Normal hematopoiesis is sustained through a carefully orchestrated balance between hematopoietic stem cell (HSC) self-renewal and differentiation. The functional importance of this axis is underscored by the severity of disease phenotypes initiated by abnormal HSC function, including myelodysplastic syndromes and hematopoietic malignancies. Major advances in the understanding of transcriptional regulation of primitive hematopoietic cells have been achieved; however, the post-transcriptional regulatory layer that may impinge on their behavior remains underexplored by comparison. Key players at this level include RNA-binding proteins (RBPs), which execute precise and highly coordinated control of gene expression through modulation of RNA properties that include its splicing, polyadenylation, localization, degradation, or translation. With the recent identification of RBPs having essential roles in regulating proliferation and cell fate decisions in other systems, there has been an increasing appreciation of the importance of post-transcriptional control at the stem cell level. Here we discuss our current understanding of RBP-driven post-transcriptional regulation in HSCs, its implications for normal, perturbed, and malignant hematopoiesis, and the most recent technological innovations aimed at RBP-RNA network characterization at the systems level. Emerging evidence highlights RBP-driven control as an underappreciated feature of primitive hematopoiesis, the greater understanding of which has important clinical implications.

Acute myeloid leukemia (AML) is fueled by leukemic stem cells (LSCs) whose determinants are challenging to discern from hematopoietic stem cells (HSCs) or uncover by approaches focused on general cell properties. We have identified a set of RNA binding proteins (RBPs) selectively enriched in human AML LSCs. Using an in vivo two-step CRISPR-Cas9 screen to assay stem cell functionality, we found 32 RBPs essential for LSCs in MLL-AF9;NrasG12D AML. Loss-of-function approaches targeting key hit RBP ELAVL1 compromised LSC-driven in vivo leukemic reconstitution and selectively depleted primitive malignant vs. healthy cells. Integrative multiomics revealed differentiation, splicing and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with mitochondrial import protein, TOMM34 being a direct ELAVL1-stabilized target whose repression impairs AML propagation. Altogether, using a stem cell-adapted in vivo CRISPR screen, this work demonstrates pervasive reliance on RBPs as regulators of LSCs and highlights their potential as therapeutic targets in AML.

Purpose: Canadian clinician-scientist trainees enrolled in dual degree programs often pursue an extended training route following completion of MD and MSc or PhD degrees. However, the proportion, plans and reasoning of trainees who intend to pursue training internationally following dual degree completion has not been investigated. In this study, we assessed the international training considerations of current clinician-scientist trainees. Methods: We designed an 11-question survey, which was sent out by program directors to all current MDPhD program and Clinician Investigator Program (CIP) trainees. Responses were collected from July 8, 2019 to August 8, 2019. Results: We received a total of 191 responses, with representation from every Canadian medical school and both MD-PhD program and CIP trainees. The majority of trainees are considering completing additional training outside Canada, most commonly post-doctoral and/or clinical fellowships. The most common reasons for considering international training include those related to quality and prestige of training programs. In contrast, the most common reasons for considering staying in Canada for additional training are related to personal and ethical reasons. Irrespective of intentions to pursue international training, the majority of trainees ultimately intend to establish a career in Canada. Conclusion: While most trainees are considering additional training outside of Canada due to prestige and quality of training, the majority of trainees intend to pursue a career as a clinician-scientist back in Canada. Trainees would likely benefit from improved guidance and mentorship on the value of international training, as well as enhanced support in facilitating cross-border mobility.

BackgroundAcute vaso-occlusive crisis (VOC) in sickle cell disease (SCD) often leads patients to seek emergency department (ED) care. The landscape of SCD ED care within Canada’s universal and publicly funded healthcare system remains largely undefined. Here, we sought to address this knowledge gap by characterizing the quality of SCD VOC ED and inpatient care in Ontario, Canada.MethodsWe used population-level health administrative data to identify patients with SCD in Ontario who presented to the ED with SCD VOC (ICD-10-CA code D57.0) from 2006 to 2018. We evaluated ED and inpatient metrics for this population and compared these with a general ED patient population and a validated Crohn’s disease cohort matched by age, sex, neighbourhood income quintile, geography, date range of visit, and ED triage score.ResultsWe identified 2,123 patients who presented to the ED with SCD VOC for a total of 18,712 unscheduled ED visits. Annual ED visit rates ranged from a mean of 2.6 to 14.2 visits per patient, with 4.6% of patients having incurred 38.4% of all SCD VOC ED visits. Subgroup analyses showed that adult patients with SCD VOC visited the ED 2.5 times more than pediatric patients, but their wait times for initial physician assessment were significantly longer (90.6 vs. 51.1 minutes, p < 0.0001) with a difference that was disproportionately larger than the reference cohorts. Males with SCD VOC experienced significantly shorter ED and inpatient stays compared to females (e.g. admission length of stay: 4.4 vs. 5.8 days, p < 0.0001), but their ED return rates within 30-days were significantly higher (0.89 vs. 0.35 times, p < 0.0001) with a difference that was disproportionately larger relative to the reference cohorts. Lower-income neighbourhoods correlated with lower acuity triage scores for SCD VOC visits and increased repeat ED visit rates at more pronounced levels than the reference cohorts. Higher acuity triage scores for adults with SCD VOC correlated with improved metrics for quality of ED and inpatient care with fewer longer-term ED visits.InterpretationPatients with SCD VOC carry a significant burden of acute care needs, with a smaller patient subset whose high ED visit rates have distinctly increased over time in Ontario, Canada. Adults, males and those from lower income quintile neighbourhoods with SCD VOC experienced poorer quality of ED and inpatient care compared to their counterparts, with differences that are disproportionately larger than those found in the general ED population and in patients with Crohn’s disease. Higher acuity triage scoring may improve and standardize the quality of ED care for adult patients with SCD VOC. Future work should address these identified care quality disparities for SCD VOC that remain present within a universal and publicly funded healthcare system.

How hematopoietic stem cells (HSCs) coordinate their divisional axis and whether this orientation is important for stem cell–driven hematopoiesis is poorly understood. Single-cell RNA sequencing data from patients with Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome, show that ARHGEF2, a RhoA-specific guanine nucleotide exchange factor and determinant of mitotic spindle orientation, is specifically downregulated in SDS hematopoietic stem and progenitor cells (HSPCs). We demonstrate that transplanted Arhgef2−/− fetal liver and bone marrow cells yield impaired hematopoietic recovery and a production deficit from long-term HSCs, phenotypes that are not the result of differences in numbers of transplanted HSCs, their cell cycle status, level of apoptosis, progenitor output, or homing ability. Notably, these defects are functionally restored in vivo by overexpression of ARHGEF2 or its downstream activated RHOA GTPase. By using live imaging of dividing HSPCs, we show an increased frequency of misoriented divisions in the absence of Arhgef2. ARHGEF2 knockdown in human HSCs also impairs their ability to regenerate hematopoiesis, culminating in significantly smaller xenografts. Together, these data demonstrate a conserved role for Arhgef2 in orienting HSPC division and suggest that HSCs may divide in certain orientations to establish hematopoiesis, the loss of which could contribute to HSC dysfunction in bone marrow failure.

<p>Table S3 shows clinical information for all AML patient specimens used in this study.</p>

<p>Table S4 shows limiting dilution analysis of secondary recipients transplanted with DMSO- or MS-444-treated bone marrow from primary mice</p>

<p>Table S6 shows altered gene sets identified by GSEA of the ELAVL1 knockout RN2c RNA-sequencing data set.</p>