bluuebunny/crossref_metadata_2025 huggingface dataset:
The 3'-5' Exonuclease, TREX1, Interacts with Poly(ADP-ribose) Polymerase-1 (PARP1) in Response to DNA Damage
Authors: Miyazaki Takuya , Kim Yong-Soo , Yoon Jeong-Heon , Wang Hongsheng , Morse Herbert C.
Published: October 2019

Abstract 1046

Background:

Recognition of nucleic acids plays a key role in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). The major 3'-5' DNA exonuclease, TREX1, degrades single- and double-stranded DNA to minimize potential immune activation by persistent self or retroelement DNA. Mutations in the human TREX1gene have been linked to four diseases: SLE; Aicardi-Goutières syndrome (AGS); retinal vasculopathy and cerebral leukodystrophy (RVCL); and familial chilblain lupus (CBL). SLE, AGS and CBL have overlapping features characterized by expression of interferon-alpha and autoantibody production, but are distinct clinical conditions. RVCL is a non-inflammatory endotheliopathy.

Methods:

As part of our efforts to determine the molecular mechanisms underlying TREX1 involvement in the pathogenesis of these diseases, we identified a novel TREX1-binding partner protein by Mass Spectrometry and Co-Immunoprecipitation (Co-IP) assays. We examined the interaction domains and the effects of mutations in TREX1 associated with these diseases.

Results:

We found that TREX1 interacted with poly(ADP-ribose) polymerase-1 (PARP1), a nuclear DNA repair enzyme involved in the DNA damage response. The interaction domain of PARP1 was mapped to the amino-terminal zinc finger domains. Subcellular localization analysis showed that TREX1 normally localized to the cytoplasm and cytomembranes, but translocated to the nucleus (with some bound to chromatin) in response to sublethal DNA damage. Co-IP assays revealed that the interaction was increased following DNA damage in concert with the post-translational PARlation of PARP1. Functional studies showed that TREX1 induced the PARlation of PARP1 in a TREX1 dose-dependent manner. Furthermore, siRNA knockdown of TREX1 promoted PARP1 cleavage and loss of PARP1 enzymatic activity, resulting in the induction of apoptosis. We further analyzed the effects of TREX1-PARP1 interaction in relation to the pathogenesis of AGS or RVCL. Co-IP assays revealed that the RVCL-related TREX1-mutant protein, V235fs, bound to PARP1 as well as wild-type TREX1, whereas the AGS-related R114H mutant protein did not bind PARP1.

Conclusion:

These findings demonstrate that TREX1 contributes to the regulation of PARP1 in the DNA damage response and that mutation-induced alterations in the function of TREX1 may be a factor in the development or progression of these autoimmune diseases by affecting PARP1 activity.

Disclosures:

No relevant conflicts of interest to declare.
Acyl-Protein Thioesterase 1 Functions in Palmitoylation/Depalmitoylation Cycles of G Proteins and Regulates Platelet Activation
Authors: Dowal Louisa M. , Dilks James R. , Fadel Nathalie A. , Aisiku Omozuanvbo R. , Merrill-Skoloff Glenn , Flaumenhaft Robert C.
Published: October 2019

Abstract 1062

Protein palmitoylation is a reversible post-translational modification that regulates both lipid-protein and protein-protein interactions. During the palmitoylation cycle, palmitoylation occurs through a thioester linkage to a cysteine residue. Depalmitoylation occurs primarily through cleavage of this bond by acyl-protein thioesterase 1 (APT1). We have previously demonstrated the presence of APT1 in platelets and showed that APT1 translocates to membranes in an activation-dependent manner. However, the function of APT1 in platelet activation is not known. To determine whether APT1 functions in platelet signal transduction we evaluated the effect of palmostatins, novel small molecule inhibitors of APT1, on platelet function. Palmostatins B and M both inhibited platelet aggregation and α -granule secretion induced through protease-activated receptor (PAR) 1 with an IC50 of 15 μM. To assess which signaling pathways were affected by APT1 inhibition, we screened palmostatins for their ability to inhibit activation induced by several agonists. Palmostatins blocked platelet aggregation induced by a PAR1 agonist, a PAR4 agonist, TxA2, or epinephrine. In contrast, palmostatins failed to inhibit aggregation induced by collagen, PMA, or ionophore. Palmostatins also inhibited α -granule exocytosis induced by a PAR1 agonist or TxA2, but not exocytosis induced by PMA or ionophore. These results suggested that palmostatins blocked proximal signaling events mediated through G protein coupled receptors (GPCRs). To evaluate this supposition, we tested the effect of palmostatin B on PAR1-mediated [Ca2+]i flux. Palmostatin B inhibited PAR1-induced Ca2+ signaling with and IC50 of 15 μM, the same concentration required for inhibition of platelet aggregation and α -granule secretion. We have recently described the platelet palmitoylome (Dowal et al., Blood, 118:e62-73) and found several components of the proximal G protein signaling pathway that are palmitoylated, including many Gα subunits. To directly assess the effect of APT1 inhibition on palmitoylation/depalmitoylation cycles on a target Gα subunit, we evaluated Gα q palmitoylation using acyl biotin exchange chemistry. Total Gα q palmitoylation decreased substantially with activation of platelets through PAR1. In the presence of palmostatin B, however, Gα q palmitoylation increased following PAR1 activation. These results demonstrate that Gα q is a substrate for APT1. Our studies demonstrate a role for palmitoylation/depalmitoylation cycles in proximal signaling pathways downstream of GPCRs and implicate APT1 as an essential regulator of G protein signaling in platelets.

