bluuebunny/crossref_metadata_2025_split huggingface dataset:
Release of Alkaline Phosphatase Caused by PIGV Mutations In Patients with Hyperphosphatasia-Mental Retardation Syndrome (HPMR), a Recently Found Second Inherited GPI Anchor Deficiency.
Authors: Murakami Yoshiko , Krawitz Peter M , Robinson Peter N , Mundlos Stefan , Maeda Yusuke , Kinoshita Taroh 
Published: October 2019

Abstract
Abstract 2031
Recent whole exome sequencing of three sibs of non-consanguineous parents demonstrated PIGV mutations in Hyperphosphatasia-Mental Retardation syndrome (HPMR), an autosomal recessive disease characterized by mental retardation and elevated serum alkaline phosphatase (ALP) levels (Eur J Med Genet, vol.53, issue2, p85, 2010). PIGV is the second mannosyltransferase essential for glycosylphosphatidylinositol (GPI) biosynthesis. Mutations found in four families caused amino acid substitutions A341E, A341V, Q256K and H385P. We have shown that these mutant PIGV proteins are unstable and the mutant cDNAs restored only subnormal GPI biosynthetic activity after transfection into PIGV deficient CHO cells. The backbone of GPI-anchor is synthesized in the endoplasmic reticulum (ER) and is transferred to the proteins which have GPI attachment signal at the C-terminal. It is known that GPI-anchored proteins are not expressed on the surface of GPI deficient cells due to degradation within the cells or secretion. ALP, a GPI anchored protein, was efficiently secreted into medium from PIGV deficient CHO cells, in which incomplete GPI bearing one mannose was accumulated. In contrast, ALP was degraded in PIGL, DPM2 or PIGX deficient CHO cells, in which GPIs lacking mannose were accumulated. Secretion of ALP required GPI transamidase that cleaves the C-terminal GPI attachment signal peptide and replaces it with GPI. It seems that GPI transamidase is activated by GPI bearing at least one mannose, cleaving the hydrophobic signal peptide and resulting in secretion of soluble ALP. It is well known that hypophosphatasia is caused by a deficiency of liver-, bone-, kidney-type alkaline phosphatase due to mutations in the tissue-nonspecific alkaline phosphatase (ALPL) gene. This inherited disorder is characterized by defective bone mineralization and some patients develop epilepsy caused by the disturbance of pyridoxal-5-phosphate (PLP) dependent metabolism of neurotransmitters due to the defect in ALPL which converts PLP to pyridoxal (PL) thereby facilitating passive uptake of this freely diffusible form at the cell surface. Some of the HPMR patients have seizures and the patients with previously reported inherited GPI deficiency caused by defective PIGM encoding the first mannosyltransferase (Nature Med. 12, p846 2006) also have seizures. This common symptom may be caused by the loss of membrane bound ALPL. On the other hand, patients with HPMR do not have the portal vein thrombosis seen in the patients with PIGM deficiency. The affected cell types and the degree of deficiencies in various GPI-anchored proteins might be the cause of the variability of the clinical features among GPI deficiencies.

Disclosures:
No relevant conflicts of interest to declare.
Organ Siderosis In a Murine Graft-Versus-Host-Model After Allogeneic Stem Cell Transplantations Unrelated to Red Cell Transfusion
Authors: Nogai Axel , Ziebig Reinhard , Muckenthaler Martina U. , Thiel Eckhard , Uharek Lutz , Notter Michael 
Published: October 2019

Abstract
Abstract 2048

Introduction:
Organ siderosis (OS) occurs frequently after allogeneic stem cell transplantations and is associated with worse survival. Although commonly attributed to transfusions of erythrocyte sediments, we present the occurrence of OS in mice suffering from Graft-versus-Host-disease (GvHD) independent of transfusions.

