Association of Functional Single Nucleotide Variation in the NLRP3 Gene with Survival Outcomes After Unrelated Bone Marrow Transplantation.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3140-3140
Author(s):  
Akiyoshi Takami ◽  
J. Luis Espinoza ◽  
Keitaro Matsuo ◽  
Yasuo Morishima ◽  
Makoto Onizuka ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Nucleotide Variation ◽  
Single Nucleotide Variation ◽  
Single Nucleotide ◽  
Hematopoietic Stem ◽  
Nlrp3 Gene ◽  
Transplant Outcomes ◽  
Functional Relevance ◽  
The Impact

Abstract Abstract 3140 NLRP3 is an intracellular trigger of IL-1β production that plays important roles in the regulation of inflammation and apoptosis. A single nucleotide variation in the 3'-untranslated region of the NLRP3 gene, rs10754558 (+29940G>C), is linked to several immunological diseases. When we examined the impact of the NLRP3 genotype in a cohort consisting of 392 pairs of patients with hematologic malignancies and their unrelated HLA 12/12 matched bone marrow donors transplanted through the Japan Donor Marrow Program, the recipient NLRP3 GG genotype was found to be associated with a significantly worse 5-year overall survival (OS) rate (34% vs. 50%, P=0.006) (Fig. 1) and a trend toward a higher transplant-related mortality (TRM) rate (39% vs. 27%, P=0.09) than the recipient CC or CG genotype. The recipient GG genotype remained statistically significant in the multivariate analysis for OS (hazard ratio [HR], 1.86; 95% confidence interval [CI], 1.22 to 2.22; P=0.004) and TRM (HR, 2.28; 95% CI, 1.20 to 4.35; P=0.01). The donor NLRP3 genotype did not significantly influence the transplant outcomes. Next, we investigated the functional relevance of the NLRP3 +29940G>C variant. When leukocytes from healthy individuals were stimulated in vitro with NLRP3 ligand, the leukocytes with the NLRP3 GG genotype produced significantly more IL-1β than those with the NLRP3 CC or CG genotype (Fig. 2). These findings substantiate the functional relevance of the NLRP3 variant, and suggest that the higher IL-1β secretion in the peri-transplant period by recipients with the NLRP3 GG genotype likely accounts for their poor transplant outcomes. NLRP3 genotyping could therefore be useful in predicting prognoses and creating therapeutic strategies for improving the final outcomes of patients who undergo allogeneic hematopoietic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Effects of G-CSF On Acute Lymphoblastic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2564-2564
Author(s):  
Jordan Basnett ◽  
Adam Cisterne ◽  
Kenneth F Bradstock ◽  
Linda J Bendall
Keyword(s):  
Bone Marrow ◽  
Disease Progression ◽  
Microarray Analysis ◽  
Environmental Changes ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Stem ◽  
Childhood All ◽  
Extracellular Matrix Components ◽  
The Impact

Abstract Abstract 2564 G-CSF is commonly used to treat chemotherapy-induced neutropenia and for the mobilization of hematopoietic stem cells for transplantation in patients with leukemia. Administration of G-CSF has profound effects on the bone marrow microenvironment including the cleavage of molecules required for the maintenance of lymphopoiesis, including CXCL12 and VLA-4. We have recently reported that G-CSF results in the dramatic suppression of B-lymphopoiesis. This, together with previous reports by ourselves, and others, showing that disruption of CXCL12 or VLA-4 slow the progression of B-lineage ALL lead us to consider that G-CSF may similarly antagonize the progression of ALL. To explore this possibility, we examined the impact of G-CSF administration on six human ALL xenografts using a NOD/SCID mouse model. Mice were engrafted without radiation and G-CSF commenced when 1% of the bone marrow consisted of ALL cells. G-CSF was administered twice daily for 10 days, at which time all animals were culled and leukemia assessed in the blood, bone marrow and spleens. Surprisingly G-CSF was found to increase disease progression in two of xenografts investigated (1345 and 0398, referred to as G-CSF responsive xenografts hereafter), while the remainder demonstrated a small reduction in leukemia, with one showing a statistical significant decrease. No evidence for a direct mitogenic effect of G-CSF could be demonstrated in any of the xenografts using exogenous G-CSF in vitro cultures in the presence or absence of human or murine stromal support. Consistent with these findings, and previous reports, little to no G-CSF receptor was detected by flow cytometry or microarray analysis of xenografts. Microarray analysis of the xenografts revealed significant differences in gene expression between the G-CSF responsive xenografts and the remainder of the samples. A total of 83 genes were expressed at a higher level and 127 genes at a lower level in the G-CSF responsive xenografts. The more highly expressed genes included cell cycle regulators (eg cyclin A1), adhesion molecules (eg ALCAM), extracellular matrix components and surface receptors. Perhaps the most interesting was the exclusive expression of the acetylcholine receptor (cholinergic receptor, nicotinic, beta 4, nAChRb4) in the G-CSF responsive cases. Analysis of a large public dataset of childhood ALL samples revealed significantly higher expression of this gene in ALL samples with rearranged MLL (p<0.03). However, small numbers of cases in all ALL subgroups had greater than an 2 fold higher expression compared to normal B cell progenitors. The role of nAChR in the response of ALL cells to micro-environmental changes induced by G-CSF remains to be determined, however, nAChR has known roles in cell proliferation and inhibition of apoptosis. Furthermore G-CSF is known to induce acetylcholine production in other tissues. In summary, G-CSF inhibited leukemia progression in the majority of patient xenografts, however, in a subset of samples G-CSF accelerated disease progression. Clinically, G-CSF administration to ALL patients has not been associated with any major adverse outcomes. However our data suggest that a small subset of patients may experience accelerated disease. Identification of features associated with adverse responses to G-CSF will permit the identification of patients for whom G-CSF may present a risk for increased disease progression. Disclosures: No relevant conflicts of interest to declare.

