Abstract 5447: A novel inhibitor of DNA methylation

Abstract We previously reported the discovery of XB05, a synthetic small molecule with antiproliferative activity. XB05 was found to be a strong inhibitor of DNMT1 activity in cell-free and cell-based assays. However, screening in the NCI 60 tumor cell lines and analysis using COMPARE indicated a novel mechanism of action for XB05, different from standard demethylating agents such as 5-azacytidine (azaC) and decitabine. Here, we report on new research to further characterize XB05 activity. We have examined the effects of XB05 on clonogenicity, promoter methylation, gene expression, and DNMT1 protein. XB05 was found to inhibit colony formation of HCT116 colon carcinoma cells in a dose-dependent manner with a sub-micromolar IC50. Western blots of nuclear extracts from HCT116 cells treated for 72 h with 100 nM XB05 or 10 µM azaC (as positive control) showed greatly decreased levels of DNMT1, suggesting that binding to XB05 can induce degradation of DNMT1 (previous research indicates direct drug-enzyme binding). Analysis of CDKN2A promoter methylation by bisulfite modification and sequencing demonstrated heavy methylation in untreated HCT116 cells. This was reversed by treatment of with 100 nM XB05 or 10 µM azaC, resulting in re-expression of the gene (which encodes tumor suppressor, p16), as shown by qRT-PCR. Experiments to assess promoter methylation using multiplex arrays and to examine gene expression changes using DNA microarrays were recently completed and data analysis is ongoing. To evaluate in vivo activity, we treated mice bearing subcutaneous HCT116 xenografts by IP injection of XB05 with multiple doses of up to 10 mg/kg. There was no evidence of toxicity as judged by body weights and gross necropsies. The tumors of mice treated with XB05, although not significantly smaller than in control animals, were characterized by extensive necrosis in the center of the tumor, leading to a “hollow” tumor in the majority (12/20) of mice. No similar necrosis was observed in vehicle-treated or azaC-treated mice, suggesting that this effect is treatment related. Because induced necrosis is seen with vascular disrupting agents, we also examined the effect of XB05 on endothelial cells in an in vitro assay, and observed reproducible inhibition of endothelial tube formation at 400 nM or higher. In summary, our new data confirm that XB05 is a potent inhibitor of DNA methylation in cultured cancer cells, and provide the first evidence of in vivo activity, which may involve effects on the tumor vasculature, as well as the tumor itself. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5447.</jats:p

Fibroblast-derived ECM induce Directional Migration.

<p>(A) Cells were seeded on ECM or fibronectin and placed in a humidified temperature controlled chamber. Fields of cells were photographed every 10 minutes for approximately 8 hours. Resultant time-lapse tracks of A549, H358, and HPL1D on WI38 ECM were followed and indicated. (B) Directionality of the cells on different substrates was calculated. N = 7. (C) The average velocity of migrated cells was calculated from the time-lapse microscopy (μm/hour) n = 7. *, p-value ≤ 0.05.</p

Single-cell transcriptomics of melanoma sentinel lymph nodes identifies immune cell signatures associated with metastasis

The sentinel lymph node (SLN) is the first lymph node encountered by a metastatic cancer cell and serves as a predictor of poor prognosis, as cases with clinically occult SLN metastases are classified as stage III with elevated rates of recurrence and diminished overall survival. However, the dynamics of immune infiltrates in SLNs remain poorly characterized. Here, using an unbiased cellular indexing of transcriptomes and epitopes by sequencing technique, we profiled 97,777 cells from SLN tissues obtained from patients with stages I/II and III cutaneous melanoma. We described the transcriptional programs of a multitude of T, B, and myeloid cell subtypes in SLNs. Based on the proportions of cell types, we determined that SLN subtypes stratified along a naive → activated axis; patients with a “high activated” signature score appeared to be undergoing a robust melanoma antigen–driven adaptive immune response and, thus, could be responsive to immunotherapy. Additionally, we identified transcriptomic signatures of SLN-infiltrating dendritic cell subsets that compromise antitumor immune responses. Our analyses provide valuable insights into tumor-driven immune changes in the SLN tissue, offering a powerful tool for the informed design of immune therapies for patients with high-risk melanoma

Characterization of fibroblasts and their ECM.

