bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Development Of The Humanized Darc Transgenic Mouse
Authors: Novitzky-Basso Igor N , Ulvmar Maria H , Bacon Andrea , Chaganti Sridhar , Moss Paul AH , Rot Antal 
Published: October 2019

Abstract
The Duffy Antigen Receptor for Chemokines (DARC) is an atypical chemokine receptor which binds CXC and CC chemokines with no measurable downstream signaling. Erythrocyte DARC serves as a chemokine sink and reservoir. DARC on endothelial cells transports chemokines thus promoting transendothelial leukocyte migration. Two common polymorphisms define the Duffy blood group, FYA and FYB, whilst the third polymorphism which is widely prevalent in West Africa, FYB(ES), abolishes erythrocyte expression of DARC. DARC is a portal of entry for plasmodium vivax suggesting that FYB(ES) was selected due to it conferring resistance to infection. The impact of the FYB(ES) polymorphism extends to a genetic association with the benign ethnic neutropenia. In order to study the mechanisms of this association we have developed a murine transgenic models of the Fy(a-b-) phenotype as encoded by FYB(ES), and Fy(a-b+) phenotype as encoded by FYB. Cloned genes were introduced by pronuclear micro-injection into fertilized oocytes of murine-DARC knockouts. The resulting embryos were transferred into pseudopregnant mice and offspring screened for transgene insertion by PCR genotyping. Gene expression in founder animals was measured by qPCR, demonstrating expression in lung, kidney, liver, spleen, skin and cerebellum. Protein expression was confirmed by immunofluorescence on frozen sections in spleen and skin in a distribution identical to human. Erythrocyte phenotyping by FACS using anti-Fy3 and anti-Fy6 monoclonal antibodies demonstrated the lack of DARC expression in mice with FYB(ES) and confirmed expression on FYB transgenic erythrocytes. We show that murine chemokines bind to human DARC and we plan to use this model to investigate the impact of FYB(ES) on in vivo chemokine functions and equilibria. These models will prove useful in the dissection of the mechanisms of ethnic neutropenia and improve the understanding in the role of DARC in chemokine homeostasis.

Disclosures:
Chaganti: Roche: Membership on an entity’s Board of Directors or advisory committees, Receipt of travel grant Other.
Critical Roles Of The IDH2 Mutation In Development and Maintenance Of Acute Myeloid Leukemia
Authors: Ogawara Yoko , Katsumoto Takuo , Aikawa Yukiko , Shima Yutaka , Kagiyama Yuki , Matsunaga Hironori , Seki Takahiko , Kitabayashi Issay 
Published: October 2019

Abstract
Mutations in genes encoding isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in numerous cancers including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and angioimmunoblastic t-cell lymphoma (AITL). The roles of mutant IDHs in tumorigenesis remain unclear because of a lack of appropriate cancer models. Here we established a mouse AML model harboring an IDH2 mutation. IDH1/2 mutations in AML frequently occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, IDH2/R140Q, NPMc, DNMT3A/R882H and FLT3/ITD cooperatively induced AML in the mouse model. When only three out of the four mutant genes were transduced, the onset of AML was delayed in any combinations. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. Gene-expression analysis indicated that IDH2/R140Q and NPMc cooperatively activate Hoxa9/Meis1 and hypoxia pathways to maintain AML cells in vivo. These two pathways are likely to be important for the IDH2/R140Q-mediated engraftment/survival of NPMc+ cells in mice. DNMT3A/R882H further upregulated the expression levels of Meis1. Furthermore, DNMT3A/R882H promoted the maintenance of cells in an undifferentiated state. Previous studies have shown that FLT3/ITD promotes cell growth and survival. Taken together, our results suggest that multiple signaling pathways are activated in this IDH-mediated AML system.
The tumor-associated mutant IDHs catalyze the formation of an oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-ketoglutarate-dependent dioxygenases that includes epigenetic regulators (TETs and KDM4A), hypoxic signaling molecule (EGLN) and others (collagen prolyl 4-hydroxylases). Because mutant IDHs act via a mechanism that is completely different from those of previously described oncogenes, it has attracted increasing attention as a new therapeutic target. Small molecules that potently and selectively inhibit the mutant IDHs induced differentiation of cancer cells in vitro. However, it remains unclear whether the mutant IDHs are valid targets for cancer therapy in vivo. Here we report that AML harboring IDH2/R140Q can be blocked by conditional deletion of IDH2/R140Q, even after leukemia has developed. Deletion of IDH2/R140Q blocked 2-HG production and the maintenance of Csf-1r+ and c-Kit+ leukemia stem cells, resulting in survival of the AML mice. These results indicate that the IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy.

