bluuebunny/crossref_metadata_2025 huggingface dataset:
Complement Component 3 (C3) Is Transcriptionally Regulated By TWIST1
Authors: Cho Min Soon , Rupaimoole Rajesha , Sood Anil , Afshar-Kharghan Vahid
Published: October 2019

C3, a central component of the complement system, is a plasma protein that is synthesized in the liver. We have found that malignant epithelial cells are also able to synthesize and secrete C3. The regulation of expression of the C3 gene in malignant cells is not well understood. We found that C3 is highly expressed in various cancer cell lines (Cancer Cell Line Encyclopedia: CCLE) and cancerous human patient tissues (The Cancer Genome Atlas: TCGA). We then explored which transcription factors were responsible for regulating C3 gene expression in ovarian cancer cells. To determine C3 transcription factors, we performed a gene promoter analysis, which identified a TWIST1-binding consensus motif on the C3 gene promoter. The TWIST proteins (TWIST1 and TWIST2) are well known transcription factors are associated with more advanced, invasive and metastatic lesions. There is significant evidence suggesting that TWIST1 promotes tumor progression.

To investigate whether TWIST1 is a transcription factor of C3 in human ovarian cancer cells, we performed a chromatin immunoprecipitation (ChIP) analysis and confirmed TWIST1 binding at the C3 promoter (Figure 1A,1B and 1c). The C3 promoter binding affinity is 5.83-fold higher in Twist antibody used pull down compared to the IgG control (n=3, p=0.001, t-test, Figure 1C). To investigate the functional effect of TWIST1 binding to C3 promoter, we performed a Luciferase reporter gene analysis, showing that a mutant TWIST1 binding site on the C3 promoter decreases luciferase reporter gene activity (control vs. C3 vs. mC3=1 vs. 3.3 vs.1.8, n=3, p=0.01, t-test, Figure 1D and 1E).

Figure 1 C3 is a target gene of TWIST1. Figure 1. C3 is a target gene of TWIST1.

Next, we manipulated expression of the Twist1 gene in ovarian cancer cells and monitored its effect on C3 gene expression. Small interfering RNAs (siRNA) against Twist1 drastically reduced C3 expression (n=3, p=0.01, t-test, Figure 1F); on the other hand, transducing these cells with lentivirus containing Twist1 increased C3 expression (n=3, p=0.01, t-test, Figure 1G). Therefore, we conclude that there is a positive correlation between expression of C3 and TWIST1 in ovarian cancer cells.

Taken together, these data provide evidence that a novel transcription factor of C3, TWIST1, upregulates C3 expression in malignant cancer cells, leading to an increase in cell proliferative potential.

Disclosures:

No relevant conflicts of interest to declare.
Histone Deacetylase 6 (HDAC6) Influences T-Cell Activation and Survival: Implications For Cancer Immunotherapy
Authors: Laino Andressa Sodre , Woods David M , Cheng Fengdong , Wang Hongwei , Sotomayor Eduardo M.
Published: October 2019

The role of histone deacetylases (HDACs) as epigenetic regulators of immune function is becoming increasingly clear. Recently, the role of specific HDACs in orchestrating T-cell maturation, survival and function has begun to emerge, giving rationale to selective therapy to direct immune responses in different disease settings, including cancer. In particular, HDAC6 has recently been characterized as a negative regulator of regulatory T-cell suppressive activity (de Zoeten, Molecular and Cellular Biology, 2011). Here we report an expanded, novel role of HDAC6 in regulating T-cell survival and activation. First, the relative expression of the eleven classic HDACs was evaluated in resting and activated T-cells from mouse and human samples. It was found that the majority of HDACs decrease in expression following activation, including HDAC6. Next, in a HDAC6KO mouse model, it was found that T-cells lacking HDAC6 had skewed survival when compared to wild-type murine T-cells. This difference seems to be the result of an increased CD4+ T-cells population in the lymph nodes, with a concomitant decrease in viable CD8+ T-cells. To determine whether this population skewing was the consequence of defects in HDAC6KO mice T-cell development, wild-type murine T-cells were treated with an isotype-selective HDAC6 inhibitor. The results seen in HDAC6KO T-cells were recapitulated when wild-type T-cells were activated and treated with HDAC6 specific inhibitors, indicating a role of HDAC6 outside of thymic development in promoting CD4+ T-cell survival at the expense of CD8+ T-cells. Interestingly, it was found that activated CD4+ T-cells displayed decreased expression of the apoptosis signaling receptor FAS after HDAC6 inhibition while no differences were observed in CD8+ T-cells under the same conditions. In addition to these results implicating HDAC6 in regulating T-cell survival, expression of surface markers was altered in both CD8+ and CD4+ T-cells, including enhanced expression of the activation molecule CD69 in stimulated T-cells treated with an isotype-selective HDAC6 inhibitor. Finally, in vivo studies in tumor-bearing HDAC6KO mice revealed a significantly delayed in tumor progression. Similar results were observed in lymphoma-bearing mice treated with HDAC6 specific inhibitors. Taken together, this data shows that HDACs are dynamic in expression with regards to T-cell activation state. More specifically, we have unveiled hereto-unexplored roles of HDAC6 in regulating T-cell survival and function, pointing at this specific HDAC as an appealing target to harness T-cell immunity in hematologic malignancies.

