scholarly journals The Population of Circulating Extracellular Vesicles Dramatically Alters after Very Premature Delivery- a Previously Unrecognised Postnatal Adaptation Process?

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1129-1129 ◽  
Author(s):  
Daniel O'Reilly ◽  
Karl Egan ◽  
Oscar Burke ◽  
Angharad Griffiths ◽  
Elaine Neary ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Board Of Directors ◽  
Extracellular Vesicles ◽  
Platelet Activation ◽  
Research Funding ◽  
Transition Period ◽  
Annexin V ◽  
Mass Spectrometry Analysis ◽  
Preterm Neonates ◽  
Advisory Committees

Abstract Introduction Following birth, the transition from intrauterine to extrauterine life is associated with major physiological changes. Many pathological processes linked with mortality and morbidity in preterm infants start at this time. Extracellular vesicles (EVs) are subcellular particles released by all known cell types and readily detectable in large numbers in all biological fluids. EVs are heterogeneous in size and origin, consisting of exosomes (endosomal origin, 30-150 nm), microvesicles (plasma membrane-derived, 50-1000nm), and apoptotic bodies (500-2000 nm). They are linked with a wide variety of processes including coagulation and cell-cell communication, and it has been hypothesized that they may affect preterm morbidities. It is unknown whether circulating EVs can change during this extrauterine transition period. Aim Here we investigate if the population of circulating EVs is altered in premature neonates during the extrauterine transition period Patients and Methods Preterm neonates were recruited through the Department of Neonatology at the Rotunda Hospital, Dublin, Ireland. Written informed consent was obtained from the parents of all participants. Blood collection was performed during routine phlebotomy. Platelet free plasma was prepared by double centrifugation at 3000g for 10 minutes. 15x Day 1 of life and 14x days 3 of life plasma samples were available from preterm neonates, 8 of which were matched Day 1 and Day 3 samples. EVs were quantified and characterised by both nanoparticle tracking analysis (NTA with a Malvern NanoSight 3000) and flow cytometry (Beckman Coulter CytoFLEX LX). Results The extrauterine transition period is characterised by a shift in plasma EVs profile. Using NTA, we observed an increase in the levels of plasma EVs (0-200nm) from Day 1 to Day 3 (Day 1; 4.0 ± 2.5 x 107/µl vs. Day 3; 7.2 ± 4.4 x 107/µl; p = 0.03). This increase in EV levels (0-200nm) was supported by flow cytometry, which also demonstrated an increase in EVs (100-900nm) from day 1 to Day 3 (Day 1; 1.1 ± 0.3 X 106/µl vs. Day 3; 4.2 ± 3.2 x 106/µl, p = 0.0009). There was a highly significant correlation between EV levels measured by NTA and flow cytometry (Spearmann rank correlation coefficient, r = 0.69, p < 0.0001), suggesting simultaneous increases in small and large EVs during the extrauterine transition period. Using flow cytometry, we also observed a change in the composition of plasma EVs during the extrauterine transition period. Flow cytometry data from Day 3 samples were characterised by the presence of a homogenous population of EVs of ~100-300nm in size, which was not observed on Day 1. The presence of this population caused a significant increase in the median side scatter height (SSC-H) value of the plasma EV population (Day 1; 1800 ± 746 vs. Day 3; 3832 ± 1633, p = 0.0013), as well as reduction in the percentage of 100nm EVs (Day 1; 73.9 ± 9.2 % vs. Day 3; 57.4 ± 12.6 %, p = 0.0005) and an increase in the percentage of 100-300nm EVs (Day 1; 19.7 ± 7.7 % vs. Day 3; 38.0 ± 12.9%, p = 0.001). EVs from Day 3 samples were characterised by higher median Red SSC-H values (Day 1; 1688 ± 2902 vs. Day 3; 3641 ± 6247, p = 0.0004) and Violet SSC-H values (Day 1; 42641 ± 21131 vs. Day 3; 97133 ± 38311, p < 0.0001), suggesting a potential change in the membrane or internal composition of EVs in the extrauterine transition period. We also observed a change in protein expression on EVs during the extrauterine transition period. Platelets and platelet activation play a physiological role in the closure of the ductus arteriosus . As such, we assessed the levels of platelet EVs (CD41+/Annexin V+), an established marker of platelet activation in vivo. The percentage of CD41+/Annexin V-EVs significantly decreased from Day 1 to Day 3 (Day 1; 6.5 ± 4.9 % vs. Day 3; 2.4 ± 1.9 %, p = 0.007), suggestive of a platelet activation event early in the extrauterine transition period. Proteomic differences between day 1 and day 3 were analysed using mass spectrometry analysis Conclusion In this study, we clearly demonstrate that the extrauterine transition period is characterised by major changes in plasma EVs. These changes include an increase in the levels of EVs, a change in the composition of EVs, and a reduction in the percentage of platelet-derived EVs. The physiological or pathophysiological causes of the changes require further elucidation. In addition, the role of this change of EV profile in the pathogenesis of important preterm morbidities needs to be clarified. Disclosures Ni Ainle: Leo Pharma: Research Funding; Actelion: Research Funding; Bayer: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Boehringer: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Proliferation and Molecular Risk Score of Low Risk Myeloma Cells Are Increased in High Risk Microenvironment Via Augmented Bioavailability of Growth Factors

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1929-1929
Author(s):  
Syed J. Mehdi ◽  
Sarah K Johnson ◽  
Sharmin Khan ◽  
Wen Ling ◽  
Randal Shelton ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Growth Factors ◽  
High Risk ◽  
Board Of Directors ◽  
Copy Number ◽  
Research Funding ◽  
Annexin V ◽  
Advisory Committees ◽  
Number Variation ◽  
Cell Dormancy

