Elucidating the Mechanisms of Leukemogenesis Driven By FBXO11 Depletion

Abstract Mutations in SKP1 and CUL1 (Zhang et. al. Oncol Lett 2018), which encode components of the SKP1-CUL1-F-BOX (SCF) ubiquitin E3-ligase complex, have previously been reported or characterized in AML. FBXO11, which encodes the substrate recognizing component, however, has not been studied in AML. We performed whole exome sequencing and RNA-seq on140 clinical AML samples and identified recurrent inactivating mutations in FBXO11. Of the components of the SCF FBXO11 complex, FBXO11 transcript expression is most significantly reduced in AML samples compared to normal. We show that loss of FBXO11 drives leukemogenesis through dysregulation of the novel target, LONP1, by reducing mitochondrial potential and promoting self-renewal. We found that UPS mutations co-occur with AML1-ETO (RUNX1-RUNX1T1) fusions and RAS mutations. Fbxo11 knockdown in mouse hematopoietic stem/progenitor cells (HSPC) cooperated with AML1-ETO to generate serially transplantable AML in mice. FBXO11 depletion in human cord-blood derived CD34+ cells (CD34+ CB), combined with AML1-ETO and a KRAS mutant, promoted stem cell maintenance and myeloid malignancy in a human xenotransplant model. Mass spectrometry analysis of FLAG-FBXO11 co-immunoprecipitating proteins in K562 cells identified mitochondrial protease, LONP1, as a top target. LONP1 protein expression did not vary with FBXO11 loss or overexpression, suggesting that LONP1 is not a degradation target of the SCF FBXO11complex. Knockdown of either FBXO11 or LONP1 resulted in myeloid bias in CD34+ CB in vitro, pointing to an activating role of FBXO11 on LONP1. Both FBXO11 and LONP1 depletion reduced mitochondrial membrane potential (MMP) in CD34+ CB and myeloid cell lines, aligning with the stemness phenotypes observed with FBXO11 depletion, as long-term hematopoietic stem cells (LT-HSCs) are characterized by low MMP (Mansell et. al. Cell Stem Cell 2021), and disruption of MMP promotes self-renewal in HSCs (Vannini et. al. Nat Commun 2016). As FBXO11 neddylates p53 to regulate transcription (Abida et. al. J. Biol. Chem 2007), we examined protein neddylation, and detected increased neddylation in immunoprecipitated LONP1 from FLAG-FBXO11-expressing K562 cells. As, neddylation regulates protein activation (Wu et. al. Nature 2005), our findings suggest that FBXO11 neddylation of LONP1 activates LONP1 to maintain mitochondrial function. Consequently, loss of FBXO11 function primes HSPC for self-renewal by reduction of MMP. To clarify the regulatory relationship between FBXO11 and LONP1, we performed RNA-seq on CD34+ CB cells expressing combinations of shRNAs targeting FBXO11 or LONP1, with overexpression of FLAG -FBXO11 or LONP1. Unsupervised clustering revealed that LONP1-overexpressing samples clustered with controls, suggesting that LONP1 requires modification by FBXO11 for functional effects. Using gene set enrichment analysis, we found that both FBXO11 and LONP1 depletion enriched for HSC and LSC (leukemic stem cell) gene sets. Knockdown of LONP1 reversed the effect of FLAG-FBXO11 overexpression, supporting a model of LONP1 being a downstream mediator of FBXO11 function. Both FBXO11 and LONP1 depletion enriched for a gene set composed of mitochondrial electron transport chain complex (ETC) genes, potentially reflecting a transcriptional response to loss of functional ETC activity, as suggested by accumulation of misfolded ETC proteins with knockdown of LONP1 (Ghosh et. al. Oncogene 2019). In this work, we demonstrate the leukemogenic effects of FBXO11 loss. We draw a novel connection between the UPS and the mitochondrial protease system with the identification of LONP1 as an FBXO11 target that regulates hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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Akt-Mediated Phosphorylation of Bmi1 Regulates Its Chromatin Association and Growth Promoting Properties.

Blood ◽

10.1182/blood.v114.22.3605.3605 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3605-3605

Author(s):

Yan Liu ◽

Fan Liu ◽

Xinyang Zhao ◽

Goro Sashida ◽

Anthony Deblasio ◽

...

