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Acute myeloid leukemia (AML) is induced by the cooperative action of deregulated genes that perturb self-renewal, proliferation, and differentiation. Internal tandem duplications (ITDs) in the FLT3 receptor tyrosine kinase are common mutations in AML, confer poor prognosis, and stimulate myeloproliferation. AML patient samples with FLT3-ITD express high levels of RUNX1, a transcription factor with known tumor-suppressor function. In this study, to understand this paradox, we investigated the impact of RUNX1 and FLT3-ITD coexpression. FLT3-ITD directly impacts on RUNX1 activity, whereby up-regulated and phosphorylated RUNX1 cooperates with FLT3-ITD to induce AML. Inactivating RUNX1 in tumors releases the differentiation block and down-regulates genes controlling ribosome biogenesis. We identified Hhex as a direct target of RUNX1 and FLT3-ITD stimulation and confirmed high HHEX expression in FLT3-ITD AMLs. HHEX could replace RUNX1 in cooperating with FLT3-ITD to induce AML. These results establish and elucidate the unanticipated oncogenic function of RUNX1 in AML. We predict that blocking RUNX1 activity will greatly enhance current therapeutic approaches using FLT3 inhibitors., (© 2017 Behrens et al.)

Disrupting mutations of the RUNX1 gene are found in 10% of patients with myelodysplasia (MDS) and 30% of patients with acute myeloid leukemia (AML). Previous studies have revealed an increase in hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells in conditional Runx1-knockout (KO) mice, but the molecular mechanism is unresolved. We investigated the myeloid progenitor (MP) compartment in KO mice, arguing that disruptions at the HSC/MPP level may be amplified in downstream cells. We demonstrate that the MP compartment is increased by more than fivefold in Runx1 KO mice, with a prominent skewing toward megakaryocyte (Meg) progenitors. Runx1-deficient granulocyte-macrophage progenitors are characterized by increased cloning capacity, impaired development into mature cells, and HSC and Meg transcription signatures. An HSC/MPP subpopulation expressing Meg markers was also increased in Runx1-deficient mice. Rescue experiments coupled with transcriptome analysis and Runx1 DNA-binding assays demonstrated that granulocytic/monocytic (G/M) commitment is marked by Runx1 suppression of genes encoding adherence and motility proteins (Tek, Jam3, Plxnc1, Pcdh7, and Selp) that support HSC-Meg interactions with the BM niche. In vitro assays confirmed that enforced Tek expression in HSCs/MPPs increases Meg output. Interestingly, besides this key repressor function of Runx1 to control lineage decisions and cell numbers in progenitors, our study also revealed a critical activating function in erythroblast differentiation, in addition to its known importance in Meg and G/M maturation. Thus both repressor and activator functions of Runx1 at multiple hematopoietic stages and lineages likely contribute to the tumor suppressor activity in MDS and AML., (© 2016 by The American Society of Hematology.)

The activity of antagonizing transcription factors represents a mechanistic paradigm of bidirectional lineage-fate control during hematopoiesis. At the megakaryocytic/erythroid bifurcation, the cross-antagonism of krueppel-like factor 1 (KLF1) and friend leukemia integration 1 (FLI1) has such a decisive role. However, how this antagonism is resolved during lineage specification is poorly understood. We found that runt-related transcription factor 1 (RUNX1) inhibits erythroid differentiation of murine megakaryocytic/erythroid progenitors and primary human CD34(+) progenitor cells. We show that RUNX1 represses the erythroid gene expression program during megakaryocytic differentiation by epigenetic repression of the erythroid master regulator KLF1. RUNX1 binding to the KLF1 locus is increased during megakaryocytic differentiation and counterbalances the activating role of T-cell acute lymphocytic leukemia 1 (TAL1). We found that corepressor recruitment by RUNX1 contributes to a block of the KLF1-dependent erythroid gene expression program. Our data indicate that the repressive function of RUNX1 influences the balance between erythroid and megakaryocytic differentiation by shifting the balance between KLF1 and FLI1 in the direction of FLI1. Taken together, we show that RUNX1 is a key player within a network of transcription factors that represses the erythroid gene expression program., (© 2015 by The American Society of Hematology.)

