RUNX1 loss renders hematopoietic and leukemic cells dependent on IL-3 and sensitive to JAK inhibition.

Disease-initiating mutations in the transcription factor RUNX1 occur as germline and somatic events that cause leukemias with particularly poor prognosis. However, the role of RUNX1 in leukemogenesis is not fully understood, and effective therapies for RUNX1-mutant leukemias remain elusive. Here, we used primary patient samples and a RUNX1-KO model in primary human hematopoietic cells to investigate how RUNX1 loss contributes to leukemic progression and to identify targetable vulnerabilities. Surprisingly, we found that RUNX1 loss decreased proliferative capacity and stem cell function. However, RUNX1-deficient cells selectively upregulated the IL-3 receptor. Exposure to IL-3, but not other JAK/STAT cytokines, rescued RUNX1-KO proliferative and competitive defects. Further, we demonstrated that RUNX1 loss repressed JAK/STAT signaling and rendered RUNX1-deficient cells sensitive to JAK inhibitors. Our study identifies a dependency of RUNX1-mutant leukemias on IL-3/JAK/STAT signaling, which may enable targeting of these aggressive blood cancers with existing agents.

The Cell Type-Specific 5hmC Landscape and Dynamics of Healthy Human Hematopoiesis and TET2-Mutant Preleukemia.

The conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is a key step in DNA demethylation that is mediated by ten-eleven translocation (TET) enzymes, which require ascorbate/vitamin C. Here, we report the 5hmC landscape of normal hematopoiesis and identify cell type-specific 5hmC profiles associated with active transcription and chromatin accessibility of key hematopoietic regulators. We utilized CRISPR/Cas9 to model TET2 loss-of-function mutations in primary human hematopoietic stem and progenitor cells (HSPC). Disrupted cells exhibited increased colonies in serial replating, defective erythroid/megakaryocytic differentiation, and in vivo competitive advantage and myeloid skewing coupled with reduction of 5hmC at erythroid-associated gene loci. Azacitidine and ascorbate restored 5hmC abundance and slowed or reverted the expansion of TET2-mutant clones in vivo. These results demonstrate the key role of 5hmC in normal hematopoiesis and TET2-mutant phenotypes and raise the possibility of utilizing these agents to further our understanding of preleukemia and clonal hematopoiesis. SIGNIFICANCE: We show that 5-hydroxymethylation profiles are cell type-specific and associated with transcriptional abundance and chromatin accessibility across human hematopoiesis. TET2 loss caused aberrant growth and differentiation phenotypes and disrupted 5hmC and transcriptional landscapes. Treatment of TET2 KO HSPCs with ascorbate or azacitidine reverted 5hmC profiles and restored aberrant phenotypes. This article is highlighted in the In This Issue feature, p. 265.

Oncogenic Gata1 causes stage-specific megakaryocyte differentiation delay.

The megakaryocyte/erythroid Transient Myeloproliferative Disorder (TMD) in newborns with Down Syndrome (DS) occurs when N-terminal truncating mutations of the hemopoietic transcription factor GATA1, that produce GATA1short protein (GATA1s), are acquired early in development. Prior work has shown that murine GATA1s, by itself, causes a transient yolk sac myeloproliferative disorder. However, it is unclear where in the hemopoietic cellular hierarchy GATA1s exerts its effects to produce this myeloproliferative state. Here, through a detailed examination of hemopoiesis from murine GATA1s ES cells and GATA1s embryos we define defects in erythroid and megakaryocytic differentiation that occur relatively late in hemopoiesis. GATA1s causes an arrest late in erythroid differentiation in vivo, and even more profoundly in ES-cell derived cultures, with a marked reduction of Ter-119 cells and reduced erythroid gene expression. In megakaryopoiesis, GATA1s causes a differentiation delay at a specific stage, with accumulation of immature, kit-expressing CD41hi megakaryocytic cells. In this specific megakaryocytic compartment, there are increased numbers of GATA1s cells in S-phase of cell cycle and reduced number of apoptotic cells compared to GATA1 cells in the same cell compartment. There is also a delay in maturation of these immature GATA1s megakaryocytic lineage cells compared to GATA1 cells at the same stage of differentiation. Finally, even when GATA1s megakaryocytic cells mature, they mature aberrantly with altered megakaryocyte-specific gene expression and activity of the mature megakaryocyte enzyme, acetylcholinesterase. These studies pinpoint the hemopoietic compartment where GATA1s megakaryocyte myeloproliferation occurs, defining where molecular studies should now be focussed to understand the oncogenic action of GATA1s.

GATA1 and cooperating mutations in myeloid leukaemia of Down syndrome.

Myeloid leukaemia of Down syndrome (ML-DS) is an acute megakaryoblastic/erythroid leukaemia uniquely found in children with Down syndrome (constitutive trisomy 21). It has a unique clinical course, being preceded by a pre-leukaemic condition known as transient abnormal myelopoiesis (TAM), and provides an excellent model to study multistep leukaemogenesis. Both TAM and ML-DS blasts carry acquired N-terminal truncating mutations in the erythro-megakaryocytic transcription factor GATA1. These result in exclusive production of a shorter isoform (GATA1s). The majority of TAM cases resolve spontaneously without the need for treatment; however, around 10% acquire additional cooperating mutations and transform to leukaemia, with differentiation block and clinically significant cytopenias. Transformation is driven by the acquisition of additional mutation(s), which cooperate with GATA1s to perturb normal haematopoiesis.

Oncogenic Drivers and Development.

In this issue of Cancer Discovery, Lopez and colleagues show that the aggressive acute leukemic phenotype caused by the chimeric transcription factor CBFA2T3-GLIS2 varies depending on the developmental stage of the cell transformed (i.e., fetal vs. adult). This is likely a general principle in pediatric cancers and begins to explain why some cancer phenotypes are more common in infants and young children, whereas others are more frequent in older individuals.See related article by Lopez et al., p. 1736.

Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6.

Precise and efficient manipulation of genes is crucial for understanding the molecular mechanisms that govern human hematopoiesis and for developing novel therapies for diseases of the blood and immune system. Current methods do not enable precise engineering of complex genotypes that can be easily tracked in a mixed population of cells. We describe a method to multiplex homologous recombination (HR) in human hematopoietic stem and progenitor cells and primary human T cells by combining rAAV6 donor delivery and the CRISPR/Cas9 system delivered as ribonucleoproteins (RNPs). In addition, the use of reporter genes allows FACS-purification and tracking of cells that have had multiple alleles or loci modified by HR. We believe this method will enable broad applications not only to the study of human hematopoietic gene function and networks, but also to perform sophisticated synthetic biology to develop innovative engineered stem cell-based therapeutics.