Disclosures:
No relevant conflicts of interest to declare.
Activation Of TLR4-MyD88 Pathway Impairs HSC Function During Acute Inflammation
Authors: Zhang Huajia , Rodriguez-Rodriguez Sonia , Wang Lin , Mumaw Christen , Zollman Amy , Liu Yunlong , Cardoso Angelo A. , Carlesso Nadia 
Published: October 2019

Abstract
Sepsis is a clinical syndrome due to a systemic inflammatory response to severe microbial infection. High mortality rates in sepsis (200,000/yr in the USA) are associated with the host’s failure to eradicate pathogens due to the lack of neutrophils, excessive pro-inflammatory cytokines, tissue damage and multiple organ failure. While most studies have focused on the late consequences of sepsis, little is known about the changes occurring in the bone marrow (BM) at early stages of hematopoiesis and how they affect the hematopoietic response to bacterial infection. Using an animal model of severe sepsis induced by Pseudomonas aeruginosa, which closely recapitulate lethal sepsis in burn patients, we have previously reported that HSC undergo a significant expansion in the BM associated with a block of myeloid differentiation. Furthermore, we found that expanded HSC were unable to generate the downstream progenitors (common myeloid progenitors and granulocytes/monocytes progenitors) necessary to produce neutrophils, and had reduced self-renewal. All these effects were TLR4-dependent. TLR4 is activated by bacterial LPS and signal through two major pathways: TRIF-dependent and MyD88-dependent. In this study, we show the different contribution of the TLR4-TRIF and the TLR4-MyD88-dependent pathways to the BM response to Pseudomonas aeruginosa LPS. LPS challenge conducted on TRIF-null and MyD88-null mice demonstrated that TRIF was involved in the expansion of the HSC pool, but did not play a major role in the myelosuppression, whereas MyD88 activation was required for LPS-induced myeloid suppression. Moreover, we found that the impaired engraftment at long-term observed in wild-type LPS-challenged HSC during transplantation was rescued by MyD88 loss of function. Taken together, our results indicate a distinct role of theTLR4-TRIF and -MyD88 pathways in the regulation of the primitive stem/progenitor pool during sepsis and provide insights for a better understanding of the molecular mechanisms leading to neutropenia.

Disclosures:
No relevant conflicts of interest to declare.
Mesenchymal Stem Cell-Derived Vesicles Reverse Hematopoietic Radiation Damage
Authors: Wen Sicheng , Goldberg Laura R , Dooner Mark S , Reagan John L , Quesenberry Peter J 
Published: October 2019

Abstract
Extracellular vesicles, including exosomes and microvesicles, have been shown to epigenetically modulate cell fate (Aliotta et al JEV, 2013). We have evaluated the effect of murine or human mesenchymal stem cell (MSC)-derived vesicles on marrow from irradiated mice or on the marrow cell line, FDC-P1, irradiated in vitro. B6.SJL mice were exposed to 0 or 100 cGy whole body irradiation and 7 days later marrow was harvested, lineage depleted and then cultured with or without the addition of murine MSC-derived vesicles for 48 hours. The cells were then engrafted into 200 cGy exposed C57BL/6J mice and engraftment was analyzed at 3, 12, 24 and 36 weeks post-transplant. Vesicle exposure led to a statistically significant increase in engraftment by the irradiated cells at 24 and 36 weeks with the average percent donor engraftment equal to 8.8 ± 4% and 9.6 ± 5% for the vesicle treated group compared to 3.7 ± 2% and 3 ± 2% for the non-vesicle treated groups at 24 weeks and 36 weeks, respectively (Figure 1). In a similar experiment, murine marrow was harvested 24 hours after 100 cGy whole body irradiation, and cultured with and without vesicles for 24 hours. There was a statistically significant increase in donor engraftment by the Lineage negative cells incubated with MSC-derived vesicles at 3 and 6 months (Figure 2). The increase in engraftment potential with vesicle exposure persisted in serial transplantation. These studies indicate that MSC-derived vesicles can reverse radiation damage to marrow when administered either 1 or 7 days after irradiation. The in vivo effect of murine MSC-derived vesicles on post-radiation blood cell count recovery was tested. C57BL/6 mice were exposed to 500 cGy total body irradiation. MSC-derived vesicles were then injected intravenously 6, 24 and 72 hours after irradiation. WBC recovery compared to non- irradiated mice was 17% in the non-vesicle group and 37% in the vesicle- injected group at 14 days; the values at 28 days were 61 and 89%, respectively, indicating that vesicle injection enhanced leukocyte recovery following radiation damage. Marrow cells from the non-irradiated, the irradiated and the irradiated plus vesicle-treated mice were then analyzed for gene expression. Twelve genes were elevated in the irradiated cells and the elevations were reversed by vesicle exposure while 17 genes were depressed in the irradiated cells and the depressions reversed by vesicle exposure. Studies with the marrow cell line, FDC-P1, showed that apoptosis in irradiated cells was reversed by exposure to MSC-derived vesicles and growth of irradiated cells was restored. Restoration of growth by in vitro exposure to MSC-derived vesicles is shown in Figure 3. In summary, these data indicate that MSC-derived vesicles have the capacity to ameliorate radiation-induced damage to marrow and may represent an important biologic for use in radiation exposures.

