bluuebunny/crossref_metadata_2025 huggingface dataset:
Oral (po) and Intravenous (iv) Clofarabine for Patients (pts) with Myelodysplastic Syndrome (MDS)
Authors: Faderl Stefan , Garcia-Manero Guillermo , Ravandi Farhad , Borthakur Gautam , Estrov Zeev , Thomas Deborah A. , Gandhi Varsha , Plunkett William , Byrd Anna , Kwari Monica , Kantarjian Hagop M.
Published: April 2020

Clofarabine (CLO) is a second generation deoxyadenosine nucleoside analog with activity in pts with acute myeloid leukemia (AML). Early reports also suggested activity in MDS, but the role of CLO in MDS has not been defined. To evaluate the activity and safety of CLO in MDS we designed two phase II studies of IV CLO and PO CLO, respectively. Pts were eligible with MDS and ≥5% blasts or IPSS intermediate-2 and high-risk, CMML, and RAEB-t by FAB. Hematopoietic growth factor support prior to and during the study was permitted. In the IV study pts were adaptively randomized (based on response) to receive CLO 15 or 30 mg/m2 IV over 1 hr. daily × 5 every 4–6 weeks. For the PO study, the starting dose of CLO was 40 mg/m2 orally daily × 5 every 4–6 weeks (oral bioavailability assumed ~ 50%, based on preclinical data), which was decreased to 30 mg/m2 orally daily × 5 after 6 pts had been treated on the higher dose. Sixty-one pts (6 RA/RARS/RCMD, 17 RAEB-1, 16 RAEB-2, 11 CMML, 11 RAEB-t) were treated. Forty-two pts (69%) had high- or intermediate-2 risk disease by IPSS. Thirty-six pts received IV CLO and 25 PO CLO. Median age was 67 yrs (range 25–89) (IV) and 70 yrs (54–86) (PO). Overall, > 80% of pts were older than 60 yrs. Approximately 1/3 of pts had secondary MDS: 13 (36%) (IV) and 8 (32%) (PO), respectively. Seventeen (47%) (IV) and 10 (40%) (PO) of pts had unfavorable cytogenetics (by IPSS definition). Thirty-nine (64%) pts failed prior hypomethylator therapy with either decitabine or azacitidine (22 [61%] pts on IV and 17 [68%] pts on PO CLO). Responses of 60 evaluable pts (one pt refused continuation of therapy on D4 of first course) are summarized in the Table:

Response (%) Route Dose (mg/m2) N CR CRp HI PO 40/30 24 7 (29) 2 (8) 3 (13) IV 15 20 7 (35) 3 (15) - 30 16 4 (25) 2 (13) - Total - 60 18 (30) 7 (12) 3 (5)

Six pts (10%) died on study (all IV CLO; most commonly related to infectious complications). All pts were evaluable for toxicities. Common AEs were nausea, vomiting, skin rash, hyperbilirubinemia and transaminase elevations. Toxicities ≥ grade 3 were rare. Acute renal failure occurred in 7 pts (2 IV [15 mg/m2], 3 IV [30 mg/m2], 2 PO). Myelosuppression and hospitalizations for neutropenic fever were common, but prolonged myelosuppression (> 42 days) was rare. In conclusion, CLO has activity in pts with higher-risk MDS. Optimal dose and schedule for PO and IV CLO remain to be defined. Lower doses of CLO are also associated with responses. Additional dose finding trials are underway to define the MTD and DLT of IV and PO CLO in high-risk MDS.
GATA1s Mutant Protein Contributes to “Down” Syndrome Megakaryoblastic Leukemia by Derepression of E2F Targets.
Authors: Klusmann Jan-Henning , Godinho Frank , Pushpanathan Thanaseelan , Reinhardt Dirk , Orkin Stuart H , Li Zhe
Published: April 2020

Somatic mutations in the hematopoietic transcription factor GATA1, leading to the production of a GATA1 variant (GATA1s) lacking its N-terminal domain, are a critical event in the development of Down syndrome (DS) associated transient myeloproliferative disease (TMD) and myeloid leukemia (ML-DS). Mice carrying Gata1s germline mutation exhibit a transient population of abnormal yolk sac/fetal liver megakaryocytic progenitors, hyperproliferating both in vitro and in vivo. However, either Gata1s mice alone or in conjunction with two DS mouse models (Ts65dn, Ts1Cje) fail to recapitulate human TMD or leukemia. Using a retroviral insertional mutagenesis screening in Gata1s fetal progenitors, we generated murine acute megakaryoblastic leukemia (AMKL) closely resembling human ML-DS. We demonstrate that retroviral integrations at the Evi1 and Plag1 loci cooperated with Gata1s in leukemogenesis. Through an integrated genomic approach, we show that restoration of full length Gata1 expression in these murine Gata1s AMKL blasts leads to downregulation of gene sets containing E2F target genes. In addition, most of these same gene sets are also enriched in Gata1s primary fetal liver-derived megakaryocytes compared to wild type megakaryocytes, in flow sorted human ML-DS leukemic blasts compared to sorted non-DS M7 leukemic blasts, and in ML-DS CMK cell line compared to other leukemic cell lines. Using a genetic approach, we could demonstrate that the hyperproliferative phenotype of Gata1s FL megakaryocytic progenitor cells can be rescued by broadly repressing the function of E2F factors. Additionally, the CMK cell line ceased to proliferate upon E2F inactivation. Thus, we discovered a new mechanism of GATA1 mediated growth regulation by controlling E2F function. GATA1s mutant protein contributes to leukemogenesis through insufficient repression of E2F targets, perturbing the coordination of proliferation and terminal differentiation. Our study reveals a previously unknown function of the N-terminal domain of GATA1 and adds a significant step towards our understanding of the role of GATA1 mutants in leukemogenesis.
OTT-MKL1 and MKL1 Inhibit Wnt Signaling.
Authors: Halene Stephanie , Cheng Ee-chun , Schulz Vincent , Tuck David , Krause Diane
Published: April 2020

