bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Number of B-Cells In the Red Pulp of the Spleen In ITP
Authors: Olsson Bob , Wadenvik Hans , Jernås Margareta , Ridell Börje
Published: October 2019

Abstract 1447

Background.

Platelets are targeted by autoantibodies and destroyed in the reticuloendothelial system, e.g. the spleen, in patients with chronic immune thrombocytopenia (ITP). However, other pathophysiologic mechanisms such as platelet destruction by cytotoxic T-cells and defective bone marrow production of platelets also contribute to the thrombocytopenia in ITP. Splenectomy normalizes the platelet count in around 70% of chronic ITP patients, however, precious little is known about the spleen micromorphology in this disease. We examined splenic pathology in patients with ITP, focusing on the intrasplenic distribution of lymphocyte subsets.

Patients and Methods.

The paraffin-embedded block containing representative material of the splenic tissue from 29 splenectomized chronic ITP patients (15 females and 14 males, mean age 45±21 (SD) years) and 11 controls, splenectomized because of traumatic splenic rupture (2 females and 9 males, mean age 48±19 (SD) years), was used to produce serial sections with a thickness of 5 μm. These sections were stained with conventional haematoxylin-eosin, and immunostained to identify CD3, CD4, CD8, CD20 and CD68 using the Dako Envision System in a Techmate Horizon Autostainer (Glostrup, Denmark). Using micromorphometry the lymphocyte subsets were enumerated in the red and white pulp of these spleens.

Results.

All ITP patients were preoperatively optimized, using corticosteroids and/or IVIg, and the immediate pre-splenectomy platelet count ranged 5–357 × 109/l.

Significantly more T-cells than B-cells were seen in the red pulp, both in ITP and control spleens (p<0.01). Conversely, more B-cells than T-cells were observed in the white pulp. There was no difference in the number of T-cells in the red and white pulp, between ITP patients and controls. However, the number of B-cells was increased in the red pulp of patients with ITP compared with the controls. Furthermore, numerous B-cells and CD8+ (cytotoxic) T-cells were located in the splenic cords, lining the sinusoids in the red pulp, a microenvironment where they readily can interact with the other blood cells, e.g. platelets.

Conclusion.

Chronic ITP is associated with an increased number of CD20+ B-cells in the red pulp of the spleen. The red pulp of the spleen also provides a microenvironment where close interaction between platelets, CD3+, CD4+, and CD8+ T-cells and B-cells can take place. Trafficking of regulatory and effector cells into this microenvironment might be a potential therapeutic target.

Disclosures:

No relevant conflicts of interest to declare.
A Single Intramuscular Injection of AAV-8 Vector Expressing MDA7/IL-24 Efficiently Suppresses Tumor Growth Mediated by Multiple Anti-Cancer Mechanisms In Lymphoma Model Mice.
Authors: Wang Nanya , Fan Bai , Miyake Noriko , Miyake Koichi , Shimada Takashi
Published: October 2019

Abstract 1474

Melanoma differentiation-associated gene-7/interleukin-24 (MDA7/IL-24) selectively induces apoptosis in cancer cells without harming normal cells. It also exerts immunomodulatory and anti-angiogenic effects, as well as potent antitumor bystander effects, making it an ideal candidate for use in a new anticancer gene therapy. To examine the feasibility of adeno-associated virus (AAV) vectors expressing MDA7/IL-24 in systemic cancer gene therapy for lymphoma, we generated an AAV type 8 vector expressing MDA7/IL-24 (AAV-IL24). In vitro studies showed that medium conditioned by AAV-IL24-transduced C2C12 cells induces tumor cell-specific apoptosis against murine lymphoma cell line (A20 cell). To assess the in vivo effects of muscle targeted AAV-mediated systemic delivery of MDA7/IL-24 we established a lymphoma murine model in which the A20 cells expressing luciferase gene was inoculated into the caudal vein of BALB/C. Using this lymphoma murine model, we can detect the tumor growth and metastases by a real-time in vivo imaging analyze system (IVIS). After single injection of AAV-IL24 (1.5×1011 vg/body) into the right quadriceps muscle of the lymphoma model mice, tumor cell growth was monitored by IVIS. ELISA analysis showed high level of IL-24 was detected in plasma of treated mice (263±16 ng/ml). Suppression of tumor growth was observed in AAV-IL24 injected mice compared to control GFP expressing AAV injected mice (1.3×108vs.2.6×108 photon/sec; p<0.05). Survival effect was also detected in AAV-IL24 mice (66±6 vs. 47±5 days; p<0.03). In addition, TUNEL analyses showed significant induction of tumor cell-specific apoptosis within the tumors and suppression of angiogenesis was also detected in AAV-IL24 treated mice. Finally, immune modulating activity of induction of Th1 cytokines (IL-6, TNFα, IFNγ) secretion was observed after AAV-IL24 injection. These results clearly demonstrate that continuous systemic delivery of MDA7/IL-24 can serve as an effective treatment for lymphoma. Thus, AAV type 8 vector-mediated systemic deliverY of MDA7/IL-24 represents a potentially promising new approach to treat lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
Targeting Histone Deacetylase 6 (HDAC6) as a Novel Approach to Overcome T-Cell Anergy.
Authors: Cheng Fengdong , Wang Hongwei , Woan Karrune , Rock-Klotz Jennifer , Wang Zi , Merino Oscar , Gill Ajay , Marante Danay , Vazquez Lianet , Seto Edward , Pinilla-Ibarz Javier , Sotomayor Eduardo M. , Villagra Alejandro
Published: October 2019

