bluuebunny/crossref_metadata_2025 huggingface dataset:
ESAP-Lenalidomide - a Highly Active Regimen in Refractory or Relapsed Hodgkin's Lymphoma.
Authors: Tempescul Adrian , Ianotto Jean-Christophe , Guillerm Gaelle , Berthou Christian
Published: October 2019

Abstract 4797

Hodgkin's disease (HD) is a commonly cured lymphoma. Unfortunately between 10 and 20% of patients are refractory or relapse after a first line of treatment. There is evidence that for these patients the best approach is salvage chemotherapy followed by high-dose chemotherapy (HDC) and autologous stem cell transplantation (ASCT). Existing salvage regimens of chemotherapy, mainly based on platines or aracytine, have a response rate between 60-85%. Usually is associated a toxicity resulting in delay of chemotherapy or dose reduction. It is very important to achieve complete remission before HDC/ABSCT.

It has been proved that 18-FDG PET has a predictive value on Hodgkin's lymphoma.

Into the pathophysiology of HD are implicated various cytokines like IL6, IL12, IL13, NFkB and angiogenic factors. Immunomodulatory drugs have an impact on microenvironment, altering cytokine secretion and the expression of adhesion molecules which can result in apoptosis of malignant B-cell.

Lenalidomide, having both immunomodulatory and antiangiogenic activity, proved to be highly active into the treatment of myeloma and some forms of non-Hodgkin's lymphoma. Into a phase two studies, Lenalidomide administrated as a single agent, proves to be also active into the refractory or relapsed Hodgkin's lymphoma.

During a period of one year, we identified eight patients with refractory ore relapsed HD. We have treated these patients with classical salvage chemotherapy regimen – ESAP- in which we associated Lenalidomide, administrated continuously during the whole period of treatment. We evaluate the response by 18-FDG PET, early, after two or three cycles of ESAP-Lenalidomide association. We obtained seven complete remissions and one very good partial remission (minimal residual disease in PET SCAN). The toxicity was essentially hematological, neutropenia and trombocytopenia.

This small study proves that Lenalidomide, a new drug with both immunomodulatory and antiangiogenic effects, associated to a classical salvage regimen of chemotherapy is highly active on refractory or relapsing Hodgkin's lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
The Plant-Derived Agent α-Bisabolol Induces Apoptosis of Leukemic Blasts Ex Vivo.
Authors: Bonifacio Massimiliano , Cavalieri Elisabetta , Rigo Antonella , de Prati Alessandra Carcereri , Pizzolo Giovanni , Vinante Fabrizio , Suzuki Hisanori
Published: October 2019

Abstract 4800

Introduction

α-bisabolol is a small, oily, non-toxic sesquiterpene alcohol that in rats crosses easily the hemato-encephalic barrier, accumulating in brain without substantial neurotoxicity. We have previously demonstated that α-bisabolol exerts strong cytotoxic effect on a number of malignant human and rat cell lines entering cells via lipid-rafts. This makes α-bisabolol a candidate molecule for anti-cancer therapy.

Materials and methods

We tested ex vivo 42 acute leukemias of lymphoid (ALL) or myeloid (AML) lineage (14 Ph-ALL, 14 Ph+ALL, 14 AML) in a 24-hour dose-response sensitivity assay to α-bisabolol (30, 60 125, 250 μM). Viable cells from freshly heparinized peripheral blood or bone marrow of healthy subjects were used as control.

Results

Low-dose α-bisabolol was highly cytotoxic against primary Ph-ALL (IC50=50±23 μM). Also Ph+ALL, including Ph+-cells resistant to imatinib mesylate, and AML cells were effectively killed (IC50=70±17 and 83±12 μM, respectively). Cytotoxicity on normal haematologic cells increased in a dose- and time-dependent manner: 60, 125, and 250 μM α-bisabolol was not cytotoxic, cytotoxic over 24, and cytotoxic over 5 hours, respectively. Normal bone marrow counterpart was less sensitive to α-bisabolol (IC50=145±11 and 95±14 μM, for CD33+ myeloid and CD34+ cells, respectively). To study the mechanism of α-bisabolol-induced apoptosis, we evaluated at single cell level the mitochondrial transmembrane potential (ΔΨm) through the lipophilic cationic fluorochrome JC-1, by comparison between leukemias and a negative control (T-lymphocytes). Microscopy observation revealed that in untreated leukemic cells, well-polarized mitochondria were marked by punctate red fluorescent staining superimposed over diffuse green fluorescence; after 3-hour incubation with 60 μM α-bisabolol this pattern was almost completely replaced by diffuse green fluorescence, thus indicating the ΔΨm disruption and then the starting of the apoptotic process. Flow cytometric analysis confirmed the same phenomenon. By contrast, normal T-lymphocytes, used as negative control, did not suffer any change in their microscopy or cytofluorimetric pattern when exposed to similar α-bisabolol concentration, and stayed vital.

Conclusions

α-bisabolol, acting via dissipation of the mitochondrial membrane potential, is an effective pro-apoptotic agent for primary leukemic cells ex vivo and could be considered as a possible candidate for treatment of both lymphocytic and myeloid acute leukemias. Interestingly, the majority of leukemias have a IC50 lower than that of their putative normal counterpart, represented by CD34+ cells.

