Aberrant Pre-mRNA Processing Due to Novel Splicing Mutations in the CBL Proto-Oncogene in Acute Myeloid Leukemia.

Ubiquitin (Ub)-dependent proteolysis is a highly controlled pathway that regulates almost all cellular processes by modulating the levels of key proteins. Several E3 Ub ligases are functionally abnormal in cancer. The E3 ubiquitin ligase, Casitas B-lineage lymphoma (CBL) protein, negatively regulates many receptor tyrosine kinases (RTKs) that are frequently over-expressed and/or constitutively activated (e.g., FLT3) in acute myeloid leukemia (AML). The CBL linker region, encoded in part by exon 8, lies between the tyrosine kinase binding and the RING finger domains and, mutations introduced into this linker region were previously shown to confer transforming ability. Herein, we describe novel splicing mutations in the c-CBL gene that lead to aberrant pre-mRNA processing in AML. DNA PCR revealed a splice site mutation whereby 14 nucleotides were deleted at the exon 8-intron 8 boundary in one CBL allele of the MOLM-13 cell line. Out of 18 patient samples, one primary AML blast sample had a 4 base pair deletion (16 bases upstream of the intron 7-exon 8 junction) which was replaced by a 12 base pair GC-rich insertion. We hypothesized that this mutation was an intronic splicing silencer element. RT-PCR/sequencing confirmed aberrant pre-mRNA processing in both the AML patient sample and MOLM-13 cells as they expressed a wild-type CBL transcript and an in-frame variant lacking exon 8. The splice variants are reminiscent of the oncogenic Cbl intragenic deletion mutation present in the murine pre-B cell line, p70Z. Such Cbl mutants are suggested to result in a structural alteration, allowing displacement of wild-type Cbl from the RTK complex thereby abrogating Cbl’s negative regulatory function. Thus it is possible that this mutant may enhance proliferation and survival of AML cells through attenuation of ubiquitin/proteosome-dependent degradation of receptor tyrosine kinases. Further investigation into the role of CBL and other players in the Ub-mediated proteolytic pathway is likely to provide additional insight into a subset of AML patients that in turn could lead to discovery of new therapeutic targets and strategies.