Patent Application
20240238274
This application is being filed electronically via EFS-Web and includes an electronically submitted Sequence Listing in .txt format. The .txt file contains a sequence listing entitled “70258102132ST25.txt” created on Apr. 25, 2022 and is 18,669 bytes in size. The Sequence Listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
The field of the invention relates to small molecule inhibitors of ubiquitin specific peptidase 22 (USP22) and the use thereof in treating diseases and disorders associated with USP22 biological activity. In particular, the field of the invention relates to small molecule inhibitors of the peptidase activity of USP22 which may be formulated as pharmaceutical compositions for treatment of cell proliferative diseases and disorders such as cancer.
The expression of ubiquitin specific peptidase 22 (USP22) is often increased in many, if not all types of human cancers. USP22 functions as a potential oncogene in tumorigenesis and progression in lung and colon cancer in part through diminishing the tumor suppressor p53 transcriptional activity and promoting cell cycle progression. Mice with genetic USP22 suppression in immune cells have better tumor rejection using multiple syngeneic tumor models including lung cancer, lymphoma, melanoma, and colon cancers. These results indicate that USP22 is an ideal therapeutic target in antitumor therapy because that, on one hand, inhibition of USP22 in tumor cells can directly induces their apoptosis and blocks cell cycle progression, on the other hand, USP22 suppression in immune cells enhances antitumor immunity.
Disclosed herein are inhibitors of ubiquitin specific peptidase 22 (USP22) and uses for treating diseases and disorders thereof. One aspect of the technology provides for a method of treating a subject in need of treatment for a disease or disorder associated with ubiquitin specific peptidase 22 (USP22) activity, the method comprising administering to the subject an effective amount of a therapeutic agent that inhibits the biological activity of USP22. In some embodiments, the disease or disorder is a cell proliferative disease or disorder. In some embodiments, the disease or disorder is a cancer. In some embodiments, the cancer may be selected from the group consisting of lung cancer, gastric carcinoma, pancreatic cancer, melanoma, lymphoma, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, mesothelioma, neuroblastoma, mantle cell lymphoma, and acute myeloid leukemia.
Another aspect of the technology provides for a method of suppressing Treg cell activity in a subject in need thereof, the method comprising administering to the subject an effective amount of a therapeutic agent that inhibits the activity of USP22. In some embodiments, the subject has an infectious disease. In some embodiments, the subject has sudden acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection.
Another aspect of the technology provides for a method for inhibiting ubiquitin specific peptidase activity (E. C. 3.4.19.12) of USP22 in a subject in need thereof, the method comprising administering to the subject an effective amount of a therapeutic agent that inhibits the biological activity of USP22.
For the disclosed methods, the therapeutic agent is an inhibitor of ubiquitin specific peptidase 22 (USP22). In some embodiments, the therapeutic agent comprises one or more compounds selected from Table Si. In some embodiments, the therapeutic agent is ii-anilino-7,8,9,10-tetrahydrobenzimidazo[1,2-b]isoquinoline-6-carbonitrile.
Pharmaceutical compositions comprising the therapeutic agents described herein and a suitable pharmaceutical carrier. In some embodiments, the therapeutic agent is ii-anilino-7,8,9,10-tetrahydrobenzimidazo[1,2-b]isoquinoline-6-carbonitrile. In some embodiments, the composition comprises an effective amount of the compound for inhibiting biological activity of USP22 when administered to a subject in need thereof. In some embodiments, the composition comprises an effective amount of the compound for suppressing Treg cell activity in a subject in need thereof. In some embodiments, the composition comprises an effective amount of the compound for inhibiting ubiquitin specific peptidase activity (E.C. 3.4.19.12) of USP22 in a subject in need thereof.
Disclosed herein are inhibitors of ubiquitin specific peptidase 22 (USP22) and uses for treating diseases and disorders thereof. Computer-based and biological approaches were used to identify small molecule specific inhibitors. As demonstrated in the Examples, treatment of regulatory T cells (Tregs), both mouse and human, with inhibitors of USP22 significantly reduced the protein expression of FoxP3, a substrate of USP22. In contrast, treatment did not further inhibit FoxP3 expression in USP22-null Tregs, indicating that the inhibitors of USP22 may be a highly specific inhibitor of USP22. In addition, treatment inhibited USP22 activity in lung cancer cells and consequently suppressed lung cancer cell growth. More importantly, treatment of lung cancer-bearing mice largely diminished the tumor mass. These results indicate that inhibitors of USP22 can be used as a potent drug in antitumor therapy. In addition, the fact that suppression of USP22 diminishes Treg suppressive functions, also allows for these inhibitors to be used to treat diseases associated to immune deficiency as well as to boost the immune response to combat infectious diseases such as SARS-CoV2 infection.
The present invention is described herein using several definitions, as set forth below and throughout the application.
The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.
As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.
As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.
As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.
Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together.).
It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”
All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.
The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”
A “subject in need thereof” as utilized herein may refer to a subject in need of treatment for a disease or disorder associated with ubiquitin specific peptidase 22 (USP22) activity and/or expression. A subject in need thereof may include a subject having a cancer that is characterized by the activity and/or expression of USP22. The disclosed compounds, pharmaceutical compositions, and methods may be utilized to treat diseases and disorders associated with USP22 activity and/or expression.
In some embodiments, a subject in need thereof may include a subject having a cancer that is treated by administering a therapeutic agent that inhibits the biological activity of USP22, and/or that inhibits dissemination of cancer cells.
The disclosed compounds, pharmaceutical compositions, and methods may be utilized to treat diseases and disorders associated with USP22 activity and/or expression which may include cell proliferative diseases and diseases and disorders such as cancers. Suitable cancers for treatment by the disclosed compounds, pharmaceutical compositions, and methods may include, but are not limited to lung cancer, gastric carcinoma, pancreatic cancer, melanoma, lymphoma, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, mesothelioma, neuroblastoma, mantle cell lymphoma, and acute myeloid leukemia.
