bluuebunny/crossref_metadata_2025 huggingface dataset:
Small Molecule Inhibition of SIRT Activity: A Novel Therapeutic Approach for Acute Myeloid Leukemia
Authors: Nawrocki Steffan T , Espitia Claudia M , Kelly Kevin R. , Bornmann William G. , Carew Jennifer S
Published: October 2019

Abstract 84

New therapeutic strategies are urgently needed to improve clinical outcomes for patients with acute myeloid leukemia (AML), which is an extremely aggressive disease with very few long-term survivors. The sirtuin deacetylases (SIRTs) are critical regulators of genes that are essential for longevity, cell growth, tumor suppression, and apoptosis. Elevated SIRT expression has been reported in several types of cancer and may promote pathogenesis and drug resistance by increasing the lifespan and survival capacity of malignant cells. Our preliminary analysis of SIRT expression indicated that SIRT1 was consistently expressed at significantly higher levels in AML cell lines and primary AML blasts as compared with normal controls. In order to investigate the potential role of SIRT1 as a regulator of AML pathogenesis, we utilized shRNA to stably knockdown its expression in MV4-11 and KG-1 AML cells. Cells with targeted SIRT1 knockdown displayed an altered gene expression profile as compared with non-targeted controls. Moreover, antagonizing SIRT1 expression significantly impeded the progression of AML in a xenograft mouse model. A number of deacetylase inhibitors have been clinically evaluated for cancer therapy. However, disrupting SIRT function as an anticancer strategy remains to be rigorously investigated as none of these previously studied drugs significantly inhibit the activity of this class of NAD+-dependent deacteylases. Tenovin-6 is a novel small molecule SIRT inhibitor. We investigated the efficacy and pharmacodynamic effects of tenovin-6 in AML cell lines, primary blasts from patients with AML, and mouse models. Treatment with tenovin-6 induced apoptosis and dramatically diminished AML clonogenic survival. Tenovin-6 promoted a dose-dependent increase in the acetylated levels of the SIRT-regulated gene p53 in AML cells and triggered the induction of several p53 transcriptional targets including p21 and PUMA. Targeted knockdown of PUMA with shRNA significantly reduced the pro-apoptotic effects of tenovin-6, indicating that it is a critical mediator of its anti-leukemic activity. Notably, administration of tenovin-6 to mice implanted with AML cells was well-tolerated and led to a highly significant reduction in disease burden and increase in overall survival. Our collective findings demonstrate that SIRT1 is a promising novel therapeutic target in AML. Further investigation aimed to elucidate the safety, efficacy, and mechanism of action of tenovin-6 is warranted.

Disclosures:

No relevant conflicts of interest to declare.
Changes in Survival in Acute Myeloblastic Leukemia by Racial and Ethnic Group: Greater Improvement for Non-Hispanic Whites and Increase in the Disparity for Minority Patients in the Early 21st Century
Authors: Pulte Dianne , Redaniel Maria Theresa , Brenner Hermann , Jansen Lina , Jeffreys Mona
Published: October 2019

Abstract 844

Background:

Acute myeloblastic leukemia (AML) is a potentially curable disease but treatment is intense, prolonged, and expensive. Survival has improved for adults age less than 75 with AML in the early 21st century1, but it is unknown whether people of all ethnic and racial backgrounds have benefitted equally. Here, we examine changes in survival in patients with AML by racial and ethnic group between 1993–97 and 2003–07.

Methods:

Using cancer registry data from the Surveillance, Epidemiology and End Results (SEER9) Program, we assessed trends in 5-year relative survival for patients with AML by racial and ethnic group, including non-Hispanic whites (nHw), African-Americans (AA), Hispanics, and Asian-Americans (Asian) for the periods 1993–97 and 2003–07. Modelled period analysis was used to obtain the most up-to-date measurements of survival. Because survival varies greatly with age, survival for patients age 15–54 was calculated separately.

Results:

Median age at diagnosis was highest for nHw, lowest for Hispanics. A statistically significant improvement in survival was seen for nHw patients with AML between 1993–97 and 2003–07. Better survival was observed for other racial/ethnic groups, but the differences were small and non-significant (table 1). When only patients age 15–54 were considered, this effect was enhanced and a trend towards shorter survival was observed for AA patients (table 2). In 1993–97, five year relative survival rates were lower for nHw than for other racial or ethnic groups overall and for patients age 15–54 (tables 1 and 2). In 2003–07, nHw had a 5-year relative survival rate that was higher than AA overall and higher than any other ethnic group for ages 15–54 (table 2).

Conclusions:

Significant disparities in survival exist for patients with AML between nHw and people of other ethnic or racial groups and these disparities are increasing, especially in younger patients. The disparity was more notable for patients age 15–54, suggesting that lack of access to aggressive treatment, particularly transplant, may be a factor in the growing disparity. Further research is needed to pinpoint where on the cancer continuum factors determining survival, including diagnosis, treatment, and biological prognostic markers, are worse for people from minority ethnic groups, and hence where interventions may be the most effective.

