bluuebunny/crossref_metadata_2025 huggingface dataset:
c-Jun N-Terminal Kinase (JNK) Activation Failure Confers a New Mechanism of Anthracycline Resistance in Acute Myeloid Leukemia (AML).
Authors: Lagadinou Eleni D. , Ziros Panagiotis G. , Tsopra Olga A. , Dimas Kostas , Pantazis Panagiotis , Spyridonidis Alexandros , Zoumbos Nicholas C.
Published: April 2020

Chemotherapy resistance remains a major challenge in the treatment of acute myeloid leukemia (AML). Besides the P-glycoprotein efflux of chemotherapeutics, additional cellular factors may contribute to drug resistance in AML. c- Jun N-terminal Kinase (JNK) is a protein kinase activated after exposure of cells to chemotherapeutic agents. Recently, studies in solid tumours have associated chemoresistance with failure of cancer cells to activate JNK. We asked whether drug resistance in AML is also attributed to intrinsic failure of the AML blasts to activate JNK. In vitro treatment of U937 AML cell line with anthracyclines induced a rapid and robust JNK phosphorylation and apoptosis. In contrast, the anthracyline-resistant derivative U937R cells showed no JNK activation after exposure to anthracyclines, also at doses that resulted in high accumulation of the drug within the cells. Inhibition of JNK in drug-sensitive U937 cells made them resistant to anthracyclines. First, JNK1-siRNA transfected U937 cells exhibited a 50.4% and 61.3% reduced daunorubicin- (DNR, 1μM 24hr) and doxorubicin- (DOX, 1.5 μM 24hr) induced apoptosis respectively; as compared to empty vector or untransfected U937 cells (P< 0.001). Second, pretreatment of U937 cells with the 420116 cell-permeable JNK inhibitor (1 μM) reduced to a less but yet significant extent DNR-induced apoptosis as compared to cells treated with a negative control peptide (P = 0.013<0.05). On the other hand, selective restoration of the inactive JNK pathway in resistant U937R cells by transfection with a mutant form of the SEK1/MKK4 upstream activator of JNK sensitized U937R cells to anthracyclines, compared to empty vector transfected cells (3.3-fold increase in DNR-induced apoptosis, 1μM DNR, 24hr and 3.1-fold increase in DOX-induced apoptosis, 1.5μM DOX, 24hr). Furthermore, we assessed the activation of JNK pathway in 29 primary AML bone marrow samples after short term (30-60 min) in vitro exposure to DNR (1 μM) and correlated it with clinical data. We found a strong correlation between the in vitro pharmacodymanic changes of phospho-JNK levels in AML primary blasts and the response of the AML patients to standard induction chemotherapy (P = 0.012). In addition, the drug-induced JNK activation pattern correlated with AML evolving from antecedent MDS (P = 0.017) and patient age (P = 0.046). In summary, our in vitro and in vivo results suggest that JNK activation failure is another mechanism of anthracycline resistance in AML. Elucidating the ultimate mechanisms leading to JNK suppression in chemoresistant AML may be of major therapeutic value.
The Coexpression of AML1-ETO9a Isoform and AML1-ETO in Acute Myeloid Leukemia M2 Subtype and Its Clinical Significance.
Authors: Zhang Hong , Chen Zi-xing , He Jun , Miao Yu-qin , Cen Jian-nong , Qiu Qiao-chen
Published: April 2020

Previous studies on either animal model or clinical settings have demonstrated that the expression of AML1/ETO9a (AE9a), a truncated version of AML1-ETO (AE) generated by alternative splicing during transcription, in patients with acute myeloid leukemia M2 subtype may have clinical significance. Moreover, the simultaneously expression of both AE and AE9a may even be more meaningful to the prognosis of the patients. To validate this hypothesis, we have measured the mRNA level of both AE and AE9a in each individual M2 patient by quantitative real-time RT-PCR, and correlated the ratio of AE/AE9a mRNA levels with the clinical outcome and prognosis in these patients. In 35 newly-diagnosed AML-M2 patients with t(8;21), expression of AE9a was detected in 30 cases (85.71%). The median value of the percentages of AE9a and AE within overall transcripts of the fusion gene in these patients were 18.16% and 81.84%, respectively (p<0.05). A positive correlation between the mRNA level of AE9a and AE was shown in 45 bone marrow samples from 19 patients during the follow-up, and the correlation coefficient was 0.900(P<0.01). The expression level of both AE9a and AE decreased in 16 patients after one course of standard conventional chemotherapy. Among them, a reduced percentage of AE9a mRNA level was prominent in 11 (68.75%) patients, while the percentage of mRNA level of AE decreased in 5 (31.25%) cases. The complete remission rate after first course of chemotherapy was 81.82%(9/11)in 11 patients, and 20.00%(1/5)in 5 patients, suggesting a higher CR rate in those patients who exhibited more conspicuous decrease in mRNA level of AE9a (P<0.05). The percentage of AE9a mRNA in the total fusion transcripts rose in all of the 5 relapsing cases. In a retrospective study on 92 patients of AML-M2 including 50 cases with t(8;21) and 42 case with either normal karyotype or other chromosome abnormalities, expression of AE9a has been found in these 50 cases by conventional RT-PCR. Between the AE9a positive and negative groups, no statistically significant difference had been found in complete remission rate, relapsing rate and the first CR period over a follow-up with medium time of 13 months (4–76 mths). However, 22 months of medium survival in AE9a positive group (38 cases) was remarkably shorter than 70 months in AE9a negative group (25 cases). Our results indicated that the mRNA of AE9a at a relatively lower level co-expressed with AE in most AML-M2 patients with t(8;21) translocation. The expression level of AE9a rather than AE may play more crucial role in development and progress of leukemia. AE9a positive patients have distinctly shorter survival time than the negative cases, predicting a worse prognosis.
Novel C-KIT Transcripts Identified in Mast Cell Leukemia: An Update of the Full Transcript and It’s Distribution.
Authors: Ozer Ozden , Zhao Yi D. , Ostler Kelly R. , Akin Cem , Anastasi John , Vardiman James , Godley Lucy
Published: April 2020

