bluuebunny/crossref_metadata_2025_split huggingface dataset:
AK2 Regulates Hematopoietic Cell Survival, Proliferation and Differentiation Along the T-Cell and Neutrophil Lineages
Authors: Six Emmanuelle M. , Lagresle-Peyrou Chantal , Chappedelaine Corinne Demerens-de , Trimouillas Justine , Rieux-Laucat Frédéric , Cassinat Bruno , Chomienne Christine , Fischer Alain , Cavazzana-Calvo Marina 
Published: October 2019

Abstract
Abstract 1315
AK2 deficiency is responsible for Reticular Dysgenesis (RD), a rare form of human Severe Combined Immunodeficiency which is characterized by the absence of blood neutrophils and T lymphocytes. AK2 which is located in the mitochondria interspace, plays an important role in energy metabolism and in efficient energy transfer through conversion of ADP into ATP and AMP and regulation of cellular adenine nucleotides homeostasis.
We modelled the RD pathology using a lentiviral-mediated RNA interference strategy to knock-down AK2 expression. Human CD34+ progenitors invalidated for AK2 are deeply affected in their proliferative ability during the process of T-cell differentiation. Between 3 to 7 days after initiation of T-cell differentiation on the OP9-Delta1 stroma, we observed an increased cell apoptosis through disruption of the mitochondrial membrane potential, associated with a 2-fold decreased in the percentage of proliferative cells. In the presence of shAK2, the apparition of CD7+ T-cell precursors was profoundly reduced - as compared to the shControl - showing that the process of differentiation itself was affected.
We also demonstrated that AK2 knock-down inhibits neutrophil differentiation using both G-CSF-mediated differentiation of CD34+ progenitors and ATRA-mediated differentiation of the HL60 cell line. In the HL60 system, we showed that the survival defect induced by the shAK2 could be rescued by expression of the anti-apoptotic protein Bcl-2, while this latter was not sufficient to restore a normal differentiation process.
These data suggest a novel mechanism in which AK2 regulates not only the process of differentiation but also survival and proliferation of hematopoietic cell lineages.

Disclosures:
No relevant conflicts of interest to declare.
Mitochondrial Dysfunction As a Potential Source of Reactive Oxygen Species in Cellular Models of Shwachman-Diamond Syndrome
Authors: Henson Adrianna , Moore Joseph B , Liu Johnson M. , Ellis Steven 
Published: October 2019

Abstract
Abstract 1343
Shwachman-Diamond Syndrome (SDS) is an inherited bone marrow failure syndrome linked to defects in ribosome synthesis. The heterogeneous array of clinical findings associated with this disease state most commonly includes exocrine pancreas insufficiency, neutropenia, and metaphyseal chondroplasia. Patients also show a predisposition for progression to myelodysplastic syndromes and acute myelogenous leukemia. Mutations in the gene SBDS are known to be responsible for most cases of SDS. Initial studies of the yeast ortholog of SBDS, Sdo1, revealed that this family of proteins is involved in the maturation of 60S subunits. Other studies have suggested that SBDS is a multifunctional protein affecting processes other than ribosome synthesis. Most recently it has been shown that reactive oxygen species are dysregulated in TF-1 erythroleukemic cells depleted of SBDS leading to increased cell death (Pediatr Blood Cancer. 2010 Dec 1;55(6): 1138–44). In an effort to elucidate potential sources of increased reactive oxygen species we investigated mitochondrial function in yeast and human models of SDS. Yeast cells lacking Sdo1 fail to grow on media containing only respiratory carbon sources, indicative of a defect in mitochondrial energy metabolism. Related studies in human TF-1 cells revealed that cells depleted of SBDS exhibit reduced oxygen consumption relative to controls. Given that the largest producer of reactive oxygen species is the mitochondrial electron transport chain, perturbation of respiratory function in cells depleted of SBDS family members could be a potential source of elevated reactive oxygen species. To investigate the potential molecular mechanisms underlying these respiratory deficient phenotypes we carried out a proteomic analysis comparing yeast cells depleted of Sdo1 with controls. Our data reveal that cells lacking Sdo1 overexpress Por1, an ortholog of human VDAC1. VDAC1 is a voltage dependent anion channel of the mitochondrial outer membrane that is thought to be an essential component of the mitochondrial permeability pore. Both over and under expression of VDAC1 have been shown to disrupt mitochondrial function and lead to enhanced apoptosis. Current efforts are focused on possible changes in VDAC1 expression and the role they play in the respiratory deficient phenotype in human SDS models. These studies continue to shed further insight into the molecular mechanisms underlying SDS pathophysiology.

