bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterisation of Nilotinib Transport in Chronic Myeloid Leukaemia Cells.
Authors: Davies Andrea , Giannoudis Athina , Lucas Claire M. , Harris Robert J. , Manley Paul W. , Pirmohamed Munir , Clark Richard E. 
Published: April 2020

Abstract
We have previously shown that imatinib is a substrate for both the efflux transporter P-glycoprotein (ABCB1; MDR-1 gene product) and the influx transporter human Organic Cation Transporter 1 (hOCT1; SLCA22). High hOCT1 and low ABCB1 expression levels in chronic myeloid leukaemia (CML) patients correlate with improved clinical outcome (Wang L. et al. Clinical Pharmacology and Therapeutics; epub June 13th 2007). The second generation tyrosine kinase inhibitor nilotinib is 30-fold more potent than imatinib and is effective in imatinib-resistant patients. However, its cellular transport has not been characterised. The CML cell line KCL22 was used as it has low basal hOCT1 expression. Drug transport studies of 14C-radiolabelled nilotinib (kind gift from Novartis) were performed using high expressing hOCT1 transfected KCL22 cells, with or without OCT1 and ABCB1 inhibitors. Further vectorial ABCB1 efflux studies were performed on confluent monolayers of ABCB1 transfected Type II Madin Darby canine kidney (MDCKII) cells. Nilotinib efflux was not modulated in the presence of the ABCB1 inhibitors PSC-833 (10μM), tariquidar (500nM) and verapamil (500μM), and no evidence of transport via ABCB1 was seen in the transfected ABCB1 MDCKII cells. Nilotinib influx was not modulated in hOCT1 over-expressing cells, or in the presence of OCT1 inhibitors prazosin (100μM) and amantadine (500μM). Nilotinib uptake was also not modulated by the hOCT1 substrate tetraethylamine (TEA) (5μM), however nilotinib inhibited TEA uptake. The distribution coefficient (logD) of nilotinib was 2.1, compared with 0.81 for imatinib, demonstrating high lipophilicity for nilotinib. Flow cytometry revealed greater suppression of phospho-CrKL (a surrogate marker for BCR-ABL blockade) with nilotinib after 1 hour when compared with imatinib, regardless of hOCT1 expression, indicating that nilotinib is effective at BCR-ABL suppression even at low hOCT1 levels, in contrast to imatinib. Nilotinib was not a substrate for the ABCB1 and hOCT1 transporters; however it may be a potential inhibitor of hOCT1. Therefore the level of expression of ABCB1 and hOCT1 in CML is unlikely to be an indicator of the clinical outcome for patients receiving nilotinib, in contrast to what we have shown for imatinib. Nilotinib may therefore be effective in patients who have low hOCT1 expression before start of therapy.
Kruppel-Like Factor 4 Is Essential for Inflammatory Monocyte Differentiation In Vivo.
Authors: Alder Jonathan K. , Georgantas Robert W. , Hildreth Richard L. , Kaplan Ian M. , Morisot Sebastien , Yu Xiaobing , McDevitt Michael , Civin Curt I. 
Published: April 2020

Abstract
Several members of the Kruppel-like factor (KLF) family of transcription factors play important roles in differentiation, survival, and trafficking of blood and immune cell types. We demonstrate here that hematopoietic cells from KLF4−/− fetal livers (FL) contained normal numbers of functional hematopoietic progenitor cells, were radioprotective, and performed as well as KLF4+/+ cells in competitive repopulation assays. However, hematopoietic “KLF4−/− chimeras” generated by transplantation of KLF4−/− FL cells into lethally irradiated wild-type (wt) mice completely lacked circulating inflammatory (CD115+Gr1+) monocytes, and had reduced numbers of resident (CD115+Gr1−) monocytes. While the numbers and function of peritoneal macrophages were normal in KLF4−/− chimeras, bone marrow monocytic cells from KLF4−/− chimeras expressed lower levels of key trafficking molecules and were more apoptotic. Thus, our in vivo loss-of-function studies demonstrate that KLF4, previously shown to mediate proinflammatory signaling in human macrophages in vitro, is essential for differentiation of mouse inflammatory monocytes, and is involved in the differentiation of resident monocytes. In addition, inducible expression of KLF4 in the HL60 human acute myeloid leukemia cell line stimulated monocytic differentiation. The inflammation-selective effects of loss-of-KLF4 and the gain-of-KLF4-induced monocytic differentiation in HL60 cells identify KLF4 as a key regulator of monocytic differentiation and a potential target for translational immune modulation.
