bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kras Is Critical for CD8 T Cell Antiviral Function
Authors: Chen Yuhong , Zheng Yongwei , You Xiaona , Xin Gang , Yu Mei , Fu Guoping , Zhou Fen , Zhu Wen , Cui Weiguo , Zhang Jing , Wen Renren , Wang Demin 
Published: October 2019

Abstract
Small GTPases regulate multiple signaling pathways and individual Ras member can have distinct biological function. To overcome embryonic lethality of Kras-deficient mice, we generated and examined mice with hematopoietic- and T cell-specific deletion of Kras. In VavCreKrasfl/fl mice with hematopoietic deletion of Kras, thymic T-cell development was normal based on the presence of normal populations of total, CD4- CD8-, CD4+ CD8+, CD4+ and CD8+ thymocytes. The populations of splenic CD4+ and CD8+ T cells were also comparable between VavCreKrasfl/fl relative to control mice. In addition, no consistent defects in the 3 H-thymidine incorporation rate of Kras-deficient splenic CD4+ or CD8+ T cells in response to anti-CD3 or anti-CD3 plus IL-2 was detected. Nonetheless, we studied the effect of Kras deficiency on CD8 T-cell immune response to acute infection of the Armstrong strain of lymphocytic choriomeningitis virus (LCMV). Sub-lethally irradiated Rag1-deficient mice transplanted with bone marrow (BM) cells from VavCreKrasfl/fl or control mice were subjected to LCMV infection. Infection-induced expansion of CD8 T cells and generation of LCMV epitope gp33-specific CD8 T cells were markedly reduced in the recipients that received the BM from VavCreKrasfl/fl relative to control mice. Following in vitro stimulation with the LCMV epitope gp33, the induction of IFNg-expressing CD8 T cells from LCMV-infected recipients that received the BM from VavCreKrasfl/fl mice was dramatically reduced. Further, BM chimeric mice with CD8 T cell-specific deficiency generated by transplantation of lethally irradiated CD8 T cell-depleted CD45.1 congenic mice with a mixture of BM cells from VavCreKrasfl/fl mice and BM cells from CD8 T cell-deficient mice exhibited an impaired CD8 T-cell immune response to LCMV infection. Lastly, we examined the role of Kras in TCR signaling. The level of total TCR-activated Ras (Ras-GTP) was markedly reduced in Kras-deficient relative to control CD8 T cells. Importantly, TCR-induced ERK1/2 activation was impaired in Kras-deficient relative to control CD8 T cells. Consistently, TCR-induced activation of Raf-1 and MEK1/2 was markedly reduced in mutant CD8 T cells. However, TCR-induced JNK and p38 activation as well as Ca2+ flux were normal in Kras-deficient CD8 T cells. Of note, TCR-induced activation of Ca2+ flux, JNK and p38 as well as ERK1/2, MEK1/2 and Raf1 was normal in Kras-deficient relative to control CD4 cells. Taken together, these data demonstrate that Kras is dispensable for T cell development or TCR-induced proliferation of CD4 or CD8 T cells in vitro, but regulates TCR-induced activation of the Raf-1/MEK/ERK pathway in CD8 but not CD4 T cells and intrinsically controls CD8 T-cell immune response to viral infection.

Disclosures
No relevant conflicts of interest to declare.
SFRP4-Mediated Inhibition of Canonical Wnt-Signaling in DLBCL
Authors: Koch Raphael , Chapuy Bjoern 
Published: October 2019

