bluuebunny/crossref_metadata_2025 huggingface dataset:
Heme-Regulated eIF2α Kinase in Erythropoiesis and Oxidative Stress
Authors: Chen Jane-Jane
Published: October 2019

Abstract SCI-23

Besides serving as a prosthetic group for hemoglobin, heme also regulates translation by inhibiting the kinase activity of heme-regulated translational inhibitor (HRI). HRI is the only known protein kinase that senses the intracellular heme concentration and does so through its two heme-binding domains. HRI is activated in heme deficiency by autophosphorylation, and phosphorylates the α-subunit of eukaryotic initiation factor 2 (eIF2α), which impairs the recycling of eIF2 for further translational initiation and results in cessation of protein synthesis. In this manner, HRI serves as a feedback inhibitor and coordinates the translation of globin mRNAs with the intracellular heme concentration to ensure that globin proteins are not made in excess of the heme available for the formation of hemoglobin. Excess of globin chains or heme is cytotoxic. In the absence of HRI, uncontrolled protein synthesis in heme deficiency results in globin aggregation and precipitation in red blood cells and their precursors. HRI is responsible for the physiological adaptation that produces hypochromic, microcytic erythrocytes in iron deficiency. The expression of HRI was increased during late stages of erythropoiesis with higher expression in Ter119high erythroblasts and reticulocytes, correlating with the active synthesis and regulation of globins at these stages of erythroid differentiation. In addition to inhibiting global protein synthesis, the second important function of eIF2α phosphorylation is to reprogram translation and the subsequent transcription of genes required for stress response. In mammalian cells, translation of the transcription factor ATF4 mRNA is upregulated specifically by eIF2α phosphorylation via upstream open reading frames in the 5’UTR. Activation of the HRI-ATF4 stress response pathway in nucleated erythroid precursors is required for adaptation to acute and chronic oxidative stress. Furthermore, this HRI-dependent ATF4 pathway is also operative and necessary for erythroid differentiation, especially under stress conditions. In chronic iron deficiency, HRI is necessary for adaptive gene expression for erythroid differentiation as well as for iron heme and redox homeostasis. Beyond heme deficiency and oxidative stress, HRI is also activated by osmotic shock and heat shock. HRI deficiency in mice exacerbates erythropoietic protoporphyria and renders β-thalassemia intermedia embryonically lethal. Hri−/− mice also develop ineffective erythropoiesis during iron/heme deficiency and are severely compromised upon phenylhydrazine-induced acute hemolytic anemia. Thus, translational regulation by HRI plays a critical role in the manifestation of red cell diseases in mice and may be a significant modifier of such diseases in humans.

Disclosures:

No relevant conflicts of interest to declare.
HSC Transplantation for Hemoglobinopathies: Allogeneic or Autologous Gene-Modified HSC?
Authors: Cavazzana-Calvo Marina , Payen Emmanuel , Negre Olivier , Hacein-Bey-Abina Salima , Leboulch Philippe
Published: October 2019

Abstract SCI-46

The hematopoietic stem cell (HSC) transplantation pioneered by the Pesaro group is now applied wildly worldwide. The European Group for Blood and Marrow Transplantation (EBMT) has established the hemoglobinopathy registry, which now contains detailed epidemiological data on over 3000 patients. Today, allogeneic transplantation remains the only definitive curative therapy for thalassemia and other hemoglobinopathies. The development of iron chelation did not change this position. However, this has not settled the debate on how this curative but potentially lethal treatment stands vis-à-vis a medical noncurative therapy for adult and advanced disease patients. Nevertheless, despite the substantial improvements, a number of problems remain unsolved, such as the absence of an HLA genoidentical donor for all the diseased patients especially when they come from Outreach countries and the long-term morbidity due to HSCT (i.e., infectious complications, acute and chronic graft-versus-host disease, sterility). In this context and despite the unsolved debate, the development of gene therapy is highly justified. Sadelain and Leboulch pioneered the use of lentiviruses in human globin gene therapy, showing that anemia in mice affected with diseases mimicking the human pathological conditions can be abbreviated significantly by using lentiviruses containing a normal human β-globin gene. These pre-clinical studies set the basis for the current human clinical trial in Paris and for the upcoming trials. In our gene therapy trial, the human β-globin gene sequence has been modified by mutating a single amino acid at position 87 of the β-globin sequence. The major reason for using the b87 globin gene in lentiglobin in patients with β-thalassemia is that Hbb87 expression can easily be distinguished from that of normal Hb (HbA). In this trial, the patients received a full dose of Busulfex in the aim to obtain a myeloablation allowing the successful intake of gene modified autologous HSC. Two patients with thalassemia have been treated to date. One out of the two patients obtained a clinical benefit and is stably transfusion independent more than 3 years after the last transfusion. He currently has approximately 9 to 10 g percent of Hb in his blood, approximately one third being Hbb87, one third human HbF, and one third HbE. The patient is healthy up to now, with a full-time job. Integration site profile data will be discussed in detail during the meeting. Additional patients affected with thalassemia are currently being recruited in the Paris trial.

