A computer readable form of the Sequence Listing XML containing the file named “3513285.0365_Sequence_Listing.xml,” which is 2,666 bytes in size (as measured in MICROSOFT WINDOWS® EXPLORER) and are herein incorporated by reference. This Sequence Listing consists of SEQ ID NOs: 1-2.
The present disclosure provides compositions and methods related to USP15 inhibitors. More particularly, the present disclosure is directed to methods of regulating the CRL4CRBN-USP15 pathway (previously referred to as the CRL4CRBN-p97 pathway) and glutamine synthetase levels and methods of treating diseases such as cancer via administration of USP15 inhibitors.
Cancer and the CRL4CRBN-USP15 (Previously CRL4CRBN-p97) Pathway
Cancer is the second leading cause of death in the United States. Despite recent advances in many cancer therapies, drug resistance to available medicines remains one of the biggest challenges in the effort to cure cancer. For example, multiple myeloma (MM) accounts for 1.8% of all new cancer cases in the United States with a 5-year survival rate of only 55.6%. Ubiquitylation of protein substrates is catalyzed by the ubiquitin-specific E1, E2, and E3 ligases and can be reversed by deubiquitinating enzymes (DUBs). The key components of the ubiquitin-proteasome system (UPS), including over 600 E3 ligases, ˜100 DUBs and the proteasome has recently emerged as therapeutic targets for treatment of cancer and other diseases. However, there are fundamental gaps in the knowledge and understanding of how E3s and DUBs control degradation of individual protein substrates.
Thalidomide was prescribed to pregnant women as a sedative to treat morning sickness in the late 1950s. It was withdrawn from the market in the early 1960s, when it was found to be a cause of severe birth defects. Recently, thalidomide and its immunomodulatory derivatives (IMiDs) lenalidomide and pomalidomide, have been used for the treatment of multiple myeloma (MM) and other hematologic malignancies. Other thalidomide analogs, now known as the Cereblon E3 ligase modulators (CELMoDs), including CC-122, CC-220 and CC-885, have been developed to target pathogenic proteins for degradation via a molecular glue mechanism. For example, CC-885 induces degradation of the translation termination factor GSPT1, a CRL4CRBN neo-substrate. In addition, CRBN is also a target for the development of proteolysis-targeting chimaera (PROTAC) technology, which relies on linking a drug that binds to a protein of interest to IMiDs. Many CRBN-based PROTACs, including the potent BRD4 protein degrader dBET1, have been developed for the degradation of target proteins in cancer and other human diseases. Despite recent advances in the field, many questions apart from clinical efficacy of IMiDs remain unknown. For example, CRBN is required for the action of IMiDs, but CRBN protein levels do not correlate with intrinsic sensitivity or resistance to IMiDs in MM cell lines, suggesting that other factors play a critical role in regulating the mechanisms underlying sensitivity and/or resistance to IMiDs.
However, the exact mechanisms of action of thalidomide that bring about its harmful and beneficial effects are not well understood. Recently, it was reported that cereblon (CRBN) protein is a primary target of thalidomide teratogenicity and that CRBN is key target of antitumor activities, and the CRL4CRBN-p97 pathway was only recently discovered.
Although the immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide have revolutionized the treatment of patients with multiple myeloma (MM) and other hematologic malignancies, almost all patients eventually develop resistance to IMiDs. Cereblon (CRBN) protein is a direct target for thalidomide teratogenicity and antitumor activity of IMiDs. The binding of IMiDs to CRBN promotes the recruitment of neo-substrates including Ikaros (IKZF1) and Aiolos (IKZF3) and casein kinase 1a (CK1a), leading to their ubiquitylation and subsequent degradation. CRBN is required for antitumor activity of IMiDs, but its expression levels do not correlate with intrinsic sensitivity or resistance to IMiDs in MM cells, suggesting that another factor is essential for regulating the mechanisms of IMiD sensitivity and resistance. It was discovered the CRL4CRBN-p97 pathway was required for degradation of GS and neo-substrates (such as GS, IKZF1, IKZF3, CK1α, RNF166, GSPT1 and BRD4). The crystal structure of CRBN bound to DDB1 and IMiDs revealed that the glutarimide rings of IMiDs bind a hydrophobic pocket in the thalidomide-binding domain of CRBN, whereas the phthalimide portion is largely exposed to solvent, critical for the recruitment of neo-substrates to the complex. Multiple neo-substrates such as ZNF692, SALL4 and RNF166 have been reported to be targeted for IMiD-induced ubiquitylation and subsequent degradation by the proteasome. Despite these developments, molecular control of the CRL4CRBN-p97 pathway to manage degradation of target proteins is poorly understood.
CRBN-based platforms have been used to develop small molecules to target pathogenic proteins for degradation via molecular glue and PROTAC mechanisms. However, the most fundamental question in the field is how the CRL4CRBN-p97 pathway manages to degrade target proteins.
