bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitotic Spindle Dysfunction Promotes Genomic Instability In Marrow Failure
Authors: Lombardo Katie , Stumpff Jason , Parkhurst Susan , Wordeman Linda , Shimamura Akiko
Published: October 2019

Abstract 880

Shwachman-Diamond syndrome (SDS) is an autosomal recessively inherited disorder associated with bone marrow failure and leukemia predisposition. The majority of patients harbor biallelic mutations in the SBDS gene. The SBDS protein has been implicated in several cellular functions including ribosome biogenesis and microtubule stabilization during mitosis. We have previously found that SBDS deficiency results in multipolar spindles, centrosome amplification and aneuploidy, implicating a role for SBDS in cell division. The mechanism by which SBDS functions to ensure proper spindle assembly and DNA segregation during mitosis remains unknown. Here we present evidence that SBDS functions to promote mitotic spindle stability both by directly modifying microtubule dynamics and through a microtubule crosslinking activity. Importantly, the microtubule stabilizing effects of SBDS appear to be essential for the growth and differentiation of hematopoietic progenitor cells. Specifically, we found that SBDS deficiency resulted in shortened mitotic spindle length and decreased spindle acetylation, a marker of microtubule stability. The loss of microtubule stability in the absence of SBDS function may be due to changes in microtubule dynamics or reduction in microtubule crosslinking activity, as we found that addition of recombinant purified wild-type SBDS to polymerized microtubules in vitro increases their polymerization rate and strongly promotes microtubule bundling. Interestingly, recombinant patient-derived missense mutant SBDS proteins showed a marked decrease in their microtubule bundling ability. To assess whether spindle destabilization contributes to marrow failure, we modeled hematopoiesis in the absence of SBDS in vitro. When SBDS expression was knocked down in human CD34+ cells, proliferation, differentiation, and hematopoietic progenitor colony formation were impaired, consistent with published data on primary marrow from SDS patients. The addition of taxol at concentrations that significantly impaired hematopoiesis in control CD34+ cells resulted in stable to improved hematopoiesis in the SBDS-deficient CD34+ cells. Based on these data, we hypothesize that spindle destabilization by SBDS loss promotes genomic instability, which in turn, contributes to marrow failure and leukemia predisposition.

Disclosures:

No relevant conflicts of interest to declare.
Cytokines Instruct Hematopoietic Lineage Choice
Authors: Schroeder Timm
Published: October 2019

Abstract SCI-13

The molecular control of multipotent cell lineage choice is one of the central questions in stem cell biology. Since decades, it is controversial whether cell extrinsic factors can actively influence lineage decisions of hematopoietic cells (instructive model). Alternatively, they may only allow the survival and proliferation of already unilineage-restricted cells, which have chosen one lineage by cell-autonomous mechanisms (selective / permissive model). One major reason for this longstanding controversy is the fact that hematopoiesis is usually followed by analyzing populations of cells - rather than individual cells - at very few time points of an experiment and without knowing their individual identities. However, the alternative lineage choice models can only be distinguished by constantly observing individual cell fates throughout differentiation.We therefore applied novel computer aided culture, imaging and cell tracking approaches to follow the fate of individual cells over many days. Analysis of primary mouse multipotent hematopoietic cell differentiation finally provided proof for the lineage instructive action of hematopoietic cytokines. This finding is of fundamental importance for our understanding of the molecular control of lineage choice. It shows that cell extrinsic signaling influences the - currently unknown - cell intrinsic molecular lineage choice and commitment machinery. Our novel approaches now allow identification of the signaling pathways mediating lineage instruction and their targets with the required precision. Moreover, we are developing technology for the quantification of lineage specific transcription factor proteins in individual living cells. This novel kind of continuous, live and quantitative molecular and cellular data is used for modeling the control of cell fates through integration of cell extrinsic signals with cell intrinsic molecular states.

Disclosures:

No relevant conflicts of interest to declare.
Origin of the Myeloma Stem Cell
Authors: Matsui William
Published: October 2019

Abstract SCI-4

Increasing evidence suggest that most human cancers consist of phenotypically and functionally heterogeneous cell types. Moreover, in some tumors, phenotypic-functional relationships exist and the capacity for clonogenic growth and self-renewal may be restricted to specific cell types. In multiple myeloma, malignant plasma cells make up the vast majority of tumor cells, phenotypically characterize the disease, and are responsible for clinical symptoms. However, myeloma plasma cells also appear terminally differentiated similar to their normal counterparts and incapable of long-term proliferation. Similar to other laboratories, we have identified minor populations of B cells that share the identical immunoglobulin gene rearrangements as the malignant plasma cells in most myeloma patients. We have further studied the functional capacity of these clonotypic B cells and found that they have ability to produce ectopic tumor growth both in vitro and in immunodeficient mice that can recapitulate human disease.1,2 Moreover, these cells are relatively resistant to several agents currently used to treat multiple myeloma suggesting that they have the capacity to persist following treatment and are responsible for tumor regrowth and disease relapse. In order to inhibit these drug resistant cancer stem cells, we have studied cellular pathways that regulate the self-renewal of normal adult stem cells and found that several, including the developmental pathway Hedgehog, are potential therapeutic targets.3 Finally, as these concepts are translated to the clinic, several challenges have emerged including the ability to detect activity against myeloma stem cells that make up only a minority of the overall tumor burden.4 To this end, we have begun to develop biomarker strategies to serially quantify myeloma stem cells in patients undergoing treatment and will examine their utility as clinical endpoints.

