Hematopoietic Stem Cell Signatures Research Articles

IMMUNOPHENOTYPIC AND MOLECULAR HETEROGENEITY ANALYSIS OF HUMAN HEMATOPOIETIC STEM AND PROGENITORS

Hematopoietic stem cells (HSCs) have the capacity to differentiate into all hematopoietic lineages, and at the same time self-renew to maintain the HSC pool. HSCs have been thoroughly investigated using immunophenotypic-, molecular- and functional-analysis resulting in the development of protocols for high-purity prospective isolation of human HSCs. However, within the current state-of-the-art HSC populations, 90% of the cells are not bona fide HSCs, confounding molecular analysis of HSCs. Thus, identification of novel immunophenotypic markers to delineate the HSC population would improve our understanding of HSC biology. To identify cell-surface markers with the potential to discriminate between functionally different cells within the HSC population, we performed antibody screens measuring the expression of 340 markers on human cord blood (CB) and bone marrow (BM). Candidate markers that divide the HSC population were included in CITE-seq experiments together with conventional HSC and progenitor markers for combined analysis of single-cell immunophenotype-/RNA sequencing analysis. This allowed us to correlate the molecular signature of each single-cell with expression of 40 cell-surface proteins, in CD34+ and CD34+CD38- populations of CB and BM. Following sequencing the cells were clustered based on molecular signature. Distinct groups with HSC, progenitor and early committed progenitor profiles were identified. Interestingly, comparison between CB and BM showed differences in progenitor proportions while the fraction of cells with HSC signature remained intact. Additionally, three novel HSC marker-candidates were discovered which are being validated. Together, using CITE-seq we can describe the immunophenotypic- and molecular-heterogeneity of the HSC and progenitor populations and identify three novel cell-surface marker candidates for prospective isolation of HSCs.

Gfi1b regulates the level of Wnt/\u03b2-catenin signaling in hematopoietic stem cells and megakaryocytes

Gfi1b is a transcriptional repressor expressed in hematopoietic stem cells (HSCs) and megakaryocytes (MKs). Gfi1b deficiency leads to expansion of both cell types and abrogates the ability of MKs to respond to integrin. Here we show that Gfi1b forms complexes with β-catenin, its co-factors Pontin52, CHD8, TLE3 and CtBP1 and regulates Wnt/β-catenin-dependent gene expression. In reporter assays, Gfi1b can activate TCF-dependent transcription and Wnt3a treatment enhances this activation. This requires interaction between Gfi1b and LSD1 and suggests that a tripartite β-catenin/Gfi1b/LSD1 complex exists, which regulates Wnt/β-catenin target genes. Consistently, numerous canonical Wnt/β-catenin target genes, co-occupied by Gfi1b, β-catenin and LSD1, have their expression deregulated in Gfi1b-deficient cells. When Gfi1b-deficient cells are treated with Wnt3a, their normal cellularity is restored and Gfi1b-deficient MKs regained their ability to spread on integrin substrates. This indicates that Gfi1b controls both the cellularity and functional integrity of HSCs and MKs by regulating Wnt/β-catenin signaling pathway.

The Role of Intracellular Calcium Signaling in Hematopoietic Stem Cells

The Role of Intracellular Calcium Signaling in Hematopoietic Stem Cells

Identification of High Risk Group for Leukemic Transformation in Higher Risk MDS Patients Using Targeted RNA-Sequencing: Hematopoietic Stem Cell Signature As a High Risk Profile for Leukemic Transformation

