Methods of treating, ameliorating, and / or preventing cardiovascular disease

Abstract

The present disclosure generally relates to methods for the treatment, amelioration, and / or prevention of vascular inflammatory disorders. In some aspects, the methods of the present disclosure comprise administration of an EZH2 inhibitor in a therapeutically effective amount to a subject in need thereof in order to treat, ameliorate, and / or prevent a vascular inflammatory disorder. In some aspects, the methods of the present disclosure comprise administration of an EZH2 inhibitor in a therapeutically effective amount to a subject in need thereof in order to treat, ameliorate, and / or prevent a cardiovascular disease.

Description

[0001] Attorney Docket No. 047162-7516WOl(02762)

[0002] METHODS OF TREATING, AMELIORATING, AND / OR PREVENTING CARDIOVASCULAR DISEASE

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 739,864, filed December 30, 2024. which is incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under HL75092 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Vascular inflammation, which is controlled mainly by the endothelial cells that line all blood vessels, is a primary cause of coronary, peripheral, and cerebral artery disease, pulmonary arterial hypertension, lung and kidney injury, diabetic vasculopathy, transplant rejection, intravascular coagulation due to sepsis, and many others. Furthermore, atherosclerotic cardiovascular disease (ASCVD), the leading cause of mortality w orldw ide, is initiated and driven by endothelial cell inflammatory activation. However, treatment of such inflammation and inflammatory disorders with anti-inflammatory therapies generally limit immune defense against pathogens and increase risk of serious infections, and those are serious drawbacks to such treatment options.

[0008] Therefore, new therapeutic options for the treatment of inflammatory disorders, such as those disorders comprising vascular inflammation are needed. The present disclosure addresses this need.

[0009] SUMMARY

[0010] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0011] Method of treating, ameliorating, and 'or preventing vascular inflammatory disease

[0012] In some aspects, the present invention is directed to a method of treating, ameliorating, and / or preventing vascular inflammatory' disease in a subject in need thereof.

[0013] In some embodiments, the method comprises administering an effective amount of an EZH2 inhibitor.

[0014] 1

[0015] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0016] In some embodiments, the EZH2 inhibitor comprises tazemetostat.

[0017] In some embodiments, the vascular inflammatory disease is a cardiovascular disease. In some embodiments, the cardiovascular disease is atherosclerosis, pulmonary arterial hypertension (PAH), a peripheral artery disease (PAD), or diabetic PAD.

[0018] In some embodiments, treatment or amelioration of the subject decreases and / or reverses progression of the atherosclerosis.

[0019] In some embodiments, the vascular inflammatory disease comprises plaque formation in the subject.

[0020] In some embodiments, the plaque is an atherosclerotic plaque.

[0021] In some embodiments, treatment or amelioration of the subject reduces the rate of plaque progression in the subject.

[0022] In some embodiments, treatment or amelioration of the subject increases the amount of stable plaque as compared to the amount of unstable plaque in the subject.

[0023] In some embodiments, treatment or amelioration of the subject increases the thickness of fibrous plaque caps in the subject.

[0024] In some embodiments, treatment or amelioration of the subject decreases the size of the necrotic core of plaque in the subject.

[0025] Method of treating, ameliorating, and / or preventing cardiovascular disease

[0026] In some aspects, the present invention is directed to a method of treating, ameliorating, and / or preventing a cardiovascular disease in a subject in need thereof.

[0027] In some embodiments, the method comprises administering an effective amount of an EZH2 inhibitor.

[0028] In some embodiments, the EZH2 inhibitor comprises tazemetostat.

[0029] In some embodiments, the cardiovascular disease is ischemic heart disease, congestive heart failure, hypertension, valvular heart disease, general atherosclerosis, hypercholesterolemia, atherosclerosis, atherosclerotic cardiovascular disease (ASCVD), thrombosis, or any combination thereof.

[0030] In some embodiments, the cardiovascular disease is atherosclerosis or ASCVD. In some embodiments, treatment or amelioration of the subject decreases and / or reverses progression of the atherosclerosis or ASCVD.

[0031] In some embodiments, the cardiovascular disease comprises plaque formation in the subject.

[0032] In some embodiments, the plaque is an atherosclerotic plaque.

[0033] 2

[0034] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0035] In some embodiments, treatment or amelioration of the subject reduces the rate of plaque progression in the subject.

[0036] In some embodiments, treatment or amelioration of the subject increases the amount of stable plaque as compared to the amount of unstable plaque in the subject.

[0037] In some embodiments, treatment or amelioration of the subject increases the thickness of fibrous plaque caps in the subject.

[0038] In some embodiments, treatment or amelioration of the subject decreases the size of the necrotic core of plaque in the subject.

[0039] Method of treating, ameliorating, and / or preventing ASCVD

[0040] In some aspects, the present invention is directed to a method of treating, ameliorating, and / or preventing atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof.

[0041] In some embodiments, the method comprises administering an effective amount of an EZH2 inhibitor.

[0042] In some embodiments, the EZH2 inhibitor comprises tazemetostat.

[0043] In some embodiments, the ASCVD comprises plaque formation in the subject.

[0044] In some embodiments, treatment or amelioration of the subject reduces the rate of plaque progression in the subject.

[0045] In some embodiments, treatment or amelioration of the subject increases the amount of stable plaque as compared to the amount of unstable plaque in the subject.

[0046] In some embodiments, treatment or amelioration of the subject increases the thickness of fibrous plaque caps in the subject.

[0047] In some embodiments, treatment or amelioration of the subject decreases the size of the necrotic core of plaque in the subject.

[0048] Method of treating, ameliorating, and / or preventing vascular inflammation

[0049] In some aspects, the present invention is directed to a method of treating, ameliorating, and / or preventing vascular inflammation in a subject in need thereof.

[0050] In some embodiments, the method comprises administering an effective amount of an EZH2 inhibitor.

[0051] In some embodiments, the EZH2 inhibitor comprises tazemetostat.

[0052] Method of treating, ameliorating, and or preventing ALI

[0053] 3

[0054] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0055] In some aspects, the present invention is directed to a method of treating, ameliorating, and / or preventing acute lung injury (ALI) in a subject in need thereof.

[0056] In some embodiments, the method comprises administering an effective amount of an EZH2 inhibitor.

[0057] In some embodiments, the EZH2 inhibitor comprises tazemetostat.

[0058] BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0060] FIG. 1A-FIG. 1H present schematics and results related to Poly comb Repressive Complex 2 (PRC2) mediation of protocadherin gamma (Pcdhg) inhibition of Klf2 and Klf4 expression. FIG. 1A presents a schematic representation of bulk RNAseq analysis of Control siRNA (hereafter, “si”), Pcdhg si and Pcdhg+Klf2 / 4 triple si human umbilical vein endothelial cells (HUVECs) that identified Pcdhg-dependent, K112 / 4 independent differentially expressed genes (DEGs). FIG. IB presents a graphical representation of upstream regulator analysis of DEGs using EnrichR to identify common upstream regulator}' factors. FIG. 1C presents a graphical representation of mRNA levels of Ezh2 and Suzl2 from RNAseq analysis. FIG. ID presents an immunoblot for Ezh2 and Klf4 in Control or Pcdhg si HUVECs exposed to laminar shear stress (LSS) (N=3 independent experiments), and further presents graphical representations of quantitation of Ezh2 and Klf4 normalized to GAPDH loading control. FIG. IE presents a graphical representation of H3K27me3 ChlP-PCR control. FIG. IF shows the signal at the Klf2 promoter. Suzl2 si was used as an internal control to confirm specificity’ of PRC2-dependentH3K27me3 (N=3 independent experiments). FIGs. 1G-1H present a graphical representation of H3K27ac ChlPseq signal on the Klf2 promoter in Control si and Pcdhg si HUVECs, showing a significantly higher peak call after Pcdhg si, confirmed by increased H3K27ac (FIG. 1G) and decreased H3K27me3 (FIG. 1H) ChlP-PCRs (N=3 independent experiments).

[0061] FIG. 2A-FIG. 2E present schematics and results related to PRC2 suppression of protective endothelial cell (EC) hemodynamic responses. FIG. 2A presents a schematic representation of bulk RNAseq analysis of HUVECs exposed to LSS or oscillatory' shear stress (OSS) and the volcano plot of the DEGs. FIG. 2B presents a graphical representation of analysis of enriched pathways / processes between LSS and OSS. FIG. 2C presents a graphical representation of upstream regulator analysis of DEGs using EnrichR that identified common 4

[0062] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0063] upstream regulatory factors. FIG. 2D presents images of aortic arch region prepared en face and immunostained with H3K27me3 to test PRC2 activity in the LSS region (greater curvature, outer part of the tissue, protected from atherosclerosis) and disturbed shear stress (DSS) region (lesser curvature, susceptible to atherosclerosis, center of the tissue) (N=3 animals). FIG. 2D further presents graphical representations of quantitation of H3K27me3 and corresponding Klf4 levels in these regions. FIG. 2E presents images of Klf4 staining in HUVECs after depletion of PRC2 by Suzl2 si or Ezh2 si then exposed to LSS or OSS (N=3 independent experiments), as well as a graphical representation of quantitation of Klf4.

[0064] FIG. 3A-FIG. 3D present data and results related to high PRC2 activity (H3K27me3) in inflammatory ASCVD. FIG. 3A presents analysis of Ezh2, Suzl2 and Pcdhg in ECs from published single-cell (sc) RNAseq data from deidentified human ASCVD patients comparing regions of plaque (Plaque) to an adjacent plaque-free region of the same artery (Normal) (Alsaigh et al., Communications Biology, 2022). Tumor necrosis factor a (TNFa) and Klf2 / 4 were used as internal controls for plaque ECs and normal ECs respectively (N=3 donors). FIG. 3B presents images of human coronary artery sections from de-identified elderly donors stained with H&E and for H3K27me3, comparing regions of plaque (Plaque region) to the regions of the same artery without evident plaque (Normal region). Sections were counter stained with Erg to mark ECs (N=3 donors). FIG. 3B further presents a graphical representation of quantitation ofHK27me3 level in ECs. FIG. 3C presents images of artery sections from deidentified human ASCVD patients containing atherosclerotic plaques and non-diseased controls stained with H3K27me3. Sections were counter stained with Erg to mark ECs (N=8 donors). FIG. 3C further presents a graphical representation of quantitation of H3K27me3 level in ECs. FIG. 3D presents images of mouse atherosclerotic carotids from a Partial Carotid Artery (PCA) Ligation model of accelerated atherosclerosis stained with H3K27me3. Sections were counter stained with 4',6-diamidino-2-phenylindole (DAPI) to mark nuclei (N=3 animals). RCA: Right Carotid Artery (control), LCA: Left Carotid Artery. FIG. 3D further presents a graphical representation of quantitation of H3K27me3 levels.

[0065] FIG. 4A-FIG. 4H present schematics, data, and results related to pharmacological inhibition of PRC2 protecting from vascular inflammation. FIGs. 4A-4B present results on the effect of pharmacological inhibition of PRC2 activity using Tazemetostat (Tz) in vitro in HUVECs exposed to Static (no flow) or LSS (FIG. 4A) and in vivo in mouse thoracic aorta prepared en face (FIG. 4B) on Klf4 levels (N=3 independent experiments). FIGs. 4A-4B further present graphical representations of quantitation of Klf4 level in ECs. FIG. 4C presents an immunoblot for the inflammatory marker VCAM1 in HUVECs, untreated or 5

[0066] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0067] treated with the inflammatory cytokine II 10 (1 ng / ml) for 16 h with or without Tz (N=2 independent experiments). FIG. 4C further presents a graphical representation of quantitation of vascular cell adhesion molecule 1 (VCAM1) normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control. FIG. 4D presents images of THP-1 monocyte binding to HUVECs exposed to LSS or OSS for 16 h with or without Tz treatment (N=3 independent experiments). FIG. 4D further presents a graphical representation of quantitation of THP1 monocytes per field per condition (THP1 binding index). FIG. 4E presents a schematic of PRC2-dependent gene expression regulation combined with hypothesis-driven analysis of Pcdhg-dependent genes to determine common enriched processes / pathways. FIG.

[0068] 4F presents a volcano plot from bulk RNAseq analysis of Control si and Suzl2 si HUVECs that was used to identify PRC2-regulated genes. FIG. 4G presents a heat map of the EC targets of the Notch pathway in Suzl2 si compared to Control si from the RNAseq analysis. FIG. 4H presents images related to inhibition of the Notch pathway with RIN1, a pharmacological inhibitor of Notch-RBPJ interaction (RBPJi) in HUVECs exposed to LSS for 16h, with or without PRC2 inhibition, and immunoblotted for proteins, as indicated (N=3 independent experiments). FIG. 4H further presents graphical representations of quantitation of Klf2 and Klf4 normalized to GAPDH loading control.

[0069] FIG. 5A-FIG. 5F present schematics and results related to interventional Tazemetostat (Tz) treatment slowing of ASCVD progression in mice. FIG. 5A presents a schematic representation of ASCVD intervention by oral administration of Tazemetostat (Tz) in hypercholesterolemic Apoe- / - mouse model. 8-10 week-old Apoe- / - mice that were maintained on High Fat Diet (HFD) for 12 w eeks, switched to chow diet containing vehicle or Tz for an additional 6 weeks, starved overnight, and analyzed for vessel histology. FIG. 5B presents images of whole aortas that were stained with Oil Red O to mark lipid-rich regions and imaged en face (N=4 animals). FIG. 5B further presents a graphical representation of quantitation of percent Oil Red O positive area. FIG. 5C and FIG. 5D present images of sections from aortic roots (FIG. 5C) and brachiocephalic artery (BCA) (FIG. 5D) that were stained with Oil Red O (N=3 animals). FIG. 5C and FIG. 5D further present graphical representations of quantitation of atherosclerotic plaque area (per animal), necrotic core (NC) area (per plaque) and fibrous cap (FC) thickness (per plaque). FIG. 5E and FIG. 5F present images of atherosclerotic plaques in aortic root sections (FIG. 5E) and BCA sections (FIG.

[0070] 5F) that were stained for the macrophage / monocyte marker CD68, for alpha smooth muscle actin (SMA) to mark smooth muscle cells (SMCs) and with DAPI to stain nuclei (N=3 animals). FIG. 5E and FIG. 5F further present graphical representations of quantitation of 6

[0071] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0072] CD68 mean fluorescence intensity (MFI).