Disclosures:

No relevant conflicts of interest to declare.
Role of Gas6 in Cancer-Induced Venous Thrombosis
Authors: Aghourian Meghedi , Lemarie Catherine A , Blostein Mark D
Published: October 2019

Abstract 108

Venous thrombosis is the most common morbid complication related to cancer and its treatments. The onset of cancer has been shown to predispose patients to hemostatic disorders mainly because of the pro-thrombotic milieu generated by the cancer. Growth arrest specific 6 (gas6) is a vitamin K dependent secreted protein that is expressed by different cell types. Gas6 and its receptor Axl are overexpressed in different cancers. Gas6 null mice have been shown to be protected against life-threatening thrombosis. Because of the association of gas6 with cancer and thrombosis, the objective of the present study is to elucidate the role of gas6 in cancer-induced venous thrombosis. Thus, we hypothesize that the absence of gas6 in a mouse model of cancer would reduce venous thrombosis. Wild type (WT) and gas6 null (−/−) mice received an intravenous injection of M27 murine lung cancer cells. After 2 weeks, WT and gas6−/− mice developed end stage cancer. Venous thrombosis was induced using 10% ferric chloride. Thrombus surface area was evaluated using ultrasonography, 30 minutes after ferric chloride application. We found that thrombi induced in mice with cancer were smaller than those induced in WT mice, by both ultrasound (p<0.05) and histologically (p<0.05). To elucidate the role of gas6 in cancer-induced thrombosis, we co-cultured murine endothelial cells, prepared from lungs of both WT and gas6−/− mice, with M27 cells. RNA was extracted from endothelial cells and we used a whole genome microarray analysis to identify differential gene expression induced by cancer in the presence or absence of gas6. Microarray analysis revealed that 28 genes were differentially expressed in WT or gas6−/− cells in the presence of cancer. Of interest, we found that thrombospondin 2 was downregulated by cancer cells in WT endothelial cells and even further decreased in gas6−/− cells. We confirmed these results using real-time qPCR and western blot analysis (p<0.05). These results suggest the possibility that gas6, through the downregulation of thrombospondin 2, has a pathophysiologic role in cancer-induced thrombosis.

Disclosures:

No relevant conflicts of interest to declare.
Thrombopoietin Can Induce Tolerogenic Responses Via the Modulation of Thymic and Splenic T Lymphocyte Populations in a Murine Model of Immune Thrombocytopenia (ITP)
Authors: Semple John W. , Zhang Xu , Hu Yu , Guo Li , Speck Edwin R. , Ni Heyu , Aslam Rukhsana
Published: October 2019

Abstract 1088

Clinical trials using thrombopoietin (TPO) mimetics have clearly shown their benefit in raising platelet counts in patients with immune thrombocytopenia (ITP), however, little is known whether TPO may affect immunologic responses. It has been demonstrated that, like other therapies, TPO treatment can rescue the peripheral CD4+CD25+FoxP3+ T regulatory cell (Tregs) deficiency that is commonly observed in patients with ITP (Bao W et al Blood 116;4639, 2010). Of interest, an earlier murine study also suggested that TPO could affect the production of T cells, particularly CD4+ T cells (Zhao JZ et al Haematologica 83:572, 1998). Using a murine model of ITP that demonstrates both antibody and T cell mediated thrombocytopenia, we studied T cell responses in ITP mice treated with TPO. ITP was induced in severe combined immunodeficient (SCID) mice by administration of 2×104 splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61 and the mice where either treated or not with weekly subcutaneous injections of 1ug of murine TPO. Mice were bled weekly to count platelets and obtain sera for anti-platelet antibody production and at 3 weeks after the splenocyte transfer, they were sacrificed and their spleens and thymi were harvested. Splenocytes and thymocytes were isolated and examined by flow cytometry to quantify CD4+CD25+FoxP3+ (Tregs) and CD8+CD25+FoxP3+ (Tc-regs). We observed that TPO treatment caused an expected significant increase in platelet counts, however, the treatment also caused a significant reduction in the titre of serum anti-platelet antibodies. This was associated with significant changes in the proportions of both CD4+ Tregs and CD8+ Tc-regs within the thymus and spleen. TPO treatment rescued the peripheral splenic Treg and Tc-reg deficiencies and the cells ability to functionally suppress the proliferation of conventional CD4+CD25- T cells. The data suggests that in addition to its megakaryocyte-stimulating properties, TPO treatment has additional tolerance-inducing effects associated with anti-platelet antibody reduction and CD4+ Treg and CD8+ Tc-reg stimulation.

Disclosures:

No relevant conflicts of interest to declare.
The Immature Platelet Fraction Is Sensitive to the Platelet Size and a Useful Parameter for Screening for Macrothrombocytopenia
Authors: Miyazaki Koji , Koike Yukako , Danbara Mikio , Horie Ryouichi , Higashihara Masaaki
Published: October 2019

Abstract 1097

The immature platelet fraction (IPF) is a useful parameter indicating thrombopoietic activity to differentiate the causes of thrombocytopenia. We previously reported that the percentage of IPF (%IPF) is negatively correlated to the platelet count among ITP patients, and not among myelodysplastic syndrome (MDS) patients. We also noticed that some MDS patients exhibited extremely high %IPF values, which were dissociated from the percentages of reticulated platelets (%RP) measured by flow cytometry. Such discrepancies were also observed in hereditary macrothrombocytopenias, which are sometimes difficult to be distinguished from ITP, because ITP also exhibits increased number of reticulated platelets in a slightly larger size. Once misdiagnosed, a hereditary macrothrombocytopenia patient might be subjected to an invasive treatment such as splenectomy. In order to avoid such mistreatments, a clear marker to differentiate macrothrombocytopenia is desperately needed. In this study, we investigated the IPF values of 16 individuals from 12 families with various hereditary macrothrombocytopenia in order to clarify whether the IPF could be a useful marker to distinguish macrothrombocytopenia from ITP, and examined the IPF during EDTA aggregation and cold-storage to elucidate how platelet size may affect the IPF value.

