Patent Application
20220280511
The present invention relates to the use of CDK4/6 inhibitors for the treatment of psoriasis, a pharmaceutical composition for the treatment of psoriasis comprising said CDK4/6 inhibitors, a method for the preparation of said pharmaceutical composition, a method for the therapeutic treatment of a living being against psoriasis, a method for the screening of active agents against psoriasis, and the use in vitro of a CDK4/6 inhibitor for the suppression of the cellular IκBζ expression.
Psoriasis is a long-lasting autoinflammatory skin disease, affecting 150 million individuals or 2-3% of the population worldwide. It is characterized by keratinocyte hyperproliferation and massive infiltration of immune cells into the skin, leading to the development of erythema, microabscesses, scales and increased skin thickness. The cause of psoriasis is not fully understood, however certain risk factors favor the development of psoriasis, including genetic predisposition, chronic infections, psychological factors such as stress, excessive alcohol consumption, cigarette smoking, and obesity etc. Importantly, psoriasis patients often develop several co-morbidities, such as arthritis, cardiovascular disease or diabetes, turning this autoinflammatory skin disorder into a life-threatening disease.
It is believed that psoriasis evolves from an abnormal activation of keratinocytes and dendritic cells, leading to the secretion of several cytokines, such as interleukin-36 (IL-36), interleukin-23 (IL-23) and interleukin-1α (IL-1β), which trigger the massive recruitment and activation of macrophages, neutrophils and T-cell subsets into the affected skin area. Most importantly, IL-17A-, IL-22- and TNFα-producing Th17 cells are recruited to the psoriatic lesions. Subsequently, these cytokines induce the hyperproliferation and dedifferentiation of keratinocytes, thereby further promoting the recruitment of immune cells and the development of chronic psoriatic skin lesions.
So far, no cure is available for psoriasis even though several treatment options do exist. Topical agents are typically used for mild disease, phototherapy for moderate disease, and systemic agents for severe disease. Topical agents include corticosteroid preparations, retinoids, vitamin D analogues, coal tar, moisturizers and emollients. Systemically applicable agents are essentially provided to suppress the immune system. They include methotrexate and cyclosporin A. Further systemic treatments are based on the administration of hydroxycarbamide, fumarates and retinoids.
However, conventional medicinal psoriasis treatment is characterized by an unspecific mode of action of the administered agents. This non-specificity results in a low curative effect and eventually the development of adverse side effects.
Up-to-date therapies comprise several monoclonal antibodies (MABs) targeting cytokines or their cognate receptor, that promote disease progression. These antibodies are directed against IL-17 (i.e. secukinumab, ixekizumab and brodalumab), TNF-α (i.e infliximab, adalimumab and etanercept) or IL-23 (i.e. risankizumab, guselku-mab and tildrakizumab). These neutralizing antibodies have been recently approved by the U.S. Food and Drug Administration (FDA) for treatment of psoriasis.
The systemic administration of neutralizing antibodies is associated with several disadvantages such as high costs, difficult application routes and systemic side effects such as upper respiratory tract infections, see Wasilewska et al., A new era in treatment of psoriasis and other skin diseases, Postepy Dermatol Alergol 33, 247-252 (2016). Importantly, therapeutic resistances have been described based on the development of anti-drug antibodies (ADA) against the administered monoclonal antibodies.
Against this background the objective underlying the invention is to provide a novel kind of medical treatment of psoriasis where the disadvantages of the ap-proaches in the art are avoided or at least reduced. In particular, such an agent should be provided which specifically addresses the molecular mechanisms underlying the development of psoriasis, thereby resulting in an improved curative effect while minimizing potential side effects.
This object is met by the provision of a CDK4/6 inhibitor for use in the treatment of psoriasis.
A “CDK4/6 inhibitor” according to the invention refers to any agent that specifically and inhibits the function of ‘cyclin-dependent kinases’ (CDK) 4 and 6. Examples of CDK4/6 inhibitors include abemaciclib (LY2835219), palbociclib (PD-0332991), ribociclib (LEE 011).
According to the invention “treatment” of psoriasis refers to the targeted reduction of a living being's symptoms of psoriasis.
While the invention allows the targeted treatment of psoriasis in any kind of living being, the treatment of a human being is preferred.
In an embodiment of the invention the CDK4/6 inhibitor may be used as the only active agent, however, in another embodiment it can also be used in combination with additional active agents against psoriasis.
The findings of the inventors were surprising and could not be expected, for the following reasons.
CDK4 and CDK6, in complex with cyclin D1, cyclin D2 or cyclin D3, represent well known cell-cycle regulating kinases that can phosphorylate RB, leading to the release of E2F transcription factors and G1-S cell cycle transition. Consistently, amplification of CDK4 and CDK6 as well as an overexpression of cyclin D proteins are frequently observed events in cancer, leading to the excessive proliferation of tumor cells. ATP-competitive CDK4/6 inhibitors, such as palbociclib and abemaciclib, have been developed for anti-cancer therapy and were recently approved for treatment of breast cancer patients; see Vidula, N. & Rugo, H. S. Cyclin-Dependent Kinase 4/6 Inhibitors for the Treatment of Breast Cancer: A Review of Preclinical and Clinical Data. Clin Breast Cancer 16, 8-17 (2016).
However, the state of the art does not provide any hints indicating that CDK4/6 inhibitors may have any beneficial effects in an inflammatory disease such psoriasis.
While documents U.S. Pat. No. 9,616,062 and WO 2010/132725 fantasize an ad-vantageous role of CDK4/6 inhibitors in psoriasis in a non-substituted manner and without the provision of any experimental evidences highly ranked scientific literature does not share this view. To the contrary, experts even point to the opposite direction and propose proinflammatory effects of CDK4/6 inhibitors.
For instance, Deng et al., CDK4/6 Inhibition Augments Antitumor Immuni-ty by Enhancing T-cell Activation, Cancer Discov 8(2), 216-233 (2017), demonstrate by means of an experimental cancer model that the administration of CDK4/6 inhibitors such as palbociclib and trilaciclib (G1T28) results in increased levels of TH1 cytokines and tumor-infiltrating T cells.