The OTT-MKL1 fusion gene product is generated as a result of t(1;22) in a subset of acute megakaryoblastic leukemia predominantly encountered in young children. Due to myelofibrosis and the age at presentation, patient samples are scarce. We generated Human Erythroid Leukemia (HEL) cell derived cell lines with tet-inducible OTT, MKL1 and OTT-MKL1 to further elucidate the function of the respective proteins. HEL cells can be induced to differentiate down the megakaryocyte lineage by TPA. Induction with doxycycline resulted in transcription and translation of the respective genes within hours. While overexpression of MKL1 led to enhancement of megakaryocytic differentiation, both OTT and OTT-MKL1 overexpression led to cell death over the course of several days by apoptosis as evident by staining for Annexin V and morphology. The apoptotic cell death was greatly enhanced by concomitant induction of differentiation by TPA. We performed microarray analysis comparing uninduced and 8-hour tet-induced samples in the presence or absence of TPA. While overexpression of OTT had only a minimal effect on the transcriptome of the HEL cells, both MKL1 and OTT-MKL1 significantly affected the gene expression of many genes. Using a false discovery rate cut-off of p < 0.05, and assessing only those genes whose expression changed by greater than 2-fold, OTT-MKL1 and MKL1 induced the upregulation of 157 and 168 genes, respectively, and the downregulation of 56 and 62 genes, respectively. Only 20 genes were upregulated by both OTT-MKL1 and MKL1, and 12 genes were downregulated greater than 2-fold by both OTT-MKL1 and MKL1. GeneGo analysis comparing OTT-MKL1 over-expressing versus non-expressing cells revealed over-representation of the Wnt pathway. Among the differentially expressed genes implicated in the Wnt pathway were Frat1 and Frat2, which have been shown to inhibit GSK3β and lead to β-catenin nuclear accumulation, and thus stimulation of the Wnt pathway. At the same time, inhibitory NLK was upregulated and several down-stream targets of the Wnt pathway were downregulated. Spenito, the homolog of OTT in Drosophila, is known to have promoter-specific activating and inhibiting effects on Wnt target genes. We thus performed reporter assays to study the effects of OTT, MKL1 and OTT-MKL1 on the Wnt pathway. Using the TOP-/FOP-FLASH reporter system in 293T cells, there was a dose-dependent inhibition of β-catenin-mediated activation of the Tcf/Lef binding site promoter by MKL1 and OTT-MKL1. Full length OTT showed a minimal stimulatory effect only at low doses., while N-terminal OTT, lacking the SPOC domain and a dominant negative form of MKL1 lacking the transactivation domain each enhanced β-catenin induced Tcf/Lef mediated transcriptional activation. Studies to define the domains of OTT and MKL1 and the underlying mechanisms in hematopoietic cells are underway. These results suggest that MKL1 and OTT-MKL1 inhibit canonical Wnt signaling by inhibiting β-catenin induced transcription.
Activation of the Unfolded Protein Response (UPR) Induces CEBPA mRNA Decay in AML Cells.
Authors: Eyholzer Marianne , Schardt Julian , Shan Deborah , Mueller Beatrice U , Pabst Thomas
Published: April 2020

The myeloid key transcription factor CEBPA (CCAAT-enhancer binding protein alpha) is crucial for normal neutrophil differentiation, and cebpa-deficient mice lack mature granulocytes. In addition, deregulation of CEBPA function is a frequent event in AML patients. The endoplasmatic reticulum (ER) is involved in proper protein folding. Alterations in calcium levels or insufficient protein folding capacity leads to ER stress, thereby inducing rescue pathways that are commonly summarized as the unfolded protein response (UPR). The UPR aims either to re-establish ER homeostasis or to induce cell death. So far, induction of ER stress has been associated with a number of non-hematopoietic diseases. Here, we analyzed whether activated ER stress in leukemic cell lines and in AML patients affects the myeloid key regulator CEBPA. We induced ER stress in the leukemic HL60 cell line by treatment with 0.4mg/ml of Calcimycin (deregulation of calcium homeostasis) or with 3mg/ml of Tunicamycin (block of protein glycosylation). Consistently, we observed a rapid decrease of CEBPA mRNA to 10% after 6 hours of treatment. In accordance, CEBPA protein was no more detectable after 8 hours. Experiments in HL60 cells with Actinomycin D and Cycloheximide as well as competition experiments with various constructs of wild-type CEBPA sequences suggest a decay mechanism involving one or several elements in the 3’UTR of the CEBPA mRNA. We excluded microRNA-124a, a previously identified CEBPA regulator acting through the 3’UTR, to be involved. The precise elements mediating ER stress induced CEBPA mRNA decay remain to be elucidated. We also determined the expression of CEBPA mRNA and mediators of UPR - such as CHOP and GRP78 as well as alternative splicing of the XBP1 mRNA - in leukemic cells from 76 AML patients of all FAB subtypes. We found that AML patients with induced ER stress (13%) tended to have two-fold lower CEBPA mRNA expression, thus underlining the in vitro results reported above. In conclusion, our experiments indicate activated ER stress to be involved in a significant subgroup of AML patients. Moreover, it suggests that activation of the UPR in myeloid leukemic cells efficiently induces 3’UTR mediated decay of CEBPA mRNA expression, thereby contributing to the block in myeloid differentiation in these leukemias.
BTG1 Controls Glucocorticoid Sensitivity in Acute Lymphoblastic Leukemia by Regulating Nuclear Receptor-Mediated Transcription.
Authors: van Galen Joost C. , Kuiper Roland P. , van Emst Liesbeth , Levers Marloes R. , van Reijmersdal Simon V. , van Kessel Ad Geurts , Hoogerbrugge Peter M. , van Leeuwen Frank N.
Published: April 2020

