bluuebunny/crossref_metadata_2025_split huggingface dataset:
Understanding the Role of Mutations in SF3B1 and Splicing in Chronic Lymphocytic Leukemia
Authors: Wu Catherine J. 
Published: October 2019

Abstract
Abstract SCI-15
A major challenge in the treatment of chronic lymphocytic leukemia (CLL) is the tremendous clinical heterogeneity of this disease, ranging from indolent disease kinetics to a rapidly fatal course despite aggressive therapy. While some prognostic markers have been established, the advent of deep-sequencing technologies has enabled identification of most or all genetic alterations underlying malignancy, which is leading to new genetic prognostic markers across cancers. We recently sequenced the exomes of 91 CLLs with matched germline controls and identified nine recurrently mutated genes, six pathways involved in CLL pathogenesis (1), as well as new prognostic factors. From this study, a striking finding was the high frequency of mutations in SF3B1, part of the catalytic core of the spliceosome. We found that 14 independent SF3B1 mutations (15% of 91 CLL samples) localized to a single region of the gene (with unknown function) with 50 percent being identical K700E mutations. This finding has been since verified by other investigators. SF3B1 mutations were significantly associated with del(11q), a cytogenetic abnormality that is associated with aggressive disease, and whose minimally deleted region contains the critical DNA damage response gene ATM. Importantly, a Cox multivariate regression model identified mutated SF3B1 as an independent predictive marker of poor prognosis. Mechanistically, CLL samples harboring SF3B1 mutation demonstrated altered pre-mRNA splicing, with increased intron retention in known spliceosome target genes. Finally, SF3B1-K700E was also identified as a recurring mutation in myelodysplasia. In short, mutations in SF3B1 are positively selected in CLL, alter splicing patterns, and represent a novel oncogenic mechanism in hematologic malignancies. Our genetic and clinical evidence suggests that altered pre-mRNA splicing is a novel cancer pathway of importance to CLL and to blood malignancies in general. An ongoing focus of investigation in our group is trying to understand the CLL-promoting activity of SF3B1 mutation by linking alterations in transcripts with functional changes in B-cell biology. Precise identification of driver transcripts and their functional characterization will enable the development of novel therapeutics that can target this pathway more efficaciously and with minimal toxicity. This is especially important in the treatment of a clinically aggressive subgroup of CLL for which effective therapies are presently unavailable. Spliceosome targeting drugs have been identified—discovered through medicinal screens of natural products with tumoricidal properties, and only later found to target the SF3b complex—and may have promise for this subset of patients. Elucidation of this novel cancer pathway has broad implications for cancer biology, from the vantage of both understanding disease mechanism and the potential for therapeutic targeting.

Disclosures:
No relevant conflicts of interest to declare.
Links Between Defective DNA Strand Break Repair and Genome Instability in Fanconi Anemia
Authors: West Stephen C. 
Published: October 2019

Abstract
Abstract SCI-2
All living organisms feature DNA repair pathways that safeguard the integrity of the genome, and mutations in proteins that mediate key events in DNA repair have been linked to genome instability and tumorigenesis. Homologous recombination provides an important DNA repair pathway that is needed for the restoration and restart of broken replication forks, for the rejoining of chromosome/chromatid breaks, and for the processing of DNA cross-links. Mutations in genes that encode a variety of recombination proteins are linked to breast cancers and to heritable diseases such as Bloom syndrome (BS) and Fanconi anemia (FA). In recent work, we purified the BLM protein (defective in BS), the BRCA2 (FANCND1) and PALB2 (FANCN) tumor suppressors (mutated in some cases of FA), and the newly discovered FANCP protein, also known as SLX4, and have initiated structure-function analyses to elucidate their molecular functions. How these proteins process DNA, and how they are regulated and controlled to direct the outcome of recombinational repair is now revealing unexpected insights that extend our understanding of efficient DNA repair and tumor avoidance.