Abstract
Introduction: Wnt-signaling enhances proliferation in a variety of malignancies including diffuse large B-cell lymphoma (DLBCL) and exosomes, small secreted microvesicles, are known to carry active wnt-molecules to mediate wnt-signaling. We found consistent suppression of SFRP4, an inhibitor of canonical wnt-signaling in DLBCL, elucidated it's mechanism of action and addressed it´s impact on regulating proliferation of diffuse large B-cell lymphomas. We conducted a screen of chemical wnt-inhibitors and assessed wnt-addiction in DLBCL cell lines.
Material & Methods: The Cancer Cell Line Encyclopedia (CCLE) was used to assess the transcript abundace of SFRP4 in 18 DLBCL cell lines. SFRP4-expression was confirmed using western blot and immunofluorescence in representative DLBCL cell lines. Purified exosomes of DLBCL cell lines, recombinant human SFRP4 and SFRP4-shRNA were used to address the biological mechanism of SFRP4 action. 5-Azacytidine was used to restore SFRP4-expression. Furthermore shRNA mediated knockdown of beta-catenin and pharmacological inhibition of canonical wnt-signaling using XAV-939, IWP-2, C59 and UM206 were used to address wnt-addiction in DLBCL cell lines.
Results: We found SFRP4 to be significantly suppressed in a variety of malignancies, including DLBCL, with promotor methylation as a known mechanism of suppression. SFRP4 was able to suppress exosomal mediated canonical wnt-signaling. SFRP4 inhibited proliferation in DLBCL cell lines and enhanced cytotoxicity of doxorubicin. Treatment with 5-Azacytidine restored expression of SFRP4, induced decline of beta-catenin and suppressed cell proliferation. Knock down of beta-catenin and pharmacological perturbation of canonical wnt-signaling identified wnt-addiction in beta-catenin expressing cell lines in the absence of a MYC translocation or amplification.
Conclusions: SFRP4 suppresses proliferation of DLBCL cell lines by antagonizing exosomal mediated canonical wnt-signaling. Pharmacological inhibition of canonical wnt-signaling significantly decreased proliferation of DLBCL cell lines in the absence of genomic alterations involving MYC.

Disclosures
No relevant conflicts of interest to declare.
Pcbp1 and Ncoa4 Regulate the Flux of Iron through Ferritin in Developing Erythroid Cells
Authors: Ryu Moon-Suhn , Wohlschlegel James , Philpott Caroline C. 
Published: October 2019

Abstract
In general human cell models, Poly(rC)-binding proteins (Pcbp) mediate the delivery and integration of iron into the iron storage protein. Conversely, nuclear receptor coactivator 4 (Ncoa4) initiates the liberation of ferritin iron by localizing the protein to autophagosomes. The erythron is the major consumer of body iron. Whether these regulators of ferritin iron are functional in developing red cells remains unclear, and thus was investigated. Both Pcbp1 and Ncoa4 were readily detectable in immunoprecipitates of erythroid ferritin. The peak of Pcbp1-binding to ferritin preceded the stage of maximal Ncoa4-ferritin interaction, which coincided with the induction of β-globin, during erythroid maturation. Both Pcbp1 and Ncoa4 deficiency resulted in lower iron incorporation into heme and hemoglobin by differentiation, without compromised Alas2 gene expression. In Pcbp1-deficent cells, less iron loading into ferritin accompanied the erythropoietic defects. Ncoa4-lacking cells exhibited a massive accumulation of iron in ferritin. No apparent interaction between Pcbp2 and ferritin was detectable in erythroid cells. However, Pcbp2 depletion affected ferritin mineralization in a manner opposite to Pcbp1 deficiency. This was associated with an increase in Pcbp1-ferritin complex formation detected by coimmunoprecipitation. Supplemental iron led to decreases in the amount of both Pcbp1 and Ncoa4 interacting with ferritin. The iron-responsiveness of Ncoa4's ferritin-binding activity was primarily due to post-transcriptional changes in protein abundance. The down-regulation of Ncoa4 protein by high iron required intact lysosome activity, and the expression of an E3 ubiquitin protein ligase, Herc2. Collectively, the present studies provide evidence of the metallochaperone activity of Pcbp1 for ferritin mineralization, and the involvement of Ncoa4 in the mobilization process of stored iron in erythroid cells. Furthermore, the impaired hemoglobinization by insufficiency of these genes identifies ferritin as an essential source of iron for heme production during terminal erythroid differentiation.

