Guanosine Nucleotide Exchange Factor Research Articles

The mevalonate pathway contributes to breast primary tumorigenesis and lung metastasis.

The mevalonate pathway plays an important role in breast cancer and other tumor types. However, many issues remain obscure as yet regarding its mechanism of regulation and action. In the present study, we report that the expression of mevalonate pathway enzymes is mediated by theRHO guanosine nucleotide exchange factors VAV2 and VAV3 in a RAC1- and sterol regulatory element-binding factor (SREBF)-dependent manner in breast cancer cells. Furthermore, in vivo tumorigenesis experiments indicated that the two most upstream steps of this metabolic pathway [3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR)] are important for primary tumorigenesis, angiogenesis, and cell survival in breast cancer cells. HMGCR, but not HMGCS1, is also important for the extravasation and subsequent fitness of breast cancer cells in the lung parenchyma. Genome-wide expression analyses revealed that HMGCR influences the expression of gene signatures linked to proliferation, metabolism, and immune responses. The HMGCR-regulated gene signature predicts long-term tumor recurrence but not metastasis in cohorts of nonsegregated and chemotherapy-resistant breast cancer patients. These results reveal a hitherto unknown, VAV-catalysis-dependent mechanism involved in the regulation of the mevalonate pathway in breast cancer cells. They also identify specific mevalonate-pathway-dependent processes that contribute to the malignant features of breast cancer cells.

Obscurin Rho GEF domains are phosphorylated by MST-family kinases but do not exhibit nucleotide exchange factor activity towards Rho GTPases in vitro.

Obscurin is a giant muscle protein (>800 kDa) featuring multiple signalling domains, including an SH3-DH-PH domain triplet from the Trio-subfamily of guanosine nucleotide exchange factors (GEFs). While previous research suggests that these domains can activate the small GTPases RhoA and RhoQ in cells, in vitro characterization of these interactions using biophysical techniques has been hampered by the intrinsic instability of obscurin GEF domains. To study substrate specificity, mechanism and regulation of obscurin GEF function by individual domains, we successfully optimized recombinant production of obscurin GEF domains and found that MST-family kinases phosphorylate the obscurin DH domain at Thr5798. Despite extensive testing of multiple GEF domain fragments, we did not detect any nucleotide exchange activity in vitro against 9 representative small GTPases. Bioinformatic analyses show that obscurin differs from other Trio-subfamily GEFs in several important aspects. While further research is necessary to evaluate obscurin GEF activity in vivo, our results indicate that obscurin has atypical GEF domains that, if catalytically active at all, are subject to complex regulation.

Spatially resolved in silico modeling of NKG2D signaling kinetics suggests a key role of NKG2D and Vav1 Co-clustering in generating natural killer cell activation.

Natural Killer (NK) cells provide key resistance against viral infections and tumors. A diverse set of activating and inhibitory NK cell receptors (NKRs) interact with cognate ligands presented by target host cells, where integration of dueling signals initiated by the ligand-NKR interactions determines NK cell activation or tolerance. Imaging experiments over decades have shown micron and sub-micron scale spatial clustering of activating and inhibitory NKRs. The mechanistic roles of these clusters in affecting downstream signaling and activation are often unclear. To this end, we developed a predictive in silico framework by combining spatially resolved mechanistic agent based modeling, published TIRF imaging data, and parameter estimation to determine mechanisms by which formation and spatial movements of activating NKG2D microclusters affect early time NKG2D signaling kinetics in a human cell line NKL. We show co-clustering of NKG2D and the guanosine nucleotide exchange factor Vav1 in NKG2D microclusters plays a dominant role over ligand (ULBP3) rebinding in increasing production of phospho-Vav1(pVav1), an activation marker of early NKG2D signaling. The in silico model successfully predicts several scenarios of inhibition of NKG2D signaling and time course of NKG2D spatial clustering over a short (~3 min) interval. Modeling shows the presence of a spatial positive feedback relating formation and centripetal movements of NKG2D microclusters, and pVav1 production offers flexibility towards suppression of activating signals by inhibitory KIR ligands organized in inhomogeneous spatial patterns (e.g., a ring). Our in silico framework marks a major improvement in developing spatiotemporal signaling models with quantitatively estimated model parameters using imaging data.

