The Effect of Rapamycin, 17-AAG and the Combination on the Bone Marrow Microenvironment in Multiple Myeloma (MM).

Abstract The bone marrow microenvironment in MM is characterized by the presence of upregulated osteoclast activity (OCL) and increased angiogenesis. We have recently demonstrated that the HSP90 inhibitor 17-AAG (provided by the NCI) and the mTOR inhibitor rapamycin (LC Laboratories, MA) have synergistic inhibitory activity on MM cells. The objective of this study was to determine the effect of rapamycin, 17-AAG and the combination on OCL formation and angiogenesis. Rapamycin (0.01–100nM), 17-AAG (10–1000nM) and the combination was tested using an in vitro human OCL formation assay and a human angiogenesis assay (AngioKit, TCS Cellworks, UK). Nonadherent human marrow mononuclear cells (1 x 105/100 μL) were plated in 96-well plates in the presence or absence of DMSO, rapamycin, 17-AAG or the combination. RANKL (100ng/ml) and MCSF (20ng/ml) were added to all wells except control media and MCSF. After 3 weeks, cells were fixed, and the number of OCL-like multinucleated cells were scored. To test the effect of the agents on early OCL precursors, we added the inhibitory agents on days 1, 7 or 14 of the culture. The AngioKit is comprised of human endothelial cells in a 24 well plate. The endothelial cells proliferate and then migrate through the matrix to form tubular structures and anastomosing tubules by 2 weeks. Two control wells were treated with VEGF (+ve control) and two with suramin (−ve control). The optimized medium and test samples were replaced on days 4, 7, and 9 after initial treatment. On day 11, cultures were fixed and stained with antibodies to CD31 to detect vessel formation. The degree of tubule formation was quantitated using computerized image analysis (Angiosys, TCS Cellworks, UK). Single agent rapamycin (20–100nM) inhibited OCL formation by 35% as compared to control in all tested doses indicating that PI3K/mTOR is an important regulator of OCL formation. The effect was similar on day 7 and day 14 indicating that this pathway is important for early and late OCL formation. 17-AAG 100–600nM significantly inhibited OCL formation with 100nM 17-AAG inducing 12% OCL formation as compared to control, while 300 and 600nM completely abrogated OCLs (0% OCLs). This effect was similar at day 7. However, when 17-AAG was added on day 14, it only induced 50–60% reduction in OCL formation indicating that 17-AAG affects early OCL formation. The combination of the two agents completely inhibited OCL formation on day 1 and 7 and led to a 65% reduction in OCLs when added on day 14 of the culture. Rapamycin showed a marked decrease in angiogenesis (similar to the negative control suramin), even at the lowest level tested of 0.01nM. 17-AAG demonstrated some inhibition of angiogenesis at 10 nM, and completely abrogated angiogenesis at 500–1000nM. In summary, rapamycin and 17-AAG inhibit OCL formation and angiogenesis. The effect of 17-AAG was on early OCL formation while rapamycin was on both early and late OCL. These results are contradictory to previous data indicating that 17-AAG increases OCL activity in MM. Rapamycin had a significant inhibitory effect on angiogenesis even at low doses. These results demonstrate that the use of rapamycin analogues and 17-AAG in clinical trials may have a therapeutic effect, not only on MM cells, but also on the bone marrow microenvironment. Supported in part by an ASH Scholar Award and an MMRF grant.

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The BAFF Inhibitor AMG523 Blocks Adhesion and Survival of Human Multiple Myeloma Cells in the Bone Marrow Microenvironment: Clinical Implication.

Blood ◽

10.1182/blood.v108.11.3452.3452 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3452-3452 ◽

Cited By ~ 1

Author(s):

Yu-Tzu Tai ◽

Jiangchun Xu ◽

Xian-Feng Li ◽

Iris Breitkreutz ◽

Klaus Podar ◽

...