1. Matsui W, Huff CA, Wang Q, et al. Characterization of clonogenic multiple myeloma cells. Blood. 2004;103:2332–2336.

2. Matsui W, Wang Q, Barber JP, et al. Clonogenic Multiple Myeloma Progenitors, Stem Cell Properties, and Drug Resistance. Cancer Research. 2008;68:190–197.

3. Peacock CD, Wang Q, Gesell GS, et al. Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proceedings of the National Academy of Sciences, USA. 2007;104:4048–4053.

4. Huff CA, Matsui W, Smith BD, Jones RJ. The paradox of response and survival in cancer therapeutics. Blood. 2006;107:431–434.

Disclosures:

Matsui: Pfizer: Consultancy; Bristol-Meyers Squibb: Consultancy; Infinity Phamaceuticals: Consultancy, Patents & Royalties; Merck: Consultancy, Research Funding; Geron Corporation: Research Funding. Off Label Use: Rituximab in multiple myeloma.
Stability of Self-Renewal in Hematopoietic Stem Cells
Authors: Iscove Norman N.
Published: October 2019

Abstract SCI-42

For many years a distinction was drawn between prospectively separable murine HSC populations with long-term, essentially permanent reconstituting potential (LT-HSC), versus HSC populations yielding short-term engraftment lasting only 4 – 6 weeks after transplantation (ST-HSC). Recent work based on transplantation of single cells shows that highly purified populations of LT-HSC prepared by standard sorting parameters consist in fact predominantly of a distinct, newly recognized class of intermediate- term reconstituting cells (IT-HSC) whose grafts endure longer than short-term HSC but also eventually fail (1). IT-HSC are separable from long-term reconstituting cells on the basis of expression of more alpha2 integrin and less SLAM150. Crucial to recognition of the distinction between LT- and IT-HSC are the endpoints used to evaluate reconstitution. If blood erythroid or myeloid reconstitution is measured, IT reconstitution is readily distinguished by the disappearance of these elements by 16 wk post-transplant. If instead reconstitution is measured simply by presence of blood leukocytes of donor origin, which in the mouse are almost entirely lymphocytes, the distinction is not made because lymphoid elements persist even in fading IT clones to 24 wk or beyond. The observations imply a need for reinterpretation of most of the published descriptions of the biology and gene expression profiles previously attributed to LT-HSC but in fact derived from analysis of populations that consisted mainly of IT-HSC. The capacity now to separate LT- from IT-HSC creates new opportunities for probing the mechanisms that specify and sustain long term function in the former but not the latter. 1. Benveniste P, Frelin C, Janmohamed S, Barbara M, Herrington R, Hyam D, Iscove NN. Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential. Cell Stem Cell. 2010;6:48–58

Disclosures:

No relevant conflicts of interest to declare.
Chemotherapy-Resistant T Cells
Authors: Turtle Cameron J.
Published: October 2019

Abstract SCI-51

The mechanisms that maintain human T cell memory during normal and perturbed homeostasis are not fully understood. Patients undergoing multiple cycles of cytotoxic chemotherapy infrequently develop infections with viruses that are controlled by memory CD8+ T cells either during or after the period of profound lymphocytopenia, suggesting that some memory cells survive chemotherapy and provide protective immunity. We recently identified a subset of quiescent human CD161hi CD8+ T cells characterized by the capacity to rapidly efflux cytotoxic drugs and the fluorescent dye, rhodamine 123 (Rh123), which endowed them with the ability to survive chemotherapy in vitro and in vivo and to contribute to the recovery of lymphocyte numbers in patients receiving chemotherapy. Rh123 effluxing CD161hi memory CD8+ cells were identified in both the CD62L+ central memory (CM) and CD62L- effector memory (EM) pools, and were characterized by uniform expression of CD28, high levels of bcl-2, and low levels of granzyme B and perforin. CD161 and CD62L expression allowed partitioning of the CD8+ memory compartment into four phenotypic subsets with distinct transcriptional profiles and functional properties. Gene expression profiling of Rh123 effluxing CD161hi memory CD8+ cells in both CM and EM pools revealed a type 17 differentiation program with high levels of RORC, RORA, IL23R, CCR6, KLRB1, and CCL20, compared with CD161lo CM and EM cells. Transcriptional profiling also revealed alterations in the T cell receptor (TCR) signaling pathway in CD161hi cells that limits their capacity to proliferate and secrete cytokines, including IL-17, after TCR stimulation. Despite stringent regulation of TCR signaling in type 17-programmed CD161hi cells, proliferation and cytokine secretion could be induced by ligating CD28 to provide costimulation, and by IL-1β, IL-12, IL-18 or IL-23. The nature of the second and third signals provided to CD161hi T cells revealed plasticity in this subset of cells and determined whether the CD161hi progeny derived after expansion had the capacity to secrete IL-17, or had differentiated to Tc1-like cells with restored capacity to proliferate to TCR ligation alone. Thus, a surprisingly large fraction of the human CD8+ T cell memory pool is comprised of CD161hi CD8+ T cells that express a type 17 transcriptional program and possess drug efflux capacity that allows them to survive cytotoxic chemotherapy and contribute to recovery of lymphocyte numbers. The stringent regulation of CD8+CD161+ memory T cells in normal physiology also suggests that this subset may have a potential role in the pathogenesis of IL-17-mediated inflammatory diseases.