In some embodiments, a subject in need thereof may include a subject in need of treatment of infection. In some embodiments, the infection is a viral infection, such as an infection by a corona virus. In some embodiments, the subject in need thereof is in need of a treatment for infection by sudden acute respiratory syndrome coronavirus 2 (SARS-CoV2) and COVID. In some embodiments, a subject in need thereof may refer to a subject in need of augmenting the immune response to an infection. In some embodiments, a subject in need thereof may refer to a subject in need of augmenting the immune response to sudden acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection.
The disclosed compounds, pharmaceutical compositions, and methods may be utilized to treat diseases and disorders associated with USP22 activity and/or expression which may include infections and diseases and disorders such as respiratory infections, including sudden acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection.
The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects.
The disclosed compounds may be utilized to modulate the biological activity of USP22, including modulating the peptidase activity of USP22. The term “modulate” should be interpreted broadly to include “inhibiting” USP22 biological activity including peptidase activity.
Ubiquitin specific peptidase (USP22) refers to the protein also referred to by the name ubiquitin carboxyl-terminal hydrolase 22. USP22 has been shown to have enzyme activities that include catalyzing the thiol-dependent hydrolysis of ester, thioester, amide, peptide and isopeptide bonds formed by the C-terminal glycine of ubiquitin. USP22 has ENZYME entry: EC 3.4.19.12. The compounds disclosed herein may inhibit one or more of the activities of USP22 accordingly.
Human USP22 is known to have two isoforms and the disclosed compounds may inhibit one or more activities of isoform 1 and/or isoform 2.
Human USP22 Isoform 1 has the following amino acid sequence:
Isoform 2 has the following sequence:
The compounds employed in the compositions and methods disclosed herein may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the compounds are considered to be embodiments of the compositions disclosed herein. Such compositions may take any physical form which is pharmaceutically acceptable; illustratively, they can be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of a disclosed compound, which effective amount is related to the daily dose of the compound to be administered. Each dosage unit may contain the daily dose of a given compound or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose. The amount of each compound to be contained in each dosage unit can depend, in part, on the identity of the particular compound chosen for the therapy and other factors, such as the indication for which it is given. The pharmaceutical compositions disclosed herein may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures.
The compounds for use according to the methods of disclosed herein may be administered as a single compound or a combination of compounds. For example, a compound that inhibits the biological activity of ubiquitin specific peptidase 22 (USP22) may be administered as a single compound or in combination with another compound inhibits the biological activity of USP22 or that has a different pharmacological activity.
As indicated above, pharmaceutically acceptable salts of the compounds are contemplated and also may be utilized in the disclosed methods. The term “pharmaceutically acceptable salt” as used herein, refers to salts of the compounds, which are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds as disclosed herein with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. It will be appreciated by the skilled reader that most or all of the compounds as disclosed herein are capable of forming salts and that the salt forms of pharmaceuticals are commonly used, often because they are more readily crystallized and purified than are the free acids or bases.
Acids commonly employed to form acid addition salts may include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of suitable pharmaceutically acceptable salts may include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleat-, butyne-.1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, α-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.
Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.
The particular counter-ion forming a part of any salt of a compound disclosed herein is may not be critical to the activity of the compound, so long as the salt as a whole is pharmacologically acceptable and as long as the counter-ion does not contribute undesired qualities to the salt as a whole. Undesired qualities may include undesirably solubility or toxicity.
Pharmaceutically acceptable esters and amides of the compounds can also be employed in the compositions and methods disclosed herein. Examples of suitable esters include alkyl, aryl, and aralkyl esters, such as methyl esters, ethyl esters, propyl esters, dodecyl esters, benzyl esters, and the like. Examples of suitable amides include unsubstituted amides, monosubstituted amides, and disubstituted amides, such as methyl amide, dimethyl amide, methyl ethyl amide, and the like.
In addition, the methods disclosed herein may be practiced using solvate forms of the compounds or salts, esters, and/or amides, thereof. Solvate forms may include ethanol solvates, hydrates, and the like.
The pharmaceutical compositions may be utilized in methods of treating a disease or disorder associated with the biological activity of ubiquitin specific peptidase 22 (USP22). As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration.
As used herein the term “effective amount” refers to the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. The disclosed methods may include administering an effective amount of the disclosed compounds (e.g., as present in a pharmaceutical composition) for treating a disease or disorder associated with biological activity of ubiquitin specific peptidase 22 (USP22).
An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
A typical daily dose may contain from about 0.01 mg/kg to about 100 mg/kg (such as from about 0.05 mg/kg to about 50 mg/kg and/or from about 0.1 mg/kg to about 25 mg/kg) of each compound used in the present method of treatment.
Compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg of each compound individually or in a single unit dosage form, such as from about 5 to about 300 mg, from about 10 to about 100 mg, and/or about 25 mg. The term “unit dosage form” refers to a physically discrete unit suitable as unitary dosages for a patient, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient.
Oral administration is an illustrative route of administering the compounds employed in the compositions and methods disclosed herein. Other illustrative routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The route of administration may be varied in any way, limited by the physical properties of the compounds being employed and the convenience of the subject and the caregiver.
As one skilled in the art will appreciate, suitable formulations include those that are suitable for more than one route of administration. For example, the formulation can be one that is suitable for both intrathecal and intracerebral administration. Alternatively, suitable formulations include those that are suitable for only one route of administration as well as those that are suitable for one or more routes of administration, but not suitable for one or more other routes of administration. For example, the formulation can be one that is suitable for oral, transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, and/or intrathecal administration but not suitable for intracerebral administration.
The inert ingredients and manner of formulation of the pharmaceutical compositions are conventional. The usual methods of formulation used in pharmaceutical science may be used here. All of the usual types of compositions may be used, including tablets, chewable tablets, capsules, solutions, parenteral solutions, intranasal sprays or powders, troches, suppositories, transdermal patches, and suspensions. In general, compositions contain from about 0.5% to about 50% of the compound in total, depending on the desired doses and the type of composition to be used. The amount of the compound, however, is best defined as the “effective amount”, that is, the amount of the compound which provides the desired dose to the patient in need of such treatment. The activity of the compounds employed in the compositions and methods disclosed herein are not believed to depend greatly on the nature of the composition, and, therefore, the compositions can be chosen and formulated primarily or solely for convenience and economy.
