Potential Eradication of Myeloma Stem Cells by Combining ATRA with WNT and HH Signaling Pathways Inhibitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2839-2839 ◽  
Author(s):  
Guido J. Tricot ◽  
Siqing Wang ◽  
Lei Shi ◽  
Hongwei Xu ◽  
Chunjiao Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Stem Cell Population ◽  
Molecular Characteristics ◽  
Myeloma Cells ◽  
Hh Signaling ◽  
Stimulation Of

Abstract Abstract 2839 Poster Board II-815 Multiple myeloma (MM) remains largely incurable. The existence of the putative myeloma stem cell population may account for drug-resistance and relapse. Previous investigation showed that the CD138-negative (CD138-) MM cell fraction contains the putative MM stem cells. However, little is known about the molecular characteristics of MM stem cells, which makes it difficult to specifically target such cells. In this study, by comparing gene expression profiles (GEP) of CD138+ and CD138- cells isolated from 10 MM cell lines, we discovered that RARα is the top one over-expressed gene in CD138- MM stem cells. RARα has two major isoforms, RARα1 and RARalpha2. Real-time PCR detected significantly higher expression of RARalpha2 but not of RARalpha1 in CD138- MM cells compared with CD138+ cells. We recently reported that increased RARalpha2 expression at diagnosis resulted in a significantly shorter overall-survival (P = 0.003); importantly, ATRA selectively killed RARalpha2-positive myeloma cells, but not RARalpha2-negative cells. These results suggested that ATRA could be used to eradicate specifically RARalpha2-over-expressing MM stem cells. Indeed, we found that ATRA selectively killed CD138- MM stem cells (P < 0.01) from ARP-1 and OCI-MY5 human myeloma cell lines and 5T33 murine myeloma cells but spared the CD138+ tumor cells from these cell lines. WNT and Hedgehog (HH) signaling pathways were activated in CD138- stem cells. To our surprise, ATRA treatment (10-6M) further increased WNT and HH signaling pathways in CD138- myeloma cells, based on increased protein levels of β-catenin and cleaved Shh/Ihh in ATRA-treated cells. Stimulation of these signaling pathways by LiCl (5 mM) and/or SHH(20 μg/mL) partially abrogated ATRA-induced cytotoxities on CD138- MM cells, demonstrating that stimulation of WNT and HH signaling induced partial ATRA-resistance in CD138- cells. A combinational treatment of ATRA (1 μM), a COX-2 inhibitor celecoxib (shown to inhibit WNT signaling, 50 μM) and a HH signaling inhibitor cyclopamine (10 μM) induced synergistic effects on cell death and growth inhibition of CD138- and RARalpha2-over-expressing MM cells in-vitro. In the 5T33 murine myeloma model, the combination of ATRA (20mg/Kg), celecoxib (10mg/Kg) and cyclopamine (20mg/Kg) induced synergistic inhibition of tumor growth in-vivo. Thus, our study provides novel approaches to specifically target MM stem cells. Disclosures: No relevant conflicts of interest to declare.

Stroma cells and Monocytes make Multiple Myeloma Cells Active In Bone Marrow Microenviroment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5333-5333
Author(s):  
Hiroshi Ikeda ◽  
Tadao Ishida ◽  
Toshiaki Hayashi ◽  
Yuka Aoki ◽  
Yasuhisa Shinomura
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Cell Contact ◽  
Drug Induced ◽  
Myeloma Cells

Abstract The Bone marrow (BM) microenvironment plays crucial role in pathogenesis of multiple myeloma　(MM). Paracrine secretion of cytokines in BM stromal cells promotes multiple myeloma cell proliferation and protects against drug-induced cytotoxicity. In current study, monocytes, component of BM cells, can directly promote mesenchymal stem cells osteogenic differentiation through cell contact interactions. Down-regulation of inhibitors such as DKK1 drives the differentiation of mesechymal stem cells into osteoblasts. In this study, we examined the role of monocytes as a potential niche component that supports myeloma cells. We investigated the proliferation of MM cell lines cultured alone or co-cultured with BM stromal cells, monocytes, or a combination of BM stromal cells and monocytes. Consistently, we observed increased proliferation of MM cell lines in the presence of either BM stromal cells or monocytes compared to cell line-only control. Furthermore, the co-culture of BM stromal cells plus monocytes induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, BMSCs and monocytes decreased the rate of apoptosis of myeloma cells. Our results therefore suggest that highlights the role of monocyte as an important component of the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

