GRN163L, a Novel and Potent Telomerase Inhibitor, Inhibits Myeloma Cell Growth In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 639-639
Author(s):  
Masood A. Shammas ◽  
Hemanta Koley ◽  
Pierfrancesco Tassone ◽  
Paola Neri ◽  
Alexei Protopopov ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Apoptotic Cell ◽  
Myeloma Cell ◽  
Telomerase Activity ◽  
Hsp90 Inhibitor ◽  
Telomere Shortening ◽  
Myeloma Cells

Abstract Telomerase activity is either low or completely absent in most normal somatic cells; while it is elevated in most cancer cells providing unlimited proliferative potential by preventing telomere shortening. The inhibitors of telomerase, therefore, induce telomere shortening leading to apoptotic cell death in tumor cells while having little or no effect on normal diploid cells. We have evaluated the in vitro and in vivo efficacy of thio-phosphoramidate oligonucleotide specifically targeting the RNA component of telomerase (GRN163L) with demonstrated nuclear uptake by >99% cells without the transfection enhancer. Delivery of GRN163L (1 μM) to MM cells (INA6 and ARP) was specifically associated with complete loss of telomerase activity as early as 6 hrs following exposure and was accompanied by a reduction in myeloma cell growth and survival. Treatment of INA6 cells with GRN163L for three weeks induced 96±4% and 100% cell death at 0.5 and 1 μM concentrations, respectively. ARP cells, which express higher levels of telomerase activity and have longer telomeres, showed 67±4% cell death at 5 weeks with 0.5 μM inhibitor and 82±3% and 100% cell death at 4 and 5 weeks, respectively, with 2 μM GRN163L. The apoptotic cell death was confirmed in 51% INA6 cells at two weeks and in >80% ARP cells at four weeks. Apoptosis was associated with reduction in mean Telomere Fluorescence Intensity (TFI) on interphase chromosomes from 87.1±6.2 in control oligo treated INA6 cells to 36.2±2 (2.4 fold) in GRN163L treated cells. Moreover, GRN163L treatment was also associated with a similar reduction in number of chromosomes with detectable telomeres, indicating development of telomere-free ends. We have confirmed in vivo efficacy of GRN163L in a SCID-hu murine model of multiple myeloma. Following growth of GFP-transduced myeloma cells in the fetal bone chip introduced into the mice, GRN163L was injected on alternate days. In two independent experiments significant reduction in tumor cell growth, as measured by reduction in human myeloma related protein, and better survival than mice injected with control oligo was observed. We have now evaluated efficacy of combination of GRN163L with other novel agents. We have observed synergistic activity with Hsp90 inhibitor 17AAG on myeloma cell death. Addition of 17AAG (0.05 μM) to myeloma cells pre-treated with GRN163L (1 μM) for one week led to complete growth arrest within four days compared to continued growth of cells not pre-treated with GRN-163. These data provide the preclinical rationale for clinical evaluation of GRN163L in myeloma and in combination with Hsp90 inhibitor.

scholarly journals Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 11-13 ◽  
Author(s):  
XG Zhang ◽  
B Klein ◽  
R Bataille
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
S Phase ◽  
Myeloma Cells ◽  
Severity Of The Disease

Abstract It has recently been demonstrated that interleukin-6 (IL-6) is a potent myeloma-cell growth factor in the majority of patients with multiple myeloma (MM). Using an anti-bromodeoxyuridine monoclonal antibody (MoAb) to specifically count myeloma cells in the S-phase (ie, labeling index, LI), we demonstrate that the IL-6 responsiveness of myeloma cells in vitro is directly correlated with their LI in vivo. Myeloma cells from all 13 patients with high LIs in vivo (greater than or equal to 1%) responded in vitro to IL-6, the strongest response occurring in cells from five patients with plasma-cell leukemia. In contrast, the cells of only two of eight patients with low myeloma-cell LIs in vivo (less than 1%) responded to IL-6 in vitro. After seven days of culturing with 1,000 U/mL recombinant IL-6 (rIL-6), the median LI value in the first group of patients (in vivo LI greater than or equal to 1%) was 11%, ie 11 times higher (P less than .01) than the median LI value (1%) in the second group of patients (in vivo LI less than 1%). Thus, the in vitro IL-6 responsiveness of myeloma cells is directly related to their in vivo proliferative status, and hence to the severity of the disease.