Inhibition of HDAC8 Reactivates p53 and Abrogates Leukemia Stem Cell Activity in CBFβ-SMMHC Associated Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 363-363
Author(s):  
Jing Qi ◽  
Qi Cai ◽  
Sandeep Singh ◽  
Ling Li ◽  
Hongjun Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Cell Activity ◽  
Disease Free Survival ◽  
Hematopoietic Stem ◽  
The Impact ◽  
Acute Myeloid

Abstract The inv(16)-created CBFβ-SMMHC fusion protein inhibits differentiation of hematopoietic stem and progenitor cells (HSPCs) and creates pre-leukemic populations predisposed to acute myeloid leukemia (AML) transformation. However, the molecular mechanism underlying the leukemogenic function of CBFβ-SMMHC has been elusive. Given the low TP53 mutation rate in AML, alternative mechanisms disrupting p53 function are expected. We showed thatCBFβ-SMMHC impairs p53 acetylation and p53 target gene activation through formation of an aberrant protein complex with p53 and HDAC8 (Blood, 120: A772; 122(21): 224). We now show that CBFβ-SMMHC binds to p53 and HDAC8 independently through distinct regions and that HDAC8 mediates the deacetylation of p53 associated with CBFβ-SMMHC. In addition, we generated mice carrying a floxed Hdac8 (Hdac8f) allele and crossed with Cbfb56M/+/Mx1-Cre (Kuo YH et al, Cancer Cell 2006). Deletion of Hdac8 signifiacntly (p<0.0001) reduced the incidence of AML and prolonged disease-free survival. Pharmacologic inhibition of HDAC8 activity with HDAC8-selective inhibitors (HDAC8i) reactivates p53 and selectively induces apoptosis of inv(16)+ AML CD34+ cells while sparing normal HSPCs. To test the effect of HDAC8i on LSC engraftment and leukemia-initiating capacity, we generated Cbfb56M/+/Mx1-Cre mice with a Cre-reporter line expressing tdTomato fluorescence protein following Cre-mediated recombination. AML cells (dTomato+/cKit+) treated with HDAC8i (22d) ex vivo showed reduced engraftment (p=0.025) and enhanced survival (p=0.025) in transplanted mice. To examine whether HDAC8i 22d treatment affects the engraftment capacity on surviving cells, we transplanted equal number (2 x 106) of AML cells treated with either 22d or vehicle in another cohort of mice (n=4). We show that HDAC8i 22d treatment reduced the engraftment of dTomato+/cKit+ AML cells and enhanced survival, suggesting that the engraftment capacity is altered in addition to reducing AML cell survival. We next performed preclinical studies to determine the efficacy of in vivo administration of HDAC8i 22d. AML transplanted mice were randomized into two groups, one group treated with vehicle and the other treated with HDAC8i 22d for 2 weeks. Flow cytometry analysis revealed significantly reduced frequency (p=0.0097) and number (p=0.0101) of dTomato+/cKit+ AML cells in the bone marrow and spleen of 22d treated mice compared to vehicle treated group. To further assess the impact on LSC activity, we transplanted bone marrow cells from these treated mice into secondary recipients and analyzed for AML engraftment. Significant reduction in the frequency (p<0.0001) and the number (p=0.0006) of dTomato+/cKit+ AML cells was observed in the bone marrow and spleen. Furthermore, HDAC8i 22d treated transplants showed no signs of leukemia while vehicle treated transplants are moribund with aggressive AML. These results indicate that HDAC8 inhibition by 22d treatment effectively eliminates engraftment and leukemia-initiating capacity of AML LSCs. In conclusion, our studies identify a novel post-translational p53-inactivating mechanism and demonstrate selective HDAC8 inhibition as a promising approach to target inv(16)+ AML LSCs. Disclosures No relevant conflicts of interest to declare.