Disclosures:

No relevant conflicts of interest to declare.
The Role Of Intraplatelet Reactive Oxygen Species In The Regulation Of Platelet Glycoprotein Ibα Ectodomain Shedding
Authors: Zhang Pingping , Du Juan , Zhao Lili , Wang Xiujuan , Zhang Yiwen , Yan Rong , Dai Jin , Liu Guanglei , Zhang Feng , Dai Kesheng
Published: October 2019

Background

Glycoprotein (GP) Ibα contains binding sites for von Willebrand factor (VWF), α-thrombin, P-selectin, and Mac-1 at the extracellular N-terminal 282 residues. Particularly, the interaction of GPIbα with VWF exposed at the injured vessel wall initiates platelet adhesion, and simultaneously triggers intracellular signaling leading to integrin activation and platelet thrombus formation. Therefore, GPIbα ectodomain shedding, which down-regulates the surface expression of the functional receptor and results in the generation of glycocalicin (GC), a soluble N-terminal fragment of GPIbα, has important implications for thrombosis and hemostasis. Stimulations of platelet with either physiological or chemical compounds result in GPIbα ectodomain shedding in vitro and in vivo. The generation of ROS has been observed in platelets stimulated with physiological or chemical compounds, such as collagen, thrombin, and thromboxane A2 analog U46619. In this paper, we plan to clarify the relationship between physiological stimulation-induced ROS generation and GPIbα shedding and the regulatory mechanism of ADAM17-mediated GPIbα shedding.

Methods

Washed platelets (3×108/ml) were pre-incubated with or without N-acetylcysteine (NAC) (10 mM), dithiothreitol (DTT) (3 mM), or vehicle control (DMSO) at RT for 15 minutes, and then were incubated with A23187 (5 μM), thrombin (1 U/ml), collagen (5 ug/ml), or dibucaine (1 mM) at 37 °C (or at RT for dibucaine) for different time. Western blot and flow cytometry was used to evaluate the GPIbα ectodomain shedding. ROS levels in platelets were examined using ROS assay kit according to the manufacture’s instruction.

Results and conclusions

Collagen, thrombin, and calcium ionophore A23187 induced ROS generation, and simultaneously incurred GPIbα ectodomain shedding. ROS scavengers NAC and DTT abolished not only collagen, thrombin, and A23187 induced ROS production, but also GPIbα ectodomain shedding. Interestingly, a recognized calpain activator, dibucaine, induced both ROS production and GPIbα shedding, which were also obviously reduced by NAC and DTT. Furthermore, calpain inhibitors calpain inhibitor I and carbobenzoxy-valinyl-phenylalaninal (MDL), obviously reduced dibucaine-induced ROS production, and inhibited ROS generations induced by A23187 and thrombin. These data indicate that ROS, regulated by calpain, plays a key role in collagen, thrombin, and A23187 induced GPIbα ectodomain shedding. These findings will help to understand the negative-regulatory mechanisms of platelet function, and may suggest a novel strategy to design new class of anti-platelet drug.

Disclosures:

No relevant conflicts of interest to declare.
Cooperative Binding Of Anti-Factor VIII Inhibitors and Induced Conformational Change Detected By Hydrogen-Deuterium Exchange Mass Spectrometry
Authors: Meeks Shannon L. , Sevy Alexander M. , Healey John F. , Deng Wei , Spiegel P. Clint , Li Renhao
Published: October 2019

The development of anti-factor VIII (fVIII) antibodies (inhibitors) is a significant complication in the management of patients with hemophilia A leading to significant increases in morbidity and treatment cost. Using a panel of anti-fVIII monoclonal antibodies to different epitopes on fVIII, we recently have shown that epitope specificity, inhibitor kinetics, and time to maximum inhibition are more important than inhibitor titer in predicting response to fVIII and the combination of fVIII and recombinant factor VIIa. Thus the ability to quickly map the epitope spectrum of patient plasma using a clinically feasible assay may fundamentally change how clinicians approach the treatment of high-titer inhibitor patients. To this end, we have characterized the binding epitopes of 4 monoclonal antibodies (MAbs) targeted against fVIII C2 domain by hydrogen-deuterium exchange coupled with liquid chromatography-mass spectrometry (HDX-MS). MAbs included both classical (inhibiting binding of fVIII to von Willebrand factor and phospholipid) and non-classical inhibitors (inhibiting activation of fVIII), which target separate regions of fVIII C2 domain and have distinct inhibitory mechanisms. HDX-MS analysis showed clear differences in binding patterns between classical and non-classical inhibitors, centering on the protruding hydrophobic loop at Met2199. The binding epitopes of classical and non-classical inhibitors mapped by HDX-MS agree well with previously reported ones characterized by structural studies and mutagenesis analysis. Classical and non-classical inhibitors could be distinguished by a limited subset of C2-derived peptides, simplifying analysis significantly. In addition, HDX-MS was able to detect subtle differences in binding patterns of various classical inhibitors, based on the HDX protection pattern around the hydrophobic loop at Leu2251. Interestingly, two MAbs, G99 and 3E6, exhibited an observable shift in HDX protection when bound to C2 as a ternary complex, as opposed to when bound individually, thus providing evidence for cooperative binding of these two MAbs (Figure 1). In summary, our results demonstrate the effectiveness and robustness of the HDX-MS method in the rapid epitope mapping of fVIII inhibitors. This method can be expanded to map epitopes of inhibitors against other domains of fVIII, potentially leading to a more personalized treatment of hemophilia A patients.Figure 1Figure 1.

Disclosures:

No relevant conflicts of interest to declare.
Use Of Novel Oral Anticoagulants For Treatment Of Venous Thromboembolism (VTE) In Patients With Malignancy: Meta-Analysis Of Randomized Controlled Trials
Authors: Jiang Bo , Malik Qasim , Crevecoeur Louis Romel
Published: October 2019

Use of Novel oral anticoagulants for treatment of venous thromboembolism (VTE) in patients with malignancy: meta-analysis of randomized controlled trials

Background

Venous thromboembolism (VTE) is a frequent complication of cancers. Patients with cancer have at least a 6 fold increased risk for VTE compared to those without cancer, and the diagnosis of VTE in cancer patient is associated with a 2-4 fold decreased survival during the first year. The mainstays of anticoagulant treatment in cancer patients remain unfractionated heparin, LMWH, and the Vitamin K antagonists (VKAs), with current guideline favoring the use of LMWH. Novel oral anticoagulants (NOAs) that directly inhibit factor Xa and thrombin, including dabigatran, rivaroxaban, and apixaban, represents a milestone in the prevention and treatment of VTE. However, there have been no clinical trials to test the efficacy of NOAs in cancer patients, therefore, use of these agents in cancer population is an extrapolation of published results with general population.

