Nuclear Vav3 is required for polycomb repression complex-1 activity in B-cell lymphoblastic leukemogenesis

Abstract

Acute B-cell lymphoblastic leukemia (B-ALL) results from oligo-clonal evolution of B-cell progenitors endowed with initiating and propagating leukemia properties. The activation of both the Rac guanine nucleotide exchange factor (Rac GEF) Vav3 and Rac GTPases is required for leukemogenesis mediated by the oncogenic fusion protein BCR-ABL. Vav3 expression becomes predominantly nuclear upon expression of BCR-ABL signature. In the nucleus, Vav3 interacts with BCR-ABL, Rac, and the polycomb repression complex (PRC) proteins Bmi1, Ring1b and Ezh2. The GEF activity of Vav3 is required for the proliferation, Bmi1-dependent B-cell progenitor self-renewal, nuclear Rac activation, protein interaction with Bmi1, mono-ubiquitination of H2A(K119) (H2AK119Ub) and repression of PRC-1 (PRC1) downstream target loci, of leukemic B-cell progenitors. Vav3 deficiency results in de-repression of negative regulators of cell proliferation and repression of oncogenic transcriptional factors. 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.

Fig. 1. VAV3 expression is upregulated and predominantly nuclear in murine and human B-ALL B-cell progenitors, and its guanine nucleotide exchange activity is essential for leukemic B-cell progenitor proliferation.

A, B Confocal immunofluorescence microscopic images (A) and quantification of mean fluorescence intensity (B) showing upregulation and nuclear distribution of Vav3 in p190-BCR-ABL⁺ murine B-cell progenitors (n = 13 per group). C Representative immunoblots for Vav3, pVav3-Y174, Gapdh and Parp in the cytoplasmic and nuclear fraction of p190-BCR-ABL⁺ B-cell progenitors. Vav3 is primarily distributed in the nuclear fraction. D Representative confocal immunofluorescence images of VAV3 expression in healthy donor and B-ALL patients derived B-cell progenitors (CD34⁺/CD19⁺). Mutation landscapes from top to bottom are: first row, normal karyotype; second row, BCR-ABL fusion; BCR/EXOSC2 fusion, CDKN2A loss; CDKN2B intron 1 truncation; third row, BCR-ABL (T315I); CD36 splice site 609+1G>A; SETD2 E282fs*19; SF3B1 T663I, sub; TLL2 G891fs*3; TP53 R248Q; fourth row, BCR-ABL (F359V; T315I); CDKN2A/B loss; IKZF1 loss; MLL2 S2173*; PAX5 Y129fs*64; fifth row, JAK1 L653 (subclonal) and R724H (subclonal); JAK2 R867Q; IGH/CRLF2; CDN2A loss exon 1; CDKN2B loss exon 2; FOXP1 R544*; ZRSR2 R448_R449insSRSR. E Nuclear/cytoplasm mean fluorescence intensity (MFI) ratio for VAV3 from primary normal and human B-ALL B-cell progenitors depicted in D (n = 16–20 per group). F Representative confocal images of PLA between c-Abl and Vav3 or p-Vav3-Y174 demonstrating physical proximity between BCR-ABL and Vav3 or p-Vav3. G Representative confocal images of PLA between c-ABL and VAV3 in Ph⁺ and Ph⁺ (T315I) patients B-cell progenitors. H Deficiency of Vav3 attenuates proliferation of leukemic B-cell progenitors as quantified by in vivo BrdU uptake assay (n = 3 per group). I, J Flow cytometry dot plots (I) and quantification (J) of proliferation (BrdU uptake) in Vav3-deficient B-cell progenitors ectopically expressing structure-function Vav3 mutants (FL- full-length, CA-constitutive active, Vav3 (N369A) (n = 3 per group). K Schematic diagram of human B-ALL model in NSG mice. L Normalized transduced human chimera (EGFP⁺) showing reduced chimera levels of VAV3 shRNA transduced Ph⁺ (BCR-ABL fusion; BCR/EXOSC2 fusion, CDKN2A loss; CDKN2B intron 1 truncation) and Ph⁺ (BCR-ABL (F359V; T315I); CDKN2A/B loss; IKZF1 loss; MLL2 S2173*; PAX5 Y129fs*64) transplanted groups in bone marrow (n = 4 per group). Engraftment of human leukemia (transduced and untransduced) was higher than 90% in all mice (see Supplementary Fig. 1F). Scale bar, 10 μm. Western blot and microscopic images are representative of a minimum of two independent experiments. Data are plotted as mean ± SD in a minimum of two independent experiments. Statistical significance was determined using the unpaired Student-t or Anova tests when more than two groups were compared. *p <  0.05; **p <  0.01; ***p <  0.001.