Schaer et al., The CDK4/6 Inhibitor Abemaciclib Induces T Cell Inflamed Tumor Microenvironment and Enhances the Efficacy of PD-L1 Checkpoint Blockade, Cell Reports 22, 2978-2994 (2018), were able to demonstrate in murine syngeneic tumor models and in vitro assays that the administration of the CDK4/6 inhibitor abemaciclib results in an increased T cell activation and pro-inflammatory, intra-tumoral response.
Against this background a person skilled in the art would have expected that the administration of CDK4/6 inhibitors into a living being affected by psoriasis would even encounter an amplification of psoriasis-related inflammation which is characteristic of said disease rather than achieving any beneficial or curing effect. The art teaches away from the invention.
Therefore, the skilled person would never have taken CDK4/6 inhibitors into consideration for the treatment of psoriasis.
The inventors have surprisingly realized for the very first time that the effect of the CDK4/6 inhibitors against psoriasis is not based on an inhibition of the cell cycle progression, but on the suppression of the cellular expression of the transcriptional regulator IκBζ. This is important because IκBζ, encoded by the gene NFKBIZ, has been identified as a transcriptional key regulator in psoriasis. IκBζ is commonly overexpressed in human psoriatic skin lesions; see Johansen, C., et al., I kappa B zeta is a key driver in the development of psoriasis, Proc Natl Acad Sci USA 112, E5825-E5833 (2015), and Müller, A., et al, IkappaBzeta is a key transcriptional regulator of IL-36-driven psoriasis-related gene expression in keratinocytes, Proc Natl Acad Sci USA 115, 10088-10093 (2018).
The use of CDK4/6 inhibitors in the treatment of psoriasis as proposed by the invention thus addresses a molecular target that is specifically involved into the development of the disease. Thus, the invention allows a targeted treatment of psoriasis.
In addition, the cell cycle inhibitory activity of the CDK4/6 inhibitors is of particular advantage, since it results in an additional blocking of the keratinocyte hyperproliferation which is a hallmark of psoriasis.
Furthermore, the inventors have realized that genes which are not IκBζ-dependent remain unaffected upon CDK4/6 inhibition. These findings indicate that the inhibition of IκBζ in psoriasis treatment will be rather specific and not associated with side effects, which are commonly found upon a broad inhibition of NF-κB, e.g. by toxic IκBζ inhibitors.
Even more importantly, as the inhibition of IκBζ by CDK4/6 inhibitors blocks multiple pathways in psoriasis, i.e. IL-17, IL-23, and IL-36 signalling, targeting IκBζ might increase overall therapeutic responses as well as prevent the development of therapy resistance.
While in principle the application of the CDK4/6 inhibitors can be accom-plished via any route, including systemically, orally, intravenously, intramuscularly, etc., in a preferred embodiment of the invention the use of the CDK4/6 inhibitor in the treatment of psoriasis is carried out via a topical application on the skin.
Importantly, the inventors were able to demonstrate via two different experimental models that a topical administration of CDK4/6 inhibitors to the skin provides effective protection against psoriasis. This measure has the advantage that the treatment of psoriasis is focused on the site of the pathologic events. Any potential side effects can be further reduced herewith. The topical administration onto the skin is less expensive than a systemic application and can be easily realized by the patient. Furthermore, the risk of the development of a therapy resistance is significantly reduced.
In another embodiment of the invention the CDK4/6 inhibitor is provided as a skin-permeable formulation.
The inventors succeeded in delivering CDK4/6 inhibitors of hydrophobic nature, exemplified by ademaciclib, into the deeper layers of the skin of an experimental animal, thereby completely suppressing psoriasis-typical skin inflammation. This measure has, therefore, the advantage, that the CDK4/6 inhibitor is provided in a safe form allowing the targeting treatment of psoriasis while, at the same time, minimizing any potential side effects.
In a further embodiment of the invention the skin-permeable formulation is selected from the group consisting of: crème, gel, lotion.
This measure has the advantage that the CDK4/6 inhibitor is provided in a well-established formulation variant that allows the targeted administration onto the skin.
In a further embodiment of the invention the CDK4/6 inhibitor is an ATP-competitive CDK4/6 inhibitor and/or, preferably selected from the group consisting of: abemaciclib, palbociclib, ribociclib, trilaciclib (G1T28).
This measure has the virtue that such kind of CDK4/6 inhibitors are de-ployed which have been tested in the treatment of cancer diseases such as breast cancer. There these agents are well tolerized with nearly no adverse effects. The invention therefore takes advantage of the long-term experience made with cancer treatment by such compounds. Thus, by this embodiment the provision of particularly suited agents is ensured.
“Abemaciclib” [CAS 1231929-97-7 (Free Base); 1231930-82-7 (Mesylate Salt)], also referred to as LY2835219, Verzenio or Verzenios was originally developed for the treatment of advanced breast cancer. It was approved in the United States by the FDA on 28 Sep. 2017 for “adult patients who have hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer that has progressed after taking therapy that alters a patient's hormones.
“Palbociclib” (CAS 571190-30-2) also referred to as PD-0332991, Ibrance or Palbonix, was developed for the treatment of HR-positive and HER2-negative breast cancer. The drug was approved by the FDA in an accelerated program on 3 Feb. 2015 as the first CDK4/6 inhibitor for a treatment in combination with letrozole for patients with estrogen receptor positive (ER+) advanced breast cancer. Approvals in other coun-tries followed.
“Ribociclib” (CAS 1211441-98-3) also named LEE-011 or Kisqali was also developed for certain types of breast cancer. It was approved in the United States by the FDA in March 2017 and the European Medicines Agency in August 2017 for use in combination with an aromatase inhibitor such as letrozole to treat HR-positive, HER2-negative advanced or metastatic breast cancers.
“Trillaciclib” (CAS 1374743-00-6) also referred to as G1T28 is a small molecule, competitive inhibitor of CDK4/6, with potential antineoplastic and chemoprotec-tive activities. It is currently undergoing clinical trials. In August 2019 it has received ‘breakthrough therapy’ status for small cell lung cancer in the USA.