Introduction: Mast cell leukemia (MCL) is the rarest but most fatal form of mast cell neoplasms. Several C-KIT activating mutations have been identified in systemic mastocytosis (SM), the most frequent one being D816V. Little is known about C-KIT mutations in MCL. In 2005, at ASH we presented a novel C-KIT transcript in two patients with MCL, accounting for the sole C-KIT transcript in one of the patients. The novel form had a deletion of exons 12 and 13, encoding the N-terminal portion of the bipartite kinase domain. Here, we describe the complete C-KIT transcript, its response to stem cell factor (SCF) stimulation in vitro, and its expression in non- MCL samples.

Materials&Methods: The entire C-KIT coding region was amplified by RT-PCR from cDNA prepared from bone marrow (BM) samples of 2 patients with MCL, 2 patients with SM associated with acute myeloid leukemia, isolated CD34+ cells from healthy individuals, and 2 sets of purified mast cells from patients with SM. Several clones from each sample were fully sequenced. Its expression in patient samples was verified by Western blotting. An expression construct was made, and the mutant C-KIT was expressed in COS-1 cells. Western blotting was used to test the ability of the mutant protein to undergo autophosphorylation upon SCF stimulation.

Results: Complete sequencing demonstrated that the mutant C-KIT contained several mutations: a large deletion of amino acids 59–437, corresponding to loss of the first three immunoglobulin-like domains in the extracellular region, deletions of amino acids 510–513, deletions of the amino acids encoded by exons 12 and 13, and novel single amino acid deletions unique to each patient. The mutant C-KIT transcript was present in normal CD34+ cells and in 2 BMs with SM associated with AML. However, it was absent in purified mast cells from 2 patients with SM. In contrast, these displayed the widely recognised D816V point mutation. Western blotting from patient samples revealed a truncated protein of the predicted 59KD size. However, the novel C-KIT did not autophosphorylate upon stem cell factor stimulation.

Discussion: We have identified a novel C-KIT isoform. It was exclusively present in MCL but completely absent in purified mast cells of SM. These findings indicate that it is a marker of immaturity and aggressiveness among mast cell neoplasms, which may serve as a diagnostic and prognostic parameter. Its presence in non-neoplastic samples in similar amounts with the wild type suggest that it is not malignancy specific. However, its predominace over the wild type may correlate with a pathologic state that remains to be explored in other hematopoetic neoplasms.
The Inflammatory Subset of Monocytes Stimulates Angiogenesis at Sites of Ischemia by Altering the Balance of Pro- and Anti-Inflammatory Cytokines.
Authors: Gregory Alyssa D. , Capoccia Benjamin J. , Link Daniel C.
Published: April 2020

Our laboratory has previously shown that early delivery of monocytes to ischemic tissue by mobilization from the bone marrow to blood or by adoptive transfer enhances revascularization after acute vascular injury. There is accumulating evidence that monocytes represent a heterogeneous population, containing subsets with distinct functional properties. Herein, we show that the inflammatory subset of monocytes mediates the angiogenic response in a murine model of hindlimb ischemia induced by femoral artery excision (FAE). Inflammatory (CX3CR1loGr-1+) and resident (CX3CR1hiGr-1−) monocytes were sorted from the bone marrow of CX3CR1+/GFP mice and injected intravenously into recipient mice 24 after FAE. Revascularization was monitored by laser Doppler imaging. A marked improvement in blood flow was observed after adoptive transfer of inflammatory but not resident monocytes [ratio of blood flow in ischemic to nonischemic limb 7 days after surgery: 0.75±0.05 (inflammatory monocytes); 0.40±0.07 (resident monocytes); 0.40±0.03 (saline control); P <0.01]. Consistent with this finding, inflammatory monocytes are selectively recruited to sites of ischemia. Finally, we show that the bone marrow is a rich reservoir of inflammatory monocytes that is readily mobilized into the blood by ischemia. To begin to elucidate the mechanisms by which inflammatory monocytes stimulate angiogenesis, we determined the effect of the adoptive transfer of monocytes on the local production of key cytokines/chemokines in the ischemic tissue. Specifically, a non-biased multiplex immunoassay-based screen for soluble factors present in the cell-free muscle homogenates was performed. Data revealed decreased levels of the pro-inflammatory cytokines IL-6, KC, and mip-1beta and increased levels of the anti-inflammatory cytokine IL-10 in mice receiving an adoptive transfer of monocytes. In keeping with this observation, levels of free myoglobin were dramatically decreased in mice receiving an adoptive transfer of monocytes, indicating a reduction in inflammation-mediated tissue damage [myoglobin levels at 28 hours post-ischemia: 18,580±879 ng/mL (no adoptive transfer); 158±112 ng/mL (adoptive transfer)]. Interestingly, known regulators of angiogenesis, MCP-1, MMP-9, VEGF, and FGF-basic were also significantly increased. Taken together, these data indicate that adoptive transfer of a small number of monocytes shifts the balance of pro- and anti-inflammatory signals in the ischemic environment, which limits tissue damage and enhances revascularization. This finding has important therapeutic implications, since deliver of inflammatory monocytes to sites of ischemia may polarize the microenviroment towards tissue regeneration and angiogenesis.
Acetylation of an Ets Transcription Factor PU.1 Suppresses Its Transcriptional Activity.
Authors: Mouri Fumihiko , Tsukada Junichi , Fukamizu Akiyoshi , Tanaka Yoshiya
Published: April 2020