Impact of Infusion Rate on the Early Engraftment Following Allogeneic Intra Bone Marrow Infusion of Hematopoietic Stem Cells in a Canine DLA-Identical Reduced Intensity Transplantation Model

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5406-5406
Author(s):  
Stephanie Schaefer ◽  
Juliane Werner ◽  
Sandra Lange ◽  
Katja Neumann ◽  
Christoph Machka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Infusion Rate ◽  
Conflicts Of Interest ◽  
Conditioning Regimen ◽  
Hematopoietic Stem ◽  
Time Points ◽  
The Impact ◽  
Reduced Intensity

Abstract Introduction: Direct intra bonemarrow (IBM) infusion of hematopoietic stem cells (HSC) is assumed to improve the homing efficiency and to accelerate the early engraftment in comparison to the conventional intravenous application of HSC. Especially for transplantation of low cell numbers i.e. "weak grafts" that is generally associated with delayed engraftment. The direct infusion of HSC in close proximity to the HSC niche by intra bone marrow transplantation (IBMT) might be a promising way. Whether the HSC infusion rate might influence the homing process and therefore the outcome after IBMT is so far unknown. Aims: Herein, we analyzed in a canine DLA-identical littermate model the impact of different graft infusion rates on the hematopoietic recovery as well as on the engraftment kinetics after IBMT following reduced intensity conditioning. Methods: Recipient dogs received IBMT following a 4.5 Gy total body irradiation (TBI). From day (d) -1 until d+35 Cyclosporin A (15mg/kg) was administered orally twice a day as immunosuppression. For IBM transfusion the graft volume was reduced by buffy coat centrifugation and dogs obtained 2x25 ml simultaneously into the humerus and femur. The infusion rate of the graft was 25ml/10 min in group 1 (IBM10, n = 8) and 25 ml/60 min in group 2 (IBM60, n = 7). A 28 day follow-up is currently available for twelve dogs (IBM10 n = 7; IBM60 n = 5). The development of the peripheral blood mononuclear cell (PBMC) and granulocyte chimerism was tested weekly. Blood count, kidney and liver enzymes were monitored routinely. Results: All animals engrafted. One dog of the IBM10 group died at d+15 (infection) and was therefore not included into analysis. The median number of infused total nucleated cells were in IBM10 4.1*108/kg (range 2.3-6.0*108/kg) and in IBM60 3.2*108/kg (range 1.8-4.4*108/kg; p=0.4). The infused CD34+ numbers were median 3.2*106/kg (range: 1.2-10.0*106/kg; IBM10) and 3.6*106/kg (range: 1.5-6.8*106/kg; IBM60; p=0.7). Time of leukocyte recovery was median d+11 after IBMT in both groups (range: d+4 to d+11, IBM10; d+8 to d+14, IBM60; p= 0.5). Median leukocytes nadirs amounted to 0.2*109/l for IBM10 and 0.3*109/l for IBM60 (p= 0.08). The median duration of leukopenia (<1*109/l) were similar (6d, range: 4-11d, IBM10; 3-9d, IBM60) (p= 0.6). Median platelet nadir was 0*109/l for both cohorts (range: 0.0-7.0*109/l, IBM10; 0.0-1.0*109/l, IBM60). The period of thrombocytopenia (≤20.0*109/l) was significantly prolonged in the IBM60 group (median 10d, range) compared to 5d (range: 3-12d) in the IBM10 group (p=0.05). Donor PBMC chimerisms at d+7, d+14 and d+28 were median 22% (range: 8-34%), 50% (range: 29-53%) and 67% (range: 47-73%) in IBM10. The results of PBMC chimerism for IBM60 were 11% (range: 5-34%), 42% (range: 20-42%) and 59% (range: 44-66%) at these time points (p = n.s.). Donor granulocyte chimerisms of median 33% (range: 11-83%), 100% (range: 58-100%) and 100% (range: 82-100%) were detected at d+7, d+14 and d+28 after HSCT in IBM10, respectively. The granulocyte chimerism in IBM60 amounted to 34% (range: 3-87%), 96% (range: 94-100%) and 98% (range: 96-100%) at the above mentioned time points p=n.s. for all time points). Conclusion: Our data suggest that early granulocyte and PBMC engraftment is not influenced by modification of the HSC infusion rate. However, the period of thrombocytopenia seems to be prolonged following a 60 minutes application. Therefore, longer infusion times in an IBMT setting seem not to be beneficial following toxicity reduced conditioning regimen. Disclosures No relevant conflicts of interest to declare.

