bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeting PKCtheta Inhibits Alloreactivity and Graft-Versus-Host Disease While Preserving Graft-Versus-Leukemia and Anti-Infection Activities.
Authors: Yu Xue-Zhong , Valenzuela Javier , Iclozan Cristina , Hossain Mohammad , Hopewell Emily , Bronk Crystina , Wang Junmei , Celis Esteban , Engelman Robert , Waller Edmund K. , Blazar Bruce R. , Beg Amer 
Published: October 2019

Abstract
Abstract 2458
Poster Board II-435

Introduction:
When used as therapy for hematopoietic malignances, allogeneic hematopoietic stem cell transplantation (HCT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host-disease (GVHD) is a potentially lethal complication of allogeneic BMT. Thus, inhibition of GVHD, while preserving GVL and protective responses against infectious agents, can enhance the therapeutic potential of BMT. GVHD is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues. Protein kinase C theta isoform (PKCθ) is crucial in TCR signaling and regulates the nature of lymphocyte-specific effector responses. The aim of this study was to investigate the significance of PKCθ in donor T-cell-mediated GVHD, anti-leukemia, and antiviral infection.

Methods:
Our results were validated using clinically relevant murine bone marrow transplantation (BMT) models. GVHD was measured by recipient survival and clinical signs such as body weight loss. Leukemia/lymphoma was induced by intravenous injection of a luciferase expressing A20 clone (a B lymphoma cell line derived from BALB/c mice). Tumor invasion was quantitated using the Xenogen IVIS-200 bioluminescence imaging system. Murine cytomegalovirus (MCMV) infection was induced by intraperitoneally injecting MCMV in the recipients after GVHD was established.

Results:
Using 3 distinct mouse models of allogeneic BMT, we found that T cells lacking PKCθ are unable to undergo robust expansion and cause damage to recipient hematopoietic or epithelial tissues, demonstrating that an essential requirement for PKCθ in alloreactivity and GVHD development. In contrast, PKCθ-/- T cells retain the ability to respond to virus infection post-BMT. Mechanistically, absence of PKCθ raises the threshold for T cell activation that selectively impacts alloresponses, but T cell responses triggered by infection or following administration of antigen plus microbial adjuvant are relatively well preserved in the absence of PKCθ Furthermore, we evaluated the role of PKCθ in GVHD and GVL responses, and found that PKCθ-/- T cells have at least a 10-fold reduction in the ability to induce GVHD but only a ∼2.5-fold reduction in GVL compared to WT T cells. Thus, absence of PKCθ impacts GVHD responses more significantly than GVL responses.

Conclusion:
These findings validate PKCθ as a potentially unique therapeutic target that is required for GVHD induction but not for GVL or protective responses to infectious agents in allogeneic BMT.

Disclosures:
No relevant conflicts of interest to declare.
Niche Induced Myelodysplasia and Secondary Hematopoietic Neoplasia Caused by Deletion of Dicer1 in Osteoprogenitor Cells.
Authors: Raaijmakers Marc HGP , Mukherjee Siddhartha , Guo Shangqin , Kobayashi Tatsuya , Schoonmaker Jesse , Aung Zinmar , Ebert Benjamin L. , Al-Shahrour Fatima , Hasserjian Robert P , Vallet Sonia , Scadden Edward , Lin Charles P , Scadden David T. 
Published: October 2019

Abstract
Abstract 247

Introduction:
Mesenchymal cells are a part of virtually every tissue in metazoans and are thought to participate in organ formation and homeostasis. In the hematopoietic system, mesenchymal cells of the osteoblast lineage have revealed their role as regulators of normal stem cell and hematopoietic physiology. Whether these cells, which have been relegated a relatively non-descript role of ‘stroma', participate in processes that result in disease is relatively understudied.

Methods:
To explore this, we conditionally deleted Dicer1, the endonuclease essential for miRNA biogenesis, from osteoprogenitor cells by intercrossing transgenic mice expressing a GFP-Cre recombinase under the transcriptional control of the osteoblastic lineage specific osterix promoter to mice containing conditional (floxed) Dicer1 alleles.

