bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induction Of Tolerance To FVIII Using Nanoparticles In a Murine Model Of Hemophilia A
Authors: Allan Zhang Ai-Hong , Rossi Robert J. , Griset Aaron , Maldonado Roberto , Kishimoto Takeshi Kei , Scott David William 
Published: October 2019

Abstract
Anti-FVIII inhibitor development is currently considered the most serious side effect in the treatment of hemophilia A patients. In this study, we sought to address this problem by determining the efficacy of a novel tolerogenic protocol utilizing nanoparticle formulations provided by Selecta Biosciences. Thus, hemophilia A mice (FVIII KO, n = 6-8) were treated weekly for five weeks through intravenous route with 1 µg recombinant human FVIII (rFVIII), plus immune modulating agent coupled nanoparticles or control nanoparticles (coded F1 or F2 or F3). An additional control group was included, in which the mice were treated on the same schedule with high dose IVIG + rFVIII. As expected, all the mice in the control group (later revealed to be F3) developed high levels anti-FVIII IgG after the five treatments, with the average levels at 21.2 ± 4.4 µg/ml. Both the F1 and F2 nanoparticle treatments effectively prevented the anti-FVIII inhibitory antibody development throughout the course of the 5-week treatment. One week after the final treatment, the majority (5/7) of both F1 and F2 groups remained undetectable for anti-FVIII IgG antibodies. Consistent with this pattern, the anti-FVIII inhibitor titers for the F1, F2, F3 and IVIG groups were 42.3 ± 9.9, 14.3 ± 7.2, 92.9 ± 2.5, and 30.3 ± 5.6 Bethesda Units (BU)/ml, respectively after another 7 days (p < 0.01 for F1 and F2 vs. F3). To determine the duration of the FVIII-tolerance, the mice were challenged twice with rFVIII (1µg) 29 and 53 days after the final nanoparticle treatment. After the first FVIII challenge, the inhibitor titers for the F1, F2, F3 and IVIG groups were 81.3 ± 10.8, 33.1 ± 13.2, 84.1 ± 2.2 (p = 0.41 for F1 vs. F3; and p < 0.01 for F2 vs. F3), and 50.6 ± 9.7 BU/ml, respectively. Remarkably, the FVIII-tolerance in F2 group was sustained even after additional FVIII challenges ∼ 2 months later, when the effect in IVIG group was completely lost. A therapeutic protocol using mice pre-immunized with FVIII also led to substantial reductions in anti-FVIII titers. In conclusion, the concomitant Selecta F2 nanoparticle treatments not only significantly prevented the anti-FVIII inhibitory antibody development during the treatments, but also resulted in long-term FVIII-tolerance, suggesting a potential clinical application. (Supported in part by a grant-in-aid from Selecta Bioscience and by HL061883)

Disclosures:
Griset: Selecta Biosciences: Employment, Equity Ownership. Maldonado:Selecta Biosciences: Employment, Equity Ownership. Kishimoto:Selecta Biosciences: Employment, Equity Ownership. Scott:Selecta Biosciences: Research Funding.
Natural Course Of Serum Ferritin In Childhood Cancer Survivors: Need For Iron Removal Therapy?
Authors: Maeba Hideaki , Kuroda Rie , Fujiki Toshihiro , Mase Shintaro , Araki Raita , Ikawa Yasuhiro , Yachie Akihiro , Nishimura Ryosei 
Published: October 2019

Abstract

Background
Iron overload has been reported in adult survivors of leukemia after chemotherapy with or without allogeneic hematopoietic stem cell transplantation (allo-HSCT). Approximately 10-15% of adult survivors suffer from liver dysfunction, endocrine disorders, and/or cardiac dysfunction due to iron overload, in which free radicals produced by iron could damage tissues. Therefore phlebotomy and iron chelation therapy in adult survivors have been used prophylactically, however iron overload has not been studied extensively in childhood survivors, so that it would be a problem how to manage the childhood patients in whom serum ferritin level was high at the completion of chemotherapy. In this study, we retrospectively analyzed the serum ferritin level over time after the completion of therapy and also referred to whether iron chelation therapy and/or phlebotomy would be needed or not in childhood survivors.