Impact of Cytopenia Following Unmanipulated Haploidentical Hematopoietic Stem Cell Transplantation Using Post-Transplant Cyclophoshamide

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2281-2281
Author(s):  
Villetard Ferdinand ◽  
Stefania Bramanti ◽  
Samia Harbi ◽  
Sabine Fürst ◽  
Catherine Faucher ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Transplantation ◽  
Disease Risk ◽  
Conflicts Of Interest ◽  
Risk Index ◽  
Conditioning Regimen ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Cell Dose ◽  
The Impact

Abstract Introduction Allogeneic transplantation from a haploidentical donor (HaploSCT) is an alternative strategy in the treatment of hematologic malignancies in absence of HLA-identical donor. Recent studies reported similar outcome after HaploSCT compared to HLA-identical transplantation in different settings (Bashey, JCO 2013; Wang, Blood 2015; Gosh, JCO 2016). Although survivals seemed promising after HaploSCT, hematopoietic recovery following such a mismatched transplantation could represent a limitation. Thus, our series aims to evaluate hematological recovery after HaploSCT using a post transplantation cyclophosphamide (PT-Cy) platform. Methods This retrospective monocentric study included consecutive patients with following criteria: adults with hematological malignancies; bone marrow or peripheral blood T-replete HaploSCT from 2011 to 2015; non-myeloablative (Baltimore approach) or reduced intensity conditioning (busulfan-based) regimen; PT-Cy as part of GVHD prophylaxis. Patients with primary graft failure were excluded. Absolute neutrophil count (ANC), red cells (RCT) or platelet transfusion (PT) requirements on day 30 (D30) and day 100 (D100) were analyzed among disease-free patients. We first separately evaluated the rate of patients with significant cytopenia in each lineage (defined by ANC < 1 G/L, RCT need, PT need) and searched for impact of pre-transplantation factors on cytopenia (multivariate analyses by binary logistic regression). Then, we evaluated outcome by D30- and D100-landmark analyses according to cytopenia. Results One hundred and forty six patients with a median age of 56 years (range: 19-73) were analyzed: 142 and 117 were evaluable at D30 (4 early deaths) and D100 (17 deaths, 11 relapses), respectively. At D30, 20% of patients had ANC<1G/L, 67% needed RCT and 63% needed PT. Corresponding values at D100 were 20%, 42% and 28%, respectively (Figure 1). At D30: the use of PBSC (HR 9.5, p=0.002) was significantly associated with ANC>1G/L at D30; the use of NMAC Baltimore schema (HR 0.3, p=0.012) and CD34+ cell dose > median (HR 0.4, p=0.041) decreased PT needs while hematopoietic cell transplantation comorbidity index (HCT-CI)≥3 (HR 3.3, p=0.004) was associated with PT needs; no factor was found to significantly influence RCT. At D100: Age>60 years (HR 2.4, p=0.045), female to male HaploSCT (HR 3.3, p=0.020) and HCT-CI≥3 (HR 3.7, p=0.006) were significantly associated with higher risk of RCT need; female to male HaploSCT (HR 3.6, p=0.015) and HCT-CI≥3 (HR 6.9, p=0.001) were associated with PT needs; no factor was found to significantly influence ANC. With a median follow up of 25 months (range: 5-55), cox multivariate model with adjustment by age (continuous), disease risk index (low/intermediate vs high/very high), HCT-CI (0-2 vs ≥3), conditioning regimen (baltimore vs. busulfan-based) and graft source (bone marrow vs PBSC) showed that ANC<1 G/L was strongly associated with higher NRM (HR 2.9, p=0.011) and shorter OS (HR 3.4, p<0.001), overcoming the impact of RCT and PT needs (Figure 2A and 2B). In contrast, D100 analysis showed that PT need was the most determinant factor of increased NRM (HR 13.7, p=0.013) and poor OS (HR 7.3, p=0.003), while both D100 ANC and RCT needs did not impact outcome (Figure 2C and 2D). Discussion We found that cytopenia remain a concern after HaploSCT, leading to increased NRM and OS. The absence of ANC>1G/L at D30 as well as the need of PT at D100 may be considered as a strong post transplantation factor predicting poor outcome. Some pre-transplantation factors of cytopenia have been identified, such as CD34+ cell dose, sex mismatch and graft source. Among them, some may help for donor selection while the optimal donor for HaploSCT is still unknown. Moreover, better neutrophil recovery at D30 is achieved with the use of PBSC. CD34+ optimal cell dose in this setting remains also to be determined. In addition, post transplantation events such GVHD and/or infections should be evaluate to explore their interactions with such cytopenia, aiming to develop early therapeutic interventions. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Characterization of the Hematopoietic Supporting Activity of Osteoblasts Derived from Bone Marrow Mesenchymal Stromal Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4358-4358
Author(s):  
Manal Alsheikh ◽  
Roya Pasha ◽  
Nicolas Pineault
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Soluble Factors ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
The Impact ◽  
Myeloid Progenitors