Deubiquitylating enzymes (DUBs) play major roles in diverse cellular processes and altered DUB activity is associated with human diseases including cancer. There are approximately 100 DUBs encoded in the human genome, divided into five families based on their specific structural domains: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), ovarian tumor proteases (OTUs), Josephins, and JAB1/MPN/MOV34 metalloenzymes (JAMMs). Genetic alterations and overexpression of USP15 have been reported in many human cancers in particular GBM, breast and ovarian cancer, leukemia and lymphomas, and MM. High expression of USP15 was significantly correlated with poor survival rate within the pan-cancer patient cohort, representing a key feature of oncogene activity. At the molecular level, USP15 has been shown to deubiquitylate and stabilize protein substrates in different signaling pathways such as TGF-β, MDM2 and NF-κB. Since Usp15−/− mice were viable, targeting USP15 in cancer could achieve major advantages for an optimal therapeutic window. However, the molecular mechanism underlying the oncogenic activity of USP15 remains elusive.
Metabolic reprogramming is now recognized as a hallmark of cancer. Glutamine plays important roles in many cellular processes, including oxidative metabolism and ATP generation, biosynthesis of proteins, lipids and nucleic acids, as well as regulation of mTOR signaling pathway and autophagy. Glutamine synthetase (GS) is the only enzyme that is capable of the de novo synthesis of glutamine and detoxifies glutamate and ammonia. Mutations and deregulation of GS have been linked to human diseases, including congenital glutamine deficiency, Alzheimer's disease, and cancers in particular glioblastoma (GBM).
Recent studies highlighted the importance of glutamine metabolism in metabolic reprogramming. Activation of oncogenes such as MYC amplification and/or loss of tumor suppressor genes including p53 directly mediate the reprogramming of glutamine metabolism by selectively activating their downstream signaling or metabolic pathways. As a result, tumor cells display “glutamine addiction” and require excessive amounts of exogenous glutamine to generate building blocks and energy for their growth and survival. In striking contrast, some tumor cell lines express high levels of GS enzyme and can synthesize sufficient glutamine to fulfill glutamine requirements for nucleotide and protein synthesis.
Prior studies also demonstrated that bacterial GS is regulated by reversible adenylylation. In contrast to the well-defined regulation of bacterial GS, the molecular mechanism underlying the regulation of GS activity in mammals is poorly understood. Almost sixty years ago, it was reported that the mammalian GS is inactivated by extracellular glutamine. Subsequent work done prior to the discovery of ubiquitin-proteasome system (UPS), suggested that glutamine stimulates GS degradation through an unknown mechanism. Recently, it was reported that GS and AMPKγ are endogenous substrates of CRL4CRBN. Notably, p97 acts downstream of CRL4CRBN to promote disassembly of ubiquitylated GS subunits, which are subsequently degraded by the proteasome. It was also recently found that endogenous GS protein levels are negatively regulated by glutamine through a feedback loop involving CRL4CRBN20. However, it remains unknown how cells sense extracellular glutamine levels to control GS stability in the CRL4CRBN-p97 pathway.
The molecular events that take place at each step of the pathway are not well understood. More recently, it was shown that valosin-containing protein (VCP)/p97, is required for GS degradation. p97 extracts ubiquitylated GS subunits from the decamer so that they can be degraded by the proteasome. Many important questions about this process remain unanswered. Two questions are how cells sense glutamine levels to control GS acetylation, and whether acetylation may be a general mechanism for targeting substrates to CRBN.
Both lysine 11 and lysine 14 on GS (KxxK motif) are acetylated, resulting in CRBN binding, CRBN-dependent ubiquitylation and subsequent degradation by the proteasome. However, the current understanding of acetylation-mediated recognition between substrate and CRBN is limited, since GS is the only endogenous substrate for CRBN has been well characterized. Because CRBN is a key target of IMiDs used to treat cancer, understanding how CRBN recognizes acetylated substrates and how this is influenced by IMiDs could create new opportunities for IMiD therapy.
In the Crbn-knockout mouse model, Crbn deficiency protects mice from obesity, fatty liver, and insulin resistance caused by high fat intake, suggesting that CRBN may regulate multiple substrates involved in body metabolism and energy homeostasis. Moreover, previous acetylome studies indicated that reversible lysine acetylation plays an important role in regulating metabolic enzymes.
Histone acetylation plays important roles in regulating global chromatin architecture and gene transcription. Previous studies indicated that actyl-CoA, a downstream metabolite of carbon sources, contributes to increased histone acetylation at genes important for cellular growth in yeast and human cells. However, the link between glutamine metabolism and epigenetic regulation of gene transcription in cancer remains unknown. It was recently observed that glutamine activates p300 to acetylate GS at lysines 11 and 14, raising the possibility that glutamine and its metabolites may induce global protein acetylation, especially histone acetylation, thereby activating transcription of genes involved in cellular growth and proliferation.
