Kinase requirements in human cells: III. Altered kinase requirements inVHL−/− cancer cells detected in a pilot synthetic lethal screen

Abstract

Clear cell renal carcinomas are the most common form of kidney cancer and frequently are linked to biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene product, pVHL, has multiple functions including directing the polyubiquitylation of the HIF transcription factor. We screened 100 shRNA vectors, directed against 88 kinases, for their ability to inhibit the viability of VHL؊/؊ renal carcinoma cells preferentially compared with isogenic cells in which pVHL function was restored. shRNAs for ''hits'' identified in the primary screen were interrogated in secondary screens that included shRNA titration studies. Multiple shRNAs against CDK6, MET, and MAP2K1 (also known as MEK1) preferentially inhibited the viability of 786-O and RCC4 VHL؊/؊ cells compared with their wild-type pVHL-reconstituted counterparts. The sensitivity of pVHL-proficient cells to these shRNAs was not restored upon HIF activation, suggesting that loss of an hypoxia-inducible factor (HIF)-independent pVHL function formed the basis for selectivity. A small-molecule Cdk4/6 inhibitor displayed enhanced activity against VHL؊/؊ renal carcinoma cells, suggesting that in some cases hits from shRNA screens such as described here might translate into therapeutic targets.essential kinases ͉ shRNA screens ͉ VHL ͉ kidney cancer ͉ therapeutics

Acquired radioresistance of human tumor cells by DNA-PK/AKT/GSK3β-mediated cyclin D1 overexpression

“…DNA-PK inhibitor NU7026 was purchased from Sigma. ATM inhibitor KU55933 (Hickson et al, 2004), AKT inhibitor of API-2 (Yang et al, 2004) and Cdk4 inhibitor (Bommi-Reddy et al, 2008) were purchased from Calbiochem (San Diego, CA, USA).…”

Genetic analysis of von Hippel-Lindau disease

“…VHL analysis in C. elegans has produced strong evidence that a group of genes is regulated by pVHL in a manner independent of oxygen levels [Bishop et al, 2004]. Furthermore, multiple groups have reported microarray analyses in mammalian cell lines that indicate a role for pVHL independent of its interaction with HIF [Bluyssen et al, 2004;Bommi-Reddy et al, 2008;Jiang et al, 2003;Wykoff 2000Wykoff , 2004Zatyka et al, 2002]. Other roles of pVHL include microtubule stability [Hergovich et al, 2003], microtubule orientation [Schermer et al, 2006], cilia formation [Esteban et al, 2006;Lolkema et al, 2008;Lutz and Burk, 2006;Schermer 2006;Thoma et al, 2007], regulation of senescence [Young et al, 2008], cytokine signalling [Wu et al, 2007], collagen IV regulation [Grosfeld et al, 2007;Kurban et al, 2008], and assembly of a normal extracellular fibronectin matrix [Ohh et al, 1998].…”

198 Hsa-mir-124-3 CpG island methylation is associated with advanced tumours and disease recurrence of patients with clear cell renal cell carcinoma

“…This would be consistent with a hypothesised silencing of mir-124-3 expression due to CGI methylation. Moreover, from a kinase silencing screen in RCC cell lines deficient for VHL, CDK6 has been identified as one of the targets to be functionally associated with the viability of kidney cancer cells and, in addition, can be affected by a small molecule inhibitor (Bommi-Reddy et al, 2008). Hence, our clinical analysis emphasises the need for further functional analysis of the CDK6 signalling in RCC.…”