bluuebunny/crossref_metadata_2025 huggingface dataset:
Study On the Mechanism of Enhancing Immune Reconstitution of Keratinocyte Growth Factor in Murine Allogeneic Umbilical Cord Blood Cell Transplantation.
Authors: Chen Guanghua , Wang Yi , Depei Wu , Huang Haiwen , Wang Ying , Xu Yang , Sun Aining , Chang Weirong
Published: October 2019

Abstract 3544

Poster Board III-481

Objective

To explore the impact and mechanism of keratinocyte growth factor (KGF) infusion on immune reconstitution post murine allogeneic umbilical cord blood cell transplantation (UCBT).

Methods

Mix cord blood was obtained at E19.5 from several litters of B6 females and was used as umbilical cord blood (UCB) graft. Thirty-two BALB/c mice were randomly assigned to 4 groups with 8 mice per group in the first cohort UCBT, mice were reconstituted with PBS or 1×106, 2×106, 3×106 UCB mononuclear cells (MNCs). Twenty-four BALB/c mice were randomly assigned to 3 groups with 8 mice per group in the second cohort UCBT, mice were injected with 1×106, 2×106 or 3×106 MNCs. All mice received platelet transfusion on +8d. Sixteen BALB/c mice were randomly assigned to 2 groups with 8 mice per group in the third cohort UCBT, mice were injected s.c. with KGF or PBS before TBI. All mice were injected with 2×106 MNCs and were supported with platelet transfusion on +8d. We studied survival, splenic lymphoid cell subsets, sjTREC assay after UCBT.

Results

The survival rates at +100d of mice resconstituted with allogeneic 1×106, 2×106, 3×106 UCB MNCs were 25%, 37.5% and 37.5% in the first cohort UCBT,respectively. The survival rates at +100d of mice injected with 1×106, 2×106, 3×106 MNCs and with platelet support were 87.5%, 100.0% and 100.0%, respectively. The splenic T, NKT, NK and B cell counts in PBS treated control mice on +35d were (9.57±0.74)×106, (0.64±0.06)×106, (1.43±0.10)×106 and (19.13±1.50)×106, respectively. While in KGF treated mice on +35d were (13.47±0.74)×106, (0.89±0.03)×106, (1.79±0.04)×106 and (20.50±0.91)×106, respectively. The level of sjTREC in control mice was 182.2±10.7copies per 105 cells Gwhile that of KGF treated mice was 224.2±9.6 copied per 105 cells and was higher than that of control mice (P=0.019).

Conclusion

Peripheral blood abtained from E19.5d fetus is rich in hematopoietic stem cells. We established a murine allogeneic UCBT model with platelet support on +8d. KGF treatment could enhance immune reconstitution after UCBT.

Disclosures:

No relevant conflicts of interest to declare.
Predicting Disease Progression to Multiple Myeloma (MM) From MGUS/SMM In SWOG-0120 Observational Trial
Authors: Dhodapkar Madhav , Shaughnessy John , Hoering Antje , Nair Bijay , Bolejack Vanessa , van Rhee Frits , Crowley John , Barlogie Bart
Published: October 2019

Abstract 1932

Background:

Most MM develops through an asymptomatic precursor stage. Current analyses understanding the risk of progression to clinical MM are based on limited set of variables. In 2002, South West Oncology Group (SWOG) began a prospective observational clinical trial of patients with asymptomatic plasmaproliferative disorders (PCD) to evaluate clinical, imaging, genetic and immunologic features predictive of disease progression.

Patients and Methods:

All patients with asymptomatic PCD underwent detailed staging evaluation at study entry and were prospectively followed at 3–6 month intervals until disease progression requiring therapy. Data from clinical features at baseline were evaluated to assess the risk for progression to clinical MM (P-MM).

Results:

Between 11/2002 and 4/2010, 230 patients have been enrolled, of whom 140 were deemed to have smoldering MM (SMM) and 90 monoclonal gammopathy of undetermined significance (MGUS), based on international myeloma working group criteria. The two groups differed in terms of abnormal serum free light chain ratio (FLCR; SMM: 79%, MGUS: 52%; p<0.001) and low levels of uninvolved Ig (SMM: 77%, MGUS; 53%; p<0.001), while no significant differences were noted in the proportion of patients with elevated beta 2 microglobulin (B2M) >=3.5mg/L, albumin <3.5 g/dL, elevated C-reactive protein >8mg/L or lactate dehydrogenase (LDH). Among 13 variables potentially linked to P-MM, baseline B2M >=3.5mg/L, baseline serum albumin <3.5g/dL, serum M-spike and bone marrow plasmacytosis (as continuous variables), serum calcium >8 mg/dL, abnormal FLCR and low levels of uninvolved Ig were all associated with greater propensity of P-MM on univariate analysis. However upon multivariate analysis, abnormal serum FLCR and SMM disease type were the only surviving adverse variables predictive of disease progression.

Conclusions:

These data demonstrate in the context of a large prospective multicenter trial that abnormal FLCR is a dominant predictor of clinical malignancy in asymptomatic PCD. These findings support the need to understand the biology of abnormal FLCR and incorporate novel criteria based on imaging, genetics and host response.

