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A dual-color fluorescence-based platform to identify selective inhibitors of Akt signaling
Aranzazú Rosado1, Fabian Zanella, Beatriz Garcia
1Experimental Therapeutics Program, Centro Nacional de Investigaciones Oncologicas, Madrid, Spain.
Background:
Inhibition of Akt signaling is considered one of the most promising therapeutic strategies for many cancers. However, rational target-orientated approaches to cell based drug screens for anti-cancer agents have historically been compromised by the notorious absence of suitable control cells.
Methodology/Principal Findings:
In order to address this fundamental problem, we have developed BaFiso, a live-cell screening platform to identify specific inhibitors of this pathway. BaFiso relies on the co-culture of isogenic cell lines that have been engineered to sustain interleukin-3 independent survival of the parental Ba/F3 cells, and that are individually tagged with different fluorescent proteins. Whilst in the first of these two lines cell survival in the absence of IL-3 is dependent on the expression of activated Akt, the cells expressing constitutively-activated Stat5 signaling display IL-3 independent growth and survival in an Akt-independent manner. Small molecules can then be screened in these lines to identify inhibitors that rescue IL-3 dependence.
Conclusions/Significance:
BaFiso measures differential cell survival using multiparametric live cell imaging and permits selective inhibitors of Akt signaling to be identified. BaFiso is a platform technology suitable for the identification of small molecule inhibitors of IL-3 mediated survival signaling.
Insights
A new live-cell screening platform, BaFiso, identifies specific inhibitors of Akt signaling in cancer. This platform uses engineered cells to find small molecules that restore IL-3 dependence, aiding anti-cancer drug discovery.
Area of Science:
- Oncology
- Cell Biology
- Pharmacology
Background:
- Targeting Akt signaling is a key strategy for cancer therapy.
- A lack of suitable control cells hinders effective drug screening for anti-cancer agents.
- Developing precise screening methods is crucial for identifying novel cancer therapeutics.
Purpose of the Study:
- To develop a live-cell screening platform for identifying specific Akt signaling inhibitors.
- To overcome limitations in current cell-based drug screening for anti-cancer agents.
- To enable the discovery of small molecules targeting cancer survival pathways.
Main Methods:
- Developed BaFiso, a live-cell screening platform using co-cultured, isogenic cell lines.
- Engineered cell lines for interleukin-3 (IL-3) independent survival, dependent on either activated Akt or Stat5.
- Utilized fluorescent protein tagging for differential cell tracking and multiparametric live cell imaging.
Main Results:
- BaFiso enables the identification of specific Akt signaling inhibitors.
- The platform distinguishes between Akt-dependent and Akt-independent survival pathways.
- Screening identified small molecules that restore IL-3 dependence, indicating Akt inhibition.
Conclusions:
- BaFiso is a robust platform technology for discovering small molecule inhibitors of IL-3 mediated survival signaling.
- The platform facilitates the identification of selective Akt signaling inhibitors for cancer therapy.
- BaFiso addresses the need for suitable control cells in targeted anti-cancer drug discovery.