The Rho guanosine nucleotide exchange factors Vav2 and Vav3 modulate epidermal stem cell function

It is known that Rho GTPases control different aspects of the biology of skin stem cells (SSCs). However, little information is available on the role of their upstream regulators under normal and tumorigenic conditions in this process. To address this issue, we have used here mouse models in which the activity of guanosine nucleotide exchange factors of the Vav subfamily has been manipulated using both gain- and loss-of-function strategies. These experiments indicate that Vav2 and Vav3 regulate the number, functional status, and responsiveness of hair follicle bulge stem cells. This is linked to gene expression programs related to the reinforcement of the identity and the quiescent state of normal SSCs. By contrast, in the case of cancer stem cells, they promote transcriptomal programs associated with the identity, activation state, and cytoskeletal remodeling. These results underscore the role of these Rho exchange factors in the regulation of normal and tumor epidermal stem cells.

VAV Proteins as Double Agents in Cancer: Oncogenes with Tumor Suppressor Roles.

Simple SummaryThe role of the VAV family (comprised of VAV1, VAV2, and VAV3) in proactive pathways involved in cell transformation has been historically assumed. Indeed, the discovery of potential gain-of-function VAV1 mutations in specific tumor subtypes reinforced this functional archetype. Contrary to this paradigm, we demonstrated that VAV1 could unexpectedly act as a tumor suppressor in some in vivo contexts. In this review, we discuss recent findings in the field, where the emerging landscape is one in which GTPases and their regulators, such as VAV proteins, can exhibit tumor suppressor functions. Guanosine nucleotide exchange factors (GEFs) are responsible for catalyzing the transition of small GTPases from the inactive (GDP-bound) to the active (GTP-bound) states. RHO GEFs, including VAV proteins, play essential signaling roles in a wide variety of fundamental cellular processes and in human diseases. Although the most widespread archetype in the field is that RHO GEFs exert proactive functions in cancer, recent studies in mice and humans are providing new insights into the in vivo function of these proteins in cancer. These results suggest a more complex scenario where the role of GEFs is not so clearly defined. For example, VAV1 can unexpectedly play non-catalytic tumor suppressor functions in T-cell acute lymphoblastic leukemia (T-ALL) by controlling the levels of the active form of NOTCH1 (ICN1). This review focuses on emerging work unveiling tumor suppressor roles for these proteins that should prompt a reevaluation of the role of VAV GEF family in tumor biology.

New Functions of Vav Family Proteins in Cardiovascular Biology, Skeletal Muscle, and the Nervous System.

Simple SummaryIn this review, we provide information on the role of Vav proteins, a group of signaling molecules that act as both Rho GTPase activators and adaptor molecules, in the cardiovascular system, skeletal muscle, and the nervous system. We also describe how these functions impact in other physiological and pathological processes such as sympathoregulation, blood pressure regulation, systemic metabolism, and metabolic syndrome.Vav proteins act as tyrosine phosphorylation-regulated guanosine nucleotide exchange factors for Rho GTPases and as molecular scaffolds. In mammals, this family of signaling proteins is composed of three members (Vav1, Vav2, Vav3) that work downstream of protein tyrosine kinases in a wide variety of cellular processes. Recent work with genetically modified mouse models has revealed that these proteins play key signaling roles in vascular smooth and skeletal muscle cells, specific neuronal subtypes, and glia cells. These functions, in turn, ensure the proper regulation of blood pressure levels, skeletal muscle mass, axonal wiring, and fiber myelination events as well as systemic metabolic balance. The study of these mice has also led to the discovery of new physiological interconnection among tissues that contribute to the ontogeny and progression of different pathologies such as, for example, hypertension, cardiovascular disease, and metabolic syndrome. Here, we provide an integrated view of all these new Vav family-dependent signaling and physiological functions.