Fig. 2. Nuclear Vav3 regulates Cdkn2a and Cdkn2b expression by modulating PRC1 activity.

A Schema depicting the assays performed in Fig. 2. B Comparative transcriptome and gene-ontology [molecular and biological functions] of differentially expressed genes in WT and Vav3^−/− leukemic B-cell progenitors showing the differential regulation of genes involved in nuclear division, cell cycle regulation, G1/S phase transition, and covalent modifications of histone. C Quantitative real time PCR (Q-RT-PCR) analyses of Cdkn2a and Cdkn2b in Vav3 deficient p190-BCR-ABL⁺ B-cell progenitors in comparison to their WT counterparts (n = 4–12 per group). D Representative immunoblots for p16/Ink4A, p15/Ink4b and Actin in the whole cell lysates of WT and Vav3^−/− leukemic B-cell progenitors. E Representative immunoblots for H2AK119Ub, H3K27me3 and Parp in the nuclear fraction of WT and Vav3^−/− leukemic B-cell progenitors. F Representative immunoblots for H2AK119Ub and Parp in the nuclear fraction of WT leukemic B-cell progenitors transduced with empty vector and Vav3^−/− leukemic B-cell progenitors transduced with empty vector or Vav3 FL or Vav3N369A GEF mutant vectors. G Q-RT-PCR analyses of Cdkn2a and Cdkn2b in empty vector transduced WT and empty or Vav3 or Vav3N369A vector transduced Vav3^−/− leukemic B-cell progenitors (n = 6–8 per group). Data are presented as mean ± SD of a 2 or 3 independent experiments. Statistical significance was determined using the unpaired Student-t or Anova tests when more than two groups were compared. *p < 0.05; **p < 0.01; p < 0.001.

Fig. 3. Nuclear Vav3 interacts with components of PRC1 complex.

A Schema depicting the assays performed in Fig. 3. B–D Western blot analyses of input (B), α−Vav3 Ab immunoprecipitated (C), and α−Bmi1 Ab Immunoprecipitated (D) cytoplasmic and nuclear fractions of empty vector and p190-BCR-ABL transduced Ba/F3 cells. E Confocal microscopic images of PLA between Vav3 and Bmi1 in empty vector or p190-BCR-ABL transduced Ba/F3 cells. F Quantification of the mean fluorescence intensity of the PLA signals depicted in D (n = 16–43 per group). G Confocal microscopic images of PLA between Vav3 and Bmi1 in empty vector or p190-BCR-ABL transduced murine B-cell progenitors. H Quantification of the mean fluorescence intensity of the PLA signals depicted in G (n = 12 per group). Nuclear Vav3 and Bmi1 reside in close proximity and p190-BCR-ABL expression enhances PLA signal. I, J Confocal microscopic images of PLA between Vav3 and Bmi1 (I) and quantification (J) in empty vector transduced WT leukemic murine B-cell progenitors and empty vector/Vav3 FL/Vav3 GEF inactivating mutant lentiviral vector transduced Vav3^−/−- leukemic murine B-cell progenitors (n = 11–17 per group). K, L Confocal microscopic images of PLA between Vav3 and Bmi1 (K) and quantification (L) in WT and Rac2^−/− leukemic murine B-cell progenitors (n = 12–15 per group). Scale bar, 10 μm. Data are presented as mean ± SD of a 2 or 3 independent experiments. Statistical significance was determined using the unpaired Student-t or Anova test when more than two groups were compared. Differences in survival were examined using the log-rank P test. *p <  0.05; **p <  0.01; ***p <  0.001.