In another embodiment of the invention the CDK4/6 inhibitor is used in combination with an additional agent active against psoriasis-associated symptoms.
Such measure may result in an even stronger effectiveness of the invention in the treatment of psoriasis. In particular, due to the combination of the CDK4/6 inhibitors and the additional agent synergistic effects may come into play. According to this embodiment of the invention, any additional agent active against psoriasis-associated symptoms is, in principle, suitable. Examples of such additional active agents include corticosteroids, retinoids, vitamin D analogues, coal tar, moisturizers and emollients, etc.
In a further embodiment of the invention the additional agent is an EZH2 inhibitor.
The inventors have surprisingly realized that EZH2 inhibitors are likewise capable of treating psoriasis. Examples of EZH2 inhibitors include EPZ-6438 (tazemeto-stat), CPI-169, 3-deazaneplanocin A (DZNep), EPZ005687, El1, GSK126, UNC1999, CPI-1205, EPZ011989, EBI-2511, PF-06726304, GSK503, and GSK343. The combination of both active agents may therefore lead to a substantive action amplification resulting in an even more efficient treatment.
Another subject-matter of the invention relates to a pharmaceutical composition for the treatment of psoriasis comprising the CDK4/6 inhibitor according to the invention and a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers are well known to the skilled person. They allow a proper formulation of the active agent and serve to improve the selectivity, effectiveness, and/or safety of drug administration. Pharmaceutically acceptable carriers include, without being limited thereto, solvents, fillers, binders, lubricants, stabilizers, surfactants, suspensions, thickeners, emulsifiers, preserving agents, liposomes, micelles, microspheres, nanoparticles, etc. suitable for the particular form of dosage. Except for cases, when the medium of conventional carriers is incompatible with the active ingredient, for example, upon occurrence of any undesirable biological effects or other adverse interactions with any other ingredient(s) of the pharmaceutical composition, the use of such compositions falls within the scope of this invention. Materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, monosaccharides and oligosaccharides, as well as derivatives thereof; malt, gelatin; talc; excipients such as: cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions. In addition, the composition may contain other non-toxic compatible lubricants, for example sodium lauryl sulfate and magnesium stearate, as well as coloring agents, parting liquids, film formers, sweeteners, flavoring additives and flavorants, preserving agents and antioxidants.
In an embodiment of the invention the pharmaceutical preparation can include the CDK4/6 inhibitor as the only active agent, however, in another embodiment it can also include additional active agents against psoriasis. Pharmaceutical compositions according to the present invention can, therefore, be used both as monotherapy and administered to a patient in need of appropriate therapy in combination with one or more other therapeutic agents.
The features, advantages and characteristics of the CDK4/6 inhibitor apply likewise to the pharmaceutical composition according to the invention. Accordingly, in an embodiment of the invention the pharmaceutical composition is provided as a skin-permeable formulation. Preferably the skin-permeable formulation is selected from the group consisting of: crème, gel, lotion.
Another subject-matter of the present invention relates to a method for the preparation of a pharmaceutical preparation comprising the formulation of a CDK4/6 inhibitor into a pharmaceutically acceptable carrier, preferably the CDK4/6 inhibitor is formulated into a skin-permeable formulation, further preferably the skin-permeable formulation is selected from the group consisting of: crème, gel, lotion.
The features, advantages and characteristics of the CDK4/6 inhibitor apply likewise to the method according to the invention.
Another subject-matter of the invention relates to a method for the therapeutic treatment of a living being against psoriasis comprising the administration of the CDK4/6 inhibitor of the invention and/or the pharmaceutical composition of the invention to the living being, such as a human being.
The features, advantages and characteristics of the CDK4/6 inhibitor apply likewise to the method according to the invention.
Another subject-matter of the present invention relates to a method for the screening of active agents against psoriasis comprising the identification of a test compound's activity of a CDK4/6 inhibitor.
The findings of the inventors of the suitability of CDK4/6 inhibitors in the treatment of psoriasis allow the identification of novel active agents due to their CDK4/6-inhibitory activity. Assays to evaluate a test component's capability to inhibit the activity of CDK4/6 are well known to the skilled person, e.g. in form of competitive phosphorylation assays.
The features, advantages and characteristics of the CDK4/6 inhibitor apply likewise to the method according to the invention.
A still further subject-matter of the present invention relates to the use ex vivo of a CDK4/6 inhibitor for the suppression of the cellular IκBζ expression.
The inventors herewith provide a research tool not only allowing a better understanding of the transcriptional events up- and downstream of IκBζ expression but also the molecular mechanisms of psoriasis. According to the invention the use of the CDK4/6 inhibitor in vitro includes the addition of the CDK4/6 inhibitor to a cell culture, e.g. a keratinocytes cell culture, and the evaluation of the molecular effects, resulting e.g. in altered gene expression, cytokine induction etc.
The features, advantages and characteristics of the CDK4/6 inhibitor apply likewise to the use according to the invention.
The features explained in the following do not only apply to the respective combination indicated or the specific embodiment but also in isolated position or to other combinations or to the invention in general without leaving the scope of the invention. The embodiments serve to illustrate the invention but are not intended to restrict the scope of the invention. Reference is made to the enclosed figures.
IκBζ represents an atypical member of the IκB family that is inducibly expressed in the nucleus, leading to the activation or repression of a selective subset of NF-κB target genes. Especially in keratinocytes, IL-17A, alone or even more potently in combination with TNFα, as well as IL-36 family cytokines, trigger a NF-κB- and STAT3-dependent transcriptional upregulation of IκBζ expression. Subsequently, IκBζ induces a subset of IL-36- and IL-17-responsive target genes in keratinocytes, including CXCL2, CXCL5, CXCL8, LCN2, DEFB4 or IL1B. How IκBζ regulates these downstream target genes remains elusive though. It is assumed that IκBζ recruits epigenetic modifiers, such as TET2 or the SWI/SNF complex to the promoter sites of its target genes, leading to a change in DNA methylation or nucleosome remodeling.
