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

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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Co-Existence of LMPP-Like and GMP-Like Leukemia Stem Cells In Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.91.91 ◽

2010 ◽

Vol 116 (21) ◽

pp. 91-91

Author(s):

Nicolas Goardon ◽

Emmanuele Marchi ◽

Lynn Quek ◽

Anna Schuh ◽

Petter Woll ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Myeloid Leukemia ◽

Expression Profiles ◽

Leukemic Stem Cell ◽

Self Renewal ◽

Two Populations ◽

Acute Myeloid

Abstract Abstract 91 In normal and leukemic hemopoiesis, stem cells differentiate through intermediate progenitors into terminal cells. In human Acute Myeloid Leukemia (AML), there is uncertainty about: (i) whether there is more than one leukemic stem cell (LSC) population in any one individual patient; (ii) how homogeneous AML LSCs populations are at a molecular and cellular level and (iii) the relationship between AML LSCs and normal stem/progenitor populations. Answers to these questions will clarify the molecular pathways important in the stepwise transformation of normal HSCs/progenitors. We have studied 82 primary human CD34+ AML samples (spanning a range of FAB subtypes, cytogenetic categories and FLT3 and NPM1 mutation states) and 8 age-matched control marrow samples. In ∼80% of AML cases, two expanded populations with hemopoietic progenitor immunophenotype coexist in most patients. One population is CD34+CD38-CD90-CD45RA+ (CD38-CD45RA+) and the other CD34+CD38+CD110-CD45RA+ (GMP-like). Both populations from 7/8 patients have leukemic stem cell (LSC) activity in primary and secondary xenograft assays with no LSC activity in CD34- compartment. The two CD34+ LSC populations are hierarchically ordered, with CD38-CD45RA+ LSC giving rise to CD38+CD45RA+ LSC in vivo and in vitro. Limit dilution analysis shows that CD38-CD45RA+LSCs are more potent by 8–10 fold. From 18 patients, we isolated both CD38-CD45RA+ and GMP-like LSC populations. Global mRNA expression profiles of FACS-sorted CD38-CD45RA+ and GMP-like populations from the same patient allowed comparison of the two populations within each patient (negating the effect of genetic/epigenetic changes between patients). Using a paired t-test, 748 genes were differentially expressed between CD38-CD45RA+ and GMP-like LSCs and separated the two populations in most patients in 3D PCA. This was confirmed by independent quantitative measures of difference in gene expression using a non-parametric rank product analysis with a false discovery rate of 0.01. Thus, the two AML LSC populations are molecularly distinct. We then compared LSC profiles with those from 4 different adult marrow normal stem/progenitor cells to identify the normal stem/progenitor cell populations which the two AML LSC populations are most similar to at a molecular level. We first obtained a 2626 gene set by ANOVA, that maximally distinguished normal stem and progenitor populations. Next, the expression profiles of 22 CD38-CD45RA+ and 21 GMP-like AML LSC populations were distributed by 3D PCA using this ANOVA gene set. This showed that AML LSCs were most closely related to their normal counterpart progenitor population and not normal HSC. This data was confirmed quantitatively by a classifier analysis and hierarchical clustering. Taken together, the two LSC populations are hierarchically ordered, molecularly distinct and their gene expression profiles do not map most closely to normal HSCs but rather to their counterpart normal progenitor populations. Finally, as global expression profiles of CD38-CD45RA+ AML LSC resemble normal CD38-CD45RA+ cells, we defined the functional potential of these normal cells. This had not been previously determined. Using colony and limiting dilution liquid culture assays, we showed that single normal CD38-CD45RA+ cells have granulocyte and macrophage (GM), lymphoid (T and B cell) but not megakaryocyte-erythroid (MK-E) potential. Furthermore, gene expression studies on 10 cells showed that CD38-CD45RA+ cells express lymphoid and GM but not Mk-E genes. Taken together, normal CD38-CD45RA+ cells are most similar to mouse lymphoid primed multi-potential progenitor cells (LMPP) cells and distinct from the recently identified human Macrophage Lymphoid progenitor (MLP) population. In summary, for the first time, we show the co-existence of LMPP-like and GMP-like LSCs in CD34+ AML. Thus, CD34+ AML is a progenitor disease where LSCs have acquired abnormal self-renewal potential (Figure 1). Going forward, this work provides a platform for determining pathological LSCs self-renewal and tracking LSCs post treatment, both of which will impact on leukemia biology and therapy. Disclosures: No relevant conflicts of interest to declare.