Keyword(s):

Stem Cell ◽

Signaling Pathway ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Akt Signaling ◽

Polycomb Group ◽

Akt Pathway ◽

Akt Signaling Pathway ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 3605 Poster Board III-541 The Polycomb group (PcG) protein Bmi1 maintains silencing of the Ink4a-Arf locus and plays a key role in stem cell self-renewal and oncogenesis. The phosphoinositide 3-kinase-Akt (PI3K-Akt) signaling pathway regulates cell survival, growth, metabolism, migration and angiogenesis. In response to acute Pten loss (which results in Akt activation), mouse embryonic fibroblasts (mefs) accumulate p16Ink4a and p19Arf and undergo senescence. Similarly, Bmi1 −/− mefs undergo premature senescence and accumulate p16Ink4a and p19Arf. PTEN and Bmi1 have similar effects on hematopoiesis; Pten deletion promotes hematopoietic stem cell (HSC) proliferation, resulting in HSC depletion, whereas loss of Bmi1 impairs HSC self-renewal capability, also leading to bone marrow failure. These similarities led us to examine whether the PI3K/Akt pathway functions upstream of Bmi1 to negatively regulate its function and indeed we found that PKB/Akt phosphorylates Bmi1 in vivo, which results in its dissociation from chromatin and in de-repression of the Ink4a-Arf locus. Furthermore, activation of the PI3K/Akt pathway suppresses the ability of Bmi1 to promote cell growth and tumourigenesis and decreases the global level of histone H2A ubiquitination. PI3K/Akt signaling is not active in hematopoietic stem cells, but it is active in more committed progenitor cells. Thus, phosphorylation and inactivation of Bmi1 by Akt may limit HSC self-renewal. Our study also provides a mechanism for the upregulation of p16Ink4a and p19Arf seen in cancer cells that have activation of the PI3K/Akt signaling pathway, and identifies important crosstalk between phosphorylation and chromatin structure. Disclosures: No relevant conflicts of interest to declare.

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MiR-29a Maintains Hematopoietic Stem Cell Self-Renewal and Is Required For Myeloid Leukemogenesis

Blood ◽

10.1182/blood.v122.21.1190.1190 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1190-1190

Author(s):

Wenhuo Hu ◽

James Dooley ◽

Stephen S. Chung ◽

Safak Yalcin ◽

Yu Sup Shin ◽

...

Keyword(s):

Stem Cell ◽

Colony Formation ◽

Target Genes ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Null Mouse ◽

Cell Cycle Regulators ◽

Self Renewal ◽

Hematopoietic Stem

Abstract microRNAs (miRNAs) are important regulators of both embryonic and adult tissue stem cell self-renewal. We previously showed that ectopic expression of miR-29a, a miRNA highly expressed in HSCs as well as in human acute myeloid leukemia (AML) stem cells, in immature mouse hematopoietic cells is sufficient to induce a myeloproliferative disorder that progresses to AML. During the early phase of this disease, miR-29a induces aberrant self-renewal of committed myeloid progenitors, strongly suggesting a role for miR-29a in regulating HSC self-renewal. In order to determine the role of miR-29a in HSC function, we have evaluated our recently described miR-29a/b1 null mouse. Homozygous deletion of miR-29a/b1 resulted in reduced bone marrow cellularity and reduced colony forming capacity of hematopoietic stem and progenitor cells (HSPCs). The phenotype was mediated specifically by miR-29a since miR-29b expression was not significantly altered in HSCs and reconstitution of miR-29a/b1 null HSPCs with miR-29a, but not miR-29b, rescued in vitro colony formation defects. Self-renewal defects were observed in miR-29a deficient HSCs in both competitive and non-competitive transplantation assays, and these deficits were associated with increased HSC cell cycling and apoptosis. Gene expression studies of miR-29a deficient HSCs demonstrated widespread gene dysregulation including a number of up-regulated miR-29a target genes including DNA methylation enzymes (Dnmt3a, -3b) and cell cycle regulators (e.g. Cdk6, Tcl1, Hbp1, Pten). Knockdown of one of these targets, Dnmt3a, in miR-29a deficient HSCs resulted in partial restoration of colony formation, providing functional validation that Dnmt3a mediates part of miR-29a null HSPCs functional defects. miR-29a loss also abrogated leukemogenesis in the MLL-AF9 retroviral AML model. Together, our results demonstrate that miR-29a positively regulates HSC self-renewal and is required for myeloid leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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Chemogenomic Profiling of Complex Karyotype AML Reveals a Novel Susceptibility to G2/M Checkpoint Inhibition Mediated By HMGA2 Overexpression

Blood ◽

10.1182/blood-2018-99-111304 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3925-3925

Author(s):

Céline Moison ◽

Vincent-Philippe Lavallee ◽

Clarisse Thiollier ◽

Jean-Francois Spinella ◽

Isabel Boivin ◽

...