Fig. 4. Deficiency of Vav3 abrogates oncogenic effect of Bmi1 over-expression in leukemic B-cell progenitors.

A Wild type or Vav3^−/− LDBM cells co-transduced with p190-BCR-ABL retroviruses and empty or Bmi1 lentiviruses were transplanted into lethally irradiated C57Bl/10 mouse for the development of p190-BCR-ABL-induced B-cell acute lymphoblastic leukemia (B-ALL). B Representative Immunoblots for Bmi1, Parp and Gapdh in the cytoplasmic and nuclear extracts of B-cell progenitors derived from p190-BCR-ABL retrovirus and empty or Bmi1 lentivirus co-transduced and transplanted leukemic mice (n = 10 mice per group). C Kaplan–Meier overall survival analyses of primary recipient mice transplanted with WT or Vav3^−/− LDBM cells (10⁶ cells/mouse) co-transduced with p190-BCR-ABL retroviruses and empty or Bmi1 lentiviruses. Vav3 deficiency resulted in significant delay in chimeric mouse death. Bmi1 overexpression in WT, but not of Vav3^−/−, leukemic cells resulted in significantly decreased latency to chimeric mouse death. D Kaplan–Meier survival analyses (90-days) of secondary recipient mice transplanted with 10⁴ (dotted lines, black-WT empty; gray-Vav3^−/− empty; light blue-Vav3^−/− Bmi1), 3 × 10⁴ (dashed lines, black-WT empty; gray-Vav3^−/− empty; light blue-Vav3^−/− Bmi1), and 10⁵ (solid lines, black-WT empty; gray-Vav3^−/− empty; light blue-Vav3^−/− Bmi1) leukemic B-cell progenitors derived from primary leukemic mice. Vav3 deficiency significantly prolongs the survival. No significant difference in survival between Vav3^−/− + empty and Vav3^−/− + Bmi1 at any of the three cell doses tested was found (n = 10 mice per group). E Quantification of BrdU uptake of WT and Vav3^−/− B-cell progenitors co-expressing p190-BCR-ABL and Bmi1. Vav3 deficiency partially impairs the Bmi1 overexpression effect (n = 3 per group). F Serial plating of CFU-proB showing abrogation of CFU generating ability of Vav3 deficient empty or Bmi1-transduced p190-BCR-ABL expressing B cell progenitors. G Representative immunoblots of H2AK119Ub, and β-actin in empty or Bmi1 over-expressed WT or Vav3^−/− leukemic B-cell progenitors (n = 3 per group). H Rac activation assay in empty or Bmi1 over-expressed WT or Vav3^−/− leukemic B-cell progenitors. I CFU-proB content of empty or Bmi1 over expressing WT or Rac2^−/− leukemic B-cell progenitors (n = 6 per group). Data are presented as mean ± SD in two or three independent experiments. Statistical significance was determined using the unpaired Student-t or Anova tests when more than 2 groups were compared. *p < 0.05; **p <  0.01; ***p < 0.001.

Fig. 5. Nuclear Vav3 modulates PRC1 mediated repression of regulators of proliferation and transcriptional factors.

