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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 6

ASO-mediated exon 6 skipping sensitizes melanoma cells to BRAFV600E inhibitors in vitro and in vivo.

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ASO-mediated exon 6 skipping sensitizes melanoma cells to BRAFV600E inhi...
(A) A BRAFV600E-positive short-term culture (MM034) was transfected with MDM4-targeting and scrambled control ASOs, and colony formation was evaluated using low-density colony formation assays 10 days after seeding and exposure to 25 nM of the BRAFV600E inhibitor PLX4032. Right panel shows the quantification of the colony formation assays. Data are presented as the percentage of area occupied. (B–D) Cohorts of the PDX model of melanoma (MEL006) were established. When tumors reached an average volume of 200 mm3, they were subdivided into cohorts for various combinatorial treatments. The mice were gavaged with dabrafenib or vehicle every day and injected i.t. with scrambled ASO or MDM4 ASO every other day. (B) Tumor development was monitored by caliper measurement for the indicated period. Data represent the mean ± SEM of the indicated biological replicates. A 2-way ANOVA was used to determine statistical significance in B. Red dotted line indicates the average starting volume of the tumors in the MDM4 ASO plus BRAFi cohort. (C) IHC for MDM4, the apoptotic marker cleaved caspase 3, and the proliferative marker Ki67 in melanoma lesions exposed to the combination treatment of ASO-based exon 6 skipping with BRAFi. Scale bar: 100 μm. (D) Quantification (mean ± SD) of the IHC for which representative images are shown in C. For each tumor, 3 slides were stained and counted. Both cohorts contained 4 different tumors each (n = 12 counted slides). (E) The PDX model MEL006 was treated with BRAFi alone (n = 1), BRAFi plus scrambled ASO (n = 1), or BRAFi plus MDM4 ASO (n = 2). Data represent the mean ± SD.
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