bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Histone Deacetylase Inhibitor Entinostat (SNDX-275) Targets Hodgkin Lymphoma through a Dual Mechanism of Immune Modulation and Apoptosis Induction
Authors: Jona Adam , Khaskhely Noor , Buglio Daniela , Shafer Jessica A. , Derenzini Enrico , Bollard Catherine M , Illes Arpad , Andreef Michael , Younes Anas 
Published: October 2019

Abstract
Abstract 2850

Introduction:
Based on recent favorable in vitro and in vivo activity of several HDACi (histone deacetylase inhibitors) in HL (Hodgkin lymphoma), we investigated the in vitro activity of SNDX-275, an oral, class 1 isoform of selective HDACi in HL-derived cell lines.

Materials and methods:
Proliferation and cell death were examined by MTS assay, Annexin-V/PI and FACS analysis. For combination studies, cells were incubated with SNDX-275 (0.1-2 μM) and either ABT-737 (0.01-0.2 μM), Obatoclax (0.1-2 μM), Gemcitabine (1-20 nM) or Bortezomib (1-20 nM) for 72 hours. Gene and protein expression were measured by RT-PCR, Western blot, and immunohistochemistry. A multiplex assay was used to determine 30 cytokines and chemokines.

Results:
SNDX-275 induced cell death in a dose and time dependent manner with an IC50 of 0.4 μM. At the molecular level, SNDX-275 increased histone-3 acetylation, up-regulated p21 expression, and activated the intrinsic apoptosis pathway by down-regulating the XIAP (X-linked inhibitor of apoptosis protein), which was associated with activation of Caspase 9 and 3. Similarly to other HDACis, SNDX-275 decreased the expression of anti-apoptotic Bcl-2 and Bcl-xL, while level of Mcl-1 and pro-apoptotic Bax remained the same level. Combination studies demonstrated that SNDX-275 had more synergistic effect when combined with Bcl-2 inhibitors ABT-737 or Obatoclax and less when combined with Gemcitabine or Bortezomib. Dysregulated cytokine/chemokine production has been shown previously to contribute to HL pathology, including immune tolerance of the cancer cells. Hence, we measured the effect of SNDX-275 on pathways that may contribute to an anti-tumor immune response. Increased IL12 p40-70, IP10, and RANTES, and decreased IL13, IL4 and TARC levels were found, thus favoring Th1-type cytokines/chemokines. Recent data has demonstrated that a variety of epigenetic-modulating drugs may up-regulate the expression of CTAs (cancer testis antigens), leading to a favorable immune response. SNDX-275 was able to induce CTA expression of SSX2 and NY-ESO only in one cell line whereas MAGE-A4 was induced in both HL cell lines.

Conclusion:
Our studies demonstrate that SNDX-275 has a dual effect on apoptotic and immunomodulatory pathways in HL, which can be enhanced by the addition of agents targeting cell survival pathways. Phase II studies with SNDX-275 in HL are ongoing, future clinical studies should investigate combinations with SNDX-275.

Disclosures:
No relevant conflicts of interest to declare.
CD74 Expression by AML Cell Lines and Bone Marrow Specimens, and Augmented In Vitro Cytotoxicity of Anti-CD74 Antibody After Interferon-Gamma (IFN-γ) Treatment
Authors: Chandra Abhinav B. , Burton Jack , Stein Rhona , Chen Susan , Mishra Nidhi , Shah Taral , Enwemnwa Nneamaka N , Goldenberg David 
Published: October 2019

Abstract
Abstract 2888

Background:
CD74 (HLA-DR-associated invariant chain) is expressed alone or along with DR in a wide range of hematologic cancers and solid tumors. Humanized anti-CD74 mAb, milatuzumab (Immunomedics, Morris Plains, NJ), exhibits direct cytotoxicity for NHL, CLL and MM cell lines, and is undergoing clinical evaluation for treatment of these malignancies. CD74 is upregulated by interferons in hematologic and epithelial cancer cell lines. Here we present the results of our analysis of CD74 expression and function in AML, and the effect of CD74 upregulation by treatment with IFN-γ on the cytotoxicity of milatuzumab for AML cell lines.

Methods:
CD74 expression in bone marrow biopsy (BMB) specimens from non-M3 AML patients was evaluated by immunohistochemistry and, for the 3 human AML cell lines, by flow cytometry, with/without permeabilization and with/without IFN-γ (40 and 200 U/mL). These cell lines were also tested in proliferation assays for responses to milatuzumab, with/without IFN-γ. Also, assessment of apoptosis and cellular signaling was performed.

