scholarly journals ATRA Could Correct the Defective S1P-Mediated Cytoskeletal Reorganization in Proplatelet Formation of ITP

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 218-218 ◽  
Author(s):  
Qiu-Sha Huang ◽  
Jing Xue ◽  
Chen-Cong Wang ◽  
Ya-Zhen Qin ◽  
Lan-Ping Xu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rho Gtpases ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Therapeutic Effects ◽  
Cytoskeletal Reorganization ◽  
Mice Model ◽  
Mrna Sequencing ◽  
Healthy Donors ◽  
Proplatelet Formation

Introduction Sphingosine-1-phosphate (S1P) is now emerging as a vital lipid mediator. Activation of sphingosine kinase (SphK) produces intracellular S1P, which in turn can be secreted out of the cell and act extracellularly by binding to S1P receptors (S1PR). Recent studies suggest that the "inside-out" signaling by S1P in megakaryocytes (MKs) plays a critical role in proplatelet formation (PPF) (Blood, 2013; J EXP MED, 2012). PPF requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases which can be activated by S1P, including Rac1 and Cdc42, have been shown to be master regulators of cytoskeletal rearrangements. The pathogenesis mechanisms of immune thrombocytopenia (ITP) are not entirely understood. Our previous data indicated that impaired PPF contributed to the development of thrombocytopenia in ITP. To further explore the underlying mechanism of impaired PPF in ITP, we found that S1P-mediated microtubule reorganization is defective in PPF of ITP. All-trans retinoic acid (ATRA), which has demonstrated to be a promising option for ITP patients in our previous study (Lancet Haematology, 2017), could correct the altered microtubule reorganization and promote PPF. Methods Thirty consecutive patients with primary ITP and 20 healthy donors were enrolled in our study. MKs were isolated from bone marrow samples, and they were collected again after ITP patients received ATRA therapy. MK mRNA sequencing by microarray was used to assess the difference of gene expression between ITP and controls. Microtubule regrowth assay was performed to observe microtubule dynamic behavior. In this assay nocodazole was first used to induce complete depolymerization of microtubule network, followed by drug washout to allow microtubule regrowth over time. ATRA was added to the culture medium of MKs to determine the mechanism of ATRA in correcting impaired PPF. Additionally, ITP mice model was established to observe the therapeutic effects of ATRA in PPF. Pf4-Cre/loxP system was used to specifically knock down gene of MKs. Results S1P concentration in bone marrow from ITP patients was lower compared to healthy donors. MKs mRNA sequencing demonstrated that S1P synthetase SphK2 and S1P receptor S1PR1 gene were downregulated while S1P lyase (SPL) gene was upregulated in ITP patients, which caused abnormal S1P signaling. Furthermore, we observed that PPF capacity of MKs in patients with ITP was reduced. Pharmacological disruption of S1PR1 blocked PPF, exogenous S1P corrected impaired PPF. Collectively, deregulation of S1P signaling was associated with impaired PPF in ITP. To verify the downstream role of S1P in regulating PPF, the Rho GTPases detection of MKs revealed a decrease in Cdc42 and Rac1 levels from ITP patients. Immunofluorescence of the differentiated MKs showed that the expression and distribution of β1 tubulin were abnormal from ITP patients. Early PPs from MKs of healthy donors displayed a well-organized tubulin bundles resembling bunches of grapes. In contrast, in MKs from ITP patients, tubulin was disorganized in thick bundles. In addition, TEM analysis of the MKs showed an irregular distribution of granules, tortuous membranes and impaired proplatelet structure. In microtubule regrowth assay, MKs from ITP patients had significantly lower microtubule regrowth at 10 min post-nocodazole washout compared with controls. Together, microtubule alteration resulted in impaired PPF in ITP. We tested whether S1P pathway were required for microtubule reorganization, both SphK2-/- and S1PR1-/- mice displayed significantly reduced S1P, Cdc42 and Rac1, altered microtubule architecture and defective PPF. Taken together, abnormal S1P pathway accounted for impaired microtubule reorganization in ITP. Next, we explored the effect of ATRA on microtubules reorganization in ITP patients, our data showed that in vitro treatment with ATRA restored microtubules structure by upregulating S1P and activating Rho GTPases. In vivo studies showed that ARTA could rescue the impaired PPF in both patients and mice model with ITP. Conclusions The MKs of ITP patients displayed defective cytoskeletal reorganization regulated by S1P pathway. ATRA restored cytoskeletal structure and corrected impaired PPF by upregulating S1P and activating Rho GTPases. It sheds light on a novel mechanism of ITP pathogenesis and provides a basis for the therapeutic potential of ARTA in ITP patients. Disclosures No relevant conflicts of interest to declare.