Overexpression of GS has been implicated in the development of different types of cancer, including Hepatocellular carcinoma (HCC), breast and glioblastoma. In addition, bioinformatic analysis of cell lines from The Cancer Cell Line Encyclopedia shows that many other types of cancer such as leukemia, chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML) and prostate express higher GS mRNA levels. Interestingly, high GS expression may facilitate tumorigenesis, in that transgenic zebrafish, expressing an activated form of the Hippo pathway effector Yap1 (YAP) that reprograms glutamine metabolism through activation of GS expression, develop enlarged livers and are prone to liver tumorigenesis. However, the Hippo pathway can activate many other downstream target genes that may contribute to tumor development. Thus, the in vivo function of GS in tumorigenesis remains to be determined.
Compelling evidence suggests that cancer cells implement metabolic reprogramming by regulating transcription and post-translational modifications (PTMs) of metabolic enzymes. A recent study analyzing mRNA profiles of 1,454 metabolic enzymes across 1,981 tumors spanning 19 cancer types to identify enzymes that are consistently differentially expressed revealed that many enzymes and pathways are consistently overexpressed or downregulated across a large number of different cancer types. Interestingly, the authors also found that among the top 50 consistently overexpressed enzymes identified, 26 are essential for tumorigenesis in vitro and in vivo in breast cancer cells. However, this study only addressed changes in enzyme expression at the mRNA level, and did not seek to identify metabolic enzymes that may be dysregulated in cancer by alterations in PTMs such as ubiquitylation, acetylation, SUMOylation and phosphorylation. It has recently been demonstrated that PTMs play an essential role in regulating metabolic enzymes by multiple mechanisms, including via control of enzymatic activity, and by influencing protein stability.
Thus, it remains unknown how metabolism controls GS stability in the CRL4CRBN-p97 pathway and how modulating metabolism and these pathways could impact cancer treatment.
One aspect of the disclosure is directed to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of a USP15 inhibitor and at least one chemotherapeutic drug.
Another aspect of the disclosure is directed to methods of diagnosing and treating resistance to IMiD therapy in a subject with multiple myeloma undergoing IMiD therapy, the method comprising: a) measuring an amount of USP15 protein in the subject; b) comparing the amount of USP15 protein in the subject to a reference; c) diagnosing the subject with resistance to IMiD therapy if the amount of USP15 protein in the subject is higher than the reference; and d) administering a USP15 inhibitor to the subject.
A further aspect of the disclosure is directed to a method of regulating CRL4CRBN-USP15 pathway and glutamine synthetase levels in a subject in need thereof, the method comprising: administering to the subject in need thereof a USP15 inhibitor.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.
In this disclosure, chemical, RNA interference (RNAi), CRISPR/Cas9 gene editing, in vivo and in vitro biochemical approaches show that USP15 is a key regulator of the CRL4CRBN-USP15 pathway (historically referred to as the ‘CRL4CRBN-p97 pathway’) to control the stability of glutamine synthetase (GS) and neo-substrates, including IKZF1/3, CK1a, RNF166, GSPT1 and BRD4 (a target of CRBN-based PROTAC dBET1), all of which are crucial drug targets in many cancers. USP15 antagonizes ubiquitylation of CRL4CRBN target proteins, thereby preventing their degradation. Notably, USP15 is highly expressed in IMiD-resistant cell lines, and depletion of USP15 sensitizes these cells to lenalidomide.
USP15 protein expression can be used as a predictive biomarker of response or resistance to IMiD therapy in patients with MM and also advance the development of a class of drugs to degrade pathogenic proteins in cancer via molecular glue degraders (CRBN E3 ligase modulators: CELMoDs) and proteolysis-targeting chimaeras (PROTACs). Inhibition of USP15 represents a valuable therapeutic opportunity to potentiate IMiD/CELMoD and PROTAC therapies for the treatment of cancer (such as glioblastoma (GBM), MM, Myelodysplastic syndrome with deletion 5q (Del(5q)MDS) and acute myeloid leukemia (AML)) and other human diseases. These findings provide a rationale for the clinical developments of USP15 inhibitors in combination with IMiD/CELMoD and CRBN-based PROTAC therapies for the treatments of many types of cancer.
The USP15 inhibitor is selected from USP15 anti sense mRNA, USP15 siRNA, USP15 shRNA, USP15 miRNA, USP15 oligonucleotides and chemical inhibitors of USP15 such as PR-619. These USP15 inhibitors can further be used in combination with IMiDs (now called CELMoDs including thalidomide, lenalidomide, pomalidomide, CC-122, CC-220 and CC-885) or CRBN-based PROTACs (dBET1) to treat cancer.
It was further shown that mTORC1-mediated phosphorylation of USP15 at serine 229 (P-USP15) results in increased USP15-GS interaction.
The present disclosure is directed to methods of treating cancer comprising administering a therapeutically effective amount of a USP15 inhibitor and at least one chemotherapeutic drug to a subject in need thereof.
