Abstract 2975: Characterization of the RAC1/CDC42 inhibitor MBQ-167 to assess its utility as a tool for interrogating Rho GTPase biology

The Simulated Cell platform highlighted RAC1 as a target with great antitumor potential. RAC1 is a small Rho GTPase with roles in cell polarity and migration, and contributes to cancer metastasis. To identify good chemical tools for RAC1, we evaluated multiple RAC1 inhibitors, and selected MBQ-167 as a potent RAC/CDC42 inhibitor with publicly available efficacy data. We analyzed the in silico effects of individual and dual RAC1/CDC42 inhibitors using the Simulated Cell. In vitro we studied MBQ-167 using viability, apoptosis and scratch assays. Viability of RAC1 KO cells treated with MBQ-167 was also analyzed. Post-treatment RNA-Seq and bioinformatics analysis of pathway activity signatures revealed MBQ-167 downstream effects. The SignalChem kinase assay was used to validate off-target identification. Our simulations revealed that, beyond their role in cytoskeleton, RAC1 and CDC42 exert effects through apoptosis signaling, with a stronger contribution of RAC1. In vitro, we confirmed the activity of MBQ-167 against RAC1 and CDC42. We observed a negative impact of MBQ-167 on cell migration and an induction of apoptosis. However, RAC1 KO did not affect sensitivity to MBQ-167, suggesting that effects of MBQ-167 on cell viability are not dependent on RAC1. Post-treatment RNA-Seq analysis of MBQ-167 treated cells and calculation of the similarity scores with LINCS consensus signatures, revealed that the MBQ-167 signature is highly similar to those of various kinase KOs and inhibitors targeting PLKs, AURKA, MET, CDK1, MAPK14, FLTs, RET and PDGFRs. RAC KOs were not involved. A kinase off-target panel validated that MBQ-167 inhibits the aforementioned kinases. It inhibited PLK2 and AURKA with an IC50 of around 200 nM, similar to its primary described targets. Other kinases were also inhibited in the nanomolar range, such as CDK1, MET and PLK1. Targeting RAC proteins and CDC42 holds great anti-tumoral potential. We investigated MBQ-167 in vitro and showed that it does not exert its effects on cancer cell viability through RAC1. Besides RAC and CDC42, this compound can inhibit cancer-relevant kinases such as AURKA and PLK2. Simulations helped understanding that MBQ-167 is not a specific inhibitor and therefore not adequate as a tool for interrogating the biology of interest. Further studies are needed to find additional inhibitors against RAC/CDC42 and to understand the effects of MBQ-167 on cancer cells and as a clinical asset tested in Phase I clinical trials as a single agent against advanced breast cancer. Citation Format: Szilvia Barsi, Árpád Varga, Csaba Szántai-Kis, María Victoria Ruiz-Pérez, Iván Fekete, Imre Gáspár, Csilla Polner-Szundi, Boldizsár Elek, Magdolna Djurec, Nóra Ordasi, István Taisz, Amrik Basran, Daniel Veres. Characterization of the RAC1/CDC42 inhibitor MBQ-167 to assess its utility as a tool for interrogating Rho GTPase biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2975.