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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 8

MDM4 ASO–mediated exon 6 skipping reduces growth of DLBCL in vivo and in vitro.

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MDM4 ASO–mediated exon 6 skipping reduces growth of DLBCL in vivo and in...
(A) Left panel: cell titer analysis of a clinical sample of DLBCL (BCL13) 3 days after transfection with MDM4-targeting and scrambled ASOs. Right panel: semi-qPCR analysis of MDM4 splicing. Data represent the mean ± SD of 2 replicates (B–D) Cohorts of the PDX model of DLBCL (BCL13) were established. When tumors reached an average volume of 150 to 250 mm3 (BCL13), the cohorts were treated with the vivo MDM4 ASO (or scrambled control) upon i.t. injections of BCL13 every 2 days. BCL13 tumor size was assessed at 20 days. Data represent the mean ± SEM of 5 biological replicates for each cohort. A 2-tailed unpaired t test was used to assess statistical differences in B–D. (C) ASO-mediated exon 6 skipping decreased the PSI MDM4 index and MDM4 protein abundance in BCL13 tumors. Semi-quantitative analysis of MDM4-FL and MDM4-S isoforms in 10 dissected DLBCL tumors exposed to the MDM4-targeting or scrambled control ASOs. Lower panel show immunoblot analysis of MDM4 expression levels. Anti-actin immunoblotting was used to detect differences in sample loading. (D) IHC for the apoptotic marker cleaved caspase 3 and the proliferative marker Ki67 in DLBCL lesions exposed to the MDM4-targeting and scrambled control ASOs. Right panels show quantification of the IHC images shown in D. Three different slides from 3 different tumors (n = 3) were analyzed for each cohort, and the results are presented as mean ± SD. Scale bar: 100 μm.
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