bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyclonal Gamma Globulin Suppression and Risk Of MGUS Progression Into Multiple Myeloma
Authors: Sheqwara Jawad Z. , Alhyari Mohammad , Keating Shannon , Kuriakose Philip
Published: October 2019

Monoclonal gammopathy of undetermined significance (MGUS) is the most common form of plasma cell dyscrasia, with a prevalence of 3% in the general population above age of fifty. MGUS has a malignant evolution rate of 1% per year. Large longitudinal studies have suggested that virtually all patients diagnosed with multiple myeloma (MM) had a preceding MGUS, with 75 % having detectible Monoclonal (M) protein ≥8 years prior to diagnosis. It is important to identify the features at diagnosis that can predict neoplastic transformation to MM.

Purpose

We identified 239 patients at our institute in whom MGUS was diagnosed between 2000 and 2010. The presenting clinico-hematologic features were correlated with the frequency of evolution into MM to identify early predictors of evolution. The primary end point was progression to MM.

Results

The patients' mean age was 70.7 years. The Male/Female ratio was 0.7. The mean concentration of the M component (MC) was 0.7 g/dL. IgG was the most frequent MC (77%), followed by IgA (13%). The median ratio of MC protein to total protein was 0.5. Single or multiple background polyclonal (PC) suppression was noted in 36% of patients. PC suppression of 50% or more was noted in 20.1% of patients, 49.8% had < 50% and 30.1% had no suppression. Mean bone marrow plasma cell percentage was 4.5 percent and mean hemoglobin was 12.4 g/dL.

Eighteen of the 239 patients with MGUS progressed into MM over ten years of follow up.

Univariate comparisons of all variables between those who progressed and those who did not, showed that the initial concentration of the serum M protein, ratio of M protein to the total protein, number of PC gamma globulins suppressed, degree of PC suppression and IgM gamma globulin suppression were statistically significant risk factors that correlated with progression into MM. Fourteen out of eighteen patients with progressive disease had either PC suppression or background IgM suppression.

Conclusions

Monoclonal protein concentration, ratio of M protein to the total protein and abnormal serum free light chain ratio are simple variables that have been shown in multiple previous studies to predict the progression of MGUS into MM. In our study, we additionally found that number of PC suppressed, degree of suppression and IgM suppression are also key risk factors that can predict progression.

We believe that these variables can be potentially applied into an approach that uses a detailed risk stratification system to predict which cases of MGUS will progress into MM and to provide more intensive monitoring for patients more likely to progress.

Disclosures:

No relevant conflicts of interest to declare.
CD3 Antibody Treatment Prevents Gvhd and Allows For Rapid, Long Term, Multilineage Engraftment Of Unfractionated UCB Into Immunodeficient Mice
Authors: Wunderlich Mark , Brooks Ryan , Rhyasen Garrett , Mulloy James C.
Published: October 2019

NSG mice are currently the best hosts for human hematopoietic stem cell xenografts, due in large part to their ability to initiate robust grafts comprised of diverse human blood cell lineages. Unfortunately, NSG mice are susceptible to GVHD induced by contaminating T cells. Therefore, stem and progenitor cells must be selected with a high degree of purity. This not only complicates the procedure, but also prevents modeling of whole umbilical cord blood (WUCB) and bone marrow transplantation. Here, we have explored both in vivo and in vitro methods to prevent xenogenic GVHD utilizing the anti-T cell antibodies ATG and OKT3. IP injection of either antibody within the first 72 hours of transplant proved effective at preventing T cell expansion. Alternatively, a simple short-term incubation of the cell suspension with antibody was equally effective. ATG, but not OKT3, had significant non-specific activity resulting in reduction and even prevention of the non-T cell portion of the graft, including SCID repopulating cells, using either the in vivo or in vitro approach. However, using the OKT3 in vitro incubation protocol, we were able to initiate stable, long term, multi-lineage engraftment from whole, unselected UCB without GVHD. Additionally, WUCB transplanted mice engrafted more readily, with a wider array of human cell types in the peripheral blood as early as 2 weeks, relative to CD34+ engrafted mice. Stem cell engraftment was confirmed by successful transplant of the human graft to secondary recipients. Limiting dilution experiments showed that significant human grafts could be generated with as few as 1 million total WBCs per mouse, indicating that very large cohorts of mice can be generated with a single UCB sample. This protocol not only has the potential to significantly streamline hematopoietic xenograft studies while greatly reducing the cost and time commitments involved, but also to allow for whole cord blood transplantation modeling and generation of unique subsets of human cells in vivo.

Disclosures:

No relevant conflicts of interest to declare.
Towards a Reference Human Platelet Transcriptome: Evaluation Of Inter-Individual Correlations and Its Relationship With a Platelet Proteome
Authors: Londin Eric R. , Hatzimichael Eleftheria , Loher Phillipe , Edelstein Leonard C. , Shaw Chad , Delgrosso Kathleen , Fortina Paolo M. , Bray Paul F , McKenzie Steven E. , Rigoutsos Isidore
Published: October 2019

Next generation sequencing of RNA (RNA-seq) is an emerging technology that has so far been used successfully to profile the transcriptomes of several cell types and cell states. For the platelet transcriptome, RNA-seq descriptions exist for only a few subjects. Additionally, there have been no studies of the same individual’s transcriptome using two different technologies. As such, it has been unclear how well platelet transcriptomes correlate among different donors or across different RNA platforms, and what the transcriptomes’ relationship is with the platelet proteome.