Glucocorticoids (GCs) are global regulators of gene transcription that act by binding to the glucocorticoid receptor, a ligand-activated transcription factor belonging to the nuclear receptor family. As synthetic GCs such as prednisolone or dexamethasone specifically induce cell cycle arrest and apoptosis in lymphoid cells, these drugs are widely used in the treatment of (childhood) acute lymphoblastic leukemia (ALL). GC resistance is a strong independent predictor of poor prognosis in the treatment of childhood ALL, but the underlying mechanisms remain poorly understood. In a previous study (Kuiper et al. Leukemia 2007) we determined by array-based SNP analysis that microdeletions affecting the anti-proliferative gene B-cell translocation gene 1 (BTG1) occurred in 4 out of 33 pre-B ALL cases. In our current study we show that prednisolone causes a 3-fold induction of BTG1 mRNA expression both in pre-B ALL cell lines and primary ALL, implicating BTG1 in cellular responses to GCs. Moreover, RNA-interference-mediated knockdown of BTG1 renders RS4;11 pre-B leukemia cells refractory to prednisolone-induced apoptosis. We show by genome-wide mRNA profiling that loss of BTG1 completely abrogates prednisolone-induced gene expression by downregulation of the glucocorticoid receptor NR3C1. Moreover, we present evidence showing that BTG1 is part of a nuclear receptor complex that regulates expression of NR3C1 and consequently GC responsiveness in ALL. In conclusion, we have identified BTG1 as an important determinant of GC sensitivity in ALL.
Novel Pleiotropic Effects of Thrombin in Regulation of the Metastatic Potential of Human Rhabdomyosarcoma (RMS) Cells.
Authors: Wysoczynski Marcin , Ratajczak Mariusz Z
Published: April 2020

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescents and children that frequently infiltrates bone marrow (BM). We identified chemokines and growth factors [i.e., stromal derived factor (SDF)-1, hepatocyte growth factor (HGF)] that play important roles in RMS metastasis (Blood2002;100:2597, Cancer Res.2003;63:7926,Cancer Res.2007;67:2131). However, novel evidence indicates that the metastatic process for many tumors may be modulated by components of the coagulation cascade (e.g., thrombin, activated platelets). Thus, we became interested in the role of coagulation cascade in modulating metastasis of RMS cells. We learned that RMS cells express tissue factor (TF) and thus may activate coagulation by generating thrombin. Thrombin activated in the tumor microenvironment activates platelets that release microvesicles. These interact with RMS cells and/or directly stimulate PAR 1–4 receptors that are expressed on RMS cells. Focusing on these effects, 9 human RMS cells lines were employed for our studies. We learned that platelet-derived microvesicles (PMVs) transfer several platelet integrin receptors to RMS cells (e.g., a2b3) that are important for interaction with the endothelium, thus increasing their adhesive potential to endothelial cells, fibronectin, and fibrinogen. More importantly, RMS cells covered with PMVs showed higher metastatic potential after intravenous injection into severe combined immunodeficient (SCID) mice. This could be explained by enhanced adhesiveness of PMV-covered RMS cells to endothelial cells in small vessels. On the other hand, PMVs also directly chemoattracted RMS cells and activated MAPKp42/44 and AKT. Since we found that RMS cell lines express functional PAR 1–4 receptors, we become interested in the thromin-mediated responses of these cells. In RMS, we observed cells stimulated by thrombin phosphorylation of MAPKp42/44 and p38, but not AKT. However, we did not observe any effect from thrombin on proliferation and survival of RMS cells. To our surprise, thrombin decreased RMS chemotactic responses to SDF-1, HGF, and PMVs. Thrombin also decreased adhesion of RMS cells to fibronectin and laminin. In conclusion, we demonstrate for first time that RMS-expressed TF activates prothrombin and that thrombin is a novel, underappreciated, pro-metastatic factor for these cells. Activated in tumor proximity by thrombin, platelets release PMVs that chemoattract and transfer several platelet-derived receptors/adhesion molecules to RMS cells that are crucial for adhesion/interaction with the endothelium. Conversely, by decreasing the responsiveness of RMS cells to local chemoattractants and decreasing adhesiveness of RMS cells, thrombin promotes their release from the primary tumor into circulation. Consequently, RMS cells that are covered by PMVs release into circulation and respond to chemoattractants in distant organs for metastasis.
Monitoring of ADAMTS13 in Patients with Thrombotic Thrombocytopenic Purpura: Prediction of Response to Therapy, Risk of Relapse, and Long- Term Outcome.
Authors: Knoebl Paul , Koder Silvia , Schellongowski Peter , Distelmaier Peter , Quehenberger Peter , Pabinger Ingrid , Jilma Bernd
Published: April 2020

A severely reduced ADAMTS13 activity due to inhibitory autoantibodies is a key feature of acquired thrombotic thrombocytopenic purpura (TTP), leading to the persistence of ultralarge VWF multimers, platelet aggregation and disturbance of microcirculation.

We followed 39 patients (8 male, 31 female, mean age 38 years) with clinical signs of TTP over a period between 5 days to 16 years and observed a total of 53 episodes of TTP. ADAMTS13 was measured with a collagen-binding assay and the FRETS-VWF73 based Technozym ADAMTS-13 assay (activity and antigen, respectively). ADAMTS13 inhibitor was measured with a modified Bethesda method with both the above mentioned assays, and with the Technozym ADAMTS-13 INH ELISA.

