bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeting Mcl-1 for Multiple Myeloma (MM) Therapy: Drug-Induced Generation of Mcl-1 Fragment Mcl-1128–350 Triggers MM Cell Death Via c- Jun Upregulation
Authors: Fan Fengjuan , Tonon Giovanni , Sattler Martin , Schimming Alexander , Goldschmidt Hartmut , Opferman Joseph , Anderson Kenneth C. , Jaeger Dirk , Podar Klaus 
Published: October 2019

Abstract
Abstract 3959
Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein of the Bcl-2 family located on chromosome 1q21. Gains of 1q21 are unfavorable genetic prognostic factors for Multiple Myeloma (MM) patients. Our present data confirm significant overexpression of Mcl-1 gene in tumor versus plasma cells derived from healthy donors. Moreover, a significant link between Mcl-1 overexpression and poor prognosis was found in a data set of 590 MM patients, supporting a key role for Mcl-1 in MM survival and drug resistance. Mcl-1 is therefore a potential therapeutic target in MM. Strategies to inhibit Mcl-1 activity in MM and other malignancies may not only include induction of proteasome-dependent Mcl-1 degradation and modulation of Mcl-1 interaction with BH3-only proteins, but also induction of proteasome- independent Mcl-1 degradation. Indeed, our own and other previous data demonstrate that in contrast to full length Mcl-1 a caspase- induced 28kD Mcl-1 fragment, Mcl-1D128–350, inhibits MM cell proliferation and triggers apoptosis. We therefore performed a large-scale drug screen for agents which induce generation of Mcl-1D128–350 in MM cells. Our results show that bortezomib and staurosporine (STS) but not melphalan, doxorubicin or thalidomide induce generation of Mcl-1D128–350. We next sought to identify molecular sequelae downstream of Mcl-1D128–350 which mediate its anti-proliferative and pro-apoptotic effect in MM cells. Surprisingly, we observed nuclear accumulation of drug- induced Mcl-1D128–350, enhanced AP-1 reporter activity as well as elevated protein levels of c-Jun. Conversely, drug- induced c-Jun upregulation was blocked after introduction of point mutations (A127D, A157D) into the two highly conserved Mcl-1 caspase-cleavage sites Asp127 and Asp157. Gene profiles on MM cells transfected with either Mcl-1wt or Mcl-1D128–350 identified a group of differentially expressed genes associated with MM cell proliferation, survival and drug resistance. Consistent with these data, treatment with bortezomib or STS triggered c-Jun upregulation followed by apoptosis in Mcl-1wt/wt but not Mcl-1d/null murine embryonic fibroblasts (MEFs). Importantly, STS-induced c-Jun upregulation and cell death was restored in Mcl-1d/null MEFs transfected with a plasmid carrying Mcl-1wt. In ongoing studies we are analyzing genome sequencing data in MM cells for the existence of point mutations in the Mcl-1 caspase cleavage sites. The presence of such mutations could represent a potential new mechanism of drug resistance. Taken together, our data add another facet to the complex cellular functions of Mcl-1 in MM and derive a promising therapeutic and potential prognostic role for Mcl-1D128–350 in MM.

Disclosures:
No relevant conflicts of interest to declare.
Down-Regulation of MMSET by RNA Interference Affects Cell Viability and Tumorigenicity in t(4;14) Myeloma Plasma Cells
Authors: Qian Shenxian , Gao Da-quan , Xie Yaping , Xu Yin , Huang Xilian 
Published: October 2019

