miR-191 regulates mouse erythroblast enucleation by down-regulating Riok3 and Mxi1.

Using RNA-seq technology, we found that the majority of microRNAs (miRNAs) present in CFU-E erythroid progenitors are down-regulated during terminal erythroid differentiation. Of the developmentally down-regulated miRNAs, ectopic overexpression of miR-191 blocks erythroid enucleation but has minor effects on proliferation and differentiation. We identified two erythroid-enriched and developmentally up-regulated genes, Riok3 and Mxi1, as direct targets of miR-191. Knockdown of either Riok3 or Mxi1 blocks enucleation, and either physiological overexpression of miR-191 or knockdown of Riok3 or Mxi1 blocks chromatin condensation. Thus, down-regulation of miR-191 is essential for erythroid chromatin condensation and enucleation by allowing up-regulation of Riok3 and Mxi1.

Muscleblind-like 1 (Mbnl1) regulates pre-mRNA alternative splicing during terminal erythropoiesis.

The scope and roles of regulated isoform gene expression during erythroid terminal development are poorly understood. We identified hundreds of differentiation-associated isoform changes during terminal erythropoiesis. Sequences surrounding cassette exons of skipped exon events are enriched for motifs bound by the Muscleblind-like (MBNL) family of splicing factors. Knockdown of Mbnl1 in cultured murine fetal liver erythroid progenitors resulted in a strong block in erythroid differentiation and disrupted the developmentally regulated exon skipping of Ndel1 mRNA, which is bound by MBNL1 and critical for erythroid terminal proliferation. These findings reveal an unanticipated scope of the alternative splicing program and the importance of Mbnl1 during erythroid terminal differentiation.

An activin receptor IIA ligand trap promotes erythropoiesis resulting in a rapid induction of red blood cells and haemoglobin.

Sotatercept (ACE-011), a recombinant human fusion protein containing the extracellular domain of the human Activin receptor IIA, binds to and inhibits activin and other members of the transforming growth factor -ß (TGF-ß) superfamily. Administration of sotatercept led to a rapid and sustained increase in red blood cell (RBC) count and haemoglobin (Hb) in healthy volunteers (phase I clinical trials), but the mechanism is not fully understood. Mice treated with RAP-011 (murine ortholog of ACE-011) respond with a rapid (within 24 h) increase in haematocrit, Hb, and RBC count. These effects are accompanied by an equally rapid stimulation of late-stage erythroid precursors in the bone marrow (BM). RAP-011 also induces a significant increase in erythroid burst-forming units and erythropoietin, which could contribute to additional, sustained effects on RBC production. Further in vitro co-culture studies demonstrate that BM accessory cells are required for RAP-011 effects. To better understand which TGF-ß family ligand(s) mediate RAP-011 effects, we evaluated the impact of several of these ligands on erythroid differentiation. Our data suggest that RAP-011 may act to rescue growth differentiation factor 11/Activin A-induced inhibition of late-stage erythropoiesis. These data define the mechanism of action of a novel agent that regulates RBC differentiation and provide the rationale to develop sotatercept for the treatment of anaemia and ineffective erythropoiesis.

Targeted shRNA screening identified critical roles of pleckstrin-2 in erythropoiesis.

Differentiation of erythroblasts to mature red blood cells involves dynamic changes of the membrane and cytoskeleton networks that are not fully characterized. Using a mouse fetal liver erythroblast culture system and a targeted shRNA functional screening strategy, we identified a critical role of pleckstrin-2 in actin dynamics and protection of early stage terminal erythroblasts from oxidative damage. Knockdown of pleckstrin-2 in the early stage of terminal erythropoiesis disrupted the actin cytoskeleton and led to differentiation inhibition and apoptosis. This pro-survival and differentiation function of pleckstrin-2 was mediated through its interaction with cofilin, by preventing cofilin's mitochondrial entry when the intracellular level of reactive oxygen species was higher in the early stage of terminal erythropoiesis. Treatment of the cells with a scavenger of reactive oxygen species rescued cofilin's mitochondrial entry and differentiation inhibition induced by pleckstrin-2 knockdown. In contrast, pleckstrin-2 knockdown in late stage terminal erythroblasts had no effect on survival or differentiation but blocked enucleation due to disorganized actin cytoskeleton. Thus, our study identified a dual function of pleckstrin-2 in the early and late stages of terminal erythropoiesis through its regulations of actin dynamics and cofilin's mitochondrial localization, which reflects intracellular level of reactive oxygen species in different developmental stages.

From stem cell to red cell: regulation of erythropoiesis at multiple levels by multiple proteins, RNAs, and chromatin modifications.

This article reviews the regulation of production of RBCs at several levels. We focus on the regulated expansion of burst-forming unit-erythroid erythroid progenitors by glucocorticoids and other factors that occur during chronic anemia, inflammation, and other conditions of stress. We also highlight the rapid production of RBCs by the coordinated regulation of terminal proliferation and differentiation of committed erythroid colony-forming unit-erythroid progenitors by external signals, such as erythropoietin and adhesion to a fibronectin matrix. We discuss the complex intracellular networks of coordinated gene regulation by transcription factors, chromatin modifiers, and miRNAs that regulate the different stages of erythropoiesis.