IκBζ-deficient mice are completely protected against imiquimod (IMQ)- or IL-36-mediated psoriasis-like skin inflammation. Moreover, human psoriatic skin lesions are characterized by an upregulated expression of IκBζ; see Johansen et al. (2015; loc. cit.) and Müller et al. (2018; loc. cit.). As IκBζ lacks any enzymatic activity, it cannot be directly inhibited. Therefore, the inventors realized that small molecule inhibitors blocking the induction or downstream function of IκBζ could represent an alternative strategy for targeting IκBζ in psoriasis.
CDK4 and CDK6, in complex with cyclin D1, cyclin D2 or cyclin D3, represent well known cell-cycle regulating kinases that can phosphorylate RB, leading to the release of E2F transcription factors and G1-S cell cycle transition. Consistently, amplification of CDK4 and CDK6 as well as an overexpression of cyclin D proteins are frequently observed events in cancer, leading to the excessive proliferation of tumor cells. ATP-competitive CDK4/6 inhibitors, such as palbociclib and abemaciclib, have been developed for anti-cancer therapy and were recently approved for treatment of breast cancer patients. Interestingly, common side effects of a CDK4/6 inhibitor therapy constitute neutro-penia and leukopenia. Moreover, it was found that CDK4/6 inhibition modulates immune cell functions in kinase-dependent or independent manners. Mechanistically, it is assumed that these atypical functions of CDK4 and CDK6 derive from their recently discovered role as cofactors for immune regulatory transcription factors. Especially chromatin-associated CDK6 can co-localize at promoter regions of a subset of NF-κB, STAT3 or AP1 target genes, thereby changing the DNA-binding properties or activity of these transcription factors.
In a screen for small-molecule inhibitors of IκBζ action in keratinocytes, the inventors identified CDK4/6 inhibitors as potent suppressors of IL-36- and IL-17A/TNFα-mediated IκBζ expression. Mechanistically, CDK4/6 inhibitors suppressed the activity of STAT3, which was identified as a major transcriptional regulator of IκBζ expression in keratinocytes. STAT3 activation was conveyed by CDK4/6-mediated phosphorylation of the methyltransferase EZH2, triggering the subsequent methylation of STAT3 and induction of IκBζ expression. Importantly, topical administration of CDK4/6 or EZH2 inhibitors on the skin completely prevented experimental psoriasis by suppressing STAT3 activation and consequently IκBζ expression in keratinocytes. Moreover, as cyclin D2, cyclin D3 and EZH2 were found to be overexpressed in human psoriatic skin lesions, the inventors propose repurposing of CDK4/6 inhibitors for topical skin treatment of psoriasis patients.
HaCaT cells were maintained in DMEM with 10% FCS and antibiotics. Human primary keratinocytes were freshly isolated from foreskin and maintained in CnT-07S medium with gentamycin (CELLnTEC). Recombinant human IL-36α (aa 6-158) and mouse IL-36α (aa 6-160) were purchased from R&D Systems. Recombinant IL-17A (11340174), TNFα (11343013) and IL-1β (11340013) were ordered from Immunotools. Flagellin (vac-fla) and poly I:C (vac-pic) were purchased from Invivogen. In cell culture experiments, the cytokines were used at the following concentrations, as described previously: IL-36α (100 ng/mL), IL-17A (200 ng/mL), TNFα (10 ng/mL) and IL-1β (100 ng/mL). Flagellin was applied at 10 ng/mL and poly (I:C) was added at a final concentration of 100 ng/mL. The following inhibitors were purchased from Selleckchem: Abemaciclib mesylate (LY2835219, S17158), Palbociclib isethionate (S1579), EPZ6438 (taze-metostat, S7128) and CPI-169 (S7616). If not otherwise indicated, the inhibitors were used in cell cultures at the following concentrations: abemaciclib (16 μM), palbociclib (50 μM) and EPZ6438 (10 μM). When indicated, cells were starved for 2 h prior to cytokine treatment, by removing cell culture supplements from the growth medium.
Lentiviral particles were produced in HEK293T cells using the vector pMD2.G and a second-generation packaging system (psPAX2, Addgene). Keratinocytes were transduced in the presence of 8 μg/mL polybrene, packaging plasmids and 5 μg of the respective shRNA construct: pLKO.1-puro (sh ctrl); pLKO.1-TRCN0000009876 (shCDK4); pLKO.1-TRCN0000010473 (shCDK6); pTRIPZ-EZH2 (V2THS63066, Dharmacon); pGIPZ noncoding ctrl (Dharmacon, RHS4351); pLKO.1-TRCN0000020840 (shSTAT3, Sigma); pLKO.1-TRCN0000014683 (shRELA, Sigma), followed by puromycin selection (1 ng/mL, Invitrogen).
Luciferase constructs were generated as described; Müller et al. (2018; loc. Cit.). 1*104 HEK293T cells were transfected for 24 h using HBS buffer and CaCl2 and a mixture of 400 ng luciferase vector and 100 ng TK-Renilla vector. For expression of other factors, the following concentrations were used: 70 ng p65, 200 ng cJUN, STAT3 or EZH2 constructs and 500 ng CDK4, CDK6 or cyclin D expression constructs (not shown). For transfection of HaCaT cells, 3*105 cells were transfected for 4 h using Lipofectamine 3000 and a mixture of 800 ng luciferase vector, 200 ng TK-Renilla vector and 4 μg expression or control plasmids according to the manufacturer's instructions (Thermo Fisher). 36 h after transfection, luciferase activity was measured with the Dual Luciferase Reporter Assay Kit (Promega). Expression of the reporter constructs was calculated as the fold induction over unstimulated transfected cells from data of three independent experiments.
HEK293T cells were transfected using HBS buffer and CaCl2. HaCaT cells were transfected with Lipofectamine 3000, according to the manufacturer's instructions (Thermo Fisher). 5 μg expression constructs were incubated with 3*105 cells for 4 h. 36-48 h post transfection, cells were harvested and analysed.