Keyword(s):

Bone Marrow ◽

Cell Survival ◽

Mononuclear Cells ◽

Flow Cytometric Analysis ◽

Inhibitory Effect ◽

Bone Marrow Microenvironment ◽

Minor Role ◽

Dependent Manner ◽

Growth And Survival

Abstract We previously identified a role of B-cell activating factor (BAFF), a member of the tumor necrosis factor superfamily, in localization and survival of MM cells in the BM microenvironment (Cancer Res2006, 66:6675–82). In the present study, we examined the potential therapeutic utility of the BAFF inhibitor, AMG523, for treating human MM using MM lines, either sensitive or resistant to conventional chemotherapy, as well as freshly isolated patient MM cells, in the presence or absence of bone marrow stromal cells (BMSCs). AMG523 induces modest cytotoxicity in MM cell lines and patient MM cells, suggesting a minor role of autocrine mechanism of BAFF for MM growth and survival. In the presence of BMSCs, AMG523 significantly decreased growth and survival in dexamethasone (Dex)-sensitive MM1S, Dex-resistant MM1R, INA6 MM cells and in patient MM cells (n=7), in a dose-dependent manner (0.1–10 μg/ml). BAFF-augmented MM adhesion to BMSCs is also blocked by AMG523 at 0.1 mg/ml in MM lines (MM1S, 28PE, INA6), as well as in freshly isolated patient MM cells (n=4). BAFF protects MM cells against dex- and lenalidomide-induced cytotoxicity; conversely, AMG523 blocks BAFF-induced protection against drug-induced apoptosis. Importantly, pretreatment of AMG523 blocks BAFF-induced activation of AKT, nuclear factor kB, and ERK in MM cells, confirming its inhibitory effect on BAFF-mediated adhesion and survival. We next asked whether AMG523 enhances Dex-, bortezomib-, Lenalidomide-induced MM cell cytotoxicity. AMG523 augments the inhibitory effect of Dex and lenalidomide in patient MM cells in the presence of BMSCs. Since osteoclasts (OCLs) secrete BAFF in the bone marrow microenvironment, we further asked whether AMG523 inhibits protection by MM-OCL interaction. OCLs were derived from peripheral blood mononuclear cells from MM patients after 2-week culture with M-CSF and RANKL, and MM cells were added in the presence or absence of AMG523. OCLs significantly increased MM cell survival, evidenced by annexin V and PI staining followed by flow cytometric analysis; conversely, AMG523 blocked MM cell survival by coculture with OCLs. Taken together, our data demonstrate that the novel therapeutic AMG523 blocks the interaction between BAFF and its receptors in human MM, thereby providing the rationale for clinical trials of AMG523 to improve patient outcome in MM.

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SGX70393 Inhibits Bcr-AblT315I In Vitro and In Vivo and Completely Suppresses Resistance When Combined with Nilotinib or Dasatinib.

Blood ◽

10.1182/blood.v110.11.535.535 ◽

2007 ◽

Vol 110 (11) ◽

pp. 535-535 ◽

Cited By ~ 3

Author(s):

Thomas O’Hare ◽

Christopher A. Eide ◽

Jeffrey W. Tyner ◽

Amie S. Corbin ◽

Matthew J. Wong ◽

...

