bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of New Upregulated Genes with Possible Relevance for Multiple Myeloma Tumorigenesis
Authors: Felix Roberta Spetic , Colleoni Gisele W. B. , Caballero Otavia L. , Almeida Manuella Sampaio , Andrade Valeria C.C. , Chauffaille Maria L. L. F. , Silva Wilson A. , Begnami Maria Dirlei , Soares Fernando Augusto , Zago Marco Antonio , Vettore Andre Luiz
Published: April 2020

Serial analysis of gene expression (SAGE) allows a comprehensive profiling of gene expression within a given tissue and also an assessment of transcript abundance.

Objectives: We generated SAGE libraries from normal and neoplastic plasma cells to identify genes differentially expressed in multiple myeloma (MM).

Material and Methods: Normal plasma cells were obtained from palatine tonsils and MM SAGE library was generated from bone marrow plasma cells of MM patients.

Results: We obtained 29,918 SAGE tags from normal and 10,340 tags from tumor libraries. Computer-generated genomic analysis identified 46 upregulated genes in the MM library. Ten upregulated genes were selected for further investigation. Differential expression was validated by quantitative real-time PCR in purified plasma cells of 31 patients and three controls. P53CSV, DDX5, MAPKAPK2, RANBP2 were found to be upregulated in at least 50% of the MM cases tested. All of them were also found upregulated in MM when compared to normal plasma cells in a meta-analysis using ONCOMINE microarray database. Antibodies specific to DDX5, RANBP2 and MAPKAPK2 were used in a TMA containing 57 MM cases and confirmed the expression of these proteins in 74, 96, and 21% of the MM samples, respectively.

Conclusions: Analysis of differential expression using SAGE could identify new genes important for myeloma tumorigenesis (P53CSV, DDX5, MAPKPK2 and RANBP2) and that could potentially be useful as therapeutic targets.
CCR1 Inhibition Targets Osteoclast-Myeloma Adhesion and Exerts in Vivo Anti-Osteoclast Activity
Authors: Vallet Soni a , Vaghela Nileshwari , Fulciniti Mariateresa , Veiby Petter , Hideshima Teru , Pozzi Samantha , Santo Loredana , Mukherjee Siddhartha , Cirstea Diana , Scadden David T. , Anderson Kenneth C. , Raje Noopur
Published: April 2020

Among the components of the tumor microenvironment, osteoclasts (OC) and bone marrow stromal cells (BMSC) play a key role in stimulating multiple myeloma (MM) cell proliferation via cell-to-cell contact and cytokine secretion. In a vicious loop, MM cells promote OC development and impair osteoblasts (OB) differentiation shaping the microenvironment to support tumor growth and chemoresistance. These effects translate in osteolytic lesions that negatively affect patient quality of life and survival. CCL3 is involved in the pathogenesis of MM bone disease. Indeed, high bone marrow plasma levels of CCL3 correlate with osteolytic lesions in MM and CCL3 has a pro-osteoclastogenic effect. We have previously shown that inhibition of CCL3 receptor, CCR1, results in impaired osteoclastogenesis and blocks the proliferative advantage conferred by OCs to MM cells in vitro. (Vallet et al, Blood 2007) Here, we extend our studies and demonstrate in an in-vivo SCID-hu mouse model the anti-OC effects of MLN3897, a small molecule CCR1 inhibitor (Millennium Pharmaceuticals, Cambridge). CB17 SCID mice bearing a human fetal bone implant engrafted with INA6 MM cells were treated orally with MLN3897 for a total of 49 doses. After 4 weeks of treatment the bones were harvested and stained for TRAP activity and hematoxylin-eosin. The number of OC/400x field was significantly reduced in the treated group (2.7 vs 1.9, p<0.05), thus confirming in vivo the anti-osteoclastogenic effect of CCR1 inhibition. Although a 2-fold inhibition of tumor growth in the treated group was noted, this difference did not reach statistical significance. Our previous data suggested that CCR1 inhibition impaired OC-MM cell adhesion with subsequent reduced MM cell proliferation. Here, we observed a specific effect of CCR1 inhibition on MM cell/OC adhesion rather than BMSCs and fibronectin. Interestingly, these effects translated in anti-proliferative effects of the CCR1 inhibitor in OC-MM cells coculture with no effects in BMSCs-MM coculture, suggesting a specific inhibitory effect on the OC compartment. Ongoing studies to characterize the underlying mechanism of CCR1 inhbition on OC-MM cell adhesion will be presented. Combination approaches to study whether forced OB differentiation by the proteasome inhibitor, bortezomib, will improve BMSC sensitivity to CCR1 inhibition are being studied. These data confirm the specific in vivo anti-OC effects of CCR1 inhibition and will highlight the role of novel combinations by using anti-OC agents like MLN3897 and anabolic drugs like Bortezomib.
Diffuse Large B-Cell Lymphoma (DLBCL) Patients with Composite Histology Have Improved Early Outcome Compared to De Novo DLBCL When Treated with R-CHOP
Authors: Maurer Matthew J. , Witzig Thomas E. , Macon William R. , Syrbu Sergei I. , Cerhan James R. , Allmer Cristine , Johnston Patrick B. , Link Brian K. , Habermann Thomas M.
Published: April 2020

Background: A significant percentage of DLBCL patients present with a composite histology, often seen as a node containing both follicular lymphoma and DLBCL or DLBCL in the node and discordant indolent lymphoma in the bone marrow. Literature from the pre-rituximab era suggests DLBCL patients with transformed lymphoma or composite histology have worse outcome than de novo DLBCL. Here we report on early events in a cohort of R-CHOP treated patients.

Goal: To determine whether patients with composite lymphoma have an inferior event free survival (EFS) and overall survival (OS) compared to de novo diffuse large B-cell lymphoma when treated with R-CHOP.