[0073] FIG. 6A-FIG. 61 present schematics and results related to analysis of PRC2 in endothelial responses to flow. FIG. 6A presents a schematic representation of bulk RNAseq analysis of HUVECs exposed to LSS or OSS. FIG. 6B-FIG. 6C present EnrichR analysis that was used to identify enriched pathways / processes (FIG. 6B) and common upstream regulators of the DEGs (FIG. 6C) between LSS and OSS. FIG. 6D presents a schematic representation of bulk RNAseq analysis of Control si, Pcdhg si and Pcdhg+Klf2 / 4 triple si HUVECs that was used to identify' Pcdhg-dependent but Klf2 / 4 independent DEGs. FIG. 6E presents EnrichR analysis that was used to identify common upstream regulators of the DEGs. FIG. 6F presents a schematic representation of PRC2 showing components and function. FIG. 6G presents a graphical representation of H3K27me3 ChlP-qPCR at the Klf2 promoter. Depletion of PRC2 by Suzl2 si was used as an internal control to confirm PRC2 specificity of H3K27me3 (N=3 independent experiments). FIG. 6H-FIG. 61 present graphical representations of H3K27me3 and H3K27ac ChlP-qPCR analy sis of Control si and Pcdhg si HUVECs on the Klf2 promoter (N=3 independent experiments). Values are means ± SEM. Statistical analysis used one-way ANOVA (FIG. 6H) or unpaired two-tailed Student’s t-test (FIG. 61).

[0074] FIG. 7A-FIG. 7F present data and results related to RNAseq and ChlPseq analysis. FIG. 7A presents a Volcano plot from bulk RNAseq of HUVECs exposed to LSS vs OSS that shows shear-regulated genes. FIG. 7B presents X2KWeb analysis that was used to identify common upstream regulators of DEGs from bulk RNAseq analysis of Control si, Pcdhg si and Pcdhg+Klf2 / 4 triple si HUVECs. Red nodes represent transcription factors while gray nodes represent co-expressed DEGs. The size of the node represents centrality (number of connections with other nodes) in the network. FIG. 7C presents graphical representations of mRNA levels of Ezh2 and Suzl2 after Pcdhg depletion. FIG. 7D presents an immunoblot for Ezh2 and Klf4 in Control or Pcdhg si HUVECs under laminar shear stress (LSS) (N=3 independent experiments). The graph presents quantitation of Ezh2 and Klf4 normalized to GAPDH loading control. FIG. 7E presents a graphical representation of a Klf2 promoter region showing peaks for H3K27ac (marking promoter) and H3K27me3 that was obtained from available ChlPseq analysis on HUVECs (UCSC Genome Browser). FIG. 7F presents H3K27me3 ChlPseq analysis on Control si and Pcdhg si HUVECs, which shows a significantly higher peak call on K1I2 promoter (N=3 independent experiments). Values are means ± SEM. Statistical analysis used unpaired two-tailed Student's t-test (FIG. 7D).

[0075] FIG. 8A-FIG. 8G present schematics, data, and results related to PRC2 activity

[0076] 7

[0077] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0078] suppressing Klf2 / 4in ASCVD. FIG. 8A presents analysis of an aortic arch region prepared en face and immunostained for H3K27me3 as a marker for PRC2 activity. Greater curvature (GC) is under LSS, lesser curvature (LC) is under DSS as shown in the schematic. Graph: quantitation of H3K27me3 MFI (N=3 animals). FIG. 8B presents analysis of de-identified human coronary7artery sections from elderly donors stained with H&E (left) and H3K27me3 levels (right), comparing regions of plaque (Plaque region) to the regions of the same artery without evident plaque (Normal region). Sections were counter stained with Erg to mark ECs (N=3 donors). Graph: quantitation of HK27me3 signal intensity in ECs. FIG. 8C presents analysis of artery7sections from deidentified human CVD patients containing atherosclerotic plaques and non-diseased controls stained with H3K27me3. Sections were counter stained with Erg to mark ECs (N=8 donors). Graph: quantitation of H3K27me3 staining intensity in ECs. FIG. 8D presents analysis of carotids from mouse Partial Carotid Artery' (PCA) Ligation model of accelerated atherosclerosis stained for H3K27me3. Sections were counter stained with DAPI to mark nuclei (N=3 animals). RCA: Right Carotid Artery (control), LCA: Left Carotid Artery (plaque). Graph: quantitation of H3K27me3 staining intensity. FIG. 8E presents re-analysis of Ezh2, Suzl2 and Pcdhg in ECs from published scRNAseq data from deidentified human ASCVD patients comparing regions of plaque (Plaque) to an adjacent plaque-free region of the same artery7(Normal) (Alsaigh, T., et al., Commun Biol 5, 1084 (2022)). TNFa and K112 / 4 were used as internal controls for plaque ECs and normal ECs respectively (N=3 donors). FIG. 8F-FIG. 8G present analysis of depletion of PRC2 by Suzl2 si or Ezh2 si in HUVECs exposed to LSS (FIG. 8F) or OSS (FIG. 8G) and stained for Klf4 (N=3 independent experiments). Graph: quantitation of Klf4 staining intensity'. Values are means ± SEM. Statistical analysis used unpaired two-tailed Student’s t-test (FIG. 8B, FIG.

[0079] 8C. FIG. 8D) or one-way ANOVA (FIG. 8F, FIG. 8G). Scale bar: (FIG. 8A) 50 pm, (FIG.

[0080] 8B-FIG. 8D) 100 pm, (FIG. 8F, FIG. 8G) 20 pm.

[0081] FIG. 9A-FIG. 9D present data and results related to PRC2 association with CVD. FIG. 9A presents images of de-identified human coronary artery7sections from elderly donors stained with H3K27me3 and leukocyte common antigen CD45. comparing regions of plaque (Plaque region) to the regions of the same artery without evident plaque (Control region). Sections were counter stained with DAPI to mark nuclei (N=3 donors). FIG. 9B-FIG. 9D present re-analysis of Ezh2, Suzl2 and Pcdhg in major plaque cell types including ECs, immune cells and SMCs (FIG. 9B) from published scRNAseq data from deidentified human ASCVD patients comparing regions of plaque (Plaque) to an adjacent plaque-free region of the same artery' (Normal) (Alsaigh, T., et al., Commun Biol 5, 1084 (2022)). TNFa and Klf2 / 4

[0082] 8

[0083] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0084] used as internal controls for plaque ECs and normal ECs respectively (N=3 donors). FIG. 9C presents a feature plot showing inverse correlation between the expression of arterial marker Efnb2 with inflammatory markers Mmp2, Selp and Ccl2 in plaque ECs. FIG. 9D presents a heat map showing Efnb2 levels in Mmp2low(<0.5) vs Mmp2hlgh(>1.5) ECs from plaque.

[0085] FIG. 10A-FIG. 10C present data and results related to expression of Ezhl and Ezh2 in vascular cells. FIG. 10A and FIG. 10B presents graphical representations of analysis of mRNA levels of Ezhl (FIG. 10A) and Ezh2 (FIG. 10B) from human RNA atlas showing a comparison between ECs vs SMCs. Other relevant cell types shown, as indicated. FIG. 10C presents functional validation of Ezh2 si and Suzl2 si as confirmed by depletion of PRC2 by Suzl2 si or Ezh2 si in HUVECs exposed to OSS and stained for H3K27me3. Scale bar: (FIG.

[0086] 10C) 20 pm.

[0087] FIG. 11 A-FIG. 11H present schematics, data, and results related to PRC2-Notch-Mek5 axis regulation of Klf2 / 4. FIG. 11 A presents analysis of pharmacological inhibition of EZH2 catalytic activity with Tz in HUVECs under static (St) or LSS, immunoblotted for Klf4 and GAPDH loading control (N=3 independent experiments). Graph: quantitation of Klf4 normalized to GAPDH. FIG. 1 IB presents an immunoblot for VCAM1 in HUVECs, untreated or treated with II 1 P (1 ng / ml) for 16 h with or without Tz (N=3 independent experiments). Graph: quantitation of VC AMI normalized to GAPDH. FIG. 11C presents analysis of THP-1 monocyte binding to HUVECs under St or OSS for 16 h with or without Tz (N=3 independent experiments). Graph: quantitation of THP1 monocytes per field per condition (THP1 binding index). FIG. 1 ID presents a Venn diagram of common processes / pathways regulated by PRC2 and Pcdhg. FIG. 1 IE presents a volcano plot from bulk RNAseq analysis of Control si and Suzl2 si HUVECs that was used to identify PRC2-regulated genes. FIG. 1 IF presents a heat map of Notch pathway targets in ECs upon Suzl2 si compared to Control si from the RNAseq analysis. FIG. 11G presents analysis of Notch pathway that was blocked by pharmacological inhibition of Notch-RBPJ interaction wdth RIN1 (Notchi) in HUVECs under LSS for 16h, with or without EZH2 inhibition by Tz, and immunoblotted for proteins, as indicated (N=3 independent experiments). Graphs: quantitation of Klf2 and Klf4 normalized to GAPDH loading control. FIG. 11H presents a schematic showing the Mekk2 / 3-Mek5-Erk5-Mef2 pathway. The pathway was activated by expression of caMek5, w ith or without PRC2 inhibitor Tz or Notch inhibitor RIN1, and immunoblotted for proteins, as indicated (N=3 independent experiments). Graph: quantitation of Klf4 normalized to GAPDH loading control. Values are means ± SEM. Statistical analysis used one-way ANOVA (FIG. 11 A-FIG. 11C, FIG. 11G, FIG. 11H). Scale bar: (FIG. 11C)

[0088] 9

[0089] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0090] 100 pm.

[0091] FIG. 12A-FIG. 12G present schematics, data, and results related to interventional Tazemetostat treatment of mice with atherosclerosis. FIG. 12A presents a schematic representation of experimental design. 8-10-week-old Apoe ^ mice were fed a High Fat Diet (HFD) for 12 weeks, switched to chow diet containing vehicle or Tz for an additional 6 weeks, and vessels analyzed. FIG. 12B presents images and analysis of whole aortas that were stained with Oil Red O to mark lipid-rich regions and imaged en face (N=4 animals). Graph: quantitation of percent Oil Red O positive area. FIG. 12C and FIG. 12D present sections from aortic roots (FIG. 12C) and brachiocephalic artery (BCA) (FIG. 12D) that were stained with Oil Red O (N=3 animals). Graphs: quantitation of atherosclerotic plaque area (per animal), necrotic core (NC) area (per plaque) and fibrous cap (FC) thickness (per plaque). FIG. 12E and FIG. 12F present analysis of atherosclerotic plaques in aortic root sections (FIG. 12E) and BCA sections (FIG. 12F) that were stained for the macrophage / monocyte marker CD68, and counterstained with SMA and DAPI to mark SMCs and nuclei respectively (N=3 animals). Graph: quantitation of CD68 MFI. FIG. 12G presents analysis of aortic root sections that were stained for plaque vulnerability marker MMP9, and counterstained with CD31 and DAPI to mark ECs and nuclei respectively (N=4 animals). Graph: quantitation of MMP9 MFI. Values are means ± SEM. Statistical analysis used one-way ANOVA (FIG. 12B-FIG. 12D) or unpaired two-tailed Student’s t-test (FIG. 12E-FIG. 12G). Scale bar: (FIG. 12B) 500 pm, (FIG. 12C, FIG. 12D) 100 pm, (FIG. 12E-FIG. 12G) 200 pm.

[0092] FIG. 13A-FIG. 13C present data and results related to mouse weight and blood lipid analysis. FIG. 13A-FIG. 13C present body weights (FIG. 13 A), plasma triglycerides (TAGs) (FIG. 13B), and cholesterol content (FIG. 13C) of overnight starved mice treated as indicated (N = 5). Values are means ± SEM. Statistical analysis used one-way ANOVA.

[0093] FIG. 14A-FIG. 14E demonstrate that PRC2 inhibition by Tz decreases hypoxia-induced PAH in rodents, in accordance with some embodiments. FIG. 14A: Lung sections from mice in hypoxia (10% O2) for 21 days to induce pulmonary arterial hypertension (PAH) or in normoxia (control). Sections were stained for H3K27me3 and quantified in FIG. 14B. Graph shows MFI for H3K27me3 staining (N=4 animals). FIG. 14B: Experimental design for Tz treatment in experimental PAH. FIGs. 14D-14E: 8-10-week-old C57B1 / 6 mice were maintained in normoxia (Nx) or hypoxia (Hx) for 21 days, fed a diet containing vehicle, Tz, or Sildenaphil (Silden). At this time, the present study measured (FIG. 14D) right ventricular systolic pressure RVSP and (FIG. 14E) Fulton index [Right ventricle w-eight / (Left ventricle +

[0094] 10

[0095] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0096] septum) weight]. N=4-6 animals. Values are means ± SEM. Statistical analysis used one-way ANOVA (FIGs. 14D & 14E) or unpaired two-tailed Student’s t-test (FIG. 14B).

[0097] FIG. 15A-FIG. 15E demonstrate elevated PRC2 activity (H3K27me3) in hindlimb ischemia as a model for PAD, in accordance with some embodiments. Mice were subject to ligation of the femoral artery to induce hindlimb ischemia (HLI) as a model for peripheral artery disease (PAD). At subsequent times, blood flow in the foot was measured using laser doppler imaging or mice were sacrificed and tissue examined. FIG. 15 A: Tibialis Anterior muscle sections from ischemic (IT A) vs non-ischemic (NTA) limbs in C57BL / 6 mice 3 days after bilateral ligation and excision, stained for H3K27me3. Sections were counterstained with DAPI to mark nuclei and CD31 to mark endothelial cells. FIGs. 15B-15E: Graphs: quantitation of the number of H3K27me3 positive, CD31 positive, H3K27me3 and CD31 double positive, and % double positive of CD31 positive cells per field (N=4 animals).

[0098] Values are means ± SEM. Statistical analysis used unpaired two-tailed Student’s t-test (FIGs.

[0099] 15B-15E). Scale bar: (FIG. 15A) 50 pm.

[0100] FIG. 16A-FIG. 16D demonstrate that Tz improves recovery from HLI in diabetic mice, in accordance with some embodiments. FIG. 16A: Experimental design for use of Tz as a treatment from HLI as a model for diabetic PAD. 8-10-week-old C57B1 / 6 mice were maintained on 60% HFD (High Fat Diet) for 8 weeks to induce ty pe II diabetes, femoral artery ligation surgery performed, maintaining the 60% HFD containing vehicle or Tz for another 14 days. FIG. 16B: Longitudinal Laser Doppler imaging showing blood flow flux and progressive recovery in the limb with ligated (L) compared to the unligated (R) artery. FIG. 16C: Graph: quantitation of % blood flow in ligated compared to the unligated internal control (N=5 animals). FIG. 16D: Graph: Weights at the end of the experiment (N=5 females and 3 males). Values are means ± SEM. Statistical analysis used unpaired two-tailed Student’s t-test (FIGs. 16C-16D).

