bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular and Cellular Effects of Oncogene Cooperation in a Genetically Adequate AML Model
Authors: Reckzeh Kristian , Bereshchenko Oxana , Mead Adam J. , Rehn Matilda , Kharazi Shabnam , Jacobsen Sten Eirik W , Nerlov Claus , Cammenga Jorg 
Published: October 2019

Abstract
Abstract 1379
Biallelic CEBPA mutations and FLT3 length mutations are frequently identified in human acute myeloid leukemia (AML) with normal cytogenetics. However, the molecular and cellular mechanisms of oncogene cooperation remain unclear due to a lack of disease models.
We have generated an AML mouse model using knock-in mouse strains to study cooperation of FLT3 ITD with commonly observed biallelic C/EBPa mutations which in this model are expressed from the original promoter and thereby at the right dose and at the correct level of cellular differentiation.
Interestingly, Cebpa mutants with FLT3 ITD progressed to AML with shorter latency than cohorts with Cebpa mutations only. Before onset of leukemia, biallelic Cebpa mutations caused depletion of granulocyte-macrophage progenitors (GMPs), and expansion of megakaryocyte-erythroid progenitors (MkP, pMegE, pCFU-E) and HSCs. However, FLT3 ITD attenuated MegE progenitor frequencies while MPPs and myeloid progenitors (pGMP and GMP) were more frequently observed. These cellular changes are accompanied by an upregulation of hematopoietic stem cell and STAT5 target genes. Both MPP and GMP cells contain the potential to induce AML similar to corresponding cells in human AML samples showing that this AML model resembles human disease. By gene expression analysis in premalignant populations we further show a role of FLT3 ITD in activating genes involved in survival/transformation and chemoresistance.
This murine AML model is perfectly suited to test the effects of chemotherapy and tyrosine kinase inhibitors blocking FLT3 ITD function on specific cellular populations to investigate their specific role in relapse of leukemia after complete remission.

Disclosures:
No relevant conflicts of interest to declare.
Ectopic Expression of the Leukemogenic Protein E2A-PBX1 in Early Hematopoietic Progenitors Blocks B-Lymphoid Commitment
Authors: Woodcroft Mark , Thompson Patrick , Slany Robert K. , LeBrun David 
Published: October 2019

Abstract
Abstract 1393
The oncoprotein E2A-PBX1 is produced by t(1;19)(q23;p13) in pre-B acute lymphoblastic leukemia (ALL). Perplexingly, in vitro and in vivo models of E2A-PBX1 leukemogenesis to date generate either myeloid or T-lymphoblasts, neither of which are representative of the human disease. Furthermore, we have observed recently that lineage-depleted (lin-) murine bone marrow cells infected with an E2A-PBX1 retrovirus selectively fail to repopulate the B-lymphoid compartment on transplantation into irradiated recipients. Therefore, we have now begun to investigate the mechanism by which enforced expression of E2A-PBX1 antagonizes B-lymphopoiesis.
We show that E2A-PBX1-transduced lin- fetal liver progenitor cells (FLPs) fail to differentiate to committed CD45R+ pro-B-cells when cultured with IL-7 in the presence of OP9 stromal cells. Instead these cells manifest a relatively immature immunophenotype (CD45R-, CD11b-, CD117+), are murine stem cell factor (SCF) dependent, and undergo myeloid differentiation upon stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) or transplantation into irradiated mice. Amino acid substitutions in E2A-PBX1 that impair co-activator recruitment or DNA binding completely rescue the differentiation block, implying a critical role for target-gene regulation by E2A-PBX1. E2A-PBX1-transduced cells fail to induce the B-lymphoid commitment genes Ebf1 and Pax5, and chromatin immunoprecipitation (ChIP) analysis shows aberrant persistence of the Polycomb silencing mark H3K27me3 at these promoters. Previous studies have identified the Polycomb gene Bmi1 as a candidate E2A-PBX1 target gene. However, enforced expression of Bmi1 neither blocked induction of Ebf1 or Pax5 nor impaired B-lymphopoiesis, indicating that Bmi1 is not sufficient to mediate blocked differentiation by E2A-PBX1. Gene expression microarray analysis identified cytokines (ex., Csf2, IL1a, Il6) and transcription factors (HoxA9, Tal1) up-regulated in E2A-PBX1-transduced FLPs that are known to antagonize B-lymphoid development.
In summary, our results support the notion that enforced expression of E2A-PBX1 in early hematopoietic progenitors blocks B-lymphopoiesis, to an important degree, by inducing the transcription of genes whose products antagonize early steps of B-lymphoid commitment, including induction of Ebf1 and Pax5. HoxA9 is particularly interesting as a mediator of E2A-PBX1 effects in view of its extreme induction ratio in our system (at least 1000-fold relative to controls), its propensity to promote myeloid over lymphoid differentiation, and its well-established role in leukemogenesis. Studies to investigate a potential requirement for HoxA9 and other candidates in mediating the E2A-PBX1-driven B-lymphoid differentiation block are ongoing.

