Journal: bioRxiv

Article Title: Efficacy of Minnelide in a Next-Generation Dual-Recombinase Regulated Genetically Engineered Mouse Model of CIC::DUX4 Sarcoma

doi: 10.1101/2025.11.06.687065

Figure Lengend Snippet: A. Schematic for Minnelide inhibition of RNAP II. Minnelide directly binds xeroderma pigmentosum type B (XPB). Inhibition of XPB’s ATPase activity leads to stalling of RNAP II at gene promoters, and inhibition of transcription. Prolonged stalling of RNAP II results in altered phosphorylation patterns on RPB1, ubiquitination, then proteasomal mediated degradation of RPB1. B. Western blot of RPB1 and phosphorylated RPB1 over a time course of Minnelide treatment in human Kitra-SRS CDS cells. C. Western blot demonstrating proteosome inhibitor Epoxomicin rescues Minnelide mediated RPB1 degradation in Kitra-SRS CDS cells. D. Strategy for generating a Minnelide-resistant XPB mutant. E. Western blot demonstrating expression of XPB C342T in Kitra-SRS and ECD1 human CDS cells rescues Minnelide mediated RPB1 degradation. F. Cell-titer-glo assay demonstrates human ECD1 CDS cells expressing XPB C342T have increased resistance to Minnelide. G. Schematic for Minnelide time course in Kitra-SRS human CDS cells. H. Venn diagrams representing transcriptional changes during early, mid and late Minnelide treatment times. I. Volcano plots demonstrating transcriptional changes following Minnelide treatment as a function of time.

Article Snippet: The following antibodies were used for immunohistochemistry: HA (Cell Signaling, 3724) at 1:800, ETV4 (Proteintech 10684-1-AP) at 1:1800, CD99 (Thermo-Fisher, MA5-12287) at 1:200, WT1 (Thermo-Fisher, MA5-32215) at 1:2000, Pan-cytokeratin (abcam, ab9377) at 1:200, Desmin (abcam, ab15200) at 1:200 and Phospho-Rpb1 CTD (Ser2/Ser5) (Cell Signaling, 13546) at 1:100.

Techniques: Inhibition, Activity Assay, Phospho-proteomics, Ubiquitin Proteomics, Western Blot, Mutagenesis, Expressing, Glo Assay

Journal: bioRxiv

Article Title: Efficacy of Minnelide in a Next-Generation Dual-Recombinase Regulated Genetically Engineered Mouse Model of CIC::DUX4 Sarcoma

doi: 10.1101/2025.11.06.687065

Figure Lengend Snippet: A. Dose selection was informed by the recommended starting dose and maximum tolerated dose reported in the Phase I Minnelide clinical trial for GI carcinoma ( NCT01927965 ) 0.21 mg/kg and 0.27 mg/kg approximates the human dose of 0.67 mg/m 2 and 0.80 mg/m 2 respectively B. NSG mice were inoculated with Kitra-SRS or ECD1 human CDS cells then treated with Minnelide at 0.21 mg/kg or 0.27 mg/kg daily for 21 days. Tumor growth was significantly reduced in Minnelide-treated groups compared to controls (one-way ANOVA, *P < 0.05, ***P < 0.001, and ****P < 0.0001 at Day 21). C. Gross morphology of Kitra-SRS dissected tumors at humane endpoint or at 21 days of treatment. D . p-RPB1 Ser2/5 IHC on sections from saline and Minnelide treated Kitra-SRS CDS xenograft tumors at 14, 18 and 21 days of Minnelide treatment. E. Western blot of RPB1 expression in Kitra-SRS CDS xenograft tumors over a 21-day treatment period with 0.27mg/kg daily. Quantification of RPB1 expression relative to GAPDH loading control.

Article Snippet: The following antibodies were used for immunohistochemistry: HA (Cell Signaling, 3724) at 1:800, ETV4 (Proteintech 10684-1-AP) at 1:1800, CD99 (Thermo-Fisher, MA5-12287) at 1:200, WT1 (Thermo-Fisher, MA5-32215) at 1:2000, Pan-cytokeratin (abcam, ab9377) at 1:200, Desmin (abcam, ab15200) at 1:200 and Phospho-Rpb1 CTD (Ser2/Ser5) (Cell Signaling, 13546) at 1:100.

Techniques: Selection, Saline, Western Blot, Expressing, Control