Abstract
Abstract 3998
The recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple myeloma patients and is the worst prognostic subgroup in MM, although the basis for this poor prognosis is unknown. The t(4;14) is unusual in that it involves 2 potential target genes: fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET). MMSET is universally overexpressed in t(4;14) MM, whereas FGFR3 expression is lost in one-third of cases. Nonetheless, The involvement of MMSET in the pathogenesis of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upregulation are still unclear. Here we demonstrate a role for MMSET in t(4;14) MM cells. We found that Down-regulation of MMSET expression in KMS11 cell by RNA interference and by selective disruption of the translocated MMSET allele using gene targeting dramatically reduced DNA synthesis in KMS11 decreased to 20% of that of the control, whereas the viability of cells decreased to 2% (p_ 0.01) of that of the control and increase in apoptosis was twice that of the control. These findings were associated with reduced expression of genes (e.g. CCND2, CCNG1, MAF and VEGF). These results provide direct evidence that MMSET plays a significant cell survival and antiapoptotic role in t(4;14) MM and suggest that therapies targeting this gene could impact this particular subset of poor-prognosis patients.

Disclosures:
No relevant conflicts of interest to declare.
The Activity of MOR202, a Fully Human Anti-CD38 Antibody, Is Complemented by ADCP and Is Synergistically Enhanced by Lenalidomide in Vitro and in Vivo
Authors: Endell Jan , Boxhammer Rainer , Wurzenberger Cornelia , Ness Daniel , Steidl Stefan 
Published: October 2019

Abstract
Abstract 4018

Background:
MOR202, a HuCAL-derived fully human anti-CD38 antibody has proven to be highly effective in preclinical models of multiple myeloma (MM) and is currently evaluated in a phase I/II clinical trial. Having previously identified ADCC as potent effector mechanism of MOR202, the capability to induce killing of MM patient cells via antibody dependent cellular phagocytosis (ADCP) was assessed. Furthermore, the immunomodulatory agent lenalidomide (LEN), a commonly used agent in MM therapy was investigated for its potential to enhance the cytotoxicity of MOR202 in vitro and in vivo.

Methods:
Drug effects in presence or absence of LEN were characterized in vitro in a FACS-based phagocytosis assay using macrophages derived from M-CSF stimulated monocytes and a panel of CD38+ MM target cells. The therapeutic potential of MOR202 in vivo as a single agent and in combination with LEN was studied in a disseminated survival model of iv injected Ramos lymphoma cells. Synergy was determined using the fractional product concept.

Results:
MOR202 induced ADCP with high potency showing EC50 values between 40 and 130pM. Maximal killing was donor and cell line dependent ranging from 11 to 72%. The combination with LEN induced additive to synergistic enhancement of MOR202 activity which was mediated by several mechanisms identified to be direct cytotoxicity and increased CD38 expression levels on MM cells. In vivo, MOR202 at 1mg/kg increased median survival (MS) by 142% compared to vehicle control (MS day 48.5 vs. day 20), while LEN had no distinct effect at any of the tested doses between 25 and 100mg/kg (MS day 21–22). Intriguingly, despite the apparent lack of efficacy by LEN alone co-administration of 1mg/kg MOR202 and 100mg/kg LEN resulted in a superior increase of MS by 225% (MS day 65), thus demonstrating strong synergism. Of note, while no mouse survived beyond day 65 in any other group, 3/8 animals in the combo group were completely free of tumor until study termination on day 98.

Conclusions:
We here demonstrate ADCP to be an additional potent effector mechanism of MOR202. The combination of MOR202 and LEN enhanced ADCP-mediated killing in vitro and increased median survival in vivo in a synergistic manner. These results suggest a mechanistic rationale for MOR202 combinations with LEN including in particular LEN non-responders or relapse/refractory patients, thus warranting further evaluation in future clinical trials.

Disclosures:
Endell: MorphoSys AG: Employment. Boxhammer:MorphoSys AG: Employment. Wurzenberger:MorphoSys AG: Employment. Ness:MorphoSys AG: Employment. Steidl:MorphoSys AG: Employment.
STAT5A Regulates DNMT3 and Inactivates PTEN Tumor Suppressor Gene in CD34+/CD38− AML Cells
Authors: Takeuchi Asako , Nishioka Chie , Ikezoe Takayuki , Yang Jing , Yokoyama Akihito 
Published: October 2019

