The co-receptor Neuropilin-1 enhances proliferation in inv(16) acute myeloid leukemia via VEGF signaling

Oncogenic programs regulate the proliferation and maintenance of cancer stem cells, and can define pharmacologic dependencies. In acute myeloid leukemia (AML) with the chromosome inversion 16 (inv(16)), the fusion CBF?::MYH11 oncoprotein regulates pathways associated with leukemia stem cell activity. Here we demonstrate that expression of Neuropilin-1 (NRP1) is regulated by the fusion oncoprotein, and promotes AML expansion. Mechanistically, we show that the NRP1 locus has open chromatin in inv(16) AML, and that CBF?::MYH11 modulates the local function of the transcription factors ERG, GATA2 and RUNX1 to sustain NRP1 levels. We found that ERG activates NRP1 expression, and that CBF?::MYH11 knockdown represses ERG expression, thereby allowing the repressive activity of GATA2/RUNX1 at three NRP1 enhancers. Functionally, we demonstrate that NRP1 enhances the expansion of leukemic cells in vitro and in mice, and that this activity is dependent on its VEGFR-associated FV/FVIII domain. Finally, we show that treatment with VEGF inhibitor axitinib reduces AML cell growth and delays median leukemia latency in vivo. Our findings reveal that the NRP1/VEGF axis mediates proliferation in inv(16) AML blasts, and suggest that targeting NRP1 function could be promising in combination AML therapy. Overall design: ME-1 cells were transduced with lentiviruses expressing Scramble- or MYH11sh4-RNA in triplicates, cultured for 2 days, and replaced with media supplemented with 5µg/mL Puromycin between days 3 and 6. On day 7, live cells were isolated using Ficoll-gradient centrifugation, and total RNA was isolated with Monarch Total RNA Miniprep kit (New England Biolabs, cat. # T2010S). The RNA concentration was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific) and the RNA integrity was evaluated with RNA 6000 kit (Agilent Technologies) using a 2100 Bioanalyzer. The sequencing libraries were prepared and sequenced utilizing DNBseq™ Technology (BGI Genomics). The transcriptome profiling of NRP1-shRNA treated ME-1 cells was performed using a similar procedure on day 7 of culture, using Scramble- or NRP1sh3-RNA transduced ME-1 cells, in triplicates.