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Self-Renewal Pathways in Acute Myeloid Leukemia Stem Cells

10.5772/intechopen.94379 ◽

2020 ◽

Author(s):

Jonason Yang ◽

Nunki Hassan ◽

Sheng Xiang Franklin Chen ◽

Jayvee Datuin ◽

Jenny Y. Wang

Keyword(s):

Stem Cells ◽

Monoclonal Antibody ◽

Acute Myeloid Leukemia ◽

Hematopoietic Stem Cells ◽

Myeloid Leukemia ◽

Leukemia Stem Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Clinical Potential ◽

Acute Myeloid

Acute myeloid leukemia (AML) is a difficult-to-treat blood cancer. A major challenge in treating patients with AML is relapse, which is caused by the persistence of leukemia stem cells (LSCs). Self-renewal is a defining property of LSCs and its deregulation is crucial for re-initiating a new leukemia after chemotherapy. Emerging therapeutic agents inhibiting aberrant self-renewal pathways, such as anti-RSPO3 monoclonal antibody discovered in our recent study, present significant clinical potential that may extend beyond the scope of leukemogenesis. In this chapter, we provide an overview of normal and malignant hematopoietic stem cells, discuss current treatments and limitations, and review key self-renewal pathways and potential therapeutic opportunities in AML.

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DNA Microarray Assay Helps to Identify Functional Genes Specific for Leukemia Stem Cells

Dataset Papers in Science ◽

10.1155/2013/520285 ◽

2013 ◽

Vol 2013 ◽

pp. 1-5

Author(s):

Haojian Zhang ◽

Shaoguang Li

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Dna Microarray ◽

Kinase Inhibitors ◽

Expression Profiles ◽

Chronic Phase ◽

Leukemia Stem Cells ◽

Myeloproliferative Disease ◽

Hematopoietic Stem ◽

Microarray Assay

Chronic myeloid leukemia (CML) is a myeloproliferative disease derived from an abnormal hematopoietic stem cell (HSC) and is consistently associated with the formation of Philadelphia (Ph) chromosome. Tyrosine kinase inhibitors (TKIs) are highly effective in treating chronic phase CML but do not eliminate leukemia stem cells (LSCs), which are believed to be related to disease relapse. Therefore, one major challenge in the current CML research is to understand the biology of LSCs and to identify the molecular difference between LSCs and its normal stem cell counterparts. Comparing the gene expression profiles between LSCs and normal HSCs by DNA microarray assay is a systematic and unbiased approach to address this issue. In this paper, we present a DNA microarray dataset for CML LSCs and normal HSCs to show that the microarray assay will benefit the current and future studies of the biology of CML stem cells.

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Gpx3 Determines Competitiveness of Normal and Leukemic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1587.1587 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1587-1587 ◽

Cited By ~ 1

Author(s):

Olivier Herault ◽

Kristin J Hope ◽

Eric Deneault ◽

Matthias Trost ◽

Nadine Mayotte ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Insertional Mutagenesis ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Cell Activity ◽