Keyword(s):

Stem Cell ◽

Enrichment Analysis ◽

Transcriptomic Analysis ◽

Gene Set Enrichment Analysis ◽

Complex Karyotype ◽

Checkpoint Inhibition ◽

Gene Set Enrichment ◽

Hematopoietic Stem ◽

Gene Set ◽

Tp53 Status

Abstract Background: Acute myeloid leukemia (AML) with complex karyotype (CK) is associated with adverse prognosis with current therapies, in particular in the presence of TP53 mutations. Identification of novel therapeutic strategies is therefore urgently needed for this subgroup of patients. Methods and aims: As part of the Leucegene project, we RNA sequenced a cohort of 415 clinically annotated primary human AML specimens. Comparative transcriptomic analysis of the 68 CK AML specimens with the non-CK samples of the cohort was performed to identify features associated with the CK AML subgroup. Based on these results, we performed a chemical screen aimed at identifying active agents toward CK AML. Gene expression analysis, primary AML specimen culture and chemical screening were performed as previously described by our group (Lavallée et al., Nature Genetics, 2015; Pabst et al., Nature Methods, 2014; Simon et al., Clinical cancer research, 2017). Results: CK AML is a heterogeneous disease by definition and accordingly, Multidimensional Scaling (MDS) analysis fails to identify a distinct transcriptional signature. Nonetheless, comparative transcriptome analysis of CK and non-CK AML specimens revealed highly deregulated genes between these subgroups (Figure 1A). Among them, EDA2R, a known target to p53, is specifically downregulated in CK AML. This reflects the frequent TP53 alterations observed in CK AML. Most importantly, we found that the recently identified AML prognostic gene HMGA2 is overexpressed in the majority of these patients, independently of the TP53 status. HMGA2 is an oncofetal gene expressed in hematopoietic stem cell (HSC) -enriched populations while its expression decreases in progenitors and is nearly absent in mature cells. Interestingly, Gene Set Enrichment Analysis (GSEA) revealed an enrichment for HSC genes in AML specimens with high HMGA2 expression levels, which could reflect a stem-cell origin or a more immature state for these leukemias. Global transcriptomic analysis of CK AML also identified a G2/M checkpoint signature for HMGA2-high specimens (Figure 1B). Accordingly, we observed that chemical inhibitors of the G2/M regulators ATR, CHK1 and WEE1 are preferentially active on HMGA2-high CK specimens and that engineered overexpression of HMGA2 in different leukemia cell lines enhanced sensitivity to G2/M inhibition (Figure 1C). This specific inhibition was independent of the TP53 status. Conclusions: Here we report a comprehensive transcriptomic analysis of the CK AML specimens of the Leucegene cohort and identified aberrant expression of the HMGA2 oncogene in close to 80% of these samples. Chemical interrogation of primary AML specimens revealed a TP53-independent but HMGA2-mediated sensitization of CK AML to G2/M checkpoint inhibition. Thus, our results reveal a novel vulnerability for AML expressing high levels of HMGA2 and identify potential biological targets for these high-risk patients. Since several CHK1 and PLK1 inhibitors have been or are currently under evaluation in clinical trials, our findings suggest that HMGA2-high AML patients could benefit from this therapeutic approach. Figure 1: A/ Volcano plot representing the genes differentially expressed in CK versus non-CK AML. Samples with low group median expression (<0.1 RPKM) were removed from this representation. B/ Highest enrichment found by Gene Set Enrichment Analysis comparing HMGA2 high (RPKM>2; n=39) and HMGA2 null (RPKM=0; n=83) AML specimens. C/ Dose-response curves and IC50 values for representative compounds in K562 cells infected with control YFP vector (blue) or HMGA2-YFP expressing vector (red). Disclosures Sauvageau: ExCellThera: Employment, Equity Ownership.

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Enforcing Post-Transcriptional Circuitries to Achieve Human Hematopoietic Stem Cell Expansion

Blood ◽

10.1182/blood.v130.suppl_1.sci-46.sci-46 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. SCI-46-SCI-46

Author(s):