Increased Risk of Osteopenic Fractures in Elderly Patients with Gaucher Disease.
Authors: Weinreb Neal J. , Costantini Lisa 
Published: April 2020

Abstract
Bone disease is a common cause of morbidity in Gaucher disease (GD). 60% of adult patients (pts) have decreased bone mineral density (BMD). However, in elderly pts, statistically “normal” BMD may not indicate functionally normal bone strength. Here we report serial dual energy x-ray absorptiometry (DXA) scores and fracture (Fx) occurrence for 29 GD pts age 57–88 (9M, 20F) treated with enzyme replacement therapy (ERT) with imiglucerase, often in conjunction with bisphosphonates.
Results: Lumbar (L) spine Z-scores were mostly normal during 9.2 ± 4.9 y on ERT: median −0.50; mean (SD) −0.05 (0.41); centiles 10–90: −1.81 - 1.21. Nonetheless, new Fxs occurred in 13 of 29 (44.8%) pts: hip (6), spine (4), other location (11), multiple sites (7). Fx occurred in 5/8 (62.5%) pts with splenectomy v 8/21 (38.1%) pts without and in 7/20 (35%) N370S homozygotes v 6/9 (67%) with other genotypes. 10y Fx probability: 42% (CI 17–55). L spine T-scores were &lt; −1 for 18/29 pts, indicating persistent osteopenia/osteoporosis in 62%, and &lt; −2.68 in 25%. New Fx occurred in 8/15 (53.0%) pts with T-scores ≤ the median of −1.60 v 5/14 (35.7%) with T-scores &gt; −1.60. Pts with low T scores were predominantly women (13/15F v 7/14M) and had higher use of bisphosphonates (86.7% v. 35.7%). Fx were not restricted to pts with the lowest T-scores. Findings were similar for femoral neck T-scores.
Conclusions: Normal DXA Z-scores in elderly GD pts do not indicate low Fx risk. Low T-scores, as seen in our pts, do confer an important Fx risk. The 42% 10y Fx probability in our elderly GD pts is higher than expected for comparably aged individuals without GD. Because other risk factors such as Fx history, nutritional status, and concurrent illness were similar in our pts regardless of Fx status, cumulative pathophysiology from long-standing, untreated GD may independently increase Fx risk. Treatment with ERT may allow pts to achieve normal Z-scores. However, starting ERT late in life, even in conjunction with bisphosphonates, may not reverse established osteopenia/osteoporosis sufficiently to avoid future Fx. Therefore, detection of bone loss in young pts and early therapeutic intervention to achieve and maintain normal BMD is an important component of GD management.
Telomere Length Shortening in Langerhans Cell Histiocytosis.
Authors: Bechan Gitanjali I. , Meeker Alan K. , DeMarzo Angelo M. , Racke Frederick , Jaffe Ronald , Sugar Elizabeth , Arceci Robert J. 
