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SummaryMLL fusion proteins in leukemia induce aberrant transcriptional elongation and associated chromatin perturbations; however, the upstream signaling pathways and activators that recruit or retain MLL oncoproteins at initiated promoters are unknown. Through functional and comparative genomic studies, we identified an essential role for NF-κB signaling in MLL leukemia. Suppression of NF-κB led to robust antileukemia effects that phenocopied loss of functional MLL oncoprotein or associated epigenetic cofactors. The NF-κB subunit RELA occupies promoter regions of crucial MLL target genes and sustains the MLL-dependent leukemia stem cell program. IKK/NF-κB signaling is required for wild-type and fusion MLL protein retention and maintenance of associated histone modifications, providing a molecular rationale for enhanced efficacy in therapeutic targeting of this pathway in MLL leukemias.

The genetic programs that maintain leukemia stem cell (LSC) self-renewal and oncogenic potential have been well defined, however the comprehensive epigenetic landscape that sustains LSC cellular identity and functionality is less well established. We report that LSCs in MLL-associated leukemia reside in an epigenetic state of relative genome-wide high-level H3K4me3 and low level H3K79me2. LSC differentiation is associated with reversal of these broad epigenetic profiles, with concomitant down-regulation of crucial MLL target genes and the LSC maintenance transcriptional program that is driven by loss of H3K4me3 but not H3K79me2. The H3K4-specific demethylase KDM5B negatively regulates leukemogenesis in murine and human MLL-rearranged AML cells, demonstrating a crucial role for the H3K4 global methylome in determining leukemia stem cell fate.

The relationships between clonal architecture and functional heterogeneity in acute myeloid leukemia (AML) samples are not yet clear. We used targeted sequencing to track AML subclones identified by whole genome sequencing using a variety of experimental approaches. We found that virtually all AML subclones trafficked from the marrow to the peripheral blood, but some were enriched in specific cell populations. Subclones showed variable engraftment potential in immunodeficient mice. Xenografts were predominantly comprised of a single genetically-defined subclone, but there was no predictable relationship between the engrafting subclone and the evolutionary hierarchy of the leukemia. These data demonstrate the importance of integrating genetic and functional data in studies of primary cancer samples, both in xenograft models and in patients.