Results:
In the initial group of AML cases, 13/14 BMB specimens showed moderate to strong CD74 expression by leukemic blasts, which was mostly intracellular, usually with a perinuclear distribution. Three AML cell lines also showed moderate to strong expression of CD74, which was mostly intracellular. Without IFN-γ, surface expression of CD74 was present, but IFN-γ treatment of these 3 lines resulted in upregulation of surface CD74 by 69–117%. Much higher levels of intracellular CD74 were observed in all 3 lines (with and without IFN-γ), with IFN-γ-induced upregulation of intracellular CD74 in all 3 lines (from 85%-868%; P<0.001). In 2/3 lines, IFN-γ increased milatuzumab-mediated growth inhibition (23.7 to 44.8% and -3.9 to 30.9%, P=0.01 and P<0.05, respectively). Cytotoxicity was in part due to apoptosis, as significant increases in Annexin V binding (P=0.01) were observed after treatment with IFN-γ plus milatuzumab. Initial experiments addressing cellular signaling suggest a role for AKT, because phosphorylated AKT levels increased (P=0.06) in response to IFN-γ + milatuzumab.

Conclusions:
CD74 is expressed in AML patient specimens and in AML cell lines, with the majority of CD74 expression found intracellularly. Cell surface and cytoplasmic expression of CD74 were upregulated in AML lines after IFN-γ exposure. This increased expression resulted in increased cytotoxicity of the anti-CD74 mAb, milatuzumab, in 2/3 AML lines. This effect was through apoptosis and involved the AKT pathway. Thus, AML is another cancer type where combined IFN-γ and milatuzumab treatment may be useful.
Supported in part by NIH grant PO1-CA103985 (DMG).

Disclosures:
No relevant conflicts of interest to declare.
Enzastaurin Treatment Affects Multiple Regulatory Pathways at Transcriptome and Cellular Proteome Level of Mantle Cell Lymphoma
Authors: Weinkauf Marc , Hutter Grit , Zimmermann Yvonne , Rieken Malte , Pastore Alessandro , Hartmann Elena , Rosenwald Andreas , Hiddemann Wolfgang , Dreyling Martin H. 
Published: October 2019

Abstract
Abstract 2893

Background:
The protein kinase C beta inhibitor enzastaurin is one of the promising molecular targeted approaches currently investigated in mantle cell lymphoma (MCL), a disease still characterized by a dismal long term prognosis.

Methods:
Four well characterized MCL cell lines (Granta 519, HBL-2, Jeko-1 and Rec-1) as well as three patient samples were exposed to enzastaurin at a previously defined dose (10 μM). Cell viability as well as cell cycle activity were analyzed by tryphan blue exclusion test and flow cytometry, respectively, after 24 and 48 hours. To dissect the regulatory processes targeted by enzastaurin, the panel of MCL cell lines was screened on both protein and RNA expression levels (2D-gel electrophoresis and mass spectrometric peptide fingerprint analysis and Affymetrix microarray) after 4h enzastaurin treatment.

Results:
Enzastaurin in vitro resulted in a reduced viability and cell proliferation by 15–20% after 24h in cell lines and 9–20% in primary patient samples after 48h. This effect was related to a G2/M block of cell cycle and induction of apoptosis. Based on the proteome and transcriptome analysis of early alterations, only HSPD1 was affected on both regulation levels. Nonetheless, combined analysis of alterations on both, protein and RNA expression levels, resulted in identification of common signal pathways characterizing a more comprehensive network of affected molecular interactions mapping to distinct canonical pathways and defined cellular functions. Indicated canonical pathways included ‘calcium signalling', (CAMKK2, HDAC5, HDAC9, TP63) ‘calcium induced T-lymphocyte apoptosis' (MEF2D, NR4A1, PRKCG, TRA@), ‘NFkB signalling' (KRAS, MAP3K8, TNFAIP3, TNFRS17) and ‘molecular mechanisms of cancer' (APAF1, CDKN2D, FOS, PAK6), whereas the top ranking cellular functions were ‘cellular growth and proliferation' (CCNG2, EIF4E, PDIA3, TOP1, TPM1,), ‘cell death' (BCL6, EEF1D, PAK6, RAD50), ‘cell cycle'(AKAP9, BMF, CUL5, GADD45B, PDIA3), ‘cellular development' (APAF1, GAS7, ID1, PAX8) and ‘gene expression' (ABCG1, HOXB4, LMO4, PIM1). Alterations of these pathways were confirmed by Western Blot analysis of selected candidate proteins marker proteins of the regulated pathways.