Methods:
Prior to transplantation (day 0), C57BL/10-wildtype (wt) mice received treosulfan 2000mg/kg on day -3 to -1 and cyclophosphamide 200 mg/kg on day-1. Mice were transplanted with 5×106 bone marrow cells and 3×106 splenocytes of Balb/c (allogeneic) or C57BL/10 (syngeneic) origin. Mice were sacrificed on day+9. Liver iron content was determined by atomabsorption spectroscopy in graphit tubes. In addition, a semi-quantitative analysis of liver, kidney and spleen sections was performed in kinetic studies. Macroscopic and histological GvHD-Scores were assessed according to standard scores.

Results:
Treatment of mice without additional transplantation or transplantation of syngeneic cells did not increase the iron content of the liver compared to untreated mice (naïve: 6.7 +/− 1.3 μmol/g; chemotherapy only: 7.2 +/− 1.9; syngeneic: 7.2 +/− 2.1). In contrast, allogeneic transplantated mice suffering from GvHD developed a siderosis of the liver of 13.6 +/− 3.1 (p=0.01, Kruskal-Wallis test). Furthermore, the iron deposition was correlated with the macroscopic and histologic GvHD score.

Conclusion:
These results show the occurrence of OS in wildtype animals after allogeneic stem cell transplantation. Since the detection of iron deposition occurs early after transplantation, OS is unlikely due to increased intestinal iron absorption, but appears to be a consequence of blocked iron export out of hepatic epithelial cells and macrophages and potentially by redistribution of iron as a consequence of liver cell destruction. Further studies are needed to elucidate the role of regulatory proteins such as hepcidin and ferroportin for OS in the context of GvHD following stem cell transplantation. In view of GvHD-associated OS in transplanted patients occuring frequently independent of transfusions, the effect appears clinically relevant with treatment options remaining to be defined.

Disclosures:
No relevant conflicts of interest to declare.
CBL Mutations In Therapy-Related Leukemia and Infant Leukemia
Authors: Shiba Norio , Taki Tomohiko , Park Myoung-ja , Nagasawa Masayuki , Takita Jyunko , Kato Motohiro , Kanazawa Takashi , Sotomatsu Manabu , Arakawa Hirokazu , Hayashi Yasuhide 
Published: October 2019

Abstract
Abstract 2149
Recent reports of somatic mutations of the CBL proto-oncogene in myeloid neoplasms are intriguing, because these CBL mutations were shown to results in aberrant tyrosine kinase signaling, which would lead also to activation of RAS signaling pathways. We and others reported that CBL mutations occurred in a variety of myeloid neoplasms, including de novo acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and MDS/myeloproliferative neoplasm, especially in chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML). The importance of CBL mutations concerning leukemogenesis is substantially increased. We investigated CBL mutations in 20 therapy-related leukemia/MDS (t-Leuk/MDS) cases and 26 infant leukemia cases. Homozygous mutation of theCBL gene (P417R), which was located in the RING finger domain, was identified in one out of 20 (5%) t-Leuk/MDS cases. This patient was a 5 year-old boy, whose biopsied specimen of the buccal lymph node showed malignant lymphoma (diffuse large T cell type, MT1(+), MB1(-), UCHL1(+)). No pathogenic nucleotide changes were identified in the CBL gene in the initial sample. Subsequently, the patient was treated with chemotherapy including VP-16 (200 mg/m2) given twice weekly. Nineteen months after initial diagnosis, he was diagnosed as having therapy-related leukemia with t(5;21) and MLL gene rearrangement due to VP-16. Furthermore, CBL gene mutations were found in 3 of 26 (12%) infant leukemia cases with 11q23 translocation/MLL gene rearrangement. CBL gene mutations were located in splice site in intron 8 and the RING finger domain (Y371H, in 2 cases), SNP array analysis (Affymetrix, GeneChip) of these cases with mutated CBL gene disclosed 11q-acquired uniparental disomy in all cases, but not in cases with wild-type CBL. CBL mutation has not been reported in acute leukemia with 11q23 translocation/MLL gene rearrangement. To our knowledge, these are the first t-Leuk/MDS case and infant cases with 11q23 translocation/MLL rearrangement, suggesting that CBL is mutated in a unique subset of t-Leuk/MDS and infant leukemia which is considered to play a pathogenic role in the development of t-Leuk/MDS and infant leukemia. Further accumulation of cases with t-Leuk/MDS and infant leukemia having the CBL mutation is needed.