Disclosures:
No relevant conflicts of interest to declare.
The Gut: Role at Steady State and Variations in Disordered Conditions
Authors: Peyssonnaux Carole 
Published: October 2019

Abstract
Abstract SCI-23
As the human body cannot excrete excess iron, its absorption needs to be finely regulated at the intestinal level. Ferric iron (Fe3+) is reduced to ferrous iron (Fe2+) by brush border ferric reductases, including duodenal cytochrome b (DCYTB), before being transported across the apical membrane by divalent metal transporter 1 (DMT1), which is the principal iron importer. Depending on body iron requirements, iron can be either stored bound to ferritin or exported across the basolateral enterocyte membrane into the plasma by the sole iron exporter ferroportin (FPN). Iron absorption responds to systemic signals reflecting body iron requirements and local signals in the enterocyte. At the systemic level, hepcidin is the key circulating peptide hormone maintaining iron homeostasis. Hepcidin controls plasma iron concentration by inhibiting intestinal iron absorption and iron recycling by macrophages. Hepcidin acts by inhibiting cellular iron efflux through binding to and inducing the degradation of FPN. Hepcidin transcription is upregulated by iron repletion and downregulated by iron deficiency, ineffective erythropoiesis, and hypoxia. Hypoxia-inducible factors HIF-1 and HIF-2 are heterodimeric transcriptional factors and central mediators of cellular and systemic adaptation to hypoxia. In the presence of oxygen, the HIF-α subunit is targeted to the proteasome, while in hypoxia (or iron deficiency), HIF-α is stabilized and induces the transcription of target genes. We hypothesized that HIFs, stabilized in the hypoxic intestinal epithelium, may also play critical local roles in regulating intestinal iron absorption. We generated conditional knockout mice that lacked either Hif1a or Hif2a specifically in the intestinal epithelium and found that HIF-1α was not necessary for iron absorption, whereas HIF-2α played a crucial role in maintaining iron balance in the organism by directly regulating the transcription of the genes encoding DMT1 and DCYTB. Specific deletion of Hif2a led to a decrease in serum and liver iron levels. Alterations in HIF-2 at the intestinal level can override systemic regulation via hepcidin. Interestingly, we further demonstrated that HIF-2α contributes to iron hyperabsorption in a genetic mouse model of hereditary hemochromatosis (HH). HH is a genetic disorder characterized by abnormally low hepcidin expression and excessive iron accumulation in the liver and parenchyma. These findings suggest a prominent role of HIF-2 in the physiopathological regulation of intestinal iron absorption and may provide new therapeutic perspectives for the treatment of anemias and iron overload-associated disorders.

Disclosures:
No relevant conflicts of interest to declare.
Links Between DNA Damage and Metabolism, Pathways Causing Bone Marrow Failure in Fanconi Anemia, and Therapeutic Implications
Authors: Patel Ketan J. 
Published: October 2019

Abstract
Abstract SCI-3
Recent work from my lab has discovered that metabolism generates reactive aldehydes. These reactive molecules are potent damagers of DNA. The consequences of this are revealed by the inactivation of enzymes that detoxify these aldehydes and the Fanconi anemia DNA repair pathway in mice and vertebrate cell lines. The scientific session presentation will discuss this work and recent unpublished research on how natural aldehydes damage blood stem cells. This work has consequences for understanding how metabolism and ethanol exposure can be genotoxic, particularly in the vast population of Southeast Asians carrying a genetic defect in aldehyde catabolism (“pink flushers”). It is also relevant to the emergence of bone marrow failure and leukemia in Fanconi anemia.

Disclosures:
No relevant conflicts of interest to declare.
New α-Granule Biology Reveals How Platelets May Regulate Angiogenesis
Authors: Italiano Joseph E. , Battinelli Elisabeth M. 
Published: October 2019

Abstract
Abstract SCI-35
In addition to their “classic” role in hemostasis, platelets are now known to be major contributors in wound healing, tumor growth, and angiogenesis. Stored within the α-granules is an array of angiogenic regulatory proteins, which are deposited by the secretion reaction of surface-activated platelets into the local environment of a tumor or wound. Despite the recognized importance of platelets in regulating new blood vessel growth, our understanding of how platelets modulate the angiogenic response remains unclear. Here, we consider how new α-granule biology may provide insights into how platelets regulate angiogenesis. First, we will present data on a new form of α-granule movement. We will show that actin filament assembly can power the propulsion of α-granule movement, and this may be a novel mechanism underlying the secretion reaction. Second, we show that one mechanism by which anticoagulants may impact malignancy is by disrupting the tumor cells' ability to hijack the angiogenic potential of platelets. Third, we will review the molecular basis of transport and delivery of α-granules to assembling platelets. Finally, we will discuss the concept of using “designer platelets” as a drug delivery system for targeted agents.