Sphingolipids As a Novel Target For The Treatment Of Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3163-3163 ◽  
Author(s):  
Jagadish Kummetha Venkata ◽  
Robert K Stuart ◽  
Luciano J Costa ◽  
Ningfei An ◽  
Houjian Cai ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Myeloma Cell ◽  
Sphingolipid Metabolism ◽  
Myeloma Cells ◽  
S1p Receptors

Abstract Introduction Multiple Myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ∼10,600 deaths annually. MM remains an incurable disease and almost all patients will eventually relapse and become refractory to currently available therapeutic agents. There is an unmet need for better understanding of the disease’s molecular pathways and identifying novel therapeutic targets. Sphingolipid metabolism is being increasingly recognized as a key pathway in cancer biology. In particular, sphingosine kinases (SK1 and SK2) provide a potential site for manipulation of the ceramide / sphingosine 1-phosphate (S1P) rheostat that regulates the balance between tumor cell proliferation and apoptosis, as well as tumor sensitivity to drugs. Currently, very little is known about sphingolipid metabolism in MM. We herein for the first time provide a detailed analysis of sphingolipid metabolism in MM and demonstrate the potential of targeting SK2 for the treatment of MM. Methods We first quantified sphingolipid metabolites and sphingolipid metabolizing genes in myeloma cell lines, in freshly isolated human primary CD138+ myeloma cells, and in a publically available gene expression dataset from MM patients. We then tested the anti-myeloma activity of SK2-specific shRNA and determined the efficacy of a selective SK2 inhibitor (ABC294640) in killing myeloma cell lines and primary human myeloma cells in vitro. The mechanistic pathway of apoptosis was analyzed by immunoblotting and flowcytometry. MM cell lines stably expressing luciferase and eGFP were generated for xenograft experiments and for in vitro co-cultures with stromal cells. Results From the publically available GSE6477 microarray data set, we found that one third of the genes involved in sphingolipid metabolism were significantly different in CD138+ MM cells from newly diagnosed MM patients compared to normal individuals, including SK2 and S1P receptors. In 5 MM cell lines compared to immortalized B cells (IBC), 19 key sphingolipid metabolites were measured, and we found that ceramides were significantly reduced whereas S1P was significantly increased. mRNA analyses of 11 sphingolipid metabolizing genes including S1P receptors in 7 MMs showed that SK1, SK2, and alkaline ceramidases were significantly increased compared to IBC. Furthermore, we isolated CD138+ myeloma cells from 21 MM patients and found that 13 of the patients had higher SK2 expression in CD138+ MM cells compared to CD138-cells. These data demonstrated abnormal sphingolipid metabolism and dys-regulated SK2 in myeloma cells. We generated SK2-specific shRNA and found that SK2 shRNA down-regulated SK2 mRNA, inhibited proliferation, and induced death in myeloma cells, suggesting that SK2 is important in myeloma cell survival. We then tested the efficacy of ABC294640 (the most-advanced, non-lipid SK2 inhibitor) in 6 MM cell lines. ABC294640 inhibited myeloma cell growth with an IC50s of ∼30 μM, including steroid-resistant and doxorubicin-resistant myeloma cells. ABC294640 inhibited MM cell growth as early as 6 hours after exposure and induced apoptotic cell death as demonstrated by Annexin V staining, PARP cleavage and caspase 9 activation. ABC294640 inhibited primary human CD138+MM cells with the same efficacy as with MM cell lines, demonstrating the potential of ABC294640 for the treatment of MM. Additionally, we found that blocking S1P receptors with FTY720 (a S1PR agonist with receptor degradation) induced apoptosis in MM cells. We performed extensive mechanistic and signaling pathway analyses and found that ABC294640 inhibited Mcl-1 and C-Myc expression, but had no effects on Bcl2. Furthermore, ABC294640 induced cell death by directing Mcl-1 to proteosomal degradation. MM is dependent on the bone marrow niche microenvironment for survival and progression. We found that ABC294640 was effective in inducing apoptosis in MM cells even in the presence of stromal cells. Finally, we are currently testing the in vivo effect of ABC294640 alone and in combination with bortezomib, thalidomide and dexamethasone in MM xenograft model transplanted with MM cells stably expressing luciferase. Our early preliminary results were encouraging. Conclusion Our data demonstrate that sphingolipid metabolism is abnormal and provides an attractive target in the treatment of refractory/relapsed MM. Disclosures: Costa: Otsuka: Research Funding.