Abstract 1483

The anti-inflammatory molecules IL-10 and STAT3 are major hurdles in our efforts to harness effective anti-tumor immunity1. Previously, we have unveiled a role for histone deacetylase 11 (HDAC11) as a negative regulator of IL-10 gene transcriptional activity in antigen-presenting cells (APCs)2. In an effort to better characterize the epigenetic influences upon IL-10, we screened other HDACs and found that over-expression of HDAC6 in the macrophage cell line RAW264.7 was able to transcriptionally upregulate IL-10 in response to LPS. Next, using lentiviral vectors encoding shRNA for HDAC6, we generated stable knockdown clones of HDAC6 (HDAC6KD). In contrast to HDAC6 overexpression, HDAC6KD cells produced less IL-10 both at the mRNA and protein levels. Further analysis of HDAC6KD clones revealed elevated expression of the co-stimulatory molecule B7.2. Functionally, LPS-stimulated HDAC6KD cells were better able to activate anti-HA (hemagglutinin-influenza) transgenic CD4+ T cells to produce IL-2 and IFN-γ. More relevant to the tumor setting, anti-HA CD4+ anergic T cells isolated from animals bearing HA-expressing A20 B-cell lymphoma regained their ability to produce IL-2 and IFN-γ when cultured in vitro with HDAC6KD cells in the presence of cognate HA-antigen. These same T-cells remained anergic when cultured with control APCs displaying normal levels of HDAC6. Mechanistically, we have found that APCs lacking HDAC6 display a significant abrogation of STAT3 activation in response to LPS stimulation. Given the demonstration that HDACs, other than HDAC6, can regulate STAT3 activity3, we are actively investigating whether the effect we have observed is a result of direct HDAC6-STAT3 interaction or a diminished autocrine signaling through IL-10. Taken together, through abrogation of two well-known tolerogenic mechanisms in APCs, IL-10 and STAT3, HDAC6 inhibition represents an attractive therapeutic approach to overcome tumor-induced T-cell anergy and tip the balance towards effective anti-lymphoma immunity.

1. Cheng F., et al. Immunity 2003 Sep;19(3):425-36.

2. Villagra A., et al. Nat Immunol. 2009 Jan;10(1):92-100.

3. Yuan Z., et al. Science. 2005 Jan 14;307(5707):269-73.

Disclosures:

No relevant conflicts of interest to declare.
A Systematic Review of Diagnostic Features and Treatment Outcomes In Hemophagocytic Syndromes In the Adult Population
Authors: Gob Alan , Lazo-Langner Alejandro
Published: October 2019

Abstract 1497

Background:

The hemophagocytic syndrome (HPS) is a rare but serious condition with a poor prognosis. The syndrome is characterized by hypercytokinemia, macrophage proliferation, and systemic inflammation. In children, the disorder is most commonly a primary dysregulation of the immune system, however, in adults hemophagocytic syndrome is generally a secondary process that is associated most frequently with viral infections, solid or hematologic malignancies (especially lymphoma), and systemic inflammatory conditions. Diagnostic criteria have been extrapolated from experience in children, and prognostic factors have not been well described. It has not been established whether the adult form of HPS is identical to the secondary form in children, or a distinct clinical entity. Additionally, management focuses on controlling the underlying cause, but it is unclear whether further benefit is derived from immunosuppression; optimal management strategies remain unclear.

Objectives:

To document the clinical, biochemical, and histopathological features associated with HPS in adults. To describe treatments used and document clinical outcomes.

Methods:

We performed a systematic review of all case reports or case series of hemophagocytic syndrome between January 1990 and July 2009. We searched Medline (Ovid), Embase (Ovid), Imbiomed, Artemisa, and Lilacs for cases and case series of hemophagocytic syndrome in adults. We included in our analysis those studies that a) clearly described patient demographics, b) described the underlying diagnosis, c) provided sufficient information on diagnostic criteria, d) met the HLH Study Group diagnostic criteria, e) provided sufficient information on therapeutic approaches, and f) provided follow up and/or survival data. For each included case, we extracted demographic, biochemical, clinical, and therapeutic data. Statistical analysis was done using χ2 or unpaired Student's t tests. Potential predictors of mortality were explored using logistic regression.

Results:

A total of 85 cases met our selection criteria. Characteristics are summarized in the table. Among the 44/85 (51.7%) patients that died, mean survival time was 30 days (Figure). Only age, ALP, and –marginally- underlying etiology were significantly different between groups. Other than age, none of the variables in the multivariate analysis were found to correlate significantly with survival.

Conclusion:

Hemophagocytic syndrome in adults carries a high mortality rate. Age may be a predictor of survival. Prospective collaborative studies on HPS in adults are needed to further define the diagnostic characteristics, prognosis and treatment outcomes.

Figure. Kaplan-Meier plot of survival in adult patient with HPS.

Disclosures:

No relevant conflicts of interest to declare.
Race, Access to Care, and Outcome After Autologous Hematopoietic Stem Cell Transplantation for Multiple Myeloma.
Authors: Alexander Erin T , Stuart Robert K , Costa Luciano J
Published: October 2019

Abstract 1509

Background:

Autologous hematopoietic stem cell transplantation (AHSCT) is an essential modality in the management of younger, newly diagnosed multiple myeloma (MM) patients. Some reports have indicated inferior survival for African-American (AA) patients following diagnosis of MM, when compared to Caucasian (Ca) patients. We hypothesized that previously claimed racial differences in outcome may reflect disparity in access to care and might be negated by timely, standard, and uniform treatment.

Methods:

We reviewed a mature database of MM patients undergoing AHSCT at a single institution to describe and compare overall survival from diagnosis and from transplantation in AA and Ca patients. Additionally, we described time from diagnosis to transplantation in both cohorts as an additional surrogate of access to care.