Disclosures:

No relevant conflicts of interest to declare.
Bone Marrow-Derived Adipocytes Promote Differentiation, Proliferation and Survival of Acute Monoblastic Leukemia Cells
Authors: Jin Linhua , Tabe Yoko , Konopleva Marina , Andreeff Michael , Ohsaka Akimichi , Miida Takashi
Published: October 2019

Abstract 1501

Acute monoblastic leukemia occurs most commonly in young individuals, whereas acute monocytic leukemia is common in adults (WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 2008). Adipocytes are the prevalent stromal cell type in adult bone marrow (BM) and play an important role in the leukemic bone marrow microenvironment (Tabe et al., Blood 2004 103: 1815–22). BM stromal cells (MSCs) from elderly subjects have a reduced capacity to differentiate into osteoblasts and an increased capacity to differentiate into adipocytes, which leads to progressive accumulation of fat in the BM space with increasing age. In this study, we examined whether BM-derived adipocytes participate in the molecular events associated with proliferation, differentiation and apoptosis of monoblastic leukemia cells. To this end, we performed gene expression microarray analysis of 339 genes associated with lipid metabolism (GeneSQUARE, Kurabo, Japan) using RNA from monoblastic leukemia cell line U937 co-cultured with MSC or MSC-derived adipocytes. Five genes were upregulated (>2.0 fold) and 2 genes down-regulated in U937 cells co-cultured with adipocytes compared to U937 cultured alone. The up-regulated genes included monocyte/macrophage differentiation specific genes, such as scavenger receptor CD36, adipsin or fatty acid binding protein 4 (FABP4). CD36 and FABP4 are classical target genes of PPARγ which was also upregulated in U937 co-cultured with adipocytes. Upregulation of the inflammation-related haptoglobin gene was observed in U937 cells co-cultured with either adipocytes or MSC. These findings were confirmed by quantitative RT-PCR assay with concordant results. Down-regulated genes included monocyte chemoattractant protein-1 (MCP-1) (0.1 fold) and glycolytic enzyme hexokinase 2 (0.4 fold). In turn, U937 cells co-cultured with premature or mature adipocytes accumulated in G2 cell cycle phase compared to U937 cultured alone or co-cultured with MSC (% G2/M-phase fraction; U937 cultured alone, 14.6, co-culture with MSC 25.1, co-culture with preadipocyte 32.0, with mature adipocyte 31.9) and were protected from spontaneous cell death under serum-starved conditions (% subG1 fraction: U937 cultured alone 23.9%, co-cultured with MSC 15.7%, with preadipocytes 3.9% and with mature adipocytes 11.6%). In summary, results suggest that BM adipocytes, abundant in adult BM space, promote monocytic differentiation, proliferation and survival of monoblastic leukemia cells. We propose that these cells represent an essential component of the BM microenvironment that contributes to malignant phenotypes of adults with monocytic leukemia.

Disclosures:

No relevant conflicts of interest to declare.
The Role of Endogenous Stromal Cell-Derived Factor-1 in the Survival and Growth of Acute Myeloid Leukemia Cells
Authors: Kim Ha-Yon , Oh Yoon-Suk , Song Ik-Chan , Lee Hyo-Jin , Yun Hwan-Jung , Kim Samyong , Jo Deog-Yeon
Published: October 2019

Abstract 1502

Stromal cell-derived factor-1 (SDF-1) is constitutively expressed and produced by stromal cells in the bone marrow (BM). The protein exerts its functions by binding to its receptor CXCR4, thus guiding the homing of hematopoietic stem/progenitor cells and leukemia cells to the BM. CD34+ cells purified from normal adult peripheral blood have been shown to express and produce SDF-1, which exerted an antiapoptotic effect on CD34+ cell through an autocrine/paracrine loop. Acute myeloid leukemia (AML) cells have also been shown to express and produce SDF-1, but the role of SDF-1 in this context is largely unknown. Thus, the aim of the present study was to determine the function of SDF-1 produced by AML cells. Using siRNA technology, SDF-1 was knocked-down in AML cells and subsequent biological alterations occurring in the cells were evaluated in vitro. All AML cell lines used in this study (U937, K562, KG1a, HL-60 and MO7e) as well as primary CD34+ cells obtained from the BM of 5 patients with AML expressed SDF-1 mRNA at various levels, as revealed by semi-quantitative and real time RT-PCR analysis. SDF-1 was detected by Western blotting analysis and ELISA allowed for the detection of SDF-1 in the supernatants of cultures of AML cells grown for 3 days in a serum-free medium, indicating that AML cells, similar to normal CD34+ cells, are able to produce and secrete SDF-1. Exogenous SDF-1 (added at a concentration of up to 200 ng/mL) alone had no discernible effects on the survival or proliferation of either the cell lines or the primary CD34+ AML cells maintained in a serum-free medium, but was able to induce the internalization of CXCR4 and CXCR7. Transfection of five AML cell lines with SDF-1 siRNA diminished the SDF-1 mRNA levels by up to 90%. The knock-down cells were stable for at least 3 days. SDF-1 knock-down did not affect the serum deprivation-induced apoptosis of the cells. Cell surface expression of CXCR4 was not changed, whereas cytoplasmic CXCR4 was significantly upregulated along with the upregulation of SDF-1 mRNA. Neither cell surface nor cytoplasmic expression of CXCR7 was affected by the knock-down of SDF-1. Conversely, the knock-down of CXCR7, but not CXCR4, upregulated the SDF-1 mRNA expression in the transfected cells. No changes in the transmembrane and trnasendothelial migration of the knock-down cells were observed. In contrast, the knock-down of SDF-1 inhibited the spontaneous proliferation of the U937, K562, and KG1a cells by 20–30% during a 3-day incubation in a serum-free medium. These results indicate that endogenous SDF-1 of AML cells functions as an autocrine growth factor.