Serotonin and Its Metabolism Enhances Megakaryopoiesis and Proplatelet Formation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4753-4753
Author(s):  
Jie yu Ye ◽  
Fan yi Meng ◽  
Qianli Jiang ◽  
Su yi Li ◽  
En yu Liang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Signal Pathway ◽  
Mice Model ◽  
Actin Reorganization ◽  
Cytoskeleton Reorganization ◽  
Colony Forming Units ◽  
Proplatelet Formation ◽  
5Ht2 Receptor ◽  
Receptor Inhibitor

Serotonin (5-HT) has been recently identified as a novel growth factor. We previously demonstrated that 5-HT enhances murine megakaryopoiesis via 5-HT2 receptors and has promotor effect on hematopoiesis(Yang M et al, Stem Cells, 2007). However, the molecular mechanism remains under explored. In the terminal stage of mammalian megakaryocyte development, platelets are released from proplatelet protruding from megakaryocytes via cytoskeleton reorganization. 5-HT is shown to modulate cell migration and remolding by activating cytoskeleton reorganization, but the effects of 5-HT on proplatelet formation have not been investigated. Our results showed that 5-HT significantly promoted human CFU-MK formation and reduced apoptosis on human megakaryocytes through phosphorylation of Akt. These effects were attenuated by addition of ketanserin, a 5HT2 receptor inhibitor. 5-HT also stimulated proplatelet formation through activating the p-Erk1/2 expression and F-actin reorganization. Melatonin, the metabolism of 5-HT, promoted the recovery of platelets and the formation of bone marrow colony forming units in irradiated mice. Our findings suggested that 5-HT and melatonin plays an important role in human megakaryopoiesis. Interaction of 5-HT and 5-HTR2B induced downstream activation of PI3-k/Akt signal pathway leading to human MK cell proliferation. In addition, activation of 5-HTR2B also induced Erk1/2 phosphorylation, which then promoted cytoskeleton reorganization and subsequent proplatelet formation. We also proved that melatonin exerts a protective effect on MK and platelets in the irradiation mice model. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Role of 5-HT on Proplatelet Formation and Thrombopoietin Production

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 14-14
Author(s):  
Mo Yang ◽  
Enyu Liang ◽  
Jieyu Ye ◽  
Beng H Chong ◽  
Liang Li
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Traumatic Stress ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Mice Model ◽  
Synthesis Inhibitor ◽  
Rhodamine Phalloidin ◽  
Proplatelet Formation ◽  
Group 5

