bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism for γ-Globin Induction by SCF Is Via PKCα/ERK1/2/Nrf2 Dependent Signal Pathway To Regulate Down-Stream Transcriptional Regulators NF-Y and COUP-TFII.
Authors: Aerbajinai Wulin , Zhu Jianqiong , Gao Peter , Chin Kyung , Rodgers Griffin P.
Published: April 2020

Increased fetal hemoglobin has been identified to be associated with stress erythropoiesis. However, the mechanisms underlying γ-globin induction during the rapid expansion of erythroid progenitor cells have not been fully elucidated. Here we examined how intracellular signaling pathway modification of specific transcriptional regulators induced γ-globin expression in vitro cultured erythroid progenitor cells in the presence of erythropoietin and stem cell factor (SCF). We find that γ-globin induced by SCF is through a PKCα-dependent Ras/Raf/Erk1/2 signaling pathway involving activation of the transcription factor NF-Ya and inhibition of the repressor Coup-TFII. Specific inhibition of PKCα with Go6976 blocked both activation of Erk1/2 and p38 MAPK induced by SCF, and abrogated the SCF increased γ-globin gene expression. Activation of Erk1/2 plays a critical role in SCF modulated down-stream transcriptional regulators, involving regulation of γ-globin gene induction. SCF induced nuclear translocation of NF-Ya is required to activate Erk1/2 increased phosphorylation of endogenous Nrf2, which involves up-regulation of thioredoxin, and down-regulation of Coup-TFII. Inhibition of either PKCα or Erk1/2 prevented SCF induced recruitment of NF-Ya, RNA polymerase II and displacement of Coup-TFII repressor from γ-globin-promoter, indicating that the PKCα-Erk1/2 MAPK pathway contributes to SCF induced the γ-globin gene induction in adult erythropoiesis. Furthermore, consistent with this concept, SCF induced the γ-globin gene induction attenuated by inhibition of PKCα or Erk1/2 MAPK. Our data suggest that SCF stimulates the PKCα-Erk1/2 MAPK signaling pathway which regulate the downstream transcriptional activator NF-Ya and repressor Coup-TFII resulting in γ-globin reactivation in adult erythropoiesis. These observations provide the molecular pathways that take part in γ-globin augmentation during “stress erythropoiesis”.
Interferon-α2b-Induced Thrombocytopenia Is Caused by Inhibition of Cytoplasmic Maturation and Platelet Production, but Not Proliferation and Endomitosis in Human Megakaryocytes.
Authors: Yamane Akiko , Nakamura Takanori , Ito Mamoru , Ohnishi Yasuyuki , Ikeda Yasuo , Miyakawa Yoshitaka
Published: April 2020

Human interferon (IFN)-α is a standard treatment for the patients with chronic hepatitis C. It is well known that chronic hepatitis C can transform into liver cirrhosis and hepatocellular carcinoma, and the risk of progression to liver cirrhosis is around 40% in 5 years. Thus, it is important for chronic hepatitis C patients to receive IFN-α treatment to prevent its malignant transformation. Thrombocytopenia is one of the major adverse effects of IFN-α and often leads to a dose reduction or discontinuation of IFN-α therapy. However, there is little information how IFN-α inhibits human megakaryopoiesis. In this study, we demonstrated that IFN-α does not inhibit colony formation of megakaryocytes (CFU-MK) from human CD34-positive hematopoietic stem cells. IFN-α also does not inhibit endomitosis (DNA duplication without cytokinesis), but inhibits cytoplasmic maturation of megakaryocytes and platelet production in vitro. The inhibitory effects of IFN-α on the development of demarcation membrane in human megakaryocytes were visualized by staining primary megakaryocytes with di-8-ANEPPS dye under a confocal laser microscope. Loss of platelet production by IFN-α was analyzed by measuring proplatelet formation (PPF) and counting the human platelets produced in the supernatant of human primary megakaryocytes by flow cytometry. The expression of transcription factors regulating late stage megakaryopoiesis such as GATA-1, p45 NF-E2 and MafG was suppressed by IFN-α. To confirm these in vitro observations in vivo system, we transplanted human CD34-positive hematopoietic cells into the immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice (hu-NOG) after 2.4 Gy irradiation. Recombinant human(rh) IFN-α significantly reduced the number of human platelets but did not decrease the number of human megakaryocytes in hu-NOG mice. rhIFN-α did not change murine platelet counts in hu-NOG mice, as IFN-α has species specificity. The DNA ploidy of human megakaryocytes in the bone marrow of hu-NOG mice was not altered after treatement with rhIFN-α. We also confirmed the life time of human platelets was not shortend by rhIFN-α in hu-NOG mice, indicating IFN-α does not promote the clearance of human platelets. We have also confirmed that a novel thrombopoietin mimetics, NIP-004, prevented rhIFN-α-induced thrombotytopenia and increased the number of polyploid human megakaryocytes in hu-NOG mice, suggesting NIP-004 might be useful for the patients with hepatitis C to avoid IFN-α-induced thrombocytopenia. In conclusion, these results clarified that IFN-α induces thrombocytopenia by inhibiting the cytoplasmic maturation of megakaryocytes but not proliferation and endomitosis in human megakaryocytes.
Biological Significance of Erythropoietin and Erythropoietin Receptor Expression by Human Ovarian Cancer Cells.
Authors: Jeong Jee-Yeong , Feldman Laurie , Solar Peter , Sytkowski Arthur J.
Published: April 2020

