Preleukemic single-cell landscapes reveal mutation-specific mechanisms and gene programs predictive of AML patient outcomes.

Acute myeloid leukemia (AML) and myeloid neoplasms develop through acquisition of somatic mutations that confer mutation-specific fitness advantages to hematopoietic stem and progenitor cells. However, our understanding of mutational effects remains limited to the resolution attainable within immunophenotypically and clinically accessible bulk cell populations. To decipher heterogeneous cellular fitness to preleukemic mutational perturbations, we performed single-cell RNA sequencing of eight different mouse models with driver mutations of myeloid malignancies, generating 269,048 single-cell profiles. Our analysis infers mutation-driven perturbations in cell abundance, cellular lineage fate, cellular metabolism, and gene expression at the continuous resolution, pinpointing cell populations with transcriptional alterations associated with differentiation bias. We further develop an 11-gene scoring system (Stem11) on the basis of preleukemic transcriptional signatures that predicts AML patient outcomes. Our results demonstrate that a single-cell-resolution deep characterization of preleukemic biology has the potential to enhance our understanding of AML heterogeneity and inform more effective risk stratification strategies.

STAT1 is essential for HSC function and maintains MHCIIhi stem cells that resist myeloablation and neoplastic expansion.

Adult hematopoietic stem cells (HSCs) are predominantly quiescent and can be activated in response to acute stress such as infection or cytotoxic insults. STAT1 is a pivotal downstream mediator of interferon (IFN) signaling and is required for IFN-induced HSC proliferation, but little is known about the role of STAT1 in regulating homeostatic hematopoietic stem/progenitor cells (HSPCs). Here, we show that loss of STAT1 altered the steady state HSPC landscape, impaired HSC function in transplantation assays, delayed blood cell regeneration following myeloablation, and disrupted molecular programs that protect HSCs, including control of quiescence. Our results also reveal STAT1-dependent functional HSC heterogeneity. A previously unrecognized subset of homeostatic HSCs with elevated major histocompatibility complex class II (MHCII) expression (MHCIIhi) displayed molecular features of reduced cycling and apoptosis and was refractory to 5-fluorouracil-induced myeloablation. Conversely, MHCIIlo HSCs displayed increased megakaryocytic potential and were preferentially expanded in CALR mutant mice with thrombocytosis. Similar to mice, high MHCII expression is a feature of human HSCs residing in a deeper quiescent state. Our results therefore position STAT1 at the interface of stem cell heterogeneity and the interplay between stem cells and the adaptive immune system, areas of broad interest in the wider stem cell field.

The stem/progenitor landscape is reshaped in a mouse model of essential thrombocythemia and causes excess megakaryocyte production.

Frameshift mutations in CALR (calreticulin) are associated with essential thrombocythemia (ET), but the stages at and mechanisms by which mutant CALR drives transformation remain incompletely defined. Here, we use single-cell approaches to examine the hematopoietic stem/progenitor cell landscape in a mouse model of mutant CALR-driven ET. We identify a trajectory linking hematopoietic stem cells (HSCs) with megakaryocytes and prospectively identify a previously unknown intermediate population that is overrepresented in the disease state. We also show that mutant CALR drives transformation primarily from the earliest stem cell compartment, with some contribution from megakaryocyte progenitors. Last, relative to wild-type HSCs, mutant CALR HSCs show increases in JAK-STAT signaling, the unfolded protein response, cell cycle, and a previously undescribed up-regulation of cholesterol biosynthesis. Overall, we have identified a novel megakaryocyte-biased cell population that is increased in a mouse model of ET and described transcriptomic changes linking CALR mutations to increased HSC proliferation and megakaryopoiesis.

Mutant Calreticulin in the Myeloproliferative Neoplasms.

Mutations in the gene for calreticulin (CALR) were identified in the myeloproliferative neoplasms (MPNs) essential thrombocythaemia (ET) and primary myelofibrosis (MF) in 2013; in combination with previously described mutations in JAK2 and MPL, driver mutations have now been described for the majority of MPN patients. In subsequent years, researchers have begun to unravel the mechanisms by which mutant CALR drives transformation and to understand their clinical implications. Mutant CALR activates the thrombopoietin receptor (MPL), causing constitutive activation of Janus kinase 2 (JAK2) signaling and cytokine independent growth in vitro. Mouse models show increased numbers of hematopoietic stem cells (HSCs) and overproduction of megakaryocytic lineage cells with associated thrombocytosis. In the clinic, detection of CALR mutations has been embedded in World Health Organization and other international diagnostic guidelines. Distinct clinical and laboratory associations of CALR mutations have been identified together with their prognostic significance, with CALR mutant patients showing increased overall survival. The discovery and subsequent study of CALR mutations have illuminated novel aspects of megakaryopoiesis and raised the possibility of new therapeutic approaches.

Mutant calreticulin knockin mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage.

Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALRdel/+ mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALRdel/del mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALRdel/+ HSCs were more proliferative in vitro, but neither CALRdel/+ nor CALRdel/del displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF.

