The CBL ubiquitin ligase targets a variety of activated tyrosine kinases (TKs) for degradation. Many TKs are mutationally or autocrine activated and/or often overexpressed at the mRNA and protein levels in acute leukemias. We hypothesized that CBL is mutated in patients with acute myeloid leukemia (AML). IntroductionThe Casitas B-cell lymphoma (CBL) gene gives rise to a protein with multiadaptor function and E3 ubiquitin ligase activity that targets a variety of tyrosine kinases (TKs) for degradation. 1-3 c-CBL, and the related family member, CBL-b, contain several functional domains, including a tyrosine kinase-binding domain (TKB), a RING finger (RF) domain, a conserved linker region between the TKB and RF, and a C-terminal domain with ubiquitin ligase activity.The first described oncogenic mutant of c-CBL, the viral homolog, v-cbl, retains the TKB domain but lacks the RF and the C-terminal domains and induces B-cell lymphoma and myeloid leukemia in mice. 4,5 The transforming mutant isolated from a murine pre-B lymphoma cell line (70Z), is an in-frame 17-amino acid deletion arising from a splice site mutation, and leads to partial deletion that includes a linker region conserved in c-CBL and CBL-b that lies between the TKB and RF. 6 The murine p95cbl transforming mutant is an in-frame deletion of 111 amino acids due to a splicing mutation and results in loss of the entire RF and a portion of the linker region. 7 In the human HUT78 T-cell lymphoma cell line, c-CBL is truncated but keeps an intact linker and RF, and is nontransforming. 6,8,9 Lastly, MLL-CBL gene fusions have been reported for 2 acute myeloid leukemia (AML) patients. 10,11 To our knowledge, transforming mutations in CBL-b have not been described. Herein, we report several novel monoallelic c-CBL and CBL-b mutations in human AML that involve the linker region and the RF, respectively.

We previously identified a rearrangement of mixed-lineage leukemia (MLL) gene (also known as ALL-1, HRX, and HTRX1), consisting of an in-frame partial tandem duplication (PTD) of exons 5 through 11 in the absence of a partner gene, occurring in approximately 4%-7% of patients with acute myeloid leukemia (AML) and normal cytogenetics, and associated with a poor prognosis. The mechanism by which the MLL PTD contributes to aberrant hematopoiesis and/or leukemia is unknown. To examine this, we generated a mouse knockin model in which exons 5 through 11 of the murine Mll gene were targeted to intron 4 of the endogenous Mll locus. Mll PTD/WT mice exhibit an alteration in the boundaries of normal homeobox (Hox) gene expression during embryogenesis, resulting in axial skeletal defects and increased numbers of hematopoietic progenitor cells. Mll PTD/WT mice overexpress Hoxa7, Hoxa9, and Hoxa10 in spleen, BM, and blood. An increase in histone H3/H4 acetylation and histone H3 lysine 4 (Lys4) methylation within the Hoxa7 and Hoxa9 promoters provides an epigenetic mechanism by which this overexpression occurs in vivo and an etiologic role for MLL PTD gain of function in the genesis of AML.

Rhabdomyosarcoma is the most common pediatric soft-tissue sarcoma, which includes two major subtypes, alveolar and embryonal rhabdomyosarcoma. The mechanism of its oncogenesis is largely unknown. However, the oncogenic process of rhabdomyosarcoma involves multi-stages of signaling protein dysregulation characterized by prolonged activation of tyrosine and serine/threonine kinases. To better understand this protein dysregulation, we evaluated the phosphorylation profiles of multiple tyrosine and serine/threonine kinases to identify whether these protein kinases are activated in rhabdomyosarcoma. We applied immunohistochemistry with phospho-specific antibodies to examine phosphorylation levels of selected receptor and non-receptor tyrosine kinases, mammalian target of rapamycin (mTOR), p70S6K, and protein kinase C (PKC) isozymes on alveolar and embryonal rhabdomyosarcoma tissue microarray slides. Our results showed that the phosphorylation levels of these kinases are elevated in some rhabdomyosarcoma tissues compared to normal tissues. Phosphorylation levels of receptor and non-receptor tyrosine kinases are elevated between 26 and 68% in alveolar rhabdomyosarcoma and between 24 and 71% in embryonal rhabdomyosarcoma, respectively, compared to normal tissues. In addition, phosphorylation levels of mTOR and its downstream targets, p70S6K, S6, and 4EBP1, are increased between 50 and 72% in both subtypes of rhabdomyosarcoma. Further, phosphorylation levels of PKCa, PKCd, PKCh, and PKCf/k are upregulated between 57 and 69% in alveolar rhabdomyosarcoma and between 43 and 72% in embryonal rhabdomyosarcoma. This is the first report to create a phosphorylation profile of tyrosine and serine/threonine kinases involved in the mTOR and PKC pathways of alveolar and embryonal rhabdomyosarcoma. These protein kinases may play roles in the development or tumor progression of rhabdomyosarcomas and thus may serve as novel targets for therapeutic intervention. Most of these tumors arise in the head and neck region, genitourinary tract, and extremities. The two main histological subtypes of rhabdomyosarcoma are embryonal rhabdomyosarcoma, which comprises more than half of all rhabdomyosarcoma cases, and alveolar rhabdomyosarcoma, which is less common but more aggressive. These two subtypes arise at different primary body sites and have different age patterns.2 Clinically, the presentation of rhabdomyosarcoma is genetically heterogeneous. However, some genetic markers have been identified. Among them, approximately 75% of alveolar rhabdomyosarcoma are characterized by chromosomal translocation, most frequently, t(2;13)(q35;q14) or the variant t(1;13)(q36;q14). 3These translocations can disrupt either the PAX3

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.