Overexpression of APC Reduces Stem Cell Capacity and Leukemogenic Potential of PLZF/RAR-Positive Leukemic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 874-874
Author(s):  
Anita Seshire ◽  
Tim Beissert ◽  
Claudia Oancea ◽  
Dieter Hoelzer ◽  
Martin Ruthardt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Affinity Purification ◽  
Wnt Signaling Pathway ◽  
Direct Interaction ◽  
Molecular Therapy ◽  
Leukemic Stem Cells ◽  
Adenomatous Polyposis Coli Protein ◽  
Self Renewal

Abstract Acute myeloid leukemia (AML)-associated fusion proteins (AAFP), such as PML/RAR, PLZF/RAR or AML1/ETO have leukemogenic potential in vivo which is related to their capacity to induce a differentiation block and aberrant self renewal in early hematopoietic progenitors. The potential of the AAFP to induce the leukemic phenotype is strictly related to their capacity to oligomerize and to form high molecular weight complexes (HMW). Furthermore we recently showed that the deregulation of the Wnt-signaling seems to be a key event for the leukemogenesis by the above mentioned AAFP because it is indispensable for the aberrant self renewal of leukemic stem cells (LSC). A „tandem affinity purification” (TAP) screen of KG1 cells expressing PLZF/RAR, PML/RAR or AML-1/ETO for elucidating the composition of the related HMW revealed the „adenomatous polyposis coli” protein (APC) as an interaction partner of PLZF/RAR. APC is a key inhibitory regulator of the Wnt-signaling and is frequently inactivated by mutations in colon carcinomas. Therefore the role of the interaction between APC and PLZF/RAR for the leukemogenesis was further investigated. Here we report that i.) only PLZF/RAR strongly interacted with APC as confirmed by co-immunoprecipitation experiments; ii.) the overexpression of APC reverted the aberrant activation of the Wnt-signaling by PLZF/RAR as revealed by a reduction of the TCF/LEF mediated transcriptional activity; iii.) the overexpression of APC also reduced the self renewal potential of PLZF/RAR-positive HSC as revealed by a reduced replating efficiency of these cells in semi solid medium, as well as by a reduction of the colony number in colony forming units spleen (CFU-S) assays; iv.) APC was able to revert the leukemogenic potential of PLZF/RAR-positive LSC as revealed by the retroviral overexpression of APC in bone marrow isolated from mice with PLZF/RAR-induced AML which were inoculated into secondary recipients. In fact until now the presence of APC significantly increased the survival of these secondary recipients with respect to mock-infected controls. Taken together these data strongly suggest that the direct interaction with PLZF/RAR is an important mechanism for the functional inactivation of APC which contributes to the aberrant activation of the Wnt-signaling in PLZF/RAR-positive cells. Furthermore our data provide further evidence that the aberrantly activated Wnt-signaling pathway represents a valid target for molecular therapy approaches.

scholarly journals Silencing of long noncoding RNA HOXA11-AS inhibits the Wnt signaling pathway via the upregulation of HOXA11 and thereby inhibits the proliferation, invasion, and self-renewal of hepatocellular carcinoma stem cells

2019 ◽  
Vol 51 (11) ◽  
pp. 1-20 ◽  
Author(s):  
Jun-Cheng Guo ◽  
Yi-Jun Yang ◽  
Jin-Fang Zheng ◽  
Jian-Quan Zhang ◽  
Min Guo ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Stem Cells ◽  
Stem Cell ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Methylation Level ◽  
Wnt Signaling Pathway ◽  
The Self ◽  
Self Renewal

