Inhibition of EZH2 Depletes MLL Fusion Leukemia Stem Cells Through Restoration of p16 Expression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3510-3510
Author(s):  
Koki Ueda ◽  
Akihide Yoshimi ◽  
Masahiro Nakagawa ◽  
Satoshi Nishikawa ◽  
Victor E Marquez ◽  
...  
Keyword(s):  
Stem Cells ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
High Specificity ◽  
Prolonged Survival ◽  
Leukemia Cells ◽  
Leukemia Stem Cells ◽  
Hematopoietic Stem ◽  
Colony Forming Capacity

Abstract Abstract 3510 Leukemia stem cells (LSCs) are resistant to conventional chemotherapy and persistent LSCs after chemotherapy are supposed to be a major cause of disease relapse or refractoriness. However, information on genetic or epigenetic regulation of stem cell properties is still limited and LSC-targeted drugs have scarcely been identified or used in clinical settings so far. Epigenetic regulators are associated with many cellular processes such as cell cycle, proliferation, and apoptosis. Of note are polycomb group proteins, because they potentially control stemness including activity of cancer stem cells, and can be pharmacologically targeted by a selective inhibitor of H3K27, 3-deazaneplanocin A (DZNep). We first administrated DZNep to MLL-related leukemia mouse model in order to test whether DZNep has potential to eradicate LSCs of the leukemic mice. Remarkably, the leukemic granulocyte-macrophage progenitors (LGMPs) in MLL/AF9 positive cells were significantly decreased in number by administration of DZNep while AraC did not affect the number of LGMPs, which implied that LSCs were targeted by DZNep. These data were reproduced by transplantation assays using short hairpin RNA (shRNA)-mediated knockdown of EZH2, a major component of polycomb repressive complex 2 (PRC2) which is responsible for H3K27 tri-methylation. Significantly, DZNep administration to wild-type mice led to only mild suppression of hematopoiesis, suggesting that this agent spares normal hematopoietic stem cells while eliminating LSCs, which is consistent with a previous report that genetic depletion of EZH2 did not compromise adult hematopoiesis in mice. Serial replating assay of MLL/AF9-induced leukemia cells showed that DZNep treatment in vivo diminished their colony forming capacity. Limiting dilution transplantation assays revealed that frequency of LSCs was markedly reduced by DZNep administration. DZNep treatment or EZH2 knockdown significantly prolonged survival of MLL/AF9 and MLL/ENL leukemic mice. To elucidate a molecular mechanism underlying the effects of DZNep on LSCs, we investigated transcriptional or epigenetic changes during DZNep treatment and EZH2 knockdown. Gene expression profiling revealed that p16 was significantly upregulated by EZH2 knockdown or DZNep administration. Knockdown of p16 completely canceled the survival advantage of the leukemia mice which received DZNep in vivo and restored the colony forming capacity of leukemia cells transduced with shRNA for EZH2 in vitro. These results supported the idea that p16 upregulation derived from EZH2 attenuation is central to the LSC reduction. Next, we investigated epigenetic status around p16 promoter and transcription start site (TSS) by chromatin immunoprecipitation (ChIP) assays. In MLL/ENL leukemia cells, both H3K4 and H3K27 methylation marks were highly enriched around the TSS of p16, together with EZH2 and Bmi1, a component of PRC1. Therefore removal of EZH2 is supposed to convert the promoter of p16 from a bivalent to an active state. The results of ChIP assays also indicated that MLL/ENL fusion protein binds to p16 coding region. In order to clarify whether dependency on EZH2 is specific for MLL fusion leukemia or can be applied for other types of leukemia, we evaluated the consequence of EZH2 inhibition in several types of leukemia. DZNep or shRNA for EZH2 strongly suppressed the proliferation of leukemia cell lines and immortalized cells harboring MLL fusion genes with high specificity. Administration of DZNep or transduction of shRNA targeting EZH2 significantly prolonged survival of MLL/AF9 and MLL/ENL-induced leukemia mice while TEL/PDGFRA-AML1/ETO-induced leukemia was not sensitive to DZNep, although bone marrow (BM) cells from either mice became globally hypo-methylated on H3K27 by exposure to this drug. Serial replating assay with DZNep or EZH2-shRNA demonstrated high sensitivity to EZH2 inhibition of MLL/AF9-transduced BM cells but not of AML1/ETO-transduced BM cells, E2A/HLF-transduced BM cells, or normal c-kit+ BM cells. Thus, the anti-leukemia effect of EZH2 inhibition is thought to be specific for MLL fusion leukemia. Collectively, our findings indicate that EZH2 is a potential therapeutic target of LSCs of MLL fusion leukemia to overcome the poor prognosis, encouraging the development of inhibitors against EZH2 with high specificity. Disclosures: No relevant conflicts of interest to declare.

