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Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth
Michael Dewaele, … , Jean-Christophe Marine, Ernesto Guccione
Michael Dewaele, … , Jean-Christophe Marine, Ernesto Guccione
Published November 23, 2015
Citation Information: J Clin Invest. 2016;126(1):68-84. https://doi.org/10.1172/JCI82534.
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Research Article Oncology

Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth

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Abstract

MDM4 is a promising target for cancer therapy, as it is undetectable in most normal adult tissues but often upregulated in cancer cells to dampen p53 tumor-suppressor function. The mechanisms that underlie MDM4 upregulation in cancer cells are largely unknown. Here, we have shown that this key oncogenic event mainly depends on a specific alternative splicing switch. We determined that while a nonsense-mediated, decay-targeted isoform of MDM4 (MDM4-S) is produced in normal adult tissues as a result of exon 6 skipping, enhanced exon 6 inclusion leads to expression of full-length MDM4 in a large number of human cancers. Although this alternative splicing event is likely regulated by multiple splicing factors, we identified the SRSF3 oncoprotein as a key enhancer of exon 6 inclusion. In multiple human melanoma cell lines and in melanoma patient–derived xenograft (PDX) mouse models, antisense oligonucleotide–mediated (ASO-mediated) skipping of exon 6 decreased MDM4 abundance, inhibited melanoma growth, and enhanced sensitivity to MAPK-targeting therapeutics. Additionally, ASO-based MDM4 targeting reduced diffuse large B cell lymphoma PDX growth. As full-length MDM4 is enhanced in multiple human tumors, our data indicate that this strategy is applicable to a wide range of tumor types. We conclude that enhanced MDM4 exon 6 inclusion is a common oncogenic event and has potential as a clinically compatible therapeutic target.

Authors

Michael Dewaele, Tommaso Tabaglio, Karen Willekens, Marco Bezzi, Shun Xie Teo, Diana H.P. Low, Cheryl M. Koh, Florian Rambow, Mark Fiers, Aljosja Rogiers, Enrico Radaelli, Muthafar Al-Haddawi, Soo Yong Tan, Els Hermans, Frederic Amant, Hualong Yan, Manikandan Lakshmanan, Ratnacaram Chandrahas Koumar, Soon Thye Lim, Frederick A. Derheimer, Robert M. Campbell, Zahid Bonday, Vinay Tergaonkar, Mark Shackleton, Christine Blattner, Jean-Christophe Marine, Ernesto Guccione

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Figure 5

ASO-mediated exon 6 skipping decreases MDM4 protein abundance and melanoma growth in vivo.

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ASO-mediated exon 6 skipping decreases MDM4 protein abundance and melano...
(A–F) Cohorts of PDX models of melanoma (MEL002 and MEL010) were established. When tumors reached an average volume of 100 mm3 (MEL002) or 150 mm2 (MEL010), cohorts were treated with the vivo MDM4 morpholino (or scrambled control) upon i.t. injections every 2 days (MEL002 i.t.) or i.v. tail vein injections every 2 days (MEL010 i.v.). Tumor development of MEL002 (A) and MEL010 (E) was monitored by caliper measurement for the indicated period. Data represent the mean ± SEM of the different biological replicates. A 2-way ANOVA was used to determine statistical significance in A and E. (B) ASO-mediated exon 6 skipping decreased MDM4 protein abundance in MEL002 lesions. Semi-quantitative analysis of MDM4-FL and MDM4-S isoforms in 12 dissected melanoma lesions exposed to the MDM4-targeting or scrambled control ASOs. Top panel shows SYBR Green–based qPCR of the PSI index in the various samples. Lower panel shows immunoblot analysis of MDM4 expression levels. Anti-actin immunoblotting was used to detect differences in sample loading. A reduction of MDM4 protein levels was confirmed by IHC staining on lesions exposed to the MDM4-targeting and scrambled control ASOs for MEL002 (C) and MEL010 (F). IHC was also performed for the apoptotic marker cleaved caspase 3 and the proliferative marker Ki67 in melanoma lesions exposed to the MDM4-targeting and scrambled control ASOs for MEL002 (C) and MEL010 (F). Scale bars: 100 μm. (D) Quantification (mean ± SD) of the IHC for the images shown in C. Three images from 2 different tumors were analyzed for each cohort.
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