred by the knockdown of UBE4B and LSDl. The protein aggregation assay was performed as in (B), except with the knockdown of p53 achieved through shRNA transient transfection, and with LSDl and UBE4B shRNAs in a pR4R3-NEO vector targeting their respective 3'UTRs.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the present invention is not limited to the particular methods and components, etc., described herein, as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to a "protein" is a reference to one or more proteins, and includes equivalents thereof known to those skilled in the art and so forth.

Unless defined otherwise, 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. Specific methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

All publications cited herein are hereby incorporated by reference including all journal articles, books, manuals, published patent applications, and issued patents. In addition, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided. The definitions are not meant to be limiting in nature and serve to provide a clearer understanding of certain aspects of the present invention.

Protein quality control is essential for clearing misfolded and aggregated proteins from the cell, or its failure would lead to numerous neurodegenerative disorders. How to boost protein quality control to enhance cellular defense against proteotoxicity is not well explored. Here, we identify two genes, ufd-2 and spr-5, that when inactivated, synergistically and robustly reduce protein aggregation and neurotoxicity in C. elegans model of neurodegenerative diseases. Remarkably, the human homologues, UBE4B and LSD1, encoding a ubiquitin ligase and a lysine-specific demethylase, when inactivated, suppress aggregation of disease-associated proteins in mammalian cells. The suppression of protein aggregation is a result of activation of both proteasomal and autophagic degradation machineries. An unbiased search in this pathway reveals downstream effectors such as transcription factor p53, a shared substrate of UBE4B and LSD1, as key regulators of protein quality control. These results demonstrate a new protein quality control pathway via post- translational modifications of transcription factors, and suggest the reprogramming of protein quality control as a wide-spectrum anti -proteotoxicity strategy.

More specifically, as described herein, the present inventors took advantage of a

Caenorhabditis elegans model that expresses neuronal ALS-linked SODl mutant proteins and develops robust movement defects, and performed an unbiased forward genetic screen for potent suppressors of the behavioral defects. The present inventors identified mutations in two genes, ufd-2, encoding a ubiquitin ligase, and spr-5, encoding a lysine-specific demethylase, that synergistically attenuate the neurotoxicity of mutant human SOD1 and other misfolded proteins. Remarkably, these two post-translational lysine modifiers were found to be part of a pathway regulating protein quality control in human cells. Further analysis showed that this pathway acts through transcription factors such as p53 that mediate cellular stress responses. Together these results describe a new mechanism involving previously unrecognized players for the cell to reprogram cellular stress responses towards protein quality control.

I. Definitions

Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. 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 being 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 can be modified by the term "about."

An "agonist" is a type of modulator and refers to an agent that can activate one or more functions of the target. For example, an agonist of a protein can activate the protein in the absence of its natural or cognate ligand.

As used herein, an "antagonist" is a type of modulator and is used interchangeably with the term "inhibitor." In certain non-limiting embodiments, the term refers to an agent that can inhibit a one or more functions of the target. For example, an antagonist of an enzymatic protein can inhibit the enzymatic activity of the protein.

As used herein, the term "antibody" is used in reference to any immunoglobulin molecule that reacts with a specific antigen. It is intended that the term encompass any immunoglobulin (e.g., IgG, IgM, IgA, IgE, IgD, etc.) obtained from any source (e.g., humans, rodents, non-human primates, caprines, bovines, equines, ovines, etc.). Specific

types/examples of antibodies include polyclonal, monoclonal, humanized, chimeric, human, or otherwise-human-suitable antibodies. "Antibodies" also includes any functional fragment or derivative of any of the herein described antibodies. Functional fragments include antigen- binding fragments. In specific embodiments, antibodies may be raised against Ube4B and/or LSD 1 and used as Ube4B and/or LSD1 modulators. As used herein, the term "effective," means adequate to accomplish a desired, expected, or intended result. More particularly, a "therapeutically effective amount" as provided herein refers to an amount of a Ube4B and/or LSD1 modulator of the present invention, either alone or in combination with another therapeutic agent, necessary to provide the desired therapeutic effect, e.g., an amount that is effective to prevent, alleviate, or ameliorate symptoms of disease or prolong the survival of the subject being treated. In a specific embodiment, the term "therapeutically effective amount" as provided herein refers to an amount of a Ube4B and/or LSD1 modulator, necessary to provide the desired therapeutic effect, e.g., an amount that is effective to prevent, alleviate, or ameliorate symptoms of disease or prolong the survival of the subject being treated. In particular embodiments, the disease or condition is a protein conformation disease. As would be appreciated by one of ordinary skill in the art, the exact amount required will vary from subject to subject, depending on age, general condition of the subject, the severity of the condition being treated, the particular compound and/or composition administered, and the like. An appropriate "therapeutically effective amount" in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.

By "high stringency conditions" is meant conditions that allow hybridization comparable with that resulting from the use of a DNA probe of, for example, at least 40 nucleotides in length, in a buffer containing 0.5 M NaHP04, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at a temperature of 65°C, or a buffer containing 48% formamide, 4.8XSSC, 0.2 M Tris-Cl, pH 7.6, lXDenhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42°C Other conditions for high stringency hybridization, such as for PCR, Northern, Southern, or in situ hybridization, DNA sequencing, etc., are well-known by those skilled in the art of molecular biology. (See, for example, F. Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1998).

The term "inhibitor" is a type of modulator and is used interchangeably with the term "antagonist." The term "inhibitor" includes any type of molecule or agent that directly or indirectly inhibits the expression or activity of a target gene or protein. An inhibitor can be any type of compound, such as a small molecule, polypeptide, polynucleotide and the like including an antibody or an RNA interference compound. In certain embodiments, the target gene or protein is Ube4B and/or LSD1. The term also includes agents that have activity in addition to Ube4B and/or LSD1 inhibitory activity. As used herein, the term "modulate" indicates the ability to control or influence directly or indirectly, and by way of non-limiting examples, can alternatively mean inhibit or stimulate, agonize or antagonize, hinder or promote, and strengthen or weaken. Thus, the terms "Ube4B modulator" and "LSD1 modulator" refers to an agent that modulates the expression and/or activity of Ube4B and LSD1 , respectively. Inhibitors may be organic or inorganic, small to large molecular weight individual compounds, mixtures and combinatorial libraries of inhibitors, agonists, antagonists, and biopolymers such as peptides, nucleic acids, or oligonucleotides. A modulator may be a natural product or a naturally-occurring small molecule organic compound. In particular, a modulator may be a carbohydrate;

monosaccharide; oligosaccharide; polysaccharide; amino acid; peptide; oligopeptide;

polypeptide; protein; receptor; nucleic acid; nucleoside; nucleotide; oligonucleotide;

polynucleotide including DNA and DNA fragments, RNA and RNA fragments and the like; lipid; retinoid; steroid; glycopeptides; glycoprotein; proteoglycan and the like; and synthetic analogues or derivatives thereof, including peptidomimetics, small molecule organic compounds and the like, and mixtures thereof. A modulator identified according to the invention is preferably useful in the treatment of a disease disclosed herein.

The phrase "nucleic acid" as used herein refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single- stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.

Optional" or "optionally" means that the subsequently described event or

circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

The terms "patient," "individual," or "subj ect" are used interchangeably herein, and refer to a mammal, particularly, a human. The patient may have a mild, intermediate or severe disease or condition, he patient may be an individual in need of treatment or in need of diagnosis based on particular symptoms or family history. In some cases, the terms may refer to treatment in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates. In particular, the term also includes mammals diagnosed with a protein conformational disease, disorder or condition. By "normal subject" is meant an individual who does not have a protein conformational disease as well as an individual who has increased susceptibility for developing a protein conformational disease.

"Polypeptide" as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules. In addition, as used herein, the term "polypeptide" refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc., and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer- RNA mediated addition of amino acids to protein such as arginylation. See Proteins- Structure and Molecular Properties 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983).

By "probe," "primer," or oligonucleotide is meant a single-stranded DNA or RNA molecule of defined sequence that can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the "target"). The stability of the resulting hybrid depends upon the extent of the base-pairing that occurs. The extent of base-pairing is affected by parameters such as the degree of complementarity between the probe and target molecules and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art. Probes or primers specific for Ube4B and/or LSDl nucleic acids (for example, genes and/or mRNAs) have at least 80%-90% sequence complementarity, preferably at least 9\%-95% sequence complementarity, more preferably at least 96%-99% sequence complementarity, and most preferably 100% sequence complementarity to the region of the Ube4B and/or LSDl nucleic acid to which they hybridize. Probes, primers, and oligonucleotides may be detectably -labeled, either radioactively, or non-radioactively, by methods well-known to those skilled in the art. Probes, primers, and oligonucleotides are used for methods involving nucleic acid hybridization, such as: nucleic acid sequencing, reverse transcription and/or nucleic acid amplification by the polymerase chain reaction, single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, electrophoretic mobility shift assay (EMSA).

The terms "protein conformational disease," "protein conformational disorder" or "protein conformational condition" and the like refer to all the pathological conditions that are associated with protein misfolding and aggregation, or proteotoxicity, and specifically include neurodegenerative diseases. Specific examples of protein conformational diseases include, but are not limited to, Creutzfeldt-Jakob disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, and amyotrophic lateral sclerosis (ALS).

The terms "specifically binds to," "specific for," and related grammatical variants refer to that binding which occurs between such paired species as antibody/antigen, enzyme/substrate, receptor/agonist, and lectin/carbohydrate which may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions. When the interaction of the two species produces a non-covalently bound complex, the binding which occurs is typically electrostatic, hydrogen-bonding, or the result of lipophilic interactions. Accordingly, "specific binding" occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody/antigen or enzyme/substrate interaction. In particular, the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs. Thus, for example, an antibody typically binds to a single epitope and to no other epitope within the family of proteins. In some embodiments, specific binding between an antigen and an antibody will have a binding affinity of at least 10^"6 M. In other embodiments, the antigen and antibody will bind with affinities of at least lO^"7 M, 10^"8 M to 10^"9 M, 10^"10 M, 10^"11 M, or 10^"12 M.

By "specifically hybridizes" is meant that a probe, primer, or oligonucleotide recognizes and physically interacts (that is, base-pairs) with a substantially complementary nucleic acid (for example, a Ube4B and/or LSD1 nucleic acid) under high stringency conditions, and does not substantially base pair with other nucleic acids.

As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a subject, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, e.g., causing regression of the disease, e.g., to completely or partially remove symptoms of the disease. In a specific embodiment, the disease or condition is a protein conformational disease.

II. Ube4B and LSD1 Inhibitors

In certain embodiments, the Ube4B and/or LSD1 inhibitor is selected from the group consisting of a small molecule, a polypeptide, a nucleic acid molecule, a peptidomimetic, or a combination thereof. In a specific embodiment, the agent can be a polypeptide. The polypeptide can, for example, comprise an antibody. In another embodiment, the agent can be a nucleic acid molecule. The nucleic acid molecule can, for example, be a Ube4B and/or LSD1 inhibitory nucleic acid molecule. The Ube4B and/or LSD1 inhibitory nucleic acid molecule can comprise a short interfering RNA (siRNA) molecule, a microRNA (miRNA) molecule, or an antisense molecule.

A. RNA Interference Compositions for Targeting Ube4B and LSD1 mRNA In one aspect of the present invention, the expression of Ube4B and/or LSD1 may be inhibited by the use of RNA interference techniques (RNAi). RNAi is a remarkably efficient process whereby double-stranded RNA (dsRNA) induces the sequence-specific degradation of homologous mRNA in animals and plant cells. See Hutvagner and Zamore, 12 CURR. OPIN. GENET. DEV. 225-32 (2002); Hammond et al, 2 NATURE REV. GEN. 110-19 (2001); Sharp, 15 GENES DEV. 485-90 (2001). RNAi can be triggered, for example, by nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al, 10 MOL. CELL. 549-61 (2002); Elbashir et al, 411 Nature 494-98 (2001)), micro-RNAs (miRNA), functional small-hai in RNA (shRNA), or other dsRNAs which are expressed in-vivo using DNA templates with RNA polymerase III promoters. See, e.g., Zeng et al, 9 MOL. CELL. 1327-33 (2002);

Paddison et al., 16 GENES DEV. 948-58 (2002); Lee et al., 20 NATURE BIOTECHNOL. 500-05 (2002); Paul et al, 20 NATURE BIOTECHNOL. 505-08 (2002); Tuschl, 20 NATURE

BIOTECHNOL. 440-48 (2002); Yu et al, 99(9) PROC. NATL. ACAD. SCI. USA, 6047-52 (2002); McManus et al, 8 RNA 842-50 (2002); Sui et al, 99(6) PROC. NATL. ACAD. SCI. USA 5515- 20 (2002).

As used herein, a Ube4B and/or LSDl inhibitory nucleic acid sequence can be a siRNA sequence or a miRNA sequence. A 21-25 nucleotide siRNA or miRNA sequence can, for example, be produced from an expression vector by transcription of a short-hairpin RNA (shRNA) sequence, a 60-80 nucleotide precursor sequence, which is processed by the cellular RNAi machinery to produce either an siRNA or miRNA sequence. Alternatively, a 21-25 nucleotide siRNA or miRNA sequence can, for example, be synthesized chemically.