Disclosures:

No relevant conflicts of interest to declare.
Could the Increased Prevalence of Thrombotic Complications at High Altitude In Polycythemia Vera (PV) Be Contributed by Hypoxia?
Authors: Lee# Jasmine CH , Zangari# Maurizio , Stein Brady L , Hickman Kimberly , Wilson Andrew , Moliterno Alison , Spivak Jerry L , Gordeuk Victor R. , Prchal Josef T.
Published: October 2019

Abstract 1988

# Both authors contributed equally.

Thrombotic complications are a major cause of morbidity and mortality in polycythemia vera (PV). While the risk of PV thrombotic complications is significantly higher in patients with a previous history of thrombosis or transient ischemic attacks, advanced age (> 60 years), leukocytosis and possibly a high JAK2 V617F allelic burden, other contributing etiologies have not been excluded. It is unclear whether environmental variations including hypoxia could modify thrombosis risk. Chronic hypoxia induces many phenotypic changes, including increased erythropoietin production resulting in secondary erythrocytosis and pulmonary hypertension. Moreover, a congenital disorder of augmented hypoxia sensing at ambient oxygen tension, Chuvash polycythemia, is associated with a marked, yet unexplained, increased rate of thrombotic complications.

Salt Lake City, UT is located in a valley at ~5,000 feet of altitude and the surrounding areas from which PV patients are referred ranges from 4,300 to 6,900 feet in altitude. In this area, the average hemoglobin concentration is 0.6–1.3 gm/dL higher than the average US value, indicating a physiological response to this level of hypoxia.

To analyze the possible effect of moderate hypoxia on PV thrombotic complications, we retrospectively compared the history of thrombotic complications in PV patients from Salt Lake City and Baltimore, MD, a city with a referral area in which the elevation is <500 feet. A total of 237 PV patients were analyzed in this study; 166 were treated in Baltimore and 71 were treated in Salt Lake City, Their demographics are presented in the Table.

Patients from Salt Lake City were older than the Baltimore patients, more often male, and had longer disease duration. A history of thrombosis was present in 58% of the Salt Lake City patients compared to 27% of the Baltimore patients (P <0.0001). After adjusting for age, sex and disease duration, Salt Lake City patients had an estimated 3.9-fold increase in the odds of history of thrombosis compared to Baltimore patients (95% confidence interval of 1.9 to 7.8; P = 0.0002).

The analysis suggests that hypoxia may modify the PV phenotype. The degree of HIF-1 and HIF-2 augmentation and altered regulation of their target genes, and the potential for increased thrombotic risk due to environmental hypoxia should be evaluated prospectively in PV patients living at different altitudes.

Disclosures:

No relevant conflicts of interest to declare.
Pegylated Interferon-Alfa-2a In Clinical Responders Often Decreases JAK2 V617F Allelic Burden and/or Reverses Clonal Hematopoiesis, but These Are Not Always Parallel In PV and ET Females
Authors: Swierczek Sabina , Verstovsek Srdan , Agarwal Archana , Manshouri Taghi , Hickman Kimberly , Prchal Josef T.
Published: October 2019

Abstract 1990

Polycythemia vera (PV) and essential thrombocythemia (ET) are hematological malignancies due to clonal proliferation arising from a multilineage stem cell. Pegylated interferon-alfa-2a is a better-tolerated form of interferon shown to be effective in inducing clinical remission and reducing the JAK2V617F allelic burden in PV and ET patients. However, while the somatic JAK2V617F mutation, which is seen in the majority of PV patients, is not the PV-initiating mutation, it appears to have prognostic significance. Conventional preparations of interferon alfa have been shown in isolated instances to convert clonal to polyclonal hematopoiesis in PV patients. Our study involved 6 PV patients and 3 ET patients who achieved complete clinical remission after peg-IFN-α-2a therapy. We evaluated both the allelic burden of JAK2V617F mutation, as well as clonality of hematopoiesis by our previously developed quantitative X-chromosome-based transcriptional clonality assay.

All of the PV and ET patients had variable allelic burdens of JAK2V617F before starting the peg-IFN-α-2a therapy. The baseline allelic burden ranged from 11.8% to 93.6%, with a mean percentage of 50.9%. Peg-IFN-α-2a was administered for at least 18 months. Following peg-IFN-α-2a treatment, the allelic burden of JAK2V617F mutation decreased in all patients, with a mean of 11.37% and a range of 2.4% -31.8% (see Table); however, clonal hematopoiesis persisted in some responding patients (see Table, these results in italics). In one ET patient (not in Table), prolonged peg-IFN-α-2a therapy induced clinical remission and resumption of polyclonal hematopoiesis, with a JAK2V617F allelic burden of <1%; however, after discontinuation of peg-IFN-α-2a, the patient relapsed and her hematopoiesis reverted to clonality, yet her JAK2V617F remained at <1%.

In summary, pegylated interferon-alfa-2a induces clinical remission in a large proportion of PV and ET patients. However, at the cellular level, improvement in JAK2V617F allelic burden and resumption of normal hematopoiesis are not always parallel.

Disclosures:

Verstovsek: Incyte Corporation: Research Funding.
Biological and Therapeutic Significance of Thioredoxin-1 (Trx-1) In Diffuse Large B-Cell Lymphomas
Authors: Pham Lan V , Thompson Michael A. , Tamayo Archito , Li Changping , Powis Garth , Ford Richard J.
Published: October 2019

