Articles de revues : « Acute myeloid leukemia, Wnt/β-catenin, MSC »

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Davis, Richard E., Vivian R. Ruvolo, Zhiqiang Wang, Wencai Ma, Wendy D. Schober, James Rolke, George Tidmarsh, Michael Andreeff et Peter P. Ruvolo. « GCS-100 Induces Apoptosis of Acute Myeloid Leukemia Cells By Disrupting Galectin-Mediated Survival Signaling ». Blood 124, n^o 21 (6 décembre 2014) : 904. http://dx.doi.org/10.1182/blood.v124.21.904.904.

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Abstract Galectins are a family of b-galactoside binding proteins with effects on cell adhesion, apoptosis, cell cycle, and mRNA processing. Galectin-3 (LGALS3) is unique among galectins by having an N terminal region of roughly 130 amino acids that allows for multimerization and binding to other proteins independent of carbohydrate binding. In addition to promoting BCL2 gene expression and mitochondrial integrity, LGALS3 (along with LGALS1) positively regulates RAS signaling and thus stabilizes survival proteins dependent on ERK phosphorylation such as MCL-1. The pro-survival functions of LGALS3 and other galectins suggest that their targeting could be therapeutic for cancers including AML. Indeed, LGALS3 expression is a predictor of poor prognosis in acute myeloid leukemia (AML), as reported by Cheng and colleagues (Blood 2013) for patients with non-M3 AML and CN-AML. The modified pectin GCS-100 (La Jolla Pharmaceutical, San Diego, CA), now in a Phase II clinical trial for chronic kidney disease, binds and blocks the function of LGALS3. We report that GCS-100 suppresses the growth of AML cell lines OCI-AML3, THP-1, and HL60 in vitro as a single agent, at doses under the 250 ug/mL (i.e., within clinically-achievable concentrations). Short-term treatment of cells (i.e., < 6 hr) potently suppressed phosphorylation of AKT and ERK and reduced expression of BCL2 and MCL-1. Because LGALS3 positively regulates anti-apoptotic BCL2 family members, the Raz group has suggested targeting galectins to enhance efficacy of BH3 mimetic drugs (Harazano et al Cancer Metastasis Review 2013). We found that GCS-100 potently synergized with ABT-737 to kill OCI-AML3 cells: while 1 uM ABT-737 or 125 ug/mL GCS-100 reduced total viable cells by ~ 30% and induced apoptosis in < 20% of cells after 48 hr as single agents, their combination at those doses and time point reduced viable cells by ~ 94% and induced apoptosis in ~ 70% of cells. Suppression of LGALS3 by lentiviral shRNA reduced BCL2 gene expression as determined by qRT-PCR and augmented killing with ABT-737. Lentiviral suppression of LGALS3 protected cells from GCS-100 at doses of 250 ug/mL but reduction of the galectin failed to protect cells from higher doses of the drug (i.e., 500 ug/mL). This result suggests other galectins are likely inhibited at higher doses of the agent. We used gene expression profiling (GEP) on Illumina HT12v4 human whole-genome arrays to assess more broadly the molecular effects of inhibiting galectins in AML cell lines OCI-AML3 and THP-1 treated with 250 ug/mL or 500 ug/ml GCS-100 for 24 hr. Data were analyzed by Gene Set Enrichment Analysis (GSEA) using gene sets from the Molecular Signatures Database (www.broadinstitute.org/gsea/msigdb/). GSEA suggested that GCS-100 promotes differentiation and inhibits genes associated with proliferation. Multiple upregulated gene sets suggest that there may be a release of a differentiation block as a result of GCS-100 treatment. Furthermore, two gene sets suggest that GCS-100 behaves similar to a GSK3 inhibitor: Known pathways regulated by GSK3 in hematopoietic stem cells are mTOR and Wnt/beta Catenin. Inhibition of Wnt/beta Catenin can release a differentiation block. Consistent with GCS-100 promoting cell differentiation, lentiviral shRNA reduced LGALS3 protein > 90% in THP-1 cells and increased CD11b expression, suggesting increased differentiation, compared to cells with control shRNA. GCS-100 was tested in an in vitro model of the bone marrow microenvironment using BM-derived mesenchymal stromal cell (MSC). MSC can protect leukemia cells from a variety of clinically relevant chemotherapy drugs including AraC. GCS-100 was effective at killing AML cells despite the presence of MSC. Both THP-1 and OCI-AML3 cells exhibited > 80% and > 60% reduction of viable cells, respectively, despite the presence of MSC when treated with 250 ug/mL GCS-100 for 72 hours. In addition, GCS-100 was found to block adhesion of OCI-AML3 cells to MSC suggesting that GCS-100 could be effective in mobilizing AML cells. In summary, our findings suggest that GCS-100 can induce apoptosis in AML cells as a single agent or in combination with the BH3 mimetic ABT-737. The agent is effective even in the presence of MSC suggesting it could be efficacious in the leukemia niche. These findings suggest GCS-100 could be effective for AML therapy. Disclosures Rolke: La Jolla Pharmaceutical Company: Employment. Tidmarsh:La Jolla Pharmaceutical Company: Employment.

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Takam Kamga, Paul, Giada Dal Collo, Adriana Cassaro, Annalisa Adamo, Alessandro Gatti, Roberta Carusone, Mariano Di Trapani et al. « Inhibition of GSK-3 Signalling Enhances Sensitivity of Non-Promyelocitic Acute Myeloid Leukemia (AML) Cell to Chemotherapy ». Blood 128, n^o 22 (2 décembre 2016) : 1582. http://dx.doi.org/10.1182/blood.v128.22.1582.1582.

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Abstract Background: GSK-3 is a serine-threonine kinase involved in metabolic regulation as well as in the control of many pathways associated to cancer development, including Notch Wnt/β-catenin, Hedgehog, and AKT. It has been demonstrated that association of GSK-3 inhibitors with All-trans-retinoic acid (ATRA) significantly improves ATRA-mediated differentiation and cell death of acute promyelocytic (APL) leukaemia cells. However, little is currently known about the contribution of GSK-3 role to non-promyelocytic AML cell response to treatment with chemotherapeutic agents. Aims: In this study, we aim to validate GSK-3 signalling as potent successful therapeutic target in non-promyelocytic AML. For this purpose we tested different GSK-3 for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatments. Methods: GSK-3 expression was analyzed by Western blot or flow cytometry inAML cell lines (HL-60, THP1, U937) or primary non-promyelocyticAML blast cells (30 samples). AML cellscultured alone or in presence ofhuman bone marrow mesenchymal stromal cells (hBM-MSCs) were treated with GSK-3 inhibitors, including LiCL, AR-A014418, SB 216763, in association or not with Cytarabine (Ara-C) or Idarubicin. Cell proliferation and cell death were measured by CFSE dilution and TOPRO-3/Annexin-V staining, respectively. Results: Flow cytometry and Western blot analysis in AML samples revealed high expression levels of all GSK-3forms, including total GSK-3α, (Ser21) GSK-3α, total GSK-3β, and (Ser 21) GSK-3β; theseforms were all down-modulated when AML cells were cultured in presence of hBM-MSCs, thus suggesting that GSK-3 plays an important role in transducting micro-environmental signals in AML cells interacting with bone marrow stroma. The treatment of AML cells with increasing concentrations of each GSK-3 inhibitors decreased AML cell viability in a dose-dependent manner; interestingly, hBM-MSCs or peripheral blood mononuclear cells were less sensitive to GSK-3inhibitors. The addition of each inhibitor increased dramatically the AML cell apoptotic rate induced by the addition of Ara-C or Idarubicin in vitro. Notably, LiCl and AR-A014418 were capable of abrogating hBM-MSC-mediated AML cell resistance to apoptosis induced by Ara-C or Idarubicin. Conclusion: Overall our data clearly demonstrated that inhibition of GSK-3 reduced proliferation and chemoresistance of non promyelocytic AML cells. Thus GSK-3 inhibition represents a therapeutic strategy not only for APL but also for other AML subtypes. Disclosures Bonifacio: Ariad Pharmaceuticals: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Amgen: Consultancy.

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Roversi, Fernanda Marconi, Maura Lima Pereira Bueno, Rafael Gonçalves Barbosa Gomes, Guilherme Rossi Assis-Mendonça, Paulo Latuf Filho, Adriana Silva Santos Duarte et Sara T. Olalla Saad. « A Novel WNT5A-Mimicking Peptide Affects Leukemia Cell Survival in the Bone Marrow Microenvironment ». Blood 138, Supplement 1 (5 novembre 2021) : 2949. http://dx.doi.org/10.1182/blood-2021-148744.

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Abstract Background: The crosstalk between hematopoietic cells and bone marrow (BM) microenvironment in hematological malignancies is related to disease initiation, maintenance and relapse. BM niche sustains a protective response against currently available treatments that have shown unwanted adverse effects and high levels of toxicity for patients. WNT5a is a glycoprotein secreted by mesenchymal stromal cells (MSC) that activates the WNT non-canonical pathway in hematopoietic cells, modulating important biological processes related to neoplasia development. Aims: To investigate WNT5a mRNA expression, protein levels and methylation pattern in Myelodysplastic Syndrome (MDS) and de novo Acute Myeloid Leukemia (AML) patients and their impact on clinical outcomes, and to analyze effects of Foxy-5 (WNTResearch), a new WNT5a-mimicking compound. Methods: WNT5a mRNA expression was analyzed in mononuclear cells (MC) from 371 AML patients (212 male, median age 61 years [range: 2-87]) (Ohsu, Nature 2018). BMMSC and BM biopsies from 5 healthy donors (HD), 6 MDS and 13 AML patients at diagnosis were submitted to analysis of WNT5a mRNA expression and methylation after Azacytidine (1μM) treatment by real-time PCR, and WNT5a protein levels by immunohistochemistry and immunofluorescence (IF). A panel of myeloid leukemia cell lines (U937, HL60, THP1, KG1a, K562) were treated with Foxy-5 (1 to 100μM) for 72h in monoculture, coculture and 3D-coculture (with MSC) and evaluated for ROS production (DCFDA dye), cell proliferation (Ki-67 stain), autophagy (acridine orange dye), chemotaxis (Transwell), actin polymerization (phalloidin stain), cell cycle (PI/RNAse stain), cell viability (MTT assay), apoptosis (Annexin-V stain) and protein expression (Western blot, WB). MC and CD34+ cells from HD were submitted to cytotoxic assays. Statistical analyzes were performed using ANOVA or Mann-Whitney tests, as appropriate. Results: WNT5a gene expression was reduced in MC from AML patients with adverse cytogenetic risk compared to favorable and intermediate cytogenetic risk (fold-decrease [FD]: 42.9; 18.8, respectively) (P<.05) and in BMMSC from AML compared to HD (FD: 53.3) (P<.05). Accordingly, WNT5a gene expression in MDS and AML BMMSC treated with Azacytidine was restored (fold-increase [FI]: 3.99; 1.50, respectively). WNT5a protein expression were diminished in BMMSC from MDS and AML patients compared to HD onto a 3D-coculture (IF)(P<.05) and immunohistochemically detected in all BM hematopoietic lineages. Foxy-5 reduced ROS production in U937 (FD of mean fluorescence intensity [MFI]: 48.2; 46.6), HL60 (FD: 47.1; 115.0), KG1a (FD: 34.9; 20.7) and K562 (FD: 19.0; 24.3) at 100μM in monoculture and coculture, respectively (P<.05). Foxy-5 also significantly decreased proliferation in U937 (FD: 41.0), HL60 (FD: 18.0), THP1 (FD: 36.0) and K562 (FD: 68.0) at 100μM (P<.05), confirmed by a 3D-coculture containing these cell lines and MSC. Foxy-5 reduced monocyte differentiation and inhibited CD11b expression in U937 (FD: 16.4) and THP1 (FD: 14.4). Cell cycle progression was blocked in sub G0/G1 phase in all cell lines (P<.05) after Foxy-5 treatment, probably mediated by the reduction of cyclin D1 protein levels, as verified by WB. Further, Foxy-5 reduced AKT1/2/3 and ERK1/2 phosphorylation levels, possibly by beta-catenin inhibition, with disruption of actin polymerization (U937 (FD: 65.3), HL60 (FD: 35.9), THP1 (FD: 58.5), K562 (FD: 15.0)) at 100μM (P<.05) and consequent impairment of CXCL12-induced chemotaxis (U937 (FD: 27.9), HL60 (FD: 42.5), THP1 (FD: 82.4), K562 (FD: 45.1)) at 100μM (P<.05). In coculture, cell autophagy was reduced in U937 (FD: 27.8), HL60 (FD: 35.9), KG1a (FD: 16.4) and K562 (FD: 35.8) when treated with Foxy-5 at 100μM (P<.05). Finally, Foxy-5 treatment did not affect cytotoxicity in MC and CD34+ cells from HD. Conclusion: WNT5a downregulation in MDS and AML patients occurs probably by methylation and contributes to poor prognosis. Foxy-5, by restoring WNT5a levels, could represent a strategy to counterbalance several oncogenic processes present in leukemia by reducing ROS production and, consequently, inhibiting cell growth and differentiation, downregulating PI3K and MAPK pathways, disrupting actin polymerization and decreasing autophagy. Thus, Foxy-5 treatment may be an important approach to impair leukemia growth and maintenance and arises as a promising therapeutic target. Disclosures No relevant conflicts of interest to declare.

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Takam Kamga, Paul, Adriana Cassaro, Giada Dal Collo, Annalisa Adamo, Alessandro Gatti, Roberta Carusone, Martina Midolo et al. « Role of Wnt/β-Catenin Signalling in Acute Myeloid Leukemia (AML) Cell Response to Chemotherapy ». Blood 128, n^o 22 (2 décembre 2016) : 2753. http://dx.doi.org/10.1182/blood.v128.22.2753.2753.

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Abstract Background: Growing evidences from both preclinical and clinical investigations reveal the critical role of Wnt signalling for the development of many cancers and for their response to chemotherapy. Although recent studies suggest that aberrant Wnt signalling can be involved in the neoplastic myeloid cell growth, the contribution of the Wnt/β-catenin pathway to AML survival and chemoresistance is still unclear. Aims: In this study, we investigated the contribution of WNT/β-CATENIN signalling to AML survival and chemoresistance. For this purpose we tested different modulators of Wnt/β-Catenin pathway for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatment. Methods: AML primary blast cells(30 samples) or AML cell lines cultured alone or in presence of human bone marrow mesenchymal stromal cells (hBM-MSCs), were treated with with Cytarabine (Ara-C) or Idarubicin, in presence or absence of Wnt modulators, including ligands (Wnt3a, Wnt5a/5b), Porcupine inhibitors (IWP-2), LRP6 inhibitors (Niclosamide), or antagonists of TCF/β-catenin (PKF118-310, PNU-74654). Results: In silico analysis showed the enrichment of Wnt signalling components in AML samples. Western Blot and flow cytometry showed the presence of total β-catenin only in about 2/3 of primary samples analyzed, while . β-catenin positive samples had different degree of activation of the pathway, as revealed by the expression of active forms of β-catenin, including (Ser675)β-catenin and non-phospho-(Ser33/37/Thr41) β-catenin. Notably, we found that active forms of β-catenin increased in AML samples in co-culture with hBM-MSCs, thus suggesting that Wnt signalling could be involved in the crosstalk between bone marrow stroma and AML cells. The addition of Wnt or pharmacological inhibitors, such as IWP-2, PNU-74654 and Niclosamide, to the culture medium of β-catenin-positive AML samples, either cultured alone or in co-culture with hBM-MSCs, reduced AML cell proliferation with slight effect on cell death. When associated to Idarubicin, all Wnt inhibitors except IWP-2 synergycally induced a dramatic cell death in AML cells in both culture conditions. However, when Idarubicin was replaced by Ara-C the synergism was observed only with Niclosamide and PKF. Cell death was mainly due to apoptosis, as shown by Annexin-V staining. Conclusion: Overall our data show that Wnt inhibitors reduce proliferation and chemoresistance of AML cells in culture or co-culture with bone marrow stroma cells. Wnt/β-catenin signalling may represent a potential therapeutic strategy to improve AML treatment, overcoming bone marrow stromal-mediated anti-apoptotic and chemoresistance effects. Disclosures Bonifacio: Ariad Pharmaceuticals: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Amgen: Consultancy.

