bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual Role for CIB1 in Thrombopoiesis: CIB1 Suppresses Megakaryocyte Quantities but Supports Adhesion, Migration, and Proplatelet Formation
Authors: Kostyak John C , Naik Ulhas P
Published: October 2019

Abstract 2384

Megakaryocytes (MKs) are large polyploid cells that produce platelets through a process known as thrombopoiesis. Thrombopoietin (Tpo) is the major cytokine that regulates a variety of steps in this process, including hematopoietic stem cell (HSC) differentiation to MKs, proplatelet formation, and platelet release into the circulation. However, the molecular mechanism of thrombopoiesis is poorly understood. We have previously reported that calcium- and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in thrombopoiesis, we utilized a Cib1−/− mouse model. We observed that Cib1−/− mice have a slightly elevated number of platelets and bone marrow (BM)-derived MKs than wild-type (WT) controls (p<0.05). Rate of platelet clearance was comparable in Cib1−/− and WT mice, suggesting that the defective clearance is not the cause of the observed elevated platelet number. In order to determine if the HSC differentiation is dysregulated by the ablation of Cib1, we analyzed MK-colony forming unit production, which revealed an increase in the colony forming cells with Cib1 deletion compared to WT (p<0.05). Additionally, BM from Cib1−/− mice, cultured with Tpo for 24 hours, produced more highly polyploid MKs than WT BM (p<0.05). These results suggest that Cib1 may negatively regulate initial steps of megakaryopoiesis. Subsequent analysis of Tpo signaling revealed that activation of FAK, a known suppresser of Tpo signaling, is attenuated, as indicated by reduced FAKY925 phosphorylation in Cib1−/− BM-derived MKs treated with Tpo. Consequently, Akt and ERK1/2 activation downstream of Tpo was enhanced. These results suggested that Cib1 inhibits Tpo signaling by augmenting FAK activation. Interestingly, platelet recovery in Cib1−/− mice following platelet depletion by experimental immunothrombocytopenia was attenuated compared to WT (p<0.05). This could be due to impaired adhesion and migration of MKs on the extracellular matrix. Consistent with this notion, adhesion to fibrinogen and fibronectin and migration towards an SDF-1α gradient were significantly reduced in Cib1−/− MKs compared to WT (p<0.05). Additionally, Cib1−/− MKs formed fewer proplatelets compared to WT (p<0.05), when plated on fibrinogen. These data suggest that CIB1 plays a dual role in thrombopoiesis, initially by negatively regulating Tpo signaling, and later by supporting MK migration and proplatelet production.

Disclosures:

No relevant conflicts of interest to declare.
TET2 Favors Mesoderm and Hematopoietic Differentiation in Human Embryonic Stem Cells
Authors: Mor Barbara da Costa Reis Monte , Langlois Thierry , Droin Nathalie , Pronier Elodie , Couédic Jean-Pierre Le , Auger Nathalie , Almire Carole , Mercher Thomas , Delhommeau Francois , Najet Debili , Bernard Olivier , Solary Eric , Plo Isabelle , Vainchenker William
Published: October 2019

Abstract 2418

TET2 belongs to the TET family proteins that catalyze 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (hmc) and plays an important role in normal and malignant adult hematopoiesis. The role of TET2 in human hematopoietic development remains unknown. Here we show that TET2 is expressed at low level in human embryonic stem (hES) cell lines and that its expression increases during hematopoietic differentiation in three different hES. TET2 knockdown does not modify hmc level and pluripotent properties of ES cells. However TET2 depletion by two different shRNA skewed differentiation into neuroectoderm at the expense of endoderm and mesoderm. This was associated with a decrease or an increase in promoter methylation of neuroectoderm and meso/endoderm genes, respectively during hES specification. Subsequently, we observed a decrease in hematopoietic progenitors (CD34+CD43+) and their cloning capacities due to a marked increase in apoptosis. Alteration of hematopoietic differentiation was coupled with a profound alteration in gene expression with up and down regulated genes including the abnormal expression of neuronal genes in hematopoietic cells. Thus our results suggest that TET2 regulates embryonic development by inhibiting neuroectoderm specification and enabling hematopoietic differentiation in hES cells.

Disclosures:

No relevant conflicts of interest to declare.
Linking the Glycolytic Enzyme HK3 to Neutrophil Differentiation of APL Cells Via PU.1
Authors: Federzoni Elena A. , Valk Peter , Löwenberg Bob , Fey Martin F. , Tschan Mario P.
Published: October 2019

Abstract 2425

Objectives:

Identification and characterization of hexokinase 3 (HK3), as novel transcriptional target of the myeloid master regulator PU.1, with a role in APL cell differentiation and survival.

Materials:

HK3 and PU.1 quantitative RT-PCR in primary AML patient samples (n=170) and granulocytes isolated from healthy donors (n=20). Gene expression profiling in PU.1-knockout and PU.1-restored myeloid cell lines. Generation of NB4 and HT93 PU.1 as well as HK3 knockdown cell lines. ATRA-induced differentiation of these cell lines was assessed by CD11b and CEBPE expression. In vivo binding of PU.1 and PML-RARA to the HK3 promoter was shown by ChIP assays. Viability of NB4 cell lines was assessed by Alamar Blue staining.

