Nanoparticle Arsenic Compound Realgar Effectively Targets Myeloma Stem-Like Side Population

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4455-4455 ◽  
Author(s):  
Jana Jakubikova ◽  
Teru Hideshima ◽  
Richard W.J. Groen ◽  
Danka Cholujova ◽  
Zdenka Bujnakova ◽  
...  
Keyword(s):  
Stem Cell ◽  
Board Of Directors ◽  
Side Population ◽  
Dependent Manner ◽  
Tumor Initiating Cells ◽  
Advisory Committees ◽  
Ic50 Values ◽  
Anti Tumor Activity ◽  
Dose Dependent

Introduction Tumors contain diverse sub-clones, which can lead to tumor persistence and re-growth after therapy. Previous studies suggest that multiple myeloma (MM) sub-clones may contain tumor-initiating cells which maintain growth and proliferation as well as contribute to drug-resistance. Recently, we have confirmed the existence of MM stem cell-like cells by identifying “side population” (SP) cells as an enriched source of tumor-initiating cells with clonogenic and tumorigenic stem cell properties. Targeting this population with novel therapies represents a promising strategy. Arsenic trioxide (ATO, As2O3) shows only limited responses compared to anti-MM agents such as bortezomib and lenalidomide in relapsed and refractory MM. Here we evaluated the anti-MM activity of another arsenic compound (arsenic sulfide, As4S4), used in traditional Chinese medicine with greater efficacy and less genotoxicity than ATO, prepared by milling into realgar nanoparticles (NREA). We assessed the effect of NREA on MM stem cell-like SP fraction in the context of the bone marrow stromal cells (BMSCs) to provide the rational for its clinical evaluation. Methods and Results First, we evaluated the effect of both NREA and ATO on MM cell survival. Both drugs significantly decreased survival of 13 MM cell lines in a concentration-dependent manner. Importantly, we observed more potent anti-MM effect with NREA compared to ATO, with IC50 values of NREA 2-4 times lower than ATO. Moreover, we confirmed the effect of NREA on MM cells from patients with primary relapsed/refractory disease: NREA showed a dose-dependent response, with IC50 values in the range 1-2 μM. Significant concentration-dependent increased apoptosis was observed in NREA-treated MM cells. Similarly, decreased mitochondrial membrane potential was more significantly triggered by NREA than ATO. Consequently, NREA modulated the MM cell cycle profile: concentration-dependent treatment induced either G0/G1 (MM.1S and KMS11) or G2/M arrest (RPMI-S and OPM1) at 48h. To compare in vivo anti-tumor activity of NREA and ATO, we used our xenograft murine model of human MM. Tumor-bearing mice were intraperitoneally treated with NREA, ATO or with the respective vehicle. Treatment with NREA triggered more significant tumor growth inhibition compared to ATO at day 8 of treatment. To evaluate the effect of NREA on the SP phenotype of MM cells, we treated MM cells with subtoxic concentrations of NREA and ATO for 72 h. Analysis of MM cells for low intracellular content of Hoechst 33342 (SP fraction) revealed that NREA significantly decreased the percentage of SP cells in MM cells in a dose-dependent manner, whereas increase in SP fraction was observed in ATO-treated cells. To evaluate the effect of the bone marrow microenvironment on targeting the SP cells with NREA, MM cells were cultured alone or with BMSCs, and then analyzed for low intracellular accumulation of Hoechst 33342. Treatment by NREA, but not ATO, significantly depleted SP cells in co-cultures MM cells with BMSCs. Moreover, NREA decreased clonogenic potential, whereas ATO had no effect. Similarly, tumorigenic potential of SP cells was significantly decreased by NREA, but not by ATO, treatment. Conclusions Overall, our results demonstrate significant anti-tumor activity of NREA against MM cells in vitro. Our in vivo data further show that NREA significantly decreased tumor burden at clinically achievable concentrations of arsenic. Importantly, targeting of SP cells by NREA in the context of BMSCs provides the preclinical rationale for its clinical evaluation. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Richardson:Millennium: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

Preclinical Studies of Salinomycin In Multiple Myeloma (MM) Models: Targeting of Side Population (SP) Cells In the Context of Tumor – Microenvironment Interactions.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1574-1574
Author(s):  
Efstathios Kastritis ◽  
Jana Jakubikova ◽  
Jake Delmore ◽  
Steffen Klippel ◽  
Douglas W. McMillin ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cell ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Committees ◽  
Ic50 Values