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Elyamany, Ghaleb, Hassan Rizwan, Ariz Akhter, Mansour S. Aljabry, Sultan Alotaibi, Mohammad A. Hameed Albalawi, Meer-Taher Shabani-Rad, Tariq Mahmood Roshan et Adnan Mansoor. « “Losing the Brakes”—Suppressed Inhibitors Triggering Uncontrolled Wnt/ß-Catenin Signaling May Provide a Potential Therapeutic Target in Elderly Acute Myeloid Leukemia ». Current Issues in Molecular Biology 45, n^o 1 (9 janvier 2023) : 604–13. http://dx.doi.org/10.3390/cimb45010040.

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Dysregulated Wnt/β-catenin signal transduction is implicated in initiation, propagation, and poor prognosis in AML. Epigenetic inactivation is central to Wnt/β-catenin hyperactivity, and Wnt/β-catenin inhibitors are being investigated as targeted therapy. Dysregulated Wnt/β-catenin signaling has also been linked to accelerated aging. Since AML is a disease of old age (>60 yrs), we hypothesized age-related differential activity of Wnt/β-catenin signaling in AML patients. We probed Wnt/β-catenin expression in a series of AML in the elderly (>60 yrs) and compared it to a cohort of pediatric AML (<18 yrs). RNA from diagnostic bone marrow biopsies (n = 101) were evaluated for key Wnt/β-catenin molecule expression utilizing the NanoString platform. Differential expression of significance was defined as >2.5-fold difference (p < 0.01). A total of 36 pediatric AML (<18 yrs) and 36 elderly AML (>60 yrs) were identified in this cohort. Normal bone marrows (n = 10) were employed as controls. Wnt/β-catenin target genes (MYC, MYB, and RUNX1) showed upregulation, while Wnt/β-catenin inhibitors (CXXR, DKK1-4, SFRP1-4, SOST, and WIFI) were suppressed in elderly AML compared to pediatric AML and controls. Our data denote that suppressed inhibitor expression (through mutation or hypermethylation) is an additional contributing factor in Wnt/β-catenin hyperactivity in elderly AML, thus supporting Wnt/β-catenin inhibitors as potential targeted therapy.

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Takam Kamga, Paul, Giada Dal Collo, Adriana Cassaro, Riccardo Bazzoni, Pietro Delfino, Annalisa Adamo, Alice Bonato et al. « Small Molecule Inhibitors of Microenvironmental Wnt/β-Catenin Signaling Enhance the Chemosensitivity of Acute Myeloid Leukemia ». Cancers 12, n^o 9 (21 septembre 2020) : 2696. http://dx.doi.org/10.3390/cancers12092696.

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Wnt/β-catenin signaling has been reported in Acute Myeloid leukemia, but little is known about its significance as a prognostic biomarker and drug target. In this study, we first evaluated the correlation between expression levels of Wnt molecules and clinical outcome. Then, we studied—in vitro and in vivo—the anti-leukemic value of combinatorial treatment between Wnt inhibitors and classic anti-leukemia drugs. Higher levels of β-catenin, Ser675-phospho-β-catenin and GSK-3α (total and Ser 9) were found in AML cells from intermediate or poor risk patients; nevertheless, patients presenting high activity of Wnt/β-catenin displayed shorter progression-free survival (PFS) according to univariate analysis. In vitro, many pharmacological inhibitors of Wnt signalling, i.e., LRP6 (Niclosamide), GSK-3 (LiCl, AR-A014418), and TCF/LEF (PNU-74654) but not Porcupine (IWP-2), significantly reduced proliferation and improved the drug sensitivity of AML cells cultured alone or in the presence of bone marrow stromal cells. In vivo, PNU-74654, Niclosamide and LiCl administration significantly reduced the bone marrow leukemic burden acting synergistically with Ara-C, thus improving mouse survival. Overall, our study demonstrates the antileukemic role of Wnt/β-catenin inhibition that may represent a potential new therapeutics strategy in AML.

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Man, Cheuk Him, Tsz Kan Fung, Haixia Wan, Chae Yin Cher, August Fan, Nelson Ng, Christa Ho et al. « Suppression of SOX7 by DNA methylation and its tumor suppressor function in acute myeloid leukemia ». Blood 125, n^o 25 (18 juin 2015) : 3928–36. http://dx.doi.org/10.1182/blood-2014-06-580993.

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Zhuang, Lihui, Richard Darley, Oliver G. Ottmann, Joanna Zabkiewicz et Caroline Alvares. « Bone Marrow Stromal Cells Mediate Adhesion Based Drug Resistance in Acute Myeloid Leukaemia through Reciprocal Feedback of the β-Catenin/CD44 Signalling Axis ». Blood 132, Supplement 1 (29 novembre 2018) : 2776. http://dx.doi.org/10.1182/blood-2018-99-113811.

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Abstract Eradication of minimal residual disease is a key goal in AML treatment. It has been found that interaction between leukaemic blasts and different cells of the bone marrow niche contributes to AML drug resistance. Previously we have demonstrated that β-catenin may mediate drug resistance in both short and long-term stromal co-culture assays. β-catenin is known to correlate with poor prognosis in AML. β-catenin has dual roles as both a central effector molecule of the canonical Wnt signalling pathway and a component of adherens junction. The role of Wnt/β-catenin in AML has been well established, but the mechanism through which it mediates adhesion in AML remains unclear. Using a human:human co-culture adhesion model, β-catenin was found to be significantly up-regulated on HS5 and AML derived mesenchymal stromal cell (MSC). Lentivirus mediated β-catenin knock-down in AML cells significantly reduced their adhesion on HS5 and primary normal MSC and AML MSC (n=3, P<0.001). β-catenin knock-down also significantly reduced the proliferation of AML cells either on their own or in suspension from co-culture with primary normal MSC and AML MSC (P<0.001, n=3). In order to dissect the mechanisms underlying β-catenin mediated blast cell adhesion phenotype, KG-1a cells were chosen based on their primitive phenotype, high adhesion to human stroma and β-catenin nuclear translocation ability.KG-1a β-catenin knock-down and control cells were co-cultured with HS5 cells for 24hr and soluble factors in supernatants were harvested and analysed by Luminex bead arrays. β-catenin knock down induced reciprocal feedback signals in both KG-1a cells and HS5 cells. KG-1a catenin knockdown cells displayed increased levels of pro-inflammatory and adhesion pathways including: Galectin-3, N-CAM, NCAM1-1, CEACAM-1, osteonectin, CCL3, C5a, CCL4 and TNFα. Other soluble factors, including S100A8, TIMP-1, CD44, IL-2 and MMP3, were found to be down-regulated in KG-1a cells with β-catenin knock down and co-culture with HS5 abrogated these changes, clearly demonstrating stromal compensation in response to β-catenin knockdown. KG-1a β-catenin knock down and control cells were co-cultured with HS5 cells and suspension and adherent fractions harvested for nuclear and cytoplasmic protein extraction. Proteomic analysis of protein lysates using TMT labelling revealed that significant knock down of catenin target and adhesion molecule CD44 was observed in both cytoplasmic and nuclear fractions in both suspension and adherent KG-1a cells with beta-catenin knock down, suggesting canonical Wnt signalling is universally affected regardless of cellular context. Reduced CD44 production in catenin knockout cell lines was also confirmed in the soluble secretory factors from supernatants analysed by Luminex bead arrays, which also showed a significant reduction in soluble CD44 in β-catenin knock down lines (p<0.01). CD44 was also found to be significantly downregulated on KG-1a cell surface by flow cytometry in both suspension and adherent cells with β-catenin knock down(P<0.01), although adherent KG-1a cells showed considerably higher reduction in CD44 compared with suspension KG-1a cells (P<0.001). Proteomic data further demonstrated the upregulation of nuclear CD44 in adherent fractions, compared with suspension fractions, in both β-catenin knock down cells and control cells, suggesting activated and internalized CD44. Previous publications have shown that nuclear CD44 activates stemness factors, such as Nanog, SOX2 and Oct-4 and also PI3K/AKT and MAPK/ERK signal pathways which might contribute to the stroma mediated drug resistance we observed before. In conclusion, stromal feedback drives an AML blast adhesion phenotype through the Catenin/CD44 signalling axis, which is a contributing mechanism underlying the β-catenin-mediated stromal drug protection in AML. This finding indicates the recent development of novel Wnt inhibitors may be insufficient as a monotherapy to eradicate minimal residual disease in AML and further investigation of stromal-specific Wnt/CD44 targeting combinations is required to tackle this resistance mechanism. Disclosures Ottmann: Fusion Pharma: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy; Incyte: Consultancy, Research Funding; Takeda: Consultancy; Pfizer: Consultancy; Amgen: Consultancy.

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Zhang, Bin, Tinisha McDonald, Tessa L. Holyoake, Randall T. Moon, Dario Campana, Leonard Shultz et Ravi Bhatia. « Microenvironmental Protection of CML Stem and Progenitor Cells From Tyrosine Kinase Inhibitors Through N-Cadherin and Wnt Signaling ». Blood 120, n^o 21 (16 novembre 2012) : 912. http://dx.doi.org/10.1182/blood.v120.21.912.912.

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Abstract Abstract 912 BCR-ABL tyrosine kinase inhibitors (TKI) do not eliminate leukemia stem cells (LSC) in chronic myeloid leukemia (CML), which remain a potential source of relapse. TKI treatment effectively inhibits BCR-ABL kinase activity in CML LSC, indicating that additional kinase-independent mechanisms contribute to LSC preservation. We investigated the role of signals from the bone marrow (BM) microenvironment in protecting chronic phase (CP) CML stem/progenitor cells from TKI treatment. Culture with human BM mesenchymal stromal cells (MSC), immortalized by ectopic expression of telomerase, significantly inhibited apoptosis in CP CML CD34+CD38- primitive and CD34+CD38+ commited progenitors treated with imatinib (5μM, p<0.01), nilotinib (5μM, p<0.01), or dasatinib(150nM, p<0.05). MSC coculture reduced TKI-mediated apoptosis in both dividing and non-dividing CD34+CD38- cells, defined using CFSE labeling, and increased the percentage of undivided CD34+CD38- cells (p<0.05). MSC coculture also enhanced colony forming ability of CML CD34+CD38- (p<0.05) and CD34+CD38+ (p<0.05) cells treated with TKI. Importantly MSC coculture, with or without imatinib treatment, significantly enhanced engraftment of CML CD34+ cells in immunodeficient NSG mice, both at 4 weeks (without IM: 27.2±5% human CD45+ cells without stroma, 52.8±8% with stroma, p<0.001; with IM: 4.9±2% without stroma, 10.1±2% with stroma, p<0.05) and at 10 weeks post-transplant (without IM : 1.48±0.2% human CD45+ cells without stroma, 2.37±0.4% with stroma, p=0.07; with IM : 0.74±0.2% without stroma, 1.2±0.3% with stroma, p<0.05). CML progenitors cultured in a transwell insert over MSC showed increased apoptosis following TKI exposure compared to cells cultured in direct contact with MSC (p<0.05). An increased proportion of CML progenitors adhered to MSC after 4 days of TKI treatment (22±4% without IM, 42±5% with IM, p<0.05). Antibody or peptide (NCDH) mediated blockade of the N-Cadherin receptor reduced adhesion of CML progenitors to MSC (p<0.05), and increased apoptosis of TKI-treated CML progenitors cocultured with MSC (p<0.05), indicating an important role for N-Cadherin in MSC-mediated protection of CML progenitors from TKI. Although N-Cadherin expression measured by flow cytometry, Western blot and Q-RT-PCR was lower in CML compared to normal CD34+CD38- and CD34+CD38+ cells, we observed significantly increased N-Cadherin expression in CML cells remaining after 4 days culture with IM and MSC (p<0.05). We observed enhanced β-catenin activity in CML progenitors cocultured with MSC, with and without TKI treatment, as manifested by increased β-catenin protein levels and nuclear localization, enhanced expression of β-catenin target genes, and increased TCF/LEF reporter activity. β-catenin levels and target gene expression were increased primarily in MSC-adherent CML progenitors compared to non-adherent cells, and were significantly reduced by antibody or peptide-mediated inhibition of N-Cadherin-mediated adhesion. Using the Duolink labeling technology, we demonstrated increased protein-protein interactions between N-cadherin and β-catenin in CML progenitors cocultured with MSC, with or without TKI treatment. Finally, we showed that addition of exogenous Wnt1 protected CML progenitors from TKI treatment, whereas inhibition of Wnt receptors by DKK1, or inhibition of β-catenin signaling by the small molecule inhibitor ICG001, enhanced apoptosis of CML progenitors cocultured with MSC, suggesting an important role for exogenous Wnt signaling in MSC-mediated protection of CML progenitors from TKI-induced apoptosis. Microarray analysis of gene expression confirmed enrichment of Wnt/β-catenin and Cadherin related gene sets in CML CD34+ cells cultured on MSC with or without IM, as well as enrichment of genes related to hematopoietic stem cell (HSC) self-renewal, HSC quiescence, and cytokine signaling. In conclusion, our studies reveal an important role for exogenous Wnt-β-catenin signaling, and a close interplay between N-Cadherin and β-catenin, in microenvonment-mediated resistance of CML stem and progenitor cells to TKI treatment. These findings support further development of novel treatment strategies to eradicate residual LSC in TKI-treated CML patients through inhibition of Wnt secretion or blockade of Wnt or N-Cadherin receptors. Disclosures: No relevant conflicts of interest to declare.

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Morgan, Rhys Gareth, Lorna Pearn, Kate Liddiard, Robert Hills, Alan Burnett, Alex Tonks et Richard L. Darley. « Distinct Regulation of β- and γ-Catenin throughout Hematopoietic Development Contrasts with Their Cooperative Roles In Acute Myeloid Leukemia. » Blood 116, n^o 21 (19 novembre 2010) : 1573. http://dx.doi.org/10.1182/blood.v116.21.1573.1573.