scholarly journals Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 11-13 ◽  
Author(s):  
XG Zhang ◽  
B Klein ◽  
R Bataille
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
S Phase ◽  
Myeloma Cells ◽  
Severity Of The Disease

It has recently been demonstrated that interleukin-6 (IL-6) is a potent myeloma-cell growth factor in the majority of patients with multiple myeloma (MM). Using an anti-bromodeoxyuridine monoclonal antibody (MoAb) to specifically count myeloma cells in the S-phase (ie, labeling index, LI), we demonstrate that the IL-6 responsiveness of myeloma cells in vitro is directly correlated with their LI in vivo. Myeloma cells from all 13 patients with high LIs in vivo (greater than or equal to 1%) responded in vitro to IL-6, the strongest response occurring in cells from five patients with plasma-cell leukemia. In contrast, the cells of only two of eight patients with low myeloma-cell LIs in vivo (less than 1%) responded to IL-6 in vitro. After seven days of culturing with 1,000 U/mL recombinant IL-6 (rIL-6), the median LI value in the first group of patients (in vivo LI greater than or equal to 1%) was 11%, ie 11 times higher (P less than .01) than the median LI value (1%) in the second group of patients (in vivo LI less than 1%). Thus, the in vitro IL-6 responsiveness of myeloma cells is directly related to their in vivo proliferative status, and hence to the severity of the disease.

CSIG-22. Cd95l/Cd95 GENE DELETION DECREASES MOUSE GLIOMA TUMORIGENESIS THROUGH THE INHIBITION OF NON-CANONICAL CD95L/CD95-MEDIATED CELL GROWTH AND IMMUNOSUPPRESSION

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi37-vi38
Author(s):  
Clara Quijano-Rubio ◽  
Michael Weller
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Cell Lines ◽  
Cancer Cell ◽  
Apoptotic Cell ◽  
Immune Effector ◽  
Immunodeficient Mice ◽  
Murine Glioma

Abstract CD95 is a transmembrane receptor with potential to promote both cell death and growth. Initially described to trigger apoptosis upon ligand (CD95L) engagement, CD95 may also prompt cell proliferation, invasion and stemness. CD95 stimulation to induce cancer cell apoptosis has been proved clinically impracticable. However, in tumors expressing both CD95 and CD95L, strategically inhibiting CD95-CD95L interactions to simultaneously block cancer cell growth and apoptotic cell death in tumor microenvironment components, including CD95-expressing antitumor immune effector cells, may represent an alternative therapeutic strategy. Here we characterized the expression of CD95 and CD95L in murine glioma models in vitro and in vivo. To fully disrupt CD95-CD95L interactions, we deleted Cd95 or Cd95l by CRISPR-Cas9-mediated knockout (KO) and assessed the consequences on cell growth and tumorigenicity in immunocompetent and immunocompromised mice. CD95 expression was identified in selected murine glioma cell lines. In vitro, expression of the canonical, membrane-bound, form of CD95L was not detected but cell lines expressed a shorter non-canonical, soluble, Cd95l variant. Tumors generated upon implantation of the same cells in vivo expressed both Cd95l variants. Upon Cd95l KO, all investigated cell lines exhibited reduced growth in vitro. Cell growth reduction upon Cd95 KO in SMA-497 murine glioma cells was rescued upon Cd95 re-transfection, validating CD95 specificity of the phenotype. Cd95-overexpression in Cd95-expressing cells did not increase growth. In vivo, Cd95 or Cd95l KO cell implantation in syngeneic mice generated smaller tumors than wildtype cells, resulting in prolonged survival. While 40% Cd95l KO cell-implanted immunocompetent mice did not develop tumors, all immunodeficient mice did. Altogether, these data reveal a growth-promoting role of non-canonical CD95L-CD95 interactions in murine gliomas, which blockade through gene KO results in decreased tumorigenicity. Furthermore, our data suggest the contribution of CD95L-mediated immunosuppression to the reduction of Cd95l KO-associated tumorigenicity.