Disclosures:
No relevant conflicts of interest to declare.
Modulation of Anthracycline-Induced Cytotoxicity by Targeting the Prenylated Prenylome in Myeloid Leukemia Cells
Authors: Morgan Michael A , Onono Fredrick O , Spielmann Peter H , Subramanian Thangaiah , Scherr Michaela , Venturinin Letizia , Dallmann Iris , Ganser Arnold , Reuter Christoph W.M. 
Published: October 2019

Abstract
Abstract 1418

Background:
Deregulation of Ras/ERK signaling in myeloid leukemias makes this pathway an interesting target for drug development.

Design and Methods:
Myeloid leukemia cell lines (HL60, KG1a, THP-1, K562) were screened for idarubicin-induced apoptosis, cell cycle progression, cell-cycle dependent MAP kinase kinase (MEK-1/2) activation, and Top2 expression. Cell-cycle dependent activation of MEK/ERK signaling was blocked using farnesyltransferase inhibitor (FTI) BMS-214,662 and dual prenyltransferase inhibitor (DPI) L-778,123 to disrupt Ras signaling. Analysis of synergy was done by the method of Chou and Talalay using the CompuSyn program. Analysis of the prenylated proteome was performed using a tagging-via-substrate approach. Knock-down of K-Ras and Cdc42 protein levels was done by RNA interference (RNAi) using lentiviral shRNA constructs.

Results:
Idarubicin caused a G2/M cell-cycle arrest characterized by elevated diphosphorylated MEK-1/2 and Top2a expression levels. Although cells were most sensitive to idarubicin, DPI L-778,123 and FTI BMS-214,662 also potently inhibited leukemia cell growth with IC50 values in the micromolar and sub-micromolar range. The FTI/DPIs elicited distinct effects on Ras signaling, protein prenylation, cell cycling, Top2 expression and apoptosis. Combining these FTI/DPIs with idarubicin synergistically inhibited proliferation of leukemia cell lines (KG1a, THP-1 and K562), but the L-778,123+idarubicin combination exhibited synergistic growth inhibition over a greater range of drug concentrations. Both BMS-214,662 and L-778,123 effectively inhibited prenylation of the majority of larger molecular weight proteins (e.g. Lamins, NAP1L1, HDJ-2), and some smaller molecular weight proteins (e.g. H-Ras, N-Ras). Additionally, only DPI L-778,123 blocked prenylation of smaller molecular weight proteins such as K-Ras and Cdc42. Interestingly, combined FTI/DPI treatment synergistically inhibited cell proliferation, induced apoptosis and nearly completely blocked protein prenylation in all cell lines tested. However, RhoB and RhoC remained prenylated even upon combined FTI/DPI treatment. Inhibition of K-Ras expression by RNA interference or blockade of its post-translational prenylation led to increased BMS-214,662-induced apoptosis.

Conclusion:
Our results suggest that nearly complete inhibition of protein prenylation using an FTI+DPI combination is the most effective method to induce apoptosis and to block anthracycline induced activation of ERK signaling.

Disclosures:
Reuter: Sanofi: Honoraria; Amgen: Honoraria.
Homologous Recombination of Wild TYPE JAK2, A NOVEL EARLY STEP In the DEVELOPMENT of Myeloproliferative Neoplasm
Authors: Vilaine Mathias , Olcaydu Damla , Harutyunyan Ashot , Bergeman Jonathan , Mourad Tiab , Ramée Jean-François , Chen Jian-Min , Kralovics Robert , Hermouet Sylvie 
Published: October 2019

