BM-MSC Hhip Induced Microenvironment Protection from Chemotherapy in AML Via Ptch/Smo/Gli Pathwa

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5235-5235
Author(s):  
Xuejie Jiang ◽  
Dan Xu ◽  
Fang Chen ◽  
Ling Jiang ◽  
Guopan Yu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Drug Resistance ◽  
Western Blotting ◽  
Conflicts Of Interest ◽  
Basic Research ◽  
Gli 1 ◽  
Low Expression ◽  
Refractory Aml ◽  
Bone Morrow

Abstract Introduction. The mechanism of drug resistance is complicated in acute myeloid leukemia (AML), bone microenvironment provides protection niche for leukemia stem cells (LSC), and considered as an internal environment for AML cells to escape from chemotherapeutics cytotoxicity, but mechanism of drug resistance induced by microenvironment mesenchymal stem cell (MSC) isn't completely clear. In prophase study, it was found that Ptch/Smo/Glis pathway was the key in the network of AML drug resistance. Smo inhibition improved the survival of AML-bearing mice. Low expression of HHIP was found in refractory AML MSC and bone morrow liquid, overexpression of Smo and Gli-1 was verified in AML cells, and negatively correlated with prognosis in AML patients. Chemotherapeutic drug sensitivity was decreased in AML cells after co-cultured with AML MSC in vitro. Methods. Bone morrow samples from AML patients and normal donors were collected to culture MSC cells. HL60/ADM and Kasumi-1 cells as well as HL60、KG-1cells were treated with daunomycin (DNR) or cytosine arabinoside (Ara-C) when cocultured with MSC. Cell cycle and apoptosis were determined by flow cytometry. The expression of HHIP was determined by confocal image and western blotting. Smo and Gli-1 activity was determined by western blotting. Result. We took advantage of AML cells cocultured with MSC to imitate leukemic microenvironment in vitro. Co-culture with AML BM-MSC decreased the sensitivity to DNR or Ara-C compared with normal BM-MSC in HL60/ADM or Kasumi-1 cells. Flow cytometry analysis showed that cells in S phase and percentage of apoptosis cells were increased after co-culture with AML MSC. Western blotting also determined that low expression of HHIP was detected in refractory AML BM-MSC, Smo and Gli-1 expression were increased in HL60/ADM or Kasumi-1 cells after co-cultured with MSC. Confocal analysis also confirmed that the combination of HHIP and Ptch was decreased in AML MSC co-culture, Smo/Gli-1 pathway was activated through decreased inhibition of Shh in refractory AML cells co-cultured with MSC. Samples from AML patients also demonstrated that HHIP expression in AML BM-MSC was lower than that in normal BM-MSC, especially in refractory AML samples. HHIP expression in BM-MSC was negatively related to Smo and Gli-1 activity. Clinic data also showed that AML patients with overexpression of HHIP had a worse prognosis. Conclusion. Our study demonstrated expression of BM-MSC HHIP was negatively related to activity of Ptch/Smo/Glis in AML. Low expression of BM-MSC HHIP resulted in activating Ptch/Smo/Gli pathway, and indicated drug resisitance and bad prognosis in AML. This project is connected with basic research and clinic, and provides support for diagnosis and targeted therapy in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibition of HIF-1a By PX-478 Normalizes Blood Vessels, Improves Drug Delivery and Suppresses Progression and Dissemination in Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-3
Author(s):  
Barbara Muz ◽  
Katherine Wasden ◽  
Kinan Alhallak ◽  
Amanda Jeske ◽  
Feda Azab ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Conflicts Of Interest ◽  
Photoacoustic Imaging ◽  
Fluorescent Microscopy ◽  
Tunel Staining

