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Mutations in the VAV1 guanine nucleotide exchange factor 1 have been recently found in peripheral T cell lymphoma and nonsmall-cell lung cancer (NSCLC). To understand their pathogenic potential, we generated a gene-edited mouse model that expresses a VAV1 mutant protein that recapitulates the signalling alterations present in the VAV1 mutant subclass most frequently found in tumours. We could not detect any overt tumourigenic process in those mice. However, the concurrent elimination of the Trp53 tumour suppressor gene in them drives T cell lymphomagenesis. This process represents an exacerbation of the normal functions that wild-type VAV1 plays in follicular helper T cells. We also found that, in combination with the Kras oncogene, the VAV1 mutant version favours progression of NSCLC. These data indicate that VAV1 mutations play critical, although highly cell-type-specific, roles in tumourigenesis. They also indicate that such functions are contingent on the mutational landscape of the tumours involved.

A missense change in RRAS2 (Gln to Leu), analogous to the Gln-to-Leu mutation of RAS oncoproteins, has been identified as a long-tail hotspot mutation in cancer and Noonan syndrome. However, the relevance of this mutation for in vivo tumorigenesis remains understudied. Here we show, using an inducible knockin mouse model, that R-Ras2 triggers rapid development of a wide spectrum of tumors when somatically expressed in adult tissues. These tumors show limited overlap with those originated by classical Ras oncogenes. R-Ras2-driven tumors can be classified into different subtypes according to therapeutic susceptibility. Importantly, the most relevant R-Ras2-driven tumors are dependent on mTORC1 but independent of phosphatidylinositol 3-kinase-, MEK-, and Ral guanosine diphosphate (GDP) dissociation stimulator. This pharmacological vulnerability is due to the extensive rewiring by R-Ras2 of pathways that orthogonally stimulate mTORC1 signaling. These findings demonstrate that RRAS2 is a bona fide oncogenic driver and unveil therapeutic strategies for patients with cancer and Noonan syndrome bearing RRAS2 mutations.

VAV1 mutations have been recently found in peripheral T cell lymphoma and non-small cell lung cancer. To understand their pathogenic potential, we generated a gene-edited mouse model that expresses a Vav1 mutant protein that recapitulates the signaling alterations present the VAV1 mutant subclass most frequently found in tumors. We could not detect any overt tumorigenic process in those mice. However, the concurrent elimination of the Trp53 tumor suppressor gene in them drives T cell lymphomagenesis. This process represents an exacerbation of the normal functions that wild-type Vav1 plays in follicular helper T cells. We also found that, in combination with an oncogenic Kras mutation, the Vav1 mutant version favors progression of non-small cell lung cancer. These data indicate that VAV1 mutations play critical, although highly cell typespecific roles in tumorigenesis. They also indicate that such functions are contingent on the mutational landscape of the tumors involved.

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.

Mutations in VAV1, a gene that encodes a multifunctional protein important for lymphocytes, are found at different frequencies in peripheral T-cell lymphoma (PTCL), non-small cell lung cancer, and other tumors. However, their pathobiological significance remains unsettled. After cataloguing 51 cancer-associated VAV1 mutations, we show here that they can be classified in five subtypes according to functional impact on the three main VAV1 signaling branches, GEF-dependent activation of RAC1, GEF-independent adaptor-like, and tumor suppressor functions. These mutations target new and previously established regulatory layers of the protein, leading to quantitative and qualitative changes in VAV1 signaling output. We also demonstrate that the most frequent VAV1 mutant subtype drives PTCL formation in mice. This process requires the concurrent engagement of two downstream signaling branches that promote the chronic activation and transformation of follicular helper T cells. Collectively, these data reveal the genetic constraints associated with the lymphomagenic potential of VAV1 mutant subsets, similarities with other PTCL driver genes, and potential therapeutic vulnerabilities.

Genetic evidence suggests that three members of the VAV family (VAV1, VAV2 and VAV3) of signal transduction proteins could play important roles in rheumatoid arthritis. However, it is not known currently whether the inhibition of these proteins protects against this disease and, if so, the number of family members that must be eliminated to get a therapeutic impact. To address this issue, we have used a collection of single and compound Vav family knockout mice in experimental models for antigen-dependent (methylated bovine serum albumin injections) and neutrophil-dependent (Zymosan A injections) rheumatoid arthritis in mice. We show here that the specific elimination of Vav1 is sufficient to block the development of antigen-induced arthritis. This protection is likely associated with the roles of this Vav family member in the development and selection of immature T cells within the thymus as well as in the subsequent proliferation and differentiation of effector T cells. By contrast, we have found that depletion of Vav2 reduces the number of neutrophils present in the joints of Zymosan A-treated mice. Despite this, the elimination of Vav2 does not protect against the joint degeneration triggered by this experimental model. These findings indicate that Vav1 is the most important pharmacological target within this family, although its main role is limited to the protection against antigen-induced rheumatoid arthritis. They also indicate that the three Vav family proteins do not play redundant roles in these pathobiological processes.

Regenerative proliferation capacity and poor differentiation are histological features usually linked to poor prognosis in head and neck squamous cell carcinoma (hnSCC). However, the pathways that regulate them remain ill-characterized. Here, we show that those traits can be triggered by the RHO GTPase activator VAV2 in keratinocytes present in the skin and oral mucosa. VAV2 is also required to maintain those traits in hnSCC patient-derived cells. This function, which is both catalysis- and RHO GTPase-dependent, is mediated by c-Myc- and YAP/TAZ-dependent transcriptomal programs associated with regenerative proliferation and cell undifferentiation, respectively. High levels of VAV2 transcripts and VAV2-regulated gene signatures are both associated with poor hnSCC patient prognosis. These results unveil a druggable pathway linked to the malignancy of specific SCC subtypes.

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.

In the last years, the development of new drugs in oncology has evolved notably. In particular, drug development has shifted from empirical screening of active cytotoxic compounds to molecularly targeted drugs blocking specific biologic pathways that drive cancer progression and metastasis. Using a rational design approach, our group has developed 1A-116 as a promising Rac1 inhibitor, with antitumoral and antimetastatic effects in several types of cancer. Rac1 is over activated in a wide range of tumor types and and it is one of the most studied proteins of the Rho GTPase family. Its role in actin cytoskeleton reorganization has effects on endocytosis, vesicular trafficking, cell cycle progression and cellular migration. In this context, the regulatory activity of Rac1 affects several key processes in the course of the cancer including invasion and metastasis. The purpose of this preclinical study was to focus on the mode of action of 1A-116, conducting an interdisciplinary approach with in silico bioinformatics tools and in vitro assays. Here, we demonstrate that the tryptophan 56 residue is necessary for the inhibitory effects of 1A-116 since this compound interferes with protein-protein interactions (PPI) of Rac1GTPase involving several GEF activators. 1A-116 is also able to inhibit the oncogenic Rac1P29S mutant protein, one of the oncogenic drivers found in sun-exposed melanoma. It also inhibits numerous Rac1-regulated cellular processes such as membrane ruffling and lamellipodia formation. These results deepen our knowledge of 1A-116 inhibition of Rac1 and its biological impact on cancer progression. They also represent a good example of how in silico analyses represent a valuable approach for drug development.

The bidirectional regulation of epithelial–mesenchymal transitions (EMT) is key in tumorigenesis. Rho GTPases regulate this process via canonical pathways that impinge on the stability of cell-to-cell contacts, cytoskeletal dynamics, and cell invasiveness. Here, we report that the Rho GTPase activators Vav2 and Vav3 utilize a new Rac1-dependent and miR-200c-dependent mechanism that maintains the epithelial state by limiting the abundance of the Zeb2 transcriptional repressor in breast cancer cells. In parallel, Vav proteins engage a mir-200c-independent expression prometastatic program that maintains epithelial cell traits only under 3D culture conditions. Consistent with this, the depletion of endogenous Vav proteins triggers mesenchymal features in epithelioid breast cancer cells. Conversely, the ectopic expression of an active version of Vav2 promotes mesenchymal-epithelial transitions using E-cadherin-dependent and independent mechanisms depending on the mesenchymal breast cancer cell line used. In silico analyses suggest that the negative Vav anti-EMT pathway is operative in luminal breast tumors. Gene signatures from the Vav-associated proepithelial and prometastatic programs have prognostic value in breast cancer patients.