The IPF values were about 5 times higher in MYH9 disorders (%IPF 48.0 ± 1.8) and about 1.5 times higher in other macrothrombocytopenias (%IPF 17.0 ± 2.2) than immune thrombocytopenic patients with similar platelet counts (%IPF 9.3 ± 0.4). These results suggested that the platelet size can affect the IPF value. However it still remains the possibility that some factors specific to these macrothrombocytopenias other than the platelet size might make an influence on the IPF, because the characteristic of large platelets in hereditary macrothrombocytopenia has not been fully understood, and no one knows whether large platelets are functionally identical to normal platelets except for the size. In order to exclude the possibility, we next examined the changes of IPF values during EDTA aggregation and cold-storage. The IPF was significantly increased during storage in a time dependent manner along with forming platelet clumps. The IPF was strongly influenced by a few tiny platelet aggregates rather than other platelet indices, such as mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet distribution width (PDW).

In conclusion, the IPF is susceptible to the platelet size, and could be a useful parameter for screening of macrothrombocytopenia from ITP.

Disclosures:

No relevant conflicts of interest to declare.
CPB2 Deficiency Protects Against Polymicrobial Sepsis
Authors: Shao Zhifei , Nishimura Toshihiko , Leung Lawrence L. , Morser John
Published: October 2019

Abstract 1110

Procarboxypetidase B-2 (pro-CPB2) (also termed thrombin-activatable fibrinolysis inhibitor, TAFI) is activated by the thrombin/thrombomodulin complex to generate a basic carboxypeptidase (CPB2). Fibrin and complement C5a have been shown to be physiological CPB2 substrates, but the role of CPB2 in sepsis is unclear. We hypothesized that in polymicrobial sepsis, the CPB2 KO mice would have exacerbated disease because there would be enhanced activation of C5a, leading to an augmented inflammatory response and tissue injury. To our surprise, in a polymicrobial sepsis model in which the cecum was ligated and punctured (CLP), CPB2 deficient mice had a significant improvement in survival compared to wild type (WT) mice (p = 0.021) (Fig. 1). The median survival time of 3 days in WT mice was extended to 4 days in CPB2 KO mice (p = 0.015). Lung edema as measured by wet/dry lung weight ratio was decreased from 4.4 in the WT mice to 2.5 in the CPB2 KO mice (p = 0.0001) at 48 hr after CLP (Fig. 2). Peritoneal lavage fluid levels of protein and the amount of cell infiltration into the peritoneum at 48 hr was lower in CPB2 KO than WT mice. There was less liver damage assessed as AST and ALT in plasma in the CPB2 KO mice at 48 hr than in WT mice while there was a trend to an increase in BUN suggesting kidney damage in WT mice. There was a greater decrease in the white blood cell count of WT mice than CPB2 KO mice with a drop in lymphocyte numbers accounting for most of that decrease. Inflammatory mediators such as IL-6 and total C5a were lower in plasma from CPB2 KO mice at 6 hr after CLP than in WT plasma. The data suggest that WT mice in this model underwent multiple organ failure leading to their death and the CPB2 deficient mice were protected from the consequences of CLP. Overall these data show that lack of CPB2 protects against polymicrobial sepsis with a concomitant reduction in inflammation. Further studies are underway to determine which substrate is key to the prolonged survival of CPB2 deficient mice in this model.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation of Prosthetic Valve Function by Platelet Function Analysis and Von Willebrand Factor Indices
Authors: Wysokinska Ewa M. , Duello Katherine M. , Chen Dong , Blackshear Joseph L
Published: October 2019

Abstract 1128

Background

Dysfunctional or mismatched prosthetic heart valves can results in high shear stress related von Willebrand factor (VWF) impairment, and clinical bleeding. This mechanism is similar to that seen in aortic stenosis patients, where gastrointestinal bleeding related to VWF multimer loss is well described (Heyde syndrome). In this study, we compared the results of VWF testing of patients with normally functioning cardiac valve prostheses, dysfunctional prosthetic valves, severe native aortic stenosis, and normal controls.

Methods

A total of 75 patients, 31 female and 44 male with a median age of 79.5 years were recruited to this study. Thirty patients had severe aortic stenosis (mean gradient > 40 mm Hg); 39 patients had normally functioning prosthetic valves, 21 aortic (AVR) and 18 mitral valves (MVR); and 6 patients had with dysfunctional valves (4 aortic and 2 mitral). Whole blood and plasma samples were drawn from patients and tested for platelet function analyzer-100 collagen ADP closure time (PFA-CADP), VWF antigen (VWF:Ag), VWF activity by latex immunoassay (VWF:Ltx), VWF multimer analysis and VWF multimer densitometry ratio of large vs medium to low molecular weight multimers (>15 mers/2–15 mers).