Results:

We identified HK3 as putative transcriptional target of PU.1 by gene expression profiling in PU.1-knockout and PU.1-restored myeloid cell lines. Unlike the other three hexokinase family members (HK1, HK2 and HK4), HK3 expression is limited to hematopoietic cells, where it is mainly expressed in the myeloid lineage. We found 50-times lower HK3 mRNA levels in primary non-APL patients as compared to granulocytes from healthy donors. Most interestingly, we found that HK3 expression in APL patients with the t(15;17) translocation was 400-times lower (p<0.0001) than compared to granulocytes. Given the particularly low HK3 and PU.1 expression in APL patients, we decided to investigate the role of HK3 and its possible regulation by PU.1 during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of NB4 and HT93 APL cell lines. Firstly, we observed a 5500- and 1100-fold increase of HK3 mRNA levels during neutrophil differentiation of NB4 and HT93, respectively. This was paralleled by markedly increased HK3 protein levels. No increased HK3 levels were observed in ATRA-resistant NB4-R2 cell lines. HK1 and HK2 mRNA expression was not markedly increased during neutrophil differentiation pointing to a particular role for HK3 in neutrophil differentiation. Regulation of the liver specific HK4 was not investigated. Next, we observed significantly decreased HK3 expression, both at the mRNA and protein levels, in all PU.1 knockdown cell lines upon ATRA-induced differentiation. In vivo PU.1 binding to three different regions of the HK3 promoter was found. Given our findings in primary APL patients, we decided to test if PML-RARa binds to the HK3 promoter, and indeed, PML-RARA binds to the same promoter regions as PU.1. Knocking down HK3 in APL cell lines resulted in significantly reduced neutrophil differentiation as measured by CD11b and CEBPE expression. Also, we observed that NB4 HK3 knockdown cell lines treated with ATRA were less viable compared to control cells.

Conclusions:

Our results strongly suggest that HK3 is a novel PU.1 transcriptional target functionally involved in neutrophil differentiation and cell viability of APL cells.

Disclosures:

No relevant conflicts of interest to declare.
Bortezomib Restores Defective Apoptosis by Upregulation of Noxa in Enteropathy-Associated T-Cell Lymphoma
Authors: de Baaij Laura R , Radersma Marijn , van de Water Jolanda MW , Groot Kim BJ , Hijmering Nathalie J , Moesbergen Laura M , Visser Otto J , Oudejans Joost J , Mulder Chris JJ , Meijer Chris JLM , Cillessen Saskia AGM
Published: October 2019

Abstract 2722

Enteropathy-associated T-cell lymphoma (EATL) is a rare intestinal lymphoma that arises from intraepithelial lymphocytes. Clinical outcome of patients with EATL is very poor, due to chemotherapy-resistance and high relapse rates. Therefore, new therapeutic options for EATL are urgently needed. Studies in other types of lymphoma have shown that inhibition of apoptosis may cause chemotherapy-resistance and that restoration of defective apoptosis can induce cell death in these lymphomas. Preliminary data in EATL samples have demonstrated an increased expression of a fraction of NF-κB target genes, suggesting upregulation of NF-κB activity in EATL tumor cells. NF-κB activity can be inhibited by the proteasome inhibitor bortezomib resulting in induction of apoptosis. In the present study, we evaluated if apoptosis is inhibited in EATL cells and if Bortezomib can restore apoptosis in EATL cells.

Laser-capture microdissection was applied to 16 fresh frozen EATL samples to obtain purified tumor cells for RNA isolation. Intraepithelial lymphocytes (IEL) of healthy controls were obtained from fresh duodenal biopsies and isolated by cell sorting. RT-MLPA analysis revealed that the pro-apoptotic BH3-only gene Noxa was significantly downregulated in most EATL samples compared to healthy donor IEL. Induction with etoposide resulted in caspase-9 mediated apoptosis in EATL cells with relatively high Noxa expression, whereas in EATL cells with low Noxa expression no apoptosis was induced, suggesting an inhibition in the intrinsic apoptosis pathway. Treatment with Bortezomib resulted in induction of apoptosis in EATL cells. The lethal dose (LD50) varied between 7.5 nM and 15 nM. Bortezomib induced cell death in EATL cells was caspase-9 mediated. mRNA and protein expression analysis showed upregulation of Noxa after incubation with bortezomib.

In conclusion, our study showed that bortezomib induces apoptosis by upregulation of Noxa in EATL cells. Bortezomib therefore may be a potential drug in the treatment of patients with EATL.

Disclosures:

No relevant conflicts of interest to declare.
The Strong and the Weak - the Dual Role of JAK1 in Inflammatory Cytokine Production,
Authors: Luft Thomas , Wagner Andreas , Conzelmann Michael , Dietrich Sascha , Krämer Oliver , Schäfer Claudia , Giese Thomas , Falk Christine , Hecker Markus
Published: October 2019

Abstract 3218

Inhibition of JAK1 is an emerging clinical concept that has promise for a variety of autoimmune diseases, myeloproliferative diseases and post-transplant immunosuppression, but similarly raises concerns regarding immunosuppressive side effects.

At the example of IL-12p70 production in human monocyte-derived dendritic cells we demonstrate that JAK1 has a dual role in differentially regulating effects of weak and strong activation stimuli. We have demonstrated recently that weak NF-kB-activating stimuli (e.g. CD40L or LPS) require complementary JAK1-targeting cytokines such as IFN-g to induce IL-12p70. This pathway involves RELA, CREL, JAK1 and/or JAK2, STAT1, IRF1 and IRF8 and is inhibited by RELB and TYK2 (Conzelmann et al. Biochem Pharm. 2010, 80(12):2074–86).

Here we provide evidence for an alternative IL-12 stimulating pathway depending on strong NF-kB activating stimuli (e.g. intact E. coli or LPS plus IL-1b). siRNA silencing demonstrated that this pathway is specifically inhibited by JAK1 and the transcription factor STAT3, but is not influenced by any of the other JAK/STAT family members. Both IL-12p35 and p40 mRNA expression is directly inhibited by STAT3. Furthermore, ChIP-assays revealed that STAT3 binds directly to a combined STAT/NF-kB site at the IL-12p35 promoter without altering access of RELA and CREL. Extending the cytokine panel we found that E.coli-induced IL-6 and TNF-a production is similarly inhibited by the JAK1/STAT3 pathway whereas IL-10 expression is not affected. The observed dual effects of JAK1 are clearly confirmed by the JAK1/2 inhibitor INCB018424 (Ruxolitinib) which enhances E.coli-induced cytokines whilst strongly inhibiting cytokine production stimulated by CD40L/IFN-g.

