Hypoxia-Inducible Factor (HIF)-1alpha Inhibitionin Myeloma Cells Significantly Increases the Anti-Myeloma Effect of Lenalidomide in Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3012-3012
Author(s):  
Paola Storti ◽  
Valentina Marchica ◽  
Denise Toscani ◽  
Irma Airoldi ◽  
Sophie Maiga ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Direct Action ◽  
Severe Combined Immunodeficiency ◽  
Hypoxia Inducible Factor ◽  
Hexokinase Ii ◽  
Inhibition Of Proliferation ◽  
Vitro Effect

Abstract The immunomodulatory drugs (IMiDs®) exert an anti-myeloma effect by cereblon-dependent destruction of IKZF proteinseither through a direct action on multiple myeloma (MM) cells or through indirect immunomodulatory and anti-angiogenic effects.Previous data indicated that MM cells overexpress hypoxia inducible factor (HIF)-1α and that HIF-1α suppression significantly blocks MM-induced angiogenesis and reduces in vivo tumoral burden in MM mouse models. Interestingly, it has been recently reported that HIF-1α knock-down in MM cells potentiates the in vitro effect of Lenalidomide (LEN) on cell proliferation without changing cell viability and that LEN is not able to suppress HIF-1α expression in MM cells. These evidences give the rationale design to investigate the in vivo effect of HIF-1α stable suppression in MM cells on LEN sensitivity. Thus, in this study, we assessed the effect of LEN in vivo in combination with HIF-1α inhibition in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) subcutaneous mouse model using JJN3, a cell line known to be resistant to the cytotoxic effect of LEN. Different groups of animals were injected with JJN3-pLKO.1 (empty vector) or JJN3-anti-HIF1α, obtained by anti-HIF1α lentiviral shRNA pool. When tumors became palpable, mice were treated with LEN (5mg/kg), using the intraperitoneal route. After three weeks, we evaluated tumor volume and weight. Moreover, by immunohistochemistry on ex vivo plasmacytomas, we evaluated the expression of p27 and the microvascular density (MVD), checked by CD34 immunostaining. In addition, the expression of a p27 inhibitor, the S-phase kinase-associated protein 2 (SKP2), the expression of the HIF-1α target key mediator of glycolysis and tumoral growth, Hexokinase II (HK2), and the levels of pERK 1/2, and total Caspase-3 (Casp-3) were evaluated in the ex vivo plasmacytomas lysates by western blot. We found that LEN treatment induced a significant weight and volume reduction of the tumor burden in mice injected with JJN3 anti-HIF1α as compared to JJN3-pLKO.1. The p27 nuclear expression was significantly increased by LEN treatment in JJN3-anti-HIF1α as compared to JJN3 pLKO.1 mice and compared to JJN3-anti-HIF1α mice treated with vehicle. In addition, we demonstrated that LEN in combination with HIF-1α inhibition significantly reduced in vivo the expression of p-ERK1/2, total Casp-3, HK2 and the p27 inhibitor, SKP2. Because it is known that LEN exerts its anti-MM effect targeting the IKZF proteins, we further checked in vitro whether the effect of HIF-1α suppression and LEN treatment combination could be mediated by IKZF proteins modulation. Interestingly, after LEN (2-10µM) treatment we found that both IKZF1 and IKZF3 were not differentially expressed, whereas IRF4 was down regulated, in JJN3-anti-HIF1α as compared to JJN3 pLKO.1. Finally, regarding a possible combinatory effect on the in vivo angiogenesis, we found that both the number of CD34 positive vessels and the MVD were reduced in mice colonized by JJN3-anti-HIF1α as compared to JJN3-pLKO.1. On the other hand, LEN treatment did not further significantly reduce the number of CD34 positive vessels and the MVD. Accordingly, we did not find any significant inhibitory effect by LEN treatment on the main pro-angiogenic molecules in JJN3 anti-HIF1α as compared to JJN3 pLKO.1 even after 72 hours. Overall, our data indicate that HIF-1α suppression in MM cells significantly increases the anti-MM effect of LEN in vivo, mainly through the inhibition of proliferation signaling including the modulation of p27 pathway and the IKZF target protein IRF4, rather than to an anti-angiogenic effect. These data suggest that the combination of LEN and HIF-1α inhibition has a therapeutic rationale in MM. Disclosures No relevant conflicts of interest to declare.