Results

The 6 dysfunctional prosthetic valve patients included patient-prosthesis mismatch (2) and (one each), thrombosed mechanical aortic prosthesis, torn biological aortic prosthesis, severe tissue mitral prosthetic regurgitation, and post-mitral valve repair severe mitral regurgitation. Four of six patients had clinically significant bleeding. Most aortic stenosis and all 6 prosthetic dysfunction patients had abnormal VWF multimers which is rare in normally functioning prostheses and completely absent in normal donors. VWF:Ltx/Ag was lower in AS and MVR than normal. VWF:Ltx/Ag ratio, VWF:multimer ratio and PFA-CADP were all significantly abnormal in patients with dysfunctional prosthetic valve. VWF:multimer ratios in normally functioning prostheses tended to be intermediate between normals and than those of patients with severe AS or prosthetic valve dysfunction (Table).

Conclusions

VWF testing including quantitative von Willebrand multimer analysis and PFA-CADP reliably separate dysfunctional from normally functioning cardiac prostheses, and should be considered in the evaluation of patients with cardiac prostheses, especially those who have clinically significant bleeding.

Disclosures:

No relevant conflicts of interest to declare.
Effects of the Timing and the Type of Venous Thromboembolic Events On Survival in Patients with Pancreatic Cancer
Authors: Kesari Vivek , Shethia Maithili , Zhou Xiao , Overman Michael , Vadhan-Raj Saroj
Published: October 2019

Abstract 1151

Background:

Patients (pts) with pancreatic cancer are at high risk for venous thromboembolic events (VTE) and the occurrence of VTE can adversely affect prognosis. However, it is unclear if the type of VTE such as symptomatic vs incidental, deep vein thrombosis (DVT) vs pulmonary embolism (PE), the location of VTE [DVT of extremities vs visceral veins (abdominal/pelvic veins)] or the timing of VTE from diagnosis can influence the survival. The purpose of this study was to evaluate the incidence of different types of VTE, the impact of types and timing of VTE (early vs late) on survival.

Methods:

Medical records of 260 pts with pancreatic cancer, newly referred to MDACC in 2006, were reviewed for cancer diagnosis, patient demographics (age, gender), presence of metastasis, the date of diagnosis of VTE, timing of VTE, type of VTE, the site of VTE, the incidence of VTE during 2 years of follow up from the date of diagnosis. Clinical and laboratory parameters predictive for survival were also reviewed. All VTE episodes, including symptomatic as well as incidental VTEs were confirmed by the radiological studies using CT ANGIO, CT scan, Doppler compression ultrasound or V/Q perfusion scans. The survival time was calculated from the date of cancer diagnosis to the date of last follow up. Survival analysis was conducted using Kaplan-Meier method and Cox proportional hazard models. The stepwise selection method was employed to build a multivariate model using variables with p<0.15 in univariate analysis.

Results:

Of the 260 pts referred, 235 were confirmed to have the diagnosis of pancreatic carcinoma. During the 2-year follow-up, 80 pts (34%) had 109 episodes of VTE, including symptomatic and incidental episodes. The median age of the pts with VTE was 59 years (range: 28–86) and 51% were males. Of the 80 pts with VTE, 21 (26%) had PE, 18 (23%) had DVT of extremities, 28 (35%) had DVT of visceral veins and 13 (16%) had concurrent PE/DVT (diagnosed on the same day). Of the 80 pts, 25 (31%) had 29 recurrent episodes. Kaplan-Meier survival analysis, as shown in the table below, indicated that the pts who had early VTE (defined as VTE diagnosed within 30 days from the date of diagnosis of pancreatic cancer) vs late VTE (> 30 days) and pts with metastasis vs no metastasis had statistically poor 1 year survival (log-rank test).

Conclusions:

These findings suggest that timing of VTE is an important indicator of prognosis, regardless of whether symptomatic or incidental. Patients with VTE within 30 days of diagnosis have shorter survival.

Disclosures:

No relevant conflicts of interest to declare.
Primary Venous Thromboembolism Prophylaxis in Patients with Solid Tumors Receiving Chemotherapy: A Meta-Analysis
Authors: Phan Minh , John Sonia , Casanegra Ana Isabel , Tafur Alfonso
Published: October 2019

Abstract 1157

Background:

Venous thromboembolism [VTE] is the second highest cause of mortality among patients with cancer. However, pharmacological thromboprophylaxis for patients with solid tumor is only recommended during hospitalization. Primary outpatient thromboprophylaxis is not a widely accepted practice.

Objective:

Determine safety and efficacy of outpatient primary VTE prophylaxis in patients with solid tumors.

Data sources:

A systematic review was conducted using MEDLINE and EMBASE up to June 2012. Key search words included venous thromboembolism, malignancy, anticoagulants, and chemotherapy. Studies were considered for our meta-analysis if they included outpatient primary pharmacological thromboprophylaxis in adult patients with active solid cancer. All the information was independently reviewed by 2 of the authors [MP, SJ] and a third reviewer resolved discrepancies. The measure of association was calculated with R (R: A Language and Environment for Statistical, R Development Core Team, www.R-project.org), R META package (Version 0.8–2, Author: Guido Schwarzer). The Q statistic was calculated and a formal test of homogeneity was conducted. Random-effects model was preferred in case of heterogeneity.

Results:

A total of 1371 abstracts were reviewed and 29 manuscripts were fully abstracted. Eight randomized controlled trials including 6706 patients were analyzed. There were less VTE events with outpatient prophylaxis: odds ratio [OR] of 0.53 (95% CI, 0.40–0.70). Six studies used low or ultra-low molecular weight heparin and two studies used warfarin. In the subgroup analysis of heparin based primary prophylaxis, there was a significant reduction in VTE events [OR 0.47, 95% CI, 0.34–0.64], no significant heterogeneity [FIG 1]. In addition, there was no difference in major bleeding events between groups [OR 1.48, 95% CI, 0.89–2.46]. Five studies reported mortality data; there was significant heterogeneity between studies.