[0101] FIG. 17A-FIG. 17E demonstrate that Tz ameliorates endothelial activation in experimental lung injury from LPS, in accordance with some embodiments. FIG. 17A:

[0102] Schematic of interventional protocol in which mice are injected with LPS to induce acute lung injury, then after 8h treated with Tz. FIGs. 17B-17C: Lung sections from animals at 24h after LPS injection (16h Tz treatment) stained with H&E (FIG. 17B) to assess tissue morphology or for CD68 (FIG. 17C) to assess inflammation marked by infiltrated immune cells (7 lungs fromN=4 animals). Graph: quantitation of CD68+ cells per field of view. FIGs.

[0103] 17D-17E: Assessment of endothelial inflammation upon LPS injection, with or without Tz injection at 8h. Graphs: quantitation of endothelial Vcaml and nuclear Klf4 MFI (N=3

[0104] 11

[0105] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0106] animals). Values are means ± SEM. Statistical analysis used unpaired two-tailed Student’s t-test (FIGs. 17C-17E). Scale bar: (FIGs. 17B-17E) 100 pm.

[0107] FIG. 18A-FIG. 18B demonstrate that Tz inhibits LPS-induced endothelial permeability, in accordance with some embodiments. FIG. 18A: Interventional Tz treatment of HUVECs cultured on biotinylated fibronectin (Biotin FN) 8 hours with or without LPS, probed with FITC-avidin to measure the extent and localization of permeability. Graph: quantitation of exposed FN MFI (N=8 fields from 4 experiments). FIG. 18B: HUVECs were treated with the indicated inflammatory mediators (LPS, Il 1 or TNFoc) for 8 hours, then Tz added and followed for an additional 16h. Trans-Endothelial Electrical Resistance (TEER) as an indicator of junctional integrity was followed over this time course. Values are means ± SEM. Statistical analysis used one-way ANOVA (FIG. 18A). Tz treatment addition accelerates recovery of barrier function.

[0108] DETAILED DESCRIPTION

[0109] DEFINITIONS

[0110] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0111] Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and nucleic acid hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzy matic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical

[0112] 12

[0113] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0114] chemi stry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0115] Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Cunent Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory' Manual (Fourth Edition) by MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0116] That the disclosure may be more readily understood, select terms are defined below. Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3. from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 and so forth, as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0117] As used herein, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. By way of example, “an element’' means one element or more than one element. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B."

[0118] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0119] As used herein, to “alleviate” a disease means reducing the severity' of one or more symptoms of the disease.

[0120] As used herein, the term “cardiovascular disease” is intended to refer to any’ pathological state(s) leading to a narrowing and / or occlusion of blood vessels in the body.

[0121] 13

[0122] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0123] The cardiovascular diseases include any diseases where the patients suffering from the cardiovascular disease is at risk of having a heart attack. As such, the term cardiovascular disease encompasses, for example, ischemic heart disease, congestive heart failure, hypertension, valvular heart disease, general atherosclerosis, hypercholesterolemia, atherosclerosis, and thrombosis. The term further encompasses atherosclerotic cardiovascular disease (ASCVD). Moreover, cardiovascular diseases include those diseases related to the cardiovascular disorders of fragile plaque disorder, occlusive disorder and stenosis. For example, a cardiovascular disease resulting from a fragile plaque disorder, can be termed a fragile plaque disease. Clinical events associated with fragile plaque disease include those signs and symptoms where the ruptures of a fragile plaque with subsequent acute thrombosis or with distal embolization are hallmarks. Examples of fragile plaque disease include certain strokes and myocardial infarctions. As another example, a cardiovascular disease resulting from an occlusive disorder can be termed an occlusive disease. Clinical events associated with occlusive disease include those signs and symptoms where the progressive occlusion of an artery affects the amount of circulation that reaches a target tissue. Progressive arterial occlusion may result in progressive ischemia that may ultimately progress to tissue death if the amount of circulation is insufficient to maintain the tissues. Signs and symptoms of occlusive disease include claudication, rest pain, angina, and gangrene, as well as physical and laboratory findings indicative of vessel stenosis and decreased distal perfusion. As yet another example, a cardiovascular disease resulting from restenosis can be termed an in-stent stenosis disease. In-stent stenosis disease includes the signs and symptoms resulting from the progressive blockage of an arterial stent that has been positioned as part of a procedure like a percutaneous transluminal angioplasty, where the presence of the stent is intended to help hold the vessel in its newly expanded configuration. The clinical events that accompany instent stenosis disease are those attributable to the restenosis of the reconstructed artery.

[0124] In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0125] As used herein, the terms "effective amount” and “therapeutically effective amount” are used interchangeably and refer to the amount required to reduce or improve at least one 14

[0126] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0127] symptom or change in a clinical marker of a disease relative to an untreated patient. The effective amount of the treatment used for therapeutic treatment of the disease varies depending upon the manner of the specific disorder, condition or disease, extent of the disorder, condition or disease, and administration of the cells, as well as the age, body weight, and general health of the subject. The effective amount is capable of achieving a particular desired biological result and / or provides a therapeutic or prophylactic benefit.

[0128] ■’Parenteral’’ administration of an immunogenic composition includes, e.g., subcutaneous (s.c ), intravenous (i.v ), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

[0129] The terms “patient"’, “subject”, and “individual” are used interchangeably and are intended to include living organisms that may be subjected to treatment for a given disease, e.g., mammals. A “subject”, "‘patient”, or “individual”, as used herein, can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals, as well as simian and non-human primate mammals. Preferably, the subject is human.

[0130] As used herein, the terms “peptide,” “polypeptide.” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0131] As used herein, the term “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with other chemical components, such as carriers, stabilizers, diluents, adjuvants, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary 15

[0132] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0133] and topical administration.

[0134] The language “pharmaceutically acceptable carrier” includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene 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;

[0135] Ringer’s solution; ethyl alcohol; phosphate buffer solutions; diluent; granulating agent; lubricant; binder; disintegrating agent; wetting agent; emulsifier; coloring agent; release agent; coating agent; sweetening agent; flavoring agent; perfuming agent; preservative; antioxidant; plasticizer; gelling agent; thickener; hardener; setting agent; suspending agent; surfactant; humectant; carrier; stabilizer; and other non-toxic compatible substances employed in pharmaceutical formulations, or any combination thereof. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.

[0136] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by any degree of suppression, remission, or eradication of a disease state.

[0137] To “treat” a disease as the term is used herein, means to reduce the frequency or severity' of at least one sign or sy mptom of a disease or disorder experienced by a subject.

[0138] METHODS OF TREATING, AMELIORATING, AND / OR PREVENTING

[0139] 16

[0140] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0141] VASCULAR INFLAMMATION, VASCULAR INFLAMMATORY DISEASES, AND CARDIOVASCULAR DISEASES

[0142] As described above, vascular inflammation is a primary cause of coronary, peripheral, and cerebral artery disease, pulmonary arterial hypertension, lung and kidney injury and intravascular coagulation due to sepsis, diabetic vasculopathy, transplant rejection and many others. In spite of the availability of potent anti-inflammatory therapies, such therapies generally limit immune defense against pathogens and increase risk of serious infections. As such, targeting the vascular endothelium offers a specific means to limit vascular inflammation without compromising immune defense and thus safely and effectively treat the wide range of diseases where vascular inflammation plays a key role.

[0143] To this end, the present disclosure describes identification of polycomb repressor complex 2 (PRC2) as a suppressor of Kruppel-like factor (Klf) 2 and 4 expression in vascular endothelial cells (ECs). Klf2 / 4 are the major protective transcription factors in ECs that mediate expression of nearly 70% of the anti-inflammatory, anti-thrombotic genes that maintain vascular health. Low Klf2 / 4 is associated with multiple cardiovascular diseases including coronary artery disease, peripheral artery disease, cerebral artery and small vessel disease and pulmonary hypertension among others. PRC2 mediates epigenetic suppression of gene expression via its catalytic subunit EZH2, which methylates histone 3 on lysine 27 to create H3K27me3 marks that promote chromatin condensation to block transcription. As described herein. PRC2-dependent H3K27 methylation increases in inflamed vasculature and blocking by knockdown of PRC2 subunits or small molecule inhibition of EZH2 with an EZH2 inhibitor, such as tazemetostat (Tz), increases Klf2 / 4 levels and suppresses inflammatory genes in vitro. As described herein, Tz was tested in a hyperlipidemic mouse model using a therapeutic protocol in which mice on high fat diet with established atherosclerosis were given Tz and examined 6 weeks later. Surprisingly, Tz reduced the rate of plaque expansion by 50% but more crucially and unexpectedly resulted in a drastic shift in plaque phenotype, with ~2x thicker fibrous caps despite the overall smaller plaque size and decreased necrotic core size by -80%. As described herein, Tz treatment resulted in a shift toward stable plaques that are less vulnerable to rupture. Notably. Tz is an approved drug with an excellent safety profile, currently used for treatment of hemopoietic cancers.

[0144] As described herein, PRC2 inhibition by Tazemetostat can be used as a therapeutic approach for treating inflammatory' disorders. For instance, inflammation is an essential element of ASCVD and other inflammatory diseases, but human clinical trials have found the benefits from immune suppression are offset by increased deaths from infections. Targeting 17

[0145] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0146] endothelial inflammatory activation offers hope of limiting vascular inflammation without compromising host defense.

[0147] As such, in one aspect, the present disclosure generally relates to methods of treating, ameliorating, and / or preventing a vascular inflammatory disease and / or vascular inflammation in a subject in need thereof. In one aspect, the disclosure generally relates to a method of treating, ameliorating, and / or preventing vascular inflammation and / or a vascular inflammatory disease in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor. In one aspect, the disclosure generally relates to a method of treating, ameliorating, and / or preventing a cardiovascular disease in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor. In one aspect, the present disclosure generally relates to a method of treating, ameliorating, and / or preventing atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor.

[0148] Moreover, in one aspect, the disclosure generally relates to a method of treating, ameliorating, and / or preventing a vascular inflammatory disease and / or vascular inflammation in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor, wherein the EZH2 inhibitor comprises tazemetostat. In one aspect, the present disclosure generally relates to a method of treating, ameliorating, and / or preventing a cardiovascular disease in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor, wherein the EZH2 inhibitor comprises tazemetostat. In one aspect, the present disclosure generally relates to a method of treating, ameliorating, and / or preventing atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor, wherein the EZH2 inhibitor comprises tazemetostat. In one aspect, the present disclosure generally relates to a method of treating, ameliorating, and / or preventing vascular inflammation in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor, wherein the EZH2 inhibitor comprises tazemetostat.

[0149] EZH2 Inhibitors

[0150] In some aspects, the EZH2 inhibitor is selected from the group consisting of PF-06821497 (5,8-dichloro-2-[(4-methoxy-6-methyl-2-oxo-lH-pyridin-3-yl)methyl]-7-[(R)-methoxy(oxetan-3-yl)methyl]-3,4-dihydroisoquinolin-l-one); UNCI 999 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-py ridiny Ijmethy 1] - 1 -( 1 -methy 1 ethy 1 ) - 6- [ 6- [4-( 1 -methylethyl)- 1 -piperazinyl]-3-pyridinyl]-lH-indazole-4-carboxamide); CPI-1205 (Lirametostat) (N-[(4- 18

[0151] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0152] methoxy-6-methyl-2-oxo-lH-pyridin-3-yl)methyl]-2-methyl-l-[(lR)-l-[l -(2,2,2- trifluoroethyl)piperidin-4-yl]ethyl]indole-3-carboxamide); DS-3201b (Valemetostat) ((2R)-7- chloro-2-[4-(dimethylamino)cyclohexyl]-N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]- 2,4-dimethyl-l,3-benzodioxole-5-carboxamide); tazemetostat (EPZ-6438) (N-[(4,6-dimethyl- 2-oxo-lH-pyridin-3-yl)methyl]-3-[ethyl(oxan-4-yl)amino]-2-methyl-5-[4-(morpholin-4- ylmethyl)phenyl]benzamide); 3-Deazaneplanocin A; GSK 126 (N-[(l,2-Dihydro-4,6- dimethy l-2-oxo-3 -pyridinyl)methy 1] -3-methyl- 1 -[( 1 S)- 1 -methylpropyl] -6- [6-( 1 -piperazinyl)- 3-pyridinyl]-lH-indole-4-carboxamide); GSK 343 (N-[(l,2-Dihydro-6-methyl-2-oxo-4- propy 1-3-py ridinyl)methyl] - 1 -( 1 -methy lethyl)-6- [2-(4-methyl- 1 -piperaziny l)-4-pyridiny 1] - lH-indazole-4-carboxamide); JQEZ5 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3- pyridinyl)methyl]-l-(l-methylethyl)-6-[6-(4-methyl-l-piperazinyl)-3-pyridinyl]-lH- pyrazolo[3,4-b]pyridine-4-carboxamide); PF 06726304 acetate (5,8-Dichloro-2-[(l,2- dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-7-(3,5-dimethyl-4-isoxazolyl)-3,4-dihydro- l(2H)-isoquinolinone acetate); ZLD-1039 (3-[Ethyl(tetrahydro-2H-pyran-4-yl)amino]-N- [(2.3.5.6.7,8-hexahydro-l-methyl-3-oxo-4-isoquinolinyl)methyl]-2-methyl-5-[6-(4-methyl-l- piperazinyl)-3-pyridinyl]benzamide); and combinations thereof. In some aspects, the EZH2 inhibitor comprises or is tazemetostat.

[0153] In some aspects, the EZH2 inhibitor is UNCI 999 having the following structure:

[0154]

[0155] In some aspects, the EZH2 inhibitor is Lirametostat having the following structure:

[0156] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0157]

[0158] In some aspects, the EZH2 inhibitor is Valemetostat having the following structure:

[0159]

[0160] In some aspects, the EZH2 inhibitor is Tazemetostat having the following structure:

[0161]

[0162] In some aspects, the EZH2 inhibitor is 3-Deazaneplanocin A having the following structure:

[0163] ""

[0164]

[0165] 20

[0166] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0167] In some aspects, the EZH2 inhibitor is GSK343 having the following structure:

[0168]

[0169] In some aspects, the EZH2 inhibitor is PF 06726304 acetate having the following

[0170] structure:

[0171]

[0172] In some aspects, the EZH2 inhibitor is ZLD1039 having the following structure:

[0173]

[0174] 21

[0175] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0176] Vascular Inflammatory Disease, Vascular Inflammation, and Treatment Thereof In one aspect, the disclosure generally relates to a method of treating, ameliorating, and / or preventing a vascular inflammatory disease and / or vascular inflammation in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor. In one aspect, the present disclosure generally relates to a method of treating, ameliorating, and / or preventing vascular inflammation in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor. In one aspect, the present disclosure generally relates to a method of treating, ameliorating, and / or preventing vascular inflammation in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor, wherein the EZH2 inhibitor comprises tazemetostat. In one aspect, the present disclosure relates to a method of treating, ameliorating, and / or preventing a vascular inflammatory disease and / or vascular inflammation in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor, wherein the EZH2 inhibitor comprises tazemetostat. In some aspects, the vascular inflammatory disease is a cardiovascular disease. In some aspects, the cardiovascular disease is atherosclerosis..