Disclosures:
No relevant conflicts of interest to declare.
IRF-4 Suppresses BCR/ABL Transformation of Myeloid Cells in a DNA-Binding Independent Manner
Authors: Jo Seung-Hee , Ren Ruibao 
Published: October 2019

Abstract
Abstract 1404
Interferon regulatory factor 4 (IRF-4) is essential for B and T cell development and immune response regulation, and has both nuclear and cytoplasmic functions. IRF-4 was originally identified as a proto-oncogene resulting from a t(6;14) chromosomal translocation in multiple myeloma and its expression was shown to be essential for multiple myeloma cell survival. However, we have previously shown that IRF-4 functions as a tumor suppressor in the myeloid lineage and in early stages of B cell development. The finding of IRF-4 functioning as a tumor suppressor raises caution for developing therapies aiming to downregulate IRF-4. Further studies on the mechanisms by which IRF-4 functions as an oncoprotein and as a tumor suppressor are necessary to develop improved therapies for malignancies involving IRF-4. In this study, we found that IRF-4 suppresses BCR/ABL transformation of myeloid cells. To gain insight into the molecular pathways that mediate IRF-4 tumor suppressor function, we performed a structure-function analysis of IRF-4 as a suppressor of BCR/ABL transformation. We found that the DNA binding domain deletion mutant of IRF-4, which is localized only in the cytoplasm, is still able to inhibit BCR/ABL transformation of myeloid cells. IRF-4 also functions as a tumor suppressor in bone marrow cells deficient in MyD88, an IRF-4 interacting protein found in the cytoplasm. However, IRF-4's tumor suppressor activity is lost in IRF association domain (IAD) deletion mutants. These results demonstrate that IRF-4 suppresses BCR/ABL transformation by a novel cytoplasmic function involving its IAD domain and suggest that the oncogenic and tumor suppressor functions of IRF-4 involve distinct pathways.

Disclosures:
No relevant conflicts of interest to declare.
Pre-B Cell Receptor-Mediated Activation of BCL6 Induces Pre-B Cell Quiescence Through Transcriptional Repression of MYC
Authors: Nahar Rahul , Ramezani-Rad Parham , Mossner Maximilian , Duy Cihangir , Cerchietti Leandro , Geng Huimin , Jumaa Hassan , Ye B. Hilda , Melnick Ari , Muschen Markus 
Published: October 2019

Abstract
Abstract 1406

Background:
Pre-B cell receptor signaling is critical to induce initial expansion of the pool of differentiating B cell precursors but also mediates subsequent cell cycle exit and quiescence. Initial cell surface expression of the pre-B cell receptor induces a series of tyrosine phosphorylation events resulting in MYC and CCND2-mediated proliferation. After 2–5 divisions, however, large pre-BII (Fraction C') cells exit cell cycle to become resting, small pre-BII cells (Fraction D). While the signaling pathway resulting in pre-B cell receptor-induced proliferation is well understood, the mechanism by which pre-BII cells exit cell cycle, however, is currently unclear.

Results:
This checkpoint and cell cycle exit at the Fraction C'-D transition is critical for immunoglobulin light chain gene recombination and to prevent malignant transformation into acute lymphoblastic leukemia. Here we demonstrate that inducible activation of pre-B cell receptor signaling recapitulates the initial proliferative burst followed by cell cycle exit, which is characterized by strong upregulation of BCL6 and subsequent loss of MYC and CCND2 expression. ChIP-on-chip analysis revealed that the transcriptional repressor BCL6 is directly recruited to the MYC promoter. Inducible activation of pre-B cell receptor signaling in pre-B cells from BCL6-deficient mice failed to induce cell cycle exit and quiescence. We conclude that activation of BCL6 downstream of the pre-B cell receptor is crucial for the induction of quiescence at the Fraction C'-D checkpoint. As expected, inducible activation of BCL6 downstream of the pre-B cell receptor results in transcriptional repression of MYC and CCND2. Overexpression of MYC prevented pre-B cell receptor/BCL6-induced cell cycle exit.

Conclusion:
Hence, pre-B cell receptor signaling induces cellular quiescence of Fraction C' pre-B cells through activation of BCL6 and BCL6-mediated transcriptional repression of MYC and CCND2.