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Kinase Inhibitor ◽

Single Agent ◽

Resistance Profile ◽

Murine Bone Marrow ◽

Abl Kinase ◽

A Cell

Abstract Overview: Bcr-AblT315I is detected in the majority of CML patients who relapse after dasatinib- or nilotinib-based second-line Bcr-Abl kinase inhibitor therapy. SGX70393, an azapyridine-based Abl kinase inhibitor, is effective against Bcr-Abl and Bcr-AblT315I at low nanomolar concentrations in vitro and in cell lines. Here, we comprehensively profiled SGX70393 against native and mutant Bcr-Abl in vitro and in vivo. We also used a cell-based mutagenesis screen to evaluate the resistance profile of SGX70393 alone and in combination with imatinib, nilotinib, or dasatinib. Methods: We assessed colony formation in the presence of SGX70393 by murine bone marrow infected with retroviruses for expression of Bcr-Abl, Bcr-AblT315I, or a variety of other mutants. Toxicity was tested in clonogenic assays of normal bone marrow. SGX70393 effects on cellular tyrosine phosphorylation were measured by immunoblot and FACS in primary Bcr-AblT315I cells isolated from patients with CML or Ph+ B-ALL. In vivo activity was evaluated in a xenograft model using Ba/F3 cells expressing Bcr-AblT315I. Lastly, the resistance profile of SGX70393 was evaluated alone and in dual combinations with imatinib, nilotinib, or dasatinib in a cell-based mutagenesis assay. Results: Colony formation by murine bone marrow cells expressing Bcr-AblT315I (IC50: 180 nM) was reduced by SGX70393 in a dose dependent manner, while no toxicity was observed in colony forming assays of normal human or murine mononuclear cells at concentrations up to 2 μM. Ex vivo exposure of human Bcr-AblT315I mononuclear cells to SGX70393 decreased CrkL phosphorylation, while imatinib, nilotinib, or dasatinib had no effect. SGX70393 inhibited Bcr-AblT315I-driven tumor growth in mice and this was correlated with reduced levels of pCrkL in tumor tissue, while imatinib was ineffective. A cell-based mutagenesis screen revealed a profile of resistant clones confined to four p-loop residues and position 317. SGX70393 in combination with imatinib contracted the spectrum of resistant mutations relative to either single agent, though outgrowth could not be completely suppressed. Combining SGX70393 with low concentrations of nilotinib or dasatinib narrowed the resistance profile still further (residues 248 and 255 for nilotinib; 317 for dasatinib) and, with clinically achievable doses of either second drug, completely abrogated emergence of resistant subclones. Conclusions: SGX70393, a potent inhibitor of Bcr-AblT315I, exhibits a resistance profile centered around the p-loop and residue 317 of Bcr-Abl. Remarkably, in combination with nilotinib or dasatinib, outgrowth of resistant clones is completely suppressed. Single-agent therapy with an effective T315I inhibitor may provide a viable option for patients who relapse with Bcr-AblT315I. However, as a broader spectrum of mutations accounts for imatinib resistance, patients with acquired dasatinib or nilotinib resistance may continue to harbor residual mutant clones other than T315I. Thus, the full clinical potential of SGX70393 may be realized in combinations with a second Abl kinase inhibitor. Our findings provide the first demonstration that Abl kinase inhibitor combinations that include a T315I-targeted component such as SGX70393 have the potential to pre-empt Bcr-Abl-dependent resistance.

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Chronic Lymphocytic Leukemia-Derived Exosomes Stimulate Cells From The Microenvironment

Blood ◽

10.1182/blood.v122.21.3683.3683 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3683-3683

Author(s):

Jerome Paggetti ◽

Guy J. Berchem ◽

Etienne Moussay

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Bone Marrow Microenvironment ◽