Published: April 2020

Abstract
The purpose of this study was to analyze telomere lengths in Langerhans Cell Histiocytosis (LCH). LCH is a clonally derived, proliferative disorder of phenotypically immature CD1a+ Langerhans cells (LCs). The etiology of LCH is unknown and data supporting an immune dysregulatory disorder as well as a clonal neoplasm have been reported. Telomere shortening has been associated with cancers and premalignant lesions as well as being important in promoting chromosomal instability. We therefore conducted a study to quantitatively determine the telomere lengths in LCH. Tissue arrays of reactive lymph nodes, local, multisystem, and systemic LCH containing 9, 7, 12, and 10 specimens respectively were used. These tissue arrays included 2 specimens that had unaffected skin represented, providing an opportunity to examine non-pathologic LCs. The tissue arrays were first hybridized with a N-terminal covalently linked Cy3-labeled, telomere specific peptide nucleic acid (PNA) probe to stain telomeres. The slides were then processed for indirect immunofluorescence to identify LCs using an anti-CD1a primary antibody followed by an anti-mouse IgG fraction Alexa Fluor 488. Finally, slides were counterstained with 4′-6-diamidino-2-phenylindole (DAPI). Digital fluorescent telomere signals were visualized using a Nikon Eclipse E400 epifluorescence microscope and telomere lengths were analyzed with ImageJ software. A total of 1039 measurements were made from 38 individuals. A significant shortening of telomeres was demonstrated in LCH LCs of local, multisystem and systemic LCH in comparison to telomere lengths of reactive lymph node LCs and normal skin, which were not significantly different. To investigate whether telomere length shortening of LCH LCs is selective, we analyzed the telomere length in lymphocytes in LCH specimens and in the reactive lymph node specimens. There was no significant evidence of telomere shortening of lymphocytes in LCH compared to lymphocytes in reactive lymph nodes (p=0.1862). These data show significant telomere shortening in the LCs in all stages of disease involvement with more variability among local cases. These observations suggest that in most cases LCH may share important mechanisms of telomere shortening and survival with clonal pre-neoplastic disorders and cancer.
Protein Kinase C α Is Critically Involved in MyD88-Dependent Toll-Like Receptor 2-Mediated Activation of Dendritic Cells.
Authors: Springael Cecile , Langlet Christelle , Goldman Michel , Willems Fabienne , Aksoy Ezra 
Published: April 2020

Abstract
Introduction: Protein kinases C (PKC) isoforms are key regulators of several Toll-like receptor (TLR) signalling pathways. In the present study, we investigated the role of conventional PKCα in the MyD88-dependent TLR2-mediated activation of dendritic cells (DC).
Methods: We compared the functional and molecular responses of DC derived from wild type and PKCa deficient mice to TLR2 stimulation. We next investigated the consequences of conventional PKC (cPKC) inhibition during TLR2 stimulation in human monocyte-derived DC using the cPKC pharmacological inhibitor Gö6976. Finally, reporter gene assays were performed in human embryonic kidney (HEK) cells stably expressing TLR2 in the presence of dominant negative (DN) PKCα. Results: Bone marrow-derived DC from PKCα deficient mice showed an attenuated response to the TLR2/1 ligand Pam3CSK4 characterized by a statistically significant reduction in interleukin (IL)-12p40 and TNFα production. IL-6 synthesis was also reduced albeit not significantly. In western blot analysis, PKCα−/− DC displayed an impaired phosphorylation of the mitogen activated protein (MAP) kinases p38 and c-Jun NH2-terminal kinase (JNK) in response to Pam3CSK4. TLR2-mediated activation of the IκB kinases (IKK) α and β and inhibitor of κB (IκB) degradation were abolished in PKC deficient DC, suggesting an impaired nuclear factor (NF)-κB activation. In human monocyte-derived DC, inhibition of conventional PKC activity by the isoform specific inhibitor Gö6976 severely decreased cytokines production in response to TLR2 ligands. At the concentration of 1μM, Gö6976-treated DC exhibited impaired p38 and JNK activation. The NF-κB activation pathway was repressed with higher concentrations of the pharmacological inhibitor. Finally, in reporter gene assays, DN PKCα but not DN PKC𝛉 repressed AP-1 and NF-κB mediated promoter activity upon TLR2 engagement.
Conclusions: Our results demonstrate that PKCα is a key component of the MyD88-dependent TLR2 signalling pathway in dendritic cells and regulates the activation of NF-κB and MAP kinases pathways.
