bluuebunny/crossref_metadata_2025_split huggingface dataset:
FANCL Ubiquitinates Beta-Catenin and Enhances Its Nuclear Function
Authors: Dao Kim-Hien T. , Rotelli Michael D. , Petersen Curtis L. , Brown Brie R. , Nelson Whitney D. , Yates Jane E. , Newell Amy Hanlon , Olson Susan B. , Druker Brian J. , Bagby Grover C. 
Published: October 2019

Abstract
Abstract 1335
Fanconi anemia (FA) is associated with a hereditary predisposition to bone marrow failure. The proteins encoded by the FANC genes are primarily involved in DNA repair responses through the formation of a large, multisubunit complex that has E3 ubiquitin ligase activity (Annual Review of Genetics 2009;43:223). FA hematopoietic stem cells display defective stem cell properties and limited replicative potential. However, the molecular basis for how a FA genetic background contributes to those defects remains poorly understood. Here we provide evidence that FANCL, which has E3 ubiquitin ligase activity, enhances beta-catenin activity (Figure A) and protein expression. Beta-catenin is a nuclear effector of canonical Wnt signaling. The Wnt/beta-catenin pathway is active in normal hematopoietic stem cells in the native bone marrow environment and disruption of this signaling pathway results in defective hematopoietic stem cells (Nature 2003;423:409). To test whether FANCL positively regulates beta-catenin through its ubiquitination activity, we performed cell-based ubiquitination assays. We show that FANCL functionally ubiquitinates beta-catenin (Figure B) and that ubiquitin chain extension can occur via non-lysine-48 ubiquitin linkages. Accumulating evidence reveal diverse, non-proteolytic biological roles for proteins modified by atypical ubiquitin chains (EMBO Reports 2008;9:536). Our data suggests that FANCL may enhance the protein function of beta-catenin via ubiquitination with atypical ubiquitin chains. Importantly, we demonstrate that suppression of FANCL expression in human CD34+ cord blood stem cells reduces beta-catenin expression (Figure C) and multilineage progenitor expansion. These results demonstrate a role for the FA pathway in regulating Wnt/beta-catenin signaling. Therefore, diminished Wnt/beta-catenin signaling may be an important underlying molecular defect in FA hematopoietic stem cells leading to their accelerated loss.
A, LEF-TCF-luciferase reporter assay showing increasing beta-catenin activity in 293FT cells with increasing FANCL expression compared with vector-control (VC) (n=4). B, Immunoprecipitation of beta-catenin in cells transfected with vector-control or FANCL and probed for hemagglutinin (HA)-tagged ubiquitin shows increased ubiquitinated forms of beta-catenin with FANCL expression (n=4). C, shRNA suppression of FANCL expression in CD34+ cord blood stem cells results in decreased beta-catenin expression compared with a scramble control (Scr) by immunofluorescence analysis (three different shRNA constructs, n=3 for each construct).

Disclosures:
No relevant conflicts of interest to declare.
Knockdown of MicroRNA-10a in Acute Myeloid Leukaemia Cells Bearing the Nucleophosmin1 Mutation Causes Cell Death and Reduced Clonogenicity
Authors: Bryant Adam J , Palma Catalina A , Lutherborrow Mark , Jayaswal Vivek , Yang Yee Hwa , Ma David 
Published: October 2019