We generated RNA-seq profiles of the long RNA transcriptomes from the platelets of 10 healthy young males (5 white and 5 black). In addition to RNA-seq, we profiled the platelet messenger RNAs of the same 10 individuals using the Affymetrix GeneChip System. We observed that the abundance of platelet mRNA transcripts was highly correlated across the 10 individuals, a finding that was independent of race and of the employed technology. Additionally, our RNA-seq data showed that these high inter-individual correlations extend beyond mRNAs to several categories of non-coding RNAs. However, there was a notable exception: the category of pseudogenes exhibited a clear difference in expression by race.

Comparison of our mRNA signatures with the only publicly available quantitative platelet proteome data showed that most (87.5%) identified platelet proteins had a detectable corresponding mRNA. Interestingly, there was also a high number of mRNAs that were present in the transcriptomes of all 10 individuals but had no representation in the proteome. Spearman correlation of the relative abundances for those platelet genes that were represented by both an mRNA and a protein, revealed an unexpectedly weak correlation between the transcriptome and the proteome. Further analysis of the overlapping and non-overlapping platelet mRNAs and proteins identified groups of genes with very distinct characteristics. Gene Ontology analysis of the respective gene identifiers revealed that the gene groups corresponded to distinct cellular processes, an interesting finding that provides novel insights for platelet biology.

The very high inter-individual correlations of the transcriptome signatures across 10 different subjects representing two races together with the results of our analyses indicate that it is feasible to assemble a platelet mRNA-ome that can serve as a reference for future platelet transcriptomic studies of human health and disease.

Disclosures:

No relevant conflicts of interest to declare.
Targeting Oncogenic Interleukein-7 Receptor Signaling With N-Acetylcysteine In T-Cell Acute Lymphoblastic Leukemia
Authors: Mansour Marc R. , Reed Casie , Eisenberg Amy , Tseng Jen-Chieh , Yoda Akinori , Rodig Scott J , DeAngelo Daniel J. , Sallan Stephen E. , Weinstock David M. , Izraeli Shai , Kung Andrew L , Kentsis Alex , Look Thomas
Published: October 2019

Activating mutations of the interleukin-7 receptor (IL7R) occur in approximately 10% of patients with T-cell acute lymphoblastic leukemia. Most mutations generate a cysteine at the transmembrane domain leading to receptor homodimerization through disulfide bond formation and ligand-independent activation of STAT5. We hypothesized that the reducing agent N-acetylcysteine (NAC), a well-tolerated drug used widely in clinical practice to treat acetaminophen overdose, would disrupt disulfide bond formation, and inhibit mutant IL7R-mediated oncogenic signaling.

To first identify a suitable cell model to study mutant IL7R signaling, we sequenced exon 6 of IL7R in 21 T-ALL cell lines. We identified a 4-amino-acid insertion (p.L242_L243insLSRC) in DND-41 cells which is predicted to form IL7R homodimers through disulfide bond formation with the unpaired cysteine of neighboring mutant IL7Rs. We found that treatment with NAC at clinically achievable concentrations disrupted IL7R homodimerization in IL7R-mutant DND-41 cells in vitro (IC50 approximately 150 micromolar) and led to STAT5 dephosphorylation and cell apoptosis. These effects could be rescued in part by a constitutively active allele of STAT5, indicating the mechanism of NAC is mediated predominantly through disruption of IL7R-STAT5 signaling in these cells. In a murine xenograft model of T-ALL, intraperitoneal NAC treatment led to significant inhibition of tumor progression, indicating NAC has activity in vivo. Previous studies of NAC pharmacokinetics in humans have shown steady state plasma levels range from 200 to 900 micromolar when given on standard treatment regimens for acetaminophen overdose, well within the therapeutic range required to kill DND-41 cells in vitro. Targeting leukemogenic IL7R homodimerization with NAC offers a potentially effective, cheap and feasible therapeutic strategy that warrants testing in clinical trials.

Disclosures:

Rodig: Daiichi-Sankyo/Arqule Inc., Ventana/Roche Inc., Shape Pharmaceuticals Inc.: Consultancy; Ventana/Roche Inc.: Research Funding.
Hereditary Thrombocytosis Caused By a Novel Germ-Line Mutation In The Gelsolin Gene
Authors: Pianta Annalisa , Liu Kun , Lundberg Pontus , Shimizu Takafumi , Hao-Shen Hui , Dirnhofer Stephan , Kralovics Robert , Shapiro Amy D. , Skoda Radek C.
Published: October 2019

Hereditary thrombocytosis (HT) is a familial myeloproliferative disorder with clinical features resembling sporadic essential thrombocythemia. In some families germline mutations causing HT have been identified in the genes for thrombopoietin (THPO) and its receptor, MPL. However, in many HT families the disease-causing genetic lesion remains unknown. Here we studied a HT pedigree with 15 affected family members in 5 generations. Thrombocytosis is transmitted as an autosomal dominant trait with high penetrance (Figure 1). Genome-wide linkage analysis was performed on DNA from 21 family members using microsatellites and single nucleotide polymorphism arrays. A single co-segregating region with a LOD score of 4.3 was found on chromosome 9. All exons and splice junctions of genes within the co-segregating region were sequenced by classical DNA sequencing and by Next Generation Sequencing (Illumina). We found a candidate mutation in the gelsolin gene (GSN). This C/T transversion was not reported in any SNP database. Computational analysis predicts that the resulting Gly to Cys amino acid change will be damaging to the protein structure. Platelet biogenesis in vitro assays in DAMI cell line stably transfected with the mutant GSN showed that the candidate alteration increased the release of platelets-like particles. To study the in vivo role of the candidate mutation, we generated transgenic mice expressing the mutant GSN gene. These mice developed thrombocytosis and showed increased numbers of megakaryocytes in bone marrow (Figure 2). Thus, our genetic and functional data strongly suggest that GSN mutation is causing thrombocytosis in this family. The exact mechanism of how this newly identified genetic alteration leads to increased megakaryopoiesis needs further investigation.