Background: Our previous work confirmed that serotonin (5-HT) promotes the proliferation of hemopoietic stem cells and megakaryocytes (Yang M et al, Stem Cells, 2007; 2014). However, the mechanisms remain indefinite. Methods: Q-PCR, Flow Cytometry, Western Blot, or Immunofluorescence microscope were used in the receptor and TPO study. MTT/CCK-8, Proplatelet assay, and Flow Cytometry were also used in cell proliferation and apoptosis study. The relationship between 5-HT and TPO was studied in a traumatic stress mice model. Results: In-vitro study, there was a stimulating effect of 5-HT on proplatelet formation in human bone marrow megakaryocytes. Human BM MK progenitors cultured in serum-free medium with either 5-HT (200nM) or TPO (100 ng/ml) had more proplatelet bearing MKs than the control group (5-HT (12.3 ± 5.0)% vs. Control (6.2 ± 3.5)%, P=0.025; TPO (15.6 ± 2.5)% vs. Control, P=0.04; n=4). The 5-HT treatment group showed more mature and more in the final stage MK cells as compared to the TPO group. 5-HT2A, 2B, 2C receptors were detected in the surface of megakaryocytes. The effect of 5-HT on proplatelet formation in MK cells was via 5-HT2 receptors and this effect was reduced by 5-HT2 receptor inhibitor ketanserin. 5-HT acted on cytoskeleton reorganization in MKs via 5-HT2 receptors and ERK1/2 pathway. Using an immunofluorescence microscope with F-actin specific binder rhodamine-phalloidin staining, the polymerized actin level was lower in the control group than the 5-HT group and actin distributed diffusely throughout the cytoplasm. In contrast, the polymerization actin level was higher in the 5-HT group. Adding ketanserin and ERK1/2 inhibitor PD98059 to 5-HT treatment, the fluorescence intensity was correspondingly reduced. Our data also demonstrated that ERK1/2 was activated in MKs treated with 5-HT for 30 minutes. In a traumatic stress mice model, both of 5-HT and TPO were increased, but the increasing of TPO is posterior to 5-HT. After added LX1606, the synthesis inhibitor of 5-HT, 5-HT was reduced markedly, as well as TPO. The expression of TPO mRNA and the production of TPO protein were increased as compared with the control in this model. Conclusions: This study suggests that 5-HT promotes thrombopoiesis from two aspects: one is the direct effect on megakaryocytes. 5-HT could promote the proplatelet formation from megakaryocytes. The second is the indirect effect by promoting the production of TPO, which is a paracrine secretion to influence thrombopoiesis. Disclosures No relevant conflicts of interest to declare.

Mesenchimal Stroma Cells From Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia Show Distinct Cytogenetic and DNA-Mutation Data as Compared with Bone Marrow Leukemic Blasts.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4697-4697
Author(s):  
Olga Blau ◽  
Wolf-Karsten Hofmann ◽  
Claudia D Baldus ◽  
Gundula Thiel ◽  
Florian Nolte ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Control Analysis ◽  
Npm1 Mutation ◽  
Dna Mutation ◽  
Healthy Donors ◽  
Acute Myeloid

Abstract Abstract 4697 Bone marrow mesenchymal stroma cells (BMSC) are key components of the hematopoietic microenvironment. BMSC from patients with acute myeloid leukemia (AML) and myelodisplasic syndrome (MDS) display functional and quantitative alterations. To gain insight into these questions, we carried out cytogenetic analyses, FISH, FLT3 and NPM1 mutation examinations of both hematopoietic (HC) and BMSC derived from 53 AML and 54 MDS patients and 35 healthy donors after in vitro culture expansion. Clonal chromosomal aberrations were detectable in BMSC of 12% of patients. Using FISH we have assume that cytogenetic markers in BMSC were always distinct as the aberrations in HC from the same individual. 17% and 12% of AML patients showed FLT3 and NPM1 mutations in HC, respectively. In BMSC, we could not detect mutations of NPM1 and FLT3, independent from the mutation status of HC. For control analysis, BMSC cultures from 35 healthy donors were prepared under the same conditions. BMSC from healthy donors did show normal diploid karyotypes and absence of specific DNA-mutations of NPM1 and FLT3. Our data indicate that BMSC from MDS and AML patients are not a part of malignant clone and characterized by genetic aberrations. Lack of aberrations as detected in HC and appearance of novel clonal rearrangements in BMSC may suggest enhanced genetic susceptibility and potential involvement of BMSC in the pathogenesis of MDS and AML. Disclosures: No relevant conflicts of interest to declare.

Preliminary Study of the Autoantigens on the Membrane of Erythropoietic Cells of the Patients with BMMNC- Coomb's Test(+) Hemocytopenia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4435-4435
Author(s):  
Rong Fu ◽  
Hui Liu ◽  
Zonghong Shao ◽  
Jun Wang ◽  
Lijuan Li
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Bone Marrow Mononuclear Cell ◽  
Rt Pcr ◽  
Glycine Buffer ◽  
Healthy Donors ◽  
Preliminary Study ◽  
The Relationship ◽  
Epor Expression ◽  
Normal Controls