Patients and methods
We retrospectively analyzed the level of serum ferritin overtime in 48 childhood cancer survivors (ALL 19, AML 13, Lymphoma 5, Pediatric solid tumor 11) except allo-HSCT, who were transfused concentrated red cells in our hospital. All the patients did not receive any phlebotomy and iron chelation therapy throughout the course.

Results
The total mean concentrated blood transfusion volume was 114 ml/kg (114±16, ranges 7-672). At the completion of chemotherapy, the median serum ferritin level was 867 ng/ml (867±216, ranges 7-6558). Three years after chemotherapy, the median serum ferritin levels decreased to 281 ng/ml (281±77, ranges 7-1285). All patients did not show any symptoms related to iron overload such as liver dysfunction and glucose intolerance. Twelve out of 48 patients (25%) exceeded 1000 ng/ml of the serum ferritin at the time of completion of chemotherapy, which has been considered as the initiation of iron chelation therapy in adult patients. However all patients except one decreased the serum ferritin level below 1000 ng/ml in 3 years after chemotherapy without any iron removal therapy. Although serum ferritin level in the exceptional case was extraordinary high (6558 ng/ml) compared to other cases at the completion of chemotherapy, it declined to 1285 ng/ml spontaneously in 3 years, which was much better than expected.

Conclusions
Although 25 percent of our childhood cancer survivors showed high level of serum ferritin more than 1000 ng/ml at the time of completion of chemotherapy, almost all the cases eventually declined thereafter without any iron removal therapy probably due to the iron consumption with growth. Further study would be needed to make specialized criteria for initiating iron removal therapy for childhood cancer survivors.

Disclosures:
No relevant conflicts of interest to declare.
AK2 Deficiency In Zebrafish Recapitulates Human Reticular Dysgenesis, An Autosomal Recessive Form Of Severe Combined Immunodeficiency
Authors: Rissone Alberto , Jagadeesh Jaya , Simon Karen , Bishop Kevin , Sood Raman B. , Candotti Fabio 
Published: October 2019

Abstract
The adenylate kinase (AK) gene family consists of 7 different members that contribute to energy cell metabolism by converting ATP+AMP to 2ADP. AKs are critical players in ensuring cellular energy homeostasis in all tissues. Mutations in the AK2 gene are responsible for reticular dysgenesis (RD), an autosomal recessive form of severe combined immunodeficiency (SCID). RD is characterized by an early differentiation arrest in the granulocyte lineage and impaired lymphoid maturation and it represents less than 2% of total SCID. Affected children succumb to overwhelming infections early in life unless their immune system is successfully restored with allogeneic hematopoietic stem cells transplant (HSCT). The mechanisms underlying the pathophysiology of RD remain unclear. The phenotype of AK2 deficient animals has never been reported in the literature, but murine lines carrying homozygous inactivating retroviral insertions are embryonically lethal (our personal observations). We used the zebrafish model to perform a comprehensive study of the effects of AK2 deficiency using Morpholino oligomers injections and two different kinds of AK2 mutants (a ENU-induced T371C/L124P missense mutant and two null mutant lines generated using zinc-finger nuclease technology). In situ hybridization analyses of AK2-deficient embryos indicated that only erythroid development was affected during primitive hematopoiesis. Conversely, during definitive hematopoiesis, the loss of function of AK2 resulted in abnormalities distributed along all hematopoietic lineages suggesting an impairment of hematopoietic stem cell (HSC) development. Moreover, we observed that the AK2 deficiency induced oxidative stress and consequent apoptosis in both primitive erythroid cells and definitive HSCs. Importantly, antioxidant treatment of AK2 mutant embryos rescued the hematopoietic phenotypes as indicated by the recovered expression of HSC and lymphoid markers (such as c-myb and rag1). Overall, our data indicate that zebrafish represents a good model for studying the molecular mechanisms involved in RD and testing of new therapeutic interventions. To date, our mutant lines remain the only animal model of this rare and lethal human disease.

