PROJECT SUMMARY Acute myeloid leukemia (AML) is one of the most aggressive hematologic malignancies in adults, yet decades- old chemotherapies remain the standard of care and few targeted therapies exist owing to its molecular and clinical heterogeneity. An emerging hallmark of AML development is the epigenetic silencing of LINE1 retrotransposons which is required to maintain AML self-renewal, differentiation blockade, and genomic stability. Aberrant reactivation of LINE1 retrotransposons selectively impairs propagation of human and mouse AML cells without affecting normal hematopoiesis; however, it remains elusive how LINE1 activity inhibits myeloid leukemogenesis. Aberrant retrotransposon reactivation by cancer-targeting epigenetic inhibitors such as DNA hypomethylating agents (DMA) produces a type I interferon (IFN)-mediated ‘viral mimicry’ response in various cancer types, including leukemias, resulting in cell cycle arrest and apoptosis. We hypothesize that LINE1 retrotransposons contribute to the development of myeloid leukemia by modulating type I interferon signaling mediated by cGAS and/or RIG-I-like Receptor (RLR) sensing of LINE1 gene products. This proposal will establish the functional role of LINE1-mediated IFN signaling in myeloid leukemogenesis and determine the mechanisms by which LINE1 activates innate immune ‘viral mimicry’ pathways in AML cells. The Specific Aims of this proposal intend to 1) establish the functional role of LINE1-mediated interferon signaling in myeloid leukemogenesis and progression; and 2) identify the molecular sensors of LINE1-mediated interferon activation in AML. Ectopic LINE1 overexpression and CRISPR activation of endogenous LINE1s in human AML cells will determine whether LINE1 expression induces type I IFNs and impacts cell proliferation, myeloid differentiation, and/or apoptosis. Combining a conditional LINE1 activation transgenic mouse with the established MLL-AF9 retroviral leukemia model will determine whether activation of LINE1s induces hematopoietic-specific type I IFN to impair AML initiation and/or maintenance in vivo. Moreover, genetic ablation of cGAS, RIG-I, or MDA5 nucleic acid sensors individually or in combination in human AML cells and their corresponding knockout mouse models will determine how loss of DNA- and/or RNA-sensing pathways affects LINE1-induced inflammation and AML pathogenesis in vivo. Altogether, these stringent genetic studies will provide direct evidence to establish the functional role of LINE1-mediated IFN signaling in myeloid leukemia. Addressing these outstanding knowledge gaps will be critical to inform whether and how modulation of the retrotransposon-innate immunity crosstalk may be leveraged as a new mechanism-based therapeutic strategy to selectively eradicate AML cells.