IκBζ was cloned into the lentiviral pinducer20 plasmid (Addgene, 44012) using pENTR TOPO cloning. After lentivirus production in HEK293T cells, HaCaT cells were transduced and selected with 450 μg/mL G418 (Invivogen). Induction of IκBζ expression cells was achieved by doxycycline treatment (2 μg/ml, AppliChem) for 24 h.
Mutation of STAT3 at K49, K140 and K180 was performed by site-directed mutagenesis of the human STAT3 pcDNA3 construct from Addgene (71447), which was previously cloned into the Strep-tagged backbone (pEXPR-IBA103). Substitu-tion of the amino acid was performed with self-designed primers (not shown).
The CRISPR/Cas9 one vector system was used to generated STAT3 KO HaCaT cells according to well-known methods. The guide RNA against STAT3 (forward: 5′-CACCGACTGCTGGTCAATCTCTCCC-3′ (SEQ ID NO:1), reverse: 5′-AAACGGGAGAGATTGACCAGCAGTC-3′ (SEQ ID NO:2)) was cloned into the len-tiCRISPRv2 containing Cas9 vector (Addgene, 52961), followed by lentiviral transduction and puromycin selection.
Synchronization of the cells with a double thymidine block was performed as described before. After the second thymidine block, cells were released in normal medium. At 0, 4, 10 and 14 h after release, cells were stimulated with IL-36α and/or abemaciclib for 1 h. Propidium iodide staining was performed by flow cytometry (LSRII, Becton Dickson) to detect the cell cycle phase at the time point of cell harvest.
Western blot analysis was performed as described before. The following antibodies were used and purchased from Cell Signaling: anti-IκBζ (9244), anti-phospho-STAT3 at Tyr705 (9145), anti-phospho-STAT3 at Ser727 (9134), anti-STAT3 (12640), anti-p65 (8242), anti-EZH2 (5246), anti-pRB (phospho-RB at Ser807/811; 8516), anti-FoxM1 (5436), anti-H3 (4499), anti-CDK4 (12790), anti-CDK6 (13331), anti-CDK9 (2316), anti-cyclin D1 (2978), anti-cyclin D2 (3741), anti-cyclin D3 (2936), anti-cJUN (9165), anti-H3K27me3 (9733), anti-GAPDH (2118), anti-H3 (9715) and anti-β-actin (3700). Anti-β-Gal (sc377257) and anti-GFP (sc9996) were obtained from Santa Cruz Biotechnology. Anti-pEZH2 at T345 (61242) anti-pEZH2 at T487 (12820) were purchased from Active Motif and anti-pan-methyl-lysine antibody was purchased from Enzo (ADI-KAP-TF121-E). For detection of mouse IκB, a self-made antibody raised against peptides CSAPGSPGSDSSDFSS (SEQ ID NO:3) and CLHIRSHKQKASGQ (SEQ ID NO:4) was applied.
ChIP assays were performed as described before. After sonification, chromatin was incubated with protein G-coupled Dynabeads (Invitrogen) and 2 μg of STAT3 (Thermo Fisher, MA1-13042), CDK4 (Cell Signaling, 12790), CDK6 (Sigma, HPA002637), EZH2 (Diagenode, C15410039) or control IgG antibody (Abcam, ab46540) overnight at 4° C. The promoter region of myoglobulin (MB) served as an internal negative control [forward: 5″-CTCTGCTCCTTTGCCACAAC-3″ (SEQ ID NO:5), reverse: 5″-GAGTGCTCTTCGGGTTTCAG-3″ (SEQ ID NO:6)]. ChIP primers corresponding to the promoter region of NFKBIZ were self-designed [forward 5″-GCCTTAACTGGGCTAACAGC-3″ (SEQ ID NO:7), reverse 5″-CTGGCAAGTCCTGGAAGGAG-3″ (SEQ ID NO:8)]. Data from two independent experiments is presented as the fold enrichment, calculated over the percentage of input from the IgG control ChIP.
Cells were lysed by mechanical disruption using standard lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1× Protease inhibitor cocktail, Roche). Subsequently, lysates were sonicated for 5 minutes at high power (Bioruptor, Diagenode), followed by pre-clearing of the lysates with protein A/G PLUS agarose beads (Santa Cruz, sc-2003) for 1 h at 4° C. Precleared lysates were incubated either with antibodies specific for CDK4 (Cell Signaling, 12790), CDK6 (Sigma, HPA002637), EZH2 (Cell Signaling, 5246), STAT3 (MA1-13042, Thermo Fisher) or β-Gal antibody (sc-19119, Santa Cruz) as an IgG control, overnight at 4° C. For endogenous IPs immune complexes were precipitat-ed with protein A/G PLUS agarose beads and eluted by 6× SDS-PAGE sample buffer.
Gene Expression Analysis by qPCR
Gene expression analyses were performed as described. Relative gene expression was analyzed using self-designed primers ordered at Metabion (not shown). Relative mRNA levels were calculated by normalization to the human reference gene RPL37A or the mouse reference gene Actin using the 2-ΔΔCt method.
Mice
Experiments were conducted in accordance with the German law guide-lines of animal care. Ears of female C57BL/6 mice (8-12 weeks old) were topically treated for 6 consecutive days with 5 mg Aldara cream (containing 5% imiquimod, 3M Pharmaceutical) and 0.02 mg of abemaciclib (in 10 μL ethanol), 0.05 mg CPI-169 (in 10 μL ethanol) or vehicle control. At day 7, mice were sacrificed and analysed. For IL-36α-mediated psoriasis induction, ears of male C57BL/6 mice (8-12 weeks old) were treated by intradermal injections using 1 μg murine IL-36α (aa 8-160, R&D) or PBS control for 5 consecutive days. For application of the vehicle control, abemaciclib (0.02 mg) or CPI-169 (0.05 mg), substances were mixed with Miglyol 812 (Carl Roth) in a ratio of 1:4. Inhibitors were topically applicated 6 h before intradermal injections of IL-36α or PBS were applied. Mice were sacrificed and analysed at day 6.