Disclosures:

No relevant conflicts of interest to declare.
GRP-78 Secreted by Tumor Cells Blocks the Anti-Angiogenic Activity of Bortezomib.
Authors: Kern Johann , Untergasser Gerold , Zenzmaier Christoph , Sarg Bettina , Gastl Gunther , Gunsilius Eberhard , Steurer Michael
Published: October 2019

Abstract 4805

Anti-angiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane (CAM). Bortezomib's inhibitory effects on CAM vascularization were abrogated in the presence of distinct tumor xenografts thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy we identified GRP-78, a chaperone protein of the unfolded protein response, as being responsible for bortezomib resistance. In fact, a variety of bortezomib-resistant solid tumor cell lines (PC-3, HRT-18), but not myeloma cell lines (U266, OPM-2), were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib, but induced prosurvival signals by phosphorylation of ERK and inhibited p53-mediated expression of pro-apoptotic Bok and Noxa proteins in endothelial cells.

From our data we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment, thus demonstrating a hitherto unknown mechanism of resistance to bortezomib.

Disclosures:

No relevant conflicts of interest to declare.
Synergistic Anti-Cancer Effects of Arsenic Trioxide and Paclitaxel On Human Malignant Lymphocytes.
Authors: Wu Ying-Li , Duan Xu-Fang , Xu Han-Zhang , Chen Guo-Qiang
Published: October 2019

Abstract 4809

Arsenic trioxide (ATO), an effective drug in the treatment of acute promyelocytic leukemia (APL), and its combination with other chemotherapeutic drugs have been shown to present some activities in other malignant cells besides APL. Increasing reports showed that ATO produces mitotic arrest possibly through targeting to tubulins before apoptosis induction in human tumor cells. Aberrations in the control of cell cycle progression occur in the majority of human malignancies; hence, tubulins are promising targets for anticancer therapy. Here, we define the cellular effects with ATO, alone or in association with paclitaxel, the first identified microtubules stabilizing agent, with respect to inhibition of cell proliferation and cell cycle progression and induction of apoptosis in malignant lymphocytic cell lines Jurkat, Raji, and NAMALWA as well as fresh acute lymphocytic leukemic (ALL) cells. Our results showed that the co-treatment of ATO and PTX at their lower concentrations could significantly induce mitotic arrest followed by growth inhibition and apoptosis in these cell lines. The combined treatment of ATO with PTX also synergistically decreased the cell viability in primary ALL cells. In vivo and in vitro experiments showed that the combination treatment did not increase the microtubules polymerization compared to ATO or PTX treatment alone. Instead, this synergistic action was attributed to the increase of phosphorylation of cyclin-dependent kinase 1 (Cdk1) on Thr161 and the promotion of the dysregulated activation of Cdk1. Furthermore, we found that the ATO/PTX combination significantly enhanced the activation of spindle checkpoint by inducing the formation of the inhibitory checkpoint complex BubR1/Cdc20. These results provide a rational basis for clinical trials of the combined regimen of ATO and PTX in the treatment of lymphocytic neoplasm.

Disclosures:

No relevant conflicts of interest to declare.
The Tyrosine Kinase Inhibitor Dasatinib Enhances Glucocorticoid-Induced Apoptosis and Sensitizes Chronic Lymphocytic Leukemia Cells to Dexamethasone.
Authors: Harr Michael W , Caimi Paolo F. , McColl Karen , Zhong Fei , Patel Shilen N , Barr Paul , Distelhorst Clark W.
Published: October 2019

Abstract 4811

Glucocorticoid hormones are frequently used as part of front-line therapy to treat lymphoid neoplasms. However, relative to acute lymphoblastic leukemia and many types of B cell lymphoma, chronic lymphocytic leukemia (CLL) cells are intrinsically resistant to glucocorticoids and the mechanism governing this resistance has not been fully investigated. Here we report that that the Src inhibitor dasatinib enhances glucocorticoid-induced apoptosis in lymphoid cells and sensitizes primary CLL cells to dexamethasone. This finding evolved from the observation that the Src kinase Lck was downregulated by dexamethasone in glucocorticoid sensitive T cells in order to suppress immunoreceptor activation and signaling. In contrast, aberrantly expressed Lck was not downregulated by dexamethasone in CLL cells. However, inhibition of Lck phosphorylation by dasatinib significantly enhanced cell killing to nanomolar concentrations of dexamethasone. Collectively, these data indicate that dasatinib sensitizes primary CLL cells to glucocorticoids, and thus, the combination of these agents may have therapeutic efficacy in patients with aggressive or refractory CLL.

Disclosures:

No relevant conflicts of interest to declare.
Identification of Target Molecules and Anti-Leukemic Effects of Novel Phospha Sugar Derivative, TMPP.
Authors: Nakamura Satoki , Yamashita Mitsuji , Takemura Tomonari , Yokota Daisuke , Hirano Isao , Ono Takaaki , Shigeno Kazuyuki , Shibata Kiyoshi , Fujie Michio , Fujisawa Shinya , Shinjo Kaori , Ohnishi Kazunori
Published: October 2019

Abstract 4814

Background and Aims

We synthesized two phospha sugar derivatives, 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (TMPP) by reacting 3-methyl-1-phenyl-2-phospholene 1-oxide with bromine, and we has reported their potential as an antileukemic agent in cell lines. In this study, we have investigated that TMPP have antileukemic effects as shown in various leukemia cell lines as well as primary AML patient specimens. Moreover, we have identified the target molecules of TMPP by using docking simulation.

Methods

In human leukemia cell lines (HL60, NB4, U937, NOMO-1, CEM, MOLT4, SUP-B15, and YRK2 cells), the anti-leukemic effects were elevated by cell proliferation assay, Cell cycle analysis, Western blotting. In AML cells derived from AML patients and normal hematopoietic progenitor cells derived from healthy volunteers, the changes of viability by treatment with TMPP were evaluated. By using docking simulation, the target molecules were identified in leukemia cells treated with TMPP.

