PhD Student Univ. de Montreal Montreal, Quebec, Canada
Disclosure(s):
Jeffrey Sullivan: No financial relationships to disclose
Introduction/Rationale:
Background: Leukemia is a cancer of hematopoietic stem cells in the bone marrow. It is classified as lymphoid or myeloid, and as acute (rapid onset) or chronic (slow progression). Advances in CRISPR technology enable deeper study of leukemia biology and therapeutic targets. While AML, CML, ALL, and CLL are distinct, comparative studies highlight shared and unique features. Identifying key pathways across subtypes may yield improved treatments. Our lab has established a unique CRISPR Activation (CRISPRa) platform for gain-of-function screening applications. We hypothesize that genome-wide CRISPRa screening will uncover subtype-specific genetic dependencies and drug resistance mechanisms, offering novel therapeutic insights.
Methods:
Methods: We engineered leukemia cell lines by nucleofecting a self-selecting CRISPRa PiggyBac plasmid carrying a blasticidin resistance gene, enabling selection of CRISPRa-competent cells. Functionality was validated using lentiviral delivery of sgRNAs targeting cell surface markers. Cells were transduced with our whole-genome CRISPRa library, Sonata, at MOI 0.4, followed by puromycin selection to enrich for sgRNA-expressing cells. Post-transduction, cells were harvested at various timepoints for sequencing to track sgRNA abundance and identify growth-modulating genes.
Results:
Results: We engineered and validated four CRISPRa-competent leukemia lines: K562 (CML), Jurkat (T-ALL), THP-1 (AML), and HL60 (AML). We have completed whole-genome screening campaigns in the CML and T-ALL backgrounds, identifying hundreds of shared and context specific growth modifiers. Functional validation, pathway analysis, and potential clinical significance is ongoing, as is expansion of our screens with the AML contexts.
Conclusion:
Conclusions: Our screens reveal genes that, upon activation, influence leukemia cell growth. These findings support discovery of new therapeutic targets and enhance understanding of leukemia subtype biology, guiding future personalized treatment strategies.