Abstract
Abstract 1367
Acute Myeloid Leukaemia (AML) with a mutation in the Nucleophosmin1 gene (NPM1c+) accounts for one of the largest subtypes of AML, with an unknown etiology. MicroRNA dysregulation has now been implicated in the oncogenesis of many cancers including AML. We sought to investigate the role of microRNAs in the initiation and development of AML with the NPM1c+ mutation.
MicroRNA profiling of bone marrow samples from 28 AML patients and confirmation by qRT-PCR demonstrated a unique microRNA signature in AML-NPM1c+ samples dominated by miR-10a over-expression of 19.6-fold compared to Nucleophosmin1 wild type (NPM1) samples. Functional assessments were performed in the human OCI-AML3 cell line, which is the only cell line to harbour NPM1c+. miR-10a repression was induced by transfection with miRCURY LNA microRNA knockdown probes (Exiqon). Cell growth (MTS) assay demonstrated a significant decrease of 19% in miR-10a knockdown cells compared to the Scrambled control. AnnexinV and Caspase 3 assays assessed the effect of miR-10a knockdown on apoptosis. miR-10a knockdown increased the proportion of AnnexinV positive events when compared to control treated cells by 34.9% and 39.3% at 24 and 48 hours respectively, but had no effect on Caspase 3 expression. Proliferation (BrdU uptake) assays did not show a change, however, clonogenic assays demonstrated a 26.1% decrease in colony number in miR-10a knockdown cells compared to the control. Potential mechanisms were elucidated by determining miR-10a mRNA targets in silico and confirmed by luciferase reporter assays. These included ARNT, GTFH1, ID4, KLF4, MAPRE1, NR4A3, RB1CC1 and TFAP2C.
In this study, we have demonstrated that miR-10a was highly differentially expressed between AML-NPM1c+ cells compared to leukaemic cells bearing wild type NPM1. Knockdown of miR-10a in OCI-AML3 cells resulted in increased cell death as detected by AnnexinV binding (but not Caspase 3, indicating an effect independent of the classical apoptotic pathways) and reduced clonogenic capacity. These effects are thought to occur through miR-10a mediated modulation of ARNT, GTFH1, ID4, KLF4, MAPRE1, NR4A3, RB1CC1 and TFAP2C, all of which are associated with neoplastic transformation. Taken together, our results suggest that aberrant miR-10a over-expression in AML-NPM1c+ patients promotes cell survival.

Disclosures:
No relevant conflicts of interest to declare.
Role of CD44 in Endogenous Kras G12D-mediated Myeloproliferative Neoplasm
Authors: Du Juan , Liu Yangang , Tan Lixuan , Lo Juinn Cherng , Wang Jinyong , Ryu Myung-Joem , Zhang Jingfang , Zhang Jing 
Published: October 2019

Abstract
Abstract 1398

Abstract
Oncogenic KRAS mutations are identified in virtually all the cancer types, making KRAS one of the most frequently mutated genes in human cancers. Our lab established a mouse model of myeloproliferative neoplasm (MPN) initiated by oncogenic Kras mutation (Kras G12D) expressed from its endogenous locus. Because CD44, a cell adhesion molecule and a cell signaling regulator, was found to involve in regulating leukemic stem cells in chronic myeloid leukemia (Krause, Lazarides et al. 2006) and acute myeloid leukemia (Jin, Hope et al. 2006), we have generated compound mice expressing oncogenic Kras but deficient for CD44 (Kras G12D; CD44−/−) to examine the role of CD44 in endogenous oncogenic Kras- mediated MPN.
Our preliminary data show that loss of CD44 prolonged survival of compound Kras G12D; CD44−/− mice and greatly downregulated GM-CSF signaling in hematopoietic stem/progenitor cells. However, CD44 deficiency did not significantly affect the development of MPN, suggesting that GM-CSF signaling is not essential for Kras G12D-initiated MPN and CD44 deficiency might not affect other cytokine signaling. We are currently investigating this issue.
Because Kras G12D hematopoietic stem cells (HSCs) are required to initiate MPN phenotypes, we then examined whether loss of CD44 would affect the behavior of Kras G12D HSCs. Our preliminary results demonstrate that CD44 deficiency did not significantly alter HSC frequencies in bone marrow and spleen, nor their cell cycle profile and apoptosis status. When Kras G12D; CD44−/− bone marrow cells were transplanted into lethally irradiated mice by retro-orbital injection, ∼19% of recipient mice developed MPN, similarly to recipient mice transplanted with same number of Kras G12D cells (∼14%). These MPN mice simultaneously developed acute T-cell lymphoblastic leukemia/lymphoma. Our results suggest that CD44 is dispensable for the homing and engraftment of Kras G12D HSCs. We are currently investigating whether CD44 function is essential for the homing and engraftment of leukemic initiating cells of MPN.