IntroductionMyelodysplastic syndrome (MDS) is a heterogeneous group of myeloid disorder characterized by defective bone marrow (BM) hematopoiesis with peripheral blood cytopenias and risk for progression to acute myeloid leukemia. Accurate determination of prognosis is critical to select an appropriate therapy and to detect any case progressing to leukemic transformation which brings ominous prognosis in patients with MDS. Despite clinical risk models, additional molecular data are needed to enhance the prediction of patients' clinical courses and to aid disease management. Therefore, the present study attempted to classify the higher risk MDS (HR-MDS) patients according to their molecular risk through targeted RNA-sequencing, to correlate it with clinical risk models, and analyzed molecular risk grouping for prognostic stratification power, especially for leukemic transformation in higher-risk patients with MDS treated with hypomethylating agents (HMA) including azacitidine or decitabine.Patients and MethodsA total of 30 patients were included with HR-MDS by International Prognostic Scoring System (IPSS). Overall, 60 bone marrow samples (30 diagnosis and follow-up pairs) were subject for targeted RNA-seq using Illumina TruSight Pan-Cancer panel. After read mapping by Tophat2, gene count was measured using HTSeq followed by DEseq2 for differential gene expression quantification. All 60 samples as well as 30 samples from T-cell fraction (CD3+, as a control) were also subjected for DNA-seq targeting a panel of 84 commonly mutated genes in myeloid malignancies (Agilent SureSelect). All downstream computational and statistical analyses were performed using R and Python.ResultsThe median age was 65 years (range 40-84 years) with 16 male patients (53%). Twenty-seven (90%) and 3 (10%) patients were intermediate-2 and high risk by IPSS, respectively. According to revised IPSS (IPSS-R), the distribution of risk groups was as follows: low (n=5, 17%), intermediate (n=8, 27%), high (n=11, 37%), and very high (n=6, 20%). A total of 56 mutations were detected in the diagnostic samples from 30 patients. Frequently mutated genes were DDX41 (n=5) and TP53 (n=4). Best response to HMA (16 azacitidine and 14 decitabine) was achieved in median 4 cycles (range 3-8). Complete response (CR) including marrow CR was achieved in 18 patients (60%), and 10 patients (33%) received allogeneic hematopoietic cell transplantation. Overall survival (OS) rate was not well correlated with IPSS-R risk groups. With median follow-up duration of 28.2 months (range 3.8-95), 3-years' OS rate showed 40%, 75%, 36%, and 67% in low, intermediate, high, and very high risk, respectively. Unsupervised clustering using top 100 genes with highest variance revealed 3 distinct clusters (n=8, 9, and 13 in group 1, 2, and 3), 3-years' OS rate of which showed 73%, 57%, and 35% in group 1, 2, and 3, respectively (p=0.004 between group 3 vs group 1/2). Despite inferior long-term outcomes in the group 3, the baseline clinical variables of some patients were classified as favorable implying that clinical factor does not reflect adverse long-term outcomes: 4 out of 13 patients with low risk by IPSS-R eventually experienced adverse outcome. The 3-years' leukemic transformation rate was 0%, 33% and 57% in group 1, 2, and 3 (p=0.039 between group 3 vs group 1/2). In the multivariate analyses, besides achievement of CR, the risk group 3 by RNA-seq were identified as independent adverse prognostic factors for OS (p=0.007, HR 6.75 [1.68-27.17]) as well as leukemic transformation (p=0.013, HR 6.91 [1.49-31.95]). In the gene set enrichment analysis using MSigDB, hematopoietic stem cell genes were enriched in RNA-seq group 3, suggesting that the high-risk signature on RNA-seq is linked with stemness of hematopoietic stem cells.ConclusionRNA-seq can be utilized to identify the higher risk patients with MDS. The higher risk group by RNA-seq enriched with genes with hematopoietic stem cells, which suggests that stemness in hematopoietic stem cells is linked with resistance to HMA therapy and increasing risk of leukemic transformation in HR-MDS. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

M6A Maintains Hematopoietic Stem and Progenitor Cell Identity

N-6-methyladenosine (m6A) is one of the most abundant posttranscriptional modifications in eukaryotic mRNAs and long noncoding RNAs. We previously found a critical role for m6A in promoting human myeloid leukemia (Vu et al. Nature Medicine 2017). Targeting the RNA methylation program in leukemias has been suggested as a potential novel therapeutic strategy. However, it is unknown whether the m6A modification controls normal adult hematopoiesis and hematopoietic stem cells (HSC) function.To investigate the role of m6A in adult hematopoiesis, we crossed with the Mettl3 conditional knockout (cKO) mouse model with the interferon inducible Mx1-Cre system to abolish m6A in the hematopoietic compartment. Deletion of Mettl3 (3 weeks post pIpC injections) resulted in pancytopenia (white blood count 9.44 k/ul in WT versus 4.35 k/ul cKO) and a 55% reduction in red blood cell counts and nearly 70% loss in platelet counts. Most remarkably, METTL3 depletion resulted in a 5-fold increase in the number of overall hematopoietic stem and progenitors HSPCs (LSKs; Lin-c-kit+Sca1+). Within the HSPC compartment there was a about a 10-fold expansion in immunophenotypic long term hematopoietic stem cells LT-HSCs (Lin-c-kit+Sca1+CD150+CD48-), multipotent progenitors (MPP-2s), (Lin-c-kit+Sca1+CD150+CD48+) and MPP4s (Lin-c-kit+Sca1+CD150-CD48+). In contrast to this general increase in HSPCs, we observed a decrease in common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP) populations by 70% and 60% respectively. Altogether, these results suggest that loss of METTL3 results in a partial blockage in hematopoietic stem and progenitor cell differentiation, and an accumulation of LT-HSC and MPPs.Despite this phenotypic expansion of LT-HSCs and MPPs, their function was impaired as demonstrated by a reduction in long-term chimerism in non-competitive transplants into congenic mice compared to the remaining wildtype host cells (66% in WT vs 26% in cKO). Interestingly, the relative differentiation block and accumulation of HSCs remained after transplantation. Furthermore, Mettl3 cKO HSCs are less quiescent (79% WT vs. 44% cKO) and more proliferative based on cell cycle profiling with pyronin Y/Hoechst or Ki67/Hoechst.RNA-seq in sorted LT-HSC, MPP1, MPP2 and MPP4s from WT and Mettl3 cKO mice demonstrated that Mettl3 cKO HSCs lose the HSC signature while MPP1 and MPP2 cells exhibited gene signatures resembling WT HSCs. Interestingly, genes uniquely upregulated in Mettl3 cKO LT-HSC showed a significant enrichment of the Mettl3 KO mouse embryonic stem cells (mESCs) expression signature. To decipher METTL3 regulation of transcriptional states in individual cells along the hematopoietic hierarchy, we performed single-cell RNA-seq using sorted WT and Mettl3 cKO cKit+ cells. Surprisingly, tSNE analysis of scRNA-seq data uncovered the loss of the normal HSC cluster and the emergence of three unique HSC-like populations in Mettl3 cKO mice. Single cell analysis identified a reduction in c-MYC levels in the HSPCs that was then confirmed by immunofluorescence in sorted HSCs. More importantly, when forced into division, Mettl3 cKO HSCs exhibited increased symmetric c-MYC low division pattern compared to control (60% to 48%) based on c-MYC IF staining in paired daughter cells. We further confirmed that sorted HSCs underwent increased symmetric renewal divisions in the Mettl3 cKO compared to controls (65% to 49%) without any effect on asymmetric division based on NUMB IF staining in paired daughter cells. These data suggest that without METTL3, HSCs are partially blocked in a self-renewing state albeit with reduced fitness compared to wildtype HSCs. Overall, our studies uncovered a novel role for METTL3 and RNA methylation to maintain normal HSC identity and progenitor differentiation. Also, these studies suggest that inhibiting METTL3 could result in significant hematopoietic defects. DisclosuresNo relevant conflicts of interest to declare.