Thirty-one patients had autoimmune TTP, and 47 episodes of TTP were analyzed in these patients. In all acute episodes, ADAMTS13 activity was below the detection limit (<0.05 U/ml), but ADAMTS13:Ag levels were below 0.1 U/ml only in 55% of the episodes. Anti-ADAMTS13 antibodies were detected in all episodes. Treatment consisted of plasma exchange (89% of the episodes), immunoadsorption (6%), steroids (70%), rituximab (15%), splenectomy (11%), aspirin (74%). Median time to platelet count normalization was 20 days (range 4–91 days), not related to the ADAMTS13-inhibitor titer. Platelet counts, LDH levels, and reticulocyte counts were better predictors of treatment response. Plasma exchange did not directly influence ADAMTS13 levels or clear the inhibitors. Three patients died during the first episode (myocardial infarction), one in 2nd relapse. ADAMTS13 activity increased >0.2 U/ml in 66% of the episodes (after median 160 days). In the remaining cases anti-ADAMTS13 antibodies persisted during remissions for up to 2 years. In 3 cases the antibody reoccurred after initial normalization of ADAMTS13 activity, and clinical relapses followed. In total, 21 relapses were observed after a median of 46 months (range 1– 87), all associated with low ADAMTS13 levels. Rituximab was given in 7 cases of relapsing TTP and resulted in complete, durable clearance of the antibodies in 100%.

Determination of ADAMTS13-related parameters is useful to distinguish between autoimmune, hereditary, and secondary forms of TTP and to choose an appropriate therapy. It is also useful to predict the risk of relapse in patients with TTP in remission.
In Vitro Efficacy of Recombinant ADAMTS13 in Acquired TTP Patients with Anti-ADAMTS13 Inhibitory Antibody.
Authors: Plaimauer Barbara , Juno Claudia , Scheiflinger Friedrich
Published: April 2020

Thrombotic thrombocytopenic purpura (TTP) is associated with a severe functional deficiency of the ADAMTS13 metalloprotease resulting in accumulation of highly adhesive ultra-large von Willebrand factor (ULVWF) multimers. ULVWF promote spontaneous platelet clumping eventually leading to life-threatening widespread microvascular thrombosis in multiple organs. Inhibitory and non-inhibitory anti-ADAMTS13 autoantibodies that result in functional deficiency and/or accelerated in vivo clearance of ADAMTS13 have been detected in patients suffering from acquired idiopathic TTP. Current treatment with plasma exchange therapy is considered to remove the autoantibodies and to replenish plasma with the deficient protease. While researching therapeutic recombinant ADAMST13 (rADAMTS13) as a possible treatment modality for TTP patients, we investigated the in vitro potency of rADAMTS13 to overwhelm and neutralize circulating anti-ADAMTS13 antibodies and concurrently to reinstate plasma ADAMTS13 to normal. Mixing and incubating patient plasma with normal reagent plasma and subsequent FRETSVWF73 activity analysis was used to calculate the individual anti-ADAMTS13 inhibitor titers in 36 untreated idiopathic TTP patient plasma samples with severe ADAMTS13 deficiency (<10% of normal). The inhibitor titers (IU/ml) ranged from 1.5 to 85IU/ml (median of 3.75 IU/ml). To compare the performance of the different patient samples at equal inhibitor titers, we diluted each sample to predefined inhibitor titers (e.g. in patient samples with inhibitor titers <10IU/ml we diluted to 1, 3, 6 and 9IU/ml). Then, incrementally increasing concentrations of purified rADAMTS13 derived from stably transfected CHO cells were added to each pre-set inhibitor titer done for each of the 36 patient samples. Complex formation of rADAMTS13 and anti-ADAMTS13 patient antibody was allowed and the remaining ADAMTS13 activity was analyzed. The input of rADAMTS13 required to saturate circulating inhibitors and to elevate the activity level up to 1U/ml in the plasma was calculated by regression analysis. Our data indicate a linear correlation between supplemented rADAMTS13 and anti-ADAMTS13 antibody titer (for titers <10IU/ml) suggesting that rADAMTS13 should be further investigated as a possible treatment of acquired TTP.
Autologous Peripheral Blood Stem Cell Products from Patients with Hematologic Malignancies Have Increased Frequency of Regulatory T Cells (CD4+FoxP3+ Treg).
Authors: Kao Grace , Stevenson Kristen , Kim Elliot , Spanjaard Elena , Kim Haesook T , Ritz Jerome
Published: April 2020

Increased numbers of regulatory T cells (Treg) with immune suppressive function have been observed in patients with hematological malignancies. Many of these patients will undergo stem cell mobilization with high dose cyclophosphamide and G-CSF to prepare for autologous stem cell transplantation. Although infusion of low dose cyclophosphamide (Cy) seems to reduce the number of circulating Tregs, mobilization with high dose Cy and G-CSF was shown to increase the frequency of immunosuppresive Treg in stem cell autografts of myeloma patients. Treg populations have been defined by different phenotypic criteria, including CD4+FoxP3+ and CD4+CD25+ CD127lo. In this study, we compare the impact of donor types and stem cell mobilization regimens on CD4+FoxP3+ and CD4+CD25+ CD127lo Treg frequencies in 63 apheresis products.

Methods: Apheresis products from 23 healthy donors (17 donors received G-CSF and 6 had no stem cell mobilization) and 40 patients with hematological malignancies (1 AML, 2 HD, 25 NHL, 10 MM, 2 neuroblastoma) were evaluated. Patients with malignancies were mobilized with chemotherapy (5 RICE, 25 Cy, 1 Ara-c+Etoposide) + G-CSF (N=31), AMD3100 + G-CSF (N=5), or G-CSF alone (N=2). Samples from each product were incubated with fluorochrome-conjugated antibodies against cell surface makers, CD45, CD4, CD14, CD127, and CD25, followed by intracellular staining with anti-FoxP3 antibody. Six-color flow cytometry and sequential gating was performed for each sample to determine the frequency of CD4+Foxp3+ and CD4+CD25+CD127lo Treg population.

