bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of Hox Proteins and Interferon Regulatory Factors in Myeloid Differentiation.
Authors: Eklund Elizabeth A.
Published: October 2019

During myelopoiesis, differentiating phagocytes develop functional competence and undergo proliferation arrest and eventual programmed cell death. This process involves transcriptional regulation of genes which mediate the innate immune response, mitotic arrest, and apoptosis. A number of transcription factor families play important roles in regulating such genes, including Hox proteins and interferon regulatory factors (IRFs). Disordered expression of Hox proteins is associated with myeloid leukemogenesis. Hox proteins are homeodomain transcription factors that are organized in four paralog groups (A–D). Expression of HoxA7-11 (the ABD-HOXA genes) is characteristic of myeloid progenitors. ABD-HOXA transcription decreases with CD34+ to CD34− progression, and persistent expression of these genes is found in poor prognosis leukemia. Abd Hox proteins regulate genes that are involved in multiple aspects of myelopoiesis. For example, HoxA10 represses transcription of the genes encoding gp91phox and p67phox in myeloid progenitors (the CYBB and NCF2 genes, respectively). In contrast, HoxA9 activates transcription of these genes as differentiation proceeds. Since these are the rate-limiting NADPH oxidase components, HoxA proteins influence the innate immune response. HoxA10 activates transcription of the gene encoding Beta3 integrin, thereby further facilitating NADPH oxidase activation and influencing adhesion. HoxA10 also activates transcription of DUSP4, the gene encoding MAP kinase phosphatase 2 (Mkp2). Mkp2 antagonizes the activity of c-Jun N-terminal kinases (Jnk). Since HoxA10-activation of DUSP4 decreases during myelopoiesis, decreased Mkp2 expression in mature phagocytes facilitates apoptosis via Jnk. Interferon regulatory factors (IRF) also regulate multiple aspects of myelopoiesis. IRF1 and ICSBP/IRF8 activate transcription of the CYBB and NCF2 genes in cooperation with the ets protein PU.1. ICSBP/IRF8 also contributes to phagocyte function by activating genes encoding TLR4, IL12, and Nramp. Additionally, IRF proteins regulate cell cycle progression and proliferation. IRF2, ICSBP/IRF8, and PU.1 activate transcription of gene encoding Neurofibromin 1, thereby downregulating the proliferative response to cytokines such as GM-CSF, M-CSF, and G-CSF. ICSBP/IRF8 and PU.1 also activate the gene encoding Ink4b, thereby also influencing proliferation. In myeloid progenitors, ICSBP/IRF8 influences cell survival by repressing transcription of PTPN13, the gene encoding Fas-associated phosphatase 1 (Fap1), a Fas-antagonist. Decreased ICSBP/IRF8-induced PTPN13 repression during myelopoiesis increases susceptibility of mature phagocytes to Fas-induced apoptosis. Such studies reveal that multiple aspects of myelopoiesis are regulated by common sets of transcription factors. This may suggest therapeutic targets for myeloid leukemias or other disorders of myeloid development.
Inducing the Tryptophan Catabolic Pathway, Indoleamine 2,3-Dioxygenase (IDO), for Suppression of Graft-Versus-Host Disease (GVHD) Lethality.
Authors: Jasperson Lisa , Bucher Christoph , Panoskaltsis-Mortari Angela , Mellor Andrew , Munn David H. , Blazar Bruce R.
Published: October 2019

Abstract 3547

Poster Board III-484

During graft-versus-host disease (GVHD), donor T cells become activated and migrate to tissue sites. Previously, we demonstrated a crucial role for the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) in GVHD regulation. Here, we show that upon arrival in the colon, activated donor T cells produced IFN-γ that upregulated IDO, causing T cell anergy and apoptosis. IDO induces GCN2 kinase, upregulating a T cell stress response implicated in IDO immunosuppression. Donor T cells did not require GCN2 kinase to respond to IDO, suggesting toxic IDO metabolites, and not tryptophan depletion, were responsible for suppression. When exogenous metabolites were administered, GVHD lethality was reduced. To determine if IDO could be induced pre-transplantation for enhanced GVHD suppression, we first determined whether antigen presenting cells (APCs) or epithelial cells were primarily responsible for IDO expression and subsequent GVHD suppression. Recipients with wild-type vs. IDO−/− APCs had increased survival, regardless of epithelial cell expression of IDO, suggesting that APCs were suitable targets for inducing IDO. Administration of an agonist to toll-like receptor (TLR)-7/8, a receptor expressed primarily on APCs, induced IDO and reduced injury in the colon, and ameliorated lethality. We conclude that IDO upregulation may have therapeutic potential for preventing GVHD in the clinic.

Disclosures:

Mellor: NewLink Genetics, Inc.: Consultancy, Intellectual property interests in the therapeutic use of IDO and IDO inhibitors and receive consulting income and research support from NewLink Genetics, Inc., Research Funding. Munn:NewLink Genetics, Inc: Consultancy, Intellectual property interests in the therapeutic use of IDO and IDO inhibitors and receive consulting income and research support from NewLink Genetics, Inc, Patents & Royalties, Research Funding.
Tissue Damage and Lymphoma Derived Chemoattractive Signals Impact Regulatory T Cell Trafficking After Allogeneic Hematopoietic Cell Transplantation.
Authors: Dürr Christoph , Pfeifer Dietmar , Schmitt-Gräff Annette , Claus Rainer , Gerlach Ulrike , Graeser Ralph , Gerbitz Armin , Negrin Robert , Finke Jürgen , Zeiser Robert
Published: October 2019