Abstract Introduction: Multiple myeloma (MM) cells from patients with smoldering MM (SMM) and low-risk (LR) MM harbor genetic alterations typically seen in patients with high-risk (HR) disease. To test whether the bone marrow (BM) microenvironment plays a role in controlling growth of LR MM cells, we established an experimental model that mimics a HR microenvironment by co-culturing normal mesenchymal stem cells (MSCs) with HR MM cells. We previously have shown that MSC conditioned media (CM) promotes growth of MM cells more effectively than cell-cell contact, as adhesion to MSCs often promotes survival at the expense of proliferation. Therefore, we utilized CM and hypothesized that MSC CM is enriched with bioactive growth factors that facilitate proliferation of LR MM cells. The aim of the study was to test the effect of CM from unprimed and primed MSCs on the survival, growth, and molecular properties of LR MM cells, and identify molecular pathways that mediate these effects. Methods: Primed MSCs were prepared by co-culturing normal MSCs with BM-dependent MM lines for 5 days. MSCs were trypsinized, replated for 40 min followed by serial washing to remove MM cells. Molecularly classified CD138-selected LR MM cells from 8 newly diagnosed patients were treated with 50% primed CM or unprimed CM, or growth media (CONT) for 5 days. Growth and survival of primary MM cells was assessed by MTT assay and detection of annexin V/PI and KI67 by flow cytometry. Microarrays were performed on primed and unprimed MSCs (n=7) and on primary LR MM cells treated with primed and unprimed MSCs CM (n=3). Pathways were analyzed using Ingenuity. Ultra low depth WGS was performed to assess copy number variation. Protein arrays were performed to test levels of secreted factors in CM (n=7). Results: Growth of primary LR MM cells (n=8) was increased by primed CM 5.1±0.05 (p<0.0001) and 7.0±0.6 (p<0.0001) folds compared to unprimed CM or CONT, respectively. In contrast, unprimed MSC CM increased growth in these cells by 1.3 fold. Flow cytometry analyses revealed no differences in proportion of annexin V/PI+ cells. However, the proportion of KI67+ cells was increased from 0.95±0.1% in unprimed CM group to 4.6±1.5% in primed CM group (p<0.04). Primed MSC CM caused MM cell GEP70 score to increase resulting in change from LR to HR in 2 experiments and from an ultra LR score to an intermediate score in another. Pathway analyses on genes differentially expressed between primed CM- and unprimed CM-treated MM cells identified oxidative phosphorylation with mitochondrial dysfunction, cell cycle, mitosis and p53 as the most significantly altered pathways. Top transcription regulators included FOXO3, TP53, E2F4, MYC and E2F1, whereas mir-16-5p and let-7 were the top microRNAs. Top significantly upregulated genes (>2 fold) by primed MSC CM included proliferation-related factors (MKI67, TOP2A, CCNB1, BIRC5 and RRM2), whereas underexpressed genes (< 2 fold) involved regulators of cell dormancy including BCL2 (survival), RICTOR (mTOR), HEY1 (NOTCH), JUN (AP-1) and CXCR4 (adhesion). Four genes we reported to powerfully predict progression of SMM to MM (Khan et al., Haematologica 2015) were highly upregulated in MM cells by primed MSC CM. WGS revealed similar copy number variation in MM cells treated with unprimed and primed CM, suggesting other mechanisms produced the observed gene expression changes. IGF1 is a central MM growth factor and IGF binding proteins (IGFBPs) control its bioavailability. We recently reported that mesenchymal cells are the main source of IGFBPs in BM, with IGFBP2 being the most downregulated gene in MM bone (Schinke et al., CCR 2018). Expression and secretion of IGFBPs (particularly IGFBP2) by MSCs were significantly reduced by priming these cells with MM cells, whereas IGF1 levels remained unchanged. IGFBP2 markedly blocked IGF1-induced MM cell growth (p<0.0003). Addition of IGF1R inhibitor significantly blocked primed CM-induced MM cell growth (p<0.02). Conclusions: MSCs primed by HR MM cells mimic a HR microenvironment, reflected by reduced level of factors that restrain bioavailability of MM growth factors such as IGF1, resulting in shutdown of master regulators of cell dormancy, which then enable a MM cells to proliferate. Such a scenario is particularly applicable in SMM and LR disease where MM cells exhibit a low proliferative index and their expansion is accelerated in distinct HR BM microenvironmental niches such as focal lesions. Disclosures Epstein: University of Arkansas for Medical Sciences: Employment. Davies:Abbvie: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; MMRF: Honoraria; Janssen: Consultancy, Honoraria; TRM Oncology: Honoraria. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding.

Mechanisms Underlying the Synergistic Interaction of Filanesib with Pomalidomide and Dexamethasone (FPD) in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1801-1801 ◽  
Author(s):  
Susana Hernández-García ◽  
Laura San-Segundo ◽  
Lorena González-Méndez ◽  
Montserrat Martín-Sánchez ◽  
Luis A Corchete ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Annexin V ◽  
Triple Combination ◽  
Advisory Committees ◽  
Cell Cycle Profile

