Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ "writer" PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease.

The innate immune response involves a variety of inflammatory reactions that can result in inflammatory disease and cancer if they are not resolved and instead are allowed to persist. The effective activation and resolution of innate immune responses relies on the production and posttranscriptional regulation of mRNAs encoding inflammatory effector proteins. The RNA-binding protein HuR binds to and regulates such mRNAs, but its exact role in inflammation remains unclear. Here we show that HuR maintains inflammatory homeostasis by controlling macrophage plasticity and migration. Mice lacking HuR in myeloid-lineage cells, which include many of the cells of the innate immune system, displayed enhanced sensitivity to endotoxemia, rapid progression of chemical-induced colitis, and severe susceptibility to colitis-associated cancer. The myeloid cell-specific HuR-deficient mice had an exacerbated inflammatory cytokine profile and showed enhanced CCR2-mediated macrophage chemotaxis. At the molecular level, activated macrophages from these mice showed enhancements in the use of inflammatory mRNAs (including Tnf, Tgfb, Il10, Ccr2, and Ccl2) due to a lack of inhibitory effects on their inducible translation and/or stability. Conversely, myeloid overexpression of HuR induced posttranscriptional silencing, reduced inflammatory profiles, and protected mice from colitis and cancer. Our results highlight the role of HuR as a homeostatic coordinator of mRNAs that encode molecules that guide innate inflammatory effects and demonstrate the potential of harnessing the effects of HuR for clinical benefit against pathologic inflammation and cancer.

Key Points• SF3B1 mutations in MDS-RS have a multipotent lymphomyeloid origin.• Transplantation of SF3B1 mutated MDS-RS HSCs into immune-deficient mice results in generation of characteristic ring sideroblasts.Mutations in the RNA splicing gene SF3B1 are found in >80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS). We investigated the origin of SF3B1 mutations within the bone marrow hematopoietic stem and progenitor cell compartments in patients with MDS-RS. Screening for recurrently mutated genes in the mononuclear cell fraction revealed mutations in SF3B1 in 39 of 40 cases (97.5%), combined with TET2 and DNMT3A in 11 (28%) and 6 (15%) patients, respectively. All recurrent mutations identified in mononuclear cells could be tracked back to the phenotypically defined hematopoietic stem cell (HSC) compartment in all investigated patients and were also present in downstream myeloid and erythroid progenitor cells. While in agreement with previous studies, little or no evidence for clonal (SF3B1 mutation) involvement could be found in mature B cells, consistent involvement at the pro-B-cell progenitor stage was established, providing definitive evidence for SF3B1 mutations targeting lymphomyeloid HSCs and compatible with mutated SF3B1 negatively affecting lymphoid development. Assessment of stem cell function in vitro as well as in vivo established that only HSCs and not investigated progenitor populations could propagate the SF3B1 mutated clone. Upon transplantation into immune-deficient mice, SF3B1 mutated MDS-RS HSCs differentiated into characteristic ring sideroblasts, the hallmark of MDS-RS. Our findings provide evidence of a multipotent lymphomyeloid HSC origin of SF3B1 mutations in MDS-RS patients and provide a novel in vivo platform for mechanistically and therapeutically exploring SF3B1 mutated MDS-RS. (Blood. 2017;130(7):881-890) Medscape Continuing Medical Education onlineIn support of improving patient care, this activity has been planned and

A high proportion of patients with lower-risk del(5q) myelodysplastic syndromes will respond to treatment with lenalidomide. The median duration of transfusion-independence is 2 years with some long-lasting responses, but almost 40% of patients progress to acute leukemia by 5 years after starting treatment. The mechanisms underlying disease progression other than the well-established finding of small TP53-mutated subclones at diagnosis remain unclear. We studied a longitudinal cohort of 35 low- and intermediate-1-risk del(5q) patients treated with lenalidomide (n=22) or not (n=13) by flow cytometric surveillance of hematopoietic stem and progenitor cell subsets, targeted sequencing of mutational patterns, and changes in the bone marrow microenvironment. All 13 patients with disease progression were identified by a limited number of mutations in TP53, RUNX1, and TET2, respectively, with PTPN11 and SF3B1 occurring in one patient each. TP53 mutations were found in seven of nine patients who developed acute leukemia, and were documented to be present in the earliest sample (n=1) and acquired during lenalidomide treatment (n=6). By contrast, analysis of the microenvironment, and of hematopoietic stem and progenitor cells by flow cytometry was of limited prognostic value. Based on our data, we advocate conducting a prospective study aimed at investigating, in a larger number of cases of del(5q) myelodysplastic syndromes, whether the detection of such mutations before and after lenalidomide treatment can guide clinical decision-making.