[0177] In some aspects, treatment or amelioration of the subject decreases and / or reverses progression of the vascular inflammation and / or vascular inflammatory' disease, e.g., atherosclerosis. In some aspects, the vascular inflammation and / or vascular inflammatory disease comprises plaque formation in the subject. In some aspects, the plaque is an atherosclerotic plaque. In some aspects, treatment or amelioration of the subject reduces the rate of plaque progression in the subject. In some aspects, treatment or amelioration of the subject increases the amount of stable plaque as compared to the amount of unstable plaque in the subject. In some aspects, treatment or amelioration of the subject increases the thickness of fibrous plaque caps in the subject. In some aspects, treatment or amelioration of the subject decreases the size of the necrotic core of plaque in the subject.

[0178] Cardiovascular Diseases and Treatment Thereof

[0179] In one aspect, the present disclosure generally relates to a method of treating, ameliorating, and / or preventing a cardiovascular disease in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor. In one aspect, the present disclosure generally relates to a method of treating, ameliorating, and / or preventing a cardiovascular disease in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor, wherein the EZH2 inhibitor 22

[0180] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0181] comprises tazemetostat.

[0182] In some aspects, the cardiovascular disease is ischemic heart disease, congestive heart failure, hypertension, valvular heart disease, general atherosclerosis, atherosclerosis, atherosclerotic cardiovascular disease (ASCVD), thrombosis, or any combination thereof. In some aspects, the cardiovascular disease cardiovascular is a disease related to the cardiovascular disorders of fragile plaque disorder, occlusive disorder and stenosis. In some aspects, the cardiovascular disease is atherosclerosis or ASCVD.

[0183] In some aspects, treatment or amelioration of the subject decreases and / or reverses progression of the atherosclerosis or ASCVD. In some aspects, the cardiovascular disease comprises plaque formation in the subject. In some aspects, the plaque is an atherosclerotic plaque. In some aspects, treatment or amelioration of the subject reduces the rate of plaque progression in the subject. In some aspects, treatment or amelioration of the subject increases the amount of stable plaque as compared to the amount of unstable plaque in the subject. In some aspects, treatment or amelioration of the subject increases the thickness of fibrous plaque caps in the subject. In some aspects, treatment or amelioration of the subject decreases the size of the necrotic core of plaque in the subject.

[0184] In one aspect, the present disclosure generally relates to a method of treating, ameliorating, and / or preventing atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor. In one aspect, the present disclosure generally relates to a method of treating, ameliorating, and / or preventing atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor, wherein the EZH2 inhibitor comprises tazemetostat. In some aspects, the ASCVD comprises plaque formation in the subject. In some aspects, treatment or amelioration of the subject reduces the rate of plaque progression in the subject. In some aspects, treatment or amelioration of the subject increases the amount of stable plaque as compared to the amount of unstable plaque in the subject. In some aspects, treatment or amelioration of the subject increases the thickness of fibrous plaque caps in the subject. In some aspects, treatment or amelioration of the subject decreases the size of the necrotic core of plaque in the subject.

[0185] Pharmaceutical Compositions / Administration / Dosage / Formulations

[0186] Also provided herein are compositions comprising an EZH2 inhibitor, such as Tz, for the treatment of any of the diseases, disorders, or conditions described herein. Among the compositions are pharmaceutical compositions and formulations for administration.

[0187] 23

[0188] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0189] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after the onset of a disease or disorder contemplated herein. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0190] Administration of the compositions described herein to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat, prevent and / or ameliorate an inflammatory disorder in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat, prevent and / or ameliorate an inflammatory disorder in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound described herein is from about 1 and 5,000 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0191] Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0192] In particular, the selected dosage level depends upon a variety' of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.

[0193] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds 24

[0194] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0195] described herein employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0196] In some aspects, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity7of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated: each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the compound(s) described herein are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound.

[0197] In some aspects, the compositions described herein are formulated using one or more pharmaceutically acceptable excipients or carriers. In some aspects, the pharmaceutical compositions described herein comprise a therapeutically effective amount of a compound described herein and a pharmaceutically acceptable earner.

[0198] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or poly alcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0199] In some aspects, the compositions described herein are administered to the patient in dosages that range from one to five times per day or more. In some aspects, the compositions described herein are administered to the patient in range of dosages that include, but are not limited to, once every day, every7two, days, every7three days to once a week, and once every7two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions described herein varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to 25

[0200] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0201] be treated, gender, overall health, and other factors. Thus, administration of the compounds and compositions described herein should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physician taking all other factors about the patient into account.

[0202] The compound(s) described herein for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 350 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg. about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.

[0203] In some aspects, the dose of a compound described herein is from about 1 mg and about 2,500 mg. In some aspects, a dose of a compound described herein used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some aspects, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg. or less than about 10 mg, or less than about 5 mg. or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0204] In some aspects, a composition as described herein is a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound described herein, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, ameliorate, and / or reduce one or more symptoms of an inflammatory disorder in a patient.

[0205] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of

[0206] 26

[0207] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0208] administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

[0209] Routes of administration of any of the compositions described herein include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the compositions described herein can be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g, trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0210] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions described herein are not limited to the particular formulations and compositions that are described herein.

[0211] EXAMPLES

[0212] The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0213] Materials and Methods (Examples 1-6)

[0214] Primary cells, cell lines, and cell culture reagents

[0215] The PyMT-immortalized Mouse Aortic ECs that express the Klf2 promoter reporter (A / 2:GFP MAEC) were described previously (Coon, B. G. et al., J Cell Biol 221, (2022)).

[0216] 27

[0217] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0218] MAECs were maintained in complete EC Medium (Cell Biologies, Ml 166) (Ni, C.W. et al., Vase Cell 6, 7 (2014)). HUVECs obtained from Yale Vascular Biology and Therapeutics Core were pooled from 3 donors. They were screened for the absence of pathogens, maintained in EGM2 Endothelial Cell Growth Medium (Lonza, CC-3162) and used at passages 2-5. All cells were routinely screened for the absence of mycoplasma. SiRNA transfection was performed with Lipofectamine RNAiMAX (ThermoFisher, 13778150) in Opti-MEM medium (ThermoFisher, 31985070) using ON-TARGET plus SMARTpool siRNAs from Horizon Discovery (Dharmacon). For NICD transcriptional activity reporting, 12xCSL / RBPJ-dlEGFP (Addgene, #47684) was used. For lentiviral transduction, HEK293T cells were transfected with lentiviral vectors along with pVSV-G (Addgene, #138479) and psPAX2 (Addgene, #12260) packaging plasmids using lipofectamine 2000 (ThermoFisher, 11668019) following the manufacturer’s instructions. Supernatants were collected 48-96 h after transfection and filtered through a 0.45 pm low-protein binding filter. Primary HUVECs were infected with lentivirus for 24h, then the medium replaced with EMG2 medium. For monocyte adhesion assays, THP-1 labeled with CellTracker Deep Red (ThermoFisher.

[0219] C34565) were resuspended in HBSS supplemented with 1 mM Ca2+, 0.5 mM Mg2+, and 0.5% BSA, added to the slides with HUVECs, incubated for 20 min at 37°C, washed 3x in HBSS and fixed with 3.7% formaldehyde. Cells were counterstained with DAPI before fluorescence imaging. Drugs used were Tazemetostat (Selleckchem), TNFa (PeproTech, 300-01A) and RIN1 (RBPJ Inhibitor-1) (Selleckchem. SS3376). All antibodies were validated in knockdown / knockout depletion by immunofluorescence and / or immunoblotting.

[0220] Shear stress stimulation

[0221] All shear stress experiments were performed in parallel plate flow chambers and perfused within a pump and environmental control system as described (Conway, D. E. et al., Curr Biol 27, 2727, (2017)). Briefly, cells (Conway, 2017 #90) were seeded at 70-90% confluency on 10 pg / ml Fibronectin-coated glass slides for 48-72 h in complete medium. For shear stimulation, slides were mounted in custom-made 25x55 mm parallel plate shear chambers with 0.5 mm thick silicone gaskets and stimulated with 15 dynes / cm2for LSS or 1 ± 4 dynes / cm2for OSS in complete media. The media was maintained at 37°C and 5% CO2 with a heat gun and humidified bubbler, respectively.

[0222] Animals and tissue preparation

[0223] Mice were maintained in a light-controlled and temperature-controlled environment with free access to food and water and all efforts were made to minimize animal suffering. Pcdhghcon3mice (Lefebvre, J. L. et al. Development 135, 4141-4151 (2008); Prasad, T. et al.,

[0224] 28

[0225] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0226] Development 135, 4153-4164 (2008))and Cdh5Cre mice (Alva, J. A. et al., Dev Dyn 235, 759-767 (2006))are described elsewhere. Pcdhgfcon3mice were a kind gift from Julie Lefebvre, University of Toronto, Canada. All mice in this study were on the C57BL / 6J background. Pcdh^C0n3and Cdh5Cre mice were maintained and bred as heterozygotes.

[0227] Euthanasia was performed by an overdose of isoflurane inhalation and death was confirmed by subsequent cervical dislocation and / or removing vital organs and / or opening the chest cavity venfied by the absence of cardiovascular function. Mice were perfused through the left ventricle with PBS and then 3.7% formaldehyde followed by tissue collection, as described. The heart and spinal column, with aorta and carotids attached, were removed, and fixed under gentle agitation for an additional 24 h at 4 °C, washed 3x with PBS and taken for further. For whole aorta en face prep, the isolated aortas were bisected along the lesser curvature and the aortic arch was bisected through the greater curvature as well. For aortic arch segment prep, the aortic arch was bisected through the greater curvature. Hearts (containing aortic roots) and carotids were allowed to sink in 30% sucrose in PBS overnight at 4°C, embedded in OCT compound (optimal cutting temperature compound, Sakura. 4583) and frozen on dry ice for sectioning. Tissue blocks were cut into 8-10 pm sections using a cryostat (Leica) and sections were stored at -80°C until use.

[0228] Atherosclerosis progression and blood lipid analysis

[0229] To induce atherosclerosis, Apoe '~ mice were maintained on a high-fat diet (HFD; Clinton / Cybulsky high-fat rodent diet with regular casein and 1.25% added cholesterol; Research Diet, DI 2108c) for 12 weeks. Blood samples were collected from overnight starved mice, centrifuged at 8,000g at 4°C for 10 min and the supernatant (plasma) was separated and stored at -80 °C. Total plasma cholesterol and triglycerides (TAG) were measured using kits according to the manufacturer’s instructions (Wako Pure Chemicals, Japan). Tissues were prepared as described above and analyzed as described below.

[0230] Tissue analysis

[0231] For IF, OCT tissue sections were thawed and washed 3x with PBS to remove OCT. Cells were fixed with 3.7% formaldehyde for 15 min at ambient temperature, washed with and stored in PBS. Deidentified human specimens were deparaffinized in Histo-Clear (National Diagnostics, HS-200). Sections were progressively rehydrated before antigen retrieval for 30 min at 95°C in IX Antigen Retrieval Buffer (Dako, 51699). Samples were incubated in perm-block buffer (5% donkey serum, 0.2% BSA, 0.3% Triton X-100 in PBS) for 1 h at room temperature, incubated with primary antibodies in perm-block overnight at 4°C, washed three times in perm-block and then incubated with alexafluor-conjugated 29

[0232] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0233] secondary antibodies (ThermoFisher) at 1:1000 dilution in perm-block for 1 h at room temperature. Slides were washed 3x in perm-block and 3x in PBS before mounting in DAPI Fluoromount G (Southern Biotech; 0100-20). Images were acquired on a Leica SP8 confocal microscope with the Leica Application Suite software. Confocal stacks were flattened by maximum intensity z-proj ection in ImageJ. Post background subtraction, the mean fluorescence intensity (MFI) or nuclear intensity (with DAPI mask) was recorded. For aorta ORO staining, the whole aorta was opened longitudinally on a soft-bottomed silica dish, incubated with ORO solution (0.6% ORO in 60% isopropanol) with gentle rocking for 1 h at ambient temperature, washed in 60% isopropanol for 20 minutes, washed in dH2O 3x, mounted on slides with endothelium side up in OCT compound. Images were acquired with a digital microscopic camera (Leica DFC295). ORO staining on OCT tissue sections was done similarly. Quantitation of ORO positive area was done in ImageJ. Hematoxylin and eosin (H&E) staining on OCT tissue sections was done by Yale Research Histology Core using standard techniques. Plaque morphometric and vulnerability' analysis was performed as described (Seimon, T. A. et al., J Clin Invest 119, 886-898 (2009)). Plaque area was determined by ORO positive staining. For each plaque, the necrotic core (NC) area was defined as a clear area in the plaque that was H&E free, and the fibrous cap (FC) thickness was quantified by selecting the largest necrotic core and measuring the thinnest part of the cap.

[0234] Partial Carotid Artery (PC A) Ligation model of accelerated experimental atherosclerosis

[0235] 8-10 week-old Apoe~ ' mice maintained on HFD for 1 week were anesthetized with ketamine and xylazine, and surgery' was performed as described (Budatha, M., et al., J Am Heart Assoc 10, e021160, doi: 10.1161 / JAHA.121.021160 (2021)). Briefly. 3 out of 4 branches of the left common carotid artery' (left external carotid, internal carotid, and occipital artery') were ligated with sutures, with superior thyroid artery left intact. 2 pg mAb A9 or isotype control antibody w as injected IP once every' week for 2 w eeks. Mice were euthanized with an overdose of isoflurane and perfused through the left ventricle with PBS and then 3.7% formaldehyde. Aortas with carotid arteries were isolated and imaged whole. Carotid arteries were embedded in OCT, sectioned at 10 pm, and IHC or immunofluorescence performed as described. LCA / RCA inner diameter ratio was calculated by measuring perimeter to avoid interference from changes in vessel morphology' during mounting and handling. For testing mAb retention in vivo (half-life), 1 pg of mAbs (100 pl of 0.25 mg / ml in saline for a ~20 g mouse) were injected IP in C57B1 / 6 mice, 100 pl blood was 30

[0236] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0237] collected via the retro-orbital route at the indicated times, centrifuged at 13,000g at 4°C for 15 min and the plasma removed and immediately stored at -80°C. mAh concentration was determined using a sandwich ELISA with immobilized anti-rat IgG (100 ng) as trap and secondary anti-rat IgGHRP antibody for detection, described elsewhere herein.