Erythropoietin (Epo) is the primary regulator of erythroid cell proliferation and differentiation mediated through its specific binding to the Epo receptor (EpoR). Epo also is known to have non-hematopoietic actions, including the promotion of wound healing, tissue protection and others. Accumulating evidence demonstrating both Epo and EpoR expression in many types of human cancers has raised concern about the clinical use of Epo and other erythropoiesis stimulating agents (ESAs) in cancer-related anemia, since the presence of functional EpoR on the tumor cells suggests the potential for an adverse effect of Epo treatment by enhancing cancer growth and progression. Previously, we demonstrated that A2780 ovarian cancer cells express the EpoR gene and that long-term Epo treatment of these cells renders them resistant to paclitaxel. We now report the characterization and biological significance of Epo and EpoR expression by four ovarian cancer cell lines. Using semi-quantitative RT-PCR, restriction digestion of the PCR products and DNA sequence analysis, we demonstrated that A2780, CaOV, SKOV, and OVCAR-3 ovarian cancer cell lines examined express Epo and EpoR at the mRNA level. We demonstrated EpoR protein expression both by western blotting and by immunofluorescence, and demonstrated biologically active Epo protein expression by quantitative in vitro bioassay of the cell culture supernatants. The EpoR on A2780 cells are functional, since Epo stimulation of the cells resulted in a time-dependent 5-fold increase in phosphorylation of Erk1/2 that reached a peak in 10 min and then returned to the basal level. None of the cell lines exhibited a short-term (3-day) growth response in culture to exogenous Epo. However, addition of a neutralizing anti-Epo antibody to the cell culture resulted in partial growth inhibition of A2780 cells that was reversed by addition of excess Epo, consistent with an autocrine/paracrine mechanism of Epo growth enhancement of these cells. We also found that long-term Epo treatment of A2780 cells resulted in the development of a phenotype exhibiting enhanced Epo signaling, evidenced by a 20-fold increase in phosphorylation levels of Erk1/2. This phenotype was sustained even after the removal of Epo, suggesting a mechanism underlying the paclitaxel resistance developed after long-term Epo treatment. Our findings have implications for the clinical use of recombinant human Epo and other ESAs to correct and/or prevent anemia in ovarian and, potentially, other cancer patients. They also suggest that endogenous Epo and the EpoR may serve as novel therapeutic targets in the treatment of cancer patients.
Protective Role of Connexin 32 in Experimental Leukemogenesis.
Authors: Hirabayashi Yoko , Yoon Byung-Il , Tsuboi Isao , Huo Yan , Kodama Yukio , Kanno Jun , Ott Thomas , Trosko James E. , Inoue Tohru
Published: April 2020

Connexin (Cx) functions in the organization of cell-cell communication in multicellular organisms. Gap junctions have been implicated in the homeostatic regulation of various cellular functions, including growth control and differentiation, apoptosis, and the synchronization of electrotonic and metabolic functions. Primitive hemopoietic progenitor cells form a multicellular system, but a previous report describes that Cx32 is not expressed in the bone marrow. Thus, a question arises as to why Cx molecules are not detected in the hematopoietic tissue other than stromal cells. Based on our preliminary study that suggested a potential impairment of hematopoiesis in Cx32-knockout (KO) mice, the objectives of the present study were to determine whether Cx32 functions in the bone marrow during steady-state hematopoiesis and further to examine its possible protective roles during regeneration after chemical abrasions and during leukemogenesis after the administration of a genotoxic chemical, methyl nitrosourea (MNU). As results, the Cx32 molecule functioning in the hematopoietic stem cell (HSC) compartment during steady-state hematopoiesis was observed for the first time; the expression of Cx32 at the mRNA level determined by PCR analysis and that at the protein level determined using an anti-Cx32 antibody were observed only in the lin−c-kit+ HSC fraction using a combination of immunobead-density gradient and immunomagnetic-bead separation. Hematopoiesis was impaired in the absence of Cx32; it was delayed during regeneration after chemical abrasion with 5-fluorouracil at 150 mg/kg body weight in Cx32-KO mice. Cx32-KO mice also showed increased leukemogenicity compared with wild-type mice after MNU injection; furthermore, in a competitive assay for leukemogenicity in mice that had been lethally irradiated and repopulated with a mixed population of equal amount of bone marrow cells from Cx32-KO mice and wild-type mice, the resulting leukemias were originated predominantly from Cx32-KO bone marrow cells. The present competitive assay clearly showed that Cx32-KO bone marrow cells have a higher risk of becoming leukemogenic. The above-mentioned findings in this study imply that Cxs play an essential role in maintaining the steady-state hematopoiesis and suppressing the neoplastic change. In summary, the role of Cx32 in hematopoiesis was not previously recognized and Cx32 was expressed only in HSCs and their progenitors. The results indicate that Cx32 in wild-type mice protects HSCs from chemical abrasion and leukemogenic impacts. Our results indicate that the risk of developing leukemia in patients with X-chromosome-linked Cx32 deficiency, called Charcot-Marie-Tooth syndrome, may not be incidental.
Transcription Factor Dosage: Maximal STAT5-Induced Proliferation and Stem Cell Self-Renewal at Intermediate STAT5 Activity Levels.
Authors: Wierenga Albertus T.J. , Vellenga Edo , Schuringa Jan Jacob
Published: April 2020