PU.1, a member of the Ets family transcription factors, is expressed restrictively in hematopoietic cells including monocytes and macrophages, and plays critical roles in the inflammatory responses and the development of hematopoietic cells. CREB-binding protein (CBP) regulates transcription by acetylating not only histones but also certain transcription factors. Here, we demonstrated that a specific inhibitor of histone deacetylases, trichostatin A (TSA) inhibits PU.1 transcriptional activity in monocytes and further showed that deletion of a histone acetyltransferase (HAT) domain of CBP resulted in synergistic cooperativity between CBP and PU.1. When human monocytic cells THP-1 were treated with TSA, our immunoprecipitaion and western blot assay showed that TSA enhanced PU.1 acetylation. Next, we investigated the effect of TSA on the transcriptional regulation of PU.1-dependent gene promoters such as the human prointerleukin 1β (IL1B) gene and the human granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) gene in transient transfection studies. Two distinct luciferase reporter plasmids (Luc) for the IL1B gene promoter and the GM-CSFRα gene promoter, IL1B-Luc and GM-CSFRα-Luc were used. When these plasmids were transiently transfected into THP-1 cells, TSA suppressed LPS-induced activities for the IL1B promoter and the GM-CSFRα promoter in a dose-dependent manner. In contrast, when NF-κB luciferase reporter, NF-κB-Luc was transfected into THP-1 cells, TSA synergistically increased LPS-induced NF-κB activities. Moreover, when a PU.1 expression vector, pECEPU.1 was cotransfected into PU.1-deficient murine thymocytes EL4 along with either IL1B-Luc or GM-CSFRα-Luc. The PU.1-induced promoter activities were strongly suppressed through TSA treatment. FACS analysis further indicated that TSA suppressed LPS-induced expression of IL-1β and GM-CSFRα proteins. In addition, our EMSA data showed that TSA treatment did not affect DNA binding activity of PU.1 to the IL1B promoter. PU.1 has been shown to interact physically with CBP to transactivate their target genes. In our study, expression vectors for CBP wild-type or with a deletion of its HAT domain was cotransfected into EL4 cells along with IL1B-Luc and pECEPU.1. The HAT activity-deficient mutant showed synergistic transcriptional activity with PU.1 more strongly than the wild-type CBP. In this regard, our GST-pulldown assay showed that deletion of CBP HAT domain did not change binding affinity of CBP for PU.1. Our results propose a novel molecular mechanism by which PU.1-dependent genes is negatively regulated by HAT-induced acetylation in monocytes.
Toll-Like Receptor-Induced Expression of the NKG2D Ligand MICA on Monocytes Modulates Their Interaction with NK Cells.
Authors: Kloss Mercedes , Decker Patrice , Krusch Matthias , Baltz Katrin M. , Baessler Tina , Kanz Lothar , Steinle Alexander , Salih Helmut R.
Published: April 2020

Reciprocal interactions of NK cells with Dendritic Cells (DC) have been shown to influence activation of NK cells as well as maturation or lysis of DC and subsequent adaptive immune responses. However, little is known about the interaction of peripheral blood Monocytes with NK cells. Here we report that stimulation of Toll-like receptors (TLR) on Monocytes using LPS, Pam3Cys, R848 and PolyI:C induced mRNA and surface expression of MICA but no relevant changes of the further ligands of the activating immunoreceptor NKG2D, MICB or ULBP1-3. MICA upregulation upon stimulation with TLR ligands correlated with CD80 and HLA-DR upregulation and increased secretion of the pro-inflammatory cytokines IL-6, IL-8 and TNF by Monocytes. Interestingly, high MICA surface expression on activated Monocytes was not accompanied by a release of MICA in soluble form. The LPS-induced MICA upregulation by Monocytes was highly sensitive since it was observed with as little as 4 ng/ml LPS after 15 hours and was associated with IL-6 secretion and inhibition of apoptosis indicating that Monocytes were activated and functional. To determine whether upregulated MICA expression on Monocytes was detected by NK cells we took advantage of the fact that NKG2D is down-regulated after interacting with its ligands. While NKG2D levels on NK cells were not substantially changed in cultures with unstimulated Monocytes, a marked reduction of NKG2D was observed in the presence of activated MICA-expressing Monocytes. The modulation of NKG2D did not occur when NK cells and monocytes were separated by a transwell filter, which demonstrates that the numerous cytokines produced by TLR-activated Monocytes are not responsible for the NKG2D modulation and confirmes the role of MICA in Monocyte-NK cell interaction. Importantly, TLR-induced MICA expression on Monocytes stimulated IFN-γ production of NK cells, which could be reduced by addition of blocking anti-MICA/B F(ab’)2 fragments while isotype control had no effect. This demonstrates that the observed stimulation of NK cells was in fact specifically due to NKG2D-NKG2DL interaction. Our data indicate that NKG2D-MICA interaction provides a mechanism by which NK cells and Monocytes may communicate directly during innate immune responses to infections in humans.
Dendritic Cells as a Novel Target for Erythropoietin: Studies in Murine Models.
Authors: Lifchitz Lilach , Sagiv Sari Prutchi , Markovitz Maayan , Mittelman Moshe , Newmann Drorit
Published: April 2020