Abstract 2004

Diffuse large B cell lymphomas (DLBCL) are the most common non-Hodgkin (NHL-B) lymphoma. Current treatment is fairly successfully (∼70-80% remission with R-CHOP frontline chemotherapy), but relapse is common (∼50% after 2–3yrs) with poor salvage therapy options and short survival in relapsed/refractory (r/r) DLBCL. The greatest challenge in improving survival of DLBCL patients is overcoming chemo-resistance, which we currently have very little understanding of the basis. Elucidation of molecular pathways and tumor-encoded genes whose expression contribute to the intrinsic resistance and rapid cell growth of lymphoma cells could yield immediate clinical benefits and reveal new therapeutic targets for effective control and treatment of r/r lymphomas. Thioredoxin (Trx) family members play critical roles in the regulation of cellular redox homeostasis. Cancer cells exist in a stressed environment and rely on the Trxs for protection against stress-disregulated redox signaling. The most extensively studied member of the family is Trx-1 whose levels are increased in many human cancers, most likely in direct response to stress. Trx-1 contributes to many of the hallmarks of cancer including increased proliferation, resistance to cell death and increased angiogenesis. Trx-1 is a validated cancer drug target associated with aggressive tumor growth, resistance to standard therapy and decreased patient survival. In this study we showed that the majority of the DLBCL-derived cell lines express high basal levels of thioredoxin-1 (Trx-1) when compared to normal B cells by both Western blotting and real-time PCR. Tissue microarray (TMA) and immunohistochemistry analysis of 100 cases of DLBCL showed that 77% of the cases were positive for Trx-1 and 40% of the cases over-expressed Trx-1. Based on these observations, we hypothesize that aberrant expression of Trx-1 contributes to the development of drug resistance and cell growth and survival phenotype in DLBCL. Inhibition of Trx-1 by small interfering RNA (siRNA) inhibited DLBCL cell line growth in vitro. Down-regulation of Trx-1 also sensitized lymphoma cells to doxorubicin-induced cell growth inhibition. A doxorubicin-resistant human DLBCL cell line (McA), that was highly sensitive to doxorubicin, has been selected by continuously exposing cells to gradually increasing doses of doxorubicin. The resistant phenotype has been retained for over 6 months despite growth in drug-free medium. Western blot analysis detected increased expression of the Trx-1 in the resistant subline. These results indicate that Trx-1 plays a key role in growth and survival, as well as chemoresistance in the pathophysiology of DLBCL. Based on these data, we plan to use small molecule inhibitors in vitro as a proof of principle for possible future human phase I studies.

Disclosures:

Ford: Cell Point: Research Funding.
Protein Kinase C Delta Mediates the Activation of Protein Kinase D In Platelets
Authors: Bhavanasi Dheeraj , Kim Soochong , Goldfinger Lawrence E , Kunapuli Satya P.
Published: October 2019

Abstract 2021

Protein kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCm, PKD2 and PKCn in humans). They are part of alternate DAG receptors along with RasGRPs, Munc 13s, chimerins and DAG kinases. Members of the novel class of PKC isoforms such as PKCd, PKCh, and PKCe associate with PKD in smooth muscle cells and COS-7 cells. The mechanism of activation of PKD and the specific PKC isoforms required for its activation are not known to date. This study is aimed at investigating the pathways involved in the activation of PKD in platelets. We show that human as well as murine platelets express PKD. PKD could be activated with PAR4 agonist AYPGKF, PAR1 agonist SFLLRN and GPVI agonist convulxin. AYPGKF and SFLLRN induced PKD phosphorylation as early as 30 sec and convulxin induced PKD phosphorylation at 1 minute. PKD phosphorylation induced by AYPGKF and convulxin were sustained for 5 minutes but phosphorylation induced by SFLLRN was attenuated after 2 minutes of stimulation. AYPGKF-induced PKD phosphorylation was reduced with a calcium chelator, dimethyl BAPTA, indicating that calcium-mediated signals might play a role in activation of PKD. PKD phosphorylation in response to AYPGKF was abolished with a Gq inhibitor, YM-254890, but was not affected by Gi-coupled P2Y12 receptor antagonist ARC-69931MX, indicating that PKD phosphorylation is Gq-, but not Gi- or G 12/13-dependent. PKD phosphorylation was abolished with pan-PKC inhibitors, GF109203X and Ro31-8220, indicating that PKCs are required for PKD activation in platelets. PKD phosphorylation was significantly inhibited with a PKC delta inhibitor, rottlerin, but was not affected by the classical PKC inhibitor, Go6976, suggesting that novel PKC isoforms are important for PKD activation. In addition, 2MeSADP that fails to activate PKCd did not induce phosphorylation of PKD in platelets. Furthermore, phosphorylation of PKD induced by AYPGKF was significantly reduced in PKCd-deficient platelets compared to that of wild type platelets. Hence, we conclude that PKD is a common signaling target downstream of various agonist receptors in platelets, and Gq-mediated signals and novel PKC isoforms, in particular PKCd is required for activation of PKD.

Disclosures:

No relevant conflicts of interest to declare.
Transcriptional Regulation of Transferrin Receptor by Heme in Erythroid Cells
Authors: Schranzhofer Matthias , Lok Nam-Chun , Schifrer Manfred , Muellner Ernst W , Ponka Prem
Published: October 2019

Abstract 2049

Erythroid cells are the largest consumers of iron which is delivered to them by tansferrin (Tf) by its cognate receptor (TfR). In contrast to other cells, developing red blood cells (RBC) regulate TfR expression not only at the level of mRNA stability via the iron regulatory proteins (IRP) 1 and 2, but also by transcription (Lok & Ponka, J Biol Chem 275:24185-90, 2000). Here we provide evidence that TfR expression and cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. Incubation of erythroid cells with 5-aminolevulinic acid (ALA) increased TfR expression accompanied by increased iron incorporation into heme. This effect of ALA can be completely prevented by the inhibitors of heme biosynthesis (succinylacetone [blocks ALA dehydratase] or N-methylprotoporphyrin [blocks ferrochelatase]), indicating that the effect of ALA requires its metabolism to heme. The induction of TfR mRNA expression by ALA is mainly a result of increased mRNA synthesis since the effect of ALA can be abolished by actinomycin D. Recently, IRP2 was proposed to play a role in maintaining TfR mRNA stability in developing RBC (Cooperman et al., Blood 106:1084-91; 2005; Galy et al., Blood 106:2580-9, 2005). Importantly, we have demonstrated that ALA added to cultures of erythroid cells derived from IRP2 knock out mice restores the expression of TfR to levels observed in cells obtained from wild type mice. In conclusion, our results indicate that in erythroid cells heme serves as a positive feedback regulator that maintains high TfR levels thus ensuring adequate iron availability for hemoglobin synthesis.

