MDM4 proteine, een veelbelovend doelwit voor kankertherapie

Cutaneous melanoma arises from uncontrolled growth of melanocytes in the basal layer of the skin. It is a very aggressive disease and its incidence has been rising faster than any other cancer type since the mid 1950’s. Once the cancer has metastasized to other organs, it becomes very difficult to manage. Recently, the melanoma field was revolutionized by the upcoming of MAPK-based targeted therapeutics and immunotherapy. In spite of initial successes in overall and progression-free survival, most patients become resistant to the treatment and relapse shortly after the start of the therapy. Novel therapies are urgently needed to use alone or in combination with existing treatments, in order to induce a more durable or even curative response in patients. Murine Double Minute 4 (MDM4) is a potent oncogene that is expressed in about 65% of all melanoma patients. It mostly works as a transcriptional squelcher for p53, meaning that it keeps p53 from exerting tumor suppressive functions. Moreover, recent data indicates that MDM4 possesses p53-independent oncogenic functions as well. Therefore, rather than disrupting the interaction between p53 and MDM4, which appears to be a very challenging task, we reasoned that directly targeting MDM4 protein abundance may be a more efficient alternative. As it was not clear how MDM4 is being upregulated in human cancers, the central aim of this PhD thesis was to unravel the mechanism(s) that contribute to high MDM4 protein expression in cancer, and in particular in melanoma. We found that melanoma cells hijack an otherwise embryonic alternative splicing event to include exon 6 in the MDM4 pre-mRNA, resulting in the generation of a full-length protein-coding transcript. Antisense oligonucleotides (ASOs) were designed to specifically force exon 6 skipping, thereby creating a premature stop codon which marks the shorter transcript for nonsense-mediated decay (NMD). We show that treating melanoma cells with MDM4-targeting ASOs reactivated p53 function in vitro and resulted in reduced colony growth. Moreover, this treatment decreased tumor growth in three different patient-derived melanoma xenografts (PDX) models, indicating that MDM4 ASOs could be used in an in vivo/preclinical setting. Interestingly, it also greatly sensitized melanoma cells to MAPK-based therapeutics. At the timepoint where BRAF inhibitor-treated mice already had become resistant to the treatment and tumors were quickly regrowing, mice that received the BRAF inhibition/MDM4 ASO combination were still sensitive. This suggests that MDM4 ASOs are able to delay the development of BRAF inhibition-induced resistance. Before uncovering this alternative splicing switch event that contributes to MDM4 upregulation, our knowledge of MDM4 expression in cancer was largely based on copy number alterations and transcriptomic analyses. By looking at the ratio of the alternatively spliced transcripts of MDM4, careful in silico re-analysis of many different tumor samples revealed that we may have been dramatically underestimating how widespread MDM4 expression is in other tumor types. The innovative targeting strategy we developed could therefore potentially be applicable to a wide variety of tumors. In conclusion, in this PhD project, we unraveled an important mechanism of MDM4 upregulation in cancer. An alternative splicing-based therapy was designed and validated in melanoma cells and in PDX. As oncogenic MDM4 is predicted to be expressed in a wide variety of other tumor types, many cancers could benefit from this ASO-based therapeutic strategy.

Publication year:2016

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