Targeting Myeloma Stem Cells through Simultaneous Inhibition of Wnt and Hedgehog (Hh) Signaling Pathways

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 615-615
Author(s):  
Guido J. Tricot ◽  
Ye Yang ◽  
Chujiao Xu ◽  
Hongwei Xu ◽  
Ming Zeng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Signaling Pathways ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cyclin D3 ◽  
Expression Levels ◽  
Gli1 Expression

Abstract Abstract 615 Cancer stem cells (CSC) have been identified in a variety of tumor types, including multiple myeloma (MM). Different signaling pathways, such as Wnt, Hedgehog (Hh), Notch and Bmi, are up-regulated in CSC. MM stem cells (MMSC) are enriched in the side population and have increased aldehyde dehydrogenase 1 A1 (ALDH1A1) activity. They are non-cycling and resistant to drugs typically used to treat MM. In this study, MMSC were isolated from 10 MM cell lines (CD138− fraction) and 4 primary MM patients by selecting the CD19+ CD138− cells from bone marrows heavily involved with MM. We confirmed that the CD19+/CD138− cells were part of the malignant clone by comparing expression levels of spiked genes, such as MAF-B and Cyclin D3, in MMSC and CD138+ MM cells. We also verified that CD138− from MM cell lines and the primary CD138−CD19+ had stem cell characteristics by clonogenic in vitro assays and tumor formation in NOD/SCID mice. GEP and real-time PCR analyses indicated that RARa2 levels were significantly higher levels in MM cell line-derived and primary MMSC than in CD138+ bulk MM cells. We also showed that over-expression of RARα2 in low-expressing MM cell lines resulted in increased Wnt and Hh activity, as evidenced by higher levels of nuclear β-catenin, Cyclin D1, TCF4, LEF1, Smo and GLI1. Expression levels of these proteins were also higher in MMSC than in CD138+ MM cells. We subsequently evaluated the efficacy of inhibition of Wnt (CAY10404, a COX-2 inhibitor) and Hh (cyclopamine and itraconazole) signaling on MMSC in vitro and also in vivo using the 5TGM1 mouse model. These drugs given separately induced potent cell death and growth inhibition in MMSC. Quantitative PCR and western blot revealed that CAY10404 decreased nuclear β-catenin and Gli1 expression, while cyclopamine and itraconazole inhibited nuclear GLI1 expression. The efficacy of CAY10404 and cyclopamine was further evaluated in the 5TGM1 mouse model. The CD138− fraction of this cell line has many of the same characteristics as human CD138− MMSC. After injection of 0.5 M cells into the C57BL/KaLwRij mice through the tail vein and allowing growth for 1 week, CAY10404 (20mg/kg, I.P.) and cyclopamine (20mg/kg, I.P) were given separately three times per week. Interestingly, we observed that the mice injected with untreated CD138+ cells survived longer than untreated mice receiving CD138− cells (P < 0.05). Treatment with CAY10404 and cyclopamine significantly reduced the tumor burden measured by idiotype IgG2 protein levels and dramatically prolonged survival (P < 0.05). We conclude that activation of Wnt and Hh pathway maintains the “stemness” features of MMSC. In MMSC, these pathways are activated by increased RARα2 expression, which we have shown to be related to drug resistance. MMSC can be eradicated by a combination of Wnt and Hh inhibitors. Disclosures: No relevant conflicts of interest to declare.