Disclosures:

No relevant conflicts of interest to declare.
R-CHOP21 Vs R-CHOP14 in Diffuse Large B-Cell Lymphoma Patients: Results From a Multicentre Retrospective Study
Authors: Rigacci Luigi , Fabbri Alberto , Puccini Benedetta , Orciuolo Enrico , Pietrini Alice , Iovino Lorenzo , Petrini Mario , Lauria Francesco , Bosi Alberto
Published: October 2019

Abstract 1626

Diffuse large B cell lymphoma (DLBCL) is one of the most common types of non-Hodgkin's lymphoma. R-CHOP21 (C21) is considered the standard therapy but a large number of studies tested R-CHOP14 (C14).

The aim of our study was to evaluate retrospectively a cohort of patients (pts) treated with C21 or C14.

All pts with diagnosis of DLBCL or follicular grade IIIb lymphoma, treated with curative intent were accrued.

From January 2002 to December 2010, 123 pts were treated with C21 and 142 were treated with C14. The median age was 63 (range 19–89). The two cohorts of pts were balanced for all clinical characteristics a part for age (<65 or >64 years) with more aged pts in C21 arm (p 0.000), PS with more advanced PS (2–3) in C21 arm (0.000) and LDH value which was more frequently elevated in C14 arm (p: 0.002). After induction therapy 190 pts (71%) obtained a complete remission: 82/123 (67%) after C21 and 108/142 (75%) after C14. After a median period of observation of 31 months 81 pts relapsed, 42 (51%) in the C21 arm and 39 (36%) in the C14 arm. Considering the two therapies, C21 vs C14, no differences were reported in OS, PFS and DFS: 61% vs 68%, 59% vs 58% and 74% vs 61% respectively. In univariate analysis OS was lower in older pts (p: 0.02), advanced stage (p: 0.02), symptomatic disease (p: 0.05), elevated LDH (p: 0.001), bone marrow infiltration (p: 0.02) and intermediate or high risk IPI (p: 0.000); PFS was lower in advanced stage (p: 0.002), symptomatic disease (p: 0.009), elevated LDH (p: 0.001), bone marrow infiltration (p: 0.001) and intermediate high risk IPI (p: 0.000). In multivariate analysis OS was significantly better in low-intermediate IPI risk pts (p: 0.000) and in pts treated with C14 (p: 0.02); the PFS was better in low-intermediate IPI risk pts (p: 0.000). Considering only pts with low or low-intermediate IPI we observed that OS was significantly superior in the group treated with C14 (90% vs 64% p: 0.03), moreover in young pts (< 65 years) OS was better in pts treated with C14 (81% vs 58% p: 0.05). As expected hematological grade III/IV toxicity was more frequent in pts treated with C14, all pts but three (2%) completed the therapy without delay or dose reduction. No differences in extra-hematological toxicity were observed.

Conclusions:

In conclusion our results confirm that C14 do not improve the results of the standard C21 in the whole lymphoma population but in a subset of pts, young and low/intermediate risk pts, the C14 scheme seems to improve the OS. We will enlarge the cohort of studied patients but further prospective randomized studies are needed to verify this preliminary observations.

Disclosures:

No relevant conflicts of interest to declare.
Hematopoietic Stem Cell Gene Therapy with Lentiviral Vector in X-Linked Adrenoleukodystrophy
Authors: Aubourg Patrick , Hacein-Bey-Abina Salima , Bartholomae Cynthia , Schmidt Manfred , Kutschera Ina , Vidaud Michel , Dal-Cortivo Liliane , Mahlaoui Nizar , Frange Pierre , Bellesme Céline , Lefrère Francois , Dufayet Gaelle , Fischer Alain , van Kalle Christof , Cavazzana-Calvo Marina , Cartier Nathalie
Published: October 2019

Abstract 163FN2

The most severe form of X-linked adrenoleukodystrophy (ALD) is characterized by rapidly progressive and lethal cerebral demyelination in childhood. The progression of cerebral demyelination of ALD can be arrested by allogeneic hematopoietic stem cell (HSC) transplantation (HCT) within 12–18 months, provided the procedure is performed at an early stage of the disease. The long term beneficial effects of HCT in ALD are likely due to the progressive turn-over of brain microglia that are derived from myeloid progenitors in the bone-marrow. Despite the increased availability of cord blood, not all boys with cerebral ALD and who are candidate for HCT have a suitable HLA-matched donor. In addition, allogeneic HCT remains associated with significant mortality risk. In late 2009, we reported that HSC gene therapy with lentiviral vector was able to arrest the progression of cerebral ALD in two boys who have no HLA-matched donor to perform HCT. ALD protein expression in myeloid and lymphoid lineages as well as the identification of identical lentiviral insertion sites in myeloid and lymphoid lineages strongly suggested that multi-potent long-term repopulating hematopoietic cells were transduced. In those 2 treated patients, hematopoiesis has remained polyclonal without evidence of clonal skewing or dominance up to the last follow-up. Data on clinical efficacy, gene marking and lentiviral integration studies with a longer follow-up (4 years ½) will be presented. HSC gene therapy however failed to arrest the progression of cerebral ALD in a third treated ALD patient, 36 months after gene therapy. Hematopoiesis remained also polyclonal in this patient and biological data that could explain failure of gene therapy in this patient will be presented. At last, data on a fourth patient who has been treated more recently (12 months) by HSC gene therapy will also be presented.