Lymphocytic Leukemia ◽

Target Cells ◽

And Migration

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in the blood and the primary lymphoid organs of long-lasting, mature, but non-functional B lymphocytes. Although CLL B cells can survive for long time periods in vivo, cells are undergoing apoptosis relatively quickly in vitro. This spontaneous apoptosis and their sensitivity to drugs is strongly reduced in presence of bone marrow mesenchymal stem cells (MSC) and endothelial cells (EC), which provide anti-apoptotic stimuli to CLL cells via direct contact or secretion of soluble factors. We recently reported the first profiling of circulating miRNA obtained from plasma of CLL patients (Moussay et al., PNAS, 2011). Specific miRNAs were found at higher level in the plasma of CLL patients compared to healthy donors. Exosomes, which are small extracellular vesicles of 50-150 nm originating from endosomes, are now known to efficiently transport nucleic acids and transfer mRNA, microRNA and proteins to target cells. Therefore, exosomes constitute a new component of intercellular communication and their role in CLL remains totally unknown. The specific miRNA signature from plasma of CLL patients combined with our observations that primary CLL B cells can transfer vesicles to MSC through 0.4 µm culture inserts in vitro prompted us to investigate whether CLL B cells secrete exosomes that could modify cells of the bone marrow microenvironment to produce tumor growth promoting factors locally in order to favor their own survival. We isolated, purified and characterized exosomes derived from CLL cell lines, primary cells culture supernatants and plasma from CLL patients. Proteins, mRNA and microRNAs contents were evaluated by high-throughput methods (LC-MS, microarrays) revealing in particular the presence of oncogenic molecules. In vitro, purified CLL-exosomes were found to rapidly enter target cells (already after 1h in MSC and endothelial cells) and to transfer proteins and miRNA. Flow cytometry showed that transferred proteins were expressed at cell surface. Luciferase reporter assay confirmed that miRNAs were efficient in targeting cellular mRNA. Exosomes could also be taken up ex vivo and in vivo by mouse bone marrow cells. Functionally, CLL-exosomes activated key signaling pathways (PI3K, AKT, and MAPK) Immunoblotting indicated the rapid phosphorylation of kinases after 5 min of incubation with CLL-exosomes and the subsequent activation of the canonical NF-kB pathway. We also observed that CLL-exosomes modulated gene expression in target cells among which cytokines (BAFF, IL-6, and IL-8), chemokines (CCL2/MCP-1, CCL5/RANTES, and CXCL1), and other factors involved in cell adhesion and migration (ICAM-1 and MMP-1). These factors were also secreted in the supernatants of MSC and EC as detected by antibody arrays. Exosomes were also shown to increase MSC and EC proliferation, to stimulate actin remodeling, cell migration and to enhance EC angiogenic capabilities (tube formation and aortic ring assays). In conclusion, CLL-exosomes contain pro-oncogenic molecules and strongly affect key functions of MSC and EC which are critical component of the bone marrow microenvironment. Activation of these cells by CLL-exosomes led to release of cytokines/chemokines and oncogenic factors that could promote angiogenesis and also favor leukemic cells survival and migration. Our findings may lead to applications in both diagnosis and therapy development. Molecules identified at the surface or inside CLL-exosomes may be further used as cancer biomarkers. Finally, the description of cell-to-cell communication mechanisms will generate opportunities of innovative therapeutic strategies and confirms the crucial role of exosomes in the development of CLL. Disclosures: No relevant conflicts of interest to declare.

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Bone marrow is a reservoir for proangiogenic myelomonocytic cells but not endothelial cells in spontaneous tumors

Blood ◽

10.1182/blood-2010-02-271122 ◽

2010 ◽

Vol 116 (17) ◽

pp. 3367-3371 ◽

Cited By ~ 32

Author(s):

Andrew C. Dudley ◽

Taturo Udagawa ◽

Juan M. Melero-Martin ◽

Shou-Ching Shih ◽

Adam Curatolo ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Tumor Angiogenesis ◽

Mononuclear Cells ◽

Myeloid Cells ◽

Local Environment ◽

Circulating Endothelial Cells ◽

Peripheral Blood Mononuclear ◽

Angiogenesis Assay

Abstract The hypothesis that bone marrow–derived, circulating endothelial cells incorporate into tumor blood vessels is unresolved. We have measured the numbers of bone marrow–derived versus resident endothelial cells in spontaneous prostate cancers during different stages of tumor progression and in age-matched normal prostates. Bone marrow–derived endothelial cells were rare in dysplasia and in well differentiated cancers representing between 0 and 0.04% of the total tumor mass. Instead, approximately 99% of all tumor-associated bone marrow–derived cells were CD45+ hematopoietic cells, including GR-1+, F4-80+, and CD11b+ myeloid cells. Similar to peripheral blood mononuclear cells, these tumor-associated myeloid cells expressed matrix metalloproteinases (MMPs), consistent with their proposed catalytic role during tumor angiogenesis. Furthermore, freshly isolated CD11b+ cells stimulated tumor endothelial cell cord formation by 10-fold in an in vitro angiogenesis assay. The bone marrow is, therefore, a reservoir for cells that augment tumor angiogenesis, but the tumor endothelium is derived primarily from the local environment.