The chemotherapeutic drug can be an immunomodulatory drug (IMiD), cereblon E3 ligase modulator (CELMoD), or CRBN-based proteolysis-targeting chimaera (PROTAC). The chemotherapeutic drug can be selected from the group consisting of thalidomide, lenalidomide, pomalidomide, CC-122, CC-220, and CC-885. The CRBN-based PROTAC can be selected from the group consisting of ARV-825 (2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(4-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethoxy)phenyl)acetamide), dBET1 ((6S)-4-(4-Chlorophenyl)-N-[4-[[2-[[2-(2,6-dioxo-3-piperidinyl)-2,3-dihydro-1,3-dioxo-1H-isoindol-4-yl]oxy]acetyl]amino]butyl]-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide), dBRD9 (2-[[[4-(1,2-Dihydro-2-methyl-1-oxo-2,7-naphthyridin-4-yl)-2-6-dimethoxyphenyl]methyl]methylamino]-N-[2-[2-[2-[[2-(2,6-dioxo-3-piperidinyl)-2,3-dihydro-1,3-dioxo-1H-isoindol-4-yl]amino]ethoxy]ethoxy]ethyl]acetamide dihydrochloride), THAL-SNS-032 (N-(5-(((5-(tert-Butyl)oxazol-2-yl)methyl)thio)thiazol-2-yl)-1-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxo-6,9,12-trioxa-3-azatetradecyl)piperidine-4-carboxamide), BJS-03-123 (N-[2-[2-[2-[2-[4-[6-[(6-Acetyl-8-cyclopentyl-7,8-dihydro-5-methyl-7-oxopyrido[2,3-d]pyrimidin-2-yl)amino]-3-pyridinyl]-1-piperazinyl]ethoxy]ethoxy]ethoxy]ethyl]-2-[[2-(2,6-dioxo-3-piperidinyl)-2,3-dihydro-1,3-dioxo-1H-isoindol-4-yl]oxy]acetamide), BSJ-02-162 (N-(4-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)butyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)acetamide), BSJ-01-187 (7-cyclopentyl-2-((5-(4-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)acetamido)butyl)piperazin-1-yl)pyridin-2-yl)amino)-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide), YKL-06-102 (2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-N-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)acetamide), BETd-246 (4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-dimethylisoxazol-4-yl)-N-(3-(2-(2-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propoxy)ethoxy)ethoxy)propyl)-6-methoxy-9H-pyrimido[4,5-b]indole-2-carboxamide), TL13-149 (N-(2-(2-(2-(2-((2-(4-(3-(5-(tert-butyl)isoxazol-3-yl)ureido)phenyl)benzo[d]imidazo[2,1-b]thiazol-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)acetamide), DD-04-015 (N-(2-(2-(2-(4-(6-((5-(3-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-2-(hydroxymethyl)phenyl)-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)amino)pyridin-3-yl)piperazin-1-yl)ethoxy)ethoxy)ethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)acetamide), MT-802 (2-[2-[2-[4-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]ethoxy]ethoxy]-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]acetamide), MS4078 (2-(4-(4-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-5-isopropoxy-2-methylphenyl)piperidin-1-yl)-N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)acetamide), GSK983 ((R)—N-(6-chloro-2,3,4,9-tetrahydro-1H-carbazol-1-yl)picolinamide), MD-224 ((3′R,4'S,5′R)-6″-chloro-4′-(3-chloro-2-fluorophenyl)-N-(4-((5-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)pent-4-yn-1-yl)carbamoyl)phenyl)-2″-oxodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indoline]-5′-carboxamide), and L18I (3-(4-(3-(2-(2-(2-(4-((R)-3-(4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)propyl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione).
The cancer can be multiple myeloma, glioblastoma, deletion 5q subtype of myelodysplastic syndrome, or acute myeloblastic leukemia. The cancer can be IMiD-resistant.
The disclosure is further directed to methods of diagnosing and treating resistance to IMiD therapy in a subject with multiple myeloma undergoing IMiD therapy, the method comprising: a) measuring an amount of USP15 protein in the subject; b) comparing the amount of USP15 protein in the subject to a reference; c) diagnosing the subject with resistance to IMiD therapy if the amount of USP15 protein in the subject is higher than the reference; and d) administering a USP15 inhibitor to the subject. Measuring the amount of USP15 protein in the subject can comprise using immunohistochemical (IHC) staining of USP15 protein in bone marrow sections of the subject. The reference can be obtained from measuring an amount of USP15 protein from a different subject or subjects without multiple myeloma.
Further disclosed are methods of regulating CRL4CRBN-USP15 pathway and glutamine synthetase levels in a subject comprising administering a USP15 inhibitor to the subject.
For any of the disclosed methods, the USP15 inhibitor can be a nucleic acid or a chemical inhibitor. The nucleic acid can be selected from the group consisting of USP15 antisense mRNA, USP15siRNA, USP15shRNA, USP15 miRNA, and USP15 oligonucleotides. The chemical inhibitor can be a deubiquitinating enzyme (DUB) inhibitor. The chemical inhibitor can be selected from the group consisting of PR-619, NSC632839, and N-Ethylmaleimide (NEM). The chemical inhibitor can be mitoxantrone or an ubiquitin variant.