Introduction: Multiple myeloma (MM) is a lymphoplasmacytic malignancy localized in the bone marrow (BM) characterized by the continuous metastasis. Despite the introduction of novel therapies, MM patients relapse due to the development of drug resistance that is, at least in part, promoted by hypoxia (insufficient oxygen). MM cells develop a hypoxic phenotype, leading to cellular adaptations that cause metastasis, angiogenesis, stemness and resistance to drugs, such as carfilzomib, promoted by a hypoxia-inducible factor-1α (HIF-1α) transcription factor. Herein, we explored the mechanisms underlying HIF pathway inhibition using for the first time in MM a HIF-1α selective small molecule inhibitor, PX-478, both in vitro and in vivo. Methods: In vitro, to test the effect of PX-478 (0 - 50 µM) in combination with carfilzomib on MM cell survival exposed to normoxia (21% O2) or hypoxia (1% O2) was assessed using MTT assay. Cell adhesion to endothelial cells (HUVECs), and cell migration to stromal cells of prelabeled MM cells treated with PX-478 was measured by fluorescent spectrophotometer and flow cytometry, respectively. Tube-like formation of HUVECs as well as survival was tested in the presence of PX-478. For in vivo study, MM.1S-Luc-GFP cells were injected intravenously (i.v.) into 40 SCID mice; 3 weeks post injection the mice were divided into 4 groups and treated with vehicle (PBS), carfilzomib, PX-478, and a combination of PX-478 and carfilzomib. Tumor progression and weight was monitored weekly by bioluminescent imaging, and survival was monitored daily. At day 28, 3 mice from each group were randomly taken: (i) to test the number of circulating tumor cells (MM-GFP+) in the peripheral blood counted by flow cytometry; (ii) to test the MM apoptosis in the femurs by TUNEL staining; and (iii) to test extramedullar metastasis of MM in the kidney, spleen and the liver using immunohistochemistry. Additionally, tumor vasculature was demonstrated in the skull using photoacoustic imaging as well as tumor involvement using fluorescent microscopy. Moreover, we tested the drug delivery by injecting fluorescent large molecule (Dextran-AF405 Mw=70,000) i.v. in MM-bearing mice treated with and without PX-478. Lastly, we tested the effect of PX-478 on prelabeled MM cell retention in the blood and homing to the BM one hour post-MM injection in naïve mice. Results: We found that PX-478 reversed the hypoxia-induced resistance of MM cells to carfilzomib, inhibited metastasis-related cell processes such as adhesion and migration, and reduced MM-mediated tube-like formation of HUVECs in vitro. In vivo, in MM-bearing mice PX-478 decreased the number of MM circulating cells, suppressed tumor metastasis, improved vascularization of the tumor thus delivery of chemotherapy, and as a result re-sensitized MM cells to carfilzomib by increasing tumor apoptosis thus completely abrogating tumor growth and significantly extending mice survival. Conclusions: This is the first study to show the efficacy of PX-478 in MM demonstrating that PX-478 is acting as a pleiotropic molecule in which it inhibited many different hypoxia-induced biological processes - migration, angiogenesis and drug resistance. By overcoming these cancer adaptations, PX-478 has a clear advantage over using agents that carry an effect against one of these processes. This data provides a preclinical basis for future clinical trials testing efficacy of PX-478 in MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

scholarly journals In Vitro Regulation of FcRIα Expression on Human Basophils by IgE Antibody

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1633-1643 ◽  
Author(s):  
Donald MacGlashan ◽  
Jane McKenzie-White ◽  
Kristine Chichester ◽  
Bruce S. Bochner ◽  
Frances M. Davis ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Receptor Expression ◽  
In Vivo Studies ◽  
Whole Cell ◽  
Cell Lysates ◽  
Ige Antibody ◽  
Human Basophils