SNP Arrays Facilitate Genotyping of Non-Synonymous SNP in MDS To Identify Disease Susceptibility Loci.
Authors: Jankowska Anna M. , Przychodzen Bartlomiej P. , Gondek Lukasz P. , Maciejewski Jaroslaw P. 
Published: April 2020

Abstract
Myelodysplastic syndrome (MDS) is a clonal premalignant disease of hematopoietic stem cells characterized by cytopenias and predilection to acute myeloid leukemia (AML). While various exogenous factors (exemplified by chemotherapy-related MDS) constitute known risks for the development of MDS, it is possible that despite long latency, complex genetic traits contribute to MDS susceptibility. Such heritable factors include genes involving DNA repair, apoptosis, senescence, carcinogen catabolism and immune surveillance. Previously, disease association studies were mainly empiric and relied on rational selection of a very limited number of polymorphisms. Recent advances of SNP-array (SNP-A) technology allow for screening of a large number of SNPs. While some SNP-A utilize haplotype tags, custom arrays may specifically target known non-synonymous SNPs. We hypothesized that application of SNP-A genotyping may facilitate identification of potentially pathogenic SNPs. Such a screening approach is a hypothesis forming tool and our study is the first application of this technique to MDS. We have used the 13.9K non-synonymous Genotyping BeadChip (Illumina); DNA from 151 MDS patients (low risk: N=79, advanced: N=51, CMML1/2: N=21) and 99 controls (120 historical controls). In total, ∼2.4 mil genotypes were obtained. Genotype calls were computed and analyzed with Exemplar software. In the initial training Bonferroni correction was not applied. Instead as a hypothesis-forming approach we have ranked all SNPs according to their p value (automated analysis of multiplexed statistical evidence) and case/control ratio. We focused our search on the 100 highest ranking SNPs with a control frequency &lt;5%. Globally, in the whole group 49 SNPs showed a p value of &lt;.001, 75 SNPs present in ≤1% of controls and were found in &gt;5% of cases. For example, the AA (rs3219484) variant of MUTYH, a gene involved in oxidative DNA repair was found in 19% of MDS vs. 2.6% of controls (p=9×10−8). To limit the impact of heterogeneity, subgroups of MDS were also analyzed separately. Among many interesting SNPs found, the AG genotype of (rs8192297) ANPEP was associated with RARS (35% vs. 15% in controls; p=.007). Similarly, the GA form of rs3730947 in DNA repair gene LIG1 was found in 11% of RARS patients (p=.00045), though it was absent in other MDS subtypes and controls. Similar, “enrichment” was observed in patients with CMML1/2, showing e.g., heterozygosity of WDR35 (rs1060742), ALPK2 (rs3809975) at the frequency of 45% and 30% (13% and 4% of controls; p=.0006, p=.0001). In sum, our study constitutes the first application of SNP-A genotyping to study susceptibility loci in MDS.
A Lack of Correlation between EVI1 Expression, MDS Subtype and Karyotype Abnormalities Suggests Multiple Pathways Involved in EVI1 Impact on Hematopoietic Cells in MDS Patients.
