bluuebunny/crossref_metadata_2025 huggingface dataset:
The ETS Protein ELF2 Contributes to a Novel TAL1/SCL-Containing DNA-Binding Complex That Positively Regulates Erythroid Gene Expression and Cell Differentiation,
Authors: Cai Ying , McCormack Matthew P. , Curtis David J. , Nagarajan Lalitha , Brandt Stephen J.
Published: October 2019

Abstract 3389

The TAL1/SCL gene, originally discovered from its involvement by a recurrent chromosomal translocation in T-cell acute lymphoblastic leukemia, is important for hematopoietic stem cell and erythroid, megakaryocyte, and macrophage progenitor function and is essential for hematopoietic and vascular development. Although Tal1 can bind DNA with one of its E protein DNA binding partners, its function in erythroid cells has been thought to be mediated by a larger complex recognizing an E box-GATA or GATA sequence motif. In independent lines of investigation, a novel DNA-binding motif for TAL1 comprised of an ETS site immediately adjacent to an E box was identified by site selection analysis in K562 cells, and multiple peptides derived from a single ETS protein, E74-like factor 2 (Elf2, also known as NERF), were identified by tandem mass spectrometry analysis of Tal1-containing protein complexes purified from murine erythroleukemia (MEL) cells. To determine whether Elf2, previously shown to interact with Tal1's interaction partner LMO2, recognized this sequence element, electrophoretic mobility shift analysis was carried out using MEL cell nuclear extracts. This revealed the existence of a novel multiprotein ETS-E box DNA-binding complex containing Tal1, Elf2, Lmo2, the LIM domain-binding protein Ldb1, and Ldb1-interacting single-stranded DNA binding proteins Ssbp2 and Ssbp3. Structure-function analysis of this DNA-binding activity, which increased with chemical induction of MEL cell differentiation, showed that both the E box and ETS site were required for ternary complex formation, that preference existed for specific ETS flanking sequences, and that precise spacing between the two transcription factor binding sites was critical for complex formation. Finally, a physiological target for this complex was identified in the Ssbp3 gene, with Tal1, Elf2, and Ldb1 found to specifically co-occupy the third intron of this gene by chromatin immunoprecipitation analysis but not a similar ETS-E box element in the 11th intron. In accord, Ssbp3 expression, ETS-E box DNA-binding activity, and hemoglobin concentration increased with enforced expression of Elf2 in MEL cells, while Ssbp3 protein abundance, ETS-E box DNA-binding activity, and cellular differentiation decreased with Elf2 knockdown. These studies identify a novel TAL1- and ELF2-containing DNA-binding complex in erythroid progenitors that positively regulates gene expression and differentiation. In addition, they reveal a previously unknown role for the ETS protein ELF2 in erythropoiesis.

Disclosures:

No relevant conflicts of interest to declare.
The Efficacy of Rabbit Antithymocyte Globulin (Thymoglobulin®) with Cyclosporin As First-Line Treatment in Aplastic Anemia,
Authors: Shin Seung-Hwan , Yahng Seung-Ah , Lee Sung-Eun , Cho Byung-Sik , Eom Ki-Seong , Kim Yoo-Jin , Kim Hee-je , Lee Seok , Min Chang-Ki , Cho Seok-Goo , Kim Dong-Wook , Min Woo-Sung , Park Chong-Won , Lee Jong-Wook
Published: October 2019

Abstract 3430

Background:

Antithymocyte globulin (ATG) is the drug of choice for immunosuppressive therapy (IST) in patients with aplastic anemia (AA) unsuitable for hematopoietic stem cell transplantation. The standard ATG preparation in AA had been horse ATG because of the larger experience and the results already reported with this preparation. Due to the unavailability of the horse ATG since 2006, rabbit ATG became the only available ATG preparation in Korea. But, there are only limited data about the therapeutic efficacy of rabbit ATG as first-line IST in AA.

Method:

We retrospectively investigated the outcome of 58 evaluable patients among 62 patients with AA treated with IST using rabbit ATG as front line between March 2006 and April 2010 at our institution. 70.7% of enrolled patients were very severe (n=18) or severe AA (n=23). All patients received rabbit ATG (Thymoglobulin®, 2.5mg/kg per day for 5 days) with methylprednisolone and cyclosporine A. Response rate (RR) was assessed at 3, 6, 9, 12 and 18 months after IST.

Results:

After IST, overall RR was 27.8%, 50.8%, 52.8%, 52.8% and 56.7% after 3, 6, 9, 12 and 18 months, respectively. Complete response (CR) rates were 0.8%, 1.8%, 5.6%, 9.6% and 21.2% after 3, 6, 9, 12 and 18 months, respectively. Median time to achieve partial response (PR) and CR were 93 (range; 12∼977 days) and 381 (range; 12–614) days. Among 31 responders, 10 patients (32.3%) relapsed. Median time between response and relapse were 396 days (range; 254∼681 days).

Estimated overall survival and failure free survival at 3 years from ATG treatment were 85.8% and 42.8%, respectively. Age (>45) at the use of ATG was an independent predictor of overall survival and overall response (p=0.033 and 0.027) in univariate analysis. Other factors such as disease severity, pre-ATG hematological parameters (absolute lymphocyte count and absolute reticulocyte count) were not associated with overall survival and failure free survival.

Conclusion:

These data indicate that rabbit ATG was as effective as horse ATG based on other recently published data. But, the response time of rabbit ATG treatment is longer than that of horse ATG treatment. Especially, more than half of patients who achieved CR required time over 1 year after ATG treatment. Rabbit ATG can be effective IST regimen comparable to horse ATG, but it takes longer time to achieve sufficient response.