Abstract In vivo studies suggested the possibility of an IgE-dependent regulation of high-affinity (FcRI) IgE receptor expression on basophils. The current studies extend these observations to in vitro cultures of human basophils. Incubation of basophils for 3 to 4 weeks resulted in a slow dissociation of IgE antibody, during which time FcRI expression decreased, as measured by flow cytometry using the anti-FcRIα monoclonal antibody, 22E7, or by measuring FcRIα mass by Western blotting of whole-cell lysates. Culture of basophils with IgE resulted in upregulation of FcRIα expression by both flow cytometry and Western blotting of whole-cell lysates. Upregulation followed a linear time course during 2 weeks of culture. The relative increase in FcRIα density depended on the starting density; with starting densities of FcRIα of 10,000 to 170,000 per basophil, the upregulation varied 20- to 1.1-fold, respectively. Upregulation occurred in high-purity basophils, was not influenced by IgG at concentrations up to 1 mg/mL, and was inhibited by dimeric IgE. Heat-inactivated IgE was less effective and the monoclonal antibody CGP51901 that prevents IgE binding to FcRIα blocked the ability of IgE to induce upregulation. The dose-response curve for IgE-induced upregulation had an effective concentration50 of 230 ng/mL. Although the receptor through which IgE induces this upregulation is not yet known, several characteristics suggest that the upregulation is mediated by IgE interacting through FcRIα itself.

Preclinical Activity of Brentuximab Vedotin (SGN-35) in Primary Effusion Lymphoma (PEL),

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3728-3728 ◽  
Author(s):  
Shruti Bhatt ◽  
Brittany Ashlock ◽  
Yaso Natkunam ◽  
Juan Carlos Ramos ◽  
Enrique Mesri ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Primary Effusion Lymphoma ◽  
Large Cell ◽  
Brentuximab Vedotin ◽  
Antibody Drug Conjugate ◽  
Human Herpes Virus 8

Abstract Abstract 3728 Primary effusion lymphoma (PEL) is a distinct and aggressive subtype of non-Hodgkin lymphoma (NHL) commonly presenting with pleural, peritoneal, or pericardial malignant effusions usually without a contiguous tumor mass. PEL is most commonly diagnosed in HIV-positive patients, accounting for 4% of all NHLs in this population, yet may also develop in immunosuppressed HIV-negative individuals. While Human Herpes Virus 8 (HHV8 or Kaposi's sarcoma-associated herpesvirus) is directly implicated in the oncogenesis of this lymphoma, most PEL cases are also associated with Epstein-Barr virus and the combination of the two may facilitate transformation. The tumor cells exhibit plasmablastic features and express CD45, CD38, CD138, HHV8 and CD30. PEL is an aggressive tumor characterized by a short median survival of only 6 months with current therapeutic approaches underscoring the urgent need for development of new therapeutics. Brentuximab vedotin (SGN-35) is an antibody-drug conjugate (ADC) comprised of an anti-CD30 monoclonal antibody cAC10 conjugated by a protease-cleavable dipeptide linker to a potent cell killing agent monomethyl auristatin E (MMAE). Following binding to CD30, brentuximab vedotin is rapidly internalized and is transported to lysosomes, where the peptide linker is selectively cleaved allowing binding of the released MMAE to tubulin and leading to cell cycle arrest and apoptosis. Brentuximab vedotin was recently reported to have promising antitumor activity in CD30 expressing tumors, such as Hodgkin and Anaplastic large cell lymphomas. Since PEL tumors are reported to express CD30, we have hypothesized that brentuximab vedotin might be effective in the treatment of this NHL subtype. Initially, we have confirmed by flow cytometry the expression of CD30 on PEL cell lines (UM-PEL 1, UM-PEL 3, BC-1 and BC-3), and by review of immunohistochemistry and flow cytometry results in patients with previous diagnosis of PEL at our institution. To examine in vitro potency of brentuximab vedotin, UM-PEL 1, UM-PEL 3, BC-1 and BC-3 PEL cell lines were treated with brentuximab vedotin at concentration ranging from 0–100 micrograms/ml. Staining with YO-PRO and Propidium Iodide (PI) demonstrated dose dependent cell apoptosis and death in all the cell lines at 72 hours post treatment. In contrast, control IgG conjugated with MMAE failed to induce apoptosis and cell death of PEL cell lines confirming specific brentuximab vedotin cytotoxicity. Furthermore, brentuximab vedotin decreased proliferation of PEL cells at 48 hours leading to a complete proliferation arrest at 72 hours, as measured by MTS assay. These effects were absent after equivalent doses of control IgG conjugated drug treatment. Supportive to this, labeling of cells with PI to detect active DNA content by flow cytometry showed that bretuximab vedotin induced growth arrest in G2/M phase. To further establish the anti-tumor potential of brentuximab vedotin in vivo, we used the direct xenograft UM-PEL 1 model, established in our laboratory (Sarosiek, PNAS 2010), which mimics human PEL tumors. UM-PEL 1 bearing mice were injected intraperitoneally 3 times a week with brentuximab vedotin or control IgG conjugated MMAE for 4 weeks. Brentuximab vedotin treatment markedly prolonged overall survival of UM-PEL-1 bearing mice compared to controls (p Disclosures: No relevant conflicts of interest to declare.