Abstract Abstract 4435 Objective To observe the relationship between EPO receptor(EPOR) and autoantibodies-IgG/IgM (auto-Ab) on the membrane of erythropoietic cells of the patients with bone marrow mononuclear cell Coomb's (BMMNC-Coomb's) test(+) immuno-related pancytopenia(IRP), and then explore the probable autoantigens of auto-Ab in IRP. Methods 46 newly diagnosed IRP patients (15 with auto-Ab on erythropoietic cells and 31 without auto-Ab on erythropoietic cells) and 18 healthy donors as controls were enrolled in this study. EPOR expression on their nuclear erythrocytes were tested with flow cytometry to observe the relationship between EPOR and auto-Ab; After sorting erythropoietic cells in bone marrow, EPOR mRNA and protein Stat5,P-Stat5 were investigated by RT-PCR and Western blot to observe the production of EPOR and EPO/EPOR signal transduction; Finally, EPOR expression on the membrane were tested again after stripping auto-Ab with glycine buffer. Results (1) EPOR of auto-Ab(+) arm(1.59±0.87)% was significantly lower than that of auto-Ab (-) arm(4.58±4.09)%(P<0.01), and the latter was significantly higher than that of normal controls (2.27±1.76)%(P<0.05); EPOR of IRP patients was negatively correlated with their auto-Ab (r=-0.543,P=0.000) and its regression equation was Y(EPOR)=0.040-0.335X(auto-Ab);(2)EPOR mRNA of auto-Ab(+) arm(0.685±0.136)was significantly higher than that of auto-Ab (-) arm(0.554±0.116)(P<0.01)and normal controls (0.580±0.119)(P<0.05);(3)Protein Stat5 of auto-Ab(+) arm(1.45±0.94) was significantly higher than that of normal controls (0.54±0.36)(P<0.05); While P-Stat5 of auto-Ab(+) arm(0.42±0.18) was significantly lower than that of normal controls (0.85±0.38)(P<0.05); (4) EPOR expression became higher while auto-Ab became lower after stripping with glycine buffer. Conclusion The auto-Ab of some IRP patients might block or competitively inhibit the EPOR on the membrane of erythropoietic cells. EPOR was one of autoantigens in IRP. Disclosures: No relevant conflicts of interest to declare.