Disclosures:
No relevant conflicts of interest to declare.
Investigational NEDD8-Activating Enzyme (NAE) Inhibitor, MLN4924, Demonstrates Activity Against Primary AML Blast, Progenitor and Stem Cell Populations
Authors: Sen Siddhartha , De Leon Jeanne P. , Smith Peter G , Roboz Gail J. , Guzman Monica L. 
Published: October 2019

Abstract
Abstract 1414
Acute myelogenous leukemia (AML) has a poor prognosis. Most patients die of either primary refractory or relapsed disease. It has been proposed that relapse may result from ineffective ablation of leukemia stem cells (LSCs) by chemotherapy. Therefore, in order to improve therapy, it is imperative to identify agents that eliminate leukemic progenitor and stem cell populations in addition to blasts. We have previously demonstrated that proteasome inhibition can result in selective ablation of LSCs, without harming normal hematopoietic stem cells (HSCs). Thus, we sought to investigate the activity of a more specific inhibitor of the ubiquitin-proteasome pathway, an investigational NEDD8-Activating Enzyme (NAE) inhibitor MLN4924. Treatment of primary human AML and blast crisis CML samples with MLN4924 resulted in a dose-dependent decrease in the total viability of the blast-cell populations, with an average LD50 of 0.367 μM (0.203–0.530 95% CI; N=17). The average LD50 for phenotypically described LSCs was 0.645μM (0.304–0.986; 95%CI; N=12). Interestingly, when performing progenitor/stem functional assays, we observed a 64% and 86% decrease in colony formation after treatment with 0.25μM and 1 μM of MLN4924 (p< 0.001; N=5; p< 0.001 N=8 respectively). Furthermore, MLN4924 had little to no effect on colony formation of normal hematopoietic cells. Thus, MLN4924 may impair the activity of AML stem/progenitor cells, but not normal cells, in vitro. Importantly, primary AML samples that were resistant to standard chemotherapeutics such as cytarabine in vitro were sensitive to MLN4924. Treatment of primary AML samples with MLN4924 showed increased γH2AX foci, indicating activation of stress responses. Together, these data suggest that MLN4924 can eliminate AML blasts, progenitor and stem cell populations in vitro.

Disclosures:
Smith: Millennium Pharmaceuticals: Employment.
Comparison Between the International Standardised Approach and a Single Tube Ten Colour Flow Cytometric Assay for Detection of Minimal Residual Disease in CLL
Authors: Sartor Mary M , Bradstock Kenneth Francis , Gottlieb David 
Published: October 2019

Abstract
Abstract 1460
The eradication of minimal residual disease (MRD) in CLL predicts for improved outcome. Approaches to CLL MRD detection vary in sensitivity and cost. Historically, CD5/CD19 co-expression with demonstration of clonality has been the principal method of MRD evaluation in CLL. However this approach is limited by the identification of normal hemopoietic cells and the inability to demonstrate light chain restriction with very low B cell numbers. More recently, an international standardised approach (ISA) to the assessment of MRD in CLL that utilises 10 monoclonal antibodies in a 4 tube test system and permits a sensitivity of 0.01% (Rawstron et al, Leukemia 2007) was published. Here we compare the ISA to a 10 colour flow cytometric assay incorporating all the mononclonal antibodies utilised in the ISA in a single tube.

Method:
Peripheral blood (n=21) and bone marrow (n=7) was collected from patients at various time points post treatment for CLL (including bone marrow from 2 patients day 30 post-allogeneic stem cell transplant). Immunophenotyping was performed using a Gallios flow cytometer (Beckman Coulter). Monoclonal antibodies were used either according to the ISA (antibody/fluorochrome combination as per Rawstron et al) or in a single tube incorporating all of the following monoclonal antibodies: CD3 ECD, CD5 PercP5.5, CD19 eFluor, CD20 PE CY7, CD81 FITC, CD22 PE, CD43 APC CY7, CD79b APC, CD38 A700, CD45 CO. A whole blood or bone marrow lyse method was used and analysis was performed according to the published ISA methodology or for the single tube using templates based on the ISA approach. Results from the two methods were compared.

Results:
Levels of residual disease in the 28 samples analysed varied from <0.01 to 22%. Sixteen samples showed residual disease of <1%. Analysing all samples showed an excellent correlation between the two methods slope = 0.989, intercept =0.1 and R(2)=0.992. There was also excellent correlation for disease levels below 1.0%, (median 0.03 range <0.01–0.63%, n=16) slope = 1.15, intercept =0.007 and R(2)=0.994. Bland Altman analysis showed a mean of 0.008 +/− 0.058 (2SD) for values below 1%.