Results:

Between August 1996 and July 2010, 128 patients underwent a first AHSCT for MM at the institution. Fifty-three patients (41%) were AA and 75 (59%) were Ca. Median age at the time of diagnosis was 54.7 years for AA and 58.3 for Ca. Sixty-six (52%) of patients were female. One hundred twenty-six (98%) patients received conditioning chemotherapy with Melphalan 200 mg/m2. Median interval from diagnosis to transplant was 10 months (IQR 7.1–15.9) for AA and 9.2 months (IQR 6.7–15.6) for Ca (p=0.23). Median overall survival for the entire group was 63 months (95% C.I. 51–76) from time of diagnosis and 51.8 months (95% C.I. 39–63) from AHSCT. There was no difference in overall survival from time of AHSCT between AA and Ca, respectively 62.6 (95% C.I. 30.9–94.4) and 51.8 months (95% C.I. 33.5–70.3, Log-rank test P=0.76). Similarly, no significant difference was seen in overall survival from time of diagnosis between AA and Ca, respectively 70.4 (95% C.I. 29.6–111.3) and 59.3 months (95% C.I. 46.8–71.8), as displayed in the Figure (Log-rank test P=0.36).

Conclusion:

These findings suggest that previously reported differences in outcome between AA and CA patients with MM are related to disparities in access to healthcare and not intrinsic biological differences.

Disclosures:

No relevant conflicts of interest to declare.
Essential Function of PDGF-B Signaling In the Placental Microenvironment In Protecting Hematopoietic Progenitors From Erythroid Differentiation.
Authors: Chhabra Akanksha , Lechner Andrew J , Acharya Asha , Ueno Masaya , Van Handel Ben , Tallquist Michelle , Mikkola Hanna
Published: October 2019

Abstract 1559

The goals of hematopoiesis during embryogenesis are two-fold, to rapidly produce red blood cells to support the survival and development of the embryo, and to establish a pool of undifferentiated hematopoietic stem cells (HSC) for postnatal life. These goals are achieved by segregation of fetal hematopoiesis in multiple waves that are executed in distinct anatomical sites and microenvironmental niches. However, the microenvironmental cues that promote “stemness” vs. differentiation remain poorly understood. The placenta is a recently discovered hematopoietic organ that supports HSC generation and expansion without promoting their differentiation. The placental HSC pool is thought to reside in the placental labyrinth, which is comprised of an intricate vascular network surrounded by trophoblasts. So far, it has been unknown how a disruption of the integrity of the placental vascular labyrinth affects fetal hematopoiesis. The structure of the placental labyrinth is compromised in embryos that lack components of PDGF-B signaling. PDGF-B-/- and PDGF-Rβ-/- (receptor for PDGF-B) embryos display dilation of fetal blood vessels and reduction of trophoblast cells in the placental labyrinth. Our studies revealed that loss of PDGF-B signaling alters the unique placental hematopoietic microenvironment, resulting in active erythropoiesis in the placenta. The unexpected erythropoiesis in the placenta exhibited the same hallmarks of normal definitive erythropoiesis observed in the fetal liver as confirmed by flow cytometry for erythroid markers, morphological analysis and association of erythroblasts with macrophages. Interestingly, deletion of PDGF-Rβ in hematopoietic cells by using a Tie2-Cre strain did not induce the differentiation of placental hematopoietic progenitors into erythroid cells, implying that the ectopic definitive erythropoiesis results from the lack of PDGF-B signaling in the placental microenvironment rather than in the hematopoietic cells themselves. Our studies revealed that the erythroid differentiation in placentas of PDGF-B-/- embryos was induced by marked upregulation of Epo in placental trophoblasts. Strikingly, lentiviral overexpression of Epo specifically in the placental trophoblasts was sufficient to convert the placenta into an ectopic erythropoietic organ. These data reveal a critical function of PDGF-B signaling in protecting the integrity of the placental microenvironment that is required for preventing hematopoietic progenitors from differentiation during their residence in the placenta. Furthermore, these studies highlight the placenta as a versatile hematopoietic organ that supports HSC development during normal pregnancy but can be recruited as a site for definitive erythropoiesis during pathological conditions.

Disclosures:

No relevant conflicts of interest to declare.
Chd7 Is a Cell Autonomous Regulator of Chromatin In Hematopoietic Stem Cells.
Authors: Huang Hsuan-Ting , Kathrein Katie , Huang Yue-Hua , Gitlin Zachary , Barton Abby , Song Anhua , Zhou Yi , Zon Leonard I.
Published: October 2019

Abstract 1596

Hematopoietic stem cells (HSC) are specified during embryogenesis, and the induction process involves not only transcription factors but also epigenetic factors that modulate chromatin to regulate the hematopoietic transcriptional programs. Here, we performed a reverse genetic screen to identify all the chromatin factors that are required for HSC induction in zebrafish. The zebrafish homologs of 350 human chromatin factors were identified by reciprocal BLAST and knocked down by injecting morpholinos designed against each homolog into the single cell embryo. Morphants were then analyzed for changes in blood formation by in situ hybridization for β-globin e3 expression in primitive erythrocytes at 16 somite stage and for c-myb and runx1 in definitive stem cells at 36 hours post fertilization. From the screen, we have identified known regulators of hematopoiesis such as bmi1, in which knock down results in loss of stem cell formation. We have also identified one novel HSC regulator chd7. Chd7 is a member of the chromodomain helicase DNA-binding domain family that functions at gene enhancer elements and in ribosomal RNA synthesis. Zebrafish embryos injected with chd7 morpholino had higher levels of β-globin e3 and c-myb/runx1 expression. Additional markers such as scl, gata1, fli1, and lmo2 were also upregulated, although vascular markers flk1 and ephrinB2 were downregulated. Early mesodermal markers eve1 and ntl expression appeared normal, suggesting that the effects of chd7 knock down occurs when the mesodermal precursor cell population becomes an HSC. Transplants of chd7 deficient Tg(c-myb:GFP) blastomeres into Tg(lmo2:DsRed) blastulas resulted in more chimeric embryos compared to controls, demonstrating that the phenotype is cell autonomous. In humans, haploinsufficiency for CHD7 is the main cause of CHARGE syndrome, and it has been recognized more recently that these patients are immunodeficient, though the etiology remains unknown. Our studies indicate a new role for chd7 in hematopoiesis in which it functions to repress HSC formation during embryogenesis.