Gene induction and repression during terminal erythropoiesis are mediated by distinct epigenetic changes.

It is unclear how epigenetic changes regulate the induction of erythroid-specific genes during terminal erythropoiesis. Here we use global mRNA sequencing (mRNA-seq) and chromatin immunoprecipitation coupled to high-throughput sequencing (CHIP-seq) to investigate the changes that occur in mRNA levels, RNA polymerase II (Pol II) occupancy, and multiple posttranslational histone modifications when erythroid progenitors differentiate into late erythroblasts. Among genes induced during this developmental transition, there was an increase in the occupancy of Pol II, the activation marks H3K4me2, H3K4me3, H3K9Ac, and H4K16Ac, and the elongation methylation mark H3K79me2. In contrast, genes that were repressed during differentiation showed relative decreases in H3K79me2 levels yet had levels of Pol II binding and active histone marks similar to those in erythroid progenitors. We also found that relative changes in histone modification levels, in particular, H3K79me2 and H4K16ac, were most predictive of gene expression patterns. Our results suggest that in terminal erythropoiesis both promoter and elongation-associated marks contribute to the induction of erythroid genes, whereas gene repression is marked by changes in histone modifications mediating Pol II elongation. Our data map the epigenetic landscape of terminal erythropoiesis and suggest that control of transcription elongation regulates gene expression during terminal erythroid differentiation.

Longitudinal high-dimensional analysis identifies immune features associating with response to anti-PD-1 immunotherapy.

Response to immunotherapy widely varies among cancer patients and identification of parameters associating with favourable outcome is of great interest. Here we show longitudinal monitoring of peripheral blood samples of non-small cell lung cancer (NSCLC) patients undergoing anti-PD1 therapy by high-dimensional cytometry by time of flight (CyTOF) and Meso Scale Discovery (MSD) multi-cytokines measurements. We find that higher proportions of circulating CD8+ and of CD8+CD101hiTIM3+ (CCT T) subsets significantly correlate with poor clinical response to immune therapy. Consistently, CD8+ T cells and CCT T cell frequencies remain low in most responders during the entire multi-cycle treatment regimen; and higher killer cell lectin-like receptor subfamily G, member 1 (KLRG1) expression in CCT T cells at baseline associates with prolonged progression free survival. Upon in vitro stimulation, CCT T cells of responders produce significantly higher levels of cytokines, including IL-1ß, IL-2, IL-8, IL-22 and MCP-1, than of non-responders. Overall, our results provide insights into the longitudinal immunological landscape underpinning favourable response to immune checkpoint blockade therapy in lung cancer patients.

miR-150 blocks MLL-AF9-associated leukemia through oncogene repression.

UNLABELLED: The microRNA miR-150, a critical regulator of hematopoiesis, is downregulated in mixed-lineage leukemia (MLL). In this study, miR-150 acts as a potent leukemic tumor suppressor by blocking the oncogenic properties of leukemic cells. By using MLL-AF9-transformed cells, we demonstrate that ectopic expression of miR-150 inhibits blast colony formation, cell growth, and increases apoptosis in vitro. More importantly, ectopic expression of miR-150 in MLL-AF9-transformed cells completely blocked the development of myeloid leukemia in transplanted mice. Furthermore, gene expression profiling revealed that miR-150 altered the expression levels of more than 30 "stem cell signature" genes and many others that are involved in critical cancer pathways. In addition to the known miR-150 target Myb, we also identified Cbl and Egr2 as bona fide targets and shRNA-mediated suppression of these genes recapitulated the pro-apoptotic effects observed in leukemic cells with miR-150 ectopic expression. In conclusion, we demonstrate that miR-150 is a potent leukemic tumor suppressor that regulates multiple oncogenes. IMPLICATIONS: These data establish new, key players for the development of therapeutic strategies to treat MLL-AF9-related leukemia.

The miR-17-92 microRNA polycistron regulates MLL leukemia stem cell potential by modulating p21 expression.

Despite advances in defining the critical molecular determinants for leukemia stem cell (LSC) generation and maintenance, little is known about the roles of microRNAs in LSC biology. Here, we identify microRNAs that are differentially expressed in LSC-enriched cell fractions (c-kit(+)) in a mouse model of MLL leukemia. Members of the miR-17 family were notably more abundant in LSCs compared with their normal counterpart granulocyte-macrophage progenitors and myeloblast precursors. Expression of miR-17 family microRNAs was substantially reduced concomitant with leukemia cell differentiation and loss of self-renewal, whereas forced expression of a polycistron construct encoding miR-17-19b miRNAs significantly shortened the latency for MLL leukemia development. Leukemias expressing increased levels of the miR-17-19b construct displayed a higher frequency of LSCs, more stringent block of differentiation, and enhanced proliferation associated with reduced expression of p21, a cyclin-dependent kinase inhibitor previously implicated as a direct target of miR-17 microRNAs. Knockdown of p21 in MLL-transformed cells phenocopied the overexpression of the miR-17 polycistron, including a significant decrease in leukemia latency, validating p21 as a biologically relevant and direct in vivo target of the miR-17 polycistron in MLL leukemia. Expression of c-myc, a crucial upstream regulator of the miR-17 polycistron, correlated with miR-17-92 levels, enhanced self-renewal, and LSC potential. Thus, microRNAs quantitatively regulate LSC self-renewal in MLL-associated leukemia in part by modulating the expression of p21, a known regulator of normal stem cell function.