Remodeling Ca2+ Flux By ORP4L Is Essential for Leukemia Stem Cells (LSCs) Survival

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5257-5257
Author(s):  
Wenbin Zhong ◽  
Vesa Olkkonen ◽  
Xu Bing ◽  
Biying Zhu ◽  
Guoping Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Oxidative Phosphorylation ◽  
Mitochondrial Function ◽  
Conflicts Of Interest ◽  
Myelogenous Leukemia ◽  
Leukemia Stem Cells ◽  
Ip3 Receptor ◽  
Hematopoietic Stem

Abstract Acute myelogenous leukemia (AML) is one of the deadliest hematological malignancies and there is at present no efficient strategy to defeat it. New detailed insight into AML leukemia stem cells (LSCs) survival will facilitate the identification of targets for the development of new therapeutic approaches. Recent work has provided evidence that LSCs are defective in their ability to employ glycolysis, but are highly reliant on oxidative phosphorylation, and the maintenance of mitochondrial function is essential for LSCs survival. It is increasingly clear that Ca2+ released constitutively from endoplasmic reticulum (ER) is taken up by mitochondria to sustain optimal bioenergetics and cell survival. Here we report three striking findings: 1) oxysterol-binding protein (OSBP)-related protein 4 (ORP4L) is expressed in LSCs but not in normal hematopoietic stem cells (HSCs). 2) ORP4L is essential for LSC bioenergetics; It forms a complex with PLCβ3 and IP3 receptor 1 (ITPR1) to control Ca2+ release from the ER and subsequent cytosolic and mitochondrial parallel Ca2+ spike oscillations that sustain pyruvate dehydrogenase (PDH) activation and oxidative phosphorylation. 3) ORP4L inhibition eradicates LSCs in vitro and in vivo through impairment of Ca2+-dependent bioenergetics. These results suggest a novel role of ORP4L in governing Ca2+ release to sustain mitochondrial function and survival of LSCs and identify ORP4L as a putative new oncoprotein and therapeutic target for LSCs elimination. Disclosures No relevant conflicts of interest to declare.

scholarly journals Leukemia-Initiating Activity in T-ALL Requires Active Hif1a and Wnt Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 57-57
Author(s):  
Vincenzo Giambra ◽  
Catherine E Jenkins ◽  
Sonya H Lam ◽  
Catherine Hoofd ◽  
Miriam Belmonte ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Cell Biology ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Negative Regulator ◽  
Myelogenous Leukemia ◽  
Leukemia Stem Cells ◽  
Hematopoietic Stem

Abstract Prior work has shown that NOTCH1 is a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL) with activating NOTCH1 mutations occurring in over 50% of cases (Weng et al, Science 2004) and loss-of-function mutations in its negative regulator FBXW7 occurring in 8-15% of cases (O’Neil et al, J Exp Med 2007; Thompson et al, J Exp Med 2007). Subsequent work has shown that continued Notch signaling is required for maintenance of T-ALL leukemia stem cells (Armstrong et al, Blood 2009; Tatarek et al, Blood 2011; Giambra et al, Nat Med 2012). Several lines of evidence have substantiated genetic interactions between the Notch and Wnt signaling pathways in various contexts, and Wnt signaling has been shown to play important roles in hematopoietic stem cell biology and also in hematopoietic cancers such as acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Luis et al, Leukemia 2012). To address what role if any Wnt signaling may play in T-ALL, we generated primary murine leukemias by viral transduction of bone marrow progenitors with activated NOTCH1, then delivered a fluorescent Wnt reporter construct (7TGP; Fuerer & Nusse, PLoS ONE 2010) by lentiviral transduction, and retransplanted the leukemias to interrogate Wnt signaling activity in vivo. We report here that active Wnt signaling is restricted to minor subpopulations within bulk T-ALL tumors, and that these Wnt-active subsets are highly enriched for leukemia-initiating cell (LIC) activity. Moreover, using Ctnnb1loxP/loxP animals we show that inducible Cre-mediated deletion of β-catenin or enforced expression of a dominant-negative TCF construct severely compromises LIC activity. We also show that β-catenin levels are upregulated by hypoxia through Hif1a stabilization, and that deletion of Hif1a also severely compromises LIC activity. Interestingly, Wnt-active subsets are distributed diffusely throughout the marrow interstitial space suggesting that tumor infiltration induces formation of local hypoxic niches as opposed to taking up residence in pre-existing anatomic compartments with low oxygen tensions. Taken together, these results suggest a model in which hypoxic niches in vivo facilitate Hif1a-dependent accumulation of β-catenin which drives Wnt signaling and self-renewal of leukemia stem cells. Finally, we show using patient-derived xenografts that antagonism of Hif1a or Wnt signaling also compromises human LIC activity, suggesting that pharmacologic targeting of these pathways could have therapeutic application in patients with T-ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals CREB is a critical regulator of normal hematopoiesis and leukemogenesis