Disclosures:
No relevant conflicts of interest to declare.
Frequency of Leukemia Stem Cell Candidates Predicts Refractoriness to Conventional Chemotherapy and Adverse Clinical Outcome
Authors: Ran Dan , Taubert Isabel , Eckstein Volker , Bellos Frauke , Wuchter Patrick , Saffrich Rainer , Schubert Mario , Ho Anthony D. 
Published: October 2019

Abstract
Abstract 2160
We have shown that leukemia stem cells candidates (LSCC) can be prospectively identified by high activity of aldehyde dehydrogenase (ALDHbr) and expression of CD34 among the leukemia blasts from the marrow of patients with AML. In this study we have examined the relationship between the frequency of LSCC at diagnosis with persistence of leukemia blasts after induction chemotherapy as well as with long-term clinical outcome. Using single cell sorting, we have further separated subsets among the LSCC and correlated their individual functional properties with the respective marker constellation. The percentage of LSCC in 101 patients ranged from 0.01% to 12.90% with a median of 0.51%. Frequencies of LSCC among the leukemia

blasts at diagnosis correlated significantly with the persistence of leukemia after the first induction chemotherapy (n=79, Spearman R=0.7797, P<0.0001). During the observation period of 24 months, 21 of 60 patients with high levels of LSCC died as compared to 7 of 41 patients with low levels of LSCC (p=0.029). The overall survival (OS) probability for the patients with high levels of LSCC was significantly worse (p=0.05) than in those with low LSCC. Characterization of these LSCC at a single cell level showed that a varying proportion, i.e. 15% to 78% of their progeny cells demonstrated the same chromosomal aberrations as the original leukemia population, indicating the presence of residual normal HSC. Thus high frequencies of LSCC at the time of diagnosis predict persistence of leukemia blasts, failure to achieve CR within the first cycle and poor overall clinical outcome. Our prospective separation of LSCC permits precise characterization of the biological properties of the subsets of stem cell candidates in human AML.

Disclosures:
Ho: Genzyme: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees.
Bone Marrow Hypoplasia Induced by Conditional Knockout of the RNase III Domain of Dicer-1
Authors: Huang Tai-Chung , Yang Yi , Brayton Cory , Chou Wen-Chien , Pandey Akhilesh 
Published: October 2019

Abstract
Abstract 2226

Background:
MicroRNAs (miRs) play important roles in normal hematopoiesis as well as in hematopoietic malignancies. In generating mature miRs, Dicer-1, an RNase III in the cytosol, excises the hairpin-loop of precursor miRs and assembles mature miRs onto an RNA-Induced Silencing Complex. Among 27 exons of Dicer1, exons 21 and 22 comprise the RNase IIIa domain. Knockout of this domain was shown to be lethal in mouse embryos. In this study, we used tamoxifen to induce Cre-mediated exon deletion and our goal was to investigate the impact of Dicer1 knockout on hematopoiesis in adult mice.

Material and Methods:
Two C57BL/6 transgenic mice were crossed: ROSA26-CreERT2 and floxed Dicer1 exons 21 & 22. At the age of 8 week, three female mice were treated with tamoxifen (1 mg/day, Day 1 to 5) and two with placebo. On Day 8, mice were sacrificed for examination. DNA genotyping and qRT-PCR were used to confirm Dicer1 genomic deletion and to quantify the transcription copy number of Dicer1. Cardiocentesis was done for complete peripheral blood count. Femoral and tibial bone marrow, spleen and inguinal lymph nodes were evaluated by flow cytometry whereas formalin-perfused sternum and thymus were subjected to histopathological examination.