Mapping the Targets of Dasatinib in Chronic Lymphocytic Leukemia Reveals Distinct Roles for Abl and Btk in Drug Resistance and Adhesion, and Explains Clinical Effects On Lymph Node Reduction

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3900-3900
Author(s):  
Eric Eldering ◽  
Christian R Geest ◽  
Martin FM de Rooij ◽  
Nora Liu ◽  
Bogdan I Florea ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Lymph Node ◽  
Chronic Lymphocytic Leukemia ◽  
Broad Spectrum ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Clinical Effects ◽  
Open Label

Abstract Abstract 3900 In the lymph node (LN) microenvironment, chronic lymphocytic leukemia (CLL) cells are protected from apoptosis by upregulation of anti-apoptotic proteins. In vitro, this can be mimicked via CD40-stimulation of CLL cells, which also provides resistance to various chemotherapeutics. Novel drugs that target kinases involved in B cell signalling, including the broad spectrum kinase inhibitor dasatinib, are currently in clinical development for CLL. We have shown previously that dasatinib prevents CD40-mediated anti-apoptotic changes in CLL (Hallaert et Blood 2008). However, the kinase(s) involved remain unidentified. Here, we coupled dasatinib to an affinity matrix and pulled down its targets from CD40-stimulated CLL cells. By mass-spectrometry and Western blotting, Abl and Btk were identified as dominant targets of dasatinib. Functional analysis revealed that CD40-mediated anti-apoptotic signals and drug-resistance could be overcome both by dasatinib and the Abl inhibitor imatinib, but not by the novel Btk inhibitor PCI-32765 (ibrutinib), whereas BCR- and chemokine-controlled adhesion could be abolished by dasatinib and ibrutinib, but not by imatinib. Thus, dasatinib combines two key aspects that are clinically relevant: inhibition of Abl overrides chemoprotective survival signals, whereas inhibition of Btk impairs integrin-mediated adhesion of CLL cells in the microenvironmental niche. This combined inhibition of Abl and Btk was put to an initial test in an open-label phase 2 trial of dasatinib combined with fludarabine in twenty refractory CLL patients. As might be expected based on the in vitro data, reductions in lymph node size were observed in most patients. A LN reduction of ≥20% provided a significant improved PFS (256 days) and OS (510 days) as compared to non-responders (80 days and 158 days respectively). Details of the clinical study will be presented separately. In conclusion, in agreement with in vitro molecular studies, dasatinib seems to have clinical efficacy in heavily pretreated refractory CLL patients. Combined, these data encourage further studies on a broad-spectrum kinase inhibitor like dasatinib in combination with other classes of drugs in relapsed and refractory CLL. Disclosures: No relevant conflicts of interest to declare.