[0238] Immunoblotting

[0239] Cells were harvested and lysed in RIPA buffer (Roche) containing lx Halt Protease Inhibitor Cocktail (ThermoFisher, 78429) and lx PhosStop (Roche, 4906837001) for 30 min on ice, clarified at 13,000 g 4°C for 15 minutes, the supernatant transferred to new 1.5 ml tubes, 4x loading buffer (250mM Tris-HCl pH 6.8, 8% SDS, 40% glycerol, 20% P-mercaptoethanol, 0.008% bromophenol blue) added and the samples heated to 95°C for 5 min. Cell lysates were resolved by a 4-15% SDS Polyacrylamide gel electrophoresis, transferred to 0.2 pm nitrocellulose membranes which were blocked with 5% non-fat skim milk for Ih at ambient temperature and incubated with desired antibodies diluted in 5% BSA using a standard immunoblotting procedure and detection by ECL (Millipore). Images were quantified in ImageJ by densitometry and normalized to GAPDH or Tubulin loading controls.

[0240] RNA isolation, sequencing, and quantitative real-time PCR ( PCR)

[0241] Total RNA was extracted from cells with RNeasy Plus Mini Kit (Qiagen, 74136) according to the manufacturer's instructions. RNA was quantified by NanoDrop, and RNA integrity was measured with an Agilent Bioanalyzer. Samples were subjected to RNA sequencing using Illumina NovaSeq 6000 (HiSeq paired-end. 100 bp). The base calling data from the sequencer were transferred into FASTQ files using bcl2fastq2 conversion software (version 2.20, Illumina). PartekFlow (a start-to-finish software analysis solution for next generation sequencing data applications) was used to determine differentially expressed genes (DEGs). For qPCR analysis, reverse transcription was performed with iScript™ Reverse Transcription Supermix for RT-qPCR (Bio-Rad). qPCR was performed real-time PCR with SsoAdvanced Universal SYBR Green Supermix (Biorad). The expression of target genes w as normalized to GAPDH.

[0242] Quantification and Statistical Analysis

[0243] ImageJ software (version 1.51; National Institutes of Health, Bethesda, MD) was used for morphometric analysis. Graph preparation and statistical analysis was performed using GraphPad Prism 10.0 software (GraphPad softw are Inc.). Data w ere considered normally distributed, statistical significance was performed using Student t test for two groups comparison or one-way ANOVA with Tukey’s post hoc analysis for multiple groups comparison, as described in the figure legends. Data are presented as mean ± SEM. A p value 31

[0244] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0245] less than 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0246] Example 1: PRC2 mediates Klf2 / 4 inhibitory function of Pcdhg

[0247] Pcdhg has been indicated to be a suppressor of KH2 / 4. To assess common regulators of the genes and processes downstream of Pcdhg, an upstream regulator analysis was performed using EnrichR and X2KWeb tools, of Pcdhg si and Pcdhg si + K112 / 4 si RNAseq datasets (FIG. 1 A). This analysis identified Ezh2 and Suzl2, the components of the epigenetic transcription regulator, the Polycomb Repressive Complex (PRC) 2 as the most significant candidate mediators (FIG. IB). Of the 494 (311 up and 183 down) differentially expressed genes (DEGs) input for the analysis, 161 were identified as PRC2 targets, two-thirds (110 / 161) of which were upregulated while one-thirds (51 / 161) were downregulated, consistent with the transcriptional repression function of PRC2. Enrichment of the known inhibitors of Klf2 / 4 in ECs such as NFKB, and the PRC1 component RING1B further validated the analysis (FIG. IB). Interestingly, PRC2 component Jarid2 was also a candidate suppressor of Klf2 in the original genome-wide CRISPR screen. Analysis using X2KWeb, another tool for upstream regulator analysis, further supported the hypothesis that PRC2 is a central regulator of Pcdhg function. Most importantly, the transcripts of PRC2 components were unaffected, verifying it as a true regulator by ruling out the effect of a feedback loop (FIG. 1C). However. Ezh2 protein levels were significantly decreased with a concomitant increase in Klf2 / 4 in Pcdhg si, implying that Pcdhg maintains PRC2 activity or stability to repress its target genes, Klf2 and Klf4 (FIG. ID).

[0248] While several histone modifying complexes are known, PRC2 is the only known creator of the H3K27me3 repressive mark. ChlP-qPCR showed the presence of H3K27me3 mark on Klf2 promoter, which was absent in control IgG ChIP samples and ablated upon Suzl2 si, validating Klf2 / 4 as direct targets of PRC2 in ECs (FIG. IE). Analysis of H3K27me3 ChlPseq datasets in HUVECs from UCSC Genome Brow ser also show ed peaks for H3K27me3 mark on the promoters (marked by peak for H3K27ac) of K112 / 4. As expected, Pcdhg si led to a significantly higher peak of H3K27ac mark at Klf2 promoter in ChlP-seq analysis (FIG. IF). This was confirmed by ChlP-qPCR showing an increase in H3K27ac with concomitant decrease in H3K27me3 on Klf2 promoter (FIG. 1G, FIG. 1H). From these data, it was concluded that Pcdhg suppresses K112 / 4 via direct epigenetic repression mediated by PRC2. Since PRC2 directly suppressed K112 / 4 level, further analysis involved understanding this regulation and its relevance.

[0249] 32

[0250] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0251] Example 2: PRC2 suppresses protective EC hemodynamic response

[0252] Klf2 / 4 are key mechanosensitive regulators of EC protective hemodynamic responses, potently induced by laminar shear stress (LSS) and suppressed by disease-prone pro-inflammatory disturbed shear stress (DSS) modelled by oscillatory7shear (OSS) in vitro. This is evident from comparative RNAseq analysis of cells under LSS and OSS showing upregulation of anti-inflammatory Klf2 / 4 and Nos3 vs. inflammatory mediator E-selectin (Sele) under LSS or OSS respectively (FIG. 2A), and overrepresentation of shear stress and atherosclerosis categories in pathway / process analysis (FIG. 2B). Interestingly, this Pcdhg-independent analysis of upstream regulators of shear-sensitive genes using EnrichR similarly identified PRC2 component Suzl2 as the leading mediator, in addition to expected general transcription modulators of EC function such as TCF3, E2F4 and Sox2 (FIG. 2C). These data indicated PRC2 as a central regulator of shear responsive gene signatures in ECs, suggesting shear stress mechanotransduction is determined by the epigenetic state of ECs. This is in concordance to the widespread transcriptional regulation by PRC2 (-80% genes are directly or indirect regulated).

[0253] The effect of shear stress on PRC2 activity in vivo was examined next. En face staining of the aortic arch showed low H3K27me3 in the athero-resistant greater curvature under LSS and high activity in the nearby athero-susceptible lesser curvature under DSS (FIG. 2D). Suppression of PRC2 by depletion of its core components Suzl2 or Ezh2 (Suzl2 si or Ezh2 si) in HUVECs elevated Klf2 / 4 compared to controls upon both LSS and OSS (FIG. 2E). These data confirmed a Klf2 / 4-suppresive role of PRC2 in the endothelium indicating its pro-inflammatory7function in the vasculature.

[0254] Example 3: Increased PRC2 level and activity in inflammatory ASCVD Atherosclerotic cardiovascular disease (ASCVD) is a vascular inflammatory disease, characterized by activation of the endothelium resulting in loss of protective Klf2 / 4. Loss of EC Klf2 / 4 positively correlates with ASCVD in humans. GWAS analysis from Cardiovascular Disease Knowledge Portal (CVDKP) showed significant association of CVD with PRC2 complex components and Pcdhg. To validate and assess the directionality7of association of PRC2 with ASCVD, published scRNAseq datasets of human atherosclerotic plaques were analyzed. Interestingly, the plaque region of the diseased artery showed increased PRC2 and Pcdhg levels in the endothelium, compared to the control adjacent region of the same artery without plaque in three individuals (FIG. 3A). TNFa levels correlated 33

[0255] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0256] positively while Klf2 and Klf4 levels correlated negatively with ASCVD, serving as true internal inflammatory and anti-inflammatory control markers respectively (FIG. 3A). Ezh2 is expressed at higher levels compared to its functional homolog Ezhl in HUVECs and in the endothelium in vivo, showing that Ezh2 is the major PRC2 enzyme in ECs.

[0257] To examine if the elevation in PRC2 components in ASCVD was functional, H3K27me3 levels were assessed. Analysis of arteries from deidentified human ASCVD patients showed ~ 4-fold increase in H3K27me3 in patient ECs compared to control donors, showing positive correlation of PRC2 activity with the disease (FIG. 3B). ASCVD is a chronic disease and plaques often form in aging vessels without the manifestation of the disease. Hence, arteries from elderly patients without symptomatic disease containing age-associated atherosclerotic plaques were examined (FIG. 3C). Interestingly, these vessels showed 3-fold higher levels of H3K27me3 in ECs in the plaque region compared to nonplaque region of the same artery (FIG. 3C). Mouse models of chronic (Apoe" ' on high fat diet or HFD for 12 weeks) or acute (Partial Carotid Artery Ligation and HFD for 3 weeks) ASCVD recapitulated this outcome (FIG. 3D). These findings established elevation in PRC2 and H3K27me3 levels as ASCVD biomarkers and strongly supported its causal pro-inflammatory role in ASCVD.

[0258] Example 4: Pharmacological inhibition of PRC2 by Tazemetostat (EPZ-6438)

[0259] PRC2 is composed of methyltransferases Ezhl / Ezh2, Suzl2, Eed, Rb and accessory components. Ezh2 being the more potent methyltransferase compared to Ezh 1 , has been a subject and target of intense investigation in cancers. While ECs express both Ezhl and Ezh2, Ezh2 is expressed at higher levels and its depletion or inhibition strongly reduces H3K27me3, confirming it as the major H3K27 methyltransferase in ECs. A specific Ezh2 inhibitor.

[0260] Tazemetostat or EPZ-6438 (35-fold specificity for Ezh2 compared to Ezhl, and -100 fold specificity for Ezh2 compared to other methyltransferases) is FDA approved for cancers where Ezh2 levels and activity are increased. Similar to Ezh2 si or Suzl2 si, treatment with Tazemetostat (Tz) increased Klf4 in HUVECs under LSS (FIG. 4A) and in thoracic aorta from treated mice (FIG. 4B) as compared to the untreated controls, showing that methyltransferase activity of Ezh2 was required to suppress Klf2 / 4.

[0261] The molecular marks and functional outcome associated with EC inflammation in vitro by treatment of HUVECs with inflammatory stresses co-treated with or without Tz was examined next. Interleukin- ip (Il ip) induced expression of pro-inflammatory marker and leukocyte adhesion receptor Vcaml, which was suppressed by Tz (FIG. 4C). Consequently,

[0262] 34

[0263] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0264] Vcaml-dependent adhesion of THP-1 monocytes to ECs under OSS was also dramatically blocked by Tz treatment, showing functional suppression of inflammation upon Tz treatment (FIG. 4D). Pcdhg si increased Klf4, which was not further increased by Tz. Additionally, Pcdhg ECKO plaques showed significantly decreased H3K27me3. Together, these data placed PRC2 dow nstream of Pcdhg as an active direct KH2 / 4 suppressor promoting vascular inflammation.

[0265] Example 5: Increased Notch transcriptional activity upon PRC2i mediates Klf2 / 4 transcriptional activation

[0266] PRC2 creates the histone EI3K27me3 repressive mark associated with facultative transcriptional silencing of genes. However, inhibition of transcriptional suppressor such as PRC2 is not sufficient for transcriptional activation, which requires transcription activating co-factors to recruit RNA Polymerase 2. To understand how PRC2 suppression increases K112 / 4 and the major transcription-inducing TFs involved, RNAseq analysis of Control or Suzl2 si HUVECs was performed (FIG. 4E). Of the DEGs in Suzl2 si. 757 (87.6%) were upregulated while 107 (12.4%) were downregulated, consistent with transcnptional silencing function of PRC2 (FIG. 4E, FIG. 4F). Since PRC2 is a positive mediator of Pcdhg which functions through the Notch pathw ay to directly upregulate K1I2 / 4 transcription, hypothesis-driven comparison to identify common transcription activating processes regulated by PRC2 and Pcdhg showed enrichment of Notch pathway targets, which were similarly upregulated in both Suzl2 si and Pcdhg si (FIG. 4E, FIG. 4G). A second compelling reason was that Notch and PRC2 are closely connected, sharing several targets. K112 / 4 promoters contain both Notch-RBPJ binding sites and PRC2-deposited H3K27me3 histone mark which was indicative of co-regulation by PRC2 and Notch. Hence, the Notch pathway was analyzed further.

[0267] Ablation of PRC2 by blocking Ezh2 methyltransferase activity by Tz increased K112 / 4 levels. Further, blocking the Notch pathway by RIN1 (a specific inhibitor of NICD-RBPJ interaction) completely blocked the effect of PRC2i, without affecting the levels of PRC2 components, placing Notch transcriptional function downstream of PRC2 (FIG. 4H). To assess if PRC2i increased Notch-dependent transcription, a NICD-RBPJ reporter assay was performed w ith or without Tz treatment. RIN1 treatment w as used as a control for transcription activation function of NICD. Treatment with Tz increased NICD reporter level in control cells, which was not further increased in NICD-V5 overexpressing cells. These data showed that transcriptional activation of Klf2 / 4 promoters upon de-repression by 35

[0268] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0269] inhibiting PRC2 requires Notch or a Notch-dependent process, supporting a role of PRC2 in limiting NICD-dependent transcription of K112 / 4.

[0270] Example 6: PRC2 inhibition by Tazemetostat suppresses ASCVD progression in mouse Cooperative regulation of Klf2 / 4 via a PRC2-Notch network has immense biological and therapeutic relevance. Suppression of K112 / 4 by PRC2 in ECs indicates that PRC2 inhibition would boost Klf2 / 4 levels and protect from ASCVD. Importantly, unlike undesirable effects from Notch modulators, the specific PRC2 inhibitor, Tazemetostat, is FDA approved, orally bioavailable and very' well tolerated for long-term use in humans without significant adverse side-effects. Tazemetostat is well tolerated and orally available in mice too. permitting examination of if Tz administration was protective in ASCVD. An ASCVD disease-progression mouse model was chosen with interventional Tz treatment (FIG.

[0271] 5 A). Apoe~'~ mice were maintained on high fat diet (HFD) for 12 weeks to induce hypercholesterolemia and atherosclerotic plaques, then switched to chow diet with or without Tz, resembling human disease and in accordance with clinical practice and recommendation (FIG. 5A). Vehicle or Tz was administered as an additive to food at 150 mg / kg / day for further 6 weeks, representing optimal dosing adapted in human patients (800 mg twice a day). Analysis of atherosclerotic plaques showed -50% reduced progression in Tz treated cohort compared to the Vehicle treated cohort, showing that plaques continue to grow, albeit at a slower rate upon Tz treatment (FIG. 5B). Detailed plaque characterization at the aortic root and BCA branching showed reduced plaque size, with thicker fibrous caps and smaller necrotic cores correlated with reduced inflammation as marked by CD68+ monocytes (FIG.

