Background: Chromosomal rearrangements leading to overexpression of EVI1 (MECOM) on chromosome 3q26 define a distinct subtype of acute myeloid leukemia (AML) that is associated with chemotherapy resistance and a 2-year survival of <10%. While genetic events driving aberrant expression of EVI1 are increasingly understood, the molecular functions of EVI1 that drive leukemogenesis are unclear, which has so far precluded the development of targeted therapeutics. Aims: We aimed to elucidate transcriptional programs that are maintained by aberrant EVI1 expression and to systematically identify vulnerabilities of EVI1-driven AML. Methods: We developed a panel of mouse models that recapitulate phenotypic and transcriptional hallmarks of patients suffering from EVI1-driven AML, allow tetracycline-controllable EVI1 expression and the functional interrogation of genetic targets using CRISPR/Cas9. We mapped transcriptional programs upon acute EVI1 repression in vivo and in vitro, profiled global EVI1 chromatin occupancy in human AML cell lines and primary patient-derived AML cells and performed comparative genome-wide CRISPR/Cas9-based loss-of-function screens in murine and human EVI1-driven AML. Results: Integration of these datasets revealed a conserved core of genes that is transcriptionally regulated by EVI1 in murine and human AML, among which we identified the ETS transcription factor ERG as the only dependency that is highly selective for EVI1-driven AML. Suppression of ERG specifically triggered cellular differentiation and apoptosis of EVI1-driven leukemia cells while other AML cell lines were unaffected. Strikingly, ectopic expression of ERG was sufficient to functionally rescue loss of EVI1 in EVI1-driven AML cells, suggesting that the major oncogenic function of EVI1 in AML is the aberrant activation of ERG. Summary/Conclusion: Interfering with the EVI1/ERG regulatory axis may provide entry points for the development of rational targeted therapies that are urgently needed for this group of AML patients.

The Ets family transcription factor PU.1 and the interferon regulatory factor (IRF)4 and IRF8 regulate gene expression by binding to composite DNA sequences known as Ets/interferon consensus elements. Although all three factors are expressed from the onset of B-cell development, single deficiency of these factors in B-cell progenitors only mildly impacts on bone marrow B lymphopoiesis. Here we tested whether PU.1 cooperates with IRF factors in regulating early B-cell development. Lack of PU.1 and IRF4 resulted in a partial block in development the pre-B-cell stage. The combined deletion of PU.1 and IRF8 reduced recirculating B-cell numbers. Strikingly, all PU.1/IRF4 and ~50% of PU.1/IRF8 double deficient mice developed pre-B-cell acute lymphoblastic leukemia (B-ALL) associated with reduced expression of the established B-lineage tumor suppressor genes, Ikaros and Spi-B. These genes are directly regulated by PU.1/IRF4/IRF8, and restoration of Ikaros or Spi-B expression inhibited leukemic cell growth. In summary, we demonstrate that PU.1, IRF4 and IRF8 cooperate to regulate early B-cell development and to prevent pre-B-ALL formation.