Abstract Abstract 1574 Cancer cells with stem cell-like features are a topic of intense research because their resistance to existing drugs is considered a culprit for relapses, even in patients with complete remission defined by clinical, biochemical and imaging parameters or by sensitive molecular techniques. Salinomycin, an antibacterial and coccidiodostatic ionophore, is reported (Cell 2009;138(4):645-59) to be >100-fold more potent against breast cancer cells with stem cell-like phenotype after mesenchymal transdifferentiation due to stable transfection with shRNA against CDH1 than against the parental cells. We evaluated whether salinomycin could also exhibit a similar activity against stem cell-like cells in multiple myeloma (MM). To establish a comparative reference for such potential activity, we first tested salinomycin (0-10 uM for up to 72hrs) against a panel of 15 MM cell lines and observed IC50 values <1 uM in 10/15 cell lines tested, including >80% reduction of tumor cell viability in 6/15 cell lines tested at 0.5 uM, i.e. levels lower than the IC50 values for in vitro activity of salinomycin against breast cancer cells with (HMLE-shCDH1, IC50 ∼1 uM) or without (HMLE-shControl, IC50 >>10 uM) stem cell-like features. CD138+ purified primary tumor cells from 3 MM patients responded to salinomycin with IC50 values (105, 332 and 750 nM, respectively) in the same range as MM cell lines. In vitro combinations with bortezomib, doxorubicin, melphalan, and dexamethasone showed overall no antagonism, while evidence of additive or even synergistic effect could be identified in certain dose ranges. Because MM cell lines and primary tumor cells responded concordantly to salinomycin and with higher sensitivity than breast cancer stem cell-like cells, we hypothesized that MM cells may in general be more responsive to salinomycin than other tumors. Since tumor-stromal interactions can increase the expression of transcriptional signatures of “stemness” in MM cells, we embarked on characterizing the anti-MM properties of salinomycin using compartment-specific bioluminescence imaging (CSBLI) assays. These showed that co-culture with stromal cells did not confer resistance to salinomycin in 5 MM cell lines (MM.1S, OCI-My5, KMS-11, KMS-18, NCI-H929) and in fact enhanced its activity against 4 of them. Side population (SP) cells, defined by their ability to efflux Hoechst stain, represent a stem cell-like population which was identified in MM cell lines and could represent the functional equivalent of the mesenchymally transdifferentiated breast cancer stem cell-like cells. We observed that salinomycin reduces the SP fraction of MM cell lines at doses >20 times lower than those required for in vitro effect against the bulk <<main population>> of the respective cell lines. Interestingly, the anti-SP effect of salinomycin was more pronounced in the presence of stroma, similarly to the CSBLI studies on the entire MM cell population and consistent with our prior observation that tumor-stroma interaction enhances transcriptional signatures of ≪stemness≫ in the tumor compartment. However, when we tested the in vivo anti-MM activity of salinomycin in an orthotopic model of i.v. injected Luc+ MM cells, no anti-MM activity (in terms of tumor burden decrease or overall survival prolongation) was observed at the maximum tolerated dose (1 mg/kg i.p. daily, which is consistent with most studies reported thus far in the literature). Ex vivo treatment of KMS-11 cells with salinomycin doses (100 nM for 72 hrs) selectively targeting SP cells was followed by s.c. injection of these cells or vehicle-treated controls in sublethallly irradiated SCID/NOD mice, but no statistically significant improvement in tumor burden or overall survival was observed. Our in vitro results indicate that salinomycin exhibits intriguing in vitro anti-MM activity, not only against SP cells but also against the bulk ≪main≫ MM cell population, even in the presence of stromal support. In contrast, the in vivo activity of salinomycin is compromised by side effects in the orthotopic model of MM lesions, while short term ex vivo exposure of tumor cells is conceivably insufficient to eradicate clonogenic cells and lead to appreciable delay in tumor growth in vivo. Our studies point to intriguing features as well as notable challenges that have to overcome before salinomycin or other more selective agents of this class can be safely tested in clinical trials in MM. Disclosures: McMillin: Axios Biosciences: Equity Ownership. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

scholarly journals Anti-tumor activity of a novel proteasome inhibitor D395 against multiple myeloma and its lower cardiotoxicity compared with carfilzomib

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Xuxing Shen ◽  
Chao Wu ◽  
Meng Lei ◽  
Qing Yan ◽  
Haoyang Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitor ◽  
Osteoclast Differentiation ◽  
Dependent Manner ◽  
Serum Enzyme ◽  
Herg Channels ◽  
Anti Tumor Activity ◽  
Dose Dependent

AbstractCarfilzomib, a second-generation proteasome inhibitor, has significantly improved the survival rate of multiple myeloma (MM) patients, but its clinical application is still restricted by drug resistance and cardiotoxicity. Here, we identified a novel proteasome inhibitor, D395, and assessed its efficacy in treating MM as well as its cardiotoxicity at the preclinical level. The activities of purified and intracellular proteasomes were measured to determine the effect of D395 on the proteasome. CCK-8 and flow cytometry experiments were designed to evaluate the effects of D395 on cell growth and apoptosis. The effects of D395 and carfilzomib on serum enzyme activity, echocardiography features, cardiomyocyte morphology, and hERG channels were also compared. In our study, D395 was highly cytotoxic to MM cell lines and primary MM cells but not normal cells, and it was well tolerated in vivo. Similar to carfilzomib, D395 inhibited osteoclast differentiation in a dose-dependent manner. In particular, D395 exhibited lower cardiotoxicity than carfilzomib in all experiments. In conclusion, D395 is a novel irreversible proteasome inhibitor that has remarkable anti-MM activity and mild cardiotoxicity in vitro and in vivo.