Failure of Erythropoiesis and Megakaryocytopoiesis in RASA3 Mutant Scat Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 680-680
Author(s):  
Lionel Blanc ◽  
Babette Gwynn ◽  
Steven L. Ciciotte ◽  
Luanne L. Peters
Keyword(s):  
Bone Marrow ◽  
Positional Cloning ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Inbred Mouse Strain ◽  
Recessive Mutation ◽  
Initial Increase ◽  
Severe Anemia ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract Abstract 680 Scat (severe combined anemia and thrombocytopenia) is a spontaneous, autosomal recessive mutation coisogenic with the BALB/cBy inbred mouse strain. Homozygous scat mice present a cyclic phenotype with alternating episodes of crisis and remission. As its name implies, crisis episodes are characterized by severe anemia and thrombocytopenia, but significant lymphocyte depletion occurs as well. The first crisis episode begins in utero, lasts until postnatal day (P) 9 on average, and is associated with 10–15% mortality. Remarkably, in homozygotes that survive the first crisis, a remission phase occurs wherein the disease phenotype reverts to normal. This remission is transient, however, and is followed by a second crisis episode during which 94% of scat/scat mice die by P30. Previously we showed that the scat phenotype is transferrable via the hematopoietic stem cells and is also recapitulated in scat/scat, Hox11−/− double homozygotes in which a spleen does not develop, indicating that the splenic micro-environment plays little or no role in disease appearance or progression. Positional cloning of scat revealed a missense mutation in Rasa3 encoding a GTPase activating protein (GAP) that negatively regulates Ras function by accelerating GTP hydrolysis and converting Ras to the inactive GDP bound form. We further showed that Rasa3 is a conserved gene critical to vertebrate erythropoiesis via morpholino knockdowns in zebrafish which resulted in profound anemia. Here we report data that shed further light on RASA3 function during hematopoiesis. Overall, the data indicate that defects in RASA3 profoundly and negatively impact erythropoiesis and megakaryocytopoieis through, at least in part, a Ras-mediated mechanism. FACS analyses of scat spleen and bone marrow erythroid populations reveal a severe block in erythropoiesis during crisis periods. In the spleen, despite an initial increase in size due to expansion of Ter-119+ cells, there is ultimately a loss of compensatory erythropoiesis resulting in a return to normal cellularity and a striking loss of hemoglobinized cells as the crisis phenotype deepens. In addition, the bone marrow shows loss of Ter-119+ cells and overall cell depletion during crisis. Megakaryocyte numbers are increased in scat crisis BM and spleen. By transmission electron microscopy, scat crisis megakaryocytes display features characteristic of a significant developmental delay: a disorganized demarcation membrane system with no platelet forming areas and few granules with hypersegmented nuclei and excess rough endoplasmic reticulum. In addition to the severe anemia and thrombocytopenia, a significant lymphopenia occurs in scat crisis mice. However, the scat phenotype is not lymphocyte mediated, as the scat phenotype is completely recapitulated in mice doubly homozygous for scat and the immunodeficient mutations, scid and Rag1tm1Mom, in which B- and T-lymphocytes are completely depleted. Together these results suggest that lymphopenia is a secondary phenomenon in scat, and the severe anemia and thrombocytopenia aspect of the phenotype neither follows from nor is dependent upon loss of lymphocytes. Despite the delay observed in erythroid differentiation, some mature red cells are produced although ∼50% of these are reticulocytes. By confocal microscopy, we show that RASA3 protein localizes to the plasma membrane as well as internal membrane compartments in wild type reticulocytes, where it partially colocalizes with CD71. Western blot analyses of reticulocytes after Percoll gradient purification show that RASA3 is lost during the maturation step, both in vivo and in vitro. Interestingly, in scat, RASA3 is present in reticulocytes, but appears to be mislocalized, the protein being found in the cytosol. Preparation of ghosts from wild type and scat reticulocytes confirms that RASA3 is not attached to the membrane in scat animals during crisis. In pull-down assays active GTP-bound Ras is increased in scat crisis reticulocytes when compared to wild type, suggesting that scat is a RASA3 loss of function mutation due to its mislocalization and demonstrating a critical role for the RASA3-Ras axis during mammalian erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

Role of PDGF/PDGFR in Regulation of Thrombopoiesis: A Possible Explanation for Imatinib Mesylate-Induced Thrombocytopenia in the Treatment of CML

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3348-3348
Author(s):  
Mo Yang ◽  
Fanyi Meng ◽  
Jie yu Ye ◽  
Yue Xu ◽  
Bin Xiao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Imatinib Mesylate ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Potential Effect ◽  
Mice Model ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Bone Marrow Histology

Abstract Abstract 3348 Platelet-derived growth factor (PDGF), a platelet alpha-granule molecule, imply their potential effect in the regulation of megakaryocytopoiesis and thrombopoiesis, which also intimates the existence of an autocrine and/or paracrine loop constructed by megakaryocytes/platelets and their granular constituents. Our previous studies demonstrated the presence of functional PDGF receptors (PDGFR) on human megakaryocytes and platelets (Yang et al, Thromb Haemastasis, 1997) and CD34+ cells, and their ability to mediate a mitogenic response. PDGF promoted the ex vivo expansion of human hematopoietic stem (CD34+) and progenitor (CD41+ CD61+) cells. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in NOD/SCID mice. PDGF stimulated in vitro megakaryocytopoiesis via PDGFR and/or the indirect effect on bone marrow microenvironment to produce TPO and other cytokines. It also showed a direct stimulatory effect of PDGF on c-Fos, GATA-1 and NF-E2 expressions in megakaryocytes. We speculate that these transcription factors might be involved in the signal transduction of PDGF on the regulation of megakaryocytopoiesis. PDGF also enhanced platelet recovery in mice model with radiation-induced thrombocytopenia. Studies showed that PDGF, like thrombopoietin (TPO), significantly promoted platelet recovery and the formation of bone marrow colony-forming unit-megakaryocyte (CFU-MK) in this irradiated-mouse. An increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis were found in the bone marrow histology sections. In the M-07e apoptotic model, PDGF had a similar anti-apoptotic effect as TPO on megakaryocytes. We also demonstrated that PDGF activated the PI3k/Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression. Our findings suggested that the PDGF-initiated radioprotective effect is likely to be mediated via PDGF receptors with subsequent activation of the PI3k/Akt pathway. The study provides a possible explanation that blockage of PDGFR may reduce thrombopoiesis and play a role in imatinib mesylate-induced thrombocytopenia in the treatment of CML. Disclosures: No relevant conflicts of interest to declare.