Plasmin Cleaves Complement iC3b, Preventing CR3/4 Mediated Phagocytosis By Macrophages

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1105-1105
Author(s):  
Erica A. Peterson ◽  
Jonathan H Foley ◽  
Michael J Krisinger ◽  
Edward Conway
Keyword(s):  
Flow Cytometry ◽  
Inflammatory Responses ◽  
Conflicts Of Interest ◽  
Tissue Injury ◽  
Complement Receptors ◽  
Red Cell Membrane ◽  
Western Blots ◽  
Complement Pathways

Abstract Introduction The plasmin(ogen) and complement systems are activated at sites of tissue injury and are involved in hemostasis, wound healing, inflammation and immune surveillance. Although the mechanisms are poorly understood, dysregulation of these systems underlie the pathogenesis and progression of inflammatory and vascular diseases. We aimed to characterize the relevant molecular interactions between the plasmin(ogen) and complement pathways. The three complement pathways converge with formation of C3-convertases that cleave C3 into C3a and C3b. C3a is liberated as an anaphylatoxin while C3b participates in further formation of the C3 and C5 convertases, thereby amplifying complement activation. To dampen the system, negative regulatory mechanisms exist. C3b is degraded to iC3b by the factor I (FI)/FH complex, which in turn is degraded to C3dg by the FI/complement receptor 1 (CR1) complex. iC3b and C3dg induce cellular responses by binding to complement receptors CR3 / CR4 / CR2, and CR2, respectively. Interactions of iC3b with CR3 or CR4 induce phagocytosis by macrophages, and binding of iC3b or C3dg to CR2 promotes B-cell responses. Recent studies show that plasmin proteolyses C3b and iC3b. We further characterized the plasmin cleavage sites in iC3b and evaluated the functional consequences in vitro. Methods and Results Plasmin cleavage of iC3b was examined over a range of concentrations and times. Plasmin (50 nM) generated a 40 kDa iC3b cleavage fragment (946TLD – PSR1303) which was notable for containing both C3dg (1002HLI – PSR1303) and the C3 thioester domain, necessary for opsonic binding to surfaces. We tested the relevance of this cleavage in phagocytosis assays using immunofluorescence and flow cytometry (Figure 1). C3b bound to the surface of fluorescent (Alexa 488) zymosan particles (C3b-zym), was treated with FI/FH to generate iC3b-zym, and subsequently incubated with FI/CR1 or plasmin to yield C3dg-zym or 946TLD – PSR1303-zym, respectively. Western blots confirmed that plasmin generated 946TLD – PSR1303 from iC3b-zym. The C3 fragment-zymosan species (C3b-zym, iC3b-zym, C3dg-zym and 946TLD – PSR1303-zym) were each incubated with macrophages (PMA-differentiated THP-1 cells) for 90 minutes. Cells were washed, stained and fixed for immunofluorescence, or suspended for flow cytometry. Figure 1, panel A shows macrophages stained with CellMask (red, cell membrane) and DAPI (blue, nucleus). Fluorescent zymosan is seen in green. No phagocytosis was detected with zymosan lacking C3 (zym alone), but there was a small amount with C3b-zym. In contrast, iC3b-zym was highly effective in inducing phagocytosis by most macrophages. This effect of iC3b-zym was abolished with FI/CR1 or plasmin, i.e. little phagocytosis was detected with C3dg-zym or 946TLD – PSR1303-zym. Flow cytometry-based quantitative analyses confirmed the preceding findings (Figure 1, panel B), with a similar pattern of phagocytosis induced by the zymosan-bound fragments. No phagocytosis was detected with zymosan lacking C3. Phagocytosis of C3b-zym and iC3b-zym was 7±2% and 17±1% of cells, respectively. C3dg-zym and 946TLD – PSR1303-zym induced phagocytosis was <5%. We also evaluated the role of the complement receptors in mediating the effect of the C3b/iC3b fragments using CR3/4 and CR1 blocking antibodies. These confirmed that phagocytosis of iC3b-zym and C3b-zym is mediated by CR3/4 and CR1, respectively. Conclusions Plasmin cleaves iC3b to form a redundant complement regulatory pathway with the FI/CR1 complex, but which notably does not require a cellular cofactor. Further studies will delineate the role of this and other plasmin-generated complement fragments in modulating innate immune and inflammatory responses. Disclosures: No relevant conflicts of interest to declare.

