bluuebunny/crossref_metadata_2025_split huggingface dataset:
Loss of Function Cohesin Complex Gene Mutations Create Neomorphic Cell States Advantageous to Oncogenesis
Authors: Smith James S , Savage Kienan I , Thompson Alexander , Mills Ken I 
Published: October 2019

Abstract
Multiple large scale sequencing projects have identified a significant number of mutations in Acute Myeloid Leukaemia (AML) and high-risk Myelodysplastic syndromes (MDS) patients. Emerging from this are those affecting members of the cohesin complex, predominantly STAG2, and it has been highlighted that the poorer overall survival of this chromatin structure group is second only to patients carrying mutations affecting TP53. The Cohesin complex is an evolutionary conserved, multimeric protein complex consisting of SMC1, SMC3, RAD21 and STAG1/ STAG2. The complex plays a pivotal role during mitosis, ensuring sister chromatid cohesion however, substantial data exists highlighting roles in DNA damage repair, homologous recombination and most significantly, contribution to long-range interactions between cis-regulatory elements of the genome.
Here, we have used the CRISPR/Cas9 genome editing system to create an isogenic model featuring a premature stop codon within STAG2 at a region which presents as highly mutated in patient data sets. This stop codon generates a loss of function phenotype due to the haplo-insufficient X-linked nature of the STAG2 gene. Following model validation, the impact on genome wide distribution of the cohesin complex was assessed using ChIP-Seq for STAG1, STAG2, SMC3 & the transcriptional insulator CTCF.
A modest increase in overall CTCF binding from ~67,000 to ~72,000 high confidence peaks was observed, despite a ~7-fold decrease in enrichment, indicating decreased global insulator activity as a result of the inactivating STAG2 mutation