Disclosures:

No relevant conflicts of interest to declare.
CD137 Identifies a Population of Regulatory T Cells That Inhibit Anti-Tumor Immune Responses In Adoptive Immunotherapy
Authors: Goldstein Matthew J , Kohrt Holbrook E , Houot Roch , Varghese Bindu , Lin Jack T , Swanson Erica , Levy Ronald
Published: October 2019

Abstract 2093

Background:

Adoptive immunotherapy is a promising novel approach to the treatment of cancer. However, clinical translation of adoptively transferred CD4 T cells is limited by cotransfer of an inhibitory population of regulatory CD4 T cells (Tregs). We identified a method of isolating viable antitumor CD4 T cells while excluding Tregs based on two surface markers—CD44 and CD137.

Methods:

We have developed a model for adoptive cell therapy of lymphoma whereby anti-tumor T cells are generated in vivo through vaccination with a CpG-loaded whole cell vaccine (CpG/H11). These vaccine-induced cells can protect from lethal tumor challenge when isolated and transferred into lethally irradiated, syngeneic recipient mice. We investigated the subsets of T cells involved in the anti-tumor response through a combination of in vitro and in vivo assays.

Results:

Adoptive transfer of CD137negCD44hi CD4 T cells, but not other sub-populations, provided protection from B cell lymphoma. We demonstrate that the population of CD137posCD44hi CD4 T cells consists primarily of activated Tregs. In vitro, these CD137pos cells suppressed the proliferation of effector cells in a contact-dependent manner. We observed that this CD137pos Treg population persisted following adoptive transfer and maintained expression of FoxP3 as well as CD137. Moreover, the addition of CD137posCD44hi CD4 cells to CD137negCD44hi CD4 cells suppressed the antitumor immune response. In the presence of CD137posCD44hi CD4 T cells, homing of other T cell populations to tumor sites was disrupted. These results suggest that CD137 expression on CD4 T cells defines a population of activated Tregs that prevent antitumor immune responses.

Conclusions:

Our findings identify two surface markers that can be used to facilitate the enrichment of anti-tumor CD4 T cells while depleting an inhibitory Treg population. This approach has direct applicability to clinical trials for patients with lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
Normal Cleavage of Von Willebrand Factor by ADAMTS13 In the Absence of Factor VIII In Patients with Severe Hemophilia A
Authors: Chen Junmei , Chung Dominic W. , Le Jennie , Konkle Barbara A. , López José A.
Published: October 2019

Abstract 2114

The size of von Willebrand factor (VWF), a carrier protein for factor VIII (FVIII), is regulated by plasma metalloprotease ADAMTS13 proteolytic activity. Recently studies by Cao et al. (PNAS, 2008; 105: 7416–7421) found that under shear stress, exogenous FVIII enhanced ADAMTS13 cleavage of VWF, especially the high molecular weight multimers, in a system using purified proteins. Based on this result, the authors suggested that in the absence of FVIII, such as in patients with severe hemophilia A, VWF will have ultra-large multimers due to defects in ADAMTS13 proteolytic process, which can be corrected by infusion of FVIII. Here, we assessed VWF multimers, antigen, and ADAMTS13 activity in citrated plasma from seven patients with severe hemophilia A. The FVIII levels in six patients were less than 1% and in one was 4%. Plasma from two patients was available both pre- and post-FVIII replacement therapy (recombinant FVIII). All patients displayed VWF multimer patterns similar to those in pooled normal plasma (PNP), and the two patients receiving FVIII infusions displayed no change in VWF multimer size or pattern between their pre- and post-infusion samples. In all patients, the VWF antigen level (0.32–0.76) was below the PNP value (designated as 1), and all had increased ADAMTS13 activity (measured by the ability of plasma to cleave a small A2 peptide substrate) (1.09–1.79, PNP designated as 1), yielding an increased ratio of ADAMTS13 activity to VWF antigen in these patients (1.4–5.2 compared to PNP). We also examined cleavage of endogenous VWF by ADAMTS13 in the plasmas of the two patients studied pre- and post-infusion, yielding different FVIII levels. In this assay, we first diluted plasma 10-fold with a buffer containing 10 mM HEPES, 6.5 mM BaCl2, and 1.5 M urea, incubated at 37°C, and ADAMTS13 cleavage was stopped at different time points with EDTA. VWF multimer patterns were examined on a 1.5% agarose gel. We found that ADAMTS13 cleaved VWF efficiently in patient plasma deficient in FVIII, similar to that in PNP. The extent of cleavage was correlated with the ratio of ADAMTS13 activity to VWF antigen, rather than with the FVIII levels. In conclusion, patients with severe hemophilia A appear to have normal ADAMTS13 processing of VWF multimers in vivo and ex vivo. Further studies of the effect of FVIII and VWF levels on ADAMTS13 cleavage of VWF and clinical correlation are needed.

