WO2025059029A1 - Methods of treating vexas syndrome - Google Patents

Abstract

There is provided a method of treating VEXAS comprising administering a therapeutically effective amount of a compound means of inhibiting UBA1, or of a UBA1 inhibitor to a patient in need thereof. Also provided is a UBA1 inhibitor, or compound means of inhibiting UBA1, for use in a method of treating VEXAS syndrome comprising the administration of a therapeutically effective amount of the compound means of inhibiting UBA1, or the UBA1 inhibitor to a patient in need of the treatment. Also provided is the use of a compound means of inhibiting UBA1, or a UBA1 inhibitor in the manufacture of a medicament for the treatment of VEXAS syndrome.

Description

METHODS OF TREATING VEXAS SYNDROME

FIELD

[0001] There are provided methods in the field of treating the myeloid autoinflammatory disease VEXAS syndrome.

BACKGROUND

[0002] Protein ubiquitination plays an essential role in all aspects of normal cellular function and involves the sequential action of ubiquitin-activating (El), ubi quitin-conjugating (E2), and ubiquitin ligase (E3) enzymes. UBA1 resides on the X chromosome and encodes the El enzyme isoforms UBAla and UBAlb, which are essential for the majority of nuclear and cytosolic protein ubiquitination, respectively.

[0003] Somatic UBA1 mutations in hematopoietic cells have been associated with an aggressive, late-onset, myeloid autoinflammatory disease called VEXAS (Vacuoles, El enzyme, X-linked, Autoinflammatory, Somatic) syndrome. The clinical features of VEXAS syndrome frequently include recurrent fever, chondritis, neutrophilic dermatoses, alveolitis, vasculitis, and cytopenias. In addition, myeloid and erythroid precursors in the bone marrow of VEXAS syndrome patients often contain abnormal vacuoles. A subset of patients with VEXAS syndrome also meet diagnostic criteria for myelodysplastic syndromes, indicating that somatic UBA1 mutations may cause both autoinflammation and bone marrow failure.

[0004] VEXAS syndrome carries a poor prognosis. Therapies for VEXAS syndrome, including glucocorticoids, hypomethylating agents, JAK inhibitors, and cytokine-targeted biologies, have shown variable efficacy in controlling inflammation and rarely lead to a reduction in the UBA1 mutant clonal burden. Indeed, aside from allogeneic hematopoietic stem cell transplantation, there are currently no treatments for VEXAS syndrome with curative potential. See, e.g., Poulter et al, in which the authors conclude that further studies may lead to development of targeting treatments and particularly that “A more radical treatment approach, such as bone marrow transplant, might need to be considered early in the disease course.” Poulter et al., “Novel Somatic Mutations in UBA1 as a cause of VEXAS Syndrome” Blood, 2021, 137(26):3676-3681.

[0005] Alternative therapeutic strategies for treating VEXAS Syndrome are urgently needed in the art. SUMMARY

[0006] There is provided a method of treating VEXAS syndrome comprising administering a therapeutically effective amount of a composition comprising compound means of inhibiting UBA1 and a pharmaceutically acceptably carrier to a patient in need thereof.

[0007] In another aspect, a method of treating VEXAS syndrome comprising administering a therapeutically effective amount of a UBA1 inhibitor to a patient in need thereof is provided.

[0008] In another aspect, a UBA1 inhibitor for use in a method of treating VEXAS syndrome in a patient in need thereof is also provided.

[0009] In another aspect, the use of a UBA1 inhibitor in the manufacture of a medicament for administration in the treatment of VEXAS syndrome to a patient in need thereof is provided.

[0010] In another aspect, a composition for treating VEXAS syndrome in a patient in need thereof is provided. The composition comprises compound means for inhibiting UBA1 and a pharmaceutically acceptable carrier, wherein said composition is suitable for use in the treatment of VEXAS syndrome.

[0011] The method, UBA1 inhibitor, composition or use provided herein may make use of a UBA1 inhibitor, or compound means of inhibiting UBA1. In some embodiments, the UBA1 inhibitor or compound means of inhibiting UBA1, administered or used is TAK-243.

[0012] In some embodiments, the UBA1 inhibitor or composition comprising compound means of inhibiting UBA1 is administered in combination with an additional treatment, the additional treatment comprising i) administration of one or more of a corticosteroid, a hypomethylating agent, a JAK inhibitor, a proteosome inhibitor, or biologies targeting inflammatory cytokines ii) allogenic hematopoietic stem cell transplant.

BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG. 1A is a line graph illustrating of the proliferation of 32D cells with hemizygous expression of Ubal^WT and Ubal^M41L after three-day stimulation with IL3.

[0014] FIG. IB is a volcano plot showing differential cytokine secretion by unstimulated 32D Ubal^WT and Ubal^M4IL cells. Three biological replicates of two single cell clones per genotype were analyzed by multiplex cytokine assay in duplicate.

[0015] FIG. 1C is a line graph illustrating of the proliferation of 32D cells with hemizygous expression of Ubal^WT and Ubal^M41L after three days in the presence of DMSO, or a range of azacytidine concentrations. [0016] FIG. ID is a line graph illustrating of the proliferation of 32D cells with hemizygous expression of Ubal^WT and Ubal^M4IL after three days in the presence of DMSO, or a range of ruxolitinib concentrations.

[0017] FIG. IE is a line graph illustrating of the proliferation of 32D cells with hemizygous expression of Ubal^WT and Ubal^M41L after three days in the presence of DMSO, or a range of bortezomib concentrations.

[0018] FIG. 2A is a line graph illustrating of the proliferation of 32D cells with hemizygous expression of Ubal ^{W T} and Ubal^M4IL after three days in the presence of DMSO, or a range of TAK- 243 concentrations.

[0019] FIG. 2B is a graphical depiction of the data collected from a competition assay between 32D Ubal^WT and Ubal^M41L cells in the presence of DMSO (open symbols) or lOnM TAK-243 (closed symbols) over four days.

[0020] FIG. 2C is a bar graph illustrating the quantification of apoptotic cells by annexinV- propidium iodide (PI) flow cytometry after 16-hour treatment of 32D Ubal^{n T} and Ubal^M4IL cells with DMSO or lOnM TAK-243.

[0021] FIG. 2D shows an immunoblot for PARP1, H2AX, and H2AX pS139 proteins after 16- hour treatment of 32D Ubal^WT and Ubal^M41L cells with DMSO or lOnM TAK-243.

[0022] FIG. 2E is a line graph illustrating of the proliferation of 32D cells with hemizygous expression of Ubal^WT Ubal^M41L or Ubal^M41L'^A580S over three days in the presence of DMSO or a range of TAK-243 concentrations. A single representative experiment (mean±s.d. of three technical replicates).

[0023] FIG. 2F is a line graph illustrating the proliferation of THP1 cells with hemizygous expression of UBA1^W1 and UBAl^M4n after three days in the presence of DMSO, or a range of TAK-243 concentrations.

