Novel Transforming Mutations of CBL in Human Acute Myeloid Leukemia
Authors: Rodrigues Margret S , Reddy Mamatha M , Walz Christoph , Salgia Ravi , Podar Klaus , Band Hamid , Reiter Andreas , Griffin James D , Sattler Martin 
Published: April 2020

Acute myeloid leukemia (AML) is a genetically heterogeneous disease initiated within the bone marrow. It is characterized by multiple mutagenic events that affect proliferation and/or viability as well as differentiation. Deregulation of tyrosine kinase activity is frequently associated with AML and required for transformation. In particular, mutational activation of the FLT3 (Fms-like tyrosine kinase 3) receptor tyrosine kinase occurs in about one third of AML and FLT3 is expressed at high levels in more than 80% of AML patient cells. Recently, it has been suggested that FLT3, Kit or other receptor tyrosine kinases (RTKs) can be active in cells containing inactivating mutations within the linker region of CBL. CBL proteins function as E3 ligases that ubiquitinate and negatively regulate activated RTKs such as FLT3. Nevertheless, the in vivo role and frequency of these CBL mutations in AML remains unknown. In order to identify novel CBL mutations, we prepared cDNA from 43 AML patients and 10 healthy individuals for sequencing of the linker region. We identified two novel CBL mutations, which included a point mutation leading to a substitution in position 371 of the coding sequence and a splice site mutation (intron7/exon8) resulting in a six base pair insertion. Both mutations were located in the alpha helical structure within the linker sequence known to play an important role in promoting receptor ubiquitination. These mutations were inserted into vectors containing the c-CBL cDNA by site-directed mutagenesis. We demonstrate that the co-expression of these mutations with FLT3 induced factor independent growth and survival of BaF3 cells, similar to control cells expressing CBL with a deletion of Y371 in the linker region This region is upstream of the RING finger, which is required for its E3 ligase function. Whereas cells expressing wild-type CBL did not grow in a three day culture, the cell number increased 13.7-fold for the CBL point mutant and 14.2-fold for the splice site mutant. This result is comparable to CBL Y731 deletion containing cells (12.8-fold increase). Also, growth of cells expressing mutant CBL required expression of FLT3 and could be inhibited by the FLT3 inhibitor midostaurin. Further, both CBL mutations lead to an increase in apoptosis with increased caspase3/7 enzyme activity. The point mutation at Y371 also led to an increase in intracellular reactive oxygen species (ROS), similar to cells containing the Y371 deletion or cells transformed by FLT3-ITD. Previously, chronically elevated ROS levels have been implicated in the induction of DNA damage and therapy related drug resistance as well as activation of signaling pathways required for transformation. Overall, our in vitro data demonstrate that mutations of CBL in the linker region are sufficient to support growth and viability of BaF3 cells and that the transforming activity can be targeted by small molecule drugs against RTKs. Thus, an abnormal increase in RTK activity in AML may occur indirectly through inactivating mutations within CBL or other protein degradation pathway proteins, independent of RTK mutations. It will now be of interest to further evaluate the role of CBL in AML in the context of disease phenotype and mutational status of RTKs.