Disclosures:

No relevant conflicts of interest to declare.
Factor X/Xa Elicits Protective Signaling Responses In Endothelial Cells Directly Via PAR-2 and Indirectly Via EPCR-Dependent Recruitment of PAR-1
Authors: Rezaie Alireza R. , Bae Jong-Sup , Yang Likui
Published: October 2019

Abstract 2115

We recently demonstrated that the Gla-domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier protective response if endothelial cells were pre-incubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells, also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a crosstalk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla-domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.

Disclosures:

No relevant conflicts of interest to declare.
To Study Resistant Mechanisms and Reversal In An Imatinib Resistant Ph+ Positive Acute Lymphoblastic Leukemia Cell Line
Authors: Xing Hongyun , Gong Yuping , Liu Ting
Published: October 2019

Abstract 2135

Objective

To establish an imatinib resistant Bcr-Abl positive acute lymphoblastic leukemia (ALL) cell line in vitro and to study imatibin resistance in Ph+ ALL. The reversal of the imatinib resistance by rapamycin, the second generation tyrosine kinase inhibitor and proteasome inhibitor was studied.

Methods

Ph(+) ALL SUP-B15 cell line was cultured in gradually increasing concentrations of imatinib to generate the imatinib resistant cell line at 6 μM imatinib. The cytotoxic effect of imatinib and other drugs was analyzed by MTT assay. RT-PCR, flow cytometry, Western blot analyses of proteins, DNA sequence analysis of ABL kinase domain were used to clarify the possible mechanisms of the imatinib resistance in the SUP-B15/RI cell line.

Results

We established the imatinib resistant Ph+ ALL cell line. The fusion bcr-abl gene was 6.1 times as high as that of the parental sensitive cell, and the mdr1 gene also increased 1.7 times in SUP-B15/RI cell line by the RT-PCR detection. However, the expression of hoct1 Abcl–2 and topoIIα gene were no difference between two cell lines by the RT-PCR detection. A K362S point mutation in the Abl kinase domain of SUP-B15/RI was found. The detection of cell signaling pathway of PI3K/AKT/mTOR, RAS/RAF, NF-κBA JNK and STAT showed the expression of PTEN and 4EBP-1 was down-regulated, AKT, mTOR and P70S6K was up-regulated and the expression of other cell signaling pathways in SUP-B15/RI was similar to its parental sensitive cell line. Dasatinib, nilotinib, and bortezomib could inhibit proliferation of SUP-B15/RI cells at nM concentration. SUP-B15/RI cell line also showed partial resistance to dasatinib and nilotinib, but not bortezomib. The combination of imatinib with rapamycin had synergistic effect to the resistance cell line.

Conclusion

In vitro, we establish imatinib resistant Ph + ALL cell line. Overexpression of bcr-abl and mdr1 gene, K362S point mutation in ABL kinase domain and up-regulation of the cell signaling pathways of PI3K/AKT/mTOR, RAS/RAF in SUP-B15/RI cell line were involved in the resistance mechanisms. The SUP-B15/RI cell line was also resistant to the second generation tyrosine kinaeses dasatinib and nilotinib,not bortezomib in vitro. However, the combination of imatinib with rapamycin can partially overcome the resistance. Blockade of the ubiquitin-proteasome could be a promising pathway to overcome resistance to imatinib.

Disclosures:

No relevant conflicts of interest to declare.
The Synonymous V107V Mutation In Factor IX Is Not So Silent and May Cause Hemophilia B In Patients
Authors: Kimchi-Sarfaty Chava , Simhadri Vijaya L , Kopelman David , Friedman Adam , Edwards Nathan , Javaid Atiq , Okunji Chinyere , Komar Anton , Sauna Zuben , Katagiri Nobuko
Published: October 2019

Abstract 2197

Hemophilia B is characterized by structural and functional defects in coagulation factor IX (FIX) caused by mutations in the F9 gene. Various mutations (nonsense, missense, etc.) are known to be associated with the disease, including a synonymous V107V mutation reported recently by Knobe and colleagues (Knobe et al., Hemophilia, 2008). However the mechanism by which this synonymous mutation contributes to the disease has not yet been elucidated. Earlier we have shown that synonymous codon substitutions in the mRNA of the multidrug resistance protein (MDR1) may change the conformation of the protein and result in altered functionality (Kimchi-Sarfaty et al., Science, 2008). Here we have performed in silico analyses of the synonymous codon substitution (GTGàGTA) leading to the V107V polymorphism and found that it may change the mRNA structure, stability, codon usage, and 3D structure of the encoded protein. We hypothesize that changes in codon usage might affect the rhythm of protein translation and thus result in slightly altered FIX conformation. In vitro analyses of FIX mRNA and protein expression supported our in silico analyses. The GTGàGTA (V107V) synonymous mutation results in reduced expression levels as well as an encoded protein with a slightly different conformation compared to wild-type FIX. These results show that the V107V polymorphism is not silent and might cause mild hemophilia B. This work sheds further light on ways in which synonymous mutations impact disease.

The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination policy

Disclosures:

No relevant conflicts of interest to declare.
Ideal Amphipathic Peptdes Coupled to Nanofibrous Micropheres Reduce Hemorrhage In Vivo
Authors: Lemarie Catherine , Wu Jianqiu , Barralet Jake , Blostein Mark
Published: October 2019