Disclosures:

Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
The Novel Platelet Activation Receptor CLEC-2 Regulates Blood/Lymphatic Vessel Separation by Inhibiting Proliferation and Migration of Lymphatic Endothelial Cells
Authors: Osada Makoto , Inoue Osamu , Ding Guo , Hirashima Masanori , Suzuki-Inoue Katsue , Ozaki Yukio
Published: October 2019

Abstract 160

CLEC-2 has been described recently as playing crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis. The snake venom rhodocytin is known as a strong platelet activator, and we have shown that this effect is mediated by CLEC-2. Podoplanin, which is expressed on the surface of tumor cells, is an endogenous ligand for CLEC-2 and facilitates tumor metastasis by inducing platelet aggregation. Mice deficient in podoplanin, which is also expressed on the surface of lymphatic endothelial cells, show abnormal patterns of lymphatic vessel formation. We have recently reported on the generation and phenotype of CLEC-2-deficient mice. These mice are lethal at the embryonic/neonatal stages associated with disorganized and blood-filled lymphatic vessels and severe edema, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, CLEC-2 is expressed in both platelets and neutrophils in mice and whether CLEC-2 in platelets, but not that in other cells, plays a role in the separation has not been directly proved yet. Moreover, the mechanism how CLEC-2 regulates of blood/lymphatic vessel separation has not been elucidated to date. In the present study, we found that specific deletion of CLEC-2 from platelets mediated by PF4-Cre conferred the defect of blood/lymphatic vessel separation, identifying that CLEC-2 expressed in platelets is required to regulate lymphatic vascular development. Tube formation of lymphatic endothelial cells, but not that of vascular endothelial cells, was inhibited in the presence of platelets. Further, proliferation and migration of lymphatic endothelial cells, but not those of vascular endothelial cells, were inhibited by CLEC-2+/+ platelets, but not by CLEC-2−/− platelets. We propose that CLEC-2 regulates blood/lymphatic vessel separation by inhibiting proliferation and migration of lymphatic endothelial cells through the interaction between CLEC-2 in platelets and podoplanin in lymphatic endothelial cells. CLEC-2 may be a novel therapeutic target protein for lymphatic metastasis of tumor if the interaction of CLEC-2 and podoplanin also regulates lymphangiogenesis by tumor cells.

Disclosures:

No relevant conflicts of interest to declare.
Endoglin Identifies the First Wave of Hematopoietic Progenitors During Embryogenesis.
Authors: Borges Luciene , Iacovino Michelina , Baik June , Mayerhofer Tim , Letarte Michelle , Perlingeiro Rita
Published: October 2019

Abstract 1600

Endoglin (Eng), an ancillary receptor for several members of the Transforming Growth Factor (TGF)-beta superfamily, plays a critical role in early development. Eng-/- embryos dye around 10.5 dpc due primarily to vascular and cardiac abnormalities. Yolk sacs (YS) of 9.5 dpc Eng-/- embryos present abnormal vasculature and anemia. However, the mechanism by which endoglin leads to an anemic phenotype is unclear. Using in vitro differentiation of Eng-/- ES cells, we have previously demonstrated that endoglin is required for proper hemangioblast and primitive hematopoietic development. To test the hypothesis that endoglin might have a direct effect on blood formation, here we investigated the role of endoglin during hematopoiesis in vivo. We first evaluated YS from 9.5 dpc Eng-/- embryos and observed significantly reduced numbers of hematopoietic colony-forming cells (CFCs), in particular GEMMs and BFU-Es, when compared to both Eng+/− and Eng+/+ embryos. Real-time PCR analysis revealed decreased expression of embryonic globin, Gata-1, and SCL in the mutant YS. We then investigated the function of endoglin in wild-type mice by sorting the endoglin positive and negative population from E7.5 to 9.5 CD1 mouse embryos. When cultured on the stromal cell line OP9 in the presence of hematopoietic cytokines, only cell cultures derived from the Eng+ fraction gave rise to hematopoietic progenitors, as evidenced by colony assays and FACS analyses. Engneg cells did not produce any hematopoietic colonies. We then separated cells into 4 fractions using Eng and Flk-1, and found that from E7.5-9.5, the hematopoietic progenitor activity resided in the Eng+Flk-1+ double-positive fraction. FACS and microarray analyses of the 4 fractions in E7.5 embryos demonstrated that the Eng+Flk-1+ fraction highly expresses both endothelial and hematopoietic markers, including VE-Cadherin, Tie-2, PECAM1, Gata-1, SCL, and Lmo2, indicative of hemogenic endothelial potential. Taken together, our findings demonstrate that endoglin marks the first wave of hematopoietic progenitors during development, pointing to a defined role for endoglin in the specification of YS hemogenic endothelium as early as E7.5, in addition to its well defined role in vascular branching and remodeling.