Erythropoietin (EPO) is the major hormone that promotes the proliferation and differentiation of erythroid progenitor cells. Unexpectedly, EPO receptor (EPO-R) was also found on non-erythroid cells; thus leading to the discovery of non-erythroid effects of EPO. Our own previous contribution to that issue was in demonstrating that the immune system is a target for EPO, including both the cellular and humoral immune response types. As yet, the direct target cells for EPO as well as the molecular mechanisms underlying its function as an immunomodulator remain unknown. We first examined lymphocytes as possible candidates, and could not detect any expression of EPO-Rs on these cells. Here, we focused on dendritic cells (DCs), known to initiate immune response as antigen presenting and T cell priming cells. We employed murine bone marrow DCs (BMDCs) and splenic DCs (SDCs) models to determine EPO-R expression, and delineate in-vitro and in-vivo effects of EPO via these cells. We found that BMDCs express EPO-R mRNA, as detected by RT-PCR. In vitro stimulation of the BMDCs with recombinant human EPO (rHuEPO) activated the NFkB and MAPK signaling pathways, and induced a higher surface expression of CD80, CD86 and MHC class II. These data are reinforced by in vivo experiments, showing that rHuEPO injection into naïve mice led to an increase in the SDC population and in the cell surface expression of CD80, CD86 and MHC class II markers. These novel findings implicate the significance of the multifunctional role of EPO in the hematopoietic and immune systems, and may lead to its further clinical applications as an immunomodulator.
Role of Novel C-Type Lectin Receptor DCL-1 in Phagocytes and Dendritic Cells.
Authors: Hart Derek N.J. , Khan Seema , d’Aniello Elisabetta , McDonald Kylie J. , Kato Masato
Published: April 2020

Cell surface C-type lectins are implicated in many aspects of immunity. For example, professional phagocytes utilise cell surface C-type lectins in pathogen recognition/binding and phagocytosing the pathogens for destruction and/or antigen processing (e.g. mannose receptor, DC-SIGN, dectin-1). Some C-type lectins are involved in cell-cell adhesion and trafficking of leukocytes (e.g. selectins). DCL-1 is a small type I transmembrane C-type lectin receptor, discovered as a genetic fusion partner of DEC-205. DCL-1 protein was highly conserved amongst the human, mouse and rat orthologs. The human DCL-1 gene, composed of 6 exons, was located to a cluster of type I transmembrane C-type lectin genes on chromosomal band 2q24. Multiple expression array, RT-PCR and FACS analysis using new anti-hDCL-1 mAbs established that DCL-1 expression in leukocytes was restricted to monocytes, macrophages, granulocytes and DC, although DCL-1 mRNA was present in many tissues. Stable hDCL-1 CHO cell transfectants endocytosed FITC-conjugated anti-hDCL-1 mAb rapidly (t1/2 = 20 min) and phagocytosed anti-hDCL-1 mAb-coated microbeads, indicating that DCL-1 may act as an antigen uptake receptor. However, anti-DCL-1 mAb-coated microbead binding and subsequent phagocytic uptake by macrophages was ∼8-fold less efficient than that of anti-macrophage mannose receptor (MMR/CD206) or anti-DEC-205/CD205 mAb-coated microbeads. Confocal studies showed that DCL-1 colocalized with F-actin in filopodia, lamellipodia and podosomes in macrophages and the colocalization was not affected with cytochalasin D, whereas the MMR/CD206 and DEC-205/CD205 did not colocalize with F-actin. Furthermore, DCL-1-EGFP transiently expressed in COS-1 cells colocalized with F-actin at cellular cortex and microvilli. These data suggest that hDCL-1 is an unconventional lectin receptor that plays roles not only in endocytosis/phagocytosis, but also in myeloid cell adhesion and migration.
Hematopoietic miRNAs Expression in Myelodysplastic Syndromes.
Authors: Pons Aina , Nomdedeu Benet , Navarro Alfons , Gaya Anna , Pairet Silvia , Granell Miquel , Valera Sandra , Gel Bernat , Montserrat Emili , Monzo Mariano
Published: April 2020

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem-cell disorders that include a broad spectrum of entities characterized by ineffective hematopoiesis and risk of transformation into acute myeloid leukemia (AML). MicroRNAs (miRNA) act as negative expression regulators of important genes as those participating in cellular proliferation, apoptosis and carcinogenesis. In this study we analyzed the expression patterns of 26 miRNA hematopoietic-related genes in patients with MDS. Total RNA was extracted from 20 MDS patients (BM and PB) and 5 normal controls. Median age of patients was 70 (range, 55–82) years. Seven patients had refractory anemia with excess of blasts (RAEB-I, n=5; RAEB-II, n=2), 7 refractory cytopenia with multilineage dysplasia (RCMD), 3 RCMD with ringed sideroblasts (RCMD-RS), 1 chronic myelomonocytic leukemia (CMML), 1 refractory anemia with ring sideroblasts (RARS) and 1 MDS associated with isolated del(5q). 15 patients presented low risk IPSS (LR) (low or intermediate-1) and 5 patients had high risk IPSS (HR) (intermediate-2 and high risk). Twenty-six mature miRNAs were assessed by Stem-loop RT-PCR and Real time PCR in ABI PRISM 7500. MiRNA expression data was normalized to overall median and relative quantification was calculated with the 2−ΔΔCt method. The data were presented as log10 of relative quantity of target miRNA. Median of normal controls was used as calibrator for all samples. Data were analyzed by class comparison methods using BRB Array Tools version 3.4.0 and TIGR multiexperiment viewer. Hierarchical clustering analysis of BM categorized two clusters corresponding to BM-MDS and BM-controls. Then, miR-142-5p (p<0.001) and miR-18a (p<0.001) were differentially expressed between BM-MDS and BM-controls while miR-103 (p=0.003) was overexpressed and miR-18a (p=0.02) was underexpressed in PB-controls with respect to PB-MDS. The analysis of miR-10a and miR-103 in PB (p=0.035 and p=0.031) and miR-181a and miR-10a in BM (p=0.044 and p=0.02) allowed us to discriminate between LR and HR groups. We also found that miR-222 was overexpressed in AML regarding MDS samples (p<0.001). It has been previously described that high activity of miR-222 is necessary to maintain low p27KIP1 levels and continuous proliferation in certain cancer cell lines. In conclusion, this study confirms that miRNAs have a role in the pathogenesis of MDS and their transformation into AML.
High-Resolution Analyses of Epigenetic Aberrations in Myelodysplastic Syndrome.
Authors: Yamamoto Go , Nakamura Fumihiko , Iio Mitsuru , Kato Motohiro , Nannya Yasuhito , Sanada14 Masashi , Hangaishi Akira , Chiba Shigeru , Mori Hiraku , Kurokawa Mineo , Watanabe Toshiki , Omine Mitsuhiro , Ogawa Seishi
Published: April 2020

