bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of Cellular Aminopeptidases as Novel Therapy for AML.
Authors: Jenkins Christopher , Pepper Chris , Mills Ken , Burnett Alan 
Published: October 2019

Abstract
CHR 2797 is one of a new class of enzyme inhibitors with a pleiotropic effect against a number of human cancer cells. It is thought to inhibit the M1 family of metalloenzymes that include aminopeptidases, and is under investigation for the treatment of acute myeloid leukemia. Aminopeptidases catalyse the hydrolysis of the terminal amino acids from short chain polypeptides and they are involved in the continuous cycle of protein formation and degradation in cells. As malignant cells are thought to be more highly dependant on this protein cycling, interrupting this pathway is therefore a potential therapeutic target for novel agents. The effects of the aminopeptidase inhibitor CHR 2797 were investigated in AML cells in-vitro. Leukemic cells and cell lines were treated with CHR 2797 at a range of 0.0002 – 20μM and IC50 values were calculated from the WST-1 proliferation experiments. The AML cell lines HL60, KG1, K562 and U937 had an average IC50 of 1μM with a range between 0.01 and 10μM. Primary diagnostic AML samples (n=40) were analysed and an IC50 range of between 0.01 and >40μM were detected, with a median of 0.8μM. The effects of CHR 2797 were also analysed on normal bone marrow samples (n=10). The IC50 range was between 6.2 and >40μM with a median of 15μM, demonstrating a potential therapeutic window between the treatment of the leukemic cells and toxicity to the normal samples. The level of synergy or antagonism with conventional therapeutic agents was calculated using a combination index. Synergy was demonstrated in 70% of cell samples in combination with ARA-C, and 80% with Velcade. Synergy was also shown in 60% of cells samples with ATRA, even in non-promyelocytic leukemia types. Annexin V and cell cycle analysis confirmed apoptosis after treatment with CHR 2797 in many cases. A degree of differentiation of acute promyelocytic cells to mature myeloid cells was also stimulated with the treatment. The effects of CHR 2797 on cellular aminopeptidases were also measured. CD13 is a cell surface protein which is expressed selectively on myeloid cells and is also classified as an aminopeptidase N. Its activity can be measured by the conversion of the substrate ala-MCA to the protein MCA that can be detected on a fluorometric plate reader. CHR 2797 was demonstrated to reduce CD13 activity in a time and dose responsive manner. The reduction in activity was demonstrated immediately following addition of the drug, and a persistent effect was shown over four days of cell culture. A reduction in CD13 activity was also shown with a concentration of CHR 2797 of under 0.5μM; and with a 10μM dose the activity in many AML samples was reduced by >90%. New treatments are needed for acute myeloid leukemia to improve survival and reduce the toxicity of conventional therapy. This study demonstrates that CHR 2797 might be an effective molecular therapy for AML, either alone or in combination with other chemotherapeutic agents.
Canonical NF-kB Pathway Is Not Constitutively Activated and May Not Be the Major Bortezomib Target in Mantle Cell Lymphoma (MCL).