AbstractHepatocellular carcinoma (HCC) is a major cause of cancer-related deaths, but its molecular mechanisms are not yet well characterized. Long noncoding RNAs (lncRNAs) play crucial roles in tumorigenesis, including that of HCC. However, the role of homeobox A11 antisense (HOXA11-AS) in determining HCC stem cell characteristics remains to be explained; hence, this study aimed to investigate the effects of HOXA11-AS on HCC stem cell characteristics. Initially, the expression patterns of HOXA11-AS and HOXA11 in HCC tissues, cells, and stem cells were determined. HCC stem cells, successfully sorted from Hep3B and Huh7 cells, were transfected with short hairpin or overexpression plasmids for HOXA11-AS or HOXA11 overexpression and depletion, with an aim to study the influences of these mediators on the self-renewal, proliferation, migration, and tumorigenicity of HCC stem cells in vivo. Additionally, the potential relationship and the regulatory mechanisms that link HOXA11-AS, HOXA11, and the Wnt signaling pathway were explored through treatment with Dickkopf-1 (a Wnt signaling pathway inhibitor). HCC stem cells showed high expression of HOXA11-AS and low expression of HOXA11. Both HOXA11-AS silencing and HOXA11 overexpression suppressed the self-renewal, proliferation, migration, and tumorigenicity of HCC stem cells in vivo, as evidenced by the decreased expression of cancer stem cell surface markers (CD133 and CD44) and stemness-related transcription factors (Nanog, Sox2, and Oct4). Moreover, silencing HOXA11-AS inactivated the Wnt signaling pathway by decreasing the methylation level of the HOXA11 promoter, thereby inhibiting HCC stem cell characteristics. Collectively, this study suggested that HOXA11-AS silencing exerts an antitumor effect, suppressing HCC development via Wnt signaling pathway inactivation by decreasing the methylation level of the HOXA11 promoter.

scholarly journals Dickkopf Wnt signaling pathway inhibitor 1 regulates the differentiation of mouse embryonic stem cells in vitro and in vivo

2015 ◽  
Vol 13 (1) ◽  
pp. 720-730 ◽  
Author(s):  
LIPING OU ◽  
LIAOQIONG FANG ◽  
HEJING TANG ◽  
HAI QIAO ◽  
XIAOMEI ZHANG ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Wnt Signaling Pathway ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells

Inhibition of CML Stem Cell Renewal By the Porcupine Inhibitor WNT974

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 54-54
Author(s):  
Puneet Agarwal ◽  
Bin Zhang ◽  
Yinwei Ho ◽  
Amy Cook ◽  
Ling Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Wnt Signaling ◽  
Cyclin D1 ◽  
Biomedical Research ◽  
Cell Renewal ◽  
Self Renewal ◽  
Cd34 Cells ◽  
Tki Treatment