Abstract 3564

Poster Board III-501

Regulatory T cells (Treg) have been shown previously to reduce graft-versus-host disease (GvHD) but allow for graft-versus-leukemia (GvL) effects. It is still unclear why the adoptive Treg transfer does not paralyze every immune response to the same extent. In that context Treg recruitment and localization may account for differential immune regulation. Therefore, we studied the impact of lymphoma and inflammation derived chemoattractive signals on Treg recruitment. Luciferase transgenic Treg accumulated in B cell lymphoma (BCL) tissue after allogeneic hematopoietic cell transplantation (alloHCT) when no conventional T cells were given. Microarray based analysis of the BCL tissue revealed increased expression of the CXC chemokine ligands (CXCL) 9, 10, 12 and CCL22. In vivo antibody based neutralization or interference with receptor signaling identified CCL22 and CXCL12 as critical chemokines for Treg attraction towards BCL tissue after alloHCT. Conversely, the CXCL9/10-CXCR3 axis was not critical for Treg recruitment. Depletion of antigen presenting cells (APC) from BCL tissue abrogated CCL22/CXCL12 secretion. CD11c-DTR recipients displayed reduced Treg recruitment towards BCL, indicative for a major role of host APC in the process of Treg recruitment. Strong local inflammatory stimuli caused by subcutaneous complete Freund's adjuvant injection caused local Treg accumulation and reduced recruitment to the BCL. Infusion of conventional T cells that caused subacute GvHD related inflammation redirected Treg towards secondary lymphoid organs and abdominal GvHD target organs.

In conclusion our study demonstrates the following novel findings: First Treg are recruited towards BCL infiltrated tissues after alloHCT and this process is dependent on CCL22 and CXCL12 production. Secondly, BCL infiltrating host type APC are the major source for CCL22 and CXCL12 and their targeted deletion impacts Treg recruitment towards BCL tissue. Thirdly, strong local or systemic inflammation can redirect Treg trafficking in the presence of BCL and affect the accumulation of this cell population at the tumor site which may explain why Treg preferentially regulate GvHD reactions post allografting.

Disclosures:

No relevant conflicts of interest to declare.
Update On the Risk of Leukemia in Genetic Subgroups of Congenital Neutropenia (CN): Comparison of Patients with Known Gene Mutations (ELA2, HAX1, WASP, G6PC3, p14).
Authors: Zeidler Cornelia , Donadieu Jean , Bolyard Audrey Anna , Vandenberghe Peter , Pracht Gusal , Beaupain Blandine , Hoy Ludwig , Zimmermann Martin , Bellanne-Chantelot Christine , Link Daniel C , Klein Christoph , Germeshausen Manuela , Dale David C. , Welte Karl
Published: October 2019

Abstract 3597

Poster Board III-534

An increased risk for malignant transformation (MDS or leukemia) is well documented in patients with congenital neutropenia (CN). In this study we assessed the incidence of leukemic transformation and potential risk factors for leukemic transformation in CN patients with known gene mutations, e.g. ELA2, HAX1, G6PC3, p14, WAS or no identified mutation, respectively, by combining all available data from the European and US Branches of the Severe Chronic Neutropenia Registry (SCNIR) and the French Neutropenia Registry (FR).

Data from mutational analysis were available for 407 patients. Mutations were identified in 259 CN patients, of whom 209 patients revealed ELA2 mutations, 20 HAX1 mutations, 18 WAS, 8 G6PC3 and 4 p14 mutations. In addition, in 57 patients neither ELA2 nor HAX 1 mutation were detectable and in another 91 patients ELA2 mutations could be excluded, but further genetic evaluation is not yet completed.

Secondary malignancies occurred in 50 of the 407 CN patients. The distribution by genetic subtype is shown in the table below:

CN subtype by gene mutation Total patient number (n) MDS/Leukemia (n/%) ELA2-CN 209 31 (14. 8%) HAX1-CN 20 4 (20%) ELA2 neg + HAX1 neg 57 6 (10.5%) ELA2 neg/ HAX1 not tested 91 5 (5.5%) WAS 18 4 (22.2%) G6PC3 8 0 p14 4 0 Total 407 50 (12.3%)

All subgroups benefit from G-CSF treatment. Median G-CSF maintenance doses required during the years prior to leukemic transformation compared by genetic subtype is shown in the following table 2:

CN subtype by gene mutation Without leukemia (n) Median G-CSF dose (μg/kg/d) With leukemia (n) Median G-CSF dose (μg/kg/d) ELA2-CN 75 5.0 15 15 HAX1-CN 15 5.0 4 13.4 ELA2-/HAX1- 36 7.2 4 10.5 WAS 15 2.6 4 3.0 G6PC3 8 3.9 0 na p14 4 5.1 0 na

Conclusion

Patients with severe congenital neutropenia who have mutations in ELA2, HAX1, or WAS and also those with no recognized mutation are at risk of secondary leukemia. So far, progression to MDS leukemia has not yet been described in the small number G6PC3 or p14 CN cases in our database. ELA2-CN or HAX1-CN patients requiring higher doses of G-CSF are at greater risk. Mutational analysis is helpful to identify the genetic cause of severe congenital neutropenia but does not serve to identify patients at risk of leukemic transformation.