The level of transcription factor activity critically regulates cell fate decisions such as hematopoietic stem cell self-renewal and differentiation. Here, we describe a model in which STAT5 activity is introduced into human hematopoietic stem/progenitor cells in a dose-dependent manner by overexpression of tamoxifen-inducible STAT5A-ER fusion proteins that can be activated upon treatment with 4-OHT. For this study, we tested STAT5A(wt), STAT5A(1*6) and STAT5A(S711F)-ER fusion proteins. Introduction of STAT5A activity in cord blood-derived CD34+ cells resulted in impaired myelopoiesis and induction of erythropoiesis which was most pronounced at the highest STAT5A transactivation levels. In contrast, intermediate STAT5A activity levels resulted in the most pronounced proliferative advantage of CD34+ cells. This coincided with increased CAFC and LTC-IC frequencies that were predominantly elevated at intermediate STAT5A activity levels, but not at high STAT5A activity levels. Self-renewal of progenitors was addressed by serial replating of CFUs grown in methylcellulose, and only progenitors containing intermediate STAT5A activity levels contained self-renewal capacity. Extensive gene expression profiling in CB CD34+ cells over a range of STAT5A activation levels identified subsets of genes that were induced by low, intermediate and high STAT5A transcriptional activity. Thus, we could cluster gene expression levels of STAT5 target genes into a ‘differentiation signature’ versus a ‘proliferation/self-renewal signature’. In conclusion, our data suggest that intermediate rather than high STAT5 activity levels might be required to induce leukemic transformation.
Ligation of TLR5 by Flagellin Converts Tolerogenic Dendritic Cells into Activating Dendritic Cells through Inhibition of IL-10 Production.
Authors: Vicente-Suarez Ildefonso , Sotomayor Eduardo M.
Published: April 2020

Antigen presentation by DCs differentiated in the presence of the anti-inflammatory cytokine IL-10 has been shown to result in the development of regulatory T cells (Tregs) and tolerance induction. Whether this outcome could be reverted -or not- through manipulation of the tolerogenic DCs (tol-DCs) has not been explored in detail. Here we report for the first time that tol-DCs differentiated in the presence of IL-10 display increased expression of TLR5 as compared with bone marrow-derived DCs differentiated in the presence of GM-CSF and IL-4 (BM-DCs). The increased expression of TLR5 in tol-DCs seems to be a unique characteristic given that expression of other TLRs such as TLR4 and TLR9 was not different in tol-DCs as compared to BM-DCs. In lieu of our previous observation that the TLR5 ligand, flagellin, inhibits IL-10 production in antigen-presenting cells (APCs)1, and the unexpected finding that tol-DCs display higher expression of TLR5, we asked next whether flagellin could influence the functional properties of tol-DCs. First, unlike BM-DCs, tol-DCs stimulated with LPS (TLR4 ligand) produced high levels of IL-10. In sharp contrast, tol-DCs stimulated with flagellin do not produce IL-10 but produce significant levels of the pro-inflammatory cytokine IL-12. Second, while antigen presentation by LPS-stimulated tol-DCs results in the generation of IL-10 producing CD4+ T-cells, presentation of cognate antigen by flagellin-stimulated tol-DCs resulted in CD4+ T-cells that secrete IFN-gamma but not IL-10. This divergent T-cell outcome in response to antigen-presentation by tol-DCs stimulated with LPS (IL-10 producing T-cells) versus flagellin (IFN-gamma producing T-cells) suggests that TLR5 engagement might be a novel approach to convert tolerogenic DCs into activating DCs and effectively trigger productive immune responses in environments that are otherwise conducive to unresponsiveness due to the presence of IL-10.
Chemotherapy Alone Versus Chemotherapy Plus Radiotherapy for Early Stage Hodgkin Lymphoma.
Authors: Rehan Fareed A. , Brillant Corinne , Schulz Holger , Knauel Ina , Bohlius Julia , Specht Lena , Engert Andreas
Published: April 2020

Both chemotherapy (CT) alone and combined modality therapy (CMT) are effective modalities for the treatment of early stages of Hodgkin lymphoma (HL). However, the optimal choice of treatment is still being discussed. Different research groups reported that ABVD-like CT followed by radiotherapy is highly effective for early favourable and early unfavourable (intermediate) stage HL. A recently conducted randomized trial aimed at determining whether CMT is superior to CT alone in early stages, showed no significant difference in 5 year overall survival (OS) and event-free survival in patients treated with either 4–6 cycles of ABVD alone or 2 cycles of ABVD plus radiotherapy. Thus, this systematic review and meta-analysis was performed for a more comprehensive comparison.

Methods: Only randomized controlled trials (RCT) comparing CT alone with CMT in newly diagnosed adults with early stages of HL (CS IA, IB, IIA, IIB) were included. Medline and Cochrane Library were systematically searched for randomized controlled trials from 1975 to 2007. Patients who received CMT were considered as experimental group and patients who received ABVD-like CT alone were considered as control group. Data were collected from full text publications or abstracts and treatment effects for OS were calculated as hazard ratios (HR).

Results: A total of 590 references were screened. Four eligible RCTs were identified, including 1207 patients with early-stage HL (early favourable and early unfavourable stages). Between 1983 and 2004, 765 patients where treated with CMT and 442 patients with CT alone. The OS for the CMT group was significantly better than the OS for the CT-alone group (HR: 0.54; 95% CI [0.35 - 0.84]; p = 0.006), but there was evidence for a substantial heterogeneity between the studies (I2 = 69%, p=0.02).

Discussion: First results of this systematic review showed improved OS for patients treated with CMT in early-stage HL compared to patients treated with CT alone. The substantial heterogeneity between the trials should be clarified. Further comprehensive analyses are ongoing and will provide more detailed information.
Does FDG-PET/CT Obviate the Need for Standard Diagnostic Contrast-Enhanced CT in Patients with Lymphoma?.
Authors: Elstrom Rebecca L. , Brown Richard K.J.
Published: April 2020

Background and Significance: Positron emission tomography using 18-fluoro-2-deoxyglucose (FDG-PET) is useful in the staging and follow up of patients with lymphoma, and has been shown in several studies to be more accurate than computed tomography (CT). These studies have, however, demonstrated a continued role for CT in staging and restaging of lymphoma, and the two modalities are complementary. Increasingly, FDG-PET is performed in conjunction with a low radiation dose, non-contrast CT scan for attenuation correction and localization of lesions. Currently, many patients undergo both FDG-PET/CT and standard diagnostic, contrast enhanced CT, at a significant cost both financially and in terms of radiation exposure. In this study, we evaluated the clinical utility of performing both studies in patients with lymphoma.