Abstract Treatment of chronic myeloid leukemia (CML) with BCR-ABL tyrosine kinase inhibitors (TKIs) fails to eradicate the leukemia stem cells (LSCs) from which the disease arises. Others and we have shown that extrinsic signals from the bone marrow (BM) microenvironment play an important role in the resistance of CML LSC to TKI treatment. Our studies indicate that microenvironmental Wnt signaling may play a role for in protecting CML LSC from TKI treatment (Blood. 2013; 121(10):1824-38). Wnt secretion and activity requires their palmitoylation by the Porcupine acyltransferase (PORCN). WNT974 is a potent PORCN inhibitor that inhibits Wnt signaling and demonstrates in vivo efficacy against Wnt-dependent tumors (PNAS. 2013; 110(50):20224-9). We have investigated whether WNT974 could sensitize CML stem/progenitor cells to TKI treatment. We observed that CML CD34+ cells show enhanced increased phospho-LRP6 (ser1490) expression, β-catenin protein levels, and colony-forming cell (CFC) growth following exposure to recombinant Wnt3a (200ng/ml) compared to normal CD34+ cells (isolated from cord blood of healthy donors) indicating an enhanced Wnt signaling response. We observed significant upregulation of FZD4 and FZD5 expression in CML compared to normal CD34+ cells on qPCR analysis, which could potentially contribute to their enhanced Wnt sensitivity. CML CD34+ cells enhanced Wnt signaling following co-culture with human bone marrow mesenchymal stromal cells (MSC) as evidenced by increased β-catenin expression and nuclear translocation, and increased expression of the Wnt target genes c-Myc, Cyclin-D1, PPARδ, and Axin2. WNT974 exposure reduced Wnt secretion from MSC and inhibited Wnt signaling in CML CD34+ cells both in the absence and presence of MSC. Treatment with WNT974 (1μM) in combination with nilotinib (Nil, 1μM) resulted in significant inhibition of CML CFC growth, both in the presence and absence of MSC, and to a significantly greater extent than the normal CFC growth. Although, no difference in engraftment of normal CD34+ cells treated with individual drugs or combination in immunodeficient mice after 16 weeks was observed, treatment of CML CD34+ cells with WNT974 + Nil resulted in significantly decreased engraftment of BCR-ABL+ CML LSC. We further investigated the effects of WNT974 and Nil on CML hematopoiesis in vivo using a transgenic BCR-ABL mouse model of CML. BM cells from CML mice were transplanted into congenic wild-type FVB/N mice to generate CML-like disease in recipient mice. Three weeks after transplantation, mice were treated with Vehicle, WNT974 (5mg/kg bid p.o), Nil (50mg/kg qd p.o), or the combination for 2 weeks. Treatment with WNT974 + Nil resulted in significantly greater reduction in WBC levels, %neutrophils and myeloid cells (Gr-1+ CD11b+) in the peripheral blood of CML mice compared with Nil alone. Long-term hematopoietic stem cells (LTHSC) and committed progenitors (MPP, CMP, GMP) were significantly reduced in the BM and spleen of mice treated with combination compared to individual drugs. Next, we evaluated subsequent survival of mice after completion of 3 weeks of drug treatment. Whereas all control mice died by 30 days, mice treated with the combination of WNT974 + Nil showed significantly prolonged survival after discontinuation of treatment when compared to individual drug-treated mice (p=0.0146). To assess the effects on LSC self-renewal potential, BM cells from primary drug-treated mice were transplanted into normal secondary recipient mice. At 12 weeks post-transplantation, there was a significant reduction in LTHSC, MPP, and GMP populations in the BM of mice transplanted with cells from primary mice treated with the combination compared with individual drugs, indicating reduced LSC self-renewal. Finally, we evaluated the effect of in vivo treatment on Wnt/β-catenin signaling in leukemic cells. Significant downregulation of c-Myc, Cyclin D1, and Axin2 was seen on qPCR performed on c-Kit+ BM progenitors from mice treated with WNT974 and Nil+WNT974 compared to vehicle treated mice. Therefore, we conclude that inhibition of Wnt secretion and signaling by treatment with the PORCN inhibitor WNT974 effectively and selectively inhibits the self-renewal capacity of CML LSC. These results support the further evaluation of combinations of PORCN inhibitors with TKI treatment for selective targeting of CML LSC. Disclosures Wang: Novartis Institute of Biomedical Research: Employment. Mclaughlin:Novartis Institute of Biomedical Research: Employment.