Objectives

Determine the efficacy of novel oral anticoagulants (thrombin inhibitor; dabigatran, and direct factor Xa inhibitors; rivaroxaban and apixaban) in VTE treatment in patients with cancer.

Data source

A systematic review was conducted using MEDLINE and EMBASE up to July 2013. Key words used included venous thromboembolism, pulmonary embolism, deep venous thrombosis, cancer, dabigatran, rivaroxaban, and apixaban. Due to the fact that no randomized controlled trials (RCTs) in cancer population, we combined data that were extracted from major RCTs that include cancer patients and performed a sub-group analysis.

Results

Three randomized controlled trials (RCTs) were reviewed, and a total of 550 patients with cancers were analyzed. NOAs are not inferior to the current standard anticoagulant therapy with enoxaparin followed by an adjusted-dose vitamin K antagonist, to prevent symptomatic recurrent venous thromboembolism. Odd ratio (OR) is 0.988 (95% CI, 0.51-1.94). There is no significant heterogeneity. The major bleeding events were not analyzed due to lack of sub-group data in the trials.

Conclusion

Cancer patients have different hemodynamics and unique features that make the treatment of VTE challenging. These includes: tumor-associated pro-coagulant, venous stasis and endothelial damage from chemotherapy and catheters, an increased risk of anticoagulant-related bleeding, and the complexity of anticoagulant control because of the occurrence of urgent procedures. The development of NOAs provided new modalities to treat VTEs in cancer patients, as it showed that NOAs is non-inferior to the current standard anticoagulant therapy. However, large scale randomized controlled trials specifically targeted cancer patients are needed.

Disclosures:

No relevant conflicts of interest to declare.
Longevity Promoting Interventions Inhibit Molecular and Functional Changes In Aging Hematopoietic Stem Cells
Authors: Yalcin Safak , Hu Wenhuo , Miller Richard A , Shin Yusup , Park Christopher Y.
Published: October 2019

Recent studies strongly suggest that aging results in the decreased regenerative capacity of adult somatic stem cells, but this process is poorly understood. Hematopoietic stem cell (HSC) aging is associated with myeloid-biased differentiation, reduced capacity for self-renewal, and reduced production of lymphoid and red blood cells. These functional changes lead to alterations in peripheral blood cell counts and reduction in adaptive immunity, as well as an increased incidence of myeloid malignancies. Previous studies have demonstrated that cell-intrinsic changes underlie HSC aging and persist through transplantation. MicroRNAs (miRNAs) are small non-coding RNAs that regulate HSC self-renewal and lineage specificity by suppressing the expression of hundreds of genes. Since rapamycin treatment has previously been shown to inhibit age-associated phenotypes in old HSCs, we hypothesized that investigating the expression and function of miRNAs in HSCs purified from mouse models of longevity may yield significant insights into the molecular mechanisms that underlie the phenotypic changes observed in the aging hematopoietic system.

Thus, we have characterized the cellular and functional alterations in aged HSCs after anti-aging interventions [rapamycin treatment and calorie restriction (CR)] in old mice versus age-matched non-treated controls. We found that anti-aging interventions significantly reduce the expansion of HSC numbers, restore erythroid and B-lymphocyte numbers, reverse age-related thrombocytosis in C57BL/6 mice, and reduce reactive oxygen species (ROS) levels specifically in the HSC compartment. Moreover, the diminished function of aged HSCs in vivo was restored by anti-aging interventions. To identify the transcriptional regulatory networks involved in age-associated functional HSC alterations, we performed miRNA expression profiling studies using highly purified HSCs from young, age-matched non-treated, and treated old mice (12 weeks old or 27 months old, n=5 per group). Analysis of age-associated miRNA expression changes revealed differential expression (p<0.05 compared with internal controls) of 30 in 768 (3.9%) mouse miRNAs tested. Compellingly, one third of these age-associated miRNA expression changes were inhibited by CR and rapamycin treatment. Thus, our findings provide evidence that anti-aging interventions also inhibit HSC aging, and that these effects are evident at the level of miRNA expression. Overall, these data provide a molecular view of HSC aging and identify numerous candidate genes that may regulate HSC aging.

Disclosures:

No relevant conflicts of interest to declare.
The Role Of tet2 DNA Methylcytosine Dioxygenase In Zebrafish Early Hematopoiesis
Authors: Lin Huan-Chau , Lim Ken-Hong , Chiang Yi-Hao , Wang Wei-Ting , Lin Ching-Sung , Chang Yu-Cheng , Hsieh Ruey-Kuen , Kuo Yuan-Yeh , Chou Wen-Chien
Published: October 2019