Abstract Osteoblasts (OST) found within the endosteal niche are important regulators of Hematopoietic Stem and Progenitor Cells (HSPC) under steady state and during hematopoietic reconstitution. OST are derived from mesenchymal stromal cells (MSC) following osteogenic differentiation. MSC and OST secrete a wide array of soluble factors that sustain hematopoiesis. Recently, we showed that media conditioned with OST derived from MSC (referred as M-OST) after 6 days of osteogenic differentiation were superior to MSC conditioned media (CM) for the expansion of cord blood (CB) progenitors, and CB cells expanded with M-OST CM supported a more robust engraftment of platelets in NSG mice after transplantation. These findings raised the possibility that M-OST could be superior to MSC for the ex vivoexpansion HSPC. In this study, we set out to test the hypothesis that the growth modulatory activity of M-OST would vary as a function of their maturation status. The objectives were to first monitor the impact of M-OST differentiation and maturation status on the expression of soluble factors that promote HSPC expansion and in second, to investigate the capacity of M-OST CMs prepared from M-OST at distinct stages of differentiation to support the expansion and differentiation of HSPCs in culture. M-OST at distinct stages of differentiation were derived by culturing bone marrow MSC in osteogenic medium for various length of time (3 to 21 days). All CB CD34+ enriched (92±7% purity) cell cultures were done with serum free media conditioned or not with MSC or M-OST and supplemented with cytokines SCF, TPO and FL. We first confirmed the progressive differentiation and maturation of M-OST as a function of osteogenic culture length, which was evident by the induction of the osteogenic transcription factors Osterix, Msx2 and Runx2 mRNAs, the gradual increase in osteopontin and alkaline phosphatase positive cells and quantitative increases in calcium deposit. Next, we investigated the expression in MSC and M-OSTs of genes known to collaborate for the expansion of HSPCs by Q-PCR. Transcript copy numbers for IGFBP-2 increased swiftly during osteogenic differentiation, peaking at day-3 (˃100-fold vs MSC, n=2) and returning below MSC level by day-21. In contrast, ANGPTL members (ANGPTL-1, -2, -3 and -5) remained superior in M-OSTs throughout osteogenic differentiation with expression levels peaking around day 6 (n=2). Next, we tested the capacity of media conditioned with primitive (day-3, -6), semi-mature (day-10, -14) and mature M-OST (day-21) to support the growth of CB cells. All M-OST CMs increased (p˂0.03) the growth of total nucleated cells (TNC) after 6 days of culture compared to non-conditioned medium used as control (mean 2.0-fold, n=4). Moreover, there was a positive correlation between cell growth and M-OST maturation status though differences between the different M-OST CMs tested were not significant. The capacity of M-OST CMs to increase (mean 2-fold, n=4) the expansion of CD34+ cells was also shared by all M-OST CMs (p˂0.05), as supported by significant increases with immature day-3 (mean ± SD of 18 ± 6, p˂0.02) and mature day-21 M-OST CMs (14 ± 5, p˂0.05) vs. control (8 ± 3, n=4). Conversely, expansions of TNC and CD34+ cells in MSC CM cultures were in-between that of control and M-OST CMs cultures. Interestingly, M-OST CMs also modulated the expansion of the HSPC compartment. Indeed, while the expansion of multipotent progenitors defined as CD34+CD45RA+ was promoted in control culture (ratio of 4.5 for CD34+CD45RA+/CD34+CD45RA- cells), M-OST CMs supported greater expansion of the more primitive CD34+CD45RA- HSPC subpopulation reducing the ratio to 3.3±0.4 for M-OST cultures (cumulative mean of 10 cultures, n=2). Moreover, the expansions of CD34+CD38- cells and of the long term HSC-enriched subpopulation (CD34+CD38-CD45RA-Thy1+) in M-OST CM cultures were respectively 2.7- and 2.8-fold greater than those measured in control cultures (n=2-4). Finally, the impact of M-OST CMs on the expansion of myeloid progenitors was investigated using a colony forming assay; expansion of myeloid progenitors were superior in all M-OST CM cultures (1.6±0.2 fold, n=2). In conclusion, our results demonstrate that M-OST rapidly acquire the expression of growth factors known to promote HSPC expansion. Moreover, the capacity of M-OST CMs to support the expansion of HSPCs appears to be a property shared by M-OST at various stages of maturation. Disclosures No relevant conflicts of interest to declare.

