The transformed state in acute leukemia requires gene regulatory programs involving transcription factors and chromatin modulators. Here, we uncover an IRF8-MEF2D transcriptional circuit as an acute myeloid leukemia (AML)-biased dependency. We discover and characterize the mechanism by which the chromatin ‘reader’ ZMYND8 directly activates IRF8 in parallel with the MYC proto-oncogene through their lineage-specific enhancers. ZMYND8 is essential for AML proliferation in vitro and in vivo, and associates with MYC and IRF8 enhancer elements that we define in cell lines and patient samples. ZMYND8 occupancy at IRF8 and MYC enhancers requires BRD4, a transcription coactivator also necessary for AML proliferation. We show that ZMYND8 binds to the ET domain of BRD4 via its chromatin reader cassette, which in turn is required for proper chromatin occupancy and maintenance of leukemic growth in vivo. Our results rationalize ZMYND8 as a potential therapeutic target for modulating essential transcriptional programs in AML. Functional genomics studies directly in primary tumor cells are greatly needed to better understand the complexity of the disease in a highly relevant clinical context. We further extended ex vivo and in vivo CRISPR-based genome/epigenome editing approaches in patient-derived xenograft (PDX) and primary AML cells. We conducted CRISPR and CRISPRi “drop-out” screenings on patient cells in vivo and prioritized AML-biased gene and cis-element dependencies, including an uncharacterized MYB regulatory element. We further developed a Perturb-seq pipeline for patient cells. We perturbed known AML therapeutic targets and delineated complex regulatory networks and changes in cell cycle stages and cellular hierarchy in response to perturbations by scRNA-seq. We uncovered an unanticipated role for SETDB1, a suppressor of the interferon response, whose depletion in a PDX model resulted in the emergence of Hematopoietic stem cell (HSC) and progenitor-like population marked with high HOXA expression. We found that in these cells, myeloid differentiation could be rescued by DOT1L inhibition, nominating a combinatorial therapy to inhibit SETDB1 and DOT1L in order to suppress the HOXA cluster. Our study motivates a strategy to annotate personalized vulnerabilities in patients to predict therapeutic response in AML.