PS02.061: TRANILAST: SPECIFIC INHIBITOR OF TRPV2 IS THERAPEUTIC AGENT OF ESOPHAGEAL CANCER STEM CELLS

2018 ◽  
Vol 31 (Supplement_1) ◽  
pp. 138-138
Author(s):  
Keita Katsurahara ◽  
Atsushi Shiozaki ◽  
Michihiro Kudou ◽  
Katsutoshi Shoda ◽  
Tomohiro Arita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Esophageal Cancer ◽  
Membrane Proteins ◽  
Cancer Stem Cells ◽  
Microarray Analysis ◽  
Therapeutic Agent ◽  
Transient Receptor Potential ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Specific Inhibitor

Abstract Background Recent studies revealed that membrane proteins, such as ion transporters, are specifically activated in cancer stem cells (CSCs). Therefore, these molecules are receiving a great attention as new chemotherapeutic targets of malignant tumor. This study aimed to investigate the expression and activity of ion transport-related molecules in CSCs of esophageal squamous cell carcinoma (ESCC). Methods We sorted cells with high expression of ALDH1A1 via FACS, and then, CSCs were generated using the sphere formation assay. The gene expression profiles of CSCs were examined using a microarray analysis. Candidate genes of membrane proteins activated in CSCs were selected based on that microarray data. Anticancer effects induced by inhibition of the selected proteins were examined. Results ALDH1A1 mRNA and protein levels were certainly upregulated in CSCs compared with non-CSCs. Obtained CSCs were resistant to Cisplatin and had the ability of re-differentiation. The results of the microarray analysis revealed that expressions of 50 genes of plasma membrane proteins were changed in CSCs, and that several genes related to ion channels, including transient receptor potential cation channel subfamily V member 2 (TRPV2), were upregulated. The upregulation of TRPV2 mRNA were also validated in CSCs derived from two types of esophageal cancer cell lines using RT-PCR method. Tranilast, which is specific TRPV2 inhibitor, was more cytotoxic at lower concentration in CSCs than in non-CSCs, and effectively decreased the number of tumorspheres. Further, Tranilast significantly decreased the cell population with high ALDH1A1 expression in esophageal cancer cells. Conclusion The results of the present study suggest that TRPV2 is involved in the maintenance of CSCs, and Tranilast, which is specific inhibitor of TRPV2, becomes a promising targeted therapeutic agent against ESCC. Disclosure All authors have declared no conflicts of interest.

scholarly journals Therapeutic Targeting of Cancer Stem Cells in Lung, Head and Neck, and Bladder Cancers

Cancers ◽  
2021 ◽  
Vol 13 (20) ◽  
pp. 5098
Author(s):  
Sarah E. Mudra ◽  
Pritam Sadhukhan ◽  
M. Talha Ugurlu ◽  
Shorna Alam ◽  
Mohammad O. Hoque
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Acquired Resistance ◽  
Therapy Resistance ◽  
Stem Cell Population ◽  
Molecular Characteristics ◽  
Targeted Agents ◽  
Regulatory Pathways ◽  
Lineage Differentiation ◽  
Solid Malignancies

Resistance to cancer therapy remains a significant obstacle in treating patients with various solid malignancies. Exposure to current chemotherapeutics and targeted agents invariably leads to therapy resistance, heralding the need for novel agents. Cancer stem cells (CSCs)—a subpopulation of tumor cells with capacities for self-renewal and multi-lineage differentiation—represent a pool of therapeutically resistant cells. CSCs often share physical and molecular characteristics with the stem cell population of the human body. It remains challenging to selectively target CSCs in therapeutically resistant tumors. The generation of CSCs and induction of therapeutic resistance can be attributed to several deregulated critical growth regulatory signaling pathways such as WNT/β-catenin, Notch, Hippo, and Hedgehog. Beyond growth regulatory pathways, CSCs also change the tumor microenvironment and resist endogenous immune attack. Thus, CSCs can interfere with each stage of carcinogenesis from malignant transformation to the onset of metastasis to tumor recurrence. A thorough review of novel targeted agents to act against CSCs is fundamental for advancing cancer treatment in the setting of both intrinsic and acquired resistance.

scholarly journals Expression profiles of protein tyrosine kinase genes in human embryonic stem cells

Reproduction ◽  
2008 ◽  
Vol 136 (4) ◽  
pp. 423-432 ◽  
Author(s):  
Mi-Young Son ◽  
Janghwan Kim ◽  
Hyo-Won Han ◽  
Sun-Mi Woo ◽  
Yee Sook Cho ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Signaling Pathways ◽  
Human Embryonic Stem Cells ◽  
Tyrosine Kinases ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Degenerate Primers ◽  
Protein Tyrosine ◽  
Hesc Lines

