Functional analysis of the APL oncogene TBLR1-RARβ, and a potential MEK2 cooperating mutation

Abstract

Acute Promyelocytic Leukaemia (APL) is a blood malignancy characterised by the accumulation of non-functional, immature promyelocytes due to a differentiation block in haematopoiesis. APL is associated with chromosomal translocations that create fusion genes that may drive the leukaemia development. TBLR1-RARβ is a rare and novel fusion gene, recently reported in APL patients by the Wood group and others. APLs with this fusion are unresponsive to standard APL therapy and thus require further study to identify alternative treatments. Currently, there are no TBLR1-RARβ animal models. In addition, TBLR1-RARβ patients harbour MEK2R231L, a variant of unknown significance (VUS) that has not been functionally characterised but represents a potentially druggable target. MEK2 encodes a protein kinase of the RAS/RAF/MEK/ERK signalling pathway that regulates key cellular processes and its aberrant activity is associated with many cancers. In this project, transgenic zebrafish were created to investigate TBLR1-RARβ and MEK2R231L activity in vivo. None of the transgenic lines developed malignancy symptoms by 8 months post fertilisation. Preliminary analyses of a subset of non-sick zebrafish (assessed by flow cytometry, and marrow and blood smears) revealed haematopoiesis to be largely normal in TBLR1-RARβ and TBLR1-RARβ MEK2R231L zebrafish. Given that the latency of other zebrafish leukaemia models can be as much as one to two years, the TBLR1-RARβ-based zebrafish generated here may require a longer incubation period for disease symptoms to develop. Assessment of sick zebrafish in the future should reveal whether preliminary suggestions of blood perturbation accurately reflect a true impact of the transgenes. A zebrafish model of TBLR1-RARβ-driven leukaemia would enable the opportunity to explore the molecular basis of the disease and perform therapeutic drug screens. Characterisation of MEK2R231L by Western blot analyses showed that variant activity results in increased RAS/RAF/MEK/ERK signalling pathway activation in vitro in cell lines. However, the impact of this activation could not be substantiated by the in vitro cell proliferation assays used here and will require further study to determine if MEK2R231L represents a contributing mutation in APL and a potential druggable target.