Disclosures:

No relevant conflicts of interest to declare.
Expression Cloning of Genes Enabling Erythropoietin -Independent Erythropoiesis in Vitro.
Authors: Yokoo Tomoko , Kurita Ryo , Takahashi Atsushi , Okada Michiyo , Kawano Hirotaka , Miura Yoshie , Hiramoto Takafumi , Inoue Hiroyuki , Suehiro Youko , Sugiyama Daisuke , Lu Hua , Yoshimura Akihiko , Komatsu Norio , Tani Kenzaburo
Published: October 2019

Abstract 3608

Poster Board III-544

Erythropoietin (EPO) is essential during both ontogeny and the course of erythropoiesis. In primitive (yolk sac) erythropoiesis, however, the role of EPO is not fully understood. Elucidation of such role in primitive erythropoiesis would be very helpful for the development of ex vivo red blood cell expansion system from embryonic stem (ES) cells. Recently, we reported the establishment of the ex vivo induction of hematopoietic stem cells from common marmoset ES cells using lentiviral-Tal1/Scl gene transfer in the absence of any stromal cells (Stem Cells 24: 2014-2022, 2006). This method should also be applicable to both of human ES cells and induced pluripotential stem (iPS) cells, but the efficiency will be very critical for its clinical application. In the present study, we proceed further to find out unknown factors which accelerate ex vivo proliferation and differentiation of erythroid cells, constructed the human fetal liver cDNA expression lentiviral library (Mol Cell Biochem 319: 181-187, 2008) and screened for cDNAs which confer EPO independency to an EPO-dependent cell line, UT-7/Epo ( Blood 82: 456-464, 1993). Among twenty-two candidate genes cloned after screening of 6×10∧5 cDNA, we particularly focused on two full-length genes, ribosomal protein L11 (RPL11) and retinol dehydrogenase 11 (RDH11). Two candidate gene-transduced-UT-7/Epo cells, respectively named LV-RPL11 and LV-RDH11, showed complete EPO-independent survival and proliferation, increased expression of fetal γ-globin, and decreased expression of adult β-globin compared with parental UT-7/Epo cells in the presence of EPO. Cell cycle and apoptosis analyses showed decreased apoptotic cell death and increased S/G2/M cells in LV-RPL11 and LV-RDH11 cells compared with UT-7/Epo cells in the absence of Epo. Moreover, STAT5 phosphorylation and upregulation of its target genes, c-Myc, cyclin D and Pim, were observed in LV-RPL11, LV-RDH11 cells in the absence of EPO. In conclusion, the findings suggest that RPL11 and RDH11 play a role in EPO-independent erythropoiesis and might be applicable to ex vivo expansion of red blood cells from ES/iPS cells.

Disclosures:

No relevant conflicts of interest to declare.
Mesenchymal Stem Cells Inhibit Th17 Differentiation through PGE2 Production.
Authors: Tatara Raine , Ozaki Katsutoshi , Oh Lekuni , Hatanaka Keiko , Meguro Akiko , Matsu Haruko , Sato Kazuya , Nagai Tadashi , Muroi Kazuo , Ozawa Keiya
Published: October 2019

Abstract 3633

Poster Board III-569

Mesenchymal stem cells (MSCs) possess an immunomodulatory function and show promise as a cell therapy for graft-versus-host disease (GVHD). In a phase II study in Europe, injections of MSCs caused 60-70% overall response rate, with longer survival of complete responder. In contrast to its clinical efficacy, the molecular mechanism(s) underlying immunomodulation by MSCs has not been fully established. Prostaglandin E2 (PGE2), tumor growth factor-b1 (TGF-b1), and indoleamine-2,3-dioxygenase have been reported to mediate the immunomodulatory function of MSCs, and we reported evidence that nitric oxide is also a mediator (Blood 2007, 109, 228). Th17 is a recently recognized differentiation category, in which CD4 cells produce IL-17. It has been reported that Th17 is crucial for experimental autoimmune encephalomyelitis (a model of the human disease, multiple sclerosis) and is also thought to be important in other autoimmune diseases. Regulatory T cells (Treg) are another newly recognized differentiation category, in which CD4 T cells have high levels of Foxp3 expression and suppress T cell proliferation. It has been reported that Th17 and Treg can be induced by incubation with TGF-b1 and IL-6 or IL-21, and TGF-b1 and IL-2, respectively, and that these two differentiations are in a reciprocal relationship. Whereas the role of Th17 in GVHD is still controversial, Treg has been reported to suppress GVHD in a mouse model.

To elucidate the molecular mechanism(s) of the immunomodulatory function of MSCs, we herein sought to identify the effects of MSCs on these relatively new differentiations. MSCs inhibit Th17 differentiation even in conditions in which growth is not completely inhibited. Interestingly, an inhibitor of prostaglandin production, indomethacin, and an inhibitor of indoleamine 2,3-dioxygenase, 1-methyltryptophan, partially restore Th17 differentiation, whereas inhibitors of nitric oxide synthase do not. These results suggest that PGE2 and depletion of tryptophan, but not nitric oxide, mediate inhibitory effects of MSCs on Th17. Additionally, we found that MSCs produced PGE2 when co-cultured with CD4 T cells in Th17 differentiation condition and PGE2 per se suppresses Th17 differentiation. Thus, our results suggest that MSCs block Th17 differentiation through PGE2 prodction. In contrast to Th17 differentiation, Treg differentiation was not significantly inhibited by MSCs. However, MSCs still inhibited proliferation of T cells under these conditions, and T cell proliferation was restored by the addition of indomethacin. These results suggest that MSCs inhibit proliferation but not Treg differentiation through PGE2 production. The mechanism by which PGE2 differentially regulates these differentiations is unknown and remains an area for further investigation.