Study Design and Methods: We retrospectively identified patients with lymphoma who had undergone both FDG-PET/CT and diagnostic, contrast-enhanced CT (a scan pair) for either staging or restaging following treatment. Patients were included if the two imaging studies were performed within 6 weeks of each other with no intervening anti-lymphoma therapy. We compared the results of the two studies, identifying findings that were detected in either FDG-PET/CT or diagnostic CT scan but not both. Discrepancies were considered clinically significant if they were determined to be related either to lymphoma or another disease process which potentially required intervention.

Results: Eighty-nine scan pairs which met the criteria were identified in 75 patients. Sixty-one scan pairs were performed for staging, and 28 were performed for treatment follow up. FDG-PET/CT detected additional potentially clinically relevant lesions over CT in 30 patients, of which 11 demonstrated increased clinical stage. Lymphoma therapy was changed based on FDG-PET/CT findings in 2 patients, and in one patient an occult rectal cancer was detected. In contrast, diagnostic CT detected 5 potentially clinically relevant findings, including 2 incidental findings (one definite and one possible venous thrombosis), and 3 patients with splenic lesions. Of the patients with splenic lesions, one was found on follow up to be definitely not related to lymphoma, and the nature of the splenic lesions in the other two patients remained indeterminate. No patient had a change of stage or lymphoma therapy based on diagnostic CT scan, and one patient was treated with anticoagulation based on CT findings. In the subgroup of scan pairs performed for follow up, diagnostic CT added clinically relevant information in none of the patients.

Conclusion: In our series of patients, diagnostic contrast-enhanced CT scan did not contribute to staging or restaging of lymphoma when performed concurrently with FDG-PET/CT. Two clinically important incidental findings were detected by CT alone, of which one led to intervention.
Case-Control Study Using Array-Based Genotyping Produced a Panel of Genes Associated with Risk to Acute Myelogenous Leukemia (AML).
Authors: Lee Il-Kwon , Kim Hee Nam , Kim Yeo-Kyeoung , Park Kyeong-Soo , Yang Deok-Hwan , Lee Je-Jung , Shin Myung Geun , Choi Chan , Kook Hoon , Hwang Tae Ju , Park Moo Rim , Jo Deog Yeon , Won Jong Ho , Kim Hyeoung-Joon
Published: April 2020

Multiple loci with small genetic effects are thought to be linked to AML pathogenesis. To detect such loci requires systematic screening of large number of single nucleotide polymorphisms (SNPs) within large study population. In this study we tested whether 3104 SNPs in 200 candidate genes were associated with risk to AML. Genes were considered potential candidates for their known or suspected roles in DNA repair system, pharmacogenomics and transcriptional regulation in hematopoiesis or putative pathways related to leukemogenesis. Selection of SNPs was performed using dbSNP and HapMap project databases, with emphasis on non-synonymous SNPs or haplotype tagging SNPs. To discover potential SNPs responsible for AML susceptibility, we conducted a case-control study using Affymetrix targeted genotyping 3K array. We applied this platform to analyze the samples from 309 de novo AML patients and 382 healthy controls. Genotype scorings were processed using GeneChip scanner 3000 TG(Affymetrix) and analyzed with GCOS software(Affymetrix). In total more than 255,000 SNPs were genotyped for this study. Samples with suboptimal call rates were excluded. Statistical testing were carried out using χ2, Cochran-Armitage trend, Fisher’s exact, odds ratio, haplotype estimation, LD block definition. Here we report that 23 SNPs in 16 genes are associated with elevated or reduced risk to AML. Some of associated genes were transcription factors such as ZNF23 and ZNF233. While associated genes were distributed evenly among the whole genome, four associated genes were found on chromosome 1. Among those associated SNPs, two were located in coding region, one in exon-intron boundary, while the rest of SNPs were located in introns of associated genes. Among 23 SNPs identified, two intronic SNPs from 1st intron of BAALC(Brain and acute leukemia, cytoplasmic) gene were associated with the reduced risk to AML. Haplotype estimation and linkage disequilibrium pattern of 9 SNPs including two associated SNPs will be presented. In conclusion, we identified SNPs responsible for AML susceptibility by candidate gene-based SNP array approach. These promising data when supported by further molecular validation would greatly enhance the current understanding of AML predisposition and diseases progression. Implication of polymorphic variants in AML etiopathogenesis will be presented and discussed.
Molecular Allelo-Karyotyping of Adult T-Cell Leukemia Using High SNP Genotyping Microarrays.
Authors: Muto Satsuki , Yamamoto Go , Nannya Yasuhito , Sanada Masashi , Dabaghmanesh Nazanin , Ishida Takaomi , Utsunomiya Atae , Yamada Yasuaki , Kamihira Shimeru , Yamaguchi Kazunari , Watanabe Toshiki , Ogawa Seishi
Published: April 2020