Capsules are prepared by mixing the compound with a suitable diluent and filling the proper amount of the mixture in capsules. The usual diluents include inert powdered substances (such as starches), powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol and sucrose), grain flours, and similar edible powders.
Tablets are prepared by direct compression, by wet granulation, or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants, and disintegrators (in addition to the compounds). Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (such as sodium chloride), and powdered sugar. Powdered cellulose derivatives can also be used. Typical tablet binders include substances such as starch, gelatin, and sugars (e.g., lactose, fructose, glucose, and the like). Natural and synthetic gums can also be used, including acacia, alginates, methylcellulose, polyvinylpyrrolidine, and the like. Polyethylene glycol, ethylcellulose, and waxes can also serve as binders.
Tablets can be coated with sugar, e.g., as a flavor enhancer and sealant. The compounds also may be formulated as chewable tablets, by using large amounts of pleasant-tasting substances, such as mannitol, in the formulation. Instantly dissolving tablet-like formulations can also be employed, for example, to assure that the patient consumes the dosage form and to avoid the difficulty that some patients experience in swallowing solid objects.
A lubricant can be used in the tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid, and hydrogenated vegetable oils.
Tablets can also contain disintegrators. Disintegrators are substances that swell when wetted to break up the tablet and release the compound. They include starches, clays, celluloses, algins, and gums. As further illustration, corn and potato starches, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, and carboxymethylcellulose can be used.
Compositions can be formulated as enteric formulations, for example, to protect the active ingredient from the strongly acid contents of the stomach. Such formulations can be created by coating a solid dosage form with a film of a polymer which is insoluble in acid environments and soluble in basic environments. Illustrative films include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate.
Transdermal patches can also be used to deliver the compounds. Transdermal patches can include a resinous composition in which the compound will dissolve or partially dissolve; and a film which protects the composition, and which holds the resinous composition in contact with the skin. Other, more complicated patch compositions can also be used, such as those having a membrane pierced with a plurality of pores through which the drugs are pumped by osmotic action.
As one skilled in the art will also appreciate, the formulation can be prepared with materials (e.g., actives excipients, carriers (such as cyclodextrins), diluents, etc.) having properties (e.g., purity) that render the formulation suitable for administration to humans. Alternatively, the formulation can be prepared with materials having purity and/or other properties that render the formulation suitable for administration to non-human subjects, but not suitable for administration to humans.
Disclosed are compounds, pharmaceutical compositions comprising the compounds, and methods of using the compounds and pharmaceutical compositions for treating a subject having or at risk for developing a disease or disorder associated with ubiquitin specific peptidase 22 (USP22) biological activity. The disclosed compounds may inhibit the biological activity of USP22. As such, the disclosed compounds and pharmaceutical compositions may be utilized in methods for treating a subject having or at risk for developing a disease or disorder that is associated with USP22 activity which may be cell proliferative diseases and disorders, such as cancer, or an infection associated disease or disorder, such as sudden acute respiratory syndrome, such as SARS-CoV2.
In some embodiments, the disclosed methods include treating a subject in need of treatment for a disease or disorder associated with ubiquitin specific peptidase 22 (USP22) activity. In the disclosed methods, the subject may be administered an effective amount of a therapeutic agent that inhibits the biological activity of USP22.
The disclosed methods may be performed in order to treat a cell proliferative disease or disorder, which may include cancer. Suitable cancers that may be treated by the disclosed methods may include, but are not limited to, lung cancer, gastric carcinoma, pancreatic cancer, melanoma, lymphoma, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, mesothelioma, neuroblastoma, mantle cell lymphoma, and acute myeloid leukemia.
In some embodiments, the disclosed methods may be performed in order to treat lung cancer, for example, non-small cell lung cancer (NSCLC).
In some embodiments, the disclosed methods may be performed in order to treat skin cancer, for example, melanoma.
In the disclosed methods, a subject in need thereof typically is administered a therapeutic agent that inhibits the biological activity of ubiquitin specific peptidase 22 (USP22). In some embodiments, the therapeutic agent inhibits ubiquitin specific peptidase activity (E.C.: 3.4.19.12) of USP22.
Suitable therapeutic agents for use in the disclosed methods may include, but are not limited to, a compound having a formula selected from the group consisting of:
In some embodiments of the disclosed methods, the subject is administered a compound selected from the group consisting of:
In some embodiments of the disclosed methods, the therapeutic agent administered to the subject may be the compound having the formula:
otherwise referred to as 11-Anilino-7,8,9,10-tetrahydrobenzimidazo[1,2-b]isoquinoline-6-carbonitrile.
The disclosed methods also may be performed in order to suppress Treg cell activity in a subject in need thereof. For example, in the disclosed methods the subject may be administered an effective amount of a therapeutic agent that inhibits the activity of USP22, thereby suppressing Treg cell activity in the subject.
In some embodiments, the disclosed methods may also be performed in order to augment the immune response of the subject to an infectious disease in a subject in need thereof.
In some embodiments, the disclosed methods are used to augment the immune response to sudden acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection in a subject in need thereof.
In some embodiments, the disclosed methods are used to augment the immune response of the subject to an infectious disease, in a subject in need thereof. In some embodiments, the therapeutic agent inhibits ubiquitin specific peptidase activity (E.C.: 3.4.19.12) of USP22.
In some embodiments, the disclosed methods of augmenting a subject's immune response to an infectious disease. For example, the therapeutic agent administered to a subject in a need thereof may be a compound having a formula selected from any of the compounds described herein.
Also disclosed are pharmaceutical compositions. In some embodiments, the disclosed pharmaceutical compositions comprise an effective amount of a therapeutic agent having a formula chosen from any of the compounds described herein and a suitable pharmaceutical carrier.