Results:
Two tamoxifen-treated mice presented with unkempt appearance, hunched posture and diarrhea from Day 7. Vehicle-treated control mice remained clinically normal. qRT-PCR showed lower copy number of Dicer1 exons 21 & 22 in tamoxifen-treated group than in control (0.09 vs. 1.00). In tamoxifen-treated group, the spleen weights were lower, white blood cell and platelet counts were lower and bone marrow histopathology revealed hypocellularity, with markedly reduced and immature myeloid elements, consistent with arrested or abrogated development of myelomonocytic and megakaryocytic elements.

Conclusions:
The RNase III domain of Dicer-1 is crucial for the normal differentiation of marrow myeloid series in adult mice, probably at the stage of common myeloid progenitor.

Disclosures:
No relevant conflicts of interest to declare.
A Microrna Feed-Forward Loop Amplifies GFI1N382S Transcriptional Reprogramming In Severe Congenital Neutropenia
Authors: Velu Chinavenmeni Subramani , Kazanjian Avedis , Colmenares Clemencia , Grimes H. Leighton 
Published: October 2019

Abstract
Abstract 2239
The Growth factor independent -1 (Gfi1) transcriptional repressor regulates both hematopoietic stem cell self renewal and myeloid differentiation. Humans with severe congenital neutropenia (SCN) display mutations in GFI1 that generate dominant negative acting proteins. Moreover, GFI1-mutant SCN patients and Gfi1-/- mice display a unique accumulation of myeloid progenitors. Recently we showed that Gfi1 regulation of HoxA9, Pbx1 and Meis1 underlies these phenomena, in that the Gfi1-Hox transcriptional circuit controls the accumulation of myeloid progenitors in vivo. We have also shown that Gfi1 regulates miR-21 during myelopoiesis, and that miR-21 is deregulated by Gfi1N382S expression. Our new data link these concepts by demonstrating that forced expression of miR-21 in bone marrow cells results in the accumulation of myeloid progenitors in transplant recipients. Moreover, miR-21 directly targets the Ski oncoprotein, and Ski-/- bone marrow cells show an accumulation of myeloid progenitors. Thus, Gfi1-/-, miR-21 overexpressing-, and Ski-/- myeloid progenitors accumulate in the marrow. Strikingly, Ski is dramatically reduced in miR-21 overexpressing Lin- bone marrow cells. Nearly undetectable Ski expression in Gfi1-/- bone marrow cells can be completely rescued by antagonizing miR-21 activity. Since Ski is a corepressor and Gfi1 is a transcriptional repressor, we next tested whether the two proteins physically interact. Indeed, endogenous Ski and Gfi1 can be coimmunoprecipitated. Synthetic Ski and Gfi1 proteins reveal that the interaction is mediated through Ski carboxy-terminal and Gfi1 zinc-finger domains. Chromatin immunoprecipitation reveals Ski and Gfi1 co-occupy several Gfi1 target genes (including HoxA9), which are derepressed upon Gfi1 or Ski knockdown. However, while Gfi1 binds and regulates the miR-21 gene, Ski is not bound to the miR-21 gene, and Ski knockdown has no effect upon miR-21 levels. Thus, the data point to a novel feed-forward transcriptional circuit. Gfi1N382S deregulation of miR-21 amplifies the dominant-negative effect of Gfi1N382S through miR-21 targeting of Ski, leading to further derepression of Gfi1-Ski target genes.