Abstract Introduction: Filanesib (ARRY-520) is a novel inhibitor of the "kinesin spindle protein" (KSP), which has demonstrated efficacy in heavily pretreated patients with refractory MM, (Lonial et al, ASH 2013). Our preliminary studies demonstrated synergy with standard anti-MM agents, especially with pomalidomide and dexamethasone. This set the stage for a recently activated trial being run by the Spanish MM group investigating FPD in relapsed MM patients. In this abstract we investigate the mechanisms underlying the synergy of the combination. Methods: In vitro action of FPD was evaluated in MM cell lines by MTT assay, bioluminescence, Annexin V staining, cell cycle profile analysis and TMRE staining by flow cytometry. Synergy was quantified with the Calcusyn software. In vivo efficacy was assessed in a subcutaneous plasmacytoma model of MM1S in CB17-SCID mice (The Jackson Laboratory, Bar Harbor, ME, USA). The mechanism of action was analyzed by Western blot, flow cytometry, genomic techniques, immunohistochemistry and immunofluorescence techniques. Results: The triple combination of FPD resulted in clear synergy in multiple myeloma cell lines (MM1S, OPM2, and RPMI8226) with combination indices between 0.4-0.7, and abrogated the effect of the soluble cytokines IL-6 and IGF-I and the protective effect of the adhesion of plasma cells to BMSCs, HS-5 and TERT cells. FPD caused cell cycle arrest in G2/M and specific apoptosis of cells arrested in these proliferative phases (with apoptosis percentage of 5, 23, 58 and 88 for control, poma+dexa, filanesib and FPD, respectively) demonstrated by flow cytometry with DRAQ5 and Annexin-V. Thus, FPD and filanesib in monotherapy treatments induced a similar effect on the cell cycle profile (arrest in G2/M) with a concordant increase of cyclin B1 and phosphorylated Histone H3. Although a secondary increase of KSP protein levels would be expected, pomalidomide and dexamethasone induced a decrease of the levels of this protein, which was still present in the triple combination (FPD). This fact could be contributing to the potentiation observed with the combination. Attending to apoptosis mechanism, proapoptotic stimulus from the extrinsic and intrinsic apoptotic pathways were promoted by pomalidomide and dexamethasone and filanesib, and converged in the triple combination. In this regard, a decrease of MCL-1 (antiapoptotic protein) and a significant increase of the proapoptotic BCL2 family members of the intrinsic pathway like NOXA and BIMEL BIML, BIMS(this last one being the most potent proapoptotic isoform), tBID (extrinsic pathway) and Bax protein were observed. We confirmed that all these proteins were translocated into the mitochondria, resulting in a decrease of the mitochondrial membrane potential by TMRE, increase of permeability and a release of cytochrome C and AIF. These results were confirmed in vivo in a model of subcutaneous plasmacytoma in small (70 mm3) and large (2000 mm3) tumors. In this model we observed a significant reduction of tumor growth, which was correlated with a statistically significant improvement in survival. Changes induced by FPD in the gene expression profile were concordant with the in vitro results as several overexpressed genes belonging to the previous pathways were identified, such as spindle assembly checkpoint (CENP-E and CENP-F) and apoptosis (BCL2L11, gene that codifies BIM protein). Furthermore, IHC of tumors treated with FPD showed more apoptosis by TUNEL and a significant increase of monopolar spindles (2, 0, 53 and 140 per 10 high-power fields, for control, poma+dexa, filanesib and FPD, respectively). Conclusions: The synergy observed with filanesib in combination with pomalidomide and dexamethasone is the result of several coincidental mechanisms: a potentiation of the KSP inhibition with a subsequent increase in monopolar spindle formation and a simultaneous activation of the intrinsic and extrinsic pathways of apoptosis. In this regard, NOXA, BIM, BAX and tBID are probably the central players that, through different mechanisms, inhibit antiapoptotic proteins (MCL-1, BCL2 and BCL-XL) and promote mitochondrial outer membrane permeabilization and the release of apoptogenic factors such us cytochrome C and AIF. This work was funded in part by the company Array BioPharma. Disclosures Tunquist: Array BioPharma: Employment. Mateos:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy. Ocio:Jassen: Honoraria; Celgene: Honoraria, Research Funding; Pharmamar: Consultancy, Research Funding; MSD: Research Funding; Novartis: Consultancy, Research Funding; Mundipharma: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Array BioPharma: Consultancy, Research Funding.

Iron Overload Diminishes the Effectiveness of the Innate Immune Response in Thalassemia Major: a Possible Mechanism for Increased Infection Risk.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4071-4071
Author(s):  
Patrick B Walter ◽  
Paul R Harmatz ◽  
Annie Higa ◽  
David Killilea ◽  
Nancy Sweeters ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
Iron Overload ◽  
Board Of Directors ◽  
Innate Immune Response ◽  
Research Funding ◽  
Innate Immune ◽  
Healthy Controls ◽  
Advisory Committees ◽  
Fluorescent Intensity

Abstract Abstract 4071 Poster Board III-1006 Introduction Infection is the second most common cause of death in thalassemia. The innate immune system provides a first line of defense against infection and specificity depends on pattern recognition receptors (PRRs) specific to microbial pathogens. One class of PRR called the toll-like receptors (TLRs) are important for transducing the signal for bacterial Lipopolysaccharide (LPS), resulting not only in cytokine production, but also in the control of extracellular iron levels through production of neutrophil gelatinase associated Lipocalin (NGAL). However, the exact role that NGAL plays and the expression level of PRRs are unknown in thalassemia. Thus, the goal in these studies is to investigate the relationship of iron overload to the innate immune cell expression of PRRs and NGAL in thalassemia. Patients and Methods Fifteen transfusion dependent thalassemia patients (11 – 29 yrs old) participating in the combination trial of deferasirox (an oral iron chelator) and deferoxamine were enrolled (Novartis sponsored CICL670AUS24T). Fasting blood samples were obtained i) at baseline after a 72 hr washout of chelator, and ii) at 6 and 12 months on study. Five healthy controls (13 - 18 yrs old) were also enrolled. Fresh monocytes were isolated using antibody-linked magnetic microbeads (Miltenyi Biotec Inc). Highly enriched populations of CD14+ monocytes were verified by flow cytometry. The expression of TLR4, also examined by flow cytometry is reported as the mean fluorescent intensity (MFI). In patients with thalassemia, liver iron concentration (LIC) was analyzed by biomagnetic susceptibility (“SQUID”, Ferritometer®). The plasma levels of NGAL were analyzed by ELISA. Results At baseline the expression of monocyte TLR4 (mean 18.8 ± 3.5 MFI) was reduced 30% compared to the healthy controls (mean 26.9 ± 7.6 MFI, p<0.05). The expression of TLR4 over the follow-up period of 52 weeks in patients receiving intensive combination chelator therapy significantly increased 27% / year (7 MFI / year, p=0.005). Interestingly the expression of monocyte TLR4 was negatively correlated with LIC (r=-0.6, p=0.04). Finally, thalassemia patients at baseline have significantly higher levels of NGAL (80 ± 20 ng/ml) compared to controls (42 ± 15 ng/ml, p=0.01). Conclusions These preliminary studies support the hypothesis that iron burden has a negative impact on the innate immune response in thalassemia as demonstrated by the decreased expression of TLR4. After intensive chelation, the levels of TLR4 increased, indicating that decreased iron overload with chelation may improve innate immune responsiveness. Finally, the iron transport protein NGAL is significantly elevated in thalassemia possibly acting to prevent essential iron uptake by pathogenic bacteria. Disclosures: Harmatz: Novartis: Research Funding; Apotex : Membership on an entity's Board of Directors or advisory committees; Ferrokin: Membership on an entity's Board of Directors or advisory committees. Vichinsky:Novartis: Consultancy, Research Funding.