Disclosures:

No relevant conflicts of interest to declare.
Noncanonical Wnt5a Is Induced by HTLV-1 bZIP Factor, and Supports Proliferation and Migration of Adult T-Cell Leukemia Cell.
Authors: Yasunaga Jun-ichirou , Ma Guangyong , Fan Jun , Matsuoka Masao
Published: October 2019

Abstract 2405

Adult T-cell leukemia (ATL) is a fatal malignancy of CD4-positive T-cells caused by human T-cell leukemia virus type1 (HTLV-1). HTLV-1 bZIP factor (HBZ) is encoded in the minus strand of HTLV-1 provirus, and is consistently expressed in HTLV-1-infected cells. It has been shown that HBZ promotes proliferation of T-cells, and HBZ-expressing transgenic mice develop T-cell lymphomas and systemic inflammations, suggesting that HBZ has the critical roles in pathogenesis of HTLV-1. However, the molecular mechanisms of HBZ-induced oncogenesis have not been clarified yet. The Wnt signaling is a highly conserved cellular signaling pathway in eukaryotes and the Wnt ligands have been found to trigger multiple pathways of Wnt signaling. The most well-studied cascade is the Wnt/β-catenin pathway (also known as the canonical Wnt pathway), which is β-catenin-dependent and mainly controls cell differentiation, proliferation, and apoptosis. Aberrant activation of the canonical Wnt pathway has been linked to many cancers. In contrast, it is known that the noncanonical Wnt pathway antagonizes the canonical pathway, while a representative noncanonical Wnt ligand, Wnt5a, is suggested to have both oncogenic and tumor suppressive properties in several malignancies. The significance of those Wnt pathways in HTLV-1-associated oncogenesis is still obscure. In this study, we found that HBZ markedly suppressed the canonical Wnt/β-catenin pathway. As a mechanism of HBZ-mediated Wnt suppression, we found that HBZ targets LEF1 and TCF1, the key transcription factors of the pathway, and impairs its DNA-binding ability. In contrast, HTLV-1 Tax protein interacted with Dvl and DAPLE, which are the regulators of the canonical Wnt signaling, and activated the pathway. Indeed, the canonical Wnt activation is not observed in ATL cells, and enforced activation of β-catenin in ATL cells inhibited cellular proliferation, implying that activation of the canonical Wnt pathway might have suppressive effect on ATL cells. On the other hand, HBZ induces the transcription of the noncanonical Wnt5a by enhancing its promoter activity. In addition, Wnt5a transcription level in ATL cells is significantly higher than in HTLV-1-uninifected control cells. Knockdown of Wnt5a impairs both proliferation and migration of ATL cells, suggesting that HBZ-induced Wnt5a supports development and progression of ATL. Our results implicate novel roles of HBZ in ATL leukemogenesis through dysregulation of both the canonical and noncanonical Wnt pathways.

Disclosures:

No relevant conflicts of interest to declare.
Expression of CD56 Is an Independent Prognostic Factor to Predict Relapse in Acute Myeloid Leukemia with t(8;21): Results of Japan Adult Leukemia Study Group (JALSG) AML97 Protocol.
Authors: Iriyama Noriyoshi , Hatta Yoshihiro , Takeuchi Jin , Ogawa Yoshiaki , Ohtake Shigeki , Sakura Toru , Mitani Kinuko , Ishida Fumihiro , Takahashi Masatomo , Maeda Tomoya , Izumi Tohru , Sakamaki Hisashi , Miyawaki Shuichi , Honda Sumihisa , Miyazaki Yasushi , Taki Tomohiko , Taniwaki Masafumi , Naoe Tomoki
Published: October 2019

Abstract 2511

Background:

Although prognosis of acute myeloid leukemia (AML) with t(8;21) is better than other types of AML, outcome of the patients has not been satisfied. Previously, aberrant antigen expression has been reported as risk factor for AML with t(8;21). However, in the reported series, number of cases was not large enough and chemotherapy regimens were variable. We investigated the association of prognosis and several biomarkers including immunophenotype, WBC count, age, and performance status for large number of AML patients with t(8;21) uniformly treated in JALSG AML97 regimen.

Patients and Methods:

Seven hundred eighty-nine eligible AML patients were evaluated for the multicenter JALSG AML97 study. Adult patients with de novo AML except for APL, ages 15–64 years, were registered consecutively from 103 institutions that participated in JALSG from December 1997 to July 2001. One hundred forty-four patients with AML with t(8;21) were analyzed in this study with a median 1205 days of observation term from diagnosis. Complete remission (CR), relapse-free survival (RFS), and overall survival (OS) rates were analyzed by Fisher's exact test and log-rank test. Factors that would affect clinical outcome were analyzed by multivariate Cox proportional hazard regression model.

Results:

AML with t(8;21) frequently expressed CD19, CD34, and CD56 compared to other subtypes of AML. CD11b was rarely expressed. Expression of CD19 favorably affected on CR rate (96% in CD19 positive and 87% in negative patients, p<0.05). Univariate analysis showed WBC>20×109/L, CD19 negativity, and CD56 positivity were adverse factors for RFS. CD56 expression was the only independent adverse factor for RFS by multivariate analysis (73.7% in CD56 negative and 48.2% in CD56 positive patients at 3 yrs) although its expression did not affect on OS. There was no difference of age, sex, WBC count, presence or absence of Auer rod, performance status, or CD15 expression between CD56 positive and negative cases. Expression of CD19 was more common in CD56 negative patients (50% in CD56 negative and 30.6% in CD56 positive patients, p<0.05).

Conclusions:

We demonstrated that the expression of CD56 was a distinctive adverse factor in a large number of AML patients with t(8;21) treated with JALSG AML97 regimen. CD56 positive AML patients with t(8;21) are possible candidates for hematopoietic stem cell transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Preimplantation Genetic Diagnosis for Benign Hematological Disorders Combined with HLA Matching and Stem Cells Development.
Authors: Altarescu Gheona , Elstein Deborah , Zimran Ari , Geva Talia Eldar , Lahad Ephrat Levy , Renbaum Paul
Published: October 2019