Results: A strong correlation was found between CD4+Foxp3+ and CD4+CD25+CD127lo Treg subsets (r=0.67, p<0.001).The percentage of both Treg populations was not different between mobilized or non-mobilized products from healthy donors (p=0.94) or between NHL and MM patients (p=0.81). Higher CD4+FoxP3+ Treg frequency was observed in autologous grafts from patients compared to healthy donors (p<0.001). The median CD4+FoxP3+ Treg frequency among mobilized products from patients and healthy donors were 8.5% (range, 1.8–38.9) and 4.3% (range, 1.8–15.0), respectively. Autografts from patients who received AMD3100+G-CSF compared to those who received chemotherapy + G-CSF also revealed higher CD4+FoxP3+ Treg content (median 11.3 vs. 7.5, p=0.04). Similar analysis using CD4+CD25+CD127lo Treg subset did not demonstrate any statistical significance.

Conclusions: Although correlation was observed between CD4+FoxP3+ and CD4+CD25+CD127lo subsets, CD4+FoxP3+ is a more sensitive marker for the Treg population in stem cell products. Higher CD4+FoxP3+ Treg frequency was observed in grafts from patients with hematological malignancies compared to healthy donors. The addition of a novel mobilization agent, AMD3100, to Cy mobilization may further increase the Treg content of autologous stem cell products.
Compared to Adult Peripheral Blood T Cells, Cord Blood T Cells Show Enhanced Immunological Recognition of Chronic Lymphocytic Leukemia Tumor Cells.
Authors: Ramsay Alan G. , Xing Dong-Xia , Decker William K. , Burks Jared K. , Wierda William G. , Gribben John G. , Shpall Elizabeth J.
Published: April 2020

Following allogeneic stem cell transplantation (SCT) and donor lymphocyte infusion (DLI) from adult peripheral blood (APB), chronic lymphocytic leukemia (CLL) cells are good targets of a graft-versus-leukemia effect. However, some patients eligible for this treatment do not have a suitable allogeneic donor and CLL B cells have been shown to be dysfunctional antigen-presenting cells (APCs) for allogeneic APB T cells. As a result, allogeneic APB T cells show suppressed immunological synapse formation with CLL cells. Umbilical cord blood (CB) is a promising source of hematopoietic cells for allogeneic transplantation and can be obtained from matched unrelated donors with greater tolerance for incompletely HLA-matched recipients. Moreover, we have successfully expanded CB T cells ex vivo (anti-CD3/CD28 beads and rIL-2) using a protocol that retains a naïve and diverse immune population including central memory cells. In this present study we used confocal microscopy to visualize F-actin polymerization to assess immunological synapse formation of CB T cells compared to APB T cells with CLL B cells with and without superantigen as APCs. Our results identify the ability of unexpanded and expanded CB CD4 and CD8 T cells to form F-actin immune synapses with CLL B cells and of note, CB was more effective than unexpanded or expanded APB T cells (p<0.05). Of interest, the expansion protocol maintained immune synapse formation with a trend towards increased F-actin polymerization. As control, we examined the ability of unexpanded and expanded T cells to form F-actin synapses with allogeneic healthy B cells with or without superantigen as APCs and found no significant difference between CB and APB as a source of T cells. Our results demonstrate that CB T cells have an enhanced ability to recognize CLL B cells as allogeneic APCs compared to APB T cells and provide important and exciting pre-clinical data for the potential use of expanded CB T cells in the setting of CB transplantation in CLL.
In Vivo Development of Murine NK Cells Early after Congeneic BMT: Effects of MHC Molecules, and the Administration of hIL-15.
Authors: Barao-Silvestre Isabel , Redelman Doug , Ames Erik , Weiss Jonathan , Sun Kai , Welniak Lisbeth , Ortaldo John , Wiltrout Robert , Murphy William
Published: April 2020

Natural Killer (NK) cells are lymphocytes of the innate immune system that are able to kill a variety of tumors and pathogen-infected cells without prior sensitization. NK cells are the first lymphoid cells to repopulate following bone marrow transplantation (BMT). The present study shows that murine NK cells developing in vivo after BMT are phenotypically and functionally distinguishable from mature adult NK cells. Using a congeneic BMT mouse model (CD45.1/H-2b → C57BL/6, CD45.2/H-2b) we demonstrated here that donor derived BM-NK progenitors (CD122+NK1.1-CD3-)were present in the BM and periphery (spleen), at day 14 after BMT, and developed into NK cells expressing high levels of CD122, NKG2D, CD94, DX5 and CD43. However, surface expression of Ly49 inhibitory receptors was altered with Ly49C/I being under-expressed (9.8 %± 3.2 %), and Ly49G2 being highly expressed (62 %± 3.1 %) when compared to NK cells from non-transplanted adult mice (Ly49C/I, 43.1 %± 7.6 % and Ly49G2, 44.3 %± 13.5 %). At 28 days after BMT, there was a normalization with regard to Ly49 phenotype. The developing NK cells manifested functional deficits compared to non-transplanted adult NK, even when stimulated in vitro with Interleukin (IL)-2, as evidenced by decreased interferongamma (IFN-g) production and cytolytic activity. In order to examine the effects of major histocompatibility complex (MHC) molecules on NK development independently of allelic variation of Ly49s, we performed syngeneic BMT using B10D2 mice (H-2d). We observed the same pattern of Ly49s expression during NK development demonstrating that the acquisition of these receptors early after BMT is not determined by the MHC haplotype. Because IL-15 plays a crucial role in NK cell development, we administrated human (h) IL-15, via hydrodynamic injection of the respective gene, at day 11 after BMT, to accelerate NK maturation. This method resulted in a high but transient production of hIL-15 (736 ± 41 pg/ml) in the serum for up to 2 weeks. A subsequent but temporary increase in the numbers of donor derived NK cells (3 fold) occurred within 72 hours, in both BM and periphery, following hIL-15 gene delivery. Despite these augmenting effects, the developing NK cells still exhibited poor functionality compared to non-transplanted adult mice. These studies describe the possibility of NK development in the spleen, and indicate that normal surface expression of Ly49C/I and Ly49G2 early after BMT is time dependent, but MHC independent. Moreover, administration of hIL-15 is not sufficient to induce NK functional maturation early after BMT despite increased NK numbers.
Pharmacological Inhibition of EGFR Signaling Enhances G-CSF Induced Hematopoietic Stem Cell Mobilization.
Authors: Ryan Marnie A. , Xing Ellen , Schleimer David , Nattamai Kalpana , Daria Deidre , Liu Wei , Jansen Michael , Ratner Nancy , Le Cras Timothy , Van Zant Gary , Zheng Yi , Geiger Hartmut
Published: April 2020

