bluuebunny/crossref_metadata_2025 huggingface dataset:
AML1/ETO and PML/RAR α Can Immortalize Committed Myeloid Progenitor Cells In-Vitro but Not Expand Them In-Vivo.
Authors: Hosen Naoki , Passegue Emmanuelle , Weissman Irving L.
Published: October 2019

For most leukemia the target cells of transforming mutations are still unknown. Here, we studied the developmental origin of t(8;21)-acute myeloid leukemia (AML), t(15;17)-acute promyelocytic leukemia (APL), and t(9;22)-chronic myeloid leukemia (CML). Purified mouse hematopoietic stem cells (HSCs) and various committed myeloid progenitor cells were retrovirally transduced with AML1/ETO, PML/RARα, or p210 BCR/ABL and subjected to in-vitro serial replating assay and in-vivo transplantation. Myeloid progenitors were efficiently immortalized in-vitro by AML1/ETO or PML/RARα as assayed in serial replating assays. However, following transplantation into lethally irradiated mice, neither AML1/ETO- nor PML/RARα-transduced myeloid progenitors were expanded in-vivo, although cells carrying the fusion gene DNA persisted well beyond their non-transduced control progenitors. In addition, 10 months after the transplant with transduced myeloid progenitor cells, PML/RARα but not AML1/ETO mRNA expression was still detected in committed myeloid progenitors, although PML/RARα-expressing cells were still not expanded. This finding demonstrates the ability of PML/RARα to increase the lifespan of committed myeloid progenitor cells both in-vitro and in-vivo and suggest that t(15;17)-APL could possibly evolve from persisting progenitor-derived cells. In contrast, BCR/ABL-expressing myeloid progenitors disappeared within 3 months post transplantation. Analysis of mice transplanted with transduced-HSCs demonstrated that AML1/ETO induced the accumulation of the most immature subset of HSCs (Lin-/c-kit+/Sca-1+/Flk-2-), while in contrast PML/RARα induced HSCs disappearance in most (5 out of 6) cases. In addition, we demonstrate that p210 BCR/ABL could induce the development of a CML-like disease from transduced HSCs (6 out of 16 cases), which is compatible with previous report. Together, these results suggest the existence of novel pre-leukemic stem cells (pre-LSCs) entities. Slowly expanding pre-LSCs could be generated from HSCs transformed by the expression of AML1/ETO. Persisting pre-LSCs could be generated from myeloid progenitors transformed by the expression of PML/RARα. These pre-LSC populations could mediate the early phases of t(8;21)-AML and t(15;17)-APL pathogenesis and could represent novel key targets for anti-leukemia therapies.
Comprehensive In Vivo Analysis of p300 Hematopoietic Tumor Suppressor Functions.
Authors: Kimbrel Erin A. , Rebel Vivienne I. , Kung Andrew L.
Published: October 2019

Characterization of chromosomal translocations in leukemias has led to a better understanding of the causes of disease, as well as to new cures. The transcriptional co-activators p300 and CBP are not only targets of translocations, but perturbation of their normal function is central to the transforming effects of most translocation oncogenes in acute myelogenous leukemia. In our prior studies, we demonstrated that genetic inactivation of p300 or CBP function resulted in perturbed hematopoietic differentiation, and the development of hematologic malignancies. However, given the large size and many functional domains of p300/CBP, it has been difficult to determine which specific domains and activities are necessary for them to suppress hematologic tumor formation. We have now developed a novel forward-genetic mouse model that allows us to systematically evaluate p300 structure-function and determine which functional domain mediates hematologic tumor suppression in vivo. By systematically mutating functional domains within p300 and expressing the mutants in embryonic stem (ES) cells, we have been able to generate a series of chimeric mice lines, each expressing a particular mutation in p300. Our current work entails the careful examination of the peripheral blood and bone marrow for each mouse line in order to determine the importance of specific p300 domains for the suppression of hematologic malignancies that arise in the absence of p300. Moreover, this study is helping us gain insight into the mechanisms responsible for the establishment and maintenance of normal, functional stem and progenitor cell populations. This information will be important for elucidating the transcriptional programs that are likely disrupted in leukemias, thus revealing molecular mechanisms that can be used to direct the development of new cures.
Exploiting the Tr3 Interaction with Bcl-2 as a Novel Therapeutic Strategy of Cancer and Leukemia Therapy.
Authors: Zhai Dayong , Sergienko Eduard , Kitada Shinichi , Luciano Frederic , Satterswait Arnold C. , Zhang Xiaokun , Reed John C.
Published: October 2019

TR3 (Nur77) is an orphan member of the retinoid/steroid family of nuclear receptors, which translocates from nucleus to mitochondria where it binds Bcl-2 and converts Bcl-2 from an anti-apoptotic to a pro-apoptotic protein (Li, et al. Science289: 1159, 2000; Lin, et al CELL116: 527, 2004). Because Bcl-2 is pathologically over-expressed in many ca cancers and leukemias, contributing to chemoresistance, we sought to exploit the TR3 interaction with Bcl-2 as a novel therapeutic approach. Mutagenesis studies identified a 9 amino-acid peptide from TR3 that mimics the effects of the full-length protein in terms of binding Bcl-2 and inducing conformational changes that covert Bcl-2 into a killer. A cell-permeable version of this TR3 peptide induced apoptosis of cultured leukemia cell lines (CEM; Jurkat), such that gene transfer-mediated over-expression of Bcl-2 enhanced sensitivity to the TR3 peptide. In contrast, Bcl-2 over-expression rendered these leukemia cells resistant to other inducers of apoptosis, such as the broad-spectrum kinase inhibitor Staurosporine. Mutant TR3 peptides that fail to bind Bcl-2 did not induce apoptosis. To identify chemical compounds that mimic the pro-apoptotic TR3 peptide, a Fluorescence Polarization Assay (FPA) was developed in which FITC-conjugated TR3 peptide binding to Bcl-2 or other appropriate anti-apoptotic Bcl-2-family proteins was measured in high-throughput mode. This high-throughput screening (HTS) assay performs well in 384 well format, with Z′ > 0.75. A chemical library of ~70,000 compounds was screened to identify compounds that displace the TR3 peptide, thus yielding hits that may serve as a potential starting point for drug discovery. Characterization of the properties of these compounds and their analogs will be described. (Supported by NIH grants R01-GM60554 and U54-HG003916).
Isolation of Human Bone Marrow Mesenchymal Stem Cells by a Novel Monoclonal Antibody, ZUC3.
Authors: Lai Xiaoyu , Huang He , Huang Li , Zeng Fenfang
Published: October 2019

