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: 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.
Fig 3: 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 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 ß-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 interacts with components of PRC1 complex.A Schema depicting the assays performed in Fig. 3. B–D Western blot analyses of input (B), a-Vav3 Ab immunoprecipitated (C), and a-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.
Supplier Page from Abcam for Anti-VAV3 (phospho Y173) antibody [EPR1068A]