For any of the disclosed methods, the subject can be a mammal. The subject can also be a human, mouse, or rat.
As used in this application, including the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the content clearly dictates otherwise, and are used interchangeably with “at least one” and “one or more.”
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the preceding description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
To identify participants in regulating GS degradation in the CRL4CRBN-p97 pathway, GS-interacting proteins were sought. Previously, two independent mass spectrometry (MS) analyses of immunoprecipitation (IP) of endogenous USP15 prepared from LN-229 cells or two leukemia cell lines (MV-4-11 and Kasumi-1) revealed that GS was one of the top USP15-interacting proteins. To validate these proteomic data, immunoprecipitation experiments were used to find that USP15Flag interacts with GSMyc (
Previous in-depth analysis of the ubiquitinome using stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative MS from Jurkat cells treated with the proteasome inhibitor MG-132 or the DUB inhibitor PR-619 for 4 h revealed that while increased ubiquitylation of GS was observed in cells treated with MG132, PR-619 treatment decreased GS ubiquitylation, suggesting that GS is rapidly ubiquitylated and degraded in PR-619-treated cells, leading to a decrease in abundance of both native and ubiquitylated forms of GS. To test this possibility, the effects of two DUB inhibitors NSC632839 and PR-619 on GS ubiquitylation and abundance were evaluated. Consistent with this proteomic study, inhibition of DUB activity by NSC632839 or PR-619 promoted glutamine-induced ubiquitylation of GS (
Next, the molecular mechanism by which USP15 might control GS stability via the CRL4CRBN-p97 pathway was studied using RNA interference (RNAi) and CRISPR/Cas9 gene editing approaches. A previous study using stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative MS to characterize changes in the ubiquitinome of Jurkat cells treated with the proteasome inhibitor MG-132 or the DUB inhibitor PR-619 for 4 h revealed that while increased ubiquitylation of GS was observed in cells treated with MG132, PR-619 treatment decreased the ubiquitination of GS, suggesting that GS is rapidly ubiquitylated and degraded in PR-619-treated cells, leading to a decrease in abundance of both native and ubiquitylated forms of GS.
First the USP15-GS interaction was disrupted by depletion of USP15 using doxycycline (Dox)-inducible shRNA in TRIPZ system. Consistent with the results obtained with the DUB inhibitors, a similar result was obtained upon shRNA knockdown of USP15. Depletion of USP15 by shRNA induced GS degradation (
Since degradation of GS is dependent on the UPS, it was next sought to examine the effect of USP15 depletion on GS ubiquitylation. Indeed, a significant increase in ubiquitylated GS forms was detected in USP15−/− 293FT cells (
GBM is the most frequent adult primary malignant brain tumors, and it is the second leading cause of cancer mortality in adults under 35 years of age. GBM and many other tumors expressing high GS levels can synthesize glutamine de novo, grow and proliferate in the absence of exogenous glutamine. Interestingly, USP15 amplification confers poor prognosis in patients with GBM. The above data provide compelling evidence that USP15 is a key component of CRL4CRBN-p97 pathway to regulate GS stability. It was found that depletion of USP15 by shRNAs in LN229 cells resulted in a marked reduction in the steady-state level of GS (
Several groups reported that the molecular glue degrader CELMoDs and the PROTAC degrader dBET1 degrade CRL4CRBN neo-substrates or target proteins via molecular glue and PROTAC mechanisms, respectively. Notably, the CRL4CRBN-p97 pathway was uncovered, which is required for degradation of both endogenous substrate GS and neo-substrates of CRL4CRBN. It was next sought to investigate a potential role for USP15 in regulating ubiquitylation of CRL4CRBN neo-substrates. As shown in
Since USP15 is a deubiquitylating enzyme, it was next tested whether it could remove polyubiquitin chains from neo-substrates of CRL4CRBN in vitro. To achieve this goal, ubiquitin-binding TUBE2 resin was utilized to purify polyubiquitylated neo-substrates from cellular extracts of USP15-KO cells treated with the CELMoDs lenalidomide and CC-885 in the presence of p97 or proteasome inhibitor, and then performed the in vitro deubiquitylation assays using rUSP15. USP15 directly deubiquitylates polyubiquitylated CK1α forms (
To gain further insight into the molecular mechanisms underlying IMiD sensitivity and resistance in MM, the USP15 protein levels were analyzed in lenalidomide (Len)-sensitive and Len-resistant MM cell lines by immunoblotting with antibodies against USP15 and core components of the CRL4CRBN-p97 pathway. While the protein levels of p97 and DDB1 remained unchanged in all tested lenalidomide-sensitive and resistant cell lines, the protein levels of CRBN and CUL4A in Len-resistant cell lines expressed at least equal or higher, compared with those in Len-sensitive cell lines (
Glutamine activates the highly conserved, atypical Serine/Threonine kinase mammalian Target of Rapamycin Complex 1 (mTORC1), which regulates protein translation, cell growth and autophagy. The core components of mTORC1 consist of mTOR, Raptor, and mLST8. Glutamine may directly activate USP15 through mTORC1. IP experiments were used to determine that glutamine significantly stimulated the binding of endogenous GS to endogenous USP15, and correlated with enhanced serine-phosphorylated, but not threonine-phosphorylated USP15 (
To map the phosphorylation sites of USP15, which has 32 potential serine/threonine phosphorylation sites reported in ‘PhosphoSitePlus’ with the most prevalent serine 229, USP15Flag was immune-purified from 293FT cells treated with DMSO or 250 nM Torin1 for 16 h and analyzed by MS analysis. Known domains of USP15 are shown in
Based on the results of the other Examples, the CRL4CRBN-p97 pathway is hereinafter called the CRL4CRBN-USP15 pathway.