The JAK Inhibitor INCB20 Induces Antiproliferative and Apoptotic Effects in Human Myeloma Cells In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3357-3357
Author(s):  
Renate Burger ◽  
Steven Legouill ◽  
Yu-Tzu Tai ◽  
Reshma Shringarpure ◽  
Klaus Podar ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Jak Inhibitor ◽  
Synthetic Compound ◽  
Akt Phosphorylation ◽  
Ic50 Values ◽  
Dose Dependent

Abstract In multiple myeloma (MM), IL-6 plays an important role for tumor cell growth, survival, and drug resistance. Janus kinases (JAKs) are protein tyrosine kinases and constitutively associated with the gp130 chain of the IL-6 receptor complex. Their activation is one of the first steps in cytokine receptor-mediated signaling and critical for virtually all subsequent downstream signaling cascades. INCB20 is a small-molecule synthetic compound which, in biochemical assays, potently inhibited all four JAKs with IC50 values between 0.3 nM and 1.2 nM (for comparison, IC50 of AG490, another JAK inhibitor, was &gt;50 μM). Consistent with the central role of JAKs in gp130-mediated signaling, INCB20 inhibited IL-6 induced phosphorylation of SHP-2, STAT1, STAT3, ERK1/2, and AKT in MM1.S cells. In contrast, AKT phosphorylation induced by IGF-1 remained unchanged. Evaluation of the cellular efficacy of INCB20 was performed using the IL-6 dependent INA -6 cell line. Growth of INA-6 cells was inhibited in a dose-dependent manner with an IC50 of approx. 0.5 μM, as measured by [3H]-thymidine uptake and an MTS-based assay (for comparison, the cellular IC50 of AG490 was 15–20 μM). This correlated with an increase in the percentage of apoptotic cells, as evaluated by Apo2.7 staining after 48 hours. Importantly, INA-6 growth was inhibited in the presence of bone marrow stromal cells accompanied by a decrease in phospho-STAT3 levels. Furthermore, in a subcutaneous INA-6-SCID model, INCB20 inhibited tumor growth (and phosphorylated STAT3) in a dose-dependent manner. Our studies provide the conceptual basis for the use of JAK inhibitors as a therapeutic approach in MM.

Genome-Wide RNA Tomography of the Hematopoietic Stem Cell Niche in Zebrafish Reveals Unexpected Functional Macrophage-Stem Cell Interactions

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3882-3882
Author(s):  
Elliott J Hagedorn ◽  
Julie R Perlin ◽  
Clara Mao ◽  
Brian Li ◽  
Christopher D'Amato ◽  
...  
Keyword(s):  
Stem Cell ◽  
Board Of Directors ◽  
Cell Interactions ◽  
Cell Populations ◽  
Rna Seq ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership

Abstract The challenges of visualizing the mammalian bone marrow have precluded a rigorous analysis of the dynamic cell-cell interactions that control hematopoietic stem and progenitor cell (HSPC) engraftment. The transparent zebrafish embryo provides an unparalleled opportunity to directly visualize HSPC-niche cell interactions in live animals. To identify genes expressed in the zebrafish caudal hematopoietic tissue (CHT) - an embryonic niche akin to the mammalian fetal liver - we employed a new technique called tomo-seq (RNA tomography). By pairing cryosectioning with RNA-seq, this technology permits spatial analysis of transcriptome-wide gene expression. Using tomo-seq we identified ~300 genes showing enriched expression in the CHT. In situ hybridization for 75 of 107 tested genes confirmed CHT expression. In parallel we performed RNA-seq on isolated cell populations, including endothelial cells, macrophages, neutrophils and erythrocytes, sorted from whole embryos. By cross-referencing these datasets we determined the cell types in which many of the 300 CHT-enriched genes were expressed. This analysis revealed several cell surface adhesion receptors enriched on macrophages in the CHT, including the integrin heterodimers itgam/itgb2, itgae/itgb7, itga4/itgb1b and itga4/itgb7. We examined whether known ligands for any of these integrins were present on HSPCs. In situ hybridization to vcam1 (ligand for itga4/itgb1b)showed punctate HSPC-like staining in the CHT. We then generated a vcam1:GFP promoter fusion, which we found was expressed in HSPCs. Using spinning disk confocal microscopy we imaged HSPCs and macrophages in the CHT and observed direct and specific physical interactions that preceded the engraftment of HSPCs. In a grooming-like behavior that lasts for 30-45 minutes, the HSPC is engaged by the macrophage, which moves all over the surface of the cell, before disengaging the HSPC, which then remains in the CHT. Between 48-72 hours post fertilization (hpf), 20% of HSPCs were engaged in this behavior with a macrophage. To evaluate the specificity of these interactions we established in vitro co-cultures using purified cell populations. In co-cultures between macrophages (mpeg1:mCherry) and HSPCs (cd41:GFP) we observed cell-cell interactions that were strikingly similar to those observed in vivo. In macrophage-HSPC co-cultures, 25% of cells were found to interact, whereas only 5% of cells were found to interact in macrophage-erythrocyte co-cultures. To functionally evaluate the macrophage-HSPC interactions in vivo, we depleted macrophages from zebrafish embryos at 55 hpf using clodronate liposomes and observed circulating HSPCs with a significant reduction in HSPC engraftment in the CHT (11/15 embryos, compared to the control where 14/14 embryos showed normal CHT engraftment). Together these studies establish a role for macrophages in promoting the niche engraftment of HSPCs. The results of this work could have important implications for the design of new therapies to improve engraftment during stem cell transplantation. Disclosures Zon: Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Marauder Therapeutics: Equity Ownership, Other: Founder.

scholarly journals TNFR2 As a Target to Improve CD19-Directed CART Cell Fitness and Antitumor Activity in Large B Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 901-901
Author(s):  
Claudia Manriquez Roman ◽  
Michelle J. Cox ◽  
Reona Sakemura ◽  
Kun Yun ◽  
Mohamad M. Adada ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Cell Activation ◽  
B Cell Lymphoma ◽  
Death Receptors ◽  
Target Cells ◽  
Advisory Committees ◽  
Anti Tumor Activity

Abstract Introduction: It has become increasingly apparent that chimeric antigen receptor T (CART) cell activation and differentiation level is an important determinant of CART cell fate and response to therapy. In this study, we aimed to 1) measure levels of activation-induced surface death receptors and ligands on CART cells; 2) investigate how CART cell activation could impact their fitness and clinical responses, and 3) identify cell-based targets to modulate CART cell activation, apoptosis, and cytotoxicity to improve anti-tumor activity. Methods: We performed flow cytometric studies on ex-vivo stimulated, clinically annotated CART products of patients with large B cell lymphoma from the pivotal ZUMA-1 clinical trial that led to FDA-approved Axicabtagene ciloleucel (Axi-Cel). We investigated possible correlations of a number of surface death receptors and ligands with T cell differentiation status and post-infusion CART cell expansion, utilizing samples from ZUMA-1 patients who achieved a complete response as a best outcome ('responders') vs patients who achieved stable or progressive disease('non-responders'). CART cell effector functions in vitro were measured, and CART apoptosis was assessed using Annexin V. For in vitro and in vivo functional studies, we used CART19 generated from healthy donors (HD CART19) as indicated in the specific experiment. CRISPR/Cas9 was employed during CART cell production to disrupt specific genes. A xenograft model of lymphoma was used to investigate the in vivo antitumor activity of CART19. Results: Following an ex vivo stimulation of Axi-Cel products with CD19 + target cells, we observed upregulation of death receptors and ligands in CART19 from non-responders, compared to responders. We also observed a possible association between such upregulated surface markers with CART cell differentiation as measured by CCR7 expression. In an extended in vitro co-culture assay, where HD CART19 cells were repeatedly stimulated through the CAR, we found that tumor necrosis factor α receptor 2 (TNFR2), unlike other death receptors and ligands, was persistently elevated, suggesting a possible role for TNFR2 in long-term antigen-dependent CART19 dysfunction (Figure 1A). We further found that HD CART19 upregulate TNFR2, but not TNFR1, upon CAR stimulation (Figure 1B). While non-specific TCR activation (CD3 stimulation) of HD CART19 cells protected them from activation-induced apoptosis, antigen-specific activation through the CAR resulted in significant initiation of apoptosis within 2 hours of stimulation (Figure 1C). Having identified a possible association between TNFR2 and CART19 dysfunction, we aimed to study the impact of TNFR2 knockout on HD CART19 functions. Using CRISPR/Cas9 during CART cell manufacturing, we generated TNFR2 k/o HD CART19 cells with a knockout efficiency of around 50%, where the expression levels of TNFR2 in activated CART19 cells were reduced, compared to control CART19 cells (with non-targeting gRNA CRISPR/Cas9, Figure 1D). TNFR2 k/o CART19 cells demonstrated reduced early activation surface markers compared to control CART19, as measured by CD25 and CD69 surface expression (Figure 1E), reduced apoptosis initiation as measured by the Annexin V assay (Figure 1F), and enhanced antigen-specific proliferation and cytotoxicity (Figure 1G). Finally, in an in vivo xenograft model of CD19 + lymphoma, TNFR2 k/o CART19 resulted in enhanced CART cell expansion and anti-tumor activity (Figure 1H). Conclusions: Our results indicate that TNFR2 plays a role in early activation and apoptosis initiation of CART19 following CAR stimulation with CD19 + target cells and present TNFR2 knockout as a strategy to enhance CART19 anti-tumor activity. Figure 1 Figure 1. Disclosures Cox: Humanigen: Patents & Royalties. Sakemura: Humanigen: Patents & Royalties. Ding: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Genentech: Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; TG Therapeutics: Research Funding; Tolero Pharmaceuticals: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees. Scholler: Kite: Current Employment. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Mattie: Kite: Current Employment. Kim: Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment. Filosto: Kite, a Gilead Company: Current Employment; Tusk Therapeutics: Patents & Royalties: or other intellecular property; Gilead Sciences: Other: stock or other ownership . Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