CD138 Regulates Myeloma Cell Survival, Proliferation, BM Engraftment and Tumor Organization

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2974-2974
Author(s):  
David R Fooksman ◽  
Amitabha Mazumder ◽  
Mark McCarron
Keyword(s):  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Early Stage ◽  
Serum Starvation ◽  
High Dose ◽  
Rapid Reduction ◽  
Myeloma Cells

Abstract Multiple myeloma is the 2nd most common blood cancer in adults with a median survival time of 5 years despite high-dose chemotherapy and bone marrow transplantation interventions. Syndecan-1 or CD138, is a heparan-sulfate coated glycoprotein, which is highly expressed on the surface of plasma cells and myeloma cells, important for adhesion and accumulating survival signals. Expression of CD138 is heterogeneous in myeloma tumors, in vivo and in vitro leading some to speculate it may distinguish stem-like subpopulations. While this role is highly disputed, we investigated the effect of CD138 expression on tumor pathology in vivo. To characterize CD138neg and CD138high subpopulations, we used GFP+ Vk*myc myeloma model from Leif Bergsagel, which develops myeloma tumors in BM and spleen of C57Bl/6 mice. We found CD138high populations were more proliferative in vivo based on EdU incorporation experiments. We transferred equal numbers of sorted subpopulations into hosts and found that CD138high cells generated larger tumors in the BM than CD138neg cells after 12 weeks. Analysis of these tumor-bearing mice revealed that all tumors contained both subpopulations, indicating that these two subsets are hierarchically equivalent. We find that in mice with small tumors, the majority of cells (80% or more) are CD138high cells, while in large tumors, the level drops (to 30-50% of tumor) with higher composition of CD138neg cells. We also find lower CD138 levels on myeloma cells found in the blood compared to BM. Using intravital two-photon time-lapse imaging in the tibial BM, we find that tumor cells from smaller, early stage tumors are physically arrested within the BM parenchyma, while in larger, more advanced tumors, myeloma cells are more motile and active. CD138neg cells were more apoptotic based on ex vivo Annexin V staining following serum starvation. Interestingly, serum starvation led to rapid reduction in CD138 surface expression. Taken together, we propose a model where CD138 expression regulates localization and survival in the BM niche, but is downregulated from the plasma membrane when tumor size outgrows the necessary resources, allowing myeloma cells to migrate and metastasize to distant new locations. Disclosures No relevant conflicts of interest to declare.

scholarly journals Furosemide and Platelet Functions

1977 ◽  
Author(s):  
I.S. Chohan ◽  
I. Singh ◽  
J. Vermvlen ◽  
M. Verstraete
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Metabolic Effects ◽  
Platelet Factor ◽  
Metabolic Products ◽  
Vitro Effect ◽  
Primary Platelet ◽  
Platelet Functions

Furosemide inhibits primary platelet aggregation by adenosine-5'-diphosphate and prolongs the latent period before Thrombofax- or collagen-induced platelet aggregation, both in vitro and ex vivo. Furosemide also inhibits the release of platelet factor 4 and 14C-serotonin. The inhibitory concentrations of furosemide in vitro range between 0.5 and 2.5 mM. The ex vivo effects were obtained after an intravenous injection of 40 mg furosemide.The furosemide concentrations required for ex vivo inhibition are hundred fold lower than those required for in vitro effect. This suggests in vivo metabolic effects of this drug, such as, calcium ions shifts, or action of other metabolic products of furosemide. In support of this concept platelet aggregation was found more disturbed six hours than 10 minutes after injection of the drug.