Conclusion:
In summary, the combined approach of RNA and protein analysis revealed the targeted signal pathways after Enzastaurin exposure. These data will allow a more rationally designed combination of biologicals to finally improve the clinical outcome of MCL.

Disclosures:
Dreyling: Eli Lilly: Support of in vitro studies of Enzostaurin in MCL.
Myeloma Cells Induce Osteoblast Suppression through Sclerostin Secretion
Authors: Colucci Silvia , Brunetti Giacomina , Oranger Angela , Mori Giorgio , Sardone Francesca , Liso Vincenzo , Curci Paola , Miccolis Rosanna M , Rinaldi Erminia , Specchia Giorgina , Passeri Giovanni , Zallone Alberta , Rizzi Rita , Grano Maria 
Published: October 2019

Abstract
Abstract 2961
Reduced osteoblast activity contributes to the development of multiple myeloma-bone disease. Wingless-type (Wnt) signalling pathway is critical in osteoblastogenesis, and it is negatively regulated by molecules such as frizzled-related proteins (sFRPs), Dickkopf proteins (DKKs) and sclerostin. Myeloma cells are known to induce inhibition of osteoblastogenesis through Wnt antagonists such as DKK-1 and sFRP-2 and -3 whereas the role of sclerostin, an osteocyte-expressed negative regulator of bone formation, has not been yet investigated. We provide novel evidence showing sclerostin expression by myeloma cells from patients with multiple myeloma-bone disease and human myeloma cell lines (HMCLs). By means of a co-culture system of bone marrow stromal cells (BMSCs) and HMCLs, we demonstrated that sclerostin expression by myeloma cells and HMCLs is responsible for reduced expression of major osteoblastic specific proteins namely bone-specific alkaline phosphatase, collagen-type I, bone sialoprotein II and osteocalcin as well as decreased mineralized nodule formation and attenuated expression of member of the AP-1 transcription factor family (i.e. Fra-1, Fra-2 and Jun-D). The addition of a neutralizing anti-sclerostin antibody to our co-culture system can restore the above parameters, through the intranuclear accumulation of β-catenin in BMSCs. On the other hand, we demonstrated that sclerostin is also involved in inducing increased receptor activator of nuclear factor-k B ligand (RANKL) and decreased osteoprotegerin (OPG) expression in osteoblasts, contributing to the enhanced osteoclast activity occurring in patients with multiple myeloma-bone disease. Our data suggest that myeloma cells contribute to the suppression of bone formation through sclerostin secretion.

Disclosures:
No relevant conflicts of interest to declare.
Effect of Thalidomide with Melphalan and Prednisone on Health-Related Quality of Life (HRQoL) in Elderly Patients with Newly Diagnosed Multiple Myeloma: A Prospective Analysis In a Randomized Trial
Authors: Verelst S. , Termorshuizen F. , Groot C. Uyl-de , Schaafsma M. R , Ammerlaan R. , Wittebol S. , Sinnige H. , Zweegman S. , Kooy M. van Marwijk , van der Griend R. , Lokhorst H. , Sonneveld P. , Wijermans P. 
Published: October 2019

Abstract
Abstract 2972
Thalidomide (T) with melphalan/prednisone (MPT) was defined as a standard treatment in elderly patients with multiple myeloma (MM) based on 5 randomized trials. Treatment with MPT not only showed improved response rate, significantly better time to response as well as quality of response but also a significant improvement of event free survival (EFS), progression free survival (PFS) and overall survival (OS). In one of these trials, HOVON 49, a prospective Health related – Quality of life (HRQoL) was initiated in order to asses the impact of T on QoL. (Wijermans et al, J Clin Oncol 28:3160-6). Patients aged >65 years with newly diagnosed symptomatic MM were randomized to receive 8 cycles of MP or MPT, followed by T maintenance in MPT arm. 284 patients were included in this HRQoL side study (MP: n=149, MPT: n=135). HRQoL was assessed with the EORTC core QoL Questionnaire (QLQ-C30) and the myeloma-specific module (QLQ-MY24) at baseline and at pre-determined intervals during treatment. Treatment-related toxicity WHO grade 2–4 occurred in 60% of MP and 87% of MP-T treated patients. Most frequently found were neutropenia-related infections, neurotoxicity (mostly T induced peripheral neuropathy) and myelotoxicity.
The QLQ-C30 subscales Physical Function (p=0.044) and Constipation (p<.001) showed a treatment related improvement during induction in favour of the MP arm. During T maintenance, the scores for the QLQ-MY24- Paraesthesia became significantly higher in the MPT arm (p<.001). The QLQ-C30 subscales Pain (p=0.12), Insomnia (p=0.068), Appetite loss (p=0.074) and the QLQ-MY24- item Sick (p=0.086) scored marginally better during T maintenance. The overall QoL scale QLQ-C30-HR-QOL showed a significant time trend towards more favourable mean values during protocol treatment but did not reveal differences between MP and MPT. For the QLQ-C30 subscales Emotional function and Future perspectives, difference in favour of the MPT arm from the start of treatment was observed (p=0.018and p=0.045 respectively) with no significant ‘time × arm' interaction, indicating a persistent better patient perspective with MPT treatment. Since the first questionnaire was filled out prior to treatment but after randomization we hypothesise that the awareness of being treated with a medication that holds out a prospect of better treatment outcome may in itself be associated with improved feeling of well-being and hope for the future.
This prospective study shows that the higher frequency of adverse effects associated with MPT does not translate into a negative effect on HRQoL and that MPT holds a better patient perspective.