Disclosures:
No relevant conflicts of interest to declare.
PRKD2 Serine-Threonine Kinase, a Downstream Effector Of GABP, Plays Essential Roles For The Maintenance Of HSCs
Authors: Yang Zhong-Fa , Junling Wang , Rosmarin Alan G. 
Published: October 2019

Abstract
Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. GABP is an ets-related transcription factor that controls critical genes in myeloid and lymphoid development, and has been implicated in control of HSC growth. GABP is an obligate multimeric transcription factor that includes the DNA-binding ets component, GABPa, along with various GABPb partner proteins. We conditionally deleted Gabpa in mouse bone marrow and found that Gabpa cells have a profound growth disadvantage due to cell cycle arrest in HSCs. We identified Protein Kinase D2 (PRKD2) as a candidate effector of GABP. PRKD2 is a diacyl glycerol- and Protein Kinase C-activated serine-threonine kinase, because deletion of Gabpa markedly reduced PRKD2 expression in normal HSCs and progenitor cells. In a Prkd2ki/ki mouse model, in which two functionally essential phosphorylation serines were inactivated genetically, their bone marrow long term HSCs reduced dramatically and the short term HSCs increased accordingly. Mice transplanted with a 1:1 mixture of Prkd2ki/ki and wild type bone marrow cells demonstrated the decreased proportion of the Prkd2ki/ki bone marrow cells with the corresponding increase of the wild type cells. Although ectopic expression of the human Chronic Myeloid Leukemia (CML) fusion oncogene BCR-ABL in wild type bone marrow cells induced rapid CML development, expression of BCR-ABL in Prkd2ki/ki bone marrow cells failed to develop CML in transplanted recipient mice. Analysis of the peripheral blood, bone marrow and spleen of these mice revealed that the BCR-ABL+, Prkd2ki/ki cells did not express myeloid or lymphoid specific cell surface antigens CD11b, Gr1, B220, or CD3e. They demonstrated an immature blast-like microscopic morphology, and recipient mice transplanted with these cells died before the onset of CML development. We conclude that the phosphorylation activated Prkd2 is required for the maintenance of HSC pool and the development of mature hematopoietic lineages from HSCs. These findings suggest that PRKD2 kinase mediate key downstream events of both PKC and transcription factor GABP, and that PRKD2 may serve as a novel therapeutic target in leukemia.

Disclosures:
No relevant conflicts of interest to declare.
Molecular Integration Of HoxB4 and STAT3 For Self-Renewal Of Hematopoietic Stem Cells: A Model Of Molecular Convergence For Stemness
Authors: Oh Il-Hoan , Tae-Min Kim , Shim Jae-Seung 
Published: October 2019

Abstract
Multiple transcription factors (TFs) that regulate the self-renewal/stem cell state of hematopoietic stem cells (HSCs) have been identified, but understanding the molecular interplay of these TFs for their functional coordination remains a challenging issue.
In this study, we investigated the functional integration and transcriptional coordination of STAT3 and HoxB4, which are TFs known to have similar effects on the self-renewal of HSCs. We found that while STAT3 (STAT3-C) or HoxB4 similarly enhanced the in vitro self-renewal and in vivo repopulating activities of HSCs, simultaneous transduction of both STAT3-C and HoxB4 did not have any additive enhancing effects. In contrast, the overexpression of HoxB4 caused a ligand-independent Tyr-phosphorylation in STAT3, and the inhibition of the STAT3 activity in HoxB4-overexpressing bone marrow cells significantly abrogated the enhancing effects of HoxB4 on both the bone marrow repopulation and maintenance of the undifferentiated state, revealing a molecular integration of these two TFs for HSC self-renewal. Expression microarray analysis revealed a significant overlap of the transcriptomes regulated by STAT3 and HoxB4 in undifferentiated hematopoietic cells. Moreover, a gene set enrichment analysis (GSEA) for TFs that can recapitulate the transcriptional changes induced by HoxB4 or STAT3 showed significant overlap in the candidate TFs. Interestingly, among these identified TFs were the puripotency-related genes, Oct-4 and Nanog. These results indicate the functional integration of tissue-specific TFs for HSC self-renewal and provide insights into the functional convergence of various TFs towards a conserved transcription program for the stem cell state.