A Dendritic Cell Based Vaccination Strategy Geared towards Eradication of Leukemic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2046-2046
Author(s):  
Hetty J Bontkes ◽  
Jurjen Ruben ◽  
Willemijn van den Ancker ◽  
Theresia M Westers ◽  
G. Ossenkoppele ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Tumor Antigens ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Vaccination Strategy ◽  
Leukemic Stem Cells ◽  
Cell Immunity

Abstract Abstract 2046 Poster Board II-23 Introduction: In the majority of cases, initial remission of acute myeloid leukemia (AML) is reached but unfortunately relapse rates remain high and therefore novel treatments are needed. It is thought that recurrent AML originates from chemotherapy resistant quiescent leukemic stem cells (LSC). The application of immunotherapeutic approaches to eradicate LSC remaining after first line chemotherapy may contribute to improved disease outcome. Vaccination strategies have often used dendritic cells (DC) ex vivo pulsed with tumor-derived whole lysates or peptides as modalities to present a broad range of tumor antigens to T cells to stimulate effective anti-tumor T-cell immunity in vivo. It is likely that certain proteins expressed by LSC have a distinct antigenicity as compared to more mature AML blasts and thus provide targets for specific T-cells. Even without identification of specific antigens, LSC can be a useful source of tumor antigens in DC vaccination-based immunotherapy. CD34+CD38- LSC can be identified using malignant stem cell associated cell surface markers including CLL-1 and lineage markers such as CD7, CD19 and CD56. However, the low frequency of these cells precludes the use of LSC derived apoptotic cells or lysates for DC loading. Alternatively, mRNA isolated from LSC can be amplified and subsequently transfected into DC. Materials and Methods: We have made use of the CD38- AML derived cell line MUTZ-3 which contains a subpopulation of CD34+CLL1+ cells which resembles the phenotype of a putative LSC. CLL1+CD34+ and CLL1-CD34- cells were isolated by FACS sorting and total RNA was isolated. mRNA was converted to cDNA and amplified by PCR using the SMART system. Subsequently, mRNA was in vitro transcribed from the amplified cDNA. Mature monocyte derived DC (MoDC) were generated from healthy donor blood and transfected with amplified CLL1+CD34+ derived mRNA and used to stimulate autologous CD8β+ T-cells. After three weekly re-stimulations with CLL1+CD34+ mRNA transfected DC, specificity of the T-cells was analyzed by intracellular IFNγ staining upon 5 hour stimulation with autologous immature MoDC transfected with GFP mRNA, mRNA amplified from unsorted, CLL1+CD34+ or CLL1-CD34- MUTZ-3 subpopulations. Results: Amplification of CLL1 and survivin (also expressed by MUTZ-3) transcripts was confirmed by RT-PCR. After 3 weekly re-stimulations with CLL1+CD34+ amplified RNA transfected DC, 0.04% (range 0.01-0.12%) of the T-cells were positive for IFNγ upon a 5 hr re-stimulation with GFP transfected DC. 0.44% (range 0.04-0.69%) of the T-cells responded to DC transfected with unsorted MUTZ-3 amplified mRNA (p<0.00005 versus GFP control, 2-sided student's T-test), 0.51% (range 0.24-1.35%) responded to DC transfected with CLL1+CD34+ amplified mRNA (p<0.005 versus GFP control) and 0.46% (range 0.24-0.94%) responded to DC transfected with CLL1-CD34- amplified mRNA (p<0.0001 versus GFP control). Conclusion: We show that MoDC transfected with RNA amplified from one MUTZ-3 sub-population resembling the phenotype of LCS cells are capable of inducing T-cells which recognize both cells transfected with mRNA from the LSC resembling MUTZ-3 subset as well as the CLL1-CD34- subset. We are currently testing the efficacy and feasibility of this approach in an autologous setting in vitro. CD8β+ T-cells are stimulated with autologous MoDC from AML patients transfected with amplified mRNA isolated from their own LSC enriched populations. The capacity of these T-cells to kill autologous AML blasts and LSC is subsequently analysed in a 6-colour FACS based cytotoxicity assay. Disclosures: No relevant conflicts of interest to declare.

Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1919-1919
Author(s):  
Iman Hatem Fares ◽  
Jalila Chagraoui ◽  
Jana Krosl ◽  
Denis-Claude Roy ◽  
Sandra Cohen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. Disclosures: No relevant conflicts of interest to declare.

Direct and Continuous Inhibition of ADAM17 Using a Novel Selective Inhibitor Restores Functional Platelet Yield From Human Pluripotent Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2323-2323
Author(s):  
Shinji Hirata ◽  
Ryoko Jono-Ohnishi ◽  
Satoshi Nishimura ◽  
Naoya Takayama ◽  
Sou Nakamura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Imaging System ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Human Platelets ◽  
P38 Map Kinase

Abstract Abstract 2323 Platelet transfusion is therapeutically important for patients with thrombocytopenia and/or bleeding disorders. Problems associated with a lack of donors and unknown infections in the blood have not been fully resolved, however. In that context, human induced pluripotent stem cells (hiPSCs) are a potentially abundant source of infection-free platelets. The pluripotent state of hiPSCs and their differentiation depend upon appropriate culture conditions defined in part by oxygen and temperature. We therefore initially examined whether temperatures at or below 24°C, which are required for preservation of platelet concentrates ex vivo, allow hiPSC differentiation to generate platelets. We found that only at 37°C were platelets generated. But at 37°C in vitro, platelets are subject to degradation exemplified by the shedding of GPIbα, a receptor for von Willebrand factor (vWF), which is caused by a disintegrin and metalloprotease (ADAM) 17. We therefore developed KP-457, a novel ADAM17 inhibitor that has a reverse hydroxamic acid structure and has been found safe in rats and dogs. Although inhibition of p38 MAP kinase, putatively upstream of ADAM17, reportedly inhibits GPIbα shedding in stored human platelets, even at 37°C, administration of the p38 inhibitor SB203580 induces cytotoxicity during differentiation, leading to a loss of platelet yield from hiPSCs. By contrast, KP-457 significantly protected GPIbα expression in platelets from hiPSCs and in aged human platelets in culture at 37°C. Moreover, iPSC-derived platelets generated in the presence of KP-457 displayed improved hemostatic function when studied using an imaging system that enables characterization of single-platelet kinetics during thrombus formation after laser-induced injury in vivo. We propose this new drug could markedly improve the maintenance of functional platelets generated in culture, particularly those derived from hiPSCs. Disclosures: No relevant conflicts of interest to declare.

Preclinical activity of ofatumumab (OFA) in mantle cell lymphoma (MCL) in vitro, ex vivo, and in vivo models.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e18537-e18537
Author(s):  
Matthew John Barth ◽  
Gopichand Pendurti ◽  
Cory Mavis ◽  
Natalie Czuczman ◽  
Jospeh J. Skitzki ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
High Dose ◽  
Cell Transplant ◽  
In Vivo Models