Disclosures:
No relevant conflicts of interest to declare.
Prohibitin Serves as a Novel Target Mitochondrial Protein of Phenyl-Chloroethyl Ureas Involved In Their Antiproliferative Effect on Multiple Myeloma Cells
Authors: Kim Hye-Ran , Eom Ha-Young , Kim Dong Min , Kim Il-Chul , Kumar Manian Rajesh , Lee Sun-Woo , Shin Jong-Hee , Suh Soon-Pal , Ryang Dong-Wook , Shin Myung-Geun 
Published: October 2019

Abstract
Abstract 3002

Background:
Prohibitin (PHB) is localized to the mitochondria where it might have a role in the maintenance of mitochondrial function. The diverse function of PHB, together with the emerging evidence that its function can be modulated specifically in certain diseases, implies that PHB would be a potential target for new therapeutics.

Materials and Methods:
We analyzed mitochondrial proteins and develop new anti-proliferative agents targeting multiple myeloma (MM) cells. Mitochondria were isolated from primary leukemia and MM cell lines (RPMI8226, ARH77, U266 and IM9) by density-gradient ultracentrifugation using swelling buffer and sucrose buffer. Dysregulated mitochondrial protein was identified using 2-DE and mass spectrometry (MALDI-TOF/TOF technology).

Results:
Out of 38 remarkable deregulated mitochondrial proteins in MM cell lines, prohibitin (PHB) (gi4505773) was highly expressed in primary MM and leukemia cells, which was confirmed by Western blot, immunohistochemistry and immunofluorecenct study in the primary bone marrow cells and sections. Potent chemical substances that can alkylate PHB, two molecules of phenyl-chloroethyl urea family such as cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU), were synthesized independently from the reaction with 2-ethylisocyanate and 4-cyclohexylaniline and 4-iodoaniline, respectively. They were characterized by 1H NMR and 13C NMR. Time and dose dependent manner of proliferation suppression when treated with CCEU and ICEU was observed in MM and leukemia cells. Moreover, notable morphological transformation of MM cells was observed when treated with 10 – 100 umol of CCEU and ICEU for 24 hours. The half of maximal inhibitory concentration (IC50) was 25umol of most MM cell lines. Cell cycle analysis of CCEU-and-ICEU-treated- MM cells showed a remarkable increase of the sub-G1 phase. Immunoblotting experiment revealed the change of cytoplasmic and nucleoplasmic PHB. Expression of cleaved caspase3 and poly ADP-ribose polymerases were also observed to have increased in MM cell lines.

Conclusions:
By analyzing mitochondrial protein in leukemia and MM cell, we discovered a new molecular marker, PHB, characteristically overexpressed in leukemia and MM cells and developed new anti-cancer agents such as CCEU and ICEU that target against PHB in MM cells.

