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Updated: Jun 15, 2025
Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter
Published on: March 27, 2020
CRISPR screen of venetoclax response-associated genes identifies transcription factor ZNF740 as a key functional
Lixia Zhang1,2,3, Xinyue Zhou1,2, Sajesan Aryal1,2
1Division of Hematology/Oncology, Department of Medicine, University of Alabama at Birmingham Heersink School of Medicine, Birmingham, AL, USA.
Abstract:
BCL-2 inhibitors such as venetoclax offer therapeutic promise in acute myeloid leukemia (AML) and other cancers, but drug resistance poses a significant challenge. It is crucial to understand the mechanisms that regulate venetoclax response. While correlative studies have identified numerous genes linked to venetoclax sensitivity, their direct impact on the drug response remains unclear. In this study, we targeted around 1400 genes upregulated in venetoclax-sensitive primary AML samples and carried out a CRISPR knockout screen to evaluate their direct effects on venetoclax response. Our screen identified the transcription factor ZNF740 as a critical regulator, with its expression consistently predicting venetoclax sensitivity across subtypes of the FAB classification. ZNF740 depletion leads to increased resistance to ventoclax, while its overexpression enhances sensitivity to the drug. Mechanistically, our integrative transcriptomic and genomic analysis identifies NOXA as a direct target of ZNF740, which negatively regulates MCL-1 protein stability. Loss of ZNF740 downregulates NOXA and increases the steady state protein levels of MCL-1 in AML cells. Restoring NOXA expression in ZNF740-depleted cells re-sensitizes AML cells to venetoclax treatment. Furthermore, we demonstrated that dual targeting of MCL-1 and BCL-2 effectively treats ZNF740-deficient AML in vivo. Together, our work systematically elucidates the causal relationship between venetoclax response signature genes and establishes ZNF740 as a novel transcription factor regulating venetoclax sensitivity.
Insights
The transcription factor ZNF740 regulates venetoclax sensitivity in acute myeloid leukemia (AML). Loss of ZNF740 increases resistance, while its overexpression enhances sensitivity by controlling NOXA and MCL-1 levels.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Venetoclax is a promising BCL-2 inhibitor for acute myeloid leukemia (AML) and other cancers.
- Drug resistance to venetoclax is a significant clinical challenge, necessitating a deeper understanding of response mechanisms.
- Previous studies identified genes correlated with venetoclax sensitivity, but their direct causal roles remain largely unelucidated.
Purpose of the Study:
- To systematically identify genes that directly regulate venetoclax response in AML using a CRISPR knockout screen.
- To elucidate the molecular mechanisms by which identified regulators impact venetoclax sensitivity.
- To establish ZNF740 as a novel therapeutic target for overcoming venetoclax resistance.
Main Methods:
- Conducted a large-scale CRISPR knockout screen targeting approximately 1400 genes upregulated in venetoclax-sensitive AML samples.
- Integrated transcriptomic and genomic analyses to identify direct gene targets and regulatory pathways.
- Performed in vivo studies to evaluate the efficacy of dual targeting strategies in ZNF740-deficient AML models.
Main Results:
- Identified the transcription factor ZNF740 as a critical regulator of venetoclax sensitivity, consistently predicting response across AML subtypes.
- Demonstrated that ZNF740 depletion confers venetoclax resistance, while its overexpression enhances sensitivity.
- Uncovered that ZNF740 directly targets NOXA, which negatively regulates MCL-1 stability, thereby controlling venetoclax response.
Conclusions:
- ZNF740 is a novel transcription factor that causally regulates venetoclax sensitivity in AML by modulating the NOXA-MCL-1 axis.
- Restoring NOXA expression or dual targeting of MCL-1 and BCL-2 can re-sensitize ZNF740-deficient AML to venetoclax.
- ZNF740 represents a potential therapeutic target for improving venetoclax efficacy in AML patients.
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