Flow Cytometry
Sample preparation was performed as described. The following anti-mouse antibodies from BioLegend were used: anti-CD45 FITC (103107), anti-CD11b PacificBlue (101223), anti-Ly6G PE (127607), anti-F4/80 APC (123115), anti-CD11c Pacific Blue (117322), anti-MHC-II APC (107613), anti-CD172a PE (144011) and anti-Silec-H PE (129605). Anti-PDCA-1 APC (17-2092-80) and anti-αβTCR PE (HM3628) were purchased from Invitrogen, and anti-γδ-TCR APC (17-5711-82) from Sigma. Data was acquired on a LSRII flow cytometer (Becton Dickson).
Histology
Ear sections from mice were fixed in 10% formalin (Carl Roth) and subsequently embedded in paraffin. 5-μm sections were prepared and incubated with the following antibodies from Cell Signaling: pSTAT3 (9145), pRB (8516) and H3K27me3 (9733). For staining of human skin samples, EZH2 antibody (Cell Signaling, 5246) was used. Antigen retrieval was performed in 1 mM EDTA pH 8.0 for pSTAT3, and 10 mM citrate buffer pH 6.0+0.5% Triton X-100 for EZH2, H3K27me3 and pRB. After incubation with peroxidase-coupled secondary antibodies, sections were stained with DAB substrate.
Gene expression data originated from the GEO data set GSE13355; see Nair, R. P., et al., Genome-wide scan reveals association of psoriasis with IL-23 and NF-kappaB pathways, Nat Genet 41, 199-204 (2009); and Swindell, W. R., et al., Genome-wide expression profiling of five mouse models identifies similarities and differences with human psoriasis. Plos One 6, e18266 (2011). Pre-normalized gene expression values from each sample was directly taken from the GEO profile data set GDS4602. The following reporters were taken for analysis: EZH2: ID 203358_s_at, CCND1: ID 208711_s_at, CCND2: ID 200953_s_at and CCND3: ID 201700_s_at.
Due to a lack of enzyme activity, direct inhibition of IκBζ is not feasible. Key regulators in psoriasis constitute IL-17 and IL-36 family members, which predominantly trigger a pro-inflammatory response in keratinocytes that is dependent on IκBζ. Thus, the inventors performed an unbiased screen for small-molecule inhibitors that are able to block induction of IκBζ expression in response to either IL-36α or IL-17A. Previously, it was shown that IL-17-induced IκBζ expression is strongly increased in combination with TNFα; see Johansen (2015; loc. cit.) and Müller (2018; loc. cit.). Intriguingly, the inventors found that two CDK4/6 inhibitors, abemaciclib (
To explore whether these effects were due to a CDK4/6 inhibitor-mediated G1-cell cycle arrest, the inventors repeated the experiments in synchronized and single cell cycle phase-arrested keratinocytes. IL-36α treatment triggered IκBζ induction largely in all phases of the cell cycle, which was completely suppressed by abemaciclib (
The inventors hypothesized that CDK4/6 are not involved in the direct regulation of IκBζ target gene expression but rather trigger the expression of IκBζ, which in turn induces a secondary, IκBζ-dependent gene expression in stimulated keratinocytes. To test this hypothesis, the inventors overexpressed a doxycycline-inducible version of IκBζ in IL-36α- or IL17A/TNFα-stimulated HaCaT cells in the presence or absence of abemaciclib (
Beside their known involvement in cell cycle regulation, CDK4 and CDK6 have previously been described to function as transcriptional cofactors for STAT3, NF-κB or AP-1. As the inventors revealed a CDK4/6-dependent induction of IκBζ on the transcriptional level, the inventors next explored the responsible transcription factor. Of note, binding sites for all three transcription factors were previously identified at the NFKBIZ promoter region. Interestingly, expression of both CDK4 and CDK6 increased the STAT3-mediated induction of NFKBIZ promoter activity, whereas no synergistic effects could be observed when CDK4/6 were co-overexpressed with NF-κB p65 or cJUN (
Previous publications reported that CDK6 acts as a cofactor for STAT3, independently of its kinase function. Therefore, the inventors tested if a kinase-dead mutant of CDK6 (CDK6 DN) could still synergize with STAT3 in driving the expression of the NFKBIZ luciferase reporter construct. Surprisingly, the kinase-dead mutant was not able to cooperate with STAT3 anymore, whereas a hyperactive version of CDK6 (CDK6 S178P) further increased the activity of the NFKBIZ promoter in a STAT3-dependent manner (
Next, the inventors explored the mechanism of how CDK4/6 regulate STAT3-mediated expression of IκBζ. Of note, in chromatin immunoprecipitation (ChIP) analyses CDK4/6 was found to localize to the NFKBIZ promoter region, which depended on the presence of STAT3 (
Previously, EZH2, a methyltransferase that directs H3K27me3 in conjunc-tion with the PRC2 complex, was found to be important in the differentiation and function of keratinocytes. Moreover, it was revealed that EZH2 can methylate STAT3 at lysine 49, 140 or 180, thereby modulating STAT3 activity by affecting the subcellular localization or phosphorylation status of STAT3 at tyrosine 705. We hypothesized that CDK4/6 might phosphorylate EZH2 in keratinocytes, thus enabling EZH2-mediated methylation and activation of STAT3. Pulldown assays in HEK293T cells validated an interaction of CDK4 and CDK6 with EZH2 (
In primary human keratinocytes, expression of EZH2 itself was induced by IL-36α (
These data suggest that CDK4/6-mediated phosphorylation of EZH2 at threonine 345 represents a regulatory switch, leading to the interaction of EZH2 with STAT3 and subsequent STAT3 activation. Accordingly, whereas wildtype EZH2 synergis-tically induced the expression of the NFKBIZ luciferase promoter in cooperation with CDK4/6 and STAT3, an EZH2 mutant lacking the CDK4/6-directed phosphorylation site (EZH2 T345A) abrogated CDK4/6- and STAT3-mediated NFKBIZ promoter-driven luciferase expression (
As reported before, EZH2 can methylate STAT3 at lysine K49, K140 or K180, thereby changing its transcription factor function or subcellular localization. Thus, the inventors immunoprecipitated STAT3 in STAT3- and EZH2-overexpressing HEK293T cells in the presence or absence of abemaciclib, and analyzed the methylation status of STAT3 using a pan-methyl-lysine-specific antibody. Simultaneous overexpression of EZH2 and STAT3 induced methylation of STAT3, as expected, whereas CDK4/6 inhibition abrogated lysine methylation of STAT3 (
