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Anaplastic thyroid cancer is among the most aggressive of human cancers, and currently there are few effective treatments for most patients. YM155, first identified as a survivin inhibitor, was highlighted in a high-throughput screen performed by the National Cancer Institute, killing anaplastic thyroid cancer cells in vitro and in vivo. However, there was no association between survivin expression and response to YM155 in clinical trials, and YM155 has been mostly abandoned for development despite favorable pharmacokinetic and toxicity profiles. Currently, alternative mechanisms are being explored for YM155 by a number of groups. In this study, anaplastic thyroid cancer patient samples show overexpression of topoisomerase Top2α compared to benign thyroid samples and to differentiated thyroid cancers. Anaplastic thyroid cancer cell lines that overexpress Top2α are more sensitive to YM155. We created a YM155-resistant cell line, which showed decreased expression of Top2α and is re-sensitized with Top2α overexpression. Molecular modeling predicts binding for YM155 in the Top2α ATP binding site and identifies key amino acids for YM155-Top2α interaction. A Top2α mutant abrogates the effect of YM155, confirming the contribution of Top2α to YM155 mechanism of action. Our results suggest a novel mechanism of action for YM155 and may represent a new therapeutic approach for the treatment of anaplastic thyroid cancer.

Supplementary Figure from YM155 Induces DNA Damage and Cell Death in Anaplastic Thyroid Cancer Cells by Inhibiting DNA Topoisomerase IIα at the ATP-Binding Site

Anaplastic thyroid cancer (ATC) is among the most aggressive of human cancers, and currently there are few effective treatments for most patients. YM155, first identified as a survivin inhibitor, was highlighted in a high-throughput screen performed by the National Cancer Institute, killing ATC cells in vitro and in vivo. However, there was no association between survivin expression and response to YM155 in clinical trials, and YM155 has been mostly abandoned for development despite favorable pharmacokinetic and toxicity profiles. Currently, alternative mechanisms are being explored for YM155 by a number of groups. In this study, ATC patient samples show overexpression of topoisomerase Top2α compared with benign thyroid samples and to differentiated thyroid cancers. ATC cell lines that overexpress Top2α are more sensitive to YM155. We created a YM155-resistant cell line, which shows decreased expression of Top2α and is resensitized with Top2α overexpression. Molecular modeling predicts binding for YM155 in the Top2α ATP-binding site and identifies key amino acids for YM155–Top2α interaction. A Top2α mutant abrogates the effect of YM155, confirming the contribution of Top2α to YM155 mechanism of action. Our results suggest a novel mechanism of action for YM155 and may represent a new therapeutic approach for the treatment of ATC.

Atherosclerosis (ASVD) is characterized by the buildup of lipids and fibrous elements in large arteries creating a plaque. As it progresses, apoptotic cells (AC) accumulate forming a necrotic core making the plaque unstable and prone to rupture. Rupture can lead to artery occlusion and moreover to stroke or myocardial infarction (MI). A main contributor to a large necrotic core is the inability of macrophages to clear accumulated AC and as a result undergo secondary necrosis. This clearance process, referred to as efferocytosis, is impaired in advanced atherosclerotic lesions. It has been shown that the process that leads to a thin fibrous cap and a large necrotic core are accelerated by a decrease in glutathione (GSH) availability by a deletion of the modifier subunit of Glutamate Cysteine Ligase (GCLM) in mice. Additionally, it has also been shown that a single nucleotide polymorphism (SNP) in the promoter region of human GCLM (-588 C/T) has an increased risk for MI. Our objective is to identify the role and mechanism of the GCLM SNP during the formation of the necrotic core in atherosclerosis. We hypothesize that proper function of GCLM and GSH is important in macrophages for adequate function and clearance of AC in ASVD. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from blood donors, genotyped to test for the presence of SNP, and used for RNA sequencing. Efferocytic index was measured using labelled AC in GCLM WT, Het, and KO murine bone marrow derived macrophages (BMDM) and PBMCs. Percent positive macrophages containing fluorescently labelled AC were quantified for each sample. Cells were collected for total protein, surface receptor expression, and mRNA analysis. Results: Efferocytosis was affected in macrophages with defective GCLM as compared to control. RNA analysis has showed a decrease in efferocytosis receptors such as MerTK, confirmed by cell surface expression. Conclusion: These results suggest that loss of GCLM and GSH in macrophages impairs adequate efferocytosis.

