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Supplementary Figure S1 shows histone post-translational modifications (B) and qRT-PCR validation (C) of RNA-seq data for vehicle (DMSO) and verticillin A treated OVCAR8 cells. Supplementary Figure S2 shows Verticillin A-eNP synthesis procedure (A), scanning electron micrograph of verticillin A expansile nanoparticles (B) and calibration curve for verticillin A (C). Supplementary Figure S3 shows structure of verticillin A (A), 1H-NMR (B) and UPLC (C) data for estimating purity of verticillin A, Supplementary Figure S4 shows mitochondrial toxicity assay for verticillin A

Extracts from the seeds of milk thistle, Silybum marianum, are known commonly as silibinin and silymarin and possess anticancer actions on human prostate carcinoma in vitro and in vivo. Seven distinct flavonolignan compounds and a flavonoid have been isolated from commercial silymarin extracts. Most notably, two pairs of diastereomers, silybin A and silybin B and isosilybin A and isosilybin B, are among these compounds. In contrast, silibinin is composed only of a 1:1 mixture of silybin A and silybin B. With these isomers now isolated in quantities sufficient for biological studies, each pure compound was assessed for antiproliferative activities against LNCaP, DU145, and PC3 human prostate carcinoma cell lines. Isosilybin B was the most consistently potent suppressor of cell growth relative to either the other pure constituents or the commercial extracts. Isosilybin A and isosilybin B were also the most effective suppressors of prostate-specific antigen secretion by androgen-dependent LNCaP cells. Silymarin and silibinin were shown for the first time to suppress the activity of the DNA topoisomerase IIα gene promoter in DU145 cells and, among the pure compounds, isosilybin B was again the most effective. These findings are significant in that isosilybin B composes no more than 5% of silymarin and is absent from silibinin. Whereas several other more abundant flavonolignans do ultimately influence the same end points at higher exposure concentrations, these findings are suggestive that extracts enriched for isosilybin B, or isosilybin B alone, might possess improved potency in prostate cancer prevention and treatment.

Supplementary Figure 2 from Milk Thistle and Prostate Cancer: Differential Effects of Pure Flavonolignans from Silybum marianum on Antiproliferative End Points in Human Prostate Carcinoma Cells

Supplementary Figure Legends from Milk Thistle and Prostate Cancer: Differential Effects of Pure Flavonolignans from Silybum marianum on Antiproliferative End Points in Human Prostate Carcinoma Cells

Supplementary Figure 1B from Milk Thistle and Prostate Cancer: Differential Effects of Pure Flavonolignans from Silybum marianum on Antiproliferative End Points in Human Prostate Carcinoma Cells

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy in women worldwide and the fifth most common cause of cancer-related deaths among U.S. women. New therapies are needed to treat HGSOC, particularly because most patients develop resistance to current first-line therapies. Many natural product and fungal metabolites exhibit anticancer activity and represent an untapped reservoir of potential new agents with unique mechanism(s) of action. Verticillin A, an epipolythiodioxopiperazine alkaloid, is one such compound, and our recent advances in fermentation and isolation are now enabling evaluation of its anticancer activity. Verticillin A demonstrated cytotoxicity in HGSOC cell lines in a dose-dependent manner with a low nmol/L IC50. Furthermore, treatment with verticillin A induced DNA damage and caused apoptosis in HGSOC cell lines OVCAR4 and OVCAR8. RNA-Seq analysis of verticillin A–treated OVCAR8 cells revealed an enrichment of transcripts in the apoptosis signaling and the oxidative stress response pathways. Mass spectrometry histone profiling confirmed reports that verticillin A caused epigenetic modifications with global changes in histone methylation and acetylation marks. To facilitate in vivo delivery of verticillin A and to monitor its ability to reduce HGSOC tumor burden, verticillin A was encapsulated into an expansile nanoparticle (verticillin A-eNP) delivery system. In an in vivo human ovarian cancer xenograft model, verticillin A-eNPs decreased tumor growth and exhibited reduced liver toxicity compared with verticillin A administered alone. This study confirmed that verticillin A has therapeutic potential for treatment of HGSOC and that encapsulation into expansile nanoparticles reduced liver toxicity.