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1182-1192 ◽  
Author(s):  
Jerry C. Cheng ◽  
Kentaro Kinjo ◽  
Dejah R. Judelson ◽  
Jenny Chang ◽  
Winston S. Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Myeloproliferative Disease ◽  
Hematopoietic Stem ◽  
Element Binding Protein ◽  
Responsive Element

AbstractThe cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation.

scholarly journals Tafazzin (TAZ) Regulates the Differentiation of AML Cells By Reducing Levels of the Phospholipid Phosphatidylethanolamine

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 788-788
Author(s):  
Ayesh K Seneviratne ◽  
Juan J Aristizabal Henao ◽  
G Wei Xu ◽  
Rose Hurren ◽  
Sejin Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Oxidative Phosphorylation ◽  
Hematopoietic Stem Cells ◽  
Cell Fate ◽  
Respiratory Chain ◽  
Conflicts Of Interest ◽  
Leukemia Cells ◽  
Mitochondrial Enzymes ◽  
Hematopoietic Stem

Abstract AML cells have unique mitochondrial characteristics with an increased reliance on mitochondrial metabolism and oxidative phosphorylation. To identify new biological vulnerabilities in the mitochondria of AML, we conducted a CRISPR knockout screen. CAS9-overexpressing human OCI-AML2 leukemia cells were transduced with a library of 91,320 sgRNAs in barcoded lentiviral vectors targeting 17,237 nuclear-encoded genes. Cells were harvested, genomic DNA was isolated, and the relative abundance of sgRNAs were determined by sequencing barcodes 14 days after puromycin selection. We focused on the sgRNAs targeting the 1050 mitochondrial proteins to identify targets in the mitochondrial proteome whose knockout reduced AML growth and viability. The cardiolipin remodeling enzyme tafazzin (TAZ) was among the top 1% of mitochondrial hits. Using individual sgRNA, we confirmed that knockout of TAZ reduced the growth of CAS9-OCI-AML2 cells by >70%, thus validating the findings from our screen. We also knocked down TAZ with 2 independent shRNA and demonstrated reductions in growth and viability of a panel of AML cells: OCI-AML2 (>80%), TEX (>50%), K562 (>50%), and U937 (>40%). Moreover, TAZ knockdown significantly reduced the engraftment of TEX leukemia cells in vivo by 80%, indicating that TAZ-knockdown reduces AML growth in vivo and can target leukemia initiating cells. In contrast, knockdown of TAZ in mouse models did not impair normal hematopoiesis nor reduced the abundance of hematopoietic stem cells, although more subtle defects in the hematopoietic stem cells might explain transient episodes of neutropenia seen in Barth's syndrome, a congenital condition associated with X-linked TAZ mutations. TAZ is responsible for the majority of Cardiolipin (CL) remodeling under physiological conditions. As expected the knockdown of TAZ in both AML and normal mouse hematopoietic cells increased the substrate (monlysocardiolipin) to product (CL) ratio of TAZ. CL is required for the proper localization, and efficient function of, respiratory chain enzymes. However, in AML cells, knockdown of TAZ did not alter respiratory chain complex activity, basal oxygen consumption, or respiratory chain reserve capacity. Recent studies have shown that mitochondrial pathways can regulate cell fate and differentiation independent of their effects on oxidative phosphorylation. Therefore, we examined changes in AML cell differentiation after TAZ knockdown. Knockdown of TAZ promoted the differentiation of AML cells as evidenced by increased non-specific esterase staining and increased CD11b expression on the cell surface. In breast cancer cells decreasing phosphatidylethanolamine (PE) levels, induced the differentiation of these cells. As TAZ regulates phospholipid remodeling, therefore we measured levels of PE and phosphatidylserine (PS) after TAZ-knockdown by spot densitometry. Interestingly, knockdown of TAZ in OCI-AML2 cells decreased PE and increased PS lipid levels. To determine whether alterations in PE and PS phospholipids are functionally important for differentiation of AML cells, we treated AML cells with MMV007285, an inhibitor of the phosphatidylserine decarboxylase (PISD), an enzyme that converts PS to PE. MMV007285 mimicked the effects of TAZ-knockdown and increased differentiation of OCI-AML2 and 8227 AML cells. In summary, the cardiolipin remodeling enzyme TAZ regulates the differentiation of AML cells by controlling levels of PS and PE, thereby highlighting a new mechanism by which phospholipids and mitochondrial enzymes regulate AML cell fate and differentiation. Moreover, PISD inhibition may be a novel therapeutic strategy to selectively promote the differentiation of AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Leukemia Stem Cells Depend on Regulated Protein Synthesis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2222-2222
Author(s):  
Yi Huang ◽  
Eda Gozel Kapti ◽  
Toby Thomas ◽  
Yuanyuan Ji ◽  
Dheepthi P. Ramasamy ◽  
...  
Keyword(s):  
Stem Cells ◽  
Protein Synthesis ◽  
High Risk ◽  
Conflicts Of Interest ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Rapamycin Treatment ◽  
Sequencing Studies