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into peripheral blood by the cytokine G-CSF has become the preferred source of HSPCs for clinical stem cell transplants. However, up to 10% of donors fail to mobilize sufficient numbers of stem cells impeding autologous transplants or significantly delaying transplant recovery time. Consequently, novel regimens are warranted to increase the number of stem cells in peripheral blood upon mobilization. Using a forward genetic approach in the mouse, we map the epidermal growth factor receptor (EGFR) to a genetic region on murine chromosome 11 modifying G-CSF-mediated HSPC mobilization. Expression levels of EGFR in HSPCs were inversely correlated with HSPC mobilization, implying a negative role for EGFR signaling in mobilization. Genetic reduction of EGFR activity (waved2 mice) or treatment with the EGFR inhibitor erlotinib increased stem cell mobilization up to 5-fold in combination with G-CSF. Increased mobilization due do alteration of EGFR activity correlated with reduced activity of Cdc42 and consequently, inhibition of Cdc42 activity in vivo by a specific Cdc42 inhibitor similarly enhanced mobilization. Our findings reveal a novel signaling pathway regulating stem cell mobilization and thus provide new rationale for targeted pharmacological approaches to further improve HSPC mobilization and thus transplantation outcomes.
Donor Bone Marrow Derived IL-17 Expressing Cells Exacerbate Chronic Graft-Versus-Host Disease in a Murine Bone Marrow Transplantation.
Authors: Kuroda Rie , Nishimura Ryosei , Sato Katsuaki , Maeba Hideaki , Naka Kazuhito , Niida Yo , Araki Raita , Iwakura Yoichiro , Koizumi Shoichi , Yachie Akihiro
Published: April 2020

Th17 is a newly identified T cell lineage that secretes the proinflamatory cytokine IL-17. Th17 cells have been shown to play a crucial role in mediating autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), arthritis, and colitis. However, recent study showed that donor Th17 cell ameliorated acute graft-versus-host disease (GVHD) due to the downregulation of Th1 differentiation in a murine BMT model. The role of IL-17 on developing chronic GVHD, which is pathologically similar to autoimmue diseases, remains unanswered. To this end, we compared the development of chronic GVHD between the lethally irradiated mice (Balb/c, H-2d) given IL-17 knockout(IL- 17KO, C57BL/6 background, H-2b) bone marrow (BM) cells and wild type BM cells with low dose WT splenocytes to induce sublethal acute GVHD and chronic GVHD subsequently. Up to day 60 after BMT no significant differences in clinical symptoms of GVHD including body weight changes were observed between the mice co-injected with IL-17 KO BM cells plus WT splenocytes and WT BM cells plus WT splenocytes. After day 60 the mice receiving WT BM cells plus WT splenocytes experienced weight loss accompanied by skin inflammatory changes, while mice receiving IL-17KO BM plus WT splenocytes showed minimal signs of GVHD as well as mice receiving IL-17 KO BM or WT BM alone. Recovery of body weight on day 160 after BMT was statistically different between two groups (p=0.035). Taken together, IL-17 was exacerbation factor of chronic GVHD, but not acute GVHD. Next we further evaluated the percentage of Th17 cells derived from BM cells (not from infused splenocytes) in the spleen on day 160 after BMT. Percentages of IL-17 expressing cells in the mice receiving WT BM plus WT splenocytes in spleen were significantly higher than those of WT BM alone receiving mice in CD4 (p=0.03) subpopulations. In conclusion, Th17 cells critically involved in the pathogenesis of chronic GVHD. Neutralizing IL-17 would be potent strategy for preventing chronic GVHD.

Figure Figure
Economic Evaluation of Thalidomide Combined with Melphalan and Prednisone in Previously Untreated Multiple Myeloma in Scotland
Authors: Deniz Baris , Facon Thierry , Singer Ian , Micallef-Eynaud Paul , Joseph Ian , Shearer Arran , Caro J. Jaime
Published: April 2020

INTRODUCTION: Thalidomide (Thalomid®/Thalidomide Pharmion®) combined with melphalan and prednisone (MPT) yields improved progression-free and overall survival compared to MP alone (Facon et. al., Lancet2007; 370:1191). This study was designed to estimate the life-time health and cost consequences of MPT versus MP in Scottish patients with previously untreated multiple myeloma.

METHODS: A Markov model was developed to determine cost and health outcomes for a cohort of patients receiving a course of MPT or MP. The disease course was conceptualized by 4 mutually exclusive health states: pre-progression without adverse events, pre-progression with adverse event, progressive disease, and death. Probabilities of moving between these states (i.e. natural progression plus efficacy and safety of the treatments) were derived from a long-term randomized clinical trial, IFM 99-06 (Facon et. al., Lancet2007; 370:1191). Both patient cohorts remained on the assigned treatment for a maximum of twelve 6-week cycles, until progression or treatment-limiting toxicity. Treatment duration and the average daily dose were modelled to match IFM 99-06. During treatment, each cohort was exposed to adverse event risks associated with therapy estimated from IFM 99-06. Health state utilities associated with adverse events and disease states were obtained from the literature. Thalidomide cost was set at UK list price; routine disease-management costs by disease-state (progressive disease and remission state) reflect clinical practice in Scotland. As recommended by the UK treasury, costs and health outcomes were discounted at 3.5% per annum to adjust to present values. Univariate and multivariate sensitivity analyses were performed around key model parameters.