Abstract 2204

We have previously shown that ideal amphipathic peptides (IAP) composed simply of leucine and lysine residues can augment the function of the procoagulant enzyme, factor IX in vitro (Biochem J 2008). We have also shown that surface attachment of IAP can promote hemostasis and reduce bleeding in vivo. We extend these observations by coupling IAPs to a nanomaterial called nanofibrousmicrospheres (NFM). NFMs have a large surface area to volume ratio thereby providing greater surface area and better access to bleeding surfaces especially those that are internal and difficult to reach. Initially we demonstrate, by fluorescently labeling IAP with dansyl chloride, that IAP attaches to NFM. When free dansyl IAP is added to NFM, 90% binds to the surface of NFM via a non-covalent linkage. Both a murine liver laceration model and a murine tail transection model were established to evaluate the in vivo hemostatic properties of IAP attached to NFM (IAP-NFM). In a murine liver laceration model, where the liver is cut with a Surgicutt template, application of IAP-NFM significantly reduces bleeding times by 40% as compared to application of NFM alone (p<0.01), or no treatment (p<0.01). As well, comparison of IAP-NFM to QuikClot, a current commercially used product for control of hemorrhagic injuries, show a greater reduction in bleeding times after application of IAP-NFM as compared to QuikClot. In addition, histologic examination of the lacerated liver after treatment reveal that IAP-NFM elicited little tissue injury whereas widespread hepatocyte injury was caused by QuikClot, due to its known exothermic reaction on tissue. The bleeding time in a murine tail transection model was also reduced by approximately 30% by IAP-NFM, as compared to NFM alone (p<0.01), or no treatment (p<0.05). In conclusion, IAP attachment to NFM results in a biocompatible material that is procoagulant and can promote hemostasis in vivo with the advantage that it exhibits little tissue injury after its application.

Disclosures:

No relevant conflicts of interest to declare.
Association of Quantitative and Qualitative Abnormalities of Von Willebrand Factor and Risk of Death In Hemodialysis Patients
Authors: Holden Rachel , Tuttle Angie , MacLeod Francis , Burbidge Toni , Hegadorn Carol , James Paula D.
Published: October 2019

Abstract 2217

In order to evaluate the possible role of abnormalities of von Willebrand factor in the hemostatic defects seen in indivdiuals with chronic kidney disease (CKD), a cohort study was performed evaluating pre- and post-dialysis levels of von Willebrand factor (VWF), VWF multimer profiles and levels of its cleaving protease, ADAMTS-13. There were 57 subjects (31 males, 26 females) enrolled with CKD with a mean age of 75 years (range 60 – 90). Subjects with known vascular disease were recruited; 49 (86%) had documented ischemic heart disease, 16 (29%) had cerebrovascular disease and 17 (31%) had peripheral vascular disease. A little over half had diabetes mellitus (30 subjects or 54%), 37 (67%) were on antiplatelet therapy and 7 (13%) were chronically anticoagulated with warfarin. Blood samples were drawn immediately pre- and again post-dialysis and all results were compared with a group of age-matched normal controls (Table 1). As has been previously reported, VWF antigen levels (VWF:Ag) and VWF functional activity as measured by the ristocetin cofactor assay (VWF:RCo) were higher in the pre-dialysis samples compared with controls, and both levels were increased even further following dialysis. Additionally, the percentage of high molecular weight VWF multimers (% HMWM) were significantly increased in the pre-dialysis samples compared with controls. This is a novel finding, and the level of % HMWM seen in the subjects is similar to what has been reported in individuals with Thrombotic Thrombocytopenic Purpura (TTP). This difference decreased following dialysis, potentially due to the effect of shear stress on VWF and the resultant proteolytic processing, however still remained significantly higher when compared with controls. ADAMTS-13 functional activity was lower in the subjects compared with controls, providing a possibly explanation for the increase in % HMWM. IL-6 levels are higher in subjects compared with controls. IL-6, which is an inflammatory cytokine known to be increased in patients with CKD, has been previously reported as a marker of inactivation of ADAMTS-13. Two years after enrollment, follow up of the subjects revealed that 22 had died, 17 from documented cardiovascular events. Higher VWF levels at the time of enrollment significantly correlated with risk of death (p=0.041) during the study period. The increase in % HMWM suggests that a “TTP-like phenotype” may also be playing a role. Taken together, these data suggest that both quantitative and qualitative abnormalities of VWF contribute to the risk of thrombotic death in chronic kidney disease.

Disclosures:

No relevant conflicts of interest to declare.
BPI A645G SNP but Not NOD2 Genotype Predicts for Acute Lung Injury After Allogeneic Stem Cell Transplantation
Authors: Wermke Martin , Maiwald Stefanie , Thiede Christian , Schetelig Johannes , Ehninger Gerhard , Platzbecker Uwe , Bornhäuser Martin , Wassmuth Ralf
Published: October 2019

Abstract 2324

Introduction:

Infectious as well as non-infectious pulmonary complications are key determinants of non-relapse mortality (NRM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Single nucleotide polymorphisms (SNP) in two genes coding for nucleotide-binding oligomerization domain containing 2 (NOD2) and bactericidal/permeability-increasing protein (BPI) have been shown to predict for the occurrence of GvHD and chronic pulmonary complications after allo-HSCT. We aimed at determining the effect of SNPs in these genes on the occurrence of acute lung injury (ALI) after allo-HSCT.

Patients and Methods:

We retrospectively analyzed a cohort of 272 patients undergoing allo-HSCT and their respective related and unrelated donors. ALI was functionally defined as a drop of the oxygenation index (OI = partial pressure of oxygen in arterialized capillary blood/inhaled oxygen concentration) below 300. Genotyping for NOD2 SNP 8, 12 and 13 and BPI SNP A645G was done using Real Time-PCR. The incidence of ALI was assessed by univariate competing events statistics and by a multivariate model according to Fine and Gray.