Disclosures:

No relevant conflicts of interest to declare.
Factor XIII Deficiency Improves Motor Function in Mice Challenged with Experimental Autoimmune Encephalomyelitis, An Established Model of Multiple Sclerosis
Authors: Mullins Eric S. , Talmage Kathryn E. , Kombrinck Keith W. , Miller Whitney M , Palumbo Joseph S. , Degen Jay L.
Published: October 2019

Abstract 858

Multiple sclerosis (MS) is an inflammatory disease characterized by autoimmune demyelination of neurons and axonal damage within the central nervous system, leading to relapsing/remitting neurological deficits, including sensory and motor deficits. MS plaques are characterized by blood-brain barrier disruption leading to perivascular deposition of fibrin that correlates with areas of microglia activation and myelin damage. Consistent with multiple studies showing that hemostatic factors can serve as important modulators of the inflammatory response in vivo, fibrin(ogen) has been identified as a novel regulator of microglial activation and differentiation. Furthermore, fibrin(ogen) has been found, via the αMβ2 binding motif on the γ chain, to play a role in microglial activation in experimental autoimmune encephalomyelitis (EAE), a murine model of MS. However, previous work by our lab, as well as others, suggests that thrombin-mediated proteolysis plays a greater role in driving neuroinflammatory disease than by merely supporting fibrin formation. Our working hypothesis is that thrombin-mediated proteolysis drives autoimmune neuroinflammation through both fibrin(ogen)-dependent and fibrin(ogen)-independent pathways. To test this hypothesis, we have initiated studies of EAE in mice lacking selected thrombin substrates, including protease activated receptors (PARs) and factor XIII (fXIII). Clinical evaluation of loss of motor function in EAE-challenged cohorts of PAR-1-deficient, PAR-4-deficient and WT control mice revealed a similar outcome in all three cohorts, suggesting PARs may have either a modest or a secondary role in EAE-induced neuroinflammatory disease. However, mice with a constitutive deficiency of another thrombin substrate, the fibrin cross-linking transglutaminase fXIII, were found to have a significant amelioration in motor function loss when challenged with EAE. To further explore this phenotype, we have utilized cystamine, a transglutaminase inhibitor, as a pharmacologic complement to the fXIII knockout mice. Mice with cystamine placed in their drinking water during the EAE challenge period also experienced significantly less disease than control mice that did not receive cystamine. This data suggests that (pro)thrombin contributes neuroinflammatory disease by both supporting local fibrinogen polymerization and by activation of fXIII leading to fibrin stabilization. Furthermore, this line of research suggests that more detailed studies of fXIII may reveal novel strategies for limiting or reversing the devastating pathologies associated with multiple sclerosis.

Disclosures:

Mullins: Baxter: Consultancy. Palumbo:Novo Nordisk Corporation: Research Funding.
Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions
Authors: Zhao Chen , Xiu Yan , Ashton John M , Xing Lianping , Morita Yoshikazu , Jordan Craig T. , Boyce Brenda
Published: October 2019

Abstract 859

RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment.

Disclosures:

No relevant conflicts of interest to declare.
A Monoclonal Antibody Specifically Targeting CD44 Inhibits B-Cell Chronic Lymphocytic Leukemia Cell Survival In Vitro and In Vivo
Authors: Zhang Suping , Wu Christina C.N. , Fecteau Jessie-Farah , Cui Bing , Wu Rongrong , Rassenti Laura Z. , Lao Fitzgerald S. , Tawatao Rommel , Weigland Stefan , Prussak Charles , Carson Dennis A. , Kipps Thomas J.
Published: October 2019

Abstract 927

B-cell chronic lymphocytic leukemia (CLL) with a highly variable clinical course is characterized by the clonal expansion of CD5+ B cells in blood, secondary lymphoid tissues, and marrow. Survival of CLL cells are supported by cells within the tissue microenvironment and by signals from the extracellular matrix, which in part are mediated via interactions with CD44, a surface glycoprotein that is expressed at high levels by CLL B cells. While monoclonal antibody (mAb) therapy targeting CD20 has improved the survival of patients with CLL, a large number of CLL patients do not achieve durable responses or become refractory altogether to therapy with anti-CD20 mAb. In this study we evaluated the cytotoxic activity against CLL cells of a newly developed, humanized anti-CD44 mAb (RO5429083, Roche). We found that sub-microgram concentrations of this anti-CD44 mAb were directly cytotoxic for CLL cells of different patients (n = 32), but had little or no effect on the viability of lymphocytes isolated from the blood of healthy donors (n = 4). The levels of cytotoxicity induced by this anti-CD44 mAb were significantly associated with the levels of CD44 expressed by each of the CLL samples (Spearman R-0.5785, p<0.001). Furthermore, survival and downstream signaling events of CLL cells induced by hyaluronic acid (HA), the principal ligand of CD44, were inhibited by this CD44 mAb. Of note, this CD44 mAb also was equally cytotoxic for CLL cells co-cultured with mesenchymal stromal cells (MSC), which otherwise can support CLL-cell survival in vitro. We also examined whether this anti-CD44 mAb could induce clearance of human CLL cells engrafted into immune deficient RAG-2−/−/γc−/− mice. Treatment of such xenografted animals with as little as 1 mg/kg of this mAb resulted in the complete clearance of engrafted CLL cells, an effect not observed in control treated animals. Based on these pre-clinical studies, we consider this anti-CD44 mAb has high potential for providing effective treatment for patients with this disease.

