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Identifying new synthetically lethal therapeutic targets

Jun 10, 2016 2:08:28 PM No Comments

The term synthetic lethality was first used in 1922 by C.Bridges1 to describe events where two non-lethal mutations are brought together in combination to cause cell death. Synthetic lethality offers significant potential in the field of cancer research, with many groups focusing on the selective inhibition of target proteins that are lethal to only those cells harboring a mutated cancer gene.

The principle behind this has been exemplified by A.Pereira et. al. who demonstrated that PTEN deficiency sensitizes tumor cells to poly (ADP-ribose) polymerase (PARP) both in vitro and in vivo2. In Figure 1 we can see that cells that are null for PTEN are more sensitive to the PARP inhibitor Olaparib (also known as KU0059436)

 

Synthetic lethality screening in PTEN isogenic cells

Figure 1. PTEN deficiency sensitizes cells to drug-like PARP inhibitors in a cell survival assay. After 15 days of culture HCT116 PTEN -/- cells were 20 times more sensitive to the PARP inhibitor KU0058948 (red plots) than their isogenic wild type counterparts (black plots).

Similarly, tumours with mutations in BRCA genes are highly sensitive to PARP inhibition4. As a consequence the first-in-class PARP inhibitor Olaparib (trade name Lynparza) was approved in December 2014 for use as a monotherapy in ovarian cancers with a BRCA mutation.

Horizon Discovery has established a program to evaluate synthetic lethality by assessing differential effects in mutant lines versus a truly isogenic parental background. This approach can deliver a larger therapeutic window by reducing potential toxic side effects in wild type cells.

Identifying synthetic lethal relationships using siRNA

As a proof of concept study Horizon combined isogenic cell lines with an siRNA targeting over 2000 key "drugable" genes, to identify a new synthetically lethal target3. The library used was composed of ON-TARGET plus™ siRNA pools (Dharmacon), with equimolar mixes of 4 sequence independent siRNAs raised against each target gene.

Using a non-targeted siRNA molecule as a control, we found that targeted disruption of Hexokinase 2 demonstrated selective anti-proliferative effects in PIK3CA mutant cells (H1047R/+) with no measurable effects in the parental MCF10A line PI3K (+/+).


 

Figure 2. Effects of Hexokinase 2 siRNA and NT controls on cell growth in MCF10A PI3Kα mutant and isogenic control cells. Cell number 96 hours post-transfection was determined using SRB. Growth was assessed as a percentage of non-targeted control (*p,0.05; **p,0.01).

It is using this approach that Horizon identified its first-in-class kinase target, HD-001, which it has partnered with AstraZeneca to explore further.

Using CRISPR Screening to identify synthetic lethal relationships

The same principles that underpin RNAi-based synthetic lethality screening can now also apply when using CRISPR-Cas9 screening technology.

However, by contrast to the above siRNA screen, where oligos are arrayed as pools for individual genes, a CRISPR knockout library contains multiples guide RNAs targeting every gene in the genome (or a subset of genes) in a single tube. These guides are in a lentiviral backbone, meaning that following infection they are integrated into the genome of the cells and can in effect serve as a barcode for that particular gene knockout.

Massively parallel sequencing can then be used to characterize the pool, and mutant and wild type cell lines compared. In this manner gene knockouts that cause cell death in a mutant background but not wild type (synthetic lethality) will be lost from the population and not observed during sequencing.

And as well as being able to work in a pooled format, CRISPR screening offers a number of benefits over arrayed siRNA including reduced issues with incomplete knockdown and off-target effects, and improved reproducibility.

To learn more about CRISPR screening and how it is revolutionizing drug discovery watch our recorded webinar

Related material:

Read our application note on using isogenic cells to identify synthetic lethal relationships Click here
Download our poster on using siRNA-based synthetic lethal screening in TP53 mutant and wild type isogenic cells Click here
Watch our recorded webinar on how CRISPR screening is revolutionizing drug discovery Click here

 

References

  1. CB.Bridges, Amer Nat. 1922 (56) p51-63.
  2. A.Pereira et al EMBO Mol. Med. 2009 (1) p315-322.
  3. R.Foster et al PLOS ONE. 2012 (7) p1-8.
  4. H. Farmer et al Nature 2005 434 p917
#Synthetic lethality

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