bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genome-Wide Mapping and Subtype Classification of Long Non-Coding RNA in Acute Myeloid Leukemia
Authors: Chen Rong , Malouf Gabriel G , Zhang Jianping , Huang Xuelin , Weinstein John N , Spano Jean-Philippe , Molldrem Jeffrey J. , Plunkett William , Su Xiaoping 
Published: November 2019

Abstract
Background: Most efforts to characterize acute myeloid leukemia (AML) have focused so far on genetic and epigenetic aberrations, which can ultimately lead to altered protein-coding gene function. The roles of long non-coding RNAs (lncRNAs), which orchestrate cell physiology and act as key regulators of the AML oncogenic state, remain uncharacterized globally.
Material and Methods: We performed a genomic analysis of GENCODE lncRNAs in 179 clinically annotated cases of de novo AML, using The Cancer Genome Atlas (TCGA) RNA-Seq data. In addition, we described global correlations between lncRNAs and the expression of cis- and trans-acting genes. We also established lncRNA-based subtype classification based on distinct signatures and then correlated that classification with fusion transcripts and genetic alterations.
Results: Using stringent criteria (RPKM ≥1 in at least 10% of AML), we identified 2,913 expressed lncRNAs and used an integrative analysis to predict those that are potential regulators of AML oncogenic state. The expression of 1,935 (66.4%) lncRNAs showed positive correlations with the mRNA expression of their neighboring genes, while only 14 (0.4%) of the lncRNAs showed negative correlations. Gene ontology analysis using GREAT revealed enrichment of cis-neighboring genes in the PML body gene set (p=8.2x10-7). Unsupervised clustering of lncRNA-based expression showed five robust molecular clusters (C1 to C5), which were highly correlated with the mRNA-based classification. Of those, three clusters (C1, C2 and C5) were tightly associated with recurrent fusion transcripts; cluster C1 (n=16) was composed exclusively of promyelocytic leukemias, while cluster C5 (n=45) was enriched for MLL-rearranged cases (24.4%), and cluster C2 (n=31) was enriched for MYH11-CBFB or RUNX1-RUNX1T1 (55.2%) rearranged cases. Importantly, cluster C4 (n=30), which includes cytogenetically normal leukemias, was highly enriched for NPM1 (p=2.3x10-11) and FLT3 (p=1.6x10-4) mutations; conversely, cluster C3 (n=53) was highly enriched for recurrent copy-number alterations as well as RUNX1 (p=0.001) and TP53 somatic mutations (p=0.004). We further discovered a core of 37 lncRNAs significantly associated with a MLL-signature and 16 lncRNAs with a NPM1-mutated signature.
Conclusion: This study describes the first genome-wide mapping and characterization of lncRNAs in AML and proposes a robust lncRNA-based classification. This classification may serve in defining core lncRNAs that orchestrate key oncogenic states in the different clinical subtypes.

Disclosures
No relevant conflicts of interest to declare.
Mutation in Retinoic X Receptor-γ Is a Possible Mechanism of All-Trans Retinoic Acid Resistance in Acute Promyelocytic Leukemia(APL): Identifying Genetic Changes Related to Drug Resistance in APL Using Whole Exome Sequencing
Authors: Youk Jeonghwan , Koh Youngil , Park Hyunkyung , Kim Dae-Yoon , Lee Chan-Soo , Lee Junglim , Kim Hyo Jung , Yoon Hwi-Joong , Ahn Kwang-Sung , Jung Jong-Sun , Kim Inho , Park Seonyang , Yoon Sung-Soo 
Published: November 2019