Disclosures:

No relevant conflicts of interest to declare.
Cyclin D1 Over-Expressing Mantle Cell Lymphoma Cells Are Hypersensitive to Inhibition of Fatty Acid Synthase (FASN)
Authors: Dengler Michael A. , Gutekunst Matthias , Kopacz Stephanie , Horn Heike , Hofmann Ute , Schwab Matthias , Ott German , van der Kuip Heiko , Aulitzky Walter E.
Published: October 2019

Abstract 1656

Mantle cell lymphoma (MCL) is an aggressive type of B-cell lymphoma characterized by a t(11;14)(q13;q32) chromosomal translocation resulting in a constitutive over-expression of cyclin D1. Cyclin D1 is a multifunctional protein not only regulating cell cycle progression but also affecting other cellular functions including glucose and lipid metabolism. In this study, we investigated the effects of the fatty acid synthase (FASN) inhibitors orlistat and C75 on viability of different MCL cell lines including JEKO-1, MINO, Granta-519, JVM-2, and Rec-1. In all cell lines inhibition of FASN alone induced apoptosis. In contrast, normal peripheral blood lymphocytes were resistant to these compounds.

This proapoptotic effect was dependent on cyclin D1 as silencing of cyclin D1 partially rescued cells from induction of cell death following FASN inhibition. Inhibition of FASN led to a strong induction of the proapoptotic protein NOXA (PMAIP) in all MCL cell lines investigated independent of the p53 status. Pre-treatment of cells with NOXA siRNA significantly reduced induction of cell death demonstrating the predominant role of this proapoptotic protein for the observed effects.

We then analyzed the combined effects of inhibition of FASN with a panel of compounds with potential clinical relevance. Combination of FASN inhibitors with the BH3 mimetic ABT737, the proteasome inhibitor Bortezomib, and the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) led to an almost complete loss of viability. These combinatory effects were selective for MCL cells as the same treatments had almost no effects on cell viability of primary PBMCs or fibroblasts from healthy donors. Bortezomib, 2-DG, and ABT737 enhanced the proapoptotic effect of FASN inhibitors by further disturbing the balance of pro- and antiapoptotic Bcl-2 family proteins: FASN inhibitor mediated induction of NOXA was enhanced by Bortezomib whereas 2-DG significantly reduced the NOXA antagonist Mcl-1.

In summary, our results suggest that FASN inhibitors exert significant antitumoral activity especially in combination with Bcl-2 family modulators in different MCL cell lines and may therefore represent an attractive model for the design of new therapeutic approaches for this entity.

Disclosures:

No relevant conflicts of interest to declare.
Loss of EGR-1 Accelerates BCR-ABL-Driven Leukemogenesis
Authors: Maifrede Silvia , Sha Xiaojin , Liebermann Dan , Hoffman Barbara
Published: October 2019

Abstract 1671

Chronic Myelogenous Leukemia (CML) is a disease resulting from the neoplastic transformation of hematopoietic stem cells (HSC) by the translocated tyrosine kinase BCR/ABL. The BCR-ABL protein product is a constitutively active tyrosine kinase, which promotes cell survival and proliferation by means of diverse intracellular signaling pathways, thereby being the culprit for malignant transformation. The early growth response (Egr)-1 gene, a member of the Egr family of genes encoding for zinc-finger transcription factors, has been shown to be an early response gene, to mediate cellular responses to growth factors and to be a stress response gene. Both growth factor stimulation and stress in most cells causes rapid induction of Egr-1 within minutes that leads to the activation of downstream growth pathways in normal cells. Egr-1 regulates the expression of multiple genes, either directly or indirectly, that impact on cell cycle arrest, survival and/or apoptosis, which can interface with BCR-ABL signaling. Included among these genes are p53, TGFbeta, PTEN, gadd45a, gadd45b, Foxo3a, p21, FasL, and Trail.

There is a large body of evidence consistent with Egr-1 behaving as a tumor suppressor in hematopoietic cells, both in vivo & in vitro, in both humans & mice. This laboratory has shown that Egr-1 abrogates the block in M1 terminal differentiation imparted by either oncogenic c-Myc or E2F-1, suppressing their leukemia promoting function in nude mice. Since Egr-1 can be a tumor suppressor and its down-stream effectors cross-talk with BCR-ABL signaling it was asked if Egr-1 can act as a suppressor of BCR-ABL driven leukemogenesis. To assess the effect of Egr-1 on BCR-ABL driven leukemia, syngeneic wild type lethally irradiated mice were reconstituted with wild type or Egr-1 null myeloid progenitors transduced with a 210-kD BCR-ABL-expressing MSCV-retrovirus. It was observed that loss of Egr-1 accelerated the development of BCR-ABL driven leukemia in recipient mice. In vitro proliferation assays have shown enhanced proliferation capacity for BCR-ABL transduced Egr-1 null myeloid progenitors compared to wild type counterparts. In addition, flow cytometric analysis of Egr-1 null myeloid progenitors from bone marrow (BM) has shown a slightly smaller stem cell (Lineage- Sca+ and c-Kit+ [LSK]) population as compared to wild type progenitors from BM. Therefore, the accelerated induction of BCR/ABL-mediated leukemia using Egr-1 null progenitors cannot be accounted for by increased numbers of stem cells. Taken together, these results indicate that Egr-1 functions as a suppressor of BCR-ABL driven CML. Further elucidating the role of Egr-1 in the murine model of BCR-ABL driven leukemia and analysis of Egr-1 expression in human CML peripheral blood and BM could result in novel targets for diagnosis and prognosis, as well as for targeted therapeutics.