Abstract
Abstract 4087
Acute myelogenous leukemia (AML) is organized as a cellular hierarchy, initiated and maintained by a subset of self renewing leukemia stem cells (LSCs). We have recently reported that signal transducer and activator of transcription 5 (STAT5) is activated in CD34+/CD38− AML cells which contain abundant leukemia stem cells (LSCs) and inhibition of STAT5 induced apoptosis and sensitized these cells to the growth inhibition mediated by conventional chemotherapeutic agents (Int J Cancer. 2011;128:2317-25). This study attempted to identify molecules which are regulated by STAT5 and support survival of CD34+/CD38− AML cells. We lentivirally transduced a short hairpin (sh) RNA targeting STAT5 in CD34+/CD38− AML cells and compared their gene expression profiles with those of control shRNA transduced CD34+/CD38− AML cells by microarray analysis. Interestingly, DNA methyltransferase (DNMT) 3A, which catalyzes the transfer of a methyl group to DNA was differentially expressed between control and STAT5A down-regulated AML cells. In contrast, lentiviral transduction of STAT5A in CD34+/CD38− AML cells increased levels of DNMT3A. Reporter gene assays found that exposure of leukemia cells to IL-10 increased levels of the phosphorylated forms of STAT5, leading to transcriptional activation of DNMT3A. On the other hand, dephosphorylation of STAT5A by AZ960, a selective JAK2 inhibitor decreased this transcriptional activity in leukemia cells. Of note, forced expression of STAT5A in leukemia cells caused hypermethylation on the promoter region of tumor suppressor gene PTEN and down-regulated its mRNA levels as measured by methylation-specific PCR and real-time RT-PCR, respectively. Taken together, STAT5A may positively regulate levels of DNMT3A, resulting in inactivation of tumor suppressor gene by epigenetic mechanisms in LSCs.

Disclosures:
No relevant conflicts of interest to declare.
Constitutive Expression of CD40L by CAR-Modified Tumor Targeted T Cells Enhances Anti-Tumor Efficacy Both in Vitro and in Vivo
Authors: Curran Kevin J. , Seinstra Beatrijs , Nikhamin Yan , Yeh Raymond , Usachenko Yelena , Brentjens Renier J 
Published: October 2019

Abstract
Abstract 4120
T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). Recent reports have demonstrated the effectiveness of CAR modified T cells in patients with relapsed or refractory malignancies. However, CAR modified T cells have yet to demonstrate the ability to recruit an endogenous anti-tumor response which would greatly enhance their therapeutic benefit. To overcome these limitations we have developed a bi-cistronic gamma-retroviral vector allowing for constitutive co-expression of a CD19-specific CAR (19–28z) and human CD40 ligand (CD40L; CD154). The CD40 ligand/CD40 system has been demonstrated to activate dendritic cells (DCs) and alter the phenotype of B cells (upregulation of co-stimulatory and adhesion molecules and secretion of pro-inflammatory cytokines) with subsequent stimulation of CD8+ T cell activation and proliferation. We now demonstrate T cells genetically modified to constitutively express CD40L undergo enhanced proliferation and up-regulated secretion of pro-inflammatory cytokines including GM-CSF and INF-g. Furthermore, T cells modified to constitutively express CD40L, upon co-culture, will alter the phenotype of CD40+ B cell tumor cell lines by enhancing the expression co-stimulatory molecules (CD80/CD86), adhesion molecules (CD54/CD58/CD70) and death receptors (CD95; Fas). These findings were similarly evident in primary patient tumor samples (e.g. CLL cells) when co-cultured with autologous T cells modified to constitutively express CD40L. We further demonstrate maturation of monocyte derived DCs with subsequent secretion of IL-12 following co-culture with autologous T cells modified to constitutively express CD40L. T cells transduced with the bi-cistronic 19–28z/CD40L vector showed enhanced in vitro cytotoxicity against a panel of CD19+ tumor cell lines. Furthermore, infusion of 19–28z/CD40L modified T cells enhances the survival of CD19+ tumor bearing immunodeficient mice (SCID/Beige) when compared to mice treated with T cells modified to express the anti-CD19 19–28z CAR alone. We conclude that further genetic modification of CAR targeted T cells to constitutively express the co-stimulatory CD40L may enhance the anti-tumor efficacy of this adoptive T cell therapy. Our data suggests this enhanced T cell efficacy may be due to both autocrine and paracrine mediated mechanisms.

