bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of p27 by S-Phase Kinase- Associated Protein 2 Is Associated with Aggressiveness in Diffuse Large B Cell Lymphoma (DLBCL)
Authors: Seki Ritsuko , Ohshima Koichi , Fujisaki Tomoaki , Uike Naokuni , Kawano Fumio , Gondo Hisashi , Makino Shigeyoshi , Eto Tetsuya , Moriuchi Yukiyoshi , Taguchi Fumihiro , Kamimura Tomohiko , Tsuda Hiroyuki , Ogawa Ryosuke , Shimoda Kazuya , Okamura Takashi
Published: October 2019

The F-box protein S-phase kinase-associated protein 2 (Skp2) is one of the positive regulators of the cell cycle that promote ubiquitin-mediated proteolysis of the cyclin-dependent kinase inhibitor p27. In this study, we investigated the significance of Skp2 and p27 expression in diffuse large B cell lymphoma (DLBCL) treated with CHOP or CHOP-R. All patients (671) were diagnosed as having DLBCL at the twenty different hospitals and were treated with either CHOP (425) or CHOP-R (246) from 1996 to 2005. All specimens were histopathologically recomfirmed by one pathologist with expertise before entering into this study. Their clinical characteristics, including either the IPI or R-IPI factors, were evenly matched in both treatment groups. The median follow-up of living patients was 3.7 and 2.1 y for CHOP vs CHOP-R, respectively.

Survival analyses were performed using the Kaplan-Meier method and the Cox regression model.

There were 257 patients with high Skp2 expression (cutoff value >40% positive cells) (257/671,39.0%). High Skp2 expression was found in all IPI groups. Expression of p27 (P<.001) was associated with better overall survival (OS), whereas expression of Skp2 was associated with worse OS in CHOP treatment groups (P<.001). We also examined the prognostic impact of Skp2 according to various expression degree of Skp2, Skp2 expression remained a significant predictor of survival (<40%,40–79%,≥80%, P<.001). The multivariant analysis revealed that both the IPI score and the expression of Skp2 and p27 were independent predictors of OS and PFS in both treatment groups (P<.001).

The patients with high Skp2 expression in combination with low p27 expression were related to worst survival in CHOP group (Fig1A). Interestingly, even in CHOP-R group, both high Skp2 expression and low p27 expression were the strong biomarker of worse prognosis (Fig 1B). DLBCL patients with high Skp2 expression did not benefit from the addition of R to CHOP.

Conclusion: Addition of rituximab did not provide a beneficial outcome to DLBCL with high Skp2 and low p27 expression. Therapeutic strategies other than CHOP-R will be needed for DLBCL patients exhibiting a high Skp2 and low p27 expression at the time of diagnosis.

Figure Figure
p19Arf Cooperates with Shp2 in Lymphoid Leukemogenesis
Authors: Chen Xiaolan , Huang Yuhui , Yan Ling , Brault Marie , Wang Zhizhang , Arildsen Maryann T , Yang Elizabeth
Published: October 2019

Shp2 gain-of-function mutations are found in human acute lymphoblastic leukemia, as well as in juvenile myelomonocytic leukemia and acute myeloid leukemias. Genetic data from Noonan Syndrome patients bearing mutations in PTPN11/Shp2 suggest that Shp2 mutation alone is insufficient for leukemogenesis, and other genetic alterations are required. We investigated E76K Shp2-induced leukemogenesis in p19Arf+/− and p19Arf−/− backgrounds. Wild type mice reconstituted with Arf+/− or Arf−/− bone marrow cells (BMCs) transduced with E76K Shp2 developed hematologic disease with increased incidence and severity than mice reconstituted with vector-transduced Arf+/− or Arf−/− BMCs, with E76K Shp2-transduced Arf−/− BMCs causing the most aggressive disease at the highest rate. E76K Shp2 expression in Arf+/− BMCs caused myeloproliferation consisting of mainly Mac1+Gr1+ cells associated with extramedullary hematopoiesis, but E76K Shp2 expression in Arf−/− BMCs caused a shift in disease phenotype to transplantable B220+ leukemias. Consistently, E76K Shp2 transduced Arf+/− or Arf−/− BMCs from reconstituted pre-leukemic mice showed increased proB cell populations compared to vector transduced BMCs, suggesting that the E76K Shp2 expressing proB cells are the leukemia precursors. While Shp2 is well known to confer GM-CSF hypersensitivity in methylcellulose cultures, we found that E76K Shp2 transduced BMCs were also hypersensitive to the lymphoid colony-stimulating factor IL-7, and E76K Shp2 expressing lymphoid BMCs formed colonies in the absence of exogenous growth factor. These data suggest that gain-of-function Shp2 confers transformation features to B cell precursors and that Arf deletion potentiates E76K Shp2 in lymphoid leukemogenesis.
Fibrinogen Alpha and Gamma Genes and Factor VLeiden in Children with Thromboembolism: Results from Two Family-Based Association Studies
Authors: Nowak-Gottl Ulrike , Weiler Hartmut , Thedieck Sabine , Seehafer Tanja , Stoll Monika
Published: October 2019

Background: Fibrinogen, the precursor of fibrin, is an essential component of the hemostatic system. A previous large case-control study showed that genetic variation in the fibrinogen gamma gene (FGG) increased the risk for VT in adults. We investigated the association of haplotypes comprising the fibrinogen alpha (FGA) and gamma (FGG) genes, carriership of the Factor VLeiden-mutation and risk for VT in two large family-based study samples for pediatric thromboembolism.

Methods: We genotyped 7 single nucleotide polymorphisms in FGA and FGG, and the G1691A Factor VLeiden polymorphism in 244 pediatric VT and 268 thrombo-embolic stroke families. Association was assessed using the Transmission Disequilibrium Test (TDT) and corrected for multiple testing.