Disclosures:

Weigland: Roche Diagnostics GmbH: Employment. Carson:Wintherix: Equity Ownership. Kipps:Gilead Sciences: Consultancy, Research Funding; GSK: Research Funding; Genentech: Research Funding; Abbott Industries: Research Funding; Celgene: Consultancy, Research Funding; Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding.
Dimension of Residual CT Scan Mass in Hodgkin's Lymphoma (HL) Is a Negative Prognostic Factor in Patients with PET Negative After Chemo+/− Radiotherapy
Authors: Magagnoli Massimo , Marzo Katia , Balzarotti Monica , Rodari Marcello , Mazza Rita , Giordano Laura , Gandolfi Sara , Bramanti Stefania , Anastasia Antonella , Lutman Fabio Romano , Chiti Arturo , Santoro Armando
Published: October 2019

Abstract 93

BACKGROUND.

Positron emission tomography (PET) with 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) currently represents the mainstay for response assessment in HL, as defined by revised response criteria (Cheson, 2007). PET negativity is mandatory to define complete remission (CR), independently from the persistence of residual masses at computed tomography (CT scan). Nevertheless, some reports suggest a slightly worse prognosis among patients (pts) with CT scan residual masses. The aim of this study was to evaluate the unfavorable predictive value of residual CT scan masses in HL pts with PET negative at the end of treatment.

MATERIAL AND METHODS.

The present analysis was retrospectively conducted in 105 pts with negative PET at the end of first or second line treatment, at our institution, from February 2004 to February 2009. All pts had disease evaluation performed also with CT scan.

RESULTS.

Main clinical characteristics: median age 58 years, males 62, B-symptoms 25, bulky disease 41, prior radiotherapy 57. Seventy-four pts were evaluated after first line treatment program, while 31 pts after salvage therapy program. In 76 pts, residual CT scan mass (PET-/CT scan +) of at least 2.0 cm in the largest diameter was assessed. Fifty-seven had only one site residual mass, while 19 pts had more than one site. Considering the whole series, with a median follow-up of 45 months, 23 pts relapsed, nine patients died and 94 are alive without disease.

The five year disease-free survival (DSF) for PET-/CT scan- vs PET-/CT scan+ pts was 89.4% and 68.7% respectively (P=0.053). The prognostic impact of residual mass at CT scan had a correlation with the dimension of the residual itself in a continuous fashion: the larger is the mass, the lower the DFS: HR 1.03 (1.01; 1.05) p 0.007. This difference is even more pronounced when a cut-off of 4 cm in the largest diameter of the residual mass is applied: in patients with a mass diameter ≥ vs < 4 cm, DFS is 50% vs 82% respectively (HR 3.25: CI 1.5; 7.04, p 0,029 – figure 1). Among all the other prognostic factors analyzed (number of masses, first vs salvage treatment program, sex, bulky disease, B-symptoms), no correlation with DSF or overall survival (OS) emerged.

CONCLUSIONS.

In our study we observe a significant difference in DFS among PET negative pts with or without CT scan residual masses after therapy for HL. This difference is more significant when the residual mass is larger than 4 cm. Thus, despite PET is the main tool in respons-e definition, CT scan maintains an important role and can not yet be abandoned. The role of consolidation radiotherapy in these cases should be focused.

Disclosures:

No relevant conflicts of interest to declare.
L-Leucine Improves the Anemia of the 5q- Syndrome Via Activation of the mTOR (mammalian target of rapamycin) Pathway
Authors: Virgilio Maria , Payne Elspeth , Narla Anupama , Sun Hong , Paw Barry H. , Look Thomas , Ebert Benjamin L. , Berliner Nancy , Khanna-Gupta Arati
Published: October 2019

Abstract 970

The “5q- syndrome” is a subtype of myelodysplastic syndrome (MDS) characterized by a profound macrocytic anemia that is thought to arise from the heterozygous loss of the RPS14 gene on chromosome 5q. The 5q- syndrome shares many characteristics with another ribosomopathy, Diamond Blackfan anemia (DBA), wherein half the patients have a heterozygous loss of a ribosomal protein gene, with RPS19 being the most frequently mutated. Both RPS19 and RPS14 are components of the 40S small ribosomal subunit. Heterozygous loss of both genes is thought to contribute to the pathophysiology of these ribosomopathies by impairing ribosome function and thereby decreasing mRNA translational efficiency. L-Leucine, a branched-chain amino acid has been shown to be a potent stimulator of mRNA translation. In a case report, administration of L-Leucine to a DBA patient resulted in complete recovery of the anemia and improvement in appetite and growth parameters. We therefore hypothesized that L-Leucine could improve the anemia associated with heterozygous loss of RPS14. To test this hypotheisis, we used zebrafish as an in vivo model for del(5q) MDS. Using antisense morpholinos, we knocked down Rps14 expression to haploinsufficient levels and observed anemia in the resulting morphants. Treatment of these morphants with L-Leucine, but not D-Leuicne, resulted in a dramatic increase in hemoglobinization as well as an increase in total erythroid cells. This observation was further validated in vitro using human CD34+ hematopoietic progenitor cells infected with RPS14 shRNA. Flow cytometric analysis demonstrated that treatment with L-Leucine increased the total number of erythroid cells (glycophorin-A/CD71 expressing cells) compared to untreated cells. In previous studies, L-leucine has been shown to upregulate mRNA translation by activating the mTOR (mammalian target of rapamycin) pathway and its downstream targets S6Kinase (S6K1) and 4E-Binding proteins (4E-BPs). We demonstrated increased levels of phospho-S6K1 as a result of L-Leucine treatment in the rps14 zebrafish morphant embryos. This increased phosphorylation of S6K1 was inhibited by rapamycin, suggesting specificity of mTOR activation in bringing about the L-Leucine effect. In summary, we have successfully demonstrated, in both a zebrafish model and in human primary hematopoietic stem cells, that L-Leucine alleviates the anemia associated with del(5q) MDS. This effect is likely mediated by activation of the mTOR pathway resulting in increased mRNA translation and an improvement in the anemia associated with loss of RPS14. Our studies support the further evaluation of L-Leucine as a potential therapeutic agent in the treatment of the 5q- syndrome.