Chemical synthesis of siRNA or miRNA sequences is commercially available from such corporations as Dharmacon, Inc. (Lafayette, Colo.), Qiagen (Valencia, Calif), and Ambion, Inc. (Austin, Tex.). An siRNA sequence preferably binds a unique sequence within the Ube4B and/or LSDl mRNA with exact complementarity and results in the degradation of the Ube4B and/or LSDl mRNA molecule. An siRNA sequence can bind anywhere within the mRNA molecule. An miRNA sequence preferably binds a unique sequence within the

Ube4B and/or LSDl mRNA with exact or less than exact complementarity and results in the translational repression of the Ube4B and/or LSDl mRNA molecule. An miRNA sequence can bind anywhere within the mRNA molecule, but preferably binds within the 3'UTR of the mRNA molecule. Methods of delivering siRNA or miRNA molecules are known in the art. See, e.g., Oh and Park, Adv. Drug Deliv. Rev. 61(10):850-62 (2009); Gondi and Rao, J. Cell. Physiol. 220(2):285-91 (2009); and Whitehead et al, Nat. Rev. Drug Discov. 8(2)129-38 (2009).

As used herein, a Ube4B and/or LSDl inhibitory nucleic acid sequence can be an antisense nucleic acid sequence. Antisense nucleic acid sequences can, for example, be transcribed from an expression vector to produce an RNA which is complementary to at least a unique portion of the Ube4B and/or LSDl mRNA and/or the endogenous gene which encodes Ube4B and/or LSDl. Hybridization of an antisense nucleic acid molecule under specific cellular conditions results in inhibition of Ube4B and/or LSDl protein expression by inhibiting transcription and/or translation. i. Small Interfering RNA

In particular embodiments, the present invention features "small interfering RNA molecules" ("siRNA molecules" or "siRNA"), methods of making siRNA molecules and methods for using siRNA molecules (e.g., research and/or therapeutic methods). The siRNAs of this invention encompass any siRNAs that can modulate the selective degradation of Ube4B and/or LSD1 mRNA. Examples of LSD 1 and Ube4B siRNA are shown in SEQ ID NOS: 14-19 and SEQ ID NOS:20-49, respectively.

In a specific embodiment, the siRNA of the present invention may comprise double- stranded small interfering RNA molecules (ds-siRNA). A ds-siRNA molecule of the present invention may be a duplex made up of a sense strand and a complementary antisense strand, the antisense strand being sufficiently complementary to a target Ube4B or LSD1 mRNA to mediate RNAi. The siRNA molecule may comprise about 10 to about 50 or more nucleotides. More specifically, the siRNA molecule may comprise about 16 to about 30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand. The strands may be aligned such that there are at least 1, 2, or 3 bases at the end of the strands which do not align (e.g., for which no complementary bases occur in the opposing strand) such that an overhang of 1, 2 or 3 residues occurs at one or both ends of the duplex when strands are annealed.

In an alternative embodiment, the siRNA of the present invention may comprise single-stranded small interfering RNA molecules (ss-siRNA). Similar to the ds-siRNA molecules, the ss-siRNA molecule may comprise about 10 to about 50 or more nucleotides. More specifically, the ss-siRNA molecule may comprise about 15 to about 45 or more nucleotides. Alternatively, the ss-siRNA molecule may comprise about 19 to about 40 nucleotides. The ss-siRNA molecules of the present invention comprise a sequence that is "sufficiently complementary" to a target mRNA sequence to direct target-specific RNA interference (RNAi), as defined herein, e.g., the ss-siRNA has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process. In one embodiment, the ss-siRNA molecule can be designed such that every residue is complementary to a residue in the target molecule. Alternatively, substitutions can be made within the molecule to increase stability and/or enhance processing activity of the molecule. Substitutions can be made within the strand or can be made to residues at the ends of the strand. In a specific embodiment, the 5 '-terminus may be phosphorylated (e.g., comprises a phosphate, diphosphate, or triphosphate group). In another embodiment, the 3' end of an siRNA may be a hydroxyl group in order to facilitate RNAi, as there is no requirement for a 3' hydroxyl group when the active agent is a ss-siRNA molecule. In other instances, the 3' end (e.g., C3 of the 3' sugar) of ss-siRNA molecule may lack a hydroxyl group (e.g., ss-siRNA molecules lacking a 3 ' hydroxyl or C3 hydroxyl on the 3 ' sugar (e.g., ribose or deoxyribose).

In another aspect, the siRNA molecules of the present invention may be modified to improve stability under in vitro and/or in vivo conditions, including, for example, in serum and in growth medium for cell cultures. In order to enhance the stability, the 3 '-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2'- deoxythymidine is tolerated and does not affect the efficiency of RNA interference. For example, the absence of a 2' hydroxyl may significantly enhance the nuclease resistance of the siRNAs in tissue culture medium.

Furthermore, the siRNAs of the present invention may include modifications to the sugar-phosphate backbone or nucleosides. These modifications can be tailored to promote selective genetic inhibition, while avoiding a general panic response reported to be generated by siRNA in some cells. In addition, modifications can be introduced in the bases to protect siRNAs from the action of one or more endogenous enzymes.

In an embodiment of the present invention, the siRNA molecule may contain at least one modified nucleotide analogue. The nucleotide analogues may be located at positions where the target-specific activity, e.g., the RNAi mediating activity is not substantially effected, e.g., in a region at the 5 '-end and/or the 3 '-end of the RNA molecule. Particularly, the ends may be stabilized by incorporating modified nucleotide analogues. Examples of nucleotide analogues include sugar- and/or backbone-modified ribonucleotides (e.g., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In backbone-modified ribonucleotides, the phosphoester group connecting to adjacent ribonucleotides may be replaced by a modified group, e.g., a phosphothioate group. In sugar- modified ribonucleotides, the 2' OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is Ci-Ce alkyl, alkenyl or alkynyl and halo is F, CI, Br or I.

Nucleobase-modified ribonucleotides may also be utilized, e.g., ribonucleotides containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase.

Exemplary modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined.

Derivatives of siRNAs may also be utilized herein. For example, cross-linking can be employed to alter the pharmacokinetics of the composition, e.g., to increase half-life in the body. Thus, the present invention includes siRNA derivatives that include siRNA having two complementary strands of nucleic acid, such that the two strands are crosslinked. The present invention also includes siRNA derivatives having a non-nucleic acid moiety conjugated to its 3' terminus (e.g., a peptide), organic compositions (e.g., a dye), or the like. Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA.

The siRNAs of the present invention can be enzymatically produced or totally or partially synthesized. Moreover, the siRNAs can be synthesized in vivo or in vitro. For siRNAs that are biologically synthesized, an endogenous or a cloned exogenous RNA polymerase may be used for transcription in vivo, and a cloned RNA polymerase can be used in vitro. siRNAs that are chemically or enzymatically synthesized are preferably purified prior to the introduction into the cell.

Although one hundred percent (100%) sequence identity between the siRNA and the target region is preferred in particular embodiments, it is not required to practice the invention. siRNA molecules that contain some degree of modification in the sequence can also be adequately used for the purpose of this invention. Such modifications may include, but are not limited to, mutations, deletions or insertions, whether spontaneously occurring or intentionally introduced.

Moreover, not all positions of a siRNA contribute equally to target recognition. In certain embodiments, for example, mismatches in the center of the siRNA may be critical and could essentially abolish target RNA cleavage. In other embodiments, the 3' nucleotides of the siRNA do not contribute significantly to specificity of the target recognition. In particular, residues 3 ' of the siRNA sequence which is complementary to the target RNA (e.g., the guide sequence) may not critical for target RNA cleavage.

Sequence identity may be determined by sequence comparison and alignment algorithms known to those of ordinary skill in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., % homology = # of identical positions/total # of positions x 100), optionally penalizing the score for the number of gaps introduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (e.g., a local alignment). A non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul, 87 PROC. NATL. ACAD. SCI. USA 2264-68 (1990), and as modified as in Karlin and Altschul 90 PROC. NATL. ACAD. SCI. USA 5873-77 (1993). Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al, 215 J. MOL. BIOL. 403-10 (1990).

In another embodiment, the alignment may optimized by introducing appropriate gaps and determining percent identity over the length of the aligned sequences (e.g., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, 25(17) NUCLEIC ACIDS RES. 3389-3402 (1997). In another embodiment, the alignment may be optimized by introducing appropriate gaps and determining percent identity over the entire length of the sequences aligned (e.g., a global alignment). A non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

In particular embodiments, greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity, between the siRNA and the portion of the target gene may be used. Alternatively, the siRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C hybridization for 12-16 hours; followed by washing). Additional hybridization conditions include, but are not limited to, hybridization at 70°C in IxSSC or 50°C in IxSSC, 50% formamide followed by washing at 70°C in 0.3xSSC or hybridization at 70°C in 4xSSC or 50°C in 4xSSC, 50% formamide followed by washing at 67°C in IxSSC. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length can be about 5-10°C less than the melting temperature (Tra) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(°C) = 2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) = 81.5+16.6(log 10[Na⁺])+0.41(% G+C)-(600/N), where N is the number of bases in the hybrid, and [Na⁺] is the concentration of sodium ions in the hybridization buffer ([Na⁺] for lxSSC=0.165 M). Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular

Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference. The length of the identical nucleotide sequences may be at least about 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47 50 or more bases.

ii. Other Compositions for Targeting Ube4B and LSD1 DNA or mRNA Antisense molecules can act in various stages of transcription, splicing and translation to block the expression of a target gene. Without being limited by theory, antisense molecules can inhibit the expression of a target gene by inhibiting transcription initiation by forming a triple strand, inhibiting transcription initiation by forming a hybrid at an RNA polymerase binding site, impeding transcription by hybridizing with an RNA molecule being synthesized, repressing splicing by hybridizing at the junction of an exon and an intron or at the spliceosome formation site, blocking the translocation of an mRNA from nucleus to cytoplasm by hybridization, repressing translation by hybridizing at the translation initiation factor binding site or ribosome biding site, inhibiting peptide chain elongation by hybridizing with the coding region or polysome binding site of an mRNA, or repressing gene expression by hybridizing at the sites of interaction between nucleic acids and proteins. An example of an antisense oligonucleotide of the present invention is a cDNA that, when introduced into a cell, transcribes into an RNA molecule having a sequence complementary to at least part of the Ube4B or LSD1 mRNA. Furthermore, antisense oligonucleotides of the present invention include

oligonucleotides having modified sugar-phosphodiester backbones or other sugar linkages, which can provide stability against endonuclease attacks. The present invention also encompasses antisense oligonucleotides that are covalently attached to an organic or other moiety that increase their affinity for a target nucleic acid sequence. For example, intercalating agents, alkylating agents, and metal complexes can be also attached to the antisense oligonucleotides of the present invention to modify their binding specificities.

The present invention also provides ribozymes as a tool to inhibit Ube4B and/or LSD1 expression. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The characteristics of ribozymes are well-known in the art. See, e.g., Rossi, 4 CURRENT BIOLOGY 469-71 (1994). Without being limited by theory, the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage. In particular embodiments, the ribozyme molecules include one or more sequences

complementary to the target gene mRNA, and include the well-known catalytic sequence responsible for mRNA cleavage. See U.S. Patent No. 5,093,246. Using the known sequence of the target Ube4B or LSD1 mRNA, a restriction enzyme-like ribozyme can be prepared using standard techniques.

The expression of the Ube4B and/or LSD1 genes can also be inhibited by using triple helix formation. Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription can be single stranded and composed of deoxynucleotides. The base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base paring rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC⁺ triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base

complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules that are purine-rich, e.g., containing a stretch of G residues, may be chosen. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so-called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5 '-3 ',3 '-5' manner, such that they base pair first with one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

The expression of Ube4B and/or LSD1 may be also inhibited by what is referred to as "co-repression." Co-repression refers to the phenomenon in which, when a gene having an identical or similar to the target sequence is introduced to a cell, expression of both introduced and endogenous genes becomes repressed. This phenomenon, although first observed in plant system, has been observed in certain animal systems as well. The sequence of the gene to be introduced does not have to be identical to the target sequence, but sufficient homology allows the co-repression to occur. The determination of the extent of homology depends on individual cases, and is within the ordinary skill in the art.

It would be readily apparent to one of ordinary skill in the art that other methods of gene expression inhibition that selectively target a Ube4B and/or LSD1 DNA or mRNA can also be used in connection with this invention without departing from the spirit of the invention. In a specific embodiment, using techniques known to those of ordinary skill in the art, the present invention contemplates affecting the promoter region of Ube4B and/or LSD1 to effectively switch off transcription.

iii. Design and Production of the RNAi Compositions

One or more of the following guidelines may be used in designing the sequence of siRNA and other nucleic acids designed to bind to a target mRNA, e.g., shRNA, stRNA, antisense oligonucleotides, ribozymes, and the like, that are advantageously used in accordance with the present invention.