In summary, our study suggests that blockade of JAK1 specifically inhibits pro-inflammatory effects of weak, IFN-g dependent, NF-kB activating stimuli while enhancing inflammatory cytokine expression induced by strong activation stimuli. Inhibition of JAK1/2 by INCB018424 (Ruxolitinib) would therefore represent a novel immunosuppressive approach that may spare the immune defence against invading pathogens.

Disclosures:

No relevant conflicts of interest to declare.
PGE2-Independent Expression of Indoleamine 2,3-Dioxygenase-2 (IDO2) Supports the Tolerogenic Function of Monocyte-Derived Dendritic Cells,
Authors: Trabanelli Sara , Curti Antonio , Ocadlikova Darina , Evangelisti Cecilia , Salvestrini Valentina , Ciciarello Marilena , Aluigi Michela , Rossi Lara , Metz Richard , Laury-Kleintop Lisa , Prendergast George , Lemoli Roberto M.
Published: October 2019

Abstract 3231

The indoleamine 2,3-dioxygenases IDO1 and IDO2 participate in tryptophan catabolism along the kynurenine pathway and are specifically inhibited by 1-methyltryptophan (1-MT). IDO1 expression in dendritic cells (DCs) is upregulated by various maturation stimuli. In particular, PGE2 plays a crucial role in inducing IDO1-expressing DCs. While it is well established that IDO1-expressing DCs contribute to immunological tolerance by a number of mechanisms, little is known about the expression, function and regulation of IDO2 in DCs.

Here we show that immature monocyte-derived DCs (Mo-DCs) express IDO2 which, like IDO1, is also upregulated upon maturation with a prostaglandin-E2 (PGE2)-containing cytokine cocktail. However, while IDO1 upregulation is strictly dependent on PGE2 during maturation, IDO2 expression level is not affected by PGE2. We show that IDO2 expressed by Mo-DCs is enzymatically active, producing L-kynurenine, and that this activity affects T-cell functions by inhibiting allogeneic and autologous T-cell proliferation and by inducing formation of a population of T regulatory cells (Tregs). Taken together, our findings establish different regulatory pathways for IDO2 and IDO1 in Mo-DCs, where IDO2 is functionally active, and they demonstrate that the tolerogenic function of Mo-DCs relies in part on PGE2-independent expression of IDO2.

Disclosures:

Metz: New Link Genetics Corporation: Employment. Prendergast:New Link Genetics Corporation: Employment.
Toll-Like Receptor 3 Activation Is Involved in Venous Thrombosis Development,
Authors: Lemarie Catherine A , Laurance Sandrine , Blostein Mark
Published: October 2019

Abstract 3277

Venous thromboembolism (VTE) afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. Inflammation may have a role in both the genesis and resolution of venous thrombi. Although septic thrombophlebitis occurs, most DVT are sterile. Recently, the role of toll-like receptor (TLR) signaling in modulating sterile inflammation has become better defined in experimental injury models. TLR3, for example, is activated by viral long double stranded RNA and endogenous RNA released by cellular apoptosis and necrosis, and induce a proinflammatory cellular response. Short single-stranded RNA, such as those used in RNA inhibition, stimulate TLR3 in endothelial cells and inhibit neoangiogenesis. Thus, we hypothesize that TLR3 might be involved in the inflammatory development of venous thrombosis. We first investigated whether stimulation of TLR3 influences venous thrombosis in mice. Intravenous injection of polyinosine polycytidylic acid (polyI:C), a synthetic double-stranded RNA analog and TLR3 ligand, increase the size and the cellular density of thrombi after FeCl3-induced inferior vena cava injury. TLR3 stimulation with polyI:C is also associated with an increased reactive oxygen species production, as well as increased neutrophil and macrophage content. Using a specific fluorescent probe for RNA, we found that FeCl3 induces RNA release and thus increases the RNA content in the thrombus. We next investigated the effect of RNase on thrombus development. Intravenous injection of RNase decreased thrombus size, as well as reactive oxygen species production and neutrophil and macrophage content in the thrombus. In vitro incubation of endothelial cells with polyI:C induces production of pro-inflammatory cytokines GM-CSF, G-CSF, IL-6, IL-8 and interferon-γ-induced protein 10 involved in the recruitment of neutrophils and macrophages. Taken together, these results strongly suggest that RNA release and TLR3 stimulation after endothelial cell injury participate in thrombus formation by inducing a pro-inflammatory response leading to the recruitment of neutrophils and macrophages.

Disclosures:

No relevant conflicts of interest to declare.
Coagulation Factor Activity Level and Clinical Bleeding Severity in Rare Bleeding Disorders: Results From the European Network of Rare Bleeding Disorders (EN-RBD),
Authors: Peyvandi Flora , Palla Roberta , Menegatti Marzia , Siboni Simona M , Halimeh Susan , Faeser Britta , Pergantou Helen , Platokouki Heleni , Giangrande Paul , Peerlink Kathelijne , Celkan Tiraje , Ozdemir Nihal , Bidlingmaier Christoph , Ingerslev Jorgen , Giansily-Blaizot Muriel , Schved Jean-Francois , Gilmore Ruth , Gadisseur Alain P. , Benedik-Dolnicar Majda , Kitanovski Lidija , Mikovic Danijela , Musallam Khaled M , Rosendaal Frits R.
Published: October 2019

Abstract 3312

The European Network of Rare Bleeding Disorders (EN-RBD) was established to bridge the gap between knowledge and practice in the care of patients with RBDs (rare bleeding disorders frequency <1/0.5-2M). The aim of this first report was to explore the relationship between the coagulation factor activity levels and clinical bleeding severity in patients with RBDs. A total of 592 records on patients with RBDs were cross-sectionally collected over a period of three years. Data on clinical bleeding episodes were available for 495 patients and were classified into four categories according to severity. The mean age of patients was 31 years (range, 7 months-95 years), with 51% being females. On linear regression analysis, there was a strong association between coagulation factor activity level and clinical bleeding severity for fibrinogen, factor (F)FX, FXIII, and combined FV and FVIII deficiencies. A weaker association was present for FV and FVII deficiencies. There was no association between coagulation factor activity level and clinical bleeding severity for FXI. It is of clinical relevance to identify levels that are not associated with spontaneous major bleeding, which was done by constructing receiver operating characteristic curves. These levels were highest for FXIII deficiency (25 U/dl), followed by FV+VII (15 U/dl), FVII (15 U/dl), FX (5 U/dl), and FV (1 U/dl) deficiencies. For fibrinogen deficiency, a factor activity level of 20 mg/dl is needed to ensure absence of major spontaneous bleeding. There is a clear relation between coagulation factor activity level and clinical bleeding severity in RBDs, which is different for the missing clotting factor.