Disclosures:
No relevant conflicts of interest to declare.
Decoding the Chromatin Code
Authors: Jones Peter A. 
Published: October 2019

Abstract
Abstract SCI-4
Epigenetic processes are reinforced by interactions between covalent chromatin marks such as DNA methylation, histone modifications, and variants thereof. These marks ultimately specify the locations of nucleosomes, particularly with respect to transcriptional start sites and other regulatory regions. Understanding how the epigenome functions, therefore, requires a coordinated approach in order to reveal the mechanisms by which the chemical modifications interact with nucleosomal remodeling machines to ensure epigenetic inheritance and control of gene expression. We have developed a new methodology to simultaneously map nucleosomal positioning and DNA methylation on individual molecules of DNA. We used this nucleosomal mapping technology to ascertain alterations in nucleosomal positioning during the abnormal silencing of genes by promoter hypermethylation. These experiments show that the methylation of CpG islands at the transcriptional start sites of key tumor-suppressor genes results in the stable placement of nucleosomes at the transcription start site. Treatment with 5-azanucleoside results in an immediate inhibition of DNA methylation and a sequence of downstream events that ultimately result in the eviction of the nucleosomes from the transcription start site and the activation of gene expression.

Disclosures:
Jones: Eli Lilly: Consultancy.
Metabolic Regulation of Hematopoietic Stem Cells During Stress
Authors: Suda Toshio 
Published: October 2019

Abstract
Abstract SCI-42
Tissue homeostasis over the life of an organism relies on both self-renewal and multipotent differentiation of stem cells. Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Adult HSCs are kept quiescent during the cell cycle in the endosteal niche of the bone marrow. Normal HSCs maintain intracellular hypoxia, stabilize the hypoxia-inducible factor-1a (HIF-1a) protein, and generate ATP by anaerobic metabolism. In HIF-1a deficiency, HSCs became metabolically aerobic, lost cell cycle quiescence, and finally became exhausted. An increased dose of HIF-1a protein in VHL-mutated HSCs and their progenitors induced cell cycle quiescence and accumulation of HSCs in the bone marrow (BM), which were not transplantable. This metabolic balance promotes HSC maintenance by limiting the production of reactive oxygen species (ROS), but leaves HSCs susceptible to changes in redox status (1). We have performed the metabolomic analysis in HSCs. Upregulation of pyruvate dehydrogenase kinases enhanced the glycolytic pathway, cell cycle quiescence, and stem cell capacity. Thus, HSCs directly utilize the hypoxic microenvironment to maintain their slow cell cycle by HIF-1a-dependent metabolism. Downregulation of mitochondrial metabolism might be reasonable, since it reduces ROS generation. On the other hand, at the time of BM transplantation, HSCs activate oxidative phosphorylation to acquire more ATP for proliferation. Autophagy also energizes HSCs by providing amino acids during transplantation. ATG (autophagy-related) 7 is essential for transplantation and metabolic homeostasis. The relationship between mitochondrial heat shock protein, mortalin, and metabolism in HSCs will also be discussed.