Relative Reduction ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 1587 Although important efforts have been invested in the discovery of genes that regulate normal or leukemic hematopoietic stem cells (HSC) self-renewal, the number of validated candidates remains low, due largely to the unavailability of functionally pure stem cell populations. Moreover, it is often difficult to identify the normal counterpart cell from which leukemia originated, further complicating studies based on comparative gene expression. In this study, we used a series of recently characterized Hoxa9 + Meis1 acute myeloid leukemias (AML) derived from fetal liver (FL) cells (Wilhelm BT et al., submitted). These leukemias are remarkably similar in several aspects including their L-HSC frequency (between ∼1 in 100 to 350) except for one leukemia (FLA2) in which 70% of the cells show repopulation ability (i.e., L-HSC). We reasoned that comparative mRNA profiling of FLA2 to the phenotypically similar FLB1 (0.3% L-HSC) might identify genes uniquely associated with L-HSC self-renewal. We observed a 2–3-fold upregulation of Gpx3 in FLA2, which was confirmed by qRT-PCR. In accordance with this, all 14 of the tested Gpx3 promoter region CpG sequences were methylated in FLB1 and hypomethylated in FLA2 cells. The higher expression of GPx3 in FLA2 was confirmed at the protein level and reflected in elevated glutathione peroxidase activity in comparison to FLB1. Importantly, we also observed in FLA2 a relative reduction in reactive oxygen species (ROS) level (DCFDA) and a concomitant decrease in p38 MAPK activation (western blot and mass spectrometry). The correlation of Gpx3 levels with L-HSC frequency could be reflective of their functional dependence on this enzyme. FLA2 cells being difficult to manipulate ex vivo, to address this we utilized retroviruses encoding shRNAs and a GFP reporter to explore the in vivo function of FLA2 cells with downregulated Gpx3. The decrease in percentage of GFP+ donor cells when leukemia became apparent (∼19 days) from that of populations initially transplanted, was 4-fold higher following Gpx3 knockdown in comparison to shLuciferase transduction. Moreover, those shGpx3 infected FLA2 remaining at day 19 displayed a 3-fold decrease in GFP mean fluorescence intensity relative to their control counterparts. These results show that GFPhigh cells were selectively depleted, and suggest that Gpx3 is critical for the competitiveness of L-HSCs. Because redox metabolism has been implicated in HSC self-renewal, we also analyzed its expression and function in normal HSC to gain further insight into the role of GPx3 in stem cell activity. Interestingly, compared to FL-HSCs, isolated 3 and 4 week bone marrow (BM), HSCs exhibited a 39- and 6-fold decrease in Gpx3 expression, respectively. A correlation of Gpx3 levels with enhanced self-renewal was also observed in vitro as overexpression of several nuclear determinants of HSC expansion such as Hoxb4, NA10HD, Klf10 and Prdm16 promoted Gpx3 expression by 3.2 to 19.2-fold. We next infected BM cells enriched for HSCs with retroviruses carrying shRNAs to Gpx3. shRNA targeting of Gpx3 dramatically inhibited hematopoietic reconstitution. Transplantations of sublethally irradiated recipients indicated that Gpx3 knockdown significantly impaired both early and late donor-derived hematopoiesis. These results suggest that GPx3 is critical for repopulation mediated by both short and long-term repopulating cells. In reciprocal gain-of-function experiments, Lin-CD150+CD48- cells engineered to overexpress Gpx3, showed a marked competitive advantage over controls when transplanted following a 7-day ex vivo culture step. Insertional mutagenesis was ruled out as proviral integration analyses of six recipients confirmed polyclonal hematopoiesis. Moreover, some mice were in part reconstituted by the same clones, indicating that self-renewal occurred in vitro prior to transplantation. Phenotypic analysis of late-transplant hematopoietic tissues showed that Gpx3-transduced cells contributed to lymphoid and myeloid repopulation, confirming their multipotentiality. Together, these results indicate that Gpx3 enhances HSC expansion ex vivo possibly through modulation of self-renewal activity. In conclusion, a unique model of primary L-HSC was exploited to identify Gpx3 as a critical determinant for the competitiveness of L-HSCs and complementary experiments demonstrated a key role for this gene in normal HSC self-renewal. Disclosures: No relevant conflicts of interest to declare.

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Poor Outcome of RUNX1-Mutated (RUNX1-mut) Patients (Pts) with Primary, Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) and Associated Gene- and MicroRNA (miR) Expression Signatures,

Blood ◽

10.1182/blood.v118.21.3454.3454 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3454-3454

Author(s):

Jason H. Mendler ◽

Kati Maharry ◽

Michael D. Radmacher ◽

Krzysztof Mrózek ◽

Jessica Kohlschmidt ◽

...

