Identifying Novel Epigenetic Dependencies in Pre-leukemic Hematopoietic Stem Cells
The DNA methylation modifying enzymes DNMT3A and TET2 are essential for proper differentiation of hematopoietic stem cells (HSC) and are frequently mutated in blood cancers. Although their functions in regulating DNA methylation have been characterized, a specific connection between methylation patterns and altered gene expression has not been established to explain the observed disease phenotype. We hypothesize that DNMT3A- and TET2-mutant HSCs are dependent on other epigenetic regulators to corrupt normal hematopoietic pathways. If these mutations induce novel epigenetic dependencies, inhibition of these chromatin modifiers could negatively impact the propagation of these pre-leukemic HSCs and thus represent a novel therapeutic target for a range of pediatric blood cancers driven by DNMT3A and TET2 mutations.
To test this hypothesis, we employed a CRISPR-based negative selection screen on cells derived from wild-type, DNMT3A-null, and TET2-null HSCs to target 180 chromatin modifying genes. We utilized multiple screen designs in the pilot experiments and decided to proceed by pooling sgRNA plasmids in an equimolar ratio to create one lentivirus per library. Following lentiviral production, cells are transduced and genomic DNA is collected at days 2, 7 and 12. Multiple PCR reactions are performed to amplify the sgDNA cassette and the products are prepared for next generation sequencing. The results are mapped back onto the original sgRNA library, normalized to a negative control and the relative change in each sgRNA is measured over time. Positive hits obtained from this screen thus indicate genes which are potentially essential for normal hematopoiesis and/or dependent with DNMT3A and TET2.