Disclosures:

No relevant conflicts of interest to declare.
Thrombin Control Mechanisms and Thrombin Targets in Cancer Biology
Authors: Degen Jay L. , Palumbo Joseph S.
Published: October 2019

Abstract SCI-17

A link between hemostasis and cancer has been recognized for more than a century, but the last decade has seen substantial strides in understanding the mechanisms by which hemostatic system components actively contribute to the malignant phenotype. The expression of procoagulants, such as tissue factor (TF), by tumor cells has been shown to be a poor prognostic factor in clinical studies and a crucial determinant of metastasis in animal models. While TF expressed by tumor cells likely plays a multifaceted role in cancer biology, a substantial body of evidence indicates that tumor cell-associated and circulating hemostatic system components (e.g., prothrombin, fibrinogen, platelets) play a cooperative role in supporting metastasis. The capacity of tumor cells to generate thrombin has been proposed to support metastasis through several mechanisms, including tumor cell proliferation, stable adhesion, regulation of apoptosis, and escape from innate immune surveillance mechanisms. More recently, the fundamental importance of endothelial regulators of thrombin activity in metastasis was established through studies of tumor dissemination in mice expressing mutant forms of thrombomodulin (TM). Mice expressing a TM derivative with reduced thrombin affinity (TMPro) exhibited a profoundly prometastatic phenotype relative to wild-type (WT) mice. The TMPro mutation was shown to support metastasis by promoting the survival of tumor cell emboli newly localized to the lung. The impact of the TMPro mutation on metastasis was dependent on tumor cell-associated tissue factor, prothrombin, thrombin function, and platelets. In contrast, mice expressing a mutant form of TM lacking the lectin-like domain (TMLed) that were shown previously to have altered immune function but normal thrombin affinity, exhibited metastatic potential comparable to wild-type mice. These studies further highlight the importance of the hemostatic system in metastasis and demonstrate that apart from tumor cell-associated and circulating procoagulants, TM-mediated regulation of hemostatic function strongly influences tumor cell metastatic success. In addition, recent studies of inflammation-driven cancer have revealed that the role of hemostatic factors in tumor biology is not limited to later phases of malignant progression, such as metastasis. Fibrin(ogen)-mediated regulation of leukocyte function was shown to support tumor development and tumor proliferation in a murine model of inflammation-driven colon cancer. Recent advances in our understanding of the role of hemostatic factors in cancer biology demonstrate that this system of proteins can be important in multiple phases of malignant progression, and underscore the potential utility of targeting selected coagulation factors as a novel adjunct therapy in the treatment of cancer.

Disclosures:

Palumbo: Novo Nordisk Corporation: Research Funding.
Cellular Bioenergetics in Lymphoid Neoplasia
Authors: Heiden Matthew Vander
Published: October 2019

Abstract SCI-25

Many cancer cells metabolize glucose by aerobic glycolysis, a phenomenon characterized by increased glycolysis with lactate production and decreased oxidative phosphorylation. We have argued that alterations in cell metabolism associated with cancer may be selected by cancer cells to meet the distinct metabolic needs of proliferation. Unlike metabolism in differentiated cells, which is geared toward efficient ATP generation, the metabolism in cancer cells must be adapted to facilitate the accumulation of biomass. Cancer cells divert a larger fraction of their nutrient metabolism to pathways other than mitochondrial respiration regardless of oxygen availability. Nevertheless, oxygen levels still influence how nutrients are metabolized. We have found that the source of carbon used in various anabolic processes varies based on oxygen levels. Furthermore, the enzymes used to metabolize nutrients can also differ based on the cellular context. This includes regulation of isocitrate dehydrogenase, an enzyme that is mutated in some cancers. There is also strong selection for use of the M2 isoform of pyruvate kinase (PK-M2) to metabolize glucose in cancer cell lines. However, evidence from mouse models suggests that PK-M2 is dispensable for glucose metabolism by many tumors in vivo, suggesting an alternate pathway to convert phosphoenolpyruvate to pyruvate can be used to metabolize glucose. This regulation of pyruvate kinase also plays an important role in hematopoietic stem cell biology. Together, these findings argue that distinct metabolic phenotypes exist among proliferating cells, and both environmental and genetic factors influence how metabolism is regulated to support cell growth.