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Expression Profiles ◽

White Blood Cells ◽

Differentially Expressed ◽

Prognostic Impact ◽

Cell Transplant ◽

Self Renewal ◽

Hematopoietic Stem ◽

Α Subunit

Abstract Abstract 3454 RUNX1 encodes the α subunit of core binding factor, a heterodimeric transcription factor required for normal hematopoiesis. Acquired RUNX1 mutations (muts) have been associated with poor clinical outcome in AML; however, prior studies analyzed pts heterogeneous for cytogenetics, age, AML type (primary or secondary), and treatment received [including allogeneic stem cell transplant (alloSCT) in 1st complete remission (CR1)] and contained limited data regarding the potential molecular drivers of the worse outcome. We report a relatively large study testing the prognostic impact of RUNX1 muts in primary CN-AML pts (n=392) treated similarly with intensive cytarabine/anthracycline-based 1st-line therapy and without alloSCT in CR1. This cohort comprised both younger [<60 years (y); n=173] and older (≥60 y; n=219) pts. Pretreatment marrow (n=303) and blood (n=89) were analyzed centrally for RUNX1 muts by PCR and direct sequencing, and for FLT3-ITD, FLT3-TKD, MLL-PTD and NPM1, CEBPA, WT1, IDH1, IDH2 and TET2 muts. Gene and miR expression profiles were derived using microarrays. RUNX1 muts were found in 12.5% of pts (8% younger, 16% older), and were associated with lower hemoglobin (P=.01), white blood cells (WBC; P=.04), and blood blasts (P=.006). RUNX1-mut pts harbored NPM1 (P<.001) and CEBPA muts (P=.06) less frequently than RUNX1-wild-type (RUNX1-wt) pts. RUNX1-mut pts had lower CR rates (P=.005 in younger; P=.006 in older), and shorter disease-free (DFS; P=.058 in younger; P<.001 in older), overall (OS; P=.003 in younger; P<.001 in older) and event-free (EFS; P<.001 for younger and older; Figures 1 and 2) survival than RUNX1-wt pts. In multivariable models, RUNX1 muts remained associated with lower CR rate (P<.001) and shorter DFS (P<.001), OS (P<.001), and EFS (P<.001; Table) after adjustment for clinical and molecular variables.Figure 1.Figure 1. Figure 2.Figure 2. Table 1Multivariable analysis for EFS according to RUNX1-mut status in all CN-AML ptsHREFSPRUNX1, mut v wt2.271.65–3.12<.001FLT3-ITD, ITD v no ITD1.571.27–1.95<.001WT1, mut v wt1.441.02–2.01.04WBC, continuous 50 unit increase1.131.04–1.23.006Age group, ≥60y v <60y1.801.46–2.22<.001Note: A hazard ratio (HR) >1 corresponds to a higher risk for higher values of continuous variables and the 1st level listed of a dichotomous variable. To gain biological insight, RUNX1 mut-associated gene and miR expression signatures were derived in CN-AML for the first time. Older, NPM1-wt pts were analyzed since RUNX1 muts are more common in this age group and are nearly exclusive from NPM1 muts, which have their own characteristic gene-expression signature. This yielded 484 probe sets representing 278 named genes differentially expressed between RUNX1-mut (n=31) and RUNX1-wt (n=45) pts (P<.001). Genes normally expressed in hematopoietic stem (HSC) and early progenitor cells, including DNTT, BAALC, MN-1, CD109, P2RY14, FOXO1 and FLT-3 were upregulated in RUNX1-mut pts, as were components of the Wnt-signaling pathway, LRP6 and TCF4, that promote self-renewal and proliferation of HSCs. Genes upregulated (SETBP1, RBPMS, and SLC37A3) and downregulated (CCNA1 and RNASE3) in AML stem cells relative to AML progenitors were similarly deregulated in the RUNX1-mut signature. B cell lineage genes BLNK, IGHM, IRF8 and several class II MHC molecules were upregulated in RUNX1-mut pts while CEBPA, a key promoter of granulopoiesis, was downregulated. Genes implicated in chemoresistance, GAS6, PRKCE, and PTK2, were upregulated and MYCN, a promoter of both proliferation and apoptosis of myeloid cells, was downregulated in RUNX1-mut pts. Seven miRs were differentially expressed between RUNX1-mut and RUNX1-wt pts. Two members of the let-7 tumor suppressor family, which represses self-renewal and promotes differentiation of stem cells, were downregulated, as was miR-223, a positive regulator of granulopoiesis. MiRs -99a and -100 were also downregulated and miRs -211 and -595 upregulated in association with RUNX1 muts. In summary, RUNX1 muts are twice as common in older CN-AML pts than younger. They negatively impact on outcome in both younger and older pts not receiving alloSCT in CR1. RUNX1-mut blasts have molecular features of normal/malignant stem cells and B cells, which may explain their chemoresistance and guide novel therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

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Growth Factor Independence 1b (Gfi1b) Regulates The Commitment, Differentiation and Expansion Of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v122.21.2433.2433 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2433-2433

Author(s):

