Frequent CBL Mutations Associated with 11q Acquired Uniparental Disomy in Myeloproliferative Neoplasms

Abstract

Abstract Recent evidence has demonstrated that acquired uniparental disomy (aUPD) is a novel mechanism by which pathogenetic mutations in cancer may be reduced to homozygosity. As a route towards identifying novel mutations in myeloproliferative neoplasms (MPN), we performed a genome wide single nucleotide polymorphism (SNP) screen to identify aUPD in 58 patients with atypical chronic myeloid leukemia (aCML; n=30), JAK2 mutation negative myelofibrosis (MF; n=18) or JAK2 mutation negative polycythaemia vera (PV; n=10). Stretches of homozygous, copy neutral SNP calls >20Mb were seen in 10 (33%) aCML, 1 (6%) MF but absent in PV. In total seven different chromosomes were involved with 7q and 11q each affected in 3 (10%) of aCML cases and 1p, 13, 17q, 20q, 21q involved in single individuals. The 1p and 13 abnormalities were associated with homozygous mutations in MPL and FLT3, respectively. To characterize the two recurrent regions at 7q and 11q, we focused on genes encoding intracellular signal transduction components because of the known association between MPNs and deregulated tyrosine kinase signaling. No mutations were detected in MET, EPHA1, EPHB6 or BRAF but homozygous CBL missense mutations were found in all three cases with 11q aUPD. To determine the prevalence of CBL mutations, we sequenced exons 8 and 9 in an additional 574 MPN cases. A total of 27 sequence variants were identified in 26 patients of whom 3 had MF, 10 had CMML, 12 had aCML/MPD-U and one had HES/CEL. Microsatellite analysis across 11q indicated significant tracts of 11q copy neutral homozygosity in 11/26 CBL mutated cases. In two cases, CBL mutations were acquired as secondary events during progression of a pre-existing MPN. Patients with CBL mutations had a shorter overall survival and progression-free survival compared to mutation negative cases (OS: 33 months vs 39 months; PFS: 22 months vs 32 months) but the differences were not significant. Similarly there was no difference in gender, age, white cell count or percentage of eosinophils between mutation positive and mutation negative cases. CBL plays both positive and negative roles in tyrosine kinase signal transduction by acting as an adaptor and also a ubiquitin ligase. Of the 27 variants, 21 (78%) were missense substitutions (15 different mutations) in the region encoding the RING or linker domains, 5 (19%) were candidate splicing abnormalities involving exon 8 and one (3%) produced a stop codon. Functional analysis of selected mutations demonstrated that they abrogated CBL ubiquitin ligase activity and were transforming as assessed by the ability to confer a proliferative advantage in both liquid and semi-solid cultures of Ba/F3-FLT3 cells. We conclude that acquired, inactivating CBL mutations are a novel and widespread pathogenetic abnormality in morphologically-related, aggressive MPNs.