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Modeling Patient Responses to Targeted Therapy with Gene Editing & Isogenic Cell Lines

Jun 10, 2016 11:52:05 AM No Comments

The emergence of RAS mutations is a key mechanism of acquired resistance to MAPK-pathway targeted agents in a number of cancers. The preclinical evaluation of targeted agents traditionally relies on panels of genetically unrelated cell lines grown as 2D monocultures. The heterogeneous nature of these panels makes identifying genotype-specific responses a challenge. In addition, 2D assays do not accurately mimic the tumour microenvironment and so add to the difficulty in interpreting which cellular responses to targeted agents will have relevance in vivo.

Isogenic panels of colorectal cancer cell lines can effectively model KRAS-mediated resistance to inhibitors of EGFR and MEK, and can be used to stratify patient-relevant genotypes into drug responsive and non-responsive populations. Additionally, through the use of 3D culture and co-culture environments, inclusion of aspects of the tumour microenvironment can be used to more accurately assess the response of colorectal cancer cells to targeted agents.

Differential sensitivity to vemurafenib in BRAF mutant X‐MAN® Cell Lines

The response seen with the MCF10A lines mirrors the reported response to BRAF inhibitors in clinical trials, where the success of B‐Raf inhibitors has been shown to be dependant on the B‐Raf mutation status of the tumour1,2. In our cell line panel, we observed stimulation of growth at lower compound concentrations in cells expressing wild‐type B‐Raf. In the lines expressing mutant B‐Raf, in either a p53 wild‐type or null context, this effect was less apparent or absent.

Cells with mutant BRAF are resistant to paradoxical RAF activation by BRAF inhibitors.

Figure 1: Cells with mutant BRAF are resistant to paradoxical RAF activation by BRAF inhibitors. The anti‐proliferative effect of the BRAF inhibitor PLX‐4032 (vemurafenib) were evaluated in the MCF10A BRAF cell line panel in 96h proliferation assays.

Differential sensitivity to EGFR inhibitors in EGFR mutant X‐MAN Cell Lines

In isolation, the introduction of common activating EGFR mutations L858R or deletion of E746‐ A750 led to increased sensitivity to gefitinib and erlotinib; the selectivity over parental cells was most pronounced following gefitinib treatment. The introduction of the T790M mutation was sufficient to confer resistance. These results confirm that the cell line panel recapitulates clinical findings.

Cells engineered to contain knock‐in EGFR mutations show differential sensitivities to EGFR inhibitors.

Figure 2. Cells engineered to contain knock‐in EGFR mutations show differential sensitivities to EGFR inhibitors. The anti‐proliferative effects of the EGFR inhibitors gefitinib and erlotinib were evaluated in the MCF10A EGFR single mutant cell line panel in 96h proliferation assays.

Combining the activating EGFR mutations with loss of p53 further enhanced the cell response to EGFR inhibitors. This may have clinical relevance and could be worthy of further investigation.

Combining activating EGFR mutations with loss of p53 further enhanced the cell response EGFR inhibition.

Figure 3. Combining activating EGFR mutations with loss of p53 further enhanced the cell response EGFR inhibition. The anti‐proliferative effects of gefitinib were evaluated in the MCF10A EGFR & TP53 (‐/‐) mutant cell line panel in 96h proliferation assays.

Conclusions

  • Through systematic profiling of panels of X‐MAN® isogenic cell lines to targeted therapeutic agents, we have identified differential sensitivities, which can be directly attributable to introduction of a given mutation.
  • The flexibility of our gene editing technology can be exploited to introduce multiple mutations into a cell line, and has enabled us to interrogate the effects of specific oncogenic mutations either alone or within the context of other genetic alterations.
  • Results such as these can enable better patient stratification for anticancer agents, and allow incorporation of molecular markers into clinical trial design for personalised therapeutic regimens.

Download the original poster version of this data

Explore our collection of EGFR modified X-MAN Cell Lines Click here
Explore our collection of TP53 modified X-MAN Cell Lines Click here
Explore our collection of BRAF modified X-MAN Cell Lines Click here

References:

1. Pratilas and Solit, Clin Cancer Res 2010 (16) p3329, 2Poulikakos et al., Nature 2010 (464) p427

#Cell lines, #Gene editing, #Target identification

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