The Pan Histone Deacetylase Inhibitor Panobinostat and the Iso-Selective Inhibitor Romidepsin Reduce Glycoprotein VI Expression and Inhibit Platelet Activation in Response to Collagen Related Peptide

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1154-1154
Author(s):  
Mark J Bishton ◽  
Miles Prince ◽  
Ricky W Johnstone ◽  
Simon J. Harrison ◽  
Benjamin T. Kile
Keyword(s):  
Board Of Directors ◽  
Histone Deacetylase ◽  
Platelet Activation ◽  
Western Blotting ◽  
Research Funding ◽  
Histone Deacetylase Inhibitors ◽  
Surface Expression ◽  
Selective Inhibitor ◽  
Advisory Committees ◽  
Related Peptide

Abstract Abstract 1154 Histone deacetylase inhibitors (HDACi) are anti-cancer drugs able to induce chromatin remodelling, alter gene expression and affect function of non-histone proteins. We recently reported that the pan-HDACi panobinostat and the iso-selective inhibitor romidepsin induce thrombocytopenia by reducing megakaryocyte proplatelet number, without effects on platelet half life (Bishton et al Blood 2011). The effect of HDACis on platelet function remains unknown, and we postulated possible interference with the expression or function of key platelet activating proteins. Platelet Glycoprotein (GP) VI is a member of the immunoglobulin superfamily, expressed exclusively on the surface of platelets and megakaryocytes, complexed with FcR g-chain dimers, predominantly responsible for adhesion of platelets to collagen. Following interaction with sub-endothelial collagen, the Src family kinases Fyn and Lyn mediate the recruitment and autophosphorylation of Syk kinase and thereby downstream signalling and platelet activation. Following the treatment of C57BL/6 mice with 10mg/kg panobinostat or 1mg/kg romidepsin intraperitoneally (IP) daily for three days, we isolated washed murine platelets for function testing. Following stimulation with thrombin, a dose-dependant increase was seen in platelet surface expression of CD62P (P-selectin), and also the conformationally active form of the integrin αIIbbIIIa, with no difference seen between groups. When collagen related peptide (CRP) was used as a platelet agonist, and activation assessed by p-selectin and activated αIIbbIIIa expression, platelets from cohorts of mice treated with panobinostat or romidepsin failed to increase the expression of either molecule in response to CRP, compared to vehicle treated mice. Co-treatment of mice with the murine thrombopoietin mimetic, AMP-4, or the proteasome inhibitor bortezomib did not alter effects of the HDACi. Ex vivo addition of panobinostat or romidepsin to naïve platelets did not however affect platelet activation, suggesting megakaryocytes rather than platelets to be the target cell responsible for these effects. Flow cytometric analysis of the expression of GPVI on platelets showed a consistent and statistically significant decrease in the median fluorescent intensity (MFI) of staining seen in both HDACi treated groups. No equivalent changes in the surface expression of the other collagen receptor integrin α2b1 were seen. Western blotting of murine platelets confirmed this reduction in GPVI and a ∼17kDa fragment was also seen with HDACi treated platelets, suggesting GPVI degradation. Following stimulation with CRP, Western blotting of platelets with a phospho-syk antibody showed a reduction in phospho-syk levels in platelets from mice treated with HDACi, consistent with decreased downstream signalling from the GPVI receptor. Western blotting of murine megakaryocytes differentiated from murine fetal liver cells by TPO, also demonstrated a reduction in GPVI expression following HDACi exposure, again suggesting an intrinsic megakaryocyte process to be responsible. qRT-PCR on HDACi treated megakaryocytes demonstrated a mild increase in GPVI mRNA levels post romidepsin, but no changes post panobinostat compared to vehicle treated cells, confirming transcriptional repression not to be responsible for these changes. We show that HDACi cause a reduction in surface expression of GPVI expression by inducing its degradation and thus inhibiting murine platelet responses to CRP. There was no evidence of an effect on gene transcription. Our work suggests a potential beneficial anti-thrombotic effect of HDACi, mediated by reduction in both platelet number and function. These findings support the need to investigate the role of HDACi and their effect on GPVI in myeloproliferative neoplasms particularly with respect to their impact on thrombotic complications. Disclosures: Off Label Use: Panobinostat and romidepsin are histone deacetylase inhibitors. We show that both reduce platelet response to collagen and therefore may have an anti-thrombotic effect. Prince:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Johnstone:N: Research Funding. Harrison:Cellgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding.

Deregulation of TNFRSF18 (GITR) Through Promoter CpG Island Methylation Induces Tumor Proliferation in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2424-2424
Author(s):  
Yang Liu ◽  
Yong Zhang ◽  
Phong Quang ◽  
Hai T Ngo ◽  
Feda Azab ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Advisory Committees ◽  
Brdu Assay

Abstract Abstract 2424 Introduction Tumor necrosis factor receptor super families (TNFRSFs) play an important role in activation of lymphocyte and cell apoptosis. However the function of TNFRSFs in multiple myeloma (MM) remains unknown. Loss of function mutation of Fas antigen (TNFRSF6) was identified in MM cells, thus suggesting the possible role of TNFRSFs in regulating MM pathogenesis. We therefore investigated the epigenetic mechanisms that may mediate inactivation of TNFRSFs and its functional role in MM. Methods Dchip software was utilized for analyzing gene expression dataset. DNA was extracted from both primary CD138+ MM plasma cells and MM cell lines using blood & tissue DNA isolation kit (Qiagen, Inc.). Expression of GITR in primary CD138+ plasma cells was detected by Imunohistochemistry (IHC) DNA methylation was analyzed by methylated DNA immunoprecipitation (Medip) assay and bisulfate sequencing. 5'azacytidine was used to demethylate genomic DNA. Gene expression was detected by qRT-PCR and confirmed at the protein level by flow cytometry and western-blot. Over-expression of GITR was obtained in MM1.S cells by using GITR recombinant plasmid and electroporation. Apoptosis was determined using Annexin/PI staining and flow cytometry analysis. Activation of apoptotic signaling was studied by western blot. Cell survival and proliferation were analyzed by MTT and BrdU assay, respectively. Recombinant GITR-lentivirus was obtained from the supernatant of culture medium after 72 hours transfection in 293 cells. GFP positive MM cells were sorted and analyzed by flow cytometry. In vivo effect of GITR on MM tumor growth was determined by injection of GITR over-expressing MM cells in null mice. Mice skull, femur and vertebrae were isolated after 4 weeks injection. Anti-human CD138+ mAb microbead was used to detect MM cells extracted from mice tissue by flow cytometry. Results Gene-expression profiling showed down-regulation of TNFRSFs, including TNFRSF11A, TNFRSF11B, TNFRSF8, TNFRSF10C, TNFRSF9, TNFRSF21, TNFRSF1B, TNFRSF1A and TNFRSF18, compared to normal plasma cells. Moreover, Our IHC results also showed that GITR expression was positive in primary CD138+ plasma cells from 9 normal bone marrow, but negative in 9 MM samples. Importantly, we found that low GITR expression significantly correlated with MM progression. Indeed, GITR gene levels were lower in smoldering and active MM patients compared to MGUS patients and normal donors. Promoter CpG island (CGI) methylation of GITR was indentified in 5 out of 7 MM primary bone marrow (BM)-derived CD138+ cells but not in normal BM-derived plasma cells. Bisulfate sequencing and Medip assay showed that methylation of GITR was significantly associated with GITR expression in 5 MM cell lines, including MM1.S, OPM1, U266, RPMI and INA6. Promoter CGI of GITR was highly methylated leading to complete silencing of GITR in MM1.S cell line. GITR expression was significantly up-regulated in MM cells upon treatment with the 5'azacytidine. MTT and BrdU assay revealed that the proliferation and survival of MM1.S cells was disrupted in the GITR over-expressing MM1.S cells, notably with inhibition of cell proliferation compared to control vector infected cells. Moreover induction of cytotoxicity in GITR over-expressing cells was confirmed by using GFP competition assay. GITR-induced apoptosis was supported by induction of caspase 8 and 3 cleavage. The inhibition of human CD138+ plasma cell growth in the bone marrow of SCID mice using a disseminated MM xenograft model was observed in the experimental group injected with GITR expressing cells compared to the control group after 4 weeks injection. Conclusion Our findings uncovered a novel epigenetic mechanism contributing to MM pathogenesis, showing the role of GITR methylation as a key regulator of MM cell survival. Disclosures: Roccaro: Roche:. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