Disclosures
No relevant conflicts of interest to declare.
Fam210b Is Required for Optimal Cellular and Mitochondrial Iron Uptake during Erythroid Differentiation
Authors: Yien Yvette Y , Chen Caiyong , Shi Jiahai , Li Liangtao , Bauer Daniel E. , Huston Nicholas , Kingsley Paul D. , Ablain Julien , Zon Leonard I , Palis James , Orkin Stuart H. , Kaplan Jerry , Phillips John D , Lodish Harvey F. , Paw Barry 
Published: October 2019

Abstract
Red cells synthesize large quantities of heme during terminal differentiation. Central to erythropoiesis is the transport and trafficking of iron within the cell. Despite the importance of iron transport during erythroid heme synthesis, the molecules involved in intracellular trafficking of iron are largely unknown. In a screen for genes that are up-regulated during erythroid terminal differentiation, we identified FAM210B, a predicted multi-pass transmembrane mitochondrial protein as an essential component of mitochondrial iron transport during erythroid differentiation.
In zebrafish and mice, Fam210b mRNA is enriched in differentiating erythroid cells and liver (fetal and adult), which are tissues that require large amounts of iron for heme synthesis. Here, we report that FAM210B facilitates mitochondrial iron import during erythroid differentiation and is essential for hemoglobin synthesis. Zebrafish are anemic when fam210b is silenced using anti-sense morpholinos (Fig. A). CRISPR knockout of Fam210b caused a heme synthesis defect in differentiating Friend murine erythroleukemia (MEL) cells. PPIX levels in Fam210b deficient cells are normal, demonstrating that Fam210b does not participate in synthesis of the heme tetrapyrrole ring. Consistent with this result, supplementation of Fam210b deficient MEL cells with either aminolevulinic acid, the first committed substrate of the heme synthesis pathway or a chemical analog of protoporphyrin IX failed to chemically complement the heme synthesis defect. While Fam210b was not required for basal housekeeping heme synthesis, Fam210b deficientcells showed defective total cellular and mitochondrial iron uptake during erythroid differentiation (Fig. B). As a result, Fam210b deficient cells had defective hemoglobinization. Supplementation of Fam210b-/- MEL cells with non-transferrin iron chelates restored erythroid differentiation and hemoglobin synthesis; whereas, similar chemical complementation could not be achieved in the Tmem14c-/- cells, which have a primary defect in tetrapyrrole transport. (Fig. C). Our findings reveal that FAM210B is required for optimal mitochondrial iron import during erythroid differentiation for hemoglobin synthesis. It may therefore function as a genetic modifier for mitochondriopathies, anemias or porphyrias.
Figure 1. Figure 1.

Disclosures
Bauer: Biogen: Research Funding; Editas Medicine: Consultancy. Orkin:Editas Inc.: Consultancy.
A Germline Mutation in ERBB3 Predisposes to Inherited Erythroid Myelodysplasia/Erythroleukemia
Authors: Braunstein Evan M. , Li Ruijuan , Sobreira Nara , Gocke Christopher , Brodsky Robert A. , Valle David , Cheng Linzhao 
Published: October 2019