GPR84, a Proinflammatory Receptor, Sustains Wnt/β-Catenin Signaling In Leukemic Stem Cells For Maintenance Of MLL-AF9-Induced Leukemogenesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3781-3781
Author(s):  
Philipp A Dietrich ◽  
Murray D Norris ◽  
Jenny Yingzi Wang
Keyword(s):  
Stem Cells ◽  
Conflicts Of Interest ◽  
Active Form ◽  
Leukemic Stem Cells ◽  
Poor Survival ◽  
Functional Link ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Inappropriate activation of Wnt/β-catenin signaling confers hematopoietic progenitors the property of self-renewal that promotes malignant transformation in MLL-rearranged acute myeloid leukemia (AML). However, it has been noted that activation of β-catenin is observed in tumors without clear mutations in the major components of the pathway or increase in Wnt signaling. This suggests that other developmental signaling pathways may be capable of inducing activation or downstream signaling of β-catenin. Recently, a number of G protein-coupled receptors (GPCRs) have been shown to activate β-catenin signaling to recruit the key downstream components of the canonical Wnt pathway in distinct cell types, including stem cells. GPCRs, the largest family of cell-surface molecules involved in signal transmission, have emerged as crucial players in tumor growth and metastasis, and represent one of the most important drug targets in pharmaceutical development. Given the close functional link with activation of β-catenin signaling, a GPCR signaling pathway may act as the upstream regulator of β-catenin signaling in the establishment of leukemic stem cells (LSC). In this study, our microarray analysis comparing genes differentially expressed between LSC and normal hematopoietic stem cells (HSC) identified GPR84, a proinflammatory GPCR, as a potential LSC-specific candidate target. An analysis of the comprehensive patient outcome database (Oncogenomics – maintained by the National Cancer Institute) showed that high levels of GPR84 were significantly associated with poor survival in patients with leukemia (P=0.0048), implying its potential clinical relevance in predicting disease prognosis. Western blot and flow cytometric analyses confirmed the microarray results and revealed a positive correlation between GPR84 and β-catenin expression. We previously demonstrated that β-catenin was highly expressed in HSC transformed by MLL-AF9 (HSC-MLLAF9) and had lower expression in HSC transduced with leukemic oncogenes Hoxa9/Meis1 (HSC-Hoxa9/Meis1), while increased β-catenin expression was correlated with a poor survival rate in vivo. Herein, our results showed that forced expression of GPR84 induced a robust upregulation of β-catenin in HSC-Hoxa9/Meis1. Conversely, shRNA-mediated ablation of GPR84 in HSC-MLLAF9 led to highly significant downregulation of both GPR84 (P=0.0003) and β-catenin (P=0.0008). Further in vitro functional studies showed that GPR84 knockdown significantly reduced HSC-MLL-AF9 colony forming units (P=0.0006), and induced a marked reduction of cells in S-phase (P=0.0017). This deficient phenotype could be rescued by expression of a constitutively active form of β-catenin. Importantly, subsequent in vivo survival studies using leukemia transplantation mouse models showed that GPR84 knockdown significantly reduced LSC frequency and severely impaired maintenance (P<0.0001; 11 mice per cohort) of HSC-MLL-AF9 induced leukemia, a highly aggressive and drug-resistant subtype of AML. The defect in disease phenotype resulted from inhibited expression of both GPR84 and β-catenin. Furthermore, forced overexpression of GPR84 alone was not sufficient for leukemic transformation of HSC but conferred a growth advantage in vivo to HSC-Hoxa9/Meis1 cells and significantly accelerated the onset of Hoxa9/Meis1-induced AML (P=0.0039), establishing a completely malignant phenotype similar to HSC-MLL-AF9 in vivo (P=0.9986). These data support an oncogenic role of GPR84 in MLL-AF9-induced leukemogenesis. In conclusion, our studies have identified a novel β-catenin regulator that contributes to leukemia maintenance by sustaining aberrant activation of a stem cell self-renewal pathway in LSC, and drugs targeting GPR84 may represent a novel and promising strategy for improving the therapy and outcome of AML patients. Disclosures: No relevant conflicts of interest to declare.

Polycomb Repressive Complex 1 Enhances HSC Self-Renewal through Repressing the Canonical Wnt Signaling Pathway

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2378-2378
Author(s):  
Sisi Chen ◽  
Xicheng Liu ◽  
Rui Gao ◽  
Michihiro Kobayashi ◽  
Hao Yu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Wnt Signaling Pathway ◽  
Canonical Wnt Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Canonical Wnt