Disclosures:

No relevant conflicts of interest to declare.
Ga13 and Talin Coordinate integrin Inside-out and Outside-In Signaling by “time-sharing” of b3 cytoplasmic Domain
Authors: Gong Haixia , Du Xiaoping
Published: October 2019

Abstract 162

The bidirectional signaling of integrins mediates cell adhesion, spreading, retraction and migration. The binding of talin and kindlins to the cytoplasmic domain of integrin b3 subunit transmits inside-out signals to induce integrin activation. Ligand-induced outside-in signaling requires the binding of a G protein subunit, Ga13, and a tyrosine kinase, c-Src, to the b3 cytoplasmic domain. It is unclear how the short cytoplasmic domain of b3 accommodates these molecules and allows coordinated bidirectional signaling. Here we show that Ga13 and talin are mutually exclusive in binding to b3 both in vivo and in vitro. Increasing expression level of talin head or full-length talin in CHO123 cell decreases Ga13-b3 association. Ga13 also competes with talin head for GST-b3 binding in purified binding system. More importantly, talin is associated with b3 only in inside-out signaling during platelet aggregation. Following integrin ligation, however, Ga13 binds to b3, replacing talin. The Ga13 binding site located between K729-T741 within the talin binding region. However, Ga13 binding and signaling require a distinct ExE733 motif (EEE in b3) conserved in most integrin b subunits that is not required for talin binding but flanked by talin binding sequences on both sides. Interference of Ga13 binding to integrin b3 cytoplasmic domain by myristorylated b3 peptide (Myr-EEERA735) or by point-mutating the EEE motif to AAA selectively inhibits outside-in signaling, thus inhibited cell spreading on fibrinogen, accelerated RhoA activation and inhibited c-Src activity. But they have no effect on talin-dependent inside-out signaling judged by fibrinogen binding assay. In conclusion, our data suggest that the timed share of binding sites in b3 between Ga13 and talin coordinates bidirectional integrin signaling.

Disclosures:

No relevant conflicts of interest to declare.
atpif1 regulates Mitochondrial Heme Synthesis In Developing Erythroid Cells
Authors: Shah Dhvanit I , Takahasi-Makise Naoko , Schultz Iman , Pierce Eric L , Li Liangtao , Vogin Guillaume , Brigham Benjamin S , Zhou Yi , Brugnara Carlo , Campanella Michelangelo , Danial Nika , Ward Diane M , Kaplan Jerry , Paw Barry H
Published: October 2019

Abstract 163

Iron plays a key role as a cofactor in many fundamental metabolic processes, which require heme synthesis and Fe/S cluster assembly in the mitochondria. Defects in the transport of iron into the mitochondria would lead to anemias due to a deficiency in heme and hemoglobin synthesis. Here we describe a zebrafish genetic mutant, pinotage (pnttq209), which exhibits a profound hypochromic, microcytic anemia. Erythrocytes from pnt mutants have a defect in hemoglobinization and decreased red cell indices (mean corpuscular volume and hemoglobin content, hematocrit, hemoglobin concentration). Through positional cloning, we showed that the mitochondrial ATPase Inhibitory Factor 1 (atpif1), which regulates the inner mitochondrial membrane potential, is the gene disrupted in pnt. The identity of the pnt gene was verified by: (a) decreased atpif1 steady-state mRNA in pnt mutants, (b) phenocopying the anemia with anti-sense atpif1 morpholinos, (c) functional complementation of the anemia with atpif1 cRNA, and (d) a genetic polymorphism in the 3'UTR co-segregating with the mutant phenotype that destabilizes the atpif1 mRNA. Consistent with the conserved function of atpif1 in higher vertebrates, the silencing of the murine ortholog of atpif1 in Friend mouse erythroleukemia (MEL) cells showed a defect in hemoglobinization by o-dianisidine staining and reduction of 59Fe incorporation into heme in 59Fe-metabolically labeled cells. Moreover, Atpif1 knockdown destabilizes their mitochondrial membrane potential and volume. Therefore, the identification of atpif1 in pnt functionally demonstrates the role of atpif1 in regulating the proton motive gradient across the inner mitochondrial membrane for mitochondrial iron incorporation in heme biosynthesis. These results uncover a novel hematopoiesis-related function of atpif1, which will directly contribute to our understanding and potential treatment of human congenital and acquired anemias.

Disclosures:

No relevant conflicts of interest to declare.
p90RSK2 Is Essential for FLT3-ITD-, but Dispensable for BCR-ABL-Induced Myeloid Leukemia
Authors: Elf Shannon , Chung Tae-Wook , Blevins Dean P. , Williams Ifor , Khoury H. Jean , Taunton Jack , Kang Sumin
Published: October 2019

Abstract 1716

p90 ribosomal S6 kinase 2 (p90RSK2) is a serine/threonine kinase that plays an active role in diverse cellular processes, including gene expression, cell proliferation, and survival. We previously demonstrated that RSK2 signaling plays a key role in the pathogenesis and disease progression of leukemogenic FGFR3 associated hematopoietic malignancies, including FGFR3-expressing t(4;14) multiple myeloma and TEL-FGFR3-expressing t(4;12)(p16;p13) peripheral T cell lymphoma. In this study, we found that p90RSK2 is commonly activated in diverse leukemia cell lines expressing different leukemogenic tyrosine kinases, including K562 (BCR-ABL), Molm14 and Mv4;11 (FLT3-ITD), HEL (JAK2 V617F), and EOL-1(FIP1L1-PDGFR alpha). We next examined the role of RSK2 in myeloid transformation induced by BCR-ABL and FLT3-ITD due to their high frequency of occurrence in CML and AML, respectively. Interestingly, although RSK2 is activated by BCR-ABL in both stably transduced Ba/F3 cells and K562 human leukemia cells, we found that genetic deficiency of RSK2 does not affect the pathogenesis or disease progression of myeloproliferative disease induced by BCR-ABL in a murine bone marrow transplant (BMT) model using wild type or RSK2-/- donor bone marrow cells. This finding suggests that RSK2 is dispensable for BCR-ABL induced myeloproliferative disease. Moreover, targeting RSK2 by treatment with potent and highly specific RSK inhibitor fmk did not effectively induce apoptosis in K562 human leukemia cells expressing BCR-ABL, or in primary leukemia cells from BCR-ABL positive CML patients. In contrast, we found that targeting RSK2 may represent an effective therapy to treat patients with FLT3-ITD positive AML. Treatment with fmk induced significant apoptotic cell death in Molm14 and Mv4;11 human leukemia cells expressing FLT3-ITD, as well as in primary leukemia cells from FLT3-ITD positive AML patients. In consonance with these results, FLT3-ITD induced T-cell lymphoma in a BMT assay using RSK2-/- donor bone marrow cells, phenotypically distinct from the myeloproliferative disease induced by FLT3-ITD using wild type donor bone marrow cells. These results suggest that RSK2 is required for FLT3-ITD induced hematopoietic transformation, likely playing a role in pathogenesis and lineage determination. Together these findings suggest that the role of RSK2 in hematopoietic transformation may depend on different upstream oncogenic signals mediated by different leukemogenic tyrosine kinases. Our data also demonstrate that RSK2 may represent an alternative therapeutic target in the treatment of FLT3-ITD positive leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Epstein-Barr Virus and Kaposi's Sarcoma Herpesvirus Lytic Cycle Induction with Bortezomib Is a Response to ER Stress
Authors: O'Farrell Courtney , Shamay Meir , Kalu Nene , DuFresne Andrew , Biswas Sunetra , Ambinder Richard F.
Published: October 2019