Disclosures:

Vander Heiden: Agios Pharmaceuticals: Consultancy, Equity Ownership.
Therapeutic Targeting of Cancer Cell Metabolism in Lymphoid Neoplasia
Authors: Van Dang Chi
Published: October 2019

Abstract SCI-27

The MYC oncogene plays a pivotal role in human lymphoid neoplasias, specifically in lymphomas and acute leukemias, which are characterized by altered glucose metabolism, termed the Warburg effect. The Warburg effect or elevated conversion glucose to lactate by cancer cells has been a prevailing model of cancer metabolism. Since the 1980’s, genetic alterations of oncogenes and tumor suppressors have provided insights into tumorigenesis. However, whether metabolism contributes to tumorigenesis was highly debated. In 1997, we reported that the MYC oncogene product, the Myc transcription factor, regulates the lactate dehydrogenase A (LDHA)gene. Myc also activates many glycolytic enzymes, mitochondrial biogenesis, and glutamine metabolism by inducing glutaminase (GLS) and glutamine transporters, thereby providing not only ATP through the TCA cycle but also anapleurotic building blocks. Myc also induces biomass accumulation by stimulating ribosomal biogenesis. It stimulates the cell cycle machinery and DNA replication. Deregulated MYC in cancer results in enforced biomass accumulation, such that cell death occurs when bioenergetic demands exceed nutrient availability. In this regard, we have exploited this conceptual framework and targeted LDHA and GLS with small molecular inhibitors as proof-of- concept that altered cancer metabolism could be targeted for cancer therapy. Specifically, we documented that a drug-like inhibitor of LDHA could decreased tumor xenograft growth, providing evidence that metabolic therapy is feasible. We further found in a human Burkitt lymphoma model that Myc induces a genetic program that drives glutamine metabolism both under aerobic and hypoxic conditions. Inhibition of glutaminase, which converts glutamine to glutamate for its catabolism by the TCA cycle, by a drug-like molecule also diminished lymphoma xenograft growth in vivo. These studies indicate that targeting cancer cell metabolism could constitute a novel strategy to treat lymphoid neoplasias.

Disclosures:

Dang: Agios Pharmaceuticals, Inc.: Consultancy, Honoraria.
Genome-Wide Programming of Myeloid Cell Fates
Authors: Göttgens Berthold
Published: October 2019

Abstract SCI-29

Hematopoiesis represents one of the most tractable models of adult stem cell development and differentiation. Transcription factor (TF) proteins have long been recognized as major regulators of blood stem cell development as well as the subsequent differentiation into the multiple mature hematopoietic lineages. Seminal studies in multiple vertebrate model systems have identified specific TFs that control cell fate choices during myeloid differentiation (1). It remains largely unknown, however, how individual TFs are integrated into wider transcriptional regulatory networks, and how combinatorial TF interactions within these networks drive lineage specific gene expression programs. We are addressing these issues using two complementary approaches. First, we use a combination of transgenic reporter assays and network modeling approaches to reconstruct core transcription factor networks operating in early myeloid differentiation. Second, we employ genome-scale analysis of transcription factor binding sites for key hematopoietic regulators in both stem/progenitor cells and mature lineages (2,3). Integrated analysis of genome-scale datasets reveals previously unrecognized combinatorial interactions within core hematopoietic regulatory networks, which can be validated using both biochemical and mouse knockout approaches. Moreover, our studies also pinpoint novel candidate hematopoietic regulators, several of which we have validated using high throughput loss-of-function assays in zebrafish.

Disclosures:

No relevant conflicts of interest to declare.
TET2: Mechanism and Functional Consequences of Hydroxymethylation
Authors: Rao Anjana , Ko Myunggon , Pastor William , Huang Yun
Published: October 2019

Abstract SCI-32

TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Somatic TET2 mutations are frequently observed in myeloid neoplasms in humans. Bone marrow samples from patients with mutant TET2, as well as some patients with wild type TET2, display low levels of 5hmC in genomic DNA compared to healthy controls. Measurement of 5hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool to tailor therapies and assess responses to anticancer drugs. We have developed novel and specific approaches to profile the genomic localization of 5hmC and will describe their application to profiling 5hmC in mouse hematopoietic progenitor cells that express or lack Tet2.

Disclosures:

No relevant conflicts of interest to declare.
Strategies for Genome Repair in Normal and Leukemic Stem Cells
Authors: Passegué Emmanuelle
Published: October 2019

Abstract SCI-41

Blood development is organized hierarchically, starting with a rare but well-defined population of hematopoietic stem cells (HSC) that give rise to a series of committed progenitors and mature cells with exclusive functional and immunophenotypic properties. HSC are the only cells within the hematopoietic system that self-renew for life, whereas other hematopoietic cells are short-lived and committed to the transient production of mature blood cells. Under steady-state conditions, HSC are a largely quiescent, slowly cycling cell population that, in response to environmental cues, is capable of dramatic expansion and contraction to ensure proper homeostatic replacement of all blood cells. While considerable work has deciphered the molecular networks controlling HSC activity, still little is known about how these mechanisms are integrated at the cellular level to ensure life-long maintenance of a functional HSC compartment. HSC reside in hypoxic niches in the bone marrow microenvironment, and are mostly kept quiescent in order to minimize stress and the potential for damage associated with cellular respiration and cell division. Recently, we have shown that HSC can also engage specialized response mechanisms that protect them from the killing effect of environmental stresses such as ionizing radiation (IR). We demonstrated that long-lived HSC, in contrast to short-lived myeloid progenitors, have enhanced expression of pro-survival members of the bcl2 gene family and robust induction of p53-mediated DNA damage response, which ensures their specific survival and repair following IR exposure. We reasoned that HSC have other unique protective features, which allow them to contend with a variety of cellular insults and damaged cellular components while maintaining their lifelong functionality and genomic integrity. We will present some of our recent findings on the fundamental mechanisms of stress-response that preserve HSC fitness during periods of metabolic stress, and allow for survival and repair following environmental stress associated with DNA damaging agents. It is now clear that oncogenic insults in diseases such as myeloproliferative neoplasms (MPN) can transform HSC and dramatically alter their biological functions leading to the emergence of leukemia-initiating stem cells (LSC), which are left untouched by most current therapies and can thereby mediate disease relapse. We will also discuss how transformed HSC may take advantage of some deregulated features of these normal stress-response mechanisms to escape therapeutic killing.