Abstract Acute myeloid leukemia (AML) is initiated and sustained by leukemia stem cells (LSCs) which arise from progenitor cells that do not usually self-renew but become aberrantly self-renewing. It is thought that LSCs gain aberrant self-renewal potential by co-opting molecular and cellular programs from hematopoietic stem cells (HSCs) (PMID: 16862118). HSCs have been shown to require tightly regulated protein synthesis rates, where increased or decreased protein synthesis impairs self-renewal (PMID: 24670665), but it is not known if LSCs share this dependence. We have shown that human LSCs reside in the population of AML cells with the highest levels of CD99 (PMID: 28123069). In RNA-sequencing studies, we found that LSCs with high levels of CD99 are depleted for ribosomal protein transcripts. We thus reasoned that similar to HSCs, LSCs may depend on tightly regulated protein synthesis to self-renew. To test if CD99 promotes LSC function by constraining protein synthesis, we transduced c-Kit+ cells from B6-CD99 Gt(pU-21T)44lmeg (CD99 KO) or wild-type (WT) mice to express AML1-ETO9a (AE9a) and transplanted them into WT mice treated with rapamycin or vehicle. There was no difference in leukemogenesis in primary recipients, but CD99 KO-AE9a AMLs exhibited a 72% (p=0.048) increase in protein synthesis compared with WT-AE9a AMLs (Figure 1A), confirming that CD99 negatively regulates protein synthesis in AML. We next performed secondary transplants to assess LSC function, as measured by survival of secondary recipients in the absence of rapamycin treatment (Figure 1B). We furthermore performed these transplants at limiting dilution to quantify LSCs (Figure 1C). CD99 KO-AE9a vehicle treated AMLs demonstrated improved survival and a lower LSC frequency compared with WT-AE9a vehicle treated AMLs, consistent with a self-renewal defect with loss of CD99. Rapamycin treatment completely rescued this defect, leading to decreased survival and increased LSC frequency in CD99 KO-AE9a AMLs compared with vehicle. Conversely, rapamycin treatment depleted LSCs in WT-AE9a AMLs, increasing survival and decreasing LSC frequency compared with vehicle. Thus, similar to HSCs, LSCs are adversely affected by both increases or decreases in protein synthesis. MLL-AF9-induced mouse AMLs initiated in HSCs as compared with granulocyte macrophage progenitors (GMPs) exhibit increased epigenetic imprinting of HSC features resulting in disease features reminiscent of high-risk AML (PMID: 23235717). To test if HSC-derived leukemias exhibit increased dependence on regulated protein synthesis, we transduced HSCs or GMPs from CD99 KO or WT mice to express MLL-AF9 and transplanted them into WT recipients, followed by secondary transplants to assess LSC function. Loss of CD99 led to increased survival indicative of decreased LSC function in HSC-derived but not GMP-derived leukemias (Figure 1D). This suggests that HSC-derived leukemias co-opt from HSCs a more pronounced dependence on tightly regulated protein synthesis. Accordingly, WT HSC-derived leukemias exhibited decreased protein synthesis as compared with their WT GMP-derived counterparts (Figure 1E), as well as increased sensitivity to rapamycin (Figure 1F). To directly study protein synthesis in human LSCs, we transduced primary AML specimens (n=4) to express a destabilized form of GFP (dGFP) from a constitutive promoter followed by xenotransplantation (Figure 1G), allowing us to measure dGFP by flow cytometry as a surrogate for protein synthesis rates in vivo. We validated this assay by measuring protein synthesis using orthogonal O-propargyl-puromycin incorporation assays (Figure 1H). Human AML cells with low levels of dGFP demonstrated increased engraftment in secondary transplants (Figure 1I), demonstrating that human LSCs exhibit low protein synthesis rates. In conclusion, our data demonstrate that LSCs co-opt from HSCs a dependence on tightly regulated protein synthesis. This is the first description of a cellular feature co-opted from HSCs that also represents a therapeutic vulnerability. Furthermore, the types of AML that exhibit the most robust re-activation of HSC programs and increased dependence on regulated protein synthesis are also likely to represent high-risk AMLs most resistant to standard therapies. Our data suggest that such therapy resistant AMLs may be highly sensitive to strategies disrupting protein synthesis to deplete LSCs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

