The leukemia stem cell (LSC) populations of acute myeloid leukemia (AML) exhibit phenotypic, genetic, and functional heterogeneity that contribute to therapy failure and relapse. Progress toward ...understanding the mechanistic basis for therapy resistance in LSCs has been hampered by difficulties in isolating cell fractions that enrich for the entire heterogeneous population of LSCs within individual AML samples. We previously reported that CD200 gene expression is upregulated in LSC-containing AML fractions. Here, we show that CD200 is present on a greater proportion of CD45dim blasts compared with more differentiated CD45high cells in AML patient samples. In 75% (49 of 65) of AML cases we examined, CD200 was expressed on ≥10% of CD45dim blasts; of these, CD200 identified LSCs within the blast population in 9 of 10 (90%) samples tested in xenotransplantation assays. CD200+ LSCs could be isolated from CD200+ normal HSCs with the use of additional markers. Notably, CD200 expression captured both CD34– and CD34+ LSCs within individual AML samples. Analysis of highly purified CD200+ LSC-containing fractions from NPM1-mutated AMLs, which are commonly CD34–, exhibited an enrichment of primitive gene expression signatures compared with unfractionated cells. Overall, our findings support CD200 as a novel LSC marker that is able to capture the entire LSC compartment from AML patient samples, including those with NPM1 mutation.
•CD200 marks LSCs in AML patient samples expressing CD200 on ≥10% of blasts.•CD200 captures both CD34– and CD34+ LSCs from individual AML patient samples.
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Refractoriness to induction chemotherapy and relapse after achievement of remission are the main obstacles to cure in acute myeloid leukaemia (AML). After standard induction chemotherapy, patients ...are assigned to different post-remission strategies on the basis of cytogenetic and molecular abnormalities that broadly define adverse, intermediate and favourable risk categories. However, some patients do not respond to induction therapy and another subset will eventually relapse despite the lack of adverse risk factors. There is an urgent need for better biomarkers to identify these high-risk patients before starting induction chemotherapy, to enable testing of alternative induction strategies in clinical trials. The high rate of relapse in AML has been attributed to the persistence of leukaemia stem cells (LSCs), which possess a number of stem cell properties, including quiescence, that are linked to therapy resistance. Here, to develop predictive and/or prognostic biomarkers related to stemness, we generated a list of genes that are differentially expressed between 138 LSC
and 89 LSC
cell fractions from 78 AML patients validated by xenotransplantation. To extract the core transcriptional components of stemness relevant to clinical outcomes, we performed sparse regression analysis of LSC gene expression against survival in a large training cohort, generating a 17-gene LSC score (LSC17). The LSC17 score was highly prognostic in five independent cohorts comprising patients of diverse AML subtypes (n = 908) and contributed greatly to accurate prediction of initial therapy resistance. Patients with high LSC17 scores had poor outcomes with current treatments including allogeneic stem cell transplantation. The LSC17 score provides clinicians with a rapid and powerful tool to identify AML patients who do not benefit from standard therapy and who should be enrolled in trials evaluating novel upfront or post-remission strategies.
Affecting more than 5.1 million Americans in 2013, heart failure (HF) is a debilitating syndrome that causes dyspnea, reduced exercise tolerance, fatigue, and diminished quality of life. Most ...commonly diagnosed in individuals over 65 years of age, the prevalence of HF and its associated health care costs are expected to grow significantly. Aerobic exercise, resistance training, and inspiratory muscle training (IMT) are interventions that are currently used in the treatment of older adults with HF. A literature search was conducted for meta-analyses, systematic reviews, and large randomized control trials to evaluate the effect of aerobic exercise, resistance training, and IMT interventions on exercise tolerance and quality of life in individuals with HF. Overall, aerobic exercise, resistance training, and IMT appear to be safe and effective in appropriately selected individuals for improving exercise tolerance and quality of life in individuals with HF. The benefits of resistance training in isolation are not clear.
Leukemia onset is preceded by clonal expansion of hematopoietic stem cells (HSCs) bearing mutations conferring a growth advantage, a preleukemic state known as age-related clonal hematopoiesis ...(ARCH). Chr13q deletions (13q-) are the most common ARCH-related chromosomal alterations. We previously reported a patient with concurrent myelo-proliferative neoplasm and chronic lymphocytic leukemia (CLL) diseases that both originated from HSCs with 13q-. However, the role of 13q- in ARCH and/or leukemogenesis remains unclear, thus hampering the goal of developing therapies that intervene during the preleukemic stage of disease progression. We first analyzed publicly available patient sequencing datasets and determined the 13q minimum deleted region (MDR) for ARCH and CLL. DLEU2, a lncRNA gene within the MDR contains an intronic miRNA cluster, miR-15a and miR-16-1. As we previously linked miRNA to HSC and leukemia stem cell function, we first focused on these two miRNAs. Using CRISPR-Cas9, we deleted the intronic miR-15a/16-1 polycistron in human long-term (LT-) and short-term HSCs (ST-HSCs) and measured the effects at single-cell resolution within in vitro differentiation assays. miR-15a/16-1 knockout (KO) reduced multilineage differentiation of LT-HSC, but increased B cell production from ST-HSC. Within xenografts, miR-15a/16-1 KO LT-HSCs showed subtle lineage output changes at 24-week post-transplantation; quantitative self-renewal assays, frequency assessment of the primitive and progenitor compartments, and cell cycle measurements are ongoing. Importantly, there was no evidence of CLL disease, which contrasts human biology with previously published mouse models, suggesting that miR-15a/16-1 KO is insufficient to cause CLL in human cells. A full accounting of HSC and CLL preleukemic data and the progress toward the KO of full 13q regions will be reported.
In the human blood system, we previously established that miRNA-mediated gene silencing is an important gene expression control mechanism that regulates the stemness of rare multipotent long-term ...hematopoietic stem cells (LT-HSCs). The Argonaute (AGO) family of proteins are the core effectors of the miRNA-induced silencing complex, with four paralogs in humans, AGO1-4. Traditionally, these paralogs are believed to function redundantly, and AGO2 has been the primary focus of most hematological studies as it is the only paralog with slicer activity. However, our previous RNA-seq performed on blood cells across the human cord blood (CB) hierarchy shows differential expression of AGO paralogs in various hematopoietic lineages, suggesting each paralog might have a distinct function; however, their role in HSC and the functional significance of other AGO family members in the hematopoietic system remains unknown. Here, we used CRISPR/Cas9 to knockout (KO) AGO1-4 individually in human CB-derived LT- and short-term HSCs (ST-HSCs), and measured effects on proliferation, maintenance, and lineage commitment in single-cell differentiation assays in vitro and through xenotransplantation. AGO2 KO induced alterations in erythroid and B cell output recapitulating previous murine studies, although our study provides a more complete functional landscape, where we observed an increase in megakaryocytes and granulocytes, as well as significant alterations within progenitor compartments. Unexpectedly, we also find distinct phenotypes with the KO of other AGO paralogs, in particular AGO3 and AGO4, pointing toward a unique role of different AGO paralogs in lineage commitment in human HSCs and arguing against the prevailing view of redundant functions. The full insights into how individual AGO family members regulate stemness and lineage commitment of human HSCs will be presented.
Long-term hematopoietic stem cells (LT-HSC) are responsible for life-long blood production and show functional erosion due to aging or inflammatory dysregulation. However, the molecular programs ...underlying aging or LT-HSC responsiveness to emergency hematopoiesis triggered by stress stimuli such as transplantation or inflammation are poorly understood. We previously identified transcriptional, epigenetic and functional heterogeneity in human LT-HSC in the transition from quiescence to cellular activation, with inflammatory pathways implicated. Here, we observe a gradient of TNFα via NFkB pathway enrichment in single cell (sc) transcriptomes of ∼4000 cord blood LT-HSC suggesting heterogeneous priming with TNFα treatment. With a TNFα-mediated inflammatory challenge xenotransplantation model, we asked how human HSC from engrafted mice respond to and recover from inflammatory stress. Xenografts treated with two doses of TNFα at 2w and 10w post-transplantion contained significantly reduced human grafts at 20w with highly dysregulated lineage differentiation profiles compared to controls. CD34+CD38-CD45RA–CD19– cells isolated from control and TNFα treated mice at 20w were profiled with Chromium sc multiome. Two HSC subsets differing in enrichment of inflammation/immune regulators such as NFKB1 and CD83 were identified, each with divergent transcriptomic and epigenomic changes following TNFα treatment compared to control. TNFα treatment was sufficient to upregulate surface expression of CD83 in LT-HSC in vitro and in our xenotranslantation model. Overall, these studies reveal heterogeneous inflammation-priming programs in human LT-HSC that are activated by both transplantation and inflammatory stress and point to a TNFα via NFkB-mediated immune activation and resolution pathway as a novel regulatory axis for human HSC function.
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