Abstract
Background
Acute promyelocytic leukemia (APL) has the best prognosis among acute myeloid leukemia (AML). However, a subset of APL patients is not cured with all-trans retinoic acid (ATRA) combined with anthracycline-based cytotoxic chemotherapy. Some mechanisms such as increased ATRA metabolism have been suggested to acquired resistance to ATRA. However genetic mechanism of ATRA resistance has not been characterized at all. Hence, in this study, we tried to reveal genetic alterations attributable to ATRA resistance.
Methods
First, we performed whole exome sequencing (WES) using DNA of three APL patients who showed resistance to ATRA based treatment. These included two patients who failed to achieve complete remission (CR) after induction chemotherapy, and one patient who experienced relapse after CR despite of consolidation treatment. DNA extracted from bone marrow at the time of diagnosis was used for analysis, while DNA extracted from saliva at the time of CR was used as germline control. Calling of single nucleotide variants (SNV) was performed using internal pipeline called Adiscan (http://www.syntekabio.com). Annotation was performed using Polyphen-2. SNV’s found by WES were validated by direct Sanger sequencing. The frequency of those validated SNV’s was defined in a separate APL cohort.
Results
We identified 34 somatic non-synonymous SNV’s in three patients. Polyphen-2 algorithm predicted 9 among 34 SNV’s to damage protein function. Sanger sequencing revealed 8 over 9 SNV’s to be validated. These validated SNV’s include RXRG M77R, N6AMT2 A78S, TXNDC15 D198E, B3GALTL A444T, RBBP8NL E182G, TNPO3 L173W, BHMT M185I and ADAMTS5 G85D. When these 8 SNV’s were genotyped in a separate cohort, none of these SNV’s was found in the APL cohort composed of 30 ATRA sensitive patients, suggesting these SNV’s would be truly related to ATRA resistance in APL. Especially, when a simulation using amber molecular dynamics was performed, we observed 1) Increase in hydrogen bonding, 2) Decreased helix folding structure, 3) Decreased energy state in RXRG mutant case.
Conclusions
WES identified eight SNV’s which were unique in ATRA resistant cases. Among those mutations, RXRG could be a promising nonsynonymous mutation that explains the genetic mechanism of ATRA resistance.

Disclosures
No relevant conflicts of interest to declare.
Somatic Mutational Landscape of AML with Inv(16) and t(8;21) Identifies Two Distinct Patterns in Relapse Tumors
Authors: Sood Raman B. , Hansen Nancy F , Donovan Frank X , Carrington Blake , Maskeri Baishali , Young Alice , Bucci Donna , Chandrasekharaappa Settara , Mullikin James C , Bloomfield Clara D. , Marcucci Guido , Liu Paul 
Published: November 2019

Abstract
Acute myeloid leukemia (AML) is a heterogeneous disease with a wide prognostic spectrum ranging from poor to good depending upon the underlying mutations and/or cytogenetic abnormalities. Although AMLs with inv(16)/t(16:16) or t(8,21), collectively referred to as core binding factor leukemias (CBF-AMLs), are classified as prognostically favorable, such patients often succumb to their disease following relapse after an initial response to cytarabine/anthracyclin-based treatment regimens. Thus, to develop successful treatment strategies, it is critical to understand the mechanisms leading to disease relapse and target them with novel therapeutic approaches. To pursue this goal, we applied genomic approaches (whole exome sequencing and single nucleotide polymorphism arrays) on DNA from samples collected at sequential time points (i.e., diagnosis, complete remission and relapse) in seven patients with inv(16) and six patients with t(8;21). We identified mutations in several previously identified AML driver genes, such as KIT, FLT3, DNMT3A, EZH2, SMC1A, SMC3, WT1 and NRAS. Three relapse samples showed mosaicism for monosomy/disomy of the region of chromosome 3 containing GATA2. Overall, our data revealed two distinct profiles that support different mechanisms of relapse: 1) diagnosis and relapse blasts harbor the same driver gene mutations, indicating the intrinsic resistance of the major clones present at diagnosis to treatment regimen used; 2) diagnosis and relapse tumors have different driver gene mutations, indicating disease clonal evolution possibly through treatment selective pressure. Furthermore, our data has identified previously unreported putative driver genes for AML. Among these, we identified same somatic variant (R222G) in DHX15, an RNA helicase involved in splicing, in two patients at diagnosis. The variant was also detected at relapse in one of these patients. Functional validation of the mechanistic roles of wild type and mutated DHX15 in hematopoiesis and leukemogenesis, respectively, is ongoing in in vitro and in vivo models.

Disclosures
No relevant conflicts of interest to declare.
Differences of Somatic Mutations and Gene Expression in Blasts of Transient Leukemia and Acute Myeloid Leukemia of Down Syndrome
Authors: Chen Jian , Yousif Fouad , Beck Timothy , McPherson John D , Hitzler Johann K. 
Published: November 2019