Abstract

Background: Myelodisplastic syndrome (MDS) and acute myeloid leukemia (AML) are usually sporadic diseases. However, rare familial cases have helped to identify human disease genes and provided insight into hematopoiesis and leukemogenesis. Further, the ability to identify familial MDS/AML disease genes has improved due to advances in next generation sequencing technology. In addition to RUNX1, GATA2, CEBPA, germline mutations in both ETV6 and DDX41 were recently shown to predispose to myeloid hematologic malignancies.
Methods: We investigated a large family with an inherited MDS/AML marked by erythroid hyperplasia in all affected individuals. A family pedigree was consistent with an autosomal dominant mode of inheritance with high penetrance. Whole exome sequencing was performed on germline DNA of the proband and multiple unaffected family members in order to identify the inherited genetic mutation predisposing to disease. Candidate single nucleotide variants (SNVs) were obtained after excluding all known SNVs with a variant allele frequency greater than 0.01, then prioritized and screened using DNA from a second affected individual.
Results: An Ala1337Thr missense variant in the ERBB3 gene, a member of the EGFR tyrosine kinase receptor family, was identified as the potential pathologic germline mutation in this family. Further sequencing identified the presence of this variant in two additional affected individuals and its absence in eight unaffected family members, indicating that it co-segregates with disease. Functional validation of this ERBB3 variant was performed using an erythroleukemia cell line. Overexpression of the ERBB3 A1337T mutant in TF1 cells conveyed a block in erythroid differentiation compared to cells expressing unmutated ERBB3, measured by the lack of upregulation of Glycophorin A expression in the presence of erythropoietin. This activity was dependent upon the presence of Neuregulin-1β, the ERBB3 receptor ligand, and expression of the co-receptor ERBB2. In addition, ERBB3 mutated cells displayed a 1.3-fold growth advantage over unmutated ERBB3 in the presence of the Neuregulin-1β.
Conclusions: This work has identified a novel mutation that predisposes to MDS/AML and adds ERBB3 to the growing list of disease predisposition genes in hematologic malignancies. It will allow for the early diagnosis of individuals with inherited erythroid MDS/AML prior to the onset of disease. In addition, it allows for the characterization of a gene with a likely crucial role in erythropoiesis as well as hematologic disease.
Figure 1. Figure 1.

Disclosures
Brodsky: Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.
CLL Disease Severity Is Enhanced in Tcl1 Mice Deficient for Pro-Apoptotic Regulator Noxa
Authors: Slinger Erik , Wensveen Felix , Kater Arnon P. , Eldering Eric 
Published: October 2019

Abstract
Despite the development of new treatment strategies, chronic lymphocytic leukemia (CLL) remains an incurable disease. Apoptosis dysregulation caused by environmental signaling, predominantly within lymph nodes, plays a key role in CLL maintenance and treatment resistance. In earlier work, we demonstrated that expression of the pro-apoptotic Bcl-2 family memberNoxa is increased in circulating CLL cells compared to normal B-cells (1). Moreover, Noxa levels are decreased in CLL cells that reside in the lymph node while its key anti-apoptotic binding partner, Mcl-1, shows a reverse trend (2). This suggests that the decreased Noxa/MCL-1 ratio within the microenvironment may contribute to enhanced CLL survival. Here we aimed at elucidating a functional role for Noxa in CLL in the context of a murine model by crossing Noxa deficient mice (Noxa-KO) with Eμ.Tcl1-Tg mice (TCL1) mice.
We first established that also in Tcl1 mice the Noxa/Mcl-1 ratio was increased PB vs. LN cells. The phenotype and distribution of leukemic cells was similar in Tcl1 and Noxa-KO/tcl1 mice. Interestingly, the accumulation of CLL cells in the peripheral blood was accelerated in Noxa-KO/TCL1 mice. This was accompanied by a reduction of apoptotic cells in the spleen of Noxa-KO/TCL1 mice. In accordance with these findings, survival of Noxa-KO/Tcl1 mice was decreased compared to the Tcl1 mice (342 days vs. 396 days, p<0.001, Figure 1). Our data suggest a role for Noxa in CLL as a tumor suppressor, and provides a rationale for the use of compounds which may alter the Noxa/Mcl1 balance in patients.
1. W. J. Mackus et al., Chronic lymphocytic leukemia cells display p53-dependent drug-induced Puma upregulation. Leukemia19, 427-434 (2005).
2. L. A. Smit et al., Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocytic leukemia cells correlates with survival capacity. Blood109, 1660-1668 (2007).
Figure 1. Survival analysis of Noxa-KO/TCL1 mice and TCL1 mice. The Kaplan-Meier curves were analyzed by a Log-Rank test. Figure 1. Survival analysis of Noxa-KO/TCL1 mice and TCL1 mice. The Kaplan-Meier curves were analyzed by a Log-Rank test.