[0024] FIG. 3A is a schematic illustration depicting the cDNA constructs overexpressed in 32D Ubal^M41L cells.

[0025] FIG. 3B shows an immunoblot for UBA1, V5 and ubiquitin in 32D Ubal^WT and Ubal^M4IL parental cells lines and Ubal^M41L cells overexpressing UBA1 Al-40 (UBAlb), UBA1 Al- 40/C632A (UBAlb/C632A) or luciferase (Luc).

[0026] FIG. 3C is a line graph illustrating of the proliferation of 32D cells with hemizygous expression of Ubal^WT and Ubal^M41L parental lines and Ubal^t'^I4IL cells overexpressing UBA1, UBAlb/C632A, or luciferase after three days in the presence of DMSO, or a range of TAK-243 concentrations. The y-axis is % maximum proliferation.

DETAILED DESCRIPTION

[0027] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains.

[0028] Further, nomenclature used herein is as commonly used in the art as can be seen by reference to, e.g., http://www.informatics.jax.org/mgihome/nomen/gene.shtml and

[0029] As used herein, the following terms have the meanings as ascribed to them below, unless specified otherwise or obvious from context.

[0030] The term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0031] The term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” may be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. All numerical values provided herein are modified by the term about.

[0032] Ranges provided herein are understood to be shorthand for all values within the range, including fractions/decimals. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0033] As used herein, “effective amount" or “therapeutically effective amount” refers to a quantity sufficient to reduce the signs/symptoms of VEXAS syndrome, e.g., inflammation, following treatment and/or to eliminate or reduce the UBA1 mutant cells in a subject. Although not required to practice the invention, it is preferred that the UBA1 mutant clonal burden be determined by genetic sequencing assays, e.g., such as those available from Genomic Testing Cooperative, Irvine CA. When an effective amount or therapeutically effective amount is indicated, the precise amount of the indicated agents to be administered may be determined by a physician with consideration of the potency of the agent(s) and the age, weight, the extent of progression of VEXAS syndrome, and/or other condition(s) of the patient (subject). UBA1 inhibitors are anticipated to be effective to treat VEXAS syndrome at lower doses than required to treat solid tumors, thereby minimizing any potential toxicity concerns.

[0034] The term “administer”, “administering” or “administration” refers to the act of the attending physician or caregiver, prescribing the agent for administration and thereby causing the application of an agent to a subject, through ingestion, inhalation, infusion, injection, or any other means, whether self-administered or administered by a clinician or other qualified care giver.

[0035] The terms "subject" and “patient” are used interchangeably herein and indicate a human.

[0036] As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating VEXAS Syndrome and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating VEXAS Syndrome does not require that the symptoms associated therewith be completely eliminated. As used herein "treatment" or "treating," includes any beneficial or desirable effect on the symptoms or pathology of VEXAS syndrome and may include even minimal reductions in one or more measurable markers of the disease.

[0037] A “Ubiquitin-like modifier Activating enzyme 1 inhibitor” or “UBA1 inhibitor” refers to a well-established class of compounds that inhibit UBA1. Via interference with the activity of UBA1, UBA1 inhibitors modulate the ubiquitin-proteasome system. UBA1 inhibitors can thus disrupt protein degradation pathways and potentially affect cellular processes that rely on such pathways. Suitable UBA1 inhibitors are known in the art or may be identified by their ability to interfere with the enzymatic activity of UBA1 and/or kill cells that are dependent on UBA1 for survival.

[0038] Ubiquitin-like modifier activating enzyme 1 (UBA1) is the first identified El activating enzyme for ubiquitin activation. UBA1 plays a central role in ubiquitin activation and protein quality. Total loss of UBA1 is lethal, while defects in UBA1 expression or activity contribute to the pathogenesis of several neurodegenerative disorders such as spinal muscular atrophy, Huntington’s disease, Parkinson’s disease, Alzheimer’s disease, acute myeloid leukemia and amyotrophic lateral sclerosis.

[0039] UBA1 inhibitors suitable for the present invention are known in the art and include representative species TAK-243. The aforementioned UBA1 inhibitor is described in Patent or Publication No. US 20130217682 (TAK-243). At least because of its specificity for UBA1, TAK- 243 is particularly preferred.

[0040] EC50/IC50/Dose data for TAK-243 are shown in TABLE 1, below: [0041] TABLE 1

[0042] The phrase “compound means of inhibiting UBA1” or “UBA1 inhibitor means” refers to organic compounds i) having a combination of atoms that result in a molecular weight of less than 1500 g/mol ii) that inhibit UBA1 by binding one or more binding sites through direct molecular interaction between the compound and UBA1 and iii) having an IC50 of 5 pM or less. In some embodiments, the compound means of inhibiting UBA1 has an IC50 of 2.5 pM or less, or 0.25 pM or less, or 25 nM or less, or 2.5 nM or less, or 0.25 nM or 0.025 nM or even 0.0025 nM or less. Structures having this function include compounds found in US 20130217682, incorporated herein by reference in their entirety. Preferably, the compound means of inhibiting UBA1 comprises TAK-243. Functional equivalents of these compounds would be organic compounds having an IC50 of 5 pM or less. In some embodiments, functional equivalents of these compounds have an IC50 of 2.5 pM or less, or 0.25 pM or less, or 25 nM or less, or 2.5 nM or less, or 0.25 nM or 0.025 nM or even 0.0025 nM or less against UBA1 as measured by a luminescent cell viability assay such as the CellTiter-Glo™ luminescent cell viability assay. Said functional equivalents would also result in inhibition of UBA1 to a degree sufficient to increase the sensitivity of a UBAl mutant cell to the inhibition of UBA1 relative to UBA1 wild-type (WT).

[0043] A person of ordinary skill in the art would be able readily ascertain whether a given UBA1 inhibitor, or compound means of inhibiting UBA1, is capable of forming a salt. A person of ordinary skill in the art would thus understand the UBA1 inhibitors, and compound means of inhibiting UBA1, described herein to include pharmaceutically acceptable salts. Pharmaceutically acceptable salts and common methodology for preparing them are well known in the art. See, e.g., P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd 30 Revised Edition (Wiley-VCR, 2011); S. M. Berge, et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, Vol. 66, No. 1, Jan. 1977.

[0044] TAK-243 is a small molecule inhibitor of UBA1 and is known to block ubiquitin conjugation and to this disrupt monoubiquitin signaling, as well as global protein ubiquitination. TAK-243 was formerly known as MLN7243 and is also identified by CAS 1450833-55-2. The chemical name of TAK-243 is [(lR,2R,3S,4R)-2,3-dihydroxy-4-[[2-[3- (trifluoromethylsulfanyl)phenyl]pyrazolo[l,5-a]pyrimidin-7-yl]amino]cyclopentyl]methyl sulfamate. TAK-243 is currently in phase I clinical trials for treatment of acute myeloid leukemia (AML). See, https://classic .clinicaltrials.gov/ct2/show/NCT03816319. Cells exhibiting a mutation of UBA1 can be particularly sensitive to UBA1 inhibitors and so, administration of lower doses of TAK-243 (see, e.g., FIG. 3C) is expected to be efficacious for the treatment of VEXAS syndrome than are necessary for the treatment of, e.g., AML and solid malignancies.