Abstract Polycomb group (PcG) proteins are epigenetic gene silencers that have been implicated in stem cell maintenance and cancer development. Genetic and biochemical studies indicate that Polycomb group proteins exist in at least two protein complexes, Polycomb repressive complex 2 (PRC2) and Polycomb repressive complex 1 (PRC1), that act in concert to initiate and maintain stable gene repression. While studies on individual PRC1 component suggest that PRC1 plays an important role in hematopoiesis, how PRC1 regulates transcriptional repression in hematopoietic stem cells (HSCs) is largely unknown. Bmi1 and Mel18 are two major homologs of the PCGF subunit within the PRC1 complex. Bmi1 is a positive regulator of HSC self-renewal; however, the role of Mel18 in hematopoiesis has been controversial. To determine whether Bmi1 and Mel18 play redundant or distinct role in HSC self-renewal, we have generated Bmi1 and Mel18 conditional knockout mice. While acute deletion of Mel18 affects neither HSC frequency nor lineage commitment, we found that Mel18-deficent hematopoietic progenitor cells showed enhanced replating potential compared to wild type cells. To determine the role of Mel18 in HSC self-renewal, we performed serial HSC transplantation assays and found that the repopulating ability of Mel18-/- HSCs was significantly higher than WT HSCs in both primary and secondary transplantation assays, demonstrating that the loss of Mel18 enhances HSC self-renewal in vivo. We hypothesize that loss of Bmi1 and Mel18 in hematopoietic stem cells will disrupt PRC1 complex and impairs HSC self-renewal. To determine the role of PRC1 complex in HSCs, we analyzed the HSC behavior in Bmi1 and Mel18 double-deficient mice. While we found that Bmi1-deficient HSCs showed decreased repopulating potential compared to WT HSCs 16 weeks following transplantation, loss of both Bmi1 and Mel18 in HSCs resulted in even more severe self-renewal defects. In addition, loss of both Bmi1 and Mel18 resulted in decreased myeloid differentiation and increased B cell differentiation compared to WT, Mel18 KO, and Bmi1 KO mice. These data demonstrate that Bmi1 and Mel18 have non-overlapping role in HSC maintenance and lineage commitment. Given that Bmi1 plays a dominant role in the PRC1 complex, we decided to identify Bmi1 target genes in hematopoietic stem cells to understand how PRC1 complex regulates HSC self-renewal. We performed transcript profiling assays to compare gene expression in HSCs isolated from wild type and Bmi1-/- mice. The Ingenuity Pathways indicates that the canonical Wnt signaling is significantly elevated in Bmi1 null HSCs compared to WT HSCs. We confirmed the upregulation of several genes of the Wnt pathway in Bmi1 null HSCs by quantitative real-time PCR analysis. To determine whether Bmi1 can repress the activation of Wnt signaling in cells, we utilized the Top-Flash Wnt reporter system. Stimulation of 293T cells with Wnt3a activates the Wnt reporter and this activation can be efficiently repressed by Bmi1. Furthermore, we detected the association of Bmi1 with the Lef1, Tcf4, and Axin2 promoters in Baf3 cells by ChIP experiment. Thus, Bmi1 directly represses the expression of several Wnt genes in hematopoietic cells. To determine the functional significance of activation of Wnt signaling in Bmi1 null HSCs, we have generated R26StopFL Bmi1-Apcf/f-Mx1-Cre+ and Bmi1f/f-Ctnnb1f/f-Mx1-Cre+ mice. Loss of Apc in hematopoietic cells activates the Wnt signaling pathway and impairs HSC self-renewal. We found that expressing three-copies of Bmi1 from the Rosa26 locus enhanced the self-renewal capabilities of Apc deficient HSCs in transplantation assays. Ctnnb1 encodes b-catenin and loss of Ctnnb1 in HSCs diminishes Wnt signaling. Acute deletion of Bmi1 in hematopoietic compartments resulted in decreased bone marrow cellularity and enhanced apoptosis of hematopoietic stem and progenitor cells. Deletion of Ctnnb1 in Bmi1 null hematopoietic cells rescued these defects. Thus, impaired HSC self-renewal seen in Bmi1 null mice is, at least in part, due to activation of the canonical Wnt signaling pathway. Taken together, we demonstrate that PRC1 complex enhances HSC self-renewal through inhibiting the canonical Wnt signaling. Disclosures No relevant conflicts of interest to declare.

scholarly journals CARF promotes spermatogonial self-renewal and proliferation through Wnt signaling pathway