Conclusions:

Heparin based outpatient VTE prophylaxis in patients with solid tumors reduced by half the risk of VTE with no significant differences in major bleeding events. The current publications do not allow a meaningful grouped analysis of survival data, improved patient selection is necessary in order to adequately target VTE prevention strategies.

Disclosures:

No relevant conflicts of interest to declare.
Critical Role for Mouse Marginal Zone B Cells in PF4/Heparin Antibody Production
Authors: Zheng Yongwei , Yu Mei , Podd Andrew , Newman Debra K. , Wen Renren , Arepally Gowthami M , Wang Demin
Published: October 2019

Abstract 1175

Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of platelet factor 4 (PF4), heparin and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, the B-cell origin of HIT antibody production is not known. Here we show that upon challenge with PF4/heparin complexes, anti-PF4/heparin antibody production is severely impaired in B cell-specific Notch2-deficient mice (CD19CreNotch2fl/fl) that specifically lack marginal zone (MZ) B cells, and that antibody production is readily generated in wild-type mice (CD19CreNotch2+/+). As expected, Notch2-deficient mice responded normally to challenge with T cell-dependent antigen NP-CGG but not T cell-independent antigen TNP-Ficoll, in agreement with the lack of MZ B cells in the mutant mice. PF4/heparin-specific antibodies produced by wild-type mice on a C57BL/6 background were IgG2b and IgG3 isotypes. An in vitro class-switching assay showed that MZ B cells from wild-type C57BL/6 mice were capable of producing antibodies of IgG2b and IgG3 isotypes. Lastly, MZ, but not follicular (FO), B cells adoptively transferred into B cell-deficient muMT mice responded to PF4/heparin complex challenge by producing PF4/heparin-specific antibodies of IgG2b and IgG3 isotypes. Taken together, these data demonstrate that MZ B cells play a critical role in production of PF4/heparin-specific antibodies.

Disclosures:

Arepally: Teva Pharmaceuticals: Research Funding.
Generation of Human Hematopoietic Stem/Progenitor Cells From Pluripotent Stem Cells Through Modulating Retinoic Acid Signaling
Authors: Rönn Roger Emanuel , Guibentif Carolina , Moraghebi Roksana , Woods Niels-Bjarne
Published: October 2019

Abstract 1203

Hematopoietic stem and progenitor cells generated from patient derived induced pluripotent stem (iPS) cells could provide an unlimited supply of HLA matched transplantable cells for the treatment of both hematological disorders and malignancies. The goal of this project is to identify novel pathways involved in hematopoietic stem and progenitor cell generation and expansion from human ES and iPS cells. By using small molecules to modulate retinoid signaling, we report enhanced in vitro generation of hematopoietic progenitors cells (CD45/43+CD34+) and cells with an adult hematopoietic stem cell phenotype (CD45/43+CD34+CD38-CD90+CD45RA-). By inhibiting the cellular synthesis of retinoic acid (RA) and subsequent retinoid signaling we significantly increased the output of cells generated possessing an adult HSC surface phenotype by 2.7-fold (p-value: 0,024, n=7) compared to DMSO carrier controls, and increased clonogenic progenitors (CFUs) by 2.5-fold (p-value: 0,065, n=7). This improvement is consistent when using both a human ES line (Hues3) and a human iPS-line (RB9-CB1 generated from cord-blood derived endothelial cells). We also increased total blood cell output by increasing the frequency of embryoid bodies (EB) that successfully give rise to blood generating colonies. Conversely, and in support of our findings, directly adding RA was found to severely decrease the blood generation efficiency of our protocol. When comparing the colony forming potential of sorted cells gated on umbilical cord blood (CB), our system enabled the generation of CFU's at efficiencies on par with that of CB (preliminary data). Following plating equal numbers of sorted CD45/43+CD34+ blood progenitors, we saw 79 CFU/500 cells for the CB control, 65 CFU/500 cells from iPS + retinoic acid signaling inhibition, and 23 CFU/500 cells from iPS + DMSO control. The hematopoietic cells generated with our ES/iPS-2-Blood system also possess lymphoid lineage potential following plating of CD45/43+CD34+ cells into an in vitro based lymphoid assay, generating cells expressing markers of T-cells, B-cells, and NK-cells. We are currently performing transplantation experiments to assess the repopulating potential of these cells. We are also investigating additional factors that also have demonstrated significant increases in hematopoietic stem/progenitor cell output, with one additional modulator of RA signaling showing additive improvement together with the first molecule. We are further investigating the molecular mechanisms of these different factors for hematopoietic stem and progenitor cell generation with the aim to advance iPS cell technology towards treatments for hematological diseases and malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Profilin 1 Is Essential for Retention and Metabolism of Hematopoietic Stem Cells in Bone Marrow
Authors: Zheng Junke , Zhang Chengcheng
Published: October 2019

Abstract 1224

How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we show that, in a hematopoietic stem cell (HSC) -specific inducible knockout model, the cytoskeleton-modulating protein profilin 1 (pfn1) is essential for the maintenance of multiple cell fates and metabolism of HSCs. The deletion of pfn1 in HSCs led to bone marrow failure, loss of quiescence, increased apoptosis, and mobilization of HSCs in vivo. In reconstitution analyses, pfn1-deficient cells were selectively lost from mixed bone marrow chimeras. By contrast, pfn1 deletion did not significantly affect differentiation or homing of HSCs. When compared to wild-type cells, levels of expression of Hif-1a, EGR1, and MLL were lower and an earlier switch from glycolysis to mitochondrial respiration with increased ROS level was observed in pfn1-deficient HSCs. This switch preceded the detectable alteration of other cell fates. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that pfn1 maintained metabolism is required for the quiescence of HSCs. Furthermore, we demonstrated that expression of wild-type pfn1 but not the actin-binding deficient or poly-proline binding-deficient mutants of pfn1 rescued the defective phenotype of pfn1-deficient HSCs. This result indicates that actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Thus, pfn1 plays an essential role in regulating the retention and metabolism of HSCs in the bone marrow microenvironment.