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Abstract Abstract 1573 Wnt proteins are important developmental regulators and are known to play a role in maintenance of hematopoietic stem cells (HSC). Wnt signaling has also been identified as one of the most frequently dysregulated processes associated with acute myeloid leukemia (AML), though the significance of this observation is as yet poorly understood. Here we investigate the role of two Wnt signaling proteins; β-catenin and γ-catenin and their respective roles in both normal human hematopoiesis and in AML. These proteins have dual and overlapping roles as transcriptional activators of Wnt target genes in the nucleus, and as structural components of the cytoskeleton. To determine the potential scope of influence of these proteins, we first examined their expression levels and subcellular location throughout normal human hematopoiesis using multi-parameter flow cytometric analysis and confocal microscopy. As expected β-catenin was strongly expressed in human cord blood derived HSC (212 MFI ±124, n=6) and at lower levels in differentiated subsets; surprisingly however β-catenin expression was maintained in granulocytic (1182 MFI±568) and monocytic cells (284 MFI±107). Nuclear localization was independent of cytoplasmic expression level, being strongly nuclear-localized in early progenitors and predominantly cytoplasmic in differentiated cells (58%±5 nuclear-localized in CD34+ cells vs 27%±1 in granulocytes, P=0.008). The expression pattern of γ-catenin was similar to β-catenin but showed a reciprocal pattern of subcellular localization, with levels of nuclear γ-catenin strongest in differentiated cells (10%±2 in CD34+ cells vs 44%±3 in monocytes P=0.0005). These data imply complementary roles for β and γ-catenin in normal hematopoiesis and show that nuclear localization of these proteins is regulated independently and irrespective of their expression level. In AML patients, β-catenin dysregulation has been previously reported; however, we also observed frequent overexpression of γ-catenin (over 5 fold in 25% of patients). This overexpression was associated with lower remission rates (OR 1.23 per log increase, P=0.03, CI 1.02–1.49) arising from resistant disease (OR 1.57 per log increase, P=0.003, CI 1.16–2.14) in a cohort of 243 AML patients adjusted for baseline diagnostic variables. In contrast to normal hematopoiesis, we found that nuclear localization of γ-catenin correlated with nuclear localization of β-catenin in AML (R=0.5, n=59) suggesting that the capacity to independently regulate the nuclear entry/retention of these catenins is disrupted in AML. To investigate this, we examined the effect of ectopic overexpression of γ-catenin in normal cord blood derived CD34+ cells and AML cell lines. Three-fold overexpression of γ-catenin failed to induce nuclear translocation of γ- or β-catenin in normal progenitors, which exhibited no major developmental defects. In contrast, in 3 of 4 AML cell lines, overexpression of γ-catenin strongly promoted its nuclear localization (9-16 fold) and was associated with a block in agonist-induced differentiation - a phenotype previously associated with β-catenin. In accord with this, we found that as in primary AML, nuclear translocation of γ-catenin in AML cell lines was associated with translocation of β-catenin (2-22 fold). In conclusion, we propose that in normal hematopoiesis, nuclear translocation of β- and γ-catenin is tightly and independently regulated for each catenin. In contrast, most AML cells lack this regulation resulting in correlated nuclear levels of β- and γ-catenin. In addition, we found while overexpression of γ-catenin has little consequence for normal cells; in malignant cells γ-catenin facilitates nuclear translocation of β-catenin. Disclosures: No relevant conflicts of interest to declare.

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Chai-Ho, Wanxing, Martina M. Roos, Michelle Li, Pang Amara, Yurun Zhang, Tiancheng Fang, Christina Termini et John P. Chute. « Grb10 Is a Tumor Suppressor in Human Acute Myeloid Leukemia ». Blood 132, Supplement 1 (29 novembre 2018) : 1344. http://dx.doi.org/10.1182/blood-2018-99-110844.

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Abstract Acute myeloid leukemia (AML) is a common and potentially fatal hematologic malignancy. Allogeneic stem cell transplantation is the only curative therapy for most subtypes of AML, but carries a significant risk of transplant-related mortality. The development of novel therapies to eradicate AML remains a substantial area of unmet medical need. Growth factor receptor bound protein 10 (Grb10) is a member of the family of imprinted genes. Our laboratory demonstrated that in normal hematopoietic system, deletion of the maternal allele of Grb10 significantly increased hematopoietic stem cell long-term repopulating capacity (Yan et al. Cell Rep 2016). Grb10 has been shown to bind the intracellular domain of various tyrosine kinase receptors, e.g. KIT, FLT3 and RET, as well as low-density lipoprotein receptor-related protein 6, a negative regulator of the Wnt/β-catenin pathway. Analyzing RNAseq data from the Leucegene Project, we found that Grb10 is expressed on the majority of patient AML samples regardless of leukemia mutation profile. Silencing of Grb10 expression via Grb10 shRNA increased the proliferation and colony forming capacity of human AML cell lines, Kasumi-1, THP-1 and OCI-AML3 in vitro (p<0.0001 and p<0.01). Conversely, overexpression of Grb10 suppressed human AML cell growth (p<0.05). In order to determine the role of Grb10 in regulating AML growth in vivo, we transduced bone marrow lineage negative cells from mice with Grb10 maternal allele deletion (Grb10 m/+) and wild type (Grb10 +/+) mice with HoxA9-Meis1-neo-MSCV (gift from G. Savageau) and transplanted the progeny into congenic mice. Primary and secondary mice transplanted with Grb10m/+ HoxA9-Meis1 leukemia cells displayed significantly decreased survival compared to mice transplanted with Grb10+/+ HoxA9-Meis1 cells (p<0.001 and p<0.05). Furthermore, leukemia cells with Grb10 maternal allele deletion displayed an increase cell cycle progression and increased leukemia colony forming capacity. RNAseq analysis of Grb10 m/+ leukemia cells from diseased mice revealed significant dysregulation of the canonical Wnt/β catenin signaling pathway compared to Grb10 +/+ mice. RT-qPCR analysis confirmed that Wnt/β-catenin target genes, including MYC, CCND1, and SOX2 were significantly up-regulated in Grb10 knockdown human AML cells lines. Taken together, these data suggest that Grb10 is a powerful tumor suppressor in human AML, and represents a novel mechanistic target for the development of new therapies for human AML. Disclosures No relevant conflicts of interest to declare.

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Hassan, Nunki, Hangyu Yi, Lucie Gaspard-Boulinc, Franklin Chen, Jayvee Datuin, Jonason Yang et Jenny Yingzi Wang. « GADD45a Controls Self-Renewal in Acute Myeloid Leukemia Stem Cells ». Blood 136, Supplement 1 (5 novembre 2020) : 31. http://dx.doi.org/10.1182/blood-2020-142389.

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Acute myeloid leukemia (AML) is a heterogenous malignancy, where the persistence of chemo-resistant leukemia stem cells (LSCs) contributes to disease relapse. We have previously demonstrated the clinical significance of WNT/β-catenin signaling in driving AML LSCs (Science, 327:1650-1653, 2010; Cancer Cell, 38:1-16, 2020). In this study, we uncover that GADD45a (growth arrest and DNA-damage inducible protein) is an essential regulator of β-catenin signaling pathway and its loss promotes LSC function and leukemia progression. Transgenic knockout of Gadd45a led to a progressive increase in aberrant self-renewal and leukemogenesis in vivo. Gadd45a-/- leukemic cells developed a more aggressive leukemia with a shorter latency than Gadd45a+/+ cells in mice, indicating the involvement of Gadd45a loss in AML initiation and progression. Subsequent serial transplantation experiments showed that Gadd45a deletion enhanced LSC self-renewal in vivo. In agreement with our findings in murine LSCs, deletion of GADD45a by CRISPR/Cas9 in AML patient-derived xenograft (PDX) cells revealed increased engraftment and tumor burden in NSG mice. Consistent with our phenotypic observations, knockout of GADD45a increased βcatenin activity and key WNT/self-renewal target genes in human AML cells. In addition, our cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) data showed that GADD45a deletion in patient-derived LSCs was associated with cell metabolism, reactive oxygen species and tumor progression, as well as poor patient outcomes in AML. Further studies are being conducted to evaluate transcriptional mechanisms discovered by our single-cell sequencing. Taken together, this study is the first to demonstrate that GADD45a loss promotes LSC potential and consequently enhances tumor growth in murine and PDX models of AML, thus showcasing GADD45a as a promising therapeutic target in AML. References: Wang Y, Krivtsov AV, Sinha AU, et al. The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML. Science. 2010;327:1650-1653. Salik B, Yi H, Hassan N, et al. Targeting RSPO-LGR4 signaling for leukemia stem cell eradication in acute myeloid leukemia. Cancer Cell. 2020; 38:1-16. Disclosures No relevant conflicts of interest to declare.

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Hassan, Nunki, Basit Salik, Alastair Duly et Jenny Yingzi Wang. « Rspo-LGR4 Cooperates with HOXA9 to Sustain Self-Renewal in Acute Myeloid Leukemia ». Blood 134, Supplement_1 (13 novembre 2019) : 2669. http://dx.doi.org/10.1182/blood-2019-129704.

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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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Almars, Amany, Panagiota S. Chondrou, Emenike K. Onyido, Sheema Almozyan, Claire Seedhouse, Roya Babaei-Jadidi et Abdolrahman S. Nateri. « Increased FLYWCH1 Expression is Negatively Correlated with Wnt/β-catenin Target Gene Expression in Acute Myeloid Leukemia Cells ». International Journal of Molecular Sciences 20, n^o 11 (4 juin 2019) : 2739. http://dx.doi.org/10.3390/ijms20112739.

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Acute myeloid leukaemia (AML) is a heterogeneous clonal malignancy of hematopoietic progenitor cells. The Wnt pathway and its downstream targets are tightly regulated by β-catenin. We recently discovered a new protein, FLYWCH1, which can directly bind nuclear β-catenin. Herein, we studied the FLYWCH1/β-catenin pathway in AML cells using qRT-PCR, Western blot, and immunofluorescence assays. In addition, the stemness activity and cell cycle were analysed by the colony-forming unit (CFU) using methylcellulose-based and Propidium iodide/flow cytometry assays. We found that FLYWCH1 mRNA and protein were differentially expressed in the AML cell lines. C-Myc, cyclin D1, and c-Jun expression decreased in the presence of higher FLYWCH1 expression, and vice versa. There appeared to be the loss of FLYWCH1 expression in dividing cells. The sub-G0 phase was prolonged and shortened in the low and high FLYWCH1 expression cell lines, respectively. The G0/G1 arrest correlated with FLYWCH1-expression, and these cell lines also formed colonies, whereas the low FLYWCH1 expression cell lines could not. Thus, FLYWCH1 functions as a negative regulator of the Wnt/β-catenin pathway in AML.

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Chai, Li, Wei Cui, Jianchang Chang, Chunhui Di, Hesham Amin et Yupo Ma. « SALL4, a Novel Oncogene Induces Myelodysplastic Syndrome and Acute Myeloid Leukemia Via Wnt/β-Catenin Pathway. » Blood 106, n^o 11 (16 novembre 2005) : 1371. http://dx.doi.org/10.1182/blood.v106.11.1371.1371.

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Abstract SALL4, a gene homologous to the Drosophila homeotic spalt, is a zinc finger transcriptional factor essential for human development. In Drosophila, spalt is regulated by the Wnt signaling pathway, a pathway critical for hematopoietic self-renewal of stem cells. We cloned SALL4 and its isoforms (SALL4A and SALL4B). We used immunohistochemistry, to demonstrate that SALL4 was constitutively expressed in primary acute myeloid leukemia cells (French American British, FAB: M1 to M5, N=81). The SALL4 in RNA was quantitative in bone marrow cells derived from acute myeloid leukemia (AML) and compared to non-neoplastic hematopietic cells from a purified CD34 progenitor pool, normal bone marrow and peripheral blood by RT-PCR. SALL4 expression was present in the purified normal CD34+ cells, AML blasts, but absent in mature myeloid cells. We tested the leukemogenic potential of constitutive overexpression of SALL4 in a murine hematopoietic model. All transgenic mice overexpressing SALL4B using the CMV promoter developed hematopoietic disorders, including myelodysplastic (MDS)-like symptoms and an AML transformation that disseminated to peripheral tissues including the spleen, liver, kidney and lymph nodes. The myelodysplastic features in these transgenic mice were pathologically similar to human MDSs and manifested as ineffective myelopoiesis. Dysplatic features such as Pseudo-Pelger-Huet like atypical white cells, and increased immature cells were detectable in the transgenic mouse peripheral blood. A high level of apoptosis and increased immature cells were also evident in transgenic mouse marrows and day-7 colony-forming unit assays in vitro. Both SALL4A and SALL4B were able to bind to β-catenin in vitro and synergistically enhanced the Wnt/β-catenin signaling pathway in a reporter gene assay. Our data suggests that the constitutive expression of SALL4 is causal to the leukemic phenotype. Our model should provide a useful platform to analyze the interaction of SALL4 with Wnt/β-catenin pathway in leukemia stem cells.

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Wang, Lei, et Jie Jin. « The Mechanisms of Synergistically Cytotoxicity Induced by Homoharringtonine and Aclarubicin in Acute Myeloid Leukemia Cells ». Blood 120, n^o 21 (16 novembre 2012) : 4313. http://dx.doi.org/10.1182/blood.v120.21.4313.4313.

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Abstract Abstract 4313 Previous studies showed HAA regime [HHT (homoharringtonine), cytarabine and ACR (aclarubicin)] resulted in a high complete remission (CR) rate and a better overall survival (OS) rate in patients with primary acute myeloid leukemia. To confirm if a synergistically cytotoxicity was found in AML cells, we investigated the antitumor effect relationship of HHT and ACR against AML cells. Using in vitro system, we demonstrated that simultaneous exposure to HHT and ACR resulted in strong synergistic anti-proliferative effect and apoptosis inducing in AML cells. In vivo, combination of HHT and ACR may be result in a favorable survival in AML xenograft mice. The assay of microarray gene expressing profiling highlighted apparent difference in expression of PI3K gene and WNT3a gene between cells treated by HHT and cells exposure to ACR. Furthermore, decreased expression of PI3K110 and P-AKT protein were observed in AML cells treated with HHT for 3h while no significant change in the expression of two proteins was observed in 90nM of ACR-treated cells. Western Blot analysis also showed ACR could obviously inhibit WNT3a and β-catenin protein levels in AML cells after 3 hours exposure. Although HHT could not inhibit WNT3a protein, it also could apparently down-regulate expression of β-catenin in AML cells. Simultaneous decrease of PI3K signal and WNT3a signal was induced by the combination of HHT and ACR in AML cell lines and primary AML cells. To explore possible targets of synergistically cytotoxity induced by combined HHT/ACR, we silenced wnt3a expression by RNA interference. Then we found suppression of wnt3a expression could enhance the cytotoxity of HHT and AKT inhibitor. Moreover, combining ACR with AKT inhibitor resulted in a synergistically cytotoxic effect too. β-catenin is a shared molecular in both AKT pathway and WNT pathway. Up-regulating of β-catenin expression failed to reduce cell apoptosis induced by HHT plus ACR while partially decrease the growth inhibition rate caused by combining treatment. β-catenin is required for the self-renewal of AML-LSC. Our study also suggests that combining HHT and ACR may synergistically induce apoptosis in LSC-enriched cells. These results indicate that simultaneously inhibiting activity of PI3K/AKT pathway and WNT/β-catenin pathway is a possible mechanism of synergistically cytotoxity induced combinated HHT/ACR in AML cells. Disclosures: No relevant conflicts of interest to declare.

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Tong, Hongyan, Kongfei Li, Chen Mei, Chao Hu et Jie Jin. « Synergistic Effect of Sequential Combination of Decitabine and Idarubicin in Mouse Model of Human Acute Myeloid Leukemia ». Blood 120, n^o 21 (16 novembre 2012) : 1515. http://dx.doi.org/10.1182/blood.v120.21.1515.1515.