Methods: Newly diagnosed patients treated with an R-CHOP containing regimen were prospectively enrolled in our Lymphoma SPORE registry from 9/2002 through 6/2007. Pathology was centrally reviewed. All patients were followed for retreatment, disease progression, and death.

Results: 401 DLBCL patients were enrolled; 14% (57/401) had a composite histology. 33 patients had DLBCL and another histology, predominantly follicular lymphoma (n=29), in the same node. 20 patients had a non-DLBCL histology in a distinct location from the DLBCL; this was primarily indolent lymphoma in the bone marrow (n=15). 4 patients had both. 19% (75/401) of patients died during follow-up and 30% (121/401) had an event (death due to any cause, progression, or retreatment). Median follow-up for living patients was 34 months (range, 5–73). Composite DLBCL patients had higher event-free (3 year EFS = 79%) and overall (3 year OS = 93%) survival then de novo DLBCL (3 year OS = 66%, 3 year EFS 79%), p=0.05 and p=0.005 respectively. These differences remained statistically significant after adjusting for the International Prognostic Index (IPI): EFS HR = 0.53, 95% CI: 0.29–0.97, p=0.02; OS HR=0.28, 95% CI: 0.10–0.76, p=0.002. OS and EFS for composite DLBCL more closely resembled R-CHOP treated grade III follicular lymphoma (A,B) from the same cohort (3 year EFS = 81%, 3 year OS = 93%). Improved outcome for composite DLBCL was consistent whether the additional histology was in the same node or distinct from the DLBCL.

Conclusions: R-CHOP treated DLBCL patients with indolent discordant bone marrow involvement or other composite histology have improved early OS and EFS compared to de novo DLBCL. Further follow-up is needed to assess the long-term prognosis of composite DLBCL in the rituximab era.

Histology N Age > 60 Stage III/IV LDH > ULN PS > 1 >2 EN Sites 3 YR EFS 3 YR OS * Denotes statistically significant difference at p=0.05 de novo DLBCL 344 58% 56% 56% 17% 22% 66% 78% Composite DLBCL 57 65% 77%* 34%* 18% 32% 79%* 93%* Figure Figure
T-Cell Receptor Signaling Proteins, BCL10 and Phosphorylated-P65 NFKB, Are Expressed in Peripheral T-Cell Lymphomas and Associated with Clinical Outcome
Authors: Briones Javier , Espinosa Iñigo , Delgado Julio , Sierra J.
Published: April 2020

Background: Peripheral T-cell lymphomas (PTCL) are characterized by T-cell receptor (TCR) signaling alterations and NF-kB activation. Protein kinase C (PKC) is a family of proteins involved in signal transduction and mediates activation of the NF-kB pathway. In normal T cells, PKC 𝛉 plays a major role in TCR-mediated activation of a novel NF-kB pathway that involves phosphorylation of p65 at Serine 536 (P-p65Ser536). BCL10 acts along the same pathway downstream of PKC 𝛉 to activate NF-kB. We thus investigated the relationship between PKC 𝛉, BCL10 and P-p65Ser536 status in biopsy specimens from patients with PTCL.

Methods: Paraffin-embedded tissues obtained at diagnosis from 30 patients with nodal PTCL (PTCL unspecified, 21cases; angioimmunoblastic, 3; ALK− anaplastic large-cell, 6) treated with curative intention were evaluated. Expression of PKC 𝛉, BCL10, and P-p65Ser536 proteins were assessed using immunohistochemistry on a tissue microarray.

Results: Expression of PKC 𝛉 was detected in 22 of 30 cases (73%), BCL10 in 20 of 30 (67%), and P-p65Ser536 in 21 of 30 (70%). Expression of BCL10 was associated with PKC 𝛉 (18 of 22) (P<0.0001), and P-p65Ser536 (19 of 21) expression (P<0.0001). After a median follow-up of 60 months for surviving patients (range, 21–160 months), 5-year overall survival (OS) was 33%. Patients with BCL10 or P-p65Ser536 positive tumors fared better, with a 5-year OS of 48% and 45%, respectively, versus 0% for those with negative tumors (P=0.029, and P=0.04, respectively). Remarkably, BCL10 expression identified a subgroup of patients within the low Ki-67 group (<80% positive tumor cells) with a different outcome; thus, within this subgroup patients with BCL10 positive tumors had a 5-year OS of 85% while it was 0% for those with BCL10 negative tumors (P=0.011). Multivariate analysis showed that BCL10, Ki-67, and P-p65Ser536 expression were independent factors associated with OS (P=0.039, 0.029, and 0.050, respectively).

Conclusion: BCL10 is expressed in PTCL, correlates with PKC 𝛉 and P-p65Ser536 NF-kB expression and it seems to be associated with improved survival in PTCL.
Clearance Mechanisms for Chilled Platelets
Authors: Rumjantseva Viktoria , Josefsson Emma C , Grewal Prabhjit K , Wandall Hans H. , Neyeb-Hashemi Sara , Larson Goran , Marth Jamey D , Hartwig John H. , Hoffmeister Karin M.
Published: April 2020

When platelets are chilled for short times (<4 hours), βN-acetylglucosamine (βGlcNAc) residues of N-linked glycans on clustered GPIbαmolecules are recognized by the lectin domain of αMβ2 receptors on hepatic macrophages, and are removed from circulation. Clearance can be prevented by galactosylation of the exposed βGlcNAc residues. Galactosylation, however, does not improve the circulation of platelets refrigerated for longer times (>48 h-refrigerated) (Wandall et al. Blood, 2008). Investigating the clearance mechanism of 48 h-refrigerated platelets, we now demonstrate macrophage-independent hepatic removal, that is mediated by the Ashwell-Morell asialoglycoprotein receptor (AMR). This conclusion is based on the following evidence:

macrophage depletion in mice using clodronate encapsulated liposomes, a procedure that dramatically improves the circulation of 4 h chilled platelets, does not prevent the clearance of 48 h chilled platelets; streptavidin-POD staining reveals abundant long-term refrigerated and biotinylated platelets in hepatocytes; 48 h-refrigeration increases by ~1.7-fold the binding of the βgalactose-recognizing lectin RCA, but not of the βGlcNAc recognizing lectin sWGA lectin to platelets; KO mice lacking Asgr-1 or Asgr-2 subunits of the AMR support 48 h-refrigerated platelet circulation times comparable to room temperature stored platelets; Co-injection of asialofetuin, a competitive inhibitor of the asialoglycoprotein-receptors, restore the survival of 48 h-refrigerated platelets in WT mice; and hepatocyte HepG2 cells ingest fluorescently labeled long-term refrigerated platelets in culture, and asialofetuin prevents this ingestion.