Disclosures:
No relevant conflicts of interest to declare.
Multicenter, Prospective Study of Tacrolimus Plus Methotrexate As Graft-Versus-Host Disease Prophylaxis in HLA-Matched Sibling Peripheral Blood Stem Cell Transplantation: Comparison with Cyclosporine Group As Control
Authors: Shin SeungHwan , Yoon JaeHo , Yahng SeungAh , Lee SungEun , Cho ByungSik , Eom KiSung , Kim YooJin , Lee Seok , Min ChangKi , Kim HeeJe , Cho SeokGoo , Kim DongWook , Min WooSung , Park ChongWon , Lee JongWook 
Published: October 2019

Abstract
Abstract 4177

Background
Graft-versus-host disease (GVHD) is an immunologic complication after allogeneic hematopoietic stem cell transplantation (HSCT) with significant mortality and morbidities. Allogeneic peripheral blood stem cell transplantation (PBSCT) has high risk of GVHD, especially chronic GVHD compared to bone marrow transplantation. Only limited data are available comparing the efficacy of FK506 with that of cyclosporine (CsA) in human leukocyte antigen (HLA)-matched sibling PBSCT.

Methods
Thirty-nine patients with various hematologic disease received PBSCT with FK506+methotrexate (MTX) as GVHD prophylaxis were compared to ninety-four historical control with CsA+MTX GVHD prophylaxis.

Results
The 1-year cumulative incidence of grade II-IV acute GVHD was significantly lower in patients who received FK506 than those in the CsA group (10.3% vs 28.2%, p=0.036). The female donor-male recipient pair (hazard ratio; 4.828, 95% CI; 17.84-13.06, p=0.002) and CsA prophylaxis (hazard ratio; 3.279, 95% CI; 1.14–9.43, p=0.027) were significant risk factor of acute GVHD in multivariate analysis. But, there was no difference in the 3-year cumulative incidence of chronic GVHD between the FK506 and the CsA group (77.6% vs 69.6%, p=0.793). The 3-year cumulative incidence of relapse (33.9% vs 23.1%, p=0.505) and 3-year treatment-related mortality (18.9% vs 28.4%, p=0.187) of the two groups were similar. The patients in the FK506 arm had a similar event-free survival (EFS) and overall survival (OS) with patients in the CsA arm (3-year EFS; 53.2% vs 55.1%, p=0.706, 3-year OS; 60.7 vs 61.5%, p=0.610). The age over 35 years (hazard ratio; 4.12, 95% CI; 1.95–8.70, p=0.001), female donor-male recipient pair (hazard ratio; 2.66, 95% CI; 1.52–4.65, p=0.001) and advanced pre-HSCT disease status (hazard ratio; 3.13, 95% CI; 1.72–5.71, p=0.001) were significant prognostic factors associated with OS in multivariate analysis.

Conclusion
These results demonstrated that the FK506+MTX can significantly reduce grade II-IV acute GVHD compared to CsA+MTX in sibling PBSCT with similar incidence of chronic GVHD, and comparable outcome in EFS, OS, relapse rate and TRM rate between two groups.