Disclosures:

No relevant conflicts of interest to declare.
Home Azacitidine Administration in High Risk Myelodysplastic Syndromes: Favorable Results of a Pilot Study in 48 Patients
Authors: Guillevic Sylvestre , Comont Thibault , Khalifa Jonathan , Recher Christian , Adoue Daniel , Ollier Sylvie , Cougoul Pierre , Laurent Guy , Beyne-Rauzy Odile
Published: October 2019

Abstract 1719

Background:

Myelodysplastic syndromes (MDS) affect elderly patients. Azacitidine represents the gold standard treatment of high risk MDS. French health organization allows home chemotherapy administration after the first cycle of treatment. We decided to develop a home administration program of this drug. Moreover we hypothesized that this modality of treatment will improve patient's quality of life.

Patients and methods: after one cycle in hospitalization, high risk MDS patients who were agree to receive home chemotherapy were included in the study. As recommended each first day of Azacitidine cycle was still administrated at hospital. Due to drug instability a quality statement was created to control all steps of administration (including storage temperature before administration). Feasibility and safety were evaluated. Comparison between home or hospital administration was performed: number of cycles, dose, tolerance, problems of administration.

Results:

68 patients were included in the home chemotherapy program, 75% were MDS patients and 48 received Azacitidine. Age distribution was 7 patients 60/69 yrs (14%), 22 patients 70/79 yrs (46%) and 19 patients 80–89 yrs (40%). More than 2000 days of hospitalization were performed at home during a 18-month period. Similar median number of cycles and delays were observed in the two groups as well as same level of adverse events and hospitalizations between 2 cycles. In few cases administration was not possible at home due to unresolved infection, cytopenia or drug availability (excessive time between preparation and distribution). No patient was excluded of this program.

Discussion and conclusion: home administration of Azacitidine was safe and feasible. All patients agree to continue this modality of administration and high level of satisfaction was observed. Next step will consist in a randomized study to compare quality of life between home and hospital administration of the drug. For this population of patient home treatment will represent an alternative which could improve quality of life as observed for children in ALL (1) or in colorectal cancer (2).

Disclosures:

No relevant conflicts of interest to declare.
Autocrine IL-6 Production Indicates the Level of Activated STAT3 and NF-Kb of Chronic Lymphocytic Leukemia Cells
Authors: Liu Fengting , Jia Li , Farren Timothy W , Gribben John G. , Agrawal Samir G
Published: October 2019

Abstract 1784

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in survival, apoptosis and proliferation of cancer cells. Previous studies have shown that IL-6 levels are significantly elevated in most cancer cells, while elevated serum levels of IL-6 correlated with adverse prognostic features and shorter survival in patients with chronic lymphocytic leukemia (CLL). IL-6 production is controled by transcription factors NF-kB and STAT3, which are themselves controled by tyrosine kinase phosphorylation, such as that mediated by JAK2. This creates a positive feedback loop, as JAK2 can be stimulated by IL-6.

In this study we investigated the relationship between autocrine IL-6 production and activation of STAT3 and NF-kB in CLL patients. The levels of IL-6 production in cell culture medium were measured by ELISA and activation of STAT3 and NF-kB was indicated by the ratio of p-STAT3/STAT3 and p-NF-kB/NF-kB. Patients with high levels of p-STAT3/STAT3 and/or p-NF-kB/NF-kB had higher levels of IL-6 production. The level of IL-6 production significantly correlated with STAT3 activation (R=0.856, p < 0.001) and NF-kB activation (R=0.815, p < 0.001). Inhibition of constitutive STAT3 and NF-kB activation by the STAT3 phosphorylation inhibitor, DPP, and the NF-kB inhibitor, CAPE, blocked IL-6 production 40% in average (p = 0.004, p = 0.003). Analysing the level of autocrine IL-6 production and in vitro spontaneous apoptosis in CLL cells from 38 patients, we found that patients with higher levels of IL-6 production (more than median) had significantly less apoptosis compared with those below the median (23% vs. 42.2% in average, p <0.0001). In this cohort, 60% of CD38+ cases, 78% of 17p- cases and 67% of cases with a lymphocyte doubling time of less than12 months were in the high IL-6 producer group. High IL-6 production was also associated with a shorter average time to first treatment (0.5 vs. 3 years, p=0.0023).

This study demonstrates that autocrine IL6 production correlates with STAT3 and NF-kB activation and apoptosis resistance in CLL. Furthermore, higher IL-6 production is associated with biological markers of poor prognosis and earlier treatment. The IL6/JAK2/STAT3 signal pathway may offer new therapeutic targets for CLL.

Disclosures:

No relevant conflicts of interest to declare.
Increased Sclerostin Secretion in Multiple Myeloma Results in Stimulation of Osteoclastogenesis and Inhibition of Osteoblastogenesis
Authors: Eda Homare , Santo Loredana , Cirstea Diana D. , Pozzi Samantha , Canavese Miriam , Scullen Tyler A. , Vallet Sonia , Raje Noopur S.
Published: October 2019

Abstract 1819

Objectives:

Osteoblasts (OB) and osteoclasts (OC), are an integral part of the bone microenvironment, and play a crucial role in myeloma growth and survival. Their imbalance results in osteolytic disease and elucidating the mechanisms underlying osteolytic lesions is important not only for the improvement of osteolytic bone disease but also for the treatment of multiple myeloma (MM).

The osteocyte-secreted protein sclerostin, encoded by the SOST gene, is a potent inhibitor of osteoblastogenesis. It is regarded as a good target for osteoporosis treatment, but its role in MM remains to be determined. Our objective was to study the role of sclerostin in MM bone disease and determine if sclerostin directed strategies were a reasonable approach in MM.

