Saturating CRISPR gene body scans in MLL-rearranged leukemia
MLL-rearranged (MLL-r) leukemia account for more than 70% of infant ALL (acute lymphoblastic leukemia) cases and 35%-50% of infant AML (acute myeloid leukemia) cases. The status quo to the treatment of MLL-r leukemia can be summarized as: little or no curative effect. The prevalence of these hematological disorders impacting the pediatric patients and the lack of effective treatments highlight the critical need for novel therapeutic strategies. Several Phase-I MLL-r clinical trials (including a pediatric NCT02141828 trial) have demonstrated the safety of the DOT1L-specific small molecule inhibitor EPZ-5676 (Pinometostat). However, limited clinical benefits observed in these trials indicate the urge to further understand the mechanism of DOT1L targeted therapy, and perhaps the mechanisms of other therapeutic candidate genes in MLL-r leukemia. To address these needs, we propose a novel genetic screen approach i.e. saturating CRISPR gene body scan that utilizes the maximal density of sgRNA to dissect the functional elements in MLL-r leukemia essential genes including DOT1L, KDM1A, RNF20, WDR5, and MOF.
Project Goal: We expect to confirm a novel DOT1L self-regulatory mechanism, which will improve future clinical trial designs and direct critical clinical decisions to benefit pediatric patients. We also foresee de novo discovery of therapeutic pockets in MLL-r leukemia using this novel technology. Finally, we envision the saturation CRISPR protein scan developed in this proposal will innovate a new research field with a technology breakthrough that bridges the functional genomics, protein structures and computational modeling for therapeutic pocket discovery.
Project Update: We completed the development of single-cell CRISPR gene tiling (sc-Tiling) workflow and published in Nature Communications (Yang et al. 2021). This technology integrates a CRISPR gene-tiling screen with single-cell transcriptomic and protein structural analyses. Distinct from other reported single-cell CRISPR screens focused on observing gene function and gene-to-gene/enhancer-to-gene regulation, sc-Tiling enables the capacity to identify regulatory mechanisms within a gene-coding region that dictate gene activity and therapeutic response. We foresee the application of this novel research tool will facilitate identification of novel therapeutic pockets for future pharmacological development targeting MLL-r leukemia and other pediatric malignancies.