Deregulated Cellular Iron Metabolism Factors Mediate Iron Overload in Myeloma Cells and Osteoclasts, and Promote Myeloma Growth and Bone Disease,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3941-3941
Author(s):  
Rakesh Bam ◽  
Wen Ling ◽  
Sharmin Khan ◽  
Sathisha Upparahalli Venkateshaiah ◽  
Xin Li ◽  
...  
Keyword(s):  
Cell Growth ◽  
Iron Overload ◽  
Iron Metabolism ◽  
Bone Disease ◽  
Iron Chelator ◽  
Myeloma Cell ◽  
High Energy ◽  
Myeloma Cells

Abstract Abstract 3941 Iron overload is a significant clinical feature in multiple myeloma (MM) and has been implicated in osteoporosis. MM patients also frequently suffer from anemia presumably due to elevated hepcidin secretion and dysfunctional erythropoiesis. The aims of the study were to shed light on molecular mechanisms associated with iron overload in MM cells and study the effect of the novel iron chelator, Dp44mT, on MM cell growth, osteoclastogenesis and MM bone disease in vitro and in vivo. In our clinical global gene expression profiling (GEP) data the main iron transporter gene TFRC (transferrin receptor) was >3 folds higher (p<0.0001) in newly diagnosed MM cells (n=556) than normal plasma cells (n=25) while the iron exporter ferroportin was downregulated in MM cells by >4 folds (p<0.0001). Deregulated TFRC and ferroportin expression were more profound in the molecularly classified proliferation (PR) subtype. Osteoclasts which are known to have abundant mitochondria due to high energy consumption express excessive TFRC (>5 folds higher than highly proliferating MM cells). In primary MM cell-osteoclast cocultures (n=8) TFRC expression was upregulated in cocultured MM cells than baseline MM cells (p<0.03) while ferroportin was lower in cocultured osteoclasts than control osteoclasts (p<0.04). Our GEP, qRT-PCR and immunohistochemistry analyses revealed expression of hepcidin by osteoclasts but not MM cells. Hepcidin was not detected in conditioned media from osteoclasts cultured alone or cocultured with MM cells using ELISA, suggesting an autocrine role of hepcidin in maintaining excess iron in osteoclasts. In vitro, Dp44mT dose dependently inhibited growth of MM cell lines (n=3) at low nanomolar levels (IC50 at 3±0.8 nM, p<0.03, 48 hrs). In contrast, known chelators such as Deferoxamine and Deferasirox inhibited myeloma cell growth at 10–50 micromoles range. At 1nM Dp44mT also suppressed formation of multinucleated osteoclasts by 87% (p<0.001) and bone resorbing activity of mature osteoclasts on dentine slices by 94% (p<0.03). Dp44mT induced upregulation of BMP2 expression in osteoblast precursors and promoted osteoblast differentiation. In vivo, SCID-rab mice engrafted with luciferase-expressing U266 MM line (6 mice/group) or the Hg MM line (maintained through in vivo passaging, 10 mice/group) were subcutaneously treated with vehicle or Dp44mT (1 mg/kg/day) for 2–3 weeks. Using live-animal imaging, Dp44mT reduced growth of U266 cells by 3 folds from pretreatment levels (p<0.01) while in control group tumor burden was increased by 52 folds from pretreatment levels (p<0.002). Dp44mT also inhibited growth of Hg MM cells determined by measurement of circulating human immunoglobulins in mice sera (p<0.01). Osteoclasts numbers were lower by 36% (p<0.003) while osteoblasts numbers were higher by 59% (p<0.017) in myelomatous bones from hosts treated with Dp44mT than control vehicle. Our data suggest that interaction of myeloma cells with osteoclasts alters expression of distinct iron metabolism associated factors which elicit iron overload in both cell types, resulting in increased myeloma cell proliferation and osteoclast activity. This study also suggests that Dp44mT is an effective iron chelator with marked anti-MM activity. Disclosures: Barlogie: Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