Recipient CD8+ DC Delete Alloreactive Donor CTL and Promote Leukemic Relapse after Allogeneic BMT

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4279-4279
Author(s):  
Kate A Markey ◽  
Rachel D Kuns ◽  
Renee J Robb ◽  
Motoko Koyama ◽  
Kate Helen Gartlan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Antigen Presentation ◽  
Median Survival ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Leukemic Relapse

Abstract Allogeneic bone marrow transplantation (BMT) remains the therapy of choice for many haematological malignancies, but despite the curative benefit of the immunological graft-versus-leukemia (GVL) effect, relapse remains a key cause of death. We have investigated the role of recipient dendritic cells (DC) in antigen presentation to donor CD8 cytotoxic T cells (CTL) in a model of BMT where GVHD and GVL are directed to multiple minor histocompatibility antigens (mHA) and survival reflects GVL activity. C3H.Sw bone marrow and purified CD8 T cell grafts were transplanted with B6-derived MLL-AF9 induced primary acute myeloid leukemia (AML) into lethally irradiated B6.CD11c.DOG recipients (diphtheria toxin receptor (DTR), ovalbumin and GFP expression driven off the CD11c promoter) such that recipient DC can be deleted by DT administration. Surprisingly, depletion of recipient DC resulted in improved leukemic control (median survival 43 vs 31 days, P <0.001). The use of IRF8-/- BMT recipients (in which the CD8+ DC subset is absent) confirmed that recipient CD8+ DC were critical for regulating these GVL effects (median survival 43 vs 34 days, P = 0.0005). Conversely, when recipient CD8+ DC were expanded in a B6 to B6D2F1 model with bcr-abl/Nup98-HoxA9 induced primary AML, by using Flt3-L treatment for 10 days prior to BMT, GVL effects were completely eliminated, rendering relapse rate equivalent to that seen in the recipients of T cell depleted (TCD) grafts (median survival 11 days in BM+T and TCD groups where recipients were pre-treated with Flt3-L, vs. >45 days in the saline treated BM+T group). The use of B6.CD11c-Rac1 transgenic BMT recipients (who cannot process and present exogenously acquired antigen) confirmed that this effect was the result of endogenous alloantigen presentation by recipient DC and independent of cross-presentation.Using the same depletion strategies in an antigen-specific model (with donor OT-I T cells and B6.CD11c.DOG x DBA/2 F1 recipients) we confirmed that recipient DC invoked effector donor CTL activation, differentiation (CD25+ CD69+ CD62L-) and subsequent apoptosis (as measured by Annexin V; 52.4% vs. 23.9% in DC replete vs. depleted recipients, P = 0.01). There was a consequent profound contraction of the donor CTL compartment by day 10 in DC replete recipients. This contraction of the CTL compartment was associated with reduced expression of the cytolytic molecule granzyme B (MFI 1922 vs 1097, P = 0.02). Antigen presentation has a critical role in the initiation of donor T cell alloreactivity and GVL after BMT. Here we demonstrate that endogenous alloantigen presentation by recipient CD8+ DC to donor T cells leads to activation induced death of donor CTL early after BMT, which in turn facilitates leukemic relapse. This concept has critical implications for the design of therapies that target DC in the peri-transplant period and confirms that recipient DC regulate GVL effects. Disclosures No relevant conflicts of interest to declare.

scholarly journals CIC-Mutation As a Potential Molecular Mechanism of Acquired Resistance to Combined BRAF/MEK Inhibition in CNS Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3181-3181 ◽  
Author(s):  
Matteo Claudio Da Via' ◽  
Antonio Giovanni Solimando ◽  
Andoni Garitano-Trojaola ◽  
Santiago Barrio ◽  
Nadine Rodhes ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Exome Sequencing ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Point Mutations ◽  
Sequencing Depth ◽  
Resistance Development ◽  
Average Sequencing Depth