Tarik Moroy ◽

Cyrus Khandanpour ◽

Joseph Krongold

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Mouse Bone Marrow ◽

Paracrine Factors ◽

Self Renewal ◽

Hematopoietic Stem

Abstract The efficacy of bone marrow stem cell transplantation is the therapy of choice for many hematopoietic diseases, in particular leukemia and lymphoma. This therapy is critically dependent on the transfer of sufficient numbers of hematopoietic stem cells (HSCs), which possess the capacity for self-renewal and can fully reconstitute the hematopoietic system. As such, the development of techniques for the expansion of fully functional HSCs is of significant clinical interest. By transiently manipulating the factors that govern HSC homeostasis it has been proposed that HSCs can be expanded without the loss of essential stem cell characteristics. Previously we have observed that ablation of the gene encoding the transcription factor Gfi1b in-vivo results in a dramatic expansion and mobilization of hematopoietic stem cells in the bone marrow and periphery. More recent data suggest that the blood mobilization of Gfi1b deficient HSCs is very likely mediated by a deregulation of the integrin expression. These data led us to hypothesize that Gfi1b could be a potential target for ex-vivo treatment and expansion of HSCs. Indeed, when deletion of Gfi1b was induced in whole bone marrow ex-vivo, HSCs showed a significant expansion in both in absolute number and in terms of proportion of bone marrow. We followed HSCs in ex-vivo expansion cultures from mouse bone marrow by tracking expression of the surface marker CD48, which indicates whether an HSC has transitioned to a differentiation committed multi-potent progenitor. We observed that Gfi1b null HSCs expanded without up-regulating CD48 in contrast to wt HSCs. This suggests that Gf11b deficient HSCs underwent symmetric self-renewal type cell divisions at a significantly increased frequency, when compared to wt HSCs. We had previously shown that HSCs lacking Gfi1b cycle at a faster rate than control HSCs. The combination of increased cell division and preferential self-renewal of Gfi1b-/- HSCs indicates that inhibition of Gfi1b may be the ideal strategy for ex-vivo HSC expansion. As well, in accordance with this preference for self-renewal, Gfi1b null HSCs that were cultured under myeloid differentiation conditions remained primarily in an undifferentiated state as defined by a lack of the myeloid surface markers Gr1 and Mac1. These cultures also demonstrated increased long term colony forming capacity versus controls, further supporting an undifferentiated phenotype in Gfi1b-/- cells. Because the stem cell niche is a highly complex and heterogeneous environment we also investigated whether bone marrow in which Gfi1b has been deleted exerts paracrine effects that contributed to HSC expansion. Co-Culture assays demonstrated that Gfi1b-/- bone marrow was able to induce an expansion of progenitors in wild-type bone marrow of more than 10 fold compared to Gfi1b-/+ bone marrow. Interestingly cells co-cultured with Gfi1b null bone marrow also exhibited an overall proliferation advantage after short-term cultures. This suggests that not only does Gfi1b deletion induce HSC expansion via cell intrinsic mechanisms, but also points to the possibility that this occurs through paracrine factors that alter bone marrow homeostasis. Disclosures: No relevant conflicts of interest to declare.

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The Novel Leukemia Stem Cell Marker GPR56 Discriminates Leukemic Subclones with Divergent Stem Cell Properties in Human Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.1859.1859 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1859-1859

Author(s):

Caroline Pabst ◽

Anne Bergeron ◽

Vincent-Philippe Lavallée ◽

Jonathan Yeh ◽

Patrick Gendron ◽

...

Keyword(s):

Stem Cells ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Myeloid Leukemia ◽