Disclosures:
No relevant conflicts of interest to declare.
HGAL Directly Interacts with Both Myosin and Actin and Increases the Binding of Myosin to Actin
Authors: Lu Xiaoqing , Katarzyna Kazmierczak , Jiang Xiaoyu , Watt James , Moore Jeffrey R , Szczesna-Cordary Danuta , Lossos Izidore S 
Published: October 2019

Abstract
Abstract 3097
HGAL is a recently identified germinal center (GC)–specific gene whose expression by tumor cells correlates with a favorable prognosis in patients with diffuse large B-cell and classical Hodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte migration, thus potentially constraining lymphocytes to the GC and decreasing lymphoma cell migration and dissemination. The latter effect may contribute to the less aggressive clinical behavior of HGAL-expressing lymphomas. Actin filaments, usually in association with myosin, are responsible for many types of cell movements. The interaction between myosin and actin is responsible for muscle contraction, migration of nonmuscle cells and also plays a role in cell division. The movement of cells appears to be driven directly by actin polymerization and by actin-myosin interactions. In our previous work (Lu et al, Blood 2007), we demonstrated by co-immunoprecipitation and co-localization studies that HGAL interacts with myosin II and actin. However, the mechanistic consequences of this interaction are unknown. Because the interaction of actin with myosin is the key factor in cell motility and HGAL negatively regulates lymphocyte migration, we hypothesized that HGAL may decelerate cell migration by affecting the binding of myosin to actin. To confirm the direct and not interdependent interaction between myosin-HGAL and actin-HGAL, we performed in vitro co-sedimentation experiments with recombinant HGAL protein and purified actin and myosin, respectively. These studies demonstrated direct and independent binding of HGAL to both myosin and actin. We next demonstrated that the N terminal portion of the HGAL protein (aa 1–118) can bind the head region of myosin II (S1) that contains the actin and ATP binding sites, and to the rod part of the myosin molecule that is responsible for filament formation. While no effect of the HGAL on the actomyosin ATPase activity was observed, it reduced the rate of actin polymerization from G-actin to F-actin. Further binding studies using pyrene-labeled F-actin showed that HGAL increases the binding of myosin to F-actin. Taken together, our results indicate that HGAL is a cellular regulator of actin assembly that can also regulate the actin-myosin interaction. This in turn may explain the role of HGAL as a favorable prognosis marker for patients with diffuse large B-cell and classical Hodgkin lymphomas.

Disclosures:
No relevant conflicts of interest to declare.
Quantification of EBV DNA In Whole Blood In Newly Diagnosed Patients with Diffuse Large B-Cell Lymphoma
Authors: Beltran Brady , Salas Renzo , Chunga Ausberto , Sotomayor Eduardo M. , Castillo Jorge J 
Published: October 2019

Abstract
Abstract 3102

Introduction:
Epstein-Barr virus (EBV) is a well-known oncogenic virus associated with the development of several lymphoproliferative disorders. The quantification of EBV viral load in plasma and peripheral blood mononuclear cells has been reported as a useful biomarker for EBV-associated non-Hodgkin lymphoma (NHL). The role of EBV in diffuse large B-cell lymphoma (DLBCL) is unclear at this time. METHODS: In the present prospective study, using real-time quantitative polymerase chain reaction (RQ-PCR), whole blood specimens from patients with a new diagnosis of DLBCL were used to quantitatively determine EBV viral load. Consecutive patients who presented from January 1, 2009 to December 30, 2009 were selected for this study. The pathological samples from the patients showing elevated EBV DNA levels were then investigated for the presence of EBV-encoded RNA (EBER) using a chromogenic in situ hybridization (CISH) technique. Clinical data from these patients was collected and analyzed, and will be presented using descriptive statistics. Overall survival estimates were obtained using the Kaplan-Meier method.

Results:
Forty six patients with a new diagnosis of DLBCL were included in our study; from which five cases showed elevated levels of EBV DNA by RQ-PCR (10.8%). These five patients (100%) were positive for EBER expression within the tumoral cells by EBER CISH. One patient was evaluated at the end of treatment and blood EBV DNA level was undetectable. All EBV DNA-positive cases were male with a median age of 69 years (range 25–73 years); only one case was younger than 50 years. The median EBV DNA viral load was 59 copies/uL (range 0.225–43200 copies/uL). Three patients (60%) presented with advanced clinical stage (III or IV) and three patients (60%) had a primary extranodal site of involvement. Four patients (80%) received CHOP-based chemotherapy regimens. The three patients with advanced stage have deceased with a survival of <3 months. The two patients with early stage experienced a complete response with immunochemotherapy and are alive 6 and 9 months after diagnosis, respectively. The median overall survival is 4 months.

Conclusions:
These results, although preliminary, suggest that whole blood EBV DNA quantification might be useful at detecting patients with EBV-positive DLBCL. Further studies are needed to investigate the potential diagnostic and/or prognostic value of EBV DNA in DLBCL.

Disclosures:
No relevant conflicts of interest to declare.