Loss-of-function mutations in Ten-Eleven-Translocation 2 (TET2) gene have been identified in various human myeloid and lymphoid malignancies. Recently, the TET gene family (TET1, TET2, and TET3) was found to function as DNA methylcytosine dioxygenase that is able to oxidize 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). In Tet2-deficient mouse models, Tet2 has been shown to play an important role in regulating self-renewal and differentiation of hematopoietic stem cells. These Tet2-deficient mice would gradually develop a chronic myeloid neoplasm resembling human chronic myelomonocytic leukemia suggesting that TET2 may function as a tumor suppressor. In the present study, we investigated the role of tet2 in zebrafish early hematopoiesis. During zebrafish early development, the expression of tet1, tet2, and tet3 by qRT-PCR can be detected mainly after the segmentation stage (26-somite), with fluctuated expression levels thereafter. Whole-mount in situ hybridization revealed that tet2 expression was strong over aorta-gonad-mesonephros region at 48 hours post-fertilization (hpf). Morpholino oligonucleotide (MO) knock-down of tet2 increased the expression of tet1, tet3, dnmt3aa, gata-1, alpha-Hb and fli1a (48 hpf) as well as rag2 and lck (4 days post-fertilization), and the expression of spi1b and mpo decreased (48 hpf). The expression of primitive hematopoietic stem cell markers scl and lmo2, as well as dnmt3ab, beta-Hb, l-plastin, and rag1 were unaffected. The levels of 5-mC and 5-hmC measured by ELISA were also decreased after MO knock-down of tet2. The number of gata-1 expressing red blood cells increased after tet2 MO knock-down as evaluated by flow-cytometry indicating that tet2 deficiency increased erythropoiesis. These preliminary results suggest that tet2 might play a role in the epigenetic regulation of zebrafish early hematopoiesis including erythropoiesis. Recently, transcription activator-like effector nuclease (TALEN) has been shown to generate targeted genomic editing in zebrafish. To validate our observation, we therefore utilized customized TALENs pair to generate tet2 knock-out zebrafish animal model. We designed a pair of TALENs targeting first exon of tet2 and our tet2 TALENs were able to generate insertion and/or deletion in targeted region of tet2 exon 1 in 25% to 44% zebrafish embryos. We obtained a total of fifteen different tet2 mutation genotypes F1 fish, and seven of them were predicted to cause early termination of transcription. The in-cross of these F1 genotypes matched the Mendelian inheritance. The tet2-/- knock-out F2 zebrafish is not embryonic lethal and can grow to sexually mature adult fish. The detailed analysis of tet2-/- knock-out zebrafish early hematopoiesis will be presented at the meeting.

Disclosures:

No relevant conflicts of interest to declare.
Role Of Long Pentraxin 3 (PTX3) In Wound Closure Induced By Bone Marrow-Derived Mesenchymal Stromal Cells
Authors: Cappuzzello Claudia , Doni Andrea , Dander Erica , Pasqualini Fabio , Nebuloni Manuela , Bottazzi Barbara , Mantovani Alberto , Biondi Andrea , Garlanda Cecilia , D'Amico Giovanna
Published: October 2019

Although several studies have shown the capacity of mesenchymal stromal cells (MSCs) to repair and regenerate different tissues, the mechanisms underlying these processes are not understood. Long Pentraxin 3 (PTX3) is a multifunctional protein produced by MSCs and other cell subsets upon activation with inflammatory cytokines. PTX3 is involved in innate immunity, inflammation and extracellular matrix deposition. In the present study we analyzed the potential role of PTX3 in wound repair process induced by MSCs.

PTX3 knockout MSCs (PTX3-/-MSCs) were collected from bone marrow of PTX3-/- mice. After 3-5 culture passages the expression of surface markers was analyzed by flow cytometry and their osteogenic and adipogenic differentiation capacity was detected by alizarin red O and oil red S staining, respectively. The ability of PTX3-/-MSCs to abrogate T cell proliferation was evaluated by co-culturing MSCs and PBMCs previously activated with Phytohaemagglutinin. Finally, equal number of both PTX3-/-MSCs and wild type (WT) MSCs were implanted into excisional wounds created by a biopsy punch on the back of allogenic WT and PTX3-/- mice. Wound area was measured up to 14 day and calculated using an image analysis program. The wound specimens were collected at 2, 7 and 14 days and processed for histological analysis.

We demonstrated that PTX3-/-MSCs, similarly to WT MSCs, displayed typical fibroblastoid morphology, they expressed common MSC markers and were able to differentiate into adipocytes and osteoblasts. In addition, they drastically decreased the mitogen-induced proliferation of lymphocyte. Importantly, in a mouse model of wound healing, PTX3-/- MSCs showed a highly significant defect in wound closure compared to WT MSCs at each time point. Histological evaluation of skin samples treated with PTX3-/- MSCs showed a reduction of the granulation tissue and a significant increase of neutrophils (GR-1+) in the wound bed. Moreover, wounds treated with PTX3-/- MSCs were characterized by an excessive accumulation of fibrin at the 2nd day after injury. Accordingly, PTX3-/- MSCs showed a defective ability to degrade the fibrin matrix in vitro. Finally, PTX3-/- MSCs failed to close the ulcers in PTX3-/- mice.

In conclusion, we demonstrated that PTX3 deficiency does not alter the phenotype and the capacity of MSCs to differentiate into mesengetic lineages; however, the production of PTX3 represents an essential requirement for MSC ability of enhancing tissue repair.

Disclosures:

No relevant conflicts of interest to declare.
Defective Nucleotide Metabolism Contributes To p53 Activation In Diamond-Blackfan Anemia
Authors: Danilova Nadia , Elena Bibikova , Covey Todd , Nathanson David , Dimitrova Elizabeth , Anne Lindgren , Glader Bertil , Radu Caius , Sakamoto Kathleen , Lin Shuo
Published: October 2019

Diamond-Blackfan Anemia (DBA) is a rare childhood bone marrow failure disorder, characterized by the presence of red cell aplasia, congenital abnormalities, and increased levels of adenosine deaminase (ADA). Haploinsufficiency of ribosomal proteins (RPs) due to mutations in RPS19 and RPL11 occurs approximately 25% and 5% of DBA patients, respectively. The pathogenesis of DBA has been associated with activation of p53, but the mechanism of how this leads to the erythroid defect in DBA patients is not well understood.