Post-Transplant Immune Monitoring In Patients Receiving Allogeneic Stem Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5486-5486
Author(s):  
Silvia Park ◽  
Chul Won Jung ◽  
Jun Ho Jang ◽  
Eun Suk Kang ◽  
Kihyun Kim
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Immune Monitoring ◽  
Disease Relapse ◽  
Cell Secretion ◽  
Color Flow ◽  
Blood Samples ◽  
Post Transplant

Abstract Introduction There are still substantial morbidity and mortality caused by insufficient immunologic recovery after allo-HSCT. In this context, we attempt to evaluate the clinical relevance of immune monitoring in allo-HSCT recipients. Method Fifty five patients who underwent allo-HSCT between 2008 and 2012 were included. Peripheral blood samples were drawn from recipients before transplant, and on 4, 8, 12, 24, 36 and 48 weeks after transplant. Each blood samples were analyzed by multi-color flow cytometry for determining lymphocyte subsets. MNC were separated from blood specimen, and analyzed for the quantitation of Treg with the use of real-time PCR. We also examined T cell derived IFN-r by using in vitro culture, intracellular staining, and flow cytometry analysis. Results The median age was 43, and AML was the most common reason for transplantation (49.1%). Grade II or more aGVHD occurred in 36.4% of cases, and 49.1% exhibited moderate or severe cGVHD. The differences in the proportion (%) and the absolute number (/uL) of CD4+, CD8+ cells, CD4+ derived IFN-r (%), CD8+ derived IFN-r (%), and Treg (%) between the groups (Gr. II or more aGVHD (+) vs (-); moderate or severe cGVHD (+) vs (-)) were compared by Two sample t-test. Patients with Gr. II or more aGVHD showed decreased CD4+ count at 4, 8 and 12 weeks, but showed rather higher CD8+ count at 8 weeks after transplant. T-cell secretion function assessed by IFN-r (%), and Treg (%) was similar between two groups within 12 weeks after transplant. In case of cGVHD, both CD4+ and CD8+ count tended to be higher in patients with moderate or severe cGVHD, and the trends lasted for up to 48 weeks from allo-HSCT. Treg (%) was almost consistently lower throughout the period in these patients. There were 12 relapses within follow up period (median 36.1 months), and higher slope of post-transplant increase in CD8+ count and CD8 derived IFN-r were identified as protective factors for disease relapse. Conclusion In view of the results so far achieved, slow recovery of CD8 count and function might be associated with disease relapse. However, this is still a preliminary data, and warrants further evaluation. Disclosures: No relevant conflicts of interest to declare.

A Whole Genome In Vivo Crispr Screen in Primary ALL Predicts Leukaemic Relapse

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2619-2619
Author(s):  
Katherine Dormon ◽  
Elda S Latif ◽  
Matthew Bashton ◽  
Deepali Pal ◽  
Matthew Selby ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Conflicts Of Interest ◽  
Gene Set Enrichment Analysis ◽  
Whole Genome ◽  
Competitive Situation ◽  
Nsg Mice ◽  
A Genome ◽  
Crispr Screen

