Leukaemogenesis requires enhanced self-renewal, which is induced by oncogenes. The underlying molecular mechanisms remain incompletely understood. Here, we identified C/D box snoRNAs and rRNA 2'-O-methylation as critical determinants of leukaemic stem cell activity. Leukaemogenesis by AML1-ETO required expression of the groucho-related amino-terminal enhancer of split (AES). AES functioned by inducing snoRNA/RNP formation via interaction with the RNA helicase DDX21. Similarly, global loss of C/D box snoRNAs with concomitant loss of rRNA 2'-O-methylation resulted in decreased leukaemia self-renewal potential. Genomic deletion of either C/D box snoRNA SNORD14D or SNORD35A suppressed clonogenic potential of leukaemia cells in vitro and delayed leukaemogenesis in vivo. We further showed that AML1-ETO9a, MYC and MLL-AF9 all enhanced snoRNA formation. Expression levels of C/D box snoRNAs in AML patients correlated closely with in vivo frequency of leukaemic stem cells. Collectively, these findings indicate that induction of C/D box snoRNA/RNP function constitutes an important pathway in leukaemogenesis.

Ectopic expression of the oncogenic transcription factor HoxA9 is a major cause of acute myeloid leukemia (AML). Here, we demonstrate that HoxA9 is a specific substrate of granule proteases. Protease knockout allowed the comprehensive determination of genome-wide HoxA9 binding sites by chromatin immunoprecipitation sequencing in primary murine cells and a human AML cell line. The kinetics of enhancer activity and transcription rates in response to alterations of an inducible HoxA9 were determined. This permitted identification of HoxA9-controlled enhancers and promoters, allocation to their respective transcription units, and discrimination against HoxA9-bound, but unresponsive, elements. HoxA9 triggered an elaborate positive-feedback loop that drove expression of the complete Hox-A locus. In addition, it controlled key oncogenic transcription factors Myc and Myb and directly induced the cell cycle regulators Cdk6 and CyclinD1, as well as telomerase, drawing the essential blueprint for perturbation of proliferation by leukemogenic HoxA9 expression.

Chromosomal translocations that fuse the mixed lineage leukemia gene (MLL) to a variety of unrelated partner genes are frequent in pediatric leukemias. The novel combination of genetic material leads to the production of active oncoproteins that depend on the contributions of both constituents. In a search for a common function amongst the diverse group of MLL fusion partners we constructed artificial fusions joining MLL with generic transactivator and repressor domains (acidic blob, GAL4 transactivator domain, Herpes simplex VP16 activation domain, KRAB repressor domain). Of all constructs tested, only MLL-VP16 was able to transform primary bone marrow cells and to induce a block of early myeloid differentiation like an authentic MLL fusion. Interestingly, the transformation capability of the artificial MLL fusions was correlated with the transcriptional potential of the resulting chimeric protein but it was not related to the strength of the isolated transactivation domain that was joined to MLL. These results prove for the first time that a general biological function -transactivation -might be the common denominator of many MLL fusion partners.

Mixed-lineage leukemia fusion proteins activate their target genes predominantly by stimulating transcriptional elongation. A core component necessary for this activity is cyclin-dependent kinase 9. Here we explored the effectiveness of small molecules targeting this enzyme as potential therapeutics. A screen of seven compounds with anti-CDK9 activity applied to a panel of leukemia cell lines identified flavopiridol and the experimental inhibitor PC585 as superior in efficacy with inhibitory concentrations in the submicromolar range. Both substances induced rapid dephosphorylation of the RNA polymerase II C-terminal domain, accompanied by downregulation of CDK9-dependent transcripts for MYC and HOXA9. Global gene expression analysis indicated the induction of a general stress response program, culminating in widespread apoptosis. Importantly, colony-forming activity in leukemia lines and primary patient samples could be completely inhibited under conditions that did not affect native precursors from bone marrow. In vivo application in a mouse transplant model significantly delayed disease with PC585 showing also oral activity. These results suggest CDK9 inhibition as novel treatment option for mixed-lineage leukemia.

Hematopoietic stem cells require MLL1, which is one of six Set1/Trithorax-type histone 3 lysine 4 (H3K4) methyltransferases in mammals and clinically the most important leukemia gene. Here, we add to emerging evidence that all six H3K4 methyltransferases play essential roles in the hematopoietic system by showing that conditional mutagenesis of Setd1b in adult mice provoked aberrant homeostasis of hematopoietic stem and progenitor cells (HSPCs). Using both ubiquitous and hematopoietic-specific deletion strategies, the loss of Setd1b resulted in peripheral thrombo- and lymphocytopenia, multilineage dysplasia, myeloid-biased extramedullary hematopoiesis in the spleen, and lethality. By transplantation experiments and expression profiling, we determined that Setd1b is autonomously required in the hematopoietic lineages where it regulates key lineage specification components, including Cebpa, Gata1, and Klf1. Altogether, these data imply that the Set1/Trithorax-type epigenetic machinery sustains different aspects of hematopoiesis and constitutes a second framework additional to the transcription factor hierarchy of hematopoietic homeostasis.