Disclosures:

Ozawa: Alexion: Research Funding.
Role of STAT3 in Immunoregulatory Function of Human Bone Marrow-Derived Mesenchymal Stem Cells.
Authors: Yoon Jinsun , Kim Seoju , Kim Eun Shil , Kim Byoung Kook , Lee Young
Published: October 2019

Abstract 3637

Poster Board III-573

The one of the best curative treatment modality in hematologic malignancies is an allogeneic hematopoietic stem cell transplantation (HSCT). However, graft-versus-host disease (GVHD) is a major obstacle of allogeneic HSCT. BM derived human MSCs are known to have immunoregulatory effect in vitro and in vivo via inhibiting alloreactive T lymphocytes, leading to their clinical use for the prevention of GVHD in HSCT. However, the molecular mechanism of immunoregulatory effect of human MSCs is not fully understood. In this study, the signaling of immunoregulatory effect was investigated by co-culture of human MSCs with lymphocytes. The proliferation of allogeneic T cells was inhibited by MSCs. Among the STATs, STAT3 was a key molecule in MLR co-cultured with MSCs. STAT3 siRNA treated MSCs did not inhibit the lymphocyte proliferation. After MSCs were trasnsfected with STAT3 plasmid, the fraction of CD4+CD25+FOXP3+ cells (Treg cells) were increased, while the fraction of CD4+, CD8+, CD25+ was decreased. In addition, Th1-related cytokines (IL-2, IL-12 and INF-γ) and Th17-related cytokines (IL-6, IL-17 and IL-21) were down-regulated, and Th2-related cytokines (GATA-3, IL-4 and IL-10) were up-regulated in MLR co-cultured with STAT3-ablated MSCs, while vice versa in MLR co-cultured with STAT3-transfected MSCs. Furthermore, ELISA showed that concentration of Th1-related cytokine (IL-2) in the supernatant of MLR co-cultured with STAT3-ablated MSCs was higher than that of control; while concentration of Th2-related cytokine (IL-4) was lower than that of control. These results suggested that induction of Th1 to Th2 shift by MSCs might be mediated via STAT3 molecule. In summary, STAT3 may be an indispensable molecule in the immunoregulatory effect in human MSCs via modulation of regulatory T cells.

Disclosures:

No relevant conflicts of interest to declare.
In Vivo Deficiency of Both Transcription Factors, C/EBPbeta and C/EBPepsilon, Results in Lack of Cytokine Response and a High Susceptibility for Overwhelming Infections.
Authors: Thoennissen Nils Heinrich , Akagi Tadayuki , Song Jee Hoon , Iwanski Gabriela B. , Koeffler H. Phillip
Published: October 2019

Abstract 3641

Poster Board III-577

Transcription factors including the CCAAT/enhancer binding protein (C/EBP) family are essential for hematopoietic cell development including myelopoiesis. In response to cytokines and/or infection, C/EBPbeta is important for emergent neutrophil production, whereas C/EBPepsilon induces terminal differentiation of granulocytes. We recently generated C/EBPbeta and C/EBPepsilon double-knockout (KO) mice and demonstrated the dependence of the hematopoietic system on both transcription factors. In order to study inflammatory response of this transgenic in vivo model, bone marrow-derived macrophages from wild-type (WT), single- as well as double-KO mice were cultured either with or without 100 ng/mL LPS and 100 ng/mL mIFNgamma as activating stimuli. After 24 hours of exposure, RNA was extracted, and gene expression difference was interrogated by whole genome microarray (Affymetrix®, mouse genome 430 2.0). The probes were normalized with the MAS 5.0 software for processing Affymetrix oligonucleotide array data, and genes were considered to be differentially expressed with a fold change level of at least 2. Remarkably, we found more than 75 genes which were up to 20-fold downregulated in the stimulated macrophages of the double-KO mice compared to either single KO or WT mice. A pathway-based analysis indicated that these deregulated genes formed networks associated with development and function of the hematopoietic system, inflammatory response and cell-cell signaling. We confirmed the expression for some of the most highly decreased genes by RT-PCR; for example, interleukin-(IL)1beta, IL-12beta, CD38, macrophage mannose receptor (Mrc-1), toll-like receptor-2 (TLR-2), C-type lectin receptor Mincle, and insulin-like growth factor (IGF-1) were almost not detectable in the activated macrophages of the double-KO mice compared to either single-deficient or WT mice. Lacking these and other important regulators of appropriate immune responses and hematopoiesis, the double KO died between 2 - 3 months of age due to systemic infections including formation of hepatic, peritoneal and lung abscesses with Streptococcus viridians, Enterococcus species and Escherichia coli. In conclusion, phentotype as well as expression pattern of mediators of inflammatory response and hematopoiesis of the double-KO mice was distinct from either the C/EBPbeta- or C/EBPepsilon-KO mice, demonstrating that each transcription factor is important in cytokine expression and host defense, but can in part compensate for each other in the single knockout genotype.

Disclosures:

No relevant conflicts of interest to declare.
Hypomethylation of Tumor Suppressor Genes in Acute Myeloid Leukemia: Characteristic of Cell Lines with MLL Abnormalities?.
Authors: Quentmeier Hilmar , Röhrs Sonja , Dirks Wilhelm G , Meyer Claus , Marschalek Rolf , Scherr Michaela , Slany Robert K. , Wallace Andrew , Drexler Hans G.
Published: October 2019

Abstract 365

Background:

Translocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemia (AML) and in mixed phenotype acute leukemia in infancy, a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type MLL carries histone methyltransferase activity and affects specific target genes, such us HOXA cluster genes. While the more than three dozen MLL fusion proteins known today exert different specific functions, they finally induce transcription of individual target genes. Consequently, acute lymphoblastic leukemias (ALL) with MLL mutations (MLLmu) exhibit typical gene expression profiles including high-level expression of HOXA cluster genes. Aim of this study was to find a correlation between the MLL mutational status and tumor suppressor gene methylation/expression in acute leukemia cell lines.