Disclosures:

No relevant conflicts of interest to declare.
Is There An Expiration Date For Cord Blood Units In Storage?
Authors: Parmar Simrit , de Lima Marcos J , Lee Dean A. , Worth Laura , Kongtim Piyanuch , Petropoulos Demetrios , McNiece Ian K. , Shpall Elizabeth J. , Rondon Gabriela , Champlin Richard E
Published: October 2019

Little is known about the impact of storage duration of an umbilical cord blood unit (CBU) on its’ long term engraftment potency in a clinical setting. We hypothesized that the longevity of storage would not affect CBU engraftment kinetics, when stored under standard conditions. A total of 86 patients undergoing single CB transplants were identified and impact of CBU age was analyzed on transplant outcomes including engraftment and survival. Based on age of the CBU, 4 quartiles were identified: (Q1: Age < 0.7 years (median: 0.07; n=21); Q2: 0.71-1.96 years (median: 1.3 years; n=22); Q3: 1.97-3.87 years (median: 2.8 years; n=22) and Q4: 3.88-12.2 years (median: 5.3 years; n=21)) (Table 1). We also performed analysis on dichotomous distribution of Old CBU (age ≥ 5 years; n=15) vs. New CBU (age < 5 years; n=71). No significant differences in the baseline patient characteristics were seen in any of the subgroups (Table 2). Engraftment was comparable across all quartiles. When comparing quartile Q1, Q2, Q3, Q4, the distribution of outcomes was: median time to neutrophil engraftment: 24, 24, 21, 22 days and median time to platelet engraftment: 42, 54, 41, 42 days, respectively (Figure1 A, B). No differences were observed in the duration of hospitalization: median 43, 48, 43 and 44 days, respectively for the 4 quartiles. For the dichotomous comparison, median time to neutrophil engraftment was 19 vs. 22 days; median time to platelet engraftment was 41 vs. 44 days; and median duration of hospitalization was 41 vs. 46 days, respectively, for Old CBU vs. New CBU. Acute GVHD rates and overall survival rates were also similar. We conclude that old CBUs remain a viable source for stem cell transplant.

Disclosures:

No relevant conflicts of interest to declare.
Rituximab Maintenance Therapy After Autologous Stem Cell Transplantation Prolongs Progression Free Survival In Patients With Mantle Cell Lymphoma
Authors: Dietrich Sascha , Weidle Julia , Meissner Julia , Radujkovic Aleksandar , Ho Anthony D. , Dreger Peter , Witzens-Harig Mathias
Published: October 2019

Background

It was recently shown that rituximab maintenance therapy prolongs progression free (PFS) and overall survival (OS) in patients with mantle cell lymphoma (MCL) after conventional chemotherapy treatment. However, it is not clear if rituximab maintenance can further improve outcome of MCL patients after autologous stem cell transplantation (autoSCT).

Methods

All patients with MCL who underwent an autoSCT at the University Hospital of Heidelberg from 2000 onwards were selected for this analysis. MCL patients who have received rituximab maintenance therapy after autoSCT in a prospective phase-2 trial were compared with regard to PFS and OS with MCL patients who were transplanted during the same time period but did not receive rituximab maintenance therapy by multivariate cox regression analysis correcting for known confounders. For the comparison of rituximab maintenance therapy with the control group, rituximab maintenance therapy was considered as a time dependent event in order to avoid that early progression before start of rituximab maintenance therapy may compromise results.

Results

A total of 72 patients met the inclusion criteria. Twenty-two patients participated in a phase-2 trial and received rituximab maintenance therapy for two years. Median age was 60 years (30-74). Prior to autoSCT all patients received rituximab and 45 patients were treated with high dose cytarabine (HD-ARA-C). AutoSCT was performed after first line treatment in 51 patients. A complete response (CR) before autoSCT was achieved by 27 patients. Patients with and without rituximab maintenance therapy did not significantly differ with regard to age, upfront autoSCT, CR rates and HD-ARA-C treatment prior to autoSCT. However, there was a trend that patients who received rituximab maintenance therapy were transplanted more recently (p=0.06).

Median observation time after autoSCT was 56 months. Two-year PFS and OS of the control group were 65% and 80%, respectively, as compared to 90% and 88%, respectively, of the rituximab maintenance group. By univariate analysis rituximab maintenance therapy was associated with significantly better PFS (HR 0.21, p=0.014; Figure 1A) but so far not OS (Figure 1B). Multivariate adjustment for age (HR per year 0.98; p=0.79), year of transplant (HR per calendar year 1.0; p=0.96), achievement of CR prior autoSCT (HR 1.59; p=0.26), upfront autoSCT (HR 0.81; p=0.80) and high dose ARA-C treatment (HR 0.69; p=0.63) confirmed the beneficial impact of rituximab maintenance therapy (p=0.02 HR 0.23).

Rituximab may prolong PFS in MCL patients who have undergone autoSCT. Prospective studies are highly warranted to confirm our preliminary finding.

