Discovery of Potent and Selective MRCK Inhibitors with Therapeutic Effect on Skin Cancer

Abstract

The myotonic dystrophy-related Cdc42-binding kinases MRCKα and MRCKβ contribute to the regulation of actin-myosin cytoskeleton organization and dynamics, acting in concert with the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The absence of highly potent and selective MRCK inhibitors has resulted in relatively little knowledge of the potential roles of these kinases in cancer. Here, we report the discovery of the azaindole compounds BDP8900 and BDP9066 as potent and selective MRCK inhibitors that reduce substrate phosphorylation, leading to morphologic changes in cancer cells along with inhibition of their motility and invasive character. In over 750 human cancer cell lines tested, BDP8900 and BDP9066 displayed consistent antiproliferative effects with greatest activity in hematologic cancer cells. Mass spectrometry identified MRCKα S1003 as an autophosphorylation site, enabling development of a phosphorylation-sensitive antibody tool to report on MRCKα status in tumor specimens. In a two-stage chemical carcinogenesis model of murine squamous cell carcinoma, topical treatments reduced MRCKα S1003 autophosphorylation and skin papilloma outgrowth. In parallel work, we validated a phospho-selective antibody with the capability to monitor drug pharmacodynamics. Taken together, our findings establish an important oncogenic role for MRCK in cancer, and they offer an initial preclinical proof of concept for MRCK inhibition as a valid therapeutic strategy. The development of selective small-molecule inhibitors of the Cdc42-binding MRCK kinases reveals their essential roles in cancer cell viability, migration, and invasive character. .

Targeting Rho GTPase Signaling Networks in Cancer

“…Although the molecular mechanisms underlying MRCK activation are not fully understood, Cdc42/Rac1 binding, membrane localization and the release of auto-inhibitory interactions appear to be required for MRCK signaling (Leung et al, 1998;Unbekandt and Olson, 2014;Zhao and Manser, 2015). Recent identification of an autophosphorylation site in MRCKα at Ser1003 led to the development of a phospho-site specific MRCKα antibody that can be used to assess levels of MRCKα activation and will likely prove useful for the further elucidation of MRCKα activation mechanisms (Unbekandt et al, 2018). Autophosphorylation of MRCKβ at Thr1108 can be used as a readout of MRCKβ kinase activity, although attempts to develop a suitable antibody tool have so far been unsuccessful (Unbekandt et al, 2019).…”

“…In a mouse model of SCC, topical application of BDP9066 reduced MRCKα activation in the skin and resulted in a decrease in tumor volume relative to controls. In a screen of 757 human cancer cell lines from 40 different cancer types, BDP8900 and BDP9066 treatment produced consistent anti-proliferative effects, suggesting MRCK may be a valid therapeutic target in a number of cancers other than SCC (Unbekandt et al, 2018). It is important to note that the toxicity of systemic BDP8900 and BDP9066 administration has not been studied and information regarding the bioavailability of these compounds is not yet available.…”

A Novel Small-Molecule Inhibitor of MRCK Prevents Radiation-Driven Invasion in Glioblastoma

“…5B). Twice-daily dosing was chosen because, although the PK profile of BDP-9066 showed good bioavailability, rapid clearance was also observed (17). The compound was well tolerated, and PK analysis of blood taken at the time of culling confirmed its presence at micromolar concentrations ( Supplementary Fig.…”

“…This mode of invasion is characterized by an elongated cell body, actin-rich protrusions, and actinmyosin contractility at the rear of the migrating cells. Unbekandt and colleagues have recently demonstrated that MRCK can drive cancer cell migration, a process that can be inhibited by specific small-molecule inhibitors (15)(16)(17). Because CDC42 activity is upregulated in glioma (18,19) and may drive the mesenchymal mode of migration employed by infiltrating glioma cells, we investigated a potential role for MRCK-driven GBM cell invasion in the pathogenesis of GBM.…”

STAT3 Contributes to Radioresistance in Cancer

“…Except for EMT process, there are other mechanisms to explain for the increased invasive ability induced by irradiation (42,(54)(55)(56)(57)(58)(59)(60)(61), which are summarized in Table 2. But whether they have relationships with STAT3 is still unknown.…”