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Rapid identification of potential drug targets using endogenous reporter cell lines and siRNA screening

Jun 10, 2016 2:22:13 PM No Comments

We have combined our proprietary genome editing technology with large scale RNAi screening capabilities to allow identification of novel drug targets.

Here we demonstrate the utility of this approach in a large scale siRNA screen using a novel reporter cell line where endogenous hypoxia-inducible factor 1 alpha (HIF1α) is C-terminally tagged with a NanoLuc® luciferase1. This siRNA screen successfully identified both novel and previously reported regulators of HIF1α

RNAi Screening Capability

Our large scale phenotypic RNAi screening capabilities consist of:

  • An siRNA library composed of siGENOME siRNA pools (Thermo Scientific) targeting over 2000 key “druggable” genes (Figure 1A and 1B).
  • Automated liquid handling capabilities optimised in collaboration with Hamilton Robotics.
  • Automated barcode sample tracking and annotation using the BMG LABTECH plate reader.
  • Extensive experience in modelling the tumour microenvironment, including large scale hypoxic facilities.

Figure 1. The siRNA library and liquid handling capabilities. The siRNA library is composed of a number druggable protein types (A) which have been grouped into discrete functional subsets (B). Screens can incorporate either the whole library or independent subsets. (C) Our automated robotic liquid handling system. (D) Pipetting accuracy of the optimised liquid handling system across 20 plates. For the siRNA screens, 5μl siRNA is aliquoted directly to the bottom of empty 384-well plates. Fountain pen ink (measurable on a plate reader) was used to assess reproducibility. Average coefficient of variance is ≤2.3%.

 

HIF1α siRNA Screen Optimization

For the siRNA screen, we utilised a reporter cell line where endogenous HIF1α has been Cterminally tagged with a NanoLuc® luciferase. As the NanoLuc® luciferase signal is proportional to cell number, it is important to measure both viability and luciferase signal. Therefore we concurrently measured:

  • Hoescht nuclear marker to assess viability
  • NanoLuc® luciferase signal to assess HIF1α protein levels

Figure 2. An overview of Hoescht and luciferase signal stability. Both the Hoescht (A) and luciferase (B) measurements provide stable assay endpoints, making them ideal for use in large scale screens.

Transfection conditions were optimised to provide conditions that achieve optimal siRNA knockdown with minimal effects on viability (Figure 3A). Additionally, we determined that a 6h incubation under hypoxia (1% oxygen) is sufficient to induce expression of HIF1α (Figure 3B). Using this optimisation information, the reporter line was then screened against a panel of 960 siRNAs against ‘druggable’ targets using our large scale hypoxic capabilities and optimised robotic liquid handling system. The screen was carried out as illustrated in Figure 3C.

 

Figure 3. Condition optimisation and screening process. (A) Transfection optimisation. HIF1α siRNA causes a significant reduction in the luciferase signal, suggesting excellent HIF1α protein knockdown, whilst having no effect on viability. The control, PLK1 demonstrates that the reduction of luciferase signal in this case is mostly the result of reduced cell number. (B) Test for hypoxic induction of HIF1α after 6h culture at 1% oxygen (hypoxia) or 20% oxygen (normoxia). (C) Screening process. Cells were reverse transfected in 384- well plates and incubated for 42h prior to exposure to hypoxic conditions for 6h. Hoescht and luciferase signals were then measured.

 

HIF1α siRNA Screen Results

The siRNA screen identified several members of both the PI3K and MAPK pathways as regulators of HIF1α (Figure 4). In all cases, except MAPK1, there was significant reduction of reporter signal but only minimal effects of the siRNA on viability, suggesting the reduction of reporter signal is due to direct regulation of HIF1α, rather than simple reduction in cell number. Both the PI3K and MAPK pathways have been previously reported as activators of the HIF1α pathway2, supporting the validity of the siRNA screening results.

Additionally, SIRT5, PANK1 and TRIM23 were identified as repressors of HIF1α levels. Whilst TRIM23 is a previously identified repressor of HIF1α3, PANK1 and SIRT5 are novel findings.

 

Summary

We utilised the novel HIF1α protein reporter line in a large scale siRNA screen and successfully identified both previously reported and novel regulators of HIF1α.

This exemplifies the potential of our novel target identification strategy that combines our endogenous gene editing technology and large scale siRNA screening capabilities.

Click here to download the original poster version of this data

References

  • Bilton et al. The subtle side to hypoxia inducible factor (HIFα) regulation. Eur. J. Biochem. 270, 791–798 (2003)
  • Vichi et al. E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1). Proc. Natl. Acad. Sci. U.S.A. 102(6):1945-50 (2005)

 

#Cell lines, #siRNA Screening, #Endogenous reporters

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