scholarly journals Wnt/beta-catenin signalling is dispensable for adult neural stem cell homeostasis and activation

Development ◽  
2021 ◽  
Author(s):  
Sophie H. L. Austin ◽  
Rut Gabarró-Solanas ◽  
Piero Rigo ◽  
Oana Paun ◽  
Lachlan Harris ◽  
...  
Keyword(s):  
Stem Cell ◽  
In Vitro Models ◽  
Beta Catenin ◽  
Dependent Manner ◽  
Direct Role ◽  
Adult Nscs ◽  
Hippocampal Networks ◽  
Dose Dependent

Adult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/β-catenin signalling acts at different steps along the hippocampal neurogenic lineage, but whether it has a direct role in the regulation of NSCs remains unclear. Here we used Wnt/β-catenin reporters and transcriptomic data from in vivo and in vitro models to show that adult NSCs respond to Wnt/β-catenin signalling. Wnt/β-catenin stimulation instructed neuronal differentiation of NSCs in an active state and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, we found that deletion of β-catenin in NSCs did not affect their activation or maintenance of their stem cell characteristics. Together, our results indicate that whilst NSCs do respond to Wnt/β-catenin stimulation in a dose-dependent and state-specific manner, Wnt/β-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which reconciles some of the contradictions in the literature as to the role of Wnt/β-catenin signalling in adult hippocampal NSCs.

scholarly journals AVID200, a Potent Trap for TGF-β Ligands Inhibits TGF-β1 Signaling in Human Myelofibrosis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1791-1791 ◽  
Author(s):  
Lilian Varricchio ◽  
John Mascarenhas ◽  
Anna Rita Migliaccio ◽  
Maureen O'Connor-McCourt ◽  
Gilles Tremblay ◽  
...  
Keyword(s):  
Stem Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Type I Collagen ◽  
Normal Donor ◽  
Type I ◽  
Cell Transplant ◽  
Dependent Manner ◽  
Advisory Committees ◽  
Hematopoietic Stem