Abstract 1736

EBV and KSHV are associated with a variety of lymphoid malignancies. Bortezomib, a proteasome inhibitor, is among the most potent activators of EBV and KSHV lytic infection and is active at nanomolar concentrations. Studying EBV Burkitt's cell lines, KSHV primary effusion lymphoma cell lines and EBV myeloma cell lines, we found that lytic gene expression is a class effect shared by other proteasome inhibitors, such as MG-132 and epoxomicin. Proteasome inhibition results in IKB stabilization and inhibition of NFKB activity and this effect has been implicated in many of the effects of bortezomib. We studied the promoters of EBV and KSHV immediate early viral protein reporters (EBV Zta and KSHV Rta) and found that IKB super-repressor did not activate promoter reporters suggesting that other pathways must be important in activation. Therefore, we sought to better understand the drug's impact on viral gene expression in virus-associated tumor cells. ER stress has been implicated in bortezomib's antitumor effects. Thapsigargin and tunicamycin are classic activators of ER stress that do not act through proteasomal inhibition. These agents were found to be potent activators of the EBV and KSHV lytic cycle. Previous studies have identified C/EBP family members (C/EBP alpha and beta) as activators of EBV and KSHV immediate early gene expression. In other studies, C/EBP family members (C/EBP beta, CHOP10) have been implicated in regulating the ER stress response. We found that bortezomib, thapsigargin and tunicamycin increased levels of the activating C/EBP beta LAP isoform as assessed by immunoblot and by real-time RT-PCR. Treatment also led to increase in ATF4, XBP1(s), CHOP10, and ATF6 cleavage, all consistent with induction of the ER stress response. Additionally, we showed that treatment with bortezomib increased C/EBP beta binding to previously characterized binding sites in the Zta promoter and expression of C/EBP beta LAP isoform was sufficient to activate EBV immediate early lytic promoters. Finally, we demonstrated that in tumor cell lines with C/EBP beta silenced by doxycycline regulated siRNA, induction of EBV lytic induction by bortezomib and thapsigargin was blocked. These results demonstrate that both human lymphoma associated herpesviruses (EBV and KSHV) are activated into lytic cycle by bortezomib and that these effects are mediated through ER stress pathways and specifically involve C/EBP beta.

Disclosures:

No relevant conflicts of interest to declare.
Herpesviridae Viral Infections Following Rituximab Combined Chemotherapy In Patients with Diffuse Large B Cell Lymphoma
Authors: Lee Ho Sup , Park Lee Chun , Shin Seong Hoon , Ye Byeong Jin , Song Moo-Kon , Oh Sung Yong , Lee Sang Min , Lee Won Sik , Kim Yang Soo
Published: October 2019

Abstract 1748

Backgroud:

Herpesviridae family includes herpes simplex virus, varicella zoster virus, Epstein-Barr virus, and cytomegalovirus, etc. Herpesviridae viral infections(HVIs) can lead to serious complications including dissemination, secondary infection, bacterial superinfection in patients with diffuse large B cell lymphoma (DLBL) undergoing rituximab combined chemotherapy. But there was no consensus on the dose and duration of antiviral agents prophylaxis in DLBL undergoing chemotherapy.

Method:

Three hundred seven patients were newly diagnosed with DLBL and received chemotherapy between June 2004 and August 2009. HVIs was confirmed based on clinical diagnosis, serologic test or pathologic diagnosis. The characteristics of the patients were as follows: the median age was 58years (range 15–89 years) with a female-to-male ratio of 171:136. All patients received chemotherapy with rituximab combined cyclophosphamide, adriamycin, vincristine, and prednisolone.

Results:

Forty three patients (14.0%) developed HVIs at a median of 6.63 months (range 0.37 – 51.33 months) after initial chemotherapy. The estimated cumulative incidence rates (CIR) of HVIs were 10.36%, 16.18% and 18.44% at 1years, 3years, and 5years in all patients. In univariate analysis, 5years CIR of HVIs were 14.64% vs 28.05% in low and low-intermediate IPI risk vs in high-intermediate and high IPI risk (p=0.0003), 34.07% vs 8.29% in cumulative dose of steroid ≥ 3000 mg vs < 3000mg (p < 0.001), 29.58% vs 13.56% in duration of chemotherapy ≥ 6 months vs < 6 months (p=0.0250), 38.32% vs 13.41% in relapse vs none-relapse (p=0.0056), 12.83% vs 43.80% in receiving first line chemotherapy vs second line and more than second line chemotherapy (p=0.0008), and 24.90% vs 15.49% in prolonged neutropenic fever vs none-neutropenic fever (p=0.0239). In multivariate analysis, the results confirmed 5 variables as independent predictive factors for the high IPI risk (P < 0.001, hazard ratio (HR): 3.236, 95% confidence interval (CI) 1.636–6.400), prolonged neutropenic fever (P = 0.029, HR: 0.484, 95% CI 0.253–0.927), longer duration of chemotherapy (P = 0.006, HR: 0.212, 95% CI 0.069–0.648), high cumulative dose of steroid (P = 0.008, HR: 2.889, 95% CI 1.319–6.328), and receiving autologous stem cell transplantation (P < 0.001, HR: 0.146, 95% CI 0.057–0.371).