Regarding the platelet target of the Asgr-1/Asgr-2 receptors, circulation of 48 h-refrigerated platelets is markedly improved by removal of GPIbα’s N-T domain using O-sialopeptidase. We conclude that longterm refrigeration exposes galactose residues on GPIbα subunits of platelets to generate ligands recognized by hepatocyte Ashwell-Morell receptors.
Engagement of Fibrinogen and β3 Integrin Is Required for Maintenance of Platelet Intracellular and Cell Surface P-Selectin Expression
Authors: Yang Hong , Lang Sean , Zhai Zhimin , Spring Christopher M. , Reheman Adili , Chen Pingguo , Flick Matthew J. , Degen Jay L. , Li Ling , Kahr Walter H.A. , Freedman John , Ni Heyu
Published: April 2020

Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. While it has been reported that von Willebrand factor (VWF) affects P-selectin expression on endothelial cells, little information is available regarding regulation of platelet P-selectin expression. Here, we first observed that P-selectin expression was significantly decreased on platelets of fibrinogen and VWF double deficient mice. Subsequently, we identified this was due to fibrinogen, but not VWF, deficiency. The impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in a human patient with severe hypofibrinogenemia and his heterozygous parents. We demonstrated that this impairment is unlikely due to excessive P-selectin shedding, deficient fibrinogen-mediated cell surface P-selectin binding, or impaired platelet granule release, but rather is due to decreased platelet P-selectin content. This effect seems to be specific for P-selectin, as other alpha-granule proteins (e.g. thrombospondin-1, vitronectin, platelet factor 4) were not decreased in fibrinogen-deficient mouse or human platelets. We found that fibrinogen transfusion recovered the P-selectin expression in fibrinogen-deficient mice to >80% of the normal level within 48 hours, and four days post-transfusion, the P-selectin levels were nearly completely recovered. Furthermore, engagement of the C-terminus of the fibrinogen γ chain with β3 integrin was required for this process since a similar impairment of P-selectin expression was also observed in fibrinogen γΔ5 mice and β3 integrin-deficient mice. To determine whether fibrinogen affects the biogenesis of platelet alpha-granules, platelets were examined via electron microscopy. No statistically significant difference in the number of platelet alpha-granules was observed in fibrinogen-deficient or β3 integrin-deficient mice compared to controls (P>0.05). These data suggest fibrinogen may play important roles in inflammation, including immune-mediated inflammation, thrombosis and hemostasis via enhancement of platelet P-selectin expression. Furthermore, there are 2–3 fold variations in human plasma fibrinogen levels in healthy populations, and there are patients with hypo- and afibrinogenemia, hyperfibrinogenemia, and Glanzmann’s Thrombasthenia (β3 integrin deficiency). Our data suggest that platelet P-selectin expression can be affected by all these conditions. Therefore, P-selectin as a platelet activation marker should be used with caution.
Oct4 Expression and Mesenchymal Stromal Cell Plasticity
Authors: Yannarelli Gustavo , Jiang Huijie , Montanari Sonia , Helke Simone , Wang Xinghua , Keating Armand
Published: April 2020

Mesenchymal stromal cells (MSCs) have the capacity to differentiate along multiple lineages and are now in cell therapy clinical trials, especially for injured myocardium. Mechanisms mediating tissue regeneration remain unclear and are likely multifactorial. We recently showed that bone marrow-derived murine MSCs can acquire cardiac markers but retain MSCs properties when co-cultured with rat embryonic cardiomyocytes (RECs) (Rose et. al., Stem Cells. Aug7, 2008). The aim of our study was to determine whether expression of the embryonic transcription factor, Oct4 was modulated in this model of MSC plasticity. Wild-type Fvb mouse MSCs (passage 4) were co-cultured with RECs for 5 days and expression of Oct4, Nanog and Sox2, was analyzed by qRT-PCR with mouse-specific primers. Oct4 protein was assessed by immunocytochemistry (ICC) and flow cytometry (FC). The MSCs expressed Oct4, Nanog, and Sox2 transcripts. After co-culture, mRNA levels for Oct4 and Sox2 were upregulated 2.6±1.2-fold (p<0.05) and 2.4±0.4-fold (p<0.05), respectively, compared with MSCs controls, in contrast to Nanog gene expression which remained unchanged (1.2±0.3-fold; p=ns). 83±9% of MSCs nuclei were positive for Oct4 by ICC. To distinguish mouse from rat cells in co-culture, cells were stained with an anti-mouse CD44 antibody which does not cross react with rat CD44. CD44 is expressed on all MSCs and absent on RECs. Flow cytometry showed that Oct4 was over-expressed in CD44+ cells after co-culture. Our data demonstrate that these embryonic transcription factors are constitutively expressed in murine MSCs and that Oct4 and Sox2 transcript levels are increased after co-culture with RECs. Oct4, Nanog and Sox2 are known to maintain pluripotency of embryonic stem cells. Moreover, induced pluripotent stem cells can be generated from mouse fibroblasts by the introduction of Oct4 and Sox2 without the need for Nanog (Cell126:663,2006). Our data infer that embryonic transcription factors are involved in MSCs progression to different cell lineages. Our findings suggest new mechanisms that may mediate MSC plasticity.
IFN-Alpha and gm-CSF Can Reverse Immunomodulatory Effects of Imatinib
Authors: Bund Dagmar , Buhmann Raymund , Kolb Hans Jochem
Published: April 2020