To understand the molecular pathways leading to DBA, we used previously published zebrafish models of RPS19 and RPL11 deficiency, in addition to primary human fetal liver CD34+ cells transduced with RPS19 shRNA to evaluate signaling pathways upstream of p53. One of the earliest responses to RP deficiency was upregulation of rrm1, a subunit of ribonucleotide reductase (RNR), responsible for de novosynthesis of dNTPs. Since ADA expression is increased in DBA and ADA is involved in nucleotide catabolism, we hypothesized that RP deficient zebrafish and primary human fetal liver CD34+ cells have defects in nucleotide metabolism, which result in replication stress and activation of p53 through the ATR/ATM/Chk1/2 pathways.

Several genes involved in de novo nucleotide synthesis, nucleotide catabolism, and purine salvage are upregulated in RPL11 and RPS19 deficient zebrafish, including rrm1 (2-7 fold), ada (2.8 fold), xdh (2.3 fold), and hprt1 (3-fold). In contrast, genes involved in pyrimidine salvage, such as tk1 and dck were downregulated in RP deficient zebrafish (0.4 and 0.8 fold, respectively). RP deficient zebrafish also had imbalanced dNTP pools, with increased dTTP and decreased dCTP levels compared to controls. To test whether these alterations in nucleotide metabolism lead to replication stress, we evaluated DNA damage and stress-induced proteins in primary human fetal liver CD34+ cells transduced with RPS19 shRNA and found increased phosphorylation of p53 (Ser15 and Ser37), ATM (Ser1981), 53BP1 (Ser1778), Chk1 (Ser345), and Chk2 (Thr68), indicating activation of the ATR/ATM/Chk1/2/p53 pathway in RP deficient cells. To rescue the RP-deficient zebrafish, we treated them with a mixture of exogenous nucleosides. Nucleoside treatment decreased p53 expression, restored rrm1, ada, tk1, and dck to normal levels, decreased number of apoptotic cells, and increased the number of Gata1-expressing cells in RP deficient zebrafish. Our data suggest that defective nucleotide metabolism contributes to p53 upregulation in DBA, and that nucleoside supplements alleviate replication stress and may prove beneficial for patients with DBA.

Disclosures:

No relevant conflicts of interest to declare.
Paroxysmal Nocturnal Hemoglobinuria and Thrombosis Before and After Eculizumab
Authors: Muñoz-Linares Cristina , Pastrana Miguel , Ojeda Emilio , Fores Rafael , Cabero Martin , Morillo Daniel , Bautista Guiomar , Navarro Belen , Krsnik Isabel , Gil Santiago , Regidor Carmen , de LaIglesia Almudena , Bueno Jose Luis , Garcia-Marco Jose A , Cabrera Jose Rafael
Published: October 2019

Thrombosis, mainly venous but also arterial, is the leading threat in Paroxysmal Nocturnal Hemoglobinuria (PNH) patients, caused by the continuous hemolysis. It constitutes the first cause of death in all reported series. Since 1964, a total of 56 patients with PNH clone were evaluated in our Hematology Unit. According with PNH Parker´s Classification, most of the patients were Classical type (28 patients), and the remaining included in the other subsets: 21 in the setting of another bone marrow failure syndrome (BMFS) and 7 subclinical.

Since November 2007, Eculizumab (an anti-C5 antibody) is employed in the disease to abrogate the hemolysis in our patients. In the last years, sixteen patients have been treated with this drug in our series. PNH patients previously anticoagulated with warfarin because thrombosis, continued on therapy after the addition of Eculizumab. Also, patients with more than 50 % PNH clone (established by Cytometry with FLAER on granulocytes) and platelets >50 x109/L received oral anticoagulation.

The incidence and localization of thrombotic events in the patients without Eculizumab was as follows:Parker’s ClassificationClassicalBMFSSubclinical28216Thrombosis cases (%)14 (50)4 (20)2 (33)Thrombotic episodes:2675Deep calf642Multiple cerebral ischemic infarcts212 (1 death)Large Cerebrovascular311Budd-Chiari11 (1 death)Portal2 (1 death)Retinal2Cava1Pulmonary thromboembolism2 (1 death)Myocardial infarction3 (1 death)Arterial ischemia2 (1 amputation)Skin ischemic2 (vasculitis, livedo reticularis)

After introduction of Eculizumab, sixteen patients have been treated with this drug and active thrombosis resolved in all cases, as was the case of a patient with a large persistent thrombosis in the inferior cava vein despite the isolated anticoagulation therapy. Only one patient on Eculizumab therapy experienced a thrombotic event and suffered a transient ischemic attack with aphasia after a prolonged catheter ablation procedure for an atrial fibrillation. This patient had previous signs of small vessel disease in MR imaging techniques. The episode occurs despite heparin anticoagulation and anticipated additional Eculizumab dose and resolves thereafter.

Eculizumab had a clear favourable impact in preventing thrombosis complications in our series of PNH patients. Careful monitoring of LDH levels and shortening the Eculizumab interval doses it is indicated in any chirurgical or invasive procedures in these patients.

Disclosures:

Pastrana: Alexion Pharmaceuticals: Speakers Bureau. Ojeda:Alexion Pharmaceuticals: Consultancy, Speakers Bureau.
Loss Of Tet2 Collaborates With AML1-ETO In Inducing Leukemogenesis
Authors: Rasmussen Kasper D , Jia Guangshuai , Johansen Jens V. , Bernard Olivier A. , Helin Kristian
Published: October 2019

Disruption of the epigenome can contribute to both cancer initiation and tumor progression. The methylcytosine dioxygenase TET2, which is involved in the regulation DNA methylation patterns, is frequently mutated in Acute Myeloid Leukemia (AML) patients and plays a central role in normal hematopoiesis. However, Tet2–deficient mouse models do not readily develop acute leukemia suggesting that additional oncogenic events are necessary for full-blown transformation.