Disclosures:
No relevant conflicts of interest to declare.
Kinomic Profiling of Pediatric Acute Myeloid Leukemia for Detailed Cellular Insights
Authors: Mahmud Hasan , Bezrouzi Pariya , ter Elst Arja , Scherpen Frank J.G. , Kampen Kim R. , De Haas Valerie , Guryev Victor , Peppelenbosch Maikel P , Johannes Frank , Kornblau Steven M. , Bont Eveline 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) remains a life threatening malignancy in children. Considerable progress has been made in elucidating the new diagnostic and prognostic markers over the past decades. The precise etiology remains unclear. Therefore, it is essential to evaluate the activation of the components of cellular signaling pathways to understand AML signaling and to design the most successful approach for combinational therapies and new kinase inhibitors.
In this study, we used a high-throughput PepChipTMKinomics microarray system containing 976 different kinase substrates and assayed primary leukemic samples of 96 AML patients to produce an exceptionally detailed map of kinome enzymatic activities towards predefined peptide substrates. The generated profiles provide a comprehensive insight in signaling pathways active in AML patients.
As expected the activation of proteins belonging to MAPK signaling, PI3K/AKT signaling, cell cycle regulation, apoptosis and insulin signaling pathways along with the signaling receptors and immune system regulators were found. Unsupervised hierarchical cluster analysis separates the AML blast profiles based on 192 peptide activities into two clusters. Cumulative incidence of relapse (CIR) was significantly higher in the patients of cluster-2. Peptide activity patterns were independent of patient characteristics. In addition, with Gaussian network modeling, a total of 540 peptides (55%) showed at least one peptide-peptide association without a prior assumptions whereas 74 peptides (7.5%) had >39 nodes suggesting to be potential interesting signaling hubs. Among these 74 peptides, 10 peptides were identified in cluster-1 and 50 peptides were in cluster-2. Thus, this total analysis defined peptides correlated to low incidence for relapse, for examples AKT1, HGFR, RGS7 and to high incidence for relapse for instance, proteins involve in MAPK pathways (RAF1, RAC1,14-3-3 eta) and cell cycle regulation and cellular growth (c-Myc, FOXO3A, RBL1).
In conclusion, our study demonstrates the feasibility of peptide activity profiling to identify two active signaling network clusters in pediatric AML correlated to CIR. Highly correlated peptides belonging to cluster-2 provide stronger leads for selection of novel targets in future therapeutics.

Disclosures
No relevant conflicts of interest to declare.
Identification of a Dynamic Core Transcriptional Network in t(8;21) AML Regulating Differentiation Block and Self-Renewal
Authors: Ptasinska Anetta , Assi Salam , Martinez-Soria Natalia , Imperato Maria Rosaria , Piper Jason , Cauchy Pierre , Pickin Anna , James Sally , Hoogenkamp Maarten , Tenen Daniel G , Ott Sascha , Westhead David R , Cockerill Peter N , Heidenreich Olaf , Bonifer Constanze 
Published: October 2019

Abstract
One of the best-characterized chromosomal rearrangements found in AML is the t(8;21) translocation which accounts for approximately 10% of all AMLs and fuses the DNA-binding domain of the hematopoietic master regulator RUNX1 to the almost the entire ETO protein. The resulting RUNX1/ETO fusion protein lacks the transactivation domain of RUNX1 resulting in major differences in the biological activities of RUNX1 and RUNX1/ETO. RUNX1 normally recruits transcriptional activators and binds to DNA as a heterodimer with CBFb which is important for high-affinity DNA binding. The RUNX1/ETO fusion protein also interacts with CBFb but functions as a RUNX1/ETO tetramer, and like ETO itself, it also interacts with NCOR and SIN3A co-repressors. The leukemogenic fusion protein RUNX1/ETO forms a complex with other transcription factors and the binding of this complex to RUNX1 target genes causes a block in myeloid differentiation.
Here we investigate the dynamic changes in global transcription factor binding patterns that occur immediately following depletion of RUNX1/ETO. To that end, we combined digital footprinting, ChIP-sequencing for multiple factors and transcriptome analysis to identify the core transcriptional network of t(8;21) AML cells, and then characterized changes in these networks upon RUNX1/ETO knockdown. These analyses revealed a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes competing for the same genomic sites. We show by sequential ChIP that both complexes have similar transcription factor compositions, but differ in their preference for the recruitment of co-activators and co-repressors. Using a novel digital DNaseI footprinting approach we show that both t(8;21)-positive cell lines and patient-derived primary AML cells share the same pattern of binding site occupancy. Within this core transcriptional network, RUNX1/ETO-bound loci are predominantly associated with gene repression. Furthermore, loss of RUNX1/ETO establishes a new differentiation-associated transcriptional network dominated by de novo binding of C/EBPa resulting from the up-regulation of CEBPA gene expression. Our results demonstrate that the block in myeloid differentiation in t(8;21) is caused by the dynamic interference of RUNX1/ETO with cis-regulatory elements destined to bind alternate factor assemblies during myeloid differentiation including both RUNX1 and C/EBPa.