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Abstract Abstract 1515 Background: Decitabine is a prototype for epigenetic therapy in cancer which targets DNA methyltransferase. While it was known that decitabine monotherapy has been associated with a relative low rate of complete remission rates in acute myeloid leukemia and myelodysplastic syndrome. Several groups have attempted to increase the response rate with decitabine-based therapy by developing combinations. In the preliminary experiments, we investigated the effect of anti-leukemia drugs (idarubicin, daunorubicin, cytarabine, thalidomide and homoharringtonine) in combination simultaneously or sequentially with decitabine of inhibiting proliferation of acute myeloid leukemia cell lines. The result showed that sequential combination of decitabine and idarubicin induced a significant synergistic effect in cell apoptosis, inhibition of cell proliferation. With the gene chip technology, we found that the gene expression of Wnt pathway changed obviously after the treatment of DAC combined with IDA. Further we confirm that sequential combination of DAC and IDA caused depression of wnt pathway in vitro. In this study, we evaluate the anti-leukemic effect and the combination mechanism of DAC in sequential combination with IDA in vivo. Methods: The AML cell line U937 (14×107 cells per animal) were injected subcutaneously into the right flank of 6 to 8-week old NOD-SCID mice. After tumor growth, decitabine(0.5mg/kg/day) were injected intraperitoneal of a five consecutive days followed by a three days of idarubicin(0.5 mg/kg/day). The tumor growth inhibition effect was evaluated by microPET to reveal the synergistic effect of decitabine and idarubicin. Using TUNEL method and electron microscopy, we detected the apoptosis of leukemic cells from tumor bearing mice. The expression of β-catenin which is the key gene of Wnt/β-catenin pathway was also examined by immunochemistry. We then detected the expression of protein levels of the other genes(cyclinD1, c-myc, SFRP1,HDPR1 and DKK3) of Wnt/β-catenin pathway in tumor cells of tumor-bearing mice by western blot. Results: In vivo, the effect of sequential combination of decitabine and idarubicin was initially examined in a subcutaneous AML mouse model. The AML cell line U937(1×107 cell per animal) was transplanted subcutaneously into the right flank of 2-week-old female NOD-SCID mouse. The cell line developed into a rapidly growing tumor. Treatment was initiated in 7th days after injection of the leukemic cells, included with 5 days of decitabine alone, 3 days of idarubicin alone and 5 days of decitabine followed by 3 days of idarubicin. A significant inhibition of tumor growth was demonstrated with microPET in animals treated with sequential combination of decitabine and idarubicin compared with the other treatment and control group animals. The apoptosis of tumor cells was also significantly increased in the sequential combination treatment group detected by TUNEL and electron microscope. We also found that the sequential treatment group induced synergistic effect in re-expression or up-expression of the Wnt inhibitor genes (SFRP1, HDPR1 and DKK3) in tumor cells. Furthermore, the down-steam genes of wnt pathway including β-catenin, c-myc and cyclinD1 were down-regulated which suggest depression of wnt/β-catenin pathway. These results suggest that sequential combination of decitabine and idarubicin has a powerful anti-leukemic effect not only in vitro but also in vivo. Conclusion: Our results demonstrate decitabine in sequential combination with idarubicin in mouse model of AML and suggest that this combination may be of clinical value in the treatment of patients with AML. Disclosures: No relevant conflicts of interest to declare.

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Jimbo, Koji, Takaaki Konuma, Takahiro Ito, Yaeko Nakajima-Takagi, Atsushi Iwama et Arinobu Tojo. « Immunoglobulin Superfamily Member 8 Is Indispensable for Myeloid Leukemia Via Wnt/β-Catenin Signaling Pathway ». Blood 136, Supplement 1 (5 novembre 2020) : 23–24. http://dx.doi.org/10.1182/blood-2020-136045.

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Immunoglobulin superfamily member 8 (IGSF8, also known as EWI-2, PGRL, and CD316), is a cell surface protein containing 4 immunoglobulin domains. IGSF8 directly binds to the tetraspanin molecules, CD9 and CD81, and modulates cell adhesion, migration, and growth. Previous studies demonstrated that IGSF8 was associated with prognosis and metastasis in several solid tumors. However, the role of IGSF8 in normal hematopoiesis and myeloid leukemia is still unclear. First, we examined the expression levels of Igsf8 in various hematopoietic fraction of wild-type murine bone marrow cells, and found that Igsf8 is expressed in all hematopoietic lineages. To investigate hematopoietic functions of Igsf8, we generated hematopoietic cells specific Igsf8 deleted mice (Igsf8fl/fl; Vav-Cre) and tamoxifen induced Igsf8 deleted mice (Igsf8fl/fl; Rosa26-CreERT). Igsf8fl/fl, Vav-Cre (denoted as Igsf8-/-) mice represented normal maturation. Deletion of Igsf8 did not significantly affect adult hematopoiesis in peripheral blood and bone marrow. Igsf8-/- long-term hematopoietic stem cells (LT-HSCs: CD34- Flk2- c-Kit+ Sca-1+ Lineage- cells) reduced colony forming ability in vitro, and serial competitive transplantation assay showed comparable donor chimerism by 3 months, but led to decrease Igsf8-/- donor chimerism at 4 months and those after second transplantation in vivo. These results suggest that Igsf8 does not affect the adult hematopoiesis, but it can affect their proliferative and reconstitutive capacity of HSCs. To investigate the effects of Igsf8 on myeloid leukemia, we generated MLL-AF9 and NRASG12V-driven acute myelogenous leukemia (AML), or BCR-ABL and NUP98-HOXA9-driven blast crisis of chronic myelogenous leukemia (CML-BC) mice models. Igsf8-/- led to a dramatic reduction in the number of leukemic colonies formed in vitro (Figure 1A). Igsf8-/- leukemia mice showed significantly longer survival in vivo (Figure 1B). This effect was also observed by eliminating Igsf8 expression after leukemia establishment using conditionally deletion. Igsf8-/- AML cells showed decreased S phase fraction. Igsf8-/- leukemia stem cells (LSCs: c-Kit+ Lineage- cells) triggered an increment of the apoptosis, which contribute to significantly lower proportion of LSCs in spleen of Igsf8-/- leukemic mice. Given that Igsf8-/- did not affect homing ability of leukemia cells, these results indicate that Igsf8 is required for propagation of myeloid leukemia and maintenance of LSC. To understand the Igsf8-mediated regulation of myeloid leukemia, we conducted RNA sequencing analysis of LT-HSCs, and LSCs of AML and CML-BC. Gene set enrichment analysis exhibited increase apoptosis related genes and decrease Wnt/β-catenin related genes in Igsf8-/- leukemic cells, but not in LT-HSCs (Figure 1C). Increment of pro-apoptosis genes, and decrement of anti-apoptosis genes and Wnt/β-catenin target genes in Igsf8-/- AML stem cells were validated in quantitative polymerase chain reaction analysis. Further, expression levels of β-catenin protein in Igsf8-/- leukemic cells were significantly lower compared to Igsf8+/+ leukemic cells, but not in normal hematopoietic stem and progenitor cells (Figure 1D). These results suggest that Igsf8 might be critical for myeloid leukemia maintenance via Wnt/β-catenin signaling pathway. Then, we investigated the effects of IGSF8 on human myeloid leukemia. We confirmed IGSF8 expression in several human myeloid leukemia cell line and primary patient-derived leukemia cells. Knockdown of IGSF8 by small hairpin RNA in myeloid leukemia cell lines (THP-1, MV4-11, SKM-1, and K562) and primary patient-derived AML cells exhibited reduced numbers of colony forming cells in vitro. Knockdown of IGSF8 also caused decrease expression of β-CATENIN in AML cell lines. These results indicate that IGSF8 is also required for propagation of human myeloid leukemia cells. Taken together, our present study reveals that Igsf8 is indispensable for myeloid leukemia, but not adult hematopoiesis, suggesting that IGSF8 inhibition should be considered for targeting myeloid leukemia. Disclosures Jimbo: Japan Society for the Promotion of Science: Research Funding. Konuma:SGH Foundation: Research Funding; The Japanese Society of Hematology: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding. Ito:Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding.

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Griffiths, Elizabeth A., Craig M. Hooker, Michael A. McDevitt, Judith E. Karp, James G. Herman et Hetty E. Carraway. « Acute Myeloid Leukemia Is Characterized by Wnt Pathway Inhibitor Promoter Methylation. » Blood 112, n^o 11 (16 novembre 2008) : 2253. http://dx.doi.org/10.1182/blood.v112.11.2253.2253.

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Abstract The Wnt pathway contributes to a stem-cell like phenotype in a variety of cancer subtypes. Nuclear localization of non-phosphorylated, active β-catenin is a surrogate marker for Wnt pathway activation and has been associated with adverse outcome in patients with acute myeloid leukemia (AML). Wnt pathway inhibitors including APC, DKK1, DKK3, LKB1/STK11, RASSF1A, RUNX3, SFRP1, SFRP2, SFRP4, SFRP5, SOX17, and WIF1 contain extensive promoter region CpG islands. Wnt pathway inhibitors are silenced by promoter methylation in many malignancies including lung cancer, colon cancer and acute lymphoid leukemias. To determine if methylation of these Wnt pathway inhibitors is present in AML, we evaluated leukemia cell lines (i.e., K562, HNT34, KG1, KG1A, U937, and HL60) for evidence of promoter methylation. Additionally, 188 AML patient (median age 61 years) samples from the Johns Hopkins Hospital leukemia tumor bank were assessed for the presence of Wnt pathway inhibitor methylation. All samples were bisulfite treated and evaluated for promoter methylation using methylation specific PCR. Diagnostic samples from a subgroup of patients with normal cytogenetics (n=73) who received high dose induction therapy were evaluated for potential associations between methylation of individual Wnt pathway inhibitor genes or total number of methylated genes and event free or overall survival. RESULTS: Extensive promoter methylation of the Wnt pathway inhibitor genes was observed in leukemia cell lines. Of the primary leukemia samples, 85% had at least one methylated gene. Promoter methylation of Wnt inhibitors was common in these samples with the following frequencies: DKK1 (16%;30/188), DKK3 (8%;15/188), RUNX3 (27%;50/188), SFRP1 (34%;63/188), SFRP2 (66%;124/188), SFRP4 (9%;16/188), SFRP5 (54%;102/188), SOX17 (29%;54/188), and WIF1 (32%;61/188). This is among the first comprehensive evaluations of Wnt pathway inhibitor methylation in primary samples from AML patients. The frequency of methylation seen here is comparable to that observed in established tumor suppressor genes in patients with AML, including p15INK4B, SOCS1 and CDH1. In marked contrast with epithelial tumors, methylation of APC (2%;2/108) and RASSF1A (0%;0/188) was rare. LKB1/STK11 methylation was also uncommon (2%;2/108). Previous reports have associated Wnt pathway activation with poor prognosis. In our treated patients with normal cytogenetics, no correlation was observed between methylation of Wnt pathway inhibitors and event free or overall survival. In conclusion, in patients with AML (a) there is a high frequency of Wnt pathway inhibitor methylation; (b) Wnt pathway inhibitor methylation is distinct from that observed in epithelial malignancies; and (c) methylation of Wnt pathway genes does not correlate with adverse clinical outcome.

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Moore, Amy C., Joseph M. Amann, Christopher S. Williams, Emilios Tahinci, Tiffany E. Farmer, J. Andres Martinez, Genyan Yang, K. Scott Luce, Ethan Lee et Scott W. Hiebert. « Myeloid Translocation Gene Family Members Associate with T-Cell Factors (TCFs) and Influence TCF-Dependent Transcription ». Molecular and Cellular Biology 28, n^o 3 (26 novembre 2007) : 977–87. http://dx.doi.org/10.1128/mcb.01242-07.

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ABSTRACT Canonical Wnt signaling is mediated by a molecular “switch” that regulates the transcriptional properties of the T-cell factor (TCF) family of DNA-binding proteins. Members of the myeloid translocation gene (MTG) family of transcriptional corepressors are frequently disrupted by chromosomal translocations in acute myeloid leukemia, whereas MTG16 may be inactivated in up to 40% of breast cancer and MTG8 is a candidate cancer gene in colorectal carcinoma. Genetic studies imply that this corepressor family may function in stem cells. Given that mice lacking Myeloid Translocation Gene Related-1 (Mtgr1) fail to maintain the secretory lineage in the small intestine, we surveyed transcription factors that might recruit Mtgr1 in intestinal stem cells or progenitor cells and found that MTG family members associate specifically with TCF4. Coexpression of β-catenin disrupted the association between these corepressors and TCF4. Furthermore, when expressed in Xenopus embryos, MTG family members inhibited axis formation and impaired the ability of β-catenin and XLef-1 to induce axis duplication, indicating that MTG family members act downstream of β-catenin. Moreover, we found that c-Myc, a transcriptional target of the Wnt pathway, was overexpressed in the small intestines of mice lacking Mtgr1, thus linking inactivation of Mtgr1 to the activation of a potent oncogene.

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Sayitoglu, Muge, Ozden Hatirnaz, Yucel Erbilgin, Fatmahan Atalar et Ugur Ozbek. « Different Activation of WNT Signaling Pathway in B-Cell and T-Cell Acute Leukemias. » Blood 108, n^o 11 (16 novembre 2006) : 4317. http://dx.doi.org/10.1182/blood.v108.11.4317.4317.

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Abstract WNT signaling pathway proteins function as hematopoietic growth factors and regulate proliferation in normal T-cell and B-cell development. Recent experimental evidence demonstrated that oncogenic transformation in leukemias of both lymphoid and myeloid lineages is dependent on WNT signaling. Not much is known about activation of WNT signaling pathway, its ligands and receptors in hematopoiesis and leukemia pathogenesis. To define its role in leukemia, we aimed to determine mRNA levels of the critical members of WNT pathway (WNT5A, WNT10B, FZ5, β catenin, APC, TCF-1 and LEF-1) by using quantitative real time PCR in acute lymphoblastic leukemia (ALL) patients (T-cell n=42, B-cell n=46 and pre B-cell n=30) and normal hematopoietic cells (bone marrow n=6, peripheral blood n=10, and CD19+ cells from peripheral blood). These genes expressed varying levels in B-cells, preB-cells and T-cells. In the B-cell leukemia patients, WNT5A was expressed notably (OR=58.05 CI 95% 1.63–1219.55, p>0,001). WNT5A directs Ca++ dependent signaling by PKC and a G protein dependent manner which is an alternative pathway for beta-catenin mediated signaling. Also LEF-1 levels were higher in B-ALL patients and APC expression was down regulated when compared to normal tissue (OR=18.81 CI 95% 0.34–5703, p>0.001 and OR=0.212 CI 95% 0.006–8.816, p=0.001, respectively). It is known that LEF-1 blocks APC mediated β catenin nuclear export and activates transcription of various transforming genes, including cyclin, D1, c-myc, MMP7, and LEF-1 itself. WNT5A or WNT10B proteins were not found to be up regulated in preB-ALL whereas APC and LEF-1 gene expressions were increased compared to normal hematopoietic cells (OR=32.97 CI 95% 0.27–1281, 38 p>0.001 and OR=5.57 CI 95% 0.28–89.51, p=0.01, respectively). We found increased TCF-1 expression (7.4 fold) without any β catenin accumulation in T-ALL patients. It is known that TCF-1 in absence of β catenin functions as a tumor suppressor gene. WNT5A, APC and LEF-1 gene expression levels were also different between T-cell, B-cell and preB cell ALL cases. WNT5A expression had the highest levels in B-ALL compared to T-ALL cases, whereas the highest APC expression levels were observed in preB and T-ALL patients. Also LEF-1 expression levels were significantly different between preB and T-cell ALL patients. Taken together these results indicate that WNT signaling genes have abnormal expression and are active in acute lymphoblastic leukemia. This data suggests different WNT activation mechanisms exist in the leukemic transformation in different hematopoietic cells.

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Kode, Aruna, Sanil Manavalan, Ioanna Mossialou, Govind Bhagat, Murty Vundavalli, Ronald DePinho, Julie Teruya-Feldstein, Ellin Berman et Stavroula Kousteni. « Leukemogenic Transformation of Hematopoietic Cells by Constitutive Activation of Canonical Wnt Signaling in Osteoblast Precursors ». Blood 120, n^o 21 (16 novembre 2012) : 509. http://dx.doi.org/10.1182/blood.v120.21.509.509.