Disclosures:

No relevant conflicts of interest to declare.
P53 is Activated without RPL11 Upregulation in Diamond-Blackfan Anemia,
Authors: Du Hong-Yan , Elghetany M. Tarek , Alter Blanche P , Shimamura Akiko
Published: October 2019

Abstract 3432

Diamond-Blackfan anemia (DBA) is an autosomal dominantly inherited bone marrow failure syndrome characterized by red cell aplasia, physical anomalies, and cancer predisposition. DBA is caused by mutations resulting in haploinsufficiency of genes encoding ribosomal proteins. p53 is activated in the erythroid lineage following reduction of ribosomal protein expression; however the mechanism whereby ribosomal stress results in p53 activation in DBA remains unclear. RPL11 has been proposed to play a central role in p53 activation following ribosomal stress. Reduced expression of individual small ribosomal subunit proteins in a tumor cell line resulted in increased translation of RPL11. Excess free RPL11 can bind and inactivate HDM2, an E3 ubiquitin ligase targeting p53 for degradation. The recent demonstration that cellular responses to ribosomal perturbations vary widely between different tissues raised the question of whether RPL11 upregulation contributes to p53 activation following ribosomal stress in hematopoietic progenitors. To address this question, we modeled DBA in human CD34+ cells. Since RPS19 is the most commonly mutated gene in DBA, we used lentiviral vectors expressing short hairpin RNAs to knock down RPS19 expression in primary human CD34+ cells. RPS19 protein levels were reduced to about 50% of control levels in a manner reflecting the haploinsufficient state in DBA. RPS19 depletion resulted in elevated p53 protein levels and increased mRNA levels of p21, a transcriptional target of p53. Total p53 mRNA levels and p53 mRNA translational activity remained unchanged consistent with a post-transcriptional mechanism for p53 activation. Although total RPL11 mRNA levels were not diminished following RPS19 depletion, RPL11 protein levels were significantly decreased consistent with post-transcriptional downregulation. Depletion of RPS19 in human CD34+ cells did not affect polysome loading of RPL11 mRNA. Reduction of additional ribosomal proteins also accompanied RPS19 knockdown consistent with coordinate regulation of multiple ribosomal protein levels. Corticosteroids, which improve anemia in the majority of DBA patients, did not prevent p53 activation, nor did this improve RPS19 or RPL11 protein levels. Expression of p53 was also assessed in bone marrow biopsy slides from 26 DBA patients with the following genotypes: RPS19 (18), RPS24 (2), RPS26 (2), RPS10 (1), RPS17 (1), RPS7 (1), and RPL11 (1). p53 was over-expressed in all but one patient (RPS26), and was clearly over-expressed in the DBA patient harboring the RPL11 mutation. In summary, we find that p53 activation in DBA does not require upregulation of RPL11 translation or elevated RPL11 protein levels. p53 activation persists in DBA caused by RPL11 deficiency. Corticosteroids do not improve ribosomal protein levels nor do they prevent p53 activation.

Disclosures:

No relevant conflicts of interest to declare.
Cytokine/Chemokine Expression with Protein Arrays Identifies Three Subsets of T-Cell Lymphomas with Differented Immunological Profiles,
Authors: Mercadal Santiago , Martinez Antonio , Colomo Lluis , Campo Elias , Lopez-Guillermo Armando
Published: October 2019

Abstract 3473

Background:

Only a few studies have addressed the expression of cytokine/chemokine in T-cell lymphomas. The aim of this study was to analyze the cytokine/chemokine protein expression profiles and to correlate them with the pathological manifestation of T-cell lymphomas. Patients and methods: Samples at diagnosis from 19 patients were T-cell lymphoma (unspecified peripheral T-cell lymphomas (PTCL), 10 cases; angioimmunoblastic (AITL), 4; ALK-negative anaplastic large-cell (ALCL), 3 and ALK-positive ALCL, 2) and were studied by protein microarrays. The characteristics of the patients were as follows: 11/8 M/F; median age, 55 years. 50% of patients were stage IV, 69% had extranodal involvement, 79% high LDH and 66% high β2 microglobulin. The high or high-intermediate risk IPI and PIT score were 62% and 61%, respectively. We have analyzed the expression of 174 cytokines by using a highly sensitive and specific antibody array containing single band specific primary antibodies duplicate. Chemolumiscence detection was performed by a HRP-conjugated streptavidin in an image analysis system. Densitometry analysis of unsaturated pixels was performed with Image Gauge software and an endogenous housekeeping gene was used to normalize results. Unsupervised clustering analysis was then performed. Results: Three clusters were obtained. Cluster 1 included 5 ALCL, 2 PTCL and 1 AITL, with a cytokine/chemokine profile that had high expression of IL21R, MIG, PDGF AA/AB, LIGHT, IL10, MIP1γ, MIP3α and IL13. Cluster 2 was composed by 5 PTCL and 1 AITL and exhibited a profile characterized by the high expression of L-SELECTIN, MMP1, IL1RII, SIGLEC 5, IL10R, IL1β, IL18BPA, FGF7, IL13RAα2, GMCSF, SDF1β, LIF, IL1Rα and IL11. Finally, the analysis defined a cluster 3 constituted by 2 AITL and 3 PTCL with a cytokine/chemokine profile that showed the high expression of TIMP1, NT4, ITAC and IGBP6. Detailed initial features and outcome of the patients according to the clusters are detailed in the table. Cluster 1 had better survival than the other groups (p=0.01). Summary: T-cell lymphomas show three different cytokine/chemokine patterns as was determined by protein microarrays. Cluster 1 was well characterized with the presence of all ALCL and better survival.