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Repurposing tofacitinib as an anti-myeloma therapeutic to reverse growth-promoting effects of the bone marrow microenvironment

10.1101/143206 ◽

2017 ◽

Author(s):

Christine Lam ◽

Megan Murnane ◽

Hui Liu ◽

Geoffrey A. Smith ◽

Sandy Wong ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Plasma Cells ◽

Single Agent ◽

Drug Repurposing ◽

Myeloma Cell ◽

Bone Marrow Microenvironment ◽

Growth Promoting

AbstractThe myeloma bone marrow microenvironment promotes proliferation of malignant plasma cells and resistance to therapy. Interleukin-6 (IL-6) and downstream JAK/STAT signaling are thought to be central components of these microenvironment-induced phenotypes. In a prior drug repurposing screen, we identified tofacitinib, a pan-JAK inhibitor FDA-approved for rheumatoid arthritis, as an agent that may reverse the tumor-stimulating effects of bone marrow mesenchymal stromal cells. Here, we validated bothin vitro, in stromal-responsive human myeloma cell lines, andin vivo, in orthotopic disseminated murine xenograft models of myeloma, that tofacitinib showed both single-agent and combination therapeutic efficacy in myeloma models. Surprisingly, we found that ruxolitinib, an FDA-approved agent targeting JAK1 and JAK2, did not lead to the same anti-myeloma effects. Combination with a novel irreversible JAK3-selective inhibitor also did not enhance ruxolitinib effects. RNA-seq and unbiased phosphoproteomics revealed that marrow stromal cells stimulate a JAK/STAT-mediated proliferative program in myeloma plasma cells, and tofacitinib reversed the large majority of these pro-growth signals. Taken together, our results suggest that tofacitinib specifically reverses the growth-promoting effects of the tumor microenvironment through blocking an IL-6-mediated signaling axis. As tofacitinib is already FDA-approved, these results can be rapidly translated into potential clinical benefits for myeloma patients.

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Abstract 1963: Endothelial Cell-Derived Microparticles Interact with Regenerating Cells and Transfer Biological Information

Circulation ◽

10.1161/circ.118.suppl_18.s_413 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Nikos Werner ◽

Nadine Heiermann ◽

Hao Liu ◽

Fritz Horlbeck ◽

Georg Nickenig

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Peripheral Blood ◽

Intracellular Transport ◽

Mononuclear Cells ◽

Biological Information ◽

Annexin I ◽

And Migration

Apoptosis of endothelial cells (ECs) leads to the development of endothelial dysfunction, which itself is strongly associated with poor cardiovascular prognosis. EC apoptosis can be quantified using flow-cytometry-based enumeration of circulating EC-derived microparticles (EMP) within peripheral blood. Bone marrow-derived endothelial progenitor cells (EPC) contribute to EC regeneration and are an important predictor for cardiovascular mortality. We postulate that EC apoptosis with concomitant release of EMP is an important signal for regenerating cells to initiate EC repair. EMP were generated from human coronary arterial endothelial cells (HCAEC). Proteomic analysis showed that the identified proteins represent cytoskeleton/cytoskeleton-binding proteins, proteins involved in intracellular transport/signalling, and protein folding. In addition, proteins related to apoptosis, proliferation, and migration were identified. HCAEC/EPC were able to incorporate EMP in an annexin I/ phosphatidylserine receptor-dependent way. Incubation of mononuclear cells with EMP lead to an enhanced conversion into early outgrowth EPC. EMP co-incubation also changed the phenotype of cultivated Langerhans-like dendritic cells into an immature phenotype. Co-cultivation of ECs and EPC with EMP prevented TNF-alpha induced cell apoptosis. Migration of EPC was enhanced in response to EMP. Finally, we measured EPC liberation from bone marrow into peripheral blood. Intravenous treatment of C57bl6 mice with EMP enhanced the number of sca-1/flk-1 positive EPC within peripheral blood compared to control. The number of CD31+/Annexin+ EMP and CD34 + /KDR + EPC was determined in 40 patients with coronary artery disease. The number of circulating EMP correlated with EPC function (p<0.001, r=0.601). EMP and EPC seem to substantially interact in an annexin I/PSR dependent way. EMP influence EPC biology in vitro and induces EPC mobilization in vivo. We speculate that the described interaction of EMP with EPC enhance the homing process of EPC within the area of EC damage.