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Abstract Abstract 509 Osteoblasts, the bone forming cells, are implicated in the fate of healthy and malignant stem cells. They affect self-renewal and expansion of hematopoietic stem cells (HSCs) and homing of tumor cells into the bone marrow. Here we show that constitutive activation of canonical Wnt signaling in osteoblast precursors disrupts hematopoiesis in mice by shifting the differentiation potential of HSC progenitors to the myeloid lineage which results in accumulation of granulocyte/monocyte progenitors and concomitant development of acute myeloid leukemia (AML). B-lymphopoiesis is also decreased. The AML phenotype is associated with clonal evolution at the cytogenetic level since clonal abnormalities could be detected in leukemic blasts from mice with constitutive activation of the canonical Wnt target β-catenin in osteoblast precursors (βcateninosb mice). Bone marrow transplantation experiments from βcateninosb mice to wild type lethally irradiated mice resulted in development of AML within 8 weeks following transplantation, demonstrating progression towards AML. At the molecular level, cell-specific gene inactivation mouse models demonstrate that β-catenin interacts with FoxO1 in osteoblasts to induce development of AML. Downstream signaling events that confer osteoblast signaling to normal HSCs and lead to their leukemogenic transformation will be presented. Importantly, malignancy-inducing osteoblasts, detected by nuclear accumulation of β-catenin in bone marrow biopsies, were identified in > 25% of patients with myelodysplasia (MDS), acute myeloid leukemia (AML) or AML arising from a prior MDS. Specifically, 15 out of 53 patients with MDS (n=17 patients), AML (n=20 patients), or MDS that had transformed to AML (n=16) chosen at random showed nuclear localization of β-catenin in osteoblasts. Of note, 12 of the 15 (80%) patients with nuclear localization of β-catenin in osteoblasts had abnormalities of chromosome 5 and/or 7, very common cytogenetic abnormalities in patients with MDS and AML. The same signaling pathways mediating AML development in βcateninosb mice were also found to be activated in osteoblasts and hematopoietic cells from the patients with nuclear accumulation of β-catenin in osteoblasts. These findings demonstrate that genetic alterations in osteoblast precursors (1) can induce AML in mice and (2) are associated with AML development in humans. They also provide a molecular basis for the leukemogenic transformation. Disclosures: No relevant conflicts of interest to declare.

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Hu, Kaimin, Lizhen Liu, Binsheng Wang, Kangni Wu, Shan Fu, Yanjun Gu, Xiaoyu Lai, Xiaohong Yu et He Huang. « Bone Marrow Mesenchymal Stromal Cells Protect Acute Lymphocytic Leukemia Cells From Cytotoxic Agents Via MAPK/Erk and Wnt/β-Catenin Signal Pathways ». Blood 120, n^o 21 (16 novembre 2012) : 4736. http://dx.doi.org/10.1182/blood.v120.21.4736.4736.

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Abstract Abstract 4736 Acute lymphoblastic leukemia (ALL) remains one of the greatest challenges in oncology. Relapsed ALL is a leading cause of death in young people, and further improvements in outcome will required the development of therapeutic approaches directed against rational therapeutic targets. Increasing evidence indicated that the bone marrow microenvironment plays a crucial role in the pathogenesis of leukemia by promoting tumor cell growth and survival as well as drug resistance. As a pivotal component of the bone marrow microenvironment, how bone marrow derived mesenchymal stromal cells (MSCs) affect ALL cells and the underlying mechanisms remain unclear. In this study, bone marrow was obtained from healthy adult donors and MSCs were cultured and identified as previous report. Our study applied the co-culture system to mimic the bone marrow microenvironment. To study the effect of bone marrow stroma on ALL cells, three different sub-types of ALL cell lines including Sup-B15, Reh, and MOTL-4 were cultured with and without an underlying confluent layer of MSC. Survival of ALL cells was detected by CCK-8 kit and apoptosis was tested by PI/Annexin V assay. Cell cycle was evaluated by flow cytometry. In addition, Western-bloting was used in mechanism study. The results demonstrated that MSCs improved the survival of ALL cells induced by the cytotoxic agents Idarubicin (IDA) and Etoposide (Vp-16). Number of viable ALL cells were increased remarkably in presence of MSCs. In Vp-16 treatment group, the ratios of viable ALL cell were 1:1.17±0.05 (Sup-B15: Sup-B15+MSC, P<0.05), 1:1.20±0.02 (Reh: Reh+MSC, P<0.05) and 1:1.74±0.16 (MOLT-4: MOLT-4+MSC, P<0.05) respectively. In IDA treatment group, the ratios were 1:1.39±0.11 (Sup-B15: Sup-B15+MSC P<0.05), 1:1.67±0.05 (Reh: Reh+MSC, P<0.01) and 1:1.06±0.07 (MOLT-4: MOLT-4+MSC) respectively. Our data also indicated that coculture with normal MSCs protected ALL cells from apoptosis induced by IDA and Vp-16. The propotion of apoptotic cells was reduced in presence of MSCs both in Vp-16 treatment (Sup-B15: 26.46 ±0.70 vs. 22.00±5.35%, Reh: 18.16±2.09 vs. 16.94±4.34%, MOLT-4: 71.54 ±19.15 vs.62.59±35.63%) and IDA treatment (Sup-B15: 46.57±3.74 vs. 28.86±5.99, Reh: 48.58±6.73 vs. 36.49±5.33%, MOLT-4: 71.54 ±43.72±6.76 vs. 25.04±6.84, P<0.05 respectively). To clarify the mechanisms of the protective role of MSCs, we compared the expression levels of apoptosis and survival related proteins in ALL cells cultured without or with MSCs. The data indicated that MAPK/Erk,Wnt/β-catenin signal pathways were activated in ALL cells co-cultured with MSC. ALL cells showed significantly higher expression of p-Erk and β-catenin in presence of MSCs. At the same time, the cycle inhibition protein p21, mitochondria mediated apoptosis proteins, and the oncogene c-myc were down-regulated. Our study provides experimental basis for the exploring of novel ALL therapy that targets the microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Tickenbrock, Lara, Joachim Schwäble, Markus Wiedehage, Björn Steffen, Bülent Sargin, Chunaram Choudhary, Christian Brandts, Wolfgang E. Berdel, Carsten Müller-Tidow et Hubert Serve. « Flt3 tandem duplication mutations cooperate with Wnt signaling in leukemic signal transduction ». Blood 105, n^o 9 (1 mai 2005) : 3699–706. http://dx.doi.org/10.1182/blood-2004-07-2924.

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AbstractActivating Flt3 mutations occur in about 30% of patients with acute myeloid leukemia (AML), often as in-frame internal tandem duplication (ITD) at the juxtamembrane domain of the receptor. These mutations transform hematopoietic cell lines and primary mouse bone marrow. Here, we analyzed the interaction between oncogenic Flt3-ITD mutations and the Wingless-type (Wnt) signaling pathway in the myeloid progenitor cell line 32D. Microarray analyses revealed higher mRNA expression of Frizzled-4, a receptor for Wnt ligands in 32D/Flt3-ITD cells. Findings were verified by quantitative realtime reverse transcription–polymerase chain reaction (RT-PCR) and on the protein level. Compared with 32D/Flt3-WT (wild-type) cells, 32D/Flt3-ITD cells also showed greatly enhanced β-catenin protein levels, irrespective of their exposure to Wnt3a, a ligand inducing the canonical Wnt signal transduction pathway. In addition, 5 of 7 AML samples with Flt3-ITD mutations expressed high β-catenin protein levels, whereas patients with wild-type Flt3 did not. Also, Flt3-ITD induced enhanced T-cell factor (TCF)–dependent transcriptional activity and the induction of the Wnt target gene c-myc. In the presence of Flt3-WT or Flt3-ITD signaling, Wnt3a slightly increased 32D cell proliferation. However, transfection experiments with dominant-negative (dn) TCF4 revealed a strong dependence of Flt3-ITD–mediated clonogenic growth on TCF activity. Taken together, our results indicate that Flt3-ITD and Wnt-dependent signaling pathways synergize in myeloid transformation.

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Müller-Tidow, Carsten, Björn Steffen, Thomas Cauvet, Lara Tickenbrock, Ping Ji, Sven Diederichs, Bülent Sargin et al. « Translocation Products in Acute Myeloid Leukemia Activate the Wnt Signaling Pathway in Hematopoietic Cells ». Molecular and Cellular Biology 24, n^o 7 (1 avril 2004) : 2890–904. http://dx.doi.org/10.1128/mcb.24.7.2890-2904.2004.

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ABSTRACT The acute myeloid leukemia (AML)-associated translocation products AML1-ETO, PML-retinoic acid receptor alpha (RARα), and PLZF-RARα encode aberrant transcription factors. Several lines of evidence suggest similar pathogenetic mechanisms for these fusion proteins. We used high-density oligonucleotide arrays to identify shared target genes in inducibly transfected U937 cells expressing AML1-ETO, PML-RARα, or PLZF-RARα. All three fusion proteins significantly repressed the expression of 38 genes and induced the expression of 14 genes. Several of the regulated genes were associated with Wnt signaling. One of these, plakoglobin (γ-catenin), was induced on the mRNA and protein level by all three fusion proteins. In addition, primary AML blasts carrying one of the fusion proteins significantly overexpressed plakoglobin. The plakoglobin promoter was cloned and shown to be induced by AML1-ETO, with promoter activation depending on the corepressor and histone deacetylase binding domains. The induction of plakoglobin by AML fusion proteins led to downstream signaling and transactivation of TCF- and LEF-dependent promoters, including the c-myc promoter, which was found to be bound by plakoglobin in vivo after AML1-ETO expression. β-Catenin protein levels and TCF and LEF target genes such as c-myc and cyclin D1 were found to be induced by the fusion proteins. On the functional level, a dominant negative TCF inhibited colony growth of AML1-ETO-positive Kasumi cells, whereas plakoglobin transfection into myeloid 32D cells enhanced proliferation and clonal growth. Injection of plakoglobin-expressing 32D cells into syngeneic mice accelerated the development of leukemia. Transduction of plakoglobin into primitive murine hematopoietic progenitor cells preserved the immature phenotype during colony growth, suggesting enhanced self-renewal. These data provide evidence that activation of Wnt signaling is a common feature of several balanced translocations in AML.

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Cheng, Chi Keung, Libby Li, Suk Hang Cheng, Kitty Ng, Natalie P. H. Chan, Rosalina K. L. Ip, Raymond S. M. Wong, Matthew M. K. Shing, Chi Kong Li et Margaret H. L. Ng. « Secreted-frizzled related protein 1 is a transcriptional repression target of the t(8;21) fusion protein in acute myeloid leukemia ». Blood 118, n^o 25 (15 décembre 2011) : 6638–48. http://dx.doi.org/10.1182/blood-2011-05-354712.

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Abstract Secreted-frizzled related proteins (SFRPs) are modulators of the Wnt signaling pathway that is closely involved in normal and malignant hematopoiesis. Epigenetic deregulation of Wnt modulators leading to aberrant signaling has been reported in adult patients with acute myeloid leukemia (AML), but its occurrence in childhood patients with AML and the role of individual modulators are unclear. In this study, we examined SFRP1, SFRP2, SFRP4, and SFRP5 promoter methylation in 83 patients with AML (59 children and 24 adults) and found preferential SFRP1 methylation and mRNA down-regulation in the prognostically favorable subgroup of AML with t(8;21) translocation. Among the 4 genes, SFRP1 methylation independently predicted prolonged event-free and relapse-free survivals in childhood patients with nonacute promyelocytic leukemia with nonadverse cytogenetics. Mechanistically, we further demonstrated that RUNX1-ETO, the t(8;21) fusion product, specifically bound the SFRP1 promoter and repressed its transcription via a consensus RUNX binding site. In t(8;21)–leukemia cells, SFRP1 selectively inhibited canonical Wnt signaling and cellular proliferation that were associated with concomitant down-regulation of Wnt/β-catenin target genes, including CCND1 and MYC. Taken together, we identified SFRP1 as a transcriptional repression target of the t(8;21) fusion protein and demonstrated a novel mechanism of Wnt activation in a specific subtype of AML.

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Liu, Lin, Keyan Chen, Hua Fan et Feng Jin. « MiR-599 regulates the activity of acute myeloid leukemia cells through MYC-mediated Wnt/β-catenin signaling ». Annals of Translational Medicine 10, n^o 3 (février 2022) : 149. http://dx.doi.org/10.21037/atm-21-6852.

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Ji, Hongyan, Li Chen, Yuqian Xing, Shanshan Li, Jianjian Dai, Ping Zhao et Yulin Wang. « CD82 supports survival of childhood acute myeloid leukemia cells via activation of Wnt/β-catenin signaling pathway ». Pediatric Research 85, n^o 7 (12 mars 2019) : 1024–31. http://dx.doi.org/10.1038/s41390-019-0370-3.

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Liu, Ye, Wei Wang, Yuan Li et Yao Huang. « SOX30 confers a tumor suppressive effect in acute myeloid leukemia through inactivation of Wnt/β-catenin signaling ». Molecular and Cellular Probes 52 (août 2020) : 101578. http://dx.doi.org/10.1016/j.mcp.2020.101578.

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Xin, Hong, Chengliang Li et Minjuan Wang. « DIXDC1 promotes the growth of acute myeloid leukemia cells by upregulating the Wnt/β-catenin signaling pathway ». Biomedicine & ; Pharmacotherapy 107 (novembre 2018) : 1548–55. http://dx.doi.org/10.1016/j.biopha.2018.08.144.

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Golding, Michelle, Pragya Srivastava, Golda Collamat, Smitha R. James, Adam R. Karpf, Michael J. Nemeth et Elizabeth A. Griffiths. « SGI-110, a Novel Hypomethylating Agent, Induces the WNT Inhibitor Secreted Frizzled Related Protein-2 (SFRP2), and Down Regulates β-Catenin in Acute Myeloid Leukemia (AML) Cells ». Blood 120, n^o 21 (16 novembre 2012) : 1290. http://dx.doi.org/10.1182/blood.v120.21.1290.1290.

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Abstract Abstract 1290 Introduction: SGI-110 (Astex Pharmaceuticals, Inc.) is a dinucleotide hypomethylating agent whose active metabolite is decitabine (DAC). This drug demonstrates superior pharmacokinetics relative to the parent drug as a result of resistance to modification by cytidine deaminase, and is being investigated in myeloid malignancy in the phase I/II setting. We and others have demonstrated that WNT inhibitory genes including SFRP2 are epigenetically silenced in AML and that exposure to DNA methyltransferase inhibitors such as 5-Azacitidine (AZA) and DAC can re-express these genes and down-regulate β-catenin signaling in AML cell lines. We hypothesized that treatment with SGI-110 would have a similar effect upon the epigenetically silenced WNT inhibitor SFRP2 and further would down-regulate β-catenin signaling in AML cells in vitro. Methods: The AML cell lines HL60 and U937 were cultured in vitro using standard techniques and treated with phosphate buffered saline, 0.1, 1 or 5 μM SGI-110, 2μM AZA or 0.5μM DAC. Results presented are pooled data from a minimum of three biological replicates. Samples were harvested on day 5 and viable cells, DNA, RNA and protein obtained. β-catenin levels and cellular localization were quantified using imaging flow cytometry (ImageStream), DNA was extracted and bisulfite converted for analysis of gene specific and global DNA methylation by pyrosequencing (LINE-1, SFRP2), RNA was converted to cDNA for analysis by RT-PCR, and protein was obtained to confirm ImageStream results by Western blot. Nuclear translocation of β-catenin, indicative of its signaling activity, was assessed in individual cells by ImageStream using a similarity score: a log-transformed Pearson's correlation coefficient between the digitized images of immunostained β-catenin and a nuclear stain (DAPI). Shifts in the population (n=5,000) distributions of this similarity score were assessed by a resolution metric (Fishers discriminant ratio, Rd). Results: Treatment of AML cell lines with 5μM SGI-110 was toxic, and in line with previous experiments in AML cell lines, above the IC90. Treatment at the lowest dose of SGI-110 had minimal effects upon viability, methylation, and mRNA and protein expression in both cell lines tested. Treatment with SGI-110 at the 1μM dose resulted in reductions in LINE-1 methylation in HL60 cells by 21% (from 82% to 61%), compared to 8% with AZA (to 74%) and 20% with DAC (to 62%). In U937 cells, LINE-1 methylation decreased by 40% (from 67% to 27%) after SGI-110 treatment compared to a 25% reduction with AZA (to 42%) and a 30% reduction with DAC (to 36%). SFRP2 methylation in HL60 and U937 decreased from 86 and 88% at baseline to 66 and 60% with SGI-110 at the 1μM dose, compared to 68% with AZA and to 61% with DAC. Expression of SFRP2 mRNA was observed following treatment with 1μM SGI-110 and with DAC, but was limited following AZA treatment. ImageStream analysis of total cellular β-catenin in HL-60 and U937 cells demonstrated 2.4-fold and 1.2-fold reductions in total β-catenin following 1μM SGI-110 treatment. These results were similar to those seen with DAC (1.8-fold and 1.3-fold in HL-60 and U937 cells respectively). AZA treatment appeared to have a greater effect on total β-catenin in U937 cells (1.3-fold reduction) than in HL-60 cells (0.84-fold reduction). Western blot confirmed reductions in β-catenin protein. We also observed decreased nuclear translocation of β-catenin after treatment of HL-60 and U937 cells with 1 μM SGI-110 (Rd = −0.58 and −0.21 respectively; the negative sign indicates a change in cellular distribution from the nucleus to the cytoplasm). Changes were comparable to those observed with DAC (Rd = −0.75 and −0.26 in HL-60 and U937 cells respectively). AZA treatment of U937 cells resulted in a shift in cellular distribution (Rd = −0.20) similar to that for DAC and SGI-110 but had no effect on β-catenin distribution in HL-60 cells (Rd= 0.00). Conclusions: SGI-110 is a novel DNMT inhibitor which demonstrates robust effects on LINE-1 methylation, SFRP2 mRNA expression, and β-catenin level and localization consistent with epigenetically mediated re-expression of the WNT inhibitor SFRP2. Both upregulated β-catenin signaling and SFRP2 methylation have been demonstrated to correlate with inferior survival in patients with myeloid malignancies. Re-expression of epigenetically silenced WNT inhibitory genes such as SFRP2 may abrogate β-catenin signaling in AML cells. Disclosures: Karpf: Astex Pharmaceuticals: Research Funding. Griffiths:Celgene: Honoraria; Astex Pharmaceuticals: Research Funding.