Myelodysplastic syndromes are heterogeneous groups of clonal hematopoietic disorders characterized by ineffective blood cells production and predisposition to acute myeloid leukemia, and as such, it is well established that these syndromes actually represent neoplastic processes in which a series of gene mutations accumulate in blood cell precursors, leading to neoplastic expansion of dominant clones. During the past two decades, a number of genetic abnormalities have been described in MDS cases, including copy number alterations of particular chromosomal segments, mutations of Ras, p53, runx1, and Flt3 genes, and translocation involving Evi-1 family genes, TEL, MLL and Nup98 genes. On the other hand, epigenetic abnormalities are also thought to play an important role in the pathogenesis of MDS, because demethylating agents such as 5-azacydine and decitabine are often effective for high risk MDS. Unfortunately, however, only a few genes, such as INK4B gene, have been implicated in MDS pathogenesis. Especially, no genome-wide analysis of epigenetic changes in MDS has been reported. So, in the current study, we comprehensively investigated abnormalities of DNA methylation in 30 MDS specimens, using Affymetrix tiling array combined with methylated DNA immunoprecipitation (MeDIP). In this method, genomic DNA from MDS specimens was first fragmentized with ultrasonication and immuno-precipitated with anti-methylcytosine antibody (MeDIP). The immunoprecipitated DNA was then amplified by PCR and subjected to hybridization to the promorter tiling array. In this array, regulatory regions of more than 25,000 genes are tiled by 6.5 millions of oligonucleotide probes to enable sensitive detection of target sequences and approximately 59% of CpG islands in the human genomes are covered in a single array. The extent and distribution of methylation were highly variable between specimens, although some CpG islands, such as p15INK4B and HOX gene clusters, seemed to be commonly involved in different cases (Figure). In conclusion, MeDIP on chip analysis could be a powerful method for genome-wide detection of DNA methylation and facilitate our understanding of the pathogenesis of MDS.

Figure Figure
High-Resolution CGH Analysis of CD34+ Cells from Lower-Risk Myelodysplastic Patients Reveals Cryptic Copy Number Alterations and Predicts Overall and Leukemia-Free Survival.
Authors: Starczynowski Daniel T. , Vercauteren Suzanne , Telenius Adele , Sung Sandy , Tohyama Kaoru , Brooks-Wilson Angela , Spinelli John J. , Eaves Connie J. , Eaves Allen C. , Horsman Douglas E. , Lam Wan L. , Karsan Aly
Published: April 2020

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematologic malignancies characterized by peripheral cytopenias, a hypercellular marrow with ineffective hematopoiesis and a propensity to progress to acute myeloid leukemia (AML). MDS is thought to arise from a primitive (CD34+) hematopoietic cell that has acquired genetic and/or epigenetic abnormalities. Risk stratification according to the International Prognostic Scoring System (IPSS) defines patient categories that correlate with survival and the likelihood of transformation to AML. Greater than 50% of individuals diagnosed with MDS are in the lower-risk groups. The importance of cytogenetics in risk stratification has been verified in several studies, but is of limited value in patients with lower-risk subtypes because approximately 50% of these patients do not have karyotypic abnormalities detectable using standard techniques. To further our biological understanding of lower-risk subtypes of MDS, and to identify potential MDS-initiating alterations in the genome, we looked for alterations in DNA extracts from purified CD34+ marrow cells from 44 MDS lower-risk patients using a submegabase bacterial artificial chromosome (BAC) array to perform whole genome comparative genomic hybridization (CGH) analyses. These studies identified numerous cryptic structural DNA alterations that were not detectable by standard cytogenetic analysis and were also not found in 15 age-matched normal controls. Although most patients tested had a normal karyotype, 23 recurring, novel copy number alterations of a median size of 0.6 megabases were identified. These included gains at 11q24.2-qter, 17q11.2 and 17q12, and losses at 2q33.1-q33.2 and 14q12. Comparison of changes in CD34+ marrow cells with DNA from CD3+ cells isolated from the same patients showed that a recurring duplication at band 17q12 was exclusive to the CD34+ cells in 2 of 3 patients. Validation of this copy number gain at chr 17q12 by FISH confirmed duplication of the locus. In addition, whole genome array CGH analysis of CD34+ marrow cells from karyotypically normal (n = 25) and abnormal (n = 15) lower risk MDS patients revealed extensive genome alterations (involving >3 Mb) correlated with poorer overall survival, and was more frequently associated with transformation to AML as compared to IPSS stratification alone. Our studies suggest that array CGH may be useful as an ancillary test to better stratify lower-risk subtypes of MDS and, at the same time lead to the identification of early mutations that contribute to disease initiation.
Dependency of Survival on Additional Karyotype Anomalies and Bone Marrow Blast Count in Patients Suffering from Myelodysplastic Syndromes with 5q Deletion.
Authors: Knipp Sabine , Hildebrandt Barbara , Jotterand Martine , Schapira Marc , Giagounidis Aristoteles , Gattermann Norbert , Aul Carlo , Germing Ulrich
Published: April 2020