Disclosures:

Ho: Sanofi-Genzyme: Research Funding, travel grant Other.
Dysregulation Of Cyclin D1 Or Activation Of KRAS Accelerate The Development Of Monoclonal Gammopathy and Cooperate To Cause Its Progression
Authors: Chesi Marta , Garbitt Victoria , Palmer Stephen , Bergsagel P. Leif
Published: October 2019

We have previously proposed the dysregulation of the D type cyclins by chromosome translocations to the IgH locus or by not fully characterized mechanisms in hyperdyploid patients as a unifying genetic event in the pathogenesis of Monoclonal Gammopathy of Uncertain Significance (MGUS) and multiple myeloma (MM). However, no experimental data are available to evaluate the direct consequences of cyclin Ds overexpression in plasma cells. Similarly, activating mutations of KRAS have been identified in 15% of MM patients, but never in MGUS, and have been proposed to contribute to the progression of MGUS to MM, but this hypothesis has never been experimentally validated. We evaluated the contribution of CyclinD1 and KRAS to the development and progression of monoclonal gammopathy (MG) that develops spontaneously in C57BL/6 mice. We aged a cohort of EuCCND1 mice, and lslKRASV12 mice crossed to three different CRE expressing mice: IgG1CRE, CD19CRE or Vk* CRE (KRAS/CRE). A cohort of Vk* MYC mice, Vk* MYC x EuCCND1 (MYC/CD1) and triple transgenic mice (CD1/KRAS/CRE) was also analyzed. Mice were bled every 10 weeks beginning at 30 weeks, and the presence of MG was evaluated by serum protein electrophoresis. The median Time To M-Spike (mTTS) detection was 97 weeks in C57Bl/6, 63w in EuCCND1, 54w in KRAS/CRE, 54w in Vk* MYC, 38w in CD1/KRAS/CRE and 36w in MYC/CD1 mice. This indicates that acceleration of the development of MG occurs in all these strain of mice. However, we noticed that the spontaneous MG in C57BL/6 wt, EuCCND1 or KRAS/CRE rarely progresses (as determined by detection of a M-spike >7 g/L), while in the other mouse strains it frequently progresses, with a mTTS >7 g/L around 65w. A clear progression of MGUS to MM was observed only in Vk* MYC mice, and was further accelerated in MYC/CD1 mice, which are characterized by extra-medullary disease and shorter overall survival.

In conclusion, these data support our hypothesis that Cyclin Ds dysregulation in plasma cells is the initial transformation step leading to MGUS. Further oncogenic events, like activating mutations of KRAS are required to expand the monoclonal plasma cells. Among the oncogenes examined MYC provides the greatest acceleration of the MG in wildtype and EuCCND1 mice, and results in a disease with the malignant features of MM.

Disclosures:

No relevant conflicts of interest to declare.
Combined Carfilzomib and Selective PI3Kδ Inhibition (TGR1202) Results In Enhanced Myeloma Cell Apoptosis
Authors: Torre Claire , Gu Yanyan , Boise Lawrence H. , Lonial Sagar
Published: October 2019

Introduction

The PI3K signaling pathway plays a vital role in regulating cell growth, proliferation and survival in multiple myeloma (MM) as well as in many other cancers. TGR-1202, an isoform-specific PI3Kδ inhibitor, with efficacy in preclinical models of hematologic malignancies, is currently in Phase I clinical development. In multiple myeloma, PI3K signaling appears to be very important for many extracellular signals, yet inhibition with -pan PI3K inhibitors have not exhibited significant activity. However, literature reports indicate that there are several MM cell lines that express PI3K -δ, and do appear to have differential sensitivity to specific isoform inhibition as opposed to pan PI3K inhibition. In this report, we sought to evaluate the effects of TGR-1202 alone and in combination with the proteasome inhibitor carfilzomib, with the intent of further understanding the mechanism of action and evaluating the impact of the combination.

Methods

Human myeloma cell lines (MM.1S, MY5, RPMI8226, U266, KMS11, ARH-77, OPM1, OPM2, LP1, JJN3 and L363) were treated with TGR-1202 alone, carfilzomib alone, or with the combination of TGR-1202 and carfilzomib. Annexin V/PI staining and Western blot were used to identify the cellular and molecular sequelae of the combination.

Result

10 µM TGR-1202 alone did not cause significant cell death in the MM cell lines tested at 48 hours. When cells were treated with the combination of TGR-1202 and 3 nM carfilzomib, we observed enhanced apoptosis in all of the tested cell lines. In the U266 cell line, 3 nM carfilzomib and 10 µM TGR-1202 induced 16% and 14% cell apoptosis respectively. In the combination treatment, apoptosis increased to 75%. To explore the molecular mechanisms underlying the combination, we used a Western blot assay to evaluate the impact of the combination on the mTOR signaling pathway, a known reciprocal feedback loop when PI3K is blocked. TGR-1202 alone did not have an obvious effect on the mTOR signaling pathway, yet combining TGR-1202 with carfilzomib, significantly inhibited phospho-mTOR, suggesting total pathway blockade.

Conclusion

The combination of TGR-1202 with carfilzomib induces synergistic apoptosis in MM cell lines. Preliminary data suggests that this occurs through blockade of the entire reciprocal feedback loop of mTOR activation. Additional data from primary patient samples and underlying mechanisms will be presented at the meeting. These findings support the rationale for future clinical studies of TGR-1202, a selective PI3K-δ inhibitor in combination with the proteasome inhibitor carfilzomib.

Disclosures:

Boise: Onyx: Consultancy. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy.
A Systematic Review Of The Role Of Erythropoietin In The Pathophysiology Of Anemia In Elderly Patients
Authors: Sriram Swetha , Xenocostas Anargyros , Lazo-Langner Alejandro
Published: October 2019

Introduction

Anemia is a significant issue in the geriatric population, having an impact on morbidity, mortality and quality of life. However, identifying the etiology of anemia is not possible in ∼30% of patients. Postulated mechanisms of anemia of unknown etiology (AUE) include a blunted response to erythropoietin (EPO) or inadequate EPO production in response to anemia. The latter mechanism is of particular interest since it might be amenable to pharmacological intervention. Therefore, in order to explore the relationship between EPO levels and hemoglobin in elderly individuals with AUE, we conducted a systematic review of observational studies.