Results:

Overall, 115 of 272 (42.3%) patients suffered from ALI at least once during the first 100 days post allo-HSCT. Median time to onset was 11 days. Neither NOD2 polymorphisms in the donor nor in the recipient correlated with the occurrence of ALI. Further, BPI recipient genotype was not associated with an increased incidence of this complication. On the other hand, ALI was more frequent in patients receiving a transplant from a donor homozygous for the G allele of the BPI A645G SNP (Figure 1). This association was confirmed in multivariate analysis (OR 1.46, p = 0.048). Other independent risk factors for the occurrence of ALI were age (as continuous variable, OR 1.03, p = 0.002) and transplantation from an unrelated vs. related donor (OR 1.66, p = 0.018) whereas the use of intensive conditioning was a risk factor of borderline significance (OR 1.59, p = 0.077). In spite of its association with ALI, BPI donor genotype did not correlate with cumulative incidence of NRM at 100 days (G/G: 6.7%, 95% CI: 1.0 – 12.4% vs. A/A or A/G: 10.2%, 95% CI: 6.0 – 14.4%; p = 0.380) or overall survival.

Conclusion:

In contrast to other investigators we found no correlation between NOD2 genotype and the occurrence of acute pulmonary complications after allo-HSCT. On the other hand, BPI A645G SNP in the donor was identified as an independent risk factor for ALI in these patients.

Disclosures:

No relevant conflicts of interest to declare.
HLA-Haploidentical Stem Cell Transplantation for Malignant Hematological Diseases Using G-CSF Mobilized Bone Marrow and Peripheral Blood Stem Cells
Authors: Luo Yi , Tan Yamin , Lai Xiaoyu , Shi Jimin , Zheng Weiyan , Huang He
Published: October 2019

Abstract 2384

Introduction:

Allogeneic haematopoietic stem cell transplantation (HSCT) has been used to treat malignant hematological diseases. For patients who do not have a matched sibling donor or a matched unrelated donor (MUD) for transplantation, an eligible HLA-haploidentical donor can be identified rapidly in nearly all cases. However, HLA-haploidentical BMT has been associated with significant risks of complication, such as graft rejection and severe GVHD and infection.

Materials and Methods:

Thirty eight consecutive patients with malignant hematological diseases received HLA-haploidentical stem cell transplant from April 2008 to June 2010. Eligible patients were 9– 48 years of age with acute myeloid leukemia (n=10),acute lymphocytic leukemia (n=17), myelodysplastic syndrome(n=5), chronic myeloid leukemia (n=4), non Hodgkin lymphoma (n=2). Transplantation conditioning consisted of Ara-C (4 g/m2/d, i.v.) on day -10 and -9, Bu (9.6mg/kg i.v. in 12 doses) on day -8, -7 and -6, Cy (1.8 g/m2/d, i.v.) on day -5 and -4, Me-CCNU (250 mg/kg, i.v.) on day -3, and ATG (2.5 mg/kg/d i.v.) on day -5 to -2. The prophylaxis of aGVHD consisted of cyclosporine A (CsA), mycophenolate mofetil (MMF), and short-term methotrexate. Donors were primed with rhG-CSF (7.5 mg/kg per day) injected subcutaneous (s.c.). Bone marrow cells and peripheral blood stem cells(PBSC) were harvested in nineteen donors, and only PBSC were harvested in the other 19 donors.

Results:

Primary engraftment was achieved in all patients. All the patients achieved complete donor chimerism in the peripheral blood before day +28. Seven patients (18.4%) had grade I aGVHD, ten (26.3%) had grade II aGVHD, four(10.5%) had grade III aGVHD and four (10.5%) had grade IV aGVHD. II-IV aGVHD occurred in nine patients treated with bone marrow cells and PBSCs, the same number patients suffered from II-IV aGVHD treated with only PBSCs. Transplant related mortality within 100 days after transplantation was 15.8%. At a median follow-up of 10 months (range 2–28months), the overall survival was 68.4%.

Conclusion:

Haploidentical stem cell transplantation is relatively safe and efficient for the patients who have no HLA matched donors.The results require to confirm and show that G-BM combined with PBSC or G-PBSC from haploidentical family donors have equal effect.

Disclosures:

No relevant conflicts of interest to declare.
The York Platelet Syndrome: A Third Case and New Findings
Authors: White James G , Professor Regents , Gunay-Aygun Merel
Published: October 2019

Abstract 2526

Studies published in 2003 described a mother and son with a mitochondrial myopathy and abnormal platelets containing giant electron opaque organelles and large, multilayered organelles resembling targets. The condition has remained a familial disorder until recently when a new patient, a four year old female, with the same myopathy and platelet ultrastructure was discovered. Now the disorder can be considered a syndrome named after the initial family, the York Platelet Syndrome (YPS). Whole mount preparations of unfixed, unstained YPS platelets revealed that the giant opaque organelles (OO) were as inherently electron dense as delta granules (dense bodies, DB) but several times larger. Analytical electron microscopy revealed both organelles were rich in phosphorous and calcium, suggesting they might have the same origin. However, measurements of serotonin and adenine nucleotides in YPS platelets revealed normal levels. Also, staining YPS platelets for acid phosphatase with cerium as the capture ion and using analytical electron microscopy to determine its location in whole mount preparations revealed the presence of cerium in the giant OO, but not in DB. The findings suggested the OO might be giant secretory organelles. However, exposure of YPS platelets to high dose thrombin caused complete secretion of α granules and DB, but OO and TO were not released through the open canalicular system or the surface membrane. Thus, OO and TO are not secretory organelles. Studies of YPS in the new patient have confirmed and extended these findings. Staining her cells with diaminobenzidine and H202 to detect platelet peroxidase has revealed enzyme reaction product in both OO and TO, as well as in channels of the dense tubular system. Thus, the giant OO and TO organelles contain proteins and elements usually separated into vesicles in the Golgi Complex and reassembled into alpha granules, dense bodies and lysosomes in megakaryocytes. Instead proteins and elements from the rough and smooth endoplasmic reticulum (SER) develop into giant OO and TO without passing through lamellae of the Golgi complex. Involvement of the SER and DTS in formation of the giant organelles is strongly supported by YPS platelets from the new patient. Large, flat sheets and coils of SER were a common finding in the new YPS patient platelets, and present, but less frequent in the original two YPS patients. The findings strongly support the concept that the YPS in a disorder of megakaryocyte endoplasmic reticulum allowing the formation OO and TO which continue to develop in the DTS of YPS circulating platelets. It is the first such disorder to be described in human megakaryocytes and platelets.