Disclosures:
No relevant conflicts of interest to declare.
Allogeneic Stem Cell Transplantation for Patients with Chronic Myeloid Leukemia After Prior Treatment with Nilotinib or Dasatinib
Authors: Schleuning Michael , Scholten Marijke , Biezen Anja van , Nagler Arnon , Apperley Jane F. , Michallet Mauricette , Grigg Andrew , Silver Richard T. , Leitner Armin , Gratwohl Alois , Ekblom Marja , Khoury H. Jean , de Witte Theo , Olavarría Eduardo 
Published: October 2019

Abstract
Abstract 2348
Stem cell transplantation (SCT) will continue to be a treatment option for patients with chronic myeloid leukemia, despite the introduction of tyrosine kinase inhibitors (TKI). However, many patients will have received prior therapy with TKI, including Nilotinib or Dasatinib at the time of allogeneic SCT. While the use of Imatinib prior to SCT seems to have no adverse impact on the outcome of allogeneic SCT little is known on the impact of prior use of second generation TKI. Therefore we conducted a retrospective registry study and identified 56 patients with CML who received an allotransplant after having been treated with Nilotinib and/or Dasatinib. Best responses to second generation TKI were major molecular response in 11%, complete cytogenetic response in 7%, partial cytogenetic response in 18%, complete hematologic remission in 25% and no response in 34%, respectively. At SCT, 37% of the patients were in accelerated or in blast phase, 36% in CP2 or higher and 27% in first chronic phase. Graft failure occurred in two patients. The median follow-up for surviving patients is 19 months. At 24 months the estimated non-relapse mortality was 33% and the relapse incidence 15%. Probability of survival is more than 85% at 2 years in patients transplanted in CP1. In univariate analysis there was a non significant trend in favor for pretreatment with Nilotinib as compared to the other groups. However, in multivariate analysis only stage of the disease was a predictor for survival. With respect to overall survival no significant differences could be identified for the following variables: patient age, donor type, stem cell source, intensity of the conditioning, time diagnosis to transplant, in or ex vivo T-cell depletion, response to treatment with second generation TKI. Patients transplanted in blast crisis had a significant higher risk of non relapse mortality. In summary, despite the shortcomings of a retrospective study, the data reported clearly show the feasibility and efficacy of allo SCT in patients pretreated with second generation TKI and it should be emphasized that the timing of allogeneic stem cell transplantation remains crucial to avoid unacceptable high treatment related mortality.

Disclosures:
Ekblom: Bristol-Myers Squibb: Honoraria.
Identification of Somatically Acquired Gene Mutations In the Kineome and Exome In CLL
Authors: Zhang Xiaosong , Li Meng , Ouillette Peter , Diaz Luis , Reis Marcelo , Velculescu Victor , Kinzler Kenneth , Malek Sami , Vogelstein Bert 
Published: October 2019

Abstract
Abstract 2410
The pathogenesis of CLL remains incompletely understood. While acquired chromosomal aberrations have been demonstrated to influence CLL biology and clinical behavior, it remains unclear what single gene defects other than p53 or ATM mutations contribute to or cause the CLL phenotype. In particular, recurrent gene mutations that are increasingly found in other hematological malignancies have not yet been identified in CLL. Further, the discovery of stereotypical immunoglobulin receptor genes in CLL has been advanced in support of the involvement of autoantigenes in the pathogenesis of CLL and thus a pro-proliferative or anti-apoptotic gene mutation may possibly not exist in CLL.
Given that kinase genes are often the targets for recurrent gene mutations and given the prospect for identification of drugable targets, we have analyzed 515 kinase genes in a discovery panel of 23 well characterized CLL samples (in DNA from CD19+ sorted cells) and tested their somatic nature in CD3+ cell-derived DNA from the same patients. A total of 8308 different exon sequences were analyzed. We identified six individual kinase gene mutations (WEE1, NEK1, BRAF, KDR, MAP4K3 and TRPM6), each occurring once in a separate CLL case as well as >1450 non-synonymous polymorphisms but no recurrent kinase gene mutations were identified. Interestingly, mutations in all these genes but NEK1 had previously been identified in cancer genome projects of cancers of the breast, colorectum, pancreas and glioblastome multiforme and WEE1, BRAF and KDR have established roles in cancer biology.
Next we used solution exon capture of genomic DNA followed by Solexa-based next gen exome sequencing on 8 paired CLL derived DNA samples (FACS-sorted CD19+ versus CD3+ paired samples), including 2 samples with known p53 mutations. Using stringent criteria for mutation calling, we identified 69 mutated genes including the 2 cases of p53 mutations. None of the mutated genes were recurrent. Work is in progress to estimate the frequency of mutations in these genes in a wider panel of CLL cases.
In summary, our data provide practical information about the mutations state of the CLL kineome and exome with implications for CLL biology/pathogenesis. This data should motivate additional exome sequence analyses of more CLL samples to fully capture and describe the mutation load in CLL.