Abstract Myelofibrosis (MF) is caused by driver mutations which upregulate JAK/STAT signaling. The only curative treatment for MF is hematopoietic stem cell transplant. Ruxolitinib alleviates many of the symptoms in MF but does not significantly alter survival. There is, therefore, an urgent need for additional rational therapies for MF. Bone marrow fibrosis and collagen deposition are hallmarks of MF which have been attributed to megakaryocyte (MK) derived TGFβ, which also plays a role in myelo-proliferation. There are three isoforms of TGFβ (TGFβ1, β2, and β3). AVID200, which was constructed by fusing TGFβR ectodomains to IgG Fc regions, is a potent TGFβ trap with pM potency against two of the three TGFβ ligands, TGFβ1 and β3 (IC50 values of ~ 3 pM ). AVID200's IC50 for TGFβ2 is ~4,000-fold higher indicating that it has minimal activity against TGFβ2, which is desirable since TGFβ2 is a positive regulator of hematopoiesis. We explored the therapeutic potential of AVID200 by culturing MF or normal donor (ND) mononuclear cells (MNCs) first in the presence of stem cell factor and thrombopoietin (TPO) and then TPO alone in order to generate MK-enriched populations. Although the percentage of mature MKs from ND and MF MNCs was similar, the absolute number of CD41+/CD42+ MKs generated from MF MNCs was two-fold greater than ND MNCs. To determine the levels of TGFβ secreted by the MKs we screened MF and ND MNC conditioned media (CM). We observed significantly higher levels of TGFβ1 but not TGFβ2 and TGFβ3 in MF MK CM. The effects of AVID200 on MKs were then evaluated by measuring the levels of phosphorylated SMAD2. Treatment with 0.001 - 0.1 nM AVID200 decreased phosphorylation of SMAD2, suggesting that AVID200 blocks autocrine MK TGFβ signaling. The increased levels of TGFβ in MF patients promote the proliferation and deposition of collagen by mesenchymal stem cells (MSCs). Cellular proliferation of MSCs was evaluated following treatment with either recombinant TGFβ1 or ND/MF CM in the presence or absence of AVID200. In the absence of AVID200, both recombinant TGFβ1 and MK-derived CM increased the proliferation of MSCs by 1.4- and 1.6-fold respectively, which returned to basal levels with the addition of increasing concentrations of AVID200. These data indicate that AVID200 directly blocks the effect of TGFβ1 on MSCs. MF stroma is characterized by an increase in Type I collagen. We therefore examined if treatment with AVID200 interferes with the ability of TGFβ1 to induce collagen expression by MSCs. MSCs were cultured in presence of recombinant TGFβ1 alone or in combination with varying concentrations of AVID200 for 72 hours. Recombinant TGFβ1 alone induced an increase in COL1A1 mRNA expression as compared to untreated controls (p<0.01). Addition of AVID200 eliminated the TGFβ-mediated increase in COL1A1 expression in a dose dependent manner. ND and MF MK-derived CM also increased COL1A1 expression by MSCs as compared to un-treated controls (p<0.01) and that effect was eliminated by AVID200 treatment (p<0.01). We next demonstrated that TGFβ1 activated pSMAD2 in MSCs without affecting total SMAD2/3 expression and that SMAD2 phosphorylation was reduced by adding AVID200. Furthermore, AVID200 treatment decreased pSTAT3 which is associated with the ability of TGFβ to induce fibrosis. We next investigated the effect of AVID200 on MF hematopoiesis. Briefly, MNCs (which produce TGFβ) from two JAK2V617F+ MF patients were incubated with or without 50 nM of AVID200 and plated in semi-solid media. Treatment with AVID200 did not affect the overall number of colonies generated, but reduced the numbers of JAKV617F+ colonies while increasing the numbers of WT colonies: for PT1, there were 32% JAKV617F+ CFUs in untreated cultures (11 JAKV617F+/34 total colonies) versus 16% JAKV617F+ CFUs (7 JAKV617F+/42 total CFUs) in AVID200 treated cultures; for PT2 there were 100% JAKV617F+ CFUs in untreated cultures (37 JAKV617F+/37 total CFUs) versus 94% JAKV617F+ CFUs (49 JAK2V617F+/52 total CFUs) in AVID200 treated cultures. The in vivo effects of AVID200 on the development of MF in GATA1 low mice will be presented at the meeting. These data indicate that AVID200 selectively suppresses TGFβ1 signaling associated with the proliferation of MSCs and type I collagen synthesis, and depletes MF MNCs of JAK2V617F+progenitor cells. We conclude that AVID200 is a promising agent for treating MF patients which will be evaluated in a phase 1 clinical trial. Disclosures Mascarenhas: Novartis: Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding; Janssen: Research Funding; Promedior: Research Funding; Merck: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding. Iancu-Rubin:Incyte: Research Funding; Merck: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Summer Road: Research Funding; Merus: Research Funding; Janssen: Research Funding; Formation Biologics: Research Funding.

scholarly journals Feasibility and Outcomes of T-Cell Depleted Hematopoietic Stem Cell Transplantation in Patients with Relapsed or Refractory AML and High Risk MDS

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3324-3324
Author(s):  
Satyajit Kosuri ◽  
Sang Mee Lee ◽  
Hongtao Liu ◽  
Mylove Mortel ◽  
Lucy A Godley ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
High Risk ◽  
Stem Cell Transplantation ◽  
Board Of Directors ◽  
Cell Transplantation ◽  
Research Funding ◽  
Advisory Committees