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Effects of Dipyrone on Prostaglandin Production by Human Platelets and Cultured Bovine Aortic Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657338 ◽

1983 ◽

Vol 49 (02) ◽

pp. 132-137 ◽

Cited By ~ 16

Author(s):

A Eldor ◽

G Polliack ◽

I Vlodavsky ◽

M Levy

Keyword(s):

Endothelial Cells ◽

Arachidonic Acid ◽

Competitive Inhibition ◽

Inhibitory Effect ◽

Human Platelets ◽

Ionophore A23187 ◽

Aortic Endothelial Cells ◽

Bovine Aortic Endothelial Cells ◽

Aortic Endothelial

SummaryDipyrone and its metabolites 4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoan- tipyrine inhibited the formation of thromboxane A2 (TXA2) during in vitro platelet aggregation induced by ADP, epinephrine, collagen, ionophore A23187 and arachidonic acid. Inhibition occurred after a short incubation (30–40 sec) and depended on the concentration of the drug or its metabolites and the aggregating agents. The minimal inhibitory concentration of dipyrone needed to completely block aggregation varied between individual donors, and related directly to the inherent capacity of their platelets to synthesize TXA2.Incubation of dipyrone with cultured bovine aortic endothelial cells resulted in a time and dose dependent inhibition of the release of prostacyclin (PGI2) into the culture medium. However, inhibition was abolished when the drug was removed from the culture, or when the cells were stimulated to produce PGI2 with either arachidonic acid or ionophore A23187.These results indicate that dipyrone exerts its inhibitory effect on prostaglandins synthesis by platelets or endothelial cells through a competitive inhibition of the cyclooxygenase system.

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Investigation of Mitochondrial Transfer between Human Erythroblasts

10.26686/wgtn.17138759 ◽

2021 ◽

Author(s):

◽

Brittany Lewer

Keyword(s):

Bone Marrow ◽

Mitochondrial Dna ◽

Mononuclear Cells ◽

Bone Marrow Cells ◽

Qpcr Assay ◽

Dna Polymorphisms ◽

Mitochondrial Transfer ◽

Hel Cells ◽

Allele Specific

<p>The increasingly studied phenomenon of mitochondria transferring between cells contrasts the popular belief that mitochondria reside permanently within their cells of origin. Research has identified this process occurring in many tissues such as brain, lung and more recently within the bone marrow. This project aimed to investigate if mitochondria could be transferred between human erythroblasts, a context not previously studied. Tissue microenvironments can be modelled using co-culture systems. Fluorescence activated cell sorting and a highly sensitive Allele-Specific-Blocker qPCR assay were used to leverage mitochondrial DNA polymorphisms between co-cultured populations. Firstly, HL-60ρ₀ bone marrow cells, without mitochondrial DNA, deprived of essential nutrients pyruvate and uridine were co-cultured in vitro with HEL cells, a human erythroleukemia. Secondly, HEL cells treated with deferoxamine or cisplatin, were cocultured with parental HL-60 cells in vitro. Lastly, ex vivo co-cultures between erythroblasts differentiated from mononuclear cells in peripheral blood were conducted, where one population was treated with deferoxamine. Co-culture was able to improve recovery when HL-60ρ₀ cells were deprived of pyruvate and uridine. Improved recovery was similarly detected for HEL cells treated with deferoxamine after co-culture with HL-60 cells. Transfer of mitochondrial DNA did not occur at a detectable level in any co-culture condition tested. The high sensitivity of the allele-specific-blocker qPCR assay required completely pure populations to analyse, however this was not achieved using FACS techniques. In conclusion, results have not demonstrated but cannot exclude the possibility that erythroid cells transfer mitochondria to each other.</p>

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Circulating stem and progenitor cells as markers of inflammation, endothelial regeneration, and prediction of vascular complications in metabolic syndrome and type 1 and 2 diabetes mellitus

Nauchno-prakticheskii zhurnal «Patogenez» ◽

10.25557/2310-0435.2018.01.58-67 ◽

2018 ◽

pp. 58-67

Author(s):

О.В. Першина ◽

А.В. Пахомова ◽

Н.Н. Ермакова ◽

О.Ю. Рыбалкина ◽

В.А. Крупин ◽

...