Kristin Hope

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Transcriptional Control ◽

Molecular Mechanisms ◽

Rna Binding ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Self Renewal ◽

Control Of Gene Expression ◽

Hematopoietic Stem

Abstract The balance between hematopoietic stem cell (HSC) differentiation and self-renewal is central to clinical regenerative paradigms. Unravelling the precise molecular mechanisms that govern HSC fate choices will thus have far reaching consequences for the development of effective therapies for hematopoietic and immunological disorders. There is an emerging recognition that beyond transcription, HSC homeostasis is subject to post-transcriptional control by RNA-binding proteins (RBPs) that ensure precise control of gene expression by modulating mRNA splicing, polyadenylation, localization, degradation or translation. RBPs can synchronously regulate the fates of operationally similar RNAs, in what have been termed RNA regulons. We have used a variety of functional approaches, in combination with unbiased genome- and proteome-scale, methods to define the tenets that govern this regulation and to determine key downstream circuitries of stem cell-regulating RBPs whose targeting could provide the basis for novel regenerative treatments. Through loss-of-function efforts, we have identified the RBP, MSI2, as a required factor for human HSC maintenance. By contrast, at supraphysiological levels, MSI2 has a profound positive effect on human HSC self-renewal decisions. Using a combination of global profiling, including mapping MSI2's targets through cross-linking immunoprecipitation (CLIP)-seq, we show that MSI2 achieves its ex vivo self-renewal-promoting effects by directing a co-ordinated post-transcriptional repression of key targets within the aryl hydrocarbon receptor (AHR) pathway. We are currently exploring the "rules" by which MSI2 influences its downstream effects on target RNAs and how it functions, in combination with other protein interactors, to instill a putative RBP regulatory code in HSCs. HSCs exhibit highly unique epigenomes, transcriptomes and proteomes and it is this distinctive molecular landscape that provides the canvas upon which MSI2, and indeed any other HSC-specific RBP exert their post-transcriptional influence over stem cell function. As such, to decipher the bona fide RNA networks that RBPs function upon in HSCs and to understand how they influence this network to enforce self-renewal, we are working towards performing systematic studies of RBP regulons in these cells specifically. In turn these approaches are elucidating a host of RBPs and post-transcriptional control mechanisms previously unappreciated for their role in HSC control. When modulated appropriately, we can successfully tailor these post-transcriptional regulons to enforce desired HSC outputs ex vivo. In summary, approaches to elucidate key HSC-regulatory RBPs and their protein and RNA interactomes provide valuable insights into a layer of HSC control previously not well understood, and one that can be capitalized on to achieve successful HSC expansion. Disclosures No relevant conflicts of interest to declare.

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Activation of Non-Canonical Immune Signalling Pathways Drives Marrow Failure in a Murine Model of MDS

Blood ◽

10.1182/blood.v124.21.3246.3246 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3246-3246

Author(s):

Rawa Ibrahim ◽

Joanna Wegrzyn ◽

Linda Ya-Ting Chang ◽

Patricia Umlandt ◽

Jeff Lam ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Line ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Signalling Pathway ◽

Gene Set Enrichment Analysis ◽

Mouse Bone Marrow ◽

Bone Marrow Niche ◽

Hematopoietic Stem

Abstract The Myelodysplastic Syndromes (MDS) are the most common hematological malignancies arising from stem/progenitor cells. MDS is characterized by ineffective hematopoiesis in one or more lineages of the bone marrow, resulting in peripheral cytopenias and the propensity to progress to either acute myeloid leukemia (AML) or bone marrow failure (BMF). The most common cytogenetic aberration associated with MDS is deletion of the long arm of chromosome 5. Many of the molecular events involved in the development of del(5q) MDS have been elucidated including haploinsufficiency of the gene encoding the ribosomal protein RPS14, responsible for the anemia observed, and haploinsufficency of the miRNAs miR-145 and miR-146a, which together target the innate immune signaling pathway, specifically, the Toll-like receptor-4 (TLR-4)signalling pathway. It has been demonstrated that overexpression of a target of miR-146a,TRAF6, in mouse bone marrow can recapitulate the phenotype of del(5q) MDS including the cytopenias and progression to BMF or AML. However, enforced expression of TIRAP, a miR-145 target gene, results in rapid BMF independent of TRAF6. The molecular and cellular mechanisms responsible for the differential outcome of overexpression of two genes that act within the same signalling pathway remain to be fully understood. We have identified several differentially expressed cytokines, including interferon gamma (IFNγ) and interleukin-10 (IL-10), following TIRAP overexpression compared with TRAF6 overexpression. Promoter methylation analysis has shown hypermethylation of key adaptors and signal transducers that lie between TIRAP and TRAF6 in the TLR-4 signalling pathway, suggesting activation of different pathways by TIRAP and TRAF6 overexpression. Indeed, blockade of TRAF6 and MyD88 did not inhibit TIRAP induced expression of these cytokines, suggesting that IFNγ and IL-10 production occurs in a TRAF6 and MyD88 independent manner. We identified IFNγ as the critical effector cytokine responsible for TIRAP mediated marrow failure. Gene set enrichment analysis has shown an enrichment of an IFNγ signature in MDS patients with a low risk of transformation to AML compared to healthy controls. Furthermore, interferon signatures were highly enriched in MDS patients compared to patients with AML, suggesting an important role for IFNγ signaling in driving MDS progression toward marrow failure as opposed to leukemic progression. IFNγ has been shown to inhibit components of the bone marrow niche by blocking RANK signalling in stromal cells such as osteoclast progenitors. Using coculture of TIRAP expressing bone marrow cells with the RAW264.7 monocyte cell line, a cell line that is capable of differentiation into osteoclasts, we found an inhibition in the ability of these cells to form osteoclasts compared to control. This provides the first line of evidence suggesting that immune signalling defects arising from genetic perturbations in the hematopoietic stem cell compartment can result in stem cell niche dysfunction leading to marrow failure. Disclosures No relevant conflicts of interest to declare.