EZH2-dependent methylation sites of STAT3 at lysine 49, 140 and 180 were previously identified by mass spectrometric analyses in tumor cells. Thus, the inventors substituted all three lysine methylation sites with arginines and tested the STAT3 mutants for their potential in activating NFKBIZ luciferase promoter expression. Whereas mutation of STAT3 at K49 and K140 had no effect on the induction of NFKBIZ promoter expression, alone or in combination with CDK6 and EZH2 (
The inventors' findings suggest that CDK4/6 mediates the phosphorylation of EZH2 in a cyclin D-dependent manner, leading to STAT3 activation and IκBζ expression. The inventors therefore investigated a potential relevance of this pathway in skin biopsies from psoriasis patients. Human psoriatic lesions, compared to non-psoriatic lesions or unaffected skin, were characterized by a marked transcriptional upregulation of CCND2 and CCND3, encoding cyclin D2 and cyclin D3, respectively (
Next, the inventors ask if an upregulation of cyclin D2, cyclin D3 and EZH2 can be also detected in relevant psoriasis mouse models. In the standard model using the TLR7 agonist imiquimod (IMQ), psoriasis-like skin inflammation was triggered by daily application of an IMQ-containing cream on the ears for 6 days, while in a second model daily intradermal injections of IL-36α into the skin of mouse ears were employed for 5 consecutive days (for details see
Due to the inventors' finding that CDK4/6 and EZH2 inhibitors suppressed psoriasis-related, pro-inflammatory gene expression downstream of IL-36α or IL-17A/TNFα, the inventors next investigated the potential of CDK4/6 and EZH2 inhibitors to block experimental psoriasis in vivo. Moreover, the inventors reasoned that topical application of both inhibitors would be sufficient, as the epidermis constitutes the main target for CDK4/6 and EZH2 inhibition. A pre-requisite for efficient take-up of small molecule inhibitors from the skin are hydrophobic features of these substances. Thus, the inventors selected more hydrophobic inhibitors, such as abemaciclib (for CDK4/6 inhibition) or CPI-169 (see Bradley, W. D., et al., EZH2 inhibitor efficacy in non-Hodgkin's lymphoma does not require suppression of H3K27 monomethylation, Chem Biol 21, 1463-1475 (2014)) (for EZH2 inhibition) that can more easily penetrate the outer skin barrier. Psoriasis-like skin inflammation was induced in the above-mentioned psoriasis model by daily application of an IMQ-containing cream on the ears of wildtype mice for 6 days, before animals were sacrificed and analyzed at day 7. Abemaciclib, CPI-169 and ethanol as vehicle control were daily applied on the ear skin in parallel to IMQ (
Treatment of mice with the TLR7 agonist IMQ represents a standard mouse model for psoriasis, however, IMQ activates immune cells in the first instance, rather than an initial keratinocyte-derived pro-inflammatory response, as it is likely to happen in human psoriasis pathogenesis. Thus, the inventors additionally investigated the therapeutic effects of abemaciclib or CPI-169 in an IL-36-triggered psoriasis-like dermatitis mouse model (
CDK4/6 inhibitors have been developed for treatment of cancer patients in order to restrain hyperproliferation of tumor cells. Recently, it was found that CDK4/6 do not only control cell cycle progression by phosphorylation of RB, but also regulate immune cell differentiation and function. In this context, CDK4/6 have been implicated as transcriptional cofactors that activate a subset of NF-κB or STAT3 target genes. Based on the inventors' results in cultured keratinocytes, human skin biopsies and mouse models, the inventors propose repurposing of CDK4/6 inhibitors for psoriasis therapy (
NFKBIZ, the gene encoding IκBζ, has been identified as a new susceptibility locus in psoriasis; see Tsoi, L. C., et al., Enhanced meta-analysis and replication studies identify five new psoriasis susceptibility loci. Nat Commun 6, 7001 (2015). The inventors recently reported that IκBζ is overexpressed in human psoriatic lesions, whereas global IκBζ KO mice are completely protected against psoriasis-like skin inflammation in several psoriasis models; see Johansen et al. (2015; loc. cit.) and Müller et al. (2018; loc. cit.). Mechanistically, IκBζ is transcriptionally induced in keratinocytes by IL-17 and IL-36, which triggers the expression of psoriasis-relevant target genes encoding for selective chemo- and cytokines and antimicrobial proteins. Deficiency of IκBζ therefore prevents the recruitment of neutrophils and monocytes that are needed for skin inflammation. Collec-tively, the inventors' data suggest that interfering with IκBζ expression or function in keratinocytes might be a promising strategy for psoriasis therapy. As IκBζ is crucial for both IL-36 and IL-17 signaling, CDK4/6 inhibitors might be applicable for different sub-types of psoriasis.
Unfortunately, based on a lack for enzyme activity, direct pharmacological inhibition of IκBζ function remains difficult. The inventors therefore sought to block the transcriptional induction of IκBζ and identified small molecule inhibitors of CDK4/6 and EZH2 as potent suppressors of IκBζ expression in keratinocytes. CDK4/6 have been previously shown to modulate several immune-relevant transcription factors by both kinase-dependent and -independent mechanisms. In the present study, the inventors clearly demonstrate that STAT3-mediated IκBζ expression is kinase-dependent, as ATP-competitive CDK4/6 inhibitors, such abemaciclib or palbociclib, abolished IκBζ expression. Consistent with these findings, a hyperactive but not a dominant-negative version of CDK6 increased NFKBIZ promoter activity. Moreover, cyclin D2 and cyclin D3 elevated the expression of NFKBIZ and its target genes, supporting the need for CDK4/6 kinase activity.