Keyword(s):

Diabetes Mellitus ◽

Metabolic Syndrome ◽

Stem Cells ◽

Bone Marrow ◽

Endothelial Cells ◽

Progenitor Cells ◽

Vascular Complications ◽

Endothelial Regeneration

Цель исследования состояла в выявлении информативных клеточных маркеров сосудистых осложнений, регенерации микрососудистой сети и воспаления в венозной крови здоровых волонтеров, больных с метаболическим синдромом, сахарным диабетом 1 и 2 типа. Методы. Обследованы больные с метаболическим синдромом (МС), диабетом 2 типа без осложнений, диабетом 1 типа средней степени тяжести и здоровые волонтеры. Диагноз пациентов подтвержден общеклиническими, биохимическими, коагулометрическими и иммуноферментными методами исследования, для оценки экспрессии антигенов использовался многопараметрический цитометрический анализ. Результаты. При анализе экспрессии маркеров показано изменение числа эндотелиальных клеток, мезенхимальных стволовых клеток (МСК) и гемопоэтических стволовых клеток (ГСК) в крови в зависимости от патологии. Эндотелиальные клетки миелоидного (CD45CD14CD34CD309CD144CD31) и немиелоидного (CD45CD14CD34CD309CD144CD31) происхождения, CD309-эндотелиальные клетки и МСК (CD44CD73CD90CD105) предлагаются в качестве маркеров повреждения эндотелия при диабетической симптоматике. При этом ГСК (CD45CD34) могут выступать ценным диагностическим и прогностическим маркером воспаления. Заключение. Для подтверждения сосудистых повреждений и прогноза развития осложнений при диабете 1 и 2 типа в венозной крови пациентов целесообразно оценивать эндотелиальные прогениторные клетки (ЭПК) не костномозговой локализации (CD31CD309CD144) и костномозговой локализации (CD34CD309), и ЭПК c высоким регенеративным потенциалом (CD45CD34CD31CD144). Циркулирующие ЭПК, формирующие колонии in vitro (CD45CD34CD31), рекомендуется использовать в качестве дифференциального маркера состояния регенерации эндотелия при диабете 2 типа. The aim of this study was to identify mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), mature endothelial cells, and endothelial progenitor cells (EPC) in the blood of healthy volunteers, patients with metabolic syndrome, and type 1 and 2 diabetes mellitus as new, informative cellular markers of vascular complications, endothelial regeneration, and inflammation. Methods. The diagnosis was confirmed by general clinical, biochemical, coagulometeric and ELISA studies; multi-parameter cytometric assay was used for evaluation of antigen expression. Results. Changes in the count of MSC, HSC, mature endothelial cells, and endothelial progenitor cells in blood of patients with metabolic syndrome and type 1 and 2 diabetes depended on the type of pathology. We propose using endothelial cells of myeloid (CD45CD14CD34CD309CD144CD31) and non-myeloid origin (CD45CD14CD34CD309CD144CD31), CD309-endothelial cells, and MSCs with the CD44CD73CD90CD105 phenotype as nonspecific markers of endothelial damage in presence of diabetic symptoms. Furthermore, HSCs (CD45CD34) can be used as a valuable diagnostic and prognostic marker of inflammation. Conclusions. It is relevant to evaluate EPCs of non-bone marrow localization (CD31CD309CD144) and bone marrow localization (CD34CD309) and EPCs with a high regenerative potential (CD45CD34CD31CD144) in the blood of patients with type 1 and 2 diabetes to confirm the presence of vascular damage and predict development of complications. Circulating, in vitro colony-forming EPCs (CD45CD34CD31) are recommended as a differential marker for inhibition of endothelial regeneration in type 2 diabetes.

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APTO-253 Induces KLF4 to Promote Potent in Vitro Pro-Apoptotic Activity in Hematologic Cancer Cell Lines and Antitumor Efficacy As a Single Agent and in Combination with Azacitidine in Animal Models of Acute Myelogenous Leukemia (AML)

Blood ◽

10.1182/blood.v124.21.4813.4813 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4813-4813 ◽

Cited By ~ 1

Author(s):

William G Rice ◽

Avanish Vellanki ◽

Yoon Lee ◽

Jeff Lightfoot ◽

Robert Peralta ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Animal Models ◽