Results: Association analysis revealed that the FGA-H1 haplotype, and the FGG-H2 and -H3 haplotypes, were significantly associated with VT (FGA-H1, P=0.05; FGG: H2, P=0.032; H3, P=0.0216). In an independent study sample, FGA-H1 (P=0.0085) and FGG-H2 (P=0.05) were significantly associated with TS. When stratifying for FVLeiden carriership, the association between FGA and FGG and VT was more pronounced in FVLeiden-negative families (FGA-H1, P=0.0006; FGG-H2, P=0.0005). Homozygous carriership of the FGG-H2 risk haplotype resulted in the lowest fibrinogen γ′ content (γ′ levels: 22.7±13.7 vs. 26.8±12.0, P=0.013; % γ′: 7.63±3.05 vs. 9.46±3.17, P=2.3×10−5), with increasing concentrations of fibrinogen γ′ in heterozygote H2 carriers. Compound heterozygote carriers of one FGG-H2 risk and one FGG-H3 protective haplotype, showed significant increase in fibrinogen γ′ (P=0.000032), while fibrinogen levels remained unchanged. In contrast, homozygote carriers of the protective FGG-H3 haplotype showed the highest concentration of fibrinogen γ′ content (% γ′: 9.21±3.09, P=0.0031) with decreased total fibrinogen levels.

Conclusion: Our results support an important role of genetic variants in FGA and FGG in thromboembolism in children and adults. Our data further suggest that the genetic architecture of VT is complex and involves subtle influences through susceptibility and protective haplotypes in FGG and a genetic interaction with the FVLeiden-mutation.
Comparative Profiling of Red Blood Cells from the Beta-Adducin Knockout Mouse Model of Hemolytic Anemia Using Label Free Differential Proteomics
Authors: Wooden Jason M , Finney Greg L , Maccoss Michael J , Peters Luanne L. , Gilligan Diana M.
Published: October 2019

Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases. While mild to severe inherited hemolytic anemias can arise from defects in the red blood cell (RBC) membrane skeleton, fundamental questions remain unanswered surrounding the clinical variability of known red blood cell mutations. To identify candidate proteins involved in hemolytic anemia pathophysiology, we utilized a label-free comparative approach to detect differences in RBCs from normal and beta-adducin (ADD2KO) knock-out mice. For each genotype, whole blood was taken from independent biological replicates and RBCs were purified using cellulose acetate chromatography. The isolated RBCs were lysed to generate RBC ghosts whose protein complements were digested with trypsin. For each biological replicate, three replicate runs utilizing 0.5 μg digested protein were performed via microcapillary liquid chromatography coupled with tandem mass spectrometry. Using the recently developed software package CRAWDAD, we detected 7 proteins that were decreased and 31 proteins with a greater abundance in the beta-adducin knock-out RBC ghosts. Differences were detected for previously known membrane skeleton components, including the predicted absence of beta-adducin and decrease in alpha-adducin. The actin binding protein capping protein (actin filament) muscle Z-line alpha 1 was increased along with ankyrin 1, spectrin alpha, tropomyosin, and glycophorin C. Interestingly, differences were also detected for the protein oxidative stress-responsive 1 and the uncharacterized protein C10orf58 which contains a thioredoxin motif. While the protein differences span a broad range of cellular processes, some of the proteins are attractive candidates for modifiers of disease severity. This label free approach is the first demonstration of comprehensively analyzing wild type versus kockout mice that circumvents the need for metabolic or chemical labeling with isotopes. This new approach adds a valuable tool to the list of standard assays for analysis of RBCs in cases of hereditary hemolytic anemia.
Pharmacodynamics of CNTO 530 in a Murine Model of B-Thalassemia
Authors: Makropoulos Dorie A , Capocasale Renold J , Achuthanandam Ram , Wilson Krista , Volk Amy , Bugelski Peter
Published: October 2019

CNTO 530 is an erythropoietin (EPO) receptor agonist that links two EMP-1 peptides to a MIMETIBODYä scaffold. Here, we characterize the in vivo efficacy of CNTO 530 in a murine model of b-Thalassemia. Hematological analysis confirmed dysregulated erythropoiesis in these mice with decreased red blood cells (RBC), hemoglobin (Hgb), and hematocrit (Hct) indices compared to age matched C57BL/6 littermates. An increase in reticulocytes was also observed concurrent with decreased mean cell volume (MCV) and increased red cell distribution width (RDW) suggesting compensatory erythropoiesis. In addition, splenomegaly was observed consistent with increased hematopoiesis. To test the effectiveness of CNTO 530 in this model, a timecourse study was done comparing treatment with CNTO 530 to darbepoetin-a. A single subcutaneous (s.c.) dose of CNTO 530 at 10,000 U/kg dose stimulated a long-lived, approximately 4 g/dL increase in hemoglobin compared to the less than 1 g/dL, short lived increase in Hgb observed in response to darbepoetin-a at 10,000 U/kg. Mice dosed with CNTO 530 also showed an increase in RBC, Hct, and MCV where darbepoetin-a had no significant effect. Non-thalassemic littermates were also given a single s.c. dose of 10,000 U/kg of CNTO 530 or darbepoetin-a which showed these drugs indeed are efficacious in normal background animals. These results demonstrate that CNTO 530 caused a potent, reproducible and longer-lived response in Hgb than darbepoietin in a model of b-thalassemia.
A Novel Subset of CD8+, CD62L-High Tumor Infiltrating Lymphocytes (TIL) Exhibits Regulatory Function
Authors: Plautz Gregory , Wang Li-Xin
Published: October 2019