Disclosures:
No relevant conflicts of interest to declare.
B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) Specific Copy Number Alterations Are Unique For Progressive Pediatric Chronic Myeloid Leukemia (CML): A Large Cohort Study
Authors: van der Sligte Naomi E , Krumbholz Manuela , Pastorczak Agata , Scheijen Blanca , Tauer Josephine T. , Nowasz Christina , Sonneveld Edwin , de Bock Truuske H. , Meeuwsen - de Boer Tiny G. , van Reijmersdal Simon V. , Kuiper Roland P. , Bradtke Jutta , Metzler Markus , Suttorp Meinolf , De Bont Evelina S. , van Leeuwen Frank N. 
Published: October 2019

Abstract
Chronic myeloid leukemia (CML) is a rare malignancy in children and is mostly diagnosed in the chronic phase (CP). In adults, the five-year overall survival rate is 89% for patients on Imatinib and disease progression occurs in 1-3% per year (Druker 2006). Once a blast crisis (BC) has occurred, treatment options are limited with a median survival of only a few months (Cortes 2008). Therefore, early recognition of patients at risk for developing a BC is desirable. Besides the translocation t(9;22)(q34;q11), IKZF1, PAX5, and CDKN2A deletions have been reported in CML lymphoid blast crisis (LyBC) of both adult and pediatric patients (Mullighan 2008, Alpár 2012).
The aim of this study was to investigate the presence of IKZF1 deletions and other copy number alterations (CNAs) by MLPA analysis in a large cohort of pediatric CML patients at time of diagnosis in order to determine whether CNAs commonly found in pediatric ALL might predict disease progression and / or treatment response.
Between October 1991 and October 2012 a total of 86 children with newly diagnosed CML were included. The median follow up was 31 months.
Among the 86 patients, 82 patients were diagnosed in CP, 2 patients in accelerated phase (AP), and 2 patients in LyBC. Six patients experienced progression to a BC respectively a myeloid blast crisis (MyBC) (N=2) and LyBC (N=4).
At time of diagnosis, an IKZF1 deletion was detected in one patient diagnosed with CML-AP (Table A, patient no 58). IKZF1 and EBF1 deletions were detected in one patient diagnosed with CML-LyBC (Table A, patient no 22). No CNAs were detected in the 82 patients diagnosed with CML-CP.
At time of disease progression, new CNAs were detected at time of the LyBC (Table A, patient no 62, 64, and 67). Due to the absence of material no CNAs could be detected in both patients experiencing a MyBC.
In conclusion, we were able to detect CNAs in progressive CML disease (CML-AP and CML-LyBC) and not in the samples at the time of chronic phase in this large pediatric cohort of CML patients. Therefore, the investigated CNAs could not be used to predict disease progression at time of diagnosis. The CNAs detected in patients with progressive CML were similar to specific CNAs detected in pediatric B-cell precursor ALL, indicating a similar disease development (Kuiper 2010).
Additionally, our results are in accordance with existing literature, suggesting that mechanisms of disease progression in pediatric and adult CML might be similar (Brazma, 2007).

Disclosures:
No relevant conflicts of interest to declare.
Bone Marrow Beta-Catenin Expression In Chronic Myelogenous Leukemia Patients Treated With Imatinib
Authors: Zhang Xiaohui , Sweet Kendra L , Moscinski Lynn , Hazlehurst Lori A , Pinilla-Ibarz Javier , Zhang Ling 
Published: October 2019

Background
Imatinib is the standard of care for treating chronic myelogeneous leukemia (CML). However, it is rarely curative and patients often show resistance to the drug. ABL-kinase domain mutations are one mechanism of resistance in CML. Regulation of hematopoietic stem cell survival, as well as disease progression in CML has been linked to the Wnt/beta-catenin pathway. Our study investigated the expression of beta-catenin in the bone marrow (BM) of CML patients treated with imatinib as well as its possible application as a prognostic marker for response to therapy.
Piggybac Mediated Gene Transfer To Correct Hemophilia A
Authors: Staber Janice M , Pollpeter Molly , Arensdorf Angela , Sinn Patrick L , Rutkowski Thomas D , McCray Paul B 
Published: October 2019