A proposed model for the role of USP15 in regulating the stability of natural substrate GS and neo-substrates in the CRL4CRBN-USP15 pathway is illustrated in
In the present study, USP15 was identified as a GS-interacting partner based on two independent proteomic studies. The presented results provide the first direct evidence that USP15 deubiquitylates and stabilizes seven tested CRL4CRBN protein targets, including GS, IKZF1, IKZF3, CK1α, RNF166, GSPT1 and BRD4 via natural substrate, molecular glue and PROTAC mechanisms. It functions downstream of CRL4CRBN and upstream of p97 and proteasome. This accounts for intrinsic resistance of IMiDs in MM. These findings also indicate that USP15 protein expression is a potential biomarker of response or resistance to IMiD therapy in patients with MM.
USP15 was identified as a potential GS-interacting protein by two independent proteomic studies. Preliminary results demonstrated that endogenous USP15 interacts with endogenous GS, and deubiquitylates polyubiquitylated GS in vivo and in vitro (
Lenalidomide (Chem-Pacific), MLN4924 (Pevonedistat, from Active Biochem), MG132 and PR-619 (Sigma), Bortezomib (LC Laboratories), CB-5083 (Selleckchem), CC-885 (MedKoo Biosciences), Torin1, dBET1 and NSC632839 hydrochloride (Tocris) were dissolved in dimethyl sulfoxide (DMSO) at room temperature and were stored at −80° C. until use. L-Methionine sulfoximine (MSO) and cycloheximide (Sigma) were dissolved in distilled water and kept at −80° C. For DNA transfection, Fugene HD was from Promega.
HEK293 cells, 293FT cells, H1299 cells, MCF7 cells, MM.1S, U266, NCI-H929 and RPMI-8226 were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). KMS-11, ARP-1 and OPM-1 cell lines were provided by from a lab at the University of Pennsylvania, Philadelphia, PA, USA.
Anti-glutamine synthetase (E-4; sc-74430), anti-IKZF1 (E-2, sc-398265), anti-IKZF3/Aiolos (3H5-G7; sc-293421) antibodies were from Santa Cruz Biotechnology. Mouse monoclonal anti-USP15 antibody (1C10, H00009958-M01) used for Western blot was from Abnova. Anti-ubiquitin (P4D1-All, 05-944), Anti-CRBN (HPA045910), anti-Flag HRP, and anti-Myc HRP antibodies were from Sigma. Anti-p97/VCP (ab11433), anti-GSPT1 (ab49878), anti-DDB1 (ab21080), anti-3-Actin HRP conjugated (AC-15, ab49900) antibodies were from Abcam. Anti-CUL4A (2699) antibody was from Cell Signaling Technology. Anti-BRD4 Antibody (BL-149-2H5; A700-004) was from Bethyl. Anti-Flag HRP conjugated antibody (600-403-383) was from Rockland Immunochemicals.
For secondary antibodies, HRP Goat Anti-Rabbit IgG (PI-1000) and HRP Horse Anti-Mouse IgG (PI-2000) were from Vector Laboratories.
For immunoprecipitation (IP), rabbit polyclonal anti-USP15 antibody (NB110-4069) was from Novus Biologicals. Rabbit IgG Isotype Control (normal rabbit IgG; sc-2027) was from Santa Cruz Biotechnology. EZview Red anti-Flag M2 (F2426) and EZview Red Anti-c-Myc Affinity Gels (E6654) were from Sigma.
Human USP15 expression vector pcDNA3.1+/C-(K)-DYK with a C-terminal Flag tag, expressing USP15 isoform 1 (NM_001252078.2) was purchased from GenScript. Human GS expression vector pCMV6-GS-Myc-Flag (C-terminal Myc-Flag tag) were purchased from OriGene. pCMV6-GS-Myc was generated by introducing a STOP codon between Myc and Flag by using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The full-length cDNA of IKZF3 was amplified from the IKZF3 expression vector pcDNA3.2 with a N-terminal HA tag (a kind gift from Dr. Benjamin Ebert, Dana Farber Cancer Institute), and then subcloned into pCMV6-Flag-Myc tags (pCMV6-IKZF3Flag-Myc). All cDNAs cloned into mammalian expression vectors are confirmed by DNA sequencing.
