Abstract 623: Investigating potential off-target effects of the dual Rac and Cdc42 inhibitor MBQ-167
Authors: Picon Hector M. , Maldonado Maria Del Mar , Flanagan Surangani F. Dharmawardhane 
Published: September 2020

Metastasis is a major cause of death in epithelial cancers, largely because of a lack of effective treatments. The metastatic inhibitor MBQ-167 was developed to target the Rho GTPases Rac and Cdc42, which promote cell cycle progression, proliferation, survival, migration/invasion, and thus, metastasis. Our published data show that MBQ-167 inhibits the activation of Rac and Cdc42 with ~100 nM IC50, and subsequently inhibits their effector p21-activated kinase (PAK) activation by autophosphorylation. In metastatic cancer cells, MBQ-167 induces a loss of cell polarity, followed by detachment from the substrate, and subsequent apoptosis (anoikis). The objective herein was to determine the potential off-target effects of MBQ-167. Therefore, the effect of MBQ-167 on the triple negative breast cancer kinome was investigated using a Human Phospho-Kinase Array for 45 phospho proteins. MDA-MB-231 cells treated with MBQ-167 for 24 h at 200 nM demonstrated no difference in phosphorylation of off-target kinases. However, phospho (p)-CREB and p-c-Jun transcription factor levels increased by ~3-fold in the cells that remained attached following MBQ-167 treatment. This was confirmed by Western blot for p-CREB and p-c-Jun as well as their transcriptional targets (Cyclin D1, survivin, ZEB1). However, a time-course analysis via Western blot revealed that this increase in CREB and c-Jun activity is transient. Western blotting of the attached and detached cell populations of MDA-MB-231 cells following MBQ-167 treatment for 24, 48, or 96 h demonstrated decreased c-Jun/p-c-jun, and CREB and Jun targets in the detached cell population. Moreover, the detached cells demonstrated additional decreases in phospho forms of ERK1/2, Akt, mTOR, beta catenin and STAT3, which have all been implicated in Rac/PAK signaling; as well as reduced expression of the anti-apoptotic proteins Bcl2, BCL-XL, and MCl-1. Therefore, we posit that the initial upregulation of transcription factors in response to MBQ-167 is a transient reaction to its strong Rac/Cdc42/PAK inhibition. Prolonged treatment with MBQ-167 causes a massive deregulation of cancer drivers and pro-survival proteins to ultimately result in anoikis. We attempted to create a MBQ-167 resistant cell population by treating MDA-MB-231 cells with ascending concentrations of MBQ-167 over time, but were unable to recover any live cells. Therefore, we conclude that MBQ-167 is a direct and specific inhibitor of Rac and Cdc42 and that potential off-target effects of MBQ-167 are only temporary, where 100% of the metastatic cancer cells treated with MBQ-167 ultimately undergo apoptosis. Collectively, our data on the mechanism of action of MBQ-167 and its efficacy in vitro and in vivo in mouse models of metastatic cancer demonstrate that MBQ-167 is a viable anti-metastatic cancer inhibitor for further development as an experimental therapeutic.

Citation Format: Hector M. Picon, Maria Del Mar Maldonado, Surangani F. Dharmawardhane Flanagan. Investigating potential off-target effects of the dual Rac and Cdc42 inhibitor MBQ-167 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 623.