Results:
Deletion of Dicer1 from osteoprogenitor cells resulted in markedly disordered hematopoiesis, affecting multiple lineages and recapitulating key features of human myelodysplastic syndrome (MDS). These features included ineffective hematopoiesis with cytopenia, multilineage dysplasia, increased proliferation and intramedullary apoptosis of primitive hematopoietic cells, decreased B-cell progenitors, increased bone marrow vascularity and the propensity to develop hematopoietic neoplasms (myeloid sarcoma and acute monocytic leukemia-like disease). Comparative genomic hybridization of tumor and germline tissues revealed multiple genetic aberrancies in myeloid sarcomas induced by the Dicer1 deleted environment. The hematopoietic abnormalities were entirely microenvironment dependent with intact Dicer1 in hematopoietic cells. Transplantation of wild-type hematopoietic cells into the mutant environment recapitulated these abnormalities whereas, conversely, transplantation of hematopoietic cells from mutant mice into a wild-type environment resulted in complete normalization of hematopoiesis. In addition, hematopoietic abnormalities were not observed when Dicer1 was deleted from mature osteoblasts indicating a central role of osteoprogenitor cells in the regulation of hematopoiesis. Finally, gene expression profiling and cytokine arrays from osteoprogenitor cells identified candidate molecular effectors of the observed hematopoietic abnormalities.

Conclusions:
The data demonstrate that a distinctive, differentiation stage specific, stromal subset of osteolineage cells can induce a highly dysfunctional hematopoietic system, recapitulating key characteristics of human myelodysplastic syndrome. Further, they reveal that primary changes in a tissue microenvironment can initiate neoplastic disease.

Disclosures:
Scadden: Fate Therapeutics: Consultancy.
Adjudicating Bleeding Outcomes in a Large Thromboprophylaxis Trial in Critical Illness.
Authors: Arnold Donald M. , Rabbat Christian , Lauzier Francois , Zytaruk Nicole , Heels-Ansdell Diane , Cook Deborah 
Published: October 2019

Abstract
Abstract 2471
Poster Board II-448

Background:
Adjudication in clinical trials can confirm or refute eligibility, describe cointerventions, judge appropriateness of care, or assess the severity of morbidity outcomes.

Objective:
To refine the adjudication process, calibrate 4 adjudicators, and measure agreement on bleeding severity in an international trial of heparin thromboprophylaxis (PROTECT).

Methods:
Independently and blinded to each others' ratings and study drug, 4 adjudicators used web-based methods to examine 40 charts of patients assessed by local research coordinators to have either major (20 patients) or minor (20 patients) bleeding. We discussed reasons for disagreement after the first 20 charts to remediate and recalibrate. We calculated crude agreement, chance-corrected agreement (kappa), and chance-independent agreement (phi).

Results:
For 45 events in 40 patients, pair-wise crude agreement ranged from 86.7-93.3% (average 82.2%). Overall kappa was 0.81. Phi (which can only analyze pair-wise values) ranged from 0.75-0.87. We resolved all disagreements. During adjudication discussions, we 1) addressed methodological issues (e.g., handling recurrent bleeds), 2) added a category (e.g., no bleed), 3) expanded a category (e.g., a major bleed did not have to be overt if other criteria were fulfilled), and 4) divorced procedure-grounded definitions (such as the need for transfusion or therapeutic interventions) from bleeding severity criteria (e.g., the patient could still be classified as having no bleed or a minor bleed if 2 units of PRBCs were transfused for anemia).

Conclusions:
After independent quadruplicate review of 45 bleeding events, we documented satisfactory agreement for bleeding outcomes, refined the adjudication process, and calibrated adjudicators for the remainder of the trial. Henceforth, charts will be randomly allocated to pairs of adjudicators for blinded review.

Disclosures:
No relevant conflicts of interest to declare.
Cost-Effectiveness of Filgrastim and Pegfilgrastim as Primary Prophylaxis against Febrile Neutropenia in Lymphoma Patients Receiving R-CHOP Chemotherapy.
Authors: Lathia Nina , Isogai Pierre K , Walker Scott , Cheung Matthew C , Krahn Murray , DeAngelis Carlo , Mittmann Nicole 
Published: October 2019

Abstract
Abstract 2475
Poster Board II-452

Introduction:
Non-Hodgkin Lymphoma (NHL) is the fifth most common type of malignancy in Canada. The most common subtype of NHL is diffuse large B-cell lymphoma (DLBCL). Initial standard treatment for DLBCL includes combination immuno-chemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). This regimen is typically administered every 21 days for a total of 6 cycles. Febrile neutropenia (FN) is a serious toxicity of lymphoma chemotherapy and patients with this condition must be treated aggressively as it could lead to complications such as prolonged hospitalization and death. Granulocyte-colony stimulating factors such as filgrastim and pegfilgrastim are efficacious in preventing FN, yet their cost-effectiveness has not been evaluated in the publicly funded Canadian healthcare system.