Disclosures:
No relevant conflicts of interest to declare.
Evaluating Prolonged Gvhd Based On Monocyte and T-Cell Activation, Leads to Early Detection of Immunological Changes Before Gvhd Exacerbations
Authors: Kuroda Rie , Fujiki Toshihiro , Mase Shintaro , Araki Raita , Maeba Hideaki , Koizumi Shoichi , Yachie Akihiro , Nishimura Ryosei 
Published: October 2019

Abstract
Abstract 4178
We have reported that evaluating the activation markers and homing molecules not only on T-cells but also on monocytes, combined with cytokine profiling, provides useful information for management of patients with prolonged GVHD (the 53th ASH). In the cases both T-cells and monocytes were highly activated as shown in Fig.1A, we have shown that steroid response was quite effective. When only T-cells were activated, administration of calcineurin inhibitors (CI) alone was sufficient to control the disease. However steroid refractory or steroid dependent patients are presented at a uniform rate. We have employed low dose MTX for these patients, which is widely used in autoimmune diseases because of its anti-inflammatory activity. In this report, we showed the transitional change of both T-cell and monocyte activation status in patients with prolonged GVHD after initiating immunosuppressive agents, especially in some steroid dependent cases given weekly low dose MTX.

Methods
We evaluated activation markers and homing molecules not only on T-cells but also on monocytes in peripheral blood obtained from 32 childhood patients (more than 700 samples at various time points) receiving hematopoietic stem cell transplantation (HSCT) by multicolor flowcytometry. The following markers were used: CD69, CD25, and HLA-DR for T cell activation, CCR4, CCR5, CXCR3, CCR9, and CLA for homing markers. Inflammatory monocytes were defined as CD14dimCD16+ cells or CD14+CD163+ cells. In addition we combined the data of cytokine profiles secreted mainly by T cells such as soluble interleukin 2 receptor (sIL-2R), or monocytes such as neopterin, or both such as tumor necrosis factor-ƒ¿ (TNF-ƒ¿), soluble TNF-ƒ¿RI, and soluble TNF-ƒ¿RII.

Results
Representative pattern of combined T-cells and monocytes activation was shown in Fig.1A. After initiating sufficient dose of steroid, both T-cells and monocytes were fully suppressed as shown in Fig.1B. When steroid was tapered just below the maintenance dose, both CD4+CCR5+ CXCR3+ and CD8+CCR5+ inflammatory T-cells were gradually increased without hypercytokinemia, and also increased frequency of CD4+CD45RO+ T-cell subpopulation was observed without any sign of GVHD exacerbations as shown in Fig.1C. In these condition, additional steroid or extra immunosuppressive drug such as weekly low dose MTX could gradually recover to fully suppressed state as shown in Fig.1D.

Discussion and conclusions
Evaluating prolonged GVHD by both T-cells and monocytes, combined with cytokine profiling, could lead to early detection of immunological changes before GVHD symptom develops. We can choose appropriate drug use such as weekly low dose MTX for steroid-sparing.

Disclosures:
No relevant conflicts of interest to declare.
Post-Allogeneic Monitoring with Molecular Markers Detected by Pre-Transplant Next Generation Sequencing (NGS) Predicts Clinical Relapse in Patients with Chronic Myelomonocytic Leukemia (CMML)
Authors: Kröger Nicolaus , Schroeder Thomas , Zabelina Tatjana , Badbaran Anita , Bacher Ulrike , Kobbe Guido , Schnittger Susanne , Kohlmann Alexander , Haferlach Torsten 
Published: October 2019