RESULTS: The model estimated improvements in health outcomes with MPT with a median time to progression of 25 months vs. 12 months with MP. Estimated median overall survivals were 4.03 years vs. 2.88 years with MP. These results translate to a gain of 0.91 (3.24 vs. 2.32) quality-adjusted life-years (QALYs). MPT is associated with higher overall costs (£25,199 per patient) compared with MP (£8,935), over the modeled life-time, leading to an incremental cost-effectiveness ratio of £17,847 per QALY and £14,803 per life-year gained. Sensitivity analyses showed that results remained consistent through broad changes in model parameters including the addition of thromboembolic prophylaxis.

CONCLUSIONS: MPT delivers improvements in progression-free and overall survival in a life-limiting orphan disease compared to MP and economic results fall within a range considered cost-effective in Scotland.
Cost-Effectiveness of Adding Imatinib to Chemotherapy in Adult Patients with Chromosom-Positive Acute Lymphoblastic Leukemia (Ph+ALL): A Canadian Perspective
Authors: Ouagari Khalid El , Tran Kimbach , Stephens Jennifer , Botteman Marc
Published: April 2020

BACKGROUND: Ph+ALL is a rare, high-risk, aggressive form of acute leukemia, affecting primarily adults and the elderly. Patients with high-risk forms of ALL typically have extremely poor prognosis and incur high disease-related costs. Successful use of imatinib in patients with Ph+CML has led to the administration of imatinib in recent clinical studies for patients with Ph+ALL. Given the changing treatment landscape of Ph+ALL and the recent development of additional targeted treatments for this disease, an appraisal of the economic burden associated with Ph+ALL is warranted for evaluation of appropriate treatment options and the potential value associated with novel therapies. This study explores the cost effectiveness of imatinib plus conventional chemotherapy (CC) regimens versus CC alone in adult Ph+ALL patients.

METHODS: A Markov model simulated a hypothetical cohort of 1000 adult Ph+ALL patients receiving imatinib+CC or CC alone. Patients were distributed over time into three health states: alive without disease progression (DFS), alive with disease progression (DS), or dead. Probabilities of being in the states were derived from the published literature. In the absence of relevant data pertaining to Ph+ALL, assumptions about costs and utilities were derived from a cost analysis of CML. A Canadian health care payer perspective was considered, therefore only direct medical costs were included in the analysis. Patients were followed for a total of ten years in monthly intervals. All outcomes were discounted at a 5% rate per annum.

RESULTS: Based on the model framework and assumptions, the total discounted survival was 1.09 years for CC and 4.13 years for imatinib+CC. Total discounted disease free survival was 0.76 year for CC and 2.69 years for imatinib+CC. Assuming utility weights of 0.854 and 0.596 for DFS and DS, respectively, the total discounted quality adjusted life years (QALY) were estimated to be 0.84 versus 3.16 for CC and imatinib+CC, respectively. Thus, the net incremental gain in discounted quality adjusted survival was 2.32 QALYs. The monthly costs of DFS and DS were estimated at $168 and $848, respectively. The net costs associated with imatinib were $101,929. The incremental cost per QALY of imatinib+CC v. CC alone was approximately $44,048 (i.e., $101,929 divided by 2.32 QALYs).

CONCLUSIONS: Our results suggest that imatinib in the treatment of Ph+ALL is a cost-effective use of health-care resources, and should be considered in patients who are appropriate candidates for such therapy.
MicroRNA Mir-23a Regulates SDF-1alpha Expression in Human Bone Marrow-Derived Mesenchymal Stem Cells
Authors: Fierro Fernando , Poitz David , Nolta Jan A. , Bornhaeuser Martin , Ehninger Gerhard , Illmer Thomas
Published: April 2020

MicroRNAs (miRNAs) can regulate hematopoietic stem/progenitor cells (HSPC) by modulation of intrinsic cell components such as transcription factors and receptors. In addition, miRNAs could play a role in the microenvironment were HSPC host and consequently affect HSPC via extrinsic factors, which to our knowledge is an hypothesis that has not been tested. Since the chemokine stromal derived factor 1alpha (SDF-1alpha) is essential for both homing and retention of HSPC in the bone marrow, we tested if miRNAs expressed by human bone marrow-derived mesenchymal stem cells (MSC; a key source of SDF-1alpha), could potentially inhibit SDF-1alpha expression. In deed, using luciferase reporter-systems we show specific binding of miR-23a to the 3′UTR of SDF-1alpha. Consequently, transfection of MSCs with a precursor miR-23a (pre-miR-23a) leads to a 30% reduction of SDF-1alpha at both mRNA and protein levels. In contrast, inhibition of endogenous miR-23a with anti-sense oligonucleotides (anti-miR-23a), leads to a significant increase of SDF-1alpha also at both mRNA (30%) and protein (10%) levels as compared to controls (scramble pre-/anti-miR). As a result, migration of CD34+ HSPC in transwell assays is strongly affected upon overexpression or inhibition of miR-23a in MSCs (with pre-miR-23a 35% less migration; with anti-miR-23a 20% more migration, as compared to respective controls). Interestingly, transforming growth factor beta 1 (TGF-beta1) inhibits SDF-1alpha expression in MSCs in a concentration dependent manner, while miR-23a is increased under same experimental settings. Even more, silencing endogenous miR-23a significantly reduces the effect of TGF-beta1 on SDF-1alpha mRNA and protein levels, suggesting that at least in part TGF-beta1 inhibits SDF-1alpha expression via increasing miR-23a levels. This is to our knowledge the first established connection between miRNA biology and HSPC-niche related factors.
Hematopoietic Stem Cells in the Mouse Spleen
Authors: Iseki Akiko , Morita Yohei , Nakauchi Hiromitsu , Ema Hideo
Published: April 2020

The spleen is a hematopoietic organ in mice. Hematopoietic stem cells (HSCs) migrate into the spleen around embryonic day 14, and then migrate into the bone marrow (BM) around embryonic day 17. Thereafter, HSCs reside in both BM and spleen throughout the life of a mouse. The spleen is the major site of extramedullary hematopoiesis in pathological conditions. The spleen serves as an active hematopoietic organ in lethally irradiated mice for a while after transplantation with BM cells. Osteoblasts are considered to be one of the stem cell niche components. Because there are no osteoblasts in the spleen, niches in the spleen possibly functions differently from ones in the BM. The regulation of HSCs likely differs between the BM and spleen. In order to understand a role of spleen HSCs in physiological conditions, we have characterized HSCs in the spleen as compared with those in the BM.