<p>A. Microscopic analysis of fibroblasts and de-cellularized matrix. Left colum- 5X phase contrast imaging of confluent fibroblasts just prior to de-cellularazation. Middle colum- 40X phase contrast microscopy of ECM following decellularization. Right column- Immunofluorescent confocal microscopy of fibroblast-derived ECM using an NHS-ester probe conjugated to Alexa Fluor 488. (B) Protein content of ECM was quantitated after scraping of the ECM from decellularized plates. Protein amount is indicated a μg/cm², N = 3. (C) SDS-PAGE and colloidal blue stain of fibroblast-derived ECM. n = 3.</p

Fibroblast-derived ECM alters mRNA profile of A549 and H358 Cells.

<p>Heat map significantly changed genes from microarray (fold-change >1.5 and p-value ≤ 0.05). Columns represent individual gene probes; rows are the different samples and each row represents one of the biological triplicates ran on the microarray. (B) Validation of microarray data. Representative genes were chosen for quantitative real-time qRT-PCR analysis. New biological triplicates were prepared, RNA extracted and converted to cDNA, and real-time qRT-PCR was performed. All samples tested were validated and all genes were changed in the same direction as the microarray, however some amplitudes of change were slightly different.</p

Single-Cell Immune Mapping of Melanoma Sentinel Lymph Nodes Reveals an Actionable Immunotolerant Microenvironment

Abstract Purpose: Improving our understanding of the immunologic response to cancer cells within the sentinel lymph nodes (SLN) of primary tumors is expected to identify new approaches to stimulate clinically meaningful cancer immunity. Experimental Design: We used mass cytometry by time-of-flight (CyTOF), flow cytometry, and T-cell receptor immunosequencing to conduct simultaneous single-cell analyses of immune cells in the SLNs of patients with melanoma. Results: We found increased effector-memory αβ T cells, TCR clonality, and γδ T cells selectively in the melanoma-bearing SLNs relative to non–melanoma-bearing SLNs, consistent with possible activation of an antitumor immune response. However, we also observed a markedly immunotolerant environment in the melanoma-bearing SLNs indicated by reduced and impaired NK cells and increased levels of CD8+CD57+PD-1+ cells, which are known to display low melanoma killing capabilities. Other changes observed in melanoma-bearing SLNs when compared with non–melanoma-bearing SLNs include (i) reduced CD8+CD69+ T cell/T regulatory cell ratio, (ii) high PD-1 expression on CD4+ and CD8+ T cells, and (iii) high CTLA-4 expression on γδ T cells. Conclusions: Our data suggest that these immunologic changes compromise antimelanoma immunity and contribute to a high relapse rate. We propose the development of clinical trials to test the neo-adjuvant administration of anti–PD-1 antibodies prior to SLN resection in patients with stage III melanoma. See related commentary by Lund, p. 1996 </jats:sec

A rapid assessment of wastewater for genomic surveillance of SARS-CoV-2 variants at sewershed scale in Louisville, KY

AbstractIn this communication, we report on the genomic surveillance of SARS-CoV-2 using wastewater samples in Jefferson County, KY. In February 2021, we analyzed seven wastewater samples for SARS-CoV-2 genomic surveillance. Variants observed in smaller catchment areas, such as neighborhood manhole locations, were not necessarily consistent when compared to associated variant results in downstream treatment plants, suggesting catchment size or population could impact the ability to detect diversity.</jats:p

Fluorovinylsulfones and -Sulfonates as Potent Covalent Reversible Inhibitors of the Trypanosomal Cysteine Protease Rhodesain: Structure–Activity Relationship, Inhibition Mechanism, Metabolism, and In Vivo Studies

Rhodesain is a major cysteine protease of Trypanosoma brucei rhodesiense, a pathogen causing Human African Trypanosomiasis, and a validated drug target. Recently, we reported the development of α-halovinylsulfones as a new class of covalent reversible cysteine protease inhibitors. Here, α-fluorovinylsulfones/-sulfonates were optimized for rhodesain based on molecular modeling approaches. 2d, the most potent and selective inhibitor in the series, shows a single-digit nanomolar affinity and high selectivity toward mammalian cathepsins B and L. Enzymatic dilution assays and MS experiments indicate that 2d is a slow-tight binder (Ki = 3 nM). Furthermore, the nonfluorinated 2d-(H) shows favorable metabolism and biodistribution by accumulation in mice brain tissue after intraperitoneal and oral administration. The highest antitrypanosomal activity was observed for inhibitors with an N-terminal 2,3-dihydrobenzo­[b]­[1,4]­dioxine group and a 4-Me-Phe residue in P2 (2e/4e) with nanomolar EC50 values (0.14/0.80 μM). The different mechanisms of reversible and irreversible inhibitors were explained using QM/MM calculations and MD simulations