COMPOSITIONS AND METHODS FOR TREATING AND PREVENTING

HYPERTENSION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U. S. Provisional Application No.

62/325, 193, filed April 20, 2016, which is hereby incorporated by reference in its entirety.

FIELD

Provided herein are compositions and method for treating or preventing pulmonary hypertension and cancer. In particular, provided herein are compositions, methods, and uses of UCHL1 inhibition/antagonism/reduction for treating and preventing pulmonary hypertension and cancer.

BACKGROUND

Pulmonary hypertension (PH) including pulmonary arterial hypertension (PAH) is an increase of blood pressure in the pulmonary artery, pulmonary vein, or pulmonary capillaries, together known as the lung vasculature, leading to shortness of breath, dizziness, fainting, leg swelling and other symptoms. Pulmonary hypertension can be a severe disease with a markedly decreased exercise tolerance.

The signs/symptoms of pulmonary hypertension are consistent with the following: shortness of breath, chest pain, heartbeat (increased), pain (right side of the chest/abdomen), poor appetite, lightheadedness, swelling (legs/ankles), and cyanosis.

Treatment of pulmonary hypertension is determined by whether the etiology is arterial, venous, hypoxic, thromboembolic, or miscellaneous. The treatment is dependent on the etiology of PH. Current conservative treatment for PAH (Group I defined by World

Health Organization) is to optimize right ventricular function by the use of diuretics, digoxin, oxygen, and /or blood thinners as necessary. Targeted pulmonary vasoactive agents such as prostacyclin therapy, phosphodiesterase 5 inhibitors, and endothelin receptor antagonists are FDA-approved to treat patients with Group I and in some cases, Group IV

(thromoboembolic) PH. Patients with left heart failure or hypoxemic lung diseases (groups II or III pulmonary hypertension) should not routinely be treated with vasoactive agents including prostanoids, phosphodiesterase inhibitors, or endothelin receptor antagonists. To make the distinction between the various etiologies of PH, doctors at a minimum will conduct cardiac catheterization of the right heart, echocardiography, chest CT, a six-minute walk test, and pulmonary function testing. Using Group I-specific PAH treatments for other kinds of pulmonary hypertension in patients with these conditions can potentially harm the patient and waste substantial medical resources.

High dose calcium channel blockers are useful in less than 5% of IP AH patients who are vasoreactive by Swan-Ganz catheter. Unfortunately, calcium channel blockers have been largely misused, being prescribed to many patients with non-vasoreactive PAH, and furthermore, contributing to excess morbidity and mortality. The criteria for vasoreactivity have changed. Only those patients whose mean pulmonary artery pressure falls by more than 10 mm Hg to less than 40 mm Hg with an unchanged or increased cardiac output when challenged with adenosine, epoprostenol, or nitric oxide are considered vasoreactive. Of these, only half of the patients are responsive to calcium channel blockers in the long term.

A number of agents have recently been introduced for primary and secondary PH. The trials supporting the use of these agents have been relatively small, and the only measure consistently used to compare their effectivity is the "6 minute walk test". Many have no data on mortality benefit or time to progression.

The prognosis of Group I pulmonary arterial hypertension has an untreated median survival of 2-3 years from time of diagnosis, with the cause of death usually being right ventricular failure. A recent outcome study of those patients who had started treatment with bosentan (Tracleer) showed that 89% patients were alive at 2 years. With new therapies, survival rates appear to be increasing but prognosis remains dismal. Most of the FDA- approved therapies have significant toxicity and side effects that limit use and do not reverse the vascular remodeling associated with disease development. Hence, additional treatments are needed. SUMMARY

Provided herein are compositions and method for treating or preventing pulmonary hypertension and cancer. In particular, provided herein are compositions, methods, and uses of UCHL1 inhibition/antagonism/reduction for treating and preventing pulmonary hypertension and cancer.

AKT1 plays a critical role in uncontrolled cancer cell proliferation as well as the hyper-proliferative state observed in ECs and smooth muscle cells of the lung vasculature in pulmonary arterial hypertension (Tang et al, Am J Physiol Lung Cell Mol Physiol, 308(2), L208-220 2015). While development of therapies have been ongoing that target inhibition of AKT expression and activation, emerging investigations now have also focused therapeutic discovery in the form of promoting AKT degradation (Chan et al, Cell Biosci, 4(1), 59 2014; Yang, Wu, Wu, & Lin, Cell Cycle, 9(3), 487-497 2010).

A mechanism linking decreased AKT1 protein levels in human pulmonary artery endothelial cells (HPAECs) with GADD45a (well-established demethylating mediator) depletion involving reductions in ubiquitincarboxyl terminalhydrolase l(UCHLl), an established deubiqutinase (DUB), resulting in greater AKT1 protein degradation in model of acute ventilator-induced lung injury (VILI) has been reported (Mitra et al, PLoS One, 9(6), el 00169 201 1). Specifically, loss of GADD45a, in particular, significantly reduced UCHLl expression associated with site-specific UCHLl promoter methylation, resulting in increased levels of site-specific Akt ubiquitination and reduced Akt levels. Based on these acute observations, it was hypothesized that UCHLl deficiency would promote protection from the development of chronic forms of pulmonary vascular disease via sustained reductions in AKT activity.

Both, Gadd45a knockout mice ("epigenetic" model of UCHLl deficiency) and WT mice administered a UCHLl -specific inhibitor, demonstrated reduced right ventricular systolic pressures (RVSP), RV hypertrophy, and pathological and cancerous -like lung vascular remodeling when exposed to chronic hypoxia (a conventional murine model of pulmonary hypertension) compared to their controls. All three of these findings are considered surrogates for severity of PH. Both treatment-exposed conditions also revealed reduced vascular-specific UCHLl expression (predominantly in the endothelial cells, considered the initiating tissue source for the development of PAH) and decreased activated AKTl levels in the lungs. UCHLl inhibitor exposure in vitro in lung vascular endothelial cells (ECs) lead to reduced proliferation and angiogenesis after exposure to VEGF (a proliferative factor and known PH stimuli). UCHLl -Aktl is, therefore, a new candidate pathway in hypoxic PH and UCHLl inhibition represents a therapeutic target in PAH and broadly in hyper-proliferative and pro-angiogenic states such as cancers.

Accordingly, provided herein are methods to reduce UCHLl as well as UCHLl inhibition/antagonism and uses thereof in the treatment and prevention of PAH and cancer. For example, in some embodiments, a method of treating or preventing pulmonary hypertension (PAH) in a subject, comprising: administering a UCHLl inhibitor to the subject is provided. In some embodiments, the UCHLl inhibitor is selected from, for example, a nucleic acid, a small molecule, a peptide, or an antibody. In some embodiments, the small molecule is LDN-57444. In some embodiments, the subject exhibits symptoms of PAH, does not exhibit symptoms of PAH, or is at increased risk for developing PAH. In some embodiments, the method further comprises the step of identifying the presence of a rs5030732 variant in a UCHLl gene in a sample from the subject. In some embodiments, the presence of the mutation is indicative of PAH in the subject.

Further embodiments provide methods and uses of treating or preventing pulmonary hypertension (PAH) in a subject, comprising: a) identifying the presence of a rs5030732 mutation in a UCHLl gene in a sample from the subject; and b) administering a UCHLl inhibitor to the subject when the presence of the mutation is identified.

Further embodiments provide the use of a UCHLl inhibitor to treat or prevent a PAH in a subject in need thereof.

Yet other embodiments provide a method of treating or preventing cancer in a subject, comprising: administering a UCHLl inhibitor to the subject. In some embodiment, the cancer is an endothelial cancer (e.g., breast or renal cancer).

Still other embodiments provide the use of a UCHLl inhibitor to treat or prevent cancer in a subject in need thereof.

Certain embodiments provide a method of characterizing a sample from a subject, comprising: detecting the presence of mutant UCHLl gene in the sample using a reagent that specifically binds to the mutant UCHLl gene but not a wild type UCHLl gene, wherein the mutant UCHLl gene comprises a rs5030732 mutation. In some embodiments, the reagent is one or more of one or more probes that specifically bind to the mutant UCHLl gene but not the wild type UCHLl gene and one or more primers that specifically bind to the mutant

UCHLl gene but not the wild type UCHLl gene. In some embodiments, the presence of the mutant UCHLl gene is indicative of a diagnosis of PAH, an increased risk of dying from PAH, or an increased severity of PAH. In some embodiments, the sample is tissue (e.g., lung tissue, cells, blood, blood products, or urine).

Some embodiments provide a method of treating or preventing pulmonary

hypertension (PAH) or cancer in a subject, comprising: administering a HIF-2a or PDGF inhibitor to the subject.

Also provided herein is a method of preventing UCHLl ubiquitination of downstream targets in a subject (e.g., HIF-Ια, HIF-2a, or PDGF), comprising administering an inhibitor of UCHLl, HIF-Ια, HIF-2a, or PDGF to the subject.

Additional embodiments are described herein. DESCRIPTION OF THE FIGURES

Figure 1 shows increased UCHL1 expression in PAH. A. Western blots display increased UCHL1 levels in both hypoxia- and monocrotaline-exposed rats compared to their respective controls. B. Immunohistochemistry reveals increased vascular-specific staining of UCHL1 in patients with PAH compared to control patients. C. Diagram depicts the time course of the three pre-clinical animal models in the current study and the timing of

LDN57444 administration. D. Peptides from dimer and monomer bands.

Figure 2 shows that UCHL1 inhibition reduces VEGF-induced cell proliferation in vitro. A. After VEGF administration (lOng/mL), quantitative measurements of cell counts reveal reduced HPAEC proliferation over time with co-administration of LDN57444. B.

BrdU incorporation assay reveals reduced cell proliferation in vitro with co-administration of LDN57444 in HPAECs exposed to VEGF. C. Reduced tube formation was observed with HPACEs after 20h treatment with LDN57444 at 5μΜ with in presence of VEGF compared to VEGF alone. D. LDN57444 exposure with VEGF results in significantly lower numbers of node, junction, branching intervals, meshes area and segment compared to VEGF treatment alone. * or p<0.05, **p<0.01, **** pO.0001

Figure 3 shows that UCHL1 inhibition attenuates murine hypoxic PH. WT mice exposed to LDN57444 for two weeks after 3 weeks of exposure to hypoxia reveal reduced (A. and B) RVSP and (C.) RVH compared to WT mice, which received vehicle. D. WT mice, which received LDN57444, demonstrated reduced pulmonary artery medial thickness index compared to WT mice which received vehicle when exposed to hypoxia. Data presented as mean ± SE. Normoxia (n=6-8) for all groups. **** pO.001, .***** pO.00001

Figure 4 shows that UCHL1 inhibition attenuates PH in a rodent PH models.

LDN57444 administration in rats for the last two weeks of a 4-week MCT model reveal reduced (A.) RVSP and (B.) RVH compared to vehicle. Similar patterns of protection were revealed in the H-SU rodent PH model for (C.) RVSP and (D.) RVH. Data below presented as mean ± SE. (n=6-8) for all groups.*** p<0.001,**p<0.01.

Figure 5 shows that UCHL1 inhibition reduces Akt activation and increases Relinked Akt ubiquitination in murine hypoxic PH. A. While total Aktl levels do not change, hypoxia exposure upregulates phospho-Akt protein levels in mouse lungs of WT mice. B.