Results:

Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLLmu and MLLwt acute leukemia cell lines. 1.8/24 TSG were methylated in MLLmu AML cells, 6.2/24 TSG were methylated in MLLwt AML cells. Hypomethylation and expression of the tumor suppressor genes (TSG) BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLLmu acute myeloid leukemia (AML) cell lines. MLL wild-type (MLLwt) AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3 confirming epigenetic silencing of these genes in MLLwt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLLmu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene.

Conclusion:

These results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLLmu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins.

Disclosures:

No relevant conflicts of interest to declare.
The Human AF9 Homologue in Zebrafish Is Involved in Primitive Hematopoietic Development.
Authors: Germano Giuseppe , Guariento Ilaria , Tiso Natascia , Argenton Francesco , Basso Giuseppe
Published: October 2019

Abstract 3653

Poster Board III-589

Introduction

Human AF9 (alias MLLT3) gene was initially identified as one of the most common translocation partners of MLL associated with acute myeloid leukemia. More recently it has been demonstrated that AF9 can be a positive regulator of early erythroid/megakaryocytic lineage decisions. However, many biological functions of AF9 and its role in leukemia remain unclear. Here, we aimed to identify the zebrafish homologue of human AF9 and to analyze its function using the zebrafish model system.

Methods and Results

A single zebrafish af9 gene was identified by homology searching at zebrafish genome database (www.ncbi.nlm.nih.gov/). The putative af9 protein showed 60% identity and 73% similarity to human counterpart. These results from the perspective of phylogenetic and synteny analyses indicated that zebrafish af9 was the likely homologue of human AF9. We used whole-mount in situ hybridization to analyzed its spatial and temporal expression patterns during embryonic development. Af9 was expressed specifically in the posterior-lateral mesoderm at the 5-somite stage and in the intermediate cell mass (ICM, site of primitive hematopoiesis) between 18 to 24 hours post fertilization (hpf). In order to characterize the role of af9 in early hematopoietic development, we performed over-expression and loss-of-function experiments in zebrafish embryos, and then analyzed various hematopoietic markers by reverse transcriptase quantitative PCR (RT-qPCR). Inducing ectopic af9 expression by mRNA injection we found an increased expression of gata1 and hbae1 (erythroid markers) and reduced expression of runx1, scl, lmo2, c-myb (stem cell/precursor associated markers) as well as pu.1 and l-plastin (myeloid markers) at 22 hpf-old embryos. Conversely af9 loss-of-function obtained using anti-sense morpholinos showed decreased expression of gata1 and hbae1 mRNA, while pu.1 and l-plastin increased their expression in morphant embryos at 22 hpf. The same morphants did not show any appreciable change in stem cell/precursor marker expression.

Conclusion

The finding that gata1 expression is controlled by af9 suggests that this gene might promote the erythroid development in zebrafish primitive hematopoiesis. As the genetic programs of AF9 are evolutionary conserved between human and zebrafish, these findings provide new insights and opening further perspectives in leukemia research and hematopoiesis studies.

Disclosures:

No relevant conflicts of interest to declare.
Modulation of Natural Killer Cells by Therapeutic Antibody Preparations.
Authors: Hokland Marianne , Bohn Anja Bille , Harbo Thomas , Vorup-Jensen Thomas , Petersen Line
Published: October 2019

Abstract 3680

Poster Board III-616

Therapeutic antibody preparations are used in the treatment of several diseases. Thus, human immunoglobulin is administered as high dose intravenous immunoglobulin (IVIg) to patients suffering from a wide range of disorders. Moreover, various monoclonal antibodies (MoAbs) are approved for therapy in cancer and immune diseases. Natural Killer (NK) cells exert important immune effector functions. As these cells express the IgG receptor, CD16 (FcγRIIIa), it is of clinical importance to investigate how these cells react to therapeutic antibody preparations. We found that cytotoxic effector functions of NK cells were significantly inhibited by therapeutic antibody preparations in vitro. Natural cytotoxicity and antibody dependent cell-mediated cytotoxicity (ADCC) were inhibited by treating peripheral blood mononuclear cells (PBMCs) with the polyclonal immunoglobulin preparation Beriglobin, as well as the MoAb preparation Rituximab, prior to standard 51Cr-release assays compared to untreated cells. Moreover, our data showed that freshly isolated NK cells had IgG bound to the cell surface. The level of bound IgG increased following short-time exposure to both therapeutic antibody preparations, and the level of CD16, which is the key receptor for inducing ADCC, decreased. To investigate whether the NK-inhibitory effects of IVIg were demonstrable in vivo too, we analyzed blood from patients diagnosed with chronic inflammatory demyelinating polyneuropathy (CIDP), a disorder in which high-dose IVIg as monotherapy has shown striking effects. Comparing peripheral blood samples obtained from patients before treatment and 3 days following high-dose IVIg using multiparameter flow cytometry, we demonstrated a substantial decrease in the number of CD56+/CD3- NK cells. Moreover, as shown in vitro, the expression of CD16 on the NK cells was markedly reduced following high-dose IVIg treatment due to IgG engaged by the FcγRIIIa receptor (CD16). Finally, we found that NK mediated cytotoxicity decreased significantly when comparing peripheral blood samples from patients before and 3 days following IVIg treatment. In conclusion, our data presented in this study show that high-dose immunoglobulin therapy induces NK inhibitory effects in vitro as well as in vivo and thus may have important consequences for the overall therapeutic efficiency.