Ex vivo human HSC expansion requires coordination of cellular reprogramming with mitochondrial remodeling and p53 activation

AbstractThe limited number of hematopoietic stem cells (HSCs) in umbilical cord blood (UCB) units restricts their use for stem cell transplantation. Ex vivo treatment of UCB-CD34+ cells with valproic acid (VPA) increases the number of transplantable HSCs. In this study, we demonstrate that HSC expansion is not merely a result of proliferation of the existing stem cells but, rather, a result of a rapid reprogramming of CD34+CD90− cells into CD34+CD90+ cells, which is accompanied by limited numbers of cell divisions. Beyond this phenotypic switch, the treated cells acquire and retain a transcriptomic and mitochondrial profile, reminiscent of primary HSCs. Single and bulk RNA-seq revealed a signature highly enriched for transcripts characteristic of primary HSCs. The acquisition of this HSC signature is linked to mitochondrial remodeling accompanied by a reduced activity and enhanced glycolytic potential. These events act in concert with a modest upregulation of p53 activity to limit the levels of reactive oxygen species (ROS). Inhibition of either glycolysis or p53 activity impairs HSC expansion. This study indicates that a complex interplay of events is required for effective ex vivo expansion of UCB-HSCs.

The Myc/Max/Mxd Network Is a Target of Mutated Flt3 Signaling in Hematopoietic Stem Cells in Flt3-ITD-Induced Myeloproliferative Disease.

Acute myeloid leukemia (AML) has poor prognosis due to various mutations, e.g., in the FLT3 gene. Therefore, it is important to identify pathways regulated by the activated Flt3 receptor for the discovery of new therapeutic targets. The Myc network of oncogenes and tumor suppressor genes is involved in mechanisms regulating proliferation and survival of cells, including that of the hematopoietic system. In this study, we evaluated the expression of the Myc oncogenes and Mxd antagonists in hematopoietic stem cell and myeloid progenitor populations in the Flt3-ITD-knockin myeloproliferative mouse model. Our data shows that the expression of Myc network genes is changed in Flt3-ITD mice compared with the wild type. Mycn is increased in multipotent progenitors and in the pre-GM compartment of myeloid progenitors in the ITD mice while the expression of several genes in the tumor suppressor Mxd family, including Mxd1, Mxd2, and Mxd4, is concomitantly downregulated, as well as the expression of the Mxd-related gene Mnt and the transcriptional activator Miz-1. LSKCD150+CD48− hematopoietic long-term stem cells are decreased in the Flt3-ITD cells while multipotent progenitors are increased. Of note, PKC412-mediated inhibition of Flt3-ITD signaling results in downregulation of cMyc and upregulation of the Myc antagonists Mxd1, Mxd2, and Mxd4. Our data provides new mechanistic insights into downstream alterations upon aberrant Flt3 signaling and rationale for combination therapies for tyrosine kinase inhibitors with Myc antagonists in treating AML.