Beginning with the AUG start codon of the Ube4B and/or LSD1 genes, each AA dinucleotide sequence and the 3' adjacent 16 or more nucleotides are potential siRNA targets. In a specific embodiment, the siRNA is specific for a target region that differs by at least one base pair between the wild type and mutant allele or between splice variants. In dsRNAi, the first strand is complementary to this sequence, and the other strand identical or substantially identical to the first strand. siRNAs with lower G/C content (35-55%) may be more active than those with G/C content higher than 55%. Thus in one embodiment, the invention includes nucleic acid molecules having 35-55% G/C content. In addition, the strands of the siRNA can be paired in such a way as to have a 3' overhang of 1 to 4, e.g., 2, nucleotides. Thus in another embodiment, the nucleic acid molecules may have a 3' overhang of 2 nucleotides, such as TT. The overhanging nucleotides may be either RNA or DNA. In one embodiment, it may be desirable to choose a target region wherein the mismatch is a purine: purine mismatch.

Using any method known in the art, compare the potential targets to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences. One such method for such sequence homology searches is known as BLAST, which is available at National Center for Biotechnology Information website (http://www.ncbi.nih.gov). Select one or more sequences that meet the criteria for evaluation.

Another method includes selecting in the sequence of the target mRNA, a region located from about 50 to about 100 nt 3' from the start codon. In this region, search for the following sequences: AA(N19)TT or AA(N21), where N=any nucleotide. The GC content of the selected sequence should be from about 30% to about 70%, preferably about 50%. To maximize the specificity of the RNAi, it may be desirable to use the selected sequence in a search for related sequences in the genome of interest; sequences absent from other genes are preferred. The secondary structure of the target mRNA may be determined or predicted, and it may be preferable to select a region of the mRNA that has little or no secondary structure, but it should be noted that secondary structure seems to have little impact on RNAi. When possible, sequences that bind transcription and/or translation factors should be avoided, as they might competitively inhibit the binding of a siRNA, sbRNA or stRNA (as well as other antisense oligonucleotides) to the mRNA. Further general information about the design and use of siRNA may be found in "The siRNA User Guide," available at The Max-Planck- Institut fur Biophysikalishe Chemie website (http://www.mpibpc.mpg.de).

Negative control siRNAs should have the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate genome. Such negative controls may be designed by randomly scrambling the nucleotide sequence of the selected siRNA; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome.

iv. Delivery of Ube4B and/or LSD1 RNA Targeting Compositions Delivery of the compositions of the present invention (e.g., siRNAs, antisense oligonucleotides, or other compositions described herein) into a patient can either be direct, e.g., the patient is directly exposed to the compositions of the present invention or compound- carrying vector, or indirect, e.g., cells are first transformed with the compositions of this invention in vitro, then transplanted into the patient for cell replacement therapy. These two approaches are known as in vivo and ex vivo therapy, respectively. In the case of in vivo therapy, the compositions of the present invention are directly administered in vivo, where they are expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering them so that they become intracellular, by infection using a defective or attenuated retroviral or other viral vector, by direct injection of naked DNA, by coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, nanoparticles, microparticles, or

microcapsules, by administering them in linkage to a peptide which is known to enter the cell or nucleus, or by administering them in linkage to a ligand subject to receptor-mediated endocytosis which can be used to target cell types specifically expressing the receptors. Further, the compositions of the present invention can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor. See, e.g., W093/14188, WO

93/20221, WO 92/22635, WO92/20316, and WO 92/06180.

Ex vivo therapy involves transferring the compositions of the present invention to cells in tissue culture by methods well-known in the art such as electroporation, transfection, lipofection, microinjection, calcium phosphate mediated transfection, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and infection with a viral vector containing the nucleic acid sequences. These techniques should provide for the stable transfer of the compositions of this invention to the cell, so that they are expressible by the cell and preferably heritable and expressible by its cell progeny. In particular embodiments, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred compositions. The resulting recombinant cells can be delivered to a patient by various methods known in the art. Examples of the delivery methods include, but are not limited to, subcutaneous injection, skin graft, and intravenous injection.

B. Small Molecule Inhibitors of Ube4B and/or LSD1

In one aspect, a Ube4B and/or LSD1 inhibitor is a small molecule. The term "small molecule organic compounds" refers to organic compounds generally having a molecular weight less than about 5000, 4000, 3000, 2000, 1000, 800, 600, 500, 250 or 100 Daltons, preferably less than about 500 Daltons. A small molecule organic compound may be prepared by synthetic organic techniques, such as by combinatorial chemistry techniques, or it may be a naturally-occurring small molecule organic compound. Specific examples of LSD1 small molecule inhibitors can be found in PCT Publication Nos. WO 2014/100818, WO 2014/100463, WO 2014/085613, WO 2014084298, WO 2012/135113, WO

2012/042042; and U.S. Patent Applications, Publication No. 20140213657, No.

20140163041, No. 20140155339, No. 20140011857, No. 20140018393, No. 20130303545, No. 20130090386, No. 20130035377, No. 20120283266.

Nevertheless, compound libraries may be screened for Ube4B and/or LSD1 inhibitors.

A compound library is a mixture or collection of one or more putative inhibitors generated or obtained in any manner. Any type of molecule that is capable of interacting, binding or has affinity for Ube4B and/or LSD1 may be present in the compound library. For example, compound libraries screened using this invention may contain naturally-occurring molecules, such as carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, receptors, nucleic acids, nucleosides, nucleotides, oligonucleotides, polynucleotides, including DNA and DNA fragments, RNA and RNA fragments and the like, lipids, retinoids, steroids, glycopeptides, glycoproteins, proteoglycans and the like; or analogs or derivatives of naturally-occurring molecules, such as peptidomimetics and the like; and non-naturally occurring molecules, such as "small molecule" organic compounds generated, for example, using combinatorial chemistry techniques; and mixtures thereof.

A library typically contains more than one putative inhibitor or member, i.e., a plurality of members or putative inhibitors. In certain embodiments, a compound library may comprise less than about 50,000, 25,000, 20,000, 15,000, 10000, 5000, 1000, 500 or 100 putative inhibitors, in particular from about 5 to about 100, 5 to about 200, 5 to about 300, 5 to about 400, 5 to about 500, 10 to about 100, 10 to about 200, 10 to about 300, 10 to about 400, 10 to about 500, 10 to about 1000, 20 to about 100, 20 to about 200, 20 to about 300, 20 to about 400, 20 to about 500, 20 to about 1000, 50 to about 100, 50 to about 200, 50 to about 300, 50 to about 400, 50 to about 500, 50 to about 1000, 100 to about 200, 100 to about 300, 100 to about 400, 100 to about 500, 100 to about 1000, 200 to about 300, 200 to about 400, 200 to about 500, 200 to about 1000, 300 to about 500, 300 to about 1000, 300 to 2000, 300 to 3000, 300 to 5000, 300 to 6000, 300 to 10,000, 500 to about 1000, 500 to about 2000, 500 to about 3000, 500 to about 5000, 500 to about 6000, or 500 to about 10,000 putative inhibitors. In particular embodiments, a compound library may comprise less than about 50,000, 25,000, 20,000, 15,000, 10,000, 5,000, 1000, or 500 putative inhibitors.

A compound library may be prepared or obtained by any means including, but not limited to, combinatorial chemistry techniques, fermentation methods, plant and cellular extraction procedures and the like. A library may be obtained from synthetic or from natural sources such as for example, microbial, plant, marine, viral and animal materials. Methods for making libraries are well-known in the art. See, for example, E. R. Felder, Chimia 1994, 48, 512-541 ; Gallop et al, J. Med. Chem. 1994, 37, 1233-1251; R. A. Houghten, Trends Genet. 1993, 9, 235-239; Houghten et al, Nature 1991, 354, 84-86; Lam et al., Nature 1991, 354, 82-84; Carell et al, Chem. Biol. 1995, 3, 171-183; Madden et al., Perspectives in Drug Discovery and Design 2, 269-282; Cwirla et al, Biochemistry 1990, 87, 6378-6382; Brenner et al, Proc. Natl. Acad. Sci. USA 1992, 89, 5381-5383; Gordon et al, J. Med. Chem. 1994, 37, 1385-1401; Lebl et al, Biopolymers 1995, 37 177-198; and references cited therein. Compound libraries may also be obtained from commercial sources including, for example, from May bridge, ChemNavigator.com, Timtec Corporation, ChemBridge Corporation, A- Syntese-Biotech ApS, Akos-SC, G & J Research Chemicals Ltd., Life Chemicals, Interchim S.A., and Spectrum Info. Ltd.

C. Antibodies to Ube4B and/or LSD1

The term antibody is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. The term can also refer to a human antibody and/or a humanized antibody. Examples of techniques for human monoclonal antibody production include those described by Cole et al. (Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985)) and by Boerner et al. (J. Immunol. 147(l):86-95 (1991)). Human antibodies (and fragments thereof) can also be produced using phage display libraries (Hoogenboom et al., J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)). The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA 90:2551-5 (1993); Jakobovits et al, Nature 362:255-8 (1993); Bruggermann et al., Year in Immunol. 7:33 (1993)).

Various procedures known in the art may be used for the production of antibodies to Ube4B and/or LSD1 or any subunit thereof, or a fragment, derivative, homolog or analog of the protein. Antibodies of the present invention include, but are not limited to, synthetic antibodies, polyclonal antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single-chain Fvs (scFv) (including bi-specific scFvs), single chain antibodies Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. In particular, antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., molecules that contain an antigen binding site that immunospecifically binds to an antigen (e.g., one or more complementarity determining regions (CDRs) of an antibody).

Another embodiment for the preparation of antibodies according to the invention is the use of peptide mimetics. Mimetics are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al, "Peptide Turn Mimetics" in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al, Eds., Chapman and Hall, New York (1993). The underlying rationale behind the use of peptide mimetics in rational design is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule. These principles may be used to engineer second generation molecules having many of the natural properties of the targeting antibodies disclosed herein, but with altered and even improved characteristics. More specifically, under this rational design approach, peptide mapping may be used to determine "active" antigen recognition residues, and along with molecular modeling and molecular dynamics trajectory analysis, peptide mimic of the antibodies containing antigen contact residues from multiple CDRs may be prepared.

In some embodiments, an antibody specifically binds an epitope of the Ube4B or LSD1 protein. It is to be understood that the peptide regions may not necessarily precisely map one epitope, but may also contain a Ube4B or LSD1 sequence that is not immunogenic. Methods of predicting other potential epitopes to which an immunoglobulin of the invention can bind are well-known to those of skill in the art and include, without limitation, Kyte- Doolittle Analysis (Kyte, J. and Dolittle, R. F., 157 J. MOL. BlOL. 105-32 (1982)); Hopp and Woods Analysis (Hopp, T. P. and Woods, K. R, 78 PROC. NATL. ACAD. SCI. USA 3824-28 (1981); Hopp, T. J. and Woods, K. R, 20 MOL. IMMUNOL. 483-89 (1983); Hopp, T. J., 88 J. IMMUNOL. METHODS 1-18 (1986)); Jameson-Wolf Analysis (Jameson, B. A. and Wolf, H., 4 COMPUT. APPL. BlOSCl. 181-86 (1988)); and Emini Analysis (Emini et al, 140 VIROLOGY 13- 20 (1985)).

Amino acid sequence variants of the Ube4B and LSD1 antibodies of the present invention may be prepared by introducing appropriate nucleotide changes into the polynucleotide that encodes the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct.

Amino acid sequence insertions include amino-terminal and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of a polypeptide that increases the serum half-life of the antibody.

Another type of antibody variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. For example, the sites of greatest interest for substitutional mutagenesis of antibodies include the hypervariable regions, but framework region (FR) alterations are also contemplated.

A useful method for the identification of certain residues or regions of the Ube4B and LSD 1 antibodies that are preferred locations for substitution, i.e., mutagenesis, is alanine scanning mutagenesis. See Cunningham & Wells, 244 SCIENCE 1081-85 (1989). Briefly, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen. The amino acid locations demonstrating functional sensitivity to the substitutions are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed antibody variants screened for the desired activity.

Substantial modifications in the biological properties of the antibody can be accomplished by selecting substitutions that differ significantly in their effect on, maintaining (i) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (ii) the charge or hydrophobicity of the molecule at the target site, or (iii) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Conservative substitutions involve exchanging of amino acids within the same class.

Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability, particularly where the antibody is an immunoglobulin fragment such as an Fv fragment.

Another type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. Generally, the resulting variant(s), i.e., functional equivalents as defined above, selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants is by affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of Ml 3 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed.

In order to identify candidate hypervariable region sites for modification, alanine- scanning mutagenesis may be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antibody-antigen complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

It may be desirable to modify the antibodies of the present invention, i.e., create functional equivalents, with respect to effector function, e.g., so as to enhance antigen- dependent cell-mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of an antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). Caron et al, 176 J. EXP MED. 1191-95 (1992); Shopes, 148 J. IMMUNOL. 2918-22 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al, 53

CANCER RESEARCH 2560-65 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. Stevenson et al, 3 ANTI-CANCER DRUG DESIGN 219-30 (1989).