Abstract 3167

Preimplantation genetic diagnosis (PGD) for molecular disorders involves development of disease- and family-specific protocols that allow simultaneous amplification of the mutation with multiple polymorphic markers in single cells. We present the development and results of PGD for 6 non-lethal hematological disorders: dyskeratosis congenita, Gaucher type 1, Fanconi C and A, hemophilia A, and beta thalassemia. For beta thalassemia and Fanconi A we performed combined PGD for the disease and HLA matching since each family had an affected child requiring bone marrow transplant. For each of the 6 diseases we developed a fluorescent PCR protocol that included the familial mutation and at least 6 informative markers surrounding the mutation. Mechanical biopsy technique was performed and a single cell of a 6-cell embryo was analyzed. For 17 families, 43 PGD cycles were performed by blastomere analysis. The efficiency of single cell amplification was 96%. 12 healthy children were born (and 3 on-going pregnancies). PGD for dyskeratosis congenita was performed in 2 families: one carrying the Arg 631Gln mutation and the other the Arg901Trp in the TERT gene. A total of 3 cycles were performed and 1 healthy child was born. Analysis for Gaucher type 1 disease was performed in 4 families (1 couple was carriers of both Tay-Sachs and Gaucher mutations) in which the offspring were predicted to have severe type 1 Gaucher involvement based on genotypes of the following mutations in the beta-glucocerebrosidase gene: IVS2+1 and IVS12+1, 84GG and R359Q, 84GG and R496H, N370S homozygous patient and 84GG carrier with 1 affected child requiring treatment. 13 PGD cycles were performed by blastomere biopsy and 6 healthy children were born (3 children to the same couple from different cycles). Mutant embryos were used for development of stem cell lines and 1 line (N370S/84GG) was achieved. PGD for Fanconi C and A was performed in 4 couples. For Fanconi A combined PGD for the disease and HLA matching was performed in 9 cycles; 2 healthy children were born and 1 on-going pregnancy. For Hemophilia A, 3 PGD cycles were performed for 2 families resulting in birth of 1 healthy boy. PGD for thalassemia beta was performed in 5 families with a total of 10 cycles, 5 of them combined with HLA matching; 1 healthy child was born. The overall pregnancy rate/embryo transfer was 34% (highest for Gaucher, 53%, lowest for beta thalassemia, 10%). We suggest that PGD is a safe and effective method for hematological disorders to prevent birth of an affected child. Combining analysis for the specific disease with HLA matching is useful for hematological diseases requiring bone marrow transplant. Mutant embryos can be developed in stem cell lines that are a valuable source for research.

Disclosures:

No relevant conflicts of interest to declare.
Whole Genome Sequencing Reveals Novel Recurring Somatic Mutations Affecting HUWE1 and DIAPH2 Genes in Multiple Myeloma
Authors: Tomasson Michael H. , Shen Dong , Hucthagowder Vishwanathan , Schierding William , Mullins Chelsea D. , Fiala Mark , Hall Ira M , Wallis John , Fulton Robert S. , Fulton Lucinda A. , Kulkarni Shashikant , Mardis Elaine R. , Wilson Richard K , Ley Timothy J. , DiPersio John F. , Maher Christopher A. , Vij Ravi , Ding Li
Published: October 2019

Abstract 320

Multiple myeloma (MM) is an incurable malignancy of antibody secreting plasma B-cells whose etiology is still poorly understood. We used whole genome sequencing (WGS) and deep-read capture validation to thoroughly characterize the mutation landscape in four newly diagnosed MM patients and further examined mutation recurrence in 89 additional MM patients. WGS cases were selected to be racially diverse and represent both hyperdiploid and non-hyperdiploid MM with otherwise “simple” karyotypes. All studies to date have used peripheral blood as controls, however, abnormal B cells and circulating tumor cells frequently contaminate the peripheral blood of MM patients and these studies may have missed the early genetic events potentially important in disease pathogenesis. We therefore chose to use matched skin samples as normal controls. The use of skin DNA controls and deep read count capture validation of single nucleotide variants (SNV) allowed us, for the first time, to demonstrate clear separation between normal and malignant cell populations. Our analysis pipeline detected both somatic SNVs and structural variants (SV, ie. translocations, deletions, insertions). In all four patients, we observed chromosomal translocations at the Ig heavy chain locus, VDJ recombination, and Ig locus somatic hypermuation. We found somatic mutations affecting the E3 ubiquitin ligase HUWE1 in 4% (4/94) of cases, as well as recurring deletions affecting the Rho pathway regulator DIAPH2 (recurrence data will be presented). RNA sequencing was preformed for 3 of the WGS patients to explore transcriptional effects of mutations observed. Statistical analysis identified significantly mutated genes, i.e. mutation hotspots, including ROBO2, BCL6 and cadherin/catenin genes. Genes involved in cell polarity, cell adhesion and axon guidance were affected in all four WGS patients. Chromosomal translocations present in all four WGS patients involved both known oncogenes (CCND1, MYC and MAFB) and putative oncogenes. A novel translocation implicated KCNT2, encoding a sodium-activated potassium channel, as a potential oncogene in MM. Recurrent point mutations were relatively rare in MM, suggesting that SVs are more common driver mutations and/or that SNVs in MM disrupt a diverse array of genes to affect key pathways. For genomic studies in MM moving forward, our results emphasize the importance of matched normal controls uncontaminated by tumor cells, and suggest that careful analysis of SVs be included to find novel oncogenes and important clinical correlations.