Authors: Camara-Clayette Valerie , Lepelletier Yves , Moura Ivan , Vainchenker William , Hermine Olivier , Ribrag Vincent 
Published: October 2019

Abstract
Bortezomib, a proteasome inhibitor, has promising activity in MCL. Bortezomib mechanism of action is complex since multiple proteins and cellular pathways are regulated by proteasome degradation. By blocking the degradation of IKBa, the canonical NF-kB pathway is one of the major Bortezomib target in lymphoid malignancies such as multiple myeloma. NF-kB activation has not been extensively assessed in other lymphoid malignancies such as MCL. NF-kB complexes (heterodimers P50/P65) were assessed by EMSA assays in REC GRANTA 519, NCEB, JEKO, JUN and UPN1 MCL cell-lines. EBV was present in 3/6 cell lines. P50/P65 complexes were detected in all EBV positive cell lines and only in 1/3 EBV negative cell-lines (JEKO). No nuclear P65 protein was detected by immunofluorescence or Western-blot analysis in the 2 other negative EBV cell lines (REC and UPN1). NF-kB transcriptional activity was measured by a luciferase-based reporter gene assay. Spontaneous NF-kB activity was low in UPN1, REC and JEKO compared to GRANTA 519 EBV positive cell-line (3 to 4 time lower) but can be strongly activated (up to 10 fold) using MEKK cotransfection assay. This suggest that NF-kB is functional, but not constitutively activated. Stable infections with Migr1-IRES-GFP IkBM (dominant negative IKB) or empty vector, were performed in GRANTA 519 EBV positive as well as in UPN1 cell line. No differences in proliferation or apoptosis were observed in UPN1 stably infected with IkBM or with the empty vector but GRANTA 519 EBV positive showed increased apoptosis and proliferation inhibition when infected with IkBM. Likewise, no P50/P65 heterodimers complexes were detected by EMSA in 4 patients with MCL. In vitro assays showed that UPN1 and JEKO cell lines had comparable sensitivities to Bortezomib than Multiple myeloma cell lines reported in the literature (IC 50: 6 nM and 12 nM respectively). This was also true for the 3 patients lymphoma cells assessed in vitro (12 nM). Taken together these results suggest that canonical NF-kB activation pathway is not constitutively active in EBV negative MCL cell lines and patients samples and could be associated with EBV infection in some MCL cell-lines. Therefore, this strongly suggests that Bortezomib target other molecules in MCL.
Multicolour Flow Cytometric Finetuning of the Classification of Myelodysplastic Syndromes.
Authors: van de Loosdrecht Arjan A. , Westers Theresia M. , Westra Guus , Draeger Angelica , Van der Velden Vincent H. , Ossenkoppele Gert J. 
Published: October 2019

Abstract
The WHO classification of myeloid disorders contribute to a more refined classification and prognostication of myelodysplastic syndromes (MDS). The considerable differences in clinical behaviour of pure refractory anemia (RA) versus refractory cytopenia with multilineage dysplasia (RCMD) with or without ringsideroblasts are of importance in the management of patients with MDS. Flow cytometry may add additional diagnostic criteria to adequately discriminate RA from RCMD (+/− ringsideroblasts; (RS)) and may contribute in identifying Idiopathic Cytopenia of Undetermined Significance. We developed a 4-colour flow-cytometric procedure that comprises all differentiation stages of granulocytic, monocytic and erythroid lineages, instrumental for the recognition of various subpopulations within all three lineages in normal bone marrow samples. In 43 evaluable patients with MDS (RA, RARS, RCMD, RCMD-RS, MDS-U, RAEB-1 and 2), aberrant expression of differentiation antigens were demonstrated in 1 or more lineages. Flow-cytometry identified aberrancies in granulopoiesis and monocytopoiesis in 93% and 74% of the cases, respectively. In the majority of cases abnormal relations between CD13, CD16, CD11b, CD15 and HLA-DR were prominent in the granulopoiesis. In 34% of the cases a striking monocytopenia was detected, whereas in 59% abnormal surface expression of CD14, CD36 and CD33 indicating aberrant differentiation of monocytes. We defined aberrant myeloid blasts by a leukaemia associated phenotype (LAP) according to the definitions used in acute myeloid leukaemia. In 47% of the patients a LAP was detectable by demonstrating co-expression of CD5, CD7, CD19 and CD56 on CD34+ myeloid blasts. In all patients diagnosed as RA/RARS and MDS-U (n=12) according to WHO criteria, additional flow aberrancies were identified including a leukaemia associated phenotype of myeloid blasts in 41% of the cases. Only in 3 out of 28 cases with RCMD/RCMD-RS no erythroid aberrancies were detectable by flow-cytometry. In 9 normal control BM samples, no flow-cytometric abnormalities were present. It is concluded that flow-cytometry in MDS identifies aberrancies in the granulocytic and monocytic lineages and may classify patients with multi-lineage aberrancies not otherwise determined by cytology (WHO). Flow-cytometry may discriminate pure RA or MDS-U from RCMD. Since new drugs are emerging in low-risk MDS, the value of flow-cytometry might be of importance to further refine the classification in MDS. The exact role of these aberrant differentiation patterns on IPSS, clinical behaviour, impact on treatment decisions and as tool in disease monitoring have to be determined in future prospective studies.
