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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 3

SRSF3 is required for efficient inclusion of MDM4 exon 6 and melanoma growth.

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SRSF3 is required for efficient inclusion of MDM4 exon 6 and melanoma gr...
(A) PSI MDM4 index relative to nontargeting shRNA (Scr) calculated upon SR protein shRNA KD in A375 human melanoma cells. Each bar represents a single shRNA targeting the indicated SRSF family member (including 2 independent scrambled controls; details are provided in Supplemental Table 4). Data represent the mean ± SD of 3 biological replicates, and the qPCR was run in 2 technical replicates. An unpaired t test was used to assess statistical differences in A (1-tailed statistical significance was determined using the Holm-Sidak method, with α = 5.000%). (B) Binding sites from the SRSF3 mouse CLIP-seq data were aligned with BLAT to the human genome. RIP primers were designed to enrich for a negative control upstream region (yellow) and for the SRSF3-binding region (green). RIP was performed on A375 cells (right panel). SRSF3 exon 4 was used as a positive control (blue). SS, splice site. (C) PSI MDM4 indexes and MDM4 protein levels (Western blot, bottom) were evaluated following SRSF3 KD with 5 independent shRNAs. A scrambled shRNA was used as a control. (D) qPCR quantification of total MDM4, SRSF3, and p53 transcriptional targets (MDM2, p21, and BBC3) following SRSF3 KD with 5 independent shRNAs. A scrambled shRNA was used as a reference control. Data represent the mean ± SD. (E) Quantification of cell viability (top panel) and apoptosis (bottom panel) upon SRSF3 KD using 2 independent shRNAs (171 and 227) in A375 cells (see also Supplemental Table 4). Data represent the mean ± SD of 2 biological replicates.
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