Methods and Results:

Sclerostin concentration in patients' blood plasma and MM cell line supernatant stimulated by IL-6, FGF-2, TNFalpha, BMP7 and TGFbeta was detected by ELISA (ALPCO immunoassays). Increased level of sclerostin was detected in MM patient plasma (n=20, median: 4.73 ng/mL, range: 1.5–19.5 ng/mL). Plasma concentrations were significantly higher (p<0.01) when compared to sclerostin concentration in the plasma of leukemia patients (n=3), gastric cancer patients (n=40) and healthy volunteers (n=4). High sclerostin levels were not associated with extent of bone disease but rather correlated with tumor burden (High B2M, creatinine and LDH, and low Hb) suggesting an autocrine loop for sclerostin production. Because sclerostin is derived from mature OB or orteocytes, we measured levels during OB differentiation but we were unable to detect increased levels. We then measured sclerostin levels in RPMI-8226 MM cell line supernatant either alone or stimulated by cytokines D Systems).

Conclusions:

These data demonstrate that increased sclerostin levels in MM patients inhibit osteoblastogenesis and stimulate osteoclastogenesis. Taken together, sclerostin may be good target to inhibit myeloma bone disease and help restore normal bone homeostasis.

Disclosures:

Raje: Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.
Prognostication by miRNome-Profiling in Multiple Myeloma
Authors: Seckinger Anja , Meißner Tobias , Benes Vladimir , Schmidt Sabine , Blake Jonathan , Zimmermann Jürgen , Goldschmidt Hartmut , Klein Bernard , Hose Dirk
Published: October 2019

Abstract 1822

INTRODUCTION.

MicroRNAs are an abundant class of small non-protein-coding RNAs that function as negative gene regulators in diverse biological processes including cancer by affecting the stability and translation of mRNAs.

METHODS.

We determined expression of 559 miRNAs by miChip (Exiqon LNA Array probes V9.2) in CD138-purified myeloma cells from previously untreated patients (n=69), normal bone marrow plasma cells of healthy donors (pooled to n=3), and human myeloma cell lines (n=20). For normalization, an invariant-based method was applied. Gene expression profiling was performed using Affymetrix U133 2.0 DNA-microarrays.

RESULTS.

We found 29 miRNAs to be significantly up- and 35 down-regulated in myeloma cells vs. normal bone marrow plasma cells, respectively. Expression of 18 miRNAs was simultaneously significantly (P<.01) associated with event-free survival (EFS) and overall survival (OS), and allowed the delineation of prognostic groups. Of these, miRNAs miR-659 (located at 22q12.1) was significantly lower expressed in myeloma cells compared to normal bone marrow plasma cells. For this miRNA, low expression delineates a group with inferior EFS (median 19.7 months vs. not reached, P<.001) and OS (median 52.9 months vs. not reached, P<.001). This group shows a significantly higher gene expression based proliferation index. In contrast, high miR-590 expression (located at 7q11.23) delineates a group with inferior EFS (median 12.4 vs. 36 months, P<0.001) and OS (median 29.4 months vs. not reached, P<.001). By using Goeman's global test, a significant association of the predicted target gene signatures with survival could be found for both, EFS (miR-590-5p, P<.001; miR-659, P<.001) and OS (P=.009; P=.002).

CONCLUSION.

In conclusion, we demonstrate the prognostic relevance of miRNome profiling in multiple myeloma.

Disclosures:

No relevant conflicts of interest to declare.
The Translation Initiation Factor eIF4E Is Overexpressed in Multiple Myeloma and Is Critical for Myeloma Cell Proliferation and Survival
Authors: Li Shirong , Jin MeiHua , Liu Ailing , Mapara Markus Y. , Lentzsch Suzanne
Published: October 2019

Abstract 1853

Methods:

The translation initiation factor eIF4E is central to protein synthesis in general, and overexpression and/or activation of eIF4E is associated with a malignant phenotype by regulating oncogenic protein translation. Several previous publications indicate that aberrant control of protein synthesis contributes to lymphoma genesis but the exact role of protein translation in multiple myeloma (MM) is less clear. Therefore, understanding the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. The goal of this study was to determine the role and regulation of eIF4E, as well as the effects of protein translation controlling drugs in MM.

Results:

By western blot analysis as well as RT-PCR we found that eIF4E protein and mRNA levels are significantly elevated (up to 20 fold) in MM cell lines (H929, RPMI-8226, MM.1S and OPM2) and primary myeloma cells compared to normal plasma cells. Silencing of eIF4E gene expression in RPMI-8226 MM cells by a stable and inducible shRNA system significantly decreased viability of myeloma cells (by ∼ 43%) but not of HEK 293 suggesting a higher dependency of MM cells to protein translation. Next we evaluated different drugs including pomalidomide, rapamycin, pp242, 4EGI-1 and ribavirin, that are known to inhibit protein synthesis for their effects on protein translation in MM. By m7GTP pull down assays we evaluated the effects of the different drugs on eIF4E expression and activity. Rapamycin blocked the phosphorylation of 4EBP1 and eIF4E release, and subsequently inhibited eIF4G binding. The compound 4EGI-1 decreased the interaction between eIF4E and eIF4G. Pomalidomide decreased eIF4E protein expression. All drugs inhibited MM cell DNA synthesis measured by 3H-Thymidine incorporation. Treatment with pomalidomide (10uM), rapamycin (40nM), pp242 (10uM), 4EGI1 (50uM) or ribavirin (50uM) for 48h significantly decreased (p<0.05) proliferation by 43–62% indicating that drugs controlling protein translation inhibit MM growth. We also found that all drugs decreased expression of eIF4E dependent targets such as cyclin D1 and c-myc.