Disclosures:

No relevant conflicts of interest to declare.
Wnt Signaling Is Associated with Anti-Apoptosis in the Interaction Between Acute Myeloid Leukemia Cells and Stromal Cells
Authors: Minami Yosuke , Niwa Yosuke , Abe Akihiro , Hayakawa Fumihiko , Naoe Tomoki
Published: October 2019

Abstract 1366

Recent studies have revealed that strength of the Wnt signaling pathway regulates normal hematopoiesis including hematopoietic stem cells, and aberrant activation of the pathway is involved in the development of several types of leukemias. In the bone marrow microenvironment, stromal cells are rich sources of cytokines and some secreted cytokines have been observed to block induction of cell death in myeloid leukemia cells exposed to chemotherapy. Here, we examined the role of the Wnt signaling pathway on cell-adhesion, proliferation and survival of the stroma-dependent human AML cell line, TRL-01 cells, which we previously established (Ninomiya, et al. Cancer Gen Cyto, 2006). TRL-01 cells were maintained in the co-culturing with the hTERT-transduced human bone marrow stroma cell line, HTS cells, and cell death of TRL-01 cells was induced after removal of HTS cells. Treatment with the Wnt-receptor competitor, secreted Frizzled related protein (sFRP)-1, or the Rho kinase inhibitor, Y29632 (previously reported as an inhibitor of the non-canonical Wnt downstream pathway), but not with the specific inhibitor of the canonical Wnt pathway (DKK-1) induced apoptosis in dose-dependent manners in TRL-01 cells co-cultured with HTS cells. These results suggested that the non-canonical pathway of Wnt signaling might regulate survival of TRL-01 on the stromal cells. Next, we comprehensively investigated transcripts of the Wnt pathway components (10 Frizzleds, 2 LRPs, and 18 Wnts) in TRL-01 cells and HTS cells using RT-PCR. Transcripts of Wnt5A and Wnt9A were expressed in TRL-01 cells, but not in HTS cells. Moreover, the cell death of TRL-01 cells after removal of HTS cells was partially prevented by additional treatment with Wnt5A or Wnt9A not by other Wnt molecules such as Wnt5B. On the other hand, treatment with Wnt3A induced activated nuclear beta-catenin using Western-blotting, however, did not contribute to the survival of TRL-01 cells without the stromal co-culturing. Moreover, we are investigating regulation of the anti-apoptotic downstream pathway molecules in TRL-01 cells as well as effects of other inhibitors targeted for the Wnt signaling. We are also examining expressions of Wnt pathway components in primary AML bone marrow cells and other human bone marrow stroma cell lines such as HS-5 cells. These results imply association of Wnt5A and Wnt9A with anti-apoptosis in the interaction between AML cells and stromal cells, and a possible therapeutic target of AML for overcoming the resistance to chemotherapy in the bone marrow microenvironment.

Disclosures:

Naoe: Chugai Pharma: Research Funding; Novartis Pharma: Research Funding; Kyowa-Hakko Kirin: Research Funding.
Acquisition or Loss of Gene Mutations Occurs in Relapsed Pediatric Acute Myeloid Leukemia: A Comparative Analysis of Paired Matched Diagnosis and Relapse Bone Marrow Samples
Authors: Liang Der-Cherng , Lui Hsi-Che , Yang Chao-Ping , Jaing Tang-Her , Hung Iou-Jih , Yeh Ting-Chi , Shih Yu-Shu , Huang Yu-Hui , Lin Tung-Huei , Shih Lee-Yung
Published: October 2019

Abstract 1389

Background and purpose:

Relapse is the major cause of treatment failure in patients with acute myeloid leukemia (AML). The molecular pathogenesis in the relapse of AML is not well understood. A better understanding of molecular aberrations of relapsed AML will further improve the treatment outcome. In the present study, we aimed to determine the role of gene mutations in the relapse of de novo pediatric AML by comparative analysis of the paired matched diagnosis and relapse samples for the mutational status of 18 known mutated genes involved in myeloid neoplasms.

Patients and Methods:

Two hundred and six children aged below 18 with de novo AML were diagnosed at Chang Gung Children's Hospital, Taoyuan, Taiwan and MacKay Memorial Hospital, Taipei, Taiwan, between 1996 and 2011. They were treated with Taiwan Pediatric Oncology Group AML-97 Protocol (Leukemia 2006). Sixty patients had relapses of leukemia, 46 of them had paired diagnosis and relapse bone marrow samples available for examination on the 18 mutated genes, including FLT3-ITD, FLT3-TKD, C-KIT, C-FMS, NRAS, KRAS, PTPN11, JAK2V617F, RUNX1, CEBPα, NPM1, MLL-PTD, WT1, P53, DNMT3A, IDH1, IDH2 and ASXL1. Mutational analysis was performed with PCR-based assay followed by direct sequencing.

