Nuclear Vav3 is required for canonical PRC1 activity in B-cell lymphoblastic leukemogenesis (CUT&RUN)

Acute B-cell lymphoblastic leukemia (B-ALL) represents a multiclonal evolution of B-cell progenitors endowed with both tumor-initiating and propagating properties. We have previously shown that the concurrent overexpression and activation of both the Rac guanine nucleotide exchange factor (RacGEF) Vav3 and Rac GTPases is required for BCR-ABL-mediated leukemogenesis. Here, we show that upon BCR-ABL expression, Vav3 expression becomes predominantly nuclear. In this location, Vav2 interacts with BCR-ABL, Rac, and the canonical polycomb repression complex proteins Bmi1, Ring1b and Ezh2. The GEF activity of Vav3 is required for B-cell proliferation and Bmi1-dependent B-cell progenitor self-renewal, nuclear Rac activation, protein interaction with Bmi1, H2A(K119) mono-ubiquitination (H2AK119Ub), and repression of downstream target loci. Vav3 deficiency results in de-repression and repression of loci encoding negative regulators of cell proliferation and oncogenic transcriptional factors, respectively. Mechanistically, we show that Vav3 prevents the Phlpp2-sensitive and Akt (S473)-dependent phosphorylation of Bmi1 on the regulatory residue S314 that, in turn, promotes the transcriptional factor reprogramming of leukemic B-cell progenitors. These results highlight the importance of non-canonical nuclear Rho GTPase signaling in leukemogenesis. Here, we performed cleavege under target and release using nuclease for Bmi1, Ring1b and H2AK119Ub followed by library preparation and subsequent next generation sequecing (CUT&RUNseq) for global mapping of Bmi1, Ring1b and H2ak119Ub binding in WT and Vav3-/- leuekmic B-cell progenitors derived from bone maarow of WT and Vav3-/- leukmeic chimeric mice. Leukemic B-cell progenitors (FSChiB220+IgM-CD43+CD19+) were sorted, bound to Concanavalin A (Con-A) magnetic bead, permeabilized using digitonin. The Con-A bound and digitonin permeabilized cells were treated with antibodies against Bmi1, Ring1b and H2aK119Ub followed by protein A-MNase (pA-MNase) binding and targeted digestion in presence of CaCl2. The CUT&RUN released DNA fragments were extracted using QIAGEN MinElute Reaction Cleanup kits, and were used for library preparation prepared using NEBNext Ultra II DNA library preparation kit for Illumina and NEBNext multiplex Oligoes for Illumina. Libraries followed next generation paired end sequencing.