Disclosures:

No relevant conflicts of interest to declare.
Treatment with the PI3K/mTOR Inhibitor, NVP-BEZ235, Overcomes Resistance to Imatinib in Quiescent or T315I-Mutated Ph+ Leukemia Cells,
Authors: Minami Yosuke , Minami Miho , Fukushima Nobuaki , Kuwatsuka Yachiyo , Naoe Tomoki
Published: October 2019

Abstract 3488

Ph+ leukemia is effectively treated with BCR-ABL kinase inhibitors such as imatinib (IM). However, recent studies suggest that IM therapy does not eradicate Ph+ leukemia stem cells (LSCs). In order to examine the mechanisms of Ph+ LSC resistance to IM and seek strategies to overcome this, Ph+ ALL patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice and abundant spleen cells derived from leukemic NOG mice were co-cultured with S17-stromal cells using methods previously reported (Minami et al., Proc Natl Acad Sci USA, 2008). Slow-cycling (Hoechst-33342low/Pyronin-Ylow) CD34+ cells were insensitive to IM despite complete dephosphorylations of BCR-ABL and its substrate CrkL, which indicated that addiction of BCR-ABL activity is lower for survival in such quiescent cells. From comprehensive drug screening of other small compounds using this co-culturing system, we found that inhibitors of PI3K/AKT/mTOR-axis signaling, including rapamycin, were promising candidates. Aberrant activation of mTOR signaling has also been reported to be involved in some systems of leukemia initiating cells. In vitro, combination treatment with IM and rapamycin analogue, everolimus (RAD001), induced substantial cell death in the slow-cycling CD34+ population with p70-S6K dephosphorylation and decreased expression of the anti-apoptotic BCL-2 family protein, MCL-1. In the leukemic NOD/SCID mouse ALL model, in vivo RAD001-treatment showed a decrease of tumor burden including slow-cycling CD34+ cells. However, during the cell death by RAD001-treatment, however, negative feedback-loop effects were detected such as reversely increased phosphorylations of AKT (Ser473) and FOXO1/3a. In vitro, the dual PI3K/mTOR inhibitor, NVP-BEZ235 (BEZ), induced substantial cell death including slow-cycling CD34+ cells at lower doses than those required by RAD001. In biomarker analyses, BEZ-treatment inhibited not only p70-S6K phosphorylation but also phosphorylations of mTOR (Ser2481) itself, 4E-BP1, AKT (Ser473) and FOXO1/3a. At the same time, expression of MCL-1 decreased during the cell death by BEZ-treatment. In T315I-mutated BCR-ABL dependent cell lines such as murine Baf3/p210-T315I and human-derived TCC-Y/T315I, treatment with BEZ induced growth inhibition and cell death. The effects of BEZ on normal stem cells and in mouse Ph+ leukemia models are under investigation. These results suggest that treatment with BEZ might have potential for overcoming the resistance to IM due to quiescent property in Ph+ LSCs or T315I-mutation.

Disclosures:

Naoe: Zenyaku-Kogyo: Research Funding; Novartis Pharma.: Research Funding; Chugai Pharma.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Kyowa-Hakko Kirin.: Research Funding; Otsuka Pharma.: Research Funding.
Platelet Factor 4 (PF4) Levels Are Inversely Correlated with Steady-State Platelet Counts and with Nadir Counts in Chemotherapy-Induced Thrombocytopenia (CIT),
Authors: Lambert Michele , Reznikov Alisa , Xiao Liqing , Poncz Mortimer
Published: October 2019

Abstract 3527

PF4 (CXLC4) is a member of the C-X-C family of chemokines and has been shown to be a negative autocrine of megakaryopoiesis in vitro and in animal models of CIT and radiation-induced thrombocytopenia (RIT). In animal models, steady-state platelet counts, as well as duration of thrombocytopenia and nadir platelet count after chemotherapy or radiation therapy were inversely correlated with platelet PF4 levels. We designed a clinical study to examine the role of PF4 in human subjects treated with chemotherapy. We examined platelet PF4 levels in 97 pediatric patients treated for standard risk leukemia in a single institution study. All patients completed therapy since January 1, 1999. Samples were collected at the completion of therapy from all patients, and a subset of randomly selected patients had multiple samples collected to determine intra-subject variability of assessment. PF4 levels were no different in this cohort from age matched normal controls. While there was significant inter-individual variability in the cohort, there was no significant difference in an individual between samples collected up to 1 year apart. There was an inverse correlation between platelet PF4 levels and steady-state platelet count (r=-0.2107 95% CI: −0.39 to −0.01, p=0.0383), yet we found no correlation with plasma thrombopoietin (TPO) or CXCL11 (stromal derived factor-1) levels, both of which are known positive regulators of megakaryopoiesis. We then examined the effect of PF4 on CIT by reviewing medical records of these patients, extracting the nadir platelet count and transfusion records for delayed intensification. There was an inverse correlation between platelet PF4 content and nadir platelet count during delayed intensification (r=-0.2087 95% CI: -0.41 to 0.01, p=0.0316). In addition, subjects who required platelet transfusion during delayed intensification had significantly higher platelet PF4 levels when compared to those subjects who did not require transfusion (11.48 ± 8.2 IU/108 platelets versus 5.23 ± 4.7 IU/108 platelets, p=0.003). All of the patients with PF4 levels > 1SD above the mean (>18 IU/108 platelets) were transfused, while only 28% of patients with PF4 levels > 1 SD below the mean (< 4 IU/108 platelets) (overall 58% of patients were transfused). In contrast, there was no difference between plasma TPO or CXCL11 levels between those patients who did and did not require transfusion. These clinical data are not only consistent with our previously published animal studies; they show that platelet PF4 levels may be the first identified, patient-specific predictor of risk of transfusion during chemotherapy and may allow for stratified dose-intensification in patients with cancer. These studies also suggest new approaches for modifying megakaryopoiesis and developing novel strategies to moderate CIT and possibly RIT.

Disclosures:

Lambert: Cangene: Honoraria.
Sequential Acquisition of AML1-ETO and c-Kit Mutation Leads to Formation of Human t(8;21) Acute Myelogenous Leukemia Stem Cells,
Authors: Shima Takahiro , Kikushige Yoshikane , Miyamoto Toshihiro , Akashi Koichi
Published: October 2019