Abstract
Abstract 2805

Background:
Adequate expression and function of Jak2 in hematopoietic progenitors is critical for normal myelopoiesis. The JAK2 46/1 (GGCC) haplotype, a congenital particularity, predisposes to myeloproliferative neoplasm (MPN) both independently and through mutation of the JAK2 gene. The JAK2 V617F mutation and acquired homozygous status for JAK2 V617F are frequent in MPN. JAK2 V617F homozygosity is currently explained acquisition of the JAK2 V617F mutation followed by mitotic homologous recombination (HR) of JAK2 occurred between wild-type and mutant alleles, leading to uniparental disomy (UPD) of chromosome 9p (9pUPD). Here we report the cases of 2 PV patients (Na1061 and Na1253) with acquired homozygous status for the JAK2 46/1 haplotype yet their granulocytes carried <20% JAK2 V617F.

Aim:
To determine whether HR of JAK2 can precede the V617F mutation in MPN.

Methods:
Granulocyte DNA and CD3+ lymphocyte DNA were examined in parallel with qPCR assays specific for the wild type and 46/1 haplotypes using rs12343867, a JAK2 intron 14 marker, as well as 4 other single nucleotide polymorphisms (SNP) on chromosome 9p. 9pUPD clonality and length were determined using SNP array studies.

Results:
For both patients, lymphocytes were heterozygous for the 46/1 haplotype, confirming that granulocyte 46/1 homozygosity was acquired. Direct sequencing of the JAK2 and GNE genes and SNP array analyses revealed homologous recombination of part of the JAK2 gene (exons 6–19, patient Na1061) and of the complete 46/1 JAK2 haplotype (patient Na1253). Furthermore, for both patients, full length sequencing of JAK2 cDNA revealed no additional mutation. In both cases, HR of wild-type JAK2 was associated with growth advantage and high expression of recombined JAK2. For both patients, further SNP array analyses revealed partial 9pUPD concerning <30% cells, which correlated with %JAK2 V617F and was consistent with 9pUPD having occurred after JAK2 V617F (Figure 1).
The distortion of SNP allelic differences was higher at the telomeric end than in the centromeric region of chr. 9p. This indicated 2 distinct partial 9pUPDs for Na1061 and 1 partial 9pUPD for Na1253.

Conclusion:
Homologous recombination involving wild type JAK2 can precede JAK2 mutation and 9pUPD in MPN. Thus multiple paths and diverse alterations of the JAK2 gene can lead to MPN in individuals carrying the JAK2 GGCC haplotype. We propose a new model with JAK2 HR as early event, followed or not by JAK2 mutation, or/and JAK2 mutation(s) facilitating subsequent recombination resulting in 9pUPD and JAK2 V617F homozygosity.

Disclosures:
No relevant conflicts of interest to declare.
JAK2 V617F, PRMT5, and GATA-1 Form a Regulatory Loop That Controls Autophagy Via Effects on ATG3 Gene Expression
Authors: Harr Michael W , Zhao Xinyang , Klein Mary , Liu Fan , Hatlen Megan , Nimer Stephen 
Published: October 2019

Abstract
Abstract 2822
The JAK2 V617F mutation is the most prevalent genetic abnormality in Philadelphia chromosome negative myeloproliferative neoplasia. We recently discovered that JAK2 V617F phosphorylates and inactivates PRMT5, a type II arginine methyltransferase that promotes transcriptional repression. To evaluate the PRMT5 dependent and independent effects of JAK2 V617F, we performed microarray analysis of human erythroleukemia cells (homozygous for JAK2 V617F) treated with a JAK2 inhibitor or transduced with PRMT5 shRNA. We discovered 5 autophagy-related genes (ATG3, ATG7, ATG2B, ATG5, and ATG16L1) that were positively regulated by JAK2 V617F, but negatively regulated by PRMT5. PRMT5 was bound to the promoters of these genes and binding was enhanced when PRMT5 was re-activated by a JAK2 inhibitor. Sequence analysis of ATG promoters identified at least one potential GATA-1 binding site in each ATG gene. Yet, knock-down of GATA-1 decreased the expression of ATG3 (by approximately 50%), but not the other ATG genes. ChIP analysis identified binding of both GATA-1 and PRMT5 to the ATG3 promoter, which suggests that ATG3 is transcriptionally induced by GATA-1 when PRMT5 is inactivated by JAK2 V617F. But when PRMT5 was active, it was bound to the GATA-1 promoter and ATG3 expression was downregulated. As JAK2 V617F (but not wild type) induces ATG3 expression and autophagy in AML cells and ATG3 levels are elevated in CD34+ cells from polycythemia vera patients, we conclude that JAK2 V617F promotes autophagy by inactivating PRMT5 and promoting GATA-1-dependent transcription of ATG3. Targeting autophagy with JAK2 inhibitors or other agents may be efficacious in the treatment of myeloproliferative neoplasia.