Despite the presence of CTLs in the tumor microenvironment, the majority of immunogenic human colon cancer does not respond to immune checkpoint inhibitor immunotherapy, and microsatellite instable (MSI) tumors are not naturally eliminated. The molecular mechanism underlying the inactivity of tumor-infiltrating CTLs is unknown. We report here that CTLs were present in both MSI and microsatellite stable colon tumors. The expression of the H3K9me3-specific histone methyltransferase SUV39H1 was significantly elevated in human colon carcinoma compared with normal colon tissues. Using a mouse colon carcinoma model, we further determined that tumor-infiltrating CTLs in the colon tumor microenvironment have high expression of SUV39H1. To target SUV39H1 in the tumor microenvironment, a virtual chemical library was screened on the basis of the SET (suppressor of variegation 3–9, enhancer of zeste and trithorax) domain structure of the human SUV39H1 protein. Functional enzymatic activity assays identified a small molecule that inhibits SUV39H1 enzymatic activity. On the basis of the structure of this small molecule, we modified it and chemically synthesized a small molecule, termed F5446, which has an EC50 of 0.496 μmol/L for SUV39H1 enzymatic activity. H3K9me3 was enriched in the promoters of GZMB, PRF1, FASLG, and IFNG in quiescent T cells. F5446 inhibited H3K9me3, thereby upregulating expression of these effectors in tumor-infiltrating CTLs and suppressing colon carcinoma growth in a CD8+ CTL-dependent manner in vivo. Our data indicate that SUV39H1 represses CTL effector gene expression and, in doing so, confers colon cancer immune escape.

Despite the presence of cytotoxic T lymphocytes (CTLs) in the tumor microenvironment, the majority of immunogenic human colon cancers do not respond to immune checkpoint inhibitor immunotherapy and microsatellite instable (MSI) tumors are not naturally eliminated. The molecular mechanism underlying the inactivity of tumor-infiltrating CTLs is unknown. We report here that CTLs are present in both MSI and microsatellite stable (MSS) colon tumors. The expression of the H3K9me3-specific histone methyltransferase SUV39H1 is significantly elevated in human colon carcinoma as compared to normal colon tissues. Using a mouse colon carcinoma model, we further determined that tumor-infiltrating CTLs in the colon tumor microenvironment express high levels of SUV39H1. To target SUV39H1 in the tumor microenvironment, a virtual chemical library was screened based on the SET domain structure of human SUV39H1 protein. Functional enzymatic activity assays identified a small molecule that inhibits SUV39H1 enzymatic activity. Based on the structure of this small molecule, we modified it and chemically synthesized a novel small molecule, termed F5446, which has an EC50 of 0.496 μM for SUV39H1 enzymatic activity. H3K9me3 is enriched in the promoters of GZMB, PRF1, FASLG and IFNG in quiescent T cells. F5446 inhibits H3K9me3, thereby upregulating expression of these effectors in tumor-infiltrating CTLs and suppressing colon carcinoma growth in a CD8+ CTL-dependent manner in vivo. Our data indicate that SUV39H1 represses CTL effector expression and, in doing so, confers colon cancer immune escape. Citation Format: Chunwan Lu, Dafeng Yang, John D. Klement, II Kyu Oh, Natasha M. Savage, Jennifer L. Waller, Aaron H. Colby, Mark W. Grinstaff, Nicholas H. Oberlies, Cedric Pearce, Zhiliang Xie, Samuel Kulp, Christopher Coss, Mitch A. Phelps, Thomas Albers, Iryna O. Lebedyeva, Kebin Liu. The SUV39H1-H3K9me3 pathway represses cytotoxic T lymphocyte effector expression to confer colon carcinoma immune escape [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A32.