Presently, Imatinib (IM) is the first line therapy for chronic myeloid leukaemia (CML), but in most patients CML recurs after discontinuation of IM. In contrast, allogeneic stem cell transplantation (ASCT) is considered to be the only curative treatment for this malignancy curing by the immune effects of donor T-cells against CML progenitor cells. Recent reports have described that IM has inhibitory effects on both the function of T-cells and antigen presenting cells (APCs) like dendritic cells. In the present study we further characterized the immunomodulatory effects of IM on T-cells and APCs and analysed whether IFN-alpha (interferon alpha, IFN-a) alone or in combination with GM-CSF (granulocyte-macrophage colony-stimulating factor) can influence the negative IM effects in vitro. Patient derived CML cells were examined to stimulate allogeneic HLA-mismatched T-cells without and with Imatinib (1, 2 or 5 micro M). The activation profile (CD25) and the expression of the TCR-alpha (T-cell receptor) on the T-cells was determined by FACS after 5 days in presence of IM as well as the proliferative T-cell response which was evaluated using CFSE (Carboxy Fluoroscein Succinimidyl Ester). In [51Cr]-release assays the cytotoxicity of CD8+ T-cells was assessed. The cytokine profile of the culture supernatants was detected by CBA (Cytokine bead array, Beckton Dickinson, USA). We also characterized the antigen profile (HLA-DR, CD40, CD54, CD58, CD80 and CD86) of the CML cells as APCs over a 5 day culture with and without IM. The rescue of the CML cells was initiated by IFN-a or IFN-a/GM-CSF after 3 days incubation with IM. On day 6 the APC profile of the CML cells was obtained and cells were used as stimulators in terms of T-cell proliferation which was determined by CFSE. We could show that the proliferation of allogeneic T-cell was inhibited in the presence of IM in a dose-dependent manner. In addition, the T-cell activation marker CD25 and the TCR-alpha expression were significantly downregulated by different concentrations of IM. Moreover, IM impaired the cytotoxic function of allogeneic T-cells in a dose-dependent manner. Interestingly, after stimulation with CML-cells cytotoxic T-cell activity could only be induced in those cases that did not secrete IL-6. Down-regulation of the APC profile on CML-cells (adhesion/costimulatory molecules) by increasing concentrations of IM could be in part reversed by the addition of IFN-a and completely restored by the combination of IFN-a and GM-CSF. Although, the proportion of proliferating T-cells could not be increased further by the combination of IFN-a ± GM-CSF, the number of T-cells was increased in the presence of IFN-a + GM-CSF. In conclusion, the down-modulating effects of IM could be almost completely reversed by the addition of IFN-a and GM-CSF. It remains to be seen whether these findings can be successfully applied to the treatment of CML-patients.
Functional Polymorphisms in Oxidant Metabolism and DNA Repair Pathways and Risk of Leukemia and Transient Myeloproliferative Disorder in Children with Down Syndrome
Authors: Rayburg Melissa S , Kim Mi-Ok , Alonzo Todd , Gerbing Robert , Radloff Gretchen A , Davies Stella M , Gamis Alan , Perentesis John
Published: April 2020

Children with Down syndrome can have a wide range of developmental abnormalities related to the presence of additional genetic material from a small region of chromosome 21. Additional copies of the Cu/Zn superoxide dismutase gene and related genes at this locus are believed to contribute to high oxidative stress, and increased endogenous DNA damage and deficient repair observed in cells from patients with Down syndrome. Children with Down syndrome also have a markedly increased risk for the development of serious hematological disorders, including an approximately 20-fold increased risk for the development of acute myeloid leukemia (AML). In addition, in early infancy, as many as 10% may develop a transient myeloproliferative disorder (TMD). The basis for this overall increased risk is unknown, as are the risk factors influencing which specific patients with Down syndrome develop AML/TMD. We sought to test the hypothesis that functional polymorphisms in genes involved in oxidant metabolism and DNA repair contribute to the increased risk of AML and TMD in children with Down syndrome. We studied functional polymorphisms in the oxidant metabolizing enzyme NADPH quinone oxidoreductase 1 (NQO1), and in the DNA repair proteins X-ray repair cross complementing 1 protein (XRCC1) and Fanconi anemia complementation group A protein (FANCA). Genotyping was conducted in 170 patients with Down syndrome AML or TMD, and 202 healthy blood donor controls. All genotype frequencies in the control populations were consistent with those expected from Hardy-Weinberg equilibrium. We found that the variant NQO1 Pro187Ser allele, associated with reduced quinone reductase activity, occurred with a greater frequency in the Down syndrome AML/TMD patient population compared to healthy controls (OR, 1.61; 95% CI, 1.067–2.426; p=0.0231). The FANCA-1501 variant allele frequency was observed more frequently in Down syndrome AML/TMD patients compared to healthy controls (OR, 1.77; 95%CI, 1.220–2.568; p=0.0025). The variant XRCC-1 Arg399Gln allele was observed less frequently in the TMD/DS AML population as compared to controls (OR, 0.536; CI, 0.354–0.811; p=0.0031). These latter findings parallel the observation of a protective effect against AML in non-Down syndrome individuals with at least one copy of the XRCC-1 Arg399Gln allele (Seedhouse et al. Blood2002, 100:3761). Conclusion: These preliminary studies, representing the only reported data in Down Syndrome TMD/AML patients, suggest that functional polymorphic variants of the NQO1, FANCA, and XRCC1 genes involved in oxidant metabolism and DNA repair may influence the risk for developing leukemia. Studies involving future cohorts will be needed to confirm these observations.
Perfect Engraftment after Fludarabine, Cyclophosphamide Plus Thymoglobulin Conditioning Regimen for Unrelated Transplantation in Severe Aplastic Anemia: Phase II Prospective Multi-Center Study in Korea
Authors: Kang Hyoung Jin , Shin Hee Young , Park Jun Eun , Lim Young Tak , Chung Nak Gyun , Cho Bin , Kim Hack Ki , Kim Sun Young , Lee Young Ho , Yoo Keon Hee , Sung Ki Woong , Koo Hong Hoe , Im Ho Joon , Seo Jong Jin , Park Sang Kyu , Ahn Hyo Seop
Published: April 2020