Introduction: In addition to its classic role in redox chemistry, glutathione (GSH) also acts as an allosteric regulator of the calcium-sensing receptor (CaSR). CaSR has been found in a variety of cell types including endothelial cells (ECs), where its activity has been linked to EC adhesion and migration. Although a relationship between GSH and CaSR has been discovered, no link has been established between GSH-CaSR interaction and regulation of angiogenesis. We hypothesize a novel mechanism for GSH regulation of blood vessel development occurs through an extracellular interaction with CaSR as a part of physiological and pathological angiogenesis. Methods: Matrigel tube formation and wound healing assays were used to measure EC activity. RGECO1.2 was used to measure changes in intracellular calcium. Cell surface expression assay was used to measure surface CaSR expression. Cell counts and flow cytometry were used to monitor cell proliferation and cell cycle progression. Endothelial and mesenchymal markers were measured using real-time PCR. Results: Extracellular GSH increased endothelial tube length and branching in tube formation assay, while a selective CaSR inhibitor abrogated these effects. GSH also influenced EC migration as measured by wound healing assay. GSH or a CaSR agonist (R-568) reduced surface CaSR expression, with no changes in intracellular calcium. Extracellular GSH alters EC proliferation and cell cycle progression, coinciding with alterations in endothelial phenotype along the endothelial-mesenchymal spectrum. Conclusion: In addition to its classic antioxidant role, GSH appears to act extracellularly to influence EC activity. GSH caused significant increases in EC branching as measured by tube formation assay. The use of a selective CaSR inhibitor confirms the contribution of CaSR to this effect. Additionally, GSH influenced EC migration in wound healing assays. These results could be explained by GSH-CaSR-mediated alterations in endothelial differentiation. These results suggest a role for GSH in vascular development and angiogenesis, through a ligand interaction with the CaSR G-protein coupled receptor, representing a novel mechanism of action for physiological and pathological blood vessel development.

Anaplastic thyroid cancer (ATC) is one of the most lethal malignancies with a median survival time of about 4 months. Currently, there is no effective treatment, and the development of new therapies is an important and urgent issue for ATC patients. YM155 is a small molecule that was identified as the top candidate in a high-throughput screen of small molecule inhibitors performed against a panel of ATC cell lines by the National Cancer Institute. However, there were no follow-up studies investigating YM155 in ATC. Here, we determined the effects of YM155 on ATC and human primary benign thyroid cell (PBTC) survival with alamarBlue assay. Our data show that YM155 inhibited proliferation of ATC cell lines while sparing normal thyroid cells, suggesting a high therapeutic window. YM155-induced DNA damage was detected by measuring phosphorylation of γ-H2AX as a marker for DNA double-strand breaks. The formamidopyrimidine-DNA glycosylase (FPG)-modified alkaline comet assay in conjunction with reactive oxygen species (ROS) assay and glutathione (GSH)/glutathione (GSSG) assay suggests that YM155-mediated oxidative stress contributes to DNA damage. In addition, we provide evidence that YM155 causes cell cycle arrest in S phase and in the G2/M transition and causes apoptosis, as seen with flow cytometry. In this study, we show for the first time the multiple effects of YM155 in ATC cells, furthering a potential therapeutic approach for ATC.

Physiological and pathological roles for R-loop structures continue to be discovered, and studies suggest that R-loops could contribute to human disease. R-loops are nucleic acid structures characterized by a DNA:RNA hybrid and displaced single-stranded DNA that occur in connection with transcription. R-loops form naturally and have been shown to be important for a number of physiological processes such as mitochondrial replication initiation, class switch recombination, DNA repair, modulating DNA topology, and regulation of gene expression. However, subsets of R-loops or persistent R-loops lead to DNA breaks, chromosome rearrangement, and genome instability. In addition, R-loops have been linked to human diseases, specifically neurological disorders and cancer. Of the large amount of research produced recently on R-loops, this review covers evidence for R-loop involvement in normal cellular physiology and pathophysiology, as well as describing factors that contribute to R-loop regulation.