Methods

We searched Medline, EMBASE, Web of Science, Biosis Previews and Dissertations and Theses using the terms erythropoietin, anemia, elderly and diagnosis as MeSH subject headings. Additional relevant articles were identified by hand searching the meeting abstracts of the European Hematology Association (2006 - 2012) and the American Society of Hematology (2004 - 2012). For inclusion into the final review, studies needed to report data on EPO levels in elderly individuals diagnosed with anemia of unknown etiology. No meta-analysis was conducted due to the heterogeneity of the retrieved studies.

Results

The search identified 4277 potentially relevant citations, of which 31 studies were reviewed in full and 7 cohort studies (2 retrospective, 5 prospective) were included in the final review. The included studies involved 2534 participants ( 1). In general, studies found: 1) lower EPO levels in AUE compared to iron deficiency anemia and other forms of anemia; 2) a lack of correlation between EPO levels and the severity of anemia; and 3) EPO levels in AUE that are in general higher than in non-anemic patients.

Conclusion

Our findings suggest that EPO levels are generally elevated in elderly individuals with AUE, but remain inappropriately low, particularly when compared to anemia of other etiologies suggesting either a relative EPO deficiency, an abnormal EPO response or an abnormal erythroid cell response to EPO. Further research is required to elucidate the mechanisms involved, and the value of pharmacological interventions.

Disclosures:

No relevant conflicts of interest to declare.
H3K9me3-Governed Senescence In Tumor Development and Lymphoma Treatment
Authors: Kase Julia , Dörr Jan , Yu Yong , Milanovic Maja , Ji Sujuan , Spiesicke-Wegener Sandra , Lohneis Philipp , Dörken Bernd , Lee Soyoung , Schmitt Clemens A.
Published: October 2019

Introduction

Premature senescence, a terminal cell-cycle arrest condition of viable cells in response to acute cellular stresses such as oncogenic activation or DNA-damaging anticancer therapy, is characterized by S-phase entry-controlling histone H3 lysine 9 trimethylation (H3K9me3). Previously, we reported an essential, tumor-suppressive role for the H3K9 histone methyltransferase Suv39h1 in oncogene-induced senescence (OIS) as a barrier to lymphoma development in vivo (Braig-M et al., Nature, 2005). In the current study, we focused, in addition to Suv39h1, on the H3K9-active demethylases LSD1 and JMJD2C in both OIS and therapy-induced senescence (TIS).

Methods

Human diploid fibroblasts (HDFs) and mouse embryo fibroblasts (MEFs) were stably transfected with H-RasG12V to induce OIS. S-phase-promoting E2F target gene control by H3K9me3 was analyzed by chromatin immunoprecipitation. Transformation was assessed by anchorage-independent colony formation in vitro and tumor development in nude mice. To establish TIS, primary Eµ-myc transgenic mouse lymphoma cells, retrovirally transduced with bcl2 to block apoptosis, were exposed to adriamycin in vitro or cyclophosphamide in vivo. Senescence was analyzed by staining for senescence-associated b-galactosidase activity (SA-b-gal), Ki67 and BrdU incorporation. Lymphoma formation and treatment in vivo were monitored by luciferase and GFP imaging, SA-b-gal/Ki67 staining in situ, and overall survival was assessed by Kaplan Meier analysis.

Results

H3K9-active demethylases – like overexpression of a dysfunctional H3R9 mutant – blocked cellular senescence, and permitted direct transformation under oncogenic Ras. In Myc-driven lymphomas, either loss of Suv39h1 or overexpression of LSD1 or JMJD2C cancelled TIS in vitro and in vivo. Notably, H3K9me3-impaired lymphomas resembled control lymphomas in their proliferation rate and sensitivity to drug-induced apoptosis, but displayed significantly shorter progression-free and overall survival after chemotherapy. Extended data sets on LSD1 and JMJD2C expression in human diffuse large B-cell lymphoma samples and their correlation to treatment outcome will be presented at the meeting.

Conclusion

The data underscore the essential albeit dynamic role of the H3K9me3 mark in OIS and TIS, and unveil the oncogenic potential of H3K9 demethylases, thereby providing a mechanistic basis for JMJD2C- or LSD1-targeting strategies in lymphoma therapy.

Disclosures:

No relevant conflicts of interest to declare.
DUSP4 Stabilizes FOXP3 Expression In Human Regulatory T Cells
Authors: Sugimoto Naoshi , Liu Yong-Jun
Published: October 2019

Background

Naturally occurring regulatory T cells (Treg) are a subset of CD4+T lymphocytes derived from the thymus. Treg suppress immune responses through multiple mechanisms and are involved in inhibiting the development of autoimmunity. Treg constitutively express the master regulatory gene FOXP3, which is necessary to maintain their phenotype and functions. Human blood circulating FOXP3+ Treg are composed of naïve and activated subsets. CD45RA+CD45RO- naïve Treg enter the blood from the thymus and become CD45RA-CD45RO+ activated Treg after activation. Activated Treg show higher suppressive capacity than naïve Treg.

We have previously compared the gene expression profiles of the four human blood CD4+T cell subsets: naïve T helper (Th) cells, memory Th cells, naïve Treg, and activated Treg. We found that some memory Th cell signature genes (those that are highly expressed in memory Th cells compared to naïve Th cells) were also strongly expressed in activated Treg. DUSP4 (dual specificity phosphatase 4) was one such gene. The DUSP family molecules are known to act as inhibitors of T cell activation signaling, primarily by dephosphorylating the activated MAP kinases. In this study, we hypothesized that DUSP4 might be associated with the phenotype and the functions of human activated Treg.

