Identification of variations in c-Cbl, Cbl-b and Cbl-c RING finger (RF) domain. Schematic representation shows the major domains of c-Cbl, Cbl-b, and Cbl-c, primarily the tyrosine kinase binding (TKB) domain, linker sequence (L), RF domain, proline-rich region (PPP), and leucine zipper (LZ)/ubiquitin-associated domain (UBA). Tyrosine and serine residues, represented by red circles, are phosphorylated by tyrosine kinases. Genomic DNA sequencing of all exons in c-Cbl, Cbl-b, and Cbl-c revealed the presence of (black arrow) missense and (blue arrow) frame shift mutations or frame shift polymorphisms in L or RF domain, except for a case with a mutation in the TKB domain. In c-Cbl, some basepair changes occured in a homozygous state because of UPD and resulted in the substitution of cysteine or arginine residues at positions 384 shared in two patients (C384Y), 404 in three patients (C404S/Y), and 420 in three patients (R420Q/P). (*) Frame shift polymorphism.

Potential intracellular consequences of c-Cbl mutations. c-Cbl is a member of the E3 ubiquitin ligase Cbl family, which poly- or monoubiquitinate a number of important receptor tyrosine kinases (RTK), including Flt-3, c-kit, and CSF-1, for degradation. Inactivation of ubiquitination activity through mutations occurring in RING finger domain may lead to enhanced and/or prolonged hematopoietic growth factor signaling, a function which can contribute to the clinical phenotype of patients with c-Cbl mutation. However, in addition to RTK, knockout of c-Cbl ubiquitination activity may result in enhanced phosphorylation of SRC kinase, STAT5, and also c-Cbl itself. Ultimately, the elevated level of important transduction factors such as STAT5 and PI3K as well as increased SRC kinase activity can lead to aberrant proliferative responses.

A microdeletion 11q23.3 is detected in c-Cbl in a patient with chronic myelomonocytic leukemia 1, leading to a hemizygous insertion mutation in the linker sequence. BM, bone marrow.

Characteristic features of megakaryocytes in patients with c-Cbl mutations. (A) A patient with chronic myelomonocytic leukemia (CMML) with bone marrow fibrosis and c-Cbl mutation shows abnormal megakaryocytes (abnormal lobulation, hyperchromatic and raisinoid nucleus). pSTAT5 staining was positive in the nuclei of megakaryocytes and erythroid precursors in a patient with secondary acute myelogenous leukemia (B), and refractory anemia with excess blasts 1 (C); both had a c-Cbl mutation. (D) In comparison, pSTAT5 staining was negative in megakaryocytes of a patient in refractory anemia phase before acquiring c-Cbl mutation. Original magnification, ×100.

Detecting acquired, segmental uniparental disomy (UPD) using single nucleotide polymorphism array (SNP-A) technology. (A) SNP-A karyograms of both whole bone marrow (BM) cells and CD3+ lymphocytes of a patient show the somatic nature of acquired UPD in chromosome 11. The blue line represents average copy number (CN) signal intensity of SNPs on the array chip. In this instance, there are no CN variations, and thus, the blue line does not deviate from normal diploid CN. The green marks below the idiogram represent heterozygosity at particular DNA loci. In the region of UPD seen in whole BM cells, drastic reduction of heterozygous loci denotes the region of UPD (pink bar). The remaining green marks in the region of UPD delineate the presence of nonclonal cells in the sample. An abnormal chromosome 11 was not detected when metaphase cytogenetic analysis was performed on bone marrow. In CD3+ sorted lymphocytes, a normal chromosome 11 is seen. (B) Comparison of Affymetrix 250K and 6.0 arrays in the detection of UPD11q. CNAG version 3.0 analysis (top) shows clear UPD of chromosome 11 by loss of heterozygous loci. Repeated testing on the 6.0 array and analysis using Genotyping Console v2.0 software (bottom) confirms the 250K SNP-A findings. Note that the Genotyping Console output includes allele difference, loss of heterozygosity (LOH), and CN variation plots. The allele difference graph represents the genotypes for each individual SNP. Dots with a value of 1 represent SNPs with an AA genotype, whereas those with a value of −1 represent SNPs with a BB genotype. Dots at 0 represent heterozygous SNPs (AB). Complete loss of all AB SNPs indicates copy-neutral LOH. This is further shown by both the LOH and CN graphs, which show no loss in CN but clear LOH. (C) Topographic maps show regions of (red) UPD or (green) deletion in individual patients on chromosome 11q. Bars corresponding to the ideogram represent the regions affected for each patient. The c-Cbl locus was included in the common deleted region (11q23.3) among UPDs and deletions. Red and pink bars represent UPD lesions with and without c-Cbl mutations, while dark and light green bars show deletions with and without mutations. Top is 6.0 array karyogram with UPD11q and bottom is one with microdeletion 11q by 250K array analysis. c-Cbl mutations were identified in both cases.

Cbl family mutations in myeloid malignancies and unique clinical characteristics of patients with mutations. (A) Cbl family mutations are frequently observed in secondary acute myelogenous leukemia (sAML; 12%), myelodysplastic/myeloproliferative neoplasms (MDS/MPN; 10%), and MPN (10%). Homozygous mutations of c-Cbl are more frequent in sAML (7%) than MDS/MPN (4%). Cbl-b mutations and/or Cbl-c frame shift polymorphism (*) are seen in all disease phenotypes. (B) Kaplan-Meier analysis shows overall survival in all cases (n = 20; gray line). Median survival is 5 months and the survival rate is 10%. By comparing patients homozygous for Cbl family mutation (n = 10; blue line) to those heterozygous (n = 10; gold line), there is a statistically significant difference in overall survival between these two cohorts regardless of treatment or remission/relapse.

In a patient with secondary acute myelogenous leukemia (sAML) transformed from myelodysplastic syndrome refractory anemia with monosomy 7, uniparental disomy (UPD) 11q14.1qter was detected only after AML evolution by single nucleotide polymorphism assay analysis, at which time a homozygous mutation was seen in the intron 8 splice donor site of c-Cbl. This mutation results in a splice variant, leading to a longer transcript with a frame shift in RF domain.

In a patient with sAML evolved from myelodysplastic/myeloproliferative neoplasms, a chromosome 3 abnormality was seen by metaphase cytogenetics and single nucleotide polymorphism array analysis revealed a deletion in the region of Cbl-b. Direct sequencing of whole bone marrow (BM) showed a hemizygous mutation (1204-51 G>A) in intron 9, whereas this mutation is not detected in CD3+ lymphocytes. Reverse transcription polymerase chain reaction revealed the utilization of a novel splice acceptor site and mutant-specific amplification, resulting in a frame shift in the RF domain.