Targeting CIC::DUX4 sarcoma with Minnelide in a dual recombinase–initiated genetically engineered mouse model
MaKenna R. Browne, Axel V. Silver, Risha Banerjee, Brendan C. Dickson, Benigno Aquino, Kristianne M. Oristian, Jonathon Himes, Peter G. Hendrickson, David G. Kirsch
MaKenna R. Browne, Axel V. Silver, Risha Banerjee, Brendan C. Dickson, Benigno Aquino, Kristianne M. Oristian, Jonathon Himes, Peter G. Hendrickson, David G. Kirsch
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Research Article Cell biology Oncology

Targeting CIC::DUX4 sarcoma with Minnelide in a dual recombinase–initiated genetically engineered mouse model

Abstract

CIC::DUX4 sarcoma (CDS) is a lethal cancer driven by a fusion between the tumor suppressor capicua (CIC) and the pioneer transcription factor double homeobox 4 (DUX4). We previously generated 3 genetically engineered mouse models (GEMMs) of CDS with CIC::DUX4 regulated by loxP-STOP-loxP cassettes, however, mice from all 3 models developed spontaneous tumors without Cre recombinase. Here, we established a next-generation GEMM of CDS (dual-flex [dFLEx] CDS) that used a dual recombinase (Cre plus the thermostable mutant of FLP recombinase FLPE) FLEx-switch design to activate CIC::DUX4 expression and initiate sarcomagenesis in a spatially and temporally controlled manner. Because CIC::DUX4 drives sarcoma development by activating an oncogenic transcriptional program, we performed a drug screen on human-derived CDS cell lines using a library of compounds that modulate transcription. This screen identified Minnelide, an inhibitor of RNA polymerase II–mediated transcription, as a selective inhibitor of CDS. Mechanistically, Minnelide acted through xeroderma pigmentosum type B to alter phosphorylation of RPB1, the largest subunit of RNA polymerase II. Subsequently, RPB1 underwent degradation leading to apoptosis of CDS cells. Minnelide demonstrated in vivo efficacy in dFLEx CDS GEMMs and in human CDS xenografts. As Minnelide has already been demonstrated to be safe in clinical trials, these findings identify Minnelide as a potential therapeutic option to test in patients with CDS.

Authors

MaKenna R. Browne, Axel V. Silver, Risha Banerjee, Brendan C. Dickson, Benigno Aquino, Kristianne M. Oristian, Jonathon Himes, Peter G. Hendrickson, David G. Kirsch

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Figure 4

Minnelide targets XPB, leading to RPB1 degradation and transcriptional inhibition.

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Minnelide targets XPB, leading to RPB1 degradation and transcriptional i...
(A) Schematic showing Minnelide inhibition of RNAP II. Minnelide directly binds 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, and then proteasome-mediated degradation of RPB1. (B) Western blot of RPB1 expression in human CDS cells (Kitra-SRS, ECD1, X1C1, and CDS#2) after treatment with 25 nM Minnelide for 48 hours. (C) Western blot of RPB1 expression and p-RPB1 over a time course of Minnelide treatment in human ECD1 cells. (D) Quantification of p-RPB1 (Ser5) expression (E) Western blot demonstrating that the proteasome inhibitor epoxomicin partially rescued Minnelide-mediated RPB1 degradation in CDS cells (Kitra-SRS, X1C1, CDS#2). (F) Western blot demonstrating that the expression of XPB C342T in Kitra-SRS, ECD1, and X1C1 human CDS cells rescued Minnelide-mediated RPB1 degradation. GAPDH was used as the loading control for Kitra-SRS and ECD1 cells, whereas β-actin was used as the loading control for X1C1 cells. (G) CellTiter-Glo assay demonstrated that human CDS cells expressing XPB C342T had increased resistance to Minnelide. (H) γH2A.X immunofluorescence on human CDS#2 cells treated with Minnelide for 48 hours. Scale bars: 5 μm. At least 50 nuclei per condition were quantified, including nuclei sampled from 4 independent experiments. *P < 0.05, by 2-tailed exact Mann-Whitney U test. (I) DNA fiber assay on human Kitra-SRS cells treated with Minnelide (Minn) for 48 hours. Ctrl, control. At least 100 fibers per condition were quantified, including fibers sampled from 4 independent experiments. *P < 0.05, by 2-tailed, exact Mann-Whitney U test. Data are presented as the mean ± SEM.