Adult T cell leukemia/lymphoma (ATL) is a mature T-cell neoplasm in adult that is caused by human T-cell leukemia virus type 1 (HTLV-1) and highly intractable to conventional therapeutics. Since there are 1.2 million HTLV-1 carriers in Japan and more than 50,000 carriers are expected to develop ATL from now on, it is of particular importance to understand the pathogenesis of ATL. The malignant processes of T-cell transformation in ATL are initiated by HTLV-1 infection in early childhood, and the HTLV-1 infected and immortalized T-cells are thought to accumulate a series of genetic hits during the later life, ultimately giving rise to malignant ATL clones after decades of latency periods. However, little is known about the nature of these genetic hits that take place after the early immortalization processes of T-cells mediated by HTLV-1 Tax protein. So in order to clarify the genetic alterations involved in the later processes of T-cell transformation in ATL, we analyzed a total of 130 ATL samples using Affymetrix® GeneChip® 250K Nsp arrays. Combined with CNAG/AsCNAR software, these arrays allow for accurate determination of allele-specific copy numbers in extremely high-resolution (less than 12kb) to detect copy number alterations as well as allelic imbalances in ATL genomes without depending on the availability of constitutive DNA of tumor specimens (molecular allelo-karyotyping). ATL genomes show characteristic copy number profiles and unique patterns of allelic imbalances, which are distinct from acute lymphoblastic ALL and non-Hodgikin’s lymphomas and include gains of 1q arm, 2q33, 3p and 3q arms, 9p12, 17q12, and 19p13, and losses of 1p13.1, 2q end, 3q22, 4q31, 6p22, 7q31, 9p21, 10p14, 12q13, 14q24, and 19q13. Moreover, allele-specific determination of copy numbers disclosed a number of regions showing copy number neutral LOH. Numerous homozygous deletions and gene amplifications were also identified and commonly mapped to less than 500Kb regions, which facilitated identification of candidate gene targets. Interestingly, these genetic lesions involved many T-cell related genes, indicating that the de-regulation of normal T cell functions may contribute the pathogenesis of ATL. In conclusion, molecular allelo-karyotyping of ATL genomes using SNP arrays provides valuable information about the molecular targets in ATL pathogenesis.
Gas6 and Its Receptors Are Implicated in Sepsis as Modulators of Innate Immunity.
Authors: Burnier Laurent , Sugamele Rocco , Le Roy Didier , Roger Thierry , Fumeaux Thierry , Chanson Marc , Borgel Delphine , Lemke Greg , Matsushima Glenn K. , Earp H. Shelton , Carmeliet Peter , Schapira Marc , Feihl François , Calandra Thierry , Angelillo-Scherrer Anne
Published: April 2020

Gas6 downregulates the activation state of macrophages and thereby their production of proinflammatory cytokines induced by various stimuli. We aimed to determine whether Gas6 is involved in sepsis. We measured Gas6 plasma levels in 13 healthy subjects, 29 patients with severe sepsis, and 18 patients with non-infectious inflammatory diseases. Gas6 level was higher in septic patients than in control groups (P≤0.0001). The sensitivity and specificity of Gas6 levels to predict fatal outcome were 83% and 88%. We next investigated whether Gas6 affects cytokine production and outcome in experimental models of endotoxemia and peritonitis in wild-type (WT) and Gas6−/− mice. Circulating levels of Gas6 after LPS 25mg/kg i.p. peaked at 1 hour (P<0.001). Similarly, TNF-α was higher in Gas6−/− than in WT mice 1 hour after LPS (P<0.05). Furthermore, 62 anti- and pro-inflammatory cytokines were quantified in plasma after LPS injection. Their levels were globally higher in Gas6−/− plasma after LPS, 47/62 cytokines being at least 50% higher in Gas6−/− than in WT plasma after 1 hour. Mortality induced by 25mg/kg LPS was 25% in WT versus 87% in Gas6−/− mice (P<0.05). LPS-induced mortality in Gas6 receptors Axl−/−, Tyro3−/− and Merkd was also enhanced when compared to WT mice (P<0.001). In peritonitis models (cecal ligation and puncture, CLP, and i.p. injection of E. coli), Gas6 plasma levels increased and remained elevated at least 24 hours. CLP increased mortality in Gas6−/− mice. Finally, we explored the role of Gas6 in LPS-treated macrophages. We found that Gas6 was released by LPS-stimulated WT macrophages and that Gas6−/− macrophages produced more TNF-α and IL-6 than WT macrophages. Cytokine release by Gas6−/− macrophages was higher than by WT macrophages (cytokine array). Adjunction of recombinant Gas6 to the culture medium of Gas6−/− macrophages diminished the cytokine production to WT levels. In LPS-treated Gas6−/− macrophages, Akt and Erk1/2 phosphorylation was reduced whereas p38 and NFκB activation was enhanced. Thus, in septic patients, elevated Gas6 levels were associated with fatal outcome. In mice, they raised in experimental endotoxemia and peritonitis models, and correlated also with sepsis severity. However, Gas6−/− mice survival in these models was reduced compared to WT. Gas6 secreted by macrophages in response to LPS activated Akt and restrained p38 and NFκB activation, thereby dampening macrophage activation. Altogether these data suggest that, during endotoxemia, Gas6−/− mice phenotype resembles that of mice which have undergone PI3K inhibition, indicating that Gas6 is a major modulator of innate immunity.
Knockdown of Peroxiredoxin VI in K562 Cells Stably Transfected with Oxidase Components Reduces NADPH Oxidase Activity in Response to PMA and fMLP.
Authors: Ambruso Daniel R. , Thurman Gail
Published: April 2020

Introduction: An intact respiratory burst is critical to the microbicidal activity of neutrophils. The respiratory burst is initiated by assembly and activation of the NADPH oxidase complex. We recently identified a 29 kDa neutrophil protein which binds p67phox, is identified as peroxiredoxin VI by sequence and activity, translocates to the plasma membrane of neutrophils during activation and enhances superoxide anion (O2−) production in a cell-free assay exhibiting saturable kinetics. The activity of p29 peroxiredoxin (Prx) requires cysteine residues of the protein. The present studies demonstrate the requirement of p29 Prx for optimal oxidase activity in intact cells.