Results

TMPP showed inhibitory effects on leukemia cell proliferation, with mean IC50 values of 10.2 nmol/L for TMPP, indicating that inhibition appeared to be dependent on the number of bromine atoms in the structure. Further, TMPP at 8.4 nmol/L induced G2/M cell cycle block in leukemia cells, and TMPP at 17.3 nmol/L induced apoptosis in these cells. TMPP treatment reduced cell cycle progression signals (FoxM1, KIS, Cdc25B, Cyclin D1, Cyclin A, and Aurora-B) and tumor cell survival (Bcl-2, p27Kip1 and p21Cip1). Further, treatment with TMPP significantly reduced the viability of AML specimens derived from AML patients, but only slightly reduced the viability of normal ALDHhi progenitor cells. We also found that TMPP showed the strong activity against Aurora kinase B and Bcl-2 by using docking simulation.

Conclusion

We demonstrate that TMPP inhibits Aurora kinase B and Bcl-2 and shows a dominant Aurora B kinase inhibition-related cell cycle arrest and the induction of mitochondrial potential catastrophe-related apoptosis in acute leukemia cells. Moreover, we identified Aurora kinase B and Bcl-2 as the target molecules of TMPP. We conclude that TMPP has great therapeutic potential in anticancer therapy in a wide range of cancers.

Disclosures:

No relevant conflicts of interest to declare.
Effect of PDMP, a Glucosylceramide Synthase Inhibitor, On Resistance to Daunorubicin in Human Leukemia Cell Line K562/A02.
Authors: Chen Bao-An , Yin Li , Cheng Jian , Ding Jia-hua , Gao Chong , Zhao Gang , Wang Jun , Xia Guo-hua , Chen Ning-Na , Pei Xiao-Ping
Published: October 2019

Abstract 4821

Objective

Previous studies showed inhibition of glucosylceramide synthase (GCS) increased sensitivity to doxorubicin toxicity in multidrug-resistant cancer cells. This study was purposed to investigate the reversal effect of D, L-threo-1-phenyl-2- -decanoylamino-3-morpholino-1-propanol (PDMP), a glucosylceramide synthase inhibitor, on daunorubicin (DNR) resistance in K562/A02 cells.

Material and methods

The cytotoxicity and reversal effect were analyzed by MTT method. Flow cytometry was used to test DNR accumulation and apoptosis percentage. The expressions of GCS and mdr1 genes of K562 and K562/A02 cells were measured by RT-PCR and Western blot.

Results

The inhibition rates of PDMP (≤20μmol/l) on the proliferation of K562 and K562/A02 cells were below 10%. The IC50 of DNR for K562 and K562/A02 cells were 0.23±0.02μg/ml and 7.15±0.24μg/ml, respectively. PDMP increased sensitivity of K562/A02 cells to DNR toxicity in a dose-dependent manner (the IC50 was decreased to 2.76±0.25μg/ml by 20μM PDMP and to 4.23±0.08μg/ml by 10μM PDMP), but it had no effect on K562 cells. Both 10μM and 20μM PDMP increased DNR induced apoptosis compared with DNR alone (the apoptosis percentage were 8.93%±0.83%, 18.33% ± 0.86%and 5.43% ± 0.70% respectively). The mean fluorescence intensity of DNR in K562/A02 cells increased from 1550 treated by DNR alone to 1631 incubating with 10μM PDMP and higher to 1998 by 20μM PDMP. The expression of GCS gene was down-regulated by 20μM PDMP and blocking GCS also showed a dramatic decrease in mdr1 expression.

Conclusion

PDMP can increase the sensitivity to DNR toxicity in K562/A02 cells by enhancing cell apoptosis rate, intracellular DNR concentration, and down-regulating expressions of both GCS and mdr1 genes.

Disclosures:

No relevant conflicts of interest to declare.
Therapeutic Potential of Anti-CD137 Antibody in Lymphoma.
Authors: Houot Roch , Goldstein Matthew J , Kohrt Holbrook E , Myklebust June H , Alizadeh Ash A , Lin Jack T , Irish Jonathan M , Torchia James A , Kolstad Arne , Chen Lieping , Levy Ronald
Published: October 2019

Abstract 722

Immunomodulating monoclonal antibodies (mAb) directed against immune cell targets can be used to enhance antitumor immune responses. CD137 (4-1BB) is a costimulatory molecule expressed on a variety of activated immune cells, including T and NK cells. Anti-CD137 agonistic mAb has demonstrated antitumor activity in various tumor models and has now entered clinical trials for the treatment of solid tumors (BMS-663513). Here, we investigate the therapeutic potential of anti-CD137 mAb in lymphoma.

Using microarray gene expression data, we compared CD137 mRNA expression across 17 different types of cancer. We found that lymphoma tumor specimens significantly over-expressed CD137 mRNA compared to other tumors (p<0.0001). Single cell analysis of primary lymphoma samples of various histologies (follicular lymphoma [n=29], mantle cell lymphoma [n=14], and diffuse large B cell lymphoma [n=11]) revealed that CD137 was expressed by a significant proportion of tumor-infiltrating T cells, particularly in the CD8 subset (mean[range]=6.1%[2.5-11.5], 15.4%[2.7-40.9] and 18.7%[6.7-51.7], respectively). In contrast, CD137 was not expressed by the tumor cells. These results suggest that the target of anti-CD137 mAb is present and selectively expressed on cells with potential antitumor activity in patients with lymphoma.

Using a murine model, we then investigated the anti-lymphoma activity of anti-CD137 agonistic mAb in vivo. BALB/c mice, bearing large and established A20 lymphoma tumors, were treated with anti-CD137 mAb or other immunomodulating mAbs (anti-OX40, anti-CTLA4, anti-GITR) for comparison. The mAbs were given i.p. on days 5 and 10 post tumor inoculation. Anti-CD137 mAb demonstrated potent anti-lymphoma activity in vivo, and appeared to be significantly superior to the other immunomodulating mAbs tested (Figure 1). The antitumor effect of anti-CD137 mAb was not due to direct targeting of the malignant cells as A20 tumor cells do not express CD137.