BM and spleen cells were obtained from 8–10 week-old C57BL/6 mice. Competitive repopulation showed that the repopulating activity per 106 BM cells was significantly greater than that per 106 spleen cells (about 10-fold). Limiting analysis showed that the frequency of long-term repopulating cells in BM cells was significantly higher than that in spleen cells (about 10-fold). As a result, the mean activity per BM stem cell was similar to that per spleen stem cell. Similarly to BM, CD34-negative, c-Kit-positive, Sca-1-positive, lineage markers-negative (CD34−KSL) cells were highly enriched in HSCs in the spleen. The frequency of CD34−KSL cells in the spleen was significantly lower than that in the BM. These data indicate that functionally equivalent HSCs exist in the spleen but at a low frequency. Data from single cell-transplantation supported this notion. The proportion of pyronin Y-negative G0 cells among BM CD34−KSL cells was greater than that among spleen CD34−KSL cells at any one time. BrdU-uptake analysis showed that spleen CD34− KSL cells were cycling more rapidly than BM CD34−KSL cells. These data suggest that spleen HSCs contribute to hematopoiesis to some extent under physiological conditions. BM and spleen HSCs may be interchangeable via the circulation. When BM HSCs are in the spleen, they are possibly under the control of spleen niches different from BM niches.
Integrated Biochemical and Computational Approach Identifies BCL6 Direct Target Genes Controlling Multiple Pathways in Normal Germinal-Center B Cells
Authors: Saito Masumichi , Basso Katia , Sumazin Pavel , Margolin Adam A , Wang Kai , Lim Wei-Keat , Califano Andrea , Dalla-Favera Riccardo
Published: April 2020

BCL6 is a transcriptional repressor required by mature B cells for germinal center (GC) formation, whose deregulated expression, through mutations and translocations, is implicated in lymphomagenesis. The normal function of BCL6 is only partially known since a limited number of direct target genes have been identified by the analysis of cell lines derived from GC-derived lymphomas. However, the complete set of targets functionally relevant for the role of BCL6 in normal GC B cell physiology has not been completely uncovered and the possibility that BCL6 function may be altered in transformed cells has not been excluded. To address these issues, we used an integrated biochemical/computational/functional approach including:

identification of BCL6-bound promoters by genome-wide chromatin immunoprecipitation (ChIP-chip) in normal human GC B cells; a computational algorythm inferring transcriptional relationships starting from gene expression data (ARACNe, Basso et al. Nature Genetics 2005); validation of physiologic relevance of the candidate target genes by selection of those either not expressed in normal GC B cells or downregulated compared to pre- and post- GC B cells.

This approach identified a set of 1,207 genes which were then subjected to pathway analysis using several databases. The results showed that BCL6 regulates important signaling pathways via direct transcriptional repression of multiple genes acting at different levels from the cell surface (receptors), through signaling molecules to the nucleus. These pathways include:

apoptosis, by impairing the expression of both pro- and anti-apoptotic molecules including several TNF-type receptors, signaling molecules (e.g. TRADD, A20, XIAP, TOSO) and effectors (e.g. CASP8); JAK-STAT signaling, through the repression of multiple interleukin and interferon receptors and the transcription factors STAT1, STAT3 and STAT5A; B cell receptor signaling, via repression of signaling molecules (e.g. lyn, vav), several MAP kinases, and transcription factors (e.g. JUN and NF-kB components); Toll-like receptor signaling, by regulating receptors (e.g. TLR1, 7 and 9); DNA damage sensing and response (e.g. TP53BP1, ATM).

Overall, these results provide a comprehensive picture of BCL6 function suggesting that one role of BCL6 is to prevent GC centroblasts from receiving activation and differentiation signals before completion of the phase of proliferative expansion and somatic hypermutation leading to their selection based on antibody affinity maturation.
The HaemAtlas: Characterising Gene Expression in Differentiated Human Blood Cells
Authors: Watkins Nicholas A. , Tijssen Marloes R. , Gusnanto Arief , de Bono Bernard , De Subhajyoti , Miranda-Saavedra Diego , Angenent Will , Attwood Anthony , Langford Cordelia , Erber Wendy N. , Foad Nicola , Garner Stephen F , Rendon Augusto , Rice Catherine , Thijssen-Timmer Daphne , Van der Schoot C. Ellen , Dudbridge Frank , Teichmann Sarah , Gottgens Bertie , Ouwehand Willem H.
Published: April 2020

Haematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are, in part, controlled by signals resulting from ligand binding to cell surface receptors. In order to further understand haematopoiesis, we have compared gene expression profiles of human erythroblasts, megakaryocytes, B-cells, cytotoxic and helper T-cells, Natural Killer cells, granulocytes and monocytes using whole genome microarrays. A bioinformatics analysis of this data was performed focusing on transcription factors, immunoglobulin superfamily members and lineage specific transcripts. We observed that the numbers of lineage specific genes varies by two orders of magnitude, ranging from five for cytotoxic T cells to 878 for granulocytes. In addition, we have identified novel co-expression patterns for key transcription factors involved in haematopoiesis (eg. GATA3–GFI1 and GATA2–KLF1). This study represents the most comprehensive analysis of gene expression in haematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data, which is freely accessible, will be invaluable for future studies on haematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies.