CRISPR-Dependent Screening in Primary Hematopoietic Stem Cells Identifies KDM3B as Specific Susceptibility for IDH2 and TET2 Mutant Genotypes

Background and Significance

Clonal Hematopoiesis (CH) refers to the abnormal growth of genetically distinct subclones of cells initiated by specific mutations in Hematopoietic Stem Cells (HSCs). This phenomenon is quite common in populations over the age of 60, affecting over 20% of this demographic. CH is associated with an increased risk of malignant transformation to Acute Myeloid Leukemia (AML) and is also linked to higher all-cause mortality and a variety of age-related diseases, such as cardiovascular diseases. Although previous genetic studies have identified mutations associated with CH, identifying therapeutic targets within CH remains challenging. This is primarily due to a lack of in vitro platforms applicable to the study of primary hematopoietic stem and progenitor cells (HSPCs). In this study, the authors utilized a co-culture system of HSPCs and Bone Marrow Endothelial Cells (BMECs) for CRISPR/Cas9 screening to identify the critical role of the histone demethylase KDM3B in IDH2 and TET2 mutant HSPCs.

Source of the Paper

This study was co-authored by Michael R. Waarts and other researchers from institutions including the Memorial Sloan Kettering Cancer Center, Dana-Farber Cancer Institute, Icahn School of Medicine at Mount Sinai, Cincinnati Children’s Hospital Medical Center, and others. The study is published in the Cancer Discovery journal.

Detailed Research Methods

Experiment Procedure

1. Platform Establishment: First, researchers established a co-culture system of HSPCs with BMECs. Within this system, by maintaining and expanding HSPCs on BMECs for a long period, the researchers preserved the transplantability of HSPCs and produced a full spectrum of myeloid lineage cells.
2. Animal Models and Genetic Background: They used previously reported genotypic models, including dnmt3aflox/flox, asxl1flox/flox, tet2flox/flox, and idh2r140qflox/+ mice. They crossed these mice with HSC-specific Cre recombinase and fluorescent reporter genes (tdT+).
3. Competitive Culturing Experiments: Through competitive co-culture experiments, the researchers compared the performances of wild-type versus mutant HSPCs in different hematopoietic cell lineages.
4. In Vitro and In Vivo Validation: They edited genes in mutant HSPCs using CRISPR/Cas9 and small-molecule drug intervention in vitro, and validated the discovered gene dependencies in vivo through transplantation experiments.
5. CRISPR Screening: A large-scale screening was conducted using a CRISPR library against epigenetic modifiers to identify dependency genes in different mutant genotypes of HSPCs.

Experimental Details

Researchers used a CRISPR library composed of 997 single-guide RNAs (sgRNAs) targeting 191 genes for screening. They determined appropriate infection conditions through genetic manipulation and maintained cell viability and proliferative capacity. The screening process involved infecting HSPCs and co-culturing them with BMECs, followed by DNA sequencing to assess sgRNA representation.

Next, they conducted CRISPR screenings on dnmt3a-/-, asxl1-/-, tet2-/-, and idh2r140q HSPCs and evaluated genetic dependency differences between wild-type and mutant HSPCs using a scoring system. Among these genes, KDM3B exhibited significant dependency in IDH2r140q and TET2-/- cells, which was further validated in vivo, showing similar gene dependency results.

Detailed Results

1. Dependency of KDM3B in Vitro and In Vivo: Loss of KDM3B significantly reduced the competitiveness of IDH2r140q and TET2-/- cells, suggesting that KDM3B is a specific dependency gene in these mutant HSPCs. In vivo experiments further indicated that the loss of KDM3B greatly weakened the mutant cells’ survival and self-renewal capabilities.
2. Dependency in Human CH Mutant Cells: In human CH mutant cells, the loss of KDM3B showed similar dependencies, especially in TET2-mutated HSPCs. The researchers validated these findings through a series of competitive culturing and colony-forming experiments.
3. Mechanistic Study: The study indicated that the 2-HG product resulting from IDH2 mutations is a key factor for KDM3B dependency, and KDM3B enzymatic activity is critical for the survival of mutant cells. KDM3B and mutated IDH2 cooperatively regulate several key genes at the epigenetic level, including cytokine receptor genes, further enhancing the transcriptional output of these genes in mutant cells.

Conclusion and Research Value

One of the significant findings of this study was identifying the histone demethylase KDM3B as a specific dependency in HSPCs with IDH2 and TET2 mutations. The research suggests that KDM3B’s enzymatic activity is crucial for the survival of cells with IDH2 mutations, indicating a potential therapeutic target. Additionally, the study revealed downregulation of cytokine receptor signaling in cells with IDH2 mutations, providing new therapeutic strategies for patients with CH and AML with these mutations.

Research Highlights

1. Mutation-Specific Gene Dependency: Identifying KDM3B’s significant role in specific mutant genotypes of HSPCs, providing a new direction for targeted therapy.
2. Mechanistic Insights into Epigenetic Regulation: Uncovering the synergistic interaction between KDM3B and IDH2 mutation on an epigenetic level, enriching our understanding of these mechanisms.
3. Development and Application of in Vitro Platform: The study developed an effective in vitro platform that allows for the rapid and accurate identification of gene dependencies, offering a powerful tool for future research.