Telomerase Inhibition, Telomere Shortening and Apoptotic Cell Death in Multiple Myeloma Cells Following Exposure to a Novel and Potent Telomerase Inhibitor (GRN163L), Targeting RNA component of Telomerase.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 638-638 ◽  
Author(s):  
Masood A. Shammas ◽  
Hemant Koley ◽  
Alexi Protopopov ◽  
Pierfrancesco Tassone ◽  
Paola Neri ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Telomere Length ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Telomerase Activity ◽  
Telomere Shortening ◽  
Myeloma Cells ◽  
Telomerase Inhibitor

Abstract Telomeres, the specialized nucleoprotein structures at the ends of chromo-somes, shorten at each DNA replication, and if unopposed leads to chromosomal erosion and cell death. Telomere shortening below a critical length is prevented by telomerase. We have previously observed elevated telomerase activity and shortened telomeres in multiple myeloma (MM), making the telomere maintenance mechanism an important target for therapy. Based on success with other non-specific telomerase inhibitors, in this study, we evaluated the effects of a thio-phosphoramidate oligonucleotide specifically targeting the RNA component of telomerase (GRN163L), with modifications to facilitate its delivery into human cells. Nuclear uptake of GRN163L without need for transfection enhancer at 24h was confirmed in >99% MM cells using fluorescein isothiocyanate-tagged GRN163L and confocal microscopy. Next we evaluated the effects of different concentrations and length of exposure of GRN163L on telomerase activity in diff MM cell lines (ARP and INA6). Whereas control oligonucleotide did not significantly affect telomerase activity, ≥ 80% loss of telomerase activity was observed in MM cell lines at day 3 at submicromolar concentrations of GRN163L. This inhibition of telomerase activity was associated with inhibition of myeloma cell growth and survival. Treatment of INA6 cells with GRN163L for three weeks induced 96±4% and 100% cell death at 0.5 and 1 μM concentrations, respectively, while ARP cells with higher telomerase activity and longer telomeres showed 67 ± 4% cell death at 5 weeks with 0.5 μM inhibitor and 82 ± 3% and 100% cell death at 4 and 5 weeks respectively with 2 μM concentration. The cell death was predominantly apoptotic, as determined by 51% annexin V-positive INA6 cells at two weeks and >80 % annexin V positive ARP cells at four weeks. The apoptotic cell death was associated with reduction in telomere length as analyzed using Telomere-FISH. While the control oligo treated ARP cells showed mean Telomere Fluorescence Intensity (TFI) on interphase chromosomes of 17.2 ± 1.5 (range 1.3–146.4), GRN163L treated cells showed reduction of mean TFI to 13.6 ± 0.46 (range 0.74 – 65.7). Although values above 100 were observed on 2 chromosomes in control oligo treated cells, GRN163L-treated cells had no chromosome with a TFI of more than 70.0. A similar reduction in telomere length was observed for INA6 cells. Subsequently, we were also able to increase cytotoxicity of DNA damaging agents in MM cells treated with GRN163L and shortened telomeres; providing a rationale for evaluation of combination therapies. These data demonstrate GRN163L as a potent and specific telomerase inhibitor able to disrupt telomere integrity and inducing apoptotic death of multiple myeloma cells. Evaluation of this agent in a SCIDhu model of myeloma is underway prior to its clinical evaluation.

scholarly journals Pyrroline-5-Carboxylate Reductase 1: A Novel Target for Sensitizing Myeloma to Cytotoxic Agents By Inhibition of PRAS40-Mediated Protein Synthesis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1574-1574
Author(s):  
Inge Oudaert ◽  
Hatice Satilmis ◽  
Philip Vlummens ◽  
Anke Maes ◽  
Elke De Bruyne ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Protein Synthesis ◽  
Apoptotic Cell ◽  
Myeloma Cell ◽  
Tumor Burden ◽  
Rpmi 8226 ◽  
Carboxylate Reductase