Objective: Due to absence of a single definitive marker of mesenchymal stem cells (MSCs) and low incidence in human bone marrow, the primary culture of MSCs, conventionally isolated with its characteristic of adherent, were considered to be heterogeneous containing of several subpopulations, which had currently limited our understanding of their biology and therapeutic applications. In our previous study, a novel murine monoclonal antibody (McAb) ZUC3 was produced by hybridoma technology, which was specifically reactive with human MSCs, while showed negative cross-reactivity when screened against a variety of human tissues. Now, ZUC3 antigen positive MSCs population would be further identified by magnetic-activated cell sorting (MACS).

Methods: Bone marrow were taken from the iliac crest of normal healthy adult volunteers, and mononuclear cells were separated by density gradient centrifugation, then separated into positively- and negatively-labelled fractions with McAb ZUC3 by immunomagnetic activated cell sorting. The purity of positive cells was analyzed by flow cytometry, then ZUC3 antigen positive and negative cells were plated respectively in human MSCs medium consisting of 10% FBS, LG-DMEM. Characteristics of ZUC3 antigen positive cells phenotype was analyzed by flow cytometry, and proliferation and multiple differentiation potential of the cells was observed in vitro.

Results: Flow cytometric analysis showed that ZUC3 antigen expression by cultured MSCs and mononuclear cells derived from bone marrow were 91.31±2.92%, 0.96±0.28% respectively, and western blotting showed the molecular mass of antigen was about 33KD. The purity of the recovered fractions for ZUC3 by MACS was 76.82±6.32%. The positive cells have adhered to culture flask in vitro, and the quantity of adhered cells that had fibroblast-like morphology increased and proliferated during primary expansion period, while the negative cells were observed as round shape cells without any proliferation. It was demonstrated that ZUC3 antigen positive cells continued growth with spindle-shape, extending beyond 30 population doublings in long-term culture. Analyzed by flow cytometry, the culture-expanded positive cells were uniformly positive for CD29, CD44, CD105, CD106, and lack typical hematopoietic antigens such as CD14, CD34, CD45, HLA-DR, which demonstrated that ZUC3 postive cells sorted from bone marrow mononuclear cells by McAb were MSCs. With proper medium, the ZUC3 antigen positive cells could be successfully induced to differentiate into adipocytes, osteoblasts, and neuro-like cells which were positive of neuron markers such as nestin, NSE and NF-M.

Conclusion: ZUC3 McAb was a specific surface marker against human MSCs for cell sorting. The ZUC3 antigen positive cells separated from bone marrow mononuclear cells had potential capacity of high proliferation and multiple differentiation.
Inhibition of PDGFRβ by Imatinib Mesylate Suppresses Proliferation and Alters Differentiation of Human Mesenchymal Stem Cells In Vitro.
Authors: Fierro Fernando , Illmer Thomas , Jing Duhoui , Le Coutre Philip , Ehninger Gerhard , Boxberger Sabine , Bornhaeuser Martin
Published: October 2019

Recent data show that the tyrosine kinase inhibitor Imatinib mesylate (IM) also affects normal hematopoietic stem cells (HSC), T lymphocyte activation and dendritic cell function not relying on the specific inhibition of bcr-abl activity. Mesenchymal stem cells (MSC) have been identified in the bone marrow (BM) as multipotent non-hematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, tenocytes, skeletal myocytes, and cells of visceral mesoderm. MSC interact with HSC, influencing their homing and differentiation through cell-cell contact and the production of factors including chemokines We evaluated possible effects of IM in vitro on human bone marrow-derived MSC.

Screening the activity of fourty-two receptor tyrosine kinases by a phospho-receptor tyrosine kinase (RTK)-array revealed an exclusive inhibition of platelet-derived growth factor receptor (PDGFRβ) by IM which consequently affects downstream targets of PDGFRβ as Akt and Erk1/2 signalling pathways in a concentration and time dependent manner. Furthermore, perinuclear multivesicular bodies harbouring PDGFRβ were found within 18–20 hours culture of MSC in the presence of 5 μM IM. Cell proliferation and clonogenicity (evaluated as the capability to form colony forming units - fibroblasts (CFU-F)) of MSC were significantly inhibited by IM in a concentration dependent fashion. IM inhibits significantly the differentiation process of MSC into osteoblasts as evaluated by decreased alkaline phosphatase activity and reduced calcium phosphate precipitates. In contrary, differentiation of MSC into adipocytes was strongly favoured in presence of IM. All these functional deficits described, probably contribute to an observed 50% reduction in the support of clonogenic hematopoietic stem cells, as evaluated by a long term culture-initiating cells (LTC-IC)-based assay.