In some embodiments of the disclosed pharmaceutical compositions, the pharmaceutical compositions may comprise an effective amount of a compound is selected from any of the compounds described herein and a suitable pharmaceutical carrier.
In some embodiments, the disclosed pharmaceutical composition may comprise an effective amount of 11-Anilino-7,8,9,10-tetrahydrobenzimidazo[1,2-b]isoquinoline-6-carbonitrile and a suitable pharmaceutical carrier.
In some embodiments, the disclosed pharmaceutical compositions comprise an effective amount of a therapeutic agent that inhibits the biological activity of ubiquitin specific peptidase 22 (USP22).
In some embodiments, the disclosed pharmaceutical compositions comprise an effective amount of the compound for suppressing Treg cell activity.
In some embodiments, the disclosed pharmaceutical compositions comprise an effective amount of the compound for inhibiting ubiquitin specific peptidase activity (E.C. 3.4.19.12) of USP22.
In some embodiments, the disclosed pharmaceutical compositions comprise an effective amount of the compound for inhibiting the biological activity of USP22 when administered to a subject in need thereof.
In some embodiments, the disclosed pharmaceutical compositions comprise an effective amount of the compound for suppressing Treg cell activity when administered to a subject in need thereof.
In some embodiments, the disclosed pharmaceutical compositions comprise an effective amount of the compound for inhibiting ubiquitin specific peptidase activity (E.C. 3.4.19.12) of USP22 when administered to a subject in need thereof.
The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.
The highly immunosuppressive tumor microenvironment (TME) favors T regulatory (Treg) cell stability and function, while diminishing the anti-tumor activity of effector T cells. Here, we characterized previously unknown TME-specific cellular and molecular mechanisms that promote intratumoral Treg adaptation. We uncovered the critical role of FOXP3 deubiquitinases, ubiquitin specific peptidase 22 (Usp22) and 21 (Usp21) in Treg stabilization under TME. Specifically, TME stressors including elevated TGF-β, hypoxia, and nutrient deprivation upregulate Usp22 and Usp21 to maintain optimal Foxp3 expression in response to alterations in HIF, AMPK and mTOR activity. The simultaneous loss of both USPs synergizes to alter Treg metabolic signatures and impair suppressive mechanisms, resulting in enhanced anti-tumor activity. Finally, we developed the first Usp22-specific small molecule inhibitor, which significantly reduced intratumoral Treg cells and consequently enhanced anti-tumor immunity. Our findings unveil new mechanisms underlying the functional uniqueness of intratumoral Treg cells and identify Usp22 as an antitumor therapeutic target that inhibits Treg adaptability in the TME.
Tumors have long been recognized as having distinctive properties of growth, invasion, and metastasis, but their ability to evade immune recognition and destruction has recently attracted attention. While neoplastic cells have sufficient antigenicity to promote an anti-tumor immune response, tumors evade the immune system through a variety of mechanisms including the production of immune suppressive mediators and cytokines, defective antigen presentation, and recruitment of immune regulatory cells such as T regulatory (Treg) cells (1, 2). Furthermore, the disorganized vascular system and enhanced rate of proliferation observed in tumors creates a hostile microenvironment depleted of oxygen, glucose, and amino acids while enriched with cytokines and lactic acid (3). Many, if not all, of these alterations in the tumor microenvironment (TME) are known to inhibit anti-tumor immune responses through a variety of mechanisms. Particularly, these TME-derived pressures favorably alter intratumoral (it)Treg cells, resulting in heightened survival and suppressive abilities, while diminishing the anti-tumor effects of effector T (Teff) cells (4-7). Moreover, itTreg cells themselves are known to aid in metastasis, and their increased number correlates with poor clinical outcomes (1,6).
The exact composition of itTreg cells, and whether the majority of this population consists of natural (n)Treg or tumor-induced Treg cells, remains unknown and may differ between tumor types (8). However, it is likely that both populations, although epigenetically distinct, thrive in the TME and further aid in dampening anti-tumor immunity. Interestingly, itTreg cells display upregulated expression of the lineage-defining Treg transcription factor, Forkhead Box P3 (FOXP3) (9, 10), which functions to enhance Treg fitness by augmenting Treg cell stability and suppressive molecular function. Importantly, Foxp3 expression is essential for proper Treg development and function (11). However, the molecular mechanisms underlying how and which TME factors upregulate Foxp3 expression to potentiate itTreg suppressive function remain unknown.
The presence of itTreg cells plays a pivotal role in inhibiting anti-tumor immunity, and is a major hurdle for current tumor-targeting immunotherapies. As Treg depletion through a Treg-specific marker remains challenging (12, 13), the particular pathways that enhance Treg suppressive capabilities within the TME are attractive candidates for new therapeutic targets to diminish itTreg suppressive function. Although Foxp3 is uniquely important for Treg identify and function, it is an intracellular protein whose targeting would require great care as complete inhibition would likely drive significant autoimmunity (11). In addition, specifically targeting a transcription factor like FOXP3 remains technically challenging. Therefore, superior therapeutic candidates will be those that control the expression and stability of Foxp3 specifically in the TME.
Foxp3 expression and stability can be regulated from the transcriptional to the post-translational level, with each layer independently controlling the stability and overall function of Treg cells. Particularly, a newly appreciated layer of Foxp3 regulation and Treg functional modulation is through ubiquitination (14, 15). Ubiquitination of histories on the Foxp3 promoter and conserved non-coding DNA sequence (CNS) regions via E3 ubiquitin ligases results in chromatin condensation and lack of Foxp3 transcription (16). Furthermore, direct ubiquitination of the FOXP3 protein can result in proteasomal degradation. Importantly, ubiquitin may be removed from these sites by deubiquitinating enzymes (DUBs), functioning to both open the chromatin at the transcriptional level, and to stabilize FOXP3 at the protein level (14). The balance between E3 Ligases and DUBs on Foxp3 expression results in an equilibrium state that regulates Foxp3 levels within Treg cells. We and others have discovered three members of the ubiquitin specific peptidase (USP) family as direct modulators of FOXP3 deubiqutination at the transcriptional and/or post-translational level: Usp7, Usp21, and Usp22 (14,17, 18). However, the broad environmental cues and cellular regulation of these deubiquitinases remain unknown. Here, we investigate the role of the TME on the USP-FOXP3 axis, and develop the first Usp22-specific inhibitor capable of antitumor activity. Our study has identified specific TME factors selectively induce FOXP3 deubiquitinases Usp22 and Usp21, but not Usp7 expression to control Treg stability and adaptation.