To increase the serum half-life of an antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an immunoglobulin fragment) as described in, for example, U.S. Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

Polynucleotide molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-Ube4B and LSD1 antibodies of the present invention.

VI. Pharmaceutical Compositions and Administration

Accordingly, a pharmaceutical composition of the present invention may comprise an effective amount of a Ube4B and/or LSD1 inhibitor. As used herein, the term "effective," means adequate to accomplish a desired, expected, or intended result. More particularly, an "effective amount" or a "therapeutically effective amount" is used interchangeably and refers to an amount of a Ube4B and/or LSD1 inhibitor, perhaps in further combination with yet another therapeutic agent, necessary to provide the desired "treatment" (defined herein) or therapeutic effect, e.g., an amount that is effective to prevent, alleviate, treat or ameliorate symptoms of a disease or prolong the survival of the subject being treated. In particular embodiments, the pharmaceutical compositions of the present invention are administered in a therapeutically effective amount to treat patients suffering from a protein conformational disease. As would be appreciated by one of ordinary skill in the art, the exact low dose amount required will vary from subject to subject, depending on age, general condition of the subject, the severity of the condition being treated, the particular compound and/or composition administered, and the like. An appropriate "therapeutically effective amount" in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.

The pharmaceutical compositions of the present invention are in biologically compatible form suitable for administration in vivo for subjects. The pharmaceutical compositions can further comprise a pharmaceutically acceptable carrier. The term

"pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a Ube4B and/or LSD1 inhibitor is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water may be a carrier when the pharmaceutical composition is administered orally. Saline and aqueous dextrose may be carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may be employed as liquid carriers for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried slim milk, glycerol, propylene, glycol, water, ethanol and the like. The pharmaceutical composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

The pharmaceutical compositions of the present invention can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. In a specific embodiment, a pharmaceutical composition comprises an effective amount of a Ube4B and/or LSD1 inhibitor together with a suitable amount of a pharmaceutically acceptable carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

The pharmaceutical compositions of the present invention may be administered by any particular route of administration including, but not limited to oral, parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intraosseous, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, iontophoretic means, or transdermal means. Most suitable routes are oral administration or injection. In certain embodiments, subcutaneous injection is preferred.

In general, the pharmaceutical compositions comprising a Ube4B and/or LSD1 inhibitor may be used alone or in concert with other therapeutic agents at appropriate dosages defined by routine testing in order to obtain optimal efficacy while minimizing any potential toxicity. The dosage regimen utilizing a pharmaceutical composition of the present invention may be selected in accordance with a variety of factors including type, species, age, weight, sex, medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular pharmaceutical composition employed. A physician of ordinary skill can readily determine and prescribe the effective amount of the pharmaceutical composition (and potentially other agents including therapeutic agents) required to prevent, counter, or arrest the progress of the condition.

Optimal precision in achieving concentrations of the therapeutic regimen (e.g., pharmaceutical compositions comprising a Ube4B and/or LSD1 inhibitor, optionally in combination with another therapeutic agent) within the range that yields maximum efficacy with minimal toxicity may require a regimen based on the kinetics of the pharmaceutical composition's availability to one or more target sites. Distribution, equilibrium, and elimination of a pharmaceutical composition may be considered when determining the optimal concentration for a treatment regimen. The dosages of a pharmaceutical composition disclosed herein may be adjusted when combined to achieve desired effects. On the other hand, dosages of the pharmaceutical compositions and various therapeutic agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either was used alone.

In particular, toxicity and therapeutic efficacy of a pharmaceutical composition disclosed herein may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index and it may be expressed as the ratio LD50/ED50. Pharmaceutical compositions exhibiting large therapeutic indices are preferred except when cytotoxicity of the composition is the activity or therapeutic outcome that is desired. Although pharmaceutical compositions that exhibit toxic side effects may be used, a delivery system can target such compositions to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. Generally, the pharmaceutical compositions of the present invention may be administered in a manner that maximizes efficacy and minimizes toxicity.

Data obtained from cell culture assays and animal studies may be used in formulating a range of dosages for use in humans. The dosages of such compositions lie preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any composition used in the methods of the invention, the therapeutically effective dose may be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (the concentration of the test composition that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information may be used to accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

Moreover, the dosage administration of the compositions of the present invention may be optimized using a pharmacokinetic/pharmacodynamic modeling system. For example, one or more dosage regimens may be chosen and a pharmacokinetic/pharmacodynamic model may be used to determine the pharmacokinetic/pharmacodynamic profile of one or more dosage regimens. Next, one of the dosage regimens for administration may be selected which achieves the desired pharmacokinetic/pharmacodynamic response based on the particular pharmacokinetic/pharmacodynamic profile. See WO 00/67776, which is entirely expressly incorporated herein by reference.

Without further elaboration, it is believed that one skilled in the art, using the preceding description, can utilize the present invention to the fullest extent. The following examples are illustrative only, and not limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and/or methods described and claimed herein are made and evaluated, and are intended to be purely illustrative and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for herein. Unless indicated otherwise, parts are parts by weight, temperature is in degrees Celsius or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component

concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.

Materials and Methods

DNA Plasmids. For mammalian expression, SOD1 and TDP-43 were expressed in pEF-BOS and pRK5-Myc, respectively, as previously described (Ketteler and Seed, 2008; Ketteler et al, 2008; Wang et al., 2003a; Zhang et al, 2011). The UBE4B (TF308519) and LSD1 shRNA (TF316984) plasmids and the scrambled control (cat #: TR30015) were from Origene. The p53 shRNA plasmid pLVTH-sip53, and control pLVTH were from D. Trono (Addgene #12239) (Wiznerowicz and Trono, 2003). The p53 transcriptional reporter PG13- Luc, was a generous gift from B. Vogelstein (el-Deiry et al, 1993). The autophagy luciferase release plasmids Act-LC3-Gluc and Act-Glue were kindly provided by B. Seed (Ketteler and Seed, 2008), and the control pCMV-SEAP was from A. Cochrane, (Addgene #24595). For transgenic C. elegans, ufd-2 and spr-5 complementary DNAs (cDNAs) were cloned into a vector under the control of an snb-1 promoter, as previously described (Zhang et al., 2011). Additional information on the shRNA targeting sequences and vectors is given in the Supplemental Materials and Methods.

C. elegans Strains. Suppressor Screen, and Mutation Identification. The Bristol N2 C. elegans strain was used in all experiments unless otherwise specified. A list of C. elegans strains is given in the Supplemental Materials and Methods. Transgenic lines were generated according to standard procedures by injecting 20 μg/ml of expression plasmid DNA into hermaphrodite gonads. For the suppressor screen, worms were mutagenized with 47 mM ethyl methanesulfonate, and a semi-clonal strategy was used with five P0 worms in one plate. Suppressors were visually selected based on strong recovery in the movement phenotype in the F2 generation. The suppressor mutations were mapped by using single nucleotide polymorphism markers in the Hawaiian strain and then identified by whole-genome deep sequencing, followed by Sanger sequencing validations (see Supplemental Materials and Methods). C. elegans Locomotor Assay and Microscopy. The C. elegans strains were observed stereoscopically and their motility was quantified by the thrashing assay (Zhang et al, 2011): Animals were transferred from the feeding plate into M9 buffer (3 mg/ml KH2PO4, 6 mg/ml Na2HP04, 5 mg/ml NaCl and 1 mM MgS04). After 1 min of adaptation, the number of body bends or thrashes was counted for 1 min as an index of the locomotor phenotype. A thrash was counted when both the head and the tail bent away from the anteroposterior axis by more than 45^°. Videos of C. elegans locomotion were recorded using a Leica MZ165 fluorescence stereoscope. High-magnification imaging was performed on a Zeiss AxioObserver Zl with Apotome, with C. elegans immobilized by 10 mM levamisole.

Mammalian Cell Lines. Transfections. Antibodies, and Drug Treatments. See

Supplemental Materials and Methods.

Protein Aggregation Assay. The protein aggregation assay for C. elegans and mammalian cells was modified from a previously described protocol (Wang et al, 2003a) (see Supplemental Materials and Methods).

Transcriptional Activity Luciferase Assay. After a 72-h knockdown, cells were detached and transfected with firefly luciferase p53 reporter plasmid (PG13-luc), together with a thymidine kinase promoter Renilla luciferase (tk-Rluc) reporter for normalization. At 24 h post transfection, cells were lysed in passive lysis buffer (Promega), and analyzed with the Dual Luciferase Reporter System according to the manufacturer's recommendations (Promega), using an injector-equipped Synergy HI microplate reader (Bio-Tek).

Proteasome Activity Assay. Proteasome assays were performed as described previously (Kisselev and Goldberg, 2005), using the Suc-LLVY-Luciferin substrate for chymotrypsin-like activity of the proteasome (the Proteasome Glo kit, Promega). In brief, cells were detached and washed in DMEM/10, followed by several washes in cold PBS. Proteasome lysis buffer (50 mM Tris-HCl, pH 7.5, 0.025% digitonin, 250 mM sucrose, 5 mM MgCh, 0.5 mM EDTA, 2 mM ATP, and 1 mM DTT) was added to the cells and incubated on ice for 5-10 min. The ly sates were then centrifuged for 15 min at 20,000 g to isolate ("squeeze-out") the cytoplasm containing the proteasomes. The supernatant was transferred to a fresh tube, and equal amounts of protein were used in each assay.

Autophagy Assays. Autophagy was quantified with a Gaussia luciferase release assay

(Ketteler and Seed, 2008; Ketteler et al, 2008), which is based on the ATG4B-induced proteolytic cleavage of an actin-anchored fusion LC3-Gluc fusion protein (FIGS. 1 IB and 11C). ATG4B-induced proteolytic cleavage of LC3 releases the Glue fragment and enables its secretion into the cell medium. The activity of the released Glue in the medium (together with constitutively secreted SEAP) was measured by the Secrete-Pair Dual Luminescence Assay kit (GeneCopoeia). Cells, plated in 12-well dishes, were transfected with Act-LC3- Gluc or control Act-Glue plasmid together with the normalization control, CMV-SEAP. At 24 h after transfection, the DMEM/10 medium was replaced, and 100 μΐ of cell growth medium was withdrawn at 24 h, 48 h, and 72 h. The medium was centrifuged at 6000 g for 5 min to remove detached cells, followed by the luciferase analysis according to the manufacturer's recommendations (GeneCopoeia) using a microplate reader (Synergy HI, Bio-Tek).

For LC3 western blot analysis, cells were lysed in LC3 buffer (50 mM Tris-Cl, pH 8.0, with 1%SDS, 0.5% NP40, 150 mM NaCl, and 5 mM EDTA), and sonicated with a Diagenode Bioruptor device (set on High, 30-sec pulse, 30-sec pause, 7.5 min total).

Microarray Transcriptional Profiling and Quantitative RT-qPCR. Total RNA was isolated from HEK293T cells with the RNeasy Mini kit and analyzed using the Affymetrix human GENE LOST array. The microarray data were managed using the Partek Genomic Suite (Partek Inc., St. Louis) and Spotfire DecisionSite software (TIBCO Software Inc., Palo Alto, CA) and analyzed using Ingenuity Pathways Analysis software (IP A, Ingenuity Systems). In addition to RNA, total protein was isolated from the same samples by acetone precipitation and resolubilizing the flow-through lysates to verify the reduction of the UBE4B and LSD 1 proteins. For quantitative RT-qPCR validations, cDNAs were synthesized with the QuantiTect reverse transcription kit using RNA samples from both (Qiagen). Primers for quantitative RT-qPCR were from PrimerBank (Spandidos et al., 2010). RT-qPCRs were performed on a BioRad thermal cycler with iQ SYBER Green PCR mix (BioRad).

Spinal Motor Neuron Survival Assay. Embryonic Sprague Dawley rat spinal cord cultures and neuronal survival assays were previously described (Mojsilovic-Petrovic et al, 2006) (see Supplemental Materials and Methods).

Statistical Analysis. The p-values for all analyses were obtained using Student's t- tests performed in Excel or GraphPad Prism 6, unless otherwise indicated. For the microarray data, Student's t-test was used to analyze the gene expressions. For the Upstream Regulator Ingenuity Pathway Analysis, Fisher's exact test was used. For the spinal cord motor neuron toxicity assay, a one-way ANOVA with multiple comparison test was used.