Introduction: Anaplastic thyroid cancer (ATC) is a rapidly fatal disease with a median survival of less than 6 months. Although rare, ATC is responsible for a disproportionate number of thyroid cancer deaths and is highly resistant to common cancer therapies. YM155 is a small molecule drug identified in a screen by the National Cancer Institute that potently kills ATC cells in vitro and in vivo. We have previously shown the ability of YM155 to inhibit ATC cell proliferation, however, the mechanism is still unclear. This study investigates the DNA damage and accumulation of R-loops that is seen with YM155 treatment of ATC cells. R-loops are structures characterized by a DNA:RNA hybrid with a displaced single strand of DNA and can be formed by transcription-replication collision, nucleotide depletion, and other situations where transcription is stalled. Materials & Methods: Clonogenic assays were performed in four ATC cell lines (ACT1, THJ11T, THJ16T, and THJ29T) to study the effect of YM155 on cell survival. DNA damage was measured by COMET assay and by counting foci of phosphorylated histone H2AX (γH2AX). R-loops were measured via immunofluorescent microscopy with S9.6 anti-R-loop antibody and anti-nucleolin antibody in conjunction with an AI algorithm using inFormTM cell segmentation software to quantify non-nucleolar nuclear R-loop signal. Results: We show in multiple ATC cell lines that YM155 potently kills ATC cells at low nanomolar concentrations. COMET assay found significant increases in DNA damage after treatment with 10 nM YM155, with large increases in tail moment seen by 24 hours in three ATC cell lines (p &lt 0.001 for all) with no change in primary benign thyroid (BT) cells. Similar trends were seen when measuring formation of γH2AX foci, a marker for double strand DNA breaks. R-loop immunofluorescence more than doubled with 10 nM YM155 treatment (p &lt 0.001) similar to hydroxyurea, a positive control. This finding was confirmed when addition of RNAseH, which is specific for RNA in DNA:RNA hybrids, reduced R-loop signal down to baseline levels. Conclusion: YM155's ability to kill ATC cells at low concentrations while sparing BT cells coincides with increases in DNA damage and accumulation of R-loops. A variety of factors aid in the resolution of R-loops, and we have previously shown patient data exhibiting overexpression of DNA topoisomerase genes TOP1 and TOP2A. Combined with published information describing the ability of YM155 to inhibit Topoisomerase Iiα (Top2α) activity, these results suggest a newly revealed, targetable aspect of ATC pathophysiology and a potential novel role for the involvement of Top2α in the resolution of R-loops. Citation Format: Ryan Mackay, Qinqin Xu, Paul Weinberger. Accumulation of DNA:RNA hybrids (R-loops) and cell death after YM155 treatment of anaplastic thyroid cancer hints at a novel therapeutic approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1838.

Aqueous and ethanol extracts from the mycelia of Cordyceps Ophioglossoides have been used as a nutritional supplement, especially for women suffering from massive postpartum vaginal bleeding in Southwest China...

Skeletal muscle-specific liver kinase B1 (LKB1) knockout mice (skmLKB1-KO) exhibit elevated mitogen-activated protein kinase (MAPK) signaling after treadmill running. MAPK activation is also associated with inflammation-related signaling in skeletal muscle. Since exercise can induce muscle damage, and inflammation is a response triggered by damaged tissue, we therefore hypothesized that LKB1 plays an important role in dampening the inflammatory response to muscle contraction, and that this may be due in part to increased susceptibility to muscle damage with contractions in LKB1-deficient muscle. Here we studied the inflammatory response and muscle damage with in situ muscle contraction or downhill running. After in situ muscle contractions, the phosphorylation of both NF-κB and STAT3 was increased more in skmLKB1-KO vs. wild-type (WT) muscles. Analysis of gene expression via microarray and RT-PCR shows that expression of many inflammation-related genes increased after contraction only in skmLKB1-KO muscles. This was associated with mild skeletal muscle fiber membrane damage in skmLKB1-KO muscles. Gene markers of oxidative stress were also elevated in skmLKB1-KO muscles after contraction. Using the downhill running model, we observed significantly more muscle damage after running in skmLKB1-KO mice, and this was associated with greater phosphorylation of both Jnk and STAT3 and increased expression of SOCS3 and Fos. In conclusion, we have shown that the lack of LKB1 in skeletal muscle leads to an increased inflammatory state in skeletal muscle that is exacerbated by muscle contraction. Increased susceptibility of the muscle to damage may underlie part of this response.