Disclosures:

No relevant conflicts of interest to declare.
Th17 Cells and Associated Cytokines in Inflammation and Cancer
Authors: Dong Chen
Published: October 2019

Abstract SCI-5

CD4+ T cells, upon activation, differentiate into cytokine-producing effector helper T (TH) cells. In addition to TH1 and TH2 lineage cells, additional TH subsets, including TH17 and T follicular helper (Tfh) cells have been identified. TH17 cells produce IL-17, IL-17F, IL-21, and IL-22 and mediate tissue inflammation. TH17 cells play protective or pathogenic roles in cancer, depending on the context. On the other hand, Tfh cells produce IL-21 and regulate germinal center reactions. Tfh cells may play a role in some forms of lymphoma. I will discuss on the regulation and function of these two subsets of T cells in the context of cancer.

Disclosures:

Dong: Ono: Consultancy; Tempero: Consultancy; Genentech: Honoraria; GSK: Consultancy; AnaptysBio: Consultancy.
Acute Leukemia in Hispanic Americans: Incidence and Incidence Rate Differences by Nativity.
Authors: Pollyea Daniel A , Kohrt Holbrook E , Yang Juan , Chang Ellen T , Gomez Scarlett Lin , Clarke Christina A
Published: October 2019

Abstract 3160

Introduction:

Acute leukemias (AL) have a distinct incidence pattern in Hispanics versus Caucasians, with higher rates of B-cell acute lymphoblastic leukemia (ALL) and acute promyelocytic leukemia (APL), and lower rates of non-APL acute myeloid leukemia (AML). Despite the fact that incidence differentials between foreign- and native-born populations can inform the relative contributions of environmental and genetic factors in cancer etiology, no such investigation has been undertaken in Hispanics with AL.

Methods:

To better understand heritable genetic versus environmental contributors to occurrence patterns, we assessed AL incidence in a large population of California Hispanics according to birthplace. Cancer incidence data were obtained from the California Cancer Registry, part of the National Cancer Institute's Surveillance, Epidemiology, and End Results Program. Patients were included if they were living in California and diagnosed between 2000–2009 with AL. Population estimates were obtained from the 2000 Census Summary Files 3, and estimates by nativity were derived from the 20% Integrated Public-Use Microdata Sample of the censuses.

Results:

Compared to Caucasians, Hispanics had an increased incidence of B-cell ALL and APL, with incidence rate ratios (IRR) of 2.13 (95% confidence interval [CI] 1.93–2.35) and 1.33 (95% CI 1.12–1.57), respectively. There was no difference in B-cell ALL incidence by nativity, with an IRR for foreign- to native-born Hispanics of 1.07 (95% CI 0.85–1.34). However, foreign-born Hispanics had a greater incidence of APL than native-born Hispanics (IRR 1.79, 95% CI 1.11–2.94).

Conclusions:

Our findings suggest the increased B-cell ALL incidence for foreign-born Hispanics is influenced by heritable genetic factors. Furthermore, the increased incidence of APL for foreign-born Hispanics may result from environmental exposures currently not described and which require further epidemiologic investigation.

Disclosures:

No relevant conflicts of interest to declare.
Transcriptome- Based Analysis of EPO Dependent Cfu-e to Erythroblast Development
Authors: Verma Rakesh , Narayanan Aishwarya , Kuhrt David , Li Lei , Su Su , Asch Ruth , Jachimowicz Edward , Wojchowski Don M.
Published: October 2019

Abstract 3188

To provide molecular insight into erythroid developmental programs, including EPO- regulated aspects, we have employed transcriptome-based approaches to analyze the stage-wise development of purified murine bone marrow- derived CFUe, proerythroblasts and maturing erythroblasts. In vivo and ex vivo, these progenitors develop as KitposCD71highTer119neg; KitnegCD71highTer119neg; and KitnegCD71highTer119pos cohorts (designated as E1, E2, E3 stages, respectively). In the context of EPO- modulation, stage E1 cells exhibited ∼250 EPO- regulated target genes. In stage-E2 proerythroblasts, in contrast, 750 transcripts proved to be EPO- regulated while stage-E3 erythroblasts exhibited only select EPO- modulated genes (<50). At E1 and E2 stages, EPO- regulated targets included overlapping yet distinct sets, and this was reflected in functional sets of GO terms. Major EPO- targets at stage E1 included cell cycle and cell biogenesis genes, while in E2 proerythroblasts, negative regulators of protein kinases (and kinase activity) constituted a major EPO/EPOR target set. As E1 CFUe transitioned to E2 proerythroblasts, an unexpected transient narrowing in the complexity of overall expressed genes was exhibited. Stage- modulated global sets of transcripts included myeloid cell differentiation factors, cell number homeostasis factors and heme biosynthetic processes. As E2 proerythroblasts transitioned to E3 erythroblasts, functional GO sets were associated predominantly with dynamics in organelle and cellular compartments. In addition, HomoloGene and Connectivity Mapping approaches were applied to compare transcriptomes of stage E1, E2 and E3 murine bone marrow erythroid progenitors with four recently studied stages of human erythroid progenitor cell development (here, termed H1, H2, H3 and H4). High correlation of stage E1 m-CFUe with not only human H1 CFUe but also H2 proerythroblasts was observed (0.59 and 0.57 correlations). Stage E2 murine proerythroblasts best corresponded to H2 human proerythroblasts (0.36 correlation score), while E3 murine erythroblasts aligned closely with human H4 late stage erythroblasts (0.58 correlation score). These latter analyses provide novel transcriptome- based comparisons, and transcript specific insight, into conserved vs distinct features of murine and human erythroid development at CFUe to maturing erythroblast stages.