Disclosures:

No relevant conflicts of interest to declare.
Acetylation-Dependent Interaction of GATA-1 with the Potential Mitotic “Bookmarking” Protein Brd3.
Authors: Lamonica Janine M , Kadauke Stephan , Deng Wulan , Blobel Gerd
Published: October 2019

Abstract 2598

Acetylation of the transcription factor GATA-1 facilitates its ability to drive erythroid differentiation by enhancing its association with in vivo target sites. However, the underlying mechanism through which GATA-1 acetylation functions has remained elusive. To test whether GATA-1 acetylation serves to recruit essential cofactors, we performed a peptide affinity screen and identified Brd3 as an acetylated GATA-1 interacting partner. Brd3 belongs to the BET protein family that also includes Brd2, Brd4, and Brdt, and is characterized by tandem bromodomains (BD1 and BD2) and an extraterminal (ET) domain. We show that Brd3 and GATA-1 physically interact in an acetylation-dependent manner in vitro and in vivo. Mapping studies revealed that the interaction depends on BD1 of Brd3 and one of the two major acetylation sites that resides near the C terminal zinc finger of GATA-1. By ChIP-seq and ChIP-qPCR, endogenous Brd3 is recruited to virtually all GATA-1-occupied regulatory elements in erythroid cells, including both GATA-1activated and repressed genes. Although Brd3 has been reported to associate with acetylated histones along the entire length of transcribed genes, we found that Brd3 recruitment correlates poorly with histone acetylation along gene bodies. In agreement with our biochemical data, an intact BD1 is essential for the in vivo recruitment of Brd3 to GATA-1-occupied elements, further demonstrating that acetylation of GATA-1 is essential for Brd3 association in vivo. Notably, a pharmacological compound that targets acetyl lysine binding sites in BD1 and BD2 disrupts the Brd3/GATA-1 interaction in vitro, diminishes Brd3 and GATA-1 association at key erythroid genes in vivo, and impairs GATA-1 target gene expression and erythroid maturation. In concert, these findings suggest a mechanism by which the first bromodomain of Brd3 recognizes acetyl-lysines on GATA-1 to facilitate GATA-1 chromatin occupancy. These studies raise an interesting question: In contrast to the great majority of transcription factors, BET family proteins bind to chromatin during mitosis and might serve an epigenetic memory function to properly reactivate gene transcription upon exit of mitosis. We are currently investigating whether Brd3 functions by bookmarking GATA-1-bound sites throughout mitosis to aid in transcriptional memory and stability of lineage specific gene expression.

Disclosures:

No relevant conflicts of interest to declare.
Natalizumab Increases Circulating CD34+ Cells with An Impaired Functional Potential In Patients with Multiple Sclerosis without Affecting the Bone Marrow.
Authors: Saure Christian , Zohren Fabian , Schroeder Thomas , Bruns Ingmar , Cadeddu Ron Patrick , Fischer Ute , Kobbe Guido , Hartung Hans Peter , Kieseier Bernd C , Haas Rainer
Published: October 2019

Abstract 2620

Introduction:

In our previous report (Zohren et al., Blood 2008) we could show that the blockade of the heterodimer VLA-4 by the monoclonal IgG4 antibody natalizumab leads to a significant increase in circulating CD34+ cells in patients with multiple sclerosis (MS). We now extend our analysis on the influence of natalizumab on CD34+ cells comparing bone marrow (BM) and peripheral blood (PB) derived CD34+ cells of natalizumab patients with those from healthy donors.

Methods:

A total of 83 patients with MS receiving natalizumab were included. In vitro adhesion, migration and apoptosis assays as well as LTC-IC of immunomagnetically enriched CD34+ cells were conducted. Flow cytometric analyses were performed to assess phenotype and composition of the CD34+ subsets.

Results:

The median concentration of circulating CD34+ cells was significantly greater compared to normal donors (7.7/μL vs. 1.8/μ L; p= 0.0001) and remained relatively stable during a one year treatment with natalizumab. Leukocyte cell counts, the number of T cell subsets as well as the number of CD19+ B cells and CD56+ natural killer cells were in normal range in PB and BM after short- and long-term treatment with natalizumab. However, we found significantly reduced adhesion and migration abilities of circulating CD34+ cells under natalizumab treatment in comparison to G-CSF mobilized CD34+ cells of healthy donors. Moreover, the self-renewal capacity of these cells was poor. In contrast, no significant difference was seen between the BM of natalizumab patients and the BM of healthy donors with regard to cellularity and proportion of CD34+ cells. In addition, neither co-expression of CD49d nor the adhesion ability of the BM derived CD34+ cells revealed a significant difference between the two collective.

Conclusions:

Our data indicate that natalizumab mediates an increase in circulating CD34+ cells by impaired homing. These findings argue against the use of natalizumab-exposed PB CD34+ cells for transplantation.

Disclosures:

No relevant conflicts of interest to declare.