Complex signaling pathways operate in human embryonic stem cells (hESCs) and are coordinated to maintain self-renewal and stem cell characteristics in them. Protein tyrosine kinases (PTKs) participate in diverse signaling pathways in various types of cells. Because of their functions as key molecules in various cellular processes, PTKs are anticipated to have important roles also in hESCs. In this study, we investigated the roles of PTKs in undifferentiated and differentiated hESCs. To establish comprehensive PTK expression profiles in hESCs, we performed reverse transcriptase PCR using degenerate primers according to the conserved catalytic PTK motifs in both undifferentiated and differentiated hESCs. Here, we identified 42 different kinases in two hESC lines, including 5 non-receptor tyrosine kinases (RTKs), 24 RTKs, and 13 dual and other kinases, and compared the protein kinase expression profiles of hESCs and retinoic acid-treated hESCs. Significantly, up- and downregulated kinases in undifferentiated hESCs were confirmed by real-time PCR and western blotting. MAP3K3, ERBB2, FGFR4, and EPHB2 were predominantly upregulated, while CSF1R, TYRO3, SRC, and GSK3A were consistently downregulated in two hESC lines. Western blot analysis showed that the transcriptional levels of these kinases were consistent with the translational levels. The obstruction of upregulated kinases’ activities using specific inhibitors disturbed the undifferentiated status and induced the differentiation of hESCs. Our results support the dynamic expression of PTKs during hESC maintenance and suggest that specific PTKs that are consistently up- and downregulated play important roles in the maintenance of stemness and the direction of differentiation of hESCs.

Mesenchymal Stem Cells Self-Regulate Their Differentiation To Maintain the Bone Marrow Stromal System.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2335-2335
Author(s):  
Iekuni Oh ◽  
Akira Miyazato ◽  
Hiroyuki Mano ◽  
Tadashi Nagai ◽  
Kazuo Muroi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Adipocyte Differentiation ◽  
Expression Profiles ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Steady Level

Abstract Mesenchymal stem cells (MSCs) account for a very small population in bone marrow stroma as a non-hematopoietic component with multipotency of differentiation into adipocytes, osteocytes and chondrocytes. MSC-derived cells are known to have hematopoiesis-supporting and immunomodulatory abilities. Although clinical applications of MSCs have already been conducted for the suppression of graft versus host disease in allogeneic stem cell transplantation and for tissue regeneration, underlying mechanisms of the biological events are still obscure. Previously, we established a differentiation model of MSCs using a mouse embryo fibroblast cell line, C3H10T1/2 (10T1/2) (Nishikawa M et al: Blood81:1184–1192, 1993). Preadipocyte (A54) and myoblast (M1601) cell lines were cloned by treatment with 5-azacytidine. A54 cells and M1601 cells can terminally differentiate into adipocytes and myotubes, respectively, under appropriate conditions, while parent 10T1/2 cells remain undifferentiated. Moreover, A54 cells show a higher ability to support hematopoiesis compared with the other cell lines. In this study, we analyzed gene expression profiles of the three cell lines by using DNA microarray and real-time PCR to investigate molecular mechanisms for maintaining immaturity of parent 10T1/2 cells. In A54 cells, 202 genes were up-regulated, including those encoding critical factors for hematopoiesis such as SCF, Angiopoietin-1, and SDF-1 as well as genes known to be involved in adipocyte differentiation such as C/EBPα, C/EBPδ and PPAR-γ genes. These data are consistent with the hematopoiesis-supporting ability of A54 cells. During adipocyte differentiation, SCF and SDF-1 expression levels decreased in A54 cells while C/EBPα expression showed a steady level. Recently, osteoblasts have been reported to play crucial roles in “niche” for self-renewal of hematopoietic stem cells. Our results also implicate that precursor cells of non-hematopoietic components may have important roles for hematopoiesis in bone marrow. Meanwhile, in parent 10T1/2 cells, 105 genes were up-regulated, including CD90, Dlk, Wnt5α and many functionally unknown genes. Although C/EBPα expression was induced in 10T1/2 cells without differentiation under the adipocyte differentiation conditions, CD90 expression decreased, Dlk showed a steady level and Wnt5α was up-regulated. Assuming that some regulatory mechanisms are needed to keep an immature state of parent 10T1/2 cells even under the differentiation-inducible conditions, we performed following experiments. First, enforced Dlk expression in A54 cells did not inhibit terminal differentiation to adipocytes under the differentiation conditions. Second, when we cultured A54 cells in the conditioned media of parent 10T1/2 cells under the differentiation-inducible conditions, adipocyte differentiation was inhibited, suggesting that 10T1/2 cells produce some soluble molecules that can inhibit adipocyte differentiation. Since Wnt family is known to be involved in the regulation of self-renewal of several stem cells, Wnt5α may be one candidate for maintenance of “stemness” of MSCs. Taken together, the data of 10T1/2 cells suggest that MSCs can self-regulate their differentiation in the bone marrow stromal system. This concept may be important to investigate the fatty change of bone marrow in aging and in aplastic anemia.