Treatment of VEXAS Syndrome

[0045] ‘ ‘A patient in need thereof’ may be a patient exhibiting any of the following signs or symptoms:

• Systemic inflammation affecting multiple organs, including neutrophilic dermatoses and/or other painful skin rashes; chondritis and/or other pain, swelling or inflammation of cartilaginous structures; pulmonary inflammation (causing cough and/or shortness of breath); pain, swelling and/or inflammation of the joints; and vasculitis and/or other inflammation of the blood vessels.

• Patients also often exhibit recurrent fevers and extreme fatigue.

• Hematologic features can include anemia, thrombocytopenia, and/or blood clots.

• In some instances, patients with VEXAS syndrome have associated clinical diagnoses, including relapsing polychondritis, polyarteritis nodosa, Sweet syndrome, myelodysplastic syndromes (MDS), monoclonal gammopathy of unknown significance (MGUS) and/or multiple myeloma.

[0046] Typically, patients may be suspected as having VEXAS if they have two or more of the above symptoms, and particularly when the patent has an overlap of inflammatory and hematologic manifestations. Genetic testing provides a definitive diagnoses of VEXAS syndrome, via identification of one or more mutations of the UBA1 gene located in the X-chromosome. In some embodiments, the patient in need of treatment may exhibit one or more somatic mutations of UBA1, including but not limited to, mutation(s) affecting methionine 41 of exon 3 (e.g., р.M41T, p.M41V, pM41L), splice region mutations at exon 3 (c.H8-2A>C, c.H8-lG>C and с.118-9 118-2del), mutations of tyrosine 55 of exon 3 (e.g., p.Y55H), mutations of serine 56 of exon 3 (e.g., p.S56F), or amino acids of exons 12, 13, 14, 15, or 16 within the active adenylation domain (e.g., p.G477A, p.A478S, p.D585A, p.S621C). [0047] UBA1 inhibitors may be formulated as pharmaceutical compositions by any of the methods known to those of ordinary skill in the art. Such pharmaceutical compositions and methods are described, e.g., in Remington: The Science and Practice of Pharmacy(I) (A. Gennaro, et al., eds., 21st ed., Mack Publishing Co., 2005).

[0048] Typically, pharmaceutical compositions may comprise the UBA1 inhibitor and a pharmaceutically acceptable carrier. As used herein, the phrase “pharmaceutically acceptable carrier (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms “carrier” or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents. The term “carrier” may also encompass any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. Pharmaceutical compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the UBA1 inhibitor based on the weight of the total composition including the carrier.

[0049] The pharmaceutical compositions of the present application include those suitable for any acceptable route of administration. Particularly acceptable routes of administration include oral or parenteral administration.

[0050] In some embodiments, the pharmaceutical composition may be formulated for oral administration. Compositions suitable for oral administration can be presented as discrete units such as capsules, sachets, granules or tablets each containing a predetermined amount (e.g., effective amount) of the active ingredient; a powder or granules; a solution or a suspension in an aqueous liquid or a non-aqueous liquid; an oil-in-water liquid emulsion; a water-in-oil liquid emulsion; packed in liposomes; or as a bolus, etc. Soft gelatin capsules can be useful for containing such suspensions, which can beneficially increase the rate of compound absorption. In the case of tablets for oral use, carriers that are commonly used include lactose, sucrose, glucose, mannitol, and silicic acid and starches. Other acceptable excipients can include: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. For oral administration in a capsule form, useful diluents include lactose and dried com starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents can be added. Compositions suitable for oral administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia.

[0051] Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions or infusion solutions which can contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, saline (e.g., 0.9% saline solution) or 5% dextrose solution, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

[0052] The injection solutions can be in the form, for example, of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant.

[0053] In the present pharmaceutical compositions, the UBA1 inhibitor is provided in a therapeutically effective amount. As described above, the effective dose can vary, the severity of the disease, the route of administration, the sex, age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician.

[0054] In some embodiments, an effective amount of the UBA1 inhibitor or compound means of inhibiting UBA1 is an amount sufficient to achieve a concentration in the blood from 0.1-100 nM. In some embodiments, the effective amount of the UBA1 inhibitor or compound means of inhibiting UBA1 is an amount sufficient to achieve a concentration in the blood of from 0.1 nM to about 75 nM; 0.1 nM to about 50 nM; 0.1 nM to about 25 nM; from 0.1 nM to about 10 nM; from 0. InM to about 5 nM; from 0.1 nM to about 1 nM; or from about 0.1 mg/kg to about 0.5 nM.

[0055] The foregoing dosages can be administered on a daily basis (e.g., as a single dose or as two or more divided doses, e.g., once daily, twice daily, thrice daily) or non-daily basis (e.g., every other day, every two days, every three days, once weekly, twice weekly, once every two weeks, once a month).

[0056] If administered as a monotherapy, the UBA1 inhibitor, or pharmaceutical composition comprising compound means of inhibiting UBA1, may be administered parenterally, e.g., via intravenous infusion, in amounts up to 60 mg, 40 mg, 20 mg, 18 mg, 12 mg, or up to 10 mg, or up to 8 mg, or up to 4 mg. One exemplary infusion schedule is the infusion of the desired dose, twice weekly for four weeks. The infusion time is determined by the attending healthcare provider and is generally administered over a period of 5 to 60, preferably about 10 minutes.

[0057] In addition to administration as a monotherapy, the UBA1 inhibitors or pharmaceutical compositions comprising the compound means of inhibiting UBA1, may be administered in combination with other treatments for reducing the symptoms of VEXAS syndrome corticosteroids, hypomethylating agents, JAK inhibitors, proteosome inhibitors, biologies targeting inflammatory cytokines and allogenic hematopoietic stem cell transplant (HSCT).

[0058] As well known in the art, corticosteroids are synthetic drugs that closely resemble cortisol. Corticosteroids are often administered to decrease immune system response to various diseases to reduced symptoms such as inflammation. Non-limiting suitable examples of corticosteroids include cortisone, prednisone, prednisolone and methyl prednisolone. Corticosteroids are typically administered orally or parenterally, in dosages ranging from 20 to 300 mg in adults and determined by weight in children.

[0059] Hypomethylating agents (or demethylating agents) are drugs that inhibit DNA methylation, i.e., the modification of DNA nucleotides by addition of a methyl group. Currently available hypomethylating agents block the activity of DNA methyltransferase. Suitable hypomethylating agents are known in the art, and include 5-azacytidine, guadecitabine, zebularine and decitabine. Any suitable administration form and dosage may be administered. Typically, hypomethylating agents are administered parenterally, in dosages up to 50 mg per day.