Disclosures:

No relevant conflicts of interest to declare.
Escalated BEACOPP Versus ABVD-Like Chemotherapy for Hodgkin Lymphoma Patients: a Cochrane Review.
Authors: Brillant Corinne , Bauer Kathrin , Herbst Christine , Monsef Ina , Skoetz Nicole , Engert Andreas
Published: October 2019

Abstract 3705

Poster Board III-641

Introduction

The main challenge in treating Hodgkin lymphoma (HL) is to find the optimal treatment with the best efficacy and least toxicity. So far there are two different international standards for the treatment of intermediate and advanced stage HL: chemotherapy with ABVD regime and chemotherapy with escalated BEACOPP regime. This review aims to clarify advantages and disadvantages of both treatments.

Methods

MEDLINE, CENTRAL and EMBASE were systematically searched for randomised controlled trials from 1985 to 2008. Trials comparing treatment with at least 2 cycles escalated BEACOPP versus chemotherapy with at least 4 cycles of ABVD within patients in intermediate or advanced stages HL were included. Trials without any published results will be excluded from the meta-analysis. Trial selection, quality assessment and data extraction were done independently by two review authors. Time-to-event outcome were analyzed with hazard ratios (HR) and 95% confidence interval (CI) in a fixed effects model.

Results

A total of 683 references were screened. Four eligible trials with 1970 patients were identified and included in the main analysis: the HD9 and HD14 trials from Germany, the HD2000 and GSM-HD trials from Italy. The escalated BEACOPP regime was more toxic during treatment than ABVD. Progression free survival (PFS) was statistically significantly longer for escalated BEACOPP: HR was 0.53 (95% CI [0.42, 0.67], I2=0%). In the main analysis of overall survival (OS), escalated BEACOPP was statistically significantly better than ABVD: HR was 0.70 (95% CI [0.50, 0.98], I2=0%). However, this result is not robust. In the OS-analysis restricted to advanced stage trials, the HR was 0.93 (95% CI [0.52, 1.04], I2=0%). Due to the high weight of the HD9 trial (57%), the OS-results are strongly influenced by this trial. In a sensitivity OS analysis without the HD9 trial the HR was 0.83 (95% CI [0.50, 1.38]). Only one trial (HD9) had a follow-up of over 5 years; the median of the other trials was approx. 3 years.

Conclusions

This meta-analysis showed a better PFS and suggests a benefit in OS for escalated BEACOPP in comparison to ABVD-like regimen. However, many trials had a short follow-up. Longer follow-up and the inclusion of the ongoing EORTC 20012 trial should allow a more definitive answer concerning OS.

Disclosures:

No relevant conflicts of interest to declare.
RAD001 Exerts Anti-Tumor Activity in Waldenstrom Macroglobulinemia.
Authors: Vesole Steven , Jia Xiaoying , Roccaro Aldo M. , Sacco Antonio , Ngo Hai T. , Azab Feda , Azab Abdel Kareem , Melhem Molly R , Runnels Judith M. , Quang Phong , Anderson Kenneth C. , Ghobrial Irene M.
Published: October 2019

Abstract 3732

Poster Board III-668

Background

Waldenstrom Macroglobulinema (WM) is an incurable B-cell disorder characterized by the presence of IgM monoclonal gammopathy and bone marrow infiltration of lymphoplasmacytic cells. Apart from promising advances in therapeutic strategy, response rates and treatment-free survival remain inconsistent across prescribed regimens. As we have previously shown, the PI3K/Akt pathway actively mediates tumor growth, survival, migration, and cell cycle within primary WM cells. An active target of this pathway includes the downstream mammalian target of rapamycin (mTOR). By inhibiting the mTOR pathway with RAD001 (Afinitor™, Novartis Pharmaceuticals), we hoped to constructively regulate aforementioned cellular function, and also understand from a preclinical perspective its specificity of action in our microenvironmental models, both alone and the presence of monoclonal antibody against CD20 and proteasome inhibitor.

Methods

WM cell lines (BCWM1) and IgM secreting cell lines MEC1 and RL were used. Bone marrow stromal cells (BMSC) were obtained from patients with WM. Peripheral blood mononuclear cells were harvested from healthy donors. Agents tested included RAD001, Rituximab and Bortezomib. Cytotoxicity, DNA synthesis, and cell cycle were measured using MTT assay, [3H]-thymidine uptake, and flow cytometry/PI staining, respectively. Antibody-dependent cellular cytoxicity (ADCC), transwell, and Matrigel assays were preformed to measure cell lysis, SDF-1 chemotaxis-induced migration, and angiogenesis, respectively. Adhesion to fibronectin has been evaluated with WM cells and BMSC in the presence of RAD001, with and without Bortezomib.

Results

RAD001 induced cytotoxicity and inhibition of DNA synthesis with an IC50 of 1-10 nM in BCWM1 cells at 48 hours. Similar effects were seen in IgM cell lines with an effective dose of 0.1-1 nM. In contrast, at IC50 treatment level donor cells displayed no significant cytotoxicity (>85% survival). Cell cycle analysis showed corresponding G1 arrest, and angiogenesis was also inhibited. RAD001, Bortezomib, and Rituxan combinations showed synergistic cytotoxicity, which was attenuated when WM cells were co-cultured with BMSC. Migration of BCWM1 and adhesion of BCWM1 to BMSC was reduced under presence of RAD001.

Conclusions

These functional assays therefore show that RAD001 has significant and synergistic antitumor activity in WM which will inform the design of future clinical trials.