Results

Isolation of Suppressors of Mutant SODl-induced Neurotoxicity in C. elegans. To better understand the regulatory mechanisms that mitigate the increased load of misfolded proteins, we conducted a large forward genetic screen for the cellular factors that alleviate such stress and relieve cells from proteotoxic insults. This screen took advantage of a C. elegans model of ALS, in which the neuron-directed overexpression of the ALS-linked, G85R mutant human SOD1 (S0D1^G85R) protein led to its aggregation into misfolded soluble oligomers and larger insoluble aggregates (Bruijn et al, 1997; Wang et al, 2009a; 2009b). Misfolded S0D1^G85R protein is highly toxic, leading to age-dependent synaptic dysfunction, neurodegeneration, and severely impaired movement in the worms (Wang et al, 2009a). This severe locomotor defect allows us to perform a large-scale screen for genes that suppress neurodegeneration and improve worm locomotion. In these experiments, we treated homozygous transgenic S0D1^G85R C. elegans with ethyl methanesulfonate (EMS) to induce genomic mutations, and the mutagenized Po hermaphrodites were allowed to self-reproduce for two generations (FIG. 1 A). Next, in the F2 offspring, which contain both heterozygous and homozygous suppressor mutations, we selected individual C. elegans based on a salient improvement in the locomotion on a background of poorly moving populations. The potential suppressor clones were bred-through until 100% of progeny showed phenotypic improvements and were then subjected to further analysis (FIG. 1A).

After screening >10⁵ haploid genomes, we isolated hundreds of independent strains with markedly improved locomotion. Most of these strains were dismissed upon closer examination because they showed a reduction in the expression of a GFP reporter gene that had been co-injected as an internal reference and expressed independently in the pharynx, suggesting silencing of the transgene cassette. Among the few remaining suppressor strains that survived this test, one particular strain, designated Ml, showed potent suppression of the locomotion defect when compared to the parental S0D1^G85R transgene line, and it reached 76% of the locomotion robustness of the SOD1-WT transgenic line (FIG. IB). Such strong recovery of locomotion was apparently not a consequence of diminished S0D1^G85R transgene expression because S0D1^G85R mRNA and total transgene protein levels were unchanged between the parental and Ml mutant strains (FIG. 1C). Because of its particularly robust suppression of locomotor-defect phenotype, Ml was selected for further analysis and mutation mapping. Further segregation analysis of Ml indicated that more than one genetic locus, in addition to the SOD1 transgene on the Chromosome IV, was linked to the suppressor phenotype, suggesting a rare multigenic suppressor underlying the phenotype.

To map and identify genes responsible for the suppression of the locomotor defect, we carried out single nucleotide polymorphism (SNP) mapping (Davis et al, 2005), which approximately located the chromosomal regions responsible for the Ml phenotype, and subsequently performed whole-genome deep sequencing (Sarin et al, 2008). SNP mapping first localized the Ml suppressor mutations to two linkage regions: a 2.2Mb-interval on Chromosome I and an 8Mb-interval on Chromosome II (FIG. ID). Two rounds of deep sequencing were performed on the Ml strain genomic DNA, attaining 27-fold coverage. When the Ml genomic DNA sequencing data was aligned with the C. elegans reference genome at UCSC, we found over 200 variants in the two linkage regions. Next, we performed deep sequencing of the parental strain carrying only the S0D1^G85R trans gene, with 7.5-fold coverage. Comparison of the parental and Ml genomic sequences indicated that most of the non-reference variants existed prior to the EMS mutagenesis and thus were not responsible for the suppressor phenotype. Thus, our analysis pinpointed two variants as likely candidates for the suppressor mutations in Ml: In the Chromosome I linkage region, there is only one missense mutation, G1937A, resulting in a single amino acid change (R646Q) in the gene suppressor of presenilin 5 (spr-5); and on the Chromosome II, among the few remaining variants is one non-sense mutation, G2472A, which results in a premature stop (W824X) in the gene ubiquitin fusion degradation 2 (ufd-2) (FIGS. ID and IE).

To examine the role of the double mutations ufd-2(W824X) and spr-5 (R646Q) in the suppression of mutant SOD 1 -mediated neurotoxicity, we performed a series of genetic, biochemical, and behavioral analyses, ufd-2 encodes a U-box type ubiquitin ligase, and the W824X mutation results in a truncated protein lacking the C-terminal U-box (FIG. IE), spr-5 encodes a lysine-specific histone demethylase, and the R646Q substitution occurs at a highly conserved residue in the C-terminal portion of an amine oxidase-like (AOL) domain (FIG. IE). While either ufd-2(W824X) or spr-5(R646Q) alone did not recapitulate the strong locomotor defect-suppressing phenotype in the Ml strain, the double mutation ufd- 2(W824X) and spr-5 (R646Q) segregated perfectly with the Ml phenotype.

To confirm ufd-2 and spr-5 as the suppressor genes, we obtained independent null alleles of the two genes: a deletion mutation, ufd-2(tml380), that lacks the C-terminal 4/5ths of the protein (Janiesch et al., 2007) and a non-sense mutation, spr-5(by 134), that results in deletion of the C-terminal half of the protein (Eimer et al, 2002) (FIG. IE). When crossed to the mutant SOD1 strain, the single allele of ufd-2(tml380) provided only a weak, two-fold locomotor improvement, and even less improvement was seen for the single allele of spr- 5(byl34) (FIG. IF). However, combining the alleles of spr-5(byl34) and ufd-2(tml380) completely recapitulated the strong locomotor defect-suppressing phenotype observed in the Ml strain (FIG. IF). Total levels of S0D1^G85R transgene expression were similar among the WT, single-, and double-mutant strains (FIG. 1G), indicating that the suppression phenotype does not result from decreased total transgene expression. This finding, however, does not preclude the possibility that a minor fraction of soluble, oligomeric, and highly toxic SODl^G85R is decreased in the suppressor strains (Zetterstrom et al, 2007). Finally, we found that restoring the function of either ufd-2 or spr-5 alone by expressing transgenic wild-type ufd-2 or spr-5 under a neuron-specific promoter from the synaptobrevin (snb-1) gene completely blocked the protection in the Ml strain (FIG. 1H), indicating again that it was the loss of function in these two genes, and not any other background mutations, that was responsible for the suppressor phenotype. Taken together, these results establish that the synergistic loss of ufd-2 and spr-5 creates a potent novel suppressor of the neurodegenerative phenotypes in the SODl C. elegans model of ALS, which we have termed the spr-5 and ufd-2 dependent neurodegeneration suppressor (SUNS).

Neuron-specific Amelioration of Toxicity and Aggregation of Diverse Misfolded Proteins in the ufd-2;spr-5 Mutant. The improved locomotion of the SUNS mutant C.

elegans could be related to a change in the amount of toxic misfolded proteins. YFP fusions of several aggregating proteins, such as S0D1^G85R-YFP (Wang et al, 2009a), TDP-43^c25-YFP (Zhang et al., 2011), and PolyQ-YFP (Brignull et al., 2006; Morley et al, 2002), are toxic to neurons and muscles, impair the movement of C. elegans, and form large protein aggregates that are easily visualized in live animals. To determine whether the SUNS mutant reduces protein aggregation, we introduced the double-null mutations ufd-2(tml380);spr-5(byl34) into strains that pan-neuronally express S0D1^G85R-YFP, TDP-43^c25-YFP, or PolyQ-YFP. Interestingly, loss of ufd-2 and spr-5 function resulted in a marked reduction in the neuronal protein aggregation when compared to controls. The reduction in the number and intensity of the large aggregates was evident in the change in the fluorescent inclusions in the nerve ring and ventral cord regions of the SUNS-mutant C. elegans (FIGS. 2A and 2B).

To confirm that this reduction in fluorescent inclusions reflects a decrease in the levels of aggregated proteins, we performed a protein aggregation assay by differentially extracting and sedimenting worm lysates into supernatant and pellet fractions. The worm pellet fraction is enriched in sedimentable large SODl protein aggregates, whereas the supernatant fraction contains smaller aggregates and oligomeric species (Wang et al, 2009a; 2009b). In western blot analysis of both supernatant and pellet fractions, we observed a significant decrease in the levels of misfolded S0D1^G85R-YFP or TDP-43^c25-YFP in the ufd- 2(tml380);spr-5(byl34) double mutant when compared with controls, with the reduction more pronounced in the pellet fraction (FIG. 2C). Unlike untagged S0D1^G85R (FIGS. 1C and 1G), the total protein level of S0D1^G85R-YFP was reduced by ufd-2(tml380);spr-5(byl34), consistent with the observation that S0D1^G85R-YFP has a much higher fraction of its protein present in the misfolded and aggregated state than does the untagged S0D1^G85R (Wang et al., 2009a; 2009b). Taken together, these data indicate that the ufd-2;spr-5 double mutations reduce the aggregation of different proteins associated with neurodegeneration, suggesting a wide-spectrum anti-proteotoxicity effect of the suppressor.

To determine whether the anti-proteotoxicity effect in the SUNS mutant in C. elegans is organism-wide or tissue-specific, we compared transgenic strains expressing the aggregate- forming protein PolyQ-YFP in either neurons or body wall muscles (Brignull et al, 2006; Morley et al, 2002) in the presence of the ufd-2(tml380);spr-5(byl34) mutations. Although the PolyQ aggregation was markedly decreased in the neurons, we saw no change in the aggregation profile in the muscle cells carrying the ufd-2;spr-5 mutations (FIG. 2B).

Consistent with the tissue-specific aggregation changes, we found that the neuron-specific PolyQ-induced locomotor defect was substantially improved by the ufd-2;spr-5 mutations, but no such behavioral improvement was observed when the PolyQ aggregates were expressed in the C. elegans muscles (FIG. 2D). Therefore, the attenuation of aggregation resulting from the loss of ufd-2 and spr-5 in C. elegans occurs in neurons but not muscles.

The ufd-2 mutation alone improved SODl^G85R-induced locomotor defects by ~2-fold in the C. elegans thrashing assay (FIG. IF). To determine whether homologs of ufd-2 and spr-5 can modulate proteotoxicity in a different organism, we utilized an independent screen in Drosophila for proteotoxicity modifiers. We used transgenic fly models expressing the ALS-related human TDP-43^{M 7V} or FUS^R521C, which develop photoreceptor degeneration and rough-eye phenotypes (Lanson et al., 2011 ; Ritson et al, 2010), and screened an RNAi library for eye phenotype modifiers. Knockdown of CG9934, a Drosophila homolog of C. elegans ufd-2 and human UBE4B, produced a significant rescue of the TDP-43^{M 7V}-induced rough-eye phenotype, resulting in a smoother eye appearance and increased pigmentation (FIG. 2E, left panel). Similarly, knockdown of the UBE4B homolog CG9934 significantly corrected the pigmentation loss and ommatidial disarray induced by FUS^R521C (FIG. 2E, middle panel). Consistent with the observation that FUS^R521C fly strain had a more severe rough-eye phenotype than the TDP-43^{M 7V} strain, we further observed that knockdown of the fly homolog of C. elegans spr-5 and human LSD1, Su(Var)3-3, also produced a significant rescue of the degenerating eye phenotypes induced by FUS^R521C (FIG. 2E, right panel).

Taken together, these results indicate that the ufd-2- and spr-5 -mediated modulation of proteotoxicity is conserved in Drosophila.

Knockdown of Mammalian UBE4B and LSD1 Enhances Clearance of Misfolded

Proteins. Homologs of ufd-2 and spr-5 are present in all eukaryotes. Ubiquitination factor E4 B (UBE4B) and lysine-specific demethylase 1 (LSD1) are human orthologs of ufd-2 and spr-5, respectively. The human and C. elegans orthologs share 32% and 29% protein sequence identity for ufd-2/UBE4B and spr-5/LSDl, respectively, and all the major protein domains are conserved (FIG. IE). To determine whether UBE4B and LSD1 affect protein aggregation in mammals, we used a protein aggregation assay that was established and most characterized in HEK293 cells (Wang et al, 2003a; 2006). The assay utilizes an aggregation- prone, ALS -linked mutant S0D1^G85R protein as a reporter, which migrates faster on PAGE than its WT counterpart (FIG. 8 A). We knocked down UBE4B and/or LSD1 with multiple RNAi sequences, and analyzed the protein levels of S0D1^G85R in HEK293T cells. Cell lysates were subjected to ultracentrifugation to separate larger aggregates (P, FIG. 3 A) from smaller aggregates and non-aggregated S0D1^G85R (S, FIG. 3A); the WT SOD1 protein remained soluble in all tested conditions, but a significant portion of SOD 1^G85R protein was enriched in the insoluble, large-aggregate pellet fraction. Knockdown of both UBE4B and LSD1 significantly reduced the levels of misfolded or aggregated S0D1^G85R in both supernatant and pellet fractions, to 7% and 5% of the controls, respectively (FIG. 3B). The UBE4B/LSD1 knockdown did not affect the protein levels of WT SOD1. The mutant S0D1^G85R had a much larger fraction of misfolded and aggregated proteins that were sensitive to UBE4B/LSD1 -dependent clearance, similar to S0D1^G85R-YFP in C. elegans (FIGS. 2A and 2C), than does the WT SOD1 protein. In agreement with the observations in C. elegans, we found that the decrease in aggregation was not specific to S0D1^G85R but also occurred with other aggregation-prone proteins, including TDP-43 (FIGS. 8B and 8C), indicative of a general effect on misfolded proteins. Interestingly, single knockdown of either LSD1 or UBE4B also resulted in decreased steady-state levels of S0D1^G85R, with the UBE4B knockdown produced a stronger effect than that of LSD 1 (FIG. 3B). The UBE4B knockdown reduced S0D1^G85R proteins in the pellet or supernatant to 10-20% of the control, whereas the LSD1 knockdown to 50-60% of the control. However the double knockdown produced an even more pronounced decrease in the S0D1^G85R levels, consistent with the synergistic effect between UBE4B and LSD1.