Methods:
A Markov model was constructed to evaluate the cost-effectiveness (cost-utility) of filgrastim and pegfilgrastim as primary prophylaxis versus no primary prophylaxis against FN in DLBCL patients receiving induction chemotherapy. Health states included in the model were hospitalization for FN, and receiving chemotherapy with no FN. It was assumed that patients in the no primary prophylaxis arm of the model who experienced a FN episode would receive secondary prophylaxis with filgrastim for all subsequent chemotherapy cycles. The time horizon of the model was 18 weeks, the time period over which the six cycles of chemotherapy are administered. The analysis was conducted from the hospital perspective. Costs are reported in 2009 Canadian dollars and outcomes in quality-adjusted life years (QALY). One-way sensitivity analyses were done on model parameters. A probabilistic sensitivity analysis was done to evaluate overall uncertainty in the model.

Results:
In the base case analysis costs associated with the no primary prophylaxis, filgrastim and pegfilgrastim interventions were $6044, $9450 and $15899, respectively. Quality-adjusted life years associated with the three interventions were 0.198, 0.200, and 0.202 respectively (over the 18-week model time horizon). The incremental cost-effectiveness ratio (ICER) of filgrastim compared to no primary prophylaxis was estimated to be $1.7 million (M)/QALY [95% confidence interval: -14M/QALY (dominated) to $15M/QALY]; for pegfilgrastim compared to filgrastim the ICER was estimated to be $4.4M/QALY [95% confidence interval: -25M/QALY (dominated) to $22.7M/QALY]. All one-way sensitivity analyses yielded ICERs of greater than $1M/QALY.

Conclusions:
The ICERs for filgrastim and pegfilgrastim when used as primary prophylaxis against FN in DLBCL patients receiving induction chemotherapy are well above the usually accepted cost-effectiveness threshold of $50,000/QALY.

Disclosures:
Mittmann: Amgen Canada: Unrestriced educational grant.
PI3 Kinase, Phospholipase C (PLC)-γ, and RasGRPs Act Cooperatively to Activate the Ras-Extracellular-Related Kinase (ERK) Pathway in Response to Cytokines in Normal and Kras Mutant Myeloid Cells.
Authors: Diaz-Flores Ernesto , Goldschmidt Hana Leah , Depeille Philippe , Ng Victor , Roose Jeroen , Shokat Kevan , Braun Benjamin S. , Shannon Kevin 
Published: October 2019

Abstract
Abstract 2512
Poster Board II-489
Introduction: KRAS is the most common gain-of-function mutation in human cancer and occur in some myeloid malignancies. Despite extensive data implicating aberrant responsiveness to cytokines in the pathogenesis of myeloid leukemias with hyperactive Ras, the underlying mechanisms are largely unknown. We investigated the biochemical basis of hypersensitivity to granulocyte macrophage colony stimulating factor (GM-CSF), IL-3, and stem cell factor (SCF) in primary cells from Mx1-Cre, KrasG12D mice. Methods: We utilized conventional biochemical techniques, phospho-specific flow cytometry, and small molecule inhibitors to interrogate signaling downstream of the activated GM-CSF and c-Kit receptors. Results: We found that endogenous KrasG12D expression deregulates multiple effectors in primary myeloid lineage cells. Although Ras-GTP levels are markedly elevated in bone marrow cells from KrasG12D mice, these cells only fully activate the Raf/MEK/ERK cascade upon cytokine stimulation. We used a “chemical genetic” strategy to explore the biochemical mechanism linking the activated GM-CSF receptor to oncogenic K-RasG12D and ERK. Surprisingly, inhibitors of type I PI3 kinases (PI-90, PI-103) as well as a p110 γ/α isoform inhibitor (AS605240) interfered with GM-CSF and SCF induced Ras/MAPK activation in wild-type and KrasG12D myeloid cells. These effects of PI3 kinase are mediated through activation of PLC-γ and the RasGRP guanine nucleotide exchange factors. Functional studies confirmed that inhibiting PI3 kinase or PLC-γ blocked KrasG12D-mediated myeloid progenitor colony growth. This PI3 kinase/PLC-γ module also functions downstream of the activated c-kit receptor in lineage- cKit+ Sca1+ (LSK) cells in SCF-induced ERK activation. Conclusions: PI3 kinase, PLC-γ, and RasGRPs function upstream of normal and oncogenic K-Ras to efficiently activate the Raf/MEK/ERK cascade in primary hematopoietic cells. These data infer that PI3 kinase is both an important upstream regulator as well as a major downstream effector of Ras-GTP. Significance: Targeting PI3 kinase in hematologic cancers characterized by hyperactive Ras may have unanticipated biochemical and functional consequences in malignant and normal hematopoietic cells.