Selective Upregulation of FoxP3 Deubiquitinases in itTreg Cells
Since tumors create a hostile microenvironment where immune cell function is greatly altered, we began by characterizing the suppressive profiles of murine itTreg cells (
As the three FOXP3-targeting USPs aid in maintaining FOXP3 stability (16-18), we hypothesized that modulation of their expression may drive the FOXP3 upregulation in itTreg cells. Interestingly, the mRNA level of Usp22 was consistently increased within itTreg cells in comparison to the peripheral Treg cells harvested from same mouse or non-challenged controls, but Usp7 mRNA level was unchanged. In contrast, Usp21 mRNA level was only increased under B16 challenge, suggesting that Usp21 upregulation in Treg cells occurs only under certain TME conditions (
As soluble factors secreted by the tumors are known to alter immune cell function (19, 20), we investigated the role of TME-soluble factors in regulating Usp22 and Usp21 in itTreg cells. We exposed in vitro induced (i)Treg cells to media obtained from cultured tumor cells (tumor conditioned media or TCM) (
Many types of tumors secrete large amounts of TGF-β, which dampens immune responses and promotes metastasis (21, 22). Together with the fact that TGF-β is particularly important for iTreg generation and stability (23), we speculated that TGF-β could aid in enhancing Foxp3 expression in itTreg cells through induction of Usp22 and Usp21. Indeed, mRNA levels of both Foxp3-targeting USPs were increased when TGF-β was added to the media of iTreg cells, while Usp7 showed no such increase (
To further determine if TGF-β is implicated in TCM-driven Usp22 and Usp21 upregulation, we added the TGF-β inhibitor to the TCM from each of the aforementioned tumor cell lines. Indeed, the TGF-β inhibitor completely diminished the mRNA enhancement of Usp22 (
The levels of Usp7 remain unchanged under all treatment groups and displayed no correlation to the increasing level of TGF-β in the various tumor types (
To determine if tumor derived TGF-β is important in upregulating Usp22 and Usp21 within the TME proper, we determined whether the levels of Usp22 and Usp21 in itTreg cells infiltrating B16 melanomas lacking TGF-β are still upregulated. shRNA knockdown largely diminished the TGF-β expression in B16 cells (
To uncover the mechanism by which TGF-β acts on Usp22 and Usp21 transcription, we first investigated the canonical TGF-β signaling pathway, which works through the co-activating SMAD transcription factors (homologues of the Drosophila protein, mothers against decapentaplegic (Mad) and the Caenorhabditis elegans protein Sma) including SMAD2, SMAD3 and SMAD4 through specifically binding to the SMAD-binding element (SBE) (24, 25). We scanned along the promoter regions of both Usp22 and Usp21 for sequences of conserved SBE. Along the Usp22 promoter, we found three promising regions for which we made primers and assessed the SMAD binding capacity (
We have recently observed that, although Usp22-null iTreg cells polarize normally with high levels of TGF-β, sub-optimal polarization conditions resulted in a significant decrease in FOXP3 MFI and percentage relative to the WT iTreg cells (16). This suggests an important function of Usp22 in perpetuating TGF-β signaling within iTreg polarization. Indeed, Usp22-null iTreg cells display a significant deficiency in both SMAD2 and SMAD4 protein levels compared to WT iTreg cells, with no difference in their mRNA levels (
Unlike with Usp22, no SBEs were found when scanning the Usp21 promoter, implying that TGF-β induces Usp21 expression independent of SMADs. Indeed, none of the regions showed binding capacity of any of the tested SMAD proteins, confirming that Usp21 expression is not induced through canonical TGF-β signaling (
Hypoxia Selectively Induces Treg Usp22, which Supports Foxp3 Expression
Although tumor derived TGF-β was central to upregulating Treg Usp22 and Usp21 in vitro, TGF-β suppression was insufficient to abolish Usp22 upregulation in itTreg cells (
Under hypoxic conditions, hypoxia inducible factors α (HIF-α) are stabilized resulting in the activation of a transcriptional program that promotes cellular adaptation to low oxygen levels (32). HIF-α are known to have two functional binding sites on the Usp22 promoter (33), suggesting that hypoxic induction of Usp22 may be HIF-α-dependent. Indeed, incubation with hypoxia-independent HIF-α activator, dimethyloxalylglycine (dMOG), increased Usp22 mRNA level in both nTreg and iTreg cells (
In addition to oxygen, glucose levels in the TME are often decreased, in part through its enhanced uptake by tumor cells which compete with the glucose necessity of the highly glycolytic Teff cells (34, 35). Conversely, FOXP3 promotes oxidative phosphorylation over glycolysis in Treg cells, potentially giving them a functional advantage within the TME (5, 36, 37). Therefore, we hypothesized the observed Treg cell advantage in nutrient deprived environments could exist partially as a consequence of USPs mediated stabilization of Foxp3 expression. Indeed, Usp22 mRNA and protein levels were increased in Treg cells upon glucose deprivation (
Along with the competition for glucose, a scarcity of amino acids within tumors may also alter immune cell function (35). Importantly, amino acid starvation is known to enhance Treg cell induction (38). To investigate the role of USPs in amino acid starvation induced Foxp3 expression we cultured Treg cells in media lacking amino acids. Indeed, amino acid starvation led to increased expression of Usp22 and Usp21, but not Usp7, in nTreg and iTreg cells (
It is well known that AMPK functions in balance with mammalian target of rapamycin (mTOR) signaling to regulate the cellular metabolic state (39). Intriguingly, pharmacologic inhibition of mTOR also resulted in increased Usp22 and Usp21, but not Usp7, expression in nTreg cells (
It has been proposed that itTreg cells better adapt to the metabolically stressful conditions of the TME, which offers them a functional advantage over Teff cells (5, 19). Combined, our data suggests that alterations in the microenvironment can drive increased levels of Usp22 and Usp21 potentially through modulation of HIFα, AMPK, and mTOR activity to enhance Treg stability in the tumor microenvironment.
Our discoveries thus far have suggested that Usp22, and to a lesser extent Usp21, are important in maintaining FOXP3 expression and thus Treg fitness in the TME through multiple pathways. To study their combined functionality in vivo, we generated a strain of Treg-specific Usp22 and Usp21 double knockout (dKO) mice by breeding Usp21f/f mice with Usp22f/fFoxP3YFPere single knockout mice. This breeding strategy gave us the Treg-specific knockout of Usp22 (22KO), Usp21 (21KO), and the dKO, all of which were confirmed via qPCR (
Unsurprisingly, all three KO groups showed significant increase in CD44hiCD62lo activated splenic Teff cells in comparison to age matched WT mice, consistent with the development of low level, progressive inflammation with age (
Interestingly, transcriptional profiling revealed more Treg cell suppressive markers were differentially expressed in the dKO mice than in either single KO animal when compared to WT gene expression (
As we demonstrated that both Usp22 and Usp21 are regulated by metabolic alterations in the TME, it was particularly interesting to identify disruption of multiple metabolic pathways in each of the KO animals. In fact, Treg cells from dKO mice had profound changes in lipid metabolic processes, one carbon metabolism, and ribosomal biogenesis (
As Usp21 seemed to have a Foxp3-independent role in Treg function, we compared the DEGs from the 22KO and the dKO mice in order to determine the contribution of Usp21 to the dKO phenotype. Interestingly, we noticed significant changes in cell cycle pathways and effector differentiation pathways (
Collectively, these data imply that both Usp22 and Usp21 modulate Treg cell metabolism although seemingly through unique pathways to maintain Treg stability and function.
To test the importance of Treg cell Usp22 and Usp21 in tumor conditions in vivo, we used the B16 melanoma syngeneic tumor model. Mice with Treg-specific ablation of Usp22 showed increased tumor rejection compared to the deletion of Usp21. Importantly, though, mice harboring the joint deletion of both Usp22 and Usp21 in Treg cells grew the smallest tumors (
Further analysis of tumor infiltrating lymphocytes indicated a significant increase in CD4+ and CD8+ T cell frequencies in the dKO mice, with each compartment in the dKO secreting higher amounts of both IFN-γ and GZMB than WT mice (
Although the loss of USP22 alone displayed significant anti-tumor immunity, the loss of both Usp22 and Usp21 in Treg cells displayed a more vigorous anti-tumor response, as documented by the dKO mice having a dramatically increased cytokine production, the highest infiltrating T cell number, and the smallest tumor sizes. Collectively, this data suggests that Usp21 and Usp22 cooperate to maintain Foxp3 expression and Treg cell function in the TME.
Although deletion of Usp21 in addition to Usp22 in Treg cells enhances antitumor immunity, Usp22 deletion alone is sufficient in diminishing tumor burden. To assess whether pharmacologic inhibition of Usp22 could modulate Treg function, we aimed to identify Usp22-specific inhibitors. It has been suggested that in vitro purified USP22 protein lacks catalytic activity (40, 41), leading to difficulties for high-throughput screening. Therefore, we used the computer-aided drug design (CADD) to develop a Usp22-specific small molecule inhibitor (
We then used both Lipinski's Rule and Veber's Rule to filter through the Specs database and found a total of 240K compounds binding to the catalytic pocket of our Usp22 model. We then filtered the top 100 compounds ranked by docking affinity by MD and MM/PBSA methods and were left with 25 compounds (Table 1). This limited number of compounds allowed us for further biological screening. As USP22 suppression leads to dramatic reduction in FOXP3 expression levels, we utilized FOXP3 MFI reduction as a readout for the biological validation of USP22 inhibitory efficacy by each of the 25 chemicals. As indicated in Table 1, the chemical S02 (11-anilino-7,8,9,10-tetrahydrobenzimidazo[1,2-b]isoquinoline-6-carbonitrile) showed strong efficacy in downregulating FOXP3 expression. The compound S02, structure shown in
After initial screening, we ran an in vitro dose response study on compound S02, now dubbed Usp22i-S02, in both WT and Usp22-null iTreg cells (
An important aspect of a potential immunotherapeutic is its antitumor functionality paired with low immune toxicity. To determine the toxicity of Usp22i-S02 in vivo, we first determined its effects in naïve mice. We found little alteration in the weights, B cell and Teff cell percentages and proliferation, and Teff cell activation of treated mice compared to DMSO-treated control mice (
To determine the functionality of Usp22i-S02 as a potential therapeutic, we tested the inhibitor on established tumors. Following initial LLC1 tumor establishment, WT mice administered Usp22i-S02 showed striking tumor rejection compared to untreated mice, as well as a significant increase in Teff cell tumor infiltration (
As Usp22 is also an important oncogene (43, 44), we were interested in the potential dual-therapeutic function of Usp22i-S02. Indeed, administration of Usp22i-S02 to LLC1 cells in vitro resulted in decreased tumor cell counts, viability, and growth (
Emerging data suggests that the TME, which is deprived of nutrients and oxygen, likely offers a metabolic advantage to Treg cells over Teff cells to further promote an immunosuppressive microenvironment. However, the TME-specific factors and their cellular targets that potentiate Treg cell suppressive function and adaptation remain largely unidentified. Our study illustrates a previously unappreciated role of Foxp3-specific DUBs, Usp22 and Usp21, as environmentally-sensitive factors that enhance Foxp3 stability in the TME. We identified several TME factors that upregulate Usp22 and Usp21, ultimately stabilizing Foxp3: (1) tumor-secreted TGF-β; (2) hypoxia; (3) glucose-restriction; and (4) amino acid-deprivation (
As it has been well-documented that itTreg cells are more suppressive and often have high Foxp3 expression (9, 10, 46), we first confirmed these findings in various murine tumor models. Interestingly, we found that itTreg cells in these models and in lung cancer patients upregulate Usp22, and sometimes Usp21, when compared to non-tumor-residing Treg cells. Furthermore, Usp22 upregulation is correlated with higher Foxp3 expression in human lung cancer itTreg cells, suggesting that TME factors selectively induce these USPs to protect Foxp3 from ubiquitin-mediated degradation while simultaneously promoting Foxp3 transcription. The fact that we observed increased Usp22 in human itTreg cells broadens the relevance of this pathway to human tumor therapies. Although Usp7 in Treg cells is known to control Foxp3 expression and Treg suppressive function in a model of colitis, we did not observe an increase in Usp7 expression in itTregs, suggesting Usp7 may primarily regulate Treg function during homeostatic conditions.
