Upregulation of Vav3 Is Required for Leukemogenesis By BCR-ABL through Polycomb Repression Complex Dependent De-Repression of the Cdkn2a Locus

Activating mutations of Rho-family of small GTPases have been linked to lymphoproliferative disorders, although the pathogenesis mechanism involved is unknown. BCR-ABL (p190) B-cell acute lymphoblastic leukemia (B-ALL) arises from the expression of the oncofusion protein BCR-ABL in a B-cell progenitor. The transforming effect of BCR-ABL is dependent on the tyrosine kinase (TK) activity of the fusion protein that leads to autophosphorylation, recruitment of adaptor proteins, and subsequent activation of downstream signaling. TK inhibitors (TKIs) have been used as frontline treatment for Ph⁺ B-ALL patients. However, relapse is common in Ph⁺ B-ALL despite high rates of complete response with initial therapy, probably because of survival of leukemic progenitors. These BCR-ABL⁺ progenitors appear to develop additional epigenetic and genetic alterations that result in proliferative advantage frequently associated with silencing of the cyclin dependent kinase inhibitor Cdkn2a, even before mutant Cdkn2a gene deleted cells are selected during clonal evolution. Recent work by our group (Chang KH et al., Blood 2012) identified the Rho GTPase guanine nucleotide exchange factor Vav3 in BCR-ABL mediated lymphoid leukemogenesis. We showed that the deficiency of the guanosine nucleotide exchange factor Vav3 delays leukemogenesis and phenocopies the effect of Rac2 (and combined Rac2/Rac1) deficiency (Thomas EK et al., Cancer Cell 2007; Sengupta A et al., Blood 2010), a downstream effector of Vav3. Upregulated Vav3 expression and activation only partly depend on ABL TK activity, and Vav3 deficiency collaborates with tyrosine kinase inhibitors to impair leukemogenesis in vitro and in vivo through impaired proliferation and survival. On the other hand, our group has demonstrated that Bmi1 overexpression frequently found in BCR-ABL⁺ B-ALL results in B-cell progenitor reprogramming through acquisition of a stem cell-like phenotype (Sengupta A et al., Blood 2012). Bmi1 forms part of the classical polycomb repression complex 1 (PRC1) where its component Ring1A/B catalyzes histone H2A mono-ubiquitination at lysine 119, which in conjunction with the PRC2 complex activity leads to chromatin compaction and repression of target genes. Through epistasis experiments, we found that Vav3 or Rac2 deficiency abrogates the oncogenic effect of Bmi1 overexpression. Co-immunoprecipitation experiments in nuclear and cytoplasmic cell extracts demonstrated that Vav3 and Rac1/Rac2 co-immunoprecipitate with Bmi1 in the nucleus but not in the cytosol of BCR-ABL⁺ leukemic cells. Interestingly, control non-BCR-ABL expressing nuclear extracts show minimal, if any, level of co-immunoprecipitation. This co-immunoprecipitation is not directly induced by BCR-ABL since BCR-ABL does not co-immunoprecipitate with Vav3/Rac1/Rac2 but does with Bmi1, suggesting that nuclear Vav3 activity may be dissected from the TK activity of BCR-ABL. Biochemically, the overexpression of Bmi1 results in increased activation of nuclear Rac which is practically abrogated by the deficiency of Vav3 as assessed in cellular pulldown assays of primary leukemic B-cell progenitors. As expected, downstream expression of Cdkn2a is repressed by overexpression by Bmi1. Deficiency of Vav3 restores the expression of Cdkn2a to control levels. This data suggests a transcriptional regulatory role of the signaling proteins Vav3/Rac2 in the nucleus. Chromatin immunoprecipitation (ChIP)-qPCR for Bmi1, Ring1B and polycomb repressive histone marks (H2AK119 and H3K27me3) and the assay for Tn5-transposase accessible chromatin (ATAQ)-qPCR for the Cdkn2a locus in Vav3- or Rac2-deficient, BCR-ABL⁺ primary B-cell progenitors were compared with their BCR-ABL, Vav3/Rac2 expressing counterparts. These assays confirmed that Vav3 and Rac2 are essential for PRC dependent transcriptional repression of Cdkn2a through occupancy of the Cdkn2a promoter and decreased accessibility to Cdkn2a chromatin. In conclusion, our studies establish for the first time an association between nuclear Vav3/Rac and polycomb repressive activities in p190-BCR-ABL⁺ leukemogenesis through their activity on the Cdkn2a locus. Vav3 may represent a novel target for adjuvant therapy with TKI in BCR-ABL⁺ lymphoblastic leukemia.