Expression Profiles ◽

Stem Cell Marker ◽

Mouse Bone Marrow ◽

Rna Seq ◽

Hematopoietic Stem ◽

Acute Myeloid

Abstract Insights into the complex clonal architecture of acute myeloid leukemia (AML) unravelled by deep sequencing technologies have challenged the concept of AML as a hierarchically organised disease initiated and driven by rare self-renewing leukemic stem cells (LSCs). In contrast to normal human hematopoietic stem cells (HSCs), which are highly enriched in the CD34+ CD38- population, LSCs have also been found in the CD34- and the CD38+ fractions questioning the existence of a consistent LSC surface marker profile for AML. Besides, low LSC frequencies in primary samples, rapid onset of differentiation upon ex vivo culture, and genetic inter-specimen heterogeneity hamper the dissection of the molecular machinery that drives LSC self-renewal. We performed RNA-Sequencing of primary human AML samples and assessed LSC frequencies by limiting dilution analyses for 56 of these in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. By comparing gene expression profiles between high vs low LSC frequency leukemias, we identified the G-protein coupled receptor 56 (GPR56) has significantly more expressed in high LSC frequency leukemias. We validated the RNA-seq data with protein expression by FACS and found an excellent correlation. To determine whether GPR56 positive cells overlapped with the known LSC-associated phenotype CD34+ CD38-, we stained 45 AML samples with CD34, CD38, GPR56, and antibodies against other described LSC markers. Although CD34+ GPR56+ and CD34+ CD38- compartments identified the same population in some samples, we found in the majority of samples that GPR56 further subdivided the CD34+ CD38- compartment. Accordingly, not only the proportions of total GPR56+ and CD34+ GPR56+ cells were significantly higher in LSChigh versus LSClow samples, but also the proportion of GPR56+ cells within the CD34+ CD38- compartment was significantly different between the groups indicating that GPR56 might be of additional value to what is currently considered the best described LSC phenotype. The percentage of total CD34 positive cells did not correlate with LSC frequency clearly distinguishing GPR56 from CD34 or CD38, which are only suitable LSC markers when used in combination. We analysed other potential LSC markers (TIM3, CD96, CD44, CD123, CLL1 and CD47) in our RNA-Seq dataset and by FACS analysis in combination with CD34 as we did for GPR56 and none of them correlated with LSC frequency in our sample collection. To determine whether GPR56 discriminates engrafting LSCs from non-LSCs, we sorted GPR56+ and GPR56- cells within the CD34-positive and -negative compartments from selected specimens with known engraftment potential. We found that GPR56 identified the engrafting fraction in CD34positive AML samples, with a >50 fold enrichment in LSC in the CD34+GRP56+ fraction vs the CD34+GPR56- fraction within the same sample, demonstrating that GPR56 is a good LSC marker. Specimens with high molecular or cytogenetic risk such as chromosome 5 or 7 anomalies and EVI1- rearrangementexpressed high levels of both, GPR56 and CD34, while samples with coexistent FLT3 -ITD, DNMT3A, and NPM1 mutations displayed a unique CD34low GPR56high profile. Moreover, we found a divergent distribution of variant allele frequencies in GPR56+ versus GPR56- fractions identifying GPR56 as a discriminator of leukemic sub-clones with high and low NSG engrafting capacity. Analysis of engrafted cells re-sorted based on GPR56 after being harvested from mouse bone marrow revealed reduced complexity of the clonal composition. Most importantly, GPR56 positive cells differentiated to GPR56 negative cells in mice, which did not happen in the human niche, in which GPR56 positive and negative fractions represented two independently evolved subclones. In summary our work identifies GPR56 as a novel LSC marker in AML and also shows that GPR56 readily identifies a functionally distinct LSC-rich subclone in the majority of human AML patients and reveals hitherto unforeseen complexity in the interaction between human LSCs and the NSG mouse environment. Disclosures No relevant conflicts of interest to declare.

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Myeloid Leukemia Stem Cells Depend on Phospholipase C Gamma 1 (Plcg1) Signaling

Blood ◽

10.1182/blood.v128.22.1202.1202 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1202-1202

Author(s):

Tina M. Schnoeder ◽

Patricia Arreba-Tutusaus ◽

Juliane Mohr ◽

Soenke Weinert ◽

Stephanie Frey ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Myeloid Leukemia ◽

Transformed Cells ◽

Leukemia Stem Cells ◽

Leukemic Stem Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Genetic Inactivation ◽