Disclosures
No relevant conflicts of interest to declare.
Prevalence of Oligoclonal Bands in Multiple Myeloma Patients Who Achieved Better Results Than Very Good Partial Response after Treatment with Standard or High Doses Chemotherapy-Final Analysis
Authors: Vieira Luiza soares , Crusoe Edvan de queiroz , Almeida Manuella de S. Sampaio , Sousa Lais , Perez ana Lucia , Cury Priscilla , Rossato Marina , Higashi Fabiana , Dias ana Luiza , Castro anna Carolina , Chiattone Carlos , Hungria Vania T M 
Published: October 2019

Abstract
Introduction - Oligoclonal bands (OB) are monoclonal proteins distinct from those originally identified in the multiple myeloma (MM) diagnosis. Some authors consider that appearance of these bands confers a better prognosis and may be linked to immune reconstitution. There is no data of the exact prevalence of OB emergence in patients with very good partial response (VGPR) or better after different treatment schedules. Objectives - To determine the prevalence of OB in MM patients treated with or without high-dose chemotherapy that obtained at least VGPR and its prognostic value. Methods- This is a retrospective and prospective cohort study. Data were collected from records of patients that achieved at least VGPR to identify the OB emergence. Subsequently, new sample collections from the positive patients were made in order to monitor the progress and duration of the maintenance of these bands. Results-Median follow-up was 42m and 101 patients were included. Median age was 58y (29-87) and 55% were male. IgG was the most frequent component (60%). Durie-Salmon IIIA/B was identified in 92% of the population; ISS was 33% in stage I, 30% in stage II, and 31% in stage III. The prevalence of OB identified by SPE and IF was 50.5% (51 cases), with a higher prevalence in those who underwent transplantation and those who achieved complete response (p=0.00139 and p=0.0368, respectively). Progression free survival (PFS) was longer in the OB group (45.4m x 34.7m p = 0.0075). Conclusion - The OB prevalence in this population was 50.5% and oligoclonality resulted in a longer PFS.
Figure 1. Figure 1.

Disclosures
No relevant conflicts of interest to declare.
Allogeneic Myeloma GVAX with Lenalidomide Enhances Progression Free Survival through the Generation of Tumor Specific Immunity in Patients in Near Complete Remission
Authors: Borrello Ivan M. , Noonan Kimberly , Huff Carol Ann , Ferguson Anna , Sidorski Amy , Rudraraju Lakshmi , Cimbro Raffaello , Marchionni Luigi 
Published: October 2019

Abstract
Background: The depth of response to therapy in myeloma (MM) correlates with improved progression free survival. We have previously shown that lenalidomide (Len) can augment vaccine efficacy to the pneumococcal conjugate vaccine (PCV), PrevnarÒ. We extend those observations to examine whether vaccinating patients on Len in a near complete remission (nCR) (negative M-spike, IFE positive) could further deepen the clinical response and generate measurable myeloma specific immunity.
Design: Patients on a Len-containing regimen (VRD, Rd, BiRD or R) that achieved and maintained a nCR for 4-6 months were eligible for the study. Patients continued only on single agent Len and received 4 GVAX vaccinations consisting of two allogeneic MM lines: H929, U266 admixed with K562 transduced to express GM-CSF as well as PCV. Patients received 3 monthly vaccines and a boost at 6 months. Immune monitoring was performed on BM samples obtained at baseline, 3 months and 1 year
Results: To date 32 patients have been screened. 17 patients initially in a nCR were ineligible for vaccination: 3 (18%) had disease progression, 7 (42%) entered into an IFE negative CR, and 7 (42%) maintained a nCRduring the observation period but opted not to enroll. 15 patients have been enrolled and completed their vaccinations. Patient characteristics are shown in the Table. Of note, none possessed high-risk features by ISS or FISH. Median follow-up for the study is 34.0 months. Median progression free survival (PFS) of the cohort of vaccinated patients has not been reached whereas the PFS in the observation arm that remained on the multidrug Len-containing therapy was 17.9 months (p<0.001). Vaccination in the setting of a rising M-spike was less likely to induce a durable remission with a median PFS of 14.3 months (p<0.003). Laboratory analysis showed that the patients achieving a CR had greater expression of PD-1 on CD4 and CD8 cells at baseline in the BM. Furthermore, durable responses were associated with the development and persistence of MM-specific immunity.
Conclusions: Vaccination in combination with Len in patients with minimal residual disease generates potent MM-specific immunity and appears to significantly extend the PFS. Vaccination in a nCR with a poly-antigenic approach such as GVAX in combination with Len-induced immunomodulation shows promising early clinical activity that warrants further investigation as an approach to inducing and maintaining durable clinical remissions.
Table 1. Vaccination(n=15) Observation(n=16) Age 69 (55-81) 66 (40-83) FISH (high risk) 0% 0% ISS Stage III 2 (13%) 3 (19%) IFE negative 0 (0%) 7 (42%) Prior Therapies 1.8 (1-4) 1.8 (1-3)