Meis1 is an essential and rate-limiting regulator of MLL leukemia stem cell potential.

Oncogenic mutations of the MLL histone methyltransferase confer an unusual ability to transform non-self-renewing myeloid progenitors into leukemia stem cells (LSCs) by mechanisms that remain poorly defined. Misregulation of Hox genes is likely to be critical for LSC induction and maintenance but alone it does not recapitulate the phenotype and biology of MLL leukemias, which are clinically heterogeneous--presumably reflecting differences in LSC biology and/or frequency. TALE (three-amino-acid loop extension) class homeodomain proteins of the Pbx and Meis families are also misexpressed in this context, and we thus employed knockout, knockdown, and dominant-negative genetic techniques to investigate the requirements and contributions of these factors in MLL oncoprotein-induced acute myeloid leukemia. Our studies show that induction and maintenance of MLL transformation requires Meis1 and is codependent on the redundant contributions of Pbx2 and Pbx3. Meis1 in particular serves a major role in establishing LSC potential, and determines LSC frequency by quantitatively regulating the extent of self-renewal, differentiation arrest, and cycling, as well as the rate of in vivo LSC generation from myeloid progenitors. Thus, TALE proteins are critical downstream effectors within an essential homeoprotein network that serves a rate-limiting regulatory role in MLL leukemogenesis.

Leukemic transformation of hematopoietic progenitors by MLL-GAS7 in the absence of Hoxa7 or Hoxa9.

Differential expression of Hox genes is associated with normal hematopoiesis, whereas inappropriate maintenance of Hox gene expression, particularly Hoxa7 and Hoxa9, is a feature of leukemias harboring mixed-lineage leukemia (MLL) mutations. To understand the pathogenic roles of Hox genes in MLL leukemias, we assessed the impact of Hoxa7 or Hoxa9 nullizygosity on hematopoietic progenitor compartments and their susceptibility to MLL-induced leukemias. Selective reductions in the absolute numbers of committed progenitors, but not of hematopoietic stem cells, distinguished Hoxa7- and Hoxa9-deficient mice. Megakaryocytic/erythroid progenitor (MEP) reductions in Hoxa7(-/-) mice correlated with reticulocytosis and thrombocytopenia without anemia. Conversely, Hoxa9(-/-) mice displayed marked lymphopenia and substantial reductions of common lymphoid progenitors (CLPs) and lymphoid precursors, in addition to significant reductions of common myeloid progenitors (CMPs) and granulocyte/monocyte progenitors (GMPs). In retroviral transduction/transplantation assays, Hoxa7- and Hoxa9-deficient progenitors remained susceptible to transformation by MLL-GAS7, which activates MLL through a dimerization-dependent mechanism. However, Hoxa7(-/-) or Hoxa9(-/-) progenitors were less efficient in generating transformed blast colony-forming units (CFUs) in vitro and induced leukemias with longer disease latencies, reduced penetrance, and less mature phenotypes. Thus, Hoxa7 and Hoxa9 contribute to hematopoietic progenitor homeostasis but are not necessary for MLL-GAS7-mediated leukemogenesis, yet they appear to affect disease latency, penetrance, and phenotypes consistent with their critical roles as downstream targets of MLL fusion proteins.

Overexpression of the ASN1 gene enhances nitrogen status in seeds of Arabidopsis.

In wild-type Arabidopsis, levels of ASN1 mRNA and asparagine (Asn) are tightly regulated by environmental factors and metabolites. Because Asn serves as an important nitrogen storage and transport compound used to allocate nitrogen resources between source and sink organs, we tested whether overexpression of the major expressed gene for Asn synthetase, ASN1, would lead to changes in nitrogen status in the ultimate storage organ for metabolites-seeds. Transgenic Arabidopsis constitutively overexpressing ASN1 under the cauliflower mosaic virus 35S promoter were constructed (35S-ASN1). In seeds of the 35S-ASN1 lines, three observations support the notion that the nitrogen status was enhanced: (a) elevations of soluble seed protein contents, (b) elevations of total protein contents from acid-hydrolyzed seeds, and (c) higher tolerance of young seedlings when grown on nitrogen-limiting media. Besides quantitative differences, changes in the relative composition of the seed amino acid were also observed. The change in seed nitrogen status was accompanied by an increase of total free amino acids (mainly Asn) allocated to flowers and developing siliques. In 35S-ASN1 lines, sink tissues such as flowers and developing siliques exhibit a higher level of free Asn than source tissues such as leaves and stems, despite significantly higher levels of ASN1 mRNA observed in the source tissues. This was at least partially due to an enhanced transport of Asn from source to sink via the phloem, as demonstrated by the increased levels of Asn in phloem exudates of the 35S-ASN1 plants.