Conclusion:
The single tube ten colour flow cytometric assay for detection of MRD in CLL gives equivalent results to the ISA. There is a potential for improved sensitivity resulting from increased event detection since there is no need to divide the sample into multiple tubes, particularly post-treatment when cell numbers are frequently limiting. The single tube assay is also simple, rapid and cost effective.

Disclosures:
No relevant conflicts of interest to declare.
Targeting AKT Signaling in Pediatric Acute Lymphoblastic Leukemia with Sulforaphane
Authors: Suppipat Koramit , Zhu Evelyn , Lacorazza Daniel 
Published: October 2019

Abstract
Abstract 1521
Refractory and relapse acute lymphoblastic leukemia (ALL) is one of the leading causes of cancer-related death in children. Novel agents are still in need as a frontline therapy in high risk patients and salvage agent in relapse patients. Leukemic cells acquire a survival advantage over normal blood cells by losing control of the cell cycle and activating pro-survival signals. There are emerging evidences showing that the PI3K/AKT/mTOR pathway is frequently activated in both T cell acute lymphoblastic leukemia (T-ALL) and precursor B cell acute lymphoblastic leukemia (pre-B ALL) (approximately 30% of ALL patients). Thus, drugs able to inhibit AKT signaling are potential candidates for ALL treatment. Sulforaphane (SF) is a dietary compound found in cruciferous vegetables with well-known chemopreventive and chemotherapeutic function in solid tumors by inducing apoptosis and inhibiting survival and cell division. However, the anti-leukemic capacity of SF in hematologic malignancies has not been extensively investigated. In this study, we found increased cleavage of PARP, caspase-3, caspase-8 and caspase-9 by immunoblots in pre-B and T-ALL cell lines treated with SF (5–10 μM) for 24 –48 hours. In addition, we detected increased expression of p21 and cyclin B that correlated with a G2/M block and DNA damage observed in SF treated cells by analysis of DNA content and H2AXγ expression. Noteworthy, SF-treated pre-B and T-ALL cell lines exhibit lower levels of total and phosphorylated AKT in addition to decreased levels of total and phosphorylated mTOR, which was independent of p53 and PTEN activities. Lower survival signals upon SF treatment correlated with a dose- and time-dependent cytotoxicity in pre-B ALL cell lines (Nalm-6, REH and RS-4) and T-ALL cell lines (Jurkat, RPMI, DND-41 and KOPTK1) compared to EBV-transformed lymphoblasts used as a control. Interestingly, SF showed a synergistic effect with vincristine in pre-B and T-ALL cell lines, suggesting a potential use of SF in adjunctive therapy. Aiming to support future clinical trials, we confirmed that SF is effective in lymphoblasts from pediatric patients suffering with ALL, both pre-B and T, in a time- and dose-dependent manner (95% confidence interval IC50: pre-B ALL = 9.9 – 12.13 μM, T-ALL = 8.3 –11.00 μM). To investigate whether SF is effective in vivo in a xenograft mouse model, we injected nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice with Nalm-6 cells transduced with GFP-FFluc for bioluminescent imaging detection. SF treatment (2 mg i.p. daily) significantly reduced tumor burden compared to the control group (injected with vehicle). Collectively, our data show that SF has anti-leukemic properties in ALL, both in primary samples from patients and cell lines, by inducing cell death selectively in leukemic cells likely by inhibiting AKT-dependent survival signals.

Disclosures:
No relevant conflicts of interest to declare.
Thymic Dendritc Cells Generated Ex Vivo Induce Donor Regulatory T Cell Differentiation and Hasten Immune Reconstitution After Hematopoietic Stem Cell Transplantation
Authors: Dallas Mari Hashitate , Langfitt Deanna , Busby Kenneth 
Published: October 2019