Disclosures
Borrello: Celgene: Research Funding. Noonan:Celgene: Speakers Bureau.
Influence of Matched Sibling Donor's Availability and Stem Cell Transplantation on Treatment Outcome for Acute Lymphoblastic Leukemia; Study on 294 Patients
Authors: Alfraih Feras , Chebbo Wahiba , Kim Dennis Dong Hwan , Aldawsari Ghuzayel , Alsharif Fahad Z. , Alzahrani Hazza A , Mohamed Said Y , Rasheed Walid , Hanbali Amr , Ahmed Syed Osman , Shaheen Marwan , Chaudhri Naeem A. , Al Mohareb Fahad , Aljurf Mahmoud 
Published: October 2019

Abstract
Introduction
Predicting treatment outcome of acute leukemia has been an important issue. Many factors have been elucidated. We evaluated the impact of donor's availability on treatment outcome including mortality in patients with acute lymphoblastic leukemia (ALL).
Methods
A total of 294 patients with ALL were evaluated after receiving chemotherapy between year 2001 and July 2014 at our center. Patients were assessed for the need of hematopoietic stem cell transplants (HSCT), availability of HLA sibling match donor and the impact on overall outcome. Indications for transplantation were defined as (1) WBC->100,000 for T-ALL, >30 for B-ALL with, (2) cytogenetic abnormalities of t(9:22), (4:11) or (1:19), (3) relapse or primary refractory disease or (4) MRD positivity. Patients were divided into 3 categories, group A with an indication for HSCT and available donor (HSCT+/D+), group B with HSCT indication but no available donor (HSCT+/D-) and group C with no indication for HSCT regardless of donor status (HSCT-).
Results
The median age was 20 (14-63 years). 95 (32%) were female while 198 (68%) were male. 276 (86%) patients were newly diagnosed while 18 (14%) were relapsed. Immuno-phenotype was B for 191 (65%), T for 91 (31%) versus mixed lineage for 12 (4%). 33 (11%) patients were positive for Philadelphia chromosome. Median WBC at diagnosis was 23.2X109/L CNS involvement was positive in 26 (8%) patients.
With a median follow-up of 60 months for survivors (range 2 to 116.5 months), 148 (50%) patients showed HSCT+/D+, HSCT+/D- in 79 (27%) and HSCT- in 67 (23%) patients.
the 5-year OS for HSCT+/D-, HSCT+/D+ and HSCT- were 29%, 57% and 55% (p value <0.001), (figure A) while DFS was 25%, 44% and 44% (p value =0.004), respectively. No statistical significance difference was noted on NRM between the groups. On multivariate analysis for OS, donor unavailability was confirmed as an independent risk factor (p<0.001, HR 2.21).
Conclusion
Our study suggested that unavailability of donor is an independent risk factor on OS. The use of alternative donor and building national /regional unrelated donor registry is highly warranted even in countries with high probability of finding matched sibling donor.
Figure 1. Figure 1.

Disclosures
Kim: Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.