A SEARCH FOR EPIGENETIC VULNERABILITIES OF AML CELLS PERFORMED BY A CDK INHIBITOR¿BASED CRISPR SCREENING

Acute myeloid leukemia is an aggressive hematologic malignancy characterized by uncontrolled proliferation and impaired differentiation of hematopoietic progenitors. Epigenetic dysregulation plays a central role in AML pathogenesis, and targeting chromatin-modifying enzymes has emerged as a promising therapeutic strategy. Among these, the histone demethylase LSD1/KDM1A has been extensively studied, yet clinical responses to LSD1 inhibition remain variable. Work from our group demonstrated that pharmacological prolongation of the G1 phase of the cell cycle through sub-optimal doses of CDK4/6 inhibitors reprograms the chromatin landscape of fast-cycling AML cells resistant to LSD1 inhibition, rendering them more similar to slow-cycling counterparts that are intrinsically sensitive to LSD1 inhibition. This finding suggested that cell cycle dynamics critically influence the response to epigenetic therapies. The present study employed CRISPR-Cas9 dropout screenings to systematically identify chromatin-related vulnerabilities emerging upon cell cycle manipulation. Using a Cas9-expressing AML cell line, we screened more than 600 epigenetic regulators in the context of CDK targeting. The optimized pipeline reliably uncovered known dependencies, validating its robustness, and revealed novel candidates whose loss synergizes with low-dose CDK inhibitors to impair leukemic cell proliferation. Genetic and pharmacological validation provided proof of principle that, in AML models unresponsive to LSD1 inhibition, cell cycle manipulation can unmask alternative chromatin-related dependencies such as DOT1L. Extending the screening to AML models where cell cycle manipulation does not result in sensitivity to LSD1 17 inhibition further identified additional context-dependent vulnerabilities unmasked by cell cycle lengthening. Nuclear phospho-proteomics studies (ongoing) revealed that Palbociclib treatment rewires kinase signaling in the nucleus and chromatin-associated phosphorylation networks, setting the ground for the discovery of mechanistic insights into how cell cycle modulation primes AML cells for epigenetic intervention. Following a similar experimental approach, but following a different line of research in our group, we have combined the CRISPR-screening platform with metabolic drugs, to uncover functional crosstalk between chromatin regulation and metabolic pathways. Overall, our studies demonstrates that cell cycle manipulation exposes epigenetic vulnerabilities that can be exploited pharmacologically. These findings propose a generalizable framework in which chromatin reprogramming through cell cycle modulation enhances the therapeutic potential of epigenetic interventions in AML.