Antitumor Activity ◽

Single Agent ◽

Xenograft Model ◽

Myelogenous Leukemia ◽

Acute Myelogenous ◽

Xenograft Models

Abstract APTO-253, a small molecule that mediates anticancer activity through induction of the Krüppel-like factor 4 (KLF4) tumor suppressor, is being developed clinically for the treatment of acute myelogenous leukemia (AML) and high risk myelodysplastic syndromes (MDS). APTO-253 was well tolerated in a Phase I study in patients with solid tumors using a dosing schedule of days 1, 2, 15, 16 of a 28 day cycle (2T-12B-2T-12B), but recent scientific observations guided APTO-253 toward AML and high risk MDS. Indeed, suppression of KLF4 was reported as a key driver in the leukemogenesis of AML and subsets of other hematologic diseases. The vast majority (~90%) of patients with AML aberrantly express the transcription factor CDX2 in human bone marrow stem and progenitor cells (HSPC) (Scholl et al., J Clin Invest. 2007, 117(4):1037-48). The CDX2 protein binds to CDX2 consensus sequences within the KLF4 promoter, thereby suppressing KLF4 expression in HSPC (Faber et al., J Clin Invest. 2013, 123(1):299-314). Based on these observations, the anticancer activity of APTO-253 was examined in AML and other hematological cancers. APTO-253 showed potent antiproliferative activity in vitro against a panel of blood cancer cell lines, with ηM IC50values in AML (6.9 - 305 ηM), acute lymphoblastic leukemia and chronic myeloid leukemia (39 – 250 ηM), non-Hodgkin’s lymphoma (11 – 190 ηM) and multiple myeloma (72 – 180 ηM). To explore in vivo efficacy, dose scheduling studies were initially conducted in the H226 xenograft model in mice. In the H226 model, APTO-253 showed improved antitumor activity when administered for two consecutive days followed by a five day break from dosing (2T-5B) each week, i.e. on days 1,2, 8,9, 15,16, 22,23, compared to the 2T-12B-2T-12B schedule. The 2T-5B schedule was used to evaluate antitumor activity of APTO-253 in several AML xenograft models in mice. In Kasumi-1 AML and KG-1 AML xenograft models, APTO-253 showed significant antitumor activity (p = 0.028 and p=0.0004, respectively) as a single agent when administered using the 2T-5B schedule each week for four weeks compared to control animals. Mice treated with APTO-253 had no overt toxicity based on clinical observations and body weight measurements. Mice bearing HL-60 AML xenograft tumors were treated with APTO-253 for one day or two consecutive days per week for three weeks, either as a single agent or combined with azacitidine, or with azacitidine alone twice per week (on days 1,4, 8, 11, 15 and 18). APTO-253 as a single agent inhibited growth of HL-60 tumors to approximately the same extent as azacitidine. Furthermore, both once weekly and twice weekly dosing of APTO-253 in combination with azacitidine resulted in significantly enhanced antitumor activity relative to either single agent alone (p = 0.0002 and p = 0.0006 for 1X and 2X weekly APTO-253 treatment, respectively, compared to control). Likewise, using a THP-1 AML xenograft model, APTO-253 administered as a single agent using the 2T-5B per week schedule showed significant efficacy, similar to that of azacitidine, while the combination of APTO-253 and azacitidine demonstrated greatly improved antitumor effects relative to either drug alone. APTO-253 was effective and well tolerated as a single agent or in combination with azacitidine in multiple AML xenograft models, plus APTO-253 does not cause bone marrow suppression in animal models or humans. Taken together, our results indicate that APTO-253 may serve as a targeted agent for single agent use and may provide enhanced efficacy to standard of care chemotherapeutics for AML and other hematological malignancies. Disclosures Rice: Lorus Therapeutics Inc.: Employment. Vellanki:Lorus Therapeutics Inc.: Employment. Lee:Lorus Therapeutics Inc.: Employment. Lightfoot:Lorus Therapeutics Inc.: Employment. Peralta:Lorus Therapeutics Inc.: Employment. Jamerlan:Lorus Therapeutics Inc.: Employment. Jin:Lorus Therapeutics Inc.: Employment. Lum:Lorus Therapeutics Inc.: Employment. Cheng:Lorus Therapeutics Inc.: Employment.

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