Abstract Introduction Multiple myeloma (MM) remains an incurable cancer despite advances in therapy. Therefore, the search for new targets is still essential to uncover potential treatment strategies. Metabolic changes, induced by the hypoxic bone marrow, contribute to both cancer cell survival and drug resistance. In this study, we aimed to identify which metabolic changes and downstream pathways are involved in myeloma cell growth and persistence. Methods Correlation of pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) with overall survival was investigated in the gene-expression data of MM patients (MMRF CoMMpass trial). To perform a tracer study, RPMI-8226 cells were supplemented with 13C-glutamine for 48h in both normoxia and hypoxia (&lt;1% O 2, by chamber). For further in vitro investigation, 2 human MM cell lines (OPM-2 and RPMI-8226) were used. Proline concentrations in cell lysates were measured by ELISA-based proline assay kit. We used siRNA to establish a knockdown of PYCR1 and/or PYCR2. Levels of apoptosis were measured using AnnexinV and 7-AAD positivity on flow cytometry. Differential protein expression was evaluated with western blot. Proliferation was measured by assessing BrdU incorporation through flow cytometry. Pargyline was used as a PYCR1 inhibitor. All in vitro experiments were performed in hypoxic conditions. For the in vivo murine experiment, C57BL/KalwRij mice were inoculated with 1 million of eGFP+ 5TGM1 cells, and treated with vehicle, bortezomib (0.6 mpk, 2x/week, starting day 14), pargyline (100 mpk, 5x/week, starting day 1) or combination of both. Tumor burden was measured by flow cytometry when vehicle mice reached end-stage. Results Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) are 2 mitochondrial enzymes that facilitate the last step in the enzymatic conversion of glutamine to proline. High expression of both enzymes correlated with a lower overall survival in the CoMMpass trial. Moreover, MM cells from relapse/refractory patients expressed significant higher levels of PYCR1. We performed a tracer study with RPMI-8226 cells, revealing an increased conversion of 13C-glutamine to proline in hypoxia compared to normoxia. We confirmed these results by increased proline production after 48h of hypoxic culture. SiRNA-mediated knockdown of PYCR1 or both PYCR1/2 combined with bortezomib increased apoptotic cell death in OPM-2 and RPMI-8226, which we confirmed by detecting upregulation of cleaved PARP and cleaved CASPASE 3 levels. In contrast, PYCR2 knockdown combined with bortezomib did not significantly alter apoptosis. Further investigation revealed that PYCR1 knockdown reduced proliferation, and led to a decrease in p-AKT, p-p42/44 MAPK and c-MYC levels. Mechanistically, we found that PYCR1 silencing affected protein synthesis, as shown by a downregulation of p-PRAS40, p-MTOR, p-p70, p-S6, p-4EBP1 and p-EIF4e levels. Next, we evaluated whether the clinically relevant anti-hypertensive agent and PYCR1 inhibitor, pargyline, was capable of inducing myeloma cell death. In vitro, pargyline reduced proline production, MM viability and increased apoptotic cell death. Pargyline was also capable of reducing viability in CD138+ cells of primary patient samples . Finally, in vivo combination of pargyline with bortezomib significantly reduced tumor burden in the 5TGM1 model. On protein level, we also observed a significant decrease in p-4EBP1 and p-EIF4e in the freshly isolated 5TGM1 cells for the combination therapy. Conclusion Hypoxia increased glutamine-to-proline conversion in myeloma cells by stimulating PYCR activity. Knockdown of PYCR1 and PYCR1/2 increased bortezomib efficacy and inhibited proliferation. Mechanistically, PYCR1 interference reduced PRAS40-mediated protein synthesis. Pargyline, a PYCR1 inhibitor, also reduced MM viability and increased apoptosis. In vivo, pargyline combined with bortezomib significantly reduced tumor burden in the 5TGM1 model compared to both single agents. In conclusion, this study identifies PYCR1 as a novel target in MM therapy. Disclosures De Veirman: Active Biotech AB: Research Funding. OffLabel Disclosure: Pargyline is a antihypertensive agent and irreversible MAO B inhibitor that also inhibits PYCR1. Pargyline is not approved by the FDA as a PYCR1 inhibitor.

scholarly journals KUS121 attenuates the progression of monosodium iodoacetate-induced osteoarthritis in rats