[0272] 5C, FIG. 5D, FIG. 5E). This demonstrated that Tz interventional treatment not only reduced plaque progression, but also dramatically reduced plaque vulnerability. These data confirmed the role of PRC2 as a pro-inflammatory mediator in ASCVD and advocated for use of specific and safe inhibitors of PRC2 for CVD intervention.

[0273] Materials and Methods (Examples 7-12)

[0274] Primary cells, cell lines, and cell culture reagents

[0275] HUVECs pooled from 3 donors were obtained from the Yale Vascular Biology and Therapeutics Core. Cultures were screened for the absence of pathogens, maintained in EGM2 Endothelial Cell Growth Medium (Lonza, CC-3162) and used at passages 2-5. All cells were routinely screened for mycoplasma. SiRNA transfection was performed with Lipofectamine RNAiMAX (ThermoFisher, 13778150) in Opti-MEM medium

[0276] 36

[0277] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0278] (ThermoFisher, 31985070) using ON-TARGET plus SMARTpool siRNAs from Horizon Discovery (Dharmacon). For lentiviral transduction, HEK293T cells were transfected with lentiviral vectors along with pVSV-G (Addgene, #138479) and psPAX2 (Addgene, #12260) packaging plasmids using lipofectamine 2000 (ThermoFisher, 11 68019) following the manufacturer’s instructions. Supernatants were collected 48-96 h after transfection and filtered through a 0.45 pm low-protein binding filter. Primary HUVECs were infected with lentivirus for 24h, then transferred to EGM2 medium. For monocyte adhesion assays, THP-1 cells labeled with CellTracker Deep Red (ThermoFisher, C34565) in HBSS supplemented with 1 mM Ca2+, 0.5 mM Mg2+, and 0.5% BSA were added to the HUVECs, incubated for 20 min at 37°C, washed 3x in HBSS and fixed with 3.7% formaldehy de. Cells were counterstained with DAPI before fluorescence imaging. Treatments used were Tazemetostat (Selleckchem), TNFa (PeproTech, 300-01A) and RIN1 (RBPJ Inhibitor-1) (Selleckchem, SS3376). All antibodies were validated in knockdown / knockout depletion by immunofluorescence and / or immunoblotting.

[0279] Shear stress

[0280] Shear stress expenments were performed in parallel plate flow chambers and perfused within a pump and environmental control system as described (Conway, D. E. et al. Curr Biol 27, 2727 (2017)). Briefly, cells were seeded at 70-90% confluency on 10 pg / ml Fibronectin-coated glass slides for 48-72 h in complete medium. For shear stimulation, slides were mounted in custom-made 25x55 mm parallel plate shear chambers with 0.5 mm thick silicone gaskets and stimulated with 15 dynes / cm2for LSS or 1 ± 4 dynes / cm2for OSS in complete media. The media was maintained at 37°C and 5% CO2 with a heat gun and humidified bubbler, respectively.

[0281] Animals and tissue preparation

[0282] Mice were maintained in a light- and temperature-controlled environment with free access to food and water and all efforts were made to minimize animal suffering. Euthanasia was performed by overdose of inhaled isoflurane and death confirmed by subsequent cervical dislocation and / or removing vital organs and / or opening the chest cavity verified by the absence of cardiovascular function. Mice were perfused through the left ventricle with PBS and then 3.7% formaldehyde followed by tissue collection, as described (Joshi, D. et al., Nat Cardiovasc Res 3, 1035-1048 (2024)). The heart and spinal column, with aorta and carotids attached, were removed, fixed under gentle agitation for an additional 24 h at 4 °C, and washed 3x with PBS. For whole aorta en face prep, the isolated aortas were bisected along the lesser curvature and the aortic arch was bisected through the greater curvature as well. For 37

[0283] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0284] aortic arch segment preparation, the aortic arch was bisected through the greater curvature. Hearts (containing aortic roots) and carotids were allowed to sink in 30% sucrose in PBS overnight at 4°C, embedded in OCT (optimal cutting temperature compound, Sakura, 4583) and frozen on dry ice before sectioning. Tissue blocks were cut into 8-10 pm sections using a cryostat (Leica) and sections stored at -80°C until use.

[0285] Atherosclerosis progression and blood lipid analysis

[0286] To induce atherosclerosis, 8-l2-week-old4 / we / _mice were maintained on a high-fat diet (HFD; Clinton / Cybulsky high-fat rodent diet with regular casein and 1.25% added cholesterol; Research Diet, D12108c) for 12 weeks. Mice were then switched to chow diet containing either vehicle or Tazemetostat (Tz) at 150 mg / kg / day for a further 6 weeks. Tz amounts were calculated considering 3g food intake per mouse per day. Blood samples were collected in EDTA-coated tubes from overnight starved mice, centrifuged at 8,000g at 4°C for 10 min and the supernatant (plasma) was separated and stored at -80 °C. Total plasma cholesterol and triglycerides (TAG) were measured using kits according to the manufacturer’s instructions (Wako Pure Chemicals. Japan). Tissues were prepared and analyzed as described below.

[0287] Tissue analysis

[0288] For IF, OCT tissue sections were thawed and washed 3x with PBS to remove OCT. Cells were fixed with 3.7% formaldehyde for 15 min at ambient temperature, washed with and stored in PBS. Deidentified human specimens were deparaffinized in Histo-Clear (National Diagnostics, HS-200). Sections were progressively rehydrated before antigen retrieval for 30 min at 95°C in IX Antigen Retrieval Buffer (Dako, 51699). Samples were incubated in perm-block buffer (5% donkey serum, 0.2% BSA, 0.3% Triton X-100 in PBS) for 1 h at room temperature, incubated with primary antibodies in perm-block overnight at 4°C, washed three times in perm-block and then incubated with alexafluor-conjugated secondary antibodies (ThermoFisher) at 1:1000 dilution in perm-block for 1 h at room temperature. Slides w ere w ashed 3x in perm-block and 3x in PBS before mounting in DAPI Fluoromount-G (Southern Biotech; 0100-20). Images were acquired on a Leica SP8 confocal microscope equipped with the Leica Application Suite software. Confocal stacks w ere flattened by maximum intensity z-projection in ImageJ. Post background subtraction, the mean fluorescence intensity (MFI) or nuclear intensity (with DAPI mask) w as recorded. For aorta ORO staining, the whole aorta was opened longitudinally on a soft-bottomed silica dish, incubated with 0.6% ORO in 60% isopropanol with gentle rocking for 1 h at ambient temperature, washed in 60% isopropanol for 20 minutes, washed in dH2O 3x, and mounted 38

[0289] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0290] on slides with endothelium side up in OCT compound. Images were acquired with a digital microscopic camera (Leica DFC295). ORO staining on OCT tissue sections was done similarly. Quantitation of ORO positive area was done in Image! Hematoxylin and eosin (H&E) staining on OCT tissue sections was done by Yale Research Histology Core using standard techniques. Plaque morphometric and vulnerability' analysis was performed as described (Joshi, D. et al., Nat Cardiovasc Res 3, 1035-1048 (2024)). Plaque area was determined by ORO positive staining. For each plaque, the necrotic core (NC) area was defined as a clear area in the plaque that was H&E free, and the fibrous cap (FC) thickness was quantified by selecting the largest necrotic core and measuring the thinnest part of the cap.

[0291] Immunoblotting

[0292] Cells were lysed in RIP A buffer (Roche) containing lx Halt Protease Inhibitor Cocktail (ThermoFisher, 78429) and lx PhosStop (Roche, 4906837001) for 30 min on ice, clarified at 13,000 g 4°C for 15 minutes, the supernatant transferred to new 1.5 ml tubes, 4x loading buffer (250mM Tris-HCl pH 6.8, 8% SDS, 40% glycerol, 20% p-mercaptoethanol, 0.008% bromophenol blue) added and the samples heated to 95°C for 5 min. Samples were resolved by a 4-15% gradient SDS Polyacrylamide gel, and transferred to 0.2 pm nitrocellulose membranes. Nitrocellulose was blocked with 5% non-fat skim milk for Ih at ambient temperature, incubated with desired antibodies diluted in 5% BSA using a standard immunoblotting procedure and detection by ECL (Millipore). Images were quantified in ImageJ by densitometry and normalized to GAPDH or Tubulin loading controls.

[0293] RNA isolation, sequencing, and Chromatin Immuno-precipitation (ChIP), ChIP quantitative real-time PCR (ChlP-qPCR)

[0294] Total RNA was extracted from cells using the RNeasy Plus Mini Kit (Qiagen, 74136) according to the manufacturer’s instructions. RNA was quantified by NanoDrop, and RNA integrity was measured with an Agilent Bioanalyzer. Samples were sequenced using Illumina NovaSeq 6000 (HiSeq paired-end, 100 bp). The base calling data from the sequencer were transferred into FASTQ files using bcl2fastq2 conversion software (version 2.20, Illumina). PartekFlow (a start-to-fimsh software analysis solution for next generation sequencing data applications) was used to determine differentially expressed genes (DEGs).

[0295] Quantification and Statistical Analysis

[0296] Morphometric analysis was done with ImageJ software (version 1.51; National Institutes of Health, Bethesda, MD). Graph preparation and statistical analysis was performed using GraphPad Prism 10.0 software (GraphPad software Inc.). Data were considered 39

[0297] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0298] normally distributed, statistical significance was performed using Student t test for two group comparison or one-way ANOVA with Tukey’s post hoc analysis for multiple groups comparison, as described in the figure legends. Data are presented as means ± SEM. A p value less than 0.05 was considered significant.

[0299] Example 7: Polycomb Repressive Complex 2 (PRC2) in endothelial responses to FSS Initially, EC gene expression was profiled under LSS, to model physiological protective blood flow and under OSS, to model ASCVD-associated pathological blood flow. Analysis of differentially expressed genes (DEGs) (FIG. 6A) showed the expected elevation of Klf2, Klf4 and their target gene Nos3 under LSS while the inflammatory mediator E-selectin (Sele) was high under OSS (FIG. 7 A / ), confirming normal responses to these FSS patterns. Likewise, pathway / process analysis showed overrepresentation of shear stress and atherosclerosis categories (FIG. 6B). Upstream regulator analysis of DEGs using EnrichR (Chen, E. Y. et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics 14. 128 (2013)) identified the core PRC2 component SUZ12 as the leading mediator (FIG. 6C), with the PRC2 catalytic subunit EZH2 as an additional strong hit. PRC2 may thus be a key node governing EC response to FSS.

[0300] It was recently reported that Pcdhg potently suppresses Klf2 / 4 in ECs (Joshi, D. et al., Nat Cardiovasc Res 3, 1035-1048 (2024)) (FIG. 6D). ECs were therefore assessed after Pcdhg si as an independent approach. This experiment included Pcdhg si + Klf2 / 4 si conditions to distinguish direct Pcdhg targets from those affected through Klf2 / 4. EnrichR analysis of upstream regulators of the observed DEGs also identified EZH2 and SUZ12, two core components of PRC2, as the most significant hits (FIG. 6E). Of the 494 DEGs (311 up and 183 down after Pcdhg suppression). 161 were identified as PRC2 targets, two-thirds (110 / 161) of which were upregulated after Pcdhg si while one-third (51 / 161) were downregulated. As PRC2 is mainly a transcriptional repressor, these data indicated that Pcdhg activates PRC2. This analysis also identified NF -KB, a known inhibitor of K112 / 4, and the PRC1 component RING1B, further validating the approach (FIG. 6E / ). The PRC2 component Jarid2 was also identified as a suppressor of Klf2 in the original genome-wide CRISPR screen, further supporting this hypothesis (Joshi, D. et al. , Nat Cardiovasc Res 3, 1035-1048 (2024)). Analysis of these DEGs using X2KWeb (Chen, E. Y. et al. BMC Bioinformatics 14, 128 (2013)), another tool for upstream regulator analysis, similarly showed PRC2 as a principal regulator of Pcdhg function (FIG. 7B). However, transcripts of PRC2 components were unaffected by Pcdhg si, ruling out a feedback loop (FIG. 7C).

[0301] 40

[0302] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0303] Interestingly, EZH2 protein was significantly reduced with a concomitant increase in Klf4 upon Pcdhg si, suggesting an effect on PRC2 complex stability (FIG. 7D).

[0304] PRC2 is a protein complex comprising a core of EZH2, SUZ12, EED and RbAp46 / 48, plus accessory factors. EZH2 is the catalytic subunit of PRC2, which trimethylates lysine-27 on Histone H3 (H3K27me3) in promoters and enhancers of target genes to repress transcription (FIG. 6F). ChlP-qPCR with an antibody to H3K27me3 pulled down the K112 promoter, whereas control IgG did not, but this signal was lost upon Suzl2 si, validating Klf2 / 4 as direct targets of PRC2 in ECs (FIG. 6G). Analysis ofH3K27me3 ChlPseq datasets in HUVECs from the UCSC Genome Browser also showed peaks for H3K27me3 on the Klf2 / 4promoters (marked by nearby peaks for H3K27ac) (FIG. 7E). As expected, ChlP-seq with an antibody to H3K27Ac, an activating mark that is mutually exclusive with H3K27me3, showed that Pcdhg si significantly increased H3K27ac at the Klf2 promoter (FIG. 7F). ChlP-qPCR confirmed that Pcdhg si decreased H3K27me3 and increased H3K27ac on the Klf2 promoter (FIG. 6H, FIG. 61). Pcdhg suppression of Klf2 / 4 thus correlates with PRC2 epigenetic modification of their promoters.

[0305] Example 8: Elevated PRC2 activity in ASCVD suppresses protective Klf2 / 4

[0306] Next, mouse and human arteries were examined to address epigenetic H3K27 modification in vivo. In mice, the aortic arch contains adjacent regions under protective LSS (greater curvature) and disease-prone DSS (lesser curvature) where inflammatory molecules such as Vcaml, Icaml are expressed in the endothelium. En face staining of this tissue revealed high H3K27me3 in the lesser curvature relative to the greater curvature (FIG. 8A), correlating PRC2 activity' with disease susceptibility'.

[0307] Staining human arteries from deidentified elderly donors without symptomatic disease for H3K27me3 showed 4-fold higher H3K27me3 staining in plaque ECs compared to nearby unaffected regions of the same arteries (FIG. 8B). Arteries from deidentified human ASCVD patients also showed a -3-fold increase in intimal H3K27me3 compared to control donors (FIG. 8C). Plaques from a mouse model of ASCVD (Partial Carotid Artery’ Ligation and HFD for 3 weeks) again showed ~3-fold higher staining for H3k27me3 (FIG. 8D). These stains localized to both luminal ECs and to cells deeper in the vessel wall that were positive for lymphocyte common antigen, CD45 (FIG. 9A). H3K27me3 is thus elevated in multiple instances of ASCVD, in ECs as well as leukocytes in the vessel wall, concomitant with increased expression of PRC2 components in ECs and immune cells.