Experimental Procedures and Results

Human Treg cells were sorted from the peripheral blood of healthy donors and infected with shRNA virus targeting DUSP4 upon stimulation with anti-CD3 and anti-CD28 antibodies. The phenotype and the functions of DUSP4 shRNA-transfected Treg cells were analyzed by real-time quantitative PCR, flow cytometry, and in vitro suppression assay. Strikingly, shRNA knockdown of DUSP4 in Treg resulted in more than 90% reduction in the FOXP3 mRNA level. FOXP3 protein down-regulation was observed as well by intracellular FOXP3 staining. Furthermore, the in vitro suppression capacity of DUSP4 shRNA-transfected Treg decreased in proportion to the FOXP3 level. The decreased FOXP3 expression by DUSP4 knockdown was considered to be due to increased activity of ERK1/2 MAP kinase, which has significant roles in T cell activation. We are currently testing whether MAP kinase inhibitors and MAP kinase genes knockdown modify FOXP3 expression by Treg.

Conclusion

Our study suggests that DUSP4 plays a fundamental role in maintaining the phenotype and the suppressive functions of human activated Treg. DUSP4 and MAP kinases may become novel targets to control Treg function in autoimmunity, allergy, allo-immunity, and cancer immunotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Generation and Functional Properties Of FVIII-Specific Human T Regulatory Cells: A Novel Approach Using Hemophilia A patient’s T Cell Receptors
Authors: Kim Yongchan , Su Yan , Rossi Robert J. , Ettinger Ruth A. , Pratt Kathleen P. , Scott David William , Shevach Ethan
Published: October 2019

Antibody responses (inhibitors) against human factor VIII (FVIII) can obviate successful therapeutic efficacy of FVIII replacement therapy in hemophilia A patients. Novel efforts are necessary to overcome this problem: we propose that specific T regulatory cells (Tregs) can be used to prevent and reverse inhibitor formation. Tregs are the most important T subset for the maintenance of self-tolerance and homeostasis of our immune system. Clinical applications using Tregs are now considered a next-generation cellular therapy in a variety of autoimmune and inflammatory immune disorders such as type 1 diabetes, GVHD, and transplantation, and potentially as a treatment for antibody-mediated diseases such as hemophilia inhibitor formation. However, therapy with polyclonal Tregs is non-specific and a potential drawback is that it could be generally immunosuppressive. Therapy with antigen-specific Tregs (CARTs) would be highly preferable, as these have proved more potent in animal models of autoimmune diseases. We demonstrate herein the generation and functional properties of human T regulatory cells that specifically recognize FVIII. Tregs were generated that expressed the same T cell receptor (TCR) as that found on an HLA-DRB1*01:01-restricted T cell clone isolated from a hemophilia subject with a high-titer inhibitor. The T effector clone was isolated by MHC (DR0101) tetramer staining of this subject’s CD4 cells followed by single-cell sorting and expansion in cell culture. Nucleotide/amino acid sequences were identified to design retroviral FVIII-specific TCR constructs. To produce FVIII-specific human Foxp3+ Tregs, we transduced both expanded T effector cells and Tregs with a TCR expressed on an effector CD4 clone isolated from an inhibitor subject; these T cells recognize the same HLA-DRB1*01:01 restricted epitope as the “parent” effector T cell clone. Specific reactivity of the transduced cells bearing the designed TCR was validated by staining of the transduced T effector and Treg cells by DR0101 tetramers loaded with the same peptide as that recognized by the original effector T cell clone. Transduced effector T cells were expanded by stimulation with the expected FVIII peptide in vitro. Moreover, activation by this peptide in the presence of oligonucleotides mediated functional expansion and stabilization of transduced Tregs in ex vivo culture. Importantly, the expanded transduced Tregs were immunosuppressive only when stimulated by FVIII, even when FVIII-specific effector T cells were present in large excess. (Supported by NIH grant RO1 HL061883-15 to DWS and unrestricted research funding from Bayer, Pfizer and CSL Behring to KPP, and the intramural program of the NIAID to EMS).

Disclosures:

Pratt: Bayer: Research Funding; Pfizer: Research Funding; CSL Behring: Research Funding.
The Clinical Utility Of Bone Turnover Markers In The Evaluation Of Bone Disease In Patients With Haemophilia A and B
Authors: Anagnostis Panagiotis , Vakalopoulou Sofia , Timoleon-Achilleas Vyzantiadis , Charizopoulou Maria , Mandala Eudokia M. , Agapidou Alexandra , Vassiliki Violaki , Vasilia Garipidou
Published: October 2019

Aim

Hemophilia A and B has been associated with increased prevalence of low bone mineral density (BMD). However, the utility of bone turnover markers (BTM) is unknown. The aim of this study was to evaluate bone metabolism in men with hemophilia and investigate associations between BTM and bone disease.

Methods

Serum N- (NTX-I), C-terminal telopeptide of type I collagen (CTX-I) and tartrate-resistant acid phosphatase band-5b (TRAP-5b), as bone resorption markers, and osteocalcin (OC) and bone-specific alkaline phosphatase (b-ALP), as bone formation markers, were assessed.

Results

Seventy men (mean age 45.4±14.7 years) with hemophilia A (n=59) or B (n=11) were studied. Patients with low BMD had significantly higher b-ALP concentrations compared with those with normal BMD, without any differences in the other BTM. NTX-I and CTX-I concentrations were negatively associated with estradiol concentrations and hip BMD and positively with HIV infection, number of affected joints and arthropathy scores. B-ALP and OC concentrations were negatively associated with hip BMD, severity of hemophilia and fracture history, and positively with the number of affected joints and testosterone concentrations.