Conclusion:

Here we show that eIF4E, a key player in translational control, is highly expressed in MM cells and critical for MM growth and survival. Therefore our study helps to understand the function and regulatory mechanism of eIF4E in MM. Further the evaluation of drugs targeting protein translation provides the basis for the optimization of current MM treatment or to open up new strategies such as targeting protein translation in future MM therapy.

Disclosures:

Lentzsch: Celgene Corp: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; prIME Oncology: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria.
Influence of Stem Cell Mobilization After Cyclophosphamide, Thalidomide and Dexamethasone (CTD) Regimen in Patients with Newly Diagnosed Multiple Myeloma
Authors: Ahn Jae-Sook , Yang Deok-Hwan , Jung Sung-Hoon , Bae Soo-Young , Kim Yeo-Kyeoung , Kim Hyeoung-Joon , Lee Je-Jung
Published: October 2019

Abstract 1927

Backgrounds:

CTD regimen has been known as an effective induction therapy in patients with newly diagnosed MM. But, there were inconsistent results for the autologous stem cell yield for transplantation. The aim of present study was to identify the influence of CTD therapy on outcome of peripheral blood stem cell (PBSC) collection.

Methods:

Forty-eight patients received 4 cycles of CTD therapy. Stem cells were mobilized with cyclophosphamide (3.0 g/m2) and G-CSF (10 ƒÝg/kg, daily) or G-CSF alone. Patients failing to collect ≤ 4.0 × 106 CD34+ cells /kg received a second mobilization courses.

Results:

The median age at diagnosis was 56 years (range, 39–69). Median duration from start of CTD therapy to first collection was 4.6 months (range, 3.3–8.7). Forty-four patients were mobilized with cyclophosphamide following with G-CSF and 4 patients with G-CSF alone. The median day of apheresis was 3 days (range, 2–7). The response rate for CTD regimen at mobilization was 10% (5/48) of CR, 25% (12/48) of VGPR and 63% (30/48) of PR. A median number of harvested CD34+ cells was 8.6 × 106 cells/kg. At the first mobilization, 83% (40/48) of patients had been reached the minimal PBSC collection target of ≥ 2.0 × 106 CD34+ cells/kg and 71% (34/48) of patients achieved the collection ≥ 4.0 × 106 CD34+ cells/kg. At the end of second mobilization, 90%(43/48) of patients had yields of at least ≥ 2.0 × 106 CD34+ cells/kg and 77% (37/48) of patients had yields of ≥ 4.0 × 106 CD34+ cells/kg. During mobilization period, three patients were developed grade 3/4 non-hematologic adverse events.

Conclusion:

CTD regimen is an effective induction therapy in patients with newly diagnosed MM showing high response rate and acceptable rate of autologuos stem cell yield without any detrimental effect for the following stem cell collection.

Disclosures:

No relevant conflicts of interest to declare.
Increased Plasma Indoleamine 2,3-Dioxygenase Activity Correlates with the Severity of Acute Graft-Versus-Host Disease
Authors: Xu Jinhuan , Zhang Yicheng
Published: October 2019

Abstract 1985

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism, which plays an important role in the induction of immune tolerance. To evaluate the expression level of IDO in the patients receiving hematopoietic stem cell transplantation (HSCT) and to identify the possible correlation between IDO activity and acute graft-versus-host disease (aGVHD), we collected blood samples from 96 patients before and after HSCT and 10 healthy volunteers served as controls. The expression of IDO mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in peripheral blood mononuclear cells of the patients and healthy controls. Reverse-phase high-performance liquid chromatography (HPLC) was performed to analyze the level of kynurenine (Kyn) and tryptophan (Trp) in plasma. The IDO activity was calculated as the ratio of kyn to trp. Plasma IFNgama was measured by standard ELISA. RT-PCR showed that IDO mRNA was detected in 65 of 85(65/85, 76.5%) patients with aGVHD; in patients without aGVHD, only 1 of them express IDO mRNA (1/11, 9.1%); none of 10 healthy volunteers was positive for the IDO expression. The plasma IDO activity was much higher in aGVHD group than those without aGVHD(4.49¡À0.46 vs. 1.69¡À0.9, respectively; p < 0.0001) or the healthy controls(4.49¡À0.46 vs. 1.77¡À0.22, respectively; p < 0.0001). Patients with severe aGVHD (grade III/IV) had significantly higher IDO activity than those with mild aGVHD (grade I/II) (6.74¡À0.58 vs. 2.17¡À0.79, respectively; p < 0.0001). IDO activity was decreased following effective treatment of aGVHD, while fluctuation of plasma IDO was also observed upon the recurrence of aGVHD. The plasma IDO activity was correlated with the IFNgama level(r=0.8816).

Conclusion:

The IDO mRNA was expressed in blood mononuclear cells from patients with aGVHD. The plasma IDO activity was elevated in aGVHD and correlated with severity of aGVHD. In combination with plasma IFNgama, IDO may serve as a potential biomarker for the diagnosis and evaluation of aGVHD following HSCT; intervention of IDO pathway may also provide an alternative way to overcome the steroids resistant aGVHD.

Keywords:

acute graft-versus-host disease, Indoleamine 2,3-dioxygenase, hematopoietic stem cell transplantation

Disclosures:

No relevant conflicts of interest to declare.