Abstract Although paediatric acute lymphoblastic leukaemia (ALL) has a favourable prognosis, a number of cases will invariably relapse. One of the major problems associated with relapse is drug resistance, in particular to glucocorticoids, the mainstay of ALL treatment. Examining the underlying mechanisms is complicated by clonal heterogeneity within a patient and the potential impact of the leukaemic niche. To address mechanisms of drug resistance in a patient-relevant setting, we performed a genome-wide in vivo CRISPR screen in primary ALL material. To that end, we took advantage of primografted material from patient L707, who initially presented with a Dexamethasone (DEX) sensitive t(17;19) ALL, but relapsed 5 months after initial diagnosis. We transduced DEX sensitive presentation cells with the full genome GeCKOv2 CRISPR library, before transplantation into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were subsequently treated with DEX by oral gavage (15mg/kg for 5 weeks, 10mg/kg thereafter). DNA from several engrafted sites in the mouse was extracted and PCR amplified before being sequenced on the Illumina HiSeq2500. Changes in pool complexity were analysed using MaGEcK software to determine which sgRNAs were significantly enriched or depleted. By far the most significantly enriched sgRNAs were those targeting NR3C1, the gene encoding the glucocorticoid receptor. In addition, two of the top five significantly depleted sgRNAs targeted the Plexins, PLXNA1 and PLXND1. Whilst PLXNA1 is expressed at low levels, PLXND1 is highly expressed and has been linked to dexamethasone resistance. Notably, the matched relapsed material from L707 was highly DEX resistant both in tissue culture and when transplanted into NSG mice. SNP 6.0 analysis revealed a 5q deletion in the relapse, spanning 5 genes including NR3C1. Whole genome sequencing showed this was comprised of 2 deletions both targeting NR3C1, with different breakpoints for each allele. The differential gene expression between the L707 presentation and relapse established that NR3C1 was the most significant of all the genes lost at relapse, based on gene set enrichment analysis (GSEA). This contrasts with many ALL cases, where one of the downstream effectors of apoptosis is lost as opposed to NR3C1. Growth of the relapse material in vivo and in vitro was slower than the presentation in a competitive situation, but with DEX treatment the relapse phenotype began to emerge with a small percentage of cells showing a heterozygous deletion of NR3C1. These combined data strongly suggest that the NR3C1 deletion is the main driver of DEX resistance in the L707 relapse. Moreover, it proves that our in vivo CRISPR screen predicted the leukaemic relapse. These results confirm NR3C1 deletion as a driver in glucocorticoid resistance and demonstrate the power of in vivo CRISPR screens to predict mechanisms of gain of drug resistance and subsequent relapse. The parallels that can be drawn between the relapse and the CRISPR screen are striking, giving the indication that the progression from presentation to relapse may follow the same path in a patient derived xenograft setting as it did in the patient. Disclosures No relevant conflicts of interest to declare.

Interactions Between BAFF and BAFF Receptors Are Involved in Macrophage-Induced Bortezomib Resistance in Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4429-4429
Author(s):  
Jing Chen ◽  
Donghua He ◽  
Xing Guo ◽  
Qingxiao Chen ◽  
Xuanru Lin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Drug Resistance ◽  
Western Blot ◽  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Counting ◽  
Therapeutic Modality ◽  
Second Primary ◽  
Induced Apoptosis