Abstract 3584

The 8;21 translocation, one of the most general chromosomal abnormalities in acute myelogenous leukemia (AML), encodes the AML1-ETO chimeric fusion gene. Because AML1-ETO can inhibit the CBF complex to transactivate myeloid-lineage genes in a dominant negative fashion, the high level expression of this gene plays a critical role in inhibiting differentiation of target cells, which leads to progression of AML. We, however, have reported that patients maintaining a long-term remission retain AML1-ETO expression at a very low level that can be detected by nested RT-PCR. The AML1-ETO transcripts in these patients were derived from a small fraction of t(8;21)+ hematopoietic stem cells (HSCs) capable of multilineage differentiation (PNAS 2000). In fact, previous data shown that AML1/ETO knock-in or AML1/ETO transgenic mice did not develop AML. These data suggest that acquisition of the AML1-ETO fusion is not sufficient to develop t(8;21) AML. Since t(8;21) AML cells frequently possess constitutive active mutation of c-Kit, we hypothesized that the c-Kit mutation may work as a second oncogenic hit in t(8;21)+ HSCs to transform into AML. To test the hypothesis, we extensively analyzed the existence of c-Kit mutation within AML1-ETO+ HSCs from patients maintaining remission for a long-term. CD34+CD38− HSCs were purified from the bone marrow of patients in long-term remission, and were cultured in vitro to form colonies. These HSC-derived colonies were picked up, and tested for the presence ofAML1-ETO and c-Kit mutation. Five t(8;21) AML patients with c-Kit mutation were enrolled in this study. All of 1020 blastic colonies at diagnosis were positive for both AML1-ETO and c-Kit mutation. In 7187 colonies formed in the culture of remission marrow, almost 1% (89 colonies) of these colonies expressed AML1-ETO. Surprisingly, none of these colonies possessed c-Kit mutation, indicating that AML1-ETO+ clones in remission are not identical to these in t(8;21) AML. Accordingly, it is highly likely that HSCs first acquire AML1-ETO, and a fraction of these cells additionally mutated c-Kit, resulting in transformation into AML stem cells. This is the first clear-cut evidence that human HSCs transform into AML via multi-step oncogenesis in vivo.

Disclosures:

No relevant conflicts of interest to declare.
A Phase 1 Study to Assess the Absolute Bioavailability and Safety of An Oral Solution of Decitabine In Subjects with Myelodysplastic Syndromes (MDS),
Authors: Mistry Bipin , Jones Mark M , Kubiak Peter , Garcia-Manero Guillermo , Litzow Mark R. , Mesa Ruben A. , Rifkin Robert , Tarassoff Peter , Cortes Jorge E.
Published: October 2019

Abstract 3801

Purpose:

Decitabine is a deoxycytidine nucleoside derivative inhibitor of DNA-methyltransferases indicated for treatment of myelodysplastic syndromes (MDS) including previously treated and untreated, de novo and secondary MDS of all French-American-British subtypes. The aim of this study was to determine the absolute bioavailability and safety of a single oral dose of decitabine. Methods: This is an open-label, Phase 1, dose-escalation absolute bioavailability trial in which decitabine was administered orally on Cycle 1 Day 1, and followed by a 1-hour IV infusion of the currently approved regimen (20 mg/m2) on Cycle 1 Days 2–5. Serial blood samples for decitabine pharmacokinetic (PK) assessment were collected up to 6 h post-dosing during Cycle 1 on Days 1 and 2. Three subjects were recruited at each oral dose level of 30, 60, 120 and 240 mg. Subjects were evaluated for adverse events (AEs). Results: Study results are summarized in Table 1. Sixteen subjects were screened and 12 were enrolled. Nine subjects had de novo MDS and 3 had secondary MDS (IPSS score Int-2 = 6, Int-1 = 5, Low = 1). Following oral dose, Cmax was reached within 0.50 h (tmax) and after Cmax the plasma profile showed biphasic decline with terminal half-life ranging between 0.36 to 0.93 hr. Increase in oral dose did not result in proportional increase in Cmax and AUC. The PK parameters following oral doses showed very high variability. Based on comparison of dose normalized exposure, the absolute bioavailability ranged between 3.9 to 14.1% and increased proportionally with dose. The non-dose normalized bioavailabilities were calculated to find a dose at which mean oral bioavailability reaches ≥80% of the current IV regimen of 20 mg/m2. The non-dose normalized bioavailability ranged between 11.7 and 80.5% with highest oral bioavailability observed at 240 mg dose. The most common SAEs were pneumonia (n=4) and neutropenic fever (n=4). Five patients (41.7%) experienced diarrhea and nausea (all grades). Conclusion: The study demonstrates that the absolute bioavailability ranged between 3.9 and 14.1%. Oral decitabine was generally well tolerated at doses between 30–240 mg in MDS subjects and exhibited a safety profile similar to the profile following intravenous administration. The bioavailability and safety data may allow for further testing in the phase 2 setting.

Disclosures:

Mistry: Eisai Inc.: Employment. Jones:Eisai Inc.: Employment. Kubiak:Eisai Inc.: Employment. Garcia-Manero:Eisai Inc.: Research Funding. Litzow:Eisai Inc.: Research Funding. Mesa:Eisai Inc.: Research Funding. Tarassoff:Eisai Inc.: Employment. Cortes:Eisai Inc.: Research Funding.
Marked Upregulations of Survivin and Aurora-B Kinase Are Associated with Disease Progression in the Myelodysplastic Syndromes,
Authors: Yoshida Akira , Zokumasu Kouichi , Yamauchi Takahiro , Takagi Kazutaka , Kishi Shinji , Urasaki Yoshimasa , Tohyama Kaoru , Ueda Takanori
Published: October 2019

Abstract 3823

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffctive hematopoiesis. Survivin belongs to the inhibitors of apoptosis (IAP) family and inhibits apoptosis. Besides its role as IAP, Survivin appears to function as a subunit of the chromosomal passenger complex (CPC) for regulating mitosis with other CPC proteins including Aurora-B. As the CPC, Survivin and Aurora-B play an important role in the maintenance of a stable genome. Until now, there has been no report to examine whether expression of Survivin and Aurora-B kinase may be increased in CD34(+) cells prepared from the patients with MDS during disease progression. To investigate the contribution of Survivin and Aurora-B to the progression and prognosis of MDS, we evaluated the expression levels of these two genes in CD34(+) cells prepared from 57 patients with MDS and 50 patients with de novo AML using real-time quantitative polymerase chain reaction. The degree of Survivin and Aurora-B expression was highly correlated with the type of MDS, was much higher in RAEB-1, RAEB-2 and secondary AML following MDS (s-AML) compared with normal control, and increased during disease progression. Expression levels of both Survivin and Aurora-B were significantly correlated with International Prognostic Scoring System (IPSS). Furthermore, the amount of Aurora-B kinase was significantly correlated with Survivin expression in MDS and s-AML, but not in de novo AML. Interestingly, the levels of Survivin and Aurora-B were remarkably higher in patients with s-AML when compared with de novo AML. We demonstrated for the first time that high levels of Survivin and Aurora-B kinase expression are distinctive molecular feature of high-risk MDS and s-AML. Marked up-regulations of Survivin and Aurora-B Kinase may contribute to genetic instability and disease progression of MDS.

