Project Summary/Abstract Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by aberrant self-renewal and blocked differentiation of myeloid progenitor cells. Many of the oncogenic drivers of AML converge in dysregulation of epigenetic and transcriptional regulation pathways, generating de novo dependencies on these regulators. A handful of epigenetic dependencies have been identified in AML, however, single-agent inhibitors against epigenetic regulators have shown limited therapeutic efficacy in patients with AML. To improve our limited understanding of epigenetic-related synergistic genetic interactions in AML, we developed a highly efficient CRISPR-Cas12a-based method enabling us to perform double deletion genetic screening. Our preliminary studies identified and validated two pairs of novel interacting synthetic sick combinations of epigenetic regulators in AML: bromodomain containing protein 9 (BRD9) and Jumonji domain-containing protein 6 (JMJD6) as well as the lysine acetyltransferase 6 (KAT6) and JMJD6. JMJD6 is a bi-functional arginine demethylase and lysyl-hydroxylase regulating transcription enhancer activation and was identified in both synergistic pairs of epigenetic factor deletions. BRD9 is a component of chromatin remodeling SWI/SNF complex and was previously identified as an AML specific dependency. KAT6A is a histone acetyltransferase and transcriptional co-activator. We hypothesize that BRD9/JMJD6 and KAT6A/JMJD6 interact synergistically at the level of transcription and chromatin. The presence of JMJD6 in both interactions suggests that JMJD6- deficiency sets up a unique transcription and chromatin state that sensitizes AML cells to distinct epigenetic stresses. In Aim 1, we will investigate the synthetic sick interactions of BRD9/JMJD6 and KAT6A/JMJD6 in vitro and in vivo and in Aim 2, we will dissect the AML-specific synthetic sick interactions of BRD9/JMJD6 and KAT6A/JMJD6 at the molecular level. Our proposed studies will offer basic mechanistic insight into how these novel AML synthetic sick epigenetic interactions sustain AML pathogenesis. Successful completion of the proposed studies holds significant promise towards developing innovative epigenetic pathway-directed therapies and revealing fundamental biological insights into the pathogenesis of AML. 1