Abstract A19: Human models of multilineage leukemia induced by NUP98-KDM5A chimeric fusion oncogene

Abstract Acute megakaryoblastic (CD41+CD61+) leukemia (AMKL) is a subset (10%) of pediatric acute myeloid leukemia (AML) that typically affects young children (<3 yrs) and carries dismal cure rates (<40%), due to primary chemoresistance or early relapse. It was recently recognized that mutually exclusive chimeric fusion oncogenes are found in 60-70% of AMKL, including the NUP98 (nucleoporin 98)-KDM5A (histone lysine demethylase 5A) fusion, in ~10% of cases. AML subtype-specific human models are needed to identify subtype-specific biomarkers and drug targets, and to overcome the paucity of primary human samples, the genetic heterogeneity of the disease, and the clonal evolution of commercially available cell lines. We engineered human models of AMKL through overexpression of NUP98-KDM5A (N5A) in cord blood (CB)-HSPC using lentiviral transduction (vector carrying GFP reporter gene). We generated multiple de novo N5A models, from distinct genetic backgrounds (independent CB units), to identify fusion-specific features. Adoptive transfer of the fusion oncogene-carrying cells (or N5A-cells) in immunodeficient (NSG) recipient mice led to AMKL development with a latency of 32-46 weeks (λ of 1/6 to 2/3, n=3 independent experiments). Mice presented clinically overt signs of disease with leukemic cell infiltration of multiple organs (bone marrow, liver, and spleen, as well as lung and kidney) and myelofibrosis. Leukemic blasts consisted of CD45loCD34-CD41+CD61+ cells, faithfully phenocopying human AMKL. In addition, N5A overexpression led to the development of acute erythroid leukemia (AEL) (CD34-CD117+CD36+) and B-ALL (CD34+CD38+CD19+CD10+), reflecting the clinical heterogeneity of leukemia associated with NUP98 fusions and suggesting distinct cells of origin for each entity. While N5A harboring B-ALL have not yet been reported, the fusion is now emerging as a recurrent genetic aberration in AEL. Transcriptomic studies of flow-sorted GFP+ xenograft cells closely matched that of N5A pediatric samples, and showed upregulation of HOXA/B cluster genes, as compared to control HSPC. A hematopoietic stem cell signature was detected by gene set enrichment analysis (GSEA) for N5A AMKL, as well as novel cell surface markers, including p-selectin (SELP) and CD96. Thus far, cooperating mutations were not identified, suggesting that the N5A fusion is a potent oncogene. In addition, using optimized culture conditions to promote HSPC expansion, leukemic cell lines were generated. N5A-cells displayed maturation block and increased cell proliferation compared to controls, as assessed by cytology, serial tracking of increasing CD34+GFP+ cell fractions, and lack of a c-KIThi differentiated mast cell population. After 3 weeks in vitro, when control-transduced cells normally begin to die off, an immature cell population emerged from the N5A-cells and this population was sustained for >140 days. This was observed by day 25 of culture in 66% (2/3) to 92% (11/12) of the wells (n=3 expts). Progenitor frequency was markedly increased in N5A-cells (on average 1:3 vs 1:200 in CTL, at day 88 of culture; n=3 expts). These N5A engineered cell lines can be efficiently transduced with shRNA carrying lentiviral vectors and thus provide suitable substrates for chemical and genetic screening approaches. We have optimized a strategy to engineer human cell lines and xenograft models of high-fatality NUP98-KDM5A driven leukemia, identifying specific leukemia-activated genes and novel cell surface biomarkers for AMKL. These studies unraveled the unappreciated multilineage leukemic potential of N5A, indicating that engineered leukemia can be exploited to unravel novel disease subtypes, which is especially relevant for cryptic mutations such as N5A fusions. The generated cell lines pave the way to genetic- or chemical-based functional screens to identify promising drug targets. Citation Format: Sophie Cardin, Melanie Bilodeau, Louise Laramee, Loubna Jouan, Mathieu Roussy, R. Keith Humphries, Josee Hebert, Brian T. Wilhelm, Sonia Cellot. Human models of multilineage leukemia induced by NUP98-KDM5A chimeric fusion oncogene [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A19.

Functional dissection of hematopoietic stem cell populations with a stemness-monitoring system based on NS-GFP transgene expression

Hematopoietic stem cells (HSCs) in a steady state can be efficiently purified by selecting for a combination of several cell surface markers; however, such markers do not consistently reflect HSC activity. In this study, we successfully enriched HSCs with a unique stemness-monitoring system using a transgenic mouse in which green florescence protein (GFP) is driven by the promoter/enhancer region of the nucleostemin (NS) gene. We found that the phenotypically defined long-term (LT)-HSC population exhibited the highest level of NS-GFP intensity, whereas NS-GFP intensity was strongly downregulated during differentiation in vitro and in vivo. Within the LT-HSC population, NS-GFPhigh cells exhibited significantly higher repopulating capacity than NS-GFPlow cells. Gene expression analysis revealed that nine genes, including Vwf and Cdkn1c (p57), are highly expressed in NS-GFPhigh cells and may represent a signature of HSCs, i.e., a stemness signature. When LT-HSCs suffered from remarkable stress, such as transplantation or irradiation, NS-GFP intensity was downregulated. Finally, we found that high levels of NS-GFP identified HSC-like cells even among CD34+ cells, which have been considered progenitor cells without long-term reconstitution ability. Thus, high NS-GFP expression represents stem cell characteristics in hematopoietic cells, making this system useful for identifying previously uncharacterized HSCs.