Generation of Stable Cell Lines Expressing shRNAs Targeting USP15
For tetracycline- and doxycycline-inducible system, the TRIPZ inducible lentiviral shRNAs for human USP15 (clone ID #1: V2THS_5710; clone ID #2: V2THS_13437) were purchased from Horizon Discovery. The shRNAs were induced by using 2 μg/ml doxycycline for 3-4 days.
For GIPZ Lentiviral shRNA system, the GIPZ lentiviral constructs expressing nontargeting (control, CT) and human USP15 shRNAs (USP15_1 shRNA: V2LHS_196921; USP15_2 shRNA: V2LHS_5710; USP15_3 shRNA: V2LHS_13436; USP15_4 shRNA: V2LHS_13437; USP15_5 shRNA: V3LHS_336555; and USP15_6 shRNA: V3LHS_336551) were purchased from Horizon Discovery. Six lentiviruses in the GIPZ Lentiviral shRNA vectors targeting USP15 were screened to identify shRNAs that optimally suppressed USP15. Virus preparation and cell infection were performed according to the manufacturer's protocol, with minor modifications. Briefly, shRNA-encoding plasmids were co-transfected with psPAX2 (packaging plasmid) and pMD.2G (enveloping plasmid) into HEK293FT cells using Fugene HD (Promega). Virus-containing supernatants were harvested at 48 h and 72 h post transfection. The lentiviruses were precipitated using PEG-it virus precipitation solution according to the manufacturer's protocol (System Biosciences). Target cells were transduced with lentiviral particles in the presence of 8 μg/ml polybrene, followed by selection with puromycin (1 μg/ml) for multiple myeloma cell lines, and 2-4 μg/ml for 293 cells and 293FT cells) for 2 weeks. Knockdown efficiencies were analyzed by immunoblot.
Cells, cultured in a 24-well plate, were transiently transfected with 0.5 μg of human USP15 CRISPR/Cas9 KO Plasmids (catalog #sc-402416; Santa Cruz Biotechnology) using Fugene HD (Promega). Two days after transfection, a single cell was seeded in 96-well plate via serial dilutions. After 2 weeks, single clones were obtained and expanded to validate the editing of USP15 by Western blot.
The protocol for generation of stable MM cell lines expressing USP15Flag and its a catalytically inactive C298A-USP15Flag mutant, constructed in pCDH-T2AcGFP-MSCV (System Biosciences), is based on recent work (Nguyen T V, et al., Mol Cell. 2016; 61(6):809-20). Human USP15 expression vector pcDNA3.1+USP15Flag (all tagged proteins are indicated by a superscripted tag either before or after the name to indicate tagging at the N- or C-terminus), USP15 isoform 1 (NM_001252078.2), was purchased from GenScript. Briefly, WT USP15Flag and its mutant cDNAs will be amplified from pcDNA3.1+ USP15Flag and re-constructed in pCDH-T2AcGFP-MSCV.
The protocols for immunoblot analysis and IP were performed as described in T. V. Nguyen et al. (Mol Cell 61, 809-820 (2016); T. Van Nguyen et al., Molecular cell 45, 210-221 (2012)).
HEK293FT cells were cultured in 10-cm plates. Cells were starved of glutamine for 24 h and pretreated with the inhibitors before adding Q4 for 2 h. Cells were harvested by washing in cold PBS 2× and freeze down at −80° C. Cells were lysed with 1 ml BD150 (10 mM Tris [pH 7.5], 150 mM NaCl, 1% Triton X-100, and 1 mM DTT containing a protease inhibitor cocktail).
Antibodies used for the IP (immunoprecipitation) include rabbit polyclonal anti-USP15 Ab (1 μg/μl) using 4 μl and normal rabbit IgG control using 10 μl (0.4 μg/μl), which is an unconjugated, affinity purified isotype control immunoglobulin from rabbit in PBS with 0.1% sodium azide and 0.1% gelatin.
IP occurred for 2 hr. Then, 30 μl protein G beads were added for 1 h. IP buffer was used 3×, and elution occurred with 50 μl 1.5×SDS-SB.
The results were run on a 4-15% gel with anti-USP15m: M-IP1-6 3 μl 1×SDS M-Input 1-6 10 μl and anti-GSm: M-IP1-6 15 μl 1×SDS Input 1-6 (5 μl).
The results were re-run on a 4-15% gel with phospho antibodies M-IP1-6 15 μl-1×1×WCL 15 μl-1× and M-IP1-6 15 μl-M-1×WCL 15 μl-1×.
LN-229 cell lines were seeded in eight to ten 10-cm cell culture plates and cultured until they reached 80% confluence. Cells were washed with 1×PBS and lysed in 500 μl 1% NP40 lysis buffer (25 mM Tris pH 7.5, 150 mM NaCl, 1% NP40, 0.5 mM, phenylmethylsulfonylfluoride (PMSF), protease inhibitor cocktail (Roche)). Lysates were centrifuged at 13000 rpm for 10 minutes. Protein concentration was measured using Bradford assay (Bio-Rad Laboratories). For the first IP MS experiment 3 mg of protein was used and for the second 5 mg. The concentration of the USP15 antibody (NB110-40690, Novus Biologicals, UK) and of the normal Rabbit IgG control (12-370, Millipore, Germany) was 5 μg/mg of lysate. The mixture of antibody/lysate was left rotating gently at 4° C. overnight.
