ANGIOPOIETIN1 - a Novel Factor Implicated In MLL-Rearranged Acute Lymphoblastic Leukemia and Regulated In a Fusion Gene-Dependent Manner

MLL-rearranged acute lymphoblastic leukemia (ALL) is prevalent in infants, constituting 70% of the cases. The preferred MLL translocation partner is the gene AF4, resulting in t(4;11)(q21;q23), which arises in 50% of infant ALL patients. This translocation generates the fusion genes MLL/AF4 and AF4/MLL, and is associated with an aggressive clinical presentation and poor outcome. Biologically, cells expressing MLL/AF4 show resistance to stress- and chemotherapy-related apoptosis. Concordantly, we have previously shown that RNAi-mediated depletion of MLL/AF4 in the t(4;11)-positive ALL cell line SEM results in induction of cell death and impaired both clonogenicity and in vivo engraftment. In order to characterize this phenotype on a molecular level, we have performed gene expression profiling of SEM cells depleted of MLL/AF4 and corresponding controls. Expression of >1000 genes was affected, including a subset of angiogenic genes, most prominently ANGIOPOIETIN1 (ANGPT1), a proangiogenic cytokine reported to play a role in acute myeloid leukemia (AML), hematopoietic stem cell (HSC) quiescence and bone marrow (BM) niche maintenance, but to date not implicated in ALL. Here we report a novel link between ANGPT1 expression and MLL-rearranged ALL.

Gene expression profiling was performed using the Illumina HT-12 platform and data processed using BeadStudio and Genespring software suites. ANGPT1 expression was analyzed by real-time RT-PCR (qRT-PCR), and ANGPT1 protein secretion determined using enzyme-linked immunosorbent assay (ELISA). The MLL/AF4 status of cells was modulated with fusion transcript-specific siRNAs and knockdown monitored by qRT-PCR. RNAi-mediated depletion of ANGPT1 was achieved using siRNA or lentiviral shRNA constructs, and validated on transcript and protein level. Effects on cell cycle progression and proliferation in response to ANGPT1 knockdown in t(4;11)-positive cells were assessed by flow cytometry and trypan blue exclusion assay, respectively. For in vivo studies, SEM cells were sequentially transduced to express both luciferase and either non-target control shRNA (shNTC) or shANGPT1. Doubly transduced cells were selected for and FACS-sorted prior to intrafemoral transplantation into immunodeficient NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice. Disease progression was monitored using bioluminescence imaging and engraftment assessed by flow cytometry at the terminal timepoint.

ANGPT1 expression was screened in a MLL-rearranged ALL patient cohort (n=35), comprising t(4;11)-positive (n=20), t(11;19)-positive (n=10) and t(9;11)-positive patients (n=5), and in a MLL-wildtype BCP ALL patient cohort (n=8). MLL-rearranged patients showed ANGPT1 upregulation, t(4;11)-positive patients having the strongest overexpression by 232-fold when compared to ANGPT1 levels in CD19+ peripheral blood (PB) cells. A 27-fold and 13-fold upregulation was detected in t(11;19)- and t(9;11)-positive patients, respectively. Conversely, MLL-wildtype BCP ALL patients had similar ANGPT1 levels as CD19+ PB cells, with only a 2-fold increase. In addition to its high expression in t(4;11)-positive ALL, ANGPT1 levels were shown to be dependent on MLL/AF4; a reduction of ANGPT1 mRNA and protein correlated with siRNA-mediated MLL/AF4 depletion in a time-dependent manner in both cell lines and primary patient samples. This was concordant with expression array data, which indicated an up to 4-fold decrease of ANGPT1 in response to MLL/AF4 depletion. The functional role of ANGPT1 in t(4;11)-positive ALL was assessed by RNAi; sustained depletion of ANGPT1 in SEM cells resulted in cell cycle arrest and a marked decrease in proliferation. In vivo, mice transplanted with shANGPT1 expressing SEM cells showed reduced splenic infiltration and development of solid tumours at the injection site, as opposed to a systemic spread of the disease and massive splenomegaly in mice injected with shNTC expressing SEM cells.

In this study we have identified ANGPT1 as a novel player in t(4;11)-positive ALL, as defined by overexpression, MLL/AF4-dependent regulation and functional consequences in vivo and in vitro. Currently we are investigating ANGPT1-mediated signalling in t(4;11) ALL cells, as it represents an attractive potential therapeutic target.

No relevant conflicts of interest to declare.