[0060] Janus kinase (JAK) inhibitors target the JAK kinases that in turn regulate the transcription of several genes involved in inflammatory, immune and cancer conditions. JAK inhibitors are well known in the art and include, but are not limited to, abrocitinib, baricitinib, delgocitinib, fedratinib, filgotinib, oclacitinib, pacritinib, peficitinib, ruxolitinib, tofacitinib and upadacitinib. JAK inhibitors are most commonly administered orally, in dosages of up to 45 mg per day.

[0061] Proteosome inhibitors work by inducing the accumulation of misfolded or unfolded protein leading to apoptosis and cell death and are used in cancer therapy. Crawford et al., “Proteosome inhibitors in Cancer Therapy,” J. Cell Commun. Signal, 2011, 5(2): 101-110. Proteosome inhibitors are well known in the art and include, but are not limited to bortezomib, carfilzomib, NPI-0052, MLN9708, CEP-18870 and ONX0912. Proteosome inhibitors are typically administered orally, in dosages of up to 1200 mg per day.

[0062] Biologies targeting inflammatory cytokines work by blocking the cytokine or cytokine receptor to inhibit inflammatory cytokine signaling. Biologies targeting IL- la, IL-ip, IL-6 and TNF-a are particularly preferred. Such biologies are anakinra, canakinumab (commercially available as Haris, which targets interleukin- ip), tocilizumab (commercially available as Actemra, which targets interkeukin-6), siltuximab, infliximab, adalimumab, and etanercept. Biologies targeting inflammatory cytokines are typically administered parenterally, in dosages of up to 600 mg per day.

[0063] Other therapies that can be considered for combination therapy with the UBA1 inhibitors include erythropoietin stimulating agents and thrombopoietin receptor agonists, e.g., eltrombopag. Treatment regimes, dosage forms and amounts of these are well known to those of ordinary skill in the art and/or capable of being determined with routine experimentation. [0064] In embodiments wherein combination therapy is contemplated, the UBA1 inhibitors or pharmaceutical compositions comprising compound means of inhibiting UBA1 and the additional treatment for VEXAS can be administered to the subject in any order. For example, a corticosteroid, a hypomethylating agent, a JAK inhibitor, or a proteosome inhibitor can be administered prior to or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before or after), or concomitantly with the administration of the UBA1 inhibitor or pharmaceutical composition comprising compound means of inhibiting UBA1 to a subject in need of treatment. Thus, the corticosteroid, hypomethylating agent, JAK inhibitor, or proteosome inhibitor or a composition containing the same, can be administered separately, sequentially or simultaneously with the UBA1 inhibitor or pharmaceutical composition comprising compound means of inhibiting UBA1. When the corticosteroid, hypomethylating agent, JAK inhibitor, or proteosome inhibitor or a composition containing the same, and the UBA1 inhibitor or pharmaceutical composition comprising compound means of inhibiting UBA1 are administered to the subject simultaneously, the therapeutic agents can be administered in a single dosage form (e.g., tablet, capsule, or a solution for injection or infusion).

[0065] In some embodiments, the subject is administered the UBA1 inhibitor, and before, during or after the UBA1 treatment cycle, may also be administered one or more corticosteroids, hypomethylating agents, JAK inhibitors, proteosome inhibitors or a combination of these. Further, before, during or after a UBA1 treatment cycle, the subject may be the recipient of an allogenic hematopoietic stem cell transplant (HSCT).

[0066] In some embodiments, the subject is administered the UBA1 inhibitor, and before, during or after the UBA1 treatment cycle, may also be administered one or more corticosteroids. In some such embodiments, the subject is treated with a combination of a UBA1 inhibitor and cortisone, prednisone, prednisolone or methyl prednisolone.

[0067] In some embodiments, the subject is administered the UBA1 inhibitor, and before, during or after the UBA1 treatment cycle, may also be administered one or more hypomethylating agents. In some such embodiments, the subject is treated with a combination of a UBA1 inhibitor and azacytidine, guadecitabine, zebularine or decitabine. [0068] In some embodiments, the subject is administered the UBA1 inhibitor, and before, during or after the UBA1 treatment cycle, may also be administered one or more JAK inhibitors. In some such embodiments, the subject is treated with a combination of a UBA1 inhibitor and abrocitinib, baricitinib, delgocitinib, fedratinib, filgotinib, oclacitinib, pacritinib, peficitinib, ruxolitinib, tofacitinib or upadacitinib.

[0069] In some embodiments, the subject is administered the UBA1 inhibitor, and before, during or after the UBA1 treatment cycle, may also be administered one or more proteosome inhibitors. In some such embodiments, the subject is treated with a combination of a UBA1 inhibitor and bortezomib, carfilzomib, NPI-0052, MLN9708, CEP-18870 or ONX0912.

[0070] In some embodiments, the subject is administered the UBA1 inhibitor, and before, during or after the UBA1 treatment cycle, may also be administered one or more biologies targeting inflammatory cytokines. In some such embodiments, the subject is treated with a combination of a UBA1 inhibitor and anakinra, canakinumab, tocilizumab, siltuximab, infliximab, adalimumab, and etanercept.

[0071] In some embodiments, the subject is administered the UBA1 inhibitor, and before, during or after the UBA1 treatment cycle, may also be the recipient of an allogenic hematopoietic stem cell transplant (HSCT).

EXAMPLES

[0072] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Generally, techniques of cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Those of ordinary skill in the art may be aware of materials and methods similar or equivalent to those described below and any of these can be used to practice or test the provided methods and compositions.

Materials and Methods

[0073] Cell Culture [0074] 32D cells (ATCC, CRL-1 1346) were cultured in 32D cell media, with antibiotics, as follows:

• RPMI (Gibco, 11875093) with 10% fetal calf serum (FCS) (Omega Scientific, FB-02),

• lx penicillin-streptomycin-glutamine (PSG) supplement (Gibco, 10378016), and

• 2ng/mL recombinant mouse IL3 (Miltenyi Biotec, 130-096-688).

[0075] THP1 cells (ATCC, TIB-202) were cultured in THP1 cell media, with antibiotics, as follows:

• RPMI (Gibco, 11875093) with 20% fetal calf serum (FCS) (Omega Scientific, FB-02), and

• lx penicillin-streptomycin-glutamine (PSG) supplement (Gibco, 10378016).

[0076] HEK293T cells (ATCC, CRL-3216) for lentivirus production were cultured in Dulbecco’s Modified Eagle Medium (“DMEM”, Gibco, 11965118) with 10% fetal calf serum (FCS) without antibiotics. All cell lines were maintained in humidified incubators at 37°C and 5% CO2.