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Wenhao Cui ◽  
Xiaoli He ◽  
Xiaohong Zhai ◽  
Huan Zhang ◽  
Yuanwei Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Knockout Mice ◽  
Physiological Function ◽  
Wnt Signaling Pathway ◽  
Pathogenic Mutation ◽  
Self Renewal

AbstractCollaborator of ARF (CARF) regulates cell proliferative fate through both p53-dependent and -independent mechanisms. Recently, we reported a new function of CARF as a positive regulator of Wnt signaling. Despite these findings, the physiological function of CARF has not been well studied. Here, we generated CARF knockout mice and found that male CARF−/− mice exhibited significantly impaired fertility and Sertoli-cell-only (SCO) syndrome phenotypes. Further studies revealed that loss of CARF in Sertoli cells led to decreased GDNF expression, which hindered spermatogonial stem cells (SSCs) self-renewal. Meanwhile, CARF loss in undifferentiated spermatogonia impaired their proliferation. These two mechanisms together led to SCO syndrome phenotypes, which could be functionally rescued by pharmacological or genetic reactivation of Wnt signaling. Finally, we identified CARFS351F as a potential pathogenic mutation in an SCO patient. Overall, our findings reveal important roles of CARF in spermatogonial self-renewal and proliferation through the Wnt signaling pathway.

Leukemia Stem Cells Are Resistant to In Vivo, Cell Non-Autonomous Wnt Inhibition.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1025-1025
Author(s):  
Steven W. Lane ◽  
Cristina Lo Celso ◽  
Stephen M Sykes ◽  
Sebastian Shterental ◽  
Mahnaz Paktinat ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Wnt Signaling ◽  
Bone Marrow Microenvironment ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Dkk1 Expression ◽  
Hsc Niche

Abstract Abstract 1025 Poster Board I-47 Acute myeloid leukemia (AML) initiating cells reside within and utilize the bone marrow microenvironment, as a sanctuary to evade chemotherapy and to maintain self-renewal. Following treatment, these leukemia stem cells (LSC) re-emerge and reconstitute disease, leading to relapse. The canonical Wnt signaling pathway is frequently dysregulated in LSC and recent data indicates that Dkk1 (a potent endogenous Wnt inhibitor) may have a therapeutic role in treating AML. Microenvironment specific Dkk1 expression inhibits hematopoietic stem cell (HSC) Wnt and extinguishes HSC self-renewal in vivo, identifying the Wnt pathway as essential in normal HSC-niche homeostasis. We investigated the importance of bone marrow microenvironment Wnt signaling in LSC survival. AML was generated using retroviral transduction of murine bone marrow with the MLL-AF9 fusion oncogene. We then assessed the potential for niche-directed Wnt inhibition of LSC using 2.3kbColl1alpha-Dkk1 transgenic mice in which Dkk1 expression is restricted to osteoblasts. AML was observed in the Dkk1 or wild type mice with similar disease latency and phenotype. AML was also observed in secondary transplant recipients, although there was a reduction of LSC (linlowcKithighSca-1-FcGRII/III+CD34+) derived from Dkk1 mice (LSC frequency 2.8% WT vs 1.6% Dkk1, p<0.05), correlating with a subtle prolongation in disease latency (n=15, 20 days WT vs. 24 days Dkk1, p<0.001). To determine the status of Wnt signaling in MLL-AF9 AML, we generated AML in bone marrow derived from TOPGal reporter mice that harbor a Tcf/Lef responsive promoter with a LacZ reporter, and quantified LacZ expression or galactosidase protein levels. Wnt activation was increased following transformation of bone marrow with MLL-AF9 (relative TOPGal expression 1.35 empty vector vs 2.58 MLLAF9, p=0.03). To assess the effects of osteoblast-restricted Dkk1 expression in vivo, Wnt signaling was measured in LSC purified by high-speed multiparameter flow cytometry. Reporter activity (fluorescein di-β-D-galactopyranoside (FDG), Invitrogen) was unchanged in LSC from WT or Dkk1 recipients (Median fluorescent intensity 552 vs 542, p=0.85), indicating that, in contrast with normal HSC, Wnt signaling in LSC is relatively resistant to Dkk1 expression in the niche. To better understand the mechanism of LSC resistance to Dkk1, we examined the homing and micro-localization of LSC in vivo using live, 3 dimensional two photon-confocal hybrid imaging of the bone marrow microenvironment. LSC proliferate with similar kinetics in Dkk1 or WT recipients (proliferating fraction 57.7% WT vs 50.3% Dkk1 LSC p=0.48). However, when compared to HSC, LSC home with less affinity to osteoblasts and may escape the effects of osteoblast specified Dkk1 expression through residence in a niche that is physically distant from endosteum (Median distance to osteoblast 18um WT vs 20.6um Dkk1 LSC, p=0.13). Taken together, these data indicate that MLL-AF9 LSC can escape the normal HSC-niche homeostatic constraints regulated by Wnt, an observation that may have important therapeutic implications. Disclosures: Scadden: Fate Therapeutics: Consultancy. Gilliland:Merck: Employment.