Disclosures:
No relevant conflicts of interest to declare.
Aurora Kinases in Childhood Acute Leukemia: The Promise of Aurora Kinase B As Drugable Target
Authors: Segers Stefanie A. , Zwaan C. Michel , Exalto Carla , Luijendijk Mirjam W.J. , Calvert Valerie S. , Petricoin Emanuel F. , Evans William E. , Reinhardt Dirk , Pieters Rob , Den Boer Monique L. 
Published: October 2019

Abstract
Abstract 1476

AIM:
Aurora kinases (AURK) A and B are known regulators of mitosis and are overexpressed in a large number of human cancers, including leukemia. Several AURK-inhibitors have shown anti-tumor activity in vitro and in vivo. However, the efficacy of AURK inhibition in the treatment of childhood acute leukemia is unexplored. We therefore investigated the effect of targeting AURKA and AURKB in leukemic cells of children with newly diagnosed acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

Materials & Methods:
Affymetrix gene expression data of 297 ALL, 237 AML and 8 normal bone marrow (nBM) samples were analyzed for AURKA and B mRNA expression levels. Protein expression levels in 172 pediatric ALL and 10 nBM samples were determined with a reverse phase protein array. Functional studies were performed in ALL and AML cell lines, in which AURKA and B were silenced using a short hairpin RNA with a lentiviral delivery system or LNA-containing oligonucleotides. Sensitivity of leukemic cell lines to the AURKB-selective inhibitor Barasertib-hQPA (AZD1152-hQPA) was tested in vitro with an MTS assay.

Results:
AURKA and B mRNA levels were low in ALL and AML patients. In contrast, Aurora A and B proteins were expressed to a greater extent in patients (p<0.0002), especially in ALL cases with an E2A-PBX1 translocation (p<0.0001) than in nBM mononuclear cells. Silencing of AURKA by shRNA and by LNA-oligonucleotide caused no or only minor growth delay in several cell lines reflecting genetic subtypes typically found in pediatric ALL and AML. In contrast, silencing of AURKB resulted in proliferation arrest and apoptosis in these cells. Furthermore, 18 out of 20 ALL and AML cell lines tested were highly sensitive to the AURKB-selective inhibitor Barasertib-hQPA in the nanomolar range (IC50 = 19–233 nM) whereas less sensitivity was seen for other inhibitors.

Conclusion:
These data show that inhibition of AURKB but not AURKA has an anti-proliferative and pro-apoptotic effect on acute leukemic cells. Thus, targeting Aurora Kinase B may offer a new strategy to treat pediatric ALL and AML.

Disclosures:
No relevant conflicts of interest to declare.
Six Cycles of BEACOPP Tailored Based on Interim Scintigraphy Provide Favorable Outcome for Patients with Standard and Advanced Risk Hodgkin Lymphoma and Female Fertility Is Preserved: Results of a 10-Year Follow-up
Authors: Dann Eldad J , Blumenfeld Zeev , Bar-Shalom Rachel , Avivi Irit , Ben-Shachar Menachem , Goor Odelia , Libster Diana , Gaitini Diana , Rowe Jacob M. , Epelbaum Ron 
Published: October 2019

Abstract
Abstract 1564

Background:
Therapy of Hodgkin lymphoma (HL) is designed to prolong survival and minimize toxicity; however, an intense debate is ongoing over whether ABVD or BEACOPP should be used upfront and whether a long-term survival benefit is obtained with the BEACOPP regimen. The current study was aimed to personalize therapy based on individual risk factors and interim scintigraphy. Design and Methods: 124 patients with newly diagnosed HL and adverse prognostic factors were prospectively studied between 7/1999 and 8/2005. Patients with early unfavorable and advanced disease were eligible for the study. Study participants were assigned to therapy based on their International Prognostic Score (IPS). Those with an IPS of ≥3 were assigned to 2 cycles of escalated BEACOPP (EB). All others received 2 cycles of standard BEACOPP (SB). Subsequent therapy was prospectively allocated according to the results of early interim GA67 or PET/CT. Four cycles of EB or SB were administered following a positive or negative scan, respectively. Results: Complete remission (CR) rate, 10-year progression free (PFS) and overall survival (OS) were 97%, 87% and 88%, respectively, at a median follow-up of 89 months (5–144). Only 38% of patients, mostly with a bulky mediastinal mass, received radiation therapy. PFS and OS were similar in both groups. Predictive value of negative interim Ga67 or PET/CT was 87% and 93%, respectively. Fertility status was assessed in 38 females aged <40 years (Table). Twenty four of these patients received 6 cycles of SB, 8 patients had 2 cycles of EB and 4 cycles of SB, 3 patients were treated with 2 cycles of SB and 4 cycles of EB and one patient received 6 cycles of EB. Thirty six females did not have disease progression and 34 of them were found to preserve their cyclic ovarian function (COF). Twenty six females were co-treated with 6 cycles of GNRH agonist triptorelin (Decapeptyl 3.75 mg) for 6 months. 19 patients conceived during the follow-up (30 pregnancies), delivering 23 babies. Deliveries were reported for up to seven years from diagnosis. Notably, the female receiving 6 cycles of EB, co-administered with the GNRH agonist, was among those who conceived and delivered a healthy baby. Conclusion: Six cycles of tailored BEACOPP administered to HL patients with adverse prognostic factors provide encouraging long-term PFS and OS, and fertility is preserved in most females following reduction in the number of chemotherapy cycles and GNRH agonist co-administration.