Abstract
Background: Transient leukemia (TL) occurs in 30% of newborns with Down syndrome (DS) and typically resolves spontaneously. Approximately 20% of infants with TL go on to develop acute myeloid leukemia of DS (DS-AML) within the first four years of life. The blasts of both TL and DS-AML harbor somatic mutations of GATA1 . The objective of this study was to identify additional genetic events, which associated with the progression of TL to DS-AML.
Methods: Leukemic blasts of TL, DS-AML and normal T lymphocytes were sorted from blood and bone marrow samples of five patients who successively developed both disorders. In addition, blasts of one patient with subsequent relapse of DS-AML were analyzed. Mutational spectrum and gene expression and were determined by exome sequencing and RNASeq (Illumina HiSeq2000). The presence of mutations, which were identified with this approach in DS-AML blasts, was examined by droplet digital PCR in TL blasts (BioRad QX200).
Results: Blasts of TL overall harbored fewer mutations than those of DS-AML. Mutations of cohesin and RAS pathway genes were identified in a subset of DS-AML but not TL. In the patient who developed a relapse, different cohesin gene mutations were detected at initial diagnosis of AML and relapse; a minor clone present at initial diagnosis of AML emerged as the predominant clone at relapse. Concordant somatic GATA1 mutations were present in both TL and DS-AML blasts derived from the same patient. In contrast, other genetic events, which were detected in DS-AML blasts by exome sequencing, were confirmed to be absent in TL (by droplet digital PCR). The majority of differentially expressed genes showed higher expression levels in blasts of TL compared to DS-AML. They included genes encoding chemokines and related to IL1 and TGFb signaling.
Conclusions: The pathogenic sequence starting with TL and culminating in AML is uniquely initiated in children with DS by somatic mutation of GATA1. In contrast, the events associated with the transformation of TL to DS-AML resemble progression factors also found in non-DS AML. These progression events were not detectable even in minor subclones of TL suggesting they are acquired after the onset of TL.
This research was supported by funding from the Canadian Cancer Society Research Institute and Ontario Institute for Cancer Research.

Disclosures
No relevant conflicts of interest to declare.
FASN Is Critical for Maintenance of NUP98 Fusion-Induced Leukemias
Authors: Shima Yutaka , Kitabayashi Issay 
Published: November 2019

Abstract
NUP98 is a component of the nuclear pore complex (NPC), which plays an important role in molecular traffic between the cytoplasm and the nucleus. The NUP98 gene is rearranged and fused to several partner genes, such as HOXA9 and DDX10, in acute myeloid leukemia and myelodysplastic syndromes and the leukemia with NUP98 rearrangement has a poor prognosis. The role of the NUP98 moiety of NUP98 fusion proteins is not well investigated. NUP98 has two Phe-Gly (FG) repeat domains, which are characteristic of the NPC proteins. To investigate the role of the NUP98 moiety of NUP98 fusion proteins, we purified the protein complexes formed with wild-type NUP98-HOXA9 and its mutants that lack the each FG repeat domain and looked for the proteins that interact with the NUP98 moiety. Mass spectrometry analysis identified that fatty acid synthase (FASN) interacted with the FG repeat domain of NUP98-HOXA9. These data suggest two possibilities. One is that NUP98-HOXA9 may affect the activity of FASN. The other is that FASN may affect activity of NUP98-HOXA9. To test the former possibility, we tested the effect of NUP98-HOXA9 on the activity of FASN in vitro, and we found that NUP98-HOXA9 reduced the FASN activity. FASN is known to be essential for cell growth. In fact, the colony formation of the NUP98-HOXA9-immortalized mouse myeloid stem/progenitor cells was inhibited by shRNA for FASN.　Because NUP98-HOXA9 reduced FASN activity, we hypothesized that FASN activity is low in NUP98 fusion gene-transformed cells and FASN inhibition selectively inhibits the colony formation of these cells. To test this hypothesis, we examined the effect of orlistat, which is the inhibitor of FASN on the colony formation. Orlistat strongly inhibited the colony formation of NUP98-HOXA9- and NUP98-DDX10-transformed cells, but it did not severely affect, the colony-forming activities of other fusion genes-transformed cells and normal progenitor cells. Other FASN inhibitors inhibited the colony formation of NUP98-HOXA9- and NUP98-DDX10-transformed cells. NUP98-HOXA9- and NUP98-DDX10 did not affect the expression level of FASN. The inhibition of FASN did not affect the expression of Hoxa genes and Meis1 gene, which are upregulated by NUP98 fusions. These results suggest that FASN activity is low but is essential for NUP98 fusion-mediated leukemia cells, and that FASN can be a therapeutic target for NUP98 fusion gene-mediated leukemia.