Depletion experiments revealed that anti-CD137 therapy required both NK and CD8 T cells. Analysis of tumor-bearing mice showed that anti-CD137 therapy increased the number of CD8 T cells and reduced the number of Tregs at the tumor site. Anti-CD137 therapy also induced long-lasting antitumor immunity as cured mice harbored antitumor IFN-g producing memory CD8 T cells and were protected from tumor re-challenge more than 100 days after initial therapy.

In conclusion, this study demonstrates for the first time that anti-CD137 agonistic mAb has potent antitumor activity in lymphoma. These results support the evaluation of anti-CD137 mAb in clinical trials for patients with lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
Dissecting the Activation Pathway of Pro-Apoptotic BAX: Mechanistic Insights and Pharmacologic Opportunities.
Authors: Gavathiotis Evripidis , Davis Marguerite , Ruiz Denis Reyna , Bird Gregory H. , Walensky Loren D.
Published: October 2019

Abstract 768

BAX is a pro-apoptotic BCL-2 family member that lies dormant in the cytosol until converted into a killer protein in response to cellular stress. We previously identified an elusive trigger site that mediates BH3-induced direct BAX activation. The continuum of conformational changes that transforms ligand-triggered BAX into a lethal mitochondrial homooligomer remains undefined. Here, we apply NMR and chemical biology methodologies to characterize the key structural alterations and corresponding functional consequences that comprise the direct BAX activation mechanism. Our structure-function analysis reveals a cascade of dynamic changes involving the N-terminal loop, C-terminal helix, and BH3 domain, which convert BAX from an inactive monomer into a toxic oligomer. Our dissection of this seminal pro-apoptotic process highlights new pharmacologic opportunities to modulate cell death in hematologic malignancy by influencing key steps of the BAX activation pathway.

Disclosures:

Walensky: Aileron Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Fox-2A Modulates Protein 4.1R Exon 16 5' Splice Site Activation.
Authors: Huang Shu-Ching , Yang Guang , Park Jennie , Ou Alexander , Benz Edward J.
Published: October 2019

Abstract 778

Erythroid differentiation-induced Fox-2A is an important regulator for the differentiation-specific exon 16 splicing switch in protein 4.1R. Up-regulation of Fox-2A in late erythroid differentiation is critical for inclusion of exon 16, which encodes a portion of the spectrin/actin binding domain vital for maintaining the mechanical stability of red blood cell membranes. Fox-2A exerts its splicing enhancing activity in a motif-dependent manner, binding to UGCAUGs located downstream of exon 16. In this study, we investigated the mechanism by which Fox-2A modulates the expression of exon 16. The excision of introns and the joining of exons depends on the recognition and usage of 5' and 3' splice sites (5' ss and 3' ss, respectively) by the splicing machinery. Exon 16 possesses a relatively strong 3' ss but a weak 5' ss. Mutation of the weak 5' ss (GAGIGTTTGT) to a strong consensus 5' ss (GAGIGTAAGT) led to nearly total exon 16 inclusion. While mutations impairing Fox-2A binding drastically reduced exon 16 inclusion in the presence of the weak 5' ss, no effect on exon 16 inclusion was observed when the strong 5' ss was presented with these mutations. These results suggest that Fox-2A facilitates exon 16 splicing by supporting the weak 5' ss. Early recognition of the 5' ss involves base-pairing interaction with the 5' end of U1 snRNA and stabilization by U1 snRNP. Psoralen-mediated UV cross-linking assays revealed a reduction in U1 snRNA recruitment to the weak 5' ss when Fox-2A binding sites were impaired, suggesting that binding of Fox-2A could promote recruitment and stabilization of U1 snRNP to the weak 5' ss. In support for a role of Fox-2A in modulating the activation of the weak 5' ss by recruiting U1 snRNP, we demonstrated that Fox-2A directly interacts with U1 specific protein U1C in an RNA-independent manner. The C-terminal domain of Fox-2A is responsible for its association with U1C. These data suggest a novel mode for exon 16 5' ss activation in which the binding of Fox-2A to an intronic splicing enhancer element UGCAUG may stabilize the pre-mRNA-U1 snRNP complex through interactions with U1C. These could then result in spliceosome commitment complex formation and exon 16 inclusion.

Disclosures:

No relevant conflicts of interest to declare.
Association Between Genetic Variants in the Base Excision Repair Pathway and Outcomes After Hematopoietic Cell Transplant.
Authors: Arora Mukta , Lindgren Bruce , Basu Saonli , Nagaraj Sriharsha , Gross Myron , Weisdorf Daniel J , Thyagarajan Bharat
Published: October 2019

Abstract 870

Introduction:

Despite recent advances in technology and supportive care, hematopoietic cell transplant (HCT) continues to be associated with high morbidity and mortality. Since the therapies used in HCT induce DNA damage that is repaired by the base excision repair (BER) pathway we hypothesized that single nucleotide polymorphisms (SNPs) in BER genes may influence HCT outcomes.

Methods:

We evaluated the association between tagSNPs and functionally important SNPs (n= 179) in the BER pathway with transplant related mortality at one year (TRM) and disease relapse in a cohort of 470 recipients of allogeneic HCT for hematologic malignancies at the University of Minnesota between 1998 and 2007 from a HLA-identical sibling donor, HLA-matched or mismatched unrelated donor (URD) or single umbilical cord graft.