[0077] IC50 Measurements

[0078] CellTiter-Glo (Promega, G7570) was used to measure relative cell viability as per the manufacturer’s instructions at 72 hours. A SpectraMax iD3 plate reader (Molecular Devices) was used to measure luminescence. Luminescence was normalized to the highest cytokine or lowest drug concentration for each cell line. Non-linear curves were fitted to the data and EC50 or IC50 values were calculated using GraphPad Prism 9 (San Diego, CA).

[0079] Cell line generation by CRISPR-Cas9-based homology directed repair

[0080] 32D Ubal knock-in cell lines were generated using Neon™ Electroporation System (ThermoFisher Scientific, MPK5000) and the below listed reagents for CRISPR-Cas9-based homology directed repair (HDR) from Integrated DNA Technologies (Coralville, Iowa):

• Alt-R™S.p. Cas9 nuclease v3 (Catalog #1081058)

• Ubal exon 3 sgRNA (sense target, TGCTCTCTGTCTCTAGGGAA SEQ ID NO: 1)

• Ubal g.20667962A>C (p.M41L) HDR template (antisense,

AGGCTCTCGTCTATGTCTGCTTCACTGCCGTTCTTCGCCAgTCCCTAGAGACA GAGAGCAAGAATGGGTTCAGAACAACATGC SEQ ID NO: 2) • Ubal exon 15 sgRNA (antisense target, CATCTATGTTGTCCAGAGCA SEQ ID NO: 3)

• Ubal g. 20675015G>T (p.A580S) HDR template (sense,

AAATTTGGATGGTGTGGCCAATGCTCTGGACAACATAGATtCCCGTAAGTTTT GAAGGCTGGTAAAGAAGGCAGGGGCAAAAG SEQ ID NO: 4)

[0081] THP1 Ubal knock-in cell lines were generated using Neon™ Electroporation System (ThermoFisher Scientific, MPK5000) and the below listed reagents for CRISPR-Cas9-based homology directed repair (HDR) from Integrated DNA Technologies (Coralville, Iowa):

• Alt-R™S.p. Cas9 nuclease v3 (Catalog #1081058)

• UBA1 exon 3 sgRNA (sense target, TTTCCTCTATTCCTAGGGAA SEQ ID NO: 5)

• UBA1 g.47191174T>C (p.M41T) HDR template (sense,

GTGTCTCCCTAAACTTGTTCTTTTCCTCTATTCCTAGGGAAcGGCCAAGAACG GCAGTGAAGCAGACATAGACGAGGGCCTTT SEQ ID NO: 6)

[0082] For each electroporation, Cas9-sgRNA ribonucleoprotein (RNP) complexes were generated by combining 0.75mL 36mM Cas9 nuclease with 0.75mL 44mM sgRNA and incubating for 20 minutes at room temperature. During this incubation, 32D cells were washed once with lx PBS and resuspended at 2xlO7/mL in Neon™ buffer R; Homology Directed Repair (HDR) template stock (Integrated DNA Technologies, Coralville, Iowa) at lOOmM was diluted to 10.8mM in Neon™ buffer R. The entire volume of each RNP complex was combined with 2x105 32D cells (in lOmL Neon™ buffer R) and 2mL 10.8mM HDR template. From this mixture, lOmL was taken up into the Neon™ pipette tip and electroporated using the following parameters: 1350V, 20ms, 2 pulses. Following electroporation, cells were transferred into a 24-well plate with 500mL prewarmed 32D cell media as described above, without antibiotics, e.g., without the IxPSG. and incubated overnight at 37°C and 5% CO2. Cells were subsequently transferred to a 6-well plate containing 4mL 32D cell media. At 72 hours post-electroporation, genomic DNA was isolated (Lucigen QuickExtract™, QE09050) from the bulk population for PCR and sequencing. Single cell clones from two independent electroporation experiments were obtained by limiting dilution and screened by genomic DNA PCR and Sanger sequencing.

[0083] PCR and DNA Sequencing [0084] Oligonucleotides used for PCR, sequencing, and site-directed mutagenesis are provided in TABLE 3, below:

[0085] TABLE 3

[0086] All PCR reactions were performed using NEBNext Ultra II Q5® (New England Biolabs, M0544X) and a BioRad T100 thermal cycler. Oligonucleotide synthesis and Sanger sequencing was performed by Genewiz (Waltham, MA). Next generation sequencing was performed by the Massachusetts General Hospital CCIB DNA Core (Cambridge, MA).

[0087] Dose Response Assays/Cell Proliferation

[0088] Cells were harvested from culture and viability was determined by trypan blue exclusion using an EVE™ automated cell counter (NanoEnTek). Eight-point dose response assays were performed in 96-well flat bottom opaque white plates (200pL/well total volume). For IL3 assays, cells were washed three times in phosphate-buffered saline (PBS) (Coming, 21-040-CV) to remove remaining cytokine and resuspended in RPMI containing 10% FCS and lx PSG supplement. An IL3 dilution series in the same media was generated in a 12-well reservoir and added to a 96-well plate containing 104 cells/well. For drug dose response assays, cells and drug dilutions were in RPMI containing 10% FCS, lx PSG supplement, and 2ng/mL IL3. All drugs were purchased from Selleck Chemicals (TAK-243 Cat. No. S8341, azacytidine Cat. No. S1782, ruxolitinib Cat. No. S1378, bortezomib Cat. No. S 1013) and dissolved in DMSO (VWR, 97063- 136). IL3 and drug concentrations, including no cytokine and DMSO alone, were tested in triplicate in each experiment. CellTiter-Glo (Promega, G7570) was used to measure relative cell viability as per the manufacturer’s instructions at 72 hours. A SpectraMax iD3 plate reader (Molecular Devices) was used to measure luminescence. Luminescence was normalized to the highest cytokine or lowest drug concentration for each cell line. Non-linear curves were fitted to the data and EC50 or IC50 values were calculated using GraphPad Prism 9 (San Diego, CA).

[0089] Immunoblots

[0090] Cell pellets were lysed in RIPA buffer (Sigma- Aldrich, R0278) containing lx Halt protease and phosphatase inhibitor cocktail (ThermoFisher Scientific, 78446) on ice for 15 minutes with vortexing every 5 minutes. Lysates were cleared by centrifugation at 21300rcf for 10 minutes at 4°C. Cleared supernatants were transferred to fresh tubes and protein concentration was measured using the Pierce BCA protein assay kit (ThermoFisher Scientific, 23225). Protein concentrations for each sample were normalized using lysis buffer, mixed with NuPAGE LDS sample buffer (ThermoFisher Scientific, NP0007) and NuPAGE sample reducing agent (ThermoFisher Scientific, NP0004), and subsequently boiled at 70°C for 10 minutes. Samples were centrifuged at 21300rcf for 5 minutes at room temperature, resolved by SDS-PAGE using NuPAGE 4-12% BisTris protein gels (ThermoFisher Scientific, NP0336), and transferred by electrophoresis at 90- 100V for 2 hours to 0.45pm nitrocellulose membranes (Life Technologies, LC2001). Membranes were blocked in Odyssey blocking buffer (Licor, 927-50000) for 1 hour at room temperature. Membranes were incubated in primary antibodies, detailed below, overnight at 4°C in Odyssey blocking buffer. Membranes were washed three times in lx TBS-T (Cell Signaling, 9997) for 5 minutes at room temperature and then incubated in secondary antibodies, detailed below, for 1 hour at room temperature in Odyssey blocking buffer. Following secondary antibody incubation, membranes were washed three times in lx TBS-T for 5 minutes at room temperature, visualized using a ChemiDoc™ MP Imaging System (BioRad, 12003154), and quantified using Image Lab Touch Software (BioRad, 12014300).