Anti-thymocyte globulin (ATG) has been used in severe aplastic anemia (SAA) as a part of the conditioning regimen. Among the many kinds of ATG preparations, thymoglobulin had been found to be more effective in preventing graft-versus-host disease (GVHD) and rejection of organ transplants. After the promising result of the pilot study (Bone Marrow Transplant. 2004. 34; 939), phase II prospective multi-center clinical trial was performed with fludarabine, cyclophosphamide plus thymoglobulin conditioning regimen to allow good engraftment in unrelated transplantation for SAA. Twenty-eight patients underwent bone marrow (N=15) or mobilized peripheral blood (N=13) transplantation with cyclophosphamide (50 mg/kg once daily i.v. on days −9, −8, −7 & −6), fludarabine (30 mg/m^2 once daily i.v. on days −5, −4, −3 & −2) and thymoglobulin (2.5 mg/kg once daily i.v. on days −3, −2 & −1) from HLA matched unrelated donors. GVHD prophylaxis regimen was composed of cyclosporine (or tacrolimus), methotrexate, with or without low dose thymoglobulin (1.25 mg/kg once daily i.v. on days 7, 9 and 11). The median infused cell dose of nucleated cells and CD34 positive cells were 6.8×10^8/kg (1.3– 39.9×10^8/kg) and 5.2×10^6/kg (1.2–27.0×10^6/kg), respectively. The median number of days required for ANC of more than 0.5×10^9/l and 1.0×10^9/l were 14 days (10–35 days) and 15 days (11–40 days), respectively. The spontaneous platelet recovery to more than 20×10^9/l required a median of 22 days (22–182 days). Donor type hematologic recovery (donor type chimerism more than 90%) was achieved in all patients. Fourteen patient developed grade II–IV acute GVHD. The event free survival (EFS) was 73% and all events were transplantation related mortality (TRM) which included coagulopathy (N=3), PTLD (N=2), pneumonia (N=1), and myocardiac infarction (N=1). The EFS of patients who received bone marrow (65%) was not different from that of patients who received mobilized peripheral blood (82%) (P=0.37), but the EFS of patients who received immunosuppressive therapy (IST) previously (55%) was lower than that of patients who didn’t receive IST (92%), significantly (P=0.04). Fludarabine, cyclophosphamide plus thymoglobulin conditioning allows for the promising result of very good engraftment, although serious events occurred in some patients. We are now planning to start new multicenter study to decrease TRM by reducing the dose of cyclophosphamide.
Rituximab Plus Hypercvad Alternating with High Dose Methotrexate and Cytarabine for Patients with Newly Diagnosed Mantle Cell Lymphoma. A Multicenter Trial from GISL
Authors: Merli Francesco , Luminari Stefano , Ilariucci Fiorella , Stelitano Caterina , Petrini Mario , Di Renzo Nicola , Angrilli Francesco , Carella Angelo Michele , Barbolini Elisa , Federico Massimo
Published: April 2020

BACKGROUND. Rituximab plus HyperCVAD alternating with High Dose Methotrexate and Cytarabine (R-HCVAD) has been tested in patients with newly diagnosed Mantle Cell Lymphoma (MCL) with promising results (Romaguera et al. JCO 2005). In 2005 the Gruppo Italiano Studio Linfomi (GISL) started a phase II multicenter study investigating clinical activity and toxicity of R-HCVAD in a similar group of patients.

PATIENTS AND METHODS. To be included in the trial patients must have histologically confirmed diagnosis of MCL, be younger than 70 years, have adequate organ function. Chemotherapy consisted of rituximab plus fractionated cyclophosphamide, vincristine, doxorubicine, and dexamethasone(considered one cycle) alternating every 21 days with rituximab plus high dose methotrexate-cytarabine (considered one cycle) for a total of eight cycles per the MD Anderson protocol. Patients with baseline PCR positivity for t(11;14) on bone marrow (BM) had to perform PCR assessment of BM at evaluation of response and during follow-up. Only patients achieving partial response (PR) were to be addressed to HDC followed by ASCT.

RESULTS. Thirty-two patients were enrolled. There were 23 males and 9 females; median age was 54 yrs (29 to 66), 80% were in stage IV, 50% and 71% had Gastrointestinal (GI) and BM involvement, respectively; PCR for t(11;14) was positive on BM in 51% of cases. Seven patients did not complete treatment due to toxicity; of these, two patients died (one with septic shock at cycle 1, one with pulmonary aspergillosis at cycle 4), one patient had thrombosis of central line extended to right atrium at cycle 1, one had grade IV skin reaction at cycle 3, one had a severe pneumonia at cycle 1, two had persistent grade IV hematological toxicity after cycle 1 and 5, respectively. All patients had grade III–IV hematological toxicity. Response was assessed in 17 patients with 16 CR and 1 PR. PCR for t(11;14) negativity on BM was achieved in 4/9 patients after cycle 4 and in 8/9 after cycle 8. After a median follow-up of 24 months 1 patient progressed at 6 months and 1 patient relapsed after 26 months of follow-up. Two-year Failure Free Survival (FFS) was 75% (IC95% 53 to 87) and 2 year Disease Free Survival was 93%(IC95% 59–99).