Disclosures:
Off Label Use: GNRH agonist triptorelin was simultaneously administered for fertility preservation.
Clonal Evolution but Not Variant Chromosomal Translocations Is An Adverse Prognostic Marker for Cytogenetic Response and Survival in CML Patients Treated with Imatinib
Authors: Tantiworawit Adisak , Abou Mourad Yasser , Barnett Michael J , Broady Raewyn , Hogge Donna E. , Nantel Stephen H. , Narayanan Sujaatha , Nevill Thomas J. , Power Maryse M. , Shepherd John D. , Song Kevin W. , Sutherland Heather J. , Toze Cynthia L. , Forrest Donna L. 
Published: October 2019

Abstract
Abstract 1694

Background:
Clonal evolution (CE) and variant Philadelphia (Ph) chromosomal translocations (vPh) are seen in <10% of newly diagnosed CML patients. While the vPh is thought to have no prognostic significance, CE maybe associated with an inferior outcome compared to the presence of a single standard Ph for patients treated with imatinib (IM). This study aims to evaluate the prognostic significance of vPh and clonal evolution in a cohort of CML chronic phase (CP) and accelerated phase (AP) patients treated with IM.

Patients and methods:
From June 1999 to December 2008, 247 Ph+ CML CP and AP patients treated with IM (300–600 mg daily) were analyzed. Patients having had a prior allogeneic stem cell transplant were excluded. Patients were categorized into 4 groups according to karyotype at diagnosis: Group 1 (CP with CE only, n=28); Group 2 (AP, n=31); Group 3 (CP with vPh, n=20); Group 4 (CP with standard Ph, n=168). Cytogenetic response, treatment failure, event free survival (EFS) and overall survival (OS) were calculated for each group and multivariate analysis was performed to determine potential variables predictive of response and survival.

Results:
Groups 1 and 2 had lower complete cytogenetic responses (CCR) to IM and higher rates of treatment failure compared to Group 4. (Table1). At a median follow up 4.2 years, Groups 1 and 2 had lower median OS and EFS compared to Groups 3 and 4 (Table1). The 5 year probabilities of OS and EFS were 79 % and 53 % (Group 1), 77 % and 73 % (Group 2), 94 % and 87 % (Group 3), and 90 % and 83 % (Group 4). (Figures 1 and 2).
In multivariate analysis, the only significant factors predictive of CCR were Sokal score (p=.01), CE at diagnosis (p=.03) and dose of IM (p=.03). When the analysis was restricted to EFS and OS, the presence of CE at diagnosis (p<0.0001), prior treatment before IM (p=0.04), the dose of IM (p=0.01) and the response to IM (p<0.0001) were the factors associated with significant EFS and the presence of CE at diagnosis (p=0.004), the dose of IM (p=0.05) and the response to IM (p<0.0001) were predictive of OS. The presence of a vPh had no effect on EFS and OS.

Conclusion:
The presence of CE at diagnosis and/or features of AP in patients with CML treated with IM are associated with an inferior prognosis, but the presence of the vPh does not appear to have any prognostic significance. It remains to be determined whether the second generation TKI's can overcome the negative influence of CE on EFS and OS.