In summary our experiments show that IM inhibits the capacity of human MSC to proliferate and to differentiate into the osteogenic lineage, favouring adipogenesis. This effect is mainly mediated by an inhibition of PDGFRβ autophosphorylation leading to a more pronounced inhibition of PI3K/Akt compared to Erk1/2 signalling. This work confirms the role of PDGFRβ recently described for the proliferation and differentiation potential of MSC and provides a first possible explanation for the altered bone metabolism found in certain patients treated with IM.
Genomics Analysis of Freshly-Purified Human Marrow Stromal Cells (MSCs): Comparison with Hematopoietic Lineage Cells and Culture-Expanded MSCs.
Authors: Jones Elena A. , Kinsey Sally E. , English Anne , Field Sarah , Rawstron Andy , Jones Richard A. , Strazsynski Liz , Rundlett Stephen , Jain Selina , Deans Robert , Emery Paul , Ponchel Frederique , McGonagle Dennis
Published: October 2019

Gene and antigen expression profile of bone marrow MSCs in culture has been investigated, but these characteristics of MSCs in vivo are poorly understood. In this study we used microarrays to compare MSCs freshly-purified from human bone marrow aspirates (f-MSCs) with donor-matched hematopoietic lineage cells (HLCs) and with standard culture-expanded MSCs (e-MSCs). Following enrichment with Anti-Fibroblast MicroBeads, f-MSCs were purified by cell sorting as CD45lowCD235a-LNGFR+ [1] and HLCs as CD45+CD235a+LNGFR-. RNA was extracted, amplified and hybridized to an Affimetrix U133 Plus 2.0 chip. Raw fluorescence data were normalised to GAPDH for direct comparison with qPCR. Duplicate hybridizations were used to define 3 relative zones of sensitivity and select appropriate thresholds for significant changes in expression. The microarray data were validated by qPCR using several mesenchymal lineage genes (including Sox9 and Runx2) and by flow cytometry for several known MSC surface markers (including CD73, CD90, CD146, CD10, CD63 and CD130). Expression data were further searched for gene ontology using a custom-made, Perl-based program. Compared to HLCs, f-MSCs over-expressed genes encoding extracellular matrix proteins (including versican, fibronectin and collagen I), stromal cytokines/chemokines (including SDF-1, IL-7 and CXCL10), and molecules associated with cell junctions and communication with endothelium (including connexin 43, VCAM-1 and angiopoietin-1). Osteogenesis-related genes (BMPs and their receptors, alkaline phosphatase, osteonectin, osteocalcin, osteomodulin and others) were more abundant in f-MSCs than in HLCs. Some mature fat tissue products were also quite abundant in f-MSCs (particularly lipoprotein lipase and adipsin), suggesting the presence of cells committed to adipogenesis. Of note, the expression of stroma- and fat-related transcripts was significantly reduced in e-MSCs compared to f-MSCs. Novel surface antigens of f-MSCs identified by microarray, Junctional Adhesion Molecule 2 (normally expressed on vascular endothelial cells), Leptin receptor (implicated in fat and bone metabolism), and CD81 (tetraspanin family adhesion molecule) were confirmed at the protein level by 6-colour flow cytometry. Altogether, this work confirmed the CD45lowCD235a-LNGFR+ phenotype f-MSCs in human bone marrow, revealed a unique expression profile of f-MSCs in vivo and described new surface markers of f-MSCs. These data have implications for our understanding of the in vivo biology of non-hematopoietic bone marrow stromal cells and their role in hematopoiesis and skeletal remodelling in health and disease.
A Role for DNA Double Strand Breaks in the Sensitivity of AML Cells to Clofarabine: Enhanced In Vitro Sensitivity in Primary AML Cells with FLT3 Mutations.
Authors: Pallis Monica , Grundy Martin , Seedhouse Claire , Pimblett Heather , Russell Nigel
Published: October 2019

Clofarabine is a purine nucleoside analogue which has been incorporated into several therapeutic trial protocols for the treatment of leukaemias including acute myeloid leukaemia (AML). The aims of the study are to ascertain mechanisms of clofarabine action in AML using cell lines and presentation samples from patients. We measured apoptosis by TdT assay, cytochrome C release and flow cytometric assays of the mitochondrial membrane potential probe DiOC6. To study the effects of clofarabine on DNA synthesis and DNA double strand breaks, we used bromodeoxyuridine (BrdU) and H2AX assays respectively. Equitoxic doses were established that caused approximately 20% cell death in the AML cell lines HL-60 (0.3 μM), KG1 (1 μM) and MV4.11 (1 μM) after 6 hours of continuous exposure. At these doses, clofarabine induced apoptotic DNA nicks, as measured by the TdT assay, and Cytochrome C release in all three cell lines. However, clofarabine-induced mitochondrial hyperpolarisation and depolarisation were found to be cell-type specific, occurring in HL-60 but not MV4.11 or KG1 cells, i.e. mitochondrial membrane depolarisation is not an essential part of the mechanism of clofarabine-induced apoptosis. Following clofarabine treatment, DNA synthesis was reduced by 91% within 1 hour in KG1 cells and by 80% in HL-60, but by only 9% in MV4.11. Out of the three cell lines, MV4.11 cells, which have a FLT3 internal tandem duplication (ITD), were the only ones to completely repair DNA double strand breaks following clofarabine removal. In 12 samples from patients with AML, the proportion of cells which incorporated BrdU in a 45 minute assay - a measure of the rate of cell cycling - differed considerably between samples, from 0.8% to 23%, median 13%. Cell death induced by clofarabine was correlated with the cycling rate of untreated cells (P=0.016). All 12 samples showed inhibition of DNA synthesis within 60 minutes of clofarabine treatment (range 20%–92% inhibition, median 54%). Mitochondrial membrane hyperpolarisation and depolarisation were not observed in patient cells. However, DNA double strand breaks were induced by clofarabine in patient cells. Paradoxically, i.e. in contrast to the results seen with the MV4.11 cell line, toxicity was greatest in samples with a FLT3 mutation (P=0.007). In conclusion, the FLT3 mutant MV4.11 cell line effectively repairs clofarabine-induced double strand breaks. Cell death in patient cells cultured with clofarabine is correlated with the presence of a FLT3 mutation. As we have previously established that AML samples with FLT3 mutations have upregulated DNA repair activity, this paradox might be explained by cycles of attempted DNA repair frustrated by renewed clofarabine incoporation into DNA, thus increasing the toxicity of the drug.
Genetic and Biochemical Biomarkers of IMPDH Inhibition in Phase I Dose Escalation of AVN-944 for Hematologic Malignancies.
Authors: Strovel Jeffrey W. , Chakiath Marion , Natarajan Pachai , Lawrence Tammy , Zuck Karina M. , Urbani Lenore , Mitchell Beverly S. , Bol David K.
Published: October 2019