An Erythroid Differentiation Gene Expression Signature Predicts Response to Lenalidomide in Myelodysplasia.
Authors: Ebert Benjamin L. , Galili Naomi , Tamayo Pablo , Mak Raymond , Pretz Jennifer , Ladd-Acosta Christine , Stone Richard , Golub Todd R. , Raza Azra 
Published: October 2019

Abstract
Lenalidomide is an effective new agent for the treatment of Myelodysplastic Syndrome (MDS). In phase II clinical trials, patients treated with Lenalidomide achieved unprecedented rates of hematologic and cytogenetic responses, particularly in patients with interstitial deletions of chromosome 5q. More than 80% of patients with MDS do not have 5q deletions, though approximately 25% of these patients respond to Lenalidomide.3–5 Neutropenia and thrombocytopenia are the primary toxicities of Lenalidomide, and are severe in some cases. MDS is a highly heterogeneous disorder, and no diagnostic assays predict which patients without 5q deletions will respond to Lenalidomide. Using gene expression profiling, we identified a molecular signature that predicts Lenalidomide response. Biologic characterization of the predictive genes demonstrated that the response signature is comprised of a cohesive set of erythroid-specific genes: patients who respond have a lower expression of the erythroid signature in bone marrow mononuclear cells. To facilitate clinical implementation of a gene expression-based assay, we evaluated the signature using two methods, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and a bead-based detection technology. Using both methods, the erythroid gene expression signature was expressed at lower levels in patients who responded to Lenalidomide. The bead-based methodology employs ligation mediated amplification (LMA), a methodology that is capable of highly multiplexed reactions with preservation of the relative abundance of transcripts, with amplicon detection on Luminex microspheres, already in use for clinical diagnostic assays. This assay is capable of detecting a large gene expression signature (currently up to 100 genes) in a single, well-controlled assay. With further validation, LMA with fluorescent microsphere detection may become the superior technology for clinical diagnostic assays of multi-gene signatures. We tested the erythroid signature in an independent set of 12 samples using LMA with fluorescent microsphere detection. Again, all responders had a lower expression of the erythroid genes than responders. Validation in a larger cohort of patients is underway. These studies suggest that Lenalidomide-responsive patients without 5q deletions have a defect in erythroid differentiation analogous to the ineffective erythropoiesis in patients with 5q deletions, and that an erythroid gene expression signature predicts Lenalidomide activity in MDS.
Genetic Profiling of Myeloproliferative Disorders by Single Nucleotide Polymorphism Oligonucleotide Microarray.
Authors: Kawamata Norihiko , Ogawa Seishi , Levine Ross L. , Pikman Yana , Nanya Toshiki , Sanada Masashi , Miller Carl W. , Gilliland D. Gary , Koeffler H. Phillip 
Published: October 2019

Abstract
Myeloproliferative disorders (MPD) are clonal hematopoietic diseases including polycythemia vera (PV), essential thrombocytosis (ET) and myelofibrosis with myeloid metaplasia (MMM). Single-nucleotide polymorphism DNA microarray (SNP-Chip) was used to analyze 43 MPDs. All five cases of PV having homozygous JAK2V617F had uniparental disomy (UPD), thus duplicating the allele with the mutated JAK2 and eliminateing the normal allele. Concerning MMM, either the Rb1 (13q14) or NF1 (17q11) gene was deleted in five of the 9 cases that had no Jak2 mutations. Two of these cases had UPD at 1p. Sequencing of the target genes within this region of UPD revealed a point mutation of the MPL gene (S240F or W515L). Each of these cases also had a deletion of Rb suggesting that the mutated MPL may co-operate with an aberrant Rb. The 9 cases of MMM with JAK2 mutations had only infrequent (2/9 cases) additional genomic alterations. Interestingly, No genomic alterations were noted in any of the cases (10) of ET having a normal Jak2. Two of 7 ET cases with JAK2 mutations had deletion of either 5q or trisomy 9. In summary, UPD is the predominant mechanism by which MPD develops homozygous JAK2 mutations. 1p UPD is a signpost that MPL is mutated in cases of MMM having wild-type JAK2; and these cells often have loss of Rb. ET cases with no JAK2 mutations are surprisingly free of genomic alterations.