Conclusion:

High IPI risk, prolonged neutropenic fever, longer duration of chemotherapy, high cumulative dose of steroid and receiving autologous stem cell transplantation were seemed to be high risk for HVIs in patients with DLBL undergoing rituximab combined chemotherapy.

Disclosures:

No relevant conflicts of interest to declare.
An Effective Salvage Treatment Using Rituximab, Ifosfamide, Etoposide, Cytarabine, and Dexamethasone (R-IVAD) for Patients with Relapsed or Refractory Aggressive B-Cell Lymphoma
Authors: Miura Katsuhiro , Takei Kazuhiro , Kobayashi Sumiko , Kobayashi Yujin , Tanaka Toshitake , Kurita Daisuke , Iriyama Noriyoshi , Inoue Yasuyuki , Hatta Yoshihiro , Kura Yoshimasa , Sawada Umihiko , Takeuchi Jin
Published: October 2019

Abstract 1759

Background

The prognosis of patients with relapsed or refractory aggressive B-cell lymphoma after the conventional first line chemotherapy is still disappointing. Although several rituximab combined salvage regimens were reported, the optimal treatment has not yet been established. We therefore evaluated the efficacy and toxicity of a non-anthracycline based salvage regimen, consisting of rituximab, ifosfamide, etoposide, cytarabine and dexamethasone (R-IVAD) in patients with relapsed or refractory aggressive B-cell lymphoma.

Patients and methods

Patients with relapsed or refractory CD20-positive diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma grade 3 (FL G3) were consecutively enrolled in this treatment. R-IVAD consisted of rituximab (375 mg/m2 on day -2), ifosfamide (1500 mg/m2 on day 1–5), etoposide (150 mg/m2 on day 1–3), cytarabine (100 mg/m2 on day 1–3) and dexamethasone (40 mg/body on day 1–3). Treatment was given every 3 weeks up to a total of 3 courses with support of granulocyte colony stimulating factor. For responders under 65 years old with good performance status (PS), we planed peripheral stem cell collection after the third cycle of R-IVAD and consolidating high-dose chemotherapy (HDC) with cycrophosphamide (60 mg/kg on day -7,-6), etoposide (500 mg/m2 on day -6, -5, -4) and ranimustine (250 mg/m2 on day -3, -2) followed by autologous stem cell transplantation (ASCT).

Results

Thirty-two patients (25 DLBCL and 7 FL G3) with a median age of 64 years old (range 38–79) participated in this study and received an average of 2.6 cycles of R-IVAD. There were 21 relapsed and 11 primary refractory patients. The overall response (OR) and the complete response (CR) rate were 72% and 56% respectively. The OR rate of relapsed patients was significantly higher than that of primary refractory patients (86% vs. 45%, p=0.035). With a median 16 months (range 2–99) of follow up, the 2-year overall survival (OS) and the event-free survival (EFS) for all patients were 55% and 36% respectively. Of the 11 eligible patients, 10 successfully proceeded to HDC/ASCT with an average of 5.5 × 106/kg of harvested CD34-positive cells, resulting in the 2-year OS of 90%. No treatment related death was observed during the investigation. Multivariate analysis revealed that the age > 60 years, PS ≥ 2, extranodal sites ≥ 2, and primary refractory disease were independently associated with worse survival.

Conclusion

R-IVAD regimen, which efficiently mobilizes peripheral stem cells, is a safe and efficacious alternative for patients with relapsed or refractory aggressive B-cell lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
Pooled Analysis of Aids-Malignancy Consortium (AMC) Trials Evaluating Rituximab Plus Either CHOP or Infusional EPOCH Chemotherapy In HIV-Associated Non-Hodgkin's Lymphoma
Authors: Barta Stefan K , Lee Jeannette Y , Sparano Joseph A , Kaplan Lawrence D , Noy Ariela
Published: October 2019

Abstract 1797

Background:

Two consecutively performed randomized studies by the AMC evaluating chemoimmunotherapy for the treatment of HIV-associated NHL include AMC010 (Kaplan LD et al, Blood 2005: concurrent Rituximab [R] + CHOP vs. CHOP, N=150) and AMC034 (Sparano JA et al, Blood 2010: concurrent R+EPOCH vs. Sequential EPOCH → R; N=106). In AMC010, the addition of Rituximab to CHOP was associated with an increased risk of infectious death (15% vs. 2%, p=0.04) without a significant improvement in complete response (CR) rate (58% vs.47%; p=0.15), event free survival (EFS), or overall survival (OS). In AMC034, the CR rate met its primary efficacy endpoint in the concurrent arm (73%; 95% confidence intervals [CI] 58%, 85%) but not the sequential arm (55%; 95% CI 41%, 68%).

Methods:

We performed a pooled analysis of these two consecutive trials including patients treated with R-CHOP and concurrent R-EPOCH in order to determine the influence of the age-adjusted International Prognostic Index (aaIPI), CD4 count (< 100/uL vs. ≥ 100/uL), and treatment (CHOP vs. EPOCH) as variables on outcomes EFS, OS and CR.

Results:

The characteristics and outcomes of the study populations are shown in table 1. Patients treated with R-EPOCH tended to have better outcomes in both the low and high risk IPI groups. In a multivariate analysis that included pooled data from both consecutive studies, features that were significantly associated with improved EFS, OS, and CR rate included low aaIPI score and baseline CD4 count of at least 100/ul. Additionally patients treated with concurrent R-EPOCH exhibited improved EFS and OS even when adjusted for prognostic covariates including aaIPI score and CD4 count (table 2).