To determine whether this decrease in protein aggregation was a consequence of increased degradation of S0D1^G85R proteins, we performed cycloheximide chase

experiments. Cells were transfected with S0D1^G85R together with either non-targeting control shRNAs or a mix of UBE4B and LSD1 shRNAs, and the clearance of the S0D1^G85R protein was quantified. Cycloheximide (CHX) was used to block de novo translation, and the amount of S0D1^G85R remaining in the supernatant fraction at the indicated time points after the translation block, was determined by SDS/PAGE/western blotting (FIG. 3C). UBE4B and LSDl double-knockdown decreased the half-life of S0D1^G85R from 8.5h to 5h, indicating that increased clearance of the mutant protein underlies the reduction of the protein aggregates (FIGS. 3D and 3E).

Knockdown of UBE4B and LSDl Synergistically Activates p53-mediated

Transcription. To identify the downstream effectors of UBE4B and LSDl in the anti- proteotoxicity pathway, we performed a comprehensive transcriptional analysis using the cell-based SOD1 aggregation model. We treated HEK293T cells with shRNAs knocking down either UBE4B or LSDl alone, or UBE4B and LSDl simultaneously, in the presence of S0D1^G85R aggregates. After UBE4B and LSDl protein levels were confirmed to be knocked down in the same samples, total RNA was isolated and subjected to microarray profiling of the whole human transcriptome (FIG. 9A). In triplicate samples of the three identical knockdown conditions and the non-targeting controls, differentially regulated genes and pathways were analyzed in unbiased approaches to identify those that convey the

UBE4B/LSD1 -mediated activation of protein quality control systems.

The most intriguing observation in our unbiased microarray analysis did not concern individually regulated genes but instead the upstream regulators that elicited changes in a whole pathway or network. By employing the Ingenuity Pathway Analysis (IP A) algorithm to compare the predicted changes in genes targeted by upstream factors and the actual changes in these genes in our microarray profiles, we identified a number of upstream regulators whose downstream targets are significantly changed (z-score >2.0) in UBE4B and LSDl single- or double-knockdowns (FIG. 4A). Among these upstream regulators, only a few were shared by more than one experimental condition, and remarkably, p53 was the only upstream regulator common to all three conditions (FIG. 4A). In the UBE4B and LSDl double-knockdown condition, a large number of p53 target genes were affected, and importantly, a large fraction was changed in the direction that statistically suggests an activation of the p53 transcription factor (FIGS. 4B, 9B and 9C). We examined a sample of eleven p53 target genes and confirmed by RT-qPCR that their expression levels were consistent with the microarray dataset (FIG. 4C). Since UBE4B is a ubiquitin ligase that decreases the stability of p53 (Wu et al, 2011), we examined the total protein levels of p53 in the UBE4B and LSDl double-knockdown cells. We detected significantly increased p53 protein levels in the UBE4B and LSDl double-knockdown cells when compared with the mock-knockdown control, indicating a stabilization of the p53 protein (FIG. 4D). To further confirm that the single- or double-knockdowns of UBE4B and LSDl were activating p53-mediated transcription, we used a reporter construct containing p53- responding elements in the promoter of a firefly luciferase (p53RE-luc). Knockdown of either UBE4B or LSDl increased p53 transcriptional activity. However, the simultaneous knockdown of both UBE4B and LSDl resulted in an even stronger p53 transcriptional activity (FIG. 4E), consistent with the synergistic anti-proteotoxicity effects of knocking down both UBE4B and LSDl . To examine if the increased luciferase activity reflected p53- dependent transcriptional activation, we expressed MDM2, a negative regulator of p53, or β- galactosidase as a control, together with the p53 activity reporter. The introduction of MDM2 significantly reduced p53-dependent transcriptional activation of the luciferase reporter under the UBE4B and LSDl double-knockdown condition (FIG. 10), confirming the specificity of the up-regulation of p53 by UBE4B and LSDl .

LSDl demethylates p53, and loss of LSDl increases dimethyl-K370 form of p53, a post-translational modification specifically recognized and bound by p53 co-activator 53BP1 (Huang et al, 2007). To determine whether the activation of p53 was partially attributable to its enhanced interaction with 53BP1, we co-immunoprecipitated p53 and 53BP1 from HEK293T cells in which LSDl and UBE4B were previously knocked down. An increased amount of 53BP1 was pulled down by an equal amount of p53 protein in the double- knockdown cells when compared to the control, indicating an increased interaction between p53 and its co-activator, 53BP1 (FIG. 4F). In summary, these data demonstrate that p53, as a transcription factor, is significantly elevated and activated by the knockdown of UBE4B and LSDl.

Among the genes that were up-regulated by the UBE4B and LSDl double- knockdown in our microarray data set, there were a few that had been reported to be important for protein quality control, including forkhead box 03 (F OX03 a), FOX04, and proteasome 26S subunit, non-ATPase, 11 (PSMDl 1) (FIG. 9C). FOXOs are a family of transcription factors invoked in protein quality control (Zhang et al, 2011 ; Zhao et al, 2007), and PSMDl 1 is a critical regulator of proteasome activity (Vilchez et al, 2012a; 2012b). It is notable that FOX03a is transcriptionally up-regulated by p53 (Renault et al, 2011), and PSMDl 1 is transcriptionally induced by FOXOs (Vilchez et al, 2012a; 2012b). We confirmed through RT-qPCR that FOX03a, FOX04, and PSMDl 1 were all transcriptionally up-regulated when UBE4B and LSDl are knocked down (FIG. 9D), linking these positive regulators of protein quality control downstream of p53 to the UBE4B- and LSDl -dependent anti-proteotoxicity activity. To examine FOXO transcriptional activity, we co-expressed a constitutively active form, FOX03a-TM, with the FHRE-lucif erase reporter and measured the activity of this particular FOXO member. Analogous to the p53 activation, we found that the FOX03a activity is induced most strongly when both UBE4B and LSDl are simultaneously knocked down (FIG. 4H). Similar results were observed with another FOXO-family member, FOXOl (FIG. 10B). Overall, these data are consistent with the synergistic anti-proteotoxicity effects of knocking down both UBE4B and LSDl .

Knockdown of UBE4B and LSDl Activates Proteasomal Degradation and

Autophagy. To determine whether the enhanced clearance of S0D1^G85R upon the knockdown of UBE4B and LSDl reflects an increase in proteasome-mediated degradation, we examined abundance of several proteasomal subunits. Consistent with our transcriptome analysis that demonstrated an increase in the level of PSMD11 RNA, PSMD11 protein level was significantly increased in the double-knockdown cells (FIG. 5A). Both PSMD11 and PSMD4 (proteasome 26S subunit, non-ATPase, 4) are resident to the 19S regulatory proteasome particle, and we found that PSMD4 protein level was also significantly increased. By comparison, 20Sa3, a component of the 20S core proteasome particle, was only marginally increased.

To determine whether the substantial increase in the quantities of 19S regulatory subunits corresponds to augmented proteasome activity, we measured the chymotrypsin-like proteasome activity in lysates derived from HEK293T cells with single- or double- knockdown of UBE4B and LSDl using a luciferase assay (FIG. 11 A). The proteasomal activity was increased in LSDl or UBE4B single-knockdown cells, but the highest activity was observed in double-knockdown cells (FIG. 5B). This finding is consistent with the results of C. elegans suppressor studies and aggregation assays in mammalian cells, in which both UBE4B and LSDl are required for the maximal suppression. Thus, the knockdown of UBE4B and LSDl significantly increases both the subunit quantity and the activity of proteasomes.

In addition to clearance by the proteasome, we asked whether autophagy is also up- regulated in the UBE4B and LSDl double-knockdown cells. To measure autophagic activity, we employed a Gaussia luciferase (GLuc) release assay (Ketteler and Seed, 2008; Ketteler et al., 2008) that reports the autophagy-dependent ATG4B cleavage of an actin-tethered actin- LC3-GLuc-fusion protein and its subsequent release from the cell into the medium (FIGS. 5B, 1 IB, and 11C). HCT116 cells, which are amenable to this assay, were transfected with shRNA constructs to knock down UBE4B and LSDl, with the LC3-GLuc plasmid used to measure the cleavage of LC3 and constitutively secreted control (secreted embryonic alkaline phosphatase [CMV-SEAP]) for transfecti on/secretion normalization (FIGS. 11B and 11C). The LC3-dependent GLuc activity, measured over a period of 72 h, showed a 2- to 3-fold increase in ATG4B proteolytic activity at the end of the time course, demonstrating the activation of autophagy by UBE4B and the double-knockdown (FIG. 5B). The cells transfected with the non-cleavable, LC3-less fusion, the Act-GLuc construct, showed only background levels of Glue activity, similar to the levels observed in non-transfected cells.

To confirm that autophagy was activated by the UBE4B and LSD1 double- knockdown, we measured LC3-II accumulation by western blotting in cells in which LC3-II recycling was inhibited by 3-methyladenine (3-MA). An increase in LC3-II in the 3-MA inhibited cells was observed, indicating that it is an increased autophagic flux rather than a block in the LC3-II recycling that underlies the LC3 accumulation (FIG. 5C). Together, these data demonstrate that a UBE4B- and LSD 1 -dependent protein quality control pathway similar to that in C. elegans also operates in mammalian cells, since a reduction in these two enzymes synergistically promotes the removal of aggregating proteins through enhanced post-translational quality control systems involving proteasome and autophagy.

p53 Regulates Protein Quality Control. Until now, p53 has not been associated with anti-proteotoxicity activity. p53 has been shown to regulate autophagy, but in conflicting directions (Balch et al., 2008; Levine and Abrams, 2008; Prusiner, 2012). Our microarray analysis and subsequent studies establish a correlation between the activation of p53- mediated transcription and enhanced protein quality control conferred by the knockdown of UBE4B and LSD1 (FIG. 5). It has been demonstrated that p53 is a target of

polyubiquitination by UBE4B and demethylation by LSD1, and each of these functions decreases the p53 activity (Bukau and Horwich, 1998; Ciechanover and Brundin, 2003; Hartl et al, 2011; Huang et al, 2007; Mizushima et al, 2008; Wu et al, 2011). Thus, p53 has emerged as a potential effector that mediates the synergistic action of UBE4B and LSD1 in the anti-proteotoxicity pathway.

To determine whether p53 directly protects against proteotoxicity, we first used small molecule activators of p53 in the cell-based SOD 1^G85R protein aggregation assay. Tenovin-1 is a SIRTl/2 deacetylase inhibitor that promotes p53 K382 acetylation, increasing its stability and activity (Lain et al, 2008; Wolff et al, 2014). CP-31398 is another drug that activates p53 by stabilizing the p53 DNA-binding domain in an active conformation and inhibiting its ubiquitination (Foster, 1999; Parge et al, 1992; Wang et al., 2003b). At various

concentrations of the drugs, both Tenovin-1 and CP-31398 significantly reduced the amount of misfolded and aggregated S0D1^G85R proteins (FIGS. 6A, 12A and 12B). Knocking down p53 via shRNA reversed the suppression of S0D1^G85R protein aggregation by either Tenovin- 1 or CP-31398, confirming that the drugs act through p53 (FIG. 12C).

To investigate the mechanism by which Tenovin-1 and CP-31398 reduced the aggregation and misfolding of S0D1^G85R proteins, we asked whether autophagy was activated by these drug treatments. In agreement with a previous report that CP-31398 activates autophagy (Fiorini et al, 2012; Lindberg et al, 2005; Wang et al, 2002; 2003a), we observed that increasing concentrations of either Tenovin-1 or CP-31398 up-regulated LC-II protein levels (FIG. 12D), confirming the activation of autophagy.

To directly confirm the role of p53 in protein quality control, we tested whether reducing p53 genetically affected the SOD 1^G85R protein aggregation. For this purpose, we performed the aggregation assay in cells in which p53 was reduced by RNAi. We found that partial removal of p53 in HEK293T increased misfolded and aggregated S0D1^G85R in both the supernatant and pellet fractions (FIG. 6B). Next, using a human HCT116 cell line in which p53 was knocked out, we asked how the complete removal of p53 affected the clearance of misfolded S0D1^G85R. Because of a low transfection efficiency in the HCT116 cells, the S0D1^G85R reporter did not produce an appreciable pellet fraction, so we focused on analyzing the supernatant fraction. Unlike the WT SOD1 protein, whose level was not affected by the absence of p53, the S0D1^G85R mutant protein was significantly increased in the p53 knockout cells when compared to the controls, indicating that the endogenous p53 promotes the clearance of misfolded proteins (FIG. 6C).