Disclosures:

No relevant conflicts of interest to declare.
Glia Maturation Factor-Gamma Regulates Macrophage Migration Through Control Recycling of alpha5beta1 Integrin in Endosome
Authors: Aerbajinai Wulin , Liu Lunhua , Chin Kyung , Rodgers Griffin P
Published: October 2019

Abstract 3273

Macrophages play an essential role in defending against invading pathogens by migrating to the sites of infection, removing apoptotic cells, and secreting inflammatory cytokines. Macrophage migration is a critical step in the regulation of immune response and is also associated with many chronic inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, atherosclerosis and cancer. Thus, understanding the mechanisms controlling macrophage migration within different environments is of paramount importance. Although it is clear that adhesion signaling via integrin receptors and the surrounding ECM play a significant role in regulating migration of macrophages to site of inflammation, the underlying cellular and molecular mechanisms responsible for these process is still not fully characterized. Defining the molecular circuits through which integrins regulate macrophage motility is therefore important for gaining a better understanding of macrophage function. Glia maturation factor gamma (GMFG), a novel ADF/cofilin superfamily protein that is predominantly expressed in inflammatory cells, has been implicated in regulating actin reorganization. We have previously demonstrated that GMFG plays a role in regulating neutrophil chemotaxis and migration. We now examine whether GMFG has similar effects on macrophage and the cellular mechanism for these effects by using small-interfering RNA technic to knockdown GMFG in human macrophage like THP-1cells. Knockdown of endogenous GMFG results in significantly reduced chemotactic migration toward fMLP and enhanced adhesion on fibronectin compared with control siRNA transfected cells. Flow cytometry analysis shows that GMFG-knockdown cells expresses significant amount of α5β1 integrin on the surface compared with control cells. Confocal microscopy analysis exhibited that GMFG was distributed throughout the cytosol and colocalized with the multiple endocytic compartment such as early endosome marker EEA1, Rab5, Rab4 and Rab11. Importantly, we found that most of the α5β1 integrins accumulated in clusters at the rear of the GMFG-knockdown cells, along with colocalization with endosome marker EEA1, rather than localization toward the leading edge in response to fMLP stimulation in control cells. These results suggest that impaired efficient α5β1 integrin recycling cause to increased adhesion, which is responsible for reduced macrophage migration on fibronectin in GMFG depletion macrophages. Accordingly, GMFG is required for proper trafficking of endosomal α5β1 integrin to the plasma membrane and is fundamental for integrin-mediated macrophage motility.

Disclosures:

No relevant conflicts of interest to declare.
Prophylactic Program in fxiii Deficient Patients From Iran
Authors: Naderi Majid , Eshghi Peyman , Karimi Mehran , Alizadeh Shaban , Dorgalaleh Akbar
Published: October 2019

Abstract 3376

Background:

Prophylaxis is the management strategy in FXIII deficient patients. Life-long prophylactic therapy by 10–20 U/kg FXIII (every 4–6 weeks) is recommended in patients with severe FXIII deficiency to prevent life-threatening spontaneous bleeding. In this study we evaluated of the efficacy and safety of prophylaxis program in a large study group consisting of 182 FXIII deficient Iranian patients. Material and methods: A total of 182 FXIII deficient patients (98 male and 84 female, mean age 26 years old) from south and south-east Iran were selected as a study group. Continual prophylaxis was done in all patients for periods of 6–168 months (mean 66.6 months). The patients received Fibrogamin-P (Dade Behring, Marburg, Germany) 10–20 U/kg every 4–6 weeks and before availability of this concentrate in Iran the prophylaxis was done by FFP 15–20 ml/kg every 20–30 days. All of patients were followed up for any bleeding diathesis and adverse effects during the prophylaxis period. Results: A total of 189 bleeding episodes including epistaxis, gum bleeding, bleeding after dental extraction, hemarthrosis, hematoma, hematuria, wound bleeding, menorrhagia and abortion were detected in 182 patients after prophylaxis program. Life-threatening intracranial bleeding was found in 34 patients before starting prophylaxis; however, there were no reports after prophylaxis. The bleeding episodes and bleeding score were significantly decreased after prophylaxis in compare to before starting prophylaxis (P<0.05) Discussion: The prophylaxis of FXIII deficiency by FXIII concentrate is seems clinical significance in prevention of spontaneous and life-threatening bleeding and also abortion in affected women. Also it is recommended to start prophylaxis in patients as soon as possible for prevention of life-threatening bleeding episodes.

Disclosures:

No relevant conflicts of interest to declare.
Anti-Platelet Factor 4/Heparin Antibodies in Patients with Gram Negative Bacteremia
Authors: Pongas Georgios , Dasgupta Swapan , Thiagarajan Perumal
Published: October 2019

Abstract 3391

Introduction

The anti-platelet factor 4(PF4)/heparin antibodies, arising as a result of previous heparin exposure, are causally related to the procoagulant state due to platelet and monocyte activation. Formation of these antibodies with subsequent thrombocytopenia or thrombosis has also been described in patients, who have not been previously exposed to heparin. The presence of anti-PF4/heparin antibodies in individuals correlates with the severity of periodontal disease, implying that their occurrence may be triggered by periodontal pathogens. In this study, we determined the presence of anti-PF4/heparin antibodies in gram-negative bacteremic patients in a hospital setting and propose a pathophysiologic mechanism of their presence.

Method

We developed an in house ELISA for quantifying anti-PF4/heparin antibodies using therapeutic heparin and PF4 isolated from platelets. We used serum from a patient with high optical density as a standard and assigned an unit of 100 arbitrarily to construct a standard curve. We tested the sera from gram negative bacteremic patients (n= 34) in the quantitative ELISA along with normal controls (n=10). We also developed an in house ELISA for studying cross reactivity between anti-PF4/heparin antibodies and lipopolysaccharide (LPS)/PF4. We tested the sera from patients (n=5) with heparin induced thrombocytopenia in this cross reactivity ELISA. To test the interaction of LPS with PF4, we labeled PF4 with Alexa488 and measured its binding to LPS by monitoring the changes in fluorescence emission spectrum following excitation at λ480.