Inducible hkRASG12D Expression in Zebrafish Hematopoietic Cells Leads to Myeloproliferative Disease.
Authors: Le Xiuning , Langenau David M. , Keefe Matthew D. , Kutok Jeffery L. , Zon Leonard I. 
Published: October 2019

Abstract
Activating mutations in RAS family members are common in myeloproliferative disease (MPD) and acute myeloid leukemia (AML). Because the zebrafish has proven to be an excellent leukemia model and can be used in forward genetic and chemical screens to identify modulators of disease pathways, we developed a transgenic zebrafish model of RAS-induced myeloproliferative disease. Stable transgenic zebrafish lines were created in which the ubiquitous B-actin promoter drives expression of a loxed GFP transgene. Upon CRE-mediated recombination, the loxed GFP cassette is excised and the human kRASG12D transgene is expressed. When mated to hsp70-CRE transgenic zebrafish that express CRE recombinase when the animals are heated to 37C for 1 hour, a cohort of 300 double transgenic embryos was generated. In the heat-shocked group, the medial survival time was 25 days, suggesting that oncogenic RAS expression is lethal in developing zebrafish larvae. Of those animals that survived past this time window, most developed sarcomatous muscle tumors. In contrast, most of the double transgenic zebrafish that did not receive heatshock survived to adulthood. Because the hsp70 promoter is active in development and can be induced by stress, the hsp70-CRE transgenic zebrafish line exhibits low levels of CRE expression even in the absence of heatshock. In the non-heat shocked cohort, 10 of 120 double transgenic zebrafish developed MPD by 66 days of life. Histological examination and fluorescence cytometry analysis revealed an expansion of myeloid cell populations within the kidney, comprising granulocytic and monocytic cells in various stages of differentiation, mimicking myeloproliferative diseases seen in both human and mouse. To specifically induce kRASG12D expression in hematopoietic cells, kidneys were dissected from healthy double transgenic animals; heat shocked ex vivo, and then transplanted into sub-lethally irradiated recipient fish. Upon analysis of transplant animals at three months of age, flow cytometry confirmed that MPD had developed in the recipient fish, however other tumor subtypes were not observed. When kidney marrow from transplant animals having MPD were introduced into sub-lethally irradiated secondary recipients, transplant efficiency was greatly reduced. Taken together these results suggest that, as is seen in MPD in mammals, myeloid cells are not fully transformed in our model. In summary, we show that inducible hkRASG12D expression in zebrafish hematopoietic cells leads to myeloproliferative disease and suggest that this model will allow for the identification of novel pathways responsible for full transformation leading to AML.
Safety and Tolerability of Anti-IL-5 Monoclonal Antibody (Mepolizumab) Therapy in Patients with HES: A Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial.
Authors: Klion Amy D. , Rothenberg Marc E. , Murray John J. , Singh Anish , Simon Hans-Uwe 
Published: October 2019

Abstract
Mepolizumab is a fully humanized monoclonal IgG antibody which binds to interleukin 5 (IL-5) with high affinity and specificity, preventing it from binding to IL-5 receptors on the eosinophil cell surface. Since IL-5 is central in eosinophil proliferation, differentiation, mobilization, activation, and survival, yet has limited effects on other cell types, IL-5 inhibition should inhibit the production and activity of eosinophils without affecting the function of other immune cells. Reducing the blood eosinophil count is a critical therapeutic objective in patients with eosinophil-driven diseases, such as hypereosinophilic syndrome (HES). The safety and tolerability of mepolizumab has been evaluated, as a secondary endpoint, in a multicenter, randomized, double-blind, placebo-controlled, parallel-group trial. The trial enrolled 85 patients with HES (blood eosinophil count >1500/μl for ≥6 months with documented eosinophilia-related organ involvement or dysfunction and no known cause of eosinophilia), who were negative for the FIP1L1-PDGFRα gene rearrangement. Blood eosinophil levels were stabilized at <1000 cells/μL on 20–60 mg/day prednisone monotherapy during