Characterization of the spectrum of trivalent VAV1-mutation-driven tumours using a gene-edited mouse model.

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.

Cancer-associated mutations in VAV1 trigger variegated signaling outputs and T-cell lymphomagenesis.

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.

Lysine Acetylation Reshapes the Downstream Signaling Landscape of Vav1 in Lymphocytes.

Vav1 works both as a catalytic Rho GTPase activator and an adaptor molecule. These functions, which are critical for T cell development and antigenic responses, are tyrosine phosphorylation-dependent. However, it is not known whether other posttranslational modifications can contribute to the regulation of the biological activity of this protein. Here, we show that Vav1 becomes acetylated on lysine residues in a stimulation- and SH2 domain-dependent manner. Using a collection of both acetylation- and deacetylation-mimicking mutants, we show that the acetylation of four lysine residues (Lys222, Lys252, Lys587, and Lys716) leads to the downmodulation of the adaptor function of Vav1 that triggers the stimulation of the nuclear factor of activated T cells (NFAT). These sites belong to two functional subclasses according to mechanistic criteria. We have also unveiled additional acetylation sites potentially involved in either the stimulation (Lys782) or the downmodulation (Lys335, Lys374) of specific Vav1-dependent downstream responses. Collectively, these results indicate that Nε-lysine acetylation can play variegated roles in the regulation of Vav1 signaling. Unlike the case of the tyrosine phosphorylation step, this new regulatory layer is not conserved in other Vav family paralogs.

The multikinase inhibitor EC-70124 synergistically increased the antitumor activity of doxorubicin in sarcomas.

Cytotoxic drugs like doxorubicin remain as the most utilized agents in sarcoma treatment. However, advanced sarcomas are often resistant, thus stressing the need for new therapies aimed to overcome this resistance. Multikinase inhibitors provide an efficient way to target several pro-tumorigenic pathways using a single agent and may constitute a valuable strategy in the treatment of sarcomas, which frequently show an aberrant activation of pro-tumoral kinases. Therefore, we studied the antitumor activity of EC-70124, an indolocarbazole analog that have demonstrated a robust ability to inhibit a wide range of pro-survival kinases. Evaluation of the phospho-kinase profile in cell-of-origin sarcoma models and/or sarcoma primary cell lines evidenced that PI3K/AKT/mTOR, JAK/STAT or SRC were among the most highly activated pathways. In striking contrast with the structurally related drug midostaurin, EC-70124 efficiently prevented the phosphorylation of these targets and robustly inhibited proliferation through a mechanism associated to the induction of DNA damage, cell cycle arrest and apoptosis. In addition, EC-70124 was able to partially reduce tumor growth in vivo. Importantly, this compound inhibited the expression and activity of ABC efflux pumps involved in drug resistance. In line with this ability, we found that the combined treatment of EC-70124 with doxorubicin resulted in a synergistic cytotoxic effect in vitro and an increased antitumor activity of this cytotoxic drug in vivo. Altogether, these results uncover the capability of the novel multikinase inhibitor EC-70124 to counteract drug resistance in sarcoma and highlight its therapeutic potential when combined with current treatments.

FUS-CHOP Promotes Invasion in Myxoid Liposarcoma through a SRC/FAK/RHO/ROCK-Dependent Pathway.

Deregulated SRC/FAK signaling leads to enhanced migration and invasion in many types of tumors. In myxoid and round cell liposarcoma (MRCLS), an adipocytic tumor characterized by the expression of the fusion oncogene FUS-CHOP, SRC have been found as one of the most activated kinases. Here we used a cell-of-origin model of MRCLS and an MRCLS cell line to thoroughly characterize the mechanisms of cell invasion induced by FUS-CHOP using in vitro (3D spheroid invasion assays) and in vivo (chicken chorioallantoic membrane model) approaches. FUS-CHOP expression activated SRC-FAK signaling and increased the invasive ability of MRCLS cells. In addition, FAK expression was found to significantly correlate with tumor aggressiveness in sarcoma patient samples. The involvement of SRC/FAK activation in FUS-CHOP-mediated invasion was further confirmed using the SRC inhibitor dasatinib, the specific FAK inhibitor PF-573228, and FAK siRNA. Notably, dasatinib and PF573228 could also efficiently block the invasion of cancer stem cell subpopulations. Downstream of SRC/FAK signaling, we found that FUS-CHOP expression increases the levels of the RHO/ROCK downstream effector phospho-MLC2 (T18/S19) and that this activation was prevented by dasatinib or PF573228. Moreover, the ROCK inhibitor RKI-1447 was able to completely abolish invasion in FUS-CHOP-expressing cells. These data uncover the involvement of SRC/FAK/RHO/ROCK signaling axis in FUS-CHOP-mediated invasion, thus providing a rationale for testing inhibitors of this pathway as potential novel antimetastatic agents for MRCLS treatment.