Oncogenic CBL Mutants Have Transforming Potential in Cells Expressing the FLT3 Tyrosine Kinase.

CBL is a E3-ubiquitin ligase, that negatively regulates many receptor tyrosine kinases (RTKs). Translocations (t(4;11); t(11;14) involving CBL in humans and two oncogenic CBL deletion mutants, CBL-70Z and v-CBL, inducing B-cell lymphomas in mice, were described. The RTK Fms-like tyrosine kinase 3 (FLT3) is expressed by the leukemic cells of 70–90% of patients with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) and is highly implicated in the malignant transformation by harbouring activating mutations, aberrant autocrine stimulation or overexpression.

We hypothesized that CBL could play a role in regulating FLT3 activity and if mutated, could cause aberrant FLT3 signaling in hematopoietic cells. To analyze if CBL mutants could lead to transformation of FLT3-expressing cells we stably transduced Ba/F3 cells with CBL-WT, CBL-70Z or v-CBL alone or together with FLT3-WT. Coexpression of FLT3-WT and a CBL mutant, CBL-70Z or v-CBL, but not CBL-WT or expression of a CBL-construct alone, conferred IL-3 independent growth to Ba/F3 cells. In presence of FLT3 ligand FLT3-WT/CBLmutant-expressing cells showed hyperproliferation. To determine whether the proliferative advantage of FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells is mediated by FLT3 we cultivated the cells in presence of selective FLT3 inhibitors, SU5614 and PKC412. Both inhibitors were able to abrogate the IL-3 independent and FL-stimulated growth of FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells. The FLT3-WT receptors and the downstream signaling pathway, STAT5, were constitutively active in FLT3-WT/CBLmutant-expressing cells in contrast to control cells. To clarify the mechanism of transformation we performed internalization assays. The rate and speed of internalization of the FLT3-WT receptors were not altered in FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells compared to FLT3-WT cells. However, we could show that CBL-WT promotes FLT3 degradation. To investigate if CBL mutations play a role in acute leukemias we screened the cDNA of 62 AML and 6 ALL cell lines and found a CBL deletion mutant in MOLM-13 and MOLM-14 cells lacking exon 8, which comprises parts of the linker and RING finger domain. Hence, this mutation targets the same functionally important regions of the CBL protein than the CBL-70Z deletion.

In summary, CBL-70Z and v-CBL, but not CBL-WT, confer a transforming potential to cells expressing FLT3. The pro-proliferative phenotype of FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells is mediated by an increase in FLT3 tyrosine kinase activity. The CBL-70Z and v-CBL mutations were hypothesized to act in a dominant negative manner abrogating the negativ-regulatory function of CBL-WT. We identified a CBL deletion mutant in the MOLM-13 and MOLM-14 cells, that is reminiscent of CBL-70Z, indicating that CBL mutations might play a role in the pathogenesis of acute leukemias.