a run-in period of up to 6 weeks, then patients were randomized to intravenous mepolizumab 750 mg (n=43) or saline (placebo; n=42) every 4 weeks for 36 weeks (final infusion at Week 32). Overall, 98% of placebo-treated and 93% of mepolizumab-treated patients reported an adverse event during treatment. The most common of these were fatigue (26% on placebo vs 30% on mepolizumab), pruritus (21% vs 28%), headache (21% vs 23%), and arthralgia (17% vs 21%). The only significant differences between the treatment groups were in pharyngolaryngeal pain (14% on placebo vs 2% on mepolizumab; P=0.027) and pain in extremity (12% on placebo vs 2% on mepolizumab; P=0.047); none of the adverse events was significantly more frequent with mepolizumab than with placebo. A greater than 8% difference in adverse event reporting for the mepolizumab vs placebo arms was noted for upper respiratory tract infections, myalgia, rhinitis, bronchitis, and urticaria. Serious adverse events were reported in 5 patients on placebo (7 events) and 7 patients on mepolizumab (14 events, including 1 death due to a cardiac arrest), but none was considered treatment related. Withdrawals due to adverse events (4 patients on placebo; 1 patient on mepolizumab) were also considered unrelated to treatment. Laboratory tests (hematology, clinical chemistry, and urinalysis), vital signs, and ECG findings did not raise any major safety concerns. In conclusion, this study, which is currently the largest placebo-controlled trial involving exclusively patients with HES, indicates that mepolizumab is safe and well tolerated in this patient population.
Characteristics and Outcomes of 38 Patients with Acute Myeloid Leukemia Evolving from Previous Well Characterized Myeloproliferative Disorder.
Authors: Tam Constantine S. , Giles Francis , Cortes Jorge , Garcia-Manero Guillermo , Estey Elihu , Kantarjian Hagop , Verstovsek Srdan 
Published: October 2019

Abstract
Antecedent hematological disorder (AHD) is a known adverse prognostic factor for patients with AML. AHD, however, encompasses a range of diverse hematological disorders. The characteristics and outcome of AML evolving from myeloproliferative disorders (MPD) without features of dysplasia remain poorly defined. Between 08/73 and 01/06, 76 patients were treated at the MD Anderson Cancer Center for AML evolving from a previous MPD. Of these, 38 were excluded from further analyses: inadequate pathological information for confident diagnosis of MPD (n=15), concomitant myelodysplastic features (n=11), chronic myelomonocytic leukemia (n=4), and less than twelve months between MPD diagnosis and AML occurrence (n=8). Baseline characteristics of remaining 38 patients with well-characterized MPD: median age 51 years (range 24–70); male 61%; cytogenetics - diploid 86%, del20q 14%; splenomegaly 27%. Diagnoses by WHO classification: polycythemia vera 47%; myelofibrosis 24%; essential thrombocythemia 18%; chronic MPD unclassifiable 11%. MPD therapy: hydroxyurea in 87% (median 3 years exposure), alkylating agent in 16%, and radioactive phosphorus in 8% of patients. AML occurred a median of 111 months (range 22 – 376) after MPD diagnosis. Median age at AML diagnosis was 65 years (range 40–81); cytogenetics were poor risk (−5, −7, 11q23, or ≥3 abnormalities) in 50% and intermediate risk in others (including diploid in 28%). Median survival was 23 weeks after AML diagnosis, and was not significantly different between patients who received AML therapy and those who received supportive therapy only (p=0.33) (figure 1), or between patients with intermediate and poor risk cytogenetics (p=0.12). Twenty-four patients received standard or high-dose Ara-C based induction chemotherapy: complete response (CR) rate was 38%, but responses were short-lived with all patients relapsing within 37 weeks in the absence of stem cell transplantation. Two patients underwent non-myeloablative matched sibling stem cell transplant in first CR: one remain leukemia free at 70 weeks, and the second relapsed 22 weeks post transplant but responded to re-induction chemotherapy, and remains alive at 38 weeks. AML evolving from a previous MPD carries an adverse prognosis, with very poor results from conventional induction chemotherapy. Exploration of novel agents in this patient population as a first line therapy is warranted.
Figure Figure