Differential Dependence On Wnt Signaling Allows Chemical Mediated Eradication of Human Acute Leukemia without Affecting Normal Blood Stem Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3278-3278
Author(s):  
Ruth M. Risueño ◽  
Eva Szabo ◽  
Uyen Maria Dang ◽  
Marilyne Levadoux-Martin ◽  
Jae-Uk Chung ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Small Molecule ◽  
Ex Vivo ◽  
Parent Compound ◽  
Canonical Wnt Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Canonical Wnt

Abstract Abstract 3278 Specific targets of signaling pathways that control self-renewal and survival of acute myeloid leukemic stem cells (LSCs) vs. normal hematopoietic stem cells (HSCs) are largely unknown. Here, using a small molecule (CWP232228) derived from a parent compound that inhibits Wnt/TCF targets, we demonstrate reduction of primary human AML-blast growth and clonogenic capacity ex vivo, without effects on normal hematopoietic progenitors. Upon establishment of AML or normal hematopoiesis in immune-deficient recipients, in vivo administration of CWP232228 reduced leukemic disease and abolished LSC self-renewal, with no effect on normal HSC function. In vivo gene profiling and ex vivo molecular studies revealed that CWP232228 induces apoptosis and differentiation of AML-blasts via inhibition of Wnt/b-catenin signaling and activation of non-canonical Wnt signaling which phenocopies the effects of this small molecule. Our study reveals an in vivo differential dependence of AML on canonical vs. non-canonical Wnt signaling that allows therapeutic targeting of LSCs whilst sparing normal HSCs. Disclosures: Chung: Choongwae Pharma Corporation: Employment.

scholarly journals OS4 - 161 Activated Wnt Signaling for the Therapeutic Targeting of Treatment-Refractory Medulloblastoma Stem Cells

2016 ◽  
Vol 43 (S4) ◽  
pp. S2-S3
Author(s):  
B. Manoranjan ◽  
S. Mahendram ◽  
D. Bakhshinyan ◽  
M. Kameda-Smith ◽  
C. Venugopal ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Gene Signature ◽  
Beta Catenin ◽  
Self Renewal ◽  
Distinct Subgroup ◽  
Treatment Paradigm ◽  
Context Specific

Brain tumours represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant pediatric brain tumour. Current molecular Nsubgroups of MB recognize distinct disease entities of which activated Wnt signaling (monosomy 6, exon 3 mutations in CTNNB1, and Wnt gene signature) is associated with a distinct subgroup and the best overall outcome. In contrast, only non-Wnt MBs are characterized by metastatic disease, increased rate of recurrence, and poor overall survivorship. Given the excellent clinical outcome in patients with Wnt-driven MB, we aimed to convert treatment-resistant MB subgroups into an ostensibly benign tumour through selective targeting by small molecule Wnt agonists (Wnt3A), GSK3 inhibitors (CHIR99021), and transgenic lines containing a stabilized beta-catenin mutant. Activated Wnt signaling resulted in decreased in vitro self-renewal and promoted differentiation within primary human MB stem cells. The clinical relevance of these findings were demonstrated with an in vivo survival advantage in mice containing orthotopic injections of cells containing a stabilized beta-catenin mutant representative of constitutively active Wnt signaling. Xenografts generated from Wnt-activated tumours were much smaller in size, maintained a much lower rate of proliferation, and reduction in key MB stem cell self-renewal genes (Bmi1, Sox2, Msi1, FoxG1). Our work establishes activated Wnt signaling as a novel treatment paradigm in childhood MB, while providing evidence for the context-specific tumour suppressive function of the canonical Wnt pathway.