Disclosures:

Ghobrial: Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.
Vorinostat Induced Cellular Stress Disrupts the Balance Between p38 MAPK and Erk Pathways Leading to Apoptosis in WM Cells.
Authors: Sun Jenny , Tseng Hsiuyi , Xu Lian , Hunter Zachary , Ciccarelli Bryan , Fulciniti Mariateresa , Yang Guang , Gong Ping , Zhou Yangsheng , Liu Xia , Munshi Nikhil C. , Patterson Christopher J , Treon Steven
Published: October 2019

Abstract 3740

Poster Board III-676

Despite advances, Waldenstrom's Macroglobulinemia (WM) remains incurable, and novel agents are urgently needed. Histone deacetylase (HDACs) are involved in transcription regulation and signal transduction of cells through a genome wide alteration in histone modification and other proteins, leading to significant increase in cellular stress in tumor cells. We therefore examined the activity of Vorinostat, a histone deacetylase inhibitor (HDAC-I), and dissected its pro-apoptotic molecular pathways in WM cells. Vorinostat exhibited dose dependent killing of both primary, bone marrow derived WM cells, as well as BCWM.1 WM cells with an LD50 of 3.5 to 5uM using Annexin V and PI staining.

Vorinostat induced apoptosis in WM cells through activation of specific caspases at different time points. Caspase 3, 6, 8, and 9 were activated after 24 hours of Vorinostat. Even though caspase 7 is downstream of caspase 3, we observed that caspase 7 was activated earlier, starting at 6 hours. We therefore hypothesized that the regulators of caspase 7 may be affected by Vorinostat at an earlier time point. Further investigation confirmed that there was significant down-regulation of inhibitor of apoptosis (IAP) family members, including c-IAP1, c-IAP2, XIAP and Livin after 12 hours of treatment with Vorinostat, and may elude to greater sensitivity for IAPs in modulating Caspase 7 versus Caspase 3 in WM cells.

We also studied the stress pathways including Erk, JNK and P38 pathways in Vorinostat treated WM cells. Activated p38, phospho-p38, was upregulated starting at 12 hours, while phopho-Erk abruptly decreased following 24 hours of treatment with Vorinostat. There was minimal change in the activity of JNK pathway following Vorinostat treatment. The activation of the p38 pathway coincided with a reduction in c-IAP1, c-IAP2, XIAP and Livin following treatment of WM cells with Vorinostat for 12 hours.

Taken together, these studies support that stress induced apoptosis in WM cells is mediated through disruption in the balanced activity between the Erk and p38 MAPK pathways. Vorinostat induced cellular stress results in the activation of p38 MAPK pathway and a reduction of the IAP family members, leading to early activation of caspase 7. While the inhibition of Erk pathway by Vorinostat results in delayed activation of caspase 3, 6, 8, and 9 at 24 hours, the collective signaling strength of p38 activation as well as inhibition of Erk likely determines the apoptotic fate WM cells upon Vorinostat treatment.

Disclosures:

No relevant conflicts of interest to declare.
Complete Responses with Denileukin Diftitox in Cutaneous T-Cell Lymphoma Studies.
Authors: Foss Francine , Duvic Madeleine , Olsen Elise A. , Kozlovski Anna
Published: October 2019

Abstract 3745

Poster Board III-681

Cutaneous T-cell lymphoma (CTCL) is a disorder of CD4+ helper T-cells with manifestations in the skin, nodes, and, in advanced stages, blood and visceral sites. For many patients, the clinical course of the disease is chronic and progressive, despite multiple therapeutic interventions. To date, are few controlled clinical trials which demonstrate durable complete responses (CR) in advanced stage patients. Denileukin diftitox (DD) is a recombinant fusion protein targeting interleukin-2 receptor which has demonstrated efficacy in early and advanced stage CTCL with response rates of 30% and 44% in two clinical trials of CTCL pts with Stage Ib-IVA relapsed and refractory CTCL or Stage I-III disease, ≤3 prior therapies respectively. In these trials, patients (pts) were randomized to receive DD at a dose of either 9 or 18 ug/kg/d daily x 5 every 21 days. Another rollover trial enrolled pts who had progressed after prior response with DD (N=29) or were CD25- (N=36); all pts in the rollover trial were treated with DD at 18ug/kg dose. Of 263 intent-to-treat pts in these trials, 227 had CD25+ skin infiltrates by immunohistochemistry (IHC) confirmed by a reference pathologist and 36 were CD25-. Overall, 24 (9.1%) pts attained durable complete response (CR). The median age of the responders was 59, and 12 CR pts were over age 60. Of the CR group, 15 pts had early stage (I-IIa) disease and 9 had advanced stage (IIb-IV) CTCL. The mean prior therapies was 3.6 with 37% of patients having received >3 therapies. Of the 24 CR pts, 21 were CD25+ and 3 were CD25-. Two CR were pts who had previously responded to DD in an earlier clinical trial. Of all pts receiving 9 ug/kg dose (N=80), there were 6 (7.5%) CR; for all pts receiving 18 ug/kg dose (N=183), there were 18 (9.8%) CR. There was no significant difference frequency of CR between the 9 and 18 ug/kg groups (P=0.56) or between the CD25+ (N=118) and CD25- (N=36) pts treated at 18 ug/kg dose (P=0.64). CR rate was similar between the early and advanced stage patients (10.7% vs 8.9% respectively). The median time to response was 53.5 vs 41days for CD25+ pts treated in the 9 and 18 ug/kg groups respectively, and 43 days for the CD25- group. The response durations ranged from 57 days to >1325 days in the CD25+ and 190-400 days in the CD25- groups. Of the 24 CR, 7 have progressed as of the time of the analysis and 17 remain in CR. The overall median PFS at the time of analysis has not been reached (range 169-1388+ days). Of the 24 CR, 3 pts had hypersensitivity reactions and 3 had capillary leak syndrome associated with DD treatment. In summary, these studies demonstrate clinical benefit of DD with CR in both early and advanced CTCL at both 9 and 18 ug/kg doses and with durable responses in a small number of pts whose malignant lymphocytes were CD25- by ICH.