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sachiko Iwai ◽  
Hanako O. Ikeda ◽  
Hisashi Mera ◽  
Kohei Nishitani ◽  
Motoo Saito ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Apoptotic Cell ◽  
Intraperitoneal Administration ◽  
Atp Depletion ◽  
Knee Joints ◽  
Cellular Protection ◽  
Monosodium Iodoacetate

AbstractCurrently there is no effective treatment available for osteoarthritis (OA). We have recently developed Kyoto University Substances (KUSs), ATPase inhibitors specific for valosin-containing protein (VCP), as a novel class of medicine for cellular protection. KUSs suppressed intracellular ATP depletion, endoplasmic reticulum (ER) stress, and cell death. In this study, we investigated the effects of KUS121 on chondrocyte cell death. In cultured chondrocytes differentiated from ATDC5 cells, KUS121 suppressed the decline in ATP levels and apoptotic cell death under stress conditions induced by TNFα. KUS121 ameliorated TNFα-induced reduction of gene expression in chondrocytes, such as Sox9 and Col2α. KUS121 also suppressed ER stress and cell death in chondrocytes under tunicamycin load. Furthermore, intraperitoneal administration of KUS121 in vivo suppressed chondrocyte loss and proteoglycan reduction in knee joints of a monosodium iodoacetate-induced OA rat model. Moreover, intra-articular administration of KUS121 more prominently reduced the apoptosis of the affected chondrocytes. These results demonstrate that KUS121 protects chondrocytes from stress-induced cell death in vitro and in vivo, and indicate that KUS121 is a promising novel therapeutic agent to prevent the progression of OA.

scholarly journals Phosphatidylcholine Synthesis Influences the Diacylglycerol Homeostasis Required for Sec14p-dependent Golgi Function and Cell Growth

2001 ◽  
Vol 12 (3) ◽  
pp. 511-520 ◽  
Author(s):  
Annette L. Henneberry ◽  
Thomas A. Lagace ◽  
Neale D. Ridgway ◽  
Christopher R. McMaster
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Phosphatidic Acid ◽  
Metabolic Pathways ◽  
Eukaryotic Cells ◽  
Kennedy Pathway ◽  
Phosphatidylcholine Synthesis ◽  
Long Chain

Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids in eukaryotic cells and thus have major roles in the formation and maintenance of vesicular membranes. In yeast, diacylglycerol accepts a phosphocholine moiety through aCPT1-derived cholinephosphotransferase activity to directly synthesize phosphatidylcholine. EPT1-derived activity can transfer either phosphocholine or phosphoethanolamine to diacylglcyerol in vitro, but is currently believed to primarily synthesize phosphatidylethanolamine in vivo. In this study we report that CPT1- and EPT1-derived cholinephosphotransferase activities can significantly overlap in vivo such that EPT1 can contribute to 60% of net phosphatidylcholine synthesis via the Kennedy pathway. Alterations in the level of diacylglycerol consumption through alterations in phosphatidylcholine synthesis directly correlated with the level of SEC14-dependent invertase secretion and affected cell viability. Administration of synthetic di8:0 diacylglycerol resulted in a partial rescue of cells fromSEC14-mediated cell death. The addition of di8:0 diacylglycerol increased di8:0 diacylglycerol levels 20–40-fold over endogenous long-chain diacylglycerol levels. Di8:0 diacylglcyerol did not alter endogenous phospholipid metabolic pathways, nor was it converted to di8:0 phosphatidic acid.

scholarly journals Inhibition of sphingosine kinase 2 downregulates the expression of c-Myc and Mcl-1 and induces apoptosis in multiple myeloma

Blood ◽  
2014 ◽  
Vol 124 (12) ◽  
pp. 1915-1925 ◽  
Author(s):  
Jagadish Kummetha Venkata ◽  
Ningfei An ◽  
Robert Stuart ◽  
Luciano J. Costa ◽  
Houjian Cai ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Survival ◽  
Specific Inhibitor ◽  
Sphingosine Kinase ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Sphingosine Kinase 2 ◽  
Key Points

Key Points SK2 is overexpressed in myeloma cells and contributes to myeloma cell survival and proliferation. SK2-specific inhibitor promotes proteasome degradation of Mcl-1 and c-Myc and inhibits myeloma growth in vitro and in vivo.

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