scholarly journals Rspo-LGR4 Cooperates with HOXA9 to Sustain Self-Renewal in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2669-2669
Author(s):  
Nunki Hassan ◽  
Basit Salik ◽  
Alastair Duly ◽  
Jenny Yingzi Wang
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Myeloid Differentiation ◽  
Beta Catenin ◽  
Leukemic Stem Cells ◽  
Self Renewal ◽  
Differentiation Block ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is associated with high relapse rates and poor survival, with limited response to conventional cancer therapy and lacking effective targeting of highly self-renewing leukemic stem cells (LSCs). The mechanism underlying the high self-renewal activity of LSCs that determines the aggressiveness of disease remains poorly understood. Although we and others have previously demonstrated the clinical significance of aberrant WNT/β-catenin signaling in AML (Science, 327:1650-1653, 2010; Cancer Cell, 18:606-618, 2010), its pharmacologically tractable components essential for the regulation of LSC self-renewal have not yet been determined. Our studies discover, for the first time, a critical link between R-spondin (RSPO)-LGR4/HOXA9 and WNT/β-catenin pathways in AML LSCs. Microarray data analysis of 183 AML patient samples showed a significant positive correlation between expression of LGR4 and HOXA9 (r=0.546, P<0.0001). LGR4 exerted a cell-of-origin-specific function in promoting aberrant self-renewal and AML progression in vivo through cooperating with HOXA9, a poor prognostic predictor. We observed that LGR4 itself was not able to fully transform normal hematopoietic stem/progenitor cells (HSPCs), but instead cooperated with HOXA9 in HSPCs to accelerate disease onset producing a highly aggressive short latency AML in vivo. LGR4 and HOXA9 were epigenetically upregulated and their coexpression was an essential determinant of RSPO-LGR4 oncogenic activity. RSPO/WNT3 ligands could serve as stem cell growth factors to sustain myeloid differentiation block and to promote proliferation of CD34+ LSC-enriched subpopulations in primary AML patient specimens co-expressing LGR4 and HOXA9. Conversely, CRISPR/Cas9-mediated knockout of LGR4 not only suppressed RSPO/WNT3 signals and markedly decreased nuclear active β-catenin, but also reduced tumor burden in a patient-derived xenograft (PDX) mouse model of relapsed AML. Importantly, this study is the first to demonstrate that pharmacological inhibition of RSPO3-LGR4 signaling by a clinical-grade anti-RSPO3 monoclonal antibody induced LSC differentiation and consequently prevented tumor growth in AML PDX mice but did not affect normal human stem cell compartment in NSG mice. Together, our findings support a critical role for RSPO-LGR4 in the Wnt/β-catenin signaling pathway to promote AML leukemogenesis. Aberrant activation of RSPO-LGR4 is crucial for enhancing the self-renewal potential and myeloid differentiation block, which contribute to an aggressive leukemia phenotype through cooperating with HOXA9. Genetic and pharmacological targeting of this pathway impairs LSC self-renewal and survival and impedes AML development in murine models and patient-derived xenografts, highlighting the therapeutic value of targeting RSPO-LGR4 signaling in AML. References: Wang Y, et al. The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML. Science. 2010;327:1650-1653. Yeung J, et al. Beta-catenin mediates the establishment and drug resistance of MLL leukemic stem cells. Cancer Cell. 2010;18:606-618. Disclosures No relevant conflicts of interest to declare.

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