Abstract Central nervous system (CNS) involvement is an extremely rare extramedullary multiple myeloma (MM) manifestation, diagnosed in less than 1% of patients. It is considered an ultimate high-risk feature, associated with unfavorable cytogenetics, and, even with intense treatment applied, survival is short, reaching less than 12 months in most cases. In June 2017 an 81 years old male with a κ light chain MM was referred to our institution for an isolated CNS MM relapse. His cerebrospinal fluid (CSF) demonstrated a high load of clonal plasma cells, however, the patient's bone marrow infiltration was very little with a percentage of plasma cells less than 5%. Imaging, including gold standard MRI and experimental 11C-methionine PET scan, was performed, and high metabolic activity was detected supra- and infratentorially as well as in the right femur and the clivus. Following CD138+ cell purification we analyzed the specimen with M3P (v3.0) a disease specific in-house customized, next generation targeted sequencing panel for MM (Ion torrent platform). This includes most commonly mutated MM genes, actionable drug targets and drug resistance associated genes. The average sequencing depth increased 700X and spatial MM heterogeneity was detected, as the CFS cells harbored a clonal BRAFV600E mutation, absent in the bone marrow. Initial intrathecal and systemic chemotherapy with Cytarabine and Thiotepa was intolerable, thus the patient underwent a combined target inhibition with Dabrafenib/Trametinib, well known specific BRAF and a MEK 1/2 inhibitors. The patient displayed a rapid complete response (Figure. 1A), however, disease relapse occurred after three months of therapy. We obtained a sequential CFS sample and Whole Exome Sequencing (Illumina platform) was applied to pre and post therapy CFS sampling. Exome sequencing of the two time points performed an average sequencing depth of 115X; a total number of 97 non-silent coding variants (missense, nonsense, indels, splice) with an allele frequency higher than 5% were detected. In detail, 19 point mutations were acquired at relapse, including a subclonal missense mutation in CIC (p.A984P, VRF 17%), recently identified as a candidate gene contributing to MEK/BRAF resistance development. Next, we established a CIC knock-down model electroporating a specific anti-CIC siRNA into U266 MM cell line. We cultured the silenced and not-silenced cells with Trametinib and Dabrafenib, either as single agents, or in combination. As expected, we observed resistance induction to the combination of the two drugs (Row Factor 85.94%; P<0.0001, Two-way ANOVA) suggesting a critical role for this patient derived mutation for his MEK/BRAF resistance development (Figure 1C, D). In order to better clarify the landscape pathway related to CIC we analyzed expression data from 647 patients enrolled in the MMRF CoMMpass trial. Remarkably, we found a significant down-regulation of ERF and ETV6 (t-test -9.95, -9.93, P <0.001, respectively), two well characterized tumor suppressor genes correlated with the re-activation of the RAS downstream pathway (Figure 1B). This is the first report giving evidence for a potential role of point mutations in CIC as a resistance mechanism to targeted MEK/BRAF inhibition in BRAF mutated MM. The performed pathway analysis significantly extends the insights of the resistance mechanisms highlighted. Our results foster a statistically powered study to corroborate the clinical relevance. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Endogenous TGF-beta1 Mediated Osteogenic Impairment of Medullar Mesenchymal Stromal Cells in Primary Myelofibrosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1873-1873
Author(s):  
Christophe Martinaud ◽  
Christophe Desterke ◽  
Johanna Konopacki ◽  
Lisa Pieri ◽  
Rachel Golub ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Hematopoietic Cells ◽  
Primary Myelofibrosis ◽  
Osteogenic Potential ◽  
Healthy Donors