To determine whether p53 mediates the UBE4B- and LSDl -dependent clearance of the S0D1^G85R aggregates, we knocked down either UBE4B and LSDl together, p53 alone, or all three (UBE4B, LSDl, and p53) and examined the aggregation of S0D1^G85R. We applied both transient and stable shRNA knockdown by creating an inducible, stable HEK293T cell line expressing tetracycline-regulated shRNAs against UBE4B, LSDl, and p53. Both transient and stable knockdown of p53 significantly reversed the aggregate-reducing effects conferred by the UBE4B and LSDl knockdown (FIGS. 7 A and 13 A). This result was confirmed with an independent set of shRNAs against UBE4B and LSDl (FIG. 13B). Taken together, these results demonstrate that p53 is required for the UBE4B- and LSDl -dependent clearance of the S0D1^G85R aggregates, and it acts downstream of UBE4B and LSDl to positively regulate the clearance of misfolded proteins.

To confirm that p53 can modulate proteotoxicity in vivo we used the Drosophila TDP-43^{M 7V} neurodegeneration model as described earlier (FIG. 2E) (Kwiatkowski et al, 2009; Lanson et al., 2011; Neumann et al, 2006; Vance et al, 2009). Either knockdown of p53 by RNAi, or expression of a dominant negative form of Drosophila p53 (p53.R155H) (Bruijn et al, 1997; Ollmann et al, 2000; Wang et al., 2009a; 2009b) with the GMR-Gal4 driver, exacerbated the TDP-43^{M 7V}-induced eye phenotype (FIG. 7B). This aggravation of the phenotype was evident in increased loss of pigmented ommatidia, and in p53 RNAi flies, the appearance of necrotic patches, which were observed at low penetrance (FIG. 7B).

Expression of the dominant negative p53.R155H transgene on its own, in a wild-type background, did not cause any eye phenotype (Ollmann et al, 2000; Wang et al, 2009a). Together, these results indicate that endogenous p53 plays a role in reducing TDP-43^{M 7V} proteotoxicity in the Drosophila eye.

We further tested whether p53 activation would alleviate SODl^G85R-induced neurotoxicity. We employed a well-characterized SOD1 neurotoxicity assay (Davis et al, 2005; Mojsilovic-Petrovic et al, 2006), in which spinal cord primary motor neurons were prepared from rat embryos, maintained on astrocyte monolayers supplemented with neurotrophic factors, and stained with a mature motor neuron marker, the neurofilament H (NF-H) antibody SMI-32. Expression of S0D1^G85R via a neuron-specific herpes simplex virus (HSV) vector induced a significant loss of motor neurons over 5 days in contrast to the HSV-LacZ control (FIG. 7C). When treated with the p53 activator Tenovin-1, the motor neurons showed protection from SODl^G85R-induced proteotoxicity, as compared to the vehicle control. Among the various concentrations tested, 0.8 μΜ of Tenovin-1 produced a significant protection against neurotoxicity with minimal toxicity from the drug itself (FIG. 7C). These results confirm that the activation of p53 provides protection against the toxicity of misfolded proteins in neurons.

Discussion

This study presents a previously unknown pathway that mitigates the toxicity of misfolded proteins by boosting protein quality control systems. Using a C. elegans genetic screen for suppressors of neurotoxicity induced by mutant SOD1, we have identified the SUNS pathway, which is mediated by two conserved genes, ufd-2/UBE4B and spr-5/LSO\. From C. elegans to human cells, inactivation of the highly conserved lysine-modifying enzymes ufd-2/\]BE4B and spr-5/LSD\ is shown to enhance the clearance of misfolded proteins. In mammalian cells, the pathway mediated by UBE4B and LSD1 acts to improve the cellular protein quality control by increasing proteasomal and autophagic activities (FIG. 7D). Although it was initially surprising that loss of ubiquitin ligase UBE4B and lysine- specific demethylase LSD1 protects against proteotoxicity, further results reveal positive downstream effectors including transcription factors, with novel implication of p53 in anti- proteotoxicity activities. Together, these results demonstrate the capacity of a cell to reprogram its protein quality control through transcriptional regulation to defend against proteotoxicity.

A Protein Quality Control Pathway Regulated by the Lysine-modifying Enzymes

UBE4B and LSD1. We isolated the SUNS C. elegans mutant based on the potent suppression of SODl-induced neurotoxicity. The suppressor was found to significantly enhance the removal of misfolded proteins, underscoring the critical role of protein misfolding in SOD 1 -mediated neurodegeneration. The enhanced clearance also applies to other misfolded proteins, such as TDP-43, FUS, and polyglutamine-containing proteins, indicating a general improvement in protein quality control. This rare but strong suppressor requires modulation of only two genes, suggesting that it provides a major protein quality control program with a readily accessible switch. Furthermore, the synergistic cooperation of two genes, ufd-2 or spr-5, points to a common downstream pathway with integrative regulation.

Consistent with the observation that the loss of function of ufd-2 and spr-5 reduces misfolded proteins in C. elegans, inactivation of their mammalian orthologs, UBE4B and LSD1, promotes the clearance of aggregation-prone proteins in human cells, indicating the existence of a protein quality control regulatory mechanism that is functionally conserved across species. Interestingly, both genes encode lysine-modifying enzymes: UBE4B is a U- box type ubiquitin ligase, and LSD1 is a ly sine-specific protein demethylase. Both UBE4B and LSD1 are highly expressed in neurons and essential for early development in mammals (Kaneko et al, 2003; Kaneko-Oshikawa et al, 2005; Sarin et al, 2008; Wang et al, 2007; Zibetti et al., 2010).

In contrast to the conventional notion that ubiquitin ligase promotes protein degradation, our studies indicate that UBE4B negatively affects clearance of misfolded proteins and its down-regulation protects against severe proteotoxicity. In line with our observation that the down-regulation of UBE4B protects against proteotoxicity in the nervous systems of C. elegans and Drosophila, mice with elevated levels of UBE4B show autophagy defects with accumulation of ubiquitin- and p62-positive aggregates in the brain (Janiesch et al., 2007; Susaki et al, 2010). UBE4B forms a complex with an AAA-ATPase p97/VCP to ubiquitinate and degrade specific client proteins (Eimer et al, 2002; Kaneko et al, 2003; Morreale et al, 2009). p97/V CP plays an essential role in handling unfolded proteins such as at endoplasmic reticulum-associated protein degradation (Ye et al, 2001; Zetterstrom et al, 2007), and it was recently linked to familial ALS (Johnson et al, 2010; Wang et al., 2009a). Our findings thus provide a new link between p97/V CP and protein quality control, which is regulated by UBE4B.

The fact that both UBE4B and LSDl are enzymes catalyzing post-translational modifications suggests that their effects on protein quality control can be timely, energy- efficient, and integrative. The synergistic interaction between the two lysine-modifying enzymes, UBE4B and LSDl, also suggests that their downstream pathways converge to influence protein quality control. Consistent with recent studies showing enhancement of protein quality control (Denzel et al, 2014; Jackrel et al, 2014; van Ham et al, 2010; Zhang et al, 2011), the identification of the strong anti-proteotoxic effects mediated by UBE4B and LSDl demonstrates that the plasticity of the cellular protein quality control programs can be substantially augmented to yield overall protection to an organism.

p53 as a Key Switch in Protein Quality Control. Unbiased transcriptome analysis points to p53 as a central regulator of the transcriptional reprograming that mediates the effects of UBE4B and LSDl on protein quality control. Consistent with this observation, p53 has been found to have a number of direct transcriptional targets functioning in protein quality control and neuroprotection, and it also activates additional stress-response transcription factors such as FOXOs (Brignull et al, 2006; Morley et al, 2002; Renault et al, 2011). Interestingly, p53 is elevated in the central nervous system of patients with neurodegenerative conditions such as Alzheimer's disease and ALS (Kitamura et al, 1997; Martin, 2000; Wang et al, 2009a; 2009b). Our observation that the transcription factors mediate the effects of this strong suppressor is reminiscent of other signaling pathways governing protein homeostasis. For example, the heat shock response activates the expression of molecular chaperones and other protein quality control machinery via the master transcription factors the heat shock factors (Morimoto, 1998; Wang et al, 2009a; 2009b). Also, the unfolded protein response promotes the endoplasmic reticulum quality control programs through the activation of a set of the transcription factors, including XBP1, ATF4, and ATF6 (Brignull et al, 2006; Morley et al, 2002; Walter and Ron, 2011). In recurring themes, the post-translational regulation by UBE4B and LSDl activates the p53 transcription factor, which is then capable of eliciting a systematic protective program against proteotoxic stress.

p53 has a well-established role in regulating responses to DNA damage (Lanson et al., 2011; Liu, 2001; Ritson et al., 2010; Smith et al, 1994), and recently, a neuroprotective role of activated DNA damage checkpoint has been demonstrated in a tau-dependent neurodegeneration model (Khurana et al, 2012; Wang et al, 2003a; 2006). Here we propose that p53 is a versatile transcriptional switch that guards against both genotoxicity and proteotoxicity. The specific activity of p53 may be fine-tuned at the post-translational level by upstream regulators such as UBE4B and LSDl . In addition, it is known that p53 promotes apoptosis in cells with irreversible genotoxic damage (Vousden and Prives, 2009; Wu et al, 2011). p53 may also function as a dual regulator in proteotoxicity: It promotes the repair and survival of moderately damaged cells, but turns on cell death pathways in cells whose damage is irreparable. Such duality has been observed for other protein quality control systems, such as the ER stress responses (Huang et al, 2007; Walter and Ron, 2011). Thus p53 could serve as a critical regulator of cellular responses to proteotoxicity by repairing or removing damaged cells.

Taken together, these findings reveal a previously unrecognized pathway that systematically antagonizes the proteotoxicity associated with neurodegenerative diseases, and they point to potential targets for harnessing the protective capacity of the cells'

reprogrammed protein quality control to develop a wide-spectrum anti-proteotoxicity therapeutic strategy.

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Supplemental Materials and Methods

Table 1. C. elegans Strains

Some strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). NBRP:

National Bioresource Project (Japan).

Drosophila Genetics. Flies were reared on standard yeast-agar-commeal medium and crosses were performed at 25°C. Drosophila transgenic strains carrying GAL4-inducible human ALS disease-causing alleles of FUS/TLS and TDP-43 were previously described (Lanson et al, 2011; Ritson et al, 2010). Standard genetic procedures were used to generate the GMR-GAL4/CyO, tub-GAL80; UAS-FUS-hR521C/TM6B, Tb and GMR-GAL4, UAS- hTDP-43-M337V/CyO, tub-GAL80 transgenic strains. The following Drosophila strains were obtained from the Bloomington Stock Center: GAL4-inducible RNAi knockdown of CG9934 (y¹ v¹; P{y^{+t7 7} v^{+tl 8}=TRiP.JF02691 }attP2), Su(Var)3-3 (y¹ sc* v¹; P{y^{+t7 7} v^{+tl 8}=TRiP.HMS00638}attP2), or Dmp53 (y¹ v¹; P{y^{+t7 7} v^{+tl 8}=TRiP.GL01220}attP40), which are the Drosophila orthologs of the human UBE4B, LSD1, or p53, respectively; a GAL4-inducible and dominant negative (DN) form of Dmp53, p53.R155H (y¹ w¹¹¹⁸;

P{w^+mC=UAS-p53.R155H.Ex}2/T(2;3)TSTL, CyO: TM6B, Tb⁺) (Ollmann et al., 2000). The dominant effects of the reduction of CG9934, Su(Var)3-3, or Dmp53, as well as the induction of DN p53.R155H, on the degenerative eye phenotypes of GMR-GAL4;UAS-FUS-hR521C and GMR-GAL4;UAS-hTDP-43-M337V strains were assessed two weeks after the crosses were performed. Qualitative changes in pigmentation, ommatidial structure and glossiness phenotypes were monitored for enhancement or suppression.

Suppressor Mapping and Mutation Identification. The suppressor mutations were assigned to chromosomal locations through linkage mapping using single nucleotide polymorphisms between the wild-type strains N2 Bristol and CB4856 Hawaii. The unique mutations within the mapping intervals were identified by deep-sequencing and comparing the genomes of the Ml suppressor mutant and the parental strain carrying the SOD1 transgene. The genome sequencing data was analyzed with a bioinformatic pipeline containing Bowtie 2 (Langmead and Salzberg, 2012), SAMtools (Li et al, 2009), SnpEff (Cingolani et al, 2012), and the Integrative Genomics Viewer (IGV) (Robinson et al, 2011; Thorvaldsdottir et al, 2013). The identified mutations were confirmed by Sanger sequencing of the PCR-amplified loci. The phenotype-causing mutations were validated by independent alleles of the candidate genes.

Whole Genome Sequencing was performed by the Johns Hopkins Deep Sequencing & Microarray Core Facility. C. elegans genomic DNA was purified using the DNeasy Blood and Tissue Kit (Qiagen). The deep sequencing was carried out on an Illumina HiSeq platform. Bowtie 2 was used to index the C. elegans reference genome and to align the Illumina sequencing reads to this reference. Using Bowtie 2, a Sequence Alignment/Map (SAM) output was obtained and used in subsequent analyses. The SAMtools software package was used to identify variants and call SNPs and INDELS based on the SAM alignment files. Variants were called and written to a VCF (Variant Call Format) file.

