Haematopoietic stem cells (HSC) maintain lifelong blood production and increase blood cell numbers in response to chronic and acute injury. However, the mechanism(s) by which inflammatory insults are communicated to HSCs and their consequences for HSC activity remain largely unknown. Here, we demonstrate that interleukin-1 (IL-1), which functions as a key pro-inflammatory ‘emergency’ signal, directly accelerates cell division and myeloid differentiation of HSCs via precocious activation of a PU.1-dependent gene program. While this effect is essential for rapid myeloid recovery following acute injury to the bone marrow (BM), chronic IL-1 exposure restricts HSC lineage output, severely erodes HSC self-renewal capacity, and primes IL-1-exposed HSCs to fail massive replicative challenges like transplantation. Importantly, these damaging effects are transient and fully reversible upon IL-1 withdrawal. Our results identify a critical regulatory circuit that tailors HSC responses to acute needs, and likely underlies deregulated blood homeostasis in chronic inflammation conditions.

Even though hematopoietic stem cell (HSC) dysfunction is presumed in myelodysplastic syndrome (MDS), the exact nature of quantitative and qualitative alterations is unknown. We conducted a study of phenotypic and molecular alterations in highly fractionated stem and progenitor populations in a variety of MDS subtypes. We observed an expansion of the phenotypically primitive long-term HSCs (lineage ؊ /CD34 ؉ /CD38 ؊ /CD90 ؉ ) in MDS, which was most pronounced in higher-risk cases. These MDS HSCs demonstrated dysplastic clonogenic activity. Examination of progenitors revealed that lower-risk MDS is

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in AML patients’ cells. Our study provides proof-of-concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

IntroductionAcute myeloid leukemia (AML) and myelodysplastic syndromes (MDSs) are heterogeneous neoplastic diseases, and most subtypes have poor clinical outcomes. Despite the established use of poly-chemotherapy and the development of new agents that transiently reduce the tumor burden, relapse or failure to achieve durable remission continues to be the most common causes of death in most subtypes of AML and MDS. Recent experimental evidence suggests that AML arises from transformed immature hematopoietic cells after the accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells (HSCs) and committed progenitors. 1 The series of transforming events are thought to initially give rise to preleukemia stem cells (pre-LSCs), preceding the formation of fully transformed LSCs. Defining the characteristics of LSCs, and also of pre-LSCs, is critical to understanding the genesis of leukemia and to developing strategies by which these cells can be eradicated. AML is characterized by a cellular heterogeneous tumor bulk, with LSCs at the top of the hierarchy and a differentiation block at various stages during myeloid maturation. 2 To address the problem of cellular heterogeneity within the tumor and to identify relevant molecular pathways effective in LSCs and pre-LSCs, novel experimental approaches other than the examination of bulk tumor cells need to be established. Recent findings have suggested that human LSCs are contained within different phenotypic compartments and at relatively low frequencies. [3][4][5] Several surface molecules were reported to permit enrichment of LSCs in AML. 4,[6][7][8][9][10][11] However, reliable markers for human LSCs at the single-cell level have yet to be identified; and because of the challenges associated with the use of xenograft models, the search for such markers remains difficult. Moreover, although there is clear evidence for the involvement of HSCs in AML pathogenesis, studies from murine models suggest that fully transformed and transplantable LSCs may reside at a committed progenitor stage. 12-15 Here we applied a novel approach of parallel transcriptional analysis of multiple, highly fractionated stem and progenitor populations in individual patients. We isolated phenotypic long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 11, 2018. by guest www.bloodjournal.org From and committed granulocyte-monocyte progenitors (GMP) from individual patients with AML, and compared gene expression profiles of each population with their phenotypic counterparts from age-matched healthy controls (HCs). Subsequent intersection of differentially expressed genes in the different cellular compartments allowed us to identify candidate genes that are consistently...