Disease Penetrance

Abstract Several genes and signaling pathways control the fine balance between self-renewal and differentiation in hematopoietic stem cells and potentially also in leukemic stem cells (LSC). Phospholipase C family members are key mediators of calcium signaling which play an important role in differentiation and proliferation of immune cells but also contribute to malignant transformation and tumorigenesis. Plcg1 is highly expressed in hematopoietic stem- and progenitor cells and also in myeloid leukemia. Plcg1 gets activated by cell extrinsic receptor stimulation and integrates signals from the cell surface. Its influence on proliferation and differentiation of hematopoietic cells may be largely independent of other bone fide mediators of self-renewal and stem cell viability such as STAT-, MEK-ERK or AKT-signaling. To which extent Plcg1-dependent signal integration is required for function and maintenance of leukemic stem cells remained so far elusive. Genetic inactivation of Plcg1 by RNAi in human AML cell lines led to decreased proliferative capacity. Likewise, knockdown of Plcg1 in AML1-ETO (AML1ETO9a) transformed murine LSK-cells resulted in reduced colony formation and decreased re-plating capacity. In order to validate these findings and to investigate the impact of Plcg1 on myeloid leukemia stem cell function, we generated a conditional knockout mouse model for Plcg1 with Exons 3-5 being flanked with loxP sites. Excision of the respective sequence by activation of a Cre-recombinase resulted in complete loss of a functional protein and transcript. LSK-cells from Plcg1f/f and Plcg1+/+ littermate controls were retrovirally infected with two different oncogenes: either MLL-AF9 (MA9-GFP) or AML1-ETO9a in combination with KRAS (AE9a/KRAS-GFP). Primary recipient mice were injected with GFP+ LSK-cells and monitored for disease development. GFP+ Kit+ cells were isolated from leukemic mice and transduced with a Cre-recombinase, followed by plating in methylcellulose. Inactivation of Plcg1 in AE9a/KRAS transformed cells significantly reduced the number of colonies and decreased re-plating capacity to three rounds. Loss of Pclg1 in MA9 transformed LSC resulted in decreased colony numbers and colony size, however, re-plating capacity was not affected to a major extent. To assess for the requirement of Plcg1 in maintenance of fully developed leukemia, we injected equal numbers of GFP+ Kit+ cells (Plcg1-/- or Plcg1+/+) into sublethally irradiated secondary recipients. Inactivation of Plcg1 was highly deleterious for AE9a/KRAS induced AML-LSC and reduced disease penetrance by more than 85%. Depletion of Plcg1 in MA9 transformed cells delayed AML development and significantly prolonged survival of recipient mice. Leukemias that developed from Plcg1-/- donors showed complete excision of Plcg1, indicating, that Plcg1 deficient leukemia can develop in an MLL-AF9 driven background. However, when transplanting MA9 transformed Plcg1-/- or Plcg1+/+ bone marrow cells into tertiary recipient mice, loss of Plcg1 significantly delayed disease progression and reduced disease penetrance. To quantify this loss of leukemic stem cells, we performed limiting dilution analysis using purified LSCs from diseased Plcg1-/- or Plcg1+/+ MA9 secondary recipient mice. LSC frequency was markedly reduced in tertiary recipients of Plcg1-depleted LSCs (1 in 78,000 Plcg1-/- vs. 1 in 3,000 Plcg1+/+). Genetic inactivation of Plcg1 in LSCs derived from primary recipient mice (either MA9 or AE9a/KRAS driven AML) led to induction of differentiation as assessed by cell morphology and immunophenotyping, and this effect was more pronounced in AE9a/KRAS transformed cells. To investigate whether transcriptional effectors of Plcg1 signaling affect the fine balance between self-renewal in MA9- and AE9a/KRAS-driven leukemia, we performed whole transcriptome analysis (RNAseq) on sorted LSCs. Ongoing analyses address the functional difference between AML-ETO and MLL-AF9 driven disease and elucidate on distinct patterns of activated gene sets depending on the oncogenic background. Taken together, Plcg1 is required for maintenance of myeloid leukemia stem cells. Understanding of its relevance in LSC biology and function may offer the opportunity to develop this relevant signaling node as a target structure in AML. Disclosures No relevant conflicts of interest to declare.

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RNA m6A Modification Plays a Key Role in Maintaining Stem Cell Function in Normal and Malignant Hematopoiesis

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.710964 ◽

2021 ◽

Vol 9 ◽

Author(s):

Peipei Wang ◽

Mengdie Feng ◽

Guoqiang Han ◽

Rong Yin ◽

Yashu Li ◽

...

