NLK is a novel therapeutic target for PTEN deficient tumour cells

Abstract

PTEN (Phosphatase and tensin homolog) is a tumour suppressor gene commonly defective in human cancer, and is thus a potentially important therapeutic target. Targeting tumour suppressor loss-of-function is possible by exploiting the genetic concept of synthetic lethality (SL). By combining the use of isogenic models of PTEN deficiency with high-throughput RNA interference (RNAi) screening, we have identified Nemo-Like Kinase (NLK) inhibition as being synthetically lethal with PTEN deficiency. This SL is likely mediated by the transcription factor FOXO1 (Forkhead box O1), an NLK substrate, as the selectivity of NLK gene silencing for PTEN deficient cells can be reversed by FOXO1 knockdown. In addition, we provide evidence that PTEN defective cells targeted by NLK gene depletion undergo senescence, suggesting that NLK function is critical for the continued proliferation of PTEN deficient cells. Taken together, these data provide new insight into the potential of targeting of NLK to treat a range of tumourigenic conditions characterised by PTEN deficiency.

Figure 1. PTEN synthetic lethality screening.

(A) High-throughput screen (HTS) schematic. PTEN proficient and deficient HCT116 cells (Horizon Discovery) were siRNA screened as described in the Methods. Example heatmaps from luminescence measurements in 96 well plates are shown. For screening, a siGENOME SMARTPool library (Dharmacon) targeting 779 kinases and kinase-related genes was used. (B) Results from data analysis of combined triplicate screens, represented as a scatter plot of Normalised Percent Inhibition (NPI) values from HCT116 PTEN^−/− and HCT116 PTEN^+/+ screens. Blue dots corresponded to siPLK effects and red dots corresponded to siCON negative control effects. NPI values below the trend line shown were considered as candidate synthetic lethalities. (C) Validation of PTEN synthetic lethal hits from the HTS using multiple siRNAs for each gene. Cells were transfected with siRNA as per the HTS and surviving fractions calculated from CellTiterGlo luminescence measurements five days later. Surviving fraction data from HCT116 PTEN^−/− and HCT116 PTEN^+/+ cells transfected with each siRNA are shown. (D) Western blot showing silencing effects of each siRNA.

Figure 2. Validation of the PTEN/NLK synthetic lethality.

(A) Validation of PTEN synthetic lethal hits in additional isogenic models. Isogenic HCT116, DLD1 and HEC1A PTEN proficient and deficient models were transfected with siRNA as in Figure 1 and surviving fractions calculated five days later. Surviving fraction data from HCT116, HEC1A and DLD1 models is shown. * p value<0.05 Student's t test (B) Western blot showing expression of each candidate gene in three PTEN isogenic models. (C) Cell inhibitory effect of siRNA targeting NLK or PLK4 in a panel of 24 human tumour cell lines (11 PTEN proficient models and 13 PTEN deficient models – see Table 1). (D) Box plots illustrating surviving fraction data for PTEN proficient and deficient groups shown in (C). p-values were calculated with Student's t-test.

Figure 3. PTEN/NLK synthetic lethality is abrogated by FOXO1 silencing.

(A) Survival analysis of HCT116 cells transfected with NLK and/or FOXO1 siRNA. HCT116 PTEN^−/− and HCT116 PTEN^+/+ cells were transfected with siRNA targeting NLK and FOXO1 as shown and surviving fractions determined after five days. The p value (*) was calculated using Student's t test. (B) Nuclear localisation of FOXO1 is enhanced in PTEN deficient cells upon NLK silencing. HCT116 isogenic cells were co-transfected with GFP-tagged FOXO1 cDNA in addition to control (non targeting) siRNA (siCON) or NLK siRNA. Two days later cells were fixed and stained with DAPI. Green signal represents GFP-FOXO1 and blue signal represents nuclear DAPI (nuclear) staining. Arrows indicate cells with nuclear localisation of FOXO1. (C) Senescence is increased by NLK siRNA in PTEN deficient cells. Bar chart of relative relative senescence levels caused by NLK silencing are shown. HCT116-derived PTEN isogenic cell lines were reversed transfected with a pool of two validated siRNAs against NLK, as well as siCON pool#2 (Dharmacon) as non-targeting control, using RNAiMAX (Invitrogen). Seven days after transfection cells were fixed and incubated overnight at 37°C in a solution containing X-gal. (D) Representative images for β-Galactosidase staining of PTEN deficient cells. Blue staining indicates β-Galactosidase.