Tumor infiltrating lymphocytes (TIL) have demonstrated therapeutic effects in adoptive cell therapy of melanoma, and there is great interest in optimizing this response. However, the role of lymphocytes during tumor initiation and progression is complex and evolves over time. We have identified two distinct subsets of TIL that have divergent effector vs. regulatory function in murine tumor models. These subsets can be efficiently segregated in vitro based on differential expression of L-selectin (CD62L). Although initially present in small numbers, they can be activated in vitro with anti-CD3 mAb and expanded with a combination of IL-2 and IL-7 to provide sufficient numbers for adoptive transfer into secondary hosts with advanced tumors. Our initial studies demonstrated that the TIL CD62L-low subset is a mixture of CD4 and CD8 cells that individually or in combination mediate tumor-specific regression. By contrast, the CD62L-high subset, which is exclusively CD8+, does not have therapeutic efficacy. Moreover, when CD62Lhigh TIL are co-transferred with CD62L-low effector cells they abrogate their therapeutic efficacy, thus they have suppressor function. Because L-selectin is involved in lymphocyte homing to secondary lymphoid tissues, we hypothesized that the CD62L-high TIL cells might preferentially re-circulate into lymph nodes and inhibit primary sensitization of naïve T cells to tumor antigens. Tumor cells were inoculated subcutaneously, alone or with either CD62L-high TIL or CD62L-low TIL. The CD62L-high TIL actually enhanced the growth of subcutaneous tumors whereas the CD62L-low cells prevented tumor growth. More importantly, tumor-draining lymph nodes were harvested twelve days later and activated in vitro with anti-CD3 and IL-2/IL-7 for adoptive cell transfer to secondary tumor-bearing hosts. The presence of TIL suppressor cells during sensitization of tumor-draining lymph node cells partially inhibited the development of tumor-reactive effector cells. This inhibition was not tumor-antigen specific because TIL suppressor cells derived from the MCA205 fibrosarcoma were able to inhibit sensitization of B16/F10 draining lymph node effector cells. These data suggest that tumors acquire a population of CD8 CD62L-high T cells that can inhibit other effector T cells. The suppressor TIL cells also appear to modulate the tumor stroma to promote tumor growth and modulate antigen-presenting cells that migrate to draining lymph nodes thereby dampening the development of effector cells. Studies are ongoing to determine the mechanism of suppressor function.
Von Willebrand Factor-Glycoprotein Ib-Alpha Interactions Play a Role in Generating Platelet Microparticles: Data from Ex Vivo and in Vitro Studies
Authors: Artoni Andrea , Lecchi Anna , Mancuso MariaElisa , Santagostino Elena , Federici Augusto , Mannucci Pier M.
Published: October 2019

Microparticles (MP) are circulating submicroscopic cell fragments expressing procoagulant phospholipids and haemostatic proteins such as tissue factor. Several evidences support a pro-haemostatic role of MP and suggest that the axis VWF-platelet GpIb might be critical in their generation. The aim of this study was to measure the levels of platelet-derived MP (PMP) and tissue factor-expressing MP (TFMP) in relationship with VWF levels and multimeric composition as well as shear stress exploiting ex vivo and in vitro natural models. PMP and TFMP were analyzed by flow cytometry and defined as Annexin V and GpIIb/IIIa or Tissue Factor positive events falling in a gate defined by 1 μm beads. MP were enumerated adding a known number of 7 μm beads to each test tube. In the first set of experiments, levels of PMP and TFMP were measured before and after DDAVP administration in 9 patients with mild hemophilia A. FVIII:C, VWF:Ag, PMP and TFMP were measured in plasma at baseline and 30 min, 1, 2, 4, 8 and 24 h after DDAVP 0.3 μg/kg, subcutaneously. A significant increase in the levels of PMP and TFMP (p<0.05 at all time points vs baseline) was observed after DDAVP; peak levels were achieved after 2–8 h. Correlation was found between the increase of VWF:Ag and FVIII:C levels and the peak levels of MP (r=0.62 and r=0.7, respectively). We then studied 27 patients with different types of von Willebrand disease (VWD) and 15 normal controls: citrated platelet rich plasma was exposed to increasing shear stress (from 0 to 90 dyne) for 45 seconds in a cone and plate viscometer and generated PMP were then enumerated by flow cytometry. In normal controls the number of MP at 25 dyne was significantly higher than the number at 0 dyne (p<0.05). In patients with type 1 VWD and type 2B PMP significantly increased as in normal controls (p<0.05 at 25 dyne), while in type 3 patients no PMP increase was observed (p n.s. till 90 dyne). Interestingly, in type 2M patients PMP started to increased significantly at 50 dyne (p<0.05), while type 2A patients had the same behaviour of type 3 patients. This study support the hypothesis that ultralarge VWF multimers released by the endothelium interact with platelets and trigger MP generation, both in vivo and in vitro.
A Chemokine Receptor CXCR4 Plays a Crucial Role for Repairing Ischemic Tissue through Regulation of HIF-2a
Authors: Nagano Masumi , Kimura Ken-ichi , Yamashita Toshiharu , Hamada Hiromi , Ohneda Kinuko , Yoshikawa Hiroyuki , Ohneda Osamu
Published: October 2019