Disclosures:
No relevant conflicts of interest to declare.
A Bi-Specific T Cell Engaging Monoclonal Antibody (mAb) Derived From a TCR-Like Mab Specific For WT1/HLA-A0201 (ESK-BiTE) Shows a Potent Activity Against Human AML and Ph+ ALL In Mouse Models
Authors: Pankov Dmitry , Dao Tao , Wang Yiyang , Scott Andrew , Korontsvit Tatyana , Zakhaleva Victoria , Veomett Nicholas , Curcio Michael , Doubrovina Ekaterina , O'Reilly Richard J. , Liu Cheng , Scheinberg David A. 
Published: October 2019

Abstract
The Wilms’ tumor oncogene protein (WT1) is an intracellular, oncogenic transcription factor that is over-expressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8 T cell HLA-A0201epitope, is a validated target for T cell-based immunotherapy. We generated a high affinity, fully human IgG1 mAb specific for the RMF/HLA-A0201 complex. The mAb shows potent anti-leukemia activity both in vitro and in vivo in mouse models. Bi-specific T cell engaging antibodies (BiTE) have been used effectively to target cell surface proteins and kill cancers. We have developed a new, potent form of the ESK mAb that is a bi-specific T cell engaging antibody (BiTE) specific for tumor cells coexpressing the intracellular oncoprotein, WT-1 and HLA A0201. ESK-BiTE and an irrelevant control BiTE were constructed with ESK1 scFv or irrelevant ScFv on one arm, and anti-CD3 ScFv fragment as the other arm. The BiTE constructs were expressed in CHO cells. Both the ESK-BiTE and the control BiTE bind human CD3 T cells. The ESK-BiTE selectively bind to leukemia cells that express both WT1 and HLA-A0201. The ESK-BiTE activated human resting T cells and EBV-specific T cells retargeting potent cytotoxicity against WT-1+ HLA A0201+ human leukemia cells in vitro. In an NSG mouse xenograft model, injection of ESK-BiTE (20 ug/ml) twice a week, following I.V. administration of 2x107 human EBV-specific T cells, once a week, significantly inhibited the growth of a previously established disseminated HLA A0201+, WT-1+ human Ph+ ALL, BV173 expressing luciferase, as measured by bioluminescence imaging. In a second NSG mouse model mice injected I.V. with an aggressive human AML, SET-2, on day 0, were treated on day 4 with ESK-Bite for 6 days consecutively at 20 ug/day together with EBV-specific T cells given twice a week. In this setting, the ESK-BiTE and T cells resulted in undetectable leukemic growth for 14 days post-leukemia inoculation, with a minimal tumor burden detected by day 18, while all control groups showed massive increases in leukemia burden by day 14. Mice bearing SET-2 leukemia, that received ESK-BiTE and T cells also showed longer survival and delayed limb paralysis. As expected, the human T cells, which were EBV-specific, did not induce signs of GVHD in mice. Our data provide evidence that ESK-BiTE is a potent and specific therapeutic agent against aggressive human leukemias expressing WT1 and HLA-A0201. This is the first study showing efficacy of a TCR-like BiTE antibody targeting an intracellular tumor antigen expressed at low density.
Supported by the Leukemia and Lymphoma Society, NIH R01CA55349 and P01 23766.