Keyword(s):

Stem Cells ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Myeloid Leukemia ◽

Cell Function ◽

Leukemia Stem Cells ◽

Hematopoietic Stem ◽

Cellular Processes ◽

Acute Myeloid

N6-methyladenosine (m6A) is a commonly modification of mammalian mRNAs and plays key roles in various cellular processes. Emerging evidence reveals the importance of RNA m6A modification in maintaining stem cell function in normal hematopoiesis and leukemogenesis. In this review, we first briefly summarize the latest advances in RNA m6A biology, and further highlight the roles of m6A writers, readers and erasers in normal hematopoiesis and acute myeloid leukemia. Moreover, we also discuss the mechanisms of these m6A modifiers in preserving the function of hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs), as well as potential strategies for targeting m6A modification related pathways. Overall, we provide a comprehensive summary and our insights into the field of RNA m6A in normal hematopoiesis and leukemia pathogenesis.

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Vascular Pericytes Sustain Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.2394.2394 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2394-2394 ◽

Cited By ~ 2

Author(s):

Mirko Corselli ◽

Chintan Parekh ◽

Elisa Giovanna Angela Montelatici ◽

Arineh Sahghian ◽

Wenyuan Wang ◽

...

Keyword(s):

Stem Cells ◽

Adipose Tissue ◽

Stem Cell ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Cell Interactions ◽

Lymphoid Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 2394 Mesenchymal stromal (or stem-) cells (MSC) are culture-selected, heterogeneous supporting cells that can differentially regulate hematopoietic stem cell (HSC) behavior in vitro. The elusive identity of native MSC has obscured the contribution, if any, of these cells to HSC support in vivo. Having previously demonstrated that vascular pericytes (ubiquitous cells encircling endothelial cells in capillaries and microvessels) are ancestors of human MSC, we now hypothesize that pericytes are a critical component of the HSC “niche”. Consequently, pericyte isolation from total stroma would allow to develop co-culture systems for human HSC maintenance. In the present study, human cord blood CD34+ cells were cultured onto confluent human pericytes isolated from adipose tissue as CD146+CD34-CD45-CD56- cells. Co-culture of CD34+ cells on pericytes, for up to 6 weeks in the absence of any added growth factor, produced significantly i) higher numbers of CD45+ and CD34+ cells (p<0.05), ii) higher percentages of primitive CD34+CD33-CD10-CD19- progenitors (p<0.05), iii) higher percentages of single- and multi-lineage CFU (p<0.05) and iv) lower percentages of mature myeloid and lymphoid cells (p<0.05), compared to control co-cultures on unfractionated adipose stromal cells (ASC) (n=10 individual experiments, n=4 biological replicates). Most importantly, only pericytes could maintain HSC with self-renewal and long-term repopulating potential, as demonstrated by transplantation into primary and secondary NOD/SCID/IL2Rg−/− mouse recipients (n=3 individual experiments). In the latter setting, none of the mice receiving CD34+ cells co-cultured with ASC engrafted (n=10), whereas all recipients of CD34+ cells cultured in the presence of pericytes developed lympho-myeloid hematopoietic human cells (n=10). Altogether, these results support the hypothesis that pericytes maintain hematopoietic cell stemness. Conversely, unfractionated stromal cell cultures may promote HSC differentiation at the expense of self-renewal. Both tentative scenarios were explored. Co-cultures with pericytes in a transwell system revealed that cell-to-cell contact is required for HSC survival. Since Notch signaling regulates stem cell maintenance by inhibiting cell differentiation through cell-cell interactions, we hypothesized that pericytes purified from total stroma express specific Notch ligands. As shown by qPCR, the expression of Jagged-1 is 2 fold higher in pericytes compared to unfractionated ASC. Addition of a Notch inhibitor (DAPT) to pericyte/HSC co-cultures resulted in the significant reduction of CFU numbers (p<0.05) and increase in B-cell development. Furthermore, increased myeloid differentiation was observed when ASC conditioned medium was added to pericytes/HSC co-cultures. In conclusion, we demonstrate that vascular pericytes sustain HSC by promoting survival and preventing differentiation via cell-to-cell interactions involving Notch activation, whereas unfractionated stroma promotes HSC differentiation through a paracrine mechanism. We thus infer that HSC-supporting stromal cells are not confined within blood-forming organs (similar observations, not reported here, have been made on skeletal muscle pericytes). This novel concept is not easy to reconcile with normal hematopoiesis, but may be highly relevant in the context of the dissemination of malignant hematopoietic cells. Of important note, adipose tissue used in this study represents a convenient, safe and often abundant source of autologous therapeutic cells. Therefore, human fat-derived pericytes emerge as a candidate cell product for HSC ex vivo manipulation in the clinic. Disclosures: No relevant conflicts of interest to declare.

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