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Braggio, Danielle Almeida, Fernanda Costas C. de Faria, David Koller, Feng Jin, Abeba Zewdu, Gonzalo Lopez, Kara Batte et al. « Preclinical efficacy of the Wnt/β-catenin pathway inhibitor BC2059 for the treatment of desmoid tumors ». PLOS ONE 17, n^o 10 (14 octobre 2022) : e0276047. http://dx.doi.org/10.1371/journal.pone.0276047.

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Mutation in the CTNNB1 gene, leading to a deregulation of the WTN/β-catenin pathway, is a common feature of desmoid tumors (DTs). Many β-catenin inhibitors have recently been tested in clinical studies; however, BC2059 (also referred as Tegavivint), a selective inhibitor of nuclear β-catenin that works through binding TBL-1, is the only one being evaluated in a clinical study, specifically for treatment of desmoid tumor patients. Preclinical studies on BC2059 have shown activity in multiple myeloma, acute myeloid leukemia and osteosarcoma. Our preclinical studies provide data on the efficacy of BC2059 in desmoid cell lines, which could help provide insight regarding antitumor activity of this therapy in desmoid tumor patients. In vitro activity of BC2059 was evaluated using desmoid tumor cell lines. Ex vivo activity of BC2059 was assessed using an explant tissue culture model. Pharmacological inhibition of the nuclear β-catenin activity using BC2059 markedly inhibited cell viability, migration and invasion of mutated DT cells, but with lower effect on wild-type DTs. The decrease in cell viability of mutated DT cells caused by BC2059 was due to apoptosis. Treatment with BC2059 led to a reduction of β-catenin-associated TBL1 in all mutated DT cells, resulting in a reduction of nuclear β-catenin. mRNA and protein levels of AXIN2, a β-catenin target gene, were also found to be downregulated after BC2059 treatment. Taken together, our results demonstrate that nuclear β-catenin inhibition using BC2059 may be a novel therapeutic strategy for desmoid tumor treatment, especially in patients with CTNNB1 mutation.

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Sun, Clare, Christopher S. Seet, Jun Zhang, Yechen Xiao, Dewen You et Jiwang Zhang. « FLT3 Signaling Enhances Stemness in Murine MLL-AF9 Acute Myeloid Leukemia. » Blood 120, n^o 21 (16 novembre 2012) : 2980. http://dx.doi.org/10.1182/blood.v120.21.2980.2980.

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Abstract Abstract 2980 Acute myeloid leukemia (AML) is developmentally similar to normal hematopoiesis and driven by a subpopulation of leukemia stem cells (LSCs). LSCs are defined by their ability to self-renew and differentiate into more mature leukemia cells. These properties are influenced by the stem cell niche in which they reside. A number of key cytokines within this microenvironment have been identified, including CXCL12 and stem cell factor (SCF), while others have altered the lineage fate of leukemia by in vitro manipulation of growth factor conditions. Activating mutations in fms-like tyrosine kinase receptor-3 (FLT3) have suggested a role for FLT3 signaling in LSC development and function, but little is known about the role of wild type FLT3 and its ligand (FLT3-L) in the LSC niche. In our study, we examined the impact of different hematopoietic growth factors, specifically FLT3-L, on leukemia stem cell properties in a mouse model of MLL-rearranged AML. Murine CD117+ hematopoietic progenitor cells retrovirally transduced with the MLL-AF9 oncogene were selected and cultured in the presence of either FLT3-L or a combination of interleukin-3 (IL-3), interleukin-6 (IL-6), and SCF. We found that FLT3-L selectively expanded a population of CD117+/CD11blo/CD16/32lo cells with a common myeloid progenitor (CMP)-like surface phenotype. This population was present at a frequency of only 1.3% in cells cultured in IL-3/IL-6/ SCF, with remaining cells being mostly CD117+/CD11bhi/CD16/32hi of a granulocyte-macrophage progenitor (GMP)-like surface phenotype. Wright-Giemsa staining showed that cells expanded in FLT3-L also appeared small and morphologically primitive compared to IL-3/IL-6/ SCF cells. When myeloid growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 were added, FLT3-L expanded cells differentiated into a heterogeneous population of CD117+/CD11bhi/CD16/32hi and CD117−/CD11bhi/CD16/32hi cells, and regained the typical myelomonocytic blast morphology seen in MLL-AF9 AML. Flow cytometry with Hoechst 33342 and Pyronin Y staining showed that the cells expanded in FLT3-L were more quiescent, with 47.1% in G0 phase compared to only 1.5% in IL-3/IL-6/SCF expanded cells. Consistent with this, FLT3-L expanded cells showed increased resistance to daunorubicin in colony forming assays. Upon transplantation into lethally irradiated mice, cells expanded in FLT3-L produced leukemia at a remarkably higher frequency (8.7 log-fold using limiting dilution analysis) than cells expanded in IL-3/IL-6/SCF. To explain the differences observed, immunoblotting revealed selective upregulation of the canonical Wnt/β-catenin pathway in FLT3-L expanded cells. In AML, Wnt signaling has been shown to mediate leukemogenesis and LSC self-renewal and homing, and thus is a plausible mechanism for regulating the leukemia stem cell properties enhanced by FLT3-L. Our findings suggest a key role for FLT3 signaling in the development and function of LSCs in MLL-AF9 AML through upregulation of the Wnt/β-catenin pathway. Disclosures: No relevant conflicts of interest to declare.

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Li, C., H. Xin, Y. Shi et J. Mu. « Knockdown of TRIM24 suppresses growth and induces apoptosis in acute myeloid leukemia through downregulation of Wnt/GSK-3β/β-catenin signaling ». Human & ; Experimental Toxicology 39, n^o 12 (16 juillet 2020) : 1725–36. http://dx.doi.org/10.1177/0960327120938845.

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Tripartite motif-containing protein 24 (TRIM24) has currently emerged as a crucial cancer-related gene present in a wide range of human cancer types. However, the involvement of TRIM24 in acute myeloid leukemia (AML) has not been well investigated. The present study aims to investigate the significance, cellular function, and potential regulatory mechanism of TRIM24 in AML. We found that TRIM24 expression was significantly upregulated in AML compared with normal tissues. AML patients with low expression of TRIM24 had higher survival rates than those expressing TRIM24 at higher levels. High expression of TRIM24 was also detected in AML cells and its knockdown markedly restricted proliferation and promoted apoptosis in AML cells. Further investigation revealed that TRIM24 contributed to the regulation of Wnt/β-catenin signaling, which was associated with modulating the phosphorylation status of glycogen synthase kinase-3β (GSK-3β). Inactivation of GSK-3β partially reversed the TRIM24 knockdown-mediated antitumor effects observed in AML cells. Furthermore, knockdown of TRIM24 retarded the growth of AML-derived xenograft tumors in nude mice in vivo. Overall, these findings demonstrate that knockdown of TRIM24 impedes the AML tumor growth through the modulation of Wnt/GSK-3β/β-catenin signaling. These findings highlight the potential TRIM24 as an attractive anticancer target to treat AML.

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Wu, Jun, Yingying Xie et Limei Han. « miR-144-3p Derived from Bone Marrow Mesenchymal Stem Cells (BMSCs) Restrains the Drug Resistance of Acute Myeloid Leukemia (AML) ». Journal of Biomaterials and Tissue Engineering 12, n^o 8 (1 août 2022) : 1525–29. http://dx.doi.org/10.1166/jbt.2022.3065.

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This study assessed whether miR-144-3p derived from BMSCs restrains the drug resistance of AML. Our study intends to assess miR-144-3p’s role in AML drug resistance. Drug resistance AML cells were transfected with miR-144-3p mimic or NC followed by measuring miR-144-3p level, relation of miR-144-3p with Wnt, cell activity and apoptosis by flow cytometry and the expression of signal proteins by Western Blot. The action of miR-144-3p in inducting drug resistance of K562/AND was more effective. Cell apoptosis and proliferative index was increased by overexpression of miR-144-3p along with significantly down regulated Wnt. In conclusion, the malignant invasion of AML with drug resistance is increased by miR-144-3p derived from BMSCs through regulating the Wnt/β-catenin signal, indicating that miT-144-3p might be a new target for the treatment of AML.

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Golding, Michelle, Benjamin E. Paluch, Pragya Srivastava, Song Liu, Qiang Hu, Meir Wetzler, Michael Nemeth et Elizabeth A. Griffiths. « Patients Treated With Decitabine Demonstrate Changes In β-Catenin Localization From The Nucleus To The Cytoplasm In Circulating Blasts ». Blood 122, n^o 21 (15 novembre 2013) : 3956. http://dx.doi.org/10.1182/blood.v122.21.3956.3956.

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Abstract Background The DNA methyltransferase inhibitors (DNMTi) 5-azacitidine (Aza) and decitabine (Dac) are a standard of care for patients with myelodysplatic syndrome (MDS) and acute myeloid leukemia (AML). Many hypotheses exist for the mechanism of these agents, including re-expression of epigenetically silenced tumor suppressor genes and direct cytotoxicity. We and others have shown hypermethylation of WNT/β-catenin inhibitory genes in primary MDS and AML samples. Activation of WNT/β-catenin signaling has furthermore been shown to play a role in the development and relapse of AML. Inhibition of WNT/β-catenin signaling is proposed to be clinically beneficial in AML. Based on published and preliminary data demonstrating that AML cell lines exposed to Aza and Dac re-express WNT/b-catenin inhibitors and down-regulate WNT/β-catenin signaling, we hypothesized that patients with AML receiving Dac would demonstrate suppression of WNT/β-catenin signaling in circulating blasts. Methods We obtained serial peripheral blood samples from 5 patients with AML during their first cycle of Dac therapy (20 mg/m2 per day for 10 days). These patients were ineligible for high-dose induction chemotherapy based on age, performance status or comorbid illness and were of intermediate or poor risk karyotype. All patients had circulating blasts at presentation, which remained stable or declined over time. Blast populations were identified using flow cytometric selection based upon the characteristics of the diagnostic sample. Global methylation was assessed (using LINE-1 as a surrogate) by bisulfite pyrosequencing of mononuclear cell DNA extracted from the peripheral blood. To determine whether reduced methylation was associated with decreases in WNT/β-catenin signaling, we performed imaging flow cytometry (ImageStream, Amnis Corporation) on viable blasts harvested pre-treatment and on days 5-9 of the first Dac cycle (at the methylation nadir). ImageStream was used to assess nuclear localization of β-catenin, a biochemical hallmark of active WNT signaling, in CD34+ blasts. Nuclear β-catenin was quantified using a similarity score: a log-transformed Pearson's correlation coefficient between the digitized images of immunostained β-catenin and a nuclear stain (DAPI). A resolution metric (Fishers discriminant ratio, Rd) was calculated to determine shifts in the population distributions of this similarity score between CD34+ blasts at diagnosis versus after Dac therapy; a negative Rd value indicates decreased similarity scores between β-catenin and DAPI in treated versus diagnostic samples and thus decreased nuclear β-catenin. To determine whether changes in nuclear β-catenin were associated with changes in gene transcription, we performed whole genome analysis of RNA extracted from diagnostic and follow up samples (n = 5 paired samples, Illumina Human-HT12v4 Expression BeadChip). Results As expected, Dac treatment significantly reduced global DNA methylation (LINE-1 methylation) from 77.0 ± 1.6% pre-treatment to a nadir of 60.9 ± 6.8% (p< 0.01, n = 6). During the methylation nadir (days 5 to 9), the cellular distribution of β-catenin in CD34+ blasts shifted from the nucleus to the cytoplasm as indicated by an average Rd value of -0.22 (n = 5). This Rd value is similar to that observed after treating U937 cells with 0.5 µM Dac. The total amount of β-catenin in CD34+ blasts from treated patients did not change compared to their pre-treatment levels (0.98 ± 0.40 fold, n = 5) indicating that the shift in the cellular distribution of β-catenin from the nucleus towards the cytoplasm was not accompanied by decreased protein level. Comparison of samples from day 5-9 of Dac treatment to pre-treatment samples revealed 128 genes with a ≥ 1.2-fold change (75 up, 53 down, p< 0.05) in mRNA levels. We did not observe changes in expression of WNT regulatory genes, suggesting that alternative mechanisms may explain the reduction in nuclear β-catenin following Dac therapy. Of the 10 genes up-regulated more than 1.5-fold, 4 (FOS, EGR1, CLEC12A, RNASE2) are associated with myeloid differentiation. Of these, FOS and EGR1 are negatively regulated by WNT/β-catenin signaling. Conclusions Given that activation of WNT/β-catenin signaling can suppress hematopoietic differentiation, these data suggest a model in which Dac exposure reduces nuclear β-catenin, leading to increased expression of pro-differentiation genes. Disclosures: Wetzler: Teva: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Griffiths:Astex Pharmaceuticals: Research Funding; Celgene, Inc.: Honoraria; Alexion Pharmaceuticals: Honoraria.

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Agarwal, Puneet, Bin Zhang, Yinwei Ho, Amy Cook, Ling Li, Youzhen Wang, Margaret E. Mclaughlin et Ravi Bhatia. « Inhibition of CML Stem Cell Renewal By the Porcupine Inhibitor WNT974 ». Blood 126, n^o 23 (3 décembre 2015) : 54. http://dx.doi.org/10.1182/blood.v126.23.54.54.

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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.

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Sakoda, Teppei, Yoshikane Kikushige, Toshihiro Miyamoto et Koichi Akashi. « TIM-3/Gal-9 Signaling Enhances Self-Renewal Capacity of AML-LSCs through Mimicking Canonical Wnt Signaling ». Blood 128, n^o 22 (2 décembre 2016) : 1673. http://dx.doi.org/10.1182/blood.v128.22.1673.1673.