The 5q deletion syndrome is described as having a favourable outcome in MDS. However, little data is available concerning del(5q) and additional karyotype anomalies or taking into account bone marrow blast count. We screened the MDS registries of Düsseldorf, Germany and Lausanne, Switzerland in order to obtain data on patients with karyotype anomalies including del(5q). In both registries 1073 patients were karyotyped at diagnosis. 198 patients with del(5q) with or without other karyotype anomalies and 105 patients with complex karyotype anomalies without del(5q) were analysed. 106 patients showed del(5q) as single anomaly, 23 had 1 additional karyotype anomaly, 69 had 2 or more additional anomalies and were therefore classified as complex karyotype. In the group with del(5q) only, mean survival was 76 months as compared to 42 months in patients with a normal karyotype. In the group with 1 additional anomaly mean survival was 47 months (p=0.02), in the group with 2 or more additional anomalies 7 months (p=0.0005). Survival differed significantly in each subgroup when patients presenting less than 10% of bone marrow blasts where compared to patients with 10% or more. Patients with del(5q) only show a median survival of 12 months when presenting with more than 10% medullary blasts. Patients with a complex karyotype including del(5q) with less than 5% medullary blasts had a median survival of 12 months, but median survival was only 6 months in patients with elevated medullary blasts. By multivariate analyses, we found that additional karyotype anomalies, followed by an elevated medullary blast count above 10% are the most important independent prognostic parameters. Furthermore, we compared the subgroups with del(5q) and 2 or more additional karyotype anomalies (n=69) to patients with complex karyotype anomalies not including del(5q) (n=105). Median survival of the group with complex karyotype anomalies including del(5q) was 7 months as compared to 12 months in the group without del(5q) (p=0.02). 12 months after diagnosis, 30% of the group with del(5q) in a complex karyotype was alive as compared to 45% of the group with a complex karyotype without del(5q). Disease related death was noted in 77% of patients with a complex karyotype and del(5q) and 73% without del(5q). Therefore, our data show that the prognosis of survival in patients with del(5q) is highly dependent on the number of additional karyotype anomalies as well as medullary blast count. The prognosis of MDS patients with del(5q) is associated with an extremely poor prognosis when diagnosed within a complex karyotype. Although there are no substantial differences in clinical, haematological and morphological data between patients with or without del(5q), the median survival differs significantly.

Figure Figure
Human Leukocyte Antigen DR15 in Patients with Myelodysplastic Syndromes (MDS) – A Study in Chinese Patients.
Authors: Master Qiong Liao , Zhang Yan , Li Xiao , Zhang Zheng , Ying Shaoxu , Master Chunkang Chang , Pu Quan ,   (Intr. by H. Joachim Deeg)
Published: April 2020

We investigate the frequency of the human leukocyte antigen DR15 (HLA-DR15) allele in patients with myelodysplastic syndromes (MDS). We used polymerase chain reaction-sequence-specific primers (PCR-SSP) to detect HLA-DR15 in the peripheral blood of patients with MDS(n = 76) and healthy controls (n = 227).

The frequency of HLA-DR15 in MDS patients (40.8%) was significantly higher than in controls (13.7%; P < 0.01). The diagnoses of refractory anemia/refractory anemia with ring sideroblasts (RA/RARS) accounted for 77.4% (24/31) and 62.2% (28/45) of the DR15-positive and the DR15-negative patients, respectively (difference not statistically significant). Although no statistically significant difference was observed, some trends were observed: IPSS low-risk MDS (IPSS score, ≤1) accounted for 80.6% of the DR15-positive patients compared to 64.4% among the DR15-negative patients. However, the difference between the numbers of DR15-positive and DR15-negative patients with chromosomal abnormalities was not statistically significant. Nevertheless, poor risk chromosome abnormalities (according to IPSS), were present in only 1 DR15-positive patient, while such abnormalities were present in 8 DR15-negative patients. In addition, the proportions of DR15-positive and DR15-negative patients with more than 5% blasts in marrow were 19.4% and 31.1%, respectively. Peripheral blood pancytopenia occurred in 51.6% of DR15-positive, and in 40.0% of DR15-negative patients. Although the HLA-DR15 allele appeared to be present more frequently in patients less than 60 years of age, this association was not significant. The frequency of HLA-DR15 was significantly higher in patients with MDS than in healthy controls suggesting the possibility that HLA-DR15 is associated with an enhanced susceptibility to develop MDS. The fact that HLA-DR15 was predominantly noted in patients with RA/RARS and low IPSS scores, suggested that HLA-DR15 might be associated more with bone marrow failure and less with leukemic transformation.

clinical/experimental characteristics in HLA-DR15 positive or negative MDS patients Cohort HLA-DR15 positive(n=31) HLA-DR15 negative(n=45) P value RA/RARS(case/%) 24/31(77.4) 28/45(62.2) 0.161 Low risk (IPSS≤1) (case/%) 25/31(80.6) 29/45(64.4) 0.126 Karyotype abnormal(case/%) 13/31(41.9) 18/45(40.0) 0.866 Poor chromosome(case/%) 1/31(3.2) 8/45(17.8) 0.117 Blast>5%(case/%) 6/31(19.4) 14/45(31.1) 0.253 Pancytopenia(case/%) 16/31(51.6) 18/45(40.0) 0.317 Male patients(case/%) 17/31(54.8) 25/45(55.6) 0.951 age(<60 years) (case/%) 20/31(64.5) 20/45(44.4) 0.085