CONCLUSIONS. Though longer follow-up is needed R-HCVAD regimen used in our multicenter setting confirmed high efficacy in terms of response (both clinical and molecular) and FFS. However the regimen was associated to a severe toxicity profile that caused treatment discontinuation in several patients and that may limit its use in the clinical setting.
High Frequency of RPL11 Gene Mutation in Italian Patients with Diamond-Blackfan Anemia
Authors: Quarello Paola , Garelli Emanuela , Carando Adriana , Campagnoli Maria Francesca , Brusco Alfredo , Dianzani Irma , Ramenghi Ugo
Published: April 2020

Diamond Blackfan anemia (DBA, MIM#205900) is a rare, pure red blood cell aplasia of childhood due to an intrinsic defect in erythropoietic progenitors. About 40% of cases show malformations. Anemia is corrected by steroid treatment in half of the patients; non-responders need chronic transfusions or stem cell transplantation.

Defects in the RPS19 gene, encoding the ribosomal protein (RP) S19, are the main known cause of DBA and account for more than 25% of patients. Mutations in other RPs (RPS24, RPS17, RPL35a) have been reported in a minority of patients (Gazda et al., 2006; Cmejla et al., 2007; Farrar et al., 2008) and show that DBA is a disorder of ribosome synthesis. Recently, Gadza et al. (2007) reported the involvement of two new genes (RPL5, RPL11) encoding for RPs of the large subunit in a considerable percentage of DBA patients without mutations in RPS19 or RPS24 (10% and 6,5%, respectively).

Here we present the results of RPL11 mutation screening in Italian DBA patients without RPS19 or RPS24 mutations. Preliminary data of RPL5 analysis are also reported.

The human RPL11 gene includes 6 exons and spans 4622 bp on chromosome 1. We identified 12 heterozygous mutations in 80 analyzed probands (15%), 10 of which were deletions of 1–47 nucleotides causing frameshift and a premature termination; another deletion (12nt) resulted in a loss of 4 aminoacids. One acceptor splice site defect (intron 5) was also detected. RPL11 mutations were spread in all exons except for exon 1.

The human RPL5 gene spans 9888 bp on chromosome 1, and includes 8 exons. We identified one heterozygous mutation in RPL5 in 14 analyzed patients (7%); it is a single nucleotide deletion in exon 5 causing frameshift and a premature termination.

None of these sequence changes were found on the NCBI SNP dalabase (http://www.ncbi.nlm.nih.gov/SNP/).

Our mutations are expected to alter the genetic information drastically and to cause haploinsufficency. Depletion of RPs compromises ribosome biogenesis and disturbs rRNA processing at different levels (Robledo et al. 2008).

Interestingly, all mutated patients (11/13) showed one or more somatic malformations; specifically, thumb anomalies and growth retardation were present in 7/11 (64%) and 6/11 (54%) patients, respectively. The erythrocyte adenosine deaminase activity was performed in 6/13 mutated patients; all of them showed an increased activity. The majority of RPL11 mutated patients were transfusion-dependent at last follow-up (58%,7/12); the RPL5 mutated patient was in clinical remission after steroid treatment. No one of the mutated patients has so far developed neoplasia.

Our data show that RPL11 mutations are more frequent than expected (Gazda et al. 2007). They seem to confer a relevant association with a malformation phenotype as compared to RPS19 mutations.
Induction of the Unfolded Protein Response but Normal Basal Granulopoiesis in Mice Expressing G192X ELA2
Authors: Murakami Mark , Woloszynek Jill , Xia Jun , Liu Fulu , Link Daniel
Published: April 2020

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by chronic neutropenia, a block in granulocytic differentiation at the promyelocyte/myelocyte stage, and a marked propensity to develop acute myeloid leukemia. Most cases of SCN are associated with germline heterozygous mutations of ELA2, encoding neutrophil elastase (NE). To date, 59 different, mostly missense, mutations of ELA2 have been reported. A unifying mechanism by which all of the different ELA2 mutants disrupt granulopoiesis is lacking. We and others previously proposed a model in which the ELA2 mutations result in NE protein misfolding, induction of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Testing this (and other) models has been limited by the rarity of SCN and difficulty in obtaining clinical samples for testing. Herein, we report the preliminary description of a novel transgenic mouse line that expresses G192X Ela2, reproducing the G193X ELA2 mutation found in some patients with SCN. The G192X mutation was introduced into the murine Ela2 locus by homologous recombination in embryonic stem cells. Heterozygous or homozygous G192 Ela2 “knock-in” mice were healthy with no apparent developmental defect. While expression of Ela2 mRNA was normal, no mature NE protein was detected in the neutrophils of homozygous G192X Ela2 mice. However, in granulocytic precursors (mainly promyelocytes/myelocytes) a small amount of heavily glycosylated mutant NE protein was detected. Together, these observations suggest that G192X NE protein is retained in the endoplasmic reticulum (ER) and rapidly degraded. Consistent with ER stress and induction of the UPR, a significant increase in BiP/GRP78 and ATF6 mRNA expression in mutant granulocytic precursors were observed. Surprisingly, G192X Ela2 mice have normal basal granulopoiesis. The number of circulating neutrophils, granulocytic differentiation in the bone marrow, and number and cytokine responsiveness of myeloid progenitors were comparable to wild type mice. In summary, the G192X Ela2 mice appear to reproduce the NE protein misfolding and UPR activation observed in human SCN granulocytic precursors. However, expression of G192X Ela2 is not sufficient to disrupt basal granulopoiesis in mice. Studies of stress granulopoiesis are underway.
Disruption of a Functional Relationship Between PU.1 and Mir-155 during the Pathogenesis of Chronic Lymphocytic Leukemia (CLL)
Authors: Stopka Tomas , Vargova Karin , Berkova Adela , Kokavec Juraj , Trneny Marek , Zavadil Jiri
Published: April 2020