The translocation t(8;21)(q22;q22) results in expression of the AML1-ETO (AE) fusion protein and is present in ∼5-10% of AML patients. Interestingly, the combination of t(8;21)(q22;q22) and TET2 mutations, although rare, have been observed in several studies in patients groups covering both pediatric and adult de novo AML. To confirm a direct role of TET2 mutations in the development of t(8;21)(q22;q22)-rearranged leukemias we expressed the AE fusion protein in hematopoietic stem and progenitor cells (HSPC) isolated from WT or Tet2-deficient animals. Serial replating assays in semisolid media as well as growth in liquid culture suggested a cooperative effect of AE and Tet2 deficiency as indicated by a significant increase in proliferation and colony formation efficiency. We then used a bone marrow transplantation model to assess the in vivo leukemic potential of AE;Tet2-/- HSPCs. The majority of recipient mice transplanted with AE;Tet2-/- (n=5/6) HSPCs succumbed to an aggressive and transplantable AML-like condition with a median latency of 5-6 months, whereas mice receiving AE HSPCs remained healthy over a 12 month observation period (n=6/6). The morbid mice showed many signs of disease including loss of weight, anemia, splenomegaly and large number of leukemic blasts (CD45neg) in circulation. Further analysis of the bone marrow and spleen revealed a massive expansion of a linneg (CD3e, Gr-1, Mac-1, B220, Ter119), c-Kitlow-high, CD16/32pos cell population indicating a block of differentiation and expansion of an immature myeloid progenitor population from AE:Tet2-/- HSPCs. To investigate how Tet2-deficiency contributes to transformation of t(8;21)(q22;q22)-rearranged cells we generated and cultured pre-leukemic AE-transduced cells in vitro from Tet2fl/fl HPSCs harboring the inducible Rosa26-CreERT2 allele. Genome-wide analysis of methylation patterns and gene expression showed that induction of Tet2-deficiency in pre-leukemic cells leads to progressive hypermethylation of gene-regulatory elements and altered expression of several genes implicated in tumorigenesis. Thus, this study illustrates how aberrant DNA methylation patterns can contribute to disease and confirm the role of Tet2 as a tumor suppressor preventing leukemogenesis.

Disclosures:

Helin: EpiTherapeutics : Consultancy, Equity Ownership.
A Genetic Screen In Zebrafish Identified Dlst As a Potential Therapeutic Target For Human Acute T-Lymphoblastic Leukemia
Authors: Anderson Nicole M , Peng Hongling , Helman Daniel , Harrold Itrat , Sanda Takaomi , Grant Trevor , Nagykery Nicholas , Samaha Yasmina , Le Meng , Amsterdam Adam , Neuberg Donna , Mansour Marc R. , Denton Travis , Kanki John , Singh Anurag , Look A. Thomas , Feng Hui
Published: October 2019

Despite improvements in treatment for T-Acute Lymphoblastic Leukemia (T-ALL), only 70-80% of children and 40% of adults achieve long-term remission. To discover novel targets for the development of molecular therapeutics, we conducted a dominant genetic modifier screen to identify genes whose inactivation delays the onset of leukemia. We have generated a Myc-driven zebrafish model of T-ALL that recapitulates its human counterpart genetically and pathologically. Utilizing this zebrafish model of T-ALL, we conducted a genetic screen that identified a gene encoding dihydrolipoamide S-succinyltransferase (DLST), whose heterozygous loss significantly delays the onset of leukemia in zebrafish. DLST is the E2 component of α-ketogluterate dehydrogenase complex (KGDHC), a key enzyme in the TCA cycle that converts α-ketogluterate (α-KG) to Succinyl CoA. Western blot analysis revealed that DLST was upregulated in zebrafish tumor cells, T-ALL cell lines and primary human T-ALL samples. Zebrafish T-ALL cells with heterozygous inactivation of dlst have decreased cell size and slowed cell cycle progression, compared to T-ALL cells without dlst inactivation. To demonstrate that DLST is a relevant target in human T-ALL, we used two structural analogs of α-KG (α-keto-n-valeric acid sodium salt (KV) and α-keto-β-methyl-n-valeric acid (KMV)) to inhibit the KGDHC activity. Inhibition of KGDHC by both KV and KMV decreased cell viability and promoted apoptotic cell death in a panel of human T-ALL cell lines. Genetic inactivation of DLST, via shRNA knockdown, also decreased cell viability in MOLT16 human T-ALL cell line, confirming the therapeutic potential of DLST in human T-ALL treatment. In vivo treatment of our zebrafish T-ALL model with KV promoted tumor regression within seven days of treatment. Taken together our findings indicate that DLST is an important contributor to T-ALL pathologenesis, and suggest that DLST is a potential target for further therapeutic development to treat human T-ALL.

Disclosures:

No relevant conflicts of interest to declare.
Development Of Novel Alkylating Chemotherapeutics, Phenyl-Chloroethyl Urea Family, Targeting Mitochondrial Prohibitin For Selective Killing Of Leukemic Cells
Authors: Kim Hye-Ran , Kim Hwan-Young , Thi Dai Trang Nguyen , Lee Il-Kwon , Kim Hyeoung-Joon , Shin Jong-Hee , Suh Soon-Pal , Ryang Dong-Wook , Shin Myung-Geun
Published: October 2019

Background

Mitochondrial aberrations have been associated with chronic degenerative diseases, aging and cancer. Mitochondrial proteins are interesting targets for the development of selective anticancer drugs in leukemia and other malignancies. We analyzed mitochondrial proteins to develop novel anti-cancer agents targeting selective leukemic cell, especially acute myelocytic leukemia (AML) cells.