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EVI1 Is Activated During the Generation of Induced Pluripotent Stem (iPS) Cells.

Blood ◽

10.1182/blood.v114.22.5048.5048 ◽

2009 ◽

Vol 114 (22) ◽

pp. 5048-5048

Author(s):

Leopoldo Laricchia-Robbio ◽

Nuria Montserrat ◽

Alessandra Giorgetti ◽

Juan Carlos Izpisúa Belmonte

Keyword(s):

Stem Cell ◽

Conflicts Of Interest ◽

Integration Site ◽

Ips Cells ◽

The Self ◽

Genomic Locus ◽

Self Renewal ◽

Hematopoietic Stem ◽

Retroviral Integration ◽

Induced Pluripotent

Abstract Abstract 5048 EVI1 gene was first identified as a common site of retroviral integration in murine leukemia models. This gene is part of a complex genomic locus, MDS1-EVI1, that has been described as a target for retroviral integration that may lead to the emergence of a non-malignant dominant hematopoietic stem cell (HSC) clone in mice, in primates, and in humans. These studies suggested that one of the genes encoded by this locus could affect the self-renewal potential of HSC. Recent studies in mice revealed that indeed EVI1 plays an essential role in cell proliferation and it also enhances the self-renewal ability of HSC. The intense attention focused on the MDS1-EVI1 locus as retrovirus integration site prompted us to investigate whether EVI1 might have a role in somatic cell re-programming generated with retroviruses. Recent developments in stem cell research have enabled the re-programming of somatic cells to a pluripotent state using exogenous factors. Induced pluripotent stem (iPS) cells have the potential to differentiate into any cells types and that might be used in the future for clinical therapy. In order to elucidate the molecular events allowing the conversion of adult somatic into pluripotent stem cell, we evaluated EVI1 expression during this process. We found that EVI1 is activated in the early stages of re-programming and then it is silenced once the cells has been fully re-programmed. EVI1 seems to facilitate the initiation of cell re-programming by up-regulating a subset of genes previously described as potent stimulators of stem cells expansion. Disclosures No relevant conflicts of interest to declare.

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Roles of Ring1A/B on Stem Cell Potential of MOZ and Other Acute Myeloid Leukemias,

Blood ◽

10.1182/blood.v118.21.3998.3998 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3998-3998

Author(s):

Haruko Shima ◽

Mika Shino ◽

Kazutsune Yamagata ◽

Yukiko Aikawa ◽

Haruhiko Koseki ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Differentiation ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Leukemia Stem Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Functions ◽

Acute Myeloid

Abstract Abstract 3998 Leukemia and other cancers possess self-renewing stem cells that help maintain cancer. Chromosomal translocations are often involved in the development of human acute myeloid leukemia (AML). The monocytic leukemia zinc finger (MOZ) gene is one of the targets of such translocations. While MOZ is essential for the self-renewal of hematopoietic stem cells, the leukemia associated MOZ-fusion proteins enable the transformation of non–self-renewing myeloid progenitors into leukemia stem cells. Ring1A and Ring1B are catalytic subunits of the polycomb-group repressive complex 1 (PRC1) complex containing Bmi1, and PRC1 complex plays an important role in the regulation of stem cell self-renewal. Using Ring1A-null and Ring1B-conditional deficient mice, we showed that Ring1A/B are required for continuous colony forming ability that is enabled by MOZ-TIF2 and other AML-associated fusions such as MLL-AF10, AML1-ETO, and PML-RARα. Furthermore, MOZ-TIF2- and MLL-AF10-induced AML development in mice were prevented by Ring 1A/B deficiency. To clarify the mechanism of stemness regulation in AML stem cells by PRC1 complex, we compared gene expression profiles of Ring1A/B deleted and non-deleted MOZ-TIF2-induced AML cells. As expected, Ink4a/Arf, a known major target of PRC1 complex involved in stem cell functions, was derepressed by deletion of Ring1A/B. Although deletion of Ink4a/Arf in Ring1A/B deficient AML cells partially restored colony formation ability, it was not substantial to initiate leukemia in recipient mice. Among several target genes which were derepressed by deletion of Ring1A/B, we focused on “Stemness inhibitory factor (SIF)”, known to be required for cell differentiation and morphogenesis in some specific organs. Enforced expression of SIF in MOZ-TIF2-induced AML cells stimulated differentiation of AML progenitors into macrophages. On the other hand, knock-down of SIF blocked cell differentiation block and restored the immortalizing ability of MOZ-TIF2-induced AML progenitors, despite of the absence of Ring1A/B. Collectively, our data demonstrate that Ring 1A/B play crucial roles in the maintenance of AML stem cells through repression of SIF, which strongly promote differentiation of leukemia stem cells. Disclosures: No relevant conflicts of interest to declare.

