Mutant Wilms’ Tumor 1 (WT1) mRNA with Premature Termination Codons Is Sensitive to Nonsense-Mediated RNA Decay in Acute Myeloid Leukemia (AML)
Authors: Abbas Saman , Erpelinck Claudia , Goudswaard Chantal , Lowenberg B. , Valk Peter 
Published: April 2020

Abstract
The Wilms’ tumor 1 (WT1) gene encodes a Krupple-like zinc finger transcription factor, which can act as a transcriptional enhancer or repressor. Mutations in WT1 were originally identified in Wilms’ tumor, but are also present in other types of cancer. WT1 has been implicated as tumor suppressor gene when mutated in AML, but as oncogene when aberrantly high expressed. Mutations in WT1 are present in approximately 10% of AML and were recently implicated as an adverse prognostic marker for patients with AML. In AML, WT1 mutations cluster predominantly within exons 7 and 9, and appear as missense mutations or insertions-deletions mutations usually resulting in frameshifts that generate premature termination codons (PTCs). In this study, we analyzed a representative cohort of 262 AML cases by RT-PCR, WAVE dHPLC followed by sequence analyses for mutations in exons 6 to 9. A relatively low number of missense and in-frame insertion mutations were detectable in the WT1 mRNA transcripts in these AML cases (2.7%). Interestingly, we subsequently screened 351 AML cases by applying genomic DNA PCR amplification, WAVE dHPLC followed by sequence analyses of WT1 exon 7 and 9 and detected WT1 mutations in 5.7% of all cases. Mutations were absent in patients older than 60 years. WT1 mutations were present in AML patients normal karyotypes (6.3%), but also in patients carrying chromosomal aberrations, such as inv(16), t(8;21) and t(15;17). Survival analyses in AML patients younger than 60 years did not reveal any significant difference in overall (OS) and event-free survival (EFS) between patients with or without WT1 mutations. These survival analyses were performed on all AML patients, except those with favourable cytogenetics (n = 233) (OS p = 0.09 and EFS p = 0.08) or AML patients with normal karyotypes (n = 134) (OS p = 0.35 and EFS p = 0.25). Although these analyses did not reveal a significant association, a trend for adverse outcome for patients carrying WT1 mutations was apparent. Missense, in-frame insertion and nonsense insertions-deletions WT1 mutations were present in our AML cohort. The majority of the detected WT1 aberrations were insertion-deletion mutations that introduced PTCs. However, by Western blot analyses we showed that the affected AML cases did not express the expected truncated WT1 proteins, whereas full-length WT1 was expressed. Nonsense-mediated RNA decay (NMD) degrades mRNAs bearing PTCs. We hypothesized that the mutant WT1 mRNA transcripts might be sensitive to NMD. The involvement of NMD in the AML cases was confirmed by treatment of primary AML cells carrying out-of-frame WT1 mutations with the translation inhibitor emetine. Treatment with emetine stabilized the mutant WT1 mRNA transcripts to levels similar or even higher than wild-type WT1. In conclusion, AML cases with WT1 missense and in-frame insertion mutations express mutant WT1 mRNA at WT1 wild-type levels. These transcripts most probably encode proteins that affect WT1 function since mutations are located within the WT1 zinc fingers. In addition, mutant WT1 mRNA transcripts that carry PTCs are sensitive to NMD in AML. In these AML cases mutations in WT1 result in haploinsufficiency rather than expression of dominant negative truncated WT1 proteins. Of note, similar mechanisms may also be important in other types of malignancies carrying WT1 mutations.