Background: Survival in patients (pts) with relapsed/refractory (R/R) acute myeloid leukemia (AML) and high risk myelodysplastic syndrome (MDS) is dismal. Treatment options are limited; however, a proportion of these individuals can be rescued by allogeneic stem cell transplantation (allo-SCT). Historically, allo-SCT, especially for R/R myeloid diseases, has used myeloablative regimens and no T-cell depletion (TCD) to maximize graft-versus-leukemia effect, often restricting this approach to younger and fit pts with matched donors. The aim of this study was to investigate outcomes of in vivo T-cell depleted stem cell transplantation (TCD-SCT) in a high-risk AML and MDS population. Methods: We performed a retrospective analysis of 141 patients with R/R AML (n=108)/high risk MDS (RAEB or CMML, n=33) who received TCD-SCT at our center from 2002-2015. Median age was 55 years (18-71) with 37 (26%) pts older than 60. Patients underwent in vivo TCD with alemtuzumab or ATG and 117 (88%) received reduced-intensity conditioning (RIC). Alemtuzumab was generally given as 100 mg total divided over 5 days whereas rabbit ATG dosing included days -1, - 3, -5 (+/- on day -7). Alemtuzumab usually partnered with matched related (n=65; 46%) or unrelated (n=53; 38%) peripheral blood stem cell (PBSC) grafts whereas ATG mostly was a component of umbilical cord grafts combined with a CD34 selected haploidentical donor (haplo-cord) (n=23; 16%). Prognostic factors such as age, HCT-CI, CIBMTR score (Duval 2010), revised disease risk index (R-DRI), donor type and pre-transplant disease status were analyzed. Multivariate cox regression models were considered from forward selection for factors with a p value <0.1 in univariate analysis. Results: Table 1 summarizes baseline characteristics. Among the 141 R/R AML or high risk MDS pts, AML predominated (77%). Sixty six (47%) pts had primary induction failure (PIF), 42 (37%) had relapse and 33 (23%) had high risk MDS. Eighty three pts (59%) had peripheral blasts at time of TCD-SCT. Cumulative incidence (CI) of relapse for all pts was 53% and non-relapse mortality was 28% at 2 yrs. Two and 5 yr PFS rates for the group were 19% and 11%, respectively. Two and 5 yr OS rates for the group were 30% and 18%, respectively. Figure 1 shows OS by disease type. Day 100 mortality was 18%. Twenty one percent developed Grade 2-4 acute GVHD (aGVHD) (6% Grade 3-4), and only 5% developed chronic GVHD (cGVHD) requiring therapy. Figure 2 shows CI of cGVHD amongst disease types. Differences in 2yr survival outcomes were not significant among prognostic factors. Specifically, age 60+ vs younger was not prognostic (PFS 24% vs 17% p=0.4, OS 29% vs 29% p=0.7). Likewise, haplo-cord did not differ relative to matched donors in outcomes (PFS 18% vs 26% p=0.2, OS 35% vs 29% p=0.5). Conclusions: Although novel therapeutic approaches are emerging for R/R AML and high risk MDS, allo-SCT remains an established option for long-term disease control. In our analysis, outcomes after in vivo TCD-SCT in R/R AML and high-risk MDS pts treated with RIC mirror published historical results (Duval 2010, Schlenk 2010) but with low rates of cGVHD. The lack of significant difference in survival outcomes amongst age groups and donor sources suggests RIC with in vivo TCD can also be utilized as a platform in older individuals and those with alternative donors. With high relapse rates in this population, better pre-transplant disease reduction, minimal residual disease monitoring and post-transplant maintenance will be critical to increase long-term cures. Disclosures Liu: Agios: Honoraria; Arog: Other: PI of clinical trial; BMS: Research Funding; Karyopharm: Research Funding; Novartis: Other: PI of clinical trial. Larson:Novartis: Honoraria, Other: Contracts for clinical trials; Agios: Consultancy; Celgene: Consultancy. Odenike:Oncotherapy: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Astra Zeneca: Research Funding; Astex Pharmaceuticals: Research Funding; NS Pharma: Research Funding; Gilead Sciences: Research Funding; Janssen Oncology: Research Funding; Agios: Research Funding; CTI/Baxalta: Research Funding. Stock:Kite, a Gilead Company: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; UpToDate: Honoraria; Research to Practice: Honoraria. Kline:Merck: Honoraria; Merck: Research Funding. Riedell:Bayer: Honoraria, Speakers Bureau; Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Van Besien:Miltenyi Biotec: Research Funding. Bishop:Kite: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Consultancy, Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Artz:Miltenyi: Research Funding.

scholarly journals Wnt/beta-catenin signalling is dispensable for adult neural stem cell homeostasis and activation

2020 ◽  
Author(s):  
Sophie H. L. Austin ◽  
Lachlan Harris ◽  
Oana Paun ◽  
Piero Rigo ◽  
François Guillemot ◽  
...  
Keyword(s):  
Stem Cell ◽  
Beta Catenin ◽  
Dependent Manner ◽  
Direct Role ◽  
Adult Nscs ◽  
Hippocampal Networks ◽  
Dose Dependent