Gfi1b regulates the level of Wnt/b-catenin signaling in hematopoietic stem cells and megakaryocytes

Gfi1b regulates the level of Wnt/b-catenin signaling in hematopoietic stem cells and megakaryocytes

Pathogen-Induced TLR4-TRIF Innate Immune Signaling in Hematopoietic Stem Cells Promotes Proliferation but Reduces Competitive Fitness

Bacterial infection leads to consumption of short-lived innate immune effector cells, which then need to be replenished from hematopoietic stem and progenitor cells (HSPCs). HSPCs express pattern recognition receptors, such as Toll-like receptors (TLRs), and ligation of these receptors induces HSPC mobilization, cytokine production, and myeloid differentiation. The underlying mechanisms involved in pathogen signal transduction in HSCs and the resulting biological consequences remain poorly defined. Here, we show that invivo lipopolysaccharide (LPS)application induces proliferation of dormant HSCsdirectly via TLR4 and that sustained LPS exposure impairs HSC self-renewal and competitive repopulation activity. This process is mediated via TLR4-TRIF-ROS-p38, but not MyD88 signaling, and can be inhibited pharmacologically without preventing emergency granulopoiesis. Live Salmonella Typhimurium infection similarly induces proliferative stress in HSCs, in part via TLR4-TRIF signals. Thus, while direct TLR4 activation in HSCs might be beneficial for controlling systemic infection, prolonged TLR4 signaling has detrimental effects and may contribute to inflammation-associated HSPC dysfunction.

P53−/− synergizes with enhanced NrasG12D signaling to transform megakaryocyte-erythroid progenitors in acute myeloid leukemia

AbstractSomatic mutations in TP53 and NRAS are associated with transformation of human chronic myeloid diseases to acute myeloid leukemia (AML). Here, we report that concurrent RAS pathway and TP53 mutations are identified in a subset of AML patients and confer an inferior overall survival. To further investigate the genetic interaction between p53 loss and endogenous NrasG12D/+ in AML, we generated conditional NrasG12D/+p53−/− mice. Consistent with the clinical data, recipient mice transplanted with NrasG12D/+p53−/− bone marrow cells rapidly develop a highly penetrant AML. We find that p53−/− cooperates with NrasG12D/+ to promote increased quiescence in megakaryocyte-erythroid progenitors (MEPs). NrasG12D/+p53−/− MEPs are transformed to self-renewing AML-initiating cells and are capable of inducing AML in serially transplanted recipients. RNA sequencing analysis revealed that transformed MEPs gain a partial hematopoietic stem cell signature and largely retain an MEP signature. Their distinct transcriptomes suggests a potential regulation by p53 loss. In addition, we show that during AML development, transformed MEPs acquire overexpression of oncogenic Nras, leading to hyperactivation of ERK1/2 signaling. Our results demonstrate that p53−/− synergizes with enhanced oncogenic Nras signaling to transform MEPs and drive AML development. This model may serve as a platform to test candidate therapeutics in this aggressive subset of AML.

Clonality and Mixed Mutational Signature in Aged Hematopoietic Stem Cells Via Single Cell Variant Analysis