A Venn diagrams depicting intersection of genes where CUT&RUNseq signal for Bmi1, Ring1b, and H2AK119Ub was decreased in Vav3^−/− leukemic B-cell progenitors (Diffbind: Log₂ Fold Change>1, p <  0.05). B Gene-ontology analyses of molecular and biological function of the genes with reduced Bmi1, Ring1b, and H2AK119Ub binding. Genes associated with negative regulation of protein kinase activity, negative regulation of protein phosphorylation, and cell cycle G1/S transition show reduced PRC1 components binding. C Representative density map of 75 common genes with decreased binding of Bmi1, Ring1b, and H2AK119Ub in Vav3^−/− leukemic B-cell progenitors. Tracks shown are for Bmi1 binding. Diffbind analyses was performed between two CUT&RUN replicates of WT and Vav3^−/− leukemic B-cell progenitors, and one representative example density map is presented. D Venn diagrams showing intersection of genes where binding of Bmi1, Ring1b, and H2AK119Ub in Vav3^−/− leukemic B-cell progenitors was increased (Diffbind: Log₂ Fold Change>1). E Gene-ontology analyses of molecular and biological function of the genes with increased Bmi1, Ring1b, and H2AK119Ub binding. Genes associated with RNA polII transcription regulatory region-specific DNA binding, transcription factor regulator activity show increased PRC1 components binding. F Representative density map of 50 common genes with increased binding of Bmi1, Ring1b and H2AK119Ub in Vav3^−/− leukemic B-cell progenitors. Tracks shown are for Bmi1 binding. Diffbind analyses was performed between two CUT&RUN replicates of WT and Vav3^−/− leukemic B-cell progenitors, and one representative example density map is presented.

Fig. 6. Phlpp2 ectopic expression rescues B-ALL development in Vav3 deficient B-cell progenitors.

A Representative immunoblots showing enhanced phosphorylation of Bmi1 in the nuclear fraction of Vav3^−/− p190-BCR-ABL⁺ B-cell progenitors. B Representative immunoblots showing decreased chromatin bound and increased nuclear matrix Bmi1 in Vav3^−/− p190-BCR-ABL⁺ B-cell progenitors. C Ex-vivo expansion of mock, Bmi1 and Bmi1 (S314A) transduced WT and Vav3^−/− p190-BCR-ABL⁺ B-cell progenitors (n = 6 per group). D Q-RT-PCR analyses of Cdkn2a expression in empty vector/Bmi1/Bmi(S314A) transduced WT and Vav3^−/− p190-BCR-ABL⁺ B-cell progenitors (n = 4 per group). E Representative immunoblots for p-Akt (S-473), Akt and Phlpp2 in the cytoplasmic and nuclear fractions of WT and Vav3-deficient p190-BCR-ABL⁺ B-cell progenitors. Representative membrane blotting is the same as in Fig. 6a. F Representative immunoblots for p-Bmi1, pAkt-S473 and Parp in the nuclear fraction of MK2206 treated WT and Vav3^−/− leukemic B-cell progenitors. G Confocal microscopic images of PLA between Vav3 and Phlpp2 in the nucleus of leukemic B-cell progenitors. H Schematic diagram depicting co-transduction of WT and Vav3^−/− Lin⁻cKit⁺Sca1⁺ BM cells with p190-BCR-ABL retroviruses and empty or Phlpp2 lentiviruses followed by transplantation into C57bl/10 mice. I Kaplan-Meier survival analyses of primary recipient mice transplanted with WT or Vav3^−/− Lin⁻cKit⁺Sca1⁺ co-transduced with p190-BCR-ABL retroviruses and empty or Phlpp2 lentiviruses (n = 5 mice per group). Vav3 deficiency significantly prolongs the survival. Phlpp2 overexpression in Vav3^−/− progenitors restores leukemogenesis. J CFU-proB content of empty vector or Phlpp2 transduced WT and Vav3^−/− leukemic B-cell progenitors (n = 3 per group). K Representative immunoblots for p-Bmi1, Bmi1, H2AK119Ub, Phlpp2, and Parp in WT and Vav3^−/− leukemic B-cell progenitors transduced with empty or Phlpp2 lentiviruses. L Q-RT-PCR analyses of Cdkn2a expression in empty or Phlpp2 transduced WT and Vav3^−/− leukemic B cell progenitors (n = 5 per group). M Schema representing the nuclear protein complex controlling leukemogenic PRC1.4 activity. Scale bar, 10 μm. Data are presented as mean ± SD of two or three independent experiments. Statistical significance was determined using the unpaired Student-t or Anova tests when more than two groups were compared. Differences in survival were examined using the log-rank P test. *p <  0.05; **p <  0.01; ***p <  0.001.