A Sequence Variation in the Promoter Region of PTPN22 Gene Predicts Relapse After HLA-Fully-Matched Unrelated Bone Marrow Transplantation for Hematologic Malignancies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3088-3088
Author(s):  
Luis J. Espinoza ◽  
Akiyoshi Takami ◽  
Katsuya Nakata ◽  
Makoto Onizuka ◽  
Yasuo Morishima ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Promoter Region ◽  
Conflicts Of Interest ◽  
Hematologic Malignancies ◽  
Negative Regulator ◽  
Ptpn22 Gene ◽  
Transplant Outcomes ◽  
The Impact

Abstract Abstract 3088 The protein tyrosine phosphatase non-receptor 22 (PTPN22), also known as LYP, is an intracellular phosphatase ubiquitously expressed with particularly high expression in hematopoietic tissues that is a critical negative regulator of signaling through the T cell receptor and has emerged as the strongest common genetic risk factor for human autoimmunity outside the major histocompatibility complex. In this study we analyzed the impact of the polymorphism rs2488457 (1123G>C) in the promoter region of PTPN22 gene on transplant outcomes in patients undergoing unrelated HLA-matched bone marrow transplantation (BMT) through the Japan Donor Marrow Program. The PTPN22 rs2488457 genotypes were retrospectively analyzed in a cohort of 663 patients with hematologic malignancies and their unrelated donors. The presence of the C/C or C/G genotype in the donor side was associated with a significantly lower incidence of relapse compared to the donor G/G genotype (28% vs. 34% 5-years, P =0.05) (Fig.1). No difference was noted in transplant-related mortality, or graft-versus-host disease in relation to the rs2488457 polymorphism. The donor C/C or C/G genotype remained statistically significant for relapse in the multivariate analyses (hazard ratio [HR], 0.60; 95% confidence interval [CI], 0.43 to 0.85; P =0.004). These differences in relapse did not significantly affect on overall survival (OS) (48% vs. 49% 5-years, P =0.85). The recipient PTPN22 genotypes did not significantly influence the transplant outcomes. These results suggest an association of the donor C/C or C/G genotype with lower disease relapse. These could therefore be useful in selecting the donor and creating therapeutic strategies for improving the final outcome of allogeneic BMT. Disclosures: No relevant conflicts of interest to declare.

A Genetic Variant in the CD53 Gene Is Associated with Clinical Outcomes after Unrelated Bone Marrow Transplantation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1244-1244
Author(s):  
Luis Jorge Espinoza ◽  
Keitaro Matsuo ◽  
Saki Toda ◽  
Yasuo Morishima ◽  
Makoto Onizuka ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Consensus Sequence ◽  
Univariate Analysis ◽  
Taqman Probe ◽  
Healthy Individuals ◽  
Transplant Outcomes ◽  
Allele Specific ◽  
Functional Relevance ◽  
The Impact

Abstract Background: CD53, a member of the tetraspanin family of proteins, broadly expressed in hematopoietic cells, is implicated in leukocyte activation and cell survival responses to stress conditions. In a recent genome linkage scan, the single nucleotide variation, rs6679497 (C>G), in the intron of the CD53 gene was identified as an important regulator of TNF release from innate immune cells in response to inflammatory stimuli. (Bos SD: Eur J Hum Genet. 2010). Tumor necrosis factor alpha (TNF-ƒ¿) which is released pretransplant due to irradiation and cytotoxic preconditioning regimens, has been implicated in transplant complications. Methods: To assess the impact of rs6679497 variation of the CD53 gene, on transplant outcomes, we evaluated 322 of patients with hematologic malignancies transplanted through the Japan Marrow Donor Program. The functional relevance of the rs6679497 variation was investigated in a whole blood culture system using samples from healthy individuals. To investigate the effect of rs6679497 on mRNA transcription activity we performed allele-specific quantitative RT-PCR with a TaqMan probe of rs6679497 using cDNA from in vitro activated leukocytes from healthy donors with heterozygous genotypes. Results: There was a significantly lower overall survival in the recipients with the GG genotype in the univariate analysis (29% vs. 53% at 5-y; P=0.01) and multivariate analysis (relative risk=1.97; 95% confidence interval, 1.22-3.18; P=0.01, Figure 1). The donors s6679497 genotypes did not significant influence the transplant outcomes. In the allele-specific quantitative PCR, the mean ratio (G allele transcripts vs. C allele transcripts) was 1.5 which is higher than that of DNA amplicons, suggesting that the G allele has higher transcriptional activity than the C allele. Using the TF-SEARCH online tool, which predict putative transcription factor binding sites, the genomic region containing the G allele of s6679497 was found to create a novel consensus sequence corresponding to the putative binding element of GATA-1. In vitro stimulated leucocytes from healthy individuals possessing the G allele secreted higher levels of TNF-ƒ¿ than those without the G allele. Consistent with these results, flow cytometry studies revealed that monocytes from donors possessing the G allele express higher levels of CD53 on their surface (p=0.02) which may account for their higher TNF-ƒ¿ secretion. These findings substantiate the functional relevance of the rs6679497 variation, and indicate that the increased TNF-ƒ¿ secretion by individuals with the G allele likely account for their worse OS. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Human Cytomegalovirus Selectively Suppresses the Megakaryo/Thrombopoiesis of PDGFR+ and αvβ3+ Megakaryocytes Via the TPO/c-Mpl Pathway after Allo-HSCT