[0308] Examination of published scRNAseq datasets from human atherosclerotic plaques 41

[0309] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0310] also showed increased PRC2 and Pcdhg expression in plaque endothelium, compared to the control adjacent region of the same artery (Alsaigh, T., Commun Biol 5, 1084 (2022)) (FIG.

[0311] 8E). TNFa levels correlated positively, while Klf2 and Klf4 levels correlated negatively with ASCVD, serving as internal inflammatory and anti-inflammatory controls, respectively. This trend was less obvious in immune cells and SMCs (FIG. 9B). Thus, tissue staining, published scRNAseq data and GW AS studies confirmed upregulation / activation of PRC2 in plaque endothelium and perhaps other cell types.

[0312] To test whether PRC2 regulates Klf2 / 4 under flow, ECs transfected with control, Suzl2, or Ezh2 siRNA were examined under LSS and OSS. Klf4 was assessed by immunostaining due to the availability of high-quality antibodies that are lacking for K112. Suzl2 si and Ezh2 si elevated Klf4 in Ecs under both flow patterns (FIG. 8F. FIG. 8G). PRC2 thus suppresses Klf2 / 4 in ECs.

[0313] Example 9: Pharmacological inhibition of PRC2

[0314] PRC2 comprises methyltransferases EZH1 / EZH2 along with other core and accessory components. EZH2 has much higher methyltransferase activity, is expressed at higher levels (FIG. 10A, FIG. 10B), and its depletion strongly reduces H3K27me3 (FIG. 10C), confirming it as the major H3K27 methyltransferase in ECs. A specific EZH2 inhibitor, Tazemetostat or EPZ-6438 (35-fold stronger inhibition of EZH2 than EZH1, and -100 fold stronger than other methyltransferases) is an FDA-approved drug used in cancer therapy. Treatment with Tazemetostat (Tz) increased Klf4 in HUVECs under LSS (FIG. 11 A) compared to the untreated controls. Next, the effects of Tz were examined on EC inflammatory activation in vitro. HUVECs were treated with or without 111 and Tz. Induction of VCAM1, a leukocyte adhesion receptor implicated in atherosclerosis, was strongly suppressed by Tz (FIG. 1 IB). VCAMl-dependent adhesion of THP-1 monocytes to ECs that were activated by OSS was also potently blocked by Tz (FIG. 11C). EZH2 methyltransferase activity thus suppresses K112 / 4 to permit vascular inflammation.

[0315] Example 10: Increased Notch transcriptional activity upon PRC2i mediates Klf2 / 4 transcriptional activation

[0316] To understand how inhibiting PRC2 increases Klf2 / 4, RNAseq analysis of control or Suzl2 si HUVECs was performed (FIG. 1 ID - FIG. 1 IF). Of the DEGs after Suzl2 si, 757 (87.6%) were upregulated while 107 (12.4%) were downregulated, consistent with transcriptional silencing by PRC2. Since PRC2 is downstream of Pcdhg, common

[0317] 42

[0318] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0319] transcriptional processes which are regulated by both PRC2 and Pcdhg were searched (FIG.

[0320] 11D). Notch pathway targets were strongly upregulated in both Suzl2 si and Pcdhg si (FIG.

[0321] 1 IE, FIG. 1 IF), which fits well with the finding that Notch is critical for KH2 / 4 transcription (Joshi, D. et al. Nat Cardiovasc Res 3, 1035-1048 (2024)). Notably, Klf2 / 4 promoters contain both Notch-RBPJ binding sites and PRC2-deposited H3K27me3 (FIG. 7E), suggesting opposing co-regulation by PRC2 and Notch.

[0322] The present study next assessed whether upregulation of K112 / 4 after inhibiting PRC2 required Notch transcriptional activity. Notch receptors mediate gene transcription after proteolytic cleavage and release of the intracellular domain (NICD), which translocates to the nucleus and binds the transcription factor RBPJ to activate gene transcription. RIN1 (Notchi), a small molecule inhibitor of the NICD-RBPJ interaction that blocks Notch-dependent transcription, prevented Klf2 / 4 upregulation by Tz, without affecting the levels of PRC2 components (FIG. 11G). Thus, PRC2 inhibition increases Notch-dependent transcription of K112 / 4.

[0323] Example 11: Notch functions via the Mekk2 / 3-Mek5-Erk5 pathway to induce K112 / 4 The best characterized mediator of Klf2 / 4 induction by LSS is the Mekk2 / 3-Mek5-Erk5 kinase cascade, which activates Mef2 transcription factors to directly induce Klf2 / 4 (FIG. 11H). It was also found that Notch directly induces K1I2 / 4 transcription. Hence, to understand the role of PRC2, an experiment was designed to address the relationship between these players. The Erk5-Mef2 pathway was specifically activated by expressing doxycycline-inducible constitutively active Mek5 (caMek5) in HUVECs with or without inhibition of Notch (Notchi) and PRC2 (Tz). caMEk5 expression increased Klf4 ~8-fold which was enhanced ~2x by Tz and completely abolished upon Notchi (FIG. 11H). Controls show ed equivalent Mek5 levels and Erk5 activation under relevant conditions, and reduction of H3K27me3 by Tz. Klf4 is thus regulated via an integrated netw ork in which PRC2 antagonizes the Mekk2 / 3-Mek5-Erk5-Mef2 cascade, which also requires Notch.

[0324] Example 12: PRC2 inhibition by Tazemetostat inhibits ASCVD in mice

[0325] Regulation of Klf2 / 4 via a PRC2-Mef2 -Notch network implies that PRC2 inhibition or Notch activation w ould boost K112 / 4 expression and protect from ASCVD. Notch however is a widely expressed, multifunctional regulator, thus, is an unlikely target. However, the specific PRC2 inhibitor, Tazemetostat (Tz), is FDA approved, orally bioavailable and well tolerated for long-term in humans with limited adverse side-effects (ClinicalTrials.gov ID 43

[0326] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0327] NCT02875548). Tz was therefore examined in a mouse model of ASCVD using an interventional strategy to mimic human clinical practice. Apoe' ' mice were maintained on a high fat diet (HFD) for 12 weeks to induce hypercholesterolemia and ASCVD, then switched to chow diet with or without Tz. Vehicle or Tz was then administered in food for 6 additional weeks (FIG. 12A). Tz treatment slowed plaque growth by >50% (FIG. 12B). However, detailed characterization of plaques at the aortic root and brachiocephalic artery’ showed ~2x thicker fibrous caps despite being smaller and essentially no expansion of necrotic cores (FIG. 12C-FIG. 12D), a >2x reduction in CD68+ monocyte / macrophages (FIG. 12E-FIG. 12F), and similarly reduced MMP9 levels (FIG. 12G). Body weight and circulating lipids (TAGs and Cholesterol) were similar betw een vehicle and Tz treated cohorts, with expected reduction in Cholesterol upon switching to chow diet (FIG. 13A-FIG. 13C). Tz interventional treatment thus moderately reduced plaque size but dramatically reduced markers of plaque vulnerability. These data confirmed PRC2 as a pro-inflammatory mediator in ASCVD and indicated use of Tz for treating ASCVD, such as advanced ASCVD.

[0328] Example 13: Additional diseases or conditions

[0329] The present study tested the inhibition of EZH2 using Tazemetostat (Tz) in several additional diseases or conditions.

[0330] Referring to FIGs. 14A-14E, Tz decreased hypoxia-induced pulmonary arterial hypertension (PAH) in rodents.

[0331] Referring to FIGs. 1 A-15E, elevated PRC2 activity was identified in a mouse model of hindlimb ischemia (HLI). Referring to FIGs. 16A-16D, Tz w as found to improve recovery’ from HLI in diabetic mice.

[0332] Referring to FIGs. 17A-17E. in a lipopolysaccharide (LPS)-induced acute lung injury’ (ALI) mouse model, Tz ameliorated endothelial activation.

[0333] Referring to FIGs. 18A-18B, Tz treatment of human umbilical vein endothelial cells (HUVECs) inflammatory' insulted by LPS reduced inflammation-induced endothelial permeability.

[0334] Enumerated Embodiments

[0335] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0336] Embodiment 1: A method of treating, ameliorating, and / or preventing vascular inflammatory' disease in a subject in need thereof, the method comprising administering an 44

[0337] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0338] effective amount of an EZH2 inhibitor. In certain embodiments, the EZH2 inhibitor comprises tazemetostat. In certain embodiments, the EZH2 inhibitor is selected from the group consisting of PF-06821497 (5,8-dichloro-2-[(4-methoxy-6-methyl-2-oxo-lH-pyridin- 3-yl)methyl]-7-[(R)-methoxy(oxetan-3-yl)methyl]-3,4-dihydroisoquinolin-l-one); UNC1999 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-pyridinyl)methyl]-l-(l-methylethyl)-6-[6-[4- ( 1 -methylethyl)- 1 -piperaziny 1] -3-py ridiny 1] - lH-indazole-4-carboxamide); CPI- 1205 (Lirametostat) (N-[(4-methoxy-6-methyl-2-oxo-lH-pyridin-3-yl)methyl]-2-methyl-l-[(lR)-l-[l-(2,2,2-trifluoroethyl)piperidin-4-yl]ethyl]indole-3-carboxamide): DS-3201b (Valemetostat) ((2R)-7-chloro-2-[4-(dimethylamino)cyclohexyl]-N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-2,4-dimethyl-l,3-benzodioxole-5-carboxamide); tazemetostat (EPZ-6438) (N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-3-[ethyl(oxan-4-yl)amino]-2-methyl-5-[4-(morpholin-4-ylmethyl)phenyl]benzamide); 3-Deazaneplanocin A; GSK 126 (N-[(l,2-Dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-3-methyl-l-[(lS)-l-methylpropyl]-6-[6-(l -piperaziny l)-3-pyridinyl]-lH-indole-4-carboxamide); GSK 343 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-pyridinyl)methyl]-l-(l-methylethyl)-6-[2-(4-methyl-l-piperazinyl)- 4-pyridinyl]-lH-indazole-4-carboxamide); JQEZ5 (N-t(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-pyridinyl)methyl]-l-(l-methylethyl)-6-[6-(4-methyl-l-piperazinyl)-3-pyridinyl]-lH-pyrazolo[3,4-b]pyridine-4-carboxamide); PF 06726304 acetate (5,8-Dichloro-2-[(l,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-7-(3,5-dimethyl-4-isoxazolyl)-3,4-dihydro-l(2H)-isoquinolinone acetate): ZLD-1039 (3-[Ethyl(tetrahydro-2H-pyran-4-yl)amino]-N-[(2,3,5,6,7,8-hexahydro- 1 -methyl-3-oxo-4-isoquinolinyl)methyl]-2-methyl-5-[6-(4-methyl-l -piperazinyl)-3-pyridinyl]benzamide); and any combinations thereof.

[0339] Embodiment 2: The method of embodiment 1, wherein the vascular inflammatory disease is a cardiovascular disease.

[0340] Embodiment 3 : The method of embodiment 1 or embodiment 2, wherein the cardiovascular disease is atherosclerosis, pulmonary arterial hypertension (PAH), a peripheral artery disease (PAD), or diabetic PAD.

[0341] Embodiment 4: The method of embodiment 3. wherein treatment or amelioration of the subject decreases and / or reverses progression of the atherosclerosis.

[0342] Embodiment 5: The method of any one of embodiments 1-4, wherein the vascular inflammatory disease comprises plaque formation in the subject.

[0343] Embodiment 6: The method of embodiment 5, wherein the plaque is an atherosclerotic plaque.

[0344] Embodiment 7 : The method of embodiment 5 or embodiment 6, wherein treatment or 45

[0345] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0346] amelioration of the subject reduces the rate of plaque progression in the subject.

[0347] Embodiment 8: The method of any one of embodiments 5-7, wherein treatment or amelioration of the subject increases the amount of stable plaque as compared to the amount of unstable plaque in the subject.

[0348] Embodiment 9: The method of any one of embodiments 5-8, wherein treatment or amelioration of the subj ect increases the thickness of fibrous plaque caps in the subj ect.

[0349] Embodiment 10: The method of any one of embodiments 5-9. wherein treatment or amelioration of the subject decreases the size of the necrotic core of plaque in the subject.

[0350] Embodiment 11 : A method of treating, ameliorating, and / or preventing a cardiovascular disease in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor. In certain embodiments, the EZH2 inhibitor comprises tazemetostat. In certain embodiments, the EZH2 inhibitor is selected from the group consisting of PF-06821497 (5,8-dichloro-2-[(4-methoxy-6-methyl-2-oxo-lH-pyridin- 3-yl)methy l]-7-[(R)-methoxy(oxetan-3-yl)methyl] -3,4-dihy droisoquinolin- 1 -one); UNC 1999 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-pyridinyl)methyl]-l-(l-methylethyl)-6-[6-[4- ( 1 -methylethyl)- 1 -piperaziny 1J -3 -pyridiny 1] - lH-indazole-4-carboxamide); CPI- 1205 (Lirametostat) (N-[(4-methoxy-6-methyl-2-oxo-lH-pyridin-3-yl)methyl]-2-methyl-l-[(lR)-l-[l-(2,2,2-trifluoroethyl)piperidin-4-yl]ethyl]indole-3-carboxamide); DS-3201b (Valemetostat) ((2R)-7-chloro-2-[4-(dimethylamino)cyclohexyl]-N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-2,4-dimethyl-l,3-benzodioxole-5-carboxamide); tazemetostat (EPZ-6438) (N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-3-[ethyl(oxan-4-yl)amino]-2-methyl-5-[4-(morpholin-4-ylmethyl)phenyl]benzamide); 3-Deazaneplanocin A; GSK 126 (N-[(l,2-Dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-3-methyl-l-[(lS)-l-methylpropyl]-6-[6-(l -piperaziny l)-3-pyridinyl]-lH-indole-4-carboxamide); GSK 343 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-pyridinyl)methyl]-l-(l-methylethyl)-6-[2-(4-methyl-l-piperazinyl)- 4-pyridinyl]-lH-indazole-4-carboxamide); JQEZ5 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3 -pyridiny l)methyl] - 1 -(1 -methylethyl)-6- [6-(4-methyl- 1 -piperaziny 1) -3 -py ridiny 1] -lH-pyrazolo[3,4-b]pyridine-4-carboxamide); PF 06726304 acetate (5,8-Dichloro-2-[(l,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-7-(3,5-dimethyl-4-isoxazolyl)-3,4-dihydro-l(2H)-isoquinolinone acetate); ZLD-1039 (3-[Ethyl(tetrahydro-2H-pyran-4-yl)amino]-N-[(2,3,5,6,7,8-hexahydro-l-methyl-3-oxo-4-isoquinolinyl)methyl]-2-methyl-5-[6-(4-methyl-l-piperazinyl)-3-pyridinyl]benzamide); and any combinations thereof.