Conclusions:

These findings suggest that treatment outcomes may be superior with concurrent R-EPOCH compared with R-CHOP, and support the design of an ongoing phase III trial comparing concurrent R-EPOCH with R-CHOP in immunocompetent patients with diffuse, large B-cell lymphoma (NCT00118209). This analysis provides additional level 2 evidence supporting the use of concurrent R-EPOCH in patients with HIV-associated lymphoma. Acknowledgments: This study is presented on behalf of the AIDS Malignancy Consortium.

Disclosures:

No relevant conflicts of interest to declare.
Rituximab Maintenance Treatment for a Maximum of 5 Years In Follicular Lymphoma: Safety Analysis of the Randomized Phase III Trial SAKK 35/03
Authors: Taverna Christian J. , Bassi Simona , Hitz Felicitas , Mingrone Walter , Pabst Thomas , Cevreska Lidija , Giglio Auro del , Vorobiof Daniel A. , Simcock Mathew , Ghielmini Michele
Published: October 2019

Abstract 1802

Background:

Rituximab maintenance has been shown to be effective in patients with follicular lymphoma. The optimal duration of maintenance remains unknown.

Methods:

We prospectively registered 270 patients with untreated, chemotherapy resistant or relapsed follicular lymphoma. All patients received rituximab induction consisting of 4 weekly doses (375 mg/m2). Responding patients (PR and CR) were randomized to a short maintenance consisting of four doses of rituximab (375 mg/m2) every two months (arm A) or prolonged maintenance consisting of rituximab every two months for a maximum of five years or until disease progression or unacceptable toxicity (arm B). Primary endpoint is event-free survival. Here we present the safety analysis results after a median long-term maintenance period of 3.3 years.

Results:

From October 2004 to November 2007 165 patients were randomized, 82 in arm A and 83 in arm B. The median follow-up time is 3.2 years for arms A and B combined. While receiving maintenance therapy a total of 899 hematological and non-hematological adverse events were observed, 28 of grade 3 and 6 of grade 4. After randomization five patients experienced subsequent cancers. Seven grade 3 and 4 infections were reported. Two grade 3 infections occurred after 2 years of maintenance. Grade 3 and 4 neutropenia occurred in 6 (3.6 %) patients, decreased levels of IgG were observed in 24 (14.6 %) patients. In arm B, maintenance was stopped due to unacceptable toxicity in 2 patients after 16 and 42 months respectively and due to subsequent breast cancer in 1 patient after 20 months. One patient died 4 months after randomization because of ileus and consecutive peritonitis, considered to be unrelated to therapy. Sixty-three patients are on maintenance for two or more years of which 48 patients are on for three or more years. Two patients have completed the 5 years of maintenance.

Conclusions:

Rituximab maintenance beyond two years is feasible without evidence for increased toxicity. However, close follow up of patients under prolonged rituximab maintenance is still necessary. The trial has been closed for accrual but there are still patients on treatment.

Disclosures:

Taverna: Roche: Membership on an entity's Board of Directors or advisory committees. Ghielmini:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau.
Variations of Noxa Levels Caused by p38 MAPK Signaling Result In Heterogeneous Sensitivity to ABT-737 In CD40L-Stimulated CLL Cells
Authors: Tromp Jacqueline M , Geest Christian R , Elias Judith , van Laar Jacoline , Van Oers Marinus H.J. , Eldering Eric
Published: October 2019

Abstract 1825

Chronic lymphocytic leukemia (CLL) is characterized by relentless relapses. It is postulated that this might be due to microenvironment-induced resistance to cytotoxic agents. Previously we have shown that in vitro CD40 stimulation of peripheral blood derived from CLL patients can to a certain extent mimic the lymph node (LN) microenvironment and result in resistance to cytotoxic drugs. This is correlated with CD40-induced changes in apoptosis regulating Bcl-2 family members. The BH3 mimetic drug ABT-737 antagonizes the anti-apoptotic molecules Bcl-XL and Bcl-2, but not Mcl-1 or Bfl-1. At present it is unknown whether patient-specific variations in expression of apoptosis regulating proteins occur and to what extent they correlate with sensitivity or resistance to ABT-737.

In the present investigation we assessed the variability in response to ABT-737 in CD40 stimulated CLL cells. Although in all tested CD40-stimulated CLL patients Mcl-1 and Bfl-1 levels increased, there was a heterogeneous response in cell death to ABT-737 when used as a single agent. Notably, time course analyses showed variations in Noxa levels upon CD40 triggering, which differed between CLL patients. High Noxa/Mcl-1 ratios correlated with sensitivity to ABT-737. This was confirmed by Noxa knockdown as well as Mcl-1 and Bfl-1 overexpression, which resulted in resistance to ABT-737. To determine the signaling pathways involved in Noxa regulation, CLL cells were stimulated with CD40L in the presence of various kinase inhibitors. We observed that Noxa levels were specifically inhibited by the p38 MAP kinase inhibitor SB203580. Preliminary data indicate that after CD40 stimulation, phosphorylation of p38 decreased over time, correlating with a decrease in Noxa levels. Importantly however, a minority of CD40 stimulated CLL samples were sensitive to ABT-737 and showed high levels of phosphorylated p38 and increasing Noxa levels over time.

In conclusion, we demonstrate that following CD40 triggering of CLL cells the p38 MAPK signaling pathway regulates Noxa levels and that patient-specific variations in this pathway contribute to the responsiveness to ABT-737. These findings raise the question whether assessment of p38 MAPK activation status and Noxa levels in vivo could predict the response of CLL patients to ABT-737. Moreover our findings offer possible new targets for the treatment of CLL.

Disclosures:

No relevant conflicts of interest to declare.