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-25
Author(s):  
Feng-qi Liu ◽  
Fei-er Feng ◽  
Gao-chao Zhang ◽  
Yan Su ◽  
Xue-yan Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Conflicts Of Interest ◽  
Antiviral Treatment ◽  
Infection Model ◽  
Hematopoietic Stem ◽  
The Impact

Introduction Virus-induced thrombocytopenia is a severe complication in immunocompromised hosts. Among patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT), human cytomegalovirus (HCMV) infection contributes to a variety of end-organ diseases and hematological complications, leading to increased mortality. Even with antiviral treatment, HCMV remains a potentially lethal infection due to the lack of understanding of the underlying mechanisms of host-virus interactions. The key to solving this problem is to identify the factors that predispose patients to HCMV infection and carry out targeted therapy. Here, we investigated the megakaryo/thrombopoiesis process, including the thrombopoietin (TPO)/c-Mpl pathway, after HCMV infection in vivo and in vitro, screened for susceptible subsets of megakaryocytes (MKs) and explored novel therapeutic targets for HCMV infection. Methods To test whether thrombocytopenia induced by HCMV results from an impaired megakaryo/thrombopoiesis process, we studied the impact of HCMV in an in vivo model of HCMV DNAemia patients following allo-HSCT and an in vitro model of bone marrow CD34+-derived MKs infected with serum from HCMV DNAemia patients. Forty patients who had received allo-HSCT were enrolled in this study, among whom 18 recipients had HCMV DNAemia and 22 were HCMV negative, and bone marrow-derived mononuclear cells (MNCs) from patients were tested for CD41, vWF, pp65, c-Mpl, PDGFR, αvβ3 and TLR2 using flow cytometry (FCM). Transmission electron microscopy (TEM) was used to detect HCMV capsids inside MKs. Cell apoptosis was measured by Annexin V. MK ploidy was determined by FCM for propidium iodide (PI) staining. Finally, inhibitors of PDGFR (IMC-3G3 and Gleevec), αvβ3 and TLR2 were cocultured with MKs. Results Our data showed that pp65+ cells accounted for 40.59±6.12% of total CD41+vWF+ MKs from HCMV DNAemia patients, and there was a significant increase in the expression of αvβ3, PDGFR and TLR2 in pp65+ MKs compared with that in control patients. Furthermore, the percentage of PDGFR+αvβ3+ MKs emerged as an independent factor associated with HCMV infection in multivariate analysis (p = 0.008). MKs in HCMV-infected patients showed increased apoptosis and necrosis and different patterns of MK ploidy distribution compared with those in HCMV-negative patients, with a decreased proportion from 16N to 64N and a peak at 8N. Meanwhile, the expression of TPO receptor c-Mpl was lower in pp65+ MKs from HCMV DNAemia patients (0.77±0.38% in pp65+ MKs from HCMV DNAemia patients, 1.75±0.40% in pp65- MKs from HCMV DNAemia patients, 1.97±0.67% in MKs from HCMV-negative patients, and 2.06±0.29% in MKs from healthy controls, p&lt;0.01) while the TPO level in serum was increased compared with that in controls. Next, we established an in vitro HCMV infection model of CD34+-derived MKs with serum from HCMV DNAemia patients, and the laboratory HCMV strain Towne was used as a positive control. After 9 days of coculturing, the viral capsids of HCMV were observed in the nuclei of MKs (Figure 1A), and HCMV infection increased the apoptosis of MKs and shifted them to low ploidy, with a significant decrease in platelet release. As with the in vivo results, c-Mpl was downregulated in HCMV-infected MKs. The expression levels of PDGFR, TLR2 and αvβ3 on MKs were increased in coculture with HCMV DNAemia serum, and pp65-positive MKs were decreased compared with the control after treatment with inhibitors of PDGFR and αvβ3 (Figure 1B). However, neither Gleevec nor anti-TLR2 altered the HCMV infection rate. Conclusions Our study showed that HCMV could impair megakaryopoiesis throughout maturation, apoptosis, and platelet generation via the TPO/c-Mpl pathway both in vivo and in vitro. MKs with PDGFR+ and αvβ3+ phenotypes are susceptible to HCMV infection and we proposed PDGFR and αvβ3 inhibitors as potential therapeutic alternatives for allo-HSCT patients with HCMV infection. Disclosures No relevant conflicts of interest to declare.

