Ezh2 loss cooperates with loss of Runx1 during leukemogenesis and reactivates a fetal gene signature

Loss of PRC2 frequently occurs in adult AML, MDS, and pediatric ML-DS, JMML. Yet, leukemogenesis is orchestrated by the cooperation of multiple genetic lesions. Therefore, we aimed to decipher the mutational and molecular context in EZH2 loss guided transformation. Using an in vitro CRISPR-Cas9 cooperation screening, we identified two highly transforming combinations of 5 gRNAs in concert with the Ezh2 gRNA. Corresponding murine transplantation experiments resulted in high penetrance and short latency leukemia that was attenuated by removal of the Ezh2 gRNA. NGS based mutation analysis and reduced complexity screenings revealed a potent cooperation between Ezh2 and Runx1 mutations. Global gene expression analysis revealed the upregulation of fetal gene signatures including Ryk, Plag1, Hif3a and Igf2bp3 to guide Ezh2 loss mediated effects. Moreover, the interplay of Runx1 and Ezh2 mutations was found to activate myeloid and stemness genes. CRISPR-Cas9 cooperation screenings were powerful to unravel cooperating effects between the losses of Ezh2 and Runx1. Uncovered oncogenic dependencies on onco-fetal and myeloid genes may be exploited for therapeutic approaches in the future.