Targeting Autophagy Potentiates Imatinib-Induced Cell Death in Philadelphia Positive Cells Including Primary CML Stem Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1070-1070 ◽  
Author(s):  
Cristian Bellodi ◽  
Maria Rosa Lidonnici ◽  
Ashley Hamilton ◽  
Gudmundur V Helgason ◽  
Angela R Soliera ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Competitive Inhibitor ◽  
Myelogenous Leukemia ◽  
Stem Cell Population ◽  
Therapeutic Effects

Abstract Imatinib mesylate (IM), a potent ATP-competitive inhibitor of the BCR/ABL tyrosine kinase, has become standard therapy for patients with chronic myelogenous leukemia (CML). However, the main limitations of IM- and second generation tyrosine kinase inhibitor (TKI)-based therapy are the insurgence of resistance in patients and the intrinsic refractoriness of primitive Philadelphia-positive stem cells. Therefore, there is the need to develop new therapeutic approaches that, in combination with TKI, might be more effective in targeting the stem cell population and preventing the outgrowth of TKI-resistant CML cells. TKI-induced elimination of BCR/ABL-dependent intracellular signals is known to trigger apoptosis, but it is unclear whether this also activates additional cell death and/or survival pathways. We show that IM treatment induces autophagy in CML blast crisis cell lines, CML primary cells and p210BCR/ABL-expressing 32Dcl3 (32D) myeloid precursor cells, but not in 32D cells expressing v-Src or the IM-resistant T315I p210BCR/ABL mutant. IM-induced autophagy does not involve c-Abl, as it is also observed in cells co-expressing p210BCR/ABL and the IM-resistant T315I c-Abl mutant. Induction of autophagy is associated with endoplasmic reticulum-stress and is suppressed by depletion of intracellular calcium. By contrast, ectopic Bcl-2 expression does not block IM-induced autophagy. Suppression of autophagy by pharmacological inhibitors or siRNA-mediated knockdown of essential autophagy genes enhances cell death induced by IM in cell lines and primary CML cells, demonstrating that induction of autophagy has a pro-survival effect. Critically, the combination of TKI with autophagy inhibitors results in near complete elimination of phenotypically (CD34+38−) and functionally (colony forming cells) defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKI in the treatment of CML.

Mesenchymal Stem Cell Differentiation Markers Shared with Soft Tissue Sarcomas

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4755-4755
Author(s):  
Stefan Wirths ◽  
Hans-Joerg Buehring ◽  
Lothar Kanz ◽  
Joerg T Hartmann ◽  
Hans-Georg Kopp
Keyword(s):  
Stem Cells ◽  
Soft Tissue ◽  
Expression Pattern ◽  
Cell Lines ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Soft Tissue Sarcomas ◽  
Stem Cell Differentiation ◽  
Antibody Panel