Role of small non-coding microRNAs (miR) in hematopoiesis has been recently established by work demonstrating critical importance of miR-223 for granulocytic development (Johnnidis 2008) and miR-155 in lymphocytic differentiation (Thai 2007) in mice. The importance of miR-155 is further supported by several independent studies demonstrating its increased levels in human diffuse large B-cell lymphoma (Eis 2005, Kluvier 2005, Volinia 2006, Lawrie 2007, Rai 2008) and in chronic lymphocytic leukemia (CLL) (Fulci 2007, Marton 2008). PU.1 is ETS family transcription factor involved in myelo-lymphoid differentiation and is directly negatively regulated by miR-155 (Vigorito 2007). Studying primary CLL (N=27) and normal (N=13) peripheral blood mononuclear cells, we found significantly increased miR-155 levels and also demonstrate that PU.1 levels are significantly decreased. Next, we have tested genes that are targets of both miR-155 and PU.1 including CSF1R and FOS and again demonstrated their significantly decreased expression in CLL. Using gene expression profiling of CLL peripheral lymphocyte samples compared to normal controls, we have determined significant downregulation of the PU.1 mRNA and of numerous miR-155 targets previously reported to be required for myelo-lymphoid differentiation. These findings suggest that the relationship between PU.1 and miR-155 becomes disrupted in CLL. This in turn leads to overexpression of miR-155, reduced levels of PU.1 and of numerous miR-155 targets (N=2481 and N=400 respectively) and to inhibition of PU.1 regulated differentiation and proliferation programs. Next, we correlated negative cytogenetic prognostic features (p53 and ATM gene deletion detected by FISH) of the CLL patients with the miR-155 and PU.1 levels, In a relatively small-size patient cohort studied so far, the low ratios of PU.1/miR- 155 levels are similarly distributed among low, intermediate and high risk CLL patients, suggesting that inverse correlation of PU.1 and miR-155 represents a hallmark of CLL and likely represents a primary event during CLL pathogenesis.
Disruption of CTNNB1/LEF-1 Complex by Small Molecule Inhibitors Induces Apoptosis in B-CLL Cells in Vitro and in Vivo
Authors: Gandhirajan Rajesh Kumar , Gehrke Iris , Paesler Julian , Razavi Regina , Filipovich Alexandra , Erdfelder Felix , Hertweck Magdalena , Plickert Guenter , Schmitt Esther Katharina , Schlösser Axel , Hallek Michael , Kreuzer Karl Anton
Published: April 2020

Deregulated WNT signaling has been implicated in variety of tumors. Previous findings suggest critical components of WNT signaling pathway (WNTs, FZD and LEF-1) are overepxressed in B-cell chronic lymphocytic leukemia (CLL) at the m-RNA level. β-catenin (CTNNB1)/LEF-1 transcription activation complex is the key component of WNT signaling which governs the expression of WNT target genes. Critical genes like C-MYC, survivin (SURV), cyclin D1 (CCND1) and LEF-1 which are also overexpressed in CLL are downstream targets of this pathway. In the current study we attempted to inhibit CTNNB1/LEF-1 interaction in CLL cells using the novel small molecule inhibitors CGP049090 and PKF115-584 and observed the survival potential both in vivo and in vitro. Substantial overexpression of LEF-1 and dephospho-CTNNB1, in CLL cells was observed when compared to healthy CD19 positive B cells and PBMCs suggests activation of WNT signaling in CLL. The LC50 calculated from 30 CLL patients was 1.18 μM for CGP049090 and 1.10 μM for PKF115-584 by the ATP based assay. This was confirmed by the DiSC assay LC50 of 0.33 μM (CGP049090) and 0.23 μM (PKF115-584) for primary B-CLL cells (n=3). Both substances showed little effects on normal PBMCs (n=5) as seen from the LC50 values of 90.92 μM and 39.88 μM for CGP049090 and PKF115-584, respectively. Time course experiments indicate activation of caspase 8, 9, 3 & 7, PARP cleavage and paralleled by downregulation of known inhibitors of apoptosis (IAPs) MCL-1, XIAP and BCL-2 leading to apoptosis. Co-immune precipitation of CTNNB1/LEF-1 complex was inhibited in the presence of CGP049090 and PKF115-584 suggesting the specificity of the interaction. Cellular uptake of PKF115-584 and cytoplasmic co-localisation with lef-1 was observed by fluorescence microscopy. In vivo studies reveal that both substances are highly tolerable and effective in eradicating tumor growth in xenografts. The tumor inhibitory rate (IRmax) was found to be 78% and 63.8% for CGP049090 and PKF115-584, respectively. These results indicate that LEF-1 is a promising target for B-CLL therapy as CGP049090 and PKF115-584 effectively induce apoptosis within micromolar concentrations in CLL cells but do not affect healthy cells at these doses. Downregulation of WNT target genes and IAPs hasten the apoptotic mechanism. Moreover, and these inhibitors are highly effective and tolerable in vivo which identifies both compounds as promising drugs for targeted therapy in CLL.
Erythrocyte Scaffolding Protein p55 Functions as An Essential Regulator of Neutrophil Polarity
Authors: Quinn Brendan J. , Chishti Athar H.
Published: April 2020