[0091] Molecular Cloning

[0092] UBA1 cDNA (NM_003334.4) was obtained from GenScript (ClonelD Ohu24932) in pcDNA3.1, PCR amplified, and subcloned into pDONR221 (ThermoFisher Scientific, 12536017) using Gateway™ BP Clonase™ II (ThermoFisher Scientific, 11789020).

[0093] pDONR221-UBAl was modified using NEB Q5 Site-Directed Mutagenesis Kit (New England Biolabs, E0554S) to generate the following pDONR221-UBAl constructs:

• Al-40 (UBAlb) • Al-40 (UBAlb)

• A1-40/C632A (catalytically inactive UBAlb)

[0094] Lentiviral expression constructs for UBA1 variants and Renilla lucerifase were generated via Gateway™ LR Clonase™ II (ThermoFisher Scientific, 11791020) reaction between each pDONR221 plasmid and lentiviral destination plasmid pLEX307 (Addgene, 41392). All pDONR221 and pLEX307 constructs were confirmed by Sanger sequencing (Genewiz) and alignment using Benchling Biology Software (2021-2023, fatps://bencMing,cpm).

[0095] Lentivirus Production

[0096] Lentivirus was produced in a 6-well plate by transient transfection of 60-70% confluent HEK293T cells in 2mL of media using 9pl TranslT-LTl (Mirus, MIR2304) in 75pL Opti-MEM™ I Reduced Serum Medium (Gibco, 31985070) containing 1.5pg lentiviral expression plasmid, 2 pg psPAX2 packaging plasmid (Addgene, 12260), and 0.75pg pCMV-VSV-G envelope plasmid (Addgene, 8454). Lentiviral supernatants were collected at 36-48 hours post-transfection, passed through a 0.45pm syringe filter (Pall, 4614), and used immediately for transduction or frozen at - 80°C.

[0097] Lentiviral Transduction

[0098] For lentiviral transductions, 2xl0⁵ cells in 250mL 32D cell culture media were transduced in 48-well plates with 500mL lentiviral supernatant and 4mg/mL polybrene (Santa Cruz Biotechnology, sc-134220A). Plates were centrifuged at 1050rcf at 37°C for 1 hour. Cells were cultured overnight, washed three times in PBS, and cultured overnight for a second night in 32D cell media prior to addition of 2mg/mL puromycin to select for transduced cells.

[0099] Competition Assays

[0100] Isogenic 32D cells expressing UBA1 WT or M41L were harvested from culture, resuspended in fresh RPMI containing 10% FCS, lx PSG, and 2ng/mL recombinant mouse IL-3, and mixed at a ratio of 10 UBA1 M41L to 1 UBA1 WT (final cell concentration 5x104 cells/mL). Mixed cells were treated with lOnM TAK-243 or DMSO in triplicate in a 12-well plate (2mL/well). After four days in culture, genomic DNA was extracted and used for PCR amplification of UBA1 exon 3. Ubal wild-type and mutant alleles were quantified using EditR analysis of Sanger sequencing data.

[0101] Apoptosis Assays

[0102] Cells were cultured as described above and treated for 16 hours with DMSO or lOnM TAK- 243. Ann exin- V/propidium iodide staining (BioLegend, 640914) was performed according to the manufacturer’s protocol and samples were analyzed by flow cytometry using a BD LSR Fortessa. In parallel, protein lysates were prepared and analyzed by immunoblot as described above.

[0103] B rightfield Microscopy

[0104] 2xl0⁴ cells of each genotype were harvested from culture and spun down onto glass slides (ThermoFisher Scientific, 12-550-15) using a CytoSpin 4 Cytocentrifuge (ThermoFisher Scientific, A78300003) at 600rpm for 6 minutes. Cells were immediately stained using HEMA 3™ according to the manufacturer’s protocol. Brightfield images were acquired as TIFF files with an Olympus BX53 microscope with a lOOx oil objective using an Olympus DP27 CCD camera and Olympus cellSens software version 3.1.

[0105] Electron Microscopy

[0106] Cell pellets were washed with PBS and fixed in 100 mM sodium cacodylate buffer containing 2.5% glutaraldehyde, 1.25% paraformaldehyde, and 0.03% picric acid (pH 7.4). Cell pellets were subsequently washed in lOOmM sodium cacodylate buffer (pH 7.4), post-fixed for 1 hour in 1% osmium tetroxide/1.5% potassium ferrocyanide, washed twice in distilled water, washed once in maleate buffer, and incubated in 1% uranyl acetate in maleate buffer for 1 hour. Following two washes in distilled water, cell pellets were dehydrated in graded alcohols (10 minutes each; 50%, 70%, 95%, and twice in 100%), incubated in propylene oxide for I hour, and infiltrated for 16 hours in a 1 : 1 mixture of propylene oxide and TAAB Epon (TAAB Laboratories, https://taab.co.uk). Samples were subsequently embedded in TAAB Epon and polymerized at 60°C for 48 hours. Ultrathin sections (~60nm) were cut on a Reichert Ultracut-S microtome, extracted onto copper grids stained with lead citrate, and examined in a JEOL 1200EX or a TecnaiG2 Spirit BioTWIN transmission electron microscope. Images were recorded with an AMT 2k CCD camera. [0107] Antibodies

[0108] TABLE 4 - Primary antibodies

[0109] TABLE 5 - Secondary antibodies

[0110] Statistical Analysis

[0U1] All statistical analyses were performed using GraphPad Prism 9 (version 9.5.1). An unpaired t test with Welch’s correction or Mann-Whitney test was used for two sample comparisons. Multiplex cytokine data were analyzed using multiple Mann-Whitney tests corrected by the Benjamini, Krieger, and Yekutieli false discover rate (FDR) method.

[0112] Graphs and Illustrations [0113] Graphs were generated using GraphPad Prism 9 (version 9.5.1) and figures were prepared in Microsoft® PowerPoint (version 16.71). Figure 1A was prepared using BioRender. Supplemental Figure 1 was prepared using Sanger sequencing traces from Benchling Biology Software (h tgs: //bench lin com ).