Methods: K562 cells stably transfected with p47phox, p60phox, gp91phox and the low affinity fMLP receptor and expressing p22phox, p40phox and Rac were grown in tissue culture at 37°C in 5% CO2 with RPMI and 10% fetal calf serum. Three siRNA molecules (Q, A1, A2) as well as non-interfering RNA (Nsi) used as a control were obtained commercially. Transfection of K562 cells was completed with Nucleofector technology (Amaxa Biosystems, Gaithersburg, MD) using Solution L and protocol T020. Transfection was >90% by expression of a GFP plasmid; viability after the procedure was >90%. O2− was measured after stimulation with PMA (200 ng/ml) as SOD inhibitable cytochrome c reduction or in response to fMLP (1 μM) by the addition of Diogenes (National Diagnostics) and detection of chemiluminescence in a luminometer (FB12, Berthod Detection Systems, Pforzheim, Germany) as relative light units. Cell lysates were made with the addition of 10% Triton X-100 and stored at −70°C. Proteins (5–10μg of cell lysate) were separated on a 10% SDS-PAGE and blotted onto nitrocellulose. Detection of specific proteins was completed using polyclonal antibodies to p29 Prx and to other phox proteins and actin and secondary antibodies with a chemiluminescent technique.

Results: Transfection of the K561 cells with Q, A1, and A2 resulted in a knockdown of p29 Prx (25–40%) compared to Nsi. Optimal knockdown occurred in the presence of 2μM siRNA, at 48 hours after transfection. No changes in actin, p67phox, p47phox, p40phox, p22phox or gp91phox were documented with nucleofection, siRNAs or Nsi. In 5 separate experiments, knockdown of p29 Prx resulted in a decrease (p<0.05) in O2− production (PMA) or O2− associated chemiluminescence (fMLP), see Figure (columns and bars represent mean ± SEM).

Conclusion: O2− production in response to PMA or fMLP was reduced in K562 cells with a knockdown of p29. These results extend previous studies and demonstrate the requirement of p29 Prx, peroxiredoxin VI, for optimal NADPH oxidase activity within intact cells.

Figure Figure
Further Evidence That MDS and AML Cells Exhibit a Differential Capacity To Activate the DNA-Damage Response (DDR) Pathways.
Authors: Boehrer Simone , Ades Lionel , Galluzzi Lorenzo , Olaussen Ken , Braun Thorsten , Grosjean Jennifer , Fabre Claire , de Botton Stephane , Fenaux Pierre , Kroemer Guido
Published: April 2020

Background: Findings, that activation of the DDR-pathway in preneoplastic lesions of solid neoplasms slows tumorigenesis, could also underly progression of MDS to AML. We previously showed (ASH 2006, abstr n° 2649), that - in contrast to the de novo AML-derived KG-1 cells - irradiated MDS/AML-derived cell lines P39 and MOLM-13exhibit earlier and greater cell cycle arrest in G2/M and a higher percentage of apoptotic cells;upregulate the crucial checkpoint kinase Chk1-P-Ser317;decrease G2/M-arrest upon inhibition of Chk1 (and to a lesser extent upon inhibition of ATM).

Methods: We further analyzed this differential checkpoint regulation in MDS versus AML cell lines by assessing irradiation-induced apoptosis in the presence of Chk1- and ATM-inhibitors and the functional role of H2AX, situated below the crucial checkpoint kinase Chk1. Myeloid cell lines P39, MOLM-13, MV4-11, and KG-1 were g-irradiated and their ability to undergo apoptosis (DIOC/PI staining) quantified in the absence and presence of the Chk1 inhibitor UCN-01 and the ATM inhibitor KU-55933. Protein expression of activated H2AX was quantified by immunoblot analysis with an anti-H2AX-P-Ser139 antibody. Histological staining of bone marrow biopsies from patients with low and high risk MDS, and AML was made with an anti-H2AX-P-Ser139 antibody. Staining was considered positive if at least 20% of the biopsy surface exhibited 50% or more stained cells.

Results: In the presence of the ATM inhibitor irradiated KG-1 cells retained their apoptosis-resistance, and irradiated P39, MOLM-13 and MV4-11 cells exhibited the same degree of apoptosis as seen in the absence of the inhibitor. In contrast, inhibition of Chk1 (newly) established apoptosis-sensitivity in KG-1 cells (which showed about 30% of DIOC-low cells following 24 hours of irradiation with 10 Gy), while having only minor effects on the other cell lines. Determining the concomitant activation status of H2AX, which is a downstream target of Chk1, showed that irradiation induced a comparable expression of H2AX-P-Ser139 in MDS and AML cell lines. Co-incubation with the Chk1 inhibitor was associated with lower expression of activated H2AX in the irradiated MDS cell lines P39 and MOLM-13 as compared to irradiated KG-1 cells. Histological staining of marrow biopsies with an anti-H2AX-P-Ser139 antibody demonstrated that activation of H2AX is less frequent in low risk MDS (1/10 samples) than in high risk MDS (9/18 samples) and AML (4/6 samples).

Conclusion: Further analyzing the different capacities of MDS and AML cells to employ DDR-pathways, we provide additional insights into the functional relevance of ATM and Chk1 inhibition in the apoptotic DDR, and give preliminary evidence for a differential activation of H2AX in irradiated cell lines and patient samples of MDS and AML.
MDS with Isolated Neutropenia or Thrombocytopenia. Incidence and Characteristics in the Groupe Francophone des Myélodysplasies (GFM) Registry.
Authors: Park Sophie , Andrieu V. , Sapena R. , Leroux G. , Stamatoullas A. , Callat M.P. , Beyne-Rauzy O. , Mairesse J. , Chaury M.P. , Dombret Herve , Siguret V. , Vey Norbert , Lepeu G. , Guerci A. , Cheze S. , Fenaux Pierre , Dreyfus F.
Published: April 2020

Background: MDS classification is regularly refined over time. WHO classification (2001) puts emphasis on cases with anemia and isolated dyserythropoiesis, as opposed to multilineage dysplasia, while the next modifications to WHO classification will probably consider cases with isolated or predominant granulocytic or thrombopoietic involvement.

Methods: In this context, we analyzed cases of IN and IT included in the GFM registry of MDS.

Results: From Jul 2003 to Jul 2007, 1009 newly diagnosed MDS patients (including 145 therapy MDS, and excluding CMML,) from 11 centers were included in the GFM registry. 22 of them (2%) presented with neutropenia (ANC <1.5×109/l) (IN) and 24 (2.4%) with thrombocytopenia (plts<100×109/l) (IT) as only cytopenia, respectively. IN included 13 F and 9 M and IT 17 F and 7 M. Median age was 70 years (range 54-89) in IN, and 72 years (range 50-84) in IT. Median ANC was 0.89×109 (range 0.14-1.48×109) in IN, and median platelet count 62×109 (range 7-99×109) in IT. According to WHO (2001), IN included 5 RA (diagnosis based in 4 pts and 1 pt on prominent unilineage dyserythropoiesis and unilineage dysmegacaryopoiesis respectively, and elimination of other causes of anemia, as karyotype was normal in all 5 pts), 1 RCMD, 8 RAEB-I, 2 RAEB-II, 1 patient with 5q- syndrome (with Hb=12.5g/dl), 1 unclassified MDS with marrow hypocellularity (of 39 U -MDS in the registry,) and 4 therapy-related MDS. IT included 13 RCMD, 7 RAEB-I, 4 RAEB-II. Cytogenetic findings were: for IN: good risk: 15 (normal: 13, del 5q, -Y), intermediate: 2 (+8), poor: 1, cytogenetic failure: 4 ; for IT: good risk: 13 (normal: 12, del 20q:1), intermediate: 5 (del 2, del3p,+8, del 9+t(9;14), del11+del13p), poor: 2, cytogenetic failure: 4. IPSS was: For IN: low (5), Int-1 (7), Int-2 (5), High (1) patients (4 ND); For IT: low (7), Int-1 (9), Int-2 (4) and High (0) (4 ND). Median follow-up of IN and IT was 23 months (range 2-48 mos). 5 IN pts progressed to AML and 4 of them died, 1 IT pt progressed to AML and 2 died.

Conclusion: IN and IT are rare in MDS, predominate in females, contrary to overall MDS, and are widely distributed in the current WHO classification. IT was always associated with multilineage dysplasia, and/or excess of marrow blasts contrary to some IN, which were therefore more difficult to diagnose, especially as karyotype was usually normal. Follow-up in the GFM registry is still limited to evaluate the prognosis of IN and IT. These findings are to be considered if new specific MDS categories are to be individualized.
The Multiple Myeloma Research Consortium Genomics Initiative.
Authors: Trent Jeffrey , Carpten John , Reich Michael , Liefeld Ted , Keats Jonathan , Mousses Spyro , Hahn William , Trudel Suzanne , Siegel David , Rajkumar S. Vincent , Alsina Melissa , Richardson Paul , Anderson Kenneth , Auclair Daniel , Perkins Louise M. , Giusti Kathy , Fonseca Rafael , Golub Todd R.
Published: April 2020

The Multiple Myeloma Research Consortium (MMRC) Genomics Initiative is a three-year program to analyze tumor tissue from hundreds of multiple myeloma (MM) patients via gene expression profiling (GEP), comparative genomic hybridization (aCGH), and exon re-sequencing. In addition, RNAi knockdown of selected genes in MM tumor cell lines is being evaluated to identify potential new targets. All genomic data generated is scheduled for placement in an open-access Multiple Myeloma Genomics Portal pre-publication and in near real-time (www.broad.mit.edu/mmgp). Additionally, samples are also destined for drug validation and correlative science on clinical protocols as this study moves forward. This comprehensive project is spearheaded by the MMRC and conducted via collaboration with the Eli and Edythe L. Broad Institute of MIT and Harvard, the Translational Genomics Research Institute (TGen), Mayo Clinic Arizona, and The Dana-Farber Cancer Center. The study is supported by the collection from member institutions of the MMRC of bone marrow aspirates and matched peripheral blood samples from over 1000 patients. Specific genomic technologies that are currently being employed across this sample set include GEP using Affymetrix Human Genome U133A 2.0 Plus Arrays, and, in parallel, efforts to identify regions of genomic gain and loss are using Agilent Human Genome CGH arrays. In contrast to other large-scale genomic projects based on exon-sequencing of targeted gene sets, this project will be the first to perform genome-scale single molecule sequencing (SMS) of DNA from patient specimens. Results will be targeted against candidate classes of genes (e.g. kinases, phosphatases, known oncogenes and tumor suppressors), and genes from GEP or within candidate regions of copy gain or loss identified by the aCGH experiments. Mutations will be further validated in an independent set of patient specimens. Finally we will attempt to identify points of vulnerability of MM through systematic loss-of-function screens in myeloma cell lines using high-throughput RNA interference (using both shRNA and siRNA platforms). Importantly, data generated from this genomics initiative will ultimately be made public pre-publication through the established MMRC Multiple Myeloma Genomics Portal. Data from all aspects of this project (sample collection and analyte isolation, GEP, aCGH, SMS, RNAi and bioinformatics) will be described in this presentation. The power of this study is the comprehensive collection of gene expression, CGH, and genome sequencing on a single reference set of clinically annotated samples. The addition of RNAi screens makes this a very important and unique data resource, which we hope will help expedite the discovery of novel targeted agents for MM scientific community.
Lenalidomide (Len) in Combination with Rituximab (R) Demonstrated Early Evidence of Efficacy in a Phase I/II Study in Relapsed/Refractory Mantle Cell Lymphoma (MCL).
Authors: Wang Michael , Fayad Luis , Hagemeister Frederick , Neelapu Sattva , Samuels Barry , Samanigo Felipe , Pro Barbara , Yi Qing , Bell Neda , Byrne Catriona , Weaver Pam , Hartig Kim , Knight Robert , Zeldis Jerry , Kwak Larry , Romaguera Jorge
Published: April 2020

Background: Relapsed/refractory MCL is difficult to treat. R targets CD20 antigen on the surface of MCL cells while Len may target the microenvironment of MCL cells and enhance the ADCC activity of R. To test this hypothesis, we initiated a single-center, phase I/II study. We now report the data from the completed phase I trial and the ongoing phase II trial.

Methods: Eighteen eligible patients (pts) with MCL had 1–4 lines of prior therapies. None had prior thalidomide and all received R. Treatment consisted of Len given orally daily on days 1–21 of a 28-day cycle and R 375 mg/m2 by IV infusion weekly for 4 weeks only during the first cycle with the first dose on Day 1 in Cycle 1. A standard 3+3 dose escalation was used to determine MTD with Len doses at 10 mg, 15 mg, 20 mg, and 25 mg. DLT was defined as grade (G) 3 or 4 non-hematologic or G4 hematologic toxicity during the first cycle. Phase I has been completed with 15 pts. Phase II is ongoing with 3 pts enrolled at MTD. Total 80 cycles of therapy were given to 18 pts. In addition to the regular radiology evaluation, a separate radiology evaluation was performed independently. EGD and colonoscopy with random biopsies were performed to confirm all CRs.

Results: In the phase I study, median age was 73 (62–84); median prior therapies were 3 (1–4); median cycles received to date (August 18, 2007) were 4 (range 1–14). Two DLT’s occurred at 25 mg. One G3 hypercalcemia and 1 G 4 non-neutropenic fever during the first cycle. After total 80 cycles of therapy in 18 patients, except for the 2 DLT’s, common non-hematologic toxic events included fatigue (23 G1-2), pruritis (18 G1-2), myalgias (5 G1-2, 1 G3) and rash (5 G1-2). There was no DVT/PE. Grade 3 hematologic toxic events included neutropenia (11), febrile neutropenia (2), and thrombocytopenia (2). The only grade 4 hematologic toxic events were neutropenia (5). There was no grade 3–4 anemia. There were no responses at 10 mg or 15 mg. Ten pts at MTD (20 mg) including 7 in phase I plus 3 in phase II were evaluable for response. Seven out of 10 pts achieved responses including 3 CRs (30%), 4 PR’s (40%), 1 SD and 2 PD’s. Therefore, the overall response rate was 70%. Media time to response was 2 cycles (range 2–4).

Conclusions: The MTD for Len with R in relapsed/refractory MCL was 20 mg, orally daily on days 1–21 of a 28-day cycle. Early evidence of response is promising with a very favorable toxicity profile at 20 mg and is being further evaluated in the ongoing phase II trial.
Lenalidomide Response in Relapsed/Refractory Diffuse Large B-Cell Non-Hodgkin’s Lymphoma.
Authors: Lossos Izidore S. , Wiernik Peter H. , Justice Glen , Tuscano Joseph M. , Cole Craig E. , Vose Julie M. , Pietronigro Dennis , Takeshita Kenichi , Ervin-Haynes Annette , Zeldis Jerome B. , Habermann Thomas M.
Published: April 2020

Background: Patients with diffuse large B-cell lymphoma (DLBCL) not cured by first line R-CHOP chemotherapy or second line high-dose chemotherapy with autologous stem cell rescue have a poor prognosis and represent a clear unmet medical need. Lenalidomide (Revlimid®), an immunomodulatory drug, has activity in hematological malignancies including relapsed/refractory multiple myeloma, chronic lymphocytic leukemia and cutaneous T-cell lymphoma.

Aim: To determine the activity and safety of lenalidomide monotherapy in relapsed/refractory DLBCL.

Methods: Patients with relapsed/refractory DLBCL with measurable disease after at least 1 prior treatment regimen were eligible. Patients received 25 mg lenalidomide orally once daily on Days 1–21 every 28 days and continued therapy for 52 weeks as tolerated or until disease progression. Response and progression were evaluated using the IWLRC methodology. Univariate analyses using Fisher’s exact test was conducted to investigate and characterize associations of prognostic variables with response.

Results: Twenty-six patients with DLBCL lymphoma were enrolled. The median age was 66 (45–86) and 13 were female. Median time from diagnosis to lenalidomide was 2.4 (0.5–12) years and median number of prior treatment regimens was 3 (1–6). Three patients (12%) exhibited an objective complete response (1 complete response (CR) and 2 complete responses unconfirmed (CRu)). Two patients had a partial response (PR) for an overall response rate of 19% and 7 had stable disease (SD). Response to lenalidomide was associated with low disease burden (33% for < 50 cm2v 0% for ≥ 50 cm2, P=0.06) and longer time from rituximab to lenalidomide treatment (33% for ≥ 230 days v 0% for < 230 days, P=0.05). Patients with favorable values for both these prognostic factors (N = 10) had a 50% response rate [RR] (30% CR/CRu RR) compared with a 0% RR for patients with unfavorable values (N = 16; P < 0.004) and a progression free survival (ongoing) of 7.4 months v 1.9 months. Although there were no responses in the poor prognosis group, 4 patients exhibited stable disease with an ongoing median PFS of > 4.2 months (> 3.6 - > 6.2). Eight patients (31%) required at least one dose reduction with a median time to first dose reduction of 1.8 months (0.4–2.9). One patient each (4%) had Grade 4 anemia, cauda equinine syndrome, febrile neutropenia, intermittent rash, lymphopenia, neutropenia, pneumonitis and thrombocytopenia. Most common Grade 3 adverse events were neutropenia (19%), thrombocytopenia (15%) and leukopenia (12%).

Conclusion: The prognostic factors of tumor burden and time from last dose of rituximab appear to identify relapsed/refractory DLBCL patients with a high likelihood of response to lenalidomide oral monotherapy.