Effects of SLx-2119, a novel small molecule inhibitor of Rho-associated kinase ROCK (ROK), on growth of human tumor xenografts in nude mice

668 A Ser/Thr kinase ROCK (Rho-associated coiled-coil forming protein kinase) is a downstream effector of small Ras-like GTPase Rho. The Rho-ROCK signaling pathway controls cell shape, adhesion, contractility, and cell motility and invasion. Thus, testing of ROCK inhibitors in animal models of cancer was previously focused mostly on their anti-metastatic effects. The goal of the present study however, was to determine whether a novel class of ROCK-selective protein kinase inhibitors will directly affect growth of primary tumors in human tumor xenograft-bearing mice. We have developed a series of ROCK inhibitors that are potent and selective in vitro: they inhibit both isoforms of human ROCK with Ki’s for ROCK2 of 5 to 35 nM, and Ki’s for ROCK1 of 25 to 120 nM; Ki’s for other kinases (insulin receptor, Aurora A, checkpoint kinase CHK1, PKA, Abl, etc.) were 300- to 1,500-fold higher. In cell-based assays, the addition of these ROCK inhibitors to culture medium resulted in morphological changes characteristic for inhibition of Rho-ROCK signaling, i.e., disappearance of actin stress fibers and decrease in the number of focal adhesions in mouse Swiss 3T3 fibroblasts and other cell types. The inhibitors also blocked other ROCK-dependent cellular responses, i.e., lysophosphatidic acid-induced neurite retraction in N1E-115 mouse neuroblastoma cells, as well as serum-stimulated migration of HT-1080 human fibrosarcoma cells. In the latter assay, the ROCK inhibitors were more potent than a standard ROCK inhibitor Y-27632: IC50s for ROCK inhibitors were between 5 and 15 uM, whereas the IC50 for Y-27632 was 50 uM. Next, we tested whether this novel series of ROCK inhibitors has a direct effect on human tumor xenograft growth in nude mice. Oral administration of ROCK inhibitors to mice bearing staged HT-1080 human fibrosarcomas or PANC-1 human pancreatic tumors resulted in significant tumor growth delay. For example, 10 days of daily treatment with 60 mg/kg p.o. of SLx-2119 resulted in 11-12 days of post-treatment growth delay in PANC-1 tumor model. In these models ROCK inhibitors were as efficacious as cytotoxic positive controls (doxorubicin and gemcitabine). The tumor growth delay was dose-dependent and was accompanied by acceptable toxicity: mice did not show any behavior abnormalities, and their weight loss did not exceed 12% at the highest dose tested. Taken together, our results suggest that in addition to previously reported inhibition of tumor metastasis, ROCK inhibitors may also directly inhibit growth of primary tumors.