Abstract Background:B-cell-activating factor (BAFF) is a member of the TNF family that critical for maintenance of B-cell development and homeostasis. BAFF receptor (BAFF-R), B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI) are three BAFF receptors. It has been reported that BAFF is expressed by neutrophils, monocytes, dentritic cells and macrophages and modulates the proliferation, survival and drug resistance of multiple myeloma (MM) cells. Our previous study showed that, macrophages protect MM cells from drug-induced apoptosis by direct interaction with MM cells. We hypothesized that BAFF/BAFF receptors play a role in macrophage-induced bortezomib resistance in myeloma. Methods: First, the expression levels of BAFF and its three receptors in primary MM cells, MM cell lines and peripheral blood monocyte(PBMC)-induced macrophages were detected by semiquantitative real time-polymerase chain reaction (qPCR),Western blot and flow-cytometry. Also the concentration of BAFF in the supernatants of MM patients' bone marrow, MM cell lines and macrophages were determined by ELISA. Second, Primary MM cells and MM cell lines were cocultured with macrophages for the indicated time (usually 4-6h and 24h), for some experiments, we added bortezomib to the coculture system. Cell viability and apoptosis of MM cells were verified by Cell Counting Kit-8(CCK8) after treated with recombinant human (rh) BAFF, BAFF neutralizing antibody and BAFF siRNA. The interactions between BAFF and its receptors are unveiled by flow-cytometry. Then, cell survival signaling activations that may confer MM drug resistance were examined by Western blot. Results: Two receptors of BAFF, TACI and BCMA were highly expressed in various MM cell lines. The expressions of BAFF in PBMC-induced macrophages were heterogeneous. Functional studies showed that rhBAFF promoted RPMI8226 and ARP1 myeloma cells growth (P<0.05) and protected them from bortezomib-induced apoptosis (P<0.05). Then we verified macrophage-mediated MM drug resistance by directly coculturing MM cells (ARP-1, RPMI8226) with PBMC-derived macrophages from healthy donors. The macrophage-induced bortezomib resistance was attenuated by neutralizing antibodies(P<0.05) and siRNA of BAFF(P<0.01) . Next we found that in MM cells cocultured with macrophages, bortezomib-induced PARP and caspase-3 cleavages were highly repressed and phosphorylated Src ,AKT and Erk1/2 were upregulated which indicated that BAFF-mediated MM drug resistance may be through ERK1/2 and Src pathway .In addition, BAFF induced activation of NF-κB2,a pathway critical for the growth and survival of these cells. Conclusions: Our data show that macrophage might induce drug resistance of MM cells by the interaction of BAFF and BAFF receptors, leading to a reduction in caspase proteins and subsequent activation of Src and Erk1/2 kinases and NF-κB2 pathways .These studies reveal a promising unknown role for BAFF/BAFF receptors, suggesting that targeting macrophage-MM interactions may represent a promising therapeutic modality. Disclosures No relevant conflicts of interest to declare.

Expression of tumor endothelial markers (TEMs) in vitro in a canine hemangiosarcoma model of malignant angiogenesis.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e21145-e21145
Author(s):  
Jackie M. Wypij ◽  
Timothy M. Fan ◽  
Holly Pondenis
Keyword(s):  
Flow Cytometry ◽  
Protein Expression ◽  
Western Blotting ◽  
Receptor Expression ◽  
Degenerate Primers ◽  
In Vitro Expression ◽  
Endothelial Markers ◽  
Animal Tumor ◽  
Novel Model

e21145 Background: Expression of tumor endothelial markers (TEMs) in tumor-associated blood vessels presents opportunities for targeted therapy. TEM7 and TEM8 are selectively expressed and are associated with a worse prognosis in cancer patients. TEM expression is conserved across species, however species differences do exist as TEM7 is undetectable in murine tumor endothelium. Thus, further investigation of the role of TEMs in malignant angiogenesis is hindered by the lack of optimal animal tumor models. Canine hemangiosarcoma is relevant spontaneous model of malignant angiogenesis based on archetypal cell markers, endothelial functionality, growth factor/receptor expression, and angiogenic gene expression. The aims of this study were to characterize the in vitro expression of TEM7 and TEM8 in canine hemangiosarcoma as a novel model of malignant angiogenesis. Methods: Two canine hemangiosarcoma cell lines were assessed (Den and Fitz). Total RNA was isolated using standard technique, reverse transcribed to cDNA, and amplified by polymerase chain reaction (PCR) reaction using degenerate primers specific for human and murine TEM7 transcripts with forward and reverse primers. Protein expression of TEM7 and TEM8 in cell lysates was evaluated via Western blotting and cell surface expression was analyzed by flow cytometry. Polyclonal anti-TEM7 and anti-TEM8 antibodies (Sigma-Aldrich) were used with positive and negative controls. Results: Basal in vitro expression of TEM7 mRNA was confirmed in canine hemangiosarcoma, as well as in vitro protein expression of TEM7 and TEM8 via Western blotting and flow cytometry. Conclusions: The validation of TEM expression in canine hemangiosarcoma establishes TEM7 and TEM8 as promising targets for further evaluation in this novel model of malignant angiogenesis, and investigation of TEM-targeting is ongoing. This may represent a novel spontaneous animal tumor model for investigation of tumor vascular targeting.

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