Endothelial progenitor cells (EPCs) are potential therapeutic tools for treatment of ischemic tissues. We previously divided EPCs into two populations on the basis of aldehyde dehydrogenase (ALDH) activities and demonstrated that EPCs with low ALDH activity (Alde-Low EPCs) possess a greater ability for repairing ischemic tissue than those with high ALDH activity by using mouse dorsal skin flap model (Nagano et al., Blood, 2007). To explore molecular basis of the function of Alde-Low EPCs, we transduced small interfering RNAs (siRNAs) into Alde-Low EPCs to reduce the expression of hypoxia inducible transcription factors (HIF-1a or HIF-2a) and examined the ability to repair ischemic tissues by using mouse skin flap model. Injections of Alde-Low EPCs or EPCs with HIF-1a siRNAs via tail vein significantly reduced the necrotic area (4.4±4.5 and 13.2±2.8% of total ischemic area, respectively, n=3 in each group) compared to control PBS injection (30.5±3.2%, n=4). In contrast, EPCs with HIF-2a siRNA showed no positive effect on reducing necrotic area (34.1± 5.2%, n=4). These data suggest that HIF- 2a plays a major role for repairing ischemic tissues in Alde-Low EPCs. We then analyzed the expression of hypoxia-inducible genes in these cells. While the VEGF mRNA level was 30% reduced in both HIF-1a and HIF-2a siRNA treated cells, the expression of a chemokine receptor CXCR4 was reduced in HIF-2a, but not HIF-1a siRNA transduced cells. Moreover, transduction of CXCR4 siRNA into Alde-Low EPCs significantly lost the ability to repair ischemic regions in a mouse flap model (35.5± 3.5%, n=3). Collectively, these results indicate that CXCR4 plays an essential role for repairing ischemic tissue through regulation of HIF-2a.
B Cells at the Interface of Innate and Adoptive Immunity: Identification of IRF8 Target Genes in Germinal Center B Cells
Authors: Shin Dong-Mi , Morse Chang-Hoon Leeand Herbert
Published: October 2019

IRF8 is a transcription factor widely expressed in hematopoietic cells and best known for its effects on myeloid and dendritic cell (DC) differentiation and function. Recent studies showed that IRF8 is expressed at low levels in naïve B cells, at significantly increased levels in germinal center (GC) B cells but little if at all in plasma cells. In GC, IRF8 was shown to contribute to the regulation of AID and BCL6. To better understand IRF8 contributions to the biology of mature B cells and possibly to transformation, we studied three mouse and three human lymphoma cell lines of GC origin by ChIP-Chip (C-C) analyses for IRF8 and POLII targets. We identified 87 IRF8 targets that were common to mouse and human B cells and were also positive for POLII. 17/18 of these putative IRF8 targets tested by PCR ChIP analyses were validated. IRF8 is transcriptionally activated by IFNg, and many of the newly identified targets were previously known to be IFN-responsive. Analyses of the targets identified 14 that contribute to various aspects of innate immunity at the cell surface (CD37, HSP90B1, Ly86), via signal transduction (AZI2, NFKBIZ, STAT2, IRF9), or with direct anti-viral activity (ADAR, PML, S100, ZC3HAV1 and others). Another 19 are active contributors to acquired immune functions of B cells with many involved in peptide processing and antigen presentation by both MHC class I and class II molecules (B2M, CD74, TAP1, TAP2, CTSS and 20S proteosomal components) and some in signal transduction (BLK, BLNK). Still others are active in B cell differentiation and function (MS4A1 [CD20], ELF1 and GNA13). CYLD plays in both arenas as a negative regulator of RIG-I-mediated antiviral responses and by regulating proper B cell activation after BCR ligation. The regulatory contributions of IRF8 to innate immunity in DC, macrophages, granulocytes and now B cells as well as elements of acquired immunity in B cells suggest that it contributes to an evolutionary bridge that positions a single cell type to integrate critical defense mechanisms from the innate and acquired immune repertoires.
Largazole, a Novel Cyclic Peptide Histone Deacetylase Inhibitor, Demonstrates Anti-MM Effects Alone and Increases the Anti-MM Effects of Bortezomib.
Authors: Li Zhi-Wei , Hu Mengyin , Leung Crystal , Steinberg Jeffrey A , Shen Jing , Li Mingjie , Wang Cathy S , Sanchez Eric , Bowers Albert , Greshock Thomas , Miller Kenneth , Newkirk Tenaya , West Nathan , Bradner James E. , Williams Robert M , Bonavida Benjamin , Chen Haiming , Berenson James R.
Published: October 2019

Abstract 4925

Multiple myeloma (MM) remains an incurable malignancy. Therefore, there is a need for the development of new agents to improve the survival for these patients. Histone acetylation, which is controlled by the balanced activities between histone acetyltransferase (HAT) and histone deacetylase (HDAC), is a major epigenetic modification that contributes to tumorigenesis. Through inhibition of HDAC activity, HDAC inhibitors (HDACis) increase acetylation levels of histones as well as other tumor suppressor gene products; and, therefore, are potential anti-cancer agents. Based on the structures, currently available HDAC inhibitors can be divided into four groups, including hydroxamates, cyclic peptides, aliphatic acids, and benzamides. Largazole is a novel member of the cyclic peptide family of HDACis some of which have shown anti-cancer effects in preclinical studies and early clinical trials including for patients with MM especially when used in combination with the proteasome inhibitor bortezomib. In this study, we have explored the potential anti-MM activity of largazole alone and in combination with bortezomib in preclinical in vitro studies. As demonstrated using the MTS assay, this HDACi inhibits the growth of cells from the MM cell lines RPMI8226, U266 and MM1S with an IC50 of approximately 0.2 μM in all three cell lines. In addition, largazole also induces apoptotic death of MM cells as determined with Annexin V staining followed by flow cytometric analysis. Largazole was also shown to induce histone acetylation in MM cells as determined with Western blot analysis using an antibody against acetyl-histone H4. Furthermore, the combination of this HDACi and bortezomib demonstrated synergistic anti-MM effects in these cell lines. Importantly, treatment of mice with largazole shows excellent tolerability at doses that produce concentrations in vivo that are higher than those shown to produce anti-MM effects in our in vitro studies. Thus, largazole may be a potential new agent for the treatment of MM alone and in combination with bortezomib. Currently, we are evaluating the cytotoxic effects of largazole on normal peripheral blood and bone marrow mononuclear cells in vitro and in vivo using our severe combined immunodeficiency mouse models of human myeloma alone as well as in combination with several other drugs including bortezomib used in the treatment of MM. Updated results from these ongoing studies will be presented at the meeting.

Disclosures

Berenson: Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau.
Incidence and Risk Factors for Intracranial Hemorrhage in Californians with Immune Thrombocytopenia
Authors: Brunson Ann , White Richard H. , Wun Ted
Published: October 2019

Abstract 1161

Introduction:

Intracranial hemorrhage (ICH) is thought to be an uncommon event in patients with immune thrombocytopenia (ITP), with reported cumulative incidences of approximately 1%. Most studies have drawn from single institution experiences. We determined the incidence using a large population-based administrative database.

Methods:

Using the California Office of Statewide Health Planning and Development Patient Discharge Dataset, we identified all the records of Californians discharged with a diagnosis of ITP from 1991–2009. We then limited analysis to cases with ITP as the principal diagnosis. Similarly, all cases with a diagnosis of ICH (see types below) in any of the first 5 diagnosis fields were identified and merged with the ITP cohort. Univariate analysis and Cox proportional hazard modeling were then done to determine associations with ICH.

Results:

A total of 37,780 unique patients were initially identified with an ITP code, of which 13,651 were in the principal position and comprise the analysis cohort. This cohort was 60% female, 60% White, 22% Hispanic, 8% Asian, and 7% African-American with a median age of 53 years. Three hundred sixty-four cases had an ICH (cumulative incidence 2.67 %) at a median of 844 days after the first admission for ITP; 109 occurred with the first hospitalization for ITP. The median age of patients with ICH was 67 years. One hundred thirty-five cases with ICH had a splenectomy a median of 296 days prior to their hemorrhage. Of the ICH cases, 65 were known to have died (death statistics available only through 2007); 41 of these deaths were during the admission for the intracranial hemorrhage.

Discussion:

The cumulative incidence of ICH in this large cohort of Californians with ITP was 2.67%, higher than that reported from another population-based study with a smaller number of patients, and from single institution studies. The median age in this study is older than in other reports. The death rate for ICH hospitalization was 11.3% (41/364). Increasing age and certain medical co-morbidities were associated with increased hazard of ICH, as was diagnosis of lupus. Splenectomy was not associated with either increased or decreased hazard, but as this procedure was likely performed only in more severely affected but otherwise healthier patients this result is difficult to interpret. The finding of increased hazard of ICH in Hispanics and Asians with ITP is novel and unexplained. Intracranial hemorrhage may be a more common complication of ITP than previously appreciated.

Disclosures:

Wun: Daiichi Sankyo Company, Ltd. and Eli Lilly and Company: Research Funding.
Limited Effect of TET2 Mutations on Promoter DNA Methylation in Chronic Myelomonocytic Leukemia
Authors: Yamazaki Jumpei , Taby Rodolphe F , Vasanthakumar Aparna , Macrae Trisha , Ostler Kelly R , Shen Lanlan , Kantarjian Hagop M. , Estecio Marcos R , Jelinek Jaroslav , Godley Lucy , Issa Jean-Pierre
Published: October 2019

Abstract 1365

TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocytic leukemia (CMML) samples. TET2 missense or nonsense mutations were detected in 53% (16/30 patients). By contrast, only 1/30 patients had a mutation in IDH1 or IDH2, and none of them had a mutation in DNMT3A. By bisulfite pyrosequencing, global methylation measured by the LINE-1 assay and DNA methylation levels of 10 promoter CpG islands frequently abnormal in myeloid leukemia were not different between TET2 mutant and wild-type cases. This was also true for 9 out of 11 gene promoters reported by others as differentially methylated by TET2 mutations. We confirmed only two non-CpG island promoters, AIM2 and SP140, as hypermethylated in patients with mutant TET2. These were the only two gene promoters (out of 14 475 genes) previously found to be hypermethylated in TET2 mutant cases. This finding shows that hypermethylation of both AIM2 and SP140 are bona fide markers of TET2 mutant cases in CMML. On the other hand, total 5-methylcytosine levels in TET2 mutant cases were significantly higher than TET2 wild-type cases. Thus, TET2 mutations have a limited impact on promoter DNA methylation in CMML. To confirm this, we performed genome-wide analysis using a next-generation sequencing method for DNA methylation levels in three TET2 mutant cases. TET2 mutant CMMLs had an average of 230 (1.9%) promoter CpG island sites hypermethylated compared to normal blood, which is close to what is generally observed when one compares cancer to normal. By contrast, all three cases had near normal to increased levels of methylation outside CpG islands. The median methylation levels in non-promoter, non-CpG island sites was 88.7% in normal blood compared to 91.7%, 92.1% and 94.6% in the three TET2 mutant cases. Thus, TET2 mutant CMMLs escape the general hypomethylation phenomenon seen in many cancers. All together, our data suggest that TET2 mutant CMML cases may have distinct DNA methylation patterns primarily outside gene promoters.