AbstractAdult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/β-catenin signalling acts at different steps along the hippocampal neurogenic lineage and has been shown to promote the proliferation of intermediate progenitor cells. However, whether it has a direct role in the regulation of NSCs still remains unclear. Here we used Wnt/β-catenin reporters and transcriptomic data from in vivo and in vitro models to show that both active and quiescent adult NSCs respond to Wnt/β-catenin signalling. Wnt/β-catenin stimulation instructed neuronal differentiation of active NSCs and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, we found that inhibiting NSCs response to Wnt, by conditionally deleting β-catenin, did not affect their activation or maintenance of their stem cell characteristics. Together, our results indicate that whilst NSCs do respond to Wnt/β-catenin stimulation in a dose-dependent and state-specific manner, Wnt/β-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which could reconcile some of the contradictions in the literature as to the role of Wnt/β-catenin signalling in adult hippocampal NSCs.

scholarly journals Eltrombopag: More Than Just a Thrombopoietin Receptor Agonist (TPO-RA) in Immune Thrombocytopenia (ITP)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2364-2364
Author(s):  
Anwar A. Sayed ◽  
Amna Malik ◽  
Grace Ayoola ◽  
Elisa Lucchini ◽  
Sasfia Candrianita ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Granzyme B ◽  
Immunomodulatory Effect ◽  
Dependent Manner ◽  
Advisory Committees ◽  
Dose Dependent

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by a skewed proinflammatory T cell profile. Thrombopoietin-receptor agonists (TPO-RA) have largely replaced immunosuppressants in the management of this disorder, with some patients achieving remission after a period of treatment with TPO-RA. The potential immune modulatory role of TPO-RA has not been fully investigated. The two current TPO-RA licensed for use in ITP; Eltrombopag (Elt) and Romiplostim (Romi) act on different parts of the TPO-R and have similar response rates. However, patients can respond to one agent but not the other. Elt has been described to have a strong iron chelating effect, and hence we propose that it may have an additive immunomodulatory effect on the T cells, absent in Romi. We determined the immunomodulatory effect of Elt by assessing the proliferation and functionality of T-cell lines and primary T-cells. T cell proliferation was assessed using both CFSE proliferation assay and MTT cell viability assay. T cell phenotype and functionality were assessed by multicolor surface and intracellular flow cytometric staining. Cells were co-cultured with Elt and Romi in vitro and ex vivo with both Jurkat and DG75 cells lines as well as primary cells, respectively. Deferoxamine (DFX) was used as a positive control for iron-chelation, and human TPO was used as a positive control for TPO-RA. All treatment doses were based on their calculated therapeutic serum levels. Mann Whitney U and Kruskal-Wallis H statistical tests were applied where applicable, and a P value of less than 0.05 were considered significant. Elt significantly decreased Jurkat T cells proliferation in a dose-dependent manner compared to no treatment and Romi. DFX, an iron chelator, also decreased Jurkat T cell proliferation to comparable levels of Elt. Interestingly, this anti-proliferative effect of Elt was only observed on Jurkat T cells, but not DG75 B cell line. Ex vivo CFSE proliferation assay was performed on primary CD4 and CD8 T cells assessing the antiproliferative effect of Elt. Elt significantly reduced proliferation compared to no treatment. DFX exhibited a similar antiproliferative effect on primary T cells, however, less potent compared to Elt. Neither Romi nor TPO affected the proliferation of Jurkat cells, DG75 cells or primary T cells. The functionality of CD4 and CD8 T cells was assessed based on the capacity of T cells to produce intracellular TNFα, IFNγ and Granzyme B. Elt significantly reduced the percentages of TNFα+/IFNγ+ CD4+ and CD8+ T cells in a dose-dependent manner. This reduction was also observed, albeit to a lesser extent, when T cells were treated with DFX. Furthermore, Granzyme B expression in CD8+ T cells was significantly reduced when cells were treated Elt, compared to no treatment. Romi did not affect the frequency of CD8+ TNFα+/IFNγ+ populations nor the expression of Granzyme B in CD8+ T cells. CD4+ and CD8+ T cells did not express TPO-R on their surface. To confirm the immunomodulatory role of Elt in vivo, the terminally-differentiated effector (CD45RA+CD62L-) CD8+ T cells were assessed in 13 Elt-treated patients and 11 Romi-treated patients. Patients on Elt had significantly reduced frequency of effector CD8 T cells compared to Romi-treated patients (44% vs 76.8%; p<0.01). Taken together, these novel findings suggest an off target immunomodulatory nature of Elt besides its thrombopoietic effect. This dose-dependent immunomodulatory effect is not TPO-R dependent and targets T cells primarily. This study is the first to display such property of Elt and could explain why there is a differential response to Elt and Romi. We hypothesise that Elt may be more effective in patients with T cell mediated disease, whilst patients with predominantly antibody mediated disease are more likely respond to Romi. These findings can also offer an explanation for Elt effectiveness in other T cell-mediated autoimmune conditions such as Aplastic Anemia. Disclosures Cooper: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Principia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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