Human colon cancer, except for the small subset of microsatellite instable (MSI) colon cancer, does not respond to anti-PD-L1/PD-1 immune checkpoint inhibitor (ICI) immunotherapy. A well-established notion is that MSI colon cancer harbors various mutations that serve as neoantigens to generate tumor-specific cytotoxic T lymphocytes (CTLs). Because CTLs suppress tumor through inducing tumor cell apoptosis, we hypothesized that colon cancer cell intrinsic apoptosis resistance is potentially another mechanism underlying microsatellite stable (MSS) colon cancer nonresponse to ICI immunotherapy. If tumor cells are not sensitive to apoptosis induction, then tumor cells cannot be killed by CTLs regardless of how potent the CTLs are. To test this hypothesis, we analyzed MSI and MSS human colon carcinoma specimens and developed a small molecule that targets H3K9me3-mediated cell death pathways as a sensitizer for colon cancer ICI immunotherapy. As expected, we observed high level of CTL infiltration in all eight MSI colon carcinoma specimens examined. However, five of the nine MSS colon carcinoma specimens also exhibited high level of CTL infiltration, suggesting that CTL level is not the sole mechanism underlying MSS colon cancer non-response to ICI immunotherapy. Analysis of the TCGA database revealed that SUV39H1, a histone methyltransferase that catalyzes H3K9me3, is significantly elevated in human colon carcinoma specimens compared to normal human colon tissues. A SUV39H1 structure-based virtual chemical library screening in combination with functional assays identified a small-molecule SUV39H1 inhibitor. Further analysis of the top forty-three hits identified a potent SUV39H1 inhibitor. Structure modifications were then performed and a chemical synthesis procedure was developed to synthesize a novel SUV39H1 inhibitor, termed F5446. F5446 has an EC50 of 0.496 µM against SUV39H1 enzymatic activity and suppresses human colon carcinoma cell growth in a dose-dependent manner in vitro. F5446 suppresses colon carcinoma growth through upregulating key cell cycle regulators to induce cell cycle arrest at the S phase. Furthermore, F5446 also decreased H3K9me3 at the FAS promoter to upregulate Fas expression. Consequently, a sublethal dose of F5446 effectively overcame human colon carcinoma cell resistance to apoptosis induced by FasL, a ligand on activated CTLs in vitro. Treatment of colon carcinoma-bearing mice with F5446 and anti-PD-1 both significantly suppress tumor growth in vivo. Our data determined that F5446 is a novel SUV39H1 inhibitor that has the potential to be further developed as a sensitizer to overcome colon carcinoma resistance to ICI immunotherapy through activating the Fas-FasL pathway. Citation Format: Chunwan Lu, Dafeng Yang, John D. Klement, Aaron H. Colby, Mark W. Grinstaff, Cedric Pearce, Nicholas H. Oberlies, Thomas Albers, Iryna Lebedyeva, Kebin Liu. The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4966.

A biocompatible polyester dendrimer composed of the natural metabolites, glycerol and succinic acid, is described for the encapsulation of the antitumor camptothecins, 10-hydroxycamptothecin and 7-butyl-10-aminocamptothecin. The cytotoxicity of the dendrimer-drug complex toward four different human cancer cell lines [human breast adenocarcinoma (MCF-7), colorectal adenocarcinoma (HT-29), non–small cell lung carcinoma (NCI-H460), and glioblastoma (SF-268)] is also reported, and low nmol/L IC50 values are measured. Cellular uptake and efflux measurements in MCF-7 cells show an increase of 16-fold for cellular uptake and an increase in drug retention within the cell when using the dendrimer vehicle. (Cancer Res 2006; 66(24): 11913-21)

Extracts from the seeds of milk thistle, Silybum marianum, are known commonly as silibinin and silymarin and possess anticancer actions on human prostate carcinoma in vitro and in vivo. Seven distinct flavonolignan compounds and a flavonoid have been isolated from commercial silymarin extracts. Most notably, two pairs of diastereomers, silybin A and silybin B and isosilybin A and isosilybin B, are among these compounds. In contrast, silibinin is composed only of a 1:1 mixture of silybin A and silybin B. With these isomers now isolated in quantities sufficient for biological studies, each pure compound was assessed for antiproliferative activities against LNCaP, DU145, and PC3 human prostate carcinoma cell lines. Isosilybin B was the most consistently potent suppressor of cell growth relative to either the other pure constituents or the commercial extracts. Isosilybin A and isosilybin B were also the most effective suppressors of prostate-specific antigen secretion by androgen-dependent LNCaP cells. Silymarin and silibinin were shown for the first time to suppress the activity of the DNA topoisomerase IIα gene promoter in DU145 cells and, among the pure compounds, isosilybin B was again the most effective. These findings are significant in that isosilybin B composes no more than 5% of silymarin and is absent from silibinin. Whereas several other more abundant flavonolignans do ultimately influence the same end points at higher exposure concentrations, these findings are suggestive that extracts enriched for isosilybin B, or isosilybin B alone, might possess improved potency in prostate cancer prevention and treatment.