Disclosures
No relevant conflicts of interest to declare.
Haploidentical Hematopoietic Stem Cell Transplantation in Pediatric Patients: Comparison of Early Post-Transplant Outcome According to in Vitro Depletion Method
Authors: Im Ho Joon , Koh Kyung Nam , Suh Jin Kyung , Choi Eun Seok , Jang Seongsoo , Park Chan-Jeoung , Seo Jong Jin 
Published: October 2019

Abstract
Currently, haploidentical hematopoietic cell transplantation (HHCT) is considered an established option for patients who have diseases curable with HCT but who lack a suitable donor. We compared the early post-transplant outcomes of the two transplant groups using different in vitro depletion method.
Between July 2008 and June 2014, 49 pediatric patients underwent in vitro T cell-depleted HHCT. Of 49 patients, 28 received CD3-depleted stem cells (CD3-HHCT) and 21 received TCRαβ-depleted grafts (TCRαβ-HHCT). Among 28 patients of CD3-HHCT, nine had hematologic malignancy [HM, one with ALL in non-CR, six with AML (3 CR1, 2 CR2, 1 non-CR), and two with MDS-RCMD], 18 had non-malignant disease (one with Fanconi anemia, 16 with acquired SAA, and one with CDA), and one had refractory neuroblastoma. Among 21 patients of TCRαβ-HHCT, 16 had HM [five with ALL (2 CR1, 2 CR2, 1 CR3), five with AML (2 CR1, 2 CR2, 1 non-CR), one with mixed lineage leukemia in non-CR, one with MDS-RCC, two with JMML, and two with NHL (1 CR2, 1 CR3)], two had SAA, and three had solid tumors [two with RMS (1 CR2, 1 refractory), and one with Ewing sarcoma in CR3].
Of 28 patients who received CD3-HHCT, two patients failed to achieve primary engraftment, and five patients experienced graft rejection (GR) within 28 days post-transplant. No patients of TCRαβ-HHCT experienced graft failure (GF). Early GF (primary GF and GR) was more common in CD3-HHCT compared to TCRαβ-HHCT (P=0.011). The cumulative incidence of acute GVHD ≥ grade II was comparable between two groups (33.3% for CD3-HHCT and 27.0% for TCRαβ-HHCT). Extensive chronic GVHD occurred in three patients from CD3-HHCT and one from TCRαβ-HHCT. T and T4 cell counts at 2 months post-transplant were higher in TCRαβ-HHCT than that of CD3-HHCT (P=0.007 for T and P=0.074 for T4). In CD3-HHCT, four patients died of non-relapse causes (two of CMV disease, one of encephalopathy, and one of autoimmune hemolytic anemia) and one died of leukemia. In contrast, five patients from TCRαβ-HHCT died of disease but, none died of non-relapse cause.
Of 20 patients (18 from CD3-HHCT and two from TCRαβ-HHCT) with non-malignant disease, only one patient of CD3-HHCT died of TRM. As for 29 patients (10 from CD3-HHCT and 19 from TCRαβ-HHCT) with malignant disease, the EFS at 1 year for CD3-HHCT and TCRαβ-HHCT were 60.0% and 56.6%, respectively (P>0.05).
In this study, TCRαβ-depleted HHCT showed a better early post-transplant outcome in terms of engraftment, immune recovery, and NRM, compared to CD3-HHCT. However, further study is warranted to evaluate the efficacy of TCRαβ-HHCT in preventing relapse in advanced malignancy.

Disclosures
No relevant conflicts of interest to declare.