PU.1-Mediated Upregulation of M-CSFR Is Critical for MOZ-Leukemia Stem Cell Potential.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 216-216
Author(s):  
Yukiko Aikawa ◽  
Takuo Katsumoto ◽  
Daniel G. Tenen ◽  
Issay Kitabayashi
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinases ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Suicide Gene ◽  
Leukemia Stem Cells ◽  
Monocytic Leukemia ◽  
Cell Potential ◽  
Hematopoietic Stem ◽  
Stem Cell Potential

Abstract Abstract 216 Leukemias and other cancers possess self-renewing stem cells that help to maintain the cancer. The eradication of cancer stem cells is thought to be critical for successful anti-cancer therapy. However, there is little evidence for this. Using an acute myeloid leukemia (AML) model by introducing the leukemia-associated monocytic leukemia zinc finger (MOZ)-TIF2 fusion protein, we show here that AML can be cured by the ablation of leukemia stem cells. Chromosomal translocations that involve the MOZ gene are typically associated with the FAB-M4 or -M5 subtype of human AML and often predict a poor prognosis. While MOZ is essential for the self-renewal of hematopoietic stem cells, MOZ-fusion proteins enable the transformation of non–self-renewing myeloid progenitors into leukemia stem cells. The MOZ-fusion proteins interacted with PU.1 to stimulate the expression of macrophage-colony stimulating factor receptor (M-CSFR). Cells expressing high levels of M-CSFR (M-CSFR -high cells), but not those expressing low levels of M-CSFR, showed potent leukemia-initiating activity. Using transgenic mice expressing a drug-inducible suicide gene controlled by the M-CSFR promoter, AML was cured by ablation of the M-CSFR -high cells. Analysis of M-CSFR-deficient and PU.1-deficient mice showed that M-CSFR and PU.1 was essential to induce AML. Inhibitors for tyrosine kinases including M-CSFR slowed the progress of MOZ-TIF2-induced leukemia. Thus, M-CSFR -high cells contain leukemia stem cells, and the PU.1-mediated upregulation of M-CSFR is a useful therapeutic target for MOZ leukemia. Disclosures: No relevant conflicts of interest to declare.

T(15;17)-PML/RAR-Induced Leukemogenesis: Long-Term Repopulating Hematopoietic Stem Cells as the Initial Target and More Mature Progenitors as the Potential Targets for Final Leukemic Transformation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3980-3980 ◽  
Author(s):  
Claudia Oancea ◽  
Brigitte Rüster ◽  
Jessica Roos ◽  
Afsar Ali Mian ◽  
Tatjana Micheilis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Population ◽  
Conflicts Of Interest ◽  
Cell Model ◽  
Leukemic Cell ◽  
Hematopoietic Stem ◽  
Proliferation Potential

Abstract Abstract 3980 Poster Board III-916 Stem cells have been shown to play an important role in the pathogenesis and maintenance of a significant number of malignancies, including leukemias. Similar to normal hematopoiesis the AML cell population is thought to be hierarchically organized. According to this model, only a few stem cells (LSC) are able to initiate and maintain the disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARα fusion protein. Treatment of APL with all-trans retinoic acid (t-RA) as monotherapy induces CR, but not molecular remission (CMR), followed by relapse within a few months. In contrast arsenic as monotherapy induces high rates of CR and CMR followed by a long relapse-free survival. We recently have shown that in contrast to t-RA, arsenic efficiently targets PML/RAR-positive stem cells, whereas t-RA increases their proliferation. For a better characterization of LSC in APL which has to be targeted for an efficient eradication of the disease we wanted to characterize the leukemia-initiating cell and the cell population able to maintain the disease in vivo. The model was based on a classical transduction/transplantation system of murine Sca1+/lin- HSC combined with a novel approach for the enrichment of transformed cells with long-term stem cell properties. We found that PML/RAR induced leukemia from the Sca1+/lin- HSC with a frequency of 40% and a long latency of 8-12 months independently of its capacity to increase dramatically replating efficiency and CFU-S12 potential as expression of the differentiation block and proliferation potential of derived committed progenitors. Based on the hypothesis that PML/RAR exerts its leukemogenic effects on only a small proportion of the Sca1+1/lin- population, we proceeded to select and to amplify rare PML/RAR-positive cells with the leukemia-initiating potential, by a negative selection of cell populations with proliferation potential without long term stem cell-capacity (LT). Therefore we expressed PML/RAR in Sca1+/lin- cells and enriched this population for LT- (lin-/Sca1+/c-Kit+/Flk2-) and ST-HSC (lin-/Sca1+/c-Kit+/Flk2+). After a passage first in semi-solid medium for 7 days and subsequent transplantation into lethally irradiated mice, cells from the ensuing CFU-S day12 were again transplanted into sublethally recipient mice. After 12 to 36 weeks, 6/6 mice developed acute myeloid leukemia without signs of differentiation in the group transplanted with the lin-/Sca1+/c-Kit+/Flk2- population but not from that transplanted with lin-/Sca1+/c-Kit+/Flk2+ cells. This leukemia was efficiently transplanted into secondary recipients. The primary leukemic cell population gave origin to 6 clearly distinct subpopulations defined by surface marker pattern as an expression of populations with distinct differentiation status, able - after sorting - to give leukemia in sublethally irradiated recipients: Sca1+/c-Kit+/CD34- (LT-HSC), Sca1+/c-Kit+/CD34+ (ST-HSC), Sca1-/c-Kit+, B220lo/GR1+/Mac1+, B220hi/GR1+/Mac1+, B220-/Gr1-/Mac1-. Interestingly, all leukemias from the different population presented an identical phenotype. These findings strongly suggest that there is a difference between a leukemia-initiating (L-IC) and leukemia-maintaining (L-MC) cell population in the murine PML/RAR leukemia model. In contrast to the L-IC, represented by a very rare subpopulation of primitive HSC, recalling a hierarchical stem cell model, the L-MC is represented by a larger cell population with a certain grade of phenotypical heterogeneity, but a high grade of functional homogeneity recalling a stochastic cancer induction model. Disclosures: No relevant conflicts of interest to declare.

Counterpoint: In Some AMLs, Stem Cells are More Mature and Present at High Frequency.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. SCI-35-SCI-35
Author(s):  
Michael L. Cleary
Keyword(s):  
Stem Cells ◽  
Mouse Models ◽  
Conflicts Of Interest ◽  
Human Cancer ◽  
Hierarchical Organization ◽  
Leukemia Stem Cells ◽  
Aberrant Expression ◽  
Myeloid Lineage ◽  
Hematopoietic Stem ◽  
Current Paradigm

Abstract Abstract SCI-35 Leukemia stem cells (LSCs) are responsible for sustaining and propagating malignant disease and, therefore, are promising targets for therapy. The current paradigm for LSC frequency, maturation and hierarchical organization is primarily based on transplantation studies in xenograft mouse models. To circumvent potential limitations of this experimental approach, investigators have recently employed syngeneic mouse models to study LSCs. In a mouse model of AML initiated by MLL oncogenes, which are associated with the FAB-M4 or M5 subtypes of human AML, LSCs are remarkably frequent, accounting for up to one-quarter of malignant myeloid cells at late-stage disease. Even in this syngeneic setting, however, transplant assays alone markedly underestimate LSC frequency due to poor engraftment efficiency. LSCs are organized in a phenotypic and functional hierarchy, and express myeloid lineage-specific antigens, placing them downstream of the known hematopoietic progenitor compartments. Thus, LSCs in this model are not synonymous with normal upstream progenitors that are targeted for leukemia initiation, but rather constitute myeloid lineage cells that have acquired an aberrant self-renewal program as well as other biologic features of hematopoietic stem cells. Gene expression profiling confirms the downstream myeloid character of LSCs in this model, and further demonstrates the aberrant expression of a stem cell associated transcriptional subprogram. However, LSC maintenance in the self-renewing compartment of AML employs a global transcriptional program more akin to embryonic rather than adult stem cells. Expression of LSC maintenance program genes is enriched in poor prognosis human malignancies, suggesting that the frequency of aberrantly self-renewing progenitor-like cancer stem cells may be linked to prognosis in human cancer. Consistent with this possibility, LSC frequencies in different syngeneic models of Hox-associated AML can vary over three orders of magnitude, depending on the particular initiating oncogene and expression levels of Hox pathway co-regulators, and correlate with leukemia biology. Studies in a human cord blood cell transduction/transplantation model of AML further support the downstream character of MLL LSCs. These findings prompt a revision of the current paradigm that AML leukemia stem cells are always rare and solely located within the most immature bone marrow progenitor compartments. The fact that LSCs can be more analogous to precursors and employ ESC-like genetic programs for their maintenance, may allow for their selective therapeutic targeting that spares HSCs required for hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Profilin 1 Is Essential for Retention and Metabolism of Hematopoietic Stem Cells in Bone Marrow

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1224-1224
Author(s):  
Junke Zheng ◽  
Chengcheng Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Actin Binding ◽  
Wild Type ◽  
Cell Fates ◽  
Hematopoietic Stem ◽  
Multiple Cell

Abstract Abstract 1224 How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we show that, in a hematopoietic stem cell (HSC) -specific inducible knockout model, the cytoskeleton-modulating protein profilin 1 (pfn1) is essential for the maintenance of multiple cell fates and metabolism of HSCs. The deletion of pfn1 in HSCs led to bone marrow failure, loss of quiescence, increased apoptosis, and mobilization of HSCs in vivo. In reconstitution analyses, pfn1-deficient cells were selectively lost from mixed bone marrow chimeras. By contrast, pfn1 deletion did not significantly affect differentiation or homing of HSCs. When compared to wild-type cells, levels of expression of Hif-1a, EGR1, and MLL were lower and an earlier switch from glycolysis to mitochondrial respiration with increased ROS level was observed in pfn1-deficient HSCs. This switch preceded the detectable alteration of other cell fates. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that pfn1 maintained metabolism is required for the quiescence of HSCs. Furthermore, we demonstrated that expression of wild-type pfn1 but not the actin-binding deficient or poly-proline binding-deficient mutants of pfn1 rescued the defective phenotype of pfn1-deficient HSCs. This result indicates that actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Thus, pfn1 plays an essential role in regulating the retention and metabolism of HSCs in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Stem Cell Factor Sall4 Regulates Normal and Malignant Hematopoiesis In an Isoform-Specific Manner

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3728-3728
Author(s):  
Samuel Milanovich ◽  
Jeremy Allred ◽  
Jonathan Peterson ◽  
Cary Stelloh ◽  
Sridhar Rao
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem ◽  
Empty Vector ◽  
Bmi1 Expression ◽  
Colony Forming Units ◽  
Colony Forming Capacity