Abstract
Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder, affecting about 1 out of 5,000 males; those affected suffer disabling joint and muscle hemorrhages. Hemophilia A is an attractive gene therapy candidate, because even small increases in FVIII levels (5-10%) will alter the phenotype. Non-viral vector systems are used increasingly in gene targeting technologies and as tools for gene transfer applications. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Advantages of this novel nonviral vector system include a large transgene cassette capacity, ease of production and purification, and potential for site-specific integration. We hypothesize that a PB transposon vector carrying a codon-optimized human FVIII cDNA along with a hyperactive transposase (iPB7) will confer persistent gene expression and correction of the hemophilia A bleeding phenotype. We engineered PB transposon to carry a codon-optimized human FVIII B-domain deleted cDNA (coFVIII-BDD). We evaluated the in vivo gene transfer efficiency in hemophilia A mice by hydrodynamic tail-vein injection using PB coFVIII-BDD driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Factor VIII null mice received 25 micrograms each of the PB coFVIII-BDD transposon and iPB7 to determine long term expression and phenotypic correction. FVIII activity and antigen levels were measured prior to injection and then every 4 weeks for 24 weeks. Results revealed therapeutic levels (50-225%) of factor VIII activity and antigen post gene transfer with stable expression for 24 weeks in most mice. A goal of gene transfer based therapies is to develop the most efficacious expression vectors with the least toxicity. To assess endoplasmic reticulum stress in the livers of treated and untreated mice, we evaluated BiP, CHOP, and EDEM levels via q-PCR. All experimental mice, null mice, and transposon treated mice without the coFVIII-BDD cassette revealed no evidence of cell stress. These data indicate codon-optimized FVIII and the piggyBac transposon vector system may provide a safe long term gene transfer strategy. To evaluate phenotypic correction, a tail clip assay was performed at the end of the study. More than 75% of mice receiving PB coFVIII-BDD transposon and iPB7 demonstrated functional correction via tail clip. These data show that the PB vector can be used to deliver transgene expression to the liver and achieve long term expression and phenotypic correction.

Disclosures:
No relevant conflicts of interest to declare.
Notch Signaling In B-ALL Reveals PLK1 As A Potential Therapeutic Target In B-ALL: PLK1-Selective Inhibitor Poloxin Modulates Cyclin B and P53 Pathways, Leading To Growth Arrest and Apoptosis
Authors: Kannan Sankaranarayanan , Hall Mandy A , Golfman Leonard S , Zweidler-McKay Patrick A 
Published: October 2019

Abstract

Background
Notch is a well-known oncogene in T-ALL, yet appears to have tumor suppressor effects in B-ALL. These cell type-specific effects of Notch signaling mirror consequences seen in early lymphocyte development and raises the question of how Notch leads to such divergent consequences in closely related cell types. In exploring these Notch mechanisms we discovered a B-ALL specific Notch-mediated reduction in the cell cycle regulator Polo-like kinase-1 (PLK1), revealing a novel targetable kinase in B-ALL.

Approach
To explore the consequences of Notch-mediated down regulation of cell cycle regulator kinase PLK1, we targeted PLK1 kinase function with the novel PLK1-selective inhibitor poloxin in human B-ALL lines.

Results
PLK1 is highly expressed in B-ALL verses normal tissues (panel A), correlates with cyclin B expression, is expressed >2-fold higher in B-ALL with t(1;19) than other B-ALL samples, and may predict response of ALL to methotrexate. In our panel of human B-ALL cell lines poloxin induced G2/M growth arrest and decreased cell number by >80% (panel B), and decreased survival in B-ALL cells (>75% AnnexinV+, panel C). PLK1 inhibition led to tumor suppressor p53 stabilization, revealing >5-fold increase in p53 protein levels following poloxin treatment in B-ALL (panel D). Mechanistically, PLK1 inhibition leads to both cytoplasmic re-localization of cyclin B, disrupting the CDC2-cyclinB complex, as well as phosphorylation of p53 at Ser20, which destabilizes p53-MDM2 interaction and thus accumulation of p53.

Conclusions
While exploring the mechanisms of cell type-specific effects of Notch signaling in ALL, we have found a novel therapeutic target, the cell cycle regulator PLK1. Our findings reveal a novel therapeutic approach whereby PLK1-selective inhibition via poloxin induces growth arrest and apoptosis in human B-ALL via consequences on cyclin B and p53 pathways.

Disclosures:
No relevant conflicts of interest to declare.