FLT3 Splice Variant (FLT3Va) As a Potential Immunotherapeutic Target in Patients with Acute Myeloid Leukemia (AML)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1681-1681
Author(s):  
Sophia Adamia ◽  
Jeffrey Nemeth ◽  
Shruti Bhatt ◽  
Sarah R Walker ◽  
Natalie I Voeks ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Board Of Directors ◽  
Research Funding ◽  
Leukemic Cells ◽  
Advisory Committees ◽  
Gm Csf ◽  
Nsg Mice ◽  
Blocking Antibodies ◽  
Pdx Models

Abstract Alternative pre-mRNA splicing (AS) is a normal epigenetic phenomenon, a key regulator of gene expression, yields multiple transcripts and thus a variety of proteins from a single gene. Mutations in the spliceosome components resulting in aberrant splicing isoforms are common in AML, and other myeloid neoplasms, and may generate leukemia-specific neoantigens targetable with an antibody-drug conjugates (ADCs) or blocking antibodies. Our previous studies revealed that the FLT3 cell surface receptor is one of the most commonly misspliced genes in AML (54-63% of ~400 AML patients). We conducted cloning and sequencing analyses in AML cells and identified multiple aberrant splice-variants of FLT3 that resulted from either skipping of one or more exons or activation of cryptic splicing sites. Transfection of cDNA with three of these variants in TF-1 (AML cell line) cells resulted in expression of Flt3 variant proteins on the cell surface. We successfully generated rabbit polyclonal antiserum against a unique peptide sequence present in the most commonly expressed abnormal splice variant, which we termed Flt3Va. Immunoblots performed with the polyclonal antibody identified a ~160 kDa protein expressed by TF-1 cells transfected with FLT3Va, and the antibody did not react with untransfected TF-1 cell lysate. Using standard techniques, we generated rabbit hybridomas and evaluated the clones by flow cytometry and western blotting experiments. Based on these data, we selected one antibody clone (15-7) for further experiments. The 15-7 anti-Flt3Va rabbit monoclonal antibody identified Flt3Va protein expressed on the cell surface and within the cytoplasm of transfected TF-1 cells by flow cytometry and western blotting. However, no Flt3Va protein was detected in untransfected TF-1 cells or normal CD34+ bone marrow cells. The 15-7 antibody bound to 26 of 52 primary AML samples and 5 of 10 primagraft samples (PDX models) of human AML. Immunoblotting analyses of PDX models and patient samples confirmed binding to a protein of the expected size (130-160 kDa). Additionally, multi-parameter flow cytometry in 10 PDX models and 52 primary demonstrated that putative AML stem cells (as defined by the CD45dim, CD34, CD38, CD33, c-Kit cell surface expression) co-expressed Flt3Va antigen in 50% samples evaluated. An analysis of Flt3Va protein localization by live cell imaging showed a punctate distribution of Flt3Va on the cell surface. Furthermore, we observed that overexpression of Flt3Va in TF-1 cells led to GM-CSF growth factor independence. Analysis of TF-1 cells in the absence of GM-CSF and Flt3 ligand demonstrated constitutive activation of STAT5, an important mediator of Flt3 signaling, in Flt3Va overexpressing cells. In addition, Erk1/2 phosphorylation was also increased in Flt3Va overexpressing cells, another downstream effector of Flt3. In an effort to determine if Flt3Va+ cells had tumor repopulating ability, we sorted 0.3X10^6 Flt3Va+ and Flt3Va- cells from a PDX sample and injected the sorted populations or unsorted bulk tumor cells into NSG mice. The human cell engraftment in the mice was detected by the expression of human CD45, CD33, CD34, CD38, and c-kit antigens in the peripheral blood. In two experiments, mice injected with Flt3Va+ cells had detectable circulating leukemic cells by ~18 days after injection, while those injected with Flt3Va- cells had detectable circulating leukemic cells after the 4th week. These results suggest both Flt3Va+ and Flt3Va- cell populations are able to reconstitute leukemia after transplantation in NSG mice. However, Flt3Va+ may be expressed by an aggressive AML clone that facilitate early tumor engraftment. Overall, these studies suggest that Flt3Va is a leukemia-specific neoantigen and is an attractive potential immunotherapeutic target in AML. Proteins such as Flt3Va generated by alternative splicing are common in AML and may be targets for of novel blocking antibodies or ADCs, minimizing effects on normal tissues. Disclosures Adamia: Janssen: Research Funding. Nemeth:Janssen: Employment. Attar:Janssen: Employment. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding. Steensma:Millenium/Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Ariad: Equity Ownership; Genoptix: Consultancy. Weinstock:Novartis: Consultancy, Research Funding. DeAngelo:Novartis: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; Baxter: Consultancy; Celgene: Consultancy; Incyte: Consultancy; Amgen: Consultancy. Stone:Agios: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celator: Consultancy; Juno Therapeutics: Consultancy; Roche: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; ONO: Consultancy; Sunesis Pharmaceuticals: Consultancy; Merck: Consultancy; Xenetic Biosciences: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Amgen: Consultancy; Karyopharm: Consultancy; Seattle Genetics: Consultancy. Griffin:Janssen: Research Funding; Novartis: Consultancy, Research Funding.

