CBL Exon 8/9 Mutants Activate the FLT3 Pathway and Cluster in Core Binding Factor/11q Deletion Acute Myeloid Leukemia/Myelodysplastic Syndrome Subtypes

Abstract

Purpose: CBL is a negative regulator of activated receptor tyrosine kinases (RTK). In this study, we determined the frequency of CBL mutations in acute leukemias and evaluated the oncogenic potential of mutant CBL. Experimental Design: The cDNA of 300 acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) and acute lymphoblastic leukemia (ALL) patients and 82 human leukemic cell lines was screened for aberrations in the linker and RING finger domain of CBL. The oncogenic potential of identified mutants was evaluated in hematopoietic cells. Results: We identified 3 of 279 AML/MDS patients expressing CBL exon 8/9 deletion mutants. Three of four cases at diagnosis expressed deleted transcripts missing exon 8 or exon 8/9. In remission samples a weak or no expression of mutant CBL was detected. No aberrations were found in normal hematopoietic tissues. One of 116 sequenced AML/MDS cases carried a R420G missense mutation. All AML/MDS patients with identified CBL mutants belonged to the core binding factor and 11q deletion AML subtypes. Functionally, CBL negatively regulated FMSlike tyrosine kinase 3 (FLT3) activity and interacted with human FLT3 via the autophosphorylation sitesY589 and Y599 and colocalized in vivo. Expression of CBLDexon8 and CBLDexon8+9 in FLT3-WT-Ba/F3 cells induced growth factor^independent proliferation associated with autophosphorylation of FLT3 and activated the downstream targets signal transducer and activator of transcription 5 (STAT5) and protein kinase B (AKT). FLT3 ligand^dependent hyperproliferation of CBL mutant cells could be abrogated by treatment with the FLT3 PTK inhibitor PKC412 (midostaurin). Conclusion: CBL exon8/9 mutants occur in genetically defined AML/MDS subtypes and transform hematopoietic cells by constitutively activating the FLT3 pathway. This phenotype resembles the one of mutated RTKs and suggests that CBL mutant AML patients might benefit from treatment with FLT3 PTK inhibitors.CBL, a known negative regulator of activated receptor tyrosine kinases (RTK), is localized on human chromosome 11q23, a region frequently associated with chromosomal aberrations. Translocations t(4;11) and t(11;14), and mixed-lineage leukemia fusion genes involving CBL have been described in patients with leukemia and lymphoma (1 -3). CBL oncogenes were initially identified in the murine system. CBL-70Z, carrying an internal deletion of 17 amino acids, was isolated from the 70Z/ 3 mouse pre-B-cell lymphoma cell line (4). CBL-70Z deregulates the cellular tyrosine kinase machinery, as NIH3T3 serumstarved cells expressing CBL-70Z showed significantly increased endothelial growth factor receptor (EGFR) kinase activity after EGF stimulation (5). p95CBL, expressed in the murine reticulum sarcoma cell line J-774, lacks internal 111 amino acids, comprising whole exons 8 and 9 (6). CBL70Z and p95CBL mutations both target the linker and RING finger domain, which points to a mutation-sensitive region within the CBL protein. Recently the first human CBL mutation has been reported in a patient...

Rapidly fatal myeloproliferative disorders in mice with deletion of Casitas B-cell lymphoma (Cbl) and Cbl-b in hematopoietic stem cells

“…Preferential expansion of the myelomonocytic cells is concordant with clinical manifestations in human patients with CBL mutations (7)(8)(9)(10)(11). Whether the skewing might reflect a Cbl/Cbl-b-dependent restriction point that regulates differentiation of HSCs toward myelomonocytic lineage or regulation of committed myelomonocytic precursors are important questions that should be possible to address using the current model.…”

“…In the past few years, several groups have reported mutations of the CBL gene in a subset of patients with myeloid neoplasms (4)(5)(6)(7)(8)(9)(10)(11)(12)(13). A large proportion of CBL mutations is associated with myelodysplastic syndrome/myeloproliferative disorder (MDS/ MPD), a heterogeneous group of hematopoietic malignancies characterized by deregulated hematopoiesis and a high propensity to develop acute myeloid leukemia (AML).…”

Spliceosome mutations involving SRSF2, SF3B1, and U2AF35 in chronic myelomonocytic leukemia: Prevalence, clinical correlates, and prognostic relevance

“…These include mutations involving; RUNX1 [3], IDH1, and IDH2 [4], KRAS and NRAS [5], CBL [6], JAK2 [7], TET2 [8], DNMT3A [9], ASXL1 [10], UTX [9], and EZH2 [9]. Thus far, in CMML, loss-of-function mutations involving EZH2 and ASXL1 have been associated with poor outcomes [10,11].…”

Suppressor of Cytokine Signaling 6 (SOCS6) Negatively Regulates Flt3 Signal Transduction through Direct Binding to Phosphorylated Tyrosines 591 and 919 of Flt3

“…Oncogenic mutations in cancer can render the RTKs less ubiquitinated, either due to mutations of the binding sites for ubiquitin ligases on the receptors themselves or through inactivating mutations in the ubiquitin ligases (7). Cbl is the most studied ubiquitin ligase, and loss-of-function mutations in Cbl have been described in AML and shown to contribute to oncogenic transformation (8).…”