Disclosures:

Peyvandi: NovoNordisk, CSL Behring: Honoraria. Bidlingmaier:CSL Behring, Bayer Schering: Research Funding; CSL Behring, NovoNordisk, Pfizer, Bayer Schering: Membership on an entity's Board of Directors or advisory committees; Baxter, Biotest, CSL Behring, NovoNordisk, Pfizer, Bayer Schering: Honoraria. Schved:LFB, CSL Behring, Novonordisk, Bayer Schering: Research Funding.
Critical Role of Factors II and X During Coumarin Anticoagulation and Their Combined Measurement with a New Prothrombin Time Variant (Fiix-PT),
Authors: Onundarson Pall T. , Gudmundsdottir Brynja R. , Francis Charles W.
Published: October 2019

Abstract 3354

Introduction:

Vitamin K antagonists (VKA) are monitored with prothrombin time (PT) based assays that are equally sensitive to reductions in factors II, VII or X. However, previous studies suggest that the anticoagulant effect of VKA depends primarily on reductions in factors II and X and not VII.

Aim and methods:

We compared the effect of vitamin K dependent (VKD) coagulation factors on PT (Quick and Owren methods) and also on rotational thromboelastometric (ROTEM) parameters. The experiments used normal platelet poor plasma (PPP) and PPP selectively immunodepleted of individual VKD factors, with and without added platelet phospholipid or washed platelets.

Results:

The PT was equally sensitive to reductions in factors II, VII or X. However, ROTEM parameters correlated poorly with the PT in anticoagulated patients` plasmas. ROTEM experiments showed that the clotting time, maximum velocity of clot formation and the maximum clot firmness were more affected by reductions in FII or FX than by FVII or FIX concentrations which had little influence except at very low concentrations. We developed a modified PT that was sensitive only to reductions in factors II and X by using factor II and X (Fiix) depleted plasma in the PT system. The Fiix-PT (Fiix-INR) correlated well with PT (INR) but the Fiix-INR fluctuated less than the INR in anticoagulated patients reflecting its insensitivity to FVII.

Conclusion:

The ROTEM results suggest that mild to moderate reductions in factors II or X are more important in clot formation than factors VII or IX at therapeutically relevant factor concentrations. Reductions in FII and X may therefore better reflect anticoagulation with VKA than FVII or IX. FVII may be a confounding source of unwanted variation in PT-INR. The new Fiix-PT that is sensitive only to FII and FX may more accurately reflect the degree of therapeutic anticoagulation in patients treated with VKA than do the current PT assays which may overestimate the fluctuation in anticoagulation.

Disclosures:

Onundarson: See i. other: Patent application for Fiix method in process. Gudmundsdottir:Other, see i: patent applicaiton filed for Fiix method.
Altered Regulation of Imprinted Non-Coding RNA Genes in Acute Myeloid Leukemia,
Authors: Yang Ming-Yu , Chang Jan-Gowth , Lin Pai-Mei , Hsu Jui-Feng , Wu Cheng-Han , Yang Wen-Chi , Liu Yi-Chang , Hsiao Hui-Hua , Lin Sheng-Fung
Published: October 2019

Abstract 3456

Studies in large-scale genome sequencing have shown that only 2% of the mammalian genome encodes mRNAs, but the most part is transcribed as long and short non-coding RNAs (ncRNAs). The ncRNAs with gene regulatory functions are starting to be seen as a common feature of mammalian gene regulation. Genomic imprinting is a form of epigenetic regulation and imprinted genes are silenced in a parental-specific manner. Imprinted genes tend to occur in clusters and ncRNAs have been found at all well-characterized imprinted clusters. Although the exact mechanism how imprinted ncRNA regulates gene expression remains largely unknown, it is general accepted that imprinted ncRNAs binds to chromatin modifying complexes, such as PRC2, TRX, and G9a, and generates specific silencing of genomic loci both in cis and trans. Imprinting is associated with many human diseases or syndromes (e.g. Prader-Willi, Angelman, Beckwith-Wiedemann, Retts, and Silver-Russell syndromes) and various cancers (e.g. breast, prostate, and colorectal cancers), but its role in leukemogenesis remain elusive. In this present study, a panel of 24 human imprinted ncRNAs genes, including ampd3, cpa4, snuf, rasgrf1, slc22a3, lgf2, treb3c, gabrb3, c15orf2, sfmbt2, rtl1, copg2, h19, l3mbtl, ppp1r9a, tspan32, lnpp5f, impact, nr3251, nr3252, znf215, prim2, peg3as and znf264, has been mined using Bioinformatics approach. We investigated the expression of these imprinted ncRNA genes using real-time quantitative RT-PCR in 67 newly-diagnosed acute myeloid leukemia patients with normal karyotypes (AML-NK), 22 AML patients with abnormal karyotypes (AML-AK), and 39 healthy individuals. In AML-NK patients, the expression of lgf2, h19, slc22a3, copg2, and impact were significantly upregulated than in healthy individuals (p < 0.0001). In AML-AK patients, besides lgf2, h19 and impact genes, ampd3 and gabrb3 were also significantly upregulated than in healthy individuals (p < 0.0001). Expression of igf2 was almost undetectable in healthy individuals but drastically increased in all AML patients. Both lgf2 and h19 were significantly increased in both AML-NK and AML-AK patients. From our preliminary results, it is reasonable to hypothesize that loss imprinting of lgf2/h19 is critical for the leukemogenesis of AML and under NK or AK conditions different additional ncRNAs are activated and affect different imprinted gene expression and thus leading to different clinical outcomes. Based on our findings, we will further perform methylation analysis of promoter CpG sites in AML patients to investigate if hypomethylation is responsible for the upregulation of these imprinted ncRNAs. We will also carry out in vitro functional analysis to elucidate the functions and mechanisms of these imprinted ncRNAs in AML tumorigenesis. Updated results of these analysis will be presented at the meeting.