Disclosures:

No relevant conflicts of interest to declare.
Dietary Supplementation with Genistein Increases Hepcidin Expression in Wild Type Mice
Authors: Zhen Aileen W. , Volovetz Josephine , Fraenkel Paula G.
Published: October 2019

Abstract 3208

Iron overload is an important cause of morbidity and death in patients with hemoglobinopathies, transfusion-dependent anemias, and hereditary hemochromatosis. As humans have no means of excreting iron, regulation of iron homeostasis depends on limiting intestinal iron absorption and optimizing iron release from macrophages to developing erythrocytes. Hepcidin, a peptide hormone produced in the liver, modulates intestinal iron absorption and macrophage iron release via effects on ferroportin. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. We conducted a small-scale chemical screen and found that the isoflavone genistein, a major dietary component of soybeans, enhanced Hepcidin transcript levels in zebrafish embryos. Furthermore genistein treatment increased Hepcidin transcript levels and Hepcidin promoter activity in human hepatocytes (HepG2 cells) in a Stat3 and Smad4-dependent manner. To evaluate genistein's effect in a mammalian model, we placed groups of 4 four-week old male C57BL/6 mice on an iron-sufficient, low soy diet (AIN93G containing 35 mg of iron/kg) supplemented with 0, 250, or 500 mg of genistein per kg of food for 7 weeks, and then sacrificed the animals for analysis. Plasma genistein levels (mean±SE) at the time of sacrifice were 0.015±0.015, 0.52±0.173, and 2.07±0.65 micromolar, respectively. Compared to mice not treated with genistein, the 250 mg/kg dose produced a significant increase in hepatic Hepcidin (HAMP1) transcript levels (1.49±0.10 vs 0.93±0.10, p=0.01), while the 500 mg/kg dose did not. Although liver iron content, spleen iron content, and weight gain were not significantly different among the groups, the ratio of Hepcidin expression to liver iron content was significantly increased in the animals treated with genistein 250 mg/kg compared to controls (0.013±0.0009 vs 0.0074±0.00068, p=0.0068). In conclusion, genistein is the first orally administered small molecule experimental drug shown to increase Hepcidin transcript levels in vivo. Future experiments will evaluate the effects of genistein on genetic models of iron overload syndromes.

Disclosures:

No relevant conflicts of interest to declare.
Erythropoietin Levels in Patients with Sickle Cell Disease Not in Vaso-Occlusive Crisis
Authors: Pulte Dianne , Nagalla Srikanth , Caro Jaime
Published: October 2019

Abstract 3242

Background:

Previous studies of erythropoietin production in sickle cell disease have shown that patients with sickle cell disease generally have an erythropoietin level in the lower end of the expected range. However, this finding was based on a relatively small number of patients including some who were in crisis when the erythropoietin level was drawn. A crisis might affect erythropoietin level by increasing inflammation beyond background and if the patient is transfused, the erythropoietin may not be well correlated with the apparent hemoglobin or hematocrit. In this study, we examine erythropoietin levels in patients with sickle cell disease seen in the outpatient clinic and not in crisis when the level was drawn.

Methods:

Patients receiving outpatient care at the Thomas Jefferson University Cardeza Sickle Center underwent laboratory evaluation as part of routine standard of care for their disease. Hemoglobin level, creatinine, erythropoietin level, lactate dehydrogenase, and reticulocyte count were extracted anonymously from patient charts for evaluation of erythropoietin in sickle patients in the outpatient setting without crisis and correlation of level with kidney function, hemoglobin level, and ongoing hemolysis.

Results:

Forty-eight measurements of erythropoietin from 39 unique patients were extracted. Two were not included as chart review suggested that the patient was in crisis when the values were obtained. Of the remaining 36 patients, three patients had SC disease, four sickle-thalassemia, the rest sickle cell anemia. Three patients received exogenous erythropoietin for hypoplastic anemia or chronic renal failure complicating sickle cell disease. Of the remaining 30 patients, creatinine ranged from 0.2–1.8.

Erythropoietin was higher than the normal range for most patients, as would be expected in a chronic anemia. Erythropoietin level was roughly correlated with hemoglobin, but some patients had erythropoietin levels much lower than expected for their hemoglobin (figure). Erythropoietin was not well correlated with creatinine or with hemolysis. Hemoglobin was higher for patients with SC disease than for sickle cell anemia or sickle-thalassemia, but erythropoietin levels were not significantly different between disease types.