Results

Patients with bacteremia had higher titers of antiPF4/heparin antibodies compared to normal controls (26.4 ± SD 33 units, N=34 versus 6.3 ± SD 2.38 units, N=10, P=0.032). Bacterial LPS interacted with alexa488-labeled PF4 in a concentration-dependent manner, as measured by the quenching of the excitation spectrum. Patients with ant-PF4/heparin antibodies also reacted with LPS/PF4 complex in ELISA. Prior absorption of serum with PF4/heparin complex coated on ELISA plates decreased the reactivity of the serum towards PF4/LPS complex (19–46%) in two out of the five patients tested suggesting some were cross-reaction between PF4/Heparin and PF4/LPS complex.

Conclusions

PF4 forms a complex with lipopolysaccharide and this complex is immunogenic. Antibodies to PF4/LPS complex can cross-react with PF4/heparin complex raising the possibility that these antibodies may be responsible for the detection of PF4/heparin in individuals never been exposed to heparin previously. These antibodies may also be at least partly responsible for increased thrombosis associated with infection.

Disclosures:

No relevant conflicts of interest to declare.
Mechanisms of Telomere Maintenance Dysfunction in B-Chronic Lymphocytic Leukemia Through CpG Island Methylation
Authors: Auchter Morgan , Medves Sandrine , Chambeau Laetitia , Gazzo Sophie , Moussay Etienne , Ammerlaan Wim , Morjani Hamid , Géli Vincent , Palissot Valérie , Berchem Guy J. , Salles Gilles , Wenner Thomas
Published: October 2019

Abstract 3489

Telomeres are a repetitive DNA sequences associated with a protein complex named shelterin that protect chromosome ends. Two types of mechanisms maintain telomere in cancer cells. The first involves telomerase an enzyme able to copy the telomeric motif that consists of three principal subunits, including the telomerase reverse transcriptase hTERT. The second, named ALT (Alternative Lengthening of Telomere), corresponds to the recombination between telomeres that involves notably a complex formed by the topoisomerase III alpha (hTopoIIIa), BLM, RMI1 and RMI2. Little is known about the involvement of the ALT mechanism in B-chronic lymphocytic leukemia (B-CLL). In fact this leukemic disease shows low telomerase activity, shelterin defect and telomeric dysfunction.

In an effort to characterize ALT cells from 31 B-CLL patients, we analyzed their telomere length and telomerase activity. B-CLL patients showed almost no hTERT transcript (detected in three cases), low telomerase activity (detected in 7 cases) and a telomere average size ranging from 3 to 10 kb. Moreover, a strong deregulation of genes encoding three shelterin proteins, TRF1, TRF2, Pot1, and an at least two fold downregulation of hTopoIIIa gene expression in 21 cases were observed, suggesting the presence of a telomere maintenance dysfunction affecting both mechanisms, telomerase dependent and ALT. CpG island methylation has been mapped for both promoters and if hTERT shows a disseminated methylation profile in 22 patients, for hTopoIIIα we identified nine CpG upstream the minimal promoter, being methylated in 19 of our 31 analyzed patients. We then performed luciferase experiments and we showed that methylation in this 9 CpG induced a strong inhibition of hTopoIIIa transcription. Finally we correlated telomere length and hTopoIIIa methylation status as we observed that 25.4% of the hTopoIIIa promoters were methylated in patients with shorter chromosomes and only 11.1 % were methylated in patients with longer telomeres (p<0.0025).

As nearly no telomerase activity have been detected in our patients and as downregulation of hTopoIIIa could increase recombination rate between sister chromatid, methylation of hTERT and hTopoIIIa promoter CpG islands may lead to telomere dysfunction and increased genetic instability in B-CLL.

Disclosures:

No relevant conflicts of interest to declare.
NF-κB Activation in Mesenchymal Stromal Cells Mediates Leukemia Cell Chemoresistance
Authors: Jacamo Rodrigo , Chen Ye , Wang Zhiqiang , Ma Wencai , Zhang Min , Battula Venkata L , Schober Wendy D. , Davis Richard E , Konopleva Marina , Andreeff Michael
Published: October 2019

Abstract 3518

Within the bone marrow (BM) microenvironment, BM mesenchymal stromal cells (BM-MSC) provide leukemia cells with a rich environment that serves as a sanctuary and protects them from chemotherapeutic agents. Interactions between leukemia cells and BM-MSC are thought to change the behavior of both stroma and leukemia cells resulting in an increased resistance to standard drugs. To discover interaction-induced changes in BM-MSCs that might promote microenvironment-mediated chemoresistance we used genome-wide gene expression profiling to study normal-donor BM-MSCs co-cultured with REH cells, a pre-B acute lymphoblastic leukemia (ALL) cell line. Upregulated genes in normal-donor BM-MSCs by co-culture included many cytokines and chemokines and implicated activation of NF-κB in BM-MSCs. Similar results were obtained by co-culturing normal-donor BM-MSCs with OCI-AML3 cells, an acute myeloid leukemia (AML) cell line. Increased expression of NF-κB target genes in BM-MSCs was also induced by co-culture with primary cells from ALL and AML patients, suggesting that NF-κB activation is a common consequence of leukemia-stroma interaction. Blocking canonical-pathway NF-κB activation by overexpressing a super-repressor form of IκBα in BM-MSC, significantly reduced stroma-mediated chemoresistance to vincristine (VCR) of leukemia cells in vitro and in vivo using an extramedullary bone marrow model. When small-molecule IKKβ inhibitors (MLN3316 and CDDO-Me) were used in co-cultures, blocking canonical-pathway NF-κB activation in ALL cell lines (REH or RS4;11) as well as BM-MSCs, the apoptotic effects of VCR on leukemia cells were further increased.

Conclusion:

results indicate that leukemia cells activate NF-κB in stromal cells and that such activation is in turn essential to promote survival of leukemia cells during chemotherapy. Targeting NF-κB in BM-MSC may ameliorate stroma-mediated chemoresistance and help in eliminating BM-resident leukemia cells.

Disclosures:

No relevant conflicts of interest to declare.
Role of DNA Methylation of the G0/G1 Switch Gene 2 (G0S2) in the Proliferation of Myeloid Leukemia Cells
Authors: Yamada Takeshi , Park Chun Shik , Shen Ye , Lacorazza H. Daniel
Published: October 2019

Abstract 3520

The G0/G1 switch gene 2 (G0S2) was originally identified in human blood mononuclear cells as an early response gene. We recently reported that G0S2 maintains quiescence in hematopoietic stem cells (Yamada et al., Plos One, 2012). In addition to hematopoietic stem cells, G0S2 also inhibits proliferation of lymphocytes and hematopoietic progenitor cells. Methylation of the G0S2 gene has been reported in lung, and head and neck cancer, suggesting that G0S2 has potential tumor suppressor function in solid tumors. However, the role of G0S2 in hematological malignancies has not been studied yet. To evaluate the impact of DNA demetylation of the G0S2 gene in human leukemia, we cultured a panel of myeloid (HEL, K562, HL-60, Kasumi) and lymphoid human leukemia cell lines (Jurkat, DND41, and H9) with 10 μM of 5-Azacytidine (5-Aza). Treatment with 5-Aza led to a significant increase of G0S2 expression in K562 cells (24.1-fold) and HL-60 cells (4.9-fold) and reduced proliferation. We show that retroviral overexpression of G0S2 in K562 cells caused inhibition of proliferation. Conversely, gene silencing of G0S2 in 5-Aza-treated K562 cells increased proliferation. Differentiation of K562 with hemin and HL-60 with all-trans retinoic acid was associated with an increase of G0S2 expression of 6.7-fold and 9.9-fold, respectively. Taken together, gain- and loss-of function studies revealed that G0S2 regulates cell proliferation and differentiation in human myeloid leukemia cells. Since G0S2 does not have a direct known function on the cell cycle, we hypothesized that G0S2 interacts with proteins involved in the control of cell proliferation. Mass spectrometric analysis of proteins pulled down with G0S2 in hematopoietic cells revealed that nucleolin, a molecule known to regulate ribosome biogenesis, physically interacts with G0S2 resulting in a perinuclear retention of nucleolin. This interaction was confirmed in K562 cells overexpressing G0S2 by reciprocal co-immunoprecipitation. This data suggest that silencing of G0S2 in leukemic cells prevents sequestration of nucleolin in the cytosol inhibiting its pro-proliferation functions. Collectively, our studies uncovered an important inhibitory role of G0S2 in the proliferation of leukemic cells, suggesting a possible tumor suppressor function in myeloid malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Activity of a Heptad of Transcription Factors Is Associated with Stem Cell Programs and Clinical Outcome in Acute Myeloid Leukaemia
Authors: Diffner Eva , Beck Dominik , Gudgin Emma , Thoms Julie , Knezevic Kathy , Pridans Clare , Foster Sam , Goode Debbie , Lim Wheng Khong , Boelen Lies , Metzeler Klaus H. , Micklem Gos , Bohlander Stefan K , Buske Christian , Burnett Alan K , Ottersbach Katrin , Vassiliou George , Olivier Jake , Wong Jason WH , Gottgens Berthold , Huntly Brian , Pimanda John E
Published: October 2019

Abstract 3525

Leukaemic transformation is driven by aberrant transcriptional programs often in combination with abnormal proliferative signalling. These programs operate in normal hematopoiesis where they are involved in hematopoietic stem cell (HSC) proliferation and maintenance. ERG is a component of normal and leukemic stem cell signatures and high ERG expression has been proposed as a risk factor for poor prognosis in acute myeloid leukemia (AML). However, mechanisms that underlie ERG expression in AML and how its expression relates to leukemic stemness are unknown. We report that ERG expression in AML is associated with activity of the ERG+85 stem cell enhancer (SCE) and a heptad of transcription factors that combinatorially regulate genes in normal HSCs. Gene expression signatures derived from ERG+85 stem cell enhancer (Fig A) and heptad activity (Fig B) predict clinical outcome in a cytogenetically normal cohort of AML (CN-AML) patients when ERG expression alone fails. The heptad signature is an independent risk factor for poor overall and event-free survival (Fig C). There were no long-term survivors amongst patients with a heptad signature, FLT3 mutations and wild-type NPM1 (Fig D) pointing to a hitherto unappreciated link between aberrant signaling and transcriptional mediators of hematopoietic stem cell identity. In two independent cohorts, the heptad signature was as closely associated with wild-type NPM1 AML as the HOX signature was with mutant NPM1 AML (Fig E–F) suggestive of a collective role for these transcription factors in mediating the leukemic signature in the former. Taken together, these results show that key transcriptional regulators cooperate in establishing stem cell signatures in leukemic cells and that the underlying spectrum of somatic mutations contributes to the development of these signatures and modulate their influence on clinical outcome.

Disclosures:

No relevant conflicts of interest to declare.
The Etiology of Pleural Effusions Develping During Dasatinib Therapy in CML Could Be Variable: Need for Awareness and Investigations
Authors: Saikia Tapan , Gore Adwaita , Matwankar Sumaiya , Patil Priyadarshini , Jain Reetu , Dhabhar Boman
Published: October 2019

Abstract 4456

Dasatinib is a highly effective oral TKI for CML at various stages of the disease. The most common non-hematologic side-effect is pleural effusion. The exact mechanism of this pathology remains to be fully elicited, although studies report it to be an immunological phenomenon. Current literature shows dasatinib alone to be the cause of such effusions. More recently, pulmonary hypertensions have been reported on dasatinib therapy.

However, in our cohort of CML patients receiving dasatinib as a first or second line therapy, other pathologies were documented. Grade 2/3 unilateral pleural effusions were detected in 3 patients.

Case 1.

A 31 yr old male with CML-CP was on first line dasatinib 100 mg OD and CCyR was documented at 3 months. At 6 month, he was detected to have grade 1/2 bilateral pleural effusion and was managed with drug interruption and oral dexamethasone with no significant reduction of fluid. CBC was normal. Dasatinib was continued. At one year, he lost cytognetic response with emergence of complex karyotype and medullary lymphoid blast crisis was confirmed. Pleural fluid showed lymphoblasts proven by cytology and immunophenotype. He received vincristine and prednisone followed by a change to imatinib. His disease progressed further and died at 2 years from diagnosis.

Case 2.

A 39 yr old male with molecular relapse and M244V Abl Kd mutation at 8 years since diagnosis and 6.5 yrs on imatinib 400–800 mg daily was offered dasatinib 100 mg OD. After 24 months, he developed grade2 left pleural effusion which responded poorly to drug interruption, therapeutic tapping and prednisone. A few months later he developed constitutional symptoms and CT chest, bronchoscopy/BAL proved to have left lung tuberculosis. Currently, he is responding favorably to anti-tubercular therapy. He was switched back to imatinib and remains in cytogenetic remission.

Case 3.

A 21 yr old male with cytogenetic non-responder to imatinib 800 mg daily was put on dasatinib 100 mg OD. An MMR was achieved. He developed left pleural effusion after 4 years of dasatinib. Extensive investigation for any specific pathology was not productive. He was put on empiric anti-tubercular therapy with no response. It is being treated as a dasatinib induced effusion.

Conclusions.

During dasatinib therapy, the causes of pleural effusions could be varied – drug related, extramedullary blast crisis or tubercular. Radiological, cytological and microbiological investigations could be indicated after careful clinical evaluation.

Disclosures:

Saikia: Bristol -Myers Squibb: Research Funding, Speakers Bureau.
Multiple Myeloma On Top of Chronic Myelogenous Leukemia
Authors: Chalhoub Elie , Meouchy Joseph , Dakhil Shaker R.
Published: October 2019

Abstract 4457

Background:

The occurrence of multiple myeloma (MM) in patients with chronic myelogenous leukemia (CML) is an extremely rare event. There are only 10 cases reported in literature, with only 5 of them treated with imatinib. The longest was for 65 months.

Case presentation:

A 70-year-old Caucasian female presented for follow-up on her chronic myelogenous leukemia (CML). She was diagnosed 8 years prior and had a Philadelphia chromosome positive on FISH and PCR. She had been treated with Imatinib for 72 months and initially responded well, but later developed pancytopenia and hematologic and cytogenetic progression of her CML despite dose adjustments. She was switched to Nilotinib 15 months prior with partial cytogenetic response, and persistence of her pancytopenia. Her symptoms were fatigue and weight loss. She denied bleeding, fever, or back pain. Her physical exam was unremarkable. Her labs showed hemoglobin of 8.5 g/dL, WBC 2.2 K/μL with 48% segmented and platelets count of 30 K/μL.

Bone marrow biopsy and aspirate showed 15–20% clustered monoclonal plasma cells and persistence of CML. Serum IgG was 2248 mg/dL with an IgG Kappa monoclonal peak on serum protein electrophoresis. A skeletal bone survey revealed no lytic lesions. Otherwise her laboratory findings were unremarkable except for a creatinine of 1.39 mg/dL and a β-2-microglobulin of 4.6 mg/L. She was diagnosed with multiple myeloma, and started on chemotherapy with bortezomib, liposomal doxorubicin, and dexamethasone. Nilotinib was held. After 4 cycles of chemotherapy, her repeat bone marrow biopsy showed partial remission of MM, and persistence of CML. Her blood counts are improving.

Discussion:

A PubMed review of literature shows 10 reported cases of MM occurrence in patients with CML (1974–2012). Only 5 of these cases have been on Imatinib, for a duration ranging between 7 and 65 months. There were no reports about other tyrosine kinase inhibitors (TKI). This is the first reported case of MM on top of CML while on Nilotinib, and after the longest duration on Imatinib.

Conclusion:

Tyrosine kinase inhibitors (TKI) constitute a revolution in the treatment of CML. With marked improvement of survival, patients are now prone to other malignancies. Reporting more cases will help understand the long term effects of TKIs.

Disclosures:

No relevant conflicts of interest to declare.
Characterization of TPO Kinetics within 60 Days of Allogeneic Hematopoietic Stem Cell Transplantation and Its Correlation with Megakaryocyte Ploidy Distribution
Authors: Zhang Xiao-hui , Fu Hai-xia , Liu Dai-Hong , Xu Lan-Ping , Liu Kai-Yan , Huang Xiao-Jun
Published: October 2019

Abstract 4463

Background:

Thrombocytopenia is a critical complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT), but its pathogenesis has remained obscure. Thrombopoietin (TPO) has been identified as a key cytokine for both megakaryogenesis and thrombopoiesis; however, the kinetics of TPO production after allo-HSCT has not been reported. This study characterized the kinetics of TPO and its correlation with the megakaryocyte ploidy distribution pattern within 60 days of allo-HSCT.

Methods and Results:

A total of 46 consecutive patients who underwent allo-HSCT from October 2008 to December 2008 were included in the study. The TPO levels and the ploidy distribution pattern of MKs were measured using ELISA and flow cytometric analysis, respectively. The results indicate that the TPO levels and the platelet counts followed opposite trends after allo-HSCT. Multivariate analysis indicate that endogenous TPO levels before allo-HSCT, and the number of transplanted CD34+ cells were significant predisposing factors for rapid platelet engraftment (P=0.010 and 0.007, respectively). Furthermore, we found a reduction of ploidy and an increase in immature MKs in patients with higher endogenous TPO levels (> 250 pg/ml) on day 60 after allo-HSCT. Moreover, lower TPO levels (≤250 pg/ml) on day 60 after allo-HSCT were associated with significantly improved 2-year OS (P=0.032) and reduced TRM (P=0.026).

Conclusion:

Our results suggest that increased endogenous TPO levels may not be sufficient after allo-HSCT, and that the decreased ability of TPO may account for the appearance of antibodies. The use of exogenous rhTPO may promote platelet recovery after allo-HSCT. However, these possibilities need to be examined further.

Disclosures:

No relevant conflicts of interest to declare.
Comparative Efficacy of Tandem Autologous Versus Autologous Followed by Allogeneic Hematopoietic Cell Transplantation in Patients with Multiple Myeloma: A Systematic Review and Meta-Analysis
Authors: Kharfan-Dabaja Mohamed A. , Hamadani Mehdi , Reljic Tea , Bensinger William I. , Djulbegovic Benjamin , Kumar Ambuj
Published: October 2019

Abstract 4519

Background:

Despite advances in our understanding of clinical, genetic, and molecular aspects of multiple myeloma (MM) coupled with availability of more effective therapies, it remains an incurable disease. Combining cytoreduction from high-dose (chemo- or chemoradio-) therapy with adoptive immunotherapy forms the basis of an autologous-allogeneic (auto-allo) hematopoietic cell transplantation (HCT) strategy. However, when an auto-allo HCT approach is compared to tandem autologous (auto-auto) HCT, conflicting results have been reported. Accordingly, we performed a systematic review of published studies comparing auto-auto HCT with auto-allo HCT in patients with newly diagnosed MM.

Methods:

A systematic search of MEDLINE thru Nov 5, 2011, and pertinent conference proceedings, was conducted. Included studies allocated newly diagnosed MM patients to auto-allo HCT if an HLA-matched sibling donor was available versus auto-auto if sibling donor was not available (biologic randomization). Independent, dual data extraction was performed. Pooling of data from similar outcomes was done using the random-effects model.

Results:

Our search identified 152 publications, of which five (manuscript=four, abstract=one) met inclusion criteria. The five included trials enrolled 1538 patients (auto-allo=565, auto-auto=973). At least a very good partial response was assessed in one study (522 patients) and did not differ among the treatment arms [risk ratio (RR) (95% CI) = 0.97 (0.87–1.09), p=0.66]; but complete remission, assessed in five studies (1130 patients), was higher in the auto-allo HCT arm [RR(95% CI) =1.65 (1.25–2.19), p=0.0005]. Event-free survival did not differ among auto-allo HCT group versus auto-auto HCT group on per-protocol analysis [hazard ratio (HR) (95% CI) = 0.78 (0.58–1.05)), p=0.11] of three trials (409 patients), or ITT analysis [HR(95% CI) = 0.83 (0.60–1.15), p=0.26] in three trials (1229 patients). Overall survival (OS) did not differ among these treatment arms whether analyzed on per-protocol [HR(95% CI) = 0.88 (0.33–2.35), p=0.79] in two trials (214 patients), or by ITT [HR(95% CI) = 0.80 (0.48–1.32), p=0.39] analysis in three trials (1229 patients). Non-relapse mortality (NRM) was worse with auto-allo HCT [RR(95%CI) = 3.55 (2.17–5.80), p<0.00001] in four trials (1047 patients).

Conclusion:

Despite higher CR rates, there is no apparent improvement in OS with auto-allo HCT; but this approach results in higher NRM in patients with newly diagnosed MM.

Disclosures:

No relevant conflicts of interest to declare.
Long-Term Follow-up Results Over 7 Years for Survival, and Safety with Imatinib Mesylate Accompany with Allogeneic Transplantation for Chronic Myeloid Leukemia
Authors: Wang Jun , Sun Aining , Depei Wu , Qiu Huiying , Tang XiaoWen
Published: October 2019

Abstract 4529

Objective:

To observe the efficacy and safety of imatinib mesylate (IM) accompany with allogeneic transplantation for chronic myeloid leukemia (CML).

Methods:

During the period from January 2003 to August 2011,we retrospectively observed 95 patients with CML receiving IM for a minimum of 4 months before allogeneic hematopoietic stem cell transplantation (HSCT). Patients with advanced CML received IM from 3 month after transplantation for 12 months.

Results:

Among 95 enrolled patients (CML-CP 76, CML-AP 10, CML-BP 9), types of transplantation: sib-matched HSCT 64, unrelated-HSCT 19, haplo-HSCT 12. For the whole patients, 7 year overall survival (OS) is 80.5%, and disease free survival (DFS) is 74.5%. Complete hematologic response (CHR) is 93.6%, complete cytogenetic response (CCR) is 84.5%, major cytogenetic response (MCR) is 60.3% at 7 year. For CML-CP1, OS is 83.2% and CML-AP/BC is 33.3% (P<0.05). Compared patients of advanced CML achieving CP2 after IM and with no CP2,the former has better results of CCR or MCR, OS and PFS (P<0.05). The total treatment related mortality (TRM) is 16.8%. Cox multivariate regression analysis of prognostic factors indicates that status of CML and severe acute graft-versus-host disease (aGVHD III-‡W) retain independent predictive value. No increase in rates of serious adverse events was observed with continuous use of IM for up to 7 years.

Conclusions:

For chronic myeloid leukemia, combining with imatinib mesylate and allogeneic transplantation is a good strategy, with favorable long-term follow-up results and acceptable TRM, especially for the patients with advanced CML.

Disclosures:

No relevant conflicts of interest to declare.