Disclosures:
No relevant conflicts of interest to declare.
NIPA Phosphorylation and Inactivation at G2/M Is Mediated by ERK2.
Authors: Zech Michael , Illert Anna Lena , Albers Corinna , Bassermann Florian , Peschel Christian , Duyster Justus 
Published: October 2019

Abstract
Abstract 2513
Poster Board II-490
Regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome. We previously reported cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, contributing to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3-ligase. The SCF-NIPA complex targets nuclear cyclin B1 for ubiquitination in interphase while phosphorylation of NIPA in late G2 phase and mitosis inactivates the complex to allow for accumulation of cyclin B1, a critical event for proper G2/M transition. Thus, SCF-NIPA executes an important G2/M checkpoint control. We recently specified three serine residues Ser 354, 359 and 395 implicated in NIPA phosphorylation at G2/M. These data suggest a sequential NIPA phosphorylation, where initial Ser 354 and 359 phosphorylation is most crucial for SCF-NIPA inactivation by dissociating the SCF-NIPA complex. Here we aimed to find the kinase responsible in this initial most important phosphorylation step. Using in vitro kinase assays we identified both ERK1 and ERK2 to phosphorylate NIPA with high efficiency. Mutation of either Ser 354 or Ser 359 abolished ERK-dependent NIPA phosphorylation. Inhibition of ERK1/2 activity in cell lines by specific inhibitors resulted in decreased NIPA phosphorylation at G2/M. To differentiate between phosphorylation by ERK1 and ERK2, we combined cell cycle analysis with stable expression of microRNA's targeting both isoforms. To this end NIH/3T3 cells were retrovirally transduced with microRNAs targeting ERK1 and 2 and cell cycle progression was analysed by BRDU/PI labeling. Using this approach, we are able to show that ERK2 but not ERK1 mediates NIPA phosphorylation at G2/M. Furthermore, ERK2 silencing leads to a distinct phenotype in cell cycle progression with a delay of ERK2 knockdown cells at the G2/M transition. Thus, our data indicate, that the recently described divergent functions of ERK1 and ERK2 in cell cycle regulation could be in part due to the differential ability of these kinases to phosphorylate and inactivate NIPA at G2/M. Since checkpoint proteins such as NIPA are constitutively inactivated in tumor cells ERK2 might represent an interesting target to reconstitute important cell cycle checkpoint controls in malignant cells.

Disclosures:
No relevant conflicts of interest to declare.
A Sensitized Screen Uncovers a Novel Platelet Phenotype in a Loss-of-Function Jak2 Allele.
Authors: Anderson Nicole , Javed Mojib Javadi , Bailey Monica L , Huang Kai , Berberovic Zorana , Flenniken Ann , Wang Chen , Minden Mark D. , Paulson Robert , McNagny Kelly M. , Stanford William L. , Barber Dwayne L 
Published: October 2019