e18537 Background: MCL is characterized by an aggressive clinical course and inevitable development of refractory disease despite early intervention that often includes: immunotherapy (e.g., rituximab), multi-agent induction chemotherapy and consolidation with high dose chemotherapy and autologous stem cell transplant in first remission. OFA is a fully human anti-CD20 mAb targeting a novel membrane-proximal epitope on CD20. To characterize the activity of ofatumumab in MCL, we conducted pre-clinical studies in cell lines, primary tumor cells derived from MCL patients and a MCL bearing severe combined immunodeficiency (SCID) mouse model. Methods: Antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays were performed in 51Cr labeled Mino, Jeko, Rec-1 and Z-138 cells comparing RTX or OFA. Primary tumor cells were exposed ex vivo to OFA or RTX with human serum, differences in cell viability were determined by Cell Titer Glo assay. Expression of CD20 and complement inhibitory proteins (CIPs) CD55 and CD59 was determined by Imagestream analysis and Western blot. SCID mice were inoculated SQ with 10x106 Z-138 cells. Once tumors were established, mice were assigned to observation versus 4 doses of either OFA or RTX, and anti-tumor activity was measured by changes in tumor volume. Results: OFA induced higher CDC than RTX in all MCL cell lines tested (Mino: 65.9% vs. 0.5% ; Jeko 43.9% vs. 13.3% ; Rec-1 25.4% vs. 4.7% ; Z-138: 56.4% vs. 0.65%). No differences in ADCC were noted between OFA and RTX. In primary tumor cells, OFA and RTX demonstrated similar activity. CD20 levels were similar in all MCL cell lines tested. Of interest, CIP expression in MCL cell lines was higher when compared to other NHL cell lines, explaining differences observed between OFA and RTX. In vivo OFA was more effective in slowing tumor progression than RTX. Conclusions: Our data suggest OFA is more potent than RTX against MCL pre-clinical models. In addition and as expected, OFA exhibits potent CDC despite high expression of CIP. Our results support the evaluation of ofatumumab in future prospective clinical trials for patients with MCL.

PS02.174: THE ACTION OF A NOVEL RADIOSENSITISER WITHIN THE OESOPHAGEAL ADENOCARCINOMA TUMOUR MICROENVIROMENT

2018 ◽  
Vol 31 (Supplement_1) ◽  
pp. 171-171
Author(s):  
Amy Buckley ◽  
Niamh Lynam-Lennon ◽  
Susan Kennedy ◽  
Aoife Cannon ◽  
Dermott O’Toole ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Survival Rates ◽  
Oesophageal Adenocarcinoma ◽  
Naive Patient ◽  
Human Patient ◽  
Hypoxic Conditions ◽  
Treatment Naïve

Abstract Background Oesophageal Cancer (OAC) is an aggressive disease with survival rates of ∼15-20%. Current therapeutic regimes focus on neo-adjuvant therapy (chemo-radiation) prior to surgery. Unfortunately, only 20–30% of patients show a beneficial response, with 70–80% of patients as non-responders. This major clinical challenge of treatment resistance reinforces the need for the identification of novel treatments which can act as radio-sensitisers in the neo-adjuvant setting. Methods Through a drug screening approach in-vivo, we have identified a novel anti-angiogenic and anti-metabolic compound, 11B_CC8 with radiosensitising activity. The ability of 11B_CC8 to act as an anti-metabolic agent under hypoxia was evaluated using the XFe24 Seahorse analyser and the Don Whitley i2 workstation. The ability of 11B_CC8 to radiosensitise our isogenic OAC cells under hypoxic conditions was assessed by clonogenic assay. The effect of 11B_CC8 on inflammatory, metabolic and angiogenic protein secretions from OAC treatment naïve tumour conditioned media (TCM) was evaluated by multiplex ELISA. Fresh treatment naïve patient biopsies were screened for their metabolic activity using the XFe24 seahorse analyser at baseline and post- treatment with 11B_CC8. The elucidation of the possible mechanism of action of 11B_CC8 was evaluated by Mass-Spectrometry. Results Our novel anti-angiogenic and anti-metabolic agent can enhance radiosensitivity in our isogenic model of OAC under both normoxic and hypoxic (0.5% O 2) conditions. 11B_CC8 significantly reduces oxygen consumption rate (OCR) under normoxic but not hypoxic conditions. Ex-vivo, 11B_CC8 significantly reduced the secretion of IL1β (P = 0.0117). Real-time ex-vivo metabolic rate analysis of our treatment naïve OAC biopsies showed significantly elevated OCR, when compared to Extracellular Acidification rate, a measure of glycolysis (P = 0.0059). Treatment with 11B_CC8 produced a reduction in OCR (P = 0.0039). Conclusion Our novel anti-angiogenic and anti-metabolic agent can enhance radiosensitivity in-vitro under both normoxic and hypoxic conditions. Ex-vivo, treatment naïve OAC human patient samples, 11B_CC8 can significantly reduce the secretion of IL1β and altered metabolic programming, specifically oxidative phosphorylation in human explants. Disclosure All authors have declared no conflicts of interest.

scholarly journals A Deptor Inhibitor Induces Its Degradation with Resulting Anti-Myeloma Cytotoxicity in Vitro and In Vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1821-1821
Author(s):  
Mario I Vega ◽  
Yijiang Shi ◽  
Patrick Frost ◽  
Sara Huerta-Yepez ◽  
Alan Lichtenstein
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Cytotoxic Effect ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Inhibition Of Proliferation ◽  
Induction Of Apoptosis

Multiple myeloma (MM) is a hematological disorder characterized by a proliferation of malignant monoclonal plasma cells in the bone marrow (BM) and / or in extramedullary sites. Despite recent progress in OS rates, MM remains an incurable disease and most patients will relapse and require treatment. Deptor is a component of mTOR complexes and a constitutive inhibitor of their activities. It is known that the inhibition of Deptor results in the inhibition of the proliferation and induction of apoptosis in MM cells. In addition, high levels of Deptor are predictive of a poor response to conventional therapies, indicating that Deptor expression are important as a prognostic marker for patients with myeloma and is a possible therapeutic target. Our group previously identified a drug which prevents mTOR-Deptor binding (NSC126405) and induces cellular cytotoxicity in MM (Shi Y, et al 2016). In this study, we developed a new related chemical inhibitor (43 M) capable of inducing the inhibition of the mTOR / Deptor interaction and results in the negative regulation of Deptor that leads to the inhibition of proliferation and induces apoptosis in several MM cell lines. The cytotoxic effect of 43 M is not dependent of caspase activation and induces the activation of p70 and AKT (T308). This leads to the induction of apoptosis in MM cell lines and tumor cells derived from MM patients. The degradation of Deptor induced by 43 M is dependent on the proteasome complex since it was prevented in the presence of MG132. In vivo, 43 M prevents the expression of Deptor in a xenograft tumor, and delayed tumor growth and interestingly, induces the eradication of tumors in 40% of mice in a murine model of MM, without significant toxic implications. Recent studies show that Deptor expression protects MM cells against Bortezomib treatment, suggesting that anti-Deptor drugs can synergize with proteasome inhibitors (PIs). However, the combination of 43 M + Bortezomib was not synergistic, and was antagonistic in vitro. These results are probably due to the prevention of the proteasomal degradation of Deptor, suggesting a possible use of the 43 M inhibitor in MM in the absence of the current PIs. This study describes for the first time the possible role of Deptor as a therapeutic target using a chemical inhibitor capable of degrading and inducing a cytotoxic effect in MM cell lines. In addition, Deptor is reported as an important therapeutic target in an in vivo MM model. Shi Y, Daniels-Wells TR, Frost P, Lee J, Finn RS, Bardeleben C, Penichet ML, Jung ME, Gera J, Lichtenstein A. Cytotoxic Properties of a DEPTOR-mTOR Inhibitor in Multiple Myeloma Cells. Cancer Res. 2016 Oct 1;76(19):5822-5831 Disclosures No relevant conflicts of interest to declare.

scholarly journals A Novel Platelet-Targeting Fibrinolytic Strategy: Endocytosed Urokinase By Megakaryocytes Is Stored in α-Granules and Is Functional In Vivo in a Murine Model of Thrombosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3627-3627
Author(s):  
Hyunsook Ahn ◽  
John Tkaczynski ◽  
Khalil Bdeir ◽  
Victoria Stepanova ◽  
Douglas B. Cines ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Point Of Care ◽  
Ldl Receptor ◽  
Single Amino Acid ◽  
Platelet Glycoprotein ◽  
Single Chain ◽  
Injury Model