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Prognostic Analysis of Refractory Anemia in Adult Patients with Myelodysplasric Syndrome in China.
Authors: Wang Xiao-qin , Chen Zi-xing , Chen Shu-chang , Lin Guo-wei , Ji Mei-rong , Liang Jian-ying , Liu Dan-dan , Li De-gao , Ma Yan
Published: April 2020

Several reports indicated a striking difference in age, chromosome abnormalities and prognosis between Western and Eastern MDS patients, even in the same subtype. The prognostic factors in Chinese MDS patients remain unclear. To investigate the prognostic factors and survival rate in Chinese cases with refractory anemia (RA) in adult MDS based on FAB classification, and to evaluate the applicability of international prognostic scoring system (IPSS) for Chinese MDS-RA patients by comparing with the clinical features of Western cases, Three hundred and seven MDS-RA cases were registered and followed-up in Shanghai, Suzhou and Beijing from 1995 to 2006. The longest follow-up duration was 103 months, and the median follow-up duration was 26.7 months. Kaplan-Meier curve, Log-rank and COX regression model were used to analyze the prognostic factors and survival rate. The median age of 307 MDS-RA cases was 52 years. The frequency of 2 or 3 lineage cytopenias was 85.6%. Abnormal karyotype occurred in 35.7% of all MDS-RA patients. By IPSS cytogenetic risk groups, 165 cases (70.2%) were in the good IPSS cytogenetic subgroup, 44 cases (18.7%) in the intermediate IPSS cytogenetic subgroup and 26 cases (11.1%) in the poor IPSS cytogenetic subgroup. According to IPSS, 20 cases (8.5%) were categorized as low risk, 195 cases (83.0%) as intermediate–I risk and 20 cases (8.5%) as intermediate–II risk. The 1–year, 2–year, 3–year, 4–year and 5–year survival rate were 90.8%, 85.7%, 82.9%, 74.9% and 71.2%, respectively. Fifteen cases (4.9%) transformed to acute myeloid leukemia and the median transformation time was 15.9 months (range 3–102 months). Lower white blood cells count (<1.5 × 109/L), platelet count (<30 × 109/L) and cytogenetic abnormalities were identified as independent prognostic factors by multivariate analysis, while the age (≥65 years), hemoglobin level(<60g/L), IPSS cytogenetic subgroup and IPSS risk subgroup were not independent prognostic factors associated with survival time by COX regression analysis. In summary, Chinese patients were younger and had lower incidence of cytogenetic abnormalities and more severe cytopenias than that of the Western patients. However, the Chinese patients have more favorable prognosis than Western patients do. WBC count, platelet count and karyotype are major prognostic factors for prediction of survival and can be helpful in identifying patients with different prognosis and consequently designing the optimal therapeutic strategies. The present IPSS scores based on conditions of western patients may not be perfectly applicable to MDS-RA patients in Asia and need to be modified.
RNAi Screen of the Druggable Genome To Identify Modulators of Bortezomib in Myeloma.
Authors: Zhu Yuan Xiao , Yin Holly , Que Qiang , Tiedemann Rodger E. , Chng Wee-Joo , Shi Chang Xin , Mousses Spyro , Stewart Keith
Published: April 2020

Bortezomib and related proteasome inhibitors have profound pre-clinical and clinical activity in Multiple Myeloma (MM). However, the critical molecular target(s) modulated by proteosome inhibition remains unknown. The overall response rate to bortezomib in relapsed and refractory MM patients is consistently 35%–50%, suggesting specific molecular targets or resistance mechanisms are inherent in patients. None of the known effects of bortezomib has been shown to correlate with clinical responses in patients or to explain the unique sensitivity of MM (as opposed to other malignancies) to these reagents. Therefore, in this study, we have conducted a small interfering RNA (siRNA) screen of the druggable genome in human myeloma cells to identify genes that modulate sensitivity to bortezomib. KMS11 human myeloma cells were reverse transfected with 13,984 synthetic short interfering RNAs (siRNA) against each of 6992 druggable targets (2 siRNA/gene with a single-siRNA per well/384 well format). At 24 hours post transfection, bortezomib was added at IC10, 30, 60 and 90. Cell viability was measured by ATP-dependent luminescence assay 72 hours post drug treatment. The resulting data from primary screening was evaluated for multiple quality control metrics and found to exceed all expected performance parameters with >99% global transfection efficiency, <0.25 CV values across the dataset with minimal plate-to-plate and set-to-set variation observed. From analysis of the primary screening data, a core panel of 320 (4.6% of druggable targets in library) bortezomib sensitizers were identified. 187 (58%) of these were further confirmed in a secondary screening experiment by multiple (2 to 4) independent siRNA. Among these 187 genes (2.6% of the genes investigated), some known targets, such as proteasome subunits are evident. Other genes belonging to the same functional gene families (e.g. TNF receptor family, Ras family, kinases, oncogenes and transporter proteins) or functioning within the same network of signal transduction (e.g. NF-kB, Wnt and estrogen receptor pathways) are evident. Our primary screen also identified genes that attenuate bortezomib-induced cytotoxicity. Members in this group include genes implicated in the negative regulation of cell proliferation and survival, such as NF-kB inhibitors, JNK pathway activators, tumor suppressors, apoptosis inducers and phosphatases. These studies enable dissection of the molecular mechanism of bortezomib in myeloma cells and may serve to point to biomarkers for response prediction and novel chemotherapeutic combination therapies.
Restricted Oligo-Clonal Usage of Monoclonal Immunoglobulin λ Light Chain Germline in POEMS Syndrome.
Authors: Nakaseko Chiaki , Abe Daijiro , Takeuchi Masahiro , Takeda Yusuke , Tanaka Hiroaki , Oda Kayo , Ohwada Chikako , Ozawa Shinichi , Sakaida Emiko , Shimizu Naomi , Masuda Shinichi , Cho Ryuko , Nishimura Miki , Saito Yasushi , Misawa Sonoko , Kuwabara Satoshi
Published: April 2020

Background: POEMS syndrome (also known as Crow-Fukase or Takatsuki syndrome) is a rare plasma cell disorder characterized by peripheral neuropathy, a monoclonal plasma cell disorder, and other paraneoplastic features including organomegaly, endocrinopathy, skin changes, edema and effusions. High levels of vascular endothelial growth factor (VEGF) in the serum may contribute to pathology seen in this disease but exact pathogenetic mechanisms are still unclear. A unique feature of this syndrome is that the proliferating monoclonal plasma cells are virtually always λ-restricted. Here, we determined complete nucleotide sequences of monoclonal immunoglobulin λ light chain (IGL) variable regions in 10 patients with POEMS syndrome and found that Vλ germline usage is highly restricted to the Vλ1 family and oligoclonal germlines.

Materials and methods: Total RNA was extracted from bone marrow mononuclear cells of patients with POEMS syndrome and the V-J region of the IGL gene was amplified by RT-PCR using 5′ degenerative primers for the Vλ consensus leader lesion (5′-ATGGCCKGSWYYSYTCTCCTC-3′) and 3′ primers matching the consensus upstream part of the cl exon (5′-CTCCCGGGTAGAGAAGTCACT-3′). Monoclonal bands were detected by heteroduplex analysis or TA cloning and subjected to nucleotide sequencing. Sequence data were analyzed using the International ImMunoGeneTics information system (IMGT, http://imgt.cines.fr) and mutations were identified by comparison with the germline sequence databases.

Results: Of 13 POEMS syndrome patients with λ-type M protein, nucleotide sequencing of the IGL gene was successful in 10. The V-region of the IGL gene of all 10 patients was restricted to the Vλ1 family. Searching for homologies with IGL germlines revealed that only two were used, with an average homology of 90.1%. IGLV1-40*01 was used in 8 patients and IGLV1-44*01 in the remaining two. In the IGL-J region, the IGLJ3 gene was found in 9 of 10 patients and IGLJ2 in one (10%). All IGLV-J rearrangements were mutated with homologies to the germline sequence ranging from 77.66% to 96.10% (average homology: 90.1%). All CDR3 were composed of 11 amino acids, with identical acidic isoelectric point (pI) values (13.0) and similar molecular weights ranging from 1394.7 to 1552.8 (median, 1506.2).

Conclusions: IGL-M protein in POEMS syndrome belongs to the Vλ1 family and is markedly restricted to specific oligoclonal germlines. Our results suggest that the restricted use of IGL plays an important role in the pathogenesis of POEMS syndrome. These data provide an important insight for elucidating the pathogenesis of POEMS syndrome. To the best of our knowledge, this the first report of restricted oligo-clonal usage of monoclonal immunoglobulin germlines in plasma cell disorders.
ß-Catenin siRNA Successfully Suppressed the Progression of Multiple Myeloma in In Vivo Mouse Model.
Authors: Kawata Eri , Ashihara Eishi , Tanaka Ruriko , Yokota Asumi , Murotani Yoshihide , Taniguchi Miki , Kamitsuji Yuri , Kuroda Junya , Inaba Tohru , Shimazaki Chihiro , Kimura Shinya , Maekawa Taira
Published: April 2020

ß-catenin is the downstream effector of the Wnt signaling pathway and has cyclin D1 and c-myc as its target genes. Activation of Wnt signaling is greatly involved in the process of carcinogenesis, and its overexpression has reported to be positively correlated with prognosis in many types of malignant tumors, as colorectal cancer and hepatocellular carcinoma, or hematological malignancy, as acute myeloid leukemia and chronic myeloid leukemia. Multiple myeloma (MM) is a relatively common hematological malignancy and remains incurable with conventional treatments. Here, we assessed the expression of ß-catenin in MM and growth inhibitory effect of ß-catenin siRNA on MM. We first examined the expression of ß-catenin on human MM cell lines: AMO-1, EJM, IM-9, KMS-12-BM, LP-1, NCIH929, OPM-2, RPMI8226, and U266. All cell lines examined expressed a significantly higher level of ß-catenin than normal human mononuclear cells. Moreover, ß-catenin was also overexpressed in myeloma cells from patients. We then assessed the in vivo growth inhibitory effects of ß-catenin siRNA in mouse model. Six -week-old male Balb/c nu/nu mice were subcutaneously inoculated in the right flank with 5 × 106 RPMI8226 myeloma cells in 100μl PBS. Three to four weeks later, when palpable tumors (100 mm3 in diameter) developed, mice (n = 5 for all groups) were treated with either subcutaneous (around tumors) injections of group A: ß-catenin siRNA (2.5μM) / atelocollagen complex (final atelocollagen concentration 0.5%), group B: control siRNA (2.5μM) /atelocollagen complex, group C: ß-catenin siRNA (2.5μM)/PBS, group D: PBS/atelocollagen twice a week for a total of eight injections. Tumor size was measured in two dimensions using a caliper, and tumor volume (mm3) was calculated as a2 × b /2 mm3 (a;minor axis,b;major axis). The mean tumor volume of each group after two weeks treatment was 412.2 mm3 in groupA, 1317.9 mm3 in group B, 2075.9 mm3 in group C, and 1802.3 mm3 in group D, respectively, and the treatment with ß-catenin siRNA (2.5μM) / atelocollagen complex significantly reduced tumor burdens and retarded tumor growth, measured as tumor volumes (p<0.05) (Figure 1). Therefore, ß-catenin siRNA revealed growth inhibitory effects on MM in in vivo mouse model, which strongly indicates that ß-catenin is the attractive novel target in treating MM.

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