Disclosures:

No relevant conflicts of interest to declare.
Fludarabine, Cytarabine, and Attenuated-Dose Idarubicin (m-FLAI) Induction for Elderly Patients with Acute Myeloid Leukemia: Results of a Multicenter Phase II Study,
Authors: Lim Yoojoo , Koh Youngil , Yoon Sung-Soo , Park Seonyang , Kim Byoung Kook , Kim Dae-Young , Lee Jung-Hee , Lee Kyoo-Hyung , Cheong June-Won , Kim Hyuk , Joo Young Don , Won Jong-Ho , Park Sung-Kyu , Kim Se Hyung , Hong Dae Sik , Park Eunkyung , Kim Min Kyoung , Sohn Sang Kyun , Kim Sung Hyun , Lee Hong-Kee , Kim Chul Soo , Estey Elihu H. , Lee Je-Hwan , Min Yoo-Hong , Kim Inho
Published: October 2019

Abstract 3626

Introduction:

Although there have been remarkable improvements in treatment of acute myeloid leukemia (AML), the prognosis of AML in elderly patients remains poor, and the best induction chemotherapy for these patients remains yet unknown. To devise an effective induction regimen for elderly patients with AML, we conducted a phase II trial to evaluate the efficacy and safety of the modified fludarabine, cytarabine, and attenuated-dose idarubicin (m-FLAI) regimen in these patients.

Patients and Methods:

Elderly (60 years or older) AML patients who did not receive previous chemotherapy were enrolled. Patients received two consecutive cycles of m-FLAI chemotherapy as an induction. The m-FLAI regimen was comprised of fludarabine (25mg/m2, days 1–4), cytarabine (1000mg/m2, days 1–4), and attenuated-dose idarubicin (5mg/m2, days 1–3). The primary end point was complete remission (CR) rate.

Results:

A total of 108 patients (median age 68.4 years, M:F=64:44) were enrolled. CR was achieved in 62.9% of patients, and treatment-related mortality rate (TRM) was 25.8%. Median overall survival (OS) was 9.3 months, and median event-free survival (EFS) was 6.6 months. The mortality at 30 and 60 days was 18% and 24%, respectively. Performance status and comorbidity did not have prognostic value in these patients. Bone marrow expression of CD117 was related to long EFS and OS.

Conclusion:

In conclusion, m-FLAI is a safe and effective induction regimen for previously untreated AML in elderly patients. Bone marrow CD117 expression is an independent good prognostic factor in these patients. (ClinicalTrials.gov number, NCT01247493)

Disclosures:

No relevant conflicts of interest to declare.
Meta-Analysis of Diffuse Large B-Cell Lymphoma Gene Expression Identifies Novel and Recurrent Biological Connections,
Authors: Care Matthew , Barrans Sharon , Worrillow Lisa , Jack Andrew , Westhead David , Tooze Reuben
Published: October 2019

Abstract 3682

Cell of origin classification of diffuse large B-cell lymphoma (DLBCL) identifies biologically and clinically meaningful subsets. In future these classes may inform distinct treatment. However a meta-analysis to assess consistency of class and gene signature associations across multiple DLBCL gene expression datasets is lacking. Furthermore diagnostic material is routinely available in formalin-fixed paraffin embedded (FFPE) form, and there is limited evidence that for DLBCL FFPE derived RNA can produce gene expression datasets comparable to freshly extracted RNA. Using an FFPE derived data-set we develop a platform robust implementation of the cell of origin classifier. With this classifier we observe comparable separation of survival in distinct data-sets derived from fresh and FFPE material. Classification of six data-sets encompassing a total of >900 DLBCL samples with this implementation establishes very similar patterns of gene expression for each DLBCL class. When tested against 12,000 signatures derived from LLMPP, MSigDB, GeneSigDB and genome wide transcription factor motif analysis of all TRANSFAC and JASPAR matrices, this communality translates into reproducible association of ABC and GCB-DLBCL with distinct molecular pathways, chromosomal regions and transcription factor motifs. This meta-analysis thus confirms the validity of FFPE derived data, identifies the most robust DLBCL class and signature associations and provides evidence for specific transcription factor signatures in both ABC and GCB-DLBCL.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of Caspase-8 Activity Caused by Overexpression of BCL10 Contributes to the Pathogenesis of High-Grade MALT Lymphoma,
Authors: Chen Yan , Yang Yishu , Sun Min , Yan Zhuohong , Cui Xiaoli , Zhang Ge , Morris Stephan W. , Zhang Quangeng
Published: October 2019