scholarly journals The MEK/ERK Inhibitor Trametinib Reduces Fibrosis in a Transduction-Transplantation Model of Mutated Calreticulin

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 635-635 ◽  
Author(s):  
Thanh Kim Nguyen ◽  
Prasanthi Tata ◽  
Stefan Brooks ◽  
Nilamani Jena ◽  
Sarah J Morse ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Average Score ◽  
Spleen Weight ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
The Impact ◽  
Transplantation Model

Abstract Insertion or deletion mutations in calreticulin (CALR) are present in the majority of JAK2V617F-negative MPN patients. We utilized a murine retroviral transduction-transplantation model to express the 52bp CALR deletion mutation (CALRDEL) in both BALB/c and C57B/6 backgrounds. As described previously (Marty et al., Blood 2016;127:1317), recipients of CALRDEL-transduced marrow developed persistent thrombocytosis without leukocytosis or erythrocytosis by two months post-transplant. Mice were euthanized at six and nine months post-transplant to evaluate the tempo of disease progression. At six months CALRDEL mice had impressive expansion of megakaryocytes expressing the CALRDEL mutant protein in the bone marrow (BM) without fibrosis or significant splenomegaly. By nine months BM fibrosis and splenomegaly were present. Both whole BM and spleen cells were able to serially transplant the MPN phenotype into secondary recipients. When cultured in collagen-based media supplemented with thrombopoietin, CALRDEL BM cells produced an increased number of megakaryocyte colonies as compared to empty vector. The increased colony formation potential of CALRDEL bone marrow cells was limited to megakaryocytes, we found no increase in colony formation from CALRDEL hematopoietic stem and progenitor cells in methylcellulose with cytokines supporting erythroid and GM colony formation. However, CALRDEL enhanced the serial replating ability of LKS (lineageneg, c-kit+ Sca-1+) cells. Both pSTAT5 and pERK were increased in whole spleen lysates from CALRDEL mice as compared to wild-type BALB/c mice. Therefore, we tested the impact of ruxolitinib, a JAK1/2 inhibitor, and trametinib, a MAPK/ERK inhibitor, on the MPN phenotype of CALRDEL mice. At six months post-transplant mice were treated with either ruxolitinib (90mg/kg PO BID), trametinib (3mg/kg PO daily), or vehicle for 40 days. Ruxolitinib reduced pSTAT5 but caused a paradoxical increase in pERK in whole spleen lysates, while trametinib reduced pERK but not pSTAT5. Trametinib caused a transient increase in platelets and white cells. In spite of pharmacodynamic evidence of effective dosing, ruxolitinib had no significant effect on platelet or leukocyte count but did reduce hemoglobin slightly. Both ruxolitinib and trametinib reduced spleen weight. Ruxolitinib reduced the fraction of the mutant CALRDEL allele (inferred from percentage of GFP+ cells) in the spleen but not the bone marrow, while trametinib had no impact on disease allele burden in any organ. Neither ruxolitinib nor trametinib reduced the expansion of megakaryocytes in the bone marrow but trametinib significantly reduced marrow fibrosis (average score MF-2.5 for vehicle, MF-1.75 for ruxolitinib, MF-1 for trametinib). To assess the role of STAT5 in the pathogenesis of the ET-like MPN induced by the CALRDEL mutant, we transduced BM from syngeneic Balb/c donors carrying a floxed Stat5ab allele in combination with a Stat5ab null allele (Mx-Cre;Stat5abfl/-; Walz et al., Blood 2012;119:3550). Haploinsufficiency for Stat5ab significantly delayed the development of ET-like MPN and attenuated thrombocytosis, implicating JAK2-STAT5 signaling directly in the pathogenesis of this disease. In summary, this CALRDELmouse model results in an MPN phenotype resembling essential thrombocythemia followed by myelofibrosis. CALRDELresults in expansion of megakaryocytes and platelets without expansion of other myeloid cell types. Both pSTAT5 and pERK are increased in our CALRDEL model and pharmacologic inhibition of pERK results in reduction of fibrosis without reducing megakaryocytes. These studies implicate pERK as a potential anti-fibrosis therapeutic target in MPN. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document