After 12 hours 100-120 μl of 50% A agarose beads (Thermoscientific, USA) were added to the samples and were incubated while rotating at 4° C. for four further hours. (Before use, the beads were washed with 1×PBS and centrifuged at 1000 rpm for 1 min).
The mixture (beads-antibody-protein sample) was washed 5 times with 0.1% Tween-20/PBS. (Washing: 500 μl 0.1% Tween-20 PBS, mix, centrifuge for 2 min at 1000 rpm/4° C.). The 1/10 of pulldown product: 301 of 2×SDS-loading buffers was added, boiled for 5 min at 95° C., 5 μl of the sample were loaded onto a reducing SDS-PAGE using standard methods, and the rest was loaded on another 10% gel in parallel for silver staining (Silver Staining Kit, SilverXpress, Invitrogen; according to the manufacturer's instructions)
The protocol was described in T. V. Nguyen et al. (Proceedings of the National Academy of Sciences of the United States of America 114, 3565-3571 (2017)).
The assay was performed as described in T. V. Nguyen et al. (Proceedings of the National Academy of Sciences of the United States of America 114, 3565-3571 (2017)). Briefly, USP15−/− 293FT cells will be treated with IMiDs or the PROTAC dBET1 in the presence of proteasome or p97 inhibitors to accumulate polyubiquitylated target proteins, purified by TUBE2 Pull-down as described in T. V. Nguyen et al. (Proceedings of the National Academy of Sciences of the United States of America 114, 3565-3571 (2017)). The beads containing polyubiquitylated proteins were mixed in 30 μl of ubiquitylation buffer, followed by addition of 1 μg recombinant USP15 (rUSP15) protein (catalog #E-594-050, R&D Systems) and incubated at 37° C. for 0.5 h. rUSP15-treated samples were mixed with 30 μl of 2×SDS sample buffer, boiled for 5 min and subjected to Western blot analysis.
For competitive ubiquitylation/deubiquitylation assay, IKZF3FM was purified from USP15−/− 293FT cells. An in vitro competitive ubiquitylation/deubiquitylation of IKZF3FM was carried out for 1 h at 30° C. in the presence or absence of E1, E2, HAUb and recombinant CLR4CRBN complex purified from insect cells in a final volume of 30 in the presence of 50 μM lenalidomide and recombinant USP15 (rUSP15; 1 μg) were added. Reactions were stopped by mixing with equal amount of 2×SDS sample buffer and analyzed by SDS-PAGE and immunoblotting with antibodies against Flag, USP15 and CRBN.
Cells were seeded overnight in complete medium in 12-well plates, and then pre-treated with or without lenalidomide (10 μM) for 30 min, followed by addition of 100 μg/ml cycloheximide (CHX). At the indicated times following addition of CHX, samples were harvested for immunoblot analysis.
Data are presented as mean±one standard deviation (SD); p values were calculated using an unpaired two-tailed Student's t test in the Microsoft Excel software. P>0.05 was not significant whereas P<0.05 and P<0.01 means significant and very significant, respectively.
The assay was previously described (Sambrook J, Russell D W. CSH Protoc. 2006; 2006 (1). Epub 2006/01/01). Briefly, 1 μg of recombinant GST-tagged human GS protein (catalog #GLUL-4993H; Creative BioMart) and GST protein (control), immobilized on glutathione-Sepharose 4B beads (GE Healthcare), will be incubated with 1 μg recombinant His-tagged human USP15 Protein (catalog #USP15-358H, Creative BioMart) for 1 h at 4° C. in binding buffer: 20 mM Tris-HCl (pH 8.0), 200 mM KCl, 1 mM DTT, 5% glycerol, 1% Triton X-100 and proteinase inhibitors. After washing, bound proteins will be analyzed by IB.
The assay will be performed as described in a recent study (Nguyen T V, et al., Mol Cell. 2016; 61(6):809-20). Biotinylated USP15 peptides (GS-binding region) and GS peptides (USP15-binding region) will be synthesized from Biomatik.
MS analysis to identify the phosphorylation sites of USP15 was done at the Charles W. Gehrke proteomics center—University of Missouri, as described previously (Peterson T R, et al., Cell. 2009; 137(5):873-86. Epub 2009/05/19).
This application is a U.S. National Phase application of PCT/US2022/076022 (published as WO 2023/039405), filed on Sep. 7, 2022, which claims the benefit of U.S. Provisional Application No. 63/241,555, filed Sep. 8, 2021, the contents of each of which are incorporated herein in their entireties.
Filing Document | Filing Date | Country | Kind |
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PCT/US2022/076022 | 9/7/2022 | WO |
Number | Date | Country | |
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63241555 | Sep 2021 | US |