Genomic stability and integrity in Hematopoietic Stem Cells (HSCs) is maintained via DNA damage checkpoints, DNA proofreading and DNA repair (Moehrle et al., 2015; Cell Rep). Despite these mechanisms, recurring and non-recurring mutations accumulate in HSCs upon aging, which correlate with an elevated incidence of myeloproliferative diseases (Rossi, Bryder, and Weissman, Exp. Gerontol. 2007) as well as changes in clonality (Akunuru and Geiger, 2016; Trends in Mol. Med.). The rate at which such mutations accumulate in individual HSCs and the selection advantage/disadvantage that they provide is unclear and is an active area of investigation. Evolutionary theory supports a strong influence of the aged niche on the selection of HSCs clones upon aging (Rozhok, Salstrom, and DeGregori, 2014; Aging). We hypothesized that variant profiling of single HSCs based on RNA transcripts will reveal mutational signatures adapted to the selection pressure of the aging microenvironment.We performed Single cell RNA-seq of daughter cell pairs from young and aged murine HSCs (LSK, CD34-, flk2-). The Genome Analysis Toolkit (GATK; Broad Institute) RNA-seq variant/mutation calling algorithm pipeline was applied with some modifications. Only variants that were observed in both daughter cells of a given pair were selected, which significantly decreased our false discovery rate (tested by a Monte Carlo simulation). First and most interestingly, we observed no significant difference in the overall number of variants/mutations between young and aged HSCs, further supporting our recently published observations on the frequency of DNA mutations in HSCs upon aging (Moehrle et al., 2015; Cell Rep). We then used an approach that takes into account the 3’ and 5’ bases flanking a variant to generate motifs whose frequencies can be mathematically analyzed to deduce characteristic mutational patterns, termed as mutational signatures (Nik-Zainal et al., 2012; Cell). We employed a non-negative matrix factorization (nmf) and principal component analysis (pca) algorithms to generate 10 mutational signatures that explained > 95% of the variance in the dataset. We then analyzed the signatures in a pairwise fashion and selected two signatures with the highest discrimination score between young and aged HSC. Based on this, cells fell into two major groups: group 1 predominantly contained aged single cells (~90% of the cells in this group) whereas, interestingly, group 2 contained a mix of young and aged HSCs. The segregation of young and aged single HSCs counts between groups 1 and 2 was tested using Fisher’s exact test and was statistically significant (p-value 0.0029). These data indicate that while the overall mutational load is not elevated, majority of aged HSCs acquire a mutational signature distinct from young HSCs, while a proportion of aged HSCs present with a young-like HSC signature. Furthermore, our results show that even those cells that have acquired an aging signature aren’t homogeneous and show sub-clustering tendencies, providing the first hint that they may potentially evolve further into more distinct clones. In conclusion, our results show that individual HSCs reflect a mixed mutational profile reminiscent of a non-uniform accumulation of variants. As such signatures are a reflection of underlying mechanisms by which the mutations accumulate (Nik-Zainal et al., 2012; Cell), the proportion of aged HSCs sharing similar mutational signatures but distinct from the young HSCs reveal an aging signature that indicates specific mutational factors and selection pressure of the aging microenvironment. DisclosuresMulaw:NuGEN: Honoraria.

Deconstructing the Complexity of TGFβ Signaling in Hematopoietic Stem Cells: Quiescence and Beyond.

The hematopoietic system is highly dynamic and must constantly produce new blood cells every day. Mature blood cells all derive from a pool of rare long-lived hematopoietic stem cells (HSCs) that are mostly quiescent but occasionally divide and self-renew in order to maintain the stem cell pool and continuous replenishment of mature blood cells throughout life. A tight control of HSC self-renewal, commitment to differentiation and maintenance of quiescence states is necessary for lifelong blood supply. Transforming growth factor-β (TGF-β) is a critical regulator hematopoietic cell functions. It is a potent inhibitor of hematopoietic cell growth. However, TGFβ functions are more complex and largely context-dependent. Emerging evidence suggests a role in aging, cell identity and cell fate decisions. Here, we will review the role of TGF-β and downstream signaling in normal HSC functions, in HSC quiescence and beyond.

Growth factor independence 1B (GFI1B) regulates WNT signaling in hematopoietic stem cells and megakaryocytes by recruiting LSD1 to B-CATENIN target genes

Growth factor independence 1B (GFI1B) regulates WNT signaling in hematopoietic stem cells and megakaryocytes by recruiting LSD1 to B-CATENIN target genes

Kras is Required for Adult Hematopoiesis.

Previous studies indicate that Kras is dispensable for fetal liver hematopoiesis, but its role in adult hematopoiesis remains unclear. Here, we generated a Kras conditional knockout allele to address this question. Deletion of Kras in adult bone marrow (BM) is mediated by Vav-Cre or inducible Mx1-Cre. We find that loss of Kras leads to greatly reduced thrombopoietin (TPO) signaling in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), while stem cell factor-evoked ERK1/2 activation is not affected. The compromised TPO signaling is associated with reduced long term- and intermediate-term HSC compartments and a bias toward myeloid differentiation in MPPs. Although granulocyte macrophage colony-stimulating factor (GM-CSF)-evoked ERK1/2 activation is only moderately decreased in Kras(-/-) myeloid progenitors, it is blunted in neutrophils and neutrophil survival is significantly reduced in vitro. At 9-12 months old, Kras conditional knockout mice develop profound hematopoietic defects, including splenomegaly, an expanded neutrophil compartment, and reduced B cell number. In a serial transplantation assay, the reconstitution potential of Kras(-/-) BM cells is greatly compromised, which is attributable to defects in the self-renewal of Kras(-/-) HSCs and defects in differentiated hematopoietic cells. Our results demonstrate that Kras is a major regulator of TPO and GM-CSF signaling in specific populations of hematopoietic cells and its function is required for adult hematopoiesis. Stem Cells 2016;34:1859-1871.

A Pilot Study of Quantitative MRI Parametric Response Mapping of Bone Marrow Fat for Treatment Assessment in Myelofibrosis.