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Abstract (Introduction) Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs). Self-renewal capacity is one of the most important biological features of LSCs, and therefore, targeting self-renewal machineries of LSCs should be necessary for the eradication of LSCs. We have recently identified the autocrine loop consisted of LSCs-specific surface molecule TIM-3 and its ligand galectin-9 (Gal-9) in human myeloid malignancies. TIM-3/Gal-9 autocrine loop constitutively activates the nucleus accumulation of β-catenin in primary AML LSCs. However, the precise mechanism how this autocrine loop induces β-catenin accumulation has been still unknown. Here, we extended the analysis to clarify the molecular basis of the enhanced β-catenin activity in TIM-3+LSCs. (Results) We tested whether TIM-3 signaling could interact with the canonical Wnt pathway, which plays a central role in controlling the self-renewal capacity in normal hematopoietic stem cells (HSCs) through regulating intracellularβ-catenin accumulation. LDL receptor-related protein 6 (LRP6) is a key component of canonical Wnt pathway and its phosphorylation is essential for the signal transduction of Wnt signaling. Surprisingly, TIM-3 signaling induced by Gal-9 (10 ng/ml) led to phosphorylation of LRP6and accumulation of β-catenin in primary TIM-3+ AML cells. Furthermore, the phosphorylation of LRP6 induced by Gal-9 was completely abrogated in the presence of an anti-TIM-3 antibody (F38-2E2), which blocks the Gal-9 ligation to TIM-3. These results suggested that the ligation of TIM-3 by Gal-9 could activate canonical Wnt pathway independent of Wnt ligands in TIM-3+AML cells. We then tried to clarify the molecular machinery for canonical Wnt pathway activation by TIM-3/Gal-9 interaction. It has been reported that Wnt signaling promptly induces formation of the protein complex described as LRP6-signalosome preceding LRP6 phosphorylation. Therefore, we tested whether TIM-3 signaling could affect the signalosome formation. In the presence of Dickkopf-1 (Dkk-1; 200 ng/mL), which could inhibit LRP6-signalosome formation, TIM-3 signaling totally failed to phosphorylate LRP6 and to activate β-catenin accumulation, indicating that TIM-3 signaling induced Wnt signaling activation through the LRP6-signalosome formation. Since it has been shown that Src family kinases are cytoplasmic mediator of TIM-3 signaling, we focused on p120-catenin because the molecule has been identified as a substrate of Src family kinases as well as an indispensable molecule in LRP6-signalosome formation. We found that the phosphorylation of p120-catenin, a very early step of canonical Wnt signaling, was promptly induced at Tyr228 by TIM-3/Gal-9 interaction as well as Wnt ligand stimulation (Wnt3a; 200 ng/mL), leading to the enhanced β-catenin accumulation in primary TIM-3+LSCs. These results collectively suggested that the TIM-3 signaling mimicked the canonical Wnt signaling from the very early phase of its signaling cascade in LSCs. (Discussion) In this study, we showed TIM-3/Gal-9 signal "mimick" canonical Wnt signaling and lead to the aberrant accumulation of intracellular β-catenin in AML LSCs. Considering that TIM-3 is commonly expressed in LSCs of myeloid malignancies, but not in normal HSCs, TIM-3/Gal-9 signal can serve as a promising therapeutic target for the selective eradication of LSCs because inhibition of TIM-3 signaling can impair the self-renewal capacity of LSCs without affecting normal hematopoiesis. Disclosures Akashi: Bristol Meyers Squibb: Research Funding; Astellas Pharma: Research Funding; Celgene: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Shionogi & Co., Ltd: Research Funding; Sunitomo Dainippon Pharma: Consultancy.

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Dietrich, Philipp A., Murray D. Norris et Jenny Yingzi Wang. « GPR84, a Proinflammatory Receptor, Sustains Wnt/β-Catenin Signaling In Leukemic Stem Cells For Maintenance Of MLL-AF9-Induced Leukemogenesis ». Blood 122, n^o 21 (15 novembre 2013) : 3781. http://dx.doi.org/10.1182/blood.v122.21.3781.3781.

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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.

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Shahid, Akbar Muhammed, In Hwa Um, Mustafa Elshani, Ying Zhang et David James Harrison. « NUC-7738 regulates β-catenin signalling resulting in reduced proliferation and self-renewal of AML cells ». PLOS ONE 17, n^o 12 (15 décembre 2022) : e0278209. http://dx.doi.org/10.1371/journal.pone.0278209.

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Acute myeloid leukemia (AML) stem cells are required for the initiation and maintenance of the disease. Activation of the Wnt/β-catenin pathway is required for the survival and development of AML leukaemia stem cells (LSCs) and therefore, targeting β-catenin is a potential therapeutic strategy. NUC-7738, a phosphoramidate transformation of 3’-deoxyadenosine (3’-dA) monophosphate, is specifically designed to generate the active anti-cancer metabolite 3’-deoxyadenosine triphosphate (3’-dATP) intracellularly, bypassing key limitations of breakdown, transport, and activation. NUC-7738 is currently in a Phase I/II clinical study for the treatment of patients with advanced solid tumors. Protein expression and immunophenotypic profiling revealed that NUC-7738 caused apoptosis in AML cell lines through reducing PI3K-p110α, phosphorylated Akt (Ser473) and phosphorylated GSK3β (Ser9) resulting in reduced β-catenin, c-Myc and CD44 expression. NUC-7738 reduced β-catenin nuclear expression in AML cells. NUC-7738 also decreased the percentage of CD34+ CD38- CD123+ (LSC-like cells) from 81% to 47% and reduced the total number and size of leukemic colonies. These results indicate that therapeutic targeting of the PI3K/Akt/GSK3β axis can inhibit β-catenin signalling, resulting in reduced clonogenicity and eventual apoptosis of AML cells.

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Zhou, Hongsheng, Po Yee Mak, Hong Mu, Duncan H. Mak, Hiroyuki Kouji, Marina Konopleva, Jorge E. Cortes, Michael Andreeff et Bing Z. Carter. « Combination of Tyrosine Kinase Inhibitor with β-Catenin/CBP Modulator C82 Reverses TKI Resistance, Eradicates Quiescent CML Stem/Progenitors Cells, and Overcomes MSC-Associated Microenvironmental Protection ». Blood 124, n^o 21 (6 décembre 2014) : 401. http://dx.doi.org/10.1182/blood.v124.21.401.401.

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Abstract Bcr-Abl tyrosine kinase inhibitors (TKIs) are effective in inducing remissions and improving survival in patients with CML but do not eliminate CML leukemia stem cells (LSCs). Wnt/β-catenin pathway is established to be active in CML and essential for CML LSC, while adult HSCs do not require fully active β-catenin for maintenance. Furthermore, Wnt/β-catenin signaling pathway plays a critical role in TKI resistance and stromal-mediated microenvironmental protection for CML stem and progenitor cells. We propose that combinations of β-catenin inhibitors and TKIs represent a potentially effective therapy by targeting both CML LSCs and leukemia-mediated microenvironmental protection. C82 is a novel β-catenin/CBP modulator that via binding to CBP inhibits the interaction of β-catenin and CBP and thus disrupts Wnt/β-catenin/CBP mediated cell proliferation and self-renewal signaling. CML cell lines and primary CML-BC patient samples were treated with combinations of C82 with different TKIs, including imatinib (IM), nilotinib (NIL), dasatinib (DAS), and ponatinib (PON). Both TKI-sensitive KBM5 (IC50=0.50±0.06µM, EC50=0.32±0.01µM, 48h) and TKI-resistant KBM5-STIT315I (IC50=1.44±0.06µM, EC50=0.36±0.09µM, 48h) cells were sensitive to C82. C82-TKI combinations synergistically induced apoptosis (C82-NIL, CI=0.30±0.07 and C82-DAS, CI=0.20±0.01 in KBM5; C82-NIL, CI=0.24±0.09 and C82-DAS, CI=0.36±0.05 in KBM5-STIT315I at 48h; respectively) and inhibited cell grwoth in both cell lines. KBM5, KBM5-STIT315I and K562 were co-cultured with normal human bone marrow derived-MSCs. Western blot showed that CML/hMSCs co-cultures increased β-catenin, CD44, and survivin proteins in CML cell lines. C82-TKI combinations induced similar degrees of cell death and proliferation inhibition with or without hMSC co-cultures, indicating the combination strategy can overcome MSC-mediated microenvironmental chemoprotection in CML. Western blot analysis showed that C82 significantly inhibited CD44 and survivin expression which was further reduced by C82-TKI combinations in KBM5 and KBM5-STIT315I cells. C82-TKI combinations were evaluated in CML sample (n=6) from heavily-treated and TKI-resistant CML-BC patients. Four out of 6 sample harbored BCR-ABL kinase mutations, including T315I, E255K/V, and H396R. Mononuclear cells from the patients were stained with cell division tracking dye CFSE and then co-cultured with hMSCs. Flow cytometry was performed to identify CD34+CFSEbright and CD34+CFSEdim cells, as quiescent and proliferating population, respectively. When CML cells were treated without hMSC co-culture, C82-TKI combinations exerted stronger synergistic effects in CFSEbright quiescent cells (CI=0.21±0.06, 0.29±0.07, 0.48±0.15, or 0.26±0.03 for combination of C-82 with IM, NIL, DAS, or PON) compared with CFSEdim proliferating cells (CI=0.43±0.05, 0.43±0.17, 0.50±0.20, or 0.44±0.06 for combination of C-82 with IM, NIL, DAS, or PON; respectively). While under co-culture conditions, similar levels of synergy was observed in proliferating (CI=0.39±0.02, 0.23±0.02, 0.32±0.05, or 0.27±0.01 for combination of C-82 with IM, NIL, DAS, or PON) and quiescent cells (CI=0.23±0.02, 0.20±0.01, 0.39±0.10, or 0.20±0.04 for combination of C-82 with IM, NIL, DAS, or PON; respectively). C82-TKI combinations also synergistically induced cell death in CD34+38- CML cells (n=4) and yielded minimum effect on normal bone marrows CD34+ cells (n=3). Invivo studies are ongoing with immunodeficient NOD/SCID/IL2rγnull mice injected with CML-BC patient samples. An open-label, dose-escalation phase I/II study of PRI-724 (active metabolite of C82) for advanced myeloid malignancies (NCT01606579), including CML patients in combination with dasatinb, is enrolling patients at MD Anderson Cancer Center and other centers. Our data demonstrate that β-catenin/CBP signaling pathway plays a critical role in quiescent CML stem/progenitor cells and disruption of the β-catenin/CBP interaction with C82 could overcome MSC-mediated microenvironmental protection for not only proliferating but also quiescent stem/progenitor cells in CML. Combinations of β-catenin/CBP signaling pathway modulator C82 with TKIs represent a potentially promising strategy to tackle TKI resistance and eradicate CML stem/progenitors cells and should be further investigated in larger studies. Disclosures Kouji: PRISM Pharma Co., Ltd: Employment. Cortes:PRISM Pharma Co., Ltd: Clinicaltrial PI for NCT01606579 Other. Andreeff:PRISM Pharma Co., Ltd: Research Funding. Carter:PRISM Pharma Co., Ltd: Research Funding.

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Yi, Hangyu, Jianlong Wang, Maria Kavallaris et Jenny Yingzi Wang. « Lgr4-Mediated Potentiation Of Wnt/β-Catenin Signaling Promotes MLL Leukemogenesis Via An Rspo3/Wnt3a-Gnaq Pathway In Leukemic Stem Cells ». Blood 122, n^o 21 (15 novembre 2013) : 887. http://dx.doi.org/10.1182/blood.v122.21.887.887.

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Abstract Although the clinical importance of aberrant Wnt/β-catenin signaling has been recognized in various cancers, including MLL-rearranged acute myeloid leukemia (MLL AML), its key tractable pathway components have not yet been discovered in leukemic stem cells (LSC). Our studies have identified an Rspo3/Wnt3a-Lgr4-Gnaq pathway, which significantly potentiates β-catenin signaling in MLL LSC. Genetic and pharmacological targeting of this pathway impairs LSC self-renewal and survival, inhibiting MLL-AF9-induced leukemia progression in vivo. Gene expression analysis of AML patient samples (Nucleic Acids Res, 41:D1034-9, 2013) revealed an approximately 3-fold increase (p=0.00002) in expression of leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) in leukemic cells from patients with MLL AML compared to normal human hematopoietic stem cells (HSC). As recent studies have highlighted a critical link between R-spondin (Rspo)/Lgr4 and Wnt/β-catenin signaling pathways, we hypothesized that up-regulation of Lgr4 is associated with aberrant activation of β-catenin signaling in MLL LSC. We have previously demonstrated that β-catenin is highly expressed in HSC transformed by MLL-AF9 and is lower in HSC transduced with leukemic oncogenes such as Hoxa9/Meis1, while increased β-catenin expression is correlated with a poor survival rate in mice. In this study, western blots confirmed high levels of Lgr4 expression in HSC expressing MLL-AF9 compared to Hoxa9/Meis1. ShRNA-mediated stable knockdown of Lgr4 markedly reduced colony formation of HSC expressing MLL-AF9 by 55-65% (p=0.0001) and significantly prolonged mouse survival (p=0.0019) through its inhibition of endogenous β-catenin expression. This deficient phenotype could be rescued by expression of a constitutively active form of β-catenin. Furthermore, ectopic expression of Lgr4 alone was not sufficient for triggering the leukemic transformation of HSC but conferred a growth advantage in vivo to HSC expressing Hoxa9/Meis1 and significantly accelerated the onset of Hoxa9/Meis1-induced AML in mice (p=0.0011). These data support an oncogenic role of Lgr4 in promoting tumor formation through activation of β-catenin signaling. As Lgr4 has recently been identified as a receptor for the Rspo family of secreted proteins (Rspo1–Rspo4), we sought to determine if Rspo is a positive regulator of β-catenin signaling in MLL AML. We found that only the combination of Rspo3 and Wnt3a potently enhanced β-catenin signaling in HSC expressing MLL-AF9 whereas Rspo and Wnt3a alone or the combination of Wnt3a with other Rspo had no effects on β-catenin activity. Depletion of Lgr4 completely abolished Rspo3/Wnt3a-induced β-catenin signaling, suggesting Rspo3/Wnt3a potentiating β-catenin signaling through Lgr4. Next, we assessed if Lgr4 signals through G protein pathways. By testing G protein alpha inhibitors in MLL LSC, we demonstrated that G protein alpha-q (Gnaq) was required for maintenance of stem cell properties by chemical suppression of the Gnaq-activated β-catenin pathway with a Gnaq selective inhibitor, which exhibited a 3-fold decrease in colony formation (p=0.0001) and a 4-fold reduction in cell number (p=0.0009), and was sufficient to induce substantial cell differentiation and apoptosis. Treatment with Gnaq inhibitor abolished the effect of Lgr4 on β-catenin transactivation, implicating an Lgr4-Gnaq-β-catenin signaling pathway in MLL LSC. Microarray analysis of gene expression confirmed enrichment of genes related to cancer cell proliferation, migration and growth, as well as enrichment of Wnt target genes in LSC expressing Lgr4. Taken together, we report here an Rspo3/Wnt3a-Lgr4-Gnaq-β-catenin signaling circuit in MLL leukemogenesis. Interference with components of the circuit can block β-catenin signaling and perturb leukemia development. Thus, our findings provide potential therapeutic targets in treating LSC-based hematological malignancy driven by Wnt/β-catenin signaling. Disclosures: No relevant conflicts of interest to declare.

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Jiang, Xuejie, Po Yee Mak, Hong Mu, Wenjing Tao, Duncan H. Mak, Steven Kornblau, Qi Zhang et al. « Disruption of Wnt/β-Catenin Exerts Antileukemia Activity and Synergizes with FLT3 Inhibition in FLT3-Mutant Acute Myeloid Leukemia ». Clinical Cancer Research 24, n^o 10 (20 février 2018) : 2417–29. http://dx.doi.org/10.1158/1078-0432.ccr-17-1556.