Disclosures:
No relevant conflicts of interest to declare.
Suppression of Murine Platelet Activation by the β Isoform of the Catalytic Subunit of Protein Phosphatase 2B
Authors: Gushiken Francisca C. , Alrehani Nawaf , Pradhan Subhashree , Kailasam Lavanya , Rumbaut Rolando , Vijayan K. Vinod 
Published: October 2019

Abstract
Abstract 190
Signal transduction mediated by the kinases and phosphatases are critical for platelet activation at the site of vascular injury. Compared to the kinases, a role for phosphatases in platelet activation is less understood. Our previous studies have focused on the roles of serine/threonine protein phosphatase 1 (PP1) and 2A (PP2A) in regulating integrin αIIbβ3functions. However, platelets also express protein phosphatase 2B (PP2B) and its role in platelet function is unexplored. PP2B-Aα and PP2B-Aβ constitute two ubiquitous isoforms of the PP2B catalytic subunit. Due to the general concerns regarding the specificity of the PP2B inhibitors, we have utilized mice deficient in the β isoform of the catalytic subunit of PP2B (PP2B-Aβ) to explore the role of PP2B in platelet functions. Mice lacking PP2B-Aα are short lived and are not considered in this study. Loss of PP2B-Aβ did not cause any compensatory increase in the PP2B-Aα levels in platelets. Compared to the wild type (WT) platelets, PP2B-Aβ−/− platelets displayed increased aggregation in response to low doses of protease-activated receptor 4-activating peptide (PAR4-AP), ADP, collagen and collagen related peptide (CRP). Enhanced α granule secretion in response to the low doses of PAR4-AP and CRP was noticed in PP2B-Aβ−/− platelets, relative to the WT platelets. Functions regulated by the outside-in αIIbβ3 integrin signaling like adhesion to immobilized fibrinogen and fibrin clot retraction were enhanced in the PP2B-Aβ−/− platelets. These studies indicate that PP2B-Aβ negatively regulate platelet functions in vitro. Consistent with these observations, PP2B-Aβ−/− mice exhibited a shorter tail bleeding time compared to the WT mice. In a FeCl3 induced endothelial denudation injury model, PP2B-Aβ−/− mice showed decreased time to occlusion in the carotid artery, and reduced number of emboli compared to the WT mice. These studies indicate that PP2B-Aβ suppress multiple murine platelet functions that contribute to an occlusive thrombi. Unlike a positive thrombus promoting role for the PP1cγ that was noticed in our previous study, PP2B-Aβ suppressed murine platelet activation, suggesting that different subtypes of Ser/Thr phosphatases have distinct roles in murine platelet activation.

Disclosures:
No relevant conflicts of interest to declare.
Albuminuria As a Risk Factor for Development of Chronic Kidney Disease and Short-Term Mortality Following Hematopoietic Stem Cell Transplantation
Authors: Takahashi Yukie , Morito Taku , Ando Minoru , Ohashi Kazuteru , Sakamaki Hisashi 
Published: October 2019

Abstract
Abstract 1998

Background:
Microalbuminuria is a risk factor for the development of chronic kidney disease (CKD), cardiovascular disease and mortality in the general population. We studied if the incidence of microalbuminuria following hematopoietic stem cell transplantation (SCT) could be a warning sign of the future CKD and all-cause mortality.

Method:
A 1-year prospective cohort study was conducted in 29 patients (46.3±12.5 years) receiving allogeneic myeloablative SCT. Patients with prevalent CKD and those who died by 1 month after SCT were excluded from the cohort. Albumin to creatinine ratio (ACR) in a morning urine sample was consecutively measured before conditioning therapy (baseline), at the time of SCT, days 7 and 14, and 1 month after SCT. Microalbuminuria was defined as ACR ≥30mg/girl. CKD was defined as a decrease in estimated glomerular filtration rate (eGFR) less than 60 ml/min/1.73m2, sustained at least for 3 months. Cumulative incidence of CKD or mortality or both was assessed by the Kaplan-Meier method. Multivariate Cox regression analysis was used to calculate the hazard ratio (HR) of developing these outcomes within 1 year, adjusted for covariates with clinical and statistical significance.

Results:
The prevalence of microalbuminuria was 6.9% at baseline, and increased to 62.1% at the day of SCT. It varied among the time points; 62.1% on day 7, 48.3% on day 14 and 51.7% on 1 month, respectively. Mortality within 1 year was 27.6%. Cumulative incidence of CKD or mortality or both was significantly higher in the group with □emicroalbuminuria on 1 month' than in the opposite. [figure1] In addition, the presence of □emicroalbuminuria on 1 month' was significantly associated with each outcome: for CKD, HR 14.5, 95% confidence interval (CI) 2.88 to 121, P=0.0006; for death, HR 11.7, 95% CI 1.58 to 252, P=0.0138; for either CKD or death, HR 16.6, 95% CI 3.79 to 151, P=0.0001, respectively.

Conclusion:
The incidence of microalbuminuria following SCT is a promising predictor of the development of CKD and short-term mortality.

Disclosures:
No relevant conflicts of interest to declare.