Disclosures:
No relevant conflicts of interest to declare.
Loss Of BTG1 Function Promotes ETV6-RUNX1-Mediated Leukemic Transformation
Authors: Tijchon Esther J.H. , van Emst Liesbeth , Schenau Dorette van Ingen , van der Meer Laurens T. , Williams Owen , Hoogerbrugge Peter M. , Scheijen Blanca , van Leeuwen Frank N. 
Published: October 2019

Abstract

Background
Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy and is characterized by the acquisition of recurrent genetic aberrations, which include chromosomal translocations, submicroscopic deletions and point mutations. The t(12;21) ETV6-RUNX1 translocation is present in about 25% of all pediatric B-cell precursor ALL (BCP-ALL) cases and represents an early genetic event, which can already be detected in utero. However, ETV6-RUNX1 expression is insufficient to induce leukemia and requires other cooperating genetic lesions for BCP-ALL to develop.

Results
Using extensive genomic profiling of pediatric BCP-ALL we and others have demonstrated that several recurrent gene deletions can be found in ETV6-RUNX1-positive leukemia. These include focal deletions affecting the B-cell translocation gene 1 (BTG1), which is a member of the BTG/Tob family of anti-proliferation genes. BTG1 deletions are present in 9% of all BCP-ALL cases, but appear to be specifically enriched (20%) in ETV6-RUNX1-positive leukemia. BTG1 protein displays no intrinsic enzymatic activity but may act by recruiting effector molecules like protein arginine methyltransferase 1 (PRMT1) to specific transcription factors, including RUNX1 and ATF4.
To investigate whether loss of BTG1 function cooperates with ETV6-RUNX1 in leukemic transformation we developed an in vitro transformation assay. To this end, primitive fetal liver progenitors (FLPs) were purified as c-Kit+ Ter119- cells from fetal livers of embryonic day 13.5 (E13.5) of C57Bl6/J wild-type and Btg1-/- mice. After transduction with control pMSCV-IRES-GFP (pMIG) and pMIG-ETV6-RUNX1 retrovirus, cells were serially replated in methylcellulose or liquid culture in the presence of cytokines SCF, FLT3L and IL-7. We observed a proliferative growth advantage of ETV6-RUNX1 over control virus, and in BTG1-deficient FLPs as compared to wild-type FLPs. Notably, the proliferative advantage of BTG1-deficient FLPs was even further enhanced by co-expression of ETV6-RUNX1. By immunoprecipitation from FLPs, we could demonstrate that endogenous PRMT1 binds to ETV6-RUNX1, and this interaction is lost in BTG1-deficient FLPs, arguing that BTG1 is required for the interaction between PRMT1 and ETV6-RUNX1.
In summary, our data indicate that loss of BTG1 function promotes leukemic transformation induced by oncogenic fusion protein ETV6-RUNX1, which implies that BTG1 gene deletions can act as a cooperating event in ETV6-RUNX1-driven leukemogenesis.

Disclosures:
No relevant conflicts of interest to declare.
Oral Arsenic Trioxide - Based Maintenance Without Hematopoietic Stem Cell Transplantation At Second Remission In Acute Promyelocytic Leukemia - A Prospective Follow-Up Study Of 65 Patients
Authors: Gill Harinder , Au Wing-Yan , Kwong Yok Lam 
Published: October 2019

