The Lipid Metabolism as Target and Modulator of BOLD-100 Anticancer Activity: Crosstalk with Histone Acetylation.

The leading first-in-class ruthenium-complex BOLD-100 currently undergoes clinical phase-II anticancer evaluation. Recently, BOLD-100 is identified as anti-Warburg compound. The present study shows that also deregulated lipid metabolism parameters characterize acquired BOLD-100-resistant colon and pancreatic carcinoma cells. Acute BOLD-100 treatment reduces lipid droplet contents of BOLD-100-sensitive but not -resistant cells. Despite enhanced glycolysis fueling lipid accumulation, BOLD-100-resistant cells reveal diminished lactate secretion based on monocarboxylate transporter 1 (MCT1) loss mediated by a frame-shift mutation in the MCT1 chaperone basigin. Glycolysis and lipid catabolism converge in the production of protein/histone acetylation substrate acetyl-coenzymeA (CoA). Mass spectrometric and nuclear magnetic resonance analyses uncover spontaneous cell-free BOLD-100-CoA adduct formation suggesting acetyl-CoA depletion as mechanism bridging BOLD-100-induced lipid metabolism alterations and histone acetylation-mediated gene expression deregulation. Indeed, BOLD-100 treatment decreases histone acetylation selectively in sensitive cells. Pharmacological targeting confirms histone de-acetylation as central mode-of-action of BOLD-100 and metabolic programs stabilizing histone acetylation as relevant Achilles' heel of acquired BOLD-100-resistant cell and xenograft models. Accordingly, histone gene expression changes also predict intrinsic BOLD-100 responsiveness. Summarizing, BOLD-100 is identified as epigenetically active substance acting via targeting several onco-metabolic pathways. Identification of the lipid metabolism as driver of acquired BOLD-100 resistance opens novel strategies to tackle therapy failure.

Synthesis and Preclinical Evaluation of Radiolabeled [103Ru]BOLD-100.

The first-in-class ruthenium-based chemotherapeutic agent BOLD-100 (formerly IT-139, NKP-1339, KP1339) is currently the subject of clinical evaluation for the treatment of gastric, pancreatic, colorectal and bile duct cancer. A radiolabeled version of the compound could present a helpful diagnostic tool. Thus, this study investigated the pharmacokinetics of BOLD-100 in more detail to facilitate the stratification of patients for the therapy. The synthesis of [103Ru]BOLD-100, radiolabeled with carrier added (c.a.) ruthenium-103, was established and the product was characterized by HPLC and UV/Vis spectroscopy. In order to compare the radiolabeled and non-radioactive versions of BOLD-100, both complexes were fully evaluated in vitro and in vivo. The cytotoxicity of the compounds was determined in two colon carcinoma cell lines (HCT116 and CT26) and biodistribution studies were performed in Balb/c mice bearing CT26 allografts over a time period of 72 h post injection (p.i.). We report herein preclinical cytotoxicity and pharmacokinetic data for BOLD-100, which were found to be identical to those of its radiolabeled analog [103Ru]BOLD-100.

The Metallodrug BOLD-100 Is a Potent Inhibitor of SARS-CoV-2 Replication and Has Broad-Acting Antiviral Activity.

The COVID-19 pandemic has highlighted an urgent need to discover and test new drugs to treat patients. Metal-based drugs are known to interact with DNA and/or a variety of proteins such as enzymes and transcription factors, some of which have been shown to exhibit anticancer and antimicrobial effects. BOLD-100 (sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]dihydrate) is a novel ruthenium-based drug currently being evaluated in a Phase 1b/2a clinical trial for the treatment of advanced gastrointestinal cancer. Given that metal-based drugs are known to exhibit antimicrobial activities, we asked if BOLD-100 exhibits antiviral activity towards SARS-CoV-2. We demonstrated that BOLD-100 potently inhibits SARS-CoV-2 replication and cytopathic effects in vitro. An RNA sequencing analysis showed that BOLD-100 inhibits virus-induced transcriptional changes in infected cells. In addition, we showed that the antiviral activity of BOLD-100 is not specific for SARS-CoV-2, but also inhibits the replication of the evolutionarily divergent viruses Human Immunodeficiency Virus type 1 and Human Adenovirus type 5. This study identifies BOLD-100 as a potentially novel broad-acting antiviral drug.

Utilization of Cancer Cell Line Screening to Elucidate the Anticancer Activity and Biological Pathways Related to the Ruthenium-Based Therapeutic BOLD-100.