scholarly journals Characterization of Residual CLL Following Long-Term Bruton Tyrosine Kinase Inhibitor Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2620-2620
Author(s):  
Shanmugapriya Thangavadivel ◽  
Alexander Pan ◽  
Xi Chen ◽  
Chen Song ◽  
Claire Snyder ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Cell ◽  
Advisory Committees ◽  
Time Points ◽  
Bruton Tyrosine Kinase ◽  
Over Time

Abstract Introduction: The development of Bruton tyrosine kinase inhibitors (BTKi) and their introduction into clinical practice represent a major advance in the treatment of chronic lymphocytic leukemia (CLL). Ibrutinib and other second generation BTKi as monotherapies generally do not produce minimal residual disease negative (MRD-) complete remissions even with extended therapy. The reason for lack of continued elimination of CLL to a MRD- status over time is unknown, and we hypothesized that biological differences in the CLL cells or immune microenvironment might make them resistant to elimination. Methods: Samples were obtained from patients on continuous ibrutinib who hadn't relapsed at time points of 3 years on treatment and 5 years on treatment; and pre-ibrutinib. Isolated CLL cells were subject to B-cell receptor (BCR) sequencing using NEBNext Immune Sequencing Kit by New England Biolabs (NEB, Inc., USA). In a separate cohort, 10X VDJ+5'-sequencing was performed on peripheral blood mononuclear cells. Flow cytometry and ELISA were used to identify alterations in immune cell subtype and identify immune profiles associated with MRD positive (MRD+) status. Results: To identify the clonal pattern in MRD+, we performed deep sequencing of the BCR repertoire on samples from 13 patients with 3 time points each. We found that dominant clones tended to remain constant, but new clones appeared in later time points (Figure 1). MiXCR (v3.0.5) was used with default parameters to identify preprocessed reads containing CDR3 regions from B-cell heavy, kappa, and lambda chains, generating a list of unique productive and nonproductive CDR3 sequences associated with their relative abundances and specific V(D)J gene usage. Two out of three patients (patients 1 and 3) showed significant change in the clone over time. In patients 1 and 2, we saw that heavy chain clones emerge at later time points. In patient 3 alone, we observed that at 5 years there are two dominant clones. Our findings suggest that each patient shows a diverse repertoire of CLL clones and that the dominant clone does not change significantly across time points. To identify cell populations based on gene expression patterns, we performed 10X VDJ+5'-seq. Based on the expression of known markers, we identified CLL cells and other immune cell subtypes. We identified differentially expressed genes (DEGs) for CLL cells in each time points. Over time, we observed upregulation of CD79a, LTB, TAGLN2, and LGALS, genes typically associated with leukemic cell survival. Suggesting differential expression of pro-survival genes contribute to continued presence of MRD over time. T cells are known to be dysfunctional in CLL and have not previously been extensively studied in the setting of long term BTKi. We performed flow cytometry to determine the repertoire and function of T cells at 3 and 5 years of ibrutinib therapy. We found that the percentage of CD3+ T cells increases at later time points in all the 8 patients (p&lt;0.05). Although T cell numbers increase, we do see skewing of these cells towards a terminally differentiated phenotype (p&lt;0.05). We also observed significant increases in NK cells across time points (p&lt;0.05), albeit non-functional due to high expression of inhibitory receptor KLRG1 in 7 out of 8 patients (p&lt;0.05). Although overall the number of immune cells increase in long time ibrutinib therapy, they exhibit exhausted or non-functional phenotypes. Conclusion: Extended ibrutinib treatment yields a subset of patients who become MRD- whereas a large majority remain MRD+. Our findings suggest that BCR repertoire in CLL MRD might change in long term ibrutinib therapy and induce necessary genes for its survival in the microenvironment. Although T cells and NK cells are non-functional at later time points, better understanding of these subtypes may lead to new strategies and to improve antitumor function of these cells. Differentiating the biology of why certain patients attain MRD- status on BTK inhibitor is of high interest as it could provide rationale for therapy discontinuation or add on approaches. Figure 1 Figure 1. Disclosures Rogers: AbbVie Inc.: Consultancy, Research Funding; Acerta Pharma: Consultancy; AstraZeneca: Consultancy; Genentech: Consultancy, Research Funding; Innate Pharma: Consultancy; Pharmacyclics LLC: Consultancy; Janssen Pharmaceuticals, Inc: Research Funding; ovartis Pharmaceuticals Corporation: Research Funding. Bhat: Beigene: Consultancy; AstraZeneca: Consultancy; Aptitude Health: Honoraria; Onclive: Honoraria. Kittai: Bristol-Meyers Squibb: Consultancy; Abbvie: Consultancy; Janssen: Consultancy. Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Byrd: Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Newave: Membership on an entity's Board of Directors or advisory committees. Woyach: AbbVie Inc, ArQule Inc, Janssen Biotech Inc, AstraZeneca, Beigene: Other: Advisory Committee; AbbVie Inc, ArQule Inc, AstraZeneca Pharmaceuticals LP, Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company,: Consultancy; AbbVie Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company: Research Funding; Gilead Sciences Inc: Other: Data & Safety.

scholarly journals CD200 Is a Stem Cell-Specific Immunosuppressive Target in AML

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2768-2768
Author(s):  
Shelley Herbrich ◽  
Keith Baggerly ◽  
Gheath Alatrash ◽  
R. Eric Davis ◽  
Michael Andreeff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
T Cells ◽  
Stem Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Cd200 Receptor ◽  
Blast Cells ◽  
Equity Ownership