Severe congenital neutropenia (CN) is a preleukemic bone marrow failure syndrome with a 20% risk of evolving into leukemia or myelodysplastic syndrome (MDS). Patterns of acquisition of leukemia-associated mutations were investigated using next-generation deep-sequencing in 31 CN patients who developed leukemia or MDS. Twenty (64.5%) of the 31 patients had mutations in RUNX1. A majority of patients with RUNX1 mutations (80.5%) also had acquired CSF3R mutations. In contrast to their high frequency in CN patients who developed leukemia or MDS, RUNX1 mutations were found in only 9 of 307 (2.9%) patients with de novo pediatric acute myeloid leukemia. A sequential analysis at stages prior to overt leukemia revealed RUNX1 mutations to be late events in leukemic transformation. Single-cell analyses in 2 patients showed that RUNX1 and CSF3R mutations were present in the same malignant clone. Functional studies demonstrated elevated granulocyte colony-stimulating factor (G-CSF)-induced proliferation with diminished myeloid differentiation of hematopoietic CD34(+) cells coexpressing mutated forms of RUNX1 and CSF3R. The high frequency of cooperating RUNX1 and CSF3R mutations in CN patients suggests a novel molecular pathway of leukemogenesis: mutations in the hematopoietic cytokine receptor (G-CSFR) in combination with the second mutations in the downstream hematopoietic transcription fator (RUNX1). The detection of both RUNX1 and CSF3R mutations could be used as a marker for identifying CN patients with a high risk of progressing to leukemia or MDS.

The t(12;21) chromosomal translocation, targeting the gene encoding the RUNX1 transcription factor, is observed in 25% of pediatric acute lymphoblastic leukemia (ALL) and is an initiating event in the disease. To elucidate the mechanism by which RUNX1 disruption initiates leukemogenesis, we investigated its normal role in murine B-cell development. This study revealed 2 critical functions of Runx1: (1) to promote survival and development of progenitors specified to the B-cell lineage, a function that can be substituted by ectopic Bcl2 expression, and (2) to enable the developmental transition through the pre-B stage triggered by the pre-B-cell antigen receptor (pre-BCR). Gene expression analysis and genomewide Runx1 occupancy studies support the hypothesis that Runx1 reinforces the transcription factor network governing early B-cell survival and development and specifically regulates genes encoding members of the Lyn kinase subfamily (key integrators of interleukin-7 and pre-BCR signaling) and the stage-specific transcription factors SpiB and Aiolos (critical downstream effectors of pre-BCR signaling). Interrogation of expression databases of 257 ALL samples demonstrated the specific down-regulation of the SPIB and IKZF3 genes (the latter encoding AIOLOS) in t(12;21) ALL, providing novel insight into the mechanism by which the translocation blocks B-cell development and promotes leukemia.

The transcription factor runt-related transcription factor 1 (Runx1) is essential for the establishment of definitive hematopoiesis during embryonic development. In adult blood homeostasis, Runx1 plays a pivotal role in the maturation of lymphocytes and megakaryocytes. Furthermore, Runx1 is required for the regulation of hematopoietic stem and progenitor cells. However, how Runx1 orchestrates self-renewal and lineage choices in combination with other factors is not well understood. In the present study, we describe a genome-scale RNA interference screen to detect genes that cooperate with Runx1 in regulating hematopoietic stem and progenitor cells. We identify the polycomb group protein Pcgf1 as an epigenetic regulator involved in hematopoietic cell differentiation and show that simultaneous depletion of Runx1 and Pcgf1 allows sustained self-renewal while blocking differentiation of lineage marker-negative cells in vitro. We found an up-regulation of HoxA cluster genes on Pcgf1 knock-down that possibly accounts for the increase in self-renewal. Moreover, our data suggest that cells lacking both Runx1 and Pcgf1 are blocked at an early progenitor stage, indicating that a concerted action of the transcription factor Runx1, together with the epigenetic repressor Pcgf1, is necessary for terminal differentiation. The results of the present study uncover a link between transcriptional and epigenetic regulation that is required for hematopoietic differentiation.