SnpEff was then used to annotate the effects of variants on protein coding. Finally, the IGV browser was used to view the variants and the underlying sequence reads.

shRNAs. Gene knockdown in mammalian cells was achieved by transiently expressing shRNA plasmids, or stably expressing doxycycline-inducible shRNA in integrated cell lines when indicated. For the transient knockdown, we used vectors expressing a shRNA with an RFP marker (Origene, pRFP-C-RS), one with an EGFP marker (pLVTH)

(Wiznerowicz and Trono, 2003), or a vector constitutively expressing multiple shRNAs on a single vector backbone (pR4R3-NEO). For the stable knockdown, we constructed the vector pR4R3-TET-PURO and generated stable cell lines as described below.

To generate vectors with multiple shRNA expression modules on the same vector pR4R3-NEO or pR4R3-TET-PURO, we used a multi-fragment Gateway cloning system, which recombines multiple DONR constructs to a DEST plasmid. To make DONR constructs that can link multiple shRNA cassettes in tandem, we modified pP4-PlR, pPl-P2, and pP2R-P3 vectors (a gift from G. Seydoux) to contain different shRNA sequences (inserted by Agel-Hindlll digestion) under the Hl-tet (HI/TO) promoter and thus generate L4Rl-Hl/TO-shRNAi, LlL2-Hl/TO-shRNA₂, and R2L3-Hl/TO-shRNA₃ DONR vectors. The HI/TO promoter itself was derived from pTET-LKO-puro (Wieders chain et al, 2009).

To design DEST vector for constitutive shRNA expression in mammalian cells (pR4R3-NEO), we first amplified the R4-R3 Gateway cassette from the plasmid pCG150 (a gift from G. Seydoux) and inserted it into pcDNA3.1 vector (Invitrogen) by Mfel-BstBI. To generate doxycycline-inducible DEST vector (pR4R3-TET-PURO), we replaced neo gene of pR4R3-NEO with the pkg promoter-TET-Repressor-IRES-Puromycin cassette from the pTET-LKO-puro plasmid. Finally, we recombined two or three Hl/TO-shRNA plasmids into the recipient Destination vector pR4R3-tet-puro to generate final tet-inducible shRNA plasmids with multiple shRNA expression modules.

Stable mammalian cell lines were generated by linearizing the pR4R3-TET-PURO shRNA plasmid, transfecting it into HEK293T cells, and selecting for puromycin-resistant colonies. Clones were further selected for effective knockdown of UBE4B, LSDl and p53 genes upon induction with doxycycline.

Table 2. shRNA Target Sequences

Mammalian Cell Lines. Transfections. and Drug Treatments. HEK293T and HCT1 16 cell lines were grown at 37^°C / 5% CO2 in standard DMEM medium, supplemented with 10% FBS, 2 mM L-glutamine and lx non-essential amino acids (DMEM/10).

Transfections of mammalian cells were performed using Lipofectamine 2000

(Invitrogen), according to the manufacturer's recommendations. For shRNAs, transfections of HEK293T cells were performed by plating 3.2xl 0⁵ cells in 60 mm poly(ethyleneimine) (PEI, 10 μg/ml in PBS, Sigma)-pretreated dishes one day before the transfection. 4 μg of shRNA-encoding plasmids, 350 ng of SOD1 reporter (BOS-SOD1 -G85R), and 10 μΐ Lipofectamine 2000 (Invitrogen) were mixed in 500 μΐ Opti-MEM I (Invitrogen), and applied to cells in 2.5 ml Opti-MEM I. One day post transfection, medium was replaced with DMEM/10. Cells were lysed 72-96 h after the start of transfections for analysis, or transfected with additional reporter plasmids for transcriptional, proteasomal, and autophagic activity assays.

Tenovin-1 (Tocris) was resuspended at 20mM in DMSO, and CP-31398 (Tocris) at

15 mg/ml in water. 3-Methyladenine (Sigma) was resuspended at (10 μΜ) in complete DMEM, by heating to 37^°C and vigorous vortexing. All drugs were diluted in DMEM/10 prior to cell treatments.

Immunoprecipitation. Western Blots, and Antibodies. Equal amounts of proteins were electrophoresed on 15% or 4-20% Tris-Cl gels (Biorad). Proteins were transferred to nitrocellulose and immuno-probed with following antibodies: rabbit anti-GFP (Life

Technologies, 1 :2,000), rabbit anti-SODl-100 (Enzo, 1 :3,000), rabbit-anti-LSD 1 (AbCam, 1 :2,000), mouse anti-UBE4B (BD Transduction Labs, 1 :2,000), mouse anti-p53 DO-1 (Sigma, P6874, 1 :5,000), rabbit anti-p53 7F5 (Cell Signaling Technology, #9282, 1 : 1 ,000), rabbit anti-53BPl (Cell Signaling Technology, #4937, 1 : 1,000), rabbit anti-GAPDH (Pierce, 1 :5,000), mouse anti-Myc 9E10 (DSHB, Univ. of Iowa, 1 :2,000), mouse anti-Actin C4 (Santa Cruz Biotech. 1 :5,000), rabbit anti-LC3 Dl l (Cell Signaling Technology, 1 :2,000), rabbit anti-20Sa3 H-125 (Santa Cruz Biotech. 1 : 1,000), rabbit anti-S5a/PSMD4 (Enzo, 1 : 1,000), and rabbit anti-PSMDl 1 (Bethyl, 1 : 1,000). Proteins were visualized using Li-Cor anti-mouse and anti-rabbit 680 and 800 fluorescent antibodies, and visualized and quantified using Odyssey scanner and Image Studio 2.0 software (Li-Cor). For immunoprecipitation, mock-knockdown or double-knockdown HEK293T cells were lysed for 30 min at 4°C, in IP buffer (50mM Tris pH7.5, 150mM NaCl, 0.5% Triton- X100, 2mM MgCh, lOOU/ml Benzonase (Sigma, E1014), 1 :100 Proteinase Inhibitors (Sigma P8340). Insolubles were removed by centrifugation (21,000g, 30min. 4°C), and supernatants were pre-cleared with agarose-IgG beads, for lh at 4^°C. About 1.7mg of total protein was incubated (~16h, 4°C) with either ΙΟμΙ of rabbit anti-p53 antibody (Cell Signaling

Technology, 7F5) or 10μg of normal rabbit IgG (NeoMarkers, NC-100P). IgGs were captured using magnetic A/G beads (Pierce, #88803), washed 4x10 min with IP buffer, and eluted with boiling in 2x SDS loading buffer. Equal amount were loaded on 4-20% Tris- Glycine gel, transferred and visualized with Li-Cor's 680RD Detection Reagent.

Protein Aggregation Assay. C. elegans strains were collected from NGM feeding plates into M9 buffer and washed five times. Mammalian culture cells grown on 60mm plate were washed two times with cold PBS. C. elegans and mammalian cells were lysed in 200- 300 μΐ of lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA),100 mM NaCl and 0.5% NP-40, 1/lOOth protease inhibitor cocktail (Sigma, P8340) and 25 mM iodoacetamide (Sigma, 16125), sonicated on ice in Diagenode Bioruptor (High, 30 sec pulse, 30 sec pause, 5 min total). Lysates were centrifuged 5-10 min at ~130,000g (25 psi) in Airfuge (Coulter-Beckman), to separate larger pelleted aggregates (PI), from soluble proteins and smaller aggregates (SI). PI pellet was resuspended in lysis buffer and sonicated as described above, except 10 min. After centrifugation (Airfuge, -130,000 g, 5-10 min), pellet (P2) was resuspended in 100 μΐ Urea/SDS buffer (8 M Urea, 5% SDS, 40 mM Tris-Cl pH 6.8, 0.1 mM EDTA), followed by 5 min sonication.

Microarrav Transcriptome Analysis. Total RNA was isolated from 293T cells grown on 60 mm dishes using the RNeasy Mini kit coupled with an on-column genomic DNA digestion (Qiagen). The genomic DNA was further removed using Ambion's Turbo DNA- free kit according to the manufacturer's instructions. Total RNA was used in labeling reactions with the 3' IVT Express labeling kit (Affymetrix). Briefly, double stranded cDNA was synthesized using 100 ng of total RNA, and further used as template to transcriptionally label and linearly amplify cell's total RNA complement. Amplified RNA was fragmented and hybridized to the Affymetrix's human GENE LOST array chip. Fluorescent signals from the hybridized probes were detected using the Affymetrix G3000 GeneArray Scanner, and analysis was performed through the Affymetrix GeneChip Command Console version 3.4 software. The microarray data were managed and analyzed using Partek Genomic Suite (Partek) and Spotfire DecisionSite software (TIBCO Software). For the Gene Ontology analysis, the annotation file for the human genome was downloaded from the website of the Gene Ontology Consortium August, 2011.

The network analysis and upstream regulator analysis were performed using Ingenuity

Pathways Analysis Software (IP A, Ingenuity Systems). For Network and Upstream

Regulators analysis, the microarray data set containing gene identifiers and expression values was uploaded into the application. Each identifier was then mapped to its corresponding gene product in the Ingenuity Knowledge Base. The molecules with expression fold changes above the threshold (≥1.2), and the p-values <0.05, were overlaid onto a global molecular network developed from information contained in the Ingenuity Knowledge Base. The relevant networks of selected molecules were then algorithmically generated based on their connectivity. Similarly, for the Upstream Regulator analysis, experimental, microarray - derived expression patterns were analyzed and compared to the literature-derived expression patterns resulting from activation/inhibition of known, upstream regulatory molecules, such as, transcriptional factors, signal transducers, receptors, or chemical effectors. The probability of significant overlap between microarray-derived and literature-derived sets was set to <0.05. The significant agreement between the literature-predicted versus microarray- derived activation/inhibition states of an upstream regulator, or the z-score, was set >2.0. The raw data of the present microarray analysis is deposited at the Gene Expression Omnibus (GEO) repository (accession # GSE58026).

Spinal Motor Neuron Survival Assay. Spinal cords from embryonic day 15 rats were dissociated and grown on cortical astrocyte monolayers established from 1- to 3-day-old rats. The spinal cord neurons were maintained in astrocyte-conditioned medium with neurotrophic factors, including ciliary neurotrophic factor, cardiotrophin-l, brain-derived neurotrophic factor, neurotrophin 4, and gUal-derived neurotrophic factor (10 ng/ml, Aiomone Labs). At day 13 post-isolation, the spinal cord culture was treated with a test drug or vehicle for 24 h before infection with a neuron-specific HSV vector expressing mutant SOD1 that induces neuronal death. The medium was replaced with drug or vehicle three times per week. At day 5 post-infection, motor neuron survival was measured after cell fixation and immunostaining with a motor neuron-specific antibody against neurofilament H (NF-H), SMI-32 (Covance). The motor neurons were quantified blindly in four random fields of view for each condition. Supplemental References

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Claims

We claim:

1. A method for treating a protein conformational disease comprising the step of administering to a patient an effective amount of a Ube4B inhibitor and a LSDl inhibitor.

2. The method of claim 1, wherein the protein conformational disease comprises a neurodegenerative disease.

3. The method of claim 2, wherein the neurodegenerative disease is Creutzfeldt- Jakob disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, or amyotrophic lateral sclerosis (ALS).

4. The method of claim 1, wherein the inhibitor is a small molecule, an antibody or an inhibitory nucleic acid molecule.

5. The method of claim 3, wherein the inhibitory nucleic acid molecule is an siRNA, shRNA, antisense RNA or a ribozyme.

6. The method of claim 1, further comprising the step of administering a p53 agonist.

7. A method for treating a protein conformational disease comprising the step of administering to a patient an effective amount of a p53 agonist, a Ube4B inhibitor and a LSDl inhibitor.

8. A method for treating a protein conformational disease comprising the step of administering to a patient an effective amount of a p53 agonist.

9. The method of claim 8, further comprising administering an effective amount of a Ube4B inhibitor and/or a LSDl inhibitor.

10. The method of claim 5, wherein the LSDl inhibitor siRNA comprises one or more of SEQ ID NOS: 15-19.

11. The method of claim 5, wherein the Ube4B siRNA inhibitor siRNA comprises one or more of SEQ ID NOS:20-49.

12. The method of claim 5, wherein the LSDl inhibitor shRNA comprises one or more of SEQ ID NOS:4-8.

13. The method of claim 5, wherein the Ube4B shRNA inhibitor shRNA comprises one or more of SEQ ID NOS:9-13.

14. A pharmaceutical composition comprising one or more siRNA encoded by SEQ ID NOS: 15-19 and a pharmaceutical carrier.

15. A pharmaceutical composition comprising one or more siRNA encoded by SEQ ID NOS: 20-49 and a pharmaceutical carrier.

16. A pharmaceutical composition comprising one or more shRNA encoded by SEQ ID NOS:4-8 and a pharmaceutical carrier.

17. A pharmaceutical composition comprising one or more shRNA encoded by SEQ ID NOS: 9- 13 and a pharmaceutical carrier.