Abstract Title Loss of BCLAF1 impairs AML pathogenesis Background Acute myelogenous leukemia (AML) pathologically activates self-renewal programs of normal hematopoietic stem cells (HSCs) to promote proliferation and prevent differentiation. We identified that BCLAF1, a transcriptional repressor, has critical roles regulating HSC self-renewal. Both fetal and adult Bclaf1-deficient HSCs have impaired repopulation activity in competitive transplantation experiments. BCLAF1 is upregulated in pediatric and adult AML, and previous literature demonstrated that BCLAF1 suppression is detrimental to AML. We aim to define the function of BCLAF1 in AML transformation and progression, and we hypothesized that BCLAF1 is essential for AML pathogenesis. Methods Wild-type and Bclaf1-deficient hematopoietic progenitors were transduced with a retrovirus expressing MLL-AF9 to generate AML, then transferred into recipients. Hematopoietic progenitor cells from Mx-Cre:Bclaf1fl/fl mice, which inducibly delete Bclaf1 after pIpC treatment, were transduced with the MLL-AF9 construct and transplanted. Additionally, primary haploinsufficient Mx-Cre:Bclaf1fl/- and wild-type Bclaf1fl/fl AMLs were transferred into new recipients and treated with pIpC two weeks later. Haploinsufficient Mx-Cre:Bclaf1fl/- cells were also treated with Tat-Cre recombinase to delete the remaining floxed allele. Lastly, Bclaf1-depletion was performed in human K562 AML cells. Results All mice receiving transduced wild-type cells developed AML, while only 50% of mice that received Bclaf1-deficient cells developed AML. Interestingly, mice that received Bclaf1fl/fl or Mx-Cre:Bclaf1fl/fl transduced cells developed AML at similar incidences; genotyping demonstrated all Mx-Cre:Bclaf1fl/fl AMLs were haploinsufficient for Bclaf1. No Bclaf1-deficient AMLs propagated from Mx-Cre:Bclaf1fl/fl, despite 85% of hematopoietic progenitors losing both floxed alleles after pIpC treatment. AML progressed equivalently in recipients of Bclaf1fl/fl or Mx-Cre:Bclaf1fl/- AML cells, and genotyping showed all Mx-Cre:Bclaf1fl/- AMLs remained haploinsufficient. Tat-Cre treatment of Mx-Cre:Bclaf1fl/- cells generated Bclaf1-/- cells (confirmed via genotyping). These Bclaf1-deficient AML cells had reduced colony formation in semisolid culture compared to haploinsufficient (Mx-Cre:Bclaf1fl/-) AML cells. K562 cells expressing shBclaf1 were selectively depleted in culture compared to cells expressing a non-targeting shRNA. Additionally, using an inducible CRISPR-Cas9 system, K562 cells expressing a guide RNA against Bclaf1 formed fewer colonies after single-cell subcloning than cells expressing a non-targeting guide RNA. Recovered colonies predominantly expressed BCLAF1 despite loss of BCLAF1 in the bulk CRISPR-generated BCLAF1-depleted K562 cells prior to subcloning. Conclusion Collectively, these studies demonstrate that BCLAF1 has critical functions in AML and will define its functions in AML transformation and maintenance. Ongoing studies will define chromatin binding patterns of BCLAF1 and BCLAF1-mediated transcriptional programs in AML.