We have been interested in developing fibrinolytic agents that have a prolonged half-life and can selectively prevent new thrombi from forming without destabilizing established hemostatic clots. We believe that platelet-targeting of urokinase plasminogen activator (uPA) might be able to achieve this goal. Two approaches had been tried in the past: In the first, we had generated chimeric drugs consisting of a platelet-targeting scFvs fused to a thrombin activatable low molecular weight (LMW) uPA (uPA-T). Infused scFv/uPA-T bound to circulating platelets that targeted the fusion within nascent thrombi where significant amounts of thrombin are generated to activate surface-bound scFv/uPA-T. In contrast, mature thrombi are spared because scFv/uPA-T platelets are not activated on the "shell", nor do they penetrate the "core" where thrombin might be present. Murine studies affirmed αIIbβ3-directed scFv/uPA-T provided thromboprophylaxis, but therapeutic doses caused significant thrombocytopenia in murine and baboon models. We therefore considered a second approach: ectopic storage of uPA during megakaryopoiesis. We found that scuPA was stored in platelet α-granules of transgenic mice that ectopically express single-chain uPA (scuPA) and did not cause systemic fibrinolysis. Infusion of such "scuPA platelets" into wildtype mice was highly effective at preventing new thrombi from developing. We now wish to develop this strategy further by taking advantage of ongoing efforts by others to generate in vitro-grown megakaryocytes (Mks) and platelets that can be modified to express uPA near the point-of-care and then infused into patients, bypassing the need to establish uPA-expressing hematopoietic cell lines. We have previously shown that Mks express low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) during their maturation, whereas the platelets that are released do not. We now asked whether in vitro-grown Mks beginning with CD34+ hematopoietic cells would endocytose uPA as seen in other cell types. We show that Mks internalize and store scuPA (scu-Mks, Fig. 1A), as well as LMW uPA and uPA-T (not shown) after overnight incubation. Endocytosed uPA is found within membrane bound structures that partly colocalize with von Willebrand factor (VWF)-positive granules, suggesting uPA is sorted to α-granules (Fig. 1A). Uptake is blocked by receptor-associated protein (RAP), which inhibits endocytosis by LDL receptor family members, including LRP1. We then studied whether platelets with endocytosed scuPA (scuPA-Plts) prevent nascent thrombus development in immunodeficient NOD-scid IL2rγnull (NSG) mice that are also homozygous for VWFR1326H (a single amino acid substitution that switches species selectivity of VWF so that it binds human platelet glycoprotein (GP) Ib/IX receptor rather than mouse GPIb/IX). These mice show a mild bleeding diathesis in a Rose Bengal photochemical carotid artery injury model unless they are infused with human Mks, which we have shown go on to release functional platelets in the recipient animal in its pulmonary capillary bed over the ensuing several hours (Fig. 1B). Thus, when 106 Mks not exposed to uPA were infused so that ~1-10% of circulating platelets were human, thrombi developed in this model; however similar infusion of scuPA-Mks did not occlude (Fig. 1B). In tail clip studies in the same genotypic mice, where the mice were first corrected with human platelets (Fig. 1C) followed 10 min later by scuPA-Mks, rebleeding did not develop when given a similar dose of scuPA-Mks that prevented thrombosis in the photochemical injury model. These studies suggest that Mks internalize biologically relevant concentrations of uPA through a process likely to involve LRP1. Whether ex vivo loading of Mks with uPA can serve as model for point-of-care therapeutics for thromboprophylaxis and diverse other hematologic and non-hematologic indications should be explored. Disclosures No relevant conflicts of interest to declare.

scholarly journals Influence of Cyclophosphamide on L-Asparaginase-Induced Allergy in Animal Model

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5119-5119
Author(s):  
Ai Nogami-Hara ◽  
Akira Shimada ◽  
Mitsunobu Mio
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Neutralizing Antibody ◽  
Treg Cells ◽  
Mouse Serum ◽  
Ear Edema ◽  
The Right