Conclusions:

Patients with sickle cell disease who are not in crisis have erythropoietin levels that are elevated, but lower than expected for a healthy patient with chronic anemia, with a greater lack in patients with sickle cell anemia as compared to SC disease. Erythropoietin did not correlate well with kidney function as measured by creatinine or calculated glomerular filtration rate, suggesting that current measures of kidney damage may not be entirely adequate to detect early kidney disease in sickle cell disease.

Disclosures:

No relevant conflicts of interest to declare.
The Role of the IAP Livin in Megakaryocytes Differentiation and Thrombopoiesis
Authors: Abd-Elrahman Ihab , Deutsch Varda R , Pick Marjorie , Kay Sigi , Neuman Tzahi , Perlman Riki , Ben-Yehuda Dina
Published: October 2019

Abstract 3297

In this study we observe that Livin plays a role in thrombopoiesis. Livin is a member of the Inhibitor of Apoptosis Proteins (IAP) family of intracellular anti-apoptotic proteins that acts by binding and inhibiting caspases. Previously we found that Livin is unique among the IAP members as upon strong apoptotic stimuli, it is specifically cleaved by caspases to produce a truncated protein (tLivin) that has a paradoxical pro-apoptotic activity. Here we have shown that Livin is expressed in normal mature bone marrow MK and in platelets.

Objective:

The objective of this study was to evaluate the potential role of Livin in thrombopoiesis.

Methods:

We studied Livin expression in normal BM using immunohistochemistry staining. We studied the ability of primary MK generated from CD34+progenitor cells over-expressing Livin to produce platelets. While, the human cell line, LAMA-84, was induced by a phorbol myristic acid (PMA) to differentiate to megakaryocytes (MK) to evaluate the potential role of Livin in thrombopoiesis.

Results and conclusions:

Livin over-expression in CD34+ progenitor cells induced differentiation of these cells into MK and increased the ability of these primary MK to produce platelets. LAMA-84, upon differentiation into MK, produces platelets that are functional and capable of aggregating. This thrombopoiesis was accompanied by increased Livin expression and downregulation of other IAPs: XIAP and Survivin. Our results show that Livin plays a dual role in thrombopoiesis, demonstrating both an anti and pro-apoptotic role in cell activity.

Disclosures:

No relevant conflicts of interest to declare.
Spontaneous Atherosclerotic Lesions in TREM-Like Transcript-1 Deficient Mice
Authors: Gonzalez Marieli , Reyes Fiorella , Marrero Deborah , Washington A. Valance
Published: October 2019

Abstract 3312

It is well known that platelets, aside from regulating hemostasis, play an important role in inflammation-associated conditions like sepsis, viral infections, and atherosclerosis. In the latter, platelets not only form occlusive thrombi at lesions, but also play a role in the initiation of the disease by depositing activating molecules such as cytokines on the developing plaque. Although the mechanism by which platelet aggregation leads to occlusion is well-defined, the role of platelets in lesion initiation and progression is poorly understood and thus remains a gap in our knowledge. TLT-1 (Triggering Receptor Expressed in Myeloid cells (TREM)-like transcript-1) is a receptor exclusively found on megakarocytes and platelets that has a demonstrated effect in inflammatory responses. Upon platelet activation, TLT-1 is moved to the platelet surface along with p-selectin from the α-granules. Studies using the treml1−/− mouse demonstrated a predisposition to hemorrhage after an acute inflammatory challenge suggesting that TLT-1 may be a key regulatory molecule in the interface between hemostatic and inflammatory mechanisms and therefore should play a role in the development of atherosclerosis. Our original hypothesis was that evaluation of atherosclerosis lesions in the treml1−/− mouse would demonstrate fewer lesions and hence, a similar phenotype as the psel−/− mouse. Evaluation of 50 week old mice fed a chow diet surprisingly revealed spontaneous lesions in C57Bl/6 treml1−/− mice. Subsequent evaluation of cholesterol levels in treml1−/−mice on an atherogenic diet for four weeks demonstrated that they have significantly higher cholesterol levels when compared to WT mice. To evaluate atherosclerosis progression in TLT-1 deficiency, we developed the apoE−/−/treml1−/− double knockout mice and assessed lesion development after a four weeks atherogenic diet. Our results demonstrate that double null mice have exacerbated lesions when compared to the apoE −/− mice. Accordingly, 50 week old double null mice fed a chow diet also have larger atherosclerotic lesions in the aortic sinus than apoE −/− mice. These results clearly support a role for TLT-1 in dampening the progression of atherosclerosis. Current data from our laboratory suggest that TLT-1 may affect leukocyte function, therefore based on our model that TLT-1 deficiency leads to a heighted inflammatory state we hypothesized that treml1−/− mice will demonstrate greater leukocyte recruitment into the injured vessel wall, leading to greater deposition of factors that recruit more platelets and macrophages that later become foam cells. To delineate the mechanism by which TLT-1 affects atherosclerosis progression, we have evaluated neutrophil and monocyte infiltration into the vessel wall of the aortic sinus after four weeks atherogenic diet. The current state of our investigation is reported here.

Disclosures:

No relevant conflicts of interest to declare.