Disclosures
No relevant conflicts of interest to declare.
The Functional Interplay Between the t(9;22)-Associated Fusion Proteins BCR/ABL and ABL/BCR in Philadelphia Chromosome Positive Acute Lymphatic Leukemia
Authors: Mian Afsar , Rafiei Anahita , Oancea Claudia , Ottmann Oliver G. , Ruthardt Martin 
Published: November 2019

Abstract
The successful targeting of BCR/ABL by selective ABL-kinase inhibitors (AKI) such as Imatinib, Nilotinib, or Dasatinib alone is unable to eradicate the leukemic clone in Philadelphia chromosome positive (Ph+ ) leukemia. The t(9;22)(q34;q11) is a balanced translocation. Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210BCR/ABL and the m-bcr, encoding for the 185BCR/ABL fusion proteins, respectively. The constitutively activated BCR/ABL kinase is responsible for the leukemic transformation through an aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40ABL/BCR, present in 65% of patients with chronic myeloid leukemia (CML) and the p96ABL/BCR, detectable in 100% of patients with Ph+ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96ABL/BCR for the pathogenesis of Ph+ ALL. We co-expressed p96ABL/BCRand p185BCR/ ABL from a p2A peptide-linked multi-cistronic retroviral vector, which allows the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The co- expression of p96ABL/BCR enhanced the kinase activity and, as a consequence, the transformation potential of p185BCR/ABL in factor dependent progenitor cells and untransformed fibroblasts. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and primary Ph+ ALL patient-derived long-term cultures (PD-LTCs). Furthermore p96ABL/BCR negatively influenced the response to AKI in these models as shown by an increased response to AKI when p96ABL/BCR was down-regulated. Our in vitro and in vivo stem cell studies on murine fetal liver cells and adult HSCs revealed a functional hierarchy between p96ABL/BCR and p185BCR/ABL. In fact, p96ABL/BCR strongly increased stem cell capacity in replating efficiency and colony forming unit-spleen day 12 (CFU-S12) assays, whereas p185BCR/ABL showed no effect. In contrast co-expression of p96ABL/BCR and p185BCR/ABL increased significantly both serial replating potential and CFU-S12 colony formation as compared to p96ABL/BCR alone. In a syngeneic mouse model co-expression of p96ABL/BCR abolished the capacity of p185BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96ABL/BCR for the pathogenesis of Ph + ALL.

Disclosures
No relevant conflicts of interest to declare.
KLF4 Acts As a Tumor Suppressor in T-Acute Lymphoblastic Leukemia and Negatively Regulates Human T Cell Development and Homeostasis
Authors: Xu Bing , Li Peng 
Published: November 2019

Abstract
The transcription factor Kruppel-like factor 4 (KLF4) may induce tumorigenesis or suppress tumor growth in a tissue-dependent manner. We found that overexpression of KLF4 induced not only human acute T-acute lymphoblastic leukemia (T-ALL) cell lines but also primary samples from T-ALL patients to undergo apoptosis through the BCL2/BCLXL pathway in vitro. T cell-associated genes including BCL11B, GATA3, and TCF7 were negatively regulated by KLF4 overexpression. Especially, KLF4 induced SUMOylation and degradation of BCL11B. However, the KLF4-induced apoptosis in T-ALL was rescued by the in vivo microenvironment. Furthermore, the invasion capacity of T-ALL to hosts was compromised when KLF4 was overexpressed. In normal human T cells, the overexpression of KLF4 severely impaired T cell development at early stages, but the blockage of T cell development was resumed by restoration of GATA3 or ICN1. In summary, our data demonstrate that KLF4 acts as a tumor suppressor in malignant T cells and that downregulation of KLF4 may be a prerequisite for early human T cell development and homeostasis.

Disclosures
No relevant conflicts of interest to declare.
Loss of PTEN in Pediatric T-Cell Acute Lymphoblastic Leukemia Patients: Proteomic and Molecular Characterization
Authors: Bonaccorso Paola , Bugarin Cristina , Buracchi Chiara , Fazio Grazia , Paganin Maddalena , Basso Giuseppe , Biondi Andrea , Cazzaniga Giovanni , Lo Nigro Luca , Gaipa Giuseppe 
Published: November 2019