AVN-944 is an orally bioavailable inhibitor of inosine monophosphate dehydrogenase 1 and 2 (IMPDH). IMPDH2 is highly upregulated and required for the de novo production of guanine nucleotides in many malignancies, including hematologic cancers. AVN-944 is currently in a repeat-dose, dose escalation trial in patients with advanced hematologic cancers including AML, CLL, Multiple Myeloma, and lymphomas. In addition to the objective of defining the maximum tolerated dose and repeat dose pharmacology of AVN-944 (given bid for 21 days on a 28 day cycle), we have examined a comprehensive set of pharmacodynamic biomarkers. We have isolated peripheral blood samples from all patients in this trial both before and after receiving AVN-944. These samples are being used to determine the effects of the drug on GTP pools and IMPDH enzyme activity, and to correlate these changes to the genetic response of the circulating cells using gene expression detection technologies. We previously identified a set of 32 genes that respond to AVN-944 exposure in a multitude of laboratory settings including different cell types, different concentrations and timepoints. These genes relate directly to cellular pathways and functions dependent upon, or required for, guanine nucleotide biosynthesis, including purine and pyrimidine biosynthesis, mitochondrial functions, metabolic pathways and energy production, cell proliferation and the apoptotic cascade. Examination of the behavior of these genes in patient samples from this phase I trial show a dynamic response of subsets of these genes depending on

the type of malignancy, the predominant cell type in the peripheral blood, and the dose of AVN-944 received.

One set of 9 disease-related genes show changes in expression following drug administration in leukemia patients, while another set of 6 genes designed to report pharmacodynamic effects in normal cells consistently shows changes in expression in the peripheral blood lymphocytes of multiple myeloma patients. In total, 27 of 34 selected genes examined by qPCR showed changes following dosing of AVN-944 at the starting dose of 25 mg p.o. Increasing doses resulted in increases in the number of genes changing per individual, and/or an increase in the magnitude of change in a gene-dependent manner. These genetic markers, correlated with biochemical effects of the drug on protein function and guanine nucleotide levels, can be used as important biomarkers in selecting the optimal dose with which to initiate phase II studies with this drug.
Urinary Hepcidin Excretion in Patients with Myelodysplastic Syndrome (MDS) and Myelofibrosis (MF).
Authors: Winder Asher , Lefkowitz Rafi , Ghoti Hussam , Leiba Merav , Ganz Tomas , Nemeth Elizabeta , Rachmilewitz E.A.
Published: October 2019

One of the main features of patients with Myelodysplastic Syndrome (MDS) is severe anemia which requires multiple blood transfusions with consequent iron overload. Moreover, iron metabolism is impaired particularly in refractory anemia with ring sideroblasts (RARS). Hepcidin is a small, cysteine-rich cationic peptide produced by hepatocytes, secreted into plasma and excreted in the urine. Hepcidin control iron absorption in the gut, release of iron from macrophages and is a key regulator of iron metabolism. Patients with iron overload have elevated urinary hepcidin levels but patients with anemia or increased erythropoietic precursors have suppressed hepcidin. The purpose of this study was to measure hepcidin excretion in the urine of patients with MDS and Myelofibrosis (MF). The assay of urinary Hepcidin was performed by the method of Nemeth et al (J. Clin.Invest113:1271–1276, 2004) and expressed per mg urinary creatinine. The samples were obtained from 20 patients, 16 with MDS and 4 with MF. Their subtypes and age, ranging from 54 to 87 years, are shown in the table. Nine MDS patients received less than 10 units of blood and the rest, between 13 and 147 units. Seven MDS patients had ferritin levels above 1000 ng/ml and their transferrin saturation in most of them was more than 50%. The urinary excretion of hepcidin was low (less then 1 ng/mg creatinine) in 3 MDS and 3 MF patients, while in the rest, with the exception of 2 patients, the levels were less than 50 ng/mg creatinine. Although the degree of iron overload was not related directly to the subtype and to the number of blood units, in some patients mainly with RARS it was quite severe. More importantly, the urinary hepcidin excretion had a more consistent pattern of low levels in all the patients even in those with high ferritin or high transferrin saturation levels. Hepcidin concentrations correlated well with serum ferritin (r2 = 0.62 on a log-log plot) but the slope of the correlation was only 0.05 (normal ~ 1) indicating marked suppression of hepcidin relative to ferritin. Suppression of hepcidin in MDS and MF suggests that hepcidin deficiency could worsen the maldistribution of iron from the nontoxic form in macrophages to toxic forms in parenchymal cells, and that intestinal iron absorption as well as transfusional iron could contribute to their iron overload.

Age Gender Diagnosis/ MDS WHO subtype No. of transfused PC Transferrin saturation % Ferritin ng/ml Hepcidin ng/mg creat 76 F RA 7 65 422 32 76 M RA 2 28 87 10 76 M RA 0 44 1350 20 65 F RA 0 43 420 24 69 M RARS 147 87 5600 137 69 F RARA 76 45 2100 49 84 F RARS 87 57 4450 16 77 F RCMD 4 16 90 <1 75 M RCMD 4 88 307 21 77 F RCMD 65 48 2870 138 72 M RAEB I 5 44 102 <1 87 M RAEB I 62 87 3190 48 77 M RAEB II 13 20 152 20 79 M RAEB II 0 26 202 <1 75 F RAEB II 4 9 104 23 71 M RAEB II 80 53 2100 35 68 F MF 17 17 111 13 73 M MF 26 27 618 27 71 F MF 7 5 19 <1 54 F MF 0 12 42 <1
Expression of DNMT Isoforms Is Differentially Associated with Aberrant Promotor Methylation in MDS Hematopoietic Progenitor Cells during Lineage Specific Differentiation.
Authors: Hopfer Olaf J. , Komor Martina , Koehler Ina S. , Schulze Matthias , Freitag Claudia , Hoelzer Dieter , Thiel Eckhard , Hofmann Wolf-Karsten
Published: October 2019

Recent findings suggest that in myelodysplastic syndrome (MDS) several key regulatory genes are affected by aberrant promotor methylation. To explore the molecular basis of this impairment we have generated an in vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Cell harvest was at days 0, 4, 7 and 11. Promotor methylation analysis of key genes involved in the control of apoptosis (p73, survivin, DAPK), DNA-repair (hMLH1), differentiation (RARb, WT1) and cell cycle control (p14, p15, p16, CHK2) was performed by methylation specific PCR of bisulfite treated genomic DNA for each lineage at each time point. In addition, expression of DNMT1 (maintenance DNA methyltransferase), DNMT3a and DNMT3b (both de novo DNA methyltransferase) was analyzed by real time RT-PCR and correlated with gene promotor methylation at any time point. DNMT1 expression was increased during erythropoiesis in both, normal controls and MDS patients. On the other hand, expression of de novo DNMTs was elevated during thrombopoiesis at all time points. During erythropoiesis hypermethylation of p73, hMLH1 and RARb was associated with elevated DNMT1, hypermethylation of p15, p16, p73 and survivin was positively associated with increasing DNMT3 expression. Interestingly, DNMT1 was only elevated in low risk MDS, but not further increased in high risk MDS patients. Surprisingly, MDS specific survivin promotor methylation was inverse correlated with DNTM1 and DNMT3a expression. However, a negative correlation of DNMT3a with survivin expression was found in low risk MDS but not in high risk MDS. In summary our data indicate that all mammalian DNMT isoforms may be involved in the aberrant DNA-methylation phenotype in MDS. Elevated DNMT1 expression may in particular contribute to ineffective erythropoiesis in low risk MDS. DNMT3a and 3b were elevated during megakaryopoiesis and their expression was inversely correlated with MDS disease risk (IPSS). We conclude that the knowledge about distinct expression patterns of DNMT isoforms in hematopoiesis may be of help for further strategies to implicate DNMT-inhibitors in the treatment of patients with MDS.
Low Doses of Thalidomide (Thal) in Low Risk MDS with Transfusion-Dependant Anemia: The GFM THAL-SMD-200 Trial.
Authors: Tamburini J. , Elie C. , Vassilief D. , Quarre M.C. , Beyne-Rauzy O. , Gardembas M. , Aouba A. , Berthou C. , Sanhes L. , Guerci A. , Stamatoullas A. , Bauduer F. , Sotto J.J. , Legros L. , Vaultier S. , Fenaux P. , Dreyfus F. , Bouscary D.
Published: October 2019

Background: Thal has shown some efficacy in low risk MDS, but with dose-limiting toxicity requiring discontinuation in > 50% pts (Bouscary D, BJH, 2005, 131, 609 and other groups). Thus we assessed Thal at lower dose in such MDS.

Methods: From Jan 2003 to Oct 2005, we performed a prospective study of low dose Thal in 87 MDS pts with transfusion-dependant anemia (THAL-SMD-200 trial). The first 59 pts received Thal at 200 mg/d, whereas the last 28 pts received 50 mg/d. Responses (IWG) were evaluated at week 12.

Results: 48 M and 39 F pts were included. Median age 73.6 years. IPSS Low 34 pts, INT-1 44 pts, INT-2 9 pts. Mean number of RBC units transfused within 2 months of inclusion 6 (2–16). Median ANC, plts, Hb were 1960/μl (0–24160), 164 000/μL (1 000–789 000) and 8,2 (5,1–14,7) resp. 14 (16%) patients had del 5q. Thirty two (37%) pts stopped Thal before week 12, 19 (32%) and 13 (46%) patients in the 200mg/d and the 50mg/d groups resp. Reasons for interruption were: important toxicity in 23 (72%) (mainly dizziness, fatigue, sleepiness), patient’s decision in 6 (19%), progression to AML in 3 (9%) and death in 1 (3%). 3 pts developed deep venous thrombosis in the 200mg/d group (none in the 50mg/d group). 55 patients completed 12 weeks of treatment, 22 of them (40%, ie 25% of pts included) had an erythroid response, including 12 major responses (HI-EM) and 10 minor responses (HI-Em). We observed 17 (43.5% of pts who completed 12 weeks, 28% of all pts) and 5 (29.4% of pts who completed 12 weeks, 18% of all pts) erythroid responses in the 200mg/d and the 50mg/d group respectively, not statistically different (p = 0,27). Erythroid response was seen in 15 (44%) patients with IPSS low, versus 6 (14%) in INT-1 and 1 (11%) in INT-2 resp (p = 0.005), whereas no other prognostic factor of response was found. No neutrophil or platelet response was seen.

Conclusion: Thal at 200mg/d improved erythropoiesis in 40% of the pts who reached week 12 (28% of included pts). Side effects leading to treatment interruption < week 12 occurred in 32% of pts, ie a similar efficacy and somewhat better tolerance than in our experience with higher doses. Most erythroid responses occurred in IPSS Low pts. On the other hand, Thal at 50mg/d gave a similar incidence of side effects as 200mg/d, with slightly lower but not statistically different erythroid response rate, and thus brought no obvious benefit over 200mg/d. Longer follow up of pts who continued Thal will be presented.
Targeting Hepcidin with Antisense Oligonucletides Improves Anemia Endpoints in Mice.
Authors: Crosby Jeffrey R. , Gaarde William A. , Egerston Jarrett , McKay Robert , Sun Yingqing , Freier Susan , Watt Andy , Hung Gene P. , Monia Bret P.
Published: October 2019

Anemia is one of the more common blood disorders and is associated with a number of diseases, including chronic kidney disease, chronic inflammation, and certain types of cancer. Under these conditions iron is essential as it is required for erythroid progenitor cell proliferation and red cell function. Hepcidin is a liver-derived growth factor that regulates iron absorption in the GI tract and iron absorption and release in tissues. Furthermore, hepcidin overexpression has been strongly linked mechanistically as a mediator of decreased iron availability and anemia. We have utilized an antisense approach to investigate the role of hepcidin in animal models of anemia and as a potential therapeutic approach for the treatment of this disorder in humans. Second-generation 2′-O-methoxyethyl chimeric antisense oligonucleotides (ASOs) were screened in isolated primary mouse hepatocytes, followed by in vivo screening in mice, for the ability to reduce hepcidin mRNA levels. ASO treatment resulted in a reduction of hepcidin mRNA in liver which was associated with a significant increase in serum iron levels. The best hepcidin ASO was then tested in a mouse model of turpentine induced hypoferremia and anemia to determine the role of hepcidin in regulating serum iron and anemia endpoints. Mice were treated (I.P. twice/weekly) with hepcidin or control ASO for two weeks at varying doses prior to a single subcutaneous injection of turpentine. Turpentine treatment 16 hours post-injection produced a significant reduction in serum iron levels and at two weeks resulted in reduced RBC numbers, hematocrit and hemoglobin levels. Treatment with the hepcidin ASO resulted in a dose dependent improvement in all of these endpoints while the control oligonucleotide had no effect. Studies are in progress to further characterize the pharmacological activity of hepcidin ASO in additional models of anemia and results from these on-going studies will also be presented.
TKI258 (CHIR-258) Selectively Inhibits FGFR1 Fusion Genes Associated with the 8p11 Myeloproliferative Syndrome.
Authors: Chase Andrew , Grand Francis H. , Cross Nicholas C.P.
Published: October 2019

The 8p11 myeloproliferative syndrome (EMS) is an atypical stem-cell myeloproliferative disorder typically characterised by eosinophilia and associated T- or B-cell lymphoma. The disease is aggressive with transformation to acute leukemia usually occurring within 1–2 years from diagnosis and stem cell transplantation remains the only curative treatment. EMS is caused by constitutive activation of the receptor tyrosine kinase FGFR1 by fusion to unrelated partner genes, the most common being ZNF198. The partner protein promotes dimerisation and ligand-independent activation of the FGFR1 kinase in a manner analogous to BCR-ABL. To investigate the possibility of targeted therapy for EMS, we have studied the effects of TKI258 (formerly CHIR-258) on transformed cell lines and primary cells from patients with FGFR1 translocation fusion genes. TKI258 is an ATP-competitive inhibitor with activity against class III-IV receptor tyrosine kinases (which includes FGFR, VEGFR and PDGFR families) that is currently being assessed for treatment of a variety of malignancies. The growth, as assessed by the MTS assay, of Ba/F3 cells transformed to IL3 independence by the ZNF198-FGFR1 fusion gene was inhibited by TKI258 with an IC50 of 160 nM, compared to an IC50 of 995 nM for Ba/F3 cells transfected with vector only. Western blotting demonstrated dose-dependent inhibition of phosphorylation by TKI258 of ZNF198-FGFR1 and downstream signaling molecules ERK and STAT5. Primary cells from four MPD patients with FGFR1 translocations (ZNF198-FGFR1, FGFR1OP1-FGFR1, MYO18A-FGFR1 and BCR-FGFR1) and three control MPD patients without FGFR1 translocations were exposed to 0, 20 and 100 nM TKI258 in liquid culture for two weeks. All four FGFR1 patients demonstrated substantial reductions in cell numbers in both 20 and 100 nM cultures relative to control whereas no differential cell loss was seen in cells from non-FGFR1 patients. The response of colony growth to inhibition by 20 nM and 100 nM TKI258 was measured in four FGFR1-translocation patients (with and without cytokines) and in ten normal peripheral blood (NPB) controls (with cytokines). To allow sample comparison an inhibition index was calculated as the mean reduction in colony numbers at 20 nM and 100 nM TKI258 at days 7 and 14 relative to control. Four FGFR1-translocation patients showed a significant reduction in colony growth (mean inhibition index = 0.48, range = 0.24–0.60) compared with normal controls (mean index = 0.73, range = 0.48–0.87) (p < 0.01). In summary TKI258 shows significant selective activity against ZNF198-FGFR1-transformed Ba/F3 cells and against primary cells from patients with FGFR1 translocations in both liquid culture and colony assays. TKI258 is therefore a good candidate for targeted therapy for EMS patients, who currently have a poor prognosis and few treatment options.
An Inhibitor of Uroporphyrinogen Decarboxylase (URO-D) Causes Porphyria Cutanea Tarda (PCT).
Authors: Kushner James P. , Franklin Michael R. , Reilly Christopher P. , Bergonia Hector A. , Phillips John D.
Published: October 2019