The tumor suppressor p53 is often inactivated via its interaction with endogenous inhibitors mouse double minute 4 homolog (MDM4 or MDMX) or mouse double minute 2 homolog (MDM2), which are frequently overexpressed in patients with acute myeloid leukemia (AML) and other cancers. Pharmacological disruption of both of these inter-actions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in cancers with wild-type p53. Selective targeting of this pathway has thus far been limited to MDM2-only small-molecule inhibitors, which lack affinity for MDMX. We demonstrate that dual MDMX/MDM2 inhibition with a stapled a-helical peptide (ALRN-6924), which has recently entered phase I clinical testing, produces marked antileukemic effects. ALRN-6924 robustly activates p53-dependent transcription at the single-cell and single-molecule levels and exhibits biochemical and molecular biological on-target activity in leukemia cells in vitro and in vivo. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibits cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patient cells, including leukemic stem cell-enriched populations, and disrupts functional clonogenic and serial replating capacity. Furthermore, ALRN-6924 markedly improves survival in AML xenograft models. Our study provides mechanistic insight to support further testing of ALRN-6924 as a therapeutic approach in AML and other cancers with wild-type p53.

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The transcription factor PU.1 is an important regulator of hematopoiesis; precise expression levels are critical for normal hematopoietic development and suppression of leukemia. We show here that noncoding antisense RNAs are important modulators of proper dosages of PU.1. Antisense and sense RNAs are regulated by shared evolutionarily conserved cis-regulatory elements, and we can show that antisense RNAs inhibit PU.1 expression by modulating mRNA translation. We propose that such antisense RNAs will likely be important in the regulation of many genes and may be the reason for the large number of overlapping complementary transcripts with so far unknown function.[Keywords: Noncoding antisense RNA; upstream and intronic regulatory elements; coordinated expression of the target and regulator; translation stalling] Supplemental material is available at http://www.genesdev.org.

Eltrombopag (EP) is a small-molecule, nonpeptide thrombopoietin receptor (TPO-R) agonist that has been approved recently for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenic purpura. Prior studies have shown that EP stimulates megakaryopoiesis in BM cells from patients with acute myeloid leukemia and myelodysplastic syndrome, and the results also suggested that it may inhibit leukemia cell growth. In the present study, we studied the effects of EP on leukemia cell proliferation and the mechanism of its antiproliferative effects. We found that EP leads to a decreased cell division rate, a block in G 1 phase of cell cycle, and increased differentiation in human and murine leukemia cells. Because EP is species specific in that it can only bind TPO-R in human and primate cells, these findings further suggested that the antileukemic effect is independent of TPO-R. We found that treatment with EP leads to a reduction in free intracellular iron in leukemic cells in a dose-dependent manner. Experimental increase of intracellular iron abrogated the antiproliferative and differentiation-inducing effects of EP, demonstrating that its antileukemic effects are mediated through modulation of intracellular iron content. Finally, determination of EP's antileukemic activity in vivo demonstrated its ability to prolong survival in 2 mouse models of leukemia. (Blood. 2012;120(2):386-394) IntroductionSurvival in acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS) has remained poor despite recent efforts to treat patients with novel therapeutic regimens. [1][2][3][4] In addition, complications secondary to thrombocytopenia and bleeding occur frequently in AML and MDS, leading to significant morbidity and mortality. 5,6 Given that the median age of patients with AML is close to 70 years, novel antileukemia agents with limited BM toxicity are needed to improve outcomes.Eltrombopag (EP) is an oral, nonpeptide, small-molecule thrombopoietin receptor (TPO-R) agonist that has proven efficacy in treating chronic immune thrombocytopenic purpura (ITP) and hepatitis C-related thrombocytopenia. 7,8 Despite concerns that some leukemia blast cells express TPO-R, we and others have reported previously that EP does not stimulate leukemia or MDS cell growth, but may rather lead to a modest inhibition while continuing to stimulate normal megakaryopoiesis in BM samples of patients with AML or MDS. 9,10 One study using a close chemical derivative of EP found a toxic effect on myeloid leukemia cells, suggesting that the entire substance class, including EP itself, may possess antileukemic activity. 11 Studies using cell lines further suggested that the growth-inhibitory effect of EP is not related to expression levels of However, this hypothesis has not yet been formally tested, and the mechanism through which EP exerts its potential antileukemic effect is not known. The concentrations at which EP inhibits leukemia cell proliferation in vitro are clinically achievable with few side effects and hav...