Myelofibrosis (MF) is a hematologic neoplasm arising as a primary disease or secondary to other blood malignancies. Both primary and secondary MF develop progressive fibrosis of bone marrow, displacing normal hematopoietic cells to other organs and disrupting normal production of mature blood cells. Activation of JAK2 signaling in hematopoietic stem cells commonly causes MF, and ruxolitinib, a drug targeting this pathway, is the preferred treatment for many patients. However, current measures of disease status in MF do not necessarily predict response to treatment with ruxolitinib or other drugs. Bone marrow biopsies are invasive and prone to sampling error, while measurements of spleen volume only indirectly reflect status of bone marrow. Toward the goal of developing an imaging biomarker for treatment response in MF, we present preliminary results from a prospective clinical study evaluating parametric response mapping (PRM) of quantitative Dixon MRI bone marrow fat fraction maps in four MF patients treated with ruxolitinib. PRM allows voxel-wise identification of temporal changes in quantitative imaging readouts, in this case bone marrow fat. We identified heterogeneous responses of bone marrow fat among patients and within different bone marrow sites in the same patient. Changes in bone marrow fat fraction also were discordant with reductions in spleen volume, the standard imaging metric for treatment response. This study provides initial support for PRM analysis of quantitative MRI of bone marrow fat to monitor therapy in MF, setting the stage for larger studies to further develop and validate this method as a complementary imaging biomarker.

GSK3 Deficiencies in Hematopoietic Stem Cells Initiate Pre-neoplastic State that Is Predictive of Clinical Outcomes of Human Acute Leukemia

Initial pathway alternations required for pathogenesis of human acute myeloid leukemia (AML) are poorly understood. Here we reveal that removal of glycogen synthase kinase-3α (GSK-3α) and GSK-3β dependency leads to aggressive AML. Although GSK-3α deletion alone has no effect, GSK-3β deletion in hematopoietic stem cells (HSCs) resulted in a pre-neoplastic state consistent with human myelodysplastic syndromes (MDSs). Transcriptome and functional studies reveal that each GSK-3β and GSK-3α uniquely contributes to AML by affecting Wnt/Akt/mTOR signaling and metabolism, respectively. The molecular signature of HSCs deleted for GSK-3β provided a prognostic tool for disease progression and survival of MDS patients. Our study reveals that GSK-3α- and GSK-3β-regulated pathways can be responsible for stepwise transition to MDS and subsequent AML, thereby providing potential therapeutic targets of disease evolution.

Identification of Existing Bioactive Compounds That Target Acute Myeloid Leukemia Stem Cells

Current therapies for acute myeloid leukemia (AML) are able to achieve complete remission in 80-85% of patients; however large portions of these cases relapse. The 5-year survival rate is estimated at 30%. Thus, the development of more efficient approaches to treat AML is absolutely needed. Similarly to normal hematopoiesis, leukemias are organized as cellular hierarchies supported by leukemic stem cells (LSCs). LSCs are relatively resistant to chemotherapy and may act as a cancer cell reservoir that leads to relapse. The discovery of therapeutics that can eradicate these cells is essential to achieve complete patient recovery.We hypothesize that by using an in silico strategy and stem cell gene expression data we can identify bioactive compounds that target LSCs with minimal impact on normal hematopoietic stem cell (HSC) function. We had previously generated human LSC and HSC expression signatures from 16 primary AML and 4 independent pooled cord blood samples that were prognostic for patient survival (Eppert et al Nat Med 2011). By interrogating existing gene expression profiles of drug response with our LSC and HSC signatures, as well as additional novel signatures, we identified 92 molecules predicted to inhibit human LSC properties without harming HSCs. Nearly 60% of the positive hits were anti-infectives, psychotropics, hormones and steroids, anticancer or cardiovascular therapies. In support of the efficacy of this bioinformatic approach, we identified known chemotherapeutic agents such as azacitidine, daunorubicin and doxorubicin, although these were predicted to harm HSCs. We assessed the anti-LSC activity of one sub-group known to target a class of G-protein-coupled receptors (GPCR) to further validate the list of candidate compounds. Using a primary human AML sample (8227) with known LSC phenotype (CD34+), we tested 2 candidate anti-GPCR compounds (hits A and B) and observed that they were highly effective in targeting both bulk cells and LSCs. The IC50 for bulk cells was 3.4 µM and 4.2 µM for compounds A and B, respectively (A - 6 day treatment, B - 2 day treatment). The IC50 of the CD34+ cell fraction, which included stem and progenitor cells, was 5.9 µM and 4.1 µM, respectively. Thus, these compounds are effective at targeting both bulk and LSC-enriched AML cell populations. The efficacy of these predicted compounds against the primitive cells within the CD34+ population was assessed using colony formation assays on human AML. 8227 cells treated with compound B resulted in significantly fewer colonies compared to the DMSO control (3.8 ± 3.0 vs 13.3 ± 2.8; at 4.5 µM). To test whether our candidate compounds target leukemic cells and not normal blood cells, we examined 3 additional primary human AML samples and a cord blood sample. There was only a minor effect on normal cells, including stem and progenitor cells, but significant cell death in all leukemia samples (70% and more at ≥ 3 µM). Next, we will determine the effectiveness of our compounds in vivo using established xenografts of primary human AML samples and cord blood samples.In conclusion, using our in silico approach we successfully identified novel anti-LSC compounds that have efficacy in vitro with minimal toxicity on primary HSCs. Furthermore, as each predicted compound is associated with specific molecular pathways, our approach has improved our understanding of LSC biology by acting as a screen for LSC-related pathways. In the near future, we will continue investigating the 92 candidate compounds to identify, develop, and transition a novel anti-LSC compound into clinical use. DisclosuresNo relevant conflicts of interest to declare.