Hepatic iron content is increased in patients with PCT and phlebotomy-induced iron depletion corrects the clinical and biochemical phenotype. Approximately 20 percent of patients with PCT are homozygous for mutations of the hemochromatosis gene (HFE) but the cause of iron overload in most patients is unknown. Hepatic URO-D activity is markedly reduced when PCT is manifest and URO-D activity improves following iron depletion. Most patients with PCT have no mutations of the URO-D gene (sporadic PCT) but approximately 1/3 of cases are heterozygous for URO-D mutations (familial PCT) (BLOOD. 2000; 95:1565–71). To determine the mechanism by which iron overload causes PCT we created 3 murine models: Mice with one null allele of Uro-d (Uro-d+/−) and 2 null Hfe alleles (Hfe−/ −) (PNAS.2001; 98:259–64); Uro-d+/− mice treated with iron-dextran, aminolevulinic acid (ALA) and polychlorinated biphenyls (PCB) and; wild type mice treated with iron, ALA and PCB (J. Biochem. Mol. Toxicol.2001; 15:287–93). All models accumulate uroporphyrin in the liver and all have hepatic URO-D activity of 25% or less but Western blots revealed no change in URO-D protein. An iron deficient diet prevented the PCT phenotype in Uro-d+/−, Hfe−/ − animals and greatly attenuated the phenotype in animals treated with ALA and PCB (Env. Toxicol. Pharmacol.2005; 417–23). Liver homogenates from all porphyric models were heat denatured and clarified by centrifugation. The supernatants inhibited the activity of purified recombinant human URO-D (rhURO-D) by approximately 60%. The inhibitory activity was further purified by solid phase extraction and HPLC. The fraction containing the inhibitory activity did not fluoresce. Mass spectrometry of this fraction demonstrated a dominant peak with a mass of 835 Da and an absorption maximum of approximately 500 nM, the optical signature of a porphomethene. An inhibitor with identical properties was generated by partially oxidizing the uroporphyrinogen (837 Da) substrate of URO-D under UV light. Full oxidation of either the inhibitor purified from porphyric mouse liver or from partially oxidized, enzymatically generated uroporphyrinogen (either isomer I or III) yielded a compound with a mass of 831 Da and an absorption maximum of approximately 400 nM, indicating that the fully oxidized inhibitor was uroporphyrin. Tandem mass spectrometry of the 835 Da inhibitor indicated that the inhibitor was a tetrapyrrole. Collectively these data indicate that the inhibitor is a porphomethene derived form uroporphyrinogen through oxidation of a single bridge carbon between adjacent pyrrole rings. An inhibitor of rhURO-D was also identified in heat-denatured cytosol from liver biopsy samples obtained from four humans with PCT. We conclude that clinical expression of PCT requires an iron dependant oxidation reaction that generates a porphomethene inhibitor of URO-D.
Tissue Factor and Soluble Markers of Platelet and Endothelial Activation in Essential Thrombocythemia: Relationship with Thrombosis and JAK2 V617F Mutation Status.
Authors: Cervantes Francisco , Arellano-Rodrigo Eduardo , Alvarez-Larran Alberto , Reverter Juan-Carlos , Villamor Neus , Colomer Dolors
Published: October 2019

There is increasing evidence that platelet and leukocyte activation plays an important role in the thrombotic complications of patients with essential thrombocythemia (ET), but the relationship of both thrombosis occurrence and JAK2 V617F mutation status with the levels of circulating tissue factor (TF) and of soluble markers of platelet and endothelial activation is not known. In 53 ET patients (26 of whom had a previous history of thrombosis), platelet TF expression and plasma levels of TF, soluble P-selectin (sP-selectin), soluble CD40 ligand (sCD40L), von Willebrand factor antigen (VWF:Ag), soluble thrombomodulin (sTM), D-dimer, and prothrombin fragment 1+2 (F1+2), measured by ELISA, were compared with those in matched healthy individuals and correlated with thrombosis occurrence and JAK2 V617F mutation status. ET patients with thrombosis had significantly higher levels of sP-selectin than patients without thrombosis and the controls, whereas ET patients without thrombosis had significantly higher levels than the controls (99.8 ± 47.1 ng/mL versus 70.6 ± 37.8 ng/mL versus 32.4 ± 11.9 ng/mL; p= 0.0001 for all comparisons). The same applied to sCD40L levels (226.7 ± 104.7 pg/mL in patients with thrombosis, 186.4 ± 92.1 pg/mL in patients without thrombosis, and 81.3 ± 22.0 pg/mL in controls; p= 0.0001 for all comparisons). Circulating VWF:Ag and F1+2 levels were higher in ET patients than in controls, but no significant difference was observed between patients with and without thrombosis. No differences in TF platelet expression, TF and sTM plasma concentrations were found between patients and controls. A positive correlation was observed between sP-selectin and F1+2, a marker of thrombin generation (r= 0.378, p= 0.01). Patients with the JAK2 mutation (22 out of 52 assessable patients), as compared with those with the wild-type allele, had significantly higher levels of sP-selectin (p= 0.002), sCD40L (p= 0.03), TF (p= 0.016), VWF:Ag (p= 0.0001), and sTM (p= 0.032). These results support a role for soluble markers of platelet activation in the thrombosis of ET as well as their potential to identify ET patients at greater risk of thrombosis. The association between JAK2 mutation and increased levels of TF and soluble markers of platelet and endothelial activation would suggest that the mutation could promote an enhanced prethrombotic state in ET.
SGN-30 (Anti-CD30 mAb) Has a Single-Agent Response Rate of 21% in Patients with Refractory or Recurrent Systemic Anaplastic Large Cell Lymphoma (ALCL).
Authors: Forero-Torres Andres , Bernstein STeven H. , Gopal Ajay , Foss Francine , Leonard John P. , Rosenblatt Joseph D. , Bartlett Nancy L. , Cheson Bruce D. , Epner Elliot , Hernandez-Ilizaliturri Francisco J. , Proctor STephen J. , Brice Pauline , Tilly Herve , Haioun Corinne , Jerkeman Mats , Bosly Andre , Gianni Alessandro M. , Holte Harald , Mahendra Premini , Uharek Lutz , Lorenz Jennie , Barton Jeremy , Sievers Eric
Published: October 2019