Results:

The results are summarized in Table 1. Fifteen patients with one mutated gene and 4 with two mutated genes at diagnosis remained unchanged at relapse, all the mutations detected were not present in the complete remission samples. Twenty-five patients without gene mutations at diagnosis did not acquire mutations at relapse. Six patients acquired gene mutations, 5 WT1 and one P53 mutations, indicating clonal evolution during leukemia relapse. Another 7 patients lost gene mutations including 2 FLT3-ITD, 2 FLT3-TKD, one NRAS, one JAK2V617F and one IDH1genes; notably, 4 of them harboring other mutated genes at relapse, suggesting outgrowth or clonal selection of these mutated clone(s) in relapse.

Conclusion:

Our study showed that gene mutation status in the majority of pediatric AML patients remained unchanged at both diagnosis and relapse, but acquisition or loss of gene mutations may occur at relapse.

Disclosures:

No relevant conflicts of interest to declare.
Constitutional Telomerase-Associated Gene Variants in Pediatric Acute Myeloid Leukemia (AML)
Authors: Gramatges M. Monica , Sasa Ghadir S. , Qi Xiaodong , Chen Julian J-L , Plon Sharon E , Bertuch Alison A.
Published: October 2019

Abstract 1408

Background:

Telomerase is an enzyme complex that maintains telomeric DNA, the TTAGGG repeats localized to chromosome ends. Defects in telomerase result in abnormally short telomeres in somatic cells, leading to early cell senescence, chemosensitivity, and susceptibility to genomic instability. Germline telomerase mutations are associated with a spectrum of disorders including idiopathic pulmonary fibrosis, familial liver disease, aplastic anemia, myelodysplastic syndrome (MDS), and dyskeratosis congenita, which confers a 90% lifetime risk for bone marrow failure, a 200-fold risk for AML, and a 2500-fold risk for MDS. These disorders have a variable clinical phenotype, but pronounced telomere shortening is a universal finding. Of note, enrichment of specific telomerase variants is observed in adults with hematologic malignancies. Our goal is to investigate the role of germline telomerase variants in pediatric AML development and treatment-related toxicities. We hypothesize that deleterious germline telomerase variants occur in children with AML and are associated with a telomere biology disorder phenotype. Exposure to chemotherapy and/or transplant may accelerate telomere shortening and manifestation of disease phenotype in affected individuals.

Methods:

This exploratory analysis will determine (1) the frequency of germline telomerase variants in our local pediatric AML population (2) the implication of these variants on telomerase function, and (3) whether variants are associated with a telomere biology disorder phenotype. We are sequencing the exons and flanking intronic regions of four telomerase-related genes, TERT, DKC1, TERC, and TINF2, in germline pediatric AML samples (n=100), and functionally characterizing all novel variants. We are reviewing all cases blinded to telomerase variant status for clinical evidence characteristic of a telomere biology disorder.

Results:

To date, out of the first 88 subjects sequenced, 12 constitutional heterozygous variants resulting in missense changes or deletions were found in 19 subjects (21.6%). Of the variants found, 7 are novel, and several represent rare variants observed in adult populations with hematologic malignancies. Among the 62 subjects that have undergone blinded chart review, clinical characteristics of telomere biology disorders were noted in subjects both with and without variants. In subjects with variants, the most predictive characteristics were (1) nail dysplasia, skin hyperpigmentation, and oral leukoplakia, (2) persistent liver or pulmonary disease of unknown etiology, and (3) delays in chemotherapy of more than 2 months due to cytopenias.

Conclusion:

Presence of deleterious telomerase variants may be susceptibility loci in pediatric AML. Our results suggest that further investigation of the disease phenotypes associated with these variants is warranted.

Disclosures:

No relevant conflicts of interest to declare.
Gene Expression Profiling (GEP) of Whole Bone Marrow Biopsies in Complete Remission (BMB-CR) of Multiple Myeloma (MM) Patients Treated On Total Therapy Protocols – Normalization of GEP Signature in Comparison with Normal Donor BMB (BMB-NL) and Consequences for Progression-Free Survival (PFS)
Authors: Heuck Christoph , Qu Pingping , Waheed Sarah , Usmani Saad Z , van Rhee Frits , Dhodapkar Madhav V. , Epstein Joshua , Zhang Qing , Johann Donald , Barlogie Bart
Published: October 2019

Abstract 198

GEP of purified plasma cells (PC) provides superb risk discrimination for PFS and overall survival (OS). It is well established that MM engages the BM micro-environment (ME) and subjugates it toward its survival and progression. International Myeloma Working Group- and flow-based CR have been linked to PFS and OS, but do not provide guidance vis-à-vis the duration of post-transplant consolidation and maintenance therapy. We reasoned that ME may still be altered in flow-CR as it may remain MM-impregnated long after CR is established; conversely, ME normalization may happen much earlier. In a first step, 20 BMB-NL were compared with 331 untreated patients with MM (BMB-MM), leading to the discovery of a 65 gene score (GEP-65), which was then applied to 83 patients with MGUS/asymptomatic multiple myeloma (AMM) and 88 MM patients in CR. GEP-65 risk scores were similar in BMB-CR and BMB-MGUS-AMM contrasting with significantly lower values for BMB-MM. Next we examined PFS for CR patients according to whether BMB-CR signature normalized (BMB-CR-NL, n=14) or failed to normalize (BMB-CR-MM, n=74). A borderline significantly superior PFS was observed in the former group suffering 2 events versus 31 in the remainder (p=0.08). We conclude that (1) BMB-MM differs markedly from BMB-NL, (2) BMB-CR resembles BMB-MGUS-AMM, and (3) BMB-CR-NL can be distinguished from BMB-CR-MM resulting in PFS differences. Availability of several hundred BMB-CR samples and nearly 100 BMB-NL will have been evaluated at the time of the meeting to address the following: (a) can these results be validated, (b) what is the time course of BMB-CR-NL and pattern to BMB-CR-MM and BMB-MM, (c) are there MM GEP molecular subgroup- and risk-dependent differences.