SummaryRefractory anaemia with ring sideroblasts (RARS) is distinguished by hyperplastic inefficient erythropoiesis, aberrant mitochondrial ferritin accumulation and anaemia. Heterozygous mutations in the spliceosome gene SF3B1 are found in a majority of RARS cases. To explore the link between SF3B1 mutations and anaemia, we studied mutated RARS CD34+ marrow cells with regard to transcriptome sequencing, splice patterns and mutational allele burden during erythroid differentiation. Transcriptome profiling during early erythroid differentiation revealed a marked up‐regulation of genes involved in haemoglobin synthesis and in the oxidative phosphorylation process, and down‐regulation of mitochondrial ABC transporters compared to normal bone marrow. Moreover, mis‐splicing of genes involved in transcription regulation, particularly haemoglobin synthesis, was confirmed, indicating a compromised haemoglobinization during RARS erythropoiesis. In order to define the phase during which erythroid maturation of SF3B1 mutated cells is most affected, we assessed allele burden during erythroid differentiation in vitro and in vivo and found that SF3B1 mutated erythroblasts showed stable expansion until late erythroblast stage but that terminal maturation to reticulocytes was significantly reduced. In conclusion, SF3B1 mutated RARS progenitors display impaired splicing with potential downstream consequences for genes of key importance for haemoglobin synthesis and terminal erythroid differentiation.

Key Points• Sipa1 loss leads to BM niche alterations prior to the initiation of MPN.• Sipa1-deficient BM niche induces lethal MPN from normal hematopoietic cells.Mutations of signal-induced proliferation-associated gene 1 (SIPA1), a RAP1 GTPaseactivating protein, were reported in patients with juvenile myelomonocytic leukemia, a childhood myelodysplastic/myeloproliferative neoplasm (MDS/MPN). Sipa1 deficiency in mice leads to the development of age-dependent MPN. However, Sipa1 expression in bone marrow (BM) microenvironment and its effect on the pathogenesis of MPN remain unclear.We here report that Sipa1 is expressed in human and mouse BM stromal cells and downregulated in these cells from patients with MPN or MDS/MPN at diagnosis. By using the

The stem and progenitor cell compartments in low- and intermediate-risk myelodysplastic syndromes (MDS) have recently been described, and shown to be highly conserved when compared to those in acute myeloid leukemia (AML). Much less is known about the characteristics of the hematopoietic hierarchy of subgroups of MDS with a high risk of transforming to AML. Immunophenotypic analysis of immature stem and progenitor cell compartments from patients with an isolated loss of the entire chromosome 7 (isolated −7), an independent high-risk genetic event in MDS, showed expansion and dominance of the malignant −7 clone in the granulocyte and macrophage progenitors (GMP), and other CD45RA+ progenitor compartments, and a significant reduction of the LIN−CD34+CD38low/−CD90+CD45RA− hematopoietic stem cell (HSC) compartment, highly reminiscent of what is typically seen in AML, and distinct from low-risk MDS. Established functional in vitro and in vivo stem cell assays showed a poor readout for −7 MDS patients irrespective of marrow blast counts. Moreover, while the −7 clone dominated at all stages of GM differentiation, the −7 clone had a competitive disadvantage in erythroid differentiation. In azacitidine-treated −7 MDS patients with a clinical response, the decreased clonal involvement in mononuclear bone marrow cells was not accompanied by a parallel reduced clonal involvement in the dominant CD45RA+ progenitor populations, suggesting a selective azacitidine-resistance of these distinct −7 progenitor compartments. Our data demonstrate, in a subgroup of high risk MDS with monosomy 7, that the perturbed stem and progenitor cell compartments resemble more that of AML than low-risk MDS.

Early therapeutic decision-making is crucial in patients with higher-risk MDS. We evaluated the impact of clinical parameters and mutational profiles in 134 consecutive patients treated with azacitidine using a combined cohort from Karolinska University Hospital (n=89) and from King's College Hospital, London (n=45). While neither clinical parameters nor mutations had a significant impact on response rate, both karyotype and mutational profile were strongly associated with survival from the start of treatment. IPSS high-risk cytogenetics negatively impacted overall survival (median 20 vs 10 months; p<0.001), whereas mutations in histone modulators (ASXL1, EZH2) were associated with prolonged survival (22 vs 12 months, p=0.01). This positive association was present in both cohorts and remained highly significant in the multivariate cox model. Importantly, patients with mutations in histone modulators lacking high-risk cytogenetics showed a survival of 29 months compared to only 10 months in patients with the opposite pattern. While TP53 was negatively associated with survival, neither RUNX1-mutations nor the number of mutations appeared to influence survival in this cohort. We propose a model combining histone modulator mutational screening with cytogenetics in the clinical decision-making process for higher-risk MDS patients eligible for treatment with azacitidine.