Rho-associated kinase 1 inhibition is synthetically lethal with von Hippel-Lindau deficiency in clear cell renal cell carcinoma

Abstract

Clear Cell Renal Cell Carcinoma (CC-RCC) is the most lethal of all genitourinary cancers. The functional loss of the von Hippel-Lindau (VHL) gene occurs in 90% of CC-RCC, driving cancer progression. The objective of this study was to identify chemical compounds that are synthetically lethal with VHL deficiency in CC-RCC. An annotated chemical library, the Library of Pharmacologically Active Compounds (LOPAC), was screened in parallel on VHL-deficient RCC4 cells and RCC4VHL cells with re-introduced VHL cDNA. The ROCK inhibitor, Y-27632, was identified and validated for selective targeting of VHL-deficient CC-RCC in multiple genetic backgrounds by clonogenic assays. Downregulation of ROCK1 by siRNA selectively reduced the colony forming ability of VHL-deficient CC-RCC, thus mimicking the effect of Y-27632 treatment, whereas downregulation of ROCK2 had no effect. In addition, two other ROCK inhibitors, RKI 1447 and GSK 429286, selectively targeted VHL-deficient CC-RCC. CC-RCC treatment with ROCK inhibitors is cytotoxic and cytostatic based on BrdU assay, Propidium Iodide (PI) staining, and growth curves; and blocks cell migration based on transwell assay. Importantly, knockdown of Hypoxia Inducible Factor (HIF) β in the VHL-deficient CC-RCC had a protective effect against Y-27632 treatment, mimicking VHL reintroduction. On the other hand, CC-RCCVHL cells were sensitized to Y-27632 treatment in hypoxia (2% O2). These results suggest that synthetic lethality between ROCK inhibition and VHL deficiency is dependent on HIF activation. Moreover, HIF1α or HIF2α overexpression in CC-RCCVHL cells is sufficient to sensitize them to ROCK inhibition. Finally, Y-27632 treatment inhibited growth of subcutaneous 786-OT1 CC-RCC tumors in mice. Thus, ROCK inhibitors represent potential therapeutics for VHL-deficient CC-RCC.

Inhibition of Rho-Associated Kinase Suppresses Medulloblastoma Growth

“…To confirm ROCK2-specific effects on medulloblastoma growth we performed siRNA experiments, and indeed observed a significant decrease in cell viability after ROCK2 downregulation ( Figure 3B,C, Figure S2C). Comparing previously studies using RKI-1447 in cancer, reported IC 50 values from cell viability assay (WST-1, MTT) and clonogenic assay are comparable to or higher than what we observed in medulloblastoma cell lines [16,30].…”

Targeting the Mevalonate Pathway Suppresses VHL-Deficient CC-RCC through an HIF-Dependent Mechanism

“…The cell lines used in this study were a gift from Dr. Giaccia (Stanford). The identities of RCC4, RCC10, and 786-O were confirmed via STR analysis through the University of Arizona Genetics Core.786-OT1 cells are a sub-line of 786-O described in (5). Simvastatin, Pravastatin, mevalonate, GGPP, squalene (Sigma-Aldrich, St. Louis, MO), Fluvastatin, Lovastatin (Selleck Chemicals, Houston, TX), and Arachidonic acid (MP Biomedicals, Santa Ana, CA).…”

“…Previously we conducted a chemical library screen and identified Y-27632 and inhibition of its target(ROCK1) being synthetically lethal with VHL loss(5). While multiple ROCK inhibitors have shown success in topical treatments for glaucoma(6), systemic treatments were conducted only with two ROCK inhibitors.…”

Tracing Clonal Dynamics Reveals that Two- and Three-dimensional Patient-derived Cell Models Capture Tumor Heterogeneity of Clear Cell Renal Cell Carcinoma

“…Tumor organoids were established as previously described [23], and could be generated from cryopreserved dissociated primary and passaged cells. It is important to note that in contrast to previously published protocols [23,24], ccRCC-derived organoids had to be cultivated in the absence of inhibitors of the Rho-associated coiled coil containing protein kinase (ROCK), as these impaired cell proliferation due to the synthetic lethal relationship with VHL deficiency [25]. Initial validation of the organoids was achieved by cytology that ensured the presence of cells with typical features of malignancy such as multiple pleomorphic nuclei and large nucleoli (Fig.…”