TGF-β is a major player in iTreg conversion and stability and is broadly secreted by many tumor types. We found that tumor secreted TGF-β is sufficient in upregulating Usp22 through canonical TGF-β signaling. Furthermore, Usp22 partakes in a feedback loop to further upregulate itself and Foxp3 through SMAD protein stabilization. Although Usp21 was not functioning through the canonical TGF-β pathway, it is possible that the non-canonical TGF-β JNK/P38 signaling pathway could be at play (47). As TGF-β is widely implicated in Foxp3 expression and stability, and iTreg function, our data adds a new level of complexity to already known systems (23, 48). These novel mechanisms potentially function to ensure Treg cell stabilization through alternate pathways, strengthening their ability to maintain their suppressive capacity in diverse microenvironments.
However, tumor-secreted TGF-β is not the only factor capable of upregulating USPs, since Treg cells treated with EG7 TCM could not recapitulate the increase of Usp22 seen in itTreg cells isolated from EG7 tumors. Therefore, we hypothesized that other environmental factors within the TME are also implicated in Treg stabilization through USPs. As hypoxia is a major hallmark of solid tumors (3, 29), we investigated how low oxygen conditions influence Usp22 levels in Treg cells. Hypoxia induced Usp22 in a HIF-dependent manner. Also, upon Usp22 deletion, nTreg cells under hypoxic stress could not sustain stable FOXP3 expression. Our findings are in line with previous data that demonstrated heightened proliferation and suppressive capabilities of nTreg cells under hypoxic conditions (27). These data, paired with the knowledge of two functioning HIF binding sites along the Usp22 promoter, imply that hypoxia can enhance Treg suppressive function through Usp22-dependent stabilization of FOXP3 (33).
Along with a decrease in oxygen availability, the competition for nutrients that occurs within the TME influences immune cell growth, survival, and function. Classically, Treg cells are thought to have a significantly lower reliance on glycolysis than Teff cells, potentially providing another advantage (5, 34, 37). Our data identifies Usp22 as an important mediator in this process, functioning to stabilize FOXP3 under glucose- and amino acid-deprivation. In part the enhanced stability of FOXP3 appears secondary to AMPK activation, which likely occurs under glucose restriction within the TME. Interestingly, AMPK activation in Treg cells is accompanied by a shift towards oxidative metabolism, which may further enhance Treg survival in the TME (49). We show that AMPK activation is sufficient to upregulate Usp22 and Usp21, implicating their involvement in FOXP3 stabilization for Treg cell function under energy stress. The promotion of AMPK signaling via nutrient deficiency also suppresses mTOR activity within T cells (35, 50). As the balance of AMPK and mTOR signaling functions as an environmental sensor for nutrient availability, it is possible that AMPK activation primarily increases Usp22 and Usp21 expression thru inhibition of mTOR signaling. Indeed, mTOR inhibition was capable of upregulating Usp22 and Usp21 in Treg cells.
The metabolic status of an immune cell is highly important within the TME for their cell survival and function. As Treg cells can adapt to low-oxygen, low nutrient environments, this gives them a metabolic advantage compared to Teff cells. Importantly, FOXP3 is essential to this process as it is known to promote oxidative phosphorylation within Treg cells. We show that Usp22- and Usp21-deficient Treg cells have significantly altered expression of metabolic genes and impaired OCR and ECAR. In addition, RNA sequencing analysis demonstrated that loss of Usp22 and Usp21 in Treg cells resulted in the upregulation of multiple pathways associated with cell growth and proliferation. Collectively, these data raise the intriguing possibility that Usp22 and Usp21 work to promote Treg cell quiescence in nutrient-restricted environments in part through modulating Treg cell metabolic programs. Together, our data indicate that microenvironmental stress within the TME upregulates Treg USP levels, which then function to stabilize FOXP3. Enhanced FOXP3 stability further supports Treg cell adaptation to the TME; thus, identifying Usp22 and Usp21 as important environment-sensitive factors that regulate Treg cell identity, metabolism and function in the TME.
Additionally, we and others have demonstrated that both Usp22 and Usp21 are upregulated in many cancer types, such as gastric carcinoma, pancreatic cancer and melanoma, and have been correlated with poor prognosis (51, 52). Usp22 promotes oncogenic c-Myc activation as well as indirectly antagonizes the tumor suppressive function of p53, while Usp21 functions as an oncogene by stabilizing a group of transcription factors including Fra1, FoxM1 and Wnt (52-54). Importantly, Usp22 and Usp21 also function to maintain Foxp3 expression through DUB function at the transcriptional (Usp22) and post-translational (both) levels. This duality makes Usp22 and Usp21 highly attractive potential therapeutics that can target both tumor cell intrinsic and immunosuppressive pathways simultaneously. Indeed, their combined loss resulted in the most significant impairment in Treg tumor-promoting functions, suggesting that Usp22 and Usp21 play distinct roles in modulating Treg cell adaption and function in the TME.