Abstract Primary myelofibrosis (PMF) is myeloproliferative neoplasm characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in bone marrow and spleen. Mesenchymal stromal cells (MSCs) are reported to play a pivotal role in fibrosis and stromal changes are considered as a reactive counterpart of the cytokine production by clonal hematopoietic cells. The present study shows that MSCs from patients demonstrate functional abnormalities that are unexpectedly maintained ex-vivo, in culture. Material and Methods: we studied MSCs and bone marrow sections from PMF patients (n=12) as compared to healthy donors (HDs) (n=6). We tested their proliferation, immunophenotype, hematopoiesis supporting capacities, differentiation abilities, in-vivo osteogenic assays, and performed secretome and transcriptome analysis. Results: We found that PMF-MSCs exhibit similar proliferative capacity and long-term hematopoiesis supporting abilities as compare to healthy donors. They overproduce interleukin 6, VEGF, RANTES, PDGF, BMP-2 and surprisingly TGF-beta1. MSCs from fibrotic PMF patients express high levels of glycosaminoglycans. Adipocytes and chondrocytes differentiation abilities were not different as compared to HDs but PMF-MSCs exhibit an increased in vitro potential. Implementation on scaffold in nude mice confirmed, in vivo, this increased osteogenic potential. We then looked into gene expression and discovered that PMF-MSCs show an original transcriptome signature related to osteogenic lineage and TGF-beta1. Indeed, osteogenic genes such as Runx2, Dlx5, Twist1, Noggin, Sclerostin, GDF5 and Serpine1 are deregulated and suggest a potential osteoprogenitor priming of PMF-MSCs. These molecular results also advocated for a TGF-beta1 impregnation that prompted us to study its impact on PMF-MSCs osteogenic differentiation. First, we then showed that Smad2 is intrinsically over-activated in PMF-MSC and that stimulation by TGF-beta1 is associated with an increase phospho-Smad2 level and an enhancement of bone master gene regulator Runx2 expression. Then, we inhibited TGF-beta1 pathway by by SB-431542 and evidenced a specific behavior of osteogenic MSCs differentiation in patients, suggesting involvement of TGF-beta1 in osteogenic impairment. Conclusion: Altogether, our results identify a signature of PMF-MSCs and suggest that they participate in PMF osteogenic dysregulation independently from in vivo local stimulation by clonal hematopoietic cells Disclosures No relevant conflicts of interest to declare.

Secretome Analyses of Primary Bone Marrow Fibroblasts Isolated From MGUS and Multiple Myeloma Show a Stepwise Occurrence of Alterations.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1801-1801
Author(s):  
Johannes Drach ◽  
Astrid Slany ◽  
Thomas Mohr ◽  
Johannes Griss ◽  
Christoph C Zielinski ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Growth Factor ◽  
Plasma Cell ◽  
Conflicts Of Interest ◽  
Cell Clone ◽  
Serum Levels ◽  
Plasma Cell Dyscrasia ◽  
Healthy Donors ◽  
Bone Marrow Fibroblasts

Abstract Abstract 1801 Poster Board I-827 The microenvironment of tumor cells in the bone marrow was demonstrated to contribute to tumor promotion and survival. The role of bone marrow fibroblasts (BMFs) in supporting the malignant plasma cell clone in multiple myeloma (MM) has been established, but it remains unclear to which extent the BM microenvironment in general and BMFs in particular are involved in the progression of monoclonal gammopathy of undetermined significance (MGUS) to MM. Therefore we performed proteomics studies on the secretome of BMFs isolated from healthy donors, patients suffering from MGUS and patients suffering from MM. Compared to normal background, BMFs derived from MGUS secreted elevated levels of proteins indicating mitogenic activity and moderate inflammation. These proteins included periostin, IL-6, CXCL5 and CSF-1. Insulin-like growth factor II, which is normally not expressed by normal BMFs, was secreted by BMF cells derived from MGUS as well as from MM. In addition to those and other proteins, BMF cells derived from MM were found to specifically secrete stem cell growth factor, MMP-28 and stanniocalcin-1. These data indicate a step-wise alteration of BMF secretion activity related to the stage of the underlying plasma cell dyscrasia. Therefore BMF might support the progression from MGUS to MM. In order to correlate the secretion performance of BMF with blood serum levels of candidate marker proteins, Luminex assays are employed. Based upon these results, it is our aim to identify serum biomarkers which allow to assess the functional state of BMF and thus the risk for the progression of MGUS to MM. Disclosures No relevant conflicts of interest to declare.

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