Erythrocyte p55 is a prototypical member of a family of scaffolding proteins known as Membrane Associated Guanylate Kinase Homologues (MAGUKs). MAGUKs are multi-domain proteins that couple signals from specialized sites at the plasma membrane to intracellular signal transduction pathways and the cytoskeleton. P55 was originally identified in the erythrocytes as part of a ternary complex with protein 4.1R and glycophorin C, providing a critical linkage between the actin cytoskeleton and the plasma membrane. Although p55 is expressed in a variety of tissues, especially hematopoietic cells, its biological function is unclear. Here, using a p55 knockout mouse model, we show that p55 plays a prominent role in the regulation of neutrophil polarization. Neutrophils are the first respondents during infection and injury, adopting a highly polarized morphology when stimulated with chemotactic factors. G proteincoupled surface receptors recognize the external chemotactic gradient and translate it into an internal gradient of signaling molecules. At the front of the cell, accumulation of the lipid product phosphatidylinositol-3,4,5-trisphosphate (PIP3), activation of the small GTPase Rac, and polymerization of F-actin stimulates a positive feedback loop promoting pseudopod formation. Here, we show that neutrophils lacking p55 form multiple transient pseudopods at the sides and back of the cell upon stimulation. P55 is required for limiting the pseudopod in the direction of chemoattractant. As a result, these neutrophils do not migrate efficiently up a chemotactic gradient in vitro. Biochemical analysis indicates that total F-actin polymerization and total Rac activation is similar between wild type and p55 knockout neutrophils. However, we found that phosphorylation of AKT, the major kinase downstream of the phosphatidylinositol 3-kinase (PI3K)-PIP3 pathway, is almost completely blocked in p55 knockout neutrophils. This finding suggests that p55 exerts its functional effect by regulating PIP3 accumulation or its localization at the membrane, which is responsible for amplification of the frontness signal and stability of the leading edge pseudopod. Consistent with this finding, the p55 null mice are significantly more susceptible to spontaneous and induced infections. Taken together, we have identified p55 as a novel mediator of the frontness signal in neutrophils that promotes polarization and efficient chemotaxis.
Lyn Kinase Overexpression Is One of the Mechanisms of Resistance to Nilotinib In Chronic Myeloid Leukemia
Authors: Mahon Francois-Xavi er , Hayette Sandrine , Lagarde Valerie , Nicolini Franck E , Belloc Francis , Leroy Cedric , Roche Serge , Manley Paul , Belanger Coralie , Etienne Gabriel , Pasquet Jean-Max
Published: April 2020

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myeloid Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Three nilotinib-resistant cell lines were obtained from the Ph-positive cell lines AR230, LAMA84 and K562. Over expression of the BCR-ABL gene was found in two nilotinib-resistant cell lines and the multidrug resistance gene (MDR-1) was found overexpressed in one of them, i.e, LAMA84 nilotinib resistant cell. The K562/DOX cell line, that displays resistance to several drugs by over expressing Pgp, was resistant to nilotinib, and this was reversed by simultaneous incubation with either verapamil or PSC833 confirming that nilotinib is a substrate of the Pgp. In one nilotinib-resistant cell line (K562-rn), we found over expression of p53/56 Lyn kinase, both at the mRNA and protein level (10- fold), and these cells were compared to their sensitive counterpart. Lyn silencing by siRNA restored sensitivity to nilotinib. Two Src kinase inhibitors (PP1 and PP2) partially restored sensitivity to nilotinib, but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, an inhibitor of Abl and Src-family kinases, inhibited phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line which over expressed p53/56 Lyn. Of 7 nilotinib-resistant CML patients, failure of nilotinib treatment was accompanied by mutations in Bcr-Abl kinase domain in 2 patients and an increase of Lyn mRNA expression (RQ-PCR) in 2 other patients. As an approach to confirm the involvement of the Lyn signalling pathway in nilotinib-resistance, we have used the Stable Isotope Labeling with Amino acids in Cell culture (SILAC) technique. The tyrosine phosphorylated protein fraction was analyzed by tandem mass spectrometry. Peptide sequencing and quantification in the nilotinib-resistant cell line identified >350 proteins, of which several were hyper-phosphorylated; functional analysis of the different candidates is in progress.

In conclusion, mechanisms of resistance to nilotinib in imatinib-resistant cell lines resemble those operating in CML patients, and up-regulated Lyn kinase signalling can play an important role in nilotinib resistance.
Aberrant p53 Signaling Pathways in RPL11 Mutant Zebrafish
Authors: Danilova Nadia , Morimoto Kenji , Sakamoto Kathleen M , Lin Shuo
Published: April 2020

Diamond Blackfan Anemia (DBA) is a congenital syndrome characterized by decreased production of red blood cells and developmental abnormalities. This syndrome is caused by mutations in ribosomal genes (RPs), most frequently in RPS19, RPL11, and RPL5. Previously, we created RPS19 deficient zebrafish using morpholinos and demonstrated that p53 family members mediate the effects of RPS19 deficiency on development and hematopoiesis through upregulation of p53-related proteins. In this study, we found that RPL11 mutations, similar to RPS19 deficiency, leads to dysregulation of p53, its related family members, and downstream signaling pathways in zebrafish. Expression of both total p53 and Δ113Np53 is strongly upregulated in mutants with more modest upregulation of ΔNp63. The p53 protein also induced p21 and G1 cyclin expression, which resulted in cell cycle arrest. The pro-apoptotic genes, puma and bax, were also upregulated. We observed that the expression of several genes involved in hematopoiesis was altered, for example, globin gene expression was decreased. The ΔNp63 protein, which increases expression of adenosine deaminase (ADA) and is upregulated in >70% of DBA patients, was also upregulated in RPL11 zebrafish mutants. Interestingly, we also observed increased expression of the proto-oncogenes c-fos, c-jun, and c-myc in RPL11 mutants. Increased expression of proto-oncogenes may contribute to the mechanisms of transcriptional activation by p53 family members in response to ribosomal protein deficiency. Furthermore, aberrant expression of oncogenes could provide a possible mechanism for the increased risk of malignancies in DBA patients. We propose that the zebrafish is a model system to study developmental and hematologic defects in addition to signaling pathways in the pathogenesis of DBA.