Both hypoxic exposure and LDN57444 administration up-regulate ubquitinated Aktl protein levels in mouse lungs of WT mice. C. Hypoxic mice samples exposed to LDN57444 revealed higher K48-linked ubiquitinated Aktl protein levels compared to those which received vehicle. Figure 6 shows Gadd45a deficiency associated with reduced UCHL1 and Akt in murine lungs. A. Hypoxia exposure upregulates total and phospho-Akt protein levels in whole mouse lung homogenates of WT mice. B. Western blot from lung homogenates from wild type and Gadd45a ^l~ mice show reduced expression of UCHL1 protein. C.

Immunohistochemistry reveals increased UCHL1 staining in WT mice exposed to chronic hypoxia compared to normoxia mice, which appears to be predominantly within the endothelium.

Figure 7 shows that Gadd45a deletion attenuates murine hypoxic PH. While both WT and Gadd45 ^1' mice exhibit elevated RVSP, Gadd45 ^1' mice demonstrate reduced (A.) RVSP and (B.) RVH compared to WT mice when exposed to hypoxia. C. While both WT and Gadd45a ^l~ mice develop vascular remodeling after exposure to hypoxic PH, Gadd45a ^l~ mice demonstrate reduced pulmonary artery medial thickness index compared to WT mice hypoxic lungs. .**p<0.01, .**p<0.01, **** pO.0001.

Figure 8. SEQ ID NO: l (UCHL1).

Figure 9 shows that UCHL1 inhibition reduces the levels of PAH mediators HIF-2a and PDGF in murine PH. A. Both LDN5744 and chronic hypoxia (4 weeks, 10% Fi02) resulted in increased ubiquintanted Akt levels in whole lungs in mice. B. Hypoxia alone was associated with increased phospo-Akt, PDGF AA and BB levels, while LDN57444 exposure reduced all three proteins in whole lungs. C. While chronic hypoxia increased HIF-2a levels in whole lungs, LDN57444 exposure resulted in reduced levels in nomroxia and hypoxia. D. HIF-2a levels are up-regulated in IP AH lung ECs compared to control cells.

Figure 10 shows that TGF-β induces Snail (A) and Vimentin (B) gene expression levels, both associated with endothelial to mesenchymal transition (EndMT)- a cellular process contributing to the vascular remodeling in PAH, in human lung ECs while

LDN57444 reduces these levels.

Figure 11 shows genotype/phenotype with UCHL1 SNP in PAH. A. Missense coding SNP of UCHL1 is found more frequently in cases of Group I PAH compared to healthy controls. B. CA genotype is associated with increased PA elastance in human PAH compared to CC. C. Relationship between PVRi and PA elastance is influenced by both gender and rs5030732 genotype. DEFINITIONS

To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below:

As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

As used herein, the term "subject suspected of having a disease" refers to a subject that presents one or more symptoms indicative of a disease (e.g., pulmonary hypertension or cancer). A subject suspected of having a disease may also have one or more risk factors. A subject suspected of having disease has generally not been tested for the disease. However, a "subject suspected of having disease" encompasses an individual who has received a preliminary diagnosis but for whom a confirmatory test has not been done or for whom the level or severity of metabolic disease is not known.

As used herein, the term "subject diagnosed with a disease" refers to a subject who has been tested and found to have a disease (e.g., pulmonary hypertension or cancer). As used herein, the term "initial diagnosis" refers to a test result of initial disease that reveals the presence or absence of disease.

As used herein, the term "subject at risk for disease" refers to a subject with one or more risk factors for developing a specific disease (e.g., pulmonary hypertension or cancer). Risk factors include, but are not limited to, gender, age, genetic predisposition, environmental exposure, and previous incidents of disease, preexisting diseases, and lifestyle.

As used herein, the term "non-human animals" refers to all non-human animals including, but not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.

As used herein, the term "cell culture" refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro.

As used herein, the term "eukaryote" refers to organisms distinguishable from

"prokaryotes." It is intended that the term encompass all organisms with cells that exhibit the usual characteristics of eukaryotes, such as the presence of a true nucleus bounded by a nuclear membrane, within which lie the chromosomes, the presence of membrane-bound organelles, and other characteristics commonly observed in eukaryotic organisms. Thus, the term includes, but is not limited to such organisms as fungi, protozoa, and animals (e.g., humans).

As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture. The term "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.

The terms "test compound" and "candidate compound" refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function (e.g., hypertension or cancer). Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present disclosure.

As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present disclosure.

As used herein, the term "effective amount" refers to the amount of a compound (e.g. , a compound described herein) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not limited to or intended to be limited to a particular formulation or administration route.

As used herein, the term "co-administration" refers to the administration of at least two agent(s) (e.g. , UCHL1 inhibitor compound having a structure presented above or elsewhere described herein) or therapies to a subject. In some embodiments, the coadministration of two or more agents/therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various

agents/therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents/therapies are coadministered, the respective agents/therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents/therapies lowers the requisite dosage of a known potentially harmful (e.g. , toxic) agent(s).

As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo, or ex vivo.

As used herein, the term "toxic" refers to any detrimental or harmful effects on a cell or tissue as compared to the same cell or tissue prior to the administration of the toxicant.

DETAILED DESCRIPTION OF THE DISCLOSURE

Provided herein are compositions and method for treating or preventing pulmonary hypertension and cancer. In particular, provided herein are compositions, methods, and uses of UCHL1 inhibition/antagonism/reduction for treating and preventing pulmonary hypertension and cancer.

Pulmonary arterial hypertension (PAH) is a rare and fatal condition characterized by gradual occlusion of the small pulmonary arterioles leading to progressively increased pulmonary vascular resistance (PVR) with worsening right heart failure and death. Although causal mechanisms are unclear, data described herein reveal novel roles for the enzyme ubiquitin carboxy-terminal hydrolase LI (UCHL1) in AKT signaling, a well-known mediator of pulmonary vascular remodeling in PAH. The studies provide further insight into UCHL1 genetics which may contribute to these roles, providing a new therapeutic target in PAH.

Although causal mechanisms are unclear, most studies on serine/threonine kinase Akt in lung vascular remodeling have focused on its expression and activation with little work on its degradation. The critical role of deubiquitination (DUB) activity by ubiquitin carboxy- terminal hydrolase LI (UCHL1) in Akt degradation in various lung pathologies including acute lung injury, lung fibrosis, PAH is previously described or described in Example 1 below. Whole exome sequencing (WES) data in PAH patients also identified a UCHL1 missense coding SNP (rs5030732), known to increase DUB activity (Example 2). Higher minor allele frequencies (MAF=0.29) in Hispanics compared to non-Hispanic Caucasians (MAF=0.18) highlights a link between UCHL1 genetics and Hispanic PAH disparities. Epigenetic regulation of the UCHL1 promoter via a well-known demethylating protein, growth arrest and DNA damage-inducible alpha (GADD45a) has been reported. In fact, while UCHL1 DUB activity regulates a variety of proteins, Akt signaling components (but not Aktl mRNA) were the top most dysregulated pathways from whole lung genome-wide expression profiling in Gadd45a knockout mice26, a model which shows striking reductions in EC-specific Uchll .

PAH is characterized by lung vascular remodeling, sustained vasoconstriction, in situ thrombosis and increased lung vascular wall stiffness, all which directly result in elevated PVR. Histopathology reveals uncontrolled lung vascular smooth muscle (SMC) and endothelial cell (EC) hyperplasia with endothelial-to-mesenchymal transition (EndMT), a process that transforms fully differentiated lung EC to highly proliferative myofibroblasts (myoFB). EndMT remodeling contributes to plexiform lesions, pathognomonic for PAH. Recently, murine lung EC-specific hypoxia-inducible factor (HIF)-2a was reported to be a canonical genetic mediator of obliterative intimal lesions via both EC transcriptomic re- programming and paracrine effects on SMC proliferation. Notably, Akt/mTOR signaling is well-known to regulate HIF-2a downstream. Data described herein connects UCHLl activity to both the development of EndMT and HIF-2a protein regulation, linking its lung EC- specific expression to paracrine SMC pathology.

Chain-specific ubiquitin linkage of proteins regulate a host of cellular processes. In general, lysine K48-linked polyubiquitin chains target proteins for proteolytic destruction. In contrast to proteasomal degradation, K63 -linked chains typically coordinate protein activation and trafficking as demonstrated by Akt in cancer. Linkage-specific Aktl ubiquitination in lung ECs has been shown; whereby Akt K63- linked ubiquitination was associated with its activation while K48-linked ubiquitination was associated with its degradation. In contrast to the paradigm where an individual enzyme has a single function, UCHLl manifests a dual and opposing role as a ligase increasing K63-linked ubiquitination in addition to its known DUB activity. This ligase activity is enhanced by its concentration- dependent dimerization. Based on this linkage-specificity, it is contemplated that both UCHLl -mediated activities (DUB function and ligase ubiquitination) potentiate Akt signaling While dimer-associated K63-linked ubiquitination can further activate Akt, increased monomer associated DUB activity can reduce Akt degradation increasing total Akt protein levels. Based on these additive functions in Akt signaling, increased UCHLl dimer and monomer expression was shown in PAH samples (Example 1). Supporting the overall premise of the overall pathological role of UCHLl in PAH, UCHLl inhibition resulted in increased total Akt ubiquitination, reduced EC/SMC proliferation, and attenuation in rodent PH models. A novel role for growth arrest and DNA-damage-inducible, alpha (GADD45a), a tumor suppressor, has been reported in the regulation of site-specific AKT1 ubiquitination and activation via ubiquitin carboxyl-terminal esterase LI (UCHLl), a deubiquitinase. Given that Aktl is an established pro-survival candidate gene in the development of pulmonary arterial hypertension (PAH), it was contemplated that Gadd45a deficiency may also lead to protection from murine models of hypoxic pulmonary hypertension (PH) via UCHLl - mediated reductions in AKT1 activation.

UCHLl is a member of a gene family whose products hydrolyze small C-terminal adducts of ubiquitin to generate the ubiquitin monomer. Expression of UCHLl was thought to be highly specific to neurons and to cells of the diffuse neuroendocrine system and their tumors. It is abundantly present in all neurons (accounts for 1-2% of total brain protein), expressed specifically in neurons and testis/ovary. But, data as detailed above, also show significant pulmonary endothelial expression of UCHLl, which is a new finding opening the door to novel frontiers in pulmonary research.

The catalytic triad of UCH-Ll contains a cysteine at position 90, an aspartate at position 176, and a histidine at position 161 that are responsible for its hydrolase activity.

Provided herein are UCHLl inhibitors or inhibitor of downstream targets of UCHLl (e.g., HIF-2a or PDGF) inhibitors and their use in treating and preventing PAH and cancer. Further provided herein are methods and compositions for identifying variants (e.g., mutants, e.g., rs5030732 mutations) in UCHLl genes.

I. UCHLl inhibitors

The present disclosure is not limited to particular UCHLl inhibitors. Examples include, but are not limited to, a nucleic acid, a small molecule, peptide, or an antibody.

In some embodiments, the UCHLl inhibitor is a small molecule (e.g., LDN-57444; See e.g., Liu et al. (2003), Discovery of inhibitors that elucidate the role of UCH-Ll activity in the H1299 lung cancer cell line; Chem. Biol, 10 837; Tan et al. (2008), Endoplasmic reticulum stress contributes to the cell death induced by UCH-Ll inhibitor; Mol. Cell.

Biochem, 318 109; Cartier et al. (2009), Regulation of synaptic structure by ubiquitin C- terminal hydrolase LI; J. Neurosci., 29 7857; each of which is herein incorporated by reference in its entirety.). LDN-57444 has the structure

and is commercially available (e.g., from Focus Biomolecules, Plymouth Meeting, PA). Small molecules inhibitors of downstream targets of UCHLl (e.g., HIF-2a or PDGF) are (e.g., Roxadustat (FG-4592) and those described in Mohammad Ali Sadiq et al, Saudi J Ophthalmol. 2015 Oct-Dec; 29(4): 287- 291.

In some embodiments, the UCHLl or UCHLl target inhibitor is a nucleic acid. Exemplary nucleic acids suitable for inhibiting UCHLl (e.g., by preventing expression of UCHLl) include, but are not limited to, antisense nucleic acids, miRNAs, and shRNAs. In some embodiments, nucleic acid therapies are complementary to and hybridize to at least a portion (e.g., at least 5, 8, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) of SEQ ID NO: l (UCHLl mRNA; accession No. KR709885).

In some embodiments, compositions comprising oligomeric antisense compounds, particularly oligonucleotides are used to modulate the function of nucleic acid molecules encoding UCHLl , ultimately modulating the amount of UCHLl expressed. This is accomplished by providing antisense compounds that specifically hybridize with one or more nucleic acids encoding UCHLl . The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds that specifically hybridize to it is generally referred to as "antisense. " The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity that may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of UCHLl . In the context of the present disclosure, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. For example, expression may be inhibited to treat or prevent a metabolic disorder.

In some embodiments, nucleic acids are siRNAs. "RNA interference (RNAi)" is the process of sequence-specific, post-transcriptional gene silencing initiated by a small interfering RNA (siRNA). During RNAi, siRNA induces degradation of target mRNA with consequent sequence-specific inhibition of gene expression.

An "RNA interference," "RNAi," "small interfering RNA" or "short interfering RNA" or "siRNA" or "short hairpin RNA" or "shRNA" molecule, or "miRNA" is a RNA duplex of nucleotides that is targeted to a nucleic acid sequence of interest, for example, SIN3A. As used herein, the term "siRNA" is a generic term that encompasses all possible RNAi triggers. An "RNA duplex" refers to the structure formed by the complementary pairing between two regions of a RNA molecule. siRNA is "targeted" to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene. In certain embodiments, the siRNAs are targeted to the sequence encoding SIN3A. In some embodiments, the length of the duplex of siRNAs is less than 30 base pairs. In some embodiments, the duplex can be 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 or 10 base pairs in length. In some embodiments, the length of the duplex is 19 to 32 base pairs in length. In certain embodiment, the length of the duplex is 19 or 21 base pairs in length. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27 nucleotides in length. In certain embodiments, the loop is 18 nucleotides in length. The hairpin structure can also contain 3' and/or 5' overhang portions. In some embodiments, the overhang is a 3' and/or a 5' overhang 0, 1 , 2, 3, 4 or 5 nucleotides in length.

As used herein, Dicer-substrate RNAs (DsiRNAs) are chemically synthesized asymmetric 25-mer/27-mer duplex RNAs that have increased potency in RNA interference compared to traditional siRNAs. Traditional 21-mer siRNAs are designed to mimic Dicer products and therefore bypass interaction with the enzyme Dicer. Dicer has been recently shown to be a component of RISC and involved with entry of the siRNA duplex into RISC. Dicer-substrate siRNAs are designed to be optimally processed by Dicer and show increased potency by engaging this natural processing pathway. Using this approach, sustained knockdown has been regularly achieved using sub-nanomolar concentrations. (U.S. Pat. No. 8,084,599; Kim et al., Nature Biotechnology 23:222 2005; Rose et al., Nucleic Acids Res., 33:4140 2005).

The transcriptional unit of a "shRNA" is comprised of sense and antisense sequences connected by a loop of unpaired nucleotides. shRNAs are exported from the nucleus by Exportin-5, and once in the cytoplasm, are processed by Dicer to generate functional siRNAs. "miRNAs" stem-loops are comprised of sense and antisense sequences connected by a loop of unpaired nucleotides typically expressed as part of larger primary transcripts (pri- miRNAs), which are excised by the Drosha-DGCR8 complex generating intermediates known as pre-miRNAs, which are subsequently exported from the nucleus by Exportin-5, and once in the cytoplasm, are processed by Dicer to generate functional miRNAs or siRNAs. "Artificial miRNA" or an "artificial miRNA shuttle vector", as used herein interchangeably, refers to a primary miRNA transcript that has had a region of the duplex stem loop (at least about 9-20 nucleotides) which is excised via Drosha and Dicer processing replaced with the siRNA sequences for the target gene while retaining the structural elements within the stem loop necessary for effective Drosha processing. The term "artificial" arises from the fact the flanking sequences (.about.35 nucleotides upstream and .about.40 nucleotides downstream) arise from restriction enzyme sites within the multiple cloning site of the siRNA. As used herein the term "miRNA" encompasses both the naturally occurring miRNA sequences as well as artificially generated miRNA shuttle vectors.

The siRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter. The nucleic acid sequence can also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal

polyadenylation signal or a sequence of six Ts.

The present disclosure contemplates the use of any genetic manipulation for use in modulating the expression of UCHL1 or a downstream target. Examples of genetic manipulation include, but are not limited to, gene knockout (e.g., removing the UCHL1 gene from the chromosome using, for example, recombination), expression of antisense constructs with or without inducible promoters, and the like. Delivery of nucleic acid construct to cells in vitro or in vivo may be conducted using any suitable method. A suitable method is one that introduces the nucleic acid construct into the cell such that the desired event occurs (e.g., expression of an antisense construct).

Introduction of molecules carrying genetic information into cells is achieved by any of various methods including, but not limited to, directed injection of naked DNA constructs, bombardment with gold particles loaded with said constructs, and macromolecule mediated gene transfer using, for example, liposomes, biopolymers, and the like. Preferred methods use gene delivery vehicles derived from viruses, including, but not limited to, adenoviruses, retroviruses, vaccinia viruses, and adeno-associated viruses. Because of the higher efficiency as compared to retroviruses, vectors derived from adenoviruses are the preferred gene delivery vehicles for transferring nucleic acid molecules into host cells in vivo. Adenoviral vectors have been shown to provide very efficient in vivo gene transfer into a variety of solid tumors in animal models and into human solid tumor xenografts in immune-deficient mice. Examples of adenoviral vectors and methods for gene transfer are described in PCT publications WO 00/12738 and WO 00/09675 and U. S. Pat. Appl. Nos. 6,033,908, 6,019,978, 6,001,557, 5,994, 132, 5,994, 128, 5,994,106, 5,981,225, 5,885,808, 5,872,154, 5,830,730, and 5,824,544, each of which is herein incorporated by reference in its entirety.

Vectors may be administered to subject in a variety of ways. For example, in some embodiments of the present disclosure, vectors are administered into tumors or tissue associated with tumors using direct injection. In other embodiments, administration is via the blood or lymphatic circulation (See e.g. , PCT publication 99/02685 herein incorporated by reference in its entirety). Exemplary dose levels of adenoviral vector are preferably 108 to 1011 vector particles added to the perfusate.

In some embodiments, the present disclosure provides antibodies that inhibit UCHL1. Any suitable antibody (e.g. , monoclonal, polyclonal, or synthetic) may be utilized in the therapeutic methods disclosed herein. In some embodiments, the antibodies are humanized antibodies. Methods for humanizing antibodies are well known in the art (See e.g. , U.S. Patents 6,180,370, 5,585,089, 6,054,297, and 5,565,332; each of which is herein incorporated by reference).

In some embodiments, candidate UCHL1 inhibitors are screened for activity (e.g., using the methods described in Example 1 below or another suitable assay).

The present disclosure further provides pharmaceutical compositions (e.g. , comprising the compounds described above). The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g. , by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets.

Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present disclosure may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present disclosure the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.

Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present disclosure. For example, cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and poly cationic molecules, such as polylysine (WO 97/30731), also enhance the cellular uptake of oligonucleotides.

The compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years.

II. Methods of treating and preventing disorders associated with UCHL1

Provided herein are methods of treating and preventing PAH and/or cancer through the inhibition of UCHL1. In some embodiments, the subject exhibits or does not exhibit symptoms of the disease. For example, in some embodiments, UCHL1 inhibitors are administered to a subject found to be at risk for a disorder (e.g., a subject exhibiting one or more markers or symptoms of cancer and/or PAH but not meeting the diagnostic criteria for diagnosis of a disorder).

In some embodiments, the compounds and pharmaceutical compositions described herein are administered in combination with one or more additional agents, treatment, or interventions (e.g., agents, treatments, or interventions useful in the treatment of cancer and/or PAH).

Examples of agents useful in the treatment of PAH include, but are not limited to, bosetan, diuretics, digoxins, blood thinners, prostanoids, phosphodiesterase inhibitors, endothelin antagonists, high dose calcium channel blockers, or surgery to repair/replace the mitral valve or aortic valve.

In some embodiments, UCHL1 inhibitors are used to treat or prevent cancer. A non- limiting exemplary list of these diseases and conditions includes, but is not limited to, glioblastoma, pancreatic cancer, breast cancer, prostate cancer, lymphoma, skin cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small- cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma,

choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma, and the like.

Some embodiments of the present invention provide methods for administering an effective amount of a compound of the invention and at least one additional therapeutic agent (including, but not limited to, chemotherapeutic antineoplastics, apoptosis-modulating agents, antimicrobials, antivirals, antifungals, and anti-inflammatory agents) and/or therapeutic technique (e.g. , surgical intervention, and/or radiotherapies). In a particular embodiment, the additional therapeutic agent(s) is a anticancer agent. In a particular embodiment, the additional therapeutic agent(s) is a radiation therapy.

A number of suitable anticancer agents are contemplated for use in the methods of the present invention. Indeed, the present invention contemplates, but is not limited to, administration of numerous anticancer agents such as: agents that induce apoptosis;

polynucleotides (e.g., anti-sense, ribozymes, siRNA); polypeptides (e.g. , enzymes and antibodies); biological mimetics; alkaloids; alkylating agents; antitumor antibiotics;

antimetabolites; hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., antibodies conjugated with anticancer drugs, toxins, defensins), toxins; radionuclides;

biological response modifiers (e.g., interferons (e.g. , IFN-a) and interleukins (e.g. , IL-2)); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g. , all-trans-retinoic acid); gene therapy reagents (e.g. , antisense therapy reagents and nucleotides); tumor vaccines; angiogenesis inhibitors; proteosome inhibitors: NF-KB modulators; anti-CDK compounds; HDAC inhibitors; and the like. Numerous other examples of chemotherapeutic compounds and anticancer therapies suitable for coadministration with the disclosed compounds are known to those skilled in the art.

In certain embodiments, anticancer agents comprise agents that induce or stimulate apoptosis. Agents that induce apoptosis include, but are not limited to, radiation (e.g. , X- rays, gamma rays, UV); tumor necrosis factor (TNF)-related factors (e.g., TNF family receptor proteins, TNF family ligands, TRAIL, antibodies to TRAIL-R1 or TRAIL-R2); kinase inhibitors (e.g. , epidermal growth factor receptor (EGFR) kinase inhibitor, vascular growth factor receptor (VGFR) kinase inhibitor, fibroblast growth factor receptor (FGFR) kinase inhibitor, platelet-derived growth factor receptor (PDGFR) kinase inhibitor, and Bcr- Abl kinase inhibitors (such as GLEEVEC)); antisense molecules; antibodies (e.g. ,

HERCEPTIN, RITUXAN, ZEVALIN, and AVASTIN); anti-estrogens (e.g. , raloxifene and tamoxifen); anti-androgens (e.g. , flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole, and corticosteroids); cyclooxygenase 2 (COX-2) inhibitors (e.g. , celecoxib, meloxicam, NS-398, and non-steroidal anti -inflammatory drugs (NSAIDs)); antiinflammatory drugs (e.g., butazolidin, DECADRON, DELTASONE, dexamethasone, dexamethasone intensol, DEXONE, HEXADROL, hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, oxyphenbutazone, PEDIAPRED, phenylbutazone, PLAQUENIL, prednisolone, prednisone, PRELONE, and TANDEARIL); and cancer chemotherapeutic drugs (e.g. , irinotecan (CAMPTOSAR), CPT-11, fludarabine (FLUDARA), dacarbazine (DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP- 16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab,

TAXOTERE or TAXOL); cellular signaling molecules; ceramides and cytokines;

staurosporine, and the like.

In still other embodiments, the compositions and methods of the present invention provide a compound of the invention and at least one anti-hyperproliferative or antineoplastic agent selected from alkylating agents, antimetabolites, and natural products (e.g., herbs and other plant and/or animal derived compounds).

Alkylating agents suitable for use in the present compositions and methods include, but are not limited to: 1) nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin); and chlorambucil); 2) ethylenimines and

methylmelamines (e.g. , hexamethylmelamine and thiotepa); 3) alkyl sulfonates (e.g., busulfan); 4) nitrosoureas (e.g. , carmustine (BCNU); lomustine (CCNU); semustine (methyl- CCNU); and streptozocin (streptozotocin)); and 5) triazenes (e.g., dacarbazine (DTIC;

dimethyltriazenoimid-azolecarboxamide).

In some embodiments, antimetabolites suitable for use in the present compositions and methods include, but are not limited to: 1) folic acid analogs (e.g., methotrexate

(amethopterin)); 2) pyrimidine analogs (e.g. , fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorode-oxyuridine; FudR), and cytarabine (cytosine arabinoside)); and 3) purine analogs (e.g. , mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycoformycin)).

In still further embodiments, chemotherapeutic agents suitable for use in the compositions and methods of the present invention include, but are not limited to: 1) vinca alkaloids (e.g., vinblastine (VLB), vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin

(mitomycin C)); 4) enzymes (e.g. , L-asparaginase); 5) biological response modifiers (e.g., interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin (cis-DDP) and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8) substituted ureas (e.g. ,

hydroxyurea); 9) methylhydrazine derivatives (e.g. , procarbazine (N-methylhydrazine;

MIH)); 10) adrenocortical suppressants (e.g., mitotane (ο,ρ'-DDD) and aminoglutethimide); 11) adrenocorticosteroids (e.g. , prednisone); 12) progestins (e.g. , hydroxy progesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 13) estrogens (e.g. , diethylstilbestrol and ethinyl estradiol); 14) antiestrogens (e.g. , tamoxifen); 15) androgens (e.g. , testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g., flutamide): and 17) gonadotropin-releasing hormone analogs (e.g. , leuprolide).

Any oncolytic agent that is routinely used in a cancer therapy context finds use in the compositions and methods of the present invention. For example, the U.S. Food and Drug Administration maintains a formulary of oncolytic agents approved for use in the United States. International counterpart agencies to the U.S.F.D.A. maintain similar formularies. Table 1 provides a list of exemplary antineoplastic agents approved for use in the U.S. Those skilled in the art will appreciate that the "product labels" required on all U.S. approved chemotherapeutics describe approved indications, dosing information, toxicity data, and the like, for the exemplary agents.

Table 1

Anastrozole Arimidex AstraZeneca

(1,3-Benzenediacetonitrile, a, a, a', a'- Pharmaceuticals, LP, tetramethyl-5-(lH-l ,2,4-triazol-l -ylmethyl)) Wilmington, DE

Arsenic trioxide Trisenox Cell Therapeutic, Inc.,

Seattle, WA

Asparaginase Elspar Merck & Co., Inc.,

(L-asparagine amidohydrolase, type EC-2) Whitehouse Station, NJ

BCG Live TICE Organon Teknika, Corp.,

(lyophilized preparation of an attenuated strain BCG Durham, NC

of Mycobacterium bovis {Bacillus Calmette- Gukin [BCG], substrain Montreal)

bexarotene capsules Targretin Ligand Pharmaceuticals

(4-[l-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl- 2-napthalenyl) ethenyl] benzoic acid)

bexarotene gel Targretin Ligand Pharmaceuticals

Bleomycin Blenoxan Bristol-Myers Squibb Co.,

(cytotoxic glycopeptide antibiotics produced by e NY, NY

Streptomyces verticillus; bleomycin A ₂ and

bleomycin B ₂ )

Capecitabine Xeloda Roche

(5'-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]- cytidine)

Carboplatin Paraplatin Bristol-Myers Squibb

(platinum, diammine [1,1- cyclobutanedicarboxylato(2-)-0, 0']-,(SP-4-2))

Carmustine BCNU, Bristol-Myers Squibb

(l,3-bis(2-chloroethyl)-l -nitrosourea) BiCNU

Carmustine with Polifeprosan 20 Implant Gliadel Guilford Pharmaceuticals,

Wafer Inc., Baltimore, MD

Celecoxib Celebrex Searle Pharmaceuticals,

(as 4-[5-(4-methylphenyl)-3- (trifluoromethyl)- England

lH-pyrazol-l-yl]

benzenesulfonamide) Chlorambucil Leukeran GlaxoSmithKline

(4-[bis(2chlorethyl)amino]benzenebutanoic

acid)

Cisplatin Platinol Bristol-Myers Squibb (PtCl ₂ H ₆ N ₂ )

Cladribine Leustatin, R.W. Johnson

(2-chloro-2'-deoxy-b-D-adenosine) 2-CdA Pharmaceutical Research

Institute, Raritan, NJ

Cyclophosphamide Cytoxan, Bristol-Myers Squibb

(2-[bis(2-chloroethyl)amino] tetrahydro-2H- Neosar

13,2-oxazaphosphorine 2-oxide monohydrate)

Cytarabine Cytosar- Pharmacia & Upjohn

(1-b-D-Arabinofuranosylcytosine, C ₉ H1 ₃ N ₃ O5) U Company

cytarabine liposomal DepoCyt Skye Pharmaceuticals,

Inc., San Diego, CA

Dacarbazine DTIC- Bayer AG, Leverkusen,

(5-(3,3-dimethyl-l-triazeno)-imidazole-4- Dome Germany

carboxamide (DTIC))

Dactinomycin, actinomycin D Cosmege Merck

(actinomycin produced by Streptomyces n

parvullus, C62H ₈₆ Ni ₂ Oi6)

Darbepoetin alfa Aranesp Amgen, Inc., Thousand (recombinant peptide) Oaks, CA

daunorubicin liposomal DanuoXo Nexstar Pharmaceuticals,

((8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy- me Inc., Boulder, CO a-L-lyxo-hexopyranosyl)oxy] -7,8,9, 10- tetrahydro-6,8, 1 1-trihydroxy-l -methoxy-5, 12- naphthacenedione hydrochloride)

Daunorubicin HC1, daunomycin Cerubidin Wyeth Ayerst, Madison, ((1 S ,3 S )-3-Acetyl-l ,2,3,4,6, l l-hexahydro- e NJ

3,5, 12-trihydroxy- 10-methoxy-6, 11 -dioxo-1 - naphthacenyl 3-amino-2,3,6-trideoxy-(alpha)-L- arabino- hexopyranosyl)oxy] -7,8,9, 10- tetrahydro-6,8,1 l-trihydroxy-8- (hy droxy acetyl)-l -methoxy-5, 12- naphthacenedione hydrochloride)

Epoetin alfa Epogen Amgen, Inc

(recombinant peptide)

Estramustine Emcyt Pharmacia & Upjohn

(estra-l,3,5(10)-triene-3,17-diol(17(beta))-, 3- Company

[bis(2-chloroethyl)carbamate] 17-(dihy drogen

phosphate), disodium salt, monohydrate, or

estradiol 3-[bis(2-chloroethyl)carbamate] 17- (dihy drogen phosphate), disodium salt,

monohydrate)

Etoposide phosphate Etopopho Bristol-Myers Squibb

(4'-Demethylepipodophyllotoxin 9-[4,6-0-(R)- s

ethylidene-(beta)-D-glucopyranoside], 4'- (dihy drogen phosphate))

etoposide, VP- 16 Vepesid Bristol-Myers Squibb

(4'-demethylepipodophyllotoxin 9-[4,6-0-(R)- ethylidene-(beta)-D-glucopyranoside])

Exemestane Aromasin Pharmacia & Upjohn

(6-methy lenandrosta- 1 ,4-diene-3 , 17-dione) Company

Filgrastim Neupogen Amgen, Inc

(r-metHuG-CSF)

floxuridine (intraarterial) FUDR Roche

(2'-deoxy-5-fluorouridine)

Fludarabine Fludara Berlex Laboratories, Inc.,

(fluorinated nucleotide analog of the antiviral Cedar Knolls, NJ agent vidarabine, 9-b -D- arabinofuranosyladenine (ara-A))

Fluorouracil, 5-FU Adrucil ICN Pharmaceuticals, Inc.,

(5-fluoro-2,4(lH,3H)-pyrimidinedione) Humacao, Puerto Rico

Fulvestrant Faslodex IPR Pharmaceuticals, (7-alpha-[9-(4,4,5,5,5-penta Guayama, Puerto Rico fluoropentylsulphinyl) nonyl]estra-l,3,5-(10)- triene-3, 17-beta-diol)

Gemcitabine Gemzar Eli Lilly

(2'-deoxy-2', 2'-difluorocytidine

monohydrochloride (b-isomer))

Gemtuzumab Ozogamicin Mylotarg Wyeth Ay erst (anti-CD33 hP67.6)

Goserelin acetate Zoladex AstraZeneca

Implant Pharmaceuticals

Hydroxyurea Hydrea Bristol-Myers Squibb

Ibritumomab Tiuxetan Zevalin Biogen lDEC, Inc., (immunoconjugate resulting from a thiourea Cambridge MA covalent bond between the monoclonal antibody

Ibritumomab and the linker-chelator tiuxetan

[N-[2-bis(carboxymethyl)amino]-3-(p- isothiocyanatophenyl)- propyl] -[N-[2- bis(carboxymethy l)amino] -2-(methy 1) - ethyl]glycine)

Idarubicin Idamycin Pharmacia & Upjohn

(5, 12-Naphthacenedione, 9-acetyl-7-[(3-amino- Company

2,3,6-trideoxy-(alpha)-L- lyxo - hexopyranosyl)oxy]-7,8,9, 10-tetrahy dro-6,9, 11 - trihydroxyhydrochloride, (7S- cis ))

Ifosfamide IFEX Bristol-Myers Squibb

(3-(2-chloroethyl)-2-[(2- chloroethyl)amino]tetrahydro-2H- 1 ,3,2- oxazaphosphorine 2-oxide)

Imatinib Mesilate Gleevec Novartis AG, Basel,

(4- [(4-Methy 1- 1 -piperaziny l)methy 1] -N- [4- Switzerland methyl-3-[[4-(3-pyridinyl)-2- py rimidiny 1] amino] -phenyl] benzamide

methanesulfonate) Interferon alfa-2a Roferon- Hoffmann-La Roche, Inc., (recombinant peptide) A Nutley, NJ

Interferon alfa-2b Intron A Schering AG, Berlin, (recombinant peptide) (Lyophili Germany

zed

Betaseron

)

Irinotecan HC1 Camptosa Pharmacia & Upjohn

((4S)-4, 1 l -diethyl-4-hydroxy-9-[(4- piperi- r Company

dinopiperidino)carbonyloxy]-lH-pyrano[3', 4':

6,7] indolizino[l,2-b] quinoline-3, 14(4H, 12H)

dione hydrochloride trihydrate)

Letrozole Femara Novartis

(4,4'-(l H- 1 ,2,4 -Triazol- 1 -y lmethy lene)

dibenzonitrile)

Leucovorin Wellcovo Immunex, Corp., Seattle,

(L-Glutamic acid, N[4[[(2amino-5-formyl- rin, WA

1 ,4,5,6,7,8 hexahydro4oxo6- Leucovori

pteridinyl)methyl] amino] benzoyl], calcium salt n

(1 : 1))

Levamisole HC1 Ergamisol Janssen Research

((-)-( S)-2,3,5, 6-tetrahydro-6-phenylimidazo Foundation, Titusville, NJ [2, 1 -b] thiazole monohydrochloride

CiiH ₁₂ N ₂ S HC1)

Lomustine CeeNU Bristol-Myers Squibb

(l-(2-chloro-ethyl)-3-cyclohexyl-l-nitrosourea)

Meclorethamine, nitrogen mustard Mustarge Merck

(2-chloro-N-(2-chloroethyl)-N- n

methylethanamine hydrochloride)

Megestrol acetate Megace Bristol-Myers Squibb

17a( acetyloxy)- 6- methylpregna- 4,6- diene- 3,20- dione

[oxalato(2-)-0,0'] platinum)

Paclitaxel TAXOL Bristol-Myers Squibb

(5B, 20-Epoxy-l,2a, 4,7B, 10B, 13a- hexahydroxytax-1 l-en-9-one 4, 10-diacetate 2- benzoate 13-ester with (2R, 3 S)- N-benzoyl-3- phenylisoserine)

Pamidronate Aredia Novartis

(phosphonic acid (3-amino-l- hydroxypropylidene) bis-, disodium salt,

pentahydrate, (APD))

Pegademase Adagen Enzon Pharmaceuticals,

((monomethoxypoly ethylene glycol (Pegadem Inc., Bridgewater, NJ succinimidyl) 11 - 17 -adenosine deaminase) ase

Bovine)

Pegaspargase Oncaspar Enzon

(monomethoxypoly ethylene glycol succinimidyl

L-asparaginase)

Pegfilgrastim Neulasta Amgen, Inc

(covalent conjugate of recombinant methionyl

human G-CSF (Filgrastim) and

monomethoxypoly ethylene glycol)

Pentostatin Nipent Parke-Davis

Pharmaceutical Co., Rockville, MD

Pipobroman Vercyte Abbott Laboratories,

Abbott Park, IL

Plicamycin, Mithramycin Mithracin Pfizer, Inc., NY, NY

(antibiotic produced by Streptomyces plicatus)

Porfimer sodium Photofrin QLT Phototherapeutics,

Inc., Vancouver, Canada

Procarbazine Matulane Sigma Tau

Thiotepa Thioplex Immunex Corporation

(Aziridine, Ι,Γ,Γ'-phosphinothioylidynetris-, or

Tris (1-aziridinyl) phosphine sulfide)

Topotecan HC1 Hycamtin GlaxoSmithKline

((S)-10-[(dimethylamino) methyl] -4-ethy 1-4,9- dihydroxy-lH-pyrano[3', 4': 6,7] indolizino

[ 1 ,2-b] quinoline-3, 14-(4H, 12H)-dione

monohydrochloride)

Toremifene Fareston Roberts Pharmaceutical

(2-(p- [(Z)-4-chloro- 1 ,2-dipheny 1- 1 -buteny 1] - Corp., Eatontown, NJ phenoxy)-N,N-dimethylethylamine citrate (1 : 1))

Tositumomab, I 131 Tositumomab Bexxar Corixa Corp., Seattle, WA (recombinant murine immunotherapeutic

monoclonal IgG _2a lambda anti-CD20 antibody

(I 131 is a radioimmunotherapeutic antibody))

Trastuzumab Herceptin Genentech, Inc

(recombinant monoclonal IgGi kappa anti- HER2 antibody)

Tretinoin, ATRA Vesanoid Roche

(all-trans retinoic acid)

Uracil Mustard Uracil Roberts Labs

Mustard

Capsules

Valrubicin, N-trifluoroacet ladriamy cin- 14- Valstar Anthra --> Medeva valerate

((2S-cis)-2- [1,2,3,4,6,11-hexahy dro-2,5,12- trihydroxy-7 methoxy-6,l l-dioxo-[[4 2,3,6- trideoxy-3- [(trifluoroacetyl)-amino-a-L-/yxo- hexopy ranosy 1] oxy 1] -2-naphthaceny 1] -2- oxoethyl pentanoate)

Vinblastine, Leurocristine Velban Eli Lilly

Vincristine Oncovin Eli Lilly

Vinorelbine Navelbine GlaxoSmithKline

(3' ,4'-didehydro-4'-deoxy-C- norvincaleukoblastine [R-(R*,R*)-2,3- dihydroxybutanedioate (1 :2)(salt)])

Zoledronate, Zoledronic acid Zometa Novartis

((l-Hydroxy-2-imidazol-l-yl-phosphonoethyl)

phosphonic acid monohydrate)

Anticancer agents further include compounds which have been identified to have anticancer activity. Examples include, but are not limited to, 3-AP, 12-0- tetradecanoylphorbol- 13 -acetate, 17AAG, 852A, ABI-007, ABR-217620, ABT-751, ADI- PEG 20, AE-941, AG-013736, AGRO100, alanosine, AMG 706, antibody G250, antineoplastons, AP23573, apaziquone, APC8015, atiprimod, ATN-161, atrasenten, azacitidine, BB-10901, BCX-1777, bevacizumab, BG00001, bicalutamide, BMS 247550, bortezomib, bryostatin-1, buserelin, calcitriol, CCI-779, CDB-2914, cefixime, cetuximab, CG0070, cilengitide, clofarabine, combretastatin A4 phosphate, CP-675,206, CP-724,714, CpG 7909, curcumin, decitabine, DENSPM, doxercalciferol, E7070, E7389, ecteinascidin 743, efaproxiral, efl ornithine, EKB-569, enzastaurin, erlotinib, exisulind, fenretinide, flavopiridol, fludarabine, flutamide, fotemustine, FR901228, G17DT, galiximab, gefitinib, genistein, glufosfamide, GTI-2040, histrelin, HKI-272, homoharringtonine, HSPPC-96, hul4.18-interleukin-2 fusion protein, HuMax-CD4, iloprost, imiquimod, infliximab, interleukin-12, IPI-504, irofulven, ixabepilone, lapatinib, lenalidomide, lestaurtinib, leuprolide, LMB-9 immunotoxin, lonafamib, luniliximab, mafosfamide, MB07133, MDX- 010, MLN2704, monoclonal antibody 3F8, monoclonal antibody J591, motexafin, MS-275, MVA-MUC1-IL2, nilutamide, nitrocamptothecin, nolatrexed dihydrochloride, nolvadex, NS- 9, 06-benzylguanine, oblimersen sodium, ONYX-015, oregovomab, OSI-774, panitumumab, paraplatin, PD-0325901, pemetrexed, PHY906, pioglitazone, pirfenidone, pixantrone, PS- 341, PSC 833, PXD101, pyrazoloacridine, Rl 15777, RADOOl, ranpirnase, rebeccamycin analogue, rhuAngiostatin protein, rhuMab 2C4, rosiglitazone, rubitecan, S-l, S-8184, satraplatin, SB-, 15992, SGN-0010, SGN-40, sorafenib, SR31747A, ST1571, SU011248, suberoylanilide hydroxamic acid, suramin, talabostat, talampanel, tariquidar, temsirolimus, TGFa-PE38 immunotoxin, thalidomide, thymalfasin, tipifarnib, tirapazamine, TLK286, trabectedin, trimetrexate glucuronate, TroVax, UCN-1, valproic acid, vinflunine,

VNP40101M, volociximab, vorinostat, VX-680, ZD1839, ZD6474, zileuton, and zosuquidar trihydrochloride.

For a more detailed description of anticancer agents and other therapeutic agents, those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician's Desk Reference and to Goodman and Gilman's "Pharmaceutical Basis of Therapeutics" tenth edition, Eds. Hardman et al , 2002.

The present invention provides methods for administering a compound of the invention with radiation therapy. The invention is not limited by the types, amounts, or delivery and administration systems used to deliver the therapeutic dose of radiation to an animal. For example, the animal may receive photon radiotherapy, particle beam radiation therapy, other types of radiotherapies, and combinations thereof. In some embodiments, the radiation is delivered to the animal using a linear accelerator. In still other embodiments, the radiation is delivered using a gamma knife.

The source of radiation can be external or internal to the animal. External radiation therapy is most common and involves directing a beam of high-energy radiation to a tumor site through the skin using, for instance, a linear accelerator. While the beam of radiation is localized to the tumor site, it is nearly impossible to avoid exposure of normal, healthy tissue. However, external radiation is usually well tolerated by animals. Internal radiation therapy involves implanting a radiation-emitting source, such as beads, wires, pellets, capsules, particles, and the like, inside the body at or near the tumor site including the use of delivery systems that specifically target cancer cells (e.g., using particles attached to cancer cell binding ligands). Such implants can be removed following treatment, or left in the body inactive. Types of internal radiation therapy include, but are not limited to, brachy therapy, interstitial irradiation, intracavity irradiation, radioimmunotherapy, and the like.

The animal may optionally receive radiosensitizers (e.g. , metronidazole,

misonidazole, intra-arterial Budr, intravenous iododeoxyuridine (IudR), nitroimidazole, 5- substituted-4-nitroimidazoles, 2H-isoindolediones, [ [(2 -bromoethyl)-amino] methyl] -nitro- lH-imidazole-l-ethanol, nitroaniline derivatives, DNA-affinic hypoxia selective cytotoxins, halogenated DNA ligand, 1,2,4 benzotriazine oxides, 2-nitroimidazole derivatives, fluorine- containing nitroazole derivatives, benzamide, nicotinamide, acridine-intercalator, 5- thiotretrazole derivative, 3-nitro-l,2,4-triazole, 4,5-dinitroimidazole derivative, hydroxylated texaphrins, cisplatin, mitomycin, tiripazamine, nitrosourea, mercaptopurine, methotrexate, fluorouracil, bleomycin, vincristine, carboplatin, epirubicin, doxorubicin, cyclophosphamide, vindesine, etoposide, paclitaxel, heat (hyperthermia), and the like), radioprotectors (e.g. , cysteamine, aminoalkyl dihydrogen phosphorothioates, amifostine (WR 2721), IL-1, IL-6, and the like). Radiosensitizers enhance the killing of tumor cells. Radioprotectors protect healthy tissue from the harmful effects of radiation.

Any type of radiation can be administered to an animal, so long as the dose of radiation is tolerated by the animal without unacceptable negative side-effects. Suitable types of radiotherapy include, for example, ionizing (electromagnetic) radiotherapy (e.g., X-rays or gamma rays) or particle beam radiation therapy (e.g. , high linear energy radiation). Ionizing radiation is defined as radiation comprising particles or photons that have sufficient energy to produce ionization, i.e. , gain or loss of electrons (as described in, for example, U.S.

5,770,581). The effects of radiation can be at least partially controlled by the clinician. In one embodiment, the dose of radiation is fractionated for maximal target cell exposure and reduced toxicity.

In one embodiment, the total dose of radiation administered to an animal is about .01 Gray (Gy) to about 100 Gy. In another embodiment, about 10 Gy to about 65 Gy (e.g. , about 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60 Gy) are administered over the course of treatment. While in some embodiments a complete dose of radiation can be administered over the course of one day, the total dose is ideally fractionated and administered over several days. Desirably, radiotherapy is administered over the course of at least about 3 days, e.g. , at least 5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (about 1-8 weeks). Accordingly, a daily dose of radiation will comprise approximately 1-5 Gy (e.g., about 1 Gy, 1.5 Gy, 1.8 Gy, 2 Gy, 2.5 Gy, 2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy, 4 Gy, 4.2 Gy, or 4.5 Gy), or 1-2 Gy (e.g. , 1.5-2 Gy). The daily dose of radiation should be sufficient to induce destruction of the targeted cells. If stretched over a period, in one embodiment, radiation is not administered every day, thereby allowing the animal to rest and the effects of the therapy to be realized. For example, radiation desirably is administered on 5 consecutive days, and not administered on 2 days, for each week of treatment, thereby allowing 2 days of rest per week. However, radiation can be administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5 days/week, 6 days/week, or all 7 days/week, depending on the animal's responsiveness and any potential side effects. Radiation therapy can be initiated at any time in the therapeutic period. In one embodiment, radiation is initiated in week 1 or week 2, and is administered for the remaining duration of the therapeutic period. For example, radiation is administered in weeks 1-6 or in weeks 2-6 of a therapeutic period comprising 6 weeks for treating, for instance, a solid tumor. Altematively, radiation is administered in weeks 1-5 or weeks 2-5 of a therapeutic period comprising 5 weeks. These exemplary radiotherapy administration schedules are not intended, however, to limit the present invention.

In some embodiments of the present invention, a compound of the invention and one or more therapeutic agents or anticancer agents are administered to an animal under one or more of the following conditions: at different periodicities, at different durations, at different concentrations, by different administration routes, etc. In some embodiments, the compound is administered prior to the therapeutic or anticancer agent (e.g., radiation therapy), e.g. , 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks prior to the administration of the therapeutic or anticancer agent. In some embodiments, the compound is administered after the therapeutic or anticancer agent, e.g. , 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks after the administration of the anticancer agent. In some embodiments, the compound and the therapeutic or anticancer agent are administered concurrently but on different schedules, e.g., the compound is administered daily while the therapeutic or anticancer agent is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In other embodiments, the compound is administered once a week while the therapeutic or anticancer agent is administered daily, once a week, once every two weeks, once every three weeks, or once every four weeks.

IV. Diagnostic and prognostic applications

In some embodiments, the present invention provides a method of characterizing a sample from a subject, comprising: detecting the presence of mutant UCHL1 gene in the sample using a reagent that specifically binds to the mutant UCHL1 gene but not a wild type UCHL1 gene. In some embodiments, the mutant UCHL1 gene comprises a rs5030732 mutation. In some embodiments, the presence of the mutant UCHL1 gene is indicative of a diagnosis or prognosis of PAH (e.g., diagnosis of PAH, an increased risk of dying from PAH, or an increased severity of PAH). In some embodiments, the sample is tissue (e.g., lung tissue, cells, blood, blood products, or urine).

In some embodiments, the presence of a mutation (e.g., rs5030732 mutation) in a UCHL1 gene is used to determine a treatment course of action (e.g., provide more aggressive therapy when the mutation is present). The presence of UCHL1 mutations described herein is detected using a variety of nucleic acid techniques, including but not limited to: nucleic acid sequencing; nucleic acid hybridization; and, nucleic acid amplification.

A variety of nucleic acid sequencing methods are contemplated for use in the methods of the present disclosure including, for example, chain terminator (Sanger) sequencing, dye terminator sequencing, and high-throughput sequencing methods. Many of these sequencing methods are well known in the art. See, e.g., Sanger et al, Proc. Natl. Acad. Sci. USA 74:5463-5467 (1997); Maxam et al, Proc. Natl. Acad. Sci. USA 74:560-564 (1977);

Drmanac, et al, Nat. Biotechnol. 16:54-58 (1998); Kato, Int. J. Clin. Exp. Med. 2: 193-202 (2009); Ronaghi et al, Anal. Biochem. 242: 84-89 (1996); Margulies et al., Nature 437:376- 380 (2005); Ruparel et al, Proc. Natl. Acad. Sci. USA 102:5932-5937 (2005), and Harris et al., Science 320: 106-109 (2008); Levene et al, Science 299:682-686 (2003); Korlach et al, Proc. Natl. Acad. Sci. USA 105: 1 176-1 181 (2008); Branton et al, Nat. Biotechnol.

26(10): 1146-53 (2008); Eid et al, Science 323 : 133-138 (2009); each of which is herein incorporated by reference in its entirety.

Next-generation sequencing (NGS) methods share the common feature of massively parallel, high-throughput strategies, with the goal of lower costs in comparison to older sequencing methods (see, e.g., Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; each herein incorporated by reference in their entirety). NGS methods can be broadly divided into those that typically use template amplification and those that do not. Amplification-requiring methods include pyrosequencing commercialized by Roche as the 454 technology platforms (e.g., GS 20 and GS FLX), the Solexa platform commercialized by Illumina, and the Supported Oligonucleotide Ligation and Detection (SOLiD) platform commercialized by Applied Biosystems. Non-amplification approaches, also known as single-molecule sequencing, are exemplified by the HeliScope platform commercialized by Helicos Biosciences, and emerging platforms commercialized by VisiGen, Oxford Nanopore Technologies Ltd., Life Technologies/Ion Torrent, and Pacific Biosciences, respectively.

Other single molecule sequencing methods include real-time sequencing by synthesis using a VisiGen platform (Voelkerding et al, Clinical Chem., 55: 641-58, 2009; U.S. Pat. No. 7,329,492; U.S. Pat. App. Ser. No. 1 1/671956; U. S. Pat. App. Ser. No. 1 1/781 166; each herein incorporated by reference in their entirety) in which immobilized, primed DNA template is subjected to strand extension using a fluorescently-modified polymerase and florescent acceptor molecules, resulting in detectible fluorescence resonance energy transfer (FRET) upon nucleotide addition.

Illustrative non-limiting examples of nucleic acid hybridization techniques include, but are not limited to, in situ hybridization (ISH), microarray, and Southern or Northern blot. In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand as a probe to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough, the entire tissue (whole mount ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts. Sample cells and tissues are usually treated to fix the target transcripts in place and to increase access of the probe. The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away. The probe that was labeled with either radio-, fluorescent- or antigen-labeled bases is localized and quantitated in the tissue using either

autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts.

In some embodiments, mutations are detected using fluorescence in situ hybridization (FISH). In some embodiments, FISH assays utilize bacterial artificial chromosomes (BACs). These have been used extensively in the human genome sequencing project (see Nature 409: 953-958 (2001)) and clones containing specific BACs are available through distributors that can be located through many sources, e.g. , NCBI. Each BAC clone from the human genome has been given a reference name that unambiguously identifies it. These names can be used to find a corresponding GenBank sequence and to order copies of the clone from a distributor.

The present invention further provides a method of performing a FISH assay on human cells (e.g., lung cells). Specific protocols can be readily adapted for the present invention. Guidance regarding methodology may be obtained from many references including: In situ Hybridization: Medical Applications (eds. G. R. Coulton and J. de

Belleroche), Kluwer Academic Publishers, Boston (1992); In situ Hybridization: In

Neurobiology; Advances in Methodology (eds. J. H. Eberwine, K. L. Valentino, and J. D. Barchas), Oxford University Press Inc., England (1994); In situ Hybridization: A Practical Approach (ed. D. G. Wilkinson), Oxford University Press Inc., England (1992)); Kuo, et al , Am. J. Hum. Genet. 49: 1 12-1 19 (1991); Klinger, et al , Am. J. Hum. Genet. 57 :55-65 (1992); and Ward, et al. , Am. J. Hum. Genet. 52: 854-865 (1993)). There are also kits that are commercially available and that provide protocols for performing FISH assays (available from e.g., Oncor, Inc., Gaithersburg, MD). Patents providing guidance on methodology include U.S. 5,225,326; 5,545,524; 6,121,489 and 6,573,043. All of these references are hereby incorporated by reference in their entirety and may be used along with similar references in the art and with the information provided in the Examples section herein to establish procedural steps convenient for a particular laboratory.

Different kinds of biological assays are called microarrays including, but not limited to: DNA microarrays (e.g., cDNA microarrays and oligonucleotide microarrays); protein microarrays; tissue microarrays; transfection or cell microarrays; chemical compound microarrays; and, antibody microarrays. A DNA microarray, commonly known as gene chip, DNA chip, or biochip, is a collection of microscopic DNA spots attached to a solid surface (e.g. , glass, plastic or silicon chip) forming an array for the purpose of expression profiling or monitoring expression levels for thousands of genes simultaneously. The affixed DNA segments are known as probes, thousands of which can be used in a single DNA microarray. Microarrays can be used to identify mutant UCHL1 genes. Microarrays can be fabricated using a variety of technologies, including but not limiting: printing with fine-pointed pins onto glass slides; photolithography using pre-made masks; photolithography using dynamic micromirror devices; ink-jet printing; or, electrochemistry on microelectrode arrays.

Southern and Northern blotting is used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from a sample is fragmented, electrophoretically separated on a matrix gel, and transferred to a membrane filter. The filter bound DNA or RNA is subject to hybridization with a labeled probe complementary to the sequence of interest. Hybridized probe bound to the filter is detected. A variant of the procedure is the reverse Northern blot, in which the substrate nucleic acid that is affixed to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from a tissue and labeled.

Nucleic acids may be amplified prior to or simultaneous with detection. Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand

displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA). Those of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require that RNA be reversed transcribed to DNA prior to amplification (e.g., RT- PCR), whereas other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g. , the presence or absence of a UCHLlmutation into data of predictive value for a clinician. The clinician can access the predictive data using any suitable means. Thus, in some preferred embodiments, the present invention provides the further benefit that the clinician, who is not likely to be trained in genetics or molecular biology, need not understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.

The present invention contemplates any method capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, information provides, medical personal, and subjects. For example, in some embodiments of the present invention, a sample (e.g. , a biopsy or a serum sample) is obtained from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility, genomic profiling business, etc.), located in any part of the world (e.g. , in a country different than the country where the subject resides or where the information is ultimately used) to generate raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves (e.g. , a urine sample) and directly send it to a profiling center. Where the sample comprises previously determined biological information, the information may be directly sent to the profiling service by the subject (e.g. , an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems). Once received by the profiling service, the sample is processed and a profile is produced (i.e. , mutation data), specific for the diagnostic or prognostic information desired for the subject.

The profile data is then prepared in a format suitable for interpretation by a treating clinician. For example, rather than providing raw expression data, the prepared format may represent a diagnosis or risk assessment (e.g., presence or absence of UCHLl mutations) for the subject, along with recommendations for particular treatment options. The data may be displayed to the clinician by any suitable method. For example, in some embodiments, the profiling service generates a report that can be printed for the clinician (e.g., at the point of care) or displayed to the clinician on a computer monitor. In some embodiments, the information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and/or to convert the raw data to information useful for a clinician or patient. The central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the fate of the data following treatment of the subject. For example, using an electronic communication system, the central facility can provide data to the clinician, the subject, or researchers.

In some embodiments, the subj ect is able to directly access the data using the electronic communication system. The subject may chose further intervention or counseling based on the results. In some embodiments, the data is used for research use. For example, the data may be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or stage of disease or as a companion diagnostic to determine a treatment course of action.

Compositions for use in the diagnostic methods described herein include, but are not limited to, probes, amplification oligonucleotides, and the like. In some embodiments, kits include all components necessary, sufficient or useful for detecting the markers described herein (e.g., reagents, controls, instructions, etc.). The kits described herein find use in research, therapeutic, screening, and clinical applications.

In some embodiments, the present invention provides one or more nucleic acid probes or primers having 8 or more (e.g., 10 or more, 12 or more, 15 or more, 18 or more, etc.) nucleotides, and that specifically bind to mutant UCHL1 but not wild type UCHL1

EXPERIMENTAL

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present disclosure and are not to be construed as limiting the scope thereof.

Example 1

Methods

Reagents. Unless otherwise specified, all reagents were obtained from Sigma- Aldrich (St. Louis, MO). Reagents for SDS-PAGE electrophoresis and western blots supplies were purchased from Thermo Fischer Scientific (Waltham, MA) and Immobilon-P transfer membranes from Millipore (Bedford, MA). Mouse anti-ki-67, anti-UCHLl, anti-Aktl, anti- phospho-Akt (ser473), anti-Ubiquitin rabbit monoclonal antibodies and secondary anti-rabbit horseradish peroxidase (Klionsky DJ, et dk., Autophagy. 2016; 12(l): l-222)-labeled antibody for western blots were all purchased from Cell Signaling Technologies (Boston, MA).

Recombinant VEGF was purchased from Cell Signaling Technologies. LDN57444 was purchased from Calbiochem EMD Millipore (Billercia, MA) and prepared into stock solution in DMSO. iBlot2 premade transfer stack and iBlot2 membrane protein transfer instrument were purchased from Thermo Fischer Scientific.

Murine hypoxic PH model. All experimental protocols were approved by the Animal Care and Use Committee of the University of Arizona. Adult (10-12 weeks in age) male C57BL/6N or Gadd45a ^l~ (obtained from NCI) mice were utilized for all experiments. Exposure to normoxia and chronic hypoxia (5 weeks) was utilized as a model for murine PH as previously described (Tang H, et ?&., Am J Physiol Lung Cell Mol Physiol. 2016). In separate experiments, LDN57444 injections were administered daily starting at week 3 to only WT mice (0.5mg/kg/day, IP) during exposure to both normoxia and hypoxia. Mice were housed in a chamber (Plexiglas) open to room air (normoxia) or to 10% inspired O ₂ fraction for the 5-week duration (for the hypoxia-exposed conditions). Chronic hypoxia was validated via a Pro:Ox sensor (BioSpherix, Redfield, NY) attached to the chamber after administration of nitrogen to reduce the fractional concentration of O ₂ in the inspired gas. At the end of 5 weeks, hemodynamic, histologic, and molecular assessments were completed.

Rodent monocrotaline (MCT) PH model. Sprague-Dawley rats (200gm) were given a single dose of MCT injection (60mg /kg body weight). At the end of 5 weeks,

hemodynamic, histologic, and molecular assessments were completed. In a subset of rats, LDN57444 injections were administered daily starting at week 3 (0.5mg/kg/day, IP) vs vehicle.

Both mice and rats had free access to food and water throughout the duration of the experiments. At the end of the 5-week protocol, assessment of right ventricular systolic pressures (RVSP) were achieved in anesthetized animals (inj ected 125 mg/kg ketamine and 1.25 mg/kg acepromazine mixture IP). RVSP was measured by a catheter (Millar

Instruments) positioned in the RV via the external jugular vein and a 1.4-French pressure transducer (Millar Instruments, SPF 1030). RVSP was recorded and analyzed using the AcgKnowledge software (Biopac Systems, Aero Camino Goleta, CA). RVSP was used as a surrogate for pulmonary arterial systolic pressure. After pressures were recorded, animals were euthanized by exsanguination, and the heart and lungs were removed en bloc. RV hypertrophy (RVH) was determined by the ratio of the weight of the RV wall divided by the sum of the weights of the left ventricle and the septum as previously reported (Tang et al, supra; Moreno-Vinasco L, et al., Physiol Genomics. 2008;33(2):278-91). Lungs were perfused with PBS, removed, and frozen in liquid nitrogen for Western blot and real-time RT-PCR analysis, as well as fixed in a 10% normalized formalin solution overnight for morphometric analysis.

Patient Lung Samples. Paraffin-embedded sections of lung tissue from patients with IP AH and control lungs (died of other non-lung related causes) were obtained from the University of Arizona Pathology Department after approval by the University of Arizona Institutional Review Board.

Histopathological analysis. Formalin-embedded tissues from each animal were cut in paraffin sections (5 μιτι thick) and mounted onto slides. Lung sections for both animal and human slides were stained with hematoxylin and eosin (H & E) for assessment of vascular remodeling. The AxioVisionLE software on Zeiss Brightfield microscope system was used to measure vascular-wall thickness of the small pulmonary arteries. External and internal diameters as well as external and internal areas for each pulmonary artery were measured. The pulmonary arterial wall thickness was calculated by the equation: Thickness = (external vessel area - internal vessel area) ÷ (external vessel area) as previously described (Tang et al., supra). The diameter measured for each pulmonary artery was used to categorize it into two groups: the pulmonary artery with a diameter of less than 50 μιτι and the pulmonary artery with a diameter between 50 μιτι and 100 μιτι.

Western Blotting. Total protein was isolated from human pulmonary artery endothelial cell (HPAEC) or peripheral lung tissue which were lysed in 1 ^χ RIPA buffer (Bio- Rad, Hercules, CA). Protein was loaded on to pre-made 4-12% NuPage or Bolt acrylamide gels from Thermo Fischer scientific, and separated at 150 volts and later transferred to a 0.2μ PVDF membrane from iBlot2 PVDF semi-dry transfer stack using iBlot2 protein transfer equipment and immunoblotted with anti-UCHLl (1 : 1000), anti-phospho-Akt (1 : 1000), and anti-Aktl monoclonal antibody (1 : 1000). Signals were detected using Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The protein levels were normalized to β-Actin and expressed in arbitrary units.

Co-immunoprecipitation (Co-IP). Co-immunoprecipitation of endogenous Aktl was performed using mouse lung tissue homogenate in normoxia or hypoxia mouse either injected with saline or with LDN57444 (0.5mg/kg/day) for 2 weeks. Lung tissues from mice were harvested and homogenate were made in RIPA lysis buffer with halt protease and phosphatase inhibitors (Thermo Fischer Scientific). Lung homogenate were incubated overnight with 2 μg of anti-Aktl antibody in the presence of Protein G beads using a Protein G Immunoprecipitation kit (Thermo Fischer Scientific) and resulting complexes were washed, denatured and eluted according to the manufacturer's protocol.

Cell proliferation. Human pulmonary artery endothelial cells (HPAECs, Lonza,

Walkersville, MD) were cultured using EGM™-2 BulletKit™ from Lonza, which contains 2% FBS, in a humidified atmosphere at 37°C and 5% CO ₂ . The cells were at passage 5-8 when used for the experiments. Cell proliferation was assessed using cell counts. Briefly, lxlO ⁵ HPAEC normal cells were plated on 96-well cell culture plate. After 24 h incubation these cells were exposed to the presence or absence of l Ong/ml vascular endothelial growth factor (VEGF) and/or 5μΜ of LDN57444 for 24-, 48- or 72h. Cell proliferation of HPAECs was confirmed by cell counting using a Bio-Rad Cell counter; similar treatments were administered as described above and cells were trypsinized and re-suspended in medium. A hemacytometer was used to count the total cells in various treatment groups. Additionally, BrdU EC proliferation chemiluminescent assay (Cell Signaling) was also performed according to manufactures' protocol.

Angiogenesis Assay. HPAECs were cultured for 48h to 70% confluency in 100mm cell culture dish in EBM2 -Basal medium with EGM™ 2 Bullet Kit. After mixing an equal volume of basal medium with Gel trex@LDEV -free reduced growth factor basement membrane matrix (Thermo Fischer Scientific), 250 μΐ of this mixture was used to coat the surface of 24 well cell culture plates (Thermo Fischer scientific). The matrix was allowed to gel at 37 C for 15 min at in a humidified atmosphere at 37°C and 5% C02. HPAECs were then washed in calcium and magnesium-free IX PBS twice and were split into single cell suspensions in VEGF-free EBM2 basal medium and mixed with an equal volume of medium containing either vehicle or VEGF [20ng/ml] or VEGF [20ng/ml] plus LDN57444 [ΙΟμΜ] . Tube formation was visualized and captured within 20 hours by EVOS FL cell imaging system (Thermo Fischer Scientific). Image J (NIH) with Angiogenesis Analyzer was used to analyze a total 6 images from four different wells (replicates) to determine number of nodes, junctions, branching intervals, meshes, meshes area and segments.

Immunohistochemistry. Antigen retrieval was carried out by heating sections from mice or human origin in Tris-EDTA (pH = 9) or citrate buffer for 30 min, followed by blocking with 5% Horse serum for 30 min and primary antibody incubation for 1 h at room temperature with UCHL1 (1 ; 1600) or Ki-67 antibody (1 : 1000). This step was followed by incubation (30 min) with anti-rabbit IgG conjugated to a HRP -labeled polymer (Cell Signaling Technologies). Slides were then developed for 10 min with 3,3-diaminobenzidine chromogen and counterstained with hematoxylin.

RT-PCR. RNA from ECs isolated from WT murine lungs exposed to 5 weeks normoxia and hypoxia were extracted using the RNeasy Plus Mini Kit (Qiagen, Germantown, MD). RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Real-time polymerase chain reaction (RT-qPCR) was performed using Taqman assays on a CFX384 Real-Time PCR Detection System (Bio-Rad). Relative mRNA expression levels oiA tl and Gadd45a were normalized to 18S ribosomal RNA and determined by calculating the delta-delta-Ct value, according to manufacturer's guidelines. Differences in gene expression between hypoxic PH and normoxic tissue were calculated using Students t-test, with a -value < 0.05 considered significant.

Statistical analysis. Values are expressed as means ± SE. Intergroup differences were determined by the Student's unpaired /-test, while multiple group differences were assessed by a simple ANOVA with post hoc Tukey analysis used for multiple-comparison test. A value of P < 0.05 was considered statistically significant (GraphPad Software, San Diego, CA).

RESULTS

Based on the role of UCHLl in mediating Aktl degradation in acute lung injury, expression levels of UCHLl were first evaluated in rodent MCT models of PH as well as human lung samples of PAH. Westem blots revealed significantly elevated UCHLl levels in whole lungs of rats exposed to chronic hypoxia as well as monocrotaline models of PH (Figure 1A). In human lung samples, UCHLl staining was stronger in patients with PAH compared to controls and were notably specific to the lung vasculature (prominently endothelial layer) (Figure IB). These data demonstrate localized activity of UCHLl in PAH that may be specific to the vasculature and mostly in the endothelium.

To investigate the functional role of UCHLl in PAH, targeted inhibition of UCHLl activity with LDN57444 was utilized in vitro first in HPAECs. While cells exposed to VEGF demonstrated significantly elevated cell proliferation counts (Figure 2A), administration of LDN57444 significantly reduced VEGF -induced EC proliferation. In parallel, BrdU incorporation in HPAECs were also increased in presence of VEGF and was blocked significantly (Figure 2B) by LDN57444. Additional evidence of its anti-proliferative characteristics were next demonstrated using an angiogenesis tube formation assay. HPAECs exposed to LDN57444 exhibited significantly suppressed VEGF -induced tube formation (Figure 2C) due to formation of lower numbers of node, junction, segment observed in 3D culture (Figure 2D). Cumulatively, these in-vitro data demonstrate the anti-proliferative properties of UCHL1 inhibition in ECs after exposure to VEGF, a well-established PAH stimulus.

To determine the role of UCHL1 inhibition in vivo (Figure 1 C), a conventional 5- week hypoxic murine PH model was assessed. LDN57444 (vs vehicle) administration during the final two weeks to WT mice resulted in significantly reduced RVSP, (pO.0001 , Figure 3A and 3B) and RVH (pO.0001 , Figure 3C) in WT mice. Histologic examination of the lungs revealed WT mice, exposed to LDN-57444, demonstrated a reduced pulmonary artery medial thickness index (Figure 3D) compared to vehicle controls when exposed to hypoxia.

Further therapeutic benefits of UCHL1 inhibition in PAH were evidenced by the use of the MCT and H-SU rodent models of PH. Similar to mice, LDN57444 administration in the last two weeks of MCT-injected rats and in the last 4 normoxic weeks of sugen-injected rats displayed significantly lower RVSP (p <0.001 , Figure 4A and 4C, respectively) and RVH (pO.01 , Figure 4B and 4D, respectively) compared to vehicle controls.

To determine mechanistic contributions to the observed protective effects of UCHL1 inhibition, total and activated Aktl protein levels were assessed based on previous literature supporting the link between UCHL1 and Aktl (Meyer et al, FASEB J. 2009;23(5): 1325-37). Validating previously reported acute observations (Tang H, et al., Am J Physiol Lung Cell Mol Physiol. 2015;308(2):L208-20), chronic hypoxia exposure up-regulated phospho-Akt protein levels with minimal changes to total Aktl levels in whole lungs of WT mice. With UCHL1 inhibition, both total and phospho-Akt protein levels were reduced (Figure 5 A) under normoxia and chronic hypoxia.

Based on the role of UCHL1 in Aktl deubiquitination, co-IP experiments between ubiquitin and Aktl were next evaluated in whole lung homogenates from murine samples. Total ubiquitinated Aktl levels were increased in mice exposed to both chronic hypoxia as well as exposure to LDN57444 compared to normoxia (Figure 5B). Based on these findings, chain-specific modification of Aktl ubiquitination by lysine 48 (K48) was next assessed in these whole lung homogenates. Protein ubiquitination has been described to serve a variety of functions based on chain-specific modifications with K48 associated with protein degradation and lysine 63 (K63) associated with protein activation. Hypoxic mice samples exposed to LDN57444 revealed higher K48-linked ubiquitinated Aktl protein levels compared to those, which received vehicle (Figure 5C). These samples did not display significant differences in total loaded immunoprecipitated protein levels or in actin levels in the unbound (to the immunoprecipitate) protein.

Based on these therapeutic effects and observations of a downstream target of UCHLl, an evaluation upstream of a reported epigenetic modifier of UCHLl expression, Gadd45a, a well-known demethylating protein, was next assessed. In Gadd45a knockout (Gadd45a ^/~ ), mice lungs, total and activated Aktl as well as UCHLl protein levels were all reduced in normoxic samples consistent with previous reports (Meyer et al, supra). Under chronic hypoxia exposure, Gadd45a deficiency resulted in sustained reductions in total and phospho-Akt as well as UCHLl protein levels (Figures 6A and 6B). Immunohistochemistry staining of lungs of Gadd45a ^l~ mice revealed strikingly reduced UCHLl staining in the lung vasculature (Figure 6C) again predominantly in the endothelium under both normoxic and hypoxic exposure compared to WT mice.

Gadd45 ^1' mice exposed to chronic hypoxia exhibited lower elevations in both RVSP and RVH compared to WT mice exposed to hypoxia (Figures 7A and 7B). Consistent with these reductions in RVSP and RVH, histologic examination revealed significant improved vascular medial thickness and remodeling (Figure 7C) in hypoxia-exposed Gadd45a ^l~ mice compared to hypoxia-exposed WT mice.

Additional experiments were performed to assess the impact of UCHLl inhibition on downstream targets of UCHLl . Figure 9 shows that UCHLl inhibition reduces HIF-2a and PDGF levels in murine PH. A. Both LDN5744 and chronic hypoxia (4 weeks, 10% Fi0 ₂ ) resulted in increased ubiquintanted Akt levels in whole lungs in mice. B. Hypoxia alone was associated with increased phospo-Akt, PDGF AA and BB levels, while LDN57444 exposure reduced all three proteins in whole lungs. C. While chronic hypoxia increased HIF-2a levels in whole lungs, LDN57444 exposure resulted in reduced levels in nomroxia and hypoxia. D. HIF-2a levels are up-regulated in IP AH lung ECs compared to control cells.

Up-regulation of HIF-2a in lung ECs (Figure 9D), a downstream mediator of Akt/mTOR signaling, contributes to severe obliterative intimal remodeling and SMC proliferation in PAH via increased PDGF expression. The genes that actively induce both HIF activities and consequently promote development of PAH remain largely unknown. UCHLl abrogates Von Hippel-Lindau-mediated HIF-Ι α ubiquitination, the regulatory subunit of HIF-Ι α, consequently promoting cancer. Similar to HIF-Ια, reduced HIF-2a protein levels were observed in normoxic and hypoxic (5 wks, 10%FiO ₂ ) murine lungs (Figure 9B and 9C) after UCHLl inhibition (LDN57444-0.5mg/kg/day, daily for last 2 wks, IP). A similar partem of expression was observed in PDGF AA and BB levels. Together with several reports on PDGF effects in lung SMC, these data indicate that UCHLl mediates increased availability of HIF-2a and PDGF connecting EC-specific UCHLl activities to paracrine mediated SMC processes involved in vascular remodeling (e.g., proliferation).

Figure 10 shows that TGF-β induces Snail (A) and Vimentin (B) gene expression levels in human lung ECs while LDN57444 reduces these levels. This indicates that reduced EndMT (endothelial to mesenchymal transition) with UCHLl inhibition. EndMT

significantly contributes to obliterative intimal lesions in PAH defined by loss of cell-cell adhesion and the conversion of the EC phenotype to a spindle-shaped cell, like

(myo)fibroblast. Molecular changes associated with EndMT include decreases in EC markers (PEC AMI, CDH5), and increases in mesenchymal cell markers (transgelin, TAGLN). In addition, increased expression of the Snail family of transcription factors (Snail, Snai2 and Snai3) is important in the progression of EndMT. It was shown that UCHLl inhibition in lung ECs influences these changes in IP AH lung Ecs, indicating an important role for UCHLl in mediating EndMT and obliterating PAH vascular remodeling.

Akt is a canonical mediator of cell survival and proliferation; furthermore, recently Aktl knockout mice have demonstrated reduced PH in a chronic hypoxia model with reduced SMC proliferation (Tang et al, Am J Physiol Lung Cell Mol Physiol, 308(2), L208-220 2015). While most cancer studies have evaluated inhibition of Akt via reductions in its expression or inhibition of its activation (via phosphorylation), there are a paucity of investigations into the degradation of Akt (Chan et al, Cell Biosci, 4(1), 59 2014;

Dienstmann, Rodon, Serra, & Tabemero, Mol Cancer Ther, 13(5), 1021-1031 2014). This example demonstrates a therapeutic mediator of PH targeting Akt degradation via UCHLl inhibition, an established deubiquitinase in the murine model and in cells from patients with PAH. Both Gadd45a knockout mice, which have reduced UCHLl and Aktl levels, as well as WT mice, which were administered a targeted UCHLl inhibitor, demonstrated reduced RVSP, RVH, and pulmonary artery smooth muscle cell (PASMC) proliferation compared to their respective controls after exposure to chronic hypoxia. Moreover, UCHLl inhibition led to reductions in angiogenesis and EC proliferation in vitro.

Gadd45a knockout mice demonstrated reduced RVSP, RVH, and PASMC proliferation compared to WT mice after exposure to chronic hypoxia. These protective findings were associated with reduced levels of lung-specific expression of Akt in Gadd45a knockout mice compared to WT mice.

To further confirm the role of UCHL1 in Gadd45a deficient-mediated reductions in Akt and PH protection, administration of UCHL1 inhibitor, LDN-57444, in WT mice exposed to chronic hypoxia alleviated RVSP, RVH, and PASMC proliferation. Furthermore, LDN-57444 also reduced VEGF-induced angiogenesis and HPAEC proliferation in vitro.

Histology demonstrates a striking predominance of reduced vascular-specific expression profiles of UCHL1 and Aktl levels in knockout mice compared to WT mice and increased EC-specific expression in human PAH samples compared to control samples. Additionally, ECs isolated from WT mice exposed to chronic hypoxia exhibited increases in GADD45a wAAKTl.

These data underscore EC cell-specificity of these targets in PH development.

In summary, these data emphasize the role of the endothelium in the GADD45a-UCHLl-aktl axis in PH development and therapeutics.

Example 2

Regulation of Akt expression and phosphorylation has been a major focus in several studies; however, substantial gaps remain in elucidating Aktl degradation. It has been established that deubiquitination (DUB) by the DUB enzyme, ubiquitin carboxy -terminal hydrolase LI (UCHL1), is important to Akt degradation in lung pathologies including acute lung injury, lung fibrosis, and most recently, in pulmonary arterial hypertension (PAH) with increased PA vascular remodeling. These published and other data form the basis for the hypothesis that UCHL1 genetic variation modifies the novel roles of UCHL1 function contributing to PAH development.

Methods/Results: Whole-exome sequencing (WES) was performed on lung tissue- derived DNA collected at the time of transplant or autopsy from 13 patients with PAH (WHO Group I). Standard BWA, SAMTOOLS, and GATK protocols were used to align, filter, and analyze WES data. Filtered SNPs on UCHL1 were compared against referenced genome databases for case-control analysis. Of 43 SNPs identified, 42 were non-coding or intronic. A single missense coding SNP in exon 3, rs5030732 (S18Y, C>A) was observed in 4 patients with PAH (-31% of patients), all with carrier status (A/C) compared to Caucasians (MAF = 0.18, 1000 Genomes). PCR genotyping in a separate cohort of 82 PAH and 45 healthy control patients revealed the prevalence of rs5030732 of 31.7% (n=26, 23 A/C and 3 A/A) in PAH patients and 17.8% (n=8, 4 A/C and 4 A/ A, P = 0.0988) in control patients, consistent with the WES population. PAH associations between genotype and indices of PAH were tested using linear regression, adjusting for age, race, sex, indexed pulmonary vascular resistance (PVR), and PAH medication use. PA elastance remained significantly associated with rs5030732 with increases observed in AA/CA genotypes (13% +0.06 mm Hg/mL,

P=0.0225).

Figure 11 shows genotype/phenotype with UCHL1 SNP in PAH. A. Missense coding SNP of UCHL1 is found more frequently in cases of Group I PAH compared to healthy controls. B. CA genotype is associated with increased PA elastance in human PAH compared to CC. C. Relationship between PVRi and PA elastance is influenced by both gender and rs5030732 genotype. 183 Group I PAH patients were genotyped for rs5030732 from the UA cohort along with 70 controls. Both case only analysis and case-control comparisons were performed (Figure 11A). PAH associations between genotype and indices of PAH were tested using linear regression, adjusting for age, race, sex, indexed PVR, and PAH medication use in case only analysis in 67 cases (subset analysis). PA elastance was associated with SNP, AA/CA genotypes exhibiting increased values (Figures 11B/C, + SE-0.13 +0.06, P=0.0225). These genotypes exhibited PVR increases, RV function reductions (Tissue Doppler-derived S\ TAPSE) and RHC (indexed RVSW).

Conclusion: rs5030732 is a novel missense UCHL1 SNP more prevalent in PAH cases than healthy subjects. As this SNP is known to increase UCHL1 DUB activity, it is contemplated that the observed increased PA elastance in PAH patients is due, at least in part, to rs5030732-mediatd increased Akt signaling.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific preferred embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled relevant fields are intended to be within the scope of the following claims.