Disclosures:

No relevant conflicts of interest to declare.
Platelet Doubling After the First Azacitidine Cycle Is a Promising Predictor for Response in MDS, CMML and AML Patients in the Dutch Azacitidine Compassionate Patient Named Program,
Authors: Huls Geert A , van der Helm Lieke H. , Alhan Canan , Wijermans P.W. , van Marwijk Kooy Marinus , Schaafsma Martijn R. , Biemond Bart J , Beeker Aart , Hoogendoorn Mels , van Rees Bastiaan P. , de Weerdt Okke , Wegman Jurgen , Libourel Eduard J , Luykx-de Bakker Sylvia A. , Minnema Monique C. , Brouwer Rolf , Croon-de Boer Fransien , Eefting Matthias , Jie Kon-Siong G. , van der Loosdrecht Arjan A. , Koedam Jan , Veeger Nic J.G.M. , Vellenga Edo
Published: October 2019

Abstract 3841

The efficacy of azacitidine in the treatment of high risk myelodysplastic syndromes (MDS), chronic myelomonocytic leukaemia (CMML) and acute myeloid leukaemia (AML) (20–30% blasts) has been demonstrated. To investigate the efficacy of azacitidine in daily clinical practice and to identify predictors for response, we analyzed a cohort of 90 MDS, CMML and AML patients who have been treated in a Dutch compassionate patient named program.

Patients received azacitidine for a median of 5 cycles (range 1–19). The overall response rate (CR/PR/HI) was 57% in low risk MDS, 53% in high risk MDS, 50% in CMML, and 39% in AML patients. Median overall survival (OS) was 13.0 (9.8–16.2) months. In multivariate analysis we confirmed that circulating blasts (HR 0.48, 95% CI 0.24–0.99; p=.05) and poor risk cytogenetics (HR 0.45, 95% CI 0.22–0.91; p=.03) are independent predictors for OS. Interestingly, in this analysis we also identified platelet doubling after the first cycle of azacitidine as a simple and independent positive predictor for OS (HR 5.4, 95% CI 0.73–39.9; p=.10).

Of the 90 treated patients, 14 (16%) had an at least two-fold increase in platelet count after the first cycle of azacitidine, which was associated with significant better OS (p=.01, according to logrank test) (figure). Of these 14 patients 13 could be classified according the azacitidine prognostic scoring system for OS as recently proposed by Itzykson et al. (Blood:2011;117:403); 6 patients belonged to the low risk and 7 to the intermediate risk group. Median baseline platelet count of these patients was 35 x109/L (range 2–290 x109/L). Characteristics of this subgroup of patients were not significantly different from the patients without platelet doubling. Interestingly, platelet doubling was observed in all cytogenetic risk groups, in patients with and without circulating blasts, and in patients who are transfusion dependent and independent.

In conclusion, routine administration of azacitidine to patients with variable risk groups of MDS, CMML and AML is feasible and subgroups with distinct efficacy of azacitidine treatment can be identified.

Disclosures:

Wijermans: Centocor Ortho Biotech Research & Development: Research Funding.
Erythroid Lineage-Restricted Expression of Jak2V617F Is Sufficient to Induce a Myeloproliferative Disease in Mice,
Authors: Akada Hajime , Hamada Saeko , Mohi Golam
Published: October 2019

Abstract 3861

A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in most cases of Ph-negative myeloproliferative neoplasms (MPNs) including ∼95% patients with polycythemia vera (PV) and 50–60% patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). To investigate the contribution of JAK2V617F in MPNs, we generated a conditional Jak2V617F knock-in mouse (Akada et al., Blood 2010; 115: 3589–3597). Expression of Jak2V617F in all hematopoietic compartments including the hematopoietic stem cells (HSC) resulted in a PV-like disease associated with a marked expansion of erythroid progenitors in the bone marrow and spleen. Since Jak2 is essential for normal erythropoiesis and expression of Jak2V617F mutant enhances erythropoiesis, so we asked if erythroid progenitors are actual target cells for Jak2V617F mutation. To address this question, we have specifically expressed Jak2V617F in erythroid progenitors using the EpoR-Cre mice. Expression of heterozygous Jak2V617F in erythroid progenitors resulted in a polycythemia-like phenotype characterized by increase in hematocrit and hemoglobin, increased red blood cells, Epo-independent erythroid colonies, and splenomegaly. Erythroid lineage-specific expression of homozygous Jak2V617F resulted in significantly greater increase in hematocrit, hemoglobin, red blood cells, Epo-independent erythroid colonies, and splenomegaly compared to heterozygous Jak2V617F expression. These results suggest that erythroid lineage-restricted expression of Jak2V617F is sufficient to induce a polycythemia-like disease in a gene-dose dependent manner. However, transplantation of Jak2V617F-expressing erythroid progenitors (c-kithighTer119lowCD71high or c-kitlowTer119highCD71high) from the diseased mice into lethally irradiated recipients could not transfer the disease suggesting that Jak2V617F mutation does not confer self-renewal capacity to erythroid progenitors. We also observed that only Jak2V617F-expressing HSC has the unique capacity to serially transplant the myeloproliferative disease in mice. Taken together, our results suggest that HSCs are the disease-initiating cancer stem cells and erythroid progenitors are the target cells in Jak2V617F-evoked MPN.