Abstract
Abstract 1905
Dendritic cells (DCs) are the first committed cells to engraft in the thymus after hematopoietic stem cell transplantation (HSCT). The critical role of thymic DCs in ensuring efficient tolerance and selection has been well demonstrated, however its role in facilitating donor engraftment has not been reported. Here we show DCs accelerates thymic reconstitution by inducing regulatory T cells (Tregs) differentiation and enhancing T cell recovery after HSCT. Lethally irradiated CD45.2 C57BL/6 control group received 103 CD45.1 lin−sca-1+c-kit+ (LSK) hematopoietic stem cell progenitors while the DCs group received 103 CD45.1 LSK cells along with 103 CD45.2 GFP+ DCs. DCs were generated ex vivo using bone marrow from CD45.2 GFP+ CD57BL/6 mice and cultured for 7 days with GMCSF. At 4 and 7 days after HSCT, the thymus of DC group contained 1.8 and 4.2- fold higher number of thymocytes (p<0.05) and a 3.2 and 7.4-fold, respectively, higher number of donor derived thymoctyes compared to the control group (p<0.05). Moreover, thymuses of the DCs group had GFP+ CD11c+ cells present in the medulla and 5.6-fold increase in the number of donor derived FoxP3+ Tregs compared to control confirmed by immunohistochemistry (IHC). Furthermore, thymic recovery scored by a pathologist blinded to the groups found significant increase in lymphoid regeneration (H&E) and higher number of CD3+ lymphoid aggregates (IHC) in the DC group compared to control group that had severe, diffuse lymphoid depletion. Lastly, at 2 and 4 weeks after HSCT, peripheral blood of DCs group contained 2.6 and 4.8-fold, respectively, higher numbers of CD3+ cells derived from donor LSK cells compared to the control group (p<0.05). Here, we demonstrate that donor DCs efficiently migrate and home to the thymic medulla and hasten thymic recovery as demonstrated by the higher number of total thymoctyes. Furthermore, DCs facilitate thymic engraftment as shown by increase number of donor derived FoxP3+ Tregs and thymocytes. Lastly, recipients of DCs have earlier generation of de-novo donor derived CD3+ T cells in the peripheral blood. By using the GFP+ cells along with donor LSK cells, we were able to confirm that the facilitation of early thymic recovery was due to the increased engraftment of the donor cells rather than autologous recovery of the host. In conclusion, this study demonstrates that DCs committed prior to thymic entry maintains the ability to home to the medullary region and facilitate thymic recovery by enhancing Tregs differentiation. Thus, ex vivo generation of donor DCs to augment a HSC graft may

Disclosures:
No relevant conflicts of interest to declare.
Cost of Autologous and Allogeneic Stem Cell Transplantations for Hematological Disease: A Dutch Multicenter Daily Practice Study
Authors: Blommestein Hedwig M , Verelst Silvia GR , Cornelissen Jan J , Huijgens Peter C , Blijlevens Nicole MA , Groot Carin A Uyl-de 
Published: October 2019

Abstract
Abstract 2079

Background:
Peripheral blood stem cell transplantations (PBSCT) are very expensive life-saving medical procedures carried out in patients with hematological disease. The current tariffs are expected to be too low due to developments in the treatment protocols. A revision of the tariffs is urgently necessary. We calculated the daily practice cost of PBSCT for treating hematological diseases in Dutch practice, to provide a proper basis for revising hospital budgets.

Patients and methods:
From three Dutch university hospitals, we randomly selected 191 who underwent an autologous (auto) or allogeneic (allo) PBSCT in 2008 or 2009. The alloPBSCT were subcategorized into sibling, matched unrelated donor (MUD) and unrelated cord blood (UCB). We obtained data from hospital registrations to study all treatment related activities. Unit prices were based both on real costs and tariffs (base year 2010). Thereafter, the average costs per patient per PBSCT and per period were calculated. The total cost included the selection and harvesting, transplantation and 1-year follow-up.

Results:
The average cost per patient of autoPBSCT were € 45,670. The cost of sibling alloPBSCT were € 101,919. The average cost of transplantations from an unrelated donor was much higher: € 171,478 for MUD and € 254,689 for UCB alloPBSCT. Hospital days, laboratory procedures and donor search were the largest cost components and mainly responsible for differences between the four types of PBSCT. None of the patient characteristics were correlated with average cost. The costs calculated in this study are above current reimbursement. The difference is significant (p=0.043) and depending on the type of PBSCT, the shortfall varied between 2% and 100%.

Conclusion:
Average cost of AutoSCT and alloSCT lay above current reimbursement levels. Appropriate financing is necessary to guarantee the continuation of PBSCT in Dutch patients according to current indications. The costs calculated in this study provide reliable input for economic evaluations and cost-effectiveness studies.

Disclosures:
No relevant conflicts of interest to declare.