Oxidative Stress is a shared characteristic of ME/CFS and Long COVID.

More than 65 million individuals worldwide are estimated to have Long COVID (LC), a complex multisystemic condition, wherein patients of all ages report fatigue, post-exertional malaise, and other symptoms resembling myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS). With no current treatments or reliable diagnostic markers, there is an urgent need to define the molecular underpinnings of these conditions. By studying bioenergetic characteristics of peripheral blood lymphocytes in over 16 healthy controls, 15 ME/CFS, and 15 LC, we find both ME/CFS and LC donors exhibit signs of elevated oxidative stress, relative to healthy controls, especially in the memory subset. Using a combination of flow cytometry, bulk RNA-seq analysis, mass spectrometry, and systems chemistry analysis, we also observed aberrations in ROS clearance pathways including elevated glutathione levels, decreases in mitochondrial superoxide dismutase levels, and glutathione peroxidase 4 mediated lipid oxidative damage. Critically, these changes in redox pathways show striking sex-specific trends. While females diagnosed with ME/CFS exhibit higher total ROS and mitochondrial calcium levels, males with an ME/CFS diagnosis have normal ROS levels, but larger changes in lipid oxidative damage. Further analyses show that higher ROS levels correlates with hyperproliferation of T cells in females, consistent with the known role of elevated ROS levels in the initiation of proliferation. This hyperproliferation of T cells can be attenuated by metformin, suggesting this FDA-approved drug as a possible treatment, as also suggested by a recent clinical study of LC patients. Thus, we report that both ME/CFS and LC are mechanistically related and could be diagnosed with quantitative blood cell measurements. We also suggest that effective, patient tailored drugs might be discovered using standard lymphocyte stimulation assays.

Disparate pathways for extrachromosomal DNA biogenesis and genomic DNA repair.

Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. Here, we couple a CRISPR-based method for induction of ecDNA with extensive characterization of newly formed ecDNA to examine ecDNA biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with formation of a 1 Mb and 1.8 Mb ecDNA both reaching 15%. We show non-homologous end joining and microhomology mediated end joining both contribute to ecDNA formation, while inhibition of DNA-PKcs and ATM have opposing impacts on ecDNA formation. EcDNA and the corresponding chromosomal excision scar form at significantly different rates and respond differently to DNA-PKcs and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar.

A Cell Type Selective YM155 Prodrug Targets Receptor-Interacting Protein Kinase 2 to Induce Brain Cancer Cell Death.

Glioblastoma (GBM) is the most prevalent and aggressive primary central nervous system (CNS) malignancy. YM155 is a highly potent broad-spectrum anti-cancer drug that was derived from a phenotypic screen for functional inhibitors of survivin expression, but for which the relevant biomolecular target remains unknown. Presumably as a result of its lack of cell-type selectivity, YM155 has suffered from tolerability issues in the clinic. Based on its structural similarity to the GBM-selective prodrug RIPGBM, here, we report the design, synthesis, and characterization of a prodrug form of YM155, termed aYM155. aYM155 displays potent cell killing activity against a broad panel of patient-derived GBM cancer stem-like cells (IC50 = 0.7-10 nM), as well as EGFR-amplified and EGFR variant III-expressing (EGFRvIII) cell lines (IC50 = 3.8-36 nM), and becomes activated in a cell-type-dependent manner. Mass spectrometry-based analysis indicates that enhanced cell-type selectivity results from relative rates of prodrug activation in transformed versus non-transformed cell types. The prodrug strategy also facilitates transport into the brain (brain-to-plasma ratio, aYM155 = 0.56; YM155 = BLQ). In addition, we determine that the survivin-suppressing and apoptosis-inducing activities of YM155 involve its interaction with receptor-interacting protein kinase 2 (RIPK2). In an orthotopic intracranial GBM xenograft model, aYM155 prodrug significantly inhibits brain tumor growth in vivo, which correlates with cell-type selective survivin-based pharmacodynamic effects.

Targeting glioblastoma signaling and metabolism with a re-purposed brain-penetrant drug.

The highly lethal brain cancer glioblastoma (GBM) poses a daunting challenge because the blood-brain barrier renders potentially druggable amplified or mutated oncoproteins relatively inaccessible. Here, we identify sphingomyelin phosphodiesterase 1 (SMPD1), an enzyme that regulates the conversion of sphingomyelin to ceramide, as an actionable drug target in GBM. We show that the highly brain-penetrant antidepressant fluoxetine potently inhibits SMPD1 activity, killing GBMs, through inhibition of epidermal growth factor receptor (EGFR) signaling and via activation of lysosomal stress. Combining fluoxetine with temozolomide, a standard of care for GBM, causes massive increases in GBM cell death and complete tumor regression in mice. Incorporation of real-world evidence from electronic medical records from insurance databases reveals significantly increased survival in GBM patients treated with fluoxetine, which was not seen in patients treated with other selective serotonin reuptake inhibitor (SSRI) antidepressants. These results nominate the repurposing of fluoxetine as a potentially safe and promising therapy for patients with GBM and suggest prospective randomized clinical trials.

BAP1 methylation: a prognostic marker of uveal melanoma metastasis.

Uveal melanoma, the most common intraocular primary cancer in adults, is characterized by striking variability in metastatic tendencies. BAP1 deletion in the primary tumor is associated with uveal melanoma metastasis, but it cannot always be resolved by bulk DNA sequencing of heterogeneous tumors. Here, we show that assessment of BAP1 methylation is an accurate and readily clinically actionable assay to accurately identify high-risk uveal melanoma patients.

Investigations into the DNA-binding mode of doxorubicinone.

Cancer treatment is one of the major challenges facing the modern biomedical profession. Development of new small-molecule chemotherapeutics requires an understanding of the mechanism of action for these treatments, as well as the structure-activity relationship. Study of the well-known DNA-intercalating agent, doxorubicin, and its aglycone, doxorubicinone, was undertaken using a variety of spectroscopic and calorimetric techniques. It was found that, despite conservation of the planar, aromatic portion of doxorubicin, the agylcone does not intercalate; it instead likely binds to the DNA minor-groove.