Abstract Stem cells play key roles in early normal development (e.g. embryonic stem cells (ESCs)), maintenance of adult organs (e.g. hematopoietic stem cells (HSCs)) and in some cancers (e.g. leukemia stem cells). To what degree these different types of stem cells rely upon shared versus distinct transcriptional programs remains controversial. Sall4 is a zinc finger transcription factor that exists in two distinct splice isoforms, Sall4a (long) and Sall4b (short). Sall4 has been implicated in embryonic, hematopoietic and malignant stem cell transcriptional regulation. Additionally, Sall4 has been proposed as a potential means of ex-vivo hematopoietic stem cell expansion prior to transplantation. Sall4 isoform-specific differences have been described in ESCs, with Sall4b shown to be critical for maintaining ESC “stemness”. Here we investigate the role of Sall4 isoforms in pediatric acute myeloid leukemia (AML) and murine hematopoiesis to unravel shared versus unique transcriptional programs across different stem cell types. Quantitative real time PCR shows that Sall4b is the predominant Sall4 isoform in murine HSCs and lin-, Sca1+, cKit+ (LSK) cells. Sall4b expression decreases in early lineage-committed progenitors, while Sall4a expression is minimal to absent across murine HSCs and progenitors. Next, we evaluated seven pediatric AML samples and found highly variable Sall4 expression across AML cases. All samples had measurable Sall4a and Sall4b; in 3/7 cases Sall4a and Sall4b expression was similar to that of ESCs, in the other 4 cases Sall4 expression was minimal (<3% of ESCs). To study overexpression of Sall4, we used a murine stem cell retrovirus system to express Sall4a or Sall4b. Bone marrow was harvested from C57/BL6 mice and lineage-committed cells were removed by magnetic column separation. Lineage-negative bone marrow was infected with either empty vector, Sall4a or Sall4b. Transduced bone marrow was then cultured in methylcellulose media to assess colony forming capacity and proliferation in vitro or transplanted in syngeneic mice to assess engraftment and hematopoietic reconstitution in vivo. Sall4a or Sall4b overexpression caused diminished colony forming capacity and cellular proliferation in vitro compared to bone marrow transduced with empty vector (Figure 1). In bone marrow transplant assays, all mice (4/4) transplanted with Sall4b-transduced bone marrow following lethal irradiation succumbed to bone marrow failure within 10 days of transplant. Transplantation of Sall4b-transduced bone marrow into sublethally irradiated mice failed to contribute to hematopoiesis as measured by peripheral blood leukocyte GFP expression (encoded by the viral vector). Together, this data shows that Sall4b-transduced hematopoietic cells fail to engraft and reconstitute hematopoiesis in vivo. We postulated that this phenotype might be mediated through the interaction of Sall4 with Bmi1. Bmi1 is a member of the polycomb complex necessary for normal hematopoiesis, and is known to be bound by Sall4. In preliminary experiments, we have found that overexpression of Sall4 leads to decreased Bmi1 expression at 48 hours post-infection compared to bone marrow infected with empty vector.Figure 1Lin- bone marrow expressing Sall4a, Sall4b or empty vector was cultured in methylcellulose; plates were flushed and replated out to three generations. Colony forming units were assessed (A) and viable cells were counted (B) after 7-10 days in culture.Figure 1. Lin- bone marrow expressing Sall4a, Sall4b or empty vector was cultured in methylcellulose; plates were flushed and replated out to three generations. Colony forming units were assessed (A) and viable cells were counted (B) after 7-10 days in culture. In conclusion, our data shows that Sall4b is expressed in murine hematopoietic stem cells and progenitors, suggesting that Sall4b but not Sall4a influences a hematopoietic cell fate. Additionally, Sall4 expression is variable in AML specimens, implicating a potential pathogenic role in some leukemias, while others are Sall4-independent. Lastly, Sall4 overexpression is associated with decreased expression of the critical hematopoietic gene Bmi1. Together this data suggests that hematopoiesis is dependent upon appropriately regulated Sall4 expression with alterations leading to impaired proliferation and self-renewal. These effects on hematopoiesis appear to be mediated at least in part through a dose-dependent effect on Bmi1 expression. Future studies will evaluate other genes targeted by Sall4 in hematopoiesis and leukemia to define Sall4-dependent gene signatures in normal versus malignant hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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