In men with low BMD, NTX-I, TRAP-5b and b-ALP concentrations were positively correlated with the number of affected joints, whereas TRAP-5b concentrations were positively associated with arthropathy scores and negatively with the level of physical activity.

Conclusion

Increased bone metabolism exists in men with hemophilia and low BMD. Arthropathy, low physical activity and low estradiol concentrations are associated with increased bone resorption and may contribute to the pathogenesis of low BMD in these patients.

Disclosures:

No relevant conflicts of interest to declare.
MiR-150 Inhibits MLL-AF9 Associated Leukemia By Suppressing Leukemic Stem Cells
Authors: Cheruku Patali S , Bousquet Marina , Zhang Guoqing , Ge Guangtao , Ying Wei , Meng Cong , Shie Andrew , Wang Stephanie , Wong Piu , Wang Gang , Safe Stephen , Zhou Beiyan
Published: October 2019

Leukemic stem cells (LSCs) are derived from hematopoietic stem or progenitor cells and often share gene expression patterns and specific pathways. Characterization and mechanistic studies of LSCs are critical as they are responsible for the initiation and potential relapse of leukemias, however the overall framework, including epigenetic regulation, is not yet clear. We previously identified microRNA-150 (miR-150) as a critical regulator of mixed lineage leukemia (MLL) -associated leukemias by targeting oncogenes. Our additional results suggest that miR-150 can inhibit LSC survival and disease initiating capacity by suppressing more than 30% of “stem cell signature genes,” hence altering multiple cancer pathways and/or stem cell identities. MLL-AF9 cells derived from miR-150 deficient hematopoietic stem/progenitor cells displayed significant proliferating advantage and enhanced leukemic colony formation. Whereas, with ectopic miR-150 expression, the MLL-AF9 associated LSC population (defined as Lin-ckit+sca1- cells) was significantly decreased in culture. This is further confirmed by decreased blast leukemic colony formation in vitro. Furthermore, restoration of miR-150 levels in transformed MLL-AF9 cells, which often display loss of miR-150 expression in AML patients with MLL-fusion protein expressing, completely blocked the myeloid leukemia development in a transplantation mouse model. Gene profiling analysis demonstrated that an increased level of miR-150 expression down regulates 30 of 114 stem cell signature genes by more than 1.5 fold, partially mediated by the suppressive effects of miR-150 on CBL, c-Myb and Egr2 oncogenes. In conclusion, our results suggest that miR-150 is a potent MLL-AF9 leukemic inhibitor that may act by suppressing the survival and leukemic initiating potency of MLL-AF9 LSCs.

Disclosures:

No relevant conflicts of interest to declare.
Induction Of Apoptosis By The Dual PI3K/Mtor Inhibitor NVP-BEZ235 Is Impaired By a Profound G1/G0 Arrest In Acute Leukemia Which Can Be Overcome By Combination Approaches
Authors: Kampa-Schittenhelm Kerstin M , Akmut Figen , Illing Barbara , Schittenhelm Marcus M
Published: October 2019

Introduction

Constitutive phosphorylation of AKT is frequently found in acute leukemia. In a proportion of patients, activation of the PI3K/AKT pathway can be linked to gain-of-function tyrosine kinase mutations. We have previously shown that TKI only incompletely inactivate AKT signaling. PI3K/AKT pathway inhibitors are attractive for targeting this pathway. However, most inhibitors lead to G1/G0 arrest. Likely for this reason, response rates to PI3K inhibitor treatment were moderate in various neoplasms. We here demonstrate that the dual PI3K-MTORC1/2 inhibitor NVP-BEZ235, which is currently under clinical investigation in relapsed acute leukemia, mediates a profound G1/G0 arrest in cells harboring leukemia-driving FLT3 ITD or BCR-ABL1 mutations, impairing induction of apoptosis. Furthermore, combination with TKI or chemotherapy overcomes cellular resistance.

Methods

Proliferation and apoptosis assays were performed in leukemia cell lines, an isogenic Ba/F3 cell model harboring mutant-KIT, FLT3 or ABL1 isoforms and primary leukemic cells as well as healthy donor cells. Immunoblots were performed to study effects on AKT signaling pathways including downstream effectors of autophagy (p-ULK1), proliferation, cell cycle (p70S6Kinases, cyclinD, pRB) and apoptosis (caspases). Isobolograms were created to compute combination indices.

Results

NVP-BEZ235 profoundly inhibited phosphorylation of AKT and p70S6K, leading to a potent antiproliferative effect in the low nanomolar range in most cell lines as well as patient blasts. However, no meaningful induction of apoptosis was observed. Instead, a sustained G1/G0 cell cycle arrest with dephosphorylation of RB and upregulation of cyclinD, induction of protective autophagy pathways (via ULK1) and absence of cleaved caspase 3 was detected. An isogenic Ba/F3 model confirmed the low proapoptotic efficacy especially for FLT3 ITD and BCR-ABL1. In an attempt to overcome NVP-BEZ235 induced cell cycle arrest, we co-treated leukemic cells with specific TK inhibitors or chemotherapeutics such as daunorubicin in a fixed dose-dilution ratio. Combination indices revealed additive to superadditive proapoptotic effects for all tested combinations.