Results:

After adjustment for age at transplant, donor type, race, and conditioning regimen, four SNPs in OGGI, LIG3 and MUTYH1 genes (rs159153, rs3135974, rs3219463, rs3219476) were associated with increased risk of TRM whereas two SNPs in TDG gene (rs167715, rs2374327) were associated with decreased risk of TRM at one year (p≤0.01). Patients with increasing numbers of deleterious alleles in the BER pathway showed an increased cumulative incidence of TRM at one year (14% for ≤ 1 deleterious allele vs. 51% for ≥ 4 deleterious alleles; p<0.001). One SNP, rs3135974, in LIG3 gene was associated with decreased risk of disease relapse (p<0.001) post HCT.

Conclusions:

Single nucleotide polymorphisms in the BER pathway are significantly associated with both TRM and disease relapse, thus supporting the hypothesis that genetic polymorphisms in the BER pathway are involved in the pathophysiology of TRM and disease relapse. If confirmed in independent cohorts, these SNPs may indicate patients who might benefit from altered or less DNA damaging conditioning regimens.

Disclosures:

No relevant conflicts of interest to declare.
MicroRNAs Characterize Genetic Diversity and Drug Sensitivity in Pediatric Acute Lymphoblastic Leukemia.
Authors: Schotte Diana , de Menezes Renee X , Moqadam Farhad Akbari , Lange-Turenhout Ellen , Chen Caifu , Pieters Rob , Den Boer Monique L.
Published: October 2019

Abstract 89

MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate the activity of protein-coding genes, including those involved in cancer. The function of a miRNA depends on the cellular context and hence prominent miRNAs in lymphoma may not be important in acute lymphoblastic leukemia (ALL). To understand which miRNAs may be relevant in pediatric ALL, the expression levels of 397 miRNAs including seven newly cloned miRNAs were measured in seven genetic subtypes of ALL and normal hematopoietic cells. Except for BCR-ABL-positive and B-other ALL all major subtypes, i.e. T-ALL, MLL-rearranged, TEL-AML1-positive, E2A-PBX1-positive and hyperdiploid ALL, have unique miRNA signatures that differ from each other and from those in healthy hematopoietic cells. The expression of miR-383, miR-125b, miR-99a, miR-100 and let-7c was increased by a five to 1700-fold (P < 0.001) in TEL-AML1-positive cases. Hyperdiploid patients demonstrated a three to 24-fold upregulation (P < 0.001) of miR-222/222*, miR-223, miR-511 and miR-660, encoded on either chromosome X or 10 which is often duplicated in hyperdiploid cases. Some ALL subtypes shared similarities in their miRNA expression signature suggesting a common underlying biology e.g. both E2A-PBX1 and T-ALL cases demonstrated a downregulation of eight miRNAs (P ≤ 0.02) and within the TEL-AML1-positive subtype, two distinct groups were identified of which one showed an overlapping miRNA expression profile with hyperdiploid cases. Aberrant miRNA expression may result in dysregulated expression of their targeted proteins. Here we observed that the 70-fold downregulation of let-7b in MLL-rearranged ALL was associated with a 2-fold upregulation of oncoprotein c-Myc (P< 0.0001). Furthermore, a classifier built with a selection of 28 miRNAs predicted the MLL-rearranged, TEL-AML1-positive, E2A-PBX-positive and T-ALL subtypes with 100% sensitivity and specificity. Besides the genetic subtype, cellular drug resistance determines outcome of ALL. In vitro resistance of patients to vincristine, daunorubicin and L-asparginase was characterized by abnormal expression of 27 miRNAs (P < 0.05) whereas no discriminative miRNAs were found for resistance to prednisolone. Most striking was the 14- to 25-fold upregulation (P ≤ 0.002) of miR-125b, miR-99a and miR-100 in cases resistant to vincristine or daunorubicin. In conclusion, genetic subtypes and drug resistant leukemic cells display characteristic miRNA expression levels. Functional studies are indicated for discriminative miRNAs and may provide new insights into leukemogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Thienopyridine-Associated Thrombotic Thrombocytopenia Purpura: Updated Antibody Results From the SERF-TTP Study.
Authors: Zakarija Anaadriana , Luu Thanh Ha , Kwaan Hau C. , McKoy June , Weiss Ivy , Yarnold Paul , Bandarenko Nicholas , Bennett Charles L.
Published: October 2019

Abstract 892

Background:

The thienopyridines, ticlopidine and clopidogrel, have been associated with thrombotic thrombocytopenia purpura (TTP). However, few studies have reported information on antibodies to ADAMTS13 among patients with thienopyridine-associated TTP. We previously reported on two mechanistic pathways of thienopyridine-associated TTP with some overlapping features. Evaluation of ADAMTS13 autoantibodies was undertaken to improve understanding of these syndromes.

Methods:

Clinical and laboratory findings were evaluated for 30 ticlopidine-, 10 clopidogrel-associated TTP cases, and 54 cases of idiopathic TTP.

Results:

Among patients with thienopyridine-induced TTP, those with a history of ticlopidine versus clopidogrel use were more likely to present with severe thrombocytopenia (platelet < 20,000) (90% versus 13%), severe ADAMTS13-deficiency (80% versus 0%), and neutralizing antibodies to ADAMTS13 (100% versus 0%), and were less likely to have less than a two week history of thienopyridine exposure (0% versus 50%) (p<0.05 for each comparison). They were also more likely to survive following therapeutic plasma exchange (TPE) (85% versus 50%). 2 patients exposed to clopidogrel later relapsed and had similar characteristics to idiopathic TTP patients with non-deficient ADAMTS13 activity.