Abstract 3694

Mucosa-associated lymphoid tissue (MALT) lymphoma comprises approximately 8% of all non-Hodgkin lymphomas and is the most common lymphoma in the gastrointestinal tract. B-cell lymphoma/leukemia 10 (BCL10) overexpression and nuclear expression has been associated with high-grade MALT lymphomas unresponsive to H. pylori eradication treatment. To explore the molecular mechanism of BCL10 overexpression on the pathogenesis and malignant phenotype of MALT lymphoma, we generated EμSR-BCL10 transgenic mice. Results in our previous report showed that predisposition to marginal zone B-cell lymphoma (MZL) development was enhanced in these transgenic mice. MALT lymphoma precursor cells - marginal zone (MZ) B cells - are expanded in BCL10 transgenic mice. Constitutive activation of both canonical and noncanonical NF-κB pathways was identified in these MZ B cells. Elevated expression of BAFF, a NF-κB downstream gene, may be in part responsible for increased tumor cell survival in MALT lymphomas. In this continuing study, quantitative analysis of BCL10 protein levels and the extent of MZ B cell expansion in heterozygous (Tg/+) and homozygous (Tg/Tg) EμSR-BCL10 transgenic mice demonstrated a direct pathogenic effect of BCL10 protein on MALT lymphomagenesis. The activities of caspase-8 and caspase-3, but not caspase-9, were inhibited with increasing BCL10 protein levels in a dose-dependent manner. Apoptosis induced by anti-IgM was selectively inhibited in MZ B cells from transgenic mice, but no differences were observed following treatment with dexamethasone, γ-irradiation or anti-CD95, implying that overexpressed BCL10 exerts anti-apoptotic effects through the B-cell antigen receptor pathway. Expression of API2, another NF-κB downstream gene, was also elevated in a dose-dependent manner with BCL10 protein level in the MZ B cells. Overexpressed BCL10 protein co-immunoprecipitated with caspase-8 and API2, suggesting an in vivo interaction leading to inhibition of caspase-8 activity. Our current data suggests a novel effect of overexpressed BCL10 in the pathogenesis of high-grade MALT lymphoma by increasing expression of API2 and recruitment of API2 and caspase-8 to form a protein complex leading to the suppression of caspase-8 activity in MZ B cells.

Disclosures:

No relevant conflicts of interest to declare.
Efficacy of 90yttrium-Ibritumomab Tiuxetan in Extranodal Marginal-Zone Lymphoma,
Authors: Vanazzi Anna , Grana Chiara , Crosta Cristiano , Pruneri Giancarlo , Rizzo Stefania , Radice Davide , Pinto Antonello , Calabrese Liliana , Negri Mara , Paganelli Giovanni , Martinelli Giovanni
Published: October 2019

Abstract 3713

BACKGROUND:

To evaluate clinical activity of 90Y-Ibritumomab Tiuxetan in Extranodal Marginal-Zone Lymphoma.

PATIENTS AND METHODS: From May 2004 to April 2011 thirty patients affected by relapsed/refractory Marginal-Zone Lymphoma - arisen at any extranodal site - were enrolled to receive 90Y-Ibritumomab Tiuxetan at the activity of 0,4 mCi/kg. Median age was 57 years (range 36–83 ys); 16 female and 14 male. Thirtheen patients had Helicobacter Pylori-negative gastric Mucosa-Associated Lymphoid tissue Non Hodgkin Lymphoma, while 17 patients had extra-gastric Marginal-Zone Lymphoma. At time of treatment 13 out of 30 patients had disseminated disease (stage III/IV; Ann-Arbor Staging System); 4 patients bone marrow involvement. All patients had received a previous treatment with a maximum of 7. The median number of previous treatments administered - including antibiotics, CT, RT or surgery - was 1 with a maximum of 7; 14/30 had received at least 2 previous treatments, 5 of them more than 3.

RESULTS:

Toxicities were primarily hematological, as expected, and reversible; infection or febrile neutropenia was observed in 3 patients. Up to now no myelodisplastic syndrome occurred. Overall Response Rate was 90%: twenty-three patients achieved a complete response (77%); partial response occurred in 4 patients (13%), stable disease in 2 patients (7%) and 1 progression (3%). With a median follow-up of 47 months, 2 patients relapsed after complete response; 8 out of 20 complete responses are still responder after > 3 years and 5 patients after > 5 years.

CONCLUSIONS:

90Y-Ibritumomab Tiuxetan seems to be active in patients with Extranodal Marginal-Zone Lymphoma resistant or refractory to conventional systemic treatment. These results suggest as a single shot of radioimmunotherapy could represent a possible option for the treatment of relapsed Marginal Zone Lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
The Allosteric AKT Inhibitor MK-2206 Demonstrates Potent Antiproliferative Activity in Lymphoma Cells and Synergizes with the HDAC Inhibitor Vorinostat,
Authors: Buglio Daniela , Lemoine Manuela , Estrella Jaymie , Neelapu Sattva S. , Davis Richard Eric , Doyle Laurence , Little Richard F , Berry Donald , Younes Anas
Published: October 2019

Abstract 3729

The serine/threonine kinase Akt plays a critical signaling role downstream of phosphatidylinositol-3-kinase (PI3K) and is important in promoting cell survival and inhibiting apoptosis. Indeed, Akt activation and overexpression is often associated with resistance to chemotherapy or radiotherapy. Previous studies demonstrated the potential therapeutic value of targeting the PI3K pathway in lymphoma, as both the selective PI3Kδ inhibitor CAL-101, and everolimus and temsirolimus, which target PI3K and mTOR, produce clinical responses in a variety of lymphomas. We evaluated the effect of the novel allosteric Akt inhibitor, MK-2206, in a panel of lymphoma cell lines and primary lymphoma cells. We found that Akt, and activated pAkt, are highly expressed in lymphoma cells. After 72 hours of incubation, the Akt inhibitor MK-2206 demonstrated antiproliferative activity in a variety of lymphoma cell lines, with an IC50 ranging between 0.1 and 5μM. There was no correlation between pre-treatment levels of pAKT, PI3K isoforms, or PTEN protein expression and sensitivity to MK-2206. Within the diffuse large B cell lymphoma cell lines, those of GCB cell of origin were more sensitive to MK-2206, compared with the ABC-derived cell lines. Resistant cell lines tended to had weak or absent expression of p-GSK3 and p-4EBPI. Mechanistically, MK-2206 treatment decreased the level of p-Akt (Ser473), and p-Akt (Thr308), irrespective of drug sensitivity. Furthermore, MK-2206 decreased the phosphorylation level of Akt downstream targets, including p-GSK3 beta and p-PRAS40, upregulated p27. and dephosphorylated p70S6K. Moreover, MK-2206 treatment decreased HIF-1 alpha and VEGF expression. Depending on the cell of origin, the antiproliferative effect resulted from cycle arrest at the G0/G1 phase, autophagy, orapoptosis. MK-2206 showed synergistic effect in combination with the HDAC inhibitor, Vorinostat. Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, the combination of MK-2206 and Vorinostat effectively altered p53 and p27 levels, which were associated with increased PARP cleavage and induction of apoptosis. Our data demonstrate that AKT is a promising target for the treatment of lymphoma, and provide a rationale for an ongoing trial, evaluating MK-2206 for the treatment of patients with relapsed lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
Upfront Imatinib in Pediatric Chronic Myeloid Leukemia Yields Results Comparable to Stem Cell Transplant,
Authors: Ganesan Prasanth , Sagar Tenali Gnana , Kannan Krishnarathnam , Rajendranath Rejiv
Published: October 2019

Abstract 3763

Background:

Imatinib mesylate (IM) is the standard therapy in adult patients with chronic myeloid leukemia (CML). In children, the superiority of Imatinib over allogenic transplantation is not well established. Concerns exist about the long term effects of Imatinib in this population.

Methods:

Outcomes of children (≤18 years) diagnosed with chronic phase CML from 2002 to 2008 were retrospectively analysed. All received Imatinib at a dose of 260–300mg/m2/day. Progression free survival (PFS- time from starting treatment till loss of complete hematologic response (CHR), or disease acceleration), ad Overall survival (OS) were estimated by the Kaplan Meier method. Those who progressed received higher doses of Imatinb or were changed to hydroxyurea. No patient had access to second line tyrosine kinase inhibitors or to allogenic stem cell transplant.

Results:

31 patients, median age 13 (6–18)years, Males-17 (48.6%), were treated. The presenting complaints over a median symptom duration of 1 month (10 days to 12 months) were abdominal distension and/or pain (68%), fever (38%), weight loss (13%), bone pains (10%), anemic symptoms (16%). Examination revealed splenomegaly (100%), heptomegaly (47%) and lymphadenopathy (6%). Laboratory: Hemoglobin (9.3g/dL;4.2–10.9), White cell count (228,000/mm3;10,000–800,000), Platelet count (412,000/mm3, 120,000–12,80,000), peripheral eosinophilia (2%;0–10), basophilia (2%; 0–7). CHR was seen in 30/31 (97%) at a median of 2 months (1.5–8). Sokal risk category was low in 13 (41%), intermediate in 15 (48.4%) and high in 3 (9.7%). The cumulative incidence of Major and Complete cytogenetic response (MCR and CCR) among those who had not progressed by 2 years (n=23) was 82% and 70% respectively. Molecular assessment done in 11 patients who had achieved CCR by 2 years showed MMR in 6/11 (55%). After a median follow up of 49.2 months (4.4 – 86.4) disease progression occurred in 8 patients (5-year PFS 68%-median not reached) which led to death in 6 patients (5-year OS 75.8%). Among those with cytogenetic data, the PFS for those with CCR vs those without was 93.8% and 53.6 % respectively at 5 years (p=0.048). The drug was well tolerated with no discontinuation because of toxicity.

Conclusions:

Imatinib is a reasonable option in the first line therapy of pediatric CML and gives 5 year PFS and OS rates comparable to the results of upfront allogenic transplant. It is to be considered as an alternative especially in resource constrained settings where options for transplant are limited.

Disclosures:

No relevant conflicts of interest to declare.
Final Phase I/II Results of Rigosertib (ON 01910.Na) Hematological Effects in Patients with Myelodysplastic Syndrome and Correlation with Overall Survival,
Authors: Raza Azra , Greenberg Peter L. , Olnes Matthew J. , Silverman Lewis R. , Wilhelm Francois
Published: October 2019

Abstract 3822

Rigosertib is a multi-kinase inhibitor that selectively induces mitotic arrest leading to apoptosis in cancer cells and blasts, while being non-toxic to normal cells. We analyzed bone marrow (BM) response and overall survival (OS) in 60 patients (pts) with myelodysplastic syndrome (MDS), including 51 patients with refractory anemia and excess blasts (RAEB) and 9 patients with refractory cytopenia and multilineage dysplasia (RCMD) enrolled in 4 independent phase 1/2 clinical trials. These pts were treated with rigosertib administered as a continuous intravenous infusion (CIV) from 2 to 6 days weekly or every other week with BM response initially assessed per protocol by week 4 or 8 and every 8 weeks thereafter. Overall survival (OS) analyses were performed by the method of Kaplan-Meier. OS was related (p=0.04) to FAB/WHO classification (see Table 1) in all MDS pts. Eight pts had hematological improvements. OS was also related to IPSS scoring (p=0.02; Table 2) and to BM blastic response (Table 3; p=0.008) in the 51 RAEB-1,-2,-t pts and in a subset of 38 RAEB-1,-2,-t pts refractory or relapsing after treatment with hypomethylating agents (azacitidine/decitabine) (Table 4; p=0.001). A 49-week OS was found in 15 patients in this last group treated with 3-day rigosertib infusions (1800 mg/day) every other week. Rigosertib infusions were well tolerated without evidence of bone marrow myelotoxicity. These results and the predictive value of BM response to rigosertib for estimating OS survival have led to the initiation of a randomized Phase III survival trial of rigosertib 3-day CIV infusions vs best supportive care in RAEB -1, -2 and-t pts who failed or progressed after receiving hypomethylating agents.Table 1.Overall Survival by FAB/WHO Classification in 60 MDS ptsFAB/WHO ClassificationRCMDRAEB-1RAEB-2RAEB-tP valueN pts9172113Median OS (weeks)988235210.04Table 2.Overall Survival by IPSS Scoring in 51 RAEB-1, -2, -t ptsIPSS ScoringIntermediate-1Intermediate-2High RiskP valueN pts101427Median OS (weeks)Not Reached37280.02Table 3.Overall Survival by BM Blast Response in 51 RAEB-1, -2, -t ptsBM Blast Response≥ 50% Blast DecreaseStable BM ResponseProgressive DiseaseNot AssessedP valueN pts1620510Median OS (weeks)513715110.008Table 4.Overall Survival by BM Blast Response in 38 RAEB-1, -2, -t pts Refractory or Relapsing After Azacitidine/Decitabine TreatmentBM Blast Response≥ 50% Blast DecreaseStable BM ResponseProgressive DiseaseNot AssessedP valueN pts1312310Median OS (weeks)444915100.001

