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 1

dFLEx CDS mice develop tumors only after expression of Cre and FLPE.

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dFLEx CDS mice develop tumors only after expression of Cre and FLPE. (A)...
(A) Schematic of the dFLEx CDS allele before and after recombination with Cre and FLPE. PCR primer sequences used to assess recombination (Supplemental Figure 1L) are indicated with Primer 1F/R and Primer 2F/R arrows. (B) Kaplan-Meier survival curve of dFLEx CDS mice showing no spontaneous tumor development without Cre and FLPE and approximately 90% tumor penetrance after electroporation of the hind limb muscle with plasmids expressing Cre plus FLPE. (C) Gross images of a hind limb tumor. (D–J) Representative photomicrographs highlighting histologic and immunohistochemical similarity between dFLEx CDS model and clinical examples of CIC::DUX4 sarcoma. (D and E) Necrosis in tumors originating from a dFLEx CDS mouse and the foot of a 22-year-old male individual (H&E; original magnification, ×100). (F and G) Sheets of monomorphic round cells, with occasional interspersed epithelioid cells, originating from a dFLEx CDS mouse and the shoulder of an 18-year-old female individual (H&E; original magnification, ×400). (H and I) Vague fascicles of monomorphic, round-ovoid cells with mitotic activity (arrowheads) and myxoid stroma originating from dFLEx CDS mouse and the thigh of a 14-year-old male individual (H&E; original magnification, ×400). (J) Diffuse nuclear staining for WT1 in cells from a dFLEx CDS mouse and a 22-year-old male individual (IHC; original magnification, ×200). (K) IHC of tumors formed in dFLEx CDS mice after electroporation of Cre plus FLPE plasmids for HA tag and ETV4 (scale bars: 50 μm). Supplemental Figure 1A shows an expanded staining panel; representative ETV4 and HA tag images from K are included again for completeness. (L) Pearson correlation of dFLEx CDS and KRASG12D Trp53fl/fl (KP) tumor transcriptional profiles to fusion-positive human sarcoma cell lines.