Disclosures:

Foss: Eisai : Speakers Bureau. Olsen:Eisai: Research Funding. Kozlovski:Eisai Pharm: Employment.
Irgm1 Is a Negative Regulator of Interferon-Gamma Signaling in Hematopoietic Stem Cells.
Authors: King Katherine Y , Baldridge Megan T , Weksberg David C , Goodell Margaret A
Published: October 2019

Abstract 382

Hematopoietic stem cells (HSCs) are a self-renewing population of bone marrow cells that give rise to all of the cellular elements of the blood and retain enormous proliferative potential in vivo. We have a growing understanding that the controls on HSC proliferation are tied in part to regulation by the immune system—specifically, that HSC proliferation and mobilization can be stimulated by the immune cytokines interferon-alpha and interferon-gamma (IFNg). Our previous work has demonstrated that HSC quiescence and function are aberrant in mice lacking the immunity-related GTPase Irgm1 (also Lrg47). Indeed, the bone marrow of Irgm1-deficient animals at baseline mimics the bone marrow of wild type animals that have been stimulated with IFNg. We hypothesized that the HSC defects in Irgm1-deficient animals are due to overabundant IFNg signaling, and that Irgm1 normally serves to dampen the stimulatory effects of IFNg on HSCs. To test this hypothesis, we used RNA expression profiling to compare gene expression in wild type versus Irgm1-deficient mice. We found that interferon-dependent signaling is globally upregulated in the HSCs of Irgm1-deficient mice. Next we generated Irgm1-/-IFNgR1-/- and Irgm1-/-Stat1-/- double knock out animals. In contrast to the phenotype of Irgm1 single knock out mutants, the hyperproliferation and self-renewal defects in HSCs were both rescued in the double knock out animals, indicating that IFNg signaling is required for manifestation of the Irgm1-deficient phenotype. Futhermore, we found that Irgm1 is expressed in HSCs in a Stat1- and IFNgR-dependent fashion, suggesting that it forms a negative feedback loop for IFNg signaling in the HSC population. Collectively, our results indicate that Irgm1 is a powerful negative regulator of IFNg-dependent stimulation in HSCs. These findings demonstrate that IFNg provides a significant stimulus for HSC proliferation even in the absence of infection, and that IFNg-dependent signaling must be tightly regulated to preserve HSC self-renewal capacity. This study provides evidence that the Irgm1 protein can serve as a link between immunity and regulation of hematopoiesis at the level of the stem cell. We speculate that utilization of Irgm1 for its immune functions may detract from its ability to regulate HSC self-renewal capacity, thus ultimately contributing to myelosuppression and increased risk of death from chronic infections such as tuberculosis.

Disclosures:

No relevant conflicts of interest to declare.
Has the Prognostic of Primary Plasma Cell Leukemia Improved with New drugs?
Authors: Chaoui Driss , Leleu Xavier , Roussel Murielle , Royer Bruno , Rubio Marie-therese , Ducastelle Sophie , Merabet Fatiha , Garderet Laurent , Kolb Brigitte , Debarri Houria , Arkam Yazid , Harousseau Jean-Luc , Moreau Philippe
Published: October 2019

Abstract 3869

Poster Board III-805

Introduction

The outcome of patients with plasma-cell leukemia (PCL) is poor. Avet-Loiseau reported on behalf the IFM, our first experience in PCL patients and showed that the median overall survival (OS) was 8 months (Avet-Loiseau, Blood, 2001). Since 1999, novel agents such as Thalidomide, Bortezomib (Velcade) or Lenalidomide (Revlimid) have been widely used in the treatment of multiple myeloma, both at the time of relapse or part of upfront therapy.

Patients and methods

In this retrospective analysis, we have looked at the outcome of PCL patients treated within the IFM since 1999 in order to study the impact of novel agents on survival.

Results

31 cases, 20 males, 11 females, median age 55 years (34-78) were analyzed. Twenty one patients less than 65 years received high-dose therapy as part of frontline treatment : 19 autologous haematopoietic stem cell transplantation (HSCT) and 5 allogeneic transplantation. Novel agents were used part of induction therapy in 6 cases, at the time of relapse for 9 patients, for both induction and relapse in 16 cases. Thirteen patients received 1 novel agent, 11 received 2 and 7 patients received the 3 novel agents. The median number of lines of therapy was 2 (1 to 4). Bortezomib was used as up front treatment in 15 patients and at relapse for 9 patients. Overall response rate according the IMWG criterias was 70% (17/24) including 11 CR or VGPR (45%). PAD (Bortezomib, Adriamycin and Dexamethasone) and VTD (Bortezomib, Thalidomide, Dexamethasone) regimens provided the best response rates. Lenalidomide was used in 13 patients mostly at relapse. A response was obtained in 53% of patients including 2CR and 2 VGPR (30%). Nineteen patients were treated with Thalidomide-based regimens. Overall response rate was 52% (10/19) including 2 CR and 6 VGPR (31%).

Overall, for the whole group of patients, the median progression-free survival was 8 months (0-26) and the median OS was 15 months (6-108). When comparing this survival with that described in our previous experience reported before 1999, we clearly showed that the use of novel agents improved the survival of patients with PCL.

Conclusion

In this retrospective study, novel agents improved the prognosis of P-PCL. Prospective IFM phase II studies are ongoing to confirm these results.

Disclosures:

No relevant conflicts of interest to declare.