Conclusion

We provide evidence that the antileukemic activity of NVP-BEZ235 in acute leukemia with mutations in tyrosine kinases is abrogated due to induction of profound G1/G0 cell cycle arrest which can be overcome by combination with TKI or chemotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Combination Of Regulatory T Cells and Rapamycin As Treatment For Experimental Chronic Graft-Versus-Host Disease
Authors: Belle Ludovic , Ehx Gregory , Somja Joan , Binsfeld Marilene , Hannon Muriel , Caers Jo , Fransolet Gilles , Beguin Yves , Humblet Stephanie , Baron Frederic
Published: October 2019

Background

Chronic graft-versus-host disease (cGvHD) is one of the main complications of allogeneic hematopoietic cell transplantation (allo-HCT). Donor CD4+ conventional T cells (Tconv) as well as regulatory T cells (Treg) are the key-players in its pathogenesis. Rapamycin (sirolimus, rapa) is a mTor inhibitor that can suppress activation and proliferation of Tconv without inhibiting Treg.

Aims

To assess the impact of a combined treatment with Tregand rapa on experimental cGvHD.

Methods

Lethally irradiated BALB/c mice were injected with 10x106 bone marrow cells and 70x106 splenocytes from B10.D2 donor mice. Mice were divided in four groups on day -1 and treatments were started on day 20 with either PBS, rapa 1 mg/kg/Day, Treg 1.106 cells (D+20), or rapa 1 mg/kg/Day + Treg 1.106 cells (D+20). Treg (CD4+ CD25+ cells) were purified from spleen cells from donor B10.D2 mice using the CD4+ CD25+ regulatory T cell isolation kit (Miltenyi Biotec, GmbH, Germany). Treg purity (defined as CD4+ FoxP3+ cells) was ≥ 70% of total cells and > 92% of CD4+T cells.

The severity of sclerodermatous cGvHD was assessed with the following clinical scoring system. Briefly, animals were individually scored every 3 days for five parameters: weight loss (1, 10-20%; 2, > 20%), posture (1, kyphosis only at rest; 2, severe kyphosis when the animal moved), activity (1, moderate activity impairment; 2, no move unless stimulated), skin (1, erythema or scaling tail; 2, open lesion on the body surface) and hair loss (1, > 1 cm2; 2, > 2 cm2). Mice which reached a score of 8 were estimated to have terminal GvHD and were sacrificed. Terminal GvHD-free survivals between the 4 groups were compared using the Log-rank test.

Results

The effects of the treatments were evaluated 7 days after starting the treatments (D+27 post-transplantation). Numbers of total and central memory and effector memory CD4+ T cells/µL were significantly (p<0.05) decreased in rapa-, Treg-, and Treg + rapa- treated mice compared to PBS mice, while numbers of naïve CD4+ T cells/µL were significantly (p<0.05) decreased in Treg-, and Treg + rapa- treated mice. Further, proliferation of CD4+ T cells (assessed by Ki67 expression) was significantly decreased in rapa- (p<0.05) and Treg + rapa- treated mice (p<0.05). In addition, counts and proliferation of CD8+ T cells/µL were significantly (p<0.05) lower in rapa- and Treg+ rapa- treated mice compared to PBS mice.

Importantly, terminal-GvHD-free survival was significantly shorter in PBS mice than in Treg (P=0.03), rapa (P=0.04), and Treg+ rapa mice (P=0.02).

Conclusion

Our results showed that Treg and rapa administration improved cGvHD in this model of cGvHD.

Disclosures:

No relevant conflicts of interest to declare.
Reticulocyte Folate Concentration: A Tool To Monitor Immediate Folate Availability
Authors: Miller Peter J. , Agnew Elizabeth B. , Knovich Mary Ann , Farland Andrew M. , Waldron Johanna , Owen John
Published: October 2019

The utilization of folic acid during erythropoiesis is widely known and accepted in medical literature and deficiency is known to cause a characteristic megaloblastic anemia resulting from the inhibition or ineffective synthesis of DNA. Although the resultant megaloblastic anemia may take considerable time before evidence or symptoms present, there are more acute changes visualized on peripheral smear that are representative of a functional folate deficiency. In addition to erythrocyte macrocytosis, hypersegmentation of neutrophils can also be seen. Often times, despite these visualized changes measurement of serum folate and/or total red cell folate yields a result within the accepted “normal” range of the assay. It has been demonstrated that these visualized characteristics represent a functional folate deficiency and can be overcome with folic acid supplementation regardless of the measured folic acid levels indicating a yet to be understood mechanism of folate utilization. Here, we sought to measure the folic acid levels in the earliest erythrocyte progenitors in peripheral circulation, the reticulocytes.

Methods

Reticulocytes were isolated using anti-CD71 (the transferrin receptor) coated magnetic beads. After separation, a sample slide was made utilizing methylene blue for visual confirmation of reticulocyte isolation. The reticulocytes were then lysed with citric acid and a Nanodrop-1000 spectrophotometer was used to determine absorbance at 413 nanometers. This absorbance was used to determine the sample hemoglobin concentration from a simple calibration curve. The sample folic acid level was then determined using the lysing method utilizing mouse monoclonal anti-folate binding protein, paramagnetic particles coated in anti-mouse IgG, human serum albumin and milk folate binding protein. Results were calculated as nanogram of folate per gram of hemoglobin.

Results

Twenty-five samples from normal individuals, not taking folate supplements, were analyzed. The range of results was 2.51 to 17.38 with a mean level of 9.61.

Conclusion

This protocol effectively and efficiently allows for isolation of reticulocytes in numbers high enough for measurement of folate in nanogram per gram of hemoglobin. By this method we show the normal reticulocyte folate level to be approximately 3-15 ng/g of Hgb. This figure is consistent with the normal red cell folate concentration. Further analysis is planned for comparing these results in patients with a suspected functional folate deficiency but “normal” red cell folate levels. Efficient isolation of reticulocytes and measurement of folate levels will allow us to probe the underlying cause of acute folate deficiency seen in very sick patients.

Disclosures:

No relevant conflicts of interest to declare.