Despite the requirement of the kinase activity, the involvement of CDK4/6 could be separated from its classical role in cell cycle regulation and phosphorylation of RB. Thus, depletion of RB did not affect IκBζ expression. Moreover, IκBζ expression was principally induced by IL-36 stimulation in all phases of the cell cycle, except for G0-arrested cells that revealed a weaker IκBζ expression. Importantly, although IκBζ expression does not rely on CDK4/6-mediated cell cycle progression, CDK4/6 inhibitors might have also beneficial effect in psoriasis treatment by additionally blocking the keratinocyte hyperproliferation, which is a hallmark of psoriasis.
In this study, the inventors demonstrate a major role for STAT3 in driving keratinocyte-specific IκBζ expression. IκBζ expression in keratinocytes is predominantly controlled from the proximal promoter 2 of the NFKBIZ locus, containing different transcription factor binding sites than the better investigated distal promotor 1, which is more tightly controlled by NF-κB. So far, the inventors have not compared the promoter usage in distinct cell types, but is likely that the contribution of the individual promoters and STAT3 to IκBζ expression differs among different cell types. The inventors' experiments show that CDK4/6 do not directly phosphorylate STAT3 but EZH2, which induces IκBζ and IκBζ-dependent pro-inflammatory target gene expression in a STAT3-dependent manner. This finding seems surprising at the first instance, since EZH2, as part of the PRC2 complex, is mainly involved in gene repression through trimethylation of H3K27. Recently, however, EZH2 was found to induce gene expression via interaction with the SWI/SNF complex or by repressing anti-inflammatory molecules, such as SOCS3, thus delimitating the expression of pro-inflammatory cytokines. CDK4/6 phosphorylated EZH2 at T345, thereby inducing an EZH2-dependent methylation of STAT3 at K180, and subsequent induction of IκBζ expression by STAT3. EZH2 phosphorylation at T345 was previously described to be mediated by CDK1 and CDK2, leading to an EZH2-directed epigenetic silencing of genes during G2 phase. Thus, even though CDK-mediated phosphorylation of EZH2 at T345 seems to be conserved, its impact on EZH2 function might depend on the stimulus or cell cycle phase.
Upon CDK4/6-mediated phosphorylation, EZH2 preferentially interacted with STAT3, resulting in STAT3 K180 methylation and enhanced STAT3 activation. Similar observations were made in glioblastoma, where IL-6-induced STAT3 activation is controlled by EZH2-mediated trimethylation of STAT3 at K180. Thus, phosphorylation of EZH2 might induce a switch in EZH2 function from H3K27 trimethylation and transcriptional repression to non-canonical functions, including STAT3 methylation and gene activation. Whether this gene-activating function of EZH2 requires the PRC2 repressor complex or whether it is PRC2-independent remains to be resolved. In addition to its main function in transcriptional repression, non-PRC functions of EZH2 via direct binding to transcriptional regulators have been reported before. For instance, EZH2 was shown to act as a co-factor for AR or the SWI/SNF complex leading to target gene activation. Similar to other non-histone targets, however, the exact molecular events that link STAT3 methylation to STAT3 activation are currently unknown.
Regardless of the detailed mechanism of EZH2-mediated STAT3 activation, the inventors' study has important clinical implications. The inventors' results suggest that targeting of the CDK4/6-EZH2-STAT3 pathway does not only suppress cytokine-mediated induction of IκBζ and pro-inflammatory target gene expression, but also inhibits immune cell recruitment and skin inflammation. The inventors demonstrate in the IMQ- and IL-36-mediated psoriasis mouse models that both CDK4/6 and EZH2 inhibitors completely blocked the development psoriatic skin lesions. The therapeutic effect of the inhibitors concurred with a suppression of IκBζ expression and a strong inhibition of IκBζ target gene expression, including chemokines (e.g. Cxcl2, Cxcl5), cytokines (e.g. II1f9, II1b, II17a, II23a) and anti-microbial proteins (e.g. Lcn2). In contrast, genes that were not IκBζ-dependent, such as NFKBIA and TNF, remained unaffected upon CDK4/6 or EZH2 inhibition. These findings further support the view of a rather selective role of IκBζ in the control of immune responses and also indicate that inhibition of IκBζ will be associated with less side effects than a broad inhibition of NF-κB by toxic IκBζ inhibitors.
In line with the inventors previous reports showing an upregulated expression of IκBζ in psoriasis (Johansen et al. (2015; loc. cit.) and Müller et al. (2018; loc. cit.), the inventors detected an increased nuclear expression of EZH2 and elevated cyclin D2 and D3 levels, both in psoriasis mouse models and in human psoriatic skin lesions. Previous studies also found constitutively active STAT3 in the epidermis of psoriatic lesions (see Miyoshi, K., et al., Stat3 as a therapeutic target for the treatment of psoriasis: a clinical feasibility study with STA-21, a Stat3 inhibitor, J Invest Dermatol 131, 108-117 (2011). The inventors assume that the CDK4/6-EZH2-STAT3 pathway is hyperactive in psoriatic skin lesions. As inhibition of IκBζ blocks multiple signaling pathways in psoriasis, targeting IκBζ might increase overall therapy responses as well as prevent the development of therapy resistance. Due to the clinical availability of hydrophobic CDK4/6 and EZH2 inhibitors, the inventors propose formulation of these inhibitors for instance in a crème, for topical treatment of psoriatic skin lesions. Topical drug administration will also restrict potential side effects and might be especially promising for those patients who have developed resistance to current psoriasis therapies.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19200621.1 | Sep 2019 | EP | regional |
This application is a continuation of copending International Patent Application PCT/EP2020/076361 filed on 22 Sep. 2020 and designating the United States of America, which was published under PCT Article 21(2) in English, and claims priority from European Patent Application EP 19 200 621.1 filed on 30 Sep. 2019. The entire contents of these prior applications are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2020/076361 | Sep 2020 | US |
| Child | 17707741 | US |