Disclosures:
No relevant conflicts of interest to declare.
Targeting Bruton's Tyrosine Kinase As a Novel Approach to Inhibit Osteoclast Function in Multiple Myeloma
Authors: Eda Homare , Santo Loredana , Cirstea Diana D. , Pozzi Samantha , Canavese Miriam , Scullen Tyler A. , Vallet Sonia , Evans Erica , Singh Juswinder , Raje Noopur S. 
Published: October 2019

Abstract
Abstract 2882
Bone disease is a hallmark of multiple myeloma (MM) and targeting osteoclasts (OC) to alleviate bone destruction is a component of the standard of care for MM. The activation of Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases, regulates B-cell activation and development and plays an important role in antibody production. Interestingly, Btk activation also occurs downstream of RANK signaling in OCs and its activation stimulates essential calcium signaling which plays an important role in OC function. Given this dual role of BTK in both B cell activation and osteoclastogenesis, we studied its role in the context of multiple myeloma (MM). Accordingly, we examined the efficacy of a potent and specific inhibitor of Btk (AVL-292) in OCs derived from MM patient monocytes.
AVL-292 is a highly selective, covalent Btk inhibitor that potently silences Btk enzymatic activity (IC50 < 0.5nM) and inhibits primary B cell proliferation and activation (EC50 ∼ 10nM). The compound showed high selectivity towards Btk when tested in a broad kinase panel of 62 kinases and importantly did not show significant inhibition against other kinases involved in BCR signaling (Syk, Lyn). OC derived from MM patient monocytes were assayed with or without AVL-292 for OC maturation by TRAP staining and functional activity by resorptive pit formation assay. OC function was inhibited in the presence of AVL-292 as determined by a decrease in pit formation. To delineate the mechanism of action of AVL-292 against OC function, the RANK signaling proteins were detected by western blotting and intracellular Ca2+ concentration was measured by fluorescence. AVL-292 inhibited phosphorylation of the Btk substrate, PLC γ2 in OCs. This was associated with an inhibition of intracellular Ca2+ release by AVL-292 which otherwise increased with RANKL stimulation in OCs. Although AVL-292 did not demonstrate direct cytotoxicity or inhibition of proliferation of MM cells, ongoing studies are confirming its activity in the context of co-cultures with accessory cells like OCs. These data demonstrate that the novel BTK inhibitor AVL-292 inhibits OC function through inhibition of Ca2+ mobilization through RANK signaling. These results suggest inhibition of Btk with AVL-292 has therapeutic potential for the treatment of myeloma related bone disease.

Disclosures:
Evans: Avila Therapeutics: Employment, Equity Ownership. Singh:Avila Therapeutics: Employment, Equity Ownership. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.
Multiple Myeloma Sequencing Reveals Subclonality and Timing of Genetic Alterations
Authors: Lohr Jens , Stojanov Petar , Lawrence Michael S , Auclair Daniel , Carter Scott , Sougnez Carrie , Gould Joshua , Cruz-Gordillo Peter , Chapman Michael A , Getz Gad , Golub Todd R. 
Published: October 2019