Conclusion:

Ticlopidine causes TTP by a pathway involving a neutralizing autoantibody to ADAMTS13 while clopidogrel causes TTP by an ADAMTS13-independent pathway. Although ADAMTS13 autoantibodies are present in both idiopathic and ticlopidine-associated TTP, spontaneous relapses are not seen in ticlopidine-associaated TTP, suggesting that drug-dependent antibodies are present. Clopidogrel associated TTP is distinct from idiopathic TTP in that ADAMTS13 autoantibodies are absent and response to TPE is poor.

Disclosures:

No relevant conflicts of interest to declare.
HIV-Related Hodgkin Lymphoma in the Era of Highly Active Antiretroviral Therapy: Incidence and Survival in a Prospective European Multi-Cohort Study On Behalf of the Collaboration of Observational HIV Epidemiological Research in Europe (COHERE) Study Group.
Authors: Bohlius Julia , Boue Francois
Published: October 2019

Abstract 895

Introduction:

HIV-infected patients are at increased risk to develop Hodgkin Lymphoma (HL). We examined the incidence and risk factors for HL, and the prognosis of patients with HIV-related HL in the era of highly active antiretroviral therapy (HAART) in the Collaboration of Observational HIV Epidemiological Research Europe (COHERE).

Patients and Methods:

40,168 adult HIV-1 infected patients who started HAART in one of 16 prospective cohort studies in Europe were included in the present analysis. Incidence rates per 100,000 person-years, Kaplan-Meier estimates of cumulative incidence and survival, and adjusted hazard ratios (HRs) from Weibull random-effects models, with 95% confidence intervals (CIs), were calculated.

Results:

During 159,133 person-years of follow-up, 78 patients were diagnosed with HL. The crude incidence rate of HL was 50.4 per 100,000 person-years for patients who developed HL before starting HAART (17 cases) and 48.7 per 100,000 person-years in patients who were already on HAART (61 cases). Age, gender, CDC clinical stage, CD4 cell counts and HIV-1 RNA viral load at baseline (start of observation) were not significantly associated with the risk of HL. At HL diagnosis median age was 38.9 years (inter quartile range (IQR) 35.3 - 45.9 years) and the median CD4 cell count was 158 cells/μL (IQR 54 – 281 cells/μL). During a median follow-up of 18 months (IQR 4.8 - 34.8 months) 12 of 78 patients with HL died (15%), six of them during the first 6 months after diagnosis. Survival was 88% (95% CI 77% - 94%) at one year and 81% (95% CI 68% - 89%) at two years. Restricting the analysis to patients aged 16-44 years, one year survival in our population (86%, 95% CI 73% - 93%) was less compared to a European population of male Hodgkin patients of similar age (97.7%) [1]. The figure shows Kaplan-Meier plots of cumulative incidence (upper panel) and survival (lower panel).

Conclusions:

HL incidence rates were similar in HAART treated and untreated patients. In contrast to HIV-related Non-Hodgkin's Lymphoma no clear association with baseline CD4 cell count was observed.

Disclosures:

No relevant conflicts of interest to declare.
Mutation and Single-Nucleotide Polymorphism (rs16754) in Wilms Tumor-1 Gene Are Independent Prognostic Factors in Acute Myeloid Leukemia.
Authors: Ma Wanlong , Kantarjian Hagop , Zhang XI , Wang Xiuqiang , Zhang Zhong , Yeh Chen-Hsiung , Sferruzza Anthony , O'Brien Susan , Albitar Maher
Published: October 2019

Abstract 995

Poster Board I-17

Several reports have suggested that mutations in the Wilms tumor 1 gene (WT1) represent an adverse prognostic factor in acute myeloid leukemia (AML). Here we examined the associations of WT1 mutations in exons 7 and 9 and the silent R301 single-nucleotide polymorphism (SNP) in exon 7 (A903G; NCBI dbSNP reference ID: rs16754) with outcome in AML patients treated at a single institution. Peripheral blood plasma and bone marrow samples from 174 newly diagnosed AML patients were tested for WT1 mutations in exons 7 and 9 by sequencing and fragment-length analysis for the detection of small deletions/insertions. Sequencing provided information on the specific genotype of the rs16754 SNP. The findings were correlated with outcome and other laboratory findings. WT1 mutation was detected in 7 of 50 (14%) AML patients <50 years of age and in 5 of 124 (4%) patients >50. The silent R301 SNP was detected at frequencies of 4% for GG, 26% for GA, and 70% for AA genotypes, but there was no difference with age. A similar SNP genotype distribution was detected in normal control subjects. WT1 mutations were associated with higher white cell count (P=0.01) and higher percentage of blasts in bone marrow (P=0.03) and peripheral blood (P=0.009). In addition, WT1 mutation was significantly associated with FLT3 mutation (P=0.002) but not NPM1 mutation (P=0.8). WT1 mutation was also significantly associated with shorter survival (P=0.025), event-free survival (P=0.002), and complete remission duration (P=0.002) in patients <50 years of age, but not in older patients. The association with shorter survival persisted when only patients with intermediate cytogenetics were considered (P=0.03). There was no correlation between WT1 mutation and response to therapy. The most striking correlation was between the presence of the GG genotype at R301 and longer survival, irrespective of age (see survival chart below). There was no difference in survival between the AA and GA groups. To the best of our knowledge, this is the first report describing correlation between survival duration and an SNP in the WT1 gene. While we confirm that the presence of WT1 mutation is associated with poor outcome in young AML patients, our data suggest that there is no clinical value in testing patients older than 50 for WT1 mutation, but that there may be value in testing adult patients of all age groups for the SNP polymorphism at R301

Disclosures:

No relevant conflicts of interest to declare.
Platelets Play a Protective Role in Erythrocytic Stage Malaria Infection.
Authors: Foote Simon J. , McMorran Brendan J. , Drysdale Karen E. , de Graaf Carolyn , Smyth Gordon K. , Alexander Warren S.
Published: October 2019

Abstract MRG-1

Mpl-/- mice lack the thrombopoietin receptor (Mpl) and have 10% the platelet mass of normal, isogenic C57BL/6 animals. When infected with the murine malarial parasite, P. chabaudi, C57BL/6 mice survive the infection whereas Mpl-/- mice die. There is a previously, well-documented thrombocytopenia associated with infection by malarial parasites and, in mice, this is co-incidental with a rise in parasitaemia, not the onset of symptoms. We show that murine infected red cells are preferentially bound to platelets. Using the TUNEL staining technique to identify dead or dying cells we show more TUNEL positive cells in C57BL/6 mice than in the thrombocytopenic Mpl-/- mice. Parasites are more likely to be TUNEL positive if their red cell is bound by a platelet or is CD41 positive. This data provided circumstantial evidence that platelet were directly killing malarial parasites within the red cell in the mouse murine model. It was therefore predictable that aspirin should decrease the resistance to malaria and this proved to be the case. The evidence for the protective role of platelets was substantiated by experiments with human platelets and P.falciparum, the lethal human malarial parasite. Unlike the murine malarias, P.falciparum can be grown in culture (with human red cells). The addition of purified human platelets inhibits the growth of P. falciparum. This is associated with an increase in TUNEL-staining of parasites. It is inhibited by most platelet activation inhibitors (aspirin, Prostaglandin E1, sodium nitroprusside, and the P2Y1 antagonist, MRS2179 as well as administration of aspirin to the platelet donor prior to platelet isolation. Interestingly, inhibition of P2Y12 by MRS2395 does not abrogate the growth inhibitory effect of platelets. Again, infected red cells preferentially bind platelets and there is an excellent correlation between the binding of platelets in infected cells and TUNEL positivity of the resident parasite. This data points strongly to the ability of platelets to kill intraerythocytic malarial parasites. In association with the specificity of binding to infected erythrocytes, this provides a further mechanism of innate immunity, mediated by platelets aimed at controlling the growth of malarial parasites. We surmise that its major impact will be early in infection, prior to the onset of significant parasitaemia or thrombocytopenia, at which stage parasite numbers will greatly outnumber platelets and this will not be an effect strategy.

Disclosures

No relevant conflicts of interest to declare.
Dynamics of Genomic Organization.
Authors: Groudine Mark T. , Rajapakse Indika , Scalzo David , Perlman Michael , Kooperberg Charles L. , Kosak Steven
Published: October 2019

Abstract SCI-17

We have investigated the relationships between lineage-specific gene expression, and total genomic organization during hematopoiesis. First, we determined the linear chromosomal distribution of genes that are co-regulated (identified via microarray analysis) when murine hematopoietic progenitor cells (FDCP-mixA) are differentiated to the erythroid and neutrophil lineages, as well as the organization of all chromosomes (in the form of rosettes) in the three cell types. Our analysis revealed a significant tendency for co-regulated genes to be proximal, which is related to the association of homologous chromosomes and the spatial juxtaposition of lineage-specific gene domains. This led us to hypothesize that the genome—at the level of chromosomes—may self-organize to facilitate coordinate gene regulation during cellular differentiation. We tested this hypothesis by applying the approaches of distance matrices and coupled oscillators to our datasets of gene expression and chromosomal associations from the differentiation of the progenitor to the erythroid and neutrophil lineages. Our analysis revealed that coordinate gene expression undergoes a phase transition—characterized by an increase in entropy—upon commitment of the progenitor. As differentiation continues, there is a gradual loss of entropy, culminating in a highly ordered state in the differentiated cell types. The coregulated gene sets of the semi-ordered progenitor and ordered erythroid and neutrophil lineages are significantly correlated with lineage-specific chromosomal association patterns. Furthermore, by transforming the gene expression networks along the time course to corresponding chromosomal association matrices, we found that chromosomal topologies change dynamically during differentiation but, as with gene expression, result in a more highly ordered state in the differentiated cell types. Our analysis demonstrates that the networks of co-regulated gene expression and chromosomal association are mutually related during differentiation, resulting in the self-organization of lineage-specific chromosomal topologies.

Disclosures

No relevant conflicts of interest to declare.
Enucleation.
Authors: Lodish Harvey F. , Ji Peng
Published: October 2019

Abstract SCI-18

Late erythroblasts undergo terminal cell cycle exit, chromatin condensation, and extrusion of the pycnotic nucleus via an asymmetric cell division, but the signaling pathways and genes involved in these last steps of erythropoiesis are not known. Last year we showed that enucleation of mouse fetal erythroblasts requires both Rac GTPases and mDia2, a downstream effector of Rho GTPases and a formin protein required for nucleation of unbranched actin filaments. Rac and mDia2 are required for formation of the cortical actin ring that apparently powers the separation between the membrane- enveloped nucleus and forming reticulocyte. In collaboration with Drs. Tzutzuy Ramirez Hernandez and Maki Murata-Hori of the Temasek Life Sciences Laboratory in Singapore we showed by time-lapse microscopy that extrusion of the nucleus begins with microtubule- dependent “pushing” of the nucleus to one pole of the cell. Final budding off of the nucleus happens quickly – requiring only about 5 minutes. By analyzing fractionated erythroblasts at all stages of differentiation we confirmed that the nucleus undergoes gradual condensation before being extruded out of the cytoplasm. To investigate how chromatin condensation affects condensation and enucleation, we treated cultured erythroblasts with trichostatin A, a pan-histone deacetylase inhibitor, and discovered that enucleation is completely blocked. We further demonstrated that HDAC1, HDAC2 and HDAC3 are highly expressed in erythroid cells. Using a retroviral system to express shRNAs in developing erythroblasts, we found that downregulation of HDAC2 partially blocked enucleation. We also focused on the role of HDAC6 on enucleation since previous reports showed that HDAC6 forms a complex with mDia2. We confirmed the interaction between HDAC6 and mDia2 and demonstrated that HDAC6 can deacetylate mDia2. This may activate mDia2 and eventually promote enucleation. Our hypothesis is that HDACs play two distinct roles in enucleation: HDAC1, HDAC2 and HDAC3 (class I HDACs) are necessary for the condensation of the chromatin which is required for enucleation; on the other hand, HDAC6 (class II HDAC) interacts with and deacetylates mDia2 to promote enucleation.