SGN-30 is a monoclonal antibody directed against the CD30 antigen expressed on some hematologic malignancies. Based on encouraging phase I data, a multicenter phase II study was conducted treating patients with refractory or recurrent CD30-positive ALCL with an ECOG performance status of ≤ 2. Thirty-nine patients (24M, 15F) with ALCL were enrolled, with a median age of 57 (range 23–82) and a median of 3 prior therapies (range 2–5). Nine patients had previously received a stem cell transplant. Eighty-five percent of tumors were negative for ALK, a poor prognostic factor. SGN-30 was administred at 6 mg/kg/wk (90 minute infusion, premedications were not required) for 6 consecutive weeks. After 24 patients were enrolled, the dose was escalated to 12 mg/kg/wk in subsequent patients. (Patients with stable disease or objective response were eligible to receive additional cycles of SGN-30. Five patients received ≥ 2 cycles of SGN-30.) Response assessments, as determined by CT scans, were performed 2 weeks after the last infusion. Best response is shown below:

CR PR SD PD Pending Eval ORR *Both CRs have ongoing durations of >365 days; both patients received additional cycles of SGN-30. **PRs had durations of 27, 53, 139 and 167 days; two additional patients have ongoing durations of 86+ and 25+ days. ***Three SDs have ongoing durations of 96+, 365+, and 365+ days. Two additional patients had SD for 71 and 174 days. 2* 6** 5*** 24 2 21%

Three drug-related toxicities ≥ Grade 3 were reported (each was considered possibly related to SGN-30): 1) lymphopenia, 2) catheter related infection and 3) urticaria. No other significant hematologic or biochemical toxicities have been observed. There was one definitely related serious adverse event (Grade 2) in a patient who experienced a transient exacerbation of his cutaneous lesions after 2 doses of SGN-30 but achieved a partial response after continuing on study. This phase II study represents one of the largest prospectively designed trials in relapsed/refractory ALCL and demonstrates good tolerability and clinically meaningful antitumor activity of SGN-30, especially in ALK negative patients who have a particularly poor prognosis.
Penetration of Yttrium-90-Labeled Ibritumomab Tiuxetan (Zevalin™) into Primary CNS Lymphoma.
Authors: Kiewe Philipp , Korfel Agnieszka , Maza Sofiane , Jahnke Kristoph , Munz Dieter L. , Thiel Eckhard
Published: October 2019

Background: Salvage treatment has not yet been established in primary CNS lymphoma (PCNSL). The introduction of anti-CD20 monoclonal antibody rituximab expanded treatment options in systemic B-cell lymphoma. Due to its large molecular size, rituximab poorly penetrates the blood-brain-barrier (BBB) limiting its use in PCNSL. According to FDG-PET investigations, however, the BBB is initially leaky in PCNSL and reconstitutes after therapy-induced tumor shrinkage. We treated patients with relapsed/resistant PCNSL with a single course of Y-90 anti-CD20 antibody ibritumomab tiuxetan and evaluated the penetration of the antibody into the tumor.

Methods: Immunocompetent patients with histologically confirmed, therapy resistant or recurrent PCNSL after at least one pretreatment, and adequate cardiac and bone marrow function were treated with rituximab 250 mg/m2 on day -7 and day 0, followed by Y-90-ibritumomab tiuxetan 15 MBq/kg IV. In three patients single photon emission computed tomography (SPECT) target imaging with gamma-emitting 111-Indium-ibritumomab tiuxetan was performed repeatedly 4 - 168 hours after Y-90-ibritumomab tiuxetan. Response evaluation by contrast-enhanced magnetic resonance imaging (MRI) and fluorodeoxyglucose positron emission tomography (FDG-PET) was scheduled before, one month and two months after treatment as well as every three months thereafter in responders.

Results: Seven patients with a median age of 46 years (range 40–63) and a median number of 4 (range 1–6) previous therapies received antibody treatment. SPECT indicated target accumulation in the tumor starting 48 hours and still ongoing 7 days after injection of 111-Indium-ibritumomab tiuxetan. Response was observed in four of seven patients: one uncertain complete response (uCR) lasting 18+ months, two CR (0.5 and one month), and one partial response (0.5 months). Relapse usually occurred distant of target lesions. Three patients had disease progression. Toxicities were leukopenia CTC grade 3/4 (n=6), pneumonia grade 3/4 (n=2) with one fatal outcome, and thrombocytopenia grade 3/4 (n=6).

Conclusions: We provide first evidence of Y-90-ibritumomab tiuxetan penetration into PCNSL with target accumulation of the antibody in SPECT. However, responses were mostly of short duration in this heavily pretreated patient cohort with relapses occurring distant of target lesions. Hematotoxicity up to CTC grade 4 was the most common side-effect. Further investigation within a phase II study is warranted.