Abstract
Abstract 4212
Allogeneic stem cell transplantation remains the only curative treatment approach for chronic myelomonocytic leukemia (CMML), but relapse is the major cause of treatment failure. Here, we evaluated the impact of molecular mutations on outcome and the value of molecular monitoring post transplantation in patients with CMML. We screened 45 patients with a median age of 57 years (range: 24–73 years) who underwent allogeneic SCT pre transplant for molecular markers including a sensitive next-generation sequencing (NGS) technique. Of those, 7 were transformed to secondary acute myeloid leukemia, 5 were classified as MDS/MPN overlap and one patient as atypical CML. In 36 patients sufficient DNA was available for molecular analyses for ASXL1, CBL, NRAS and TET2, mutations. In particular, TET2 and CBL mutations were screened applying amplicon deep-sequencing (454 Life Sciences, Branford, CT).
In 32 patients (89%) at least one mutation could be detected: ASXL1: n=18: 50%; CBL: n=7: 19%; TET2: n=15:42%; NRAS: n= 11: 32%. Allogeneic SCT was performed after reduced intensity (66%) or standard myeloablative (33%) conditioning from related (22%) or unrelated donors (78%). The overall survival at 5 years was 46% (95% CI 28–64%) and the cumulative incidence of relapse at 2 year was 35%. Neither TET2, ASXL1, CBL nor NRAS mutations influenced overall survival significantly.
In 21 surviving patients with at least one positive marker pre transplantation, the corresponding marker was subsequently tested in peripheral blood after a median of 6 months (range 3–9 months) after transplantation.
In 6 (33%) of the patients one of the molecular markers was still detectable: ASXL1: n=3; CBL: n= 3; TET2: n=2, NRAS: n=3) and in 66% the markers were undetectable. In patients who became negative for any molecular marker the cumulative incidence of relapse at 1 year was significantly lower than in the positive tested patients. (15% versus 50%, p = 0.04).
In conclusion, this study shows that molecular monitoring post transplant with molecular markers detected also by NGS pre-transplant can help to predict the risk of relapse and may be used to guide post transplant immune-modulating therapies to prevent clinical relapse. In larger studies sequential monitoring of molecular markers after stem cell transplantation are mandatory to determine optimal timing for predicting relapse and for therapeutic intervention.

Disclosures:
Bacher: MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.
Revisiting the Role of the Stat Laboratory: Is It Safe to Perform Activated Clotting Time Determinations (ACTs) Out of the Operating Room
Authors: Plietz Michael , Leifer Aaron , Padilla Vilma , Pineda Carole , Naraine Khrishan , Greenberg Mark , Fox Amy S 
Published: October 2019

Abstract
Abstract 4256

Background:
Activated clotting time (ACT) is determined during cardiac procedures in order to assess the level of anti-coagulation. There are three main anticoagulants used during cardiac procedures:heparin, angiomax and Plavix. Heparin is often used in the cardiac OR for major procedures and is neutralized, over time, by platelet factor 4(PF4). Angiomax, bivalirudin, has a quick onset of action, but a much shorter half-life when compared to Heparin. Plavix, clopidogrel, irreversibly inhibits platelets from clotting and is used on patients in need of anticoagulation for an extended period of time. The purpose of this study was to evaluate the stability of ACT results over time utilizing different coagulation regimens. If the ACT value remains stable over time, this would enable us to remove the device from the OR and place it in a stat laboratory.

Methods:
ACTs were determined by the ISTAT (Abbott), a hand held point of care device, using kaolin activated cartridges. A single blood sample was obtained and was evaluated at intervals of 0, 5, 10, 15, and 20 minutes. In addition, temperature, age, body surface area and platelet count of patient were recorded. The time 0 specimen was used to determine the patients ACT and interval testing was performed on the remnant.

Results:
36 samples representing 22 patients had ACT tests performed using the ISTAT. There was no significant change from 0 to 20 minutes over the entire dada set. However, when divided into different anti-clotting agents there were significant changes. Patients who had only taken Angiomax had a significant difference within the first 5 minutes (p value=.0094). Patients taking Angiomax and Plavix together had no change in ACT values at 20 minutes. Patients on Heparin alone demonstrated a loss of ACT stability at 10 minutes with values both increasing and decreasing.

Conclusions:
Patients on Angiomax alone demonstrated a significant difference in ACT value within the first 5 minutes and all intervals thereafter. Based on Angiomax short half-life the ACT should be performed as soon as possible. The ACT values in patients taking both Angiomax and Plavix achieved steady state throughout the 20 minutes and had little if any decrease in value. The results for the Heparin group were unreliable at 10 minutes. As a result of these findings, performing an ACT outside the OR is feasible assuming the test can be performed by 10 minutes.

Disclosures:
No relevant conflicts of interest to declare.