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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.

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.

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 modulate the signaling output of this protein. Here, we show that Vav1 becomes acetylated in a stimulation- and SH2 domain-dependent manner. We also demonstrate that the acetylation of four residues located in the catalytic, lysine rich and SH2 domains preferentially buffers the Vav1 adaptor function that favors the stimulation of the nuclear factor of activated T cells. As a result, it modifies the signaling diversification properties normally exhibited by Vav1 in lymphocytes. This new regulatory layer is not shared by Vav family paralogs.