A mutation-created novel intra-exonic pre-mRNA splice site causes constitutive activation of KIT in human gastrointestinal stromal tumors

Abstract

We report a new mechanism of aberrant pre-mRNA splicing resulting in constitutive activation of a mis-spliced oncoprotein (KIT) leading to malignancy (gastrointestinal stromal tumor) in contrast to loss of function of misspliced proteins resulting in diverse human diseases in the literature. The mechanisms of three consecutive molecular events, deletion of noncoding and coding regions encompassing the 3 0 authentic splice site, creation of a novel intra-exonic pre-mRNA 3 0 splice acceptor site leading to in-frame loss of 27 nucleotides (nine amino acids; Lys550-Lys558), and the mechanism of constitutive activation of the mis-spliced KIT are elucidated. Loss of a peptide in a critical location unleashed the protein from autoinhibition (as evidenced by three-dimensional structural analysis), causing KIT to become constitutively activated and resulting in the GIST phenotype. We also demonstrated that only one of the following two exonic splicing enhancers is sufficient for inclusion of the KIT exon 11 in vivo: AACCCATGT (nucleotides 2-10 from the 5 0 end, which are recognized by SC35, SRp55, and SF2/ASF) or GGTTGTTGAGG (nucleotides 27-37 from the 5 0 end, which are recognized by SC35 and SRp55), suggestive of exonic enhancer redundancy.

Addiction to multiple oncogenes can be exploited to prevent the emergence of therapeutic resistance

“…Although splice site mutations have been associated with loss of function in several tumor suppressor genes, such as TP53, RB1, and BRCA1 (33-36), there have been relatively few reports of splicing mutations resulting in the gain of function of an oncogene. Examples to date include deletions at the intron 10-exon 11 boundary of the receptor tyrosine kinase c-KIT that result in loss of an auto-inhibitory region and constitutive activation of the protein (37,38), and splice acceptor site mutations in c-MET that lead to skipping of exon 14 and deletion of a juxtamembrane domain important for negative regulation (39). Intragenic deletions in β-catenin that remove all or part of exon 3 have been reported in colorectal carcinoma (40).…”

Modulation of Gene Expression and Cell-Cycle Signaling Pathways by the EGFR Inhibitor Gefitinib (Iressa) in Rat Urinary Bladder Cancer

“…A number of splicing-related mutations have been reported to be associated with malignancies, and some of these are within splice sites or splicing enhancers/silencers of cancer-related genes (21)(22)(23). To identify exon splicing, we first calculated the splicing index for each exon.…”

Molecular features and genetic markers of gastrointestinal stromal tumors

“…К наиболее частым относятся мутации кодонов W557 и / или K558, именно делеция W557-K558 коррелирует с высокой скоростью прогрессирования опухоли и метастазированием [53]. Делеции могут затрагивать акцепторные сайты сплайсинга в 10-м интроне -11-м экзоне KIT, делеции на белковом уровне K550-K558 затрагивают акцепторные сайты сплайсинга на 3'-кон-це пре-мРНК [54,55] и вызывают постоянное фосфо-рилирование ТК KIT.…”