Drug Vulnerabilities and Disease Prognosis Linked to the Stem Cell-Like Gene Expression Program Triggered by the RHO GTPase Activator VAV2 in Hyperplastic Keratinocytes and Head and Neck Cancer

We have recently shown that VAV2, a guanosine nucleotide exchange factor that catalyzes the stimulation step of RHO GTPases, is involved in a stem cell-like (SCL) regenerative proliferation program that is important for the development and subsequent maintenance of the tumorigenesis of both cutaneous (cSCC) and head and neck squamous cell carcinomas (hnSCC). In line with this, we have observed that the levels of the VAV2 mRNA and VAV2-regulated gene signatures are associated with poor prognosis in the case of human papillomavirus-negative hnSCC patients. These results suggest that the SCL program elicited by VAV2 in those cells can harbor therapeutically actionable downstream targets. We have addressed this issue using a combination of both in silico and wet-lab approaches. Here, we show that the VAV2-regulated SCL program does harbor a number of cell cycle- and signaling-related kinases that are essential for the viability of undifferentiated keratinocytes and hnSCC patient-derived cells endowed with high levels of VAV2 activity. Our results also show that the VAV2-regulated SCL gene signature is associated with poor hnSCC patient prognosis. Collectively, these data underscore the critical role of this VAV2-regulated SCL program for the viability of both preneoplastic and fully transformed keratinocytes.

Expression of DOCK9 and DOCK11 Analyzed with Commercial Antibodies: Focus on Regulation of Mutually Exclusive First Exon Isoforms.

Dedicators of cytokinesis 9 and 11 (DOCK9 and DOCK11) are members of the dedicator of cytokinesis protein family encoding the guanosine nucleotide exchange factors for Rho GTPases. Together with DOCK10, they constitute the DOCK-D or Zizimin subfamily. Two alternative full-length amino terminal isoforms of DOCK9 are known, which we will call DOCK9.1 and DOCK9.2. In order to investigate the relevance of the presence of the alternative first exon isoforms within this family, and to lay the groundwork for future studies that seek to investigate their potential role as biomarkers of disease, the expression levels of DOCK9 and DOCK11 were measured by qRT-PCR in 26 human tissues and 23 human cell lines, and by Western blot analysis, using commercial antibodies in cell lines. DOCK9.1 and DOCK9.2 were widely distributed. High levels of expression of both isoforms were found in the lungs, placenta, uterus, and thyroid gland. However, only DOCK9.1 was significantly expressed in the neural and hematopoietic tissues. The unique first exon form of DOCK11 was highly expressed in hematopoietic tissues, such as the peripheral blood leukocytes, spleen, thymus, or bone marrow, and in others such as the lungs, placenta, uterus, or thyroid gland. In contrast to tissues, the expression of DOCK9.1 and DOCK9.2 differed from one another and also from total DOCK9 in cell lines, suggesting that the amino terminal isoforms of DOCK9 may be differentially regulated. This study demonstrates the usefulness of antibodies in investigating the regulation of the expression of DOCK9.1, total DOCK9, and DOCK11.

Vav2 pharmaco-mimetic mice reveal the therapeutic value and caveats of the catalytic inactivation of a Rho exchange factor.

The current paradigm holds that the inhibition of Rho guanosine nucleotide exchange factors (GEFs), the enzymes that stimulate Rho GTPases, can be a valuable therapeutic strategy to treat Rho-dependent tumors. However, formal validation of this idea using in vivo models is still missing. In this context, it is worth remembering that many Rho GEFs can mediate both catalysis-dependent and independent responses, thus raising the possibility that the inhibition of their catalytic activities might not be sufficient per se to block tumorigenic processes. On the other hand, the inhibition of these enzymes can trigger collateral side effects that could preclude the practical implementation of anti-GEF therapies. To address those issues, we have generated mouse models to mimic the effect of the systemic application of an inhibitor for the catalytic activity of the Rho GEF Vav2 at the organismal level. Our results indicate that lowering the catalytic activity of Vav2 below specific thresholds is sufficient to block skin tumor initiation, promotion, and progression. They also reveal that the negative side effects typically induced by the loss of Vav2 can be bypassed depending on the overall level of Vav2 inhibition achieved in vivo. These data underscore the pros and cons of anti-Rho GEF therapies for cancer treatment. They also support the idea that Vav2 could represent a viable drug target.

The Warburg Micro Syndrome-associated Rab3GAP-Rab18 module promotes autolysosome maturation through the Vps34 Complex I.

Warburg micro syndrome (WMS) is a hereditary autosomal neuromuscular disorder in humans caused by mutations in Rab18, Rab3GAP1, or Rab3GAP2 genes. Rab3GAP1/2 forms a heterodimeric complex, which acts as a guanosine nucleotide exchange factor and activates Rab18. Although the genetic causes of WMS are known, it is still unclear whether loss of the Rab3GAP-Rab18 module affects neuronal or muscle cell physiology or both, and how. In this work, we characterize a Rab3GAP2 mutant Drosophila line to establish a novel animal model for WMS. Similarly to symptoms of WMS, loss of Rab3GAP2 leads to highly decreased motility in Drosophila that becomes more serious with age. We demonstrate that these mutant flies are defective for autophagic degradation in multiple tissues including fat cells and muscles. Loss of Rab3GAP-Rab18 module members leads to perturbed autolysosome morphology due to destabilization of Rab7-positive autophagosomal and late endosomal compartments and perturbation of lysosomal biosynthetic transport. Importantly, overexpression of UVRAG or loss of Atg14, two alternative subunits of the Vps34/PI3K (vacuole protein sorting 34/phosphatidylinositol 3-kinase) complexes in fat cells, mimics the autophagic phenotype of Rab3GAP-Rab18 module loss. We find that GTP-bound Rab18 binds to Atg6/Beclin1, a permanent subunit of Vps34 complexes. Finally, we show that Rab3GAP2 and Rab18 are present on autophagosomal and autolysosomal membranes and colocalize with Vps34 Complex I subunits. Our data suggest that the Rab3GAP-Rab18 module regulates autolysosomal maturation through its interaction with the Vps34 Complex I, and perturbed autophagy due to loss of the Rab3GAP-Rab18 module may contribute to the development of WMS.

The function of Vav protein in immune system

Vav-family proteins are guanosine nucleotide exchange factors(GEFs)and one of RhoGEFs family numbers.Vav proteins can influence cell signaling transduction, cytoskeleton recombination and adherence migration in special cells by RhoA, RhoG and Rac-1 pathway.There are 80 kinds of RhoGEFs in mammalian cell.Vav proteins play an important role in hematological system, nervous system, cardiovascular system and immune system.So the function of vav protein in immune system was reviewed in this paper. Key words: Vav; Guanosine nucleotide exchange factors; Immune system

The Vav GEF Family: An Evolutionary and Functional Perspective.

Vav proteins play roles as guanosine nucleotide exchange factors for Rho GTPases and signaling adaptors downstream of protein tyrosine kinases. The recent sequencing of the genomes of many species has revealed that this protein family originated in choanozoans, a group of unicellular organisms from which animal metazoans are believed to have originated from. Since then, the Vav family underwent expansions and reductions in its members during the evolutionary transitions that originated the agnates, chondrichthyes, some teleost fish, and some neoaves. Exotic members of the family harboring atypical structural domains can be also found in some invertebrate species. In this review, we will provide a phylogenetic perspective of the evolution of the Vav family. We will also pay attention to the structure, signaling properties, regulatory layers, and functions of Vav proteins in both invertebrate and vertebrate species.

SMCR8 negatively regulates AKT and MTORC1 signaling to modulate lysosome biogenesis and tissue homeostasis

ABSTRACTThe intronic hexanucleotide expansion in the C9orf72 gene is one of the leading causes of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), two devastating neurodegenerative diseases. C9orf72 forms a heterodimer with SMCR8 (Smith-Magenis syndrome chromosome region, candidate 8) protein. However, the physiological function of SMCR8 remains to be characterized. Here we report that ablation of SMCR8 in mice results in splenomegaly with autoimmune phenotypes similar to that of C9orf72 deficiency. Furthermore, SMCR8 loss leads to a drastic decrease of C9orf72 protein levels. Many proteins involved in the macroautophagy-lysosome pathways are downregulated upon SMCR8 loss due to elevated activation of MTORC1 and AKT, which also leads to increased spine density in the Smcr8 knockout neurons. In summary, our studies demonstrate a key role of SMCR8 in regulating MTORC1 and AKT signaling and tissue homeostasis.Abbreviations: ALS: amyotrophic lateral sclerosis; C9orf72: chromosome 9 open reading frame 72; FTLD: frontotemporal lobar degeneration; GEF: guanosine nucleotide exchange factor; GTPase: guanosine tri-phosphatase; KO: knockout; MTOR: mechanistic target of rapamycin kinase; SMCR8: Smith-Magenis chromosome region, candidate 8; WDR41: WD repeat domain 41; WT: wild type

Vav3-induced cytoskeletal dynamics contribute to heterotypic properties of endothelial barriers.

Through multiple cell-cell and cell-matrix interactions, epithelial and endothelial sheets form tight barriers. Modulators of the cytoskeleton contribute to barrier stability and act as rheostats of vascular permeability. In this study, we sought to identify cytoskeletal regulators that underlie barrier diversity across vessels. To achieve this, we correlated functional and structural barrier features to gene expression of endothelial cells (ECs) derived from different vascular beds. Within a subset of identified candidates, we found that the guanosine nucleotide exchange factor Vav3 was exclusively expressed by microvascular ECs and was closely associated with a high-resistance barrier phenotype. Ectopic expression of Vav3 in large artery and brain ECs significantly enhanced barrier resistance and cortical rearrangement of the actin cytoskeleton. Mechanistically, we found that the barrier effect of Vav3 is dependent on its Dbl homology domain and downstream activation of Rap1. Importantly, inactivation of Vav3 in vivo resulted in increased vascular leakage, highlighting its function as a key regulator of barrier stability.

The guanine nucleotide exchange factor Arf-like protein 13b is essential for assembly of the mouse photoreceptor transition zone and outer segment

Arf-like protein 13b (ARL13b) is a small GTPase that functions as a guanosine nucleotide exchange factor (GEF) for ARL3-GDP. ARL13b is located exclusively in photoreceptor outer segments (OS) presumably anchored to discs by palmitoylation, whereas ARL3 is an inner segment cytoplasmic protein. Hypomorphic mutations affecting the ARL13b G-domain inactivate GEF activity and lead to Joubert syndrome (JS) in humans. However, the molecular mechanisms in ARL13b mutation-induced Joubert syndrome, particularly the function of primary cilia, are still incompletely understood. Because Arl13b germline knockouts in mouse are lethal, we generated retina-specific deletions of ARL13b in which ARL3-GTP formation is impaired. In mouse retArl13b-/- central retina at postnatal day 6 (P6) and older, outer segments were absent, thereby preventing trafficking of outer segment proteins to their destination. Ultrastructure of postnatal day 10 (P10) central retArl13b-/- photoreceptors revealed docking of basal bodies to cell membranes, but mature transition zones and disc structures were absent. Deletion of ARL13b in adult mice via tamoxifen-induced Cre/loxP recombination indicated that axonemes gradually shorten and outer segments progressively degenerate. IFT88, essential for anterograde intraflagellar transport (IFT), was significantly reduced at tamArl13b-/- basal bodies, suggesting impairment of intraflagellar transport. AAV2/8 vector-mediated ARL13b expression in the retArl13b-/- retina rescued ciliogenesis.