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Mir-125a Confers Multi-Lineage Long-Term Repopulating Stem Cell Activity to Human Hematopoietic Committed Progenitors

Blood ◽

10.1182/blood.v126.23.900.900 ◽

2015 ◽

Vol 126 (23) ◽

pp. 900-900 ◽

Cited By ~ 1

Author(s):

Eric R. Lechman ◽

Karin G. Hermans ◽

Erwin M. Schoof ◽

Aaron Trotman-Grant ◽

Stephanie M Dobson ◽

...

Keyword(s):

Stem Cell ◽

B Cell ◽

Conflicts Of Interest ◽

Cell Activity ◽

Lymphoid Cells ◽

Cell Populations ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Recent studies have shown that several miRNA are differentially expressed in hematopoietic stem cells (HSC) and involved in regulating self-renewal, pointing to a new axis of epigenetic control of HSC function. Murine studies have documented a role for miR-125a in regulating HSC as miR-125a enforced expression augments self-renewal. We examined whether these attributes are evolutionarily conserved within human hematopoiesis. Lentiviral vectors over-expressing miR-125a (miR-125OE) were developed and HSC function was investigated using xenotransplantation of CD34+ CD38- human umbilical cord blood (CB) hematopoietic stem and progenitor cells (HSPCs). miR-125OE resulted in significantly increased human bone marrow (BM) chimerism at 12 and 24 weeks post-transplantation and splenomegaly. Within enlarged spleens, there were significantly increased proportions of CD34+CD19+CD10+CD20-B lymphoid cells suggesting a partial B cell differentiation block at the pro-B cell stage. In the BM, CD41+ megakaryocytes, GlyA+ erythroid and CD3+ T cell populations were significantly expanded. Within the primitive compartment, multi-lymphoid progenitors (MLP) were massively expanded by 12 weeks, followed by a combined reduction of immuno-phenotypic HSC and multi-potent progenitors (MPP) by 24 weeks. Given this loss of immuno-phenotypic HSC, we wondered whether stem cell function was compromised in vivo. Secondary transplantation with limiting dilution (LDA) revealed that stem cell frequencies were increased by 4.5 fold in miR-125OE recipients. Using lentivirus sponge-mediated inhibition of miR-125 (miR-125KD) in CD34+CD38-human CB, we were able to directly link these effects to miR-125: B cells increased at the expense of T cells; immuno-phenotypic HSC increased with a concomitant loss of MLP; and functional HSC were decreased by 2.5 fold using secondary LDA assays. Together, these data strongly suggest that miR-125a expression levels regulate human HSC self-renewal and lineage commitment. Since HSC frequency increased so substantially upon miR-125OE, we asked whether more committed cell populations might also be endowed with enhanced self-renewal. Highly purified populations of HSC, MPP and MLP and CD34+CD38+ committed progenitors were transduced and transplanted cells into xenografts. Unexpectedly, miR-125OE transduced CD34+CD38+ progenitors produced a substantial graft after 12 weeks. Control transduced CD34+CD38+ cells did not engraft and only control transduced HSC generated a disseminating graft in recipient mice. miR-125OE transduced HSC and MPP generated robust engraftment, while MLP did not. In all cases, xenografts generated by CD34+CD38+ and MPP transduced with miR-125OE showed multi-lineage repopulation. Moreover, the miR-125OE grafts from CD34+CD38+ and MPP recipients were durable as secondary transplantation generated multi-lineage grafts for at least 20 weeks in 5/7 and 6/10 recipients, respectively; no control transduced groups generated secondary grafts. Thus, the enhancement of self-renewal by enforced expression of miR-125a occurs not only in HSC, but also in MPP and to an as yet unidentified subpopulation within the CD34+38+ committed progenitor compartment. Using protein mass spectrometry, we identified and validated a miR-125a target network in CD34+ CB that normally functions to restrain self-renewal in more committed progenitors. Together, our data suggest that increased miR-125a expression can endow an HSC-like program upon a selected set of non-self-renewing hematopoietic progenitors. Our findings offer the innovative potential to use MPP with enhanced self-renewal to augment limited sources of HSC to improve clinical outcomes. Disclosures No relevant conflicts of interest to declare.

