Supplemental Figures 1-9 from Combined Targeting of the BRD4–NUT–p300 Axis in NUT Midline Carcinoma by Dual Selective Bromodomain Inhibitor, NEO2734

https://doi.org/10.1158/1535-7163.22520908

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posted on 2023-04-03, 18:23 authored by Chevaun D. Morrison-Smith, Tatiana M. Knox, Ivona Filic, Kara M. Soroko, Benjamin K. Eschle, Margaret K. Wilkens, Prafulla C. Gokhale, Francis Giles, Andrew Griffin, Bill Brown, Geoffrey I. Shapiro, Beth E. Zucconi, Philip A. Cole, Madeleine E. Lemieux, Christopher A. French

<p>Supplemental Figure S1: NEO2734 induces differentiation in 10-15 cells. Supplemental Figure S2: Caspase-mediated apoptosis plays a minimal role in the activity of GNE-781, OTX015, or NEO2734. Supplemental Figure S3: BRD4-NUT protein levels decrease slightly upon exposure to NEO2734. Supplemental Figure S4: Global H3K27Ac levels do not change upon exposure to NEO2734. Supplemental Fig. S5. BRD4-NUT foci diminish over time course in TC-797 cells. Images were taken at identical magnification (1000x) with identical exposure settings per stain. Supplemental Figure S6. Dot plots of fold change of gene expression in samples treated with the indicated compound versus that in DMSO-treated samples. Supplemental Figure S7: Proportion of differentially expressed genes overlapping BRD4-NUT megadomains is greater in genes whose expression is downregulated in the presence of p300/CBP and/or BET bromodomain inhibition. Supplemental Figure S8: Tolerability in the full 10-15 mouse xenograft study. Supplemental Figure S9: Cleaved PARP is unchanged, but BRD4-NUT is somewhat depleted in mouse xenograft tumors exposed to NEO2734.</p>

Funding

NCI

NIGMS

Genentech Inc.

NIH

Epigene Therapeutics

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DOI - Is supplement to Combined Targeting of the BRD4–NUT–p300 Axis in NUT Midline Carcinoma by Dual Selective Bromodomain Inhibitor, NEO2734

ARTICLE ABSTRACT

NUT midline carcinoma (NMC) is a rare, aggressive subtype of squamous carcinoma that is driven by the BRD4-NUT fusion oncoprotein. BRD4, a BET protein, binds to chromatin through its two bromodomains, and NUT recruits the p300 histone acetyltransferse (HAT) to activate transcription of oncogenic target genes. BET-selective bromodomain inhibitors have demonstrated on-target activity in patients with NMC, but with limited efficacy. P300, like BRD4, contains a bromodomain. We show that combining selective p300/CBP and BET bromodomain inhibitors, GNE-781 and OTX015, respectively, induces cooperative depletion of MYC and synergistic inhibition of NMC growth. Treatment of NMC cells with the novel dual p300/CBP and BET bromodomain–selective inhibitor, NEO2734, potently inhibits growth and induces differentiation of NMC cells in vitro; findings that correspond with potentiated transcriptional effects from combined BET and p300 bromodomain inhibition. In three disseminated NMC xenograft models, NEO2734 provided greater growth inhibition, with tumor regression and significant survival benefit seen in two of three models, compared with a lead clinical BET inhibitor or “standard” chemotherapy. Our findings provide a strong rationale for clinical study of NEO2734 in patients with NMC. Moreover, the synergistic inhibition of NMC growth by CBP/p300 and BET bromodomain inhibition lays the groundwork for greater mechanistic understanding of the interplay between p300 and BRD4-NUT that drives this cancer.