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Zhai, Hong, Junting Zhao, Juan Pu, Pan Zhao et Jin Wei. « LncRNA-DUXAP8 Regulation of the Wnt/β-Catenin Signaling Pathway to Inhibit Glycolysis and Induced Apoptosis in Acute Myeloid Leukemia ». Turkish Journal of Hematology 38, n^o 4 (10 décembre 2021) : 264–72. http://dx.doi.org/10.4274/tjh.galenos.2021.2020.0769.

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Wu, Shiwen, Dongqin Shen et Li Zhao. « AKAP9 Upregulation Predicts Unfavorable Prognosis in Pediatric Acute Myeloid Leukemia and Promotes Stemness Properties via the Wnt/β-Catenin Pathway ». Cancer Management and Research Volume 14 (janvier 2022) : 157–67. http://dx.doi.org/10.2147/cmar.s343033.

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Li, Hongjiao, Chenglian Xie, Yurong Lu, Kaijing Chang, Feng Guan et Xiang Li. « Exosomal miR92a Promotes Cytarabine Resistance in Myelodysplastic Syndromes by Activating Wnt/β-catenin Signal Pathway ». Biomolecules 12, n^o 10 (9 octobre 2022) : 1448. http://dx.doi.org/10.3390/biom12101448.

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Cytarabine (Ara-C) has been one of the frontline therapies for clonal hematopoietic stem cell disorders, such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but Ara-C resistance often occurs and leads to treatment failure. Exosomal microRNAs (miRNAs, miRs) as small noncoding RNA that play important roles in post-transcriptional gene regulation, can be delivered into recipient cells by exosomes and regulate target genes’ expression. miR92a has been reported to be dysregulated in many cancers, including MDS and AML. However, the effects of exosomal miR92a in hematologic malignancies have not been fully investigated. In this study, qualitative analysis showed the significantly enhanced expression of exosomal miR92a in MDS/AML plasma. Subsequent functional assays indicated that exosomal miR92a can be transported and downregulate PTEN in recipient cells and, furthermore, activate the Wnt/β-catenin signaling pathway and interfere with the Ara-C resistance of receipt MDS/AML cells in vitro and in vivo. Altogether, our findings offer novel insights into plasma exosomal miR92a participating in Ara-C resistance in MDS/AML and we propose miR92a as a potential therapeutic target for MDS/AML.

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Iasenza, Isabella Angela, Safia Safa, Frederic Barabe, Sonia Cellot, Brian T. Wilhelm et Kolja Eppert. « High-Throughput Chemical Screen on Acute Myeloid Leukemia Stem Cells Identifies Novel Anti-LSC Compounds ». Blood 138, Supplement 1 (5 novembre 2021) : 1871. http://dx.doi.org/10.1182/blood-2021-145369.

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Abstract Acute myeloid leukemia (AML) is an aggressive form of blood cancer defined by the uncontrolled proliferation and clonal expansion of immature myeloblast cells in the blood and bone marrow, leading to hematopoietic failure. Despite the use of aggressive and cytotoxic standard-of-care drugs, patients often relapse and succumb to the disease partially due to the inability of medically unfit patients to withstand the cytotoxic treatments, regrowth from minimal residual disease and the chemo-resistant nature of leukemic stem cells (LSCs) which can remain in a quiescent state and reside in a protective bone marrow niche. Hence, novel therapies targeting unique leukemic stem cell biology are highly needed to eliminate and avoid reoccurrence. High-throughput screens of human AML LSCs are not performed due to technical issues such as low LSC frequency within primary samples, an inability to purify LSCs, and the difficulty maintaining and expanding primary patient samples and LSCs in vitro. We were able to optimize conditions for a 4-week in vitro large-scale expansion (>600 million bulk) of the primary human AML sample 8227 (OCI-AML-8227), functionally validated to be enriched for LSCs in long-term xenotransplant assays (Eppert et al., 2011). These optimized conditions enabled the isolation and maintenance of the LSC-containing fraction for a chemical screen. We isolated the CD34+ LSC-containing fraction (>90% purity) and performed a high-throughput screen of 11,166 chemical molecules using a CellTiter Glo assay followed by a counter screen against normal CD34+ cord blood (CB) hematopoietic stem and progenitor cells. From this HT screen, a total of 61 hits had >70% inhibition on CD34+ 8227 cells and <30% inhibition on CD34+ CB cells. We also identified glucocorticoids, which were also identified in our prior small-scale anti-LSC screen where they were found to specifically drive human LSCs to terminally differentiate (Laverdière & Boileau, et al., 2018). We then performed dose response assays for each candidate compounds and confirmed 35 potent anti-LSC compounds with IC 50 < 1 μM. This refined the types of compounds to including anti-apoptotic inhibitors, GSK inhibitors, protease inhibitors, metabolism inhibitors, HDAC inhibitors, BET inhibitors, nucleic acid synthesis inhibitors, cell cycle inhibitors and Wnt/β-catenin inhibitors. This is interesting as some of the classes of these compounds (inhibitors of GSK, BET, nucleic acid synthesis, Wnt/β-catenin and metabolism) have been shown to target bulk and leukemic stem cells in AML in vitro and in vivo. We now aim to examine LSC eradication in a panel of genetically defined primary AMLs confirmed through in vitro and in vivo assays. Our goal is to be able to understand and establish the molecular mechanisms and biomarkers on primary functional LSCs. Disclosures No relevant conflicts of interest to declare.

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Daud, Siti Sarah, Alan K. Burnett, Richard L. Darley et Alex Tonks. « Large-Scale Integration of Gene Profiling Identifies TCF7L2/TCF4 as the Most Frequently Dysregulated Wnt Signaling Component In AML ». Blood 116, n^o 21 (19 novembre 2010) : 2480. http://dx.doi.org/10.1182/blood.v116.21.2480.2480.

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Abstract Abstract 2480 Acute myeloid leukemia (AML) represents one of the most genetically heterogeneous malignancies; however, some processes are commonly dysregulated. One of the most frequently dysregulated processes in AML is Wnt signaling. In solid cancers, aberrant Wnt signaling has been shown to promote cancer by increasing nuclear accumulation of β-catenin and with consequent activation of target genes. In AML, overexpression of β-catenin is also common; in addition however, patient studies and genetic models indicate that other components of the Wnt pathway are also commonly dysregulated and may mediate transcriptional changes independently of β-catenin. The aim of this study was to identify aberrantly regulated Wnt components and target genes in AML by interactome analysis of the AML Affymetrix GeneChip® 3` expression microarray datasets; a network building algorithm used to understand relationships between genes. Analysis and interpretation of microarray data is still both biologically and computationally challenging. To address this, we performed batch adjustment to the large scale AML dataset by merging gene expression profile (GEP) data derived from different database sources (including different array platforms). GEPs data were generated from our AML patients enrolled in two different AML NCRI-MRC UK clinical trials using two different Affymetrix platforms, HG-U133A (n=216) and HG-U133Plus2.0 (n=139). GEPs from normal CD34+ bone marrow samples were downloaded from ArrayExpress (n=26). In order to compare AML vs. normal haematopoietic GEP, all data were merged into a single dataset. Individual. CEL files were imported into Partek® Genomics Suite™ and GC-RMA normalization was applied. Linear contrasts, mixed model analysis of variance with false discovery rate correction (P<0.05) and threshold analysis (>1.5 or <1.5 fold-change) were applied to the adjusted data followed by gene enrichment analysis using MetaCore™ (GeneGo Inc). Batch adjustment was performed using Distance Weight Discrimination (DWD) method to the merged GEPs. Prior to further inferential and gene ontology testing, the DWD merged datasets showed significant reduction in the source of data bias with GEP clustered according to their biological variation rather than technical variation. As a result, we present a final list of 58 significant changes in the expression of Wnt related genes in AML. Enrichment by protein function analysis highlighted 8 Wnt transcription factors to be dysregulated (TCF7L2/TCF4, MYC, NANOG, WT1, RUNX2, p300, TCF7, SMAD2), along with 5 receptors (CD44, FZD3, FZD4, FZD5, LDLR), 3 types of phosphatases (B56G, PR61-β, PPP2R5A) and other categories of Wnt related objects (n=33). Consistent findings were seen with previously established Wnt-associated genes specific to AML (CD44, WT1, MYC) showing that data sources from DWD adjustment was effective. We sought to evaluate the significant biological and functional relationships within the genes in the final dataset by transcription factor target modeling using MetaCore™ Interactome tools. Direct network interaction uncovered TCF7L2/TCF4 as the most significantly upregulated Wnt transcription factor with concurrent high expression of its downstream Wnt responsive genes CD44, AXIN1, ID2 that were also present in the final list. Importantly, β-catenin is unlikely to contribute to this transcriptional activation due to the fact that our data showed increased transcription of β-catenin degradation complexes (or negative regulation of Wnt signalling). Specifically, RUVBL1, that directly increases β-catenin activity was significantly downregulated, whereas the other significantly overexpressed upstream genes (APC, CSNK1E, AXIN1, WT1) are known to have inhibitory effect on β-catenin-mediated transcription. In summary, by using multiple GEP data from a large AML cohort in conjunction with robust statistical adjustments, we have identified TCF7L2/TCF4 mediated transcription as the most significant Wnt-regulated process to be altered in AML compared with normal blasts. We also predict that transcription of TCF7L2/TCF4 regulated genes is likely to be independent of β-catenin, supporting observations in genetic models which indicate that β-catenin (and γ-catenin) are redundant for normal haematopoiesis and are not required for TCF-mediated transcription. Disclosures: No relevant conflicts of interest to declare.

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Beghini, Alessandro. « Core Binding Factor Leukemia : Chromatin Remodeling Moves Towards Oncogenic Transcription ». Cancers 11, n^o 12 (7 décembre 2019) : 1973. http://dx.doi.org/10.3390/cancers11121973.

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Acute myeloid leukemia (AML), the most common acute leukemia in adults, is a heterogeneous malignant clonal disorder arising from multipotent hematopoietic progenitor cells characterized by genetic and concerted epigenetic aberrations. Core binding factor-Leukemia (CBFL) is characterized by the recurrent reciprocal translocations t(8;21)(q22;q22) or inv(16)(p13;q22) that, expressing the distinctive RUNX1-RUNX1T1 (also known as Acute myeloid leukemia1-eight twenty-one, AML1-ETO or RUNX1/ETO) or CBFB-MYH11 (also known as CBFβ-SMMHC) translocation product respectively, disrupt the essential hematopoietic function of the CBF. In the past decade, remarkable progress has been achieved in understanding the structure, three-dimensional (3D) chromosomal topology, and disease-inducing genetic and epigenetic abnormalities of the fusion proteins that arise from disruption of the CBF subunit alpha and beta genes. Although CBFLs have a relatively good prognosis compared to other leukemia subtypes, 40–50% of patients still relapse, requiring intensive chemotherapy and allogenic hematopoietic cell transplantation (alloHCT). To provide a rationale for the CBFL-associated altered hematopoietic development, in this review, we summarize the current understanding on the various molecular mechanisms, including dysregulation of Wnt/β-catenin signaling as an early event that triggers the translocations, playing a pivotal role in the pathophysiology of CBFL. Translation of these findings into the clinical setting is just beginning by improvement in risk stratification, MRD assessment, and development of targeted therapies.

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Agarwal, Puneet, Bin Zhang, YinWei Ho, Amy Cook, Ling Li, Youzhen Wang, Margaret McLaughlin et Ravi Bhatia. « Inhibition of CML Stem Cell Growth By Targeting WNT Signaling Using a Porcupine Inhibitor ». Blood 124, n^o 21 (6 décembre 2014) : 3130. http://dx.doi.org/10.1182/blood.v124.21.3130.3130.

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Abstract BCR-ABL tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myeloid leukemia (CML). However, TKIs are unable to eradicate the leukemia stem cells (LSCs) from which CML arises, despite effective inhibition of BCR-ABL kinase activity in this population. There is evidence that extrinsic signals from the bone marrow (BM) microenvironment play an important role in the resistance of CML LSC to TKI treatment. Our previous studies have shown a role for Wnt signaling in protecting CML LSC from TKI treatment (Blood. 2013; 121(10):1824-38). Palmitoylation of Wnts by the Porcupine acyltransferase (PORCN) is required for their secretion and activity. LGK974, a potent PORCN inhibitor, has recently been shown to inhibit Wnt signaling and shows in vivo efficacy against several Wnt-dependent tumors (PNAS. 2013; 110(50):20224-9). In this study, we investigated whether inhibition of Wnt secretion using LGK974 could sensitize CML stem/progenitor cells to TKI treatment. CML and normal CD34+ cells and purified CD34+CD38- stem cells and CD34+CD38+ committed progenitors were treated with LGK974 with or without addition of the BCR-ABL TKI (nilotinib), and in the presence and absence of human BM mesenchymal stromal cells (MSC). CML CD34+ cells showed enhanced Wnt signaling following exposure to recombinant Wnt3a (200ng/ml) compared to normal CD34+ cells as evidenced by increased phospho-LRP6 (ser1490) and β-catenin protein levels. Analysis of all 10 frizzled receptors expression by qRT-PCR revealed a significant upregulation of FZD4 (p=0.026, n=10), and FZD5 (p=0.003, n=10), but downregulation of FZD8 (p=0.009, n=10), in CML compared to normal CD34+ cells. Co-culture with MSC was associated with increased Wnt signaling in CML CD34+ cells as detected by increased expression and nuclear translocation of β-catenin protein, and increased expression of Wnt target genes including c-Myc, Cyclin-D1, PPARδ, and Axin2. Treatment with LGK974 reduced Wnt secretion from MSC measured in a TCF/LEF reporter assay (LGK 0.5μM, 0.74 fold reduction compared to control, p=0.016, n=5, LGK 1μM, 0.62 fold reduction, p=0.007, n=5) and potently inhibited Wnt signaling in CML CD34+ cells both in the absence and presence of MSC. Treatment with LGK974 (1μM) in combination with nilotinib (1μM) resulted in significantly increased inhibition of CML CFC growth compared to nilotinib alone, both in the presence and absence of MSC (34+38- cells LGK alone 82.75±6.38% inhibition of control, Nil alone 43.12±15.32%, combination 23.17±6.48%, p<0.05, n=4; 34+38+ cells LGK alone 87.65±6.13% inhibition of control, Nil alone 26.99±4.54%, combination 16.04±0.48%, p<0.05, n=4) Importantly, LGK974 inhibited CML CFC growth to a significantly greater extent than the normal CFC growth ( normal 34+38- cells LGK alone 88.09±6.85% inhibition of control, Nil alone 89.79±10.09%, combination 70.95±9.39%, p=ns, n=4; 34+38+ cells LGK alone 86.42±8.41% inhibition of control, Nil alone 87.43±4.66%, combination 73.39±6.91%, p=ns, n=4). However, LGK974 treatment did not induce apoptosis in CML or normal progenitors. These results suggest that the effects of LGK974 on clonogenic growth could be related to the long-term inhibition of LSC self-renewal. Therefore, studies designed to evaluate the effect of in vivo administration of LGK974 in a transgenic BCR-ABL mouse model of CML are in progress. Finally, our preliminary studies indicate that in vivo treatment of control healthy mice with LGK974 (5mg/kg bid p.o.), nilotinib (50mg/kg qd p.o.), or the combination for 4 weeks (n=7 per cohort) did not result in clinical toxicity or alter the short-term and long-term hematopoietic stem cell populations. In conclusion, our results indicate that treatment with the PORCN inhibitor LGK974 effectively inhibits both autocrine and paracrine WNT signaling in CML stem cells and inhibits their clonogenic capacity in vitro, and support further evaluation of this approach to selectively target CML stem cells. Disclosures Wang: Novartis Institute for Biomedical Research: Employment. McLaughlin:Novartis Institute for Biomedical Research: Employment.

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