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Single Cell RNASeq Demonstrates That Mouse Erythroid Cells Are the Last Lineage to Emerge

Blood ◽

10.1182/blood-2018-99-110318 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1274-1274

Author(s):

Elisabeth F Heuston ◽

Bethan Psaila ◽

Stacie M Anderson ◽

NISC Comparative Sequencing Program ◽

David M. Bodine

Keyword(s):

Gene Expression ◽

Single Cell ◽

Conflicts Of Interest ◽

Gene Set Enrichment Analysis ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Transcriptional Profiles ◽

Gene Set ◽

Rna Signature

Abstract The hierarchical model of hematopoiesis posits that hematopoietic stem cells (HSC) give rise to myeloid progenitors (CMP), that can become further restricted to bipotential granulocyte/monocyte progenitors (GMP) or megakaryocyte/erythroid progenitors (MEP). We and others have shown that this model may not accurately depict hematopoiesis. Recent studies have shown that shown that populations of mouse hematopoietic stem and progenitor cells (LSK) have a strong megakaryocyte (Mk) transcriptional profile (Heuston, 2018, Epig. & Chrom.), and single cell studies have identified lineage committed cells in progenitor populations thought to be multipotent. For example, we recently reported that human MEP contain 3 populations: erythroid (Ery) primed, Mk primed, and bipotential (Psaila, 2016; Gen. Bio.). To determine when Mk and Ery cells emerge during mouse hematopoiesis, we performed single cell RNASeq on 10000 LSK, 12000 CMP, 6000 MEP and 8000 GMP cells. Clustering analysis (Satija, 2018, Nat. Biotech.) of all 4 populations identified 33 transcriptionally distinct clusters. In 30 of 33 clusters, 85% of cells were from a single defined population (e.g. MEP). LSK and CMP clusters grouped closely together. We used gene set profiling (Gene Set Enrichment Analysis, GO and KEGG) to correlate transcriptional profiles of clusters with specific hematopoietic lineages and cellular activities. In LSK, the most common transcriptional profiles correlated with active cell cycling. Mk-associated genes (Meis1, Myct1, and Fli1), were co-expressed with lymphoid genes in 56% of all LSK. Consistent with previous studies, we conclude that cells with Mk transcriptional profiles are abundant in LSK. No cells with an Ery RNA signature were observed in LSK. 23% of all CMP cells expressed Mk genes (e.g., Pf4, Itga2b, and Fli1) and were enriched for processes involved in platelet biology (p < 3E-18). 12% of CMP had an Ery RNA signature (low expression of Gata1, Klf1, and Nfe2) and decreased Mk gene expression (e.g., Gata2 and Gfi1b, [p < 3E-18]) compared to other CMP clusters. The high ratio of Gata2/Gata1 expression (1.90) suggests that this cluster contained immature Ery cells. More than 94% of all mouse MEP had Ery RNA signatures. Clusters could be distinguished by gene expression (e.g., Gata1, Klf1, Tfrc) and biological processes (ribosome synthesis and heme-biology processes [p < 4 E-10]). Based on the transcriptional profiles, we determined the most mature erythroid cells in MEP were late BFU-E. To compare the differentiation of Mk and Ery cells, we pooled our LSK, CMP, and MEP data for analysis using the Monocle software package. GMP contained only clusters expressing granulocytic or monocytic genes and were excluded from the analysis. Monocle arranges cells into trajectories based on their transcriptional profiles, with more differentiated cells positioned further from a common node (Xiaojie, 2017, bioRxiv). We found that LSK cells near the node had overlapping lymphoid and Mk transcriptional profiles. Closest to the node, we found 38% of CMP expressed a profile similar to LSK. An additional 45% of CMP formed one trajectory with lymphoid and granulocyte RNA signatures. Another 17% of CMP formed a second trajectory, with cells expressing an Mk signature closest to the node, cells with a mixed Ery/Mk signature further along the trajectory, and MEP cells with Ery-only signatures furthest from the node. To clarify the Mk/Ery divergence, we focused our analysis on the CMP populations expressing Mk RNAs (Figure1). We observed cells in G1/S phase with an immature Mk signature to the left of the node where the trajectories diverge. On the right, cells with immature Mk signatures were nearest the node and cells with a mixed Ery/Mk signature were at the end of the trajectory (upper right; Mk/Ery). Along the second trajectory, rapidly cycling G2/M Mk cells with an early endomitosis-associated RNA signature (e.g., Pf4, Gp1bb, Gp9, and Vwf) were located at the end of the trajectory (lower right; Mk early endomitosis). Our data are consistent with a model in which two waves of Mk differentiation begin in LSK and progresses to CMP. The Mk lineage is divided in CMP, producing cells that begin endomitosis and cells that have an Mk-repressing/Ery-activating cell program that gives rise to the Ery lineage. We conclude that the erythroid lineage is derived from an Mk-like precursor and is the last lineage to be specified in mouse hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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Mir-99 Regulates Normal and Leukemic Stem Cell Function