BOLD-100 (sodium trans-[tetrachlorobis(1H indazole)ruthenate(III)]) is a ruthenium-based anticancer compound currently in clinical development. The identification of cancer types that show increased sensitivity towards BOLD-100 can lead to improved developmental strategies. Sensitivity profiling can also identify mechanisms of action that are pertinent for the bioactivity of complex therapeutics. Sensitivity to BOLD-100 was measured in a 319-cancer-cell line panel spanning 24 tissues. BOLD-100's sensitivity profile showed variation across the tissue lineages, including increased response in esophageal, bladder, and hematologic cancers. Multiple cancers, including esophageal, bile duct and colon cancer, had higher relative response to BOLD-100 than to cisplatin. Response to BOLD-100 showed only moderate correlation to anticancer compounds in the Genomics of Drug Sensitivity in Cancer (GDSC) database, as well as no clear theme in bioactivity of correlated hits, suggesting that BOLD-100 may have a differentiated therapeutic profile. The genomic modalities of cancer cell lines were modeled against the BOLD-100 sensitivity profile, which revealed that genes related to ribosomal processes were associated with sensitivity to BOLD-100. Machine learning modeling of the sensitivity profile to BOLD-100 and gene expression data provided moderative predictive value. These findings provide further mechanistic understanding around BOLD-100 and support its development for additional cancer types.

The Anticancer Ruthenium Compound BOLD-100 Targets Glycolysis and Generates a Metabolic Vulnerability towards Glucose Deprivation.

Cellular energy metabolism is reprogrammed in cancer to fuel proliferation. In oncological therapy, treatment resistance remains an obstacle and is frequently linked to metabolic perturbations. Identifying metabolic changes as vulnerabilities opens up novel approaches for the prevention or targeting of acquired therapy resistance. Insights into metabolic alterations underlying ruthenium-based chemotherapy resistance remain widely elusive. In this study, colon cancer HCT116 and pancreatic cancer Capan-1 cells were selected for resistance against the clinically evaluated ruthenium complex sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (BOLD-100). Gene expression profiling identified transcriptional deregulation of carbohydrate metabolism as a response to BOLD-100 and in resistance against the drug. Mechanistically, acquired BOLD-100 resistance is linked to elevated glucose uptake and an increased lysosomal compartment, based on a defect in downstream autophagy execution. Congruently, metabolomics suggested stronger glycolytic activity, in agreement with the distinct hypersensitivity of BOLD-100-resistant cells to 2-deoxy-d-glucose (2-DG). In resistant cells, 2-DG induced stronger metabolic perturbations associated with ER stress induction and cytoplasmic lysosome deregulation. The combination with 2-DG enhanced BOLD-100 activity against HCT116 and Capan-1 cells and reverted acquired BOLD-100 resistance by synergistic cell death induction and autophagy disturbance. This newly identified enhanced glycolytic activity as a metabolic vulnerability in BOLD-100 resistance suggests the targeting of glycolysis as a promising strategy to support BOLD-100 anticancer activity.

Inhibition of DNA Repair Pathways and Induction of ROS Are Potential Mechanisms of Action of the Small Molecule Inhibitor BOLD-100 in Breast Cancer.

BOLD-100, a ruthenium-based complex, sodium trans-[tetrachloridobis (1H-indazole) ruthenate (III)] (also known as IT-139, NKP1339 or KP1339), is a novel small molecule drug that demonstrated a manageable safety profile at the maximum tolerated dose and modest antitumor activity in a phase I clinical trial. BOLD-100 has been reported to inhibit the upregulation of the endoplasmic reticulum stress sensing protein GRP78. However, response to BOLD-100 varies in different cancer models and the precise mechanism of action in high-response versus low-response cancer cells remains unclear. In vitro studies have indicated that BOLD-100 induces cytostatic rather than cytotoxic effects as a monotherapy. To understand BOLD-100-mediated signaling mechanism in breast cancer cells, we used estrogen receptor positive (ER+) MCF7 breast cancer cells to obtain gene-metabolite integrated models. At 100 µM, BOLD-100 significantly reduced cell proliferation and expression of genes involved in the DNA repair pathway. BOLD-100 also induced reactive oxygen species (ROS) and phosphorylation of histone H2AX, gamma-H2AX (Ser139), suggesting disruption of proper DNA surveillance. In estrogen receptor negative (ER-) breast cancer cells, combination of BOLD-100 with a PARP inhibitor, olaparib, induced significant inhibition of cell growth and xenografts and increased gamma-H2AX. Thus, BOLD-100 is a novel DNA repair pathway targeting agent and can be used with other chemotherapies in ER- breast cancer.