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Published on: June 17, 2022
Targeted CRISPR activation and knockout screenings identify novel doxorubicin transporters
Yufeng Li1, Minkang Tan1, Shengnan Sun1
1Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands.
Purpose:
Tissue-specific drug uptake has not been well studied, compared to the deeper understanding of drug resistance mediated by the cellular efflux system such as MDR1 proteins. It has been suggested that many drugs need active or defined transporters to pass the cell membrane. In contrast to efflux components induced after anti-cancer drugs reach the intracellular compartment, drug importers are required for initial drug responses. Furthermore, tissue-specific uptake of anti-cancer drugs may directly impact the side effects of many drugs when they accumulate in healthy tissues. Therefore, linking anti-cancer drugs to their respective drug import transporters would directly help to predict drug responses, whilst minimizing side effects.
Methods:
To identify drug transporters of the commonly used anti-cancer drug doxorubicin, we performed focused CRISPR activation and knockout genetic screens targeting all potential membrane-associated transporters and proteins. We monitored the direct uptake of doxorubicin by fluorescence-activated cell sorting (FACS) as the screening readout for identifying transporters/proteins directly involved in doxorubicin uptake.
Results:
Integrating the data from these comprehensive CRISPR screenings, we confirmed previously indicated doxorubicin exporters such as ABCB1 and ABCG2 genes, and identified novel doxorubicin importer gene SLC2A3 (GLUT3). Upregulation of SLC2A3 led to higher doxorubicin uptake and better cell killing, indicating SLC2A3 could be a new marker to predict doxorubicin drug response and minimize side effects for the personalized application of this conventional chemotherapeutic drug.
Conclusions:
Our study provides a comprehensive way for identifying drug transporters, as exemplified by the commonly used anti-cancer drug doxorubicin. The newly identified importers may have direct clinical implications for the personalized application of doxorubicin in treating distinct tumors. Our results also highlight the necessity of combining both CRISPR knockout and CRISPR activation genetic screens to identify drug transporters.
Insights
This study identified SLC2A3 (GLUT3) as a novel importer for doxorubicin, a key chemotherapy drug. Understanding drug transporters like SLC2A3 can predict treatment response and reduce side effects in cancer patients.
Area of Science:
- Pharmacology
- Genetics
- Cancer Biology
Background:
- Tissue-specific drug uptake is crucial for anti-cancer drug efficacy and side effect profiles.
- Drug importers are essential for initial cellular drug response, unlike efflux systems.
- Identifying drug transporters aids in predicting drug responses and minimizing toxicity.
Purpose of the Study:
- To identify transporters responsible for the cellular uptake of the anti-cancer drug doxorubicin.
- To explore the role of drug importers in doxorubicin's mechanism of action and tissue distribution.
- To establish a framework for predicting anti-cancer drug response and side effects based on transporter identification.
Main Methods:
- Utilized CRISPR activation and knockout genetic screens targeting membrane-associated proteins.
- Focused screens on identifying transporters involved in doxorubicin uptake.
- Employed fluorescence-activated cell sorting (FACS) to quantify doxorubicin uptake as a screening readout.
Main Results:
- Confirmed known doxorubicin exporters (ABCB1, ABCG2).
- Identified SLC2A3 (GLUT3) as a novel doxorubicin importer.
- Demonstrated that SLC2A3 upregulation enhances doxorubicin uptake and cancer cell killing.
Conclusions:
- Developed a comprehensive CRISPR-based method for identifying drug transporters.
- The novel importer SLC2A3 has potential clinical implications for personalized doxorubicin therapy.
- Combined CRISPR knockout and activation screens are essential for thorough transporter identification.