Disclosures:
No relevant conflicts of interest to declare.
Re-Expression of the AML1/ETO Target Gene LAT2/NTAL/LAB Results In Direct Interference with Myeloid Differentiation In AML1/ETO-Positive Cells
Authors: Duque-Afonso Jesus , Essig Aitomi , Solari Leticia M , Berg Tobias , Pahl Heike L. , Lübbert Michael 
Published: October 2019

Abstract
Abstract 2474

Background:
The leukemia-specific oncofusion protein AML1/ETO regulates different target genes, including the LAT2 gene (encoding the adaptor molecule LAT2/NTAL/LAB), which is epigenetically repressed by AML1/ETO as we have previously described. LAT2 is phosphorylated by c-kit and has a role in mast cell and B cell activation. To address the functional role of LAT2 during myeloid differentiation, expression studies were performed in myeloid cell lines, and LAT2 was overexpressed by retroviral gene transfer in AML1/ETO-positive Kasumi-1 cells and AML1/ETO-negative U-937 cells.

Methods:
To induce monocytic and granulocytic differentiation, the myeloid cell lines U-937, HL-60 and NB4 were treated with PMA and ATRA, respectively, and LAT2 expression measured by both Northern and Western blot. LAT2 was overexpressed in Kasumi-1 and U-937 cells by use of the retroviral vector pMYSiG-IRES-GFP. Virus was produced in 293T cells and titrated in TE671 cells. Following transduction, GFP-positive cells were sorted by fluorescence-activated cell sorting (FACS). Transduced cells were treated with PMA (2 and 10 nM for 24 and 48 hours) and ATRA (0.1 μM and 0.5 μM for 48 and 96 hours), respectively.

Results:
The AML1/ETO-negative myeloid cell lines HL-60, NB4 and U-937 readily expressed LAT2, which was further upregulated by PMA, and transiently downregulated with ATRA. In the AML1/ETO-positive Kasumi-1 and SKNO-1 cells, LAT2 expression was absent. To address the functional role of this repression, forced expression of LAT2 was achieved in Kasumi-1 and U-937 cells and resulted in effective processing of LAT2 protein (confirmed by Western blot), and a decrease in the expression of the differentiation markers CD11b and CD11c (FACS analysis) in Kasumi-1 but not U-937, with only minor effects of LAT2 overexpression upon apoptosis and cell growth arrest. Notably, during both PMA- and ATRA-induced differentiation, a striking maturation block occurred in Kasumi-1 (measured by CD11b/CD11c expression, observed at different doses and time points of these treatments), while differentiation of U-937 cells was unimpaired by overexpression of LAT2.

Conclusions:
In AML1/ETO-negative leukemia cells, LAT2 expression is differentially regulated during monocytic and granulocytic differentiation. In AML1/ETO-positive leukemia cells, in which LAT2 is repressed, LAT2 re-expression imposes a striking maturation block. Graded expression of this novel AML1/ETO target gene may therefore play an important role in maintaining the phenotypic characteristics of this leukemia subtype.

Disclosures:
No relevant conflicts of interest to declare.