BACKGROUND: L-Asparaginase (L-ASP) is one of the essential drugs for acute lymphoblastic leukemia and lymphoma. Since L-ASP proteins used clinically are derived from bacteria, they often cause allergies, including anaphylaxis. However, the characteristics of L-ASP-induced hypersensitivity are not well documented so far. Also, there is neither suitable treatment for hypersensitivity to L-ASP nor adequate experimental model of L-ASP allergy. In addition, some anti-cancer drugs, used concomitantly with L-ASP, are known to affect lymphocyte activities, although the role of such drugs on L-ASP hypersensitivity is not known. Previously, we have established mouse model of L-ASP allergy and found that a neutralizing antibody (Ab) of IgE is effective to inhibit L-ASP-induced allergic reaction. Aims: In order to establish in vitro model of L-ASP allergy, we used RBL-2H3 mast cells and evoked degranulation of the cells sensitized with L-ASP-allergic mouse serum using L-ASP as an antigen. Furthermore, we examined the effect of cyclophosphamide (CY), which reportedly impairs the functions of regulatory T (Treg) cells, on L-ASP allergy both in vivo and in vitro. Methods: Male BALB/c mice (7 week old) were sensitized with L-ASP (100 units) with Al(OH)3 gel (1 mg) on days 1 and 15. The right ears of the mice were locally sensitized on days 18, 21, 24 by i.d. injection of L-ASP (10 units). Antigen challenge was carried out on day 27 by i.d. injection (10 units). CY (75, 150, 300 mg/kg) was i.p. administrated at day -1 and day 13. Anti-IgE treatment (BD Bioscience, clone R35-92; 100 μg, i.p.) was carried out on the day before L-ASP challenge. The serum was collected on day 27. Total IgE level in the serum was measured by ELISA kit (Yamasa). RBL-2H3 cells were sensitized by the serum and stimulated by L-ASP to determine β-hexosaminidase (β-Hex) release in vitro. Results : L-ASP-sensitization induced allergic ear edema and an increase in serum IgE level in mice, both of which are augmented by an administration of CY at 150 mg/kg. L-ASP-induced allergic ear edema was inhibited by a pretreatment with anti-IgE Ab. L-ASP-induced CY-enhanced ear edema was also inhibited by anti-IgE Ab. On the other hand, at 300 mg/kg of CY, L-ASP-induced ear edema was much lower than 150 mg/kg group, though IgE concentration in the serum was as high as CY 150 mg/kg group. When RBL-2H3 cells were sensitized by anti-L-ASP serum, L-ASP challenge induced β-Hex release. Anti-L-ASP serum of CY 150 mg/kg-treated mice induced higher β-Hex release than normal anti-L-ASP sera in vitro, though that of CY 300 mg/kg-treated mice did not induce β-Hex release. After removing IgG from the serum of CY 300 mg/kg-treated mice using protein G Mag Sepharose (GE Healthcare), β-Hex release became higher than normally sensitized mice. Anti-IgE Ab treatment both ex vivo and in vitro inhibited L-ASP-induced β-Hex release from the RBL-2H3 cells sensitized by anti-L-ASP serum of CY-treated mice. Conclusions: In this study, we showed that L-ASP sensitization induced IgE production in vivo and this serum could bring about β-Hex release from RBL-2H3 cells in vitro; both allergic reactions were inhibited by an anti-IgE antibody which binds to Fc region of IgE molecule. From these results, we propose that non-anaphylactogenic neutralizing antibody of IgE, such as omalizumab, can be a candidate drug for the treatment of L-ASP hypersensitivity. Also in this study, CY induced biphasic effects on L-ASP allergy in mice, enhancing it at low dosage and attenuating it at high dosage. Since CY is reportedly impair the functions of Treg cells, CY-induced suppression of Treg may enhance Th2 responses so as to augment L-ASP hypersensitivity. Further investigations for the detection and avoidance of L-ASP hypersensitivity are under the way. Disclosures No relevant conflicts of interest to declare.

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