Disclosures:

No relevant conflicts of interest to declare.
Frequent Involvement of PVT1 in Multiple Myeloma Carrying 8q24 Rearrangement and Identification of Novel PVT1-NBEA Chimeric Gene,
Authors: Nagoshi Hisao , Taki Tomohiko , Hanamura Ichiro , Nitta Masakazu , Otsuki Takemi , Nishida Kazuhiro , Okuda Keiko , Sakamoto Natsumi , Kobayashi Satoru , Yamamoto Mio , Tsutsumi Yasuhiko , Kobayashi Tsutomu , Matsumoto Yosuke , Horiike Shigeo , Kuroda Junya , Taniwaki Masafumi
Published: October 2019

Abstract 3917

The 8q24 rearrangement has been identified in 3.5–5.0 % of multiple myeloma (MM) patients with conventional cytogenetic analyses, such as G-banding, and in 9.5–20% of MM patients with fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) techniques. Of note, 8q24 rearrangements accompany with advanced MM patients and MM cell lines more frequently. Chromosomal translocation of the immunoglobulin gene (Ig), such as t(8;14)(q24;q32) and t(8;22)(q24;q11), occur in approximately 25% in MM with 8q24 rearrangements, while non-Ig chromosomal loci, including 1p13, 1p21–22, 6p21, 6q12–15, 13q14, and 16q22, have been also identified as translocation partners where no candidate genes have been delineated so far. In this study, we precisely investigate molecular features of chromosomal 8q24 rearrangements to access pathophysiology of MM. FISH, SKY, and RT-PCR analyses were performed as described previously. DNA gain and loss assay based on oligonucleotide array (GeneChip Human Mapping 50K, 250K, or 6.0 array, Affymetrix) was performed on the genomic DNA extracted from MM cells. Oligonucleotide array data was analyzed using the CNAG3.0 or 3.3 programs. Various types of 8q24 rearrangements were detected in 12 (22.2 %) of 54 MM patients and 8 (72.7 %) of 11 MM cell lines by means of FISH procedure. A breakpoint cluster of approximately 360 kb region containing myelocytomatosis oncogene (MYC) and plasmacytoma variant translocation 1 (PVT1) genes at 8q24 was divided into three subclusters according to rearranged region, that is, PVT1 region, region which is centromerically adjacent to PVT1 (including MYC), and region which is 120kb centromeric to MYC. Seven of 12 patient-derived cells and 5 of 8 cell lines with 8q24 abnormalities showed PVT1 rearrangements with various partner loci, such as 4p16, 4q13, 13q13, 14q32, or 16q23–24 in SKY combined with FISH analyses (SKY-FISH). Oligonucleotide array combined with SKY analysis delineated the candidate genes within partner loci of 8q24 rearrangements by mapping boundary breakpoints of copy number gains and losses, identifying MMSET, EPHA5, NBEA, and WWOX as candidate genes at 4p16, 4q13, 13q13, and 16q23–24, respectively. We identified the novel chimeric gene PVT1-NBEA in AMU-MM1 cell line with t(8;13)(q24;q13). The PVT1-NBEA fusion transcript in which PVT1 exon 1 was fused to NBEA exon 3 was accompanied with overexpression of abnormal NBEA lacking its part of N-terminus. In conclusion, PVT1 rearrangement may play pivotal roles in pathophysiology of MM harboring 8q24 abnormalities.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of mTOR with Everolimus (RAD001) and Silencing by Vascular Endothelial Cell Growth Factor Specific siRNA Induces Additive Antitumor Activity in Multiple Myeloma Cells,
Authors: Koldehoff Michael , Elmaagacli Ahmet , Beelen Dietrich W.
Published: October 2019

Abstract 3984

Background:

Angiogenesis plays an important role in the pathogenesis and progression in multiple myeloma (MM). MM cells secrete vascular endothelial growth factor (VEGF), which further promotes proliferation of the tumor cells. Several RNA interference (RNAi) methodologies are rapidly being established and hold promise to specifically inhibit gene expression in mammals. RNAi is the sequence-specific, posttranscription gene silencing methods initiated by double-stranded RNAs, which are homologous to the gene being suppressed. Mammalian target of rapamycin (mTOR) is an essential part of tumour growth being capable of integrating proliferative, antiapoptotic and angiogenic signalling by connecting VEGF, hypoxia-inducible factor 1 (HIF-1) and HER family receptors. Several reports have demonstrated the anti-tumor activity of everolimus (RAD001), an mTOR inhibitor, in a variety of cancers, including various leukemias and lymphomas.

Aims:

Therefore, we evaluated the anti-myeloma effect of VEGF siRNA silencing in MM cells and whether it can be augmented by the additional application of everolimus.

Methods and Results: After transfection with VEGF siRNA we observed a reduction of VEGF expression in all studied cell lines: OPM-2, RPMI-8226, INA-6, Jurkat, Raji and Karpas-299, as well as in cells of MM- and lymphoma patients. Next, using the MM cell line OPM-2 we studied the time courses of VEGF siRNA transfection in order to investigate the knock-down efficiency of VEGF expression. The efficiency of VEGF siRNA transfection in treated OPM-2 cells showed a reduction of 75.5% VEGF protein levels after 24 h compared to the untreated OPM-2 cells (p<0.001). Further, VEGF siRNA both significantly induced apoptosis and inhibited proliferation in OPM-2 cells (p<0.001), RPMI-8226 (p<0.001), and in INA-6 (p<0.01) versus controls. To assess whether everolimus treatment of OPM-2 and RPMI-8226 affects the viability of these cells, cells were treated with various doses (1–20 nM) of everolimus for 24 hours, harvested, and analyzed for cell viability by MTT assay. Everolimus significantly decreased the viability of OPM-2 cells (IC50 = 5.9 nM) and of RPMI-8226 cells (IC50 = 0.6 nM), respectively. Everolimus and siRNA both together might to reduce the VEGF gene expression up to 33% (p<0.001) compare to siRNA VEGF (61%) alone or everolimus alone (39%), that demonstrated additive effects of everolimus and siRNA.

Conclusions:

These findings suggest that mTOR inhibition and silencing by VEGF specific siRNA may be associated with an additive antitumor activity and might be a suitable target for new therapeutic strategies using RNA interference in MM.