Disclosures:

No relevant conflicts of interest to declare.
A Subnuclear Targeting Defective Mutant of Runx1 Uncouples the Roles of Runx1 In Embryonic Development and Adult Definitive Hematopoiesis
Authors: Dowdy Christopher R , Frederick Dana , Zaidi Sayyed K. , Lian Jane B. , van Wijnen J. , Stein Janet L. , Stein Gary S.
Published: October 2019

Abstract 1387

As shown by gene ablation or replacement studies, Runx1, a master regulator of hematopoiesis, is critical for the emergence of the hematopoietic stem cell (HSC). Runx1 is also the most common target for point mutations and chromosomal translocations in human leukemia. Localization of Runx1 to distinct foci within the nucleus through a unique subnuclear targeting signal is linked to physiological roles of the protein. Many leukemia-related chromosomal translocations (e.g., 8;21 translocation that encodes for AML1-ETO protein) disrupt the Runx1 C-terminus and modify its biological activity as well as alter subnuclear localization. We hypothesize that altered subnuclear routing of leukemic proteins plays a key role and provides a common mechanism in human leukemias. We have previously developed a knock-in mouse model with a Runx1 C-terminal truncation (Runx1 Q307X), which similar to a complete ablation of Runx1 or a genetic knock-in of the leukemic AML1-ETO, causes mid gestation lethality. The Runx1 Q307X mutation also abolishes subnuclear targeting and alters Runx1-dependent myeloid gene expression. To further dissect the specific functional contributions of altered Runx1 subnuclear targeting, we developed a knock-in mouse model in which endogenous Runx1 is replaced with a triple point mutation (Runx1 HTY350-352AAA) that modifies the subnuclear targeting. In contrast to Runx1 Q307X or AML1-ETO knock-in mice, animals homozygous for Runx1 HTY350-352AAA bypass embryonic lethality. However, expression of key Runx1 target genes that regulate hematopoiesis is altered. In addition, bone marrow from Runx1 HTY350-352AAA animals shows increased proliferation ex vivo, resulting in an increase of myeloid and a decrease of B-lymphoid lineage cells. Importantly, red blood cell development, previously thought unrelated to Runx1 expression, is also modified in the Runx1 HTY350-352AAA homozygous animals, as shown by decreased mean corpuscle volume and selective effects on globin gene expression. Together, these findings show that altered subnuclear targeting uncouples the critical role of Runx1 during hematopoietic stem cell emergence and differentiation into various lineages in the adult.

Disclosures:

No relevant conflicts of interest to declare.
Genetic and Epigenetic Profiling on 7q22 Identifies ATXN7L1 As Candidate Tumor Suppressor in Acute Myelogenous Leukemia
Authors: Poetsch Anna R , Claus Rainer , Bonnet Jacques , Devys Didier , Rücker Frank G. , Tora László , Döhner Konstanze , Döhner Hartmut , Plass Christoph
Published: October 2019

Abstract 1408

About 10 % of patients with acute myeloid leukemia (AML) present with either monosomy 7 or deletions on the long arm of chromosome 7. This chromosomal aberration is associated with poor prognosis and adverse therapeutic response. Following biallelic inactivation as proposed by Knudsen's hypothesis, a “second hit” of the remaining allele might be required for loss of gene function. So far, no consistent additional genetic hits could be identified in the minimally deleted regions. Thus, epigenetic silencing might display a local alternative mechanism ultimately causing silencing of a potential tumor suppressor. Based on cytogenetic data we defined a minimally deleted region on 7q22.2 of 2–3 Mb in size to narrow down the location of the putative tumor suppressor. It is flanked by the microsatellite markers D7S1503 and D7S1841. We utilized comprehensive DNA methylation profiling in the CpGrich areas of the minimal deleted regions by high resolution DNA methylation assessment by MassARRAY to identify aberrant epigenetic patterns in these regions. In this region we quantitatively analyzed CpG island methylation in a cohort of 64 AML patients with del(7q), 11 with monosomy 7, ten with normal karyotype and five CD34+ bone marrow cell DNA from healthy donors as controls. We identified four genes (PRES, LHFPL3, ATXN7L1, and CDH28) that are hypermethylated in their promoters in this region with significant mean methylation differences from 5 to 20 % comparing AML to healthy controls. Hypermethylation occurred both in patients with chromosome 7 aberrations and in patients with normal karyotypes. To narrow down aberrantly methylated genes to functional relevant candidates, we excluded those that were not expressed in healthy CD34+ cells. ATXN7L1, a gene located on 7q22.2 and coding for a potential subunit of the histone acetyl transferase (HAT) and deubiquitinase SAGA complex, was expressed in healthy CD34+ cells and granulocytes, but downregulated upon hypermethylation (>30%) in patients. To elucidate a potential functional role of ATXN7L1, we immunopurified SAGA from HeLa cells using antibodies against a SAGA subunit, GCN5,and analyzed the coimmunoprecipated proteins by subsequent mass spectrometry. Based on the identity of the co-precipitated proteins we demonstrate that ATXN7L1 is a bona fide component of the human SAGA complex. Thus our results suggest that ATXNL1 has a similar role as the closely related protein, ATXN7 that is known to be involved in transcription regulation via USP22 dependent deubiquitination of histones. Experiments to understand how ATXN7L1 silencing may affect the leukemic phenotype are in progress.

Disclosures:

No relevant conflicts of interest to declare.
Impact of Iron Chelation Therapy on Overall Survival and AML Transformation in Lower Risk MDS Patients Treated At the Moffitt Cancer Center
Authors: Komrokji Rami S. , Ali Najla H Al , Padron Eric , Lancet Jeffrey E. , List Alan F.
Published: October 2019

Abstract 2776

Background:

Elevated serum ferritin levels and red blood cell transfusion dependence are associated with poor outcome in patients with lower risk myelodysplastic syndromes (MDS). Few retrospective and observational studies suggest that iron chelation therapy (ICT) may favorably impact outcome in lower risk MDS. The vast majority of patients in those studies were treated with deferoxamine (Desferal). Two studies reported that oral deferasirox (Exjade) significantly decreases ferritin level over time in MDS. An ongoing randomized placebo-controlled trial (TELESTO) is designed to address the impact of deferasirox on overall survival (OS) in lower risk MDS. We examined the impact of ICT predominantly deferasirox in lower risk MDS patients treated at the Moffitt Cancer Center (MCC).

Methods:

Patients were retrospectively identified from the MCC database and individual patients' records reviewed. Inclusion criteria included lower risk MDS patients defined as low or intermediate-1 (int-1) risk disease by the international prognostic scoring system (IPSS) and serum ferritin level ≥ 1000 ng/ml. Patients were divided into two comparator groups: ICT vs. no ICT. Baseline characteristics were compared between the two groups; chi square test was used for categorical variables and t-test for continuous variables. The primary endpoint was overall survival compared between the two groups using Kaplan-Meier estimates. Cox regression was used for multivariate analysis. All analyses were conducted using SPSS version 19.0 statistical software.

Results:

Between July 2001 and July 2009, 97 patients with lower risk MDS and serum ferritin ≥ 1000 ng/ml were identified. Forty five (46.4%) received ICT and 52 did not. The ICT included deferasirox in 35 patients and deferoxamine in 10 patients. The baseline characteristics between the two groups (ICT and no ICT) are summarized in (table-1). No statistically significant difference in baseline characteristics was observed except more patients in the ICT group were transfusion dependent. The median duration of follow up was 85.7 month from time of diagnosis.

The median OS was 59 months (95%CI 22–48) for patients who received ICT compared to 33.7 months (95%CI 38–80) for patients who did not receive ICT (P= 0.013). After adjustment for age and cytogenetics in Cox multivariable analysis, ICT was associated with better OS (HR 0.52, 95%CI 0.31–0.87, P= 0.013). The rate of AML transformation was 21.2% in patients who did not receive ICT compared to 15.6% in those who had ICT. (p=0.33).

Conclusion:

ICT in lower risk MDS patients with elevated serum ferritin ≥ 1000 ng/ml was associated with improved overall survival and a trend to lower AML transformation. Results of ongoing randomized clinical study with deferasirox are needed to confirm the retrospective data.

Disclosures:

Komrokji: Novartis: Honoraria, Research Funding.
T Cells of Patients with Myelodysplastic Syndrome Are Frequently Derived From the Malignant Clone
Authors: Vercauteren Suzanne , Starczynowski Daniel T. , Sung Sandy , McNeil Kelly , Salski Chris , Jensen Clara-Lynn , Bruyere Helene , Lam Wan , Karsan Aly
Published: October 2019

Abstract 2808

T cell clonality is a common finding in myelodysplastic syndromes (MDS), but is believed to be a reactive phenomenon. We compared copy number abnormalities in matched CD34+ progenitor cells and CD3+ T cells from 40 MDS patients by array comparative genomic hybridization. In 9 of 14 patients with large copy number variants, we detected the same aberration in CD34+ and CD3+ cells. Chromosome 20q (2 patients), isodicentric × chromosome (2 patients), trisomy 8 (2 patients), 11q abnormalities (1 patient), deletion 5q (1 patient) and partial trisomy 9 with trisomies 19 and 22 (1 patient) were detected in the CD34+ and CD3+ cells of these patients. The presence of deletion 20q, trisomy 8 and deletion of chromosome arm 11q in the T cells of 3 patients was confirmed by FISH. Multiplex PCR for TCR γ rearrangement was performed on 6 of the 14 MDS patients with large copy number variants. T cell clonality was detected in 3 of 4 and oligoclonality in 1 of 4 patients when the copy number variant was present in both CD34+ and CD3+ cells. In contrast, the 2 cases lacking the CD34+ copy number variant in the CD3+ population were polyclonal at the TCR γ locus. These data suggest that in a large proportion of MDS at least a proportion of the T cells are part of the malignant clone, and that CD3+ cells do not represent an appropriate patient normal control for genome-wide studies.

Disclosures:

No relevant conflicts of interest to declare.
Intrinsic Resistance to JAK2 Inhibition in Myelofibrosis
Authors: Kalota Anna , Jeschke Grace R , Carroll Martin , Hexner Elizabeth
Published: October 2019

Abstract 2825

Myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders with a variable clinical course: essential thrombocytosis (ET) and polycythemia vera (PV) are associated with excess clotting and bleeding but an overall normal or near-normal life expectancy, while myelofibrosis has a variable but generally poor prognosis. The disorders are unified by the constitutive activation of the JAK2 pathway, most commonly by a point mutation in the pseudokinase domain of JAK2 (JAK2 V617F). Small molecule tyrosine kinase inhibitors have been shown to be active in both preclinical and late-phase clinical testing, and yet notably pharmacodynamic validation of target (JAK2) inhibition has never been rigorously demonstrated in clinical studies. Clinical investigation of JAK2 inhibitors has focused on myelofibrosis, with some unexpected results: while reduction in spleen size and amelioration of constitutional symptoms are frequent, only modest changes in mutant clonal burden have been observed and complete responses by any criteria are exceedingly rare. In order to develop a feasible and informative pharmacodynamic assay for measuring JAK2 signaling, we used phospho-specific flow cytometry on whole peripheral blood to measure phosphorylation of STAT3 and STAT5, the canonical targets of JAK2 in hematopoietic cells. Treating whole blood with tyrosine kinase inhibitors currently in clinical development, we observed a dose-dependent decrease in phospho-STAT5 and STAT3 in granulocytes treated ex vivo in cells from normal donors, patients with ET, PV and to a lesser extent from patients with myelofibrosis. We found that phosphorylation of STAT3 and STAT5 in cells from patients with myelofibrosis were significantly less inhibited than in PV and ET as well as normal donors. Using quantitative RT-PCR, we found that sensitivity to inhibition did not correlate with JAK2 V617F clonal burden. To determine whether extracellular determinants conferred resistance, mixing studies showed that plasma from patients with myelofibrosis did not transfer resistance. Plasma cytokines that signal through JAK2 and/or are known to be altered in myelofibrosis were measured and no single cytokine appeared to account for the observed pattern of resistance. Taken together these observations suggest that there are cell intrinsic mechanisms that define a priori resistance to JAK2 inhibition in myelofibrosis, and the lesion is localized upstream of STAT3 and STAT5.