Authors: Cermak Jaroslav , Ing Ota Fuchs , Provaznikova Dana , Michalova Kyra , Mikulenkova Dana , Brezinova Jana , Klener Pavel 
Published: April 2020

Abstract
EVI1 proto-oncogene located on human chromosome 3q26 has been repeatedly described as an important gene involved in pathogenesis of human acute myeloid leukemia or myelodysplastic syndrome (MDS). EVI1 encodes a nuclear DNA binding protein and its abnormal expression in immature hematopoietic cells leads to altered proliferation and differentiation of both erythroid and myeloid lineages. In our study, an incidence and prognostic relevance of EVI1 expression was studied in a group of 62 patients with different subtypes of primary MDS. EVI1 expression was detected using semiquantitative RT-PCR on samples obtained repeatedly from bone marrow and peripheral blood of patients or healthy controls. The results for individual cDNAs corresponding to the amounts of individual EVI1 mRNAs were related to the amount of standard housekeeping constitutively expressed GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Abnormal EVI-1 expression was found in 12 out of 62 patients (19%). The distribution of positive patients according to the WHO classification was: RA-2, RCMD-3, 5q- syndrome-1, RAEB I.-3, RAEB II.-1, sAML -2, that represented 0% (RARS,CMML), 17% (RCMD,RAEB II.), 18% (sAML), 30%(RA,5q-syndrome) and 33% (RAEB I.) of all patients studied. Only 2 out of 12 patients with abnormal EVI-1 expression had abnormalities of chromosome 3. confirmed by mFISH, 2 patients had complex karyotype abnormalities involving chromosome 11. Five out of 50 negative patients had complex abnormalities including loss of chromosome 3. in 1 patient and abnormities of chromosome 11. in 2 patients. No significant difference in median survival and rate of leukemic transformation was observed between both the groups (25,0 months and 33% in EVI1 positive group vs. 15,3 months and 28% in EVI1 negative group, P=0,35). Nevertheless, in four EVI1 negative patients with RCMD or RAEB I. an abnormal EVI1 expression occurred repeatedly at the time of progression to RAEB I.or RAEB II. All these patients subsequently died on refractory AML. Our results suggest that abnormal EVI1 expression may play role in early MDS (eg. by impairment of erythropoiesis due to blocking of GATA-1 dependent transcription) as well as in advanced MDS (eg. by stimulation of proliferation and alteration of differentiation of myeloid precursors by inhibition of TGFβ or IFNα signalling pathway). This may be an explanation for limited prognostic relevance of abnormal EVI1 expression in MDS patients in our study. However, a novel occurrence of EVI1 expression in the course of the disease might serve as an additional adverse prognostic factor.
Different Patterns of AML1 Mutations between De Novo Myelodysplastic Syndrome and Chronic Myelomonocytic Leukemia.
Authors: Shih Lee-Yung , Liang Der-Cherng , Huang Chein-Fuang , Kuo Ming-Chung , Lin Tung-Liang , Fu Jen-Fen , Shih Yu-Shu , Wu Jin-Hou , Wang Po-Nan , Dunn Po , Tang Tzung-Chih , Chang Hung 
Published: April 2020

Abstract
Background: Transcription factor AML1/RUNX1 is essential for normal hematopoiesis. AML1 mutations have been described in radiation-associated and therapy-related myelodysplastic syndrome (MDS) and have rarely been reported in patients with chronic myelomonocytic leukemia (CMML).
Aims: We sought (1) to define the frequencies of AML1 mutations in de novo MDS and CMML, and (2) to compare the difference in mutation patterns between the two disorders.
Methods: AML1 mutations were examined on bone marrow samples obtained at initial diagnosis from 107 patients with de novo MDS (12 RCMD, 45 RAEB1 and 50 RAEB2) and 79 patients with CMML (53 CMML1 and 26 CMML2). Mutational analysis was performed by direct sequencing of all RT-PCR products amplified with 3 overlapping primer pairs which cover the entire coding sequences of AML1b gene from exon 1 through exon 8. Samples with abnormal sequencing results were subjected to repeated PCR and sequencing using genomic DNA with alternative primers.
Results: Seventeen of 107 patients with MDS (15.9%) had 20 AML1 mutations; 8 mutations were located in Runt homology domain (RHD) (exons 3–5) and 12 mutations were located in the non-RHD region (exons 6–8). The 20 AML1 mutations included 9 missense mutations, 5 nonsense mutations, 2 frameshift mutations, and 4 silent mutations. One patient has two missense mutations at RHD and another two had one missense mutation with one silent mutation. Thirty-one AML1 mutations were detected in 29 of 79 CMML patients (36.7%); 20 patients had 22 mutations located in the N-terminal part and the remaining 9 patients had one single mutation located in the C-terminal region. The patterns of the 31 mutations consisted of 8 missense mutations, 7 nonsense mutations, 15 frameshift mutations and 1 silent mutation. One CMML patient had two missense mutations in RHD and another patient had two frameshift mutations in RHD. Cloning analysis showed that the two mutations were on different alleles in both patients. The frequency of AML1 mutations was significantly higher in patients with CMML than in MDS (P=0.002). Mutations in N-terminal region occurred more frequently in CMML than in MDS (22/30 vs. 8/20, P=0.042). CMML patients had a higher frequency of frameshift mutations as compared with MDS patients (P=0.006). Except for a significantly lower platelet count in CMML patients with AML1 mutations, there were no differences in age, sex, blood counts, percentages of blasts in bone marrow or peripheral blood, subtypes of MDS or CMML, cytogenetic risk groups, time to AML transformation or overall survival between AML1(+) and AML1(−) in MDS and between AML1(+) and AML1(−) CMML patients.