Disclosures:

Dhodapkar: Celgene: Research Funding; KHK: Research Funding.
Rictor Overexpression and mTORC2 Signaling in Chronic Lymphocytic Leukemia
Authors: Jordaan Gwen , Liao Wei , Gera Joe , Sharma Sanjai
Published: October 2019

Abstract 3884

Signaling via the B-cell receptor (BCR) stimulates growth and survival of CLL leukemic cells and inhibits apoptosis by phosphorylating immunoreceptor tyrosine based activation motifs. This signaling subsequently activates PI3 Kinase/AKT, mTOR, ERK and other pathways. Activation of Akt in turn requires phosphorylation by mTOR kinase, which assembles in two complexes mTORC1 and mTORC2 and it is the mTORC2 complex that phosphorylates and activates Akt. This phosphorylation of Akt in CLL specimen's upregulates anti-apoptotic proteins such as Mcl-1, Bcl-xl and XIAP. We have identified that Rictor, a component of mTORC2 complex is over-expressed in CLL specimens as compared to normal peripheral mononuclear B cells. This over-expression was noted by real time PCR that showed 1.5 to 4 fold upregulation (n=12). Western blot analysis also showed Rictor overexpression in all the twelve CLL specimens tested. Rictor overexpression was also seen in Mantle cell lymphoma cell lines and to study its role in BCR signaling, stable Mantle cell lymphoma lines with SiRNA mediated Rictor knockdown were established. Rictor knockdown resulted in a significant decrease in Akt activation as phosphorylation (phospho S473) of Akt both in unstimulated cells and when the cells were stimulated with BCR crosslinking was decreased.

To determine the effect of Rictor and mTORC2 inhibition on CLL specimens, we tested the activity of three compounds isolated via yeast two hybrid drug screen designed to identify molecules that inhibit Rictor/mTOR interaction. When tested on CLL specimens in the presence of BCR crosslinking, these mTORC2 inhibitor compounds inhibited the downstream phosphorylation of Akt S473. Functionally the inhibitors also induced apoptosis in CLL cells with 40–60% of CLL cells undergoing apoptosis (1.0mM, cells treated for 48 hours). In comparison, rapamycin (mTORC1 inhibitor) and ppp242 (mTORC1, 2 inhibitor) were comparatively less active in CLL specimens as they were less effective in the induction of apoptosis. Western blot analysis of mTORC2 inhibitor treated cells also showed PARP cleavage and an increase in the pro-apoptotic protein BAD. Our data indicates that Rictor overexpression in CLL specimens is required for Akt phosphorylation activation and downstream BCR signaling. Inhibition of this pathway by mTORC2 inhibitors in CLL will be an effective therapeutic strategy.

Disclosures:

No relevant conflicts of interest to declare.
Mapping the Targets of Dasatinib in Chronic Lymphocytic Leukemia Reveals Distinct Roles for Abl and Btk in Drug Resistance and Adhesion, and Explains Clinical Effects On Lymph Node Reduction
Authors: Eldering Eric , Geest Christian R , de Rooij Martin FM , Liu Nora , Florea Bogdan I , Van Oers Marinus H.J. , Overkleeft Hermen S , Spaargaren Marcel , Kater Arnon P.
Published: October 2019

Abstract 3900

In the lymph node (LN) microenvironment, chronic lymphocytic leukemia (CLL) cells are protected from apoptosis by upregulation of anti-apoptotic proteins. In vitro, this can be mimicked via CD40-stimulation of CLL cells, which also provides resistance to various chemotherapeutics. Novel drugs that target kinases involved in B cell signalling, including the broad spectrum kinase inhibitor dasatinib, are currently in clinical development for CLL. We have shown previously that dasatinib prevents CD40-mediated anti-apoptotic changes in CLL (Hallaert et Blood 2008). However, the kinase(s) involved remain unidentified. Here, we coupled dasatinib to an affinity matrix and pulled down its targets from CD40-stimulated CLL cells. By mass-spectrometry and Western blotting, Abl and Btk were identified as dominant targets of dasatinib. Functional analysis revealed that CD40-mediated anti-apoptotic signals and drug-resistance could be overcome both by dasatinib and the Abl inhibitor imatinib, but not by the novel Btk inhibitor PCI-32765 (ibrutinib), whereas BCR- and chemokine-controlled adhesion could be abolished by dasatinib and ibrutinib, but not by imatinib. Thus, dasatinib combines two key aspects that are clinically relevant: inhibition of Abl overrides chemoprotective survival signals, whereas inhibition of Btk impairs integrin-mediated adhesion of CLL cells in the microenvironmental niche.

This combined inhibition of Abl and Btk was put to an initial test in an open-label phase 2 trial of dasatinib combined with fludarabine in twenty refractory CLL patients. As might be expected based on the in vitro data, reductions in lymph node size were observed in most patients. A LN reduction of ≥20% provided a significant improved PFS (256 days) and OS (510 days) as compared to non-responders (80 days and 158 days respectively). Details of the clinical study will be presented separately. In conclusion, in agreement with in vitro molecular studies, dasatinib seems to have clinical efficacy in heavily pretreated refractory CLL patients. Combined, these data encourage further studies on a broad-spectrum kinase inhibitor like dasatinib in combination with other classes of drugs in relapsed and refractory CLL.

Disclosures:

No relevant conflicts of interest to declare.