Disclosures:

Carroll: Sanofi Aventis Corporation: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.
The Depth of Renal Response Strongly Predicts Overall Surival in Patients with AL Amyloidosis
Authors: Glavey Siobhan V , Dispenzieri Angela , Gertz Morie A , Kumar Shaji , Lacy Martha Q , Buadi Francis K , Rajkumar S. Vincent , Hayman Suzanne R , Lust John A , Russell Stephen J. , Zeldenrust Steven R , Dingli David , Hall Rob L , Leung Nelson
Published: October 2019

Abstract 2868

Introduction:

Improvement in renal parameters is fundamental for the assessment of organ response to treatment in immunoglobulin light chain amyloidosis (AL). Renal response, defined as a >50% reduction in proteinuria with <25% decrease in renal function, is associated with superior overall survival (OS); however, a systematic study of the interplay of proteinuria, creatinine, and free light chain has not been performed. The intent of this study is to better understand the components of the renal response criteria.

Methods:

Patients who underwent autologous stem cell transplantation from 1995 to 2010 were eligible for this retrospective analysis if they had > 1 year of follow-up and a baseline 24 hour urine protein > 1 g/d. Those who were dialysis dependent or died before 1 year were excluded as not evaluable for renal response.

Results:

Of the 435 patients screened, 152 met criteria. On univariate analysis, >50% proteinuria reduction (p < 0.001), serum free light chain (sFLC) reduction by >50% (p = 0.002), <25% increase in serum creatinine (Scr) (p = 0.0003), and a 50% proteinuria reduction within 15 months of treatment (p = 0.04) were associated with better OS. Multivariate analysis using a proportional hazard model showed that sFLC reduction by >50% (0.006), <25% increase in serum creatinine (Scr) (p = 0.0005) and >50% proteinuria reduction (p = 0.0006) were independently associated with OS. In addition, OS was significantly better among patients achieving >85% reduction in proteinuria than those with < 85% but >50%, p = 0.03 (Figure 1). Patients with >75% reduction in sFLC were more likely to achieve >50% proteinuria reduction (p = 0.009). Most importantly, among those with > 25% increase in Scr, only those patients who had <85% proteinuria reduction had a worse OS (Figure 2a, p = 0.006). There was no difference in OS among those with >85% proteinuria reduction and a rise in Scr of > 25% (Figure 2b, p = 0.77).

Conclusion:

Renal response is an excellent prognostic marker in patients with AL. This is the first study to show the depth of renal response correlates with OS. Patients who had the highest levels of proteinuria reduction (> 85%) had a significantly better OS than those with >50% but <85%. We also found that the negative impact of >25% decrease in renal function was only pertinent in those who did not have >85% reduction in proteinuria. These findings should help better refine the definition of renal response criteria in AL patients.

Disclosures:

Leung: Binding Site: Honoraria.
Autocrine Sonic Hedgehog Protects Multiple Myeloma Cells From Apoptosis and Enhances Drug Resistance
Authors: Liu Zhiqiang , Zheng Yuhuan , Li Haiyan , Lu Yong , Weber Donna M. , He Jin , Kwak Larry W , Yi Qing
Published: October 2019

Abstract 2904

The secreted protein sonic hedgehog (SHH) and the hedgehog signaling are of great importance in proliferation and differentiation of cells in the hematopoietic system, and also play a vital role in oncogenesis of B cell malignance. However, the functions and mechanism of SHH signaling in multiple myeloma (MM) is mostly unknown. Thus far, aberrant activation of the hedgehog signaling in tumor growth promoting and/or survival capabilities as well as a paracrine model of SHH secretion have been demonstrated in MM. In the current study, we demonstrated a new autocrine SHH functioning manner in MM cells. The Shh mRNA and the SHH protein were highly expressed both in the MM cell lines and in purified CD138+ MM cells from patients using real-time PCR, Western Blot and immunohistochemistry analyses, respectively; and the SHH protein was also detected in the culture medium. Accordingly, the Hh ligand receptor PTCH1 and PTCH2 as well as the transcriptional factor GLI1 were all overexpressed in MM cells, indicating the activation of Hh signaling pathway. Autocrine SHH played a role in MM cells survival and protected MM cells from apoptosis in vitro, and autocrine SHH accelerated xenograft tumor growth in myeloma-SCID mouse model in vivo. Moreover, autocrine SHH enhanced drug resistance of MM cells, as SHH overexpressed CAG cells (SHH+CAG) had a significantly low apoptosis rate when treated with chemotherapy drugs dexamethasone or bortezomib, as compared with wild type cells (wt-CAG). On the contrary, SHH knockdown cells (SHH-CAG) had a dramatically higher apoptosis rate. Blocking autocrine SHH ligand and treating cells with dexamethasone or bortezomib significantly improved the drug killing effect. Finally, we found that upregulated BLC2 via SHH-Gli1signaling is the signaling pathway by which MM cells enhanced the drug resistance. Our study provides a new insight into the biologic function of the autocrine SHH in proliferation, survival and the drug resistance in the myeloma cells.

Disclosures:

No relevant conflicts of interest to declare.