Disclosures:

Raza: Onconova Therapeutics Inc: Research Funding. Greenberg:Onconova Therapeutics Inc: Research Funding. Olnes:Onconova Therapeutics Inc: Onconova Therapeutics Inc provided study drug. Silverman:Onconova Therapeutics Inc: Research Funding. Wilhelm:Onconova Therapeutics Inc: Employment, Equity Ownership.
Increased CD39 Expression on CD4+ T-Lymphocytes Has Clinical and Prognostic Significance in Chronic Lymphocytic Leukemia,
Authors: Herishanu Yair , Hazan-Hallevi Inbal , Kay Sigi , Deutsch Varda , Polliack Aaron , Naparstek Elizabeth , Perry Chava
Published: October 2019

Abstract 3895

Chronic lymphocytic leukemia (CLL) cells depend on their microenvironment for proliferation and survival. Ectonucleotidase CD39 has anti-inflammatory properties as it hydrolyzes pro-inflammatory extra-cellular ATP, generates anti-inflammatory adenosine and also protects regulatory T cells from ATP-induced cell death. In this study we investigated the clinical significance of CD39 expression on CD4+T-cells in 45 patients with CLL as well as its compartmental regulation and explored the possible mechanisms for its induction. Compared to healthy individuals, CD4+CD39+ lymphocytes were increased in the peripheral blood of patients with CLL (4.6%±2.28 vs. 17.3%±12.49, respectively, p=0.004), and correlated with advanced stage of disease (9.72%±5.76, 18.15%±12.03 and 25.90%±16.34, of CD4+ lymphocytes, in patients with Rai stages 0, 1+2 and 3+4, respectively, p=0.019). CD4+CD39+ cells were also higher in patients with CLL who needed therapeutic intervention (untreated; 12.99%±10.63 vs treated; 22.21%±12.88, p=0.01) and in those who were ZAP70+ or had b2-microglobulin levels>3g/L. There were more CD4+CD39+ lymphocytes in the bone marrow compartment (22.25%±16.16) than in the peripheral blood (16.60%±15.84, p=0.009). In-vitro studies showed that CD39 can be induced on CD4+cells by exposure to ATP or indirectly, following B-cell receptor (BCR) engagement (CD4+CD39+ lymphocytes increased by 1.56 fold, in the BCR engaged samples compared to their paired controls; 20.27%±11.3 vs. 13%±9.42, respectively, p=0.0006). Conclusions: Increased CD39 expression on CD4+ T-lymphocytes in CLL associates with an aggressive disease. This may reflect the ability of the leukemic cells to suppress the surrounding immune environment, and contribute to a poorer prognosis. CD39+ may also serve as a future target for the development of novel therapies with immune modulating anti–tumor agents in CLL.

Disclosures:

No relevant conflicts of interest to declare.
Non-Lineage/Stage Restricted Effects of a Gain-of-Function Mutation in Tyrosine Phosphatase Ptpn11 (Shp2) on Malignant Transformation of Hematopoietic Cells
Authors: Xu Dan , Liu Xia , Yu Wen-Mei , Meyerson Howard J. , Gerson Stanton L. , Qu Cheng-Kui
Published: October 2019

Abstract 392

SHP2, a protein tyrosine phosphatase implicated in multiple cell signaling processes, plays an essential role in hematopoietic cell development. Our previous studies have demonstrated that this phosphatase is required for erythroid, myeloid, and lymphoid development and that it functions in cytokine signaling in both catalytically-dependent and –independent manners. Notably, germline and somatic mutations (heterozygous) in PTPN11 (encoding SHP2) have been identified in 35% of the patients with juvenile myelomonocytic leukemia (JMML), a childhood myeloproliferative disorder (MPD). Furthermore, PTPN11 mutations are also found in pediatric myelodysplastic syndromes (10%), B cell lymphoblastic leukemia (B-ALL) (7%), acute myeloid leukemia (AML) (4%), and sporadic solid tumors. These mutations result in hyperactivation of SHP2 catalytic activity. In addition, PTPN11 disease mutations, especially leukemia mutations, enhance the binding of mutant SHP2 to signaling partners. Although previous studies have shown that Ptpn11 mutations induce cytokine hypersensitivity in myeloid progenitors and MPD in mice, it is unclear whether Ptpn11 mutations also play a causal role in the pathogenesis of acute leukemias. If so, the underlying mechanisms and the cell origin of leukemia initiating/stem cells (LSCs) remain to be determined. PTPN11E76K mutation is the most common and most active PTPN11 mutation found in JMML and acute leukemias. However, the pathogenic effects of this mutation have not been well characterized. We created Ptpn11E76K conditional knock-in mice. Global Ptpn11E76K/+ mutation resulted in early embryonic lethality associated with enhanced ERK signaling. Induced knock-in of this mutation in pan hematopoietic cells led to MPD as a result of aberrant activation of hematopoietic stem cells (HSCs) and myeloid progenitors. These animals subsequently progressed to acute leukemias. Intriguingly, in addition to AML, T-ALL and B-ALL were evolved. PTPN11E76K/+ mutation induced LSC development not only in stem cells but also in lineage committed progenitors as tissue-specific knock-in of Ptpn11E76K/+ mutation in myeloid, T lymphoid, and B lymphoid progenitors also resulted in AML, T-ALL, and B-ALL, respectively. Further analyses revealed that Shp2 was distributed to centrosomes and that Ptpn11E76K/+ mutation promoted LSC development partly by causing centrosome amplification and genomic instability. Thus, Ptpn11E76K mutation has non-lineage specific effects on malignant transformation of hematopoietic cells and initiates acute leukemias at various stages of hematopoiesis. This mutation may play an initiating role in the pathogenesis of pediatric acute leukemias.

Disclosures:

No relevant conflicts of interest to declare.