Disclosures:

No relevant conflicts of interest to declare.
Cell Autonomous Role of TGFβ and IL-2 Receptor in the In Vivo Generation of CD4 and CD8 Inducible Regulatory T Cells During Graft-Versus-Host Disease,
Authors: Satake Atsushi , Sawamukai Norifumi , Kambayashi Taku
Published: October 2019

Abstract 4015

FoxP3+ regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) while preserving graft-versus-tumor effects, making them an attractive target for GVHD therapy. The donor-derived Treg pool can potentially be derived from expansion of pre-existing natural Tregs (nTregs) or from de novo generation of inducible Tregs (iTregs) from donor conventional T cells (Tconvs) in the transplant recipient. Although the co-adoptive transfer of nTregs or in vitro -derived iTregs has been shown to prevent the development of GVHD, the relative contribution of these two Treg subsets in protection against GVHD has been unclear. To investigate the contribution of the different FoxP3+ Treg subsets, we used a MHC-mismatched mouse model of acute GVHD. Lethally irradiated (500cGy × 2) B6D2F1.SJL (H-2bxd) host mice were injected with T cell-depleted bone marrow cells and FACS-sorted Tconvs (WT or Foxp3-deficient) with or without FACS-sorted Tregs of C57BL/6 (H-2b) mouse origin. Weight loss in mice receiving Foxp3-deficient Tconvs alone was significantly more pronounced compared to other groups. The presence of either donor-derived nTregs or iTregs alone protected against GVHD-induced weight loss but was suboptimal compared to the presence of both donor-derived nTregs and iTregs.

Next, we sought to determine how the donor-derived Treg pool was established during acute GVHD and tracked the appearance of Tregs in the secondary lymphoid organs at different time points post transplant. On Day 8 post GVHD induction, ∼5% of the donor-derived CD4+ T cells in the spleen were FoxP3+. We found that the Treg pool was comprised equally of donor-derived nTregs and iTregs. Unexpectedly, we found a significant fraction of CD8+FoxP3+ T cells (1–3% of all CD8+ T cells) in the spleen and in the lymph nodes. These CD8+FoxP3+ T cells representing ∼70% of the iTreg pool on Day 8 post GVHD induction. These CD8+FoxP3+ T cells shared phenotypic markers with their CD4+ counterparts and displayed suppressive activity, suggesting that they were bona fide iTregs. Both CD4+ and CD8+ Tregs expanded in vivo with IL-2 treatment and required IL-2 and TGFβ receptor expression for their generation.

In summary, we found that donor derived-iTregs are generated during GVHD and contribute to suppression of acute GVHD induced-weight loss. Surprisingly, CD8+Foxp3+T cells were a major contributor to the donor derived-iTreg pool after transplantation. The generation of CD8+ and CD4+ iTregs occurred at least in part by a cell autonomous IL-2 and TGFβ receptor-dependent mechanism. Thus, our data suggest that in addition to increasing nTregs, concomitant strategies aimed at enhancing the conversion of donor-derived Tconvs to iTregs for example by engaging the IL-2 and TGFβ signaling pathways might be beneficial for the treatment of GVHD.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation of the Toxicity of Total Body Irradation As a Separate Treatment in Preparation for Hematopoietic Cell Transplantation
Authors: Giebel Sebastian , Kruzel Tomasz , Sadus-Wojciechowska Maria , Najda Jacek , Czerw Tomasz , Miszczyk Leszek , Holowiecki Jerzy
Published: October 2019

Abstract 4579

BACKGROUND:

Myeloablative regimens used for allogeneic or autologous hematopoietic cell transplantation (HCT) may be based on either chemotherapy alone or total body irradiation (TBI). TBI is generally considered highly toxic, however, it is usually combined with high-doses of cyclophosphamide or other cytostatics, which does not allow reliable assessment of its tolerance. In Maria Sklodowska-Curie Cancer Center and Institute of Oncology in Gliwice TBI is used a single treatment in the preparation for the first autologous HCT in patients with multiple myeloma (MM). Second HCT in a tandem procedure is performed after 4 months interval with high-dose melphalan as conditioning. The protocol gives opportunity to separately assess early toxicity of TBI, which is a goal of this study.

PATIENTS AND METHODS:

The clinical course of 30 patients with MM, aged 58 years (range, 50–66), including 14 men and 16 women was evaluated. The disease status prior to autoHCT was CR (n=9), VGPR (n=7), or PR (n=14). 47% patients were pre-treated with 2 or more lines of chemotherapy and the median number of preceding cycles was 8. TBI was administered at the dose of 12 Gy divided in 3 fractions on days -3, -2, -1. 6 MV X-ray beam was used with lung shielding after 6 Gy and additional irradiation of chest wall using electrons 9–15 MeV to the planned total dose. Peripheral blood was used as a source of stem cells. The incidence of adverse events was assessed using ‘Common Toxicity Criteria v4.03’.

RESULTS:

Within 100 days follow-up all patients were alive. Median time to neutrophil >0.5 ×10e9/L and platelet >50 ×10e9/L recovery was 12 days (11–21) and 14 days (9–18), respectively. Patients required the median of 1 (0–3) platelet and 0 (0–6) red blood cell transfusions. The median hospital stay since HCT was 16 days (14–21). The most frequent grade 3 or 4 adverse events were infections (43%), including neutropenic fever (23%). Other severe toxicities were rare. In particular no patient experienced hepatic or renal complications (Table 1).

CONCLUSION:

TBI as a separate treatment is well tolerated and may be safely used for conditioning in a population of patients up to 66 years old. Our observation may potentially be applied to both autologous and allogeneic transplantations.