Inhibition of smooth muscle contraction and ARF6 activity by the inhibitor for cytohesin GEFs, secinH3, in the human prostate.

Prostate smooth muscle contraction is critical for etiology and treatment of male lower urinary tract symptoms (LUTS) and is promoted by small monomeric GTPases (RhoA and Rac). GTPases may be activated by guanosine nucleotide exchange factors (GEFs). GEFs of the cytohesin family may indirectly activate Rac, or ADP ribosylation factor (ARF) GTPases directly. Here we investigated the expression of cytohesin family GEFs and effects of the cytohesin inhibitor Sec7 inhibitor H3 (secinH3) on smooth muscle contraction and GTPase activities in human prostate tissues. Of all four cytohesin isoforms, cytohesin-1 and -2 showed the highest expression in real-time PCR. Western blot and fluorescence staining suggested that cytohesin-2 may be the predominant isoform in prostate smooth muscle cells. Contractions induced by norepinephrine, the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analog U-46619 , and endothelin-1 and -3, as well as neurogenic contractions induced by electric field stimulation (EFS), were reduced by secinH3 (30 µM). Inhibition of EFS-induced contractions appeared to have efficacy similar to that of inhibition by the α1-adrenoceptor antagonist tamsulosin (300 nM). Combined application of secinH3 plus tamsulosin caused larger inhibition of EFS-induced contractions than tamsulosin alone. Pull-down assays demonstrated inhibition of the small monomeric GTPase ARF6 by secinH3, but no inhibition of RhoA or Rac1. In conclusion, we suggest that a cytohesin-ARF6 pathway takes part in smooth muscle contraction. This may open attractive new possibilities in medical treatment of male LUTS.

Association of VAV2 and VAV3 polymorphisms with cardiovascular risk factors

Hypertension, diabetes and obesity are cardiovascular risk factors closely associated to the development of renal and cardiovascular target organ damage. VAV2 and VAV3, members of the VAV family proto-oncogenes, are guanosine nucleotide exchange factors for the Rho and Rac GTPase family, which is related with cardiovascular homeostasis. We have analyzed the relationship between the presence of VAV2 rs602990 and VAV3 rs7528153 polymorphisms with cardiovascular risk factors and target organ damage (heart, vessels and kidney) in 411 subjects. Our results show that being carrier of the T allele in VAV2 rs602990 polymorphism is associated with an increased risk of obesity, reduced levels of ankle-brachial index and diastolic blood pressure and reduced retinal artery caliber. In addition, being carrier of T allele is associated with increased risk of target organ damage in males. On the other hand, being carrier of the T allele in VAV3 rs7528153 polymorphism is associated with a decreased susceptibility of developing a pathologic state composed by the presence of hypertension, diabetes, obesity or cardiovascular damage, and with an increased risk of developing altered basal glycaemia. This is the first report showing an association between VAV2 and VAV3 polymorphisms with cardiovascular risk factors and target organ damage.

Neutrophil mechanotransduction: A GEF to sense fluid shear stress.

Forces deriving from blood flow shear modulate vascular adherence and transendothelial migration of leukocytes into inflamed tissues, but the mechanisms by which shear is sensed are unclear. In this issue, Fine et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201603109) identify the guanosine nucleotide exchange factor GEF-H1 as critical for shear stress-induced transendothelial neutrophil migration.

994 Inhibition of smooth muscle contraction by the inhibitor for cytohesin familiy guanosine nucleotide exchange factors, secin H3 in the hyperplastic human prostate

994 Inhibition of smooth muscle contraction by the inhibitor for cytohesin familiy guanosine nucleotide exchange factors, secin H3 in the hyperplastic human prostate

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. DisclosuresNo relevant conflicts of interest to declare.