[0114] EXAMPLE 1 Hemizygous Ubal^M41L and ///M Expression in Myeloid Cell Lines

[0115] Isogenic single cell clones expressing Ubal^WT and Ubal^M41L were generated from the endogenous Ubal locus using CRISPR-Cas9-based homology directed repair (HDR) in an IL3- dependent 32D mouse myeloid line. Isogenic single cell clones expressing UBAl^nT and UBA1^M4IT were generated from the endogenous UBA1 locus using CRISPR-Cas9-based HDR in the THP1 human myeloid line.

[0116] Brightfield microscopy of 32D cells with hemizygous expression of Ubal^M41L showed aberrant vacuoles in the cells. Electron microscopy studies revealed that vacuoles in Ubal^M4IL cells contained endolysosomal membranes, including small vesicles, multivesicular bodies and multilamellar lysosomes. Microscopy images are not included.

[0117] As shown in FIG. 1A, there was no significant difference in IL3 -dependent cell proliferation between Ubal^WT and Ubal^M4IL 32D cell lines. Statistical significance was determined using a two-tailed unpaired t-test with Welch’s correction. FIG. 1A thus suggests that the Ubal^M4IL mutations do not lead to enhanced proliferation or survival in response to IL3 growth factor.

[0118] Given the autoinflammatory features of VEXAS syndrome, measurements were also taken of a panel of 44 cytokines in supernatants from unstimulated Ubal^WT and UbaB^i4IL 32D cell lines. The concentrations of IL lb and CXCL10 were significantly increased in supernatants from Ubal^M4IL cells compared to Ubal^WT cells; Ubal^M4IL cell supernatants also contained higher concentrations of CCL5, CCL17, CXCL9, and CXCL12 and lower concentrations of IL6 and LIF. (FIG. IB).

[0119] This inflammatory phenotype can be explained by increased activation of the NFkB and inflammasome pathways, which has been observed in VEXAS syndrome. See, FIG. IB in which data from three biological replicates of two single cell clones per genotype were analyzed by multiplex cytokine assay in duplicate is shown in a volcano plot. Statistical significance was determined using multiple Mann-Whitney tests corrected by the Benjamini, Krieger, and Yekutieli false discover rate method.

[0120] Current therapies for VEXAS syndrome, including hypomethylating agents and JAK inhibitors, have shown some efficacy in controlling autoinflammation but have infrequently led to a reduction in the UBA1 mutant clonal burden. Bourbon et al., Blood, 2021, 137(26):3682-3684. As shown in FIG. 1C-1E, the Ubal^nT and Ubal^M4IL 32D cells developed as above were similarly sensitive to the anti-proliferative effects of azacytidine (FIG. 1C) and ruxolitinib (FIG. ID). As also shown in FIG. IE, sensitivity to proteasome inhibition by bortezomib in Ubal^WT and Ubal^M41L cells was similar. These results show that azacytidine, ruxolitinib and bortezomib are not selective for Ubal^M41L mutant cells, but may be effective in combination therapy with a UBA1 inhibitor in that these agents have been successfully and safely used in the treatment of the symptoms of VEXAS.

[0121] This example establishes that the hemizygous knock-in of Ubal^M41L in a myeloid cell line provides the basis of an accurate model of key biochemical, morphological, and inflammatory features of VEXAS syndrome, including UBAlc expression, decreased protein polyubiquitination, abnormal vacuolization, and increased levels of ILip and inflammatory chemokines. The developed cell line was, or will be, used in the following Examples 2-4.

[0122] EXAMPLE 2 Effect of UBA1 Inhibitor TAK-243 on Ubal¹'^!41L and 7/ 47^Wjr Mutant Cells

[0123] Example 2A - Cell Proliferation using Ubal^ and Ubal^M4IL

[0124] Cell proliferation assays were carried out in accordance with the procedures described above. Specifically, the Ubal^wl and Ubal^M4IL cells produced according to Example 1 were cultured over three days in the presence of DMSO or a range of UBA1 inhibitor (TAK-243) concentrations. Statistical significance was determined using a Mann-Whitney test. As shown in FIG. 2A, the Ubal^M4IL cells were significantly more sensitive to the UBA1 inhibitor than the Ubal^WT cells. Statistical significance was determined using a Mann-Whitney test. TAK243 IC50 data for Example 2 A is shown below in TABLE 6:

[0125] TABLE 6 TAK243 IC50 in 32D Uba 1^WT and Ubal^M41L cells

[0126] Example 2B - Competition Assay

[0127] To test whether differential TAK-243 sensitivity could be exploited to reduce Ubal^M4IL mutant clonal burden in a mixed population, Ubal^nT and UbaI '^!4 ' cells were co-cultured for four days at -1 :10 ratio. Thereafter, the Ubal^M41L variant allele fraction (VAF) was measured after treatment with DMSO or 10 mM TAK-243. Ubal¹'^I4IL VAF was determined by quantitative analysis of Sanger sequencing data (mean±s.d. of three technical replicates). Kluesner et al., Crispr Journal. 2018;l(3):239-250. Statistical significance was determined using a two-tailed unpaired t-testwith Welch’s correction. As shown in FIG. 2B, the Ubal^M4IL VAF decreased from 0.85±0.05 to 0.003±0.006 (mean±s.d. of three technical replicates) after four days of treatment with lOnM TAK-243. FIG. 2B thus shows that TAK243 selectively eliminates the Ubal mutant cells.

[0128] 2C - Apoptosis Assays

[0129] 32D Ubal^WT and Ubal^M4IL cells were treated for 16 hours with DMSO or lOnM TAK-243 Thereafter, apoptotic cells were quantified by annexin V-propidium iodide (PI) flow cytometry. As shown in FIG. 2C, exposure to TAK-243 led to increased apoptosis in the Ubal^M4IL cells as compared to similarly treated Ubal^WT cells. Bars depict live (annexin V-/PI-), early apoptotic (annexinV+/PIlow-mid), and late apoptotic (annexinV+/PIhigh) cells (mean±s.d. of three technical replicates).

[0130] Example 2D - Immunoblots

[0131] Immunoblots were conducted as described above. Immunoblots for PARP1, H2AX, and H2AX pS139 proteins after 16-hour treatment of 32D Ubal^WT and Ubal^M41L cells with DMSO or lOnM TAK-243 are provided at FIG. 2D. Immunoblot for vinculin (VINC) was performed as a protein loading control. As shown in FIG. 2D, PARP1 has been cleaved in the Ubal^M41L cells treated with TAK243, but not the Ubal^WT cells (whether exposed to DMSO or TAK243) or Ubal^M41L cells treated with DMSO. This shows that Ubal^M41L cells treated with TAK243 are undergoing apoptosis. Similarly, the relatively larger quantities of H2AX and H2AX pS139 detected in the UhaI^M41L cells treated with TAK243, compared with the Ubal^KT cells (whether exposed to DMSO or TAK243) or Ubal^M4IL cells treated with DMSO, also indicates that the Ubal^M4IL cells treated with TAK243 are undergoing apoptosis.