Abstract
Approximately 20 percent of patients with acute promyelocytic leukemia (APL) relapse after initial remission. More than 80 percent of these patients achieve a second complete remission (CR2). Autologous hematopoietic stem cell transplantation (HSCT) at CR2 is a widely accepted post-remission strategy. However, only a few prospective trials have examined the optimal post-remission therapy for relapsed APL.
In this study, we prospectively evaluated the outcome of 65 patients with APL in their first relapse (R1) treated with oral arsenic trioxide (As2O3) and chemotherapy-based re-induction followed by oral arsenic trioxide-based maintenance. Sixty-five patients (men=37; women=28) at a median age of 39 (3-76) years were treated with oral As2O3, all-trans retinoic acid (ATRA), and ascorbic acid combined with idarubicin (6mg/m2/day for 5 days) for re-induction. On achieving CR2, 2 cycles of idarubicin consolidation (6mg/m2/day for 2 days each cycle) were given. That was followed by oral As2O3-based maintenance (2 weeks every 2 months) for 2 years.
The median duration of follow-up was 90 (21–378) months. All evaluable patients achieved CR2. Twenty-one patients (32.8%) subsequently had a second relapse (R2). On multivariate analysis, male gender was associated with an increased risk of second relapse (P=0.01). The age, white blood cell (WBC) count, platelet count, peak WBC during reinduction and the occurrence of differentiation syndrome did not impact on the risk of second relapse. The median overall survival (OS) was not reached. The 5-year and 10-year OS were 82.2% and 74.4% respectively. Significantly inferior OS was associated with male gender (P=0.02), prior oral As2O3-based maintenance at CR1 (P=0.01) and central nervous system (CNS) involvement at relapse (P<0.001). The median leukemia-free survival (LFS) after CR2 was not reached. The 2-year and 5-year LFS were 72.4% and 64.1% respectively. Male gender and relapse from prior oral As2O3-based maintenance at CR1 were associated with worse LFS (P=0.01 and P=0.04 respectively).
In conclusion, our study showed that durable remissions and long-term survival were achieved with oral As2O3-based maintenance without the need of autologous HSCT at CR2.

Disclosures:
No relevant conflicts of interest to declare.
Decitabine Is More Cost Effective Than Standard Conventional Induction Therapy In Elderly Acute Myeloid Leukemia Patients
Authors: Batty Nicolas , Wiles Samuel , Kabalan Mathew , Sharma Rohit , Shatzel Joseph , Pang Jonathan , Yi David , Alatovic Iris , Saif Sana , Narasimha Deepika , LaPenna Joseph , Troitino Anthony , Attwood Kristopher , Yin Yong , Wetzler Meir 
Published: October 2019

Abstract

Introduction
Decitabine (Dec) is not approved in the United States (US) for acute myeloid leukemia (AML) because it did not improve overall survival compared with standard conventional induction treatment. We asked what would be the cost effectiveness of Dec versus conventional induction therapy in AML patients (pts) older than 60 years of age.

Methods
The standard conventional induction including cytarabine, and daunorubicin, (AD) (N Engl J Med. 2009 361:1235-48) was compared with Dec (Haematologica. 2012 97:393-401) using a semi-Markov model compiling survival and cost data. Survival probabilities were retrieved from the literature. Data accounted for re-induction therapy with IDA-FLAG (idarubicin, fludarabine, cytarabine and granulocyte colony-stimulating factor) and consolidation therapy with high-dose cytarabine (HiDAC) but not for stem cell transplantation. The assumption-based model considered a maximum of 4 cycles of HiDAC and continuing Dec until loss of benefit. Drug costs were derived from the 2012 US market. Hospital costs accrued were evaluated in a diagnosis-related group (DRG) system. Drug dosage was estimated based on a body surface area of 1.85 m2. The quality of life (QoL) was assumed as 1 for healthy individuals; 0 for death; 0.524 for active disease; 0.91 for AML in remission on AD; 0.71 and 0.524 for AML being actively treated with Dec or AD, and 0.81 for AML in remission treated with Dec or HIDAC. QoL data were based on the literature except for pts on consolidation therapy. The latter was the mean of QoL of AML in remission and AML actively being treated.

Results
Assuming 1,000 pts for each treatment arm in a semi-Markov model over 1 year time horizon, the quality-adjusted life year (QALY) for AD vs. Dec was 0.1754 and 0.5982. The percentage survival for AD and Dec was 45.2% and 50.5%. Their costs were $127,867 and $55,777. The incremental cost-effectiveness ratio (ICER) was -$72,090/0.4228 = -$170,506/year. By sensitivity analysis, Dec was superior to AD to all parameters (Table 1).

Conclusion
Dec is a more cost-effective therapy for pts older than 60 years of age than conventional induction therapy. Given the economic pressures in the US Health System, one should consider approving Dec for newly diagnosed AML pts older than 60 years of age.

Disclosures:
No relevant conflicts of interest to declare.