Abstract Acute myeloid leukemia (AML) stem cells (LSC) are an extremely rare fraction of the overall disease (likely <0.3%), largely quiescent, and capable of both long-term self-renewal and production of more differentiated leukemic blasts. Besides their role in disease initiation, they are also hypothesized as the likely source of deadly, relapsed leukemia. Due to the quiescent nature of the LSCs, they are capable of evading the majority of chemotherapeutic agents that rely on active cell-cycling for cytotoxicity. Therefore, novel therapeutic approaches specifically engineered to eradicate LSCs are critical for curing AML. We previously introduced a novel bioinformatics approach that harnessed publically available AML gene expression data to identify genes significantly over-expressed in LSCs when compared to their normal hematopoietic stem cell (HSC) counterparts (Herbrich et al Blood 2017 130:3962). These datasets contain gene expression arrays on human AML patient samples sorted by leukemia stem, progenitor, and blast cells (with normal hematopoietic cell subsets for comparison). We have since expanded our statistical model to identify targets that are both significantly overexpressed in AML LSCs when compared to HSC as well as LSCs compared to their corresponding, more differentiated blast cells. Instead of traditional methods for multiple testing corrections, we looked at the intersection of genes that met the above criteria in 3 independently generated datasets. This resulted in a list of 30 genes, 28 of which appear to be novel markers of AML LSCs. From this list, we first chose to focus on CD200, a type-1 transmembrane glycoprotein. CD200 is broadly expressed on myeloid, lymphoid, and epithelial cells, while the CD200 receptor (CD200R) expression is strictly confined to myeloid and a subset of T cells. CD200 has been shown to have an immunosuppressive effect on macrophages and NK cells and correlates with a high prevalence FOXP3+ regulatory T cells (Coles et al Leukemia 2012; 26:2146-2148). Additionally, CD200 has been implicated as a poor prognostic marker in AML (Damiani et al Oncotarget 2015; 6:30212-30221). To date, we have screened 20 primary AML patient samples by flow cytometry, 90% of which are positive for CD200. Expression is significantly enriched in the CD34+/CD123+ stem cell compartment. To examine the role of CD200 in AML, we established two in vitro model systems. First, we used CRISPR/Cas9 to knockout the endogenous CD200 protein in Kasumi-1. Further, we induced CD200 in the OCI-AML3 cell line that had no expression at baseline. Both cell lines did not express the CD200 receptor before or after manipulation, negating any autocrine signaling. In both systems, CD200 manipulation did not affect the proliferation rate or viability of the cells. To examine the immune function of CD200 in AML, we performed a series of mixed lymphocyte reactions. We cultured normal human peripheral blood mononuclear cells (PBMCs) with the CD200+ or CD200- cells from each line both. Cells were incubated in the culture media for 4-48 hours before being harvested and measured by flow cytometry for apoptosis or intracellular cytokine production. The presence of CD200 on the cell surface reduced the rate of immune-specific apoptosis among these leukemia cells. The difference in cell killing was most likely attributable to a CD200-specific suppression of CD107a, a surrogate marker or cytotoxic activity. In the OCI-AML3 model, PBMCs co-cultured with CD200+ cells produced approximately 40% less CD107a when compared to the CD200- co-culture. Additionally, we characterized our new cell lines using RNA sequencing. By comparing the CD200+ to the CD200- cells within each line, we observed that CD200+ cells significantly downregulate genes involved in defining an inflammatory response as well as genes regulated by NF-κB in response to TNFα. This indicates that CD200 may have an undiscovered intrinsic role in suppressing the immune microenvironment of AML LSCs. In conclusion, we have expanded our novel bioinformatics approach for robustly identifying AML LSC-specific targets. Additionally, we have shown that one of these markers, CD200, has a potential role as a stem cell-specific immunosuppressive target by reducing immune-mediated apoptosis and transcriptionally suppressing inflammatory cell processes. We are extending our study to explore CD200 in primary patient samples using a CD200-blocking antibody. Disclosures Andreeff: SentiBio: Equity Ownership; Amgen: Consultancy, Research Funding; Oncolyze: Equity Ownership; Reata: Equity Ownership; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Jazz Pharma: Consultancy; Astra Zeneca: Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy. Konopleva:Stemline Therapeutics: Research Funding.

scholarly journals Secreted Mutant Calreticulins As Rogue Cytokines Trigger Thrombopoietin Receptor Activation Specifically in CALR Mutated Cells: Perspectives for MPN Therapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4-4 ◽  
Author(s):  
Christian Pecquet ◽  
Thomas Balligand ◽  
Ilyas Chachoua ◽  
Anita Roy ◽  
Gaelle Vertenoeil ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Flow Cytometry ◽  
Cell Surface ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Allele Burden ◽  
In Trans ◽  
Equity Ownership ◽  
In Cells

Abstract Background Mutant calreticulins carrying the sequence translated after a +1 frameshift at the C-terminus are major drivers of myeloproliferative neoplasms (MPNs). These mutant CALRs bind and activate TpoR/MPL in cells co-expressing TpoR and mutant CALRs, resulting in persistent JAK2-STAT5 signaling. Whether mutant CALR proteins are secreted, thus acting in trans on other cells, is not known. Aims Our objectives were to: 1) assess the direct TpoR-mutant CALR interaction both when expressed in the same or in different cells; 2) determine whether mutant CALRs are secreted; and 3) determine whether mutant CALR can act as extracellular cytokines. Methods Engineered CALR and TpoR mutants were analyzed by a combination of biochemical approaches (bioluminescence resonance energy transfer, recombinant protein production), functional assays (cell growth and transcriptional assays, flow cytometry, primary megakaryocytic clonogenic assay, analysis of CALR del52 knock-in mice) and cell imaging (confocal microscopy, flow cytometry and immuno-gold electron microscopy). Secreted CALRs were determined by ELISA using mutant specific antibodies. Results 1) Two systems provided evidence that mutant CALRs and TpoR directly interact. First, using Nano-BRET in cells co-expressing N-terminally fused TpoR or EpoR with Nano-luciferase and mutant or WT CALR C-terminally tagged with HaloTag that is bound to the 618-ligand fluorophore, we show that TpoR and mutant CALRs interact in a complex whether the two proteins are within 10 nm. The interaction does not occur between TpoR and WT CALR, or between EpoR and mutant or WT CALRs. Second, expressing mutant CALR and TpoR extracellular domain in S2 Drosophila Schneider cells showed that stable complex formation requires immature high mannose structure on TpoR. Lastly, we could detect surface expression of the TpoR/CALRdel52 complex using proximity ligation assay with anti-TpoR and anti-mutant CALR antibodies in CRISPR/Cas9 engineered UT7/Tpo cells that express endogenous mutant CALR and TpoR levels. 2) We used flow cytometry, confocal immunofluorescence and immunogold electron microscopy and could show that mutant CALRs are trafficking via cis-, medial- and trans-Golgi to the cell-surface and are secreted, independently from TpoR expression. Importantly, mutant CALRs are also secreted in CALR mutated MPN patients as determined by mutant CALR-specific ELISA assay in patient plasma (mean plasma level 24.6 ng/ml, range 0-156.5 ng/ml). In the 113 evaluated CALR mutated patients from different centers the plasma mutant CALR levels correlated with CALR mutant allele burden (P<0.001). Secreted mutant CALR can also be found in plasma from knock-in CALR del52 mice. 3) We show that recombinant mutant CALR can act as a cytokine and specifically stimulate JAK2-STAT5 pathway in cells that carry the TpoR at the surface. Using Nano-BRET, we could demonstrate that extracellular mutant Halo-tagged CALR can specifically bind in trans to the cell-surface TpoR fused with Nano-luciferase, but not to EpoR fused with Nano-luciferase. This binding and the subsequent JAK2 activation were obtained at levels of around 100-150 ng/ml only in cells exposing at the cell-surface TpoR with at least one immature N-linked sugar. This can be accomplished by co-expressing in the reporter cells non-tagged mutant CALR, which will promote cell-surface localization of partially immature TpoR. The effect of exogenous mutant CALR could involve both stabilization of the endogenous cell-surface mutant CALR-TpoR complexes and binding to unoccupied immature TpoRs. Conclusion We show that mutant CALRs directly interact with TpoR and also are secreted and can act as rogue cytokines, leading to activation of cells carrying TpoR. Activation of TpoR in trans is efficient at mutant CALR levels similar to those detected in patients when target cells co-express heterozygous mutant CALR and TpoR, where endogenous mutant CALR transports to the surface TpoR with immature glycosylation. Thus, secreted mutant CALRs is predicted to expand the MPN clone. Given that cell-surface mutant CALR in TpoR expressing cells is crucial for oncogenicity, and that mutant CALRs are also secreted correlating with allele burden, we discuss how antibodies and other immunotherapy approaches could specifically target the mutant CALR MPN clone. Disclosures Xu: MyeloPro Research and Diagnostics GmBH: Employment. Hug:MyeloPro Diagnostics and Research GmbH: Employment. Gisslinger:Janssen: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Shire: Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Kralovics:MyeloPro Diagnostics and Research GmbH: Equity Ownership. Constantinescu:Personal Genetics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; AlsaTECH: Equity Ownership; Novartis: Honoraria; MyeloPro Research and Diagnostics GmbH: Equity Ownership.