Single Cell Whole Transcriptome Analysis Reveals Distinct Molecular Signatures of Therapy-Resistant Chronic Myeloid Leukemia Stem Cells

Molecularly targeted therapy frequently induces remissions in cancer, but rarely achieves complete disease eradication, with resulting risk of disease relapse/progression. Chronic myeloid leukemia (CML) is a good example, with rare, propagating stem cells (SCs) that are incompletely eradicated by BCR-ABL directed tyrosine kinase inhibitor (TKI) therapy in most cases. Consequently, CML relapse usually occurs following treatment discontinuation. Further, some patients fail to achieve a satisfactory response to TKI therapy and are at risk of disease progression. Ultimately, the depth of response to TKIs is dictated by CML-SCs, however, it has proven challenging to characterise this crucial population of cells as they reside in the same compartment as their normal hematopoietic stem cell (HSC) counterparts from which they cannot be reliably distinguished throughout the disease course.Advances in single cell transcriptomics are opening up unprecedented opportunities to unravel heterogeneity in cell populations. However, to date, this technology has primarily been used to analyse normal tissues, partly reflecting the lack of sensitivity for detection of somatic mutations using current techniques. Herein, we developed a novel protocol allowing targeted amplification of BCR-ABL during single cell cDNA library generation using a modification of a published protocol (PMID:24385147). We then applied this method to analyse 1082 bone marrow SCs (defined as Lin-CD34+CD38-) from a cohort of 10 patients with CML and 3 normal controls.We first validated our method using the BCR-ABL +ve K562 cell line. Surprisingly, using standard methods, BCR-ABL was only detected in a small minority of cells. With our modified technique, we demonstrate robust detection of BCR-ABL in 100% of single K562 cells with parallel whole transcriptome amplification from the same cell. Further, using a plasmid Òspike-inÓ, we demonstrate sensitivity to detect a single molecule of BCR-ABL per cell, without false +ve BCR-ABL amplification in 142 cells tested. We then analysed 40 SCs from a patient with CML who was in a cytogenetic remission (CyR) following 3 months of TKI therapy. BCR-ABL was detected in 16 cells and was absent in 24 cells. With an average of 3.3 million mapped reads per single cell, we detected over 4600 genes per cell with good correlation of single cell ensemble data with bulk analysis. Using this approach we were uniquely able to compare BCR-ABL +ve and -ve cells and identify a large number of differentially expressed genes, which would not have been detected through bulk analysis. We selected a number of genes of biologic interest for validation by Q-PCR and 80% of them were confirmed as differentially expressed, a number of these are highlighted in the heatmap (see Figure).We then analysed 717 single SCs from 8 CML diagnostic samples. This analysis identified marked heterogeneity of BCR-ABL expression with frequencies comparable to the published literature (9% to 92%). Unsupervised clustering analysis revealed many genes that were differentially expressed between BCR-ABL +ve and -ve SCs e.g. CML-SCs showed upregulation of cell cycle signatures and decreased expression of HSC-affiliated genes. We next analysed 223 single SCs from a series of 5 patients in CyR following TKI initiation. BCR-ABL positive SCs could still be detected, albeit at a reduced frequency (0.5% to 45%). In contrast to the diagnostic samples, CML-SCs showed quiescence and HSC signatures that were comparable to normal HSCs. Furthermore, through analysis of serial samples from diagnosis to remission from the same patient, this single-cell approach uniquely allowed analysis of gene expression specifically of the CML-SCs which evade TKI therapy, facilitating the identification of a number of candidate gene-sets including cell surface markers and potentially ÒdruggableÓ targets. Finally, through analysis of a patient with early blast crisis transformation of CML during TKI therapy, we illustrate how single cell RNA sequencing might be applied to predict such early disease progression events.We herein describe a new approach for single cell RNA sequencing of CML-SCs that might be applied to fate-map persistent CML-SCs during and following treatment for discovery research and also to refine precision medicine in CML. This approach could be applied across a range of clonal disorders with potential broader relevance for cancer research. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.