Disclosures

Lodish: Amgen: Consultancy, Research Funding.
Factor VIII Biosynthesis and the Unfolded Protein Response.
Authors: Kaufman Randal J.
Published: October 2019

Abstract SCI-19

Factor VIII is the protein deficient in the × chromosome-linked bleeding disorder hemophilia A. Previous studies demonstrated that FVIII expression in mammalian cells is limited due to protein misfolding of the newly synthesized polypeptide in the lumen of the endoplasmic reticulum (ER). Although oxidative stress can disrupt protein folding, how protein misfolding and oxidative stress impact each other has not been explored. We have analyzed expression of FVIII to elucidate the relationship between protein misfolding and oxidative stress. Accumulation of misfolded FVIII in the lumen of the ER activates the unfolded protein response (UPR), causes oxidative stress, and induces apoptosis in vitro and in vivo in mice. Strikingly, antioxidant treatment reduces UPR activation, oxidative stress, and apoptosis, and increases FVIII secretion in vitro and in vivo. The findings indicate that reactive oxygen species are a signal generated by misfolded protein in the ER that cause UPR activation and cell death. Genetic or chemical intervention to reduce reactive oxygen species improves protein folding and cell survival and may provide an avenue to treat and/or prevent diseases of protein misfolding.

Disclosures

No relevant conflicts of interest to declare.
The NLR (NBD-LRR containing) Family of Intracellular Sensors in Inflammation and Immune Response.
Authors: Ting Jenny P-Y.
Published: October 2019

Abstract SCI-22

The NLR gene family encodes proteins that are comprised of conserved nucleotide-binding domain (NBD) and leucine rich-repeats (LRR). These genes are conserved from plants, lower animals to mammals. Family members include CIITA, NOD1, NOD2, and NLRP3. Several of these genes are genetically linked to immunodeficiency, inflammatory and immune diseases. This large gene family controls a number of innate responses, including inflammasome function leading to IL-1b/IL-18 production, cell death and type I IFN production. This talk will discuss the broad function of this family of genes, with emphasis on their regulation of inflammation and innate immunity, and their divergent roles in pathogen responses.

Disclosures

No relevant conflicts of interest to declare.
Hepcidin Regulation by the BMP Pathway.
Authors: Babitt Jodie L.
Published: October 2019

Abstract SCI-25

Systemic iron balance is regulated by the key iron regulatory hormone hepcidin. Secreted by the liver, hepcidin inhibits iron absorption from the diet and iron mobilization from body stores by decreasing cell surface expression of the iron export protein ferroportin. Iron administration increases hepcidin expression, thereby providing a feedback mechanism to limit further iron absorption, while anemia and hypoxia inhibit hepcidin expression, thereby increasing iron availability for erythropoiesis. Hepcidin excess is thought to have a role in the anemia of inflammation, while hepcidin deficiency is thought to be the common pathogenic mechanism underlying the iron overload disorder hereditary hemochromatosis, due to mutations in the genes encoding hepcidin itself (HAMP), HFE, transferrin receptor 2 (TFR2), or hemojuvelin (HFE2). Notably the precise molecular mechanisms by which iron levels are “sensed” and how this iron “signal” is transduced to modulate hepcidin expression have remained elusive. We have recently discovered that hemojuvelin is a co-receptor for the bone morphogenetic protein (BMP) signaling pathway, and that hemojuvelin-mediated BMP signals increase hepcidin expression at the transcriptional level. In addition to patients with HFE2 mutations and Hfe2 knockout mice, other genetic mouse models associated with impaired hepatic BMP signaling through a global knockout of the ligand Bmp6, or selective hepatic knockout of an intracellular mediator of BMP signaling, Smad4, also cause inappropriately low hepcidin expression and iron overload. Exogenous BMP6 administration in mice increases hepatic hepcidin expression and reduces serum iron, while BMP6 antagonists inhibit hepatic hepcidin expression, mobilize reticuloendothelial cell iron stores and increase serum iron. Not only does the BMP6-hemojuvelin-SMAD pathway regulate hepcidin expression and thereby systemic iron homeostasis, but also the BMP6-SMAD pathway itself is regulated by iron. Acute iron administration in mice increases phosphorylation of Smad proteins in the liver, and chronic changes in dietary iron modulate hepatic Bmp6 mRNA expression and phosphorylation of Smad proteins concordantly with Hamp mRNA expression. Together, these data support the paramount role of the BMP6-hemojuvelin-SMAD signaling pathway in the iron-mediated regulation of hepcidin expression and systemic iron homeostasis, and suggest that modulators of this pathway may be an alternative therapeutic strategy for treating disorders of iron homeostasis. Recent work elucidating the role of the BMP signaling pathway in hepcidin regulation and systemic iron homeostasis will be presented.

Disclosures

Babitt: Ferrumax Pharmaceuticals, Inc.: Equity Ownership.