Disclosures:

No relevant conflicts of interest to declare.
Aberrant Expression of Golgin-84 in Non-Hodgkin Lymphomas and Plasma Cell Myeloma
Authors: Chen Ling , Yang Yaling , Yin C. Cameron , Lu Gary , Chen Su , Huang Xiaoyan , Chen Jiayu , Chang Felix , Rahman Effie , Calin Steliana , Medeiros L. Jeffrey , Miranda Roberto N. , You M. James
Published: October 2019

Abstract 4633

Background:

Golgins are proteins of the Golgi complex. Several Golgins have been implicated in apoptosis. Expression of Golgin-84, a Golgin protein, is altered in apoptotic WEHI-231, a B-cell lymphoma line, suggesting that Golgin-84 may play a role in lymphoid tumorigenesis. Here, we aimed to determine the expression levels of Golgin-84 in human primary non-Hodgkin lymphomas and plasma cell myeloma.

Design:

Golgin-84 expression was investigated in non-Hodgkin lymphoma cell lines by using Western blot analysis and polyclonal antibodies. Using immunohistochemical stains, Western blotting analysis and Q-PCR, Golgin-84 expression was assessed in 5 reactive lymph nodes, 149 cases of primary non-Hodgkin lymphoma and 28 cases of primary plasma cell myeloma.

Results:

Immunohistochemical stains, Western blotting analysis and Q-PCR on 5 reactive lymph nodes demonstrated that Golgin-84 was expressed at low levels in lymphoid cells of germinal centers, mantle cells, marginal zones, and interfollicular areas. Golgin-84 was variably expressed in non-Hodgkin lymphoma cell lines tested, with the highest levels in cells from high-grade tumors (e.g. anaplastic large cell lymphoma; ALCL, Diffuse large B-cell lymphoma (DLBCL), ALCL and peripheral T-cell lymphoma unspecified (PTCL)) and the lowest levels in mantle cell lymphoma (MCL) cells. DLBCL, ALCL and PTCL frequently showed high expression of Golgin-84. Most lymphoplasmacytic lymphomas (LPL) and plasma cell myeloma (PCM) expressed high levels of Golgin-84. Expression levels of Golgin-84 were lower in MCL and low-grade B-cell non-Hodgkin lymphomas, including chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), follicular lymphoma (FL), and marginal zone lymphoma (MZL).

Conclusions:

Golgin-84 expression levels are low in lymphoid cells of normal lymph nodes. Most (>90%) cases of LPL and PCM, and at least half of cases of DLBCL, ALCL and PTCL express high levels of Golgin-84. These findings suggest that Golgin-84 may be involved in tumorigenesis or lymphoma progression, particularly in neoplasms with plasmacytic differentiation.

Disclosures:

No relevant conflicts of interest to declare.
Enforced E47 Expression Has Differential Effects on Lmo2-Induced T-Cell Leukemia
Authors: Goodings Charnise , Dave Utpal P.
Published: October 2019

Abstract 4637

Lmo2 is one of the most commonly deregulated oncogenes in human T-cell acute lymphoblastic leukemia (T-ALL). In mouse models, Lmo2 overexpression causes a differentiation block before the onset of T-ALL at a developmental stage that is similar to the block seen in E47 knockout mice. Furthermore, Lmo2 and E47 are part of an oligomeric protein complex in hematopoietic stem and progenitor cells. Since E47 knockout mice also develop T-ALL, it has been hypothesized that Lmo2 may induce T-ALL by redirecting E47 activity away from its normal target genes. We noted downregulation of many E2A targets in Lmo2-induced T-ALL. To directly test whether E47 is required in Lmo2-induced T-ALLs, we transduced four stable T-ALL lines established from Lmo2 transgenic mice with retrovirus expressing E47 fused with estrogen receptor. All 4 lines tolerated stable high- level protein expression of E47-ER with no change in from their baseline growth rates. The E47-ER fusion protein allowed forced dimerization upon treatment with 4-hydroxytamoxifen. Tamoxifen treatment increased expression CD4 and other described E2A targets in all 4 T-ALL lines; but two lines underwent G0/G1 cell cycle arrest. Our data suggest that E47 deficiency is not a universal feature of Lmo2- induced T-ALL and E47 forced expression has differential effects on T-ALL.

Disclosures:

No relevant conflicts of interest to declare.
A Comparison of INNOVANCE® PFA P2Y and VerifyNow P2Y12 Assay for the Assessment of Clopidogrel Resistance in Patients Undergoing Percutaneous Coronary Intervention
Authors: Shin Soyoung , Kim Yonggoo , Lim Jihyang , Jang Jiyoung , Kim Jayoung
Published: October 2019

Abstract 4671

Introduction:

VerifyNow P2Y12 is commonly used to measure responsiveness to clopidogrel. We sought to compare the results obtained from novel INNOVANCE® PFA P2Y and VerifyNow P2Y12 assay for the assessment of clopidogrel resistance in patients with undergoing percutaneous coronary intervention.

Methods:

255 patients undergoing percutaneous coronary intervention, preliminarily treated with 100 mg/day of aspirin followed co- administration of clopidogrel(loading dose 600 mg, maintenance dose 75 mg/day), were enrolled in this study. Platelet aggregation was measured by INNOVANCE® PFA P2Y and VerifyNow P2Y12.

Results:

INNOVANCE® PFA P2Y and VerifyNow P2Y12 assay showed moderate correlations with INNOVANCE® PFA P2Y Vs VerifyNow %inhibition: r = 0.412, p < 0.0001; INNOVANCE® PFA P2Y Vs VerifyNow platelet reactivity units(PRU): r = −0.402, p < 0.0001. The agreement between INNOVANCE® PFA P2Y and VerifyNow %inhibition was 85 % and that of INNOVANCE® PFA P2Y and VerifyNow platelet reactivity units(PRU) was 79%: The k statistics between INNOVANCE® PFA P2Y and VerifyNow %inhibition and platelet reactivity units(PRU) were 0.52 and 0.44, respectively.

Conclusions:

INNOVANCE® PFA P2Y’s sensitivity in detecting clopidogrel resistance is comparable to VerifyNow P2Y12 assay. As the PFA-100® system was already widely used, the new test cartilage may be useful tool for the assessment of clopidogrel effects. Additional Clinical correlation studies were required to validate the effectiveness of INNOVANCE® PFA P2Y for predicting long term clinical outcomes.

Disclosures:

No relevant conflicts of interest to declare.