Abstract
Signaling networks such as the PI3K/Akt/PTEN/mTOR pathway play a role in the modulation of the aggressiveness of T-Cell Acute Lymphoblastic Leukemia (T-ALL), but the significance of an aberrant activation of this pathway is poorly investigated. The master regulator of PI3K-AKT signaling is the lipid phosphatase and tensin homolog (PTEN); decreased or absent PTEN expression or activity could be associated with a constitutive activation of PI3K/Akt/PTEN/mTOR signaling pathway in T-ALLs. We investigated PTEN Exon7 mutations in diagnostic DNA samples from 67 pediatric T-ALL enrolled in AIEOP ALL2000 and R2006 protocols according to methods by Bandapalli et al. (Haematologica, 2012). We also investigated the PI3K/Akt/PTEN/mTOR pathway using Western Blotting (WB) and Flow Cytometry (FC) in parallel. WB analysis was performed using standard RIPA buffer for protein extraction. FC analysis of protein expression was applied as previously described (Gaipa G et al, Leukemia 2008), and protein levels were measured as % of positive cells compared to isotype control. Positivity or negativity by WB was established by presence or absence of a protein band, while for FC a threshold for positivity was set at ≥ 1% of positive cells. PTEN Exon 7 mutations were identified in 11 out of 67 (16.4%) of patients. According to samples availability, PI3K/Akt/PTEN/mTOR pathway was studied in 9 out of 67 patients (3 PTEN Exon 7 mutated and 6 wild type). PTEN protein resulted completely absent in all three PTEN Exon 7 mutated patients, by contrast PTEN was expressed in all 6 PTEN exon 7 wild type patients (mean by FC 46.98% ± 28.58%). This finding was fully confirmed when WB was applied to the same samples. We did not observe any statistically significant differences in p4EBP1 or mTOR levels in Exon 7 mutated patients as compared to wild type. By contrast, PTEN Exon 7 mutated blasts showed lower phospho-S6 levels compared to PTEN Exon7 wild type patients (mean 3.0% of positive cells vs 32.0%, p=ns). In conclusion, our data show a frequency of PTEN Exon 7 mutations of 16.4%, in agreement with the report by Bandapalli et al. (17.3%). Interestingly, we observed a strong association between the PTEN Exon 7 mutation and the total absence of PTEN protein in the pediatric T-ALL patients studied (data not reported so far to our knowledge). Moreover, although the differences are not statistically significant, p-S6 expression resulted consistently lower in mutated patients as compared to wild type patients. If confirmed in a larger cohort of pediatric T-ALLs, our data could open new insights in the significance of PTEN Exon 7 mutation in pediatric T-ALL and its associated functional profile.

Disclosures
No relevant conflicts of interest to declare.
Identification of Minor Histocompatibility Antigens Based on the 1000 Genomes Project for Application in Therapeutic Dendritic Cell Vaccination
Authors: Oostvogels Rimke , Lokhorst Henk M. , van Elk Maureen , van den Oudenalder Kelly , Spierings Eric , Minnema Monique C. , Mutis Tuna , Spaapen Robbert 
Published: November 2019

Abstract
Minor histocompatibility antigens (mHags) are highly immunogenic, HLA-bound polymorphic peptides playing prominent roles in the development of the graft vs host disease (GvHD) as well as the therapeutic graft vs tumor (GvT) effect of allogeneic stem cell transplantation (allo-SCT). To date, a small subset of mHags has been identified as genuinely hematopoietic system-specific. This small number of conceptually GvT-associated mHags are currently being tested in various clinical trials for the feasibility and efficacy of inducing an exclusive GvT effect without GvHD in patients with relapsing haematological malignancies after allo-SCT. Nonetheless, a broader and timely application of mHags as therapeutic tools in allo-SCT, as well as the development of appropriate GvHD prevention strategies still requires more insights that can be acquired through the identification of a much larger set of mHags presented by various HLA molecules in different ethnic populations. Recently, the introduction of genome-wide association (GWAS)-based strategies, despite a number of drawbacks, significantly accelerated the identification of mHags. To develop a strategy that can overcome the drawbacks of these earlier developed strategies, we now integrated our previously developed GWAS methodology [1,2] with the comprehensive genomic databases from the 1000 Genomes Project [3]. We show that the data set of the 1000 Genomes Project is suitable to identify all theoretical possible mHags, hereby enabling us to delineate important yet unknown characteristics of mHags. Furthermore, we demonstrate the actual power of this novel approach by the rapid and unambiguous identification of the HLA-DP4 restricted mHag UTDP4-1, which -despite extensive efforts in the past 15 years- could not be identified using any of the previously developed biochemical, molecular biological or genetic strategies. The 1000 Genomes Project-based identification of mHags thus represents a very rapid, convenient and robust method for the identification of new mHags, that will not only improve the understanding of the yet unknown facets of mHags but may also significantly enlarge the arsenal of targets for immunotherapy in haematological cancers.
Spaapen et al., J.Exp.Med. 2008 Nov 24;205(12):2863-72. Oostvogels et al., Leukemia 2013 Mar;27(3):642-9. Abecasis et al., Nature 2012 Nov 1;491(7422):56-65.

Disclosures
Lokhorst: Celgene: Research Funding; J&J: Research Funding; Genmab: Research Funding. Mutis:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding.