THE PROSURVIVAL CYTOKINE ANGIOPOIETIN-1 IS OVEREXPRESSED IN T(4;11)-POSITIVE ALL AND REGULATED IN A MLL/AF4-DEPENDENT MANNER
P Garrido Castro,1 S Bomken,1 J Vormoor,1 J Greil,2 O Heidenreich1
Background. The chromosomal translocation t(4;11)(q21;q23) marks an acute lymphoblastic leukaemia (ALL) subtype prevalent in infants and associated with poor outcome. ALL cells carrying this translocation express the fusion gene MLL/AF4; in concordance with the adverse prognosis of t(4;11)-positive paediatric ALL, cell lines expressing MLL/AF4 are associated with resistance to chemotherapy-related stress and cell death. In order to address the functional role of MLL/AF4 in leukaemogenesis and leukaemic maintenance, we employ siRNA oligonucleotides targeting the fusion site of the MLL/AF4 transcript inherent of our cell line model, the t(4;11)-positive ALL cell line SEM. As previously reported by us, sustained MLL/AF4 depletion induces cell death. Gene expression profiling of the SEM cell line transfected with siMLL/AF4 revealed a subset of differentially expressed angiogenic genes, most prominently ANGIOPOIETIN-1 (ANGPT1), a proangiogenic cytokine previously reported to play a role in proliferation and survival of acute myeloid leukaemia (AML) cells, but to date not implicated in ALL. Recently, ANGPT1 signalling has been linked to haematopoietic stem cell (HSC) quiescence and bone marrow (BM) niche maintenance; pathways activated downstream of ANGPT1 include several prosurvival cascades associated with leukaemia, such as PI3K/AKT, MAPK and STAT3/5 signalling. Methods. ANGIOPOIETIN mRNA transcript levels were analysed by real-time RT-PCR (Q-RT-PCR), and ANGPT1 protein secretion determined using enzyme-linked immunosorbent assay (ELISA). Expression of ANGIOPOIETIN receptors and integrins was detected using RT-PCR. The MLL/AF4 status of cells was modulated using fusion transcript-specific siRNA oligonucleotides; successful knock-down was monitored by Q-RT-PCR. Results. Screening of a B-cell precursor ALL cell line cohort showed ANGPT1 mRNA expression to be restricted to the t(4;11) ALL subtype, where it is upregulated up to 100-fold compared to peripheral blood B cells from healthy donors. High levels of ANGPT1 were also detected in 11q23 rearranged infant ALL patient samples. Conversely, 11q23 rearranged AML cell lines show substantially lower ANGPT1 levels. Notably, in addition to its expression being restricted to t(4;11)-positive ALL, ANGPT1 levels are dependent on MLL/AF4; reduction of ANGPT1 mRNA and protein levels correlated with siRNA-mediated MLL/AF4 depletion in a time-dependent manner. Conversely, ANGIOPOIETIN-2 (ANGPT2), the biological antagonist of ANGPT1 and an independent prognostic marker in AML, showed increased RNA levels in response to MLL/AF4 knockdown in SEM cells. Expression analyses of receptors reported to mediate ANGPT1 and ANGPT2 signalling revealed presence of the integrins ITGB1 and ITGB5 in t(4;11)-positive ALL cell lines, enabling an autocrine function of secreted ANGPT1. Conclusions. Here we report for the first time a possible implication of ANGIOPOIETIN-1 in t(4;11) ALL, as defined by its overexpression and the regulation of mRNA and protein levels by the MLL/AF4 status of cells. ANGPT1 signalling represents an attractive potential target, both in an autocrine manner and in the context of crosstalk with the BM niche, which may promote ALL cell survival. We are currently assessing the functional role of ANGPT1 in t(4;11) ALL cells. This work was supported the North of England Children's Cancer Research Fund, the Leukaemia & Lymphoma Research Fund and the Deutsche Jose Carreras Leukaemie-Stiftung.