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CRISPR-based functional genomics for dissecting therapeutic dependency in primary acute myeloid leukemia samples
Zhendong Cao1, Sixiang Yu2, Jacqueline Peng3
1Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Graduate Group in Cell and Molecular Biology, University of Pennsylvania, Philadelphia, PA, USA.
Abstract:
Cancer functional genomics enables high-throughput target discovery and mechanistic investigation, yet its application has remained largely confined to mouse models and established human cancer cell lines. Direct functional interrogation of heterogeneous primary tumors offers a powerful opportunity to evaluate therapeutic targets and uncover cancer dependencies or resistance mechanisms. Here, we developed an optimized CRISPR-based platform for functional genomics in patient-derived xenograft and primary acute myeloid leukemia (AML) samples harboring diverse pathogenic mutations. Integrated in vitro and in vivo CRISPR-Cas9 knockout and CRISPR interference (CRISPRi) dropout screens validated known AML-biased targets and identified cis-regulatory elements essential for leukemic growth. Coupling pooled CRISPR perturbations with single-cell RNA sequencing (Perturb-seq) further resolved the perturbation-induced alterations in regulatory networks, cell cycle states, and cellular hierarchies in primary AML samples. Together, these studies establish a general and robust framework for leveraging CRISPR-based functional genomics to directly dissect cancer dependencies and cellular heterogeneity in primary AML patient samples.
Insights
This study introduces a CRISPR-based functional genomics platform for direct analysis of primary acute myeloid leukemia (AML) samples. The platform identifies essential genes and regulatory elements, advancing cancer target discovery in patient tumors.
Area of Science:
- Genomics
- Molecular Biology
- Cancer Research
Background:
- Functional genomics in cancer research is crucial for target discovery but often limited to preclinical models.
- Direct analysis of primary tumors is needed to understand therapeutic targets and resistance mechanisms in heterogeneous cancer samples.
Purpose of the Study:
- To develop and validate a CRISPR-based functional genomics platform for primary acute myeloid leukemia (AML) samples.
- To identify novel cancer dependencies and cis-regulatory elements essential for AML growth.
- To resolve perturbation-induced alterations in regulatory networks and cellular heterogeneity using single-cell RNA sequencing.
Main Methods:
- Developed an optimized CRISPR-Cas9 knockout and CRISPR interference (CRISPRi) screening platform.
- Applied the platform to patient-derived xenograft and primary AML samples with diverse mutations.
- Integrated pooled CRISPR perturbations with single-cell RNA sequencing (Perturb-seq).
Main Results:
- Validated known AML-biased targets and identified essential cis-regulatory elements for leukemic growth.
- Perturb-seq revealed detailed alterations in regulatory networks, cell cycle states, and cellular hierarchies.
- Demonstrated the platform's robustness in dissecting cancer dependencies within primary AML samples.
Conclusions:
- Established a versatile CRISPR-based functional genomics framework for direct interrogation of primary cancer samples.
- The platform enables comprehensive analysis of cancer dependencies and cellular heterogeneity in AML.
- This approach facilitates high-throughput target discovery and mechanistic investigation in patient-derived tumors.