scholarly journals Outcomes Following Allogeneic Stem Cell Transplantation for AML in First Completion Remission Are Comparable between MRD Negative Patients and MRD Positive Patients Receiving Induction Only and Are Superior to MRD Positive Patients Receiving Induction and Additional Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4615-4615
Author(s):  
Jonathan A Gutman ◽  
Prashant Sharma ◽  
Enkhtsetseg Purev ◽  
Clayton Smith ◽  
Amanda Winters ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Poor Prognosis ◽  
Residual Disease ◽  
Conditioning Regimen ◽  
Optimal Timing ◽  
Advisory Committees ◽  
Positive Induction ◽  
Additional Therapy

Background: Data suggests that the presence of measurable residual disease (MRD) at the time of transplant for AML portends a poor prognosis. The timing of MRD assessment and transplant relative to the amount of pre-transplant therapy, however, may affect this prognosis. To examine further the question of optimal timing of transplant in the setting of MRD, we reviewed outcomes in AML patients treated with traditional cytotoxic induction at our center who achieved CR1 and proceeded to transplant. Methods: We analyzed outcomes in patients undergoing first transplant for AML in CR1 between January 2014 and March 2018. CR was defined according to 2017 European Leukemia Network (ELN) guidelines. Non-core binding factor patients were included if they underwent initial therapy with 7+3 chemotherapy (+/- adjunctive inhibitor during induction or as post-transplant maintenance). MRD testing modalities included cytogenetics/FISH (performed in 37 patients, positive in 10), flow cytometry performed by Hematologics Inc (performed in 49 patients, positive in 16), and/or mutation specific droplet digital PCR (ddPCR) performed at our center (performed in 33 patients, positive in 16). For three patients, other molecular methods demonstrated MRD. We compared outcomes in three groups: patients who underwent transplant in CR1 with positive MRD after induction only (MRD positive induction only) (n=15), patients who underwent transplant in CR1 with positive MRD after induction and additional therapy (MRD positive induction plus) (n=19), and patients undergoing transplant in CR1 with no MRD (MRD negative) (n=37). Patient details are summarized in Table 1. Results: CI of relapse was higher among MRD positive induction plus patients than MRD positive induction only patients (p=0.042, HR 0.301 (0.094-0.96)) and comparable among MRD positive induction only patients and MRD negative patients (p=0.987, HR 1.011 (0.29-3.58)). CI of transplant related mortality (TRM) was comparable between MRD positive induction plus patients and MRD positive induction only patients (p=0.165, HR 0.23 (95% CI 0.028-1.81)) and comparable among MRD positive induction only patients and MRD negative patients (p=0.871, HR 1.22 (95% CI 0.12-12.87)). Relapse free survival (RFS) and overall survival (OS) were comparable between MRD positive induction only patients and MRD negative patients and significantly better than for MRD positive induction plus patients (RFS (p<0.0001) and OS (p=0.0008)). (Figure 1) On multivariate analysis including MRD positive induction only, MRD positive induction plus, MRD negative, patient age, Sorror comorbidity index, donor source, conditioning regimen, use of adjunctive therapy, and ELN AML risk status, MRD positive induction plus was associated with a significantly higher risk of relapse than MRD positive induction only (p=0.014, HR 0.231 (95% CI 0.072-0.742)). No other factors were statistically significant. Because multiple strategies were used to assess for MRD, we compared MRD positive induction only to MRD positive induction plus patients using flow cytometry performed by Hematologics Inc as the only MRD assessment technique. CI of relapse trended toward lower in the MRD positive induction only group (p=0.10, HR 0.22, (95% CI 0.036-1.34). OS and RFS were significantly improved in the MRD positive induction only patients (p=0.027 and p=0.026 respectively). Conclusions: For patients achieving CR following induction and moving directly to transplant in spite of MRD positivity, outcomes were comparable to patients going to transplant in an MRD negative state and were significantly improved compared to outcomes of patients in an MRD positive state who received additional therapy following induction. Our series is small, and multiple MRD monitoring strategies were used. However, given the paucity of data on this specific question, uncertainty about whether MRD will clear with additional cytotoxic therapy following induction, and the poor prognosis of patients with persistent MRD in the induction plus group, we consider transplant following induction reasonable in this population regardless of MRD status. Larger series are necessary to more definitively answer this question. Disclosures Loken: Hematologics, Inc: Employment, Equity Ownership. Pollyea:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Diachii Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forty-Seven: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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