Blood ◽

10.1182/blood.v128.22.1469.1469 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1469-1469

Author(s):

Mona Khalaj ◽

Carolien Woolthuis ◽

Wenhuo Hu ◽

Benjamin Heath Durham ◽

Christopher Y. Park

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Cell Function ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Gene Set Enrichment Analysis ◽

Hematopoietic Stem

Abstract Acute myeloid leukemia (AML) is composed of functionally heterogeneous cells including leukemic stem cells (LSCs), which exhibit the ability to self-renew and propagate disease. It is thought that failure of common chemotherapy regimens is due to insufficient eradication of LSCs. However, the mechanisms that maintain stem cell function in the hematopoietic system are not well understood. MicroRNAs play an important role in the regulation of normal and malignant hematopoietic stem cells. Our studies showed that miR-99, a miRNA highly expressed in AML patient cell populations enriched for LSC activity, is among the most highly expressed miRNAs in hematopoietic stem cells (HSCs), suggesting that miR-99 plays a role in regulating normal HSCs as well as LSCs. To test the role of miR-99 in normal hematopoiesis, we knocked down (KD) miR-99 in mouse HSCs (Lin-cKit+Sca1+CD34-SLAM+), which resulted in ~3 fold reduced methylcellulose colony formation upon secondary plating (P=0.01), as well as accelerated granulopoiesis as demonstrated by increased Gr1+Mac1+ cells 7 days after culture initiation (P<0.01), suggesting that miR-99 functions to suppresses differentiation. Consistent with this model, transplantation assays demonstrated >10-fold reduction in long-term engraftment capacity of miR-99 KD compared to scrambled controls (P=0.0004). In addition, Ki-67/DAPI staining of stably engrafted miR-99 KD hematopoietic stem and progenitor cells (HSPCs) showed increased cell cycling, demonstrating that miR-99 also maintains HSPC quiescence. Gene set enrichment analysis (GSEA) of RNA-sequencing data generated from stably engrafted Lin-Sca-1+c-Kit+ cells revealed that miR-99 KD induces significant depletion of LT-HSC gene signatures (P<0.001) and induction of a late progenitor signature (P<0.001), providing further evidence that miR-99 normally functions to maintain HSPCs in the undifferentiated state. To test whether miR-99 maintains LSCs, we performed miR-99 KD experiments using the MLL-AF9 retroviral mouse model. miR-99 KD resulted in a significant extension in survival in secondary transplants compared to scrambled controls (median 92 days vs. 48 days, P<0.001). Evaluation of the bone marrow at the time of death revealed ~2.5 fold decrease in the frequency of LSCs (P<0.01) and ~2 fold increase in the percentage of cycling LSCs (in SG2M) (P<0.001). Analysis of RNA-seq data from miR-99 KD LSCs revealed induction of a differentiated normal progenitor signature (P<0.001) and depletion of a shared HSC/LSC gene signature (P=0.05). Giemsa staining of peripheral blood showed miR-99 KD also induced a significant increase in the number of differentiated myeloid precursors in the peripheral blood (P<0.001), reminiscent of AML differentiation-inducing agents used in the clinic such as ATRA. Consistent with a role in regulating leukemic blast differentiation, microRNA-Seq data from the 153 AML patients in the TCGA database revealed that miR-99 expression inversely correlated with their French-American-British classifications, with low expression levels associated with M4 and M5 subtypes. Compatible with a role in maintaining LSCs, miR-99 KD in a primary AML sample reduced long-term engraftment upon xenotransplantation into NSG mice, and the engrafting cells displayed increased CD14 expression. Together, these data demonstrate that similar to normal HSPCs, miR-99 maintains LSCs function. As miR-99 functions to maintain both LSCs and HSCs, we asked which miR-99 target genes mediate miR-99 KD phenotypes. To address this question, we performed a shRNA library-based forward genetic screen designed to rescue the reduced HSC function following miR-99 KD. We designed 180 shRNAs against 45 predicted miR-99 targets that we identified as upregulated upon acute miR-99 KD in mouse HSPCs. Among the conserved miR-99 targets, Hoxa1, a member of the Hox family of transcription factors, was among the top hits, with all 4 shRNAs being enriched compared to controls. Ectopic expression of Hoxa1 in MonoMac6 AML cells was sufficient to induce differentiation, a phenotype similar to miR-99 KD. These data indicate that Hoxa1 is an important downstream mediator of miR-99 function. Disclosures No relevant conflicts of interest to declare.

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