Rationale and design of ASPIRE-ICU: a prospective cohort study on the incidence and predictors of Staphylococcus aureus and Pseudomonas aeruginosa pneumonia in the ICU.

BACKGROUND: The epidemiology of ICU pneumonia caused by Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) is not fully described, but is urgently needed to support the development of effective interventions. The objective of this study is to estimate the incidence of S. aureus and P. aeruginosa ICU pneumonia and to assess its association with patient-related and contextual risk factors. METHODS: ASPIRE-ICU is a prospective, observational, multi-center cohort study nested within routine surveillance among ICU patients in Europe describing the occurrence of S. aureus and P. aeruginosa ICU pneumonia. Two thousand (2000) study cohort subjects will be enrolled (50% S. aureus colonized) in which specimens and data will be collected. Study cohort subjects will be enrolled from a larger surveillance population, in which basic surveillance data is captured. The primary outcomes are the incidence of S. aureus ICU acquired pneumonia and the incidence of P. aeruginosa ICU acquired pneumonia through ICU stay. The analysis will include advanced survival techniques (competing risks and multistate models) for each event separately as well as for the sub-distribution of ICU pneumonia to determine independent association of outcomes with risk factors.. A risk prediction model will be developed to quantify the risk for acquiring S. aureus or P. aeruginosa ICU pneumonia during ICU stay by using a composite score of independent risk factors. DISCUSSION: The diagnosis of pathogen-specific ICU pneumonia is difficult, however, the criteria used in this study are objective and comparable to those in the literature. TRIAL REGISTRATION: This study is registered on clinicaltrials.gov under identifier NCT02413242 .

STIM1 is cleaved by calpain.

Store-operated calcium entry (SOCE) is a pathway that moves Ca(2+) across the plasma membrane and is mediated by two major proteins, STIM1 and Orai1. Here, we discovered that the cytoplasmic domain of STIM1 is a target for calpains, a family of Ca(2+)-activated proteases. We found that calpain cleavage of STIM1 serves to control its cellular abundance and was noticeably increased under conditions of cellular stress and apoptosis. Dysregulation of STIM1 levels has been reported to have human disease consequences and our results suggest a mechanism for controlling STIM1 abundance.

UBC9-dependent association between calnexin and protein tyrosine phosphatase 1B (PTP1B) at the endoplasmic reticulum.

Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein, molecular chaperone, and a component of the translocon. We discovered a novel interaction between the calnexin cytoplasmic domain and UBC9, a SUMOylation E2 ligase, which modified the calnexin cytoplasmic domain by the addition of SUMO. We demonstrated that calnexin interaction with the SUMOylation machinery modulates an interaction with protein tyrosine phosphatase 1B (PTP1B), an ER-associated protein tyrosine phosphatase involved in the negative regulation of insulin and leptin signaling. We showed that calnexin and PTP1B form UBC9-dependent complexes, revealing a previously unrecognized contribution of calnexin to the retention of PTP1B at the ER membrane. This work shows that the SUMOylation machinery links two ER proteins from divergent pathways to potentially affect cellular protein quality control and energy metabolism.

Calmodulin-dependent binding to the NHE1 cytosolic tail mediates activation of the Na+/H+ exchanger by Ca2+ and endothelin.

The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is a ubiquitous plasma membrane protein that regulates intracellular pH by removing a single proton (H(+)) in exchange for one extracellular Na(+). The human protein contains a ∼500-amino acid membrane domain and a regulatory, ∼315-amino acid cytosolic domain. NHE1 is activated by a number of hormones including endothelin (ET) and by Ca(2+). The regulatory tail possesses an inhibitory calmodulin (CaM)-binding domain, and inhibition of NHE1 is relieved by binding of a Ca(2+)-CaM complex. We examined the dynamics of ET-1 and Ca(2+) regulation of binding to NHE1 in vivo. CFP was linked to the NHE1 protein cytoplasmic COOH terminus. This was stably transfected into AP-1 cells that are devoid of their own NHE1 protein. The protein was expressed and targeted properly and retained NHE1 activity comparable to the wild-type protein. We examined the in vivo coupling of NHE1 to CaM by Förster resonance energy transfer using CaM linked to the fluorescent protein Venus. CaM interaction with NHE1 was dynamic. Removal of serum reduced CaM interaction with NHE1. Addition of the Ca(2+) ionophore ionomycin increased the interaction between CaM and NHE1. We expressed an ET receptor in AP-1 cells and also found a time-dependent association of NHE1 with CaM in vivo that was dependent on ET treatment. The results are the first demonstration of the in vivo association of NHE1 and CaM through ET-dependent signaling pathways.

Modulation of STIM1 and capacitative Ca2+ entry by the endoplasmic reticulum luminal oxidoreductase ERp57.

STIM1 is an endoplasmic reticulum (ER) membrane Ca(2+) sensor responsible for activation of store-operated Ca(2+) influx. We discovered that STIM1 oligomerization and store-operated Ca(2+) entry (SOC) are modulated by the ER oxidoreductase ERp57. ERp57 interacts with the ER luminal domain of STIM1, with this interaction involving two conserved cysteine residues, C(49) and C(56). SOC is accelerated in the absence of ERp57 and inhibited in C(49) and C(56) mutants of STIM1. We show that ERp57, by ER luminal interaction with STIM1, has a modulatory role in capacitative Ca(2+) entry. This is the first demonstration of a protein involved in ER intraluminal regulation of STIM1.