[0132] Example 2E - Generation of UBA1 inhibitor binding mutant cell line

[0133] A TAK-243 binding mutant described in Barghout et al., Leukemia, 2019, 33(l):37-51, Ubal^A580S , was generated using CRISPR-HDR as. Cell proliferation assays of 32D cells with hemizygous expression of Ubal^WT, Ubal^M41L, or Ubcil^{A4A A5N}'⁴' were conducted over three days in the presence of DMSO or a range of TAK-243 concentrations. As shown in FIG. 2E, knock-in of the Ubal^M41L/A580S mutation (the dashed line to the right of the wild-type) rendered Ubal^M4IL cells resistant to TAK-243. FIG. 2E thus shows that TAK-243 mediated inhibition of proliferation of Ubal^M41L cells requires TAK-243 binding to UBA1 Stated another way, FIG. 2E shows that UBA1 is the target of TAK-243 in Ubal^M4IL cells. As shown in TABLE 7, below, the IC50 of TAK-243 was significantly higher in Ubal^M41L'^A580S cells compared with Ubal^M4IL cells (0.07±0.02nM vs. 0.004±0.001nM, respectively, mean±s.d. of three biological replicates). Statistical significance was determined using a two-tailed unpaired t-test with Welch’s correction. [0134] TABLE 7 TAK243 IC50 in 32D Ubal^WT, Ubal^M41L and ubal^M41L,A580S cells

[0135] Example 2F - Cell Proliferation using BA l⁴’ ⁴ and UBA1^M4IT

[0136] Cell proliferation assays were carried out substantially in accordance with the procedures described above. Specifically, the UBA 1^WT and UBA l^M41L cells produced according to Example 1 were cultured over three days in the presence of DMSO or a range of UBA1 inhibitor (TAK-243) concentrations. Statistical significance was determined using a Mann-Whitney test. As shown in FIG. 2F, the Ubal^M41T cells were significantly more sensitive to the UBA1 inhibitor than the UbaBⁿ cells. Statistical significance was determined using a Mann-Whitney test. TAK243 IC50 data for Example 2F is shown below in TABLE 8:

[0137] TABLE 8 TAK243 IC50 in THP1 F7M/'^:7 and UBA l''^!4n cells

[0138] Taken together, the data of Examples 2A-2F demonstrate that hemizygous expression of Ubal^M4IL in 32D cells and UBA1^M4IT in THP1 cells confers TAK-243 sensitivity via on-target inhibition of UBA1 and support the conclusion that UBA1 inhibitor TAK-243 would be an effective treatment for VEXAS syndrome.

[0139] EXAMPLE 3 - Overexpression of UBAlb promotes TAK-243 resistance in Ubal^M4IL mutant cells

[0140] cDNA constructs were prepared as described above and as shown in FIG. 3 A.

[0141] Example 3A - Immunoblots

[0142] Immunoblots were conducted as described above. Immunoblots for UBA1, V5, and ubiquitin in 32D Ubal^WT and Ubal^M4IL parental cell lines and Ubal^M4IL cells overexpressing UBAlb, UBAlb/C632A, or luciferase (Luc) are provided at FIG. 3B. Immunoblot for vinculin (VINC) was performed as a protein loading control. As shown in FIG. 3B, overexpression of UBAlb and the catalytically inactive form of UBAlb (i.e., UBAlb/C632A) are detectable by immunoblot in the Ubal^M41L cells. As shown, overexpression of UBAlb, but not the catalytically inactive form of UBAlb (i.e., UBAlb/C632A) or luciferase, in the Ubal^M41L cells restores polyubiquitination to wild-type levels.

[0143] Example 3B - Cell Proliferation Assays

[0144] Cell proliferation assays were carried out in accordance with the procedures described above. Specifically, the 32D Ubal^WT and Ubal^M41L cells produced according to Example 1 and Ubal^M4IL cells overexpressing UBAlb, catalytically inactive UBAlb or luciferase were cultured over three days in the presence of DMSO or a range ofUBAl inhibitor (TAK-243) concentrations. Statistical significance was determined using a Mann-Whitney test. As shown in FIG. 3C, overexpression of UBAlb, but not the catalytically inactive form of UBAlb (i.e., UBAlb/C632A) in the Ubal^M41L cells rendered these cells functionally equivalent to the wild-type, wherein the rightmost dashed line indicates the Ubal^M4IL cells with UBAlb overexpression, the rightmost solid line indicates the Ubal^WT cells, and the three lines on the left are the Ubal^M41L cells with nothing overexpressed, with UBAlb/C632A overexpressed, or with luciferase overexpressed. Stated another way, FIG. 3C shows that recovery of the Ubal^M4IL mutant cell is possible upon addition of UBAlb back to the mutant cell line. Specifically, a single representative experiment (FIG. 3C, mean ± s.d. of three technical replicates) and calculated IC50 values (TABLE 9, below, mean ± s.d. of four biological replicates) are shown. Statistical significance was determined using a Mann- Whitney test.

[0145] TABLE 9 TAK243 IC50 in 32D Ubal^⁷, Ubal^M41L cells with or without overexpression of UBAlb, UBAlb/C632A, or luciferase

[0146] This example shows that reduced UBA1 enzymatic activity in the setting of UBAlb loss is associated with increased sensitivity to targeted UBA1 inhibition.

Claims

WHAT IS CLAIMED IS:

1. A method of treating VEXAS syndrome comprising administering a therapeutically effective amount of a composition comprising compound means of inhibiting UBA1 and a pharmaceutically acceptable carrier to a patient in need thereof.

2. A method of treating VEXAS syndrome comprising administering a therapeutically effective amount of a UBA1 inhibitor to a patient in need of thereof.

3. A UBA1 inhibitor for use in a method of treating VEXAS syndrome in a patient in need thereof.

4. A composition for treating VEXAS syndrome in a patient in need thereof comprising compound means for inhibiting UBA1 and a pharmaceutically acceptable carrier.

5. Use of a UBA1 inhibitor in the manufacture of a medicament for administration in the treatment of VEXAS syndrome to a patient in need thereof.

6. The method, compound(s) for use or use of any one of claims 1-5, wherein the UBA1 inhibitor is TAK-243.

7. The method of claim 1 or 2, wherein the UBA1 inhibitor or the compound means of inhibiting UBA1 is administered orally or parenterally as a monotherapy.

8. The method of claim 1 or 2, wherein the UBA1 inhibitor or compound means of inhibiting UBA1 is administered in combination with an additional treatment, the additional treatment comprising i) administration of one or more of a corticosteroid, a hypomethylating agent, a JAK inhibitor, a proteosome inhibitor, or a biologic targeting inflammatory cytokines, or ii) allogenic hematopoietic stem cell transplant.