Mutations of an E3 ubiquitin ligase c-Cbl but not TET2 mutations are pathogenic in juvenile myelomonocytic leukemia

Abstract

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm characterized by excessive proliferation of myelomonocytic cells. When we investigated the presence of recurrent molecular lesions in a cohort of 49 children with JMML, neurofibromatosis phenotype (and thereby NF1 mutation) was present in 2 patients (4%), whereas previously described PTPN11, NRAS, and KRAS mutations were found in 53%, 4%, and 2% of cases, respectively. Consequently, a significant proportion of JMML patients without identifiable pathogenesis prompted our search for other molecular defects. When we applied single nucleotide polymorphism arrays to JMML patients, somatic uniparental disomy 11q was detected in 4 of 49 patients; all of these cases harbored RING finger domain c-Cbl mutations. In total, c-Cbl mutations were detected in 5 (10%) of 49 patients. No mutations were identified in Cbl-b and TET2. c-Cbl and RAS pathway mutations were mutually exclusive. Comparison of clinical phenotypes showed earlier presentation and lower hemoglobin F levels in patients with c-Cbl mutations. Our results indicate that mutations in c-Cbl may represent key molecular lesions in JMML patients without RAS/PTPN11 lesions, suggesting analogous pathogenesis to those observed in chronic myelomonocytic leukemia (CMML) patients.

Figure 1

Single nucleotide polymorphism array–based karyotyping of JMML. (A) Genomic distribution and type of lesion identified in patients with JMML by SNP-A analysis. Green bar represent amplification, red shows deletion, and blue corresponds to UPD. Red lines pinpoint the locus of genes discussed in the text, as well as the number of patients mutated at that locus. NF1 mutational status was not assessed in this cohort. (B) Increased sensitivity of SNP-A for detecting chromosomal lesions. The results of MC (25%) and by SNP-A (49%) from the JMML cohort studied are shown. (C) Representative 250-K SNP-A analysis of UPD11q by CNAG Version 3.0 (patient 16). Both the raw and averaged total copy number (CN) tracks (red dots, blue line) show a normal copy number, whereas heterozygous SNP calls and allele-specific copy number tracks (green dashes, red/green lines) show a reduction in copy number, indicating UPD. The specific localization of 11qUPD in 4 patients (patients 16, 27, 38, and 43) is indicated by the blue bars. The c-Cbl locus is indicated on the chromosome 11 idiogram with a yellow line.

Figure 2

Site of the c-Cbl mutations and predicted product of splice variant in the intron 8 splice acceptor site. (A) Localization of the c-Cbl mutations within the predicted protein product. Red arrows show the site of mutations in exon, and blue arrows show the site of splice variant. (B) In patient 48, a homozygous mutation was seen in the intron 8 splice acceptor site of c-Cbl. According to http://genome.cbs.dtu.dk/services/NetGene2, this mutation may result in a splice variant, leading to a shorter transcript in RF domain.

Figure 3

GM-CSF hypersensitivity assay. Colony counts are expressed as percentage of maximal numbers of CFU-GM (colony counts cultured with each concentration of GM-CSF/colony counts cultured with 10 ng/mL GM-CSF). The similar GM-CSF hypersensitivity was seen in JMML patients with or without c-Cbl mutation. Error bars represent SE.