[0351] Embodiment 12: The method of embodiment 11, wherein the cardiovascular disease is ischemic heart disease, congestive heart failure, hypertension, valvular heart disease,

[0352] 46

[0353] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0354] general atherosclerosis, hypercholesterolemia, atherosclerosis, atherosclerotic cardiovascular disease (ASCVD), thrombosis, or any combination thereof.

[0355] Embodiment 13 : The method of embodiment 11 or embodiment 12, wherein the cardiovascular disease is atherosclerosis or ASCVD.

[0356] Embodiment 14: The method of embodiment 13, wherein treatment or amelioration of the subject decreases and / or reverses progression of the atherosclerosis or ASCVD.

[0357] Embodiment 15: The method of any one of embodiments 11-14, wherein the cardiovascular disease comprises plaque formation in the subject.

[0358] Embodiment 16: The method of any one of embodiment 15, wherein the plaque is an atherosclerotic plaque.

[0359] Embodiment 17: The method of any one of embodiment 15 or embodiment 16. wherein treatment or amelioration of the subject reduces the rate of plaque progression in the subject.

[0360] Embodiment 18: The method of any one of embodiments 15-17, wherein treatment or amelioration of the subject increases the amount of stable plaque as compared to the amount of unstable plaque in the subject.

[0361] Embodiment 19: The method of any one of embodiments 15-18, wherein treatment or amelioration of the subj ect increases the thickness of fibrous plaque caps in the subj ect.

[0362] Embodiment 20: The method of any one of embodiments 15-19, wherein treatment or amelioration of the subject decreases the size of the necrotic core of plaque in the subject.

[0363] Embodiment 21 : A method of treating, ameliorating, and / or preventing atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor. In certain embodiments, the EZH2 inhibitor comprises tazemetostat. In certain embodiments, the EZH2 inhibitor is selected from the group consisting of PF-06821497 (5,8-dichloro-2-[(4-methoxy-6-methyl-2-oxo-lH-pyridin-3-yl)methyl]-7-[(R)-methoxy(oxetan-3-yl)methyl]-3,4-dihydroisoquinolin-l-one); UNC1999 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-pyridinyl)methyl]-l-(l-methylethyl)-6-[6-[4-(l-methylethyl)-l-piperazinyl]-3-pyridinyl]-lH-indazole-4-carboxamide); CPI-1205 (Lirametostat) (N-[(4-methoxy-6-methyl-2-oxo-lEI-pyridin-3-yl)methyl]-2-methyl-l-[(lR)-l-[l-(2,2,2-trifluoroethyl)piperidin-4-yl]ethyl]indole-3-carboxamide); DS-3201b (Valemetostat) ((2R)-7-chloro-2-[4-(dimethylamino)cyclohexyl]-N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-2,4-dimethyl-l,3-benzodioxole-5-carboxamide); tazemetostat (EPZ-6438) (N-[(4.6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-3-[ethyl(oxan-4-yl)amino]-2-methyl-5-[4-(morpholin-4-ylmethyl)phenyl]benzamide); 3- 47

[0364] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0365] Deazaneplanocin A; GSK 126 (N-[(l,2-Dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-3-methyl-l-[(lS)-l-methylpropyl]-6-[6-(l-piperazinyl)-3-pyridinyl]-lH-indole-4-carboxamide); GSK 343 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-pyridinyl)methyl]-l-(l-methylethyl)-6-[2-(4-methyl-l-piperazinyl)-4-pyridinyl]-lH-indazole-4-carboxamide); JQEZ5 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-pyridinyl)methyl]-l-(l-methylethyl)-6-[6-(4-methyl-l-piperazinyl)-3-pyridinyl]-lH-pyrazolo[3,4-b]pyridine-4-carboxamide); PF 06726304 acetate (5,8-Dichloro-2-[(l,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-7-(3,5-dimethyl-4-isoxazolyl)-3,4-dihydro-l(2H)-isoquinolinone acetate); ZLD-1039 (3-[Ethyl(tetrahydro-2H-pyran-4-yl)amino]-N-[(2,3,5,6,7,8-hexahydro-l-methyl-3-oxo-4-isoquinolinyl)methyl]-2-methyl-5-[6-(4-methyl-l-piperazinyl)-3-pyridinyl]benzamide); and any combinations thereof.

[0366] Embodiment 22: The method of embodiment 21, wherein the ASCVD comprises plaque formation in the subject.

[0367] Embodiment 23: The method of any one of embodiment 22, wherein treatment or amelioration of the subject reduces the rate of plaque progression in the subject.

[0368] Embodiment 24: The method of any one of embodiment 22 or embodiment 23, wherein treatment or amelioration of the subj ect increases the amount of stable plaque as compared to the amount of unstable plaque in the subject.

[0369] Embodiment 25: The method of any one of embodiments 22-24, wherein treatment or amelioration of the subject increases the thickness of fibrous plaque caps in the subject.

[0370] Embodiment 26: The method of any one of embodiments 22-25, wherein treatment or amelioration of the subject decreases the size of the necrotic core of plaque in the subject.

[0371] Embodiment 27: A method of treating, ameliorating, and / or preventing vascular inflammation in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor. In certain embodiments, the EZH2 inhibitor comprises tazemetostat. In certain embodiments, the EZH2 inhibitor is selected from the group consisting of PF-06821497 (5,8-dichloro-2-[(4-methoxy-6-methyl-2-oxo-lH-pyridin-3-yl)methyl]-7-[(R)-methoxy(oxetan-3-yl)methyl]-3,4-dihydroisoquinolin-l-one); UNC1999 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-pyridinyl)methyl]-l-(l-methylethyl)-6-[6-[4-(l-methylethyl)-l-piperazinyl]-3-pyridinyl]-lH-indazole-4-carboxamide); CPI-1205 (Lirametostat) (N-[(4-methoxy-6-methyl-2-oxo-lH-pyridin-3-yl)methyl]-2-methyl-l-[(lR)-l-[l-(2,2,2-trifluoroethyl)piperidin-4-yl]ethyl]indole-3-carboxamide); DS-3201b (Valemetostat) ((2R)-7-chloro-2-[4-(dimethylamino)cyclohexyl]-N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-2,4-dimethyl-l,3-benzodioxole-5-carboxamide); tazemetostat (EPZ- 48

[0372] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0373] 6438) (N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-3-[ethyl(oxan-4-yl)amino]-2-methyl-5-[4-(morpholin-4-ylmethyl)phenyl]benzamide); 3-Deazaneplanocin A; GSK 126 (N-[( 1 ,2-Dihy dro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl] -3-methyl- 1 -[( 1 S)- 1 -methylpropyl]-6-[6-(l-piperazinyl)-3-pyridinyl]-lH-indole-4-carboxamide); GSK 343 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-pyridinyl)methyl]-l-(l-methylethyl)-6-[2-(4-methyl-l-piperazinyl)-4-pyridinyl]-lH-indazole-4-carboxamide); JQEZ5 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-pyridinyl)methyl]-l-(l-methylethyl)-6-[6-(4-methyl-l-piperazinyl)-3-pyridinyl]-lH-pyrazolo[3,4-b]pyridine-4-carboxamide); PF 06726304 acetate (5,8-Dichloro-2-[(l,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-7-(3,5-dimethyl-4-isoxazolyl)-3,4-dihydro-l(2H)-isoquinolinone acetate): ZLD-1039 (3-[Ethyl(tetrahydro-2H-pyran-4-yl)amino]-N-[(2.3.5.6.7.8-hexahydro-l-methyl-3-oxo-4-isoquinolinyl)methyl]-2-methyl-5-[6-(4-methyl-l-piperazinyl)-3-pyridinyl]benzamide); and any combinations thereof.

[0374] Embodiment 28: A method of treating, ameliorating, and / or preventing acute lung injury in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor. In certain embodiments, the EZH2 inhibitor comprises tazemetostat. In certain embodiments, the EZH2 inhibitor is selected from the group consisting of PF-06821497 (5,8-dichloro-2-[(4-methoxy-6-methyl-2-oxo-lH-pyridin-3-yl)methyl]-7-[(R)-methoxy(oxetan-3-yl)methyl]-3,4-dihydroisoquinolin-l-one); UNCI 999 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-pyridinyl)methyl]-l-(l-methylethyl)-6-[6-[4-(l-methylethyl)-l-piperazinyl]-3-pyridinyl]-lH-indazole-4-carboxamide): CPI-1205 (Lirametostat) (N-[(4-methoxy-6-methyl-2-oxo-lH-pyridin-3-yl)methyl]-2-methyl-l-[(lR)-l-[l-(2,2,2-trifluoroethyl)piperidin-4-yl] ethyl] indole-3 -carboxamide); DS-3201b (Valemetostat) ((2R)-7-chloro-2-[4-(dimethylamino)cyclohexyl]-N-[(4,6-dimethyl-2-oxo-lH-pyridin-3-yl)methyl]-2,4-dimethyl-l,3-benzodioxole-5-carboxamide); tazemetostat (EPZ-6438) (N-[(4,6-dimethyl- 2-oxo-lH-pyridin-3-yl)methyl]-3-[ethyl(oxan-4-yl)amino]-2-methyl-5-[4-(morpholin-4-ylmethyl)phenyl]benzamide); 3-Deazaneplanocin A; GSK 126 (N-[(l,2-Dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-3-methyl-l -[(1 S)-l -methylpropyl]-6-[6-(l -piperazinyl)- 3-pyridinyl]-lH-indole-4-carboxamide): GSK 343 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-pyridinyl)methyl]-l-(l-methylethyl)-6-[2-(4-methyl-l-piperazinyl)-4-pyridinyl]-lH-indazole-4-carboxamide); JQEZ5 (N-[(l,2-Dihydro-6-methyl-2-oxo-4-propyl-3-pyridinyl)methyl]-l-(l-methylethyl)-6-[6-(4-methyl-l-piperazinyl)-3-pyridinyl]-lH-pyrazolo[3,4-b]pyridine-4-carboxamide); PF 06726304 acetate (5,8-Dichloro-2-[(l,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-7-(3,5-dimethyl-4-isoxazolyl)-3,4-dihydro-1 (2H)-isoquinolinone acetate): ZLD-1039 (3-[Ethyl(tetrahydro-2H-pyran-4-yl)amino]-N- 49

[0375] 56887053 3Attorney Docket No. 047162-7516WOl(02762)

[0376] [(2,3,5,6,7,8-hexahydro-l-methyl-3-oxo-4-isoquinolinyl)methyl]-2-methyl-5-[6-(4-methyl-l-piperazinyl)-3-pyridinyl]benzamide); and any combinations thereof.

[0377] The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

[0378] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0379] In sum, while this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0380] 50

[0381] 56887053 3

Claims

Attorney Docket No. 047162-7516WOl(02762)CLAIMSWhat is claimed:

1. A method of treating, ameliorating, and / or preventing a vascular inflammatory disease in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor, wherein the EZH2 inhibitor comprises tazemetostat.

2. The method of claim 1, wherein the vascular inflammatory disease is a cardiovascular disease.

3. The method of claim 1 or claim 2, wherein the cardiovascular disease is atherosclerosis, pulmonary arterial hypertension (PAH), peripheral artery7disease (PAD), or diabetic PAD.

4. The method of claim 3, wherein treatment or amelioration of the subject decreases and / or reverses progression of the atherosclerosis.

5. The method of any one of claims 1-4, wherein the vascular inflammatory' disease comprises plaque formation in the subject.

6. The method of claim 5, wherein the plaque is an atherosclerotic plaque.

7. The method of claim 5 or claim 6, wherein treatment or amelioration of the subject reduces the rate of plaque progression in the subject.

8. The method of any one of claims 5-7, wherein treatment or amelioration of the subject increases the amount of stable plaque as compared to the amount of unstable plaque in the subject.

9. The method of any one of claims 5-8, wherein treatment or amelioration of the subject increases the thickness of fibrous plaque caps in the subject.

10. The method of any one of claims 5-9, wherein treatment or amelioration of the subject decreases the size of the necrotic core of plaque in the subject.

11. A method of treating, ameliorating, and / or preventing a cardiovascular disease in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor, wherein the EZH2 inhibitor comprises tazemetostat.

12. The method of claim 11, wherein the cardiovascular disease is ischemic heart disease,5156887053 3Attorney Docket No. 047162-7516WOl(02762)congestive heart failure, hypertension, valvular heart disease, general atherosclerosis, hypercholesterolemia, atherosclerosis, atherosclerotic cardiovascular disease (ASCVD), thrombosis, or any combination thereof.

13. The method of claim 11 or claim 12, wherein the cardiovascular disease is atherosclerosis or ASCVD.

14. The method of claim 13, wherein treatment or amelioration of the subject decreases and / or reverses progression of the atherosclerosis or ASCVD.

15. The method of any one of claims 11-14, wherein the cardiovascular disease comprises plaque formation in the subject.

16. The method of claim 15. wherein the plaque is an atherosclerotic plaque.

17. The method of claim 15 or claim 16, wherein treatment or amelioration of the subject reduces the rate of plaque progression in the subject.

18. The method of any one of claims 15-17, wherein treatment or amelioration of the subject increases the amount of stable plaque as compared to the amount of unstable plaque in the subject.

19. The method of any one of claims 15-18, wherein treatment or amelioration of the subject increases the thickness of fibrous plaque caps in the subject.

20. The method of any one of claims 15-19, wherein treatment or amelioration of the subject decreases the size of the necrotic core of plaque in the subject.

21. A method of treating, ameliorating, and / or preventing atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor, wherein the EZH2 inhibitor comprises tazemetostat.

22. The method of claim 21, wherein the ASCVD comprises plaque formation in the subject.

23. The method of claim 22, wherein treatment or amelioration of the subject reduces the rate of plaque progression in the subject.

24. The method of claim 22 or claim 23, wherein treatment or amelioration of the subject 5256887053 3Attorney Docket No. 047162-7516WOl(02762)increases the amount of stable plaque as compared to the amount of unstable plaque in the subject.

25. The method of any one of claims 22-24, wherein treatment or amelioration of the subject increases the thickness of fibrous plaque caps in the subject.

26. The method of any one of claims 22-25, wherein treatment or amelioration of the subject decreases the size of the necrotic core of plaque in the subject.

27. A method of treating, ameliorating, and / or preventing vascular inflammation in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor, wherein the EZH2 inhibitor comprises tazemetostat.

28. A method of treating, ameliorating, and / or preventing acute lung injury in a subject in need thereof, the method comprising administering an effective amount of an EZH2 inhibitor, wherein the EZH2 inhibitor comprises tazemetostat.5356887053 3

Images