Abstract
Abstract 2516
Poster Board II-493
Our group has undertaken a concerted effort to develop novel heritable mouse strains via ethylnitrosourea (ENU)-based random mutagenesis. We have complemented this strategy to perform a dominant sensitized screen to identify mouse mutants with altered recovery following treatment with 5-fluoruracil (5-FU). We isolated a heritable line with elevated platelets as well as an overshoot in platelet numbers when exposed to 5-FU. Affected mice also displayed megakaryocytosis and centrilobular hepatic lipidosis.
The mutation was mapped to a 8.7Mb region on mouse Chromosome 19, which contained the candidate gene Jak2. The entire coding region of Jak2 was sequenced and a mutation in exon 19 was found. The causative mutation was located at nucleotide 2740 and leads to an A to T transversion in exon 19 of JAK2 and causes a substitution of a Lys to a premature stop codon at amino acid 914. The resulting protein product terminates within the JH1 kinase domain of JAK2.
JAK2 914 is distinct from the JAK2 null allele characterized by the Ihle and Pfeffer labs. For example, JAK2−/− embryos die at E12.5, whereas JAK2914/914 embryos survive until E13.5, suggesting that the truncated protein product may serve as a scaffold. Differences were also observed in clonogenic potential of both strains. For example, JAK2+/− mice had increased CFU-C whereas JAK2914/+ were normal. Splenic BFU-E and CFU-C were increased in JAK2+/− mice while both cell types were decreased in JAK2914/+. In contrast, bone marrow CFU-E were increased in both strains of mice and splenic CFU-E were normal in JAK2+/− and JAK2914/+. In addition, both mouse strains display elevated platelets when animals are housed under resting conditions.
The hepatic phenotype is phenocopied by Leptin Receptor null mice that are hyperphagic, poorly cold-adapted, sterile and have enhanced fat conversion resulting in lipidosis.
In conclusion, a sensitized screen has shown that JAK2 regulates the response to the chemotherapeutic drug, 5-fluorouracil. These findings have significance to potential myelosuppressive effects of the clinical use of 5-FU in colorectal and pancreatic cancer.

Disclosures:
No relevant conflicts of interest to declare.
Exo1 Independent DNA Mismatch Repair Pathway in Hematopoiesis.
Authors: Desai Amar , Qing Yulan , Liu Min , Kenyon Jonathan , Nwafor Uchenna , Gerson Stanton L. 
Published: October 2019

Abstract
Abstract 2526
Poster Board II-503
The DNA mismatch repair pathway (MMR) is a fundamental process in cells that functions to correct mispaired bases and insertion/deletion loops caused by errors in replication. Failure in MMR can lead to the accumulation of mutations and carcinogenesis, notably hereditary nonpolyposis colorectal cancer (HNPCC). In hematopoiesis, loss of MMR results in methylating agent resistance and an HSC repopulation defect. Our research focused on the significance of Exonuclease 1, the enzyme responsible for excising mispaired bases in MMR. Interestingly Exo1−/− mice display a much milder pathogenic phenotype when compared to the MMR deficient MSH2−/− mice; characterized by lower mutation rates, lower levels of microsatellite instability, and in humans no association with HNPCC. These findings led us to hypothesize that the limited phenotype observed in Exo1−/− mice is due to (a) complementation through another exonuclease occurs which restores the proficiency of the MMR pathway or (b) an alternative pathway exists which allows for exonuclease independent repair.
To test this hypothesis we derived primary MEFs from WT, Exo1−/− and MSH2−/− mice and performed temozolomide sensitivity assays. We observed that Exo1−/− MEFs are similarly sensitive as WT cells when treated with the drug, confirming proficient MMR, while MSH2−/− MEFs display a decreased sensitivity. Additionally, comet assays with the same cell populations show a persistence of DNA single strand breaks in the Exo1−/− and WT cells 24 hours post temozolomide treatment, consistent with proficient MMR activity, while MSH2−/− MEFs show no such damage. To confirm functional MMR in hematopoiesis of Exo1−/− mice we conducted a competitive repopulation study. We found that Exo1−/− and WT marrow engraft at similar levels 8 and 16 weeks post transplantation. We subsequently treated these mice with 80 mg/kg temozolomide to determine if Exo1−/− marrow progenitors would confer a competitive survival advantage post treatment. Consistent with our hypothesis we found that the ratio of marrow cells remained approximately 50:50 (Exo1:WT) in contrast to data obtained from MSH2−/− mice in which the MSH2−/− cells display a strong 95:5 (MSH2:WT) advantage post drug treatment. This strongly suggests functional MMR in the Exo1 deficient mice. We next coimmunoprecipitated the MMR complex by pulling down Mlh1 in Exo1−/− and WT MEFs. Exo1, a key exonuclease in mismatch repair, does not modulate hematopoietic function as do the other MMR proteins and appears to be complemented by an as yet undefined exonuclease.

Disclosures:
No relevant conflicts of interest to declare.