Abstract Malignant tumors are hypothesized to harbor small populations of self-renewing cancer stem cells. Targeting these cells may be the decisive step to overcome treatment resistance and achieve tumor eradication in cancer patients. Advanced soft tissue sarcomas (STS) are rare tumors with a dismal prognosis and a small number of systemic treatment options. STS may originate from mesenchymal stem cells (MSC); the latter have mainly been isolated from adult bone marrow (BM) as non-hematopoietic, self-renewing cells whose in vitro progeny comprises osteoblasts, chondroblasts, myocytes, and adipocytes. While in vitro expression profiles of MSC have been investigated extensively, the in vivo counterparts of MSC are still hypothetical. To target rare human cell BM populations including MSC, an exclusive antibody panel was developed. The target antigens include platelet-derived growth factor receptor-β (CD140b), HER-2/erbB2 (CD340), the recently described W8B2 antigen as well as several surface antigens identified by novel antibodies. To define the expression pattern of MSC-markers in STS, three STS cell lines were tested for expression of these antigens. In addition, snap-frozen primary STS sections were analyzed by immunohistochemistry using the same antibody panel. All cell lines revealed expression of selected markers including CD340, W8B2, and CD140b. Several MSC markers were restricted to a subpopulation of cells. In addition, leiomyosarcoma cells displayed a different expression pattern as compared to liposarcoma and Ewing’s sarcoma cells. Results of immunohistochemistry analysis of primary leiomyosarcoma tumor samples correlated strongly with expression patterns established by FACS analysis. However, important cytoarchitectural features regarding selected markers were revealed by immunohistochemistry: while primary leiomyosarcomas displayed uniform expression of W7C6, HEK3D6, CD10, and CD318, other markers such as CD34, W5C5, and 57D2 were expressed by tumor endothelia only. Moreover, a population of perivascular tumor cells was found to express the MSC-markers W4A5, W8B2, CD140b, W3D5, and W5C4. Novel MSC-markers are expressed by subpopulations in STS cell lines as well as in primary sarcoma tissue. Further studies on the functional significance of these phenotypical studies are underway and may help to identify novel specific targets recognizing the self-renewing STS-compartment.

Serum/Glucocorticoid-Regulated Kinase 1 (SGK1) Is a Prominent Target Gene of the Transcriptional Response to Cytokines In Multiple Myeloma and Supports the Growth of Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1915-1915
Author(s):  
Unn-Merete Fagerli ◽  
Thorsten Stühmer ◽  
Toril Holien ◽  
Randi Utne Holt ◽  
Ove Bruland ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factors ◽  
Cell Lines ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Transcriptional Response ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Growth And Survival ◽  
Stat3 Phosphorylation

Abstract Abstract 1915 Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Stability of Self-Renewal in Hematopoietic Stem Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. SCI-42-SCI-42
Author(s):  
Norman N. Iscove
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Single Cells ◽  
Gene Expression Profiles ◽  
Short Term ◽  
Blood Leukocytes ◽  
Hematopoietic Stem

Abstract Abstract SCI-42 For many years a distinction was drawn between prospectively separable murine HSC populations with long-term, essentially permanent reconstituting potential (LT-HSC), versus HSC populations yielding short-term engraftment lasting only 4 – 6 weeks after transplantation (ST-HSC). Recent work based on transplantation of single cells shows that highly purified populations of LT-HSC prepared by standard sorting parameters consist in fact predominantly of a distinct, newly recognized class of intermediate- term reconstituting cells (IT-HSC) whose grafts endure longer than short-term HSC but also eventually fail (1). IT-HSC are separable from long-term reconstituting cells on the basis of expression of more alpha2 integrin and less SLAM150. Crucial to recognition of the distinction between LT- and IT-HSC are the endpoints used to evaluate reconstitution. If blood erythroid or myeloid reconstitution is measured, IT reconstitution is readily distinguished by the disappearance of these elements by 16 wk post-transplant. If instead reconstitution is measured simply by presence of blood leukocytes of donor origin, which in the mouse are almost entirely lymphocytes, the distinction is not made because lymphoid elements persist even in fading IT clones to 24 wk or beyond. The observations imply a need for reinterpretation of most of the published descriptions of the biology and gene expression profiles previously attributed to LT-HSC but in fact derived from analysis of populations that consisted mainly of IT-HSC. The capacity now to separate LT- from IT-HSC creates new opportunities for probing the mechanisms that specify and sustain long term function in the former but not the latter. 1. Benveniste P, Frelin C, Janmohamed S, Barbara M, Herrington R, Hyam D, Iscove NN. Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential. Cell Stem Cell. 2010;6:48–58 Disclosures: No relevant conflicts of interest to declare.

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