However, loss of Usp22 in Treg cells resulted in enhanced anti-tumor immunity relative to the loss of Usp21, suggesting a dominance of Usp22 in itTreg cells. Therefore, specifically targeting Usp22 may be sufficient in eliminating the advantage Treg cells have over Teff Within the TME. To test this, we developed and tested the first ever Usp22-specific inhibitor. Administration of the inhibitor resulted in a dramatic decrease in itTreg number, resulting in strong in vivo anti-tumor effects. Our data demonstrate that Usp22 is a targetable protein, and that the inhibitor Usp22i-S02 has the potential of being incorporated into tumor immune therapies. Furthermore, many current therapeutics focus on promoting Teff cell function, as such the addition of Usp22 inhibition with current therapies could further enhance anti-tumor immunity through synergistic effects.
EG7 Lymphoma, LLC 1 lung carcinoma, and B 16-F10 melanoma cell lines were provided by the Zhang laboratory at Northwestern and used for tumor models as previously reported (14). The cells lines were cultured in DMEM with 10% FBS, and were tested for mycoplasma using LookOut Mycoplasma PCR detection kit (Sigma, MP0035-1KT). Cultured cancer cells were trypsinized and washed once with PBS. LLC1 lung carcinoma tumor cells were subcutaneously administered to the right flank of 8- to 10-week-old mice at 1×106 tumor cells per mouse, and B16 melanoma at 5×104 tumor cells per mouse. Tumors were measured every 2-3 days by measuring along 3 orthogonal axes (x, y and z) and tumor volume was calculated as (xyz)/2. The tumor size limit agreed by IRB was 2 cm3.
In Vitro iTreg Cell TCM and TGF-β Assays
Previously generated iTreg cells were washed and rested for 7 hours in OPTImem media containing 5 ng/ml of IL-2 to maintain survival. OPTImem was used to avoid any TGF-β contamination found in serum. After resting, the cells were incubated in OPTImem containing IL-2 with or without the addition of 20 ng/ml TGF-β or the various tumor cell medias (B16, LLC1, and EG7). TCM was obtained by plating B16, EG7, or LLC1 cell lines at 50% confluency for 16 hours. TCM was then mixed 50:50 with fresh OPTImem and incubated on iTreg cells for 24 hours. TGF-β inhibitor LY 3200882 (Med Chem Express: Cat. No.: HY-103021) was added at 25 μg/mL where indicated.
nTreg cells were isolated as described above and cultured at 37° C. in either normoxic (21% O2) or in a hypoxic condition (1% O2) for 24 hours. Hypoxia was induced using (Name of hypoxia chamber and company). T cell medium was incubated at 37° C. at normoxia or hypoxia for 3 hours prior to usage. Cells were then collected and RNA was extracted as described above. For iTreg cells, cells were isolated and polarized as described above. Subsequently, cells were rested in optiMEM overnight and then plated in optiMEM containing 5 ng/ml IL-2 in either normoxic or hypoxic conditions. optiMEM media was incubated at 37° C. at normoxia or hypoxia overnight prior to usage. Hypoxia stability assay was conducted as described above but cells were cultured in normoxia or hypoxia for 72 hours, then collected and stained for FOXP3 for flow cytometry.
nTreg cells were isolated as described above and cultured in either normal T cell medium, T cell medium lacking glucose (Thermo Fisher Catalog #11879020), or T cell medium lacking amino acids including glutamine (US Biological Catalog #R9010-02) substituted with dialyzed FBS (GIBCO Catalog #A3382001) for 24 hours at 1×105 cells per well. T cell media included with 2000U of IL-2 and CD3/CD28 beads as described above. iTreg cells were isolated and polarized as described above for 3 days. Following polarization, iTreg cells were cultured in normal T cell media or T cell media lacking glucose or amino acids for 24 hours. Both nTreg and iTreg cells were then collected and RNA was extracted as described above. For stability assays, cells were cultured as described above for 48 hours, then collected and stained for FOXP3 for flow cytometry.
All nTreg and iTreg cells were plated as described above DMOG (Sigma Catalog #D3695) was administered to the cells in relevant experiments at 1 mM for 24 hours. Oligomycin (Sigma Catalog #75351) was administered at 1 μM to the media of the cells in relevant experiments for 24 hours. Torin 1 (Millipore Catalog #475991) was administered to the relevant cells at 250 nM for 24 hours. FOXP3 protein level was assessed via flow cytometry following 48 hours of treatment of inhibitors described above. In vitro administration of Usp22i-S02 was at 10 ug/mL.
LLC 1 cells were transplanted into 6-to-8-week-old C57BL/6 male mice. Subcutaneous injections were performed in the right flank of mice in a final volume of 100 μL using 1{circumflex over ( )}6 cells per injection. The USP22i-S02 was injected intraperitoneally (i.p) at a concentration of 20 mg/kg/time, in 100 μL of oil, twice a day for 5 days beginning on the day of the LLC1 cells injection. Control animals received 100 μL of oil alone. Subcutaneous tumor diameters were measured daily with calipers until any tumor in the mouse cohort reached 2.5 cm in its largest diameter. Cells were processed and analyzed as stated above.
No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. All statistical analyses were computed with GraphPad and tests used for each experiment are listed in the fig. legends. ANOVAs with multiple comparisons between rows were corrected with Tukey's test to determine statistical significance. Two-tailed unpaired t tests were performed with Welch's correction.
Immunotherapy 6, 1295-1311 (2014).
Our studies identify 11-anilino-7,8,9,10-tetrahydrobenzimidazo[1,2-b]isoquinoline-6-carbonitrile, or USP22i-S02, as a USP22-specific inhibitor. This inhibitor appears to be an ideal antitumor therapeutic drug because: (i) it inhibits Treg suppressive functions and (ii) inhibit tumor cell expression of PD-L 1, both of which enhances antitumor immune response. In addition, (iii) USP22i-S02 can directly inhibit tumor cell proliferation through USP22 suppression.
This application claims benefit of priority to U.S. Patent Application Ser. No. 63/201,330, filed Apr. 23, 2021, the contents of which are incorporated by reference in its entirety.
This invention was made with government support under CA232347 and CA220801 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/26159 | 4/25/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63201330 | Apr 2021 | US |