Organellar calcium buffers.

Ca(2+) is an important intracellular messenger affecting many diverse processes. In eukaryotic cells, Ca(2+) storage is achieved within specific intracellular organelles, especially the endoplasmic/sarcoplasmic reticulum, in which Ca(2+) is buffered by specific proteins known as Ca(2+) buffers. Ca(2+) buffers are a diverse group of proteins, varying in their affinities and capacities for Ca(2+), but they typically also carry out other functions within the cell. The wide range of organelles containing Ca(2+) and the evidence supporting cross-talk between these organelles suggest the existence of a dynamic network of organellar Ca(2+) signaling, mediated by a variety of organellar Ca(2+) buffers.

Higher plant calreticulins have acquired specialized functions in Arabidopsis.

BACKGROUND: Calreticulin (CRT) is a ubiquitous ER protein involved in multiple cellular processes in animals, such as protein folding and calcium homeostasis. Like in animals, plants have evolved divergent CRTs, but their physiological functions are less understood. Arabidopsis contains three CRT proteins, where the two CRTs AtCRT1a and CRT1b represent one subgroup, and AtCRT3 a divergent member. METHODOLOGY/PRINCIPAL FINDINGS: Through expression of single Arabidopsis family members in CRT-deficient mouse fibroblasts we show that both subgroups have retained basic CRT functions, including ER Ca2+-holding potential and putative chaperone capabilities. However, other more general cellular defects due to the absence of CRT in the fibroblasts, such as cell adhesion deficiencies, were not fully restored. Furthermore, in planta expression, protein localization and mutant analyses revealed that the three Arabidopsis CRTs have acquired specialized functions. The AtCRT1a and CRT1b family members appear to be components of a general ER chaperone network. In contrast, and as recently shown, AtCRT3 is associated with immune responses, and is essential for responsiveness to the bacterial Pathogen-Associated Molecular Pattern (PAMP) elf18, derived from elongation factor (EF)-Tu. Whereas constitutively expressed AtCRT1a fully complemented Atcrt1b mutants, AtCRT3 did not. CONCLUSIONS/SIGNIFICANCE: We conclude that the physiological functions of the two CRT subgroups in Arabidopsis have diverged, resulting in a role for AtCRT3 in PAMP associated responses, and possibly more general chaperone functions for AtCRT1a and CRT1b.

ERp57 modulates STAT3 signaling from the lumen of the endoplasmic reticulum.

ERp57 is an endoplasmic reticulum (ER) resident thiol disulfide oxidoreductase. Using the gene trap technique, we created a ERp57-deficient mouse model. Targeted deletion of the Pdia3 gene, which encodes ERp57, in mice is embryonic lethal at embryonic day (E) 13.5. Beta-galactosidase reporter gene analysis revealed that ERp57 is expressed early on during blastocyst formation with the highest expression in the inner cell mass. In early stages of mouse embryonic development (E11.5) there is a relatively low level of expression of ERp57. As the embryos developed, ERp57 became highly expressed in both the brain and the lungs (E15.5 and E18.5). The absence of ERp57 has no impact on ER morphology; expression of ER-associated chaperones and folding enzymes, ER stress, or apoptosis. ERp57 has been reported to interact with STAT3 (signal transducer and activator of transcription)-DNA complexes. We show here that STAT3-dependent signaling is increased in the absence of ERp57 and this can be rescued by expression of ER-targeted ERp57 but not by cytoplasmic-targeted protein, indicating that ERp57 affects STAT3 signaling from the lumen of the ER. ERp57 effects on STAT3 signaling are enhanced by ER luminal complex formation between ERp57 and calreticulin. In conclusion, we show that ERp57 deficiency in mouse is embryonic lethal at E13.5 and ERp57-dependent modulation of STAT3 signaling may contribute to this phenotype.

Endoplasmic reticulum proteins in cardiac development and dysfunction.

An understanding of cardiac pathologies and the molecular mechanisms thereof is essential for the development of therapies for cardiovascular disease, a common cause of death in Western societies. Investigations into heart diseases have shown that the endoplasmic reticulum and its diverse functions may lie at the center of many cardiac pathologies. Animal models have demonstrated that in numerous cases, faulty endoplasmic reticulum activity is manifested in defective cardiogenesis or impaired heart function. These findings suggest that the endoplasmic and sarcoplasmic reticulum membranes may represent functionally independent organelles responsible for specialized functions in the heart. This review addresses the molecular pathways linking endoplasmic reticulum function and malfunction with impaired cardiac phenotypes. The endoplasmic reticulum affects cardiac development and function through Ca2+-dependent pathways, its catalytic role in the proper folding and targeting of membrane-bound and secretory proteins, and its response to cellular stress events, particularly hypoxic conditions. These pathways present potential novel targets for treatment of cardiac disease.