Rho GDP/GTP exchange factors (GEFs) are enzymes that promote the activation of Rho GTPases in both normal and cancer cells. Due to this, it is widely assumed that they can represent potential anticancer drug targets. However, we still have very little information about the actual role of these enzymes in human cancer, the therapeutic effectiveness of inhibiting their enzyme activities, and the side effects that such inhibition elicits in healthy tissues. Our group is trying to address these issues using as main tool the three known members of the Vav GEF family, the proteins Vav1, Vav2 and Vav3. To this end, we are utilizing a multifaceted approach based on the use of genetically modified mice, genome wide expression profiling techniques, cross-species transcriptomal comparisons, and patient-derived cell models. In my talk, I will present recent data demonstrating that the endogenous wild type and mutant versions of these proteins play proactive roles in mature T cell leukemia and a variety of epithelial tumors. Furthermore, I will discuss data from our “pharmaco-mimetic” knock-in mice demonstrating that the inhibition of GEFs does have a negative impact on the fitness of tumor cells in vivo.

RHO GDP/GTP exchange factors, including VAV1, are considered key protumorigenic factors. Against this paradigm, we have found that VAV1 plays tumor suppressor roles by buffering NOTCH1 signals in thymocytes. The silencing of this pathway contributes to the pathogenesis of T cell acute lymphoblastic leukemia of the early cortical, TLX+ subtype.

Lymphocyte migration, which is essential for effective immune responses, belongs to the so-called amoeboid migration. The lymphocyte migration is up to 100 times faster than between mesenchymal and epithelial cell types. Migrating lymphocytes are highly polarized in three well-defined structural and functional zones: uropod, medial zone, and leading edge (LE). The actiomyosin-dependent driving force moves forward the uropod, whereas massive actin rearrangements protruding the cell membrane are observed at the LE. These actin rearrangements resemble those observed at the immunological synapse driven by clathrin, a protein normally involved in endocytic processes. Here, we used cell lines as well as primary lymphocytes to demonstrate that clathrin and clathrin adaptors colocalize with actin at the LE of migrating lymphocytes, but not in other cellular zones that accumulate both clathrin and actin. Moreover, clathrin and clathrin adaptors, including Hrs, the clathrin adaptor for multivesicular bodies, drive local actin accumulation at the LE. Clathrin recruitment at the LE resulted necessary for a complete cell polarization and further lymphocyte migration in both 2D and 3D migration models. Therefore, clathrin, including the clathrin population associated to internal vesicles, controls lymphocyte migration by regulating actin rearrangements occurring at the LE.

Coronin 1A (Coro1A) is involved in cytoskeletal and signaling events, including the regulation of Rac1 GTPase– and myosin II–dependent pathways. Mutations that generate truncated or unstable Coro1A proteins cause immunodeficiencies in both humans and rodents. However, in the case of the peripheral T-cell–deficient (Ptcd) mouse strain, the immunodeficiency is caused by a Glu-26-Lys mutation that targets a surface-exposed residue unlikely to affect the intramolecular architecture and stability of the protein. Here we report that this mutation induces pleiotropic effects in Coro1A protein, including the exacerbation of Coro1A-dependent actin-binding and -bundling activities; the formation of large meshworks of Coro1AE26K-decorated filaments endowed with unusual organizational, functional, and staining properties; and the elimination of Coro1A functions associated with both Rac1 and myosin II signaling. By contrast, it does not affect the ability of Coro1A to stimulate the nuclear factor of activated T-cells (NF-AT). Coro1AE26K is not a dominant-negative mutant, indicating that its pathological effects are derived from the inability to rescue the complete loss of the wild-type counterpart in cells. These results indicate that Coro1AE26K behaves as either a recessive gain-of-function or loss-of-function mutant protein, depending on signaling context and presence of the wild-type counterpart in cells.

Hypertension-associated cardiorenal diseases represent one of the heaviest burdens for current health systems. In addition to hemodynamic damage, recent results have revealed that hematopoietic cells contribute to the development of these diseases by generating proinflammatory and profibrotic environments in the heart and kidney. However, the cell subtypes involved remain poorly characterized. Here we report that CD39+ regulatory T (TREG) cells utilize an immunosuppression-independent mechanism to counteract renal and possibly cardiac damage during angiotensin II (AngII)-dependent hypertension. This mechanism relies on the direct apoptosis of tissue-resident neutrophils by the ecto-ATP diphosphohydrolase activity of CD39. In agreement with this, experimental and genetic alterations in TREG/TH cell ratios have a direct impact on tissue-resident neutrophil numbers, cardiomyocyte hypertrophy, cardiorenal fibrosis, and, to a lesser extent, arterial pressure elevation during AngII-driven hypertension. These results indicate that TREG cells constitute a first protective barrier against hypertension-driven tissue fibrosis and, in addition, suggest new therapeutic avenues to prevent hypertension-linked cardiorenal diseases.