Abstract
Abstract 2897
Multiple myeloma is considered to be a homogenous disease within a given patient, and current classification systems and treatment algorithms are based on this assumption. We have asked if there is genetic heterogeneity of multiple myeloma within a patient and if this heterogeneity can be quantified.
To address this question, we have used massively parallel whole exome and whole genome sequencing of tumors and matched normal controls of 64 patients with multiple myeloma. We present an analytic strategy to distinguish potential driver mutations based on their clonality. We demonstrate that in some patients there are many somatic mutations that are only present in a subclonal fraction of the malignant plasma cells, and the subclonal fraction comprises up to 50%. These mutations are therefore less likely to confer a selective clonal advantage and are less attractive therapeutic targets because they only affect a small fraction of the myeloma cells. As an example, we found KRAS to be one of the most prevalent mutated genes in multiple myeloma, and KRAS mutations are significantly more likely clonal than subclonal, while other mutations in other genes are predominantly subclonal.
We also used this approach to investigate how copy number variations are related to somatic mutations, i.e. to define the temporal sequence of these events. This question is particularly relevant for hyperdiploidy in multiple myeloma, since this is associated with trisomies of odd numbered chromosomes. However, these trisomies do not occur with the same frequency in all odd numbered chromosomes and some hyperdiploid samples are also associated with trisomies of various even numbered chromosomes. It is unclear if these trisomies occur as a single catastrophic event, or rather in a sequential fashion. By assuming a constant rate of somatic mutations and utilizing this rate as a “timer” for chromosomal duplications we demonstrate that trisomies of odd-numbered chromosomes appear to occur early in a distinct order, whereas trisomies of even-numbered chromosomes and chromosome 1q occur late.
Our analyses allow us to determine which somatic mutations occurred before chromosomal duplication and may therefore give insight in the time course of pathogenic genetic alterations in multiple myeloma. Our work may also play an important role in prioritizing somatic mutations for therapeutic targeting in multiple myeloma.

Disclosures:
No relevant conflicts of interest to declare.
CD8+ CD26++ Lymphocytes Are T Memory Cells with T Cell Repopulating Capacity and Are Depleted in the Course of Haematopoietic Progenitor Cell Aphaeresis
Authors: Hildebrandt Martin , Daemen Kerstin , Stevanovic-Meyer Maja , Falk Christine 
Published: October 2019

Abstract
Abstract 2989
The glycoprotein dipeptidyl peptidase IV (DPP IV, CD26) possesses an enzymatic activity which is crucial for the turnover of chemokines including CCL5/RANTES, CXCL12/SDF-1a, CCL8/MCP-2 and CCL11/Eotaxin. CD26 is primarily expressed on subsets of human lymphocytes as well as on murine splenocytes, thymocytes and decidual lymphocytes. A subpopulation of memory T cells shows a high surface expression of CD26 that has been shown to be related to resolved viral infections, production of interleukin-2, and tissue invasion. Furthermore, CD26 affects the turnover of adenosine, since human CD26 anchors adenosine deaminase (ADA) to the lymphocyte surface.
We had shown previously that, in the course of haematopoietic progenitor cell (HPC) aphaeresis, a distinct subset of CD8+CD26++ lymphocytes was enriched in the aphaeresis products to a varying extent. High numbers of these cells in HPC autografts were associated with a poor clinical outcome after autologous HPC transplantation. A phenotypic characterization of CD26++ cells was performed with samples of peripheral blood from healthy donors using multicolor flow cytometric analysis.
The characterization of CD26++/CD8+ cells identified these cells as a homogeneous population with a distinct T memory cell phenotype (CD45RO+, CD161++, IL18Ralpha++, CCR7−), consistent with a population of T cells with high rhodamine efflux, chemoresistance and T cell repopulating capacity described before. Conversely, gating of CD45RO+, CD161++, IL18Ralpha++, CCR7− cells yielded a population of cells that were exclusively CD8+ and CD26++.
We conclude that CD8+ CD26++ cells define T memory cells with chemoresistance and repopulating capacity as defined by the phenotype: CD45RO+ CD161++ IL18Ralpha++ CCR7−. The observation that the enrichment of these cells in the course of progenitor cell apheresis is associated with poor clinical outcome after PBSCT could be explained by a depletion of these cells from the peripheral blood which could be hazardous. Technical improvements of the apheresis procedure are feasible. Furthermore, the characterization of T memory cells with repopulating capacity as CD8+ CD26++ allows a simple approach for their isolation fur further processing. The quantification of CD8+ CD26++ cells has now been integrated into the routine immunomonitoring to further assess this facet of post-transplantation immune reconstitution and possible correlations with the clinical course after allogeneic stem cell transplantation.

Disclosures:
No relevant conflicts of interest to declare.