Conclusion: Our results showed that AML1 mutations occurred frequently in both de novo MDS and CMML but with different mutation patterns.
Microarray Analysis of PU.1-Induced Growth Arrest and Apoptosis of Myeloma Cell Lines.
Authors: Ueno Shikiko , Tatetsu Hiro , Harada Naoko , Hata Hiroyuki , Iino Tadafumi , Niiro Hiroaki , Akashi Koichi , Tenen Daniel G. , Mitsuya Hiroaki , Okuno Yutaka 
Published: April 2020

Abstract
PU.1 is an Ets family transcription factor, which is important for differentiation of granulocytes, monocytes/macrophages, and B cells. In the Friend leukemia model, it is reported that the failure of PU.1 down-regulation in erythroblasts reportedly results in differentiation arrest that leads to erythroleukemia. In conditional knockout mice of the 3.5 kb length of enhancer region located in14 kb 5′ of the PU.1 gene, PU.1 is down-regulated in myeloid cells and B cells down to 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like disease. In addition, a deletion of the 3.5 kb enhancer region, which also contains the suppressor region for PU.1 in T cells, results in ectopic expression of PU.1 in T cells, which leads to T cell lymphoma in those mice. Taken together, the failure of up-regulation or down-regulation of PU.1 in certain differentiation stages for each lineage appears to cause differentiation arrest and hematological malignancies. We recently reported that PU.1 is down-regulated in a majority of myeloma cell lines through the methylation of the promoter and enhancer region located in17 kb 5′ of human PU.1 gene which is homologous to that in14 kb 5′ of murine PU.1 gene. Conditionally expressed PU.1 induced cell growth arrest and apoptosis of those PU.1 low-negative myeloma cell lines, U266 and KMS12PE, suggesting that down-regulation of PU.1 is necessary for myeloma cell growth. In addition, we reported that PU.1 is expressed in normal plasma cells and PU.1 is down-regulated in myeloma cells of some myeloma patients. Myeloma patients with low-to-negative PU.1 expression (lower 25th percentile of PU.1 expression level distribution among 30 patients we examined) may have poor prognosis compared to those with high PU.1 expression, although more patient samples have to be examined to define the significance of the relationship of PU.1 expression levels and prognosis. To elucidate the mechanisms of PU.1 induced cell growth arrest and apoptosis of myeloma cells, we next performed DNA microarray analysis to compare gene expression levels before and after PU.1 induction. We utilized Illumina Sentrix® Human-6 Expression BeadChip. Of 47296 genes, 479 genes were up-regulated (&gt;2fold) and 1697 genes down-regulated (&lt;0.5 fold) either day 1 or 3 after PU.1 induction in U266 cells. Among apoptosis related genes, TRAIL was highly up-regulated in both U266 and KMS12PE cell lines. Stably expressed siRNA for TRAIL partially inhibited apoptosis of U266 cells expressing PU.1, suggesting that TRAIL is related to PU.1 induced cell death of U266 cells. Among cell-cycle related genes, p21WAF1/CIP1 was found up-regulated in U266 cells, which was confirmed with protein levels. We are now examining the roles of the observed up-regulated genes in both U266 and KMS12PE myeloma cell lines.