Materials and Methods

Mitochondria were isolated from primary AML cells and AML cell lines (THP-1 and KG-1) by density-gradient ultracentrifugation using swelling buffer and sucrose buffer. Dysregulated mitochondrial protein were identified using 2-DE and mass spectrometry (MALDI-TOF/TOF technology). For in vivo experiments, AML cell grafts were established in 6-week-old Balb/c mice by subcutaneous injection of 1x107 THP-1 AML cells in the right flank at day 0.

Results

Out of these deregulated proteins, totally 12 and 20 proteins were observed in up- or down-regulated spots, respectively. Interestingly, prohibitin (gi4505773) was highly expressed in all type of leukemic cells, especially primary AML cells (Fig. 1), which is confirmed by immunoblot and immunofluorecenct methods. Aberrant expressed prohibitin induced growth suppression and repressed E2F-mediated transcription. We synthesized potent chemical substances that can alkylate PHB, two molecules of phenyl-chloroethyl urea family such as cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU) from the reaction with 2-ethylisocyanate and 4-cyclohexylaniline and 4-iodoaniline, respectively. They were characterized by 1H NMR and 13C NMR. Time and dose dependent manner of proliferation suppression when treated with CCEU and ICEU was observed in primary AML and AML cell lines. Notably morphological transformation of AML cells was observed when treated with 10 - 100 umol of CCEU and ICEU for 24 hours. The half maximal inhibitory concentration (IC50) was 25umol of most AML cell lines. Cell cycle analysis of CCEU-and-ICEU-treated- AML cells showed a remarkable increase of the sub-G1 phase. Immunoblotting experiment revealed the change of cytoplasmic and nucleoplasmic PHB. The increment expression of cleaved caspase3 and poly ADP-ribose polymerases were also observed in AML cell lines. Moreover, CCEU and ICEU selectively killed AML cells in mouse model (Fig. 2).

Conclusion

This study showed the development of novel alkylating chemotherapeutics, phenyl-chloroethyl urea family (CCEU and ICEU), targeting mitochondrial prohibitin for selective killing of leukemic cells. Selective eradication effects of these novel chemotherapeutics were further confirmed using in vivo mice model.

Disclosures:

No relevant conflicts of interest to declare.
Second Hematologic Malignancies After Radioiodine Exposure In Patients With Thyroid Cancer and The Relationship With Radioiodine Dosage: A Nationwide Population-Based Study
Authors: Liu Chia-Jen , Hung Man-Hsin , Teng Chung-Jen , Hu Yu-Wen , Hung Yi-Ping , Gau Jyh-Pyng , Chiou Tzeon-Jye , Tzeng Cheng-Hwai
Published: October 2019

Background

Second primary malignancies are one of the most important late effects after radioactive iodine (RAI) exposure, which is widely used in the diagnosis and treatment for thyroid cancers. However, dose-response correlation between RAI and second hematologic malignancies (SHM) was not established. Therefore, we conducted a nationwide population-based study to investigate the risk of SHM among thyroid cancer patients and the association between RAI dosage and development of SHM, including leukemia.

Material and methods

We recruited patients with newly diagnosed thyroid cancer aged 20 years or older without antecedent cancer from the Taiwan National Health Insurance database between 1997 and 2010. The standardized incidence ratios (SIR) of cancers were calculated to compare the cancer incidence of thyroid cancer patients to the general population according to gender, calendar year, and age in 5-year intervals by the corresponding stratum-specific person-time accrued in the cohort. The association of cumulative RAI dosage and development of SHM, including leukemia, was estimated using time-dependent analysis with 2-year latent period.

Results

After exclusion of first-year follow-up, 692 second primary malignancies developed among 20,235 patients with thyroid cancer, with a follow-up of 134,178 person-years. Fifty SHM developed with a SIR of 2.37 (95% confidence interval 1.76-3.13). The significant increased risk of SHM included: leukemia (SIR, 2.74; 95% CI, 1.65-4.28), non-Hodgkin lymphoma (SIR, 2.38; 95% CI, 1.55-3.48). As a time-dependent covariate with a 2-year latent period, RAI significantly increased the risk of leukemia with a clear dose-response correlation (age- and sex-adjusted hazard ratio per 100 mCi increase, 1.10; 95% CI, 1.01-1.19, P = 0.032). This effect was not seen in other second primary malignancies.

Conclusions

Our study reveals increased risk of SHM among patients with thyroid cancer. RAI increases the risk of leukemia with a clear dose-response relationship.

Disclosures:

No relevant conflicts of interest to declare.
Genomic Heterogeneity In Pediatric Acute Lymphoblastic Leukemia
Authors: Yaniv Isaac , Jeison Marta , Hameiri-Grossman Michal , Mardoukh Jacque , Sharon Nir Adam , Elitzur Sarah , Gilad Gil , Avrahami Gali , Stark Batia , Avigad Smadar
Published: October 2019

Pediatric B cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is characterized by recurrent chromosomal aneuploidies and translocations, which are important for diagnosis and risk stratification. Classical cytogenetic and FISH are routinely used for the diagnosis of most of these alterations, particularly those involving balanced translocations, and numerical changes. Recently, genome wide analysis, such as aCGH and SNP technologies have identified copy number alterations (CNA) in genes involved in lymphoid differentiation: IKZF1, PAX5, EBF1 BTG1; and in cell cycle control: CDNK2A/B, RB1 and ETV6. Multiplex ligation–dependent probe amplification (MLPA) technique has been used for the identification of those focal genetic aberrations in a single assay but it cannot reliably detect aberrations present in less than 50% of cells. Cytoscan HD SNP array (Affymetrix) can overcome this limitation and is the only array which enables independent confirmation of CNA with SNP allelic information. Thus minor genetic subclones missed by other methods, can be detected.

The aim of this work was to compare between SNP array and MLPA platforms in a routine diagnostic setting.

Samples of 39 pediatric BCP-ALL patients well characterized by classical cytogenetic and FISH at diagnosis, were studied by MLPA P335 (MRCHolland) and SNP array (Affymetrix). In total 273 genomic loci were evaluated, 13 (5%) were not informative, of them 7 were not covered by the MLPA kit and 6 gave not evaluable MLPA results. 231/260 (89%) of the loci showed concordance, 39/260 (15%) with CNA and 192/260 (74%) normal. A total of 68 CNA were detected, 29 were discordant and 39 concordant.

IKZF1 deletion was identified by both methods in 5 cases (13%). However, a deletion was identified in 3 additional samples, only by cytoscan. In these cases, the deletion was present in a minor subpopulation (in 45% or less of the cells), while the main cytogenetic aberration was present in the majority of cells (60%-90%).

We conclude that although MLPA is a useful technique, it is not sensitive enough to detect small populations. In this small cohort, 3 IKZF1 deletions were missed since they were present only in a minor subclone. Interestingly, one of these patients relapsed, and the relapse clone harbored the IKZF1 deletion.

Conclusion

Genome-wide sensitive methods identify different subpopulations in each patient, thus detecting previously unrecognized genetic heterogeneities.

Disclosures:

No relevant conflicts of interest to declare.
The Combination Of 2-DG and Metformin Inhibits The mTORC1 Pathway and Suppresses Aggressive B Cell Lymphoma Growth and Survival
Authors: Bi Chengfeng , Fu Kai , Jiang Chunsun , Huang Xin , Chan Wing Chung , McKeithan Timothy
Published: October 2019

mTORC1 (mammalian target of rapamycin complex 1) plays a central role in integrating nutrient and growth factor inputs to control cell growth in all eukaryotes and is commonly deregulated in human cancers. Inhibition of mTORC1 is a promising strategy in lymphoma therapy. However, only a few drugs, such as rapamycin and its analogs (rapalogs), have been approved for treatment in a limited number of cancer types, due to their incomplete and nonspecific inhibition of mTORC1 as well as their limited effects toward the 4EBP1 pathway. 4EBP1 pathway regulates protein translation which is considered to be crucial in cancer cell survival and proliferation. In this study, we used the glycolysis inhibitor 2-deoxyglucose (2-DG) together with the mitochondrial respiratory inhibitor metformin to treat aggressive B cell lymphoma cells in vitro and in vivo. We found that the combined treatment inhibited mTORC1 and its major downstream targets, including 4EBP1. As a result, combined treatment significantly inhibited tumor cell growth and survival by the inhibition of 5’ cap-dependent translation involving lymphoma associated oncogenes such as MCL-1, BCL-XL and Cyclin D1. Moreover, the combination of 2-DG and metformin suppressed tumor growth in B cell lymphoma xenograft mouse models. Although the combined treatment dramatically decreased cellular ATP levels, mTORC1 inhibition was independent of AMPK activity but instead resulted from inhibitory effects on Rag-GTPases, which are upstream activators of mTORC1. Given that both 2-DG and metformin have been used in clinical diagnosis or treatment for decades, the combination of the two drugs hold promise as a new strategy to treat aggressive B cell lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
Excessive Early Mortality Drives Poor Long Term Outcomes Among Older Patients With Classical Hodgkin Lymphoma: A Population Study
Authors: Costa Luciano J.
Published: October 2019

Background

Classical Hodgkin Lymphoma (cHL) is a highly curable malignancy in young adults. Most of the information available on natural history and management of cHL comes from series and clinical trials in young patients. There is limited information on the outcomes of older patients with cHL

Methods

We analyzed the characteristics and outcomes of a large contemporary cohort of cHL patients ≥ 65 years diagnosed in the US and reported to the Surveillance Epidemiology and End Results program (SEER-18). Inclusion criteria consisted of diagnosis of cHL as first malignant neoplasm, year of diagnosis 2000-2010, known stage and known race. Cases reported from death certificate or autopsy only were excluded. Information retrieved contained age at diagnosis, year of diagnosis, race, sex, histological subtype, stage, presence of extra-nodal disease and survival time. Characteristics of cHL in patients ≥ 65 years (older) were compared to those < 65 years (younger). We subsequently estimated relative survival (RS) for different stages and age strata among older patients comparing with outcomes in younger patients.

Results

There were 20815 cases of cHL reported during the period with median follow up of 48 months, including 2884 (13.8%) cases in older patients. Older patients were more likely to present with extra-nodal disease (4.7% vs. 2.2%, P< 0.001), advanced stage (52.6% vs. 37.3%, P<0.001) and with lymphocyte depleted cHL (3.3% vs. 0.9%) or mixed cellularity cHL (22.5% vs. 11.3%, P<0.001) than younger patients. Additionally, older cHL patients with early stage disease were less likely to receive radiation therapy (35.6% vs. 48.2%, P<0.001). RS at one year (64% vs. 95.7%, P<001) and 5-years (49% vs. 89%, P<0.001) was much inferior in older than in younger patients, even when stratified by early (I and II) and advanced (III and IV) stages (Figure). RS deteriorated quickly with increasing age at diagnosis. One year RS was 73.9%, 56.3% and 43.3% and 5-years RS was 57.9%, 41.6% and 30.4% for patients 65-74, 75-84 and 85+ years, respectively. Even with adjustment for histology, sex, year of diagnosis and race, patients 65-74 years (HR = 4.81, 95%CI 4.41-5.26, P<0.001) 75-84 years (HR = 9.60, 95% CI 8.78-10.49, P<0.001) and 85+ years (HR = 16.19, 95% CI 14.15-18.51, P<0.001) were at a much higher risk of death than younger cHL patients.

Conclusion

cHL has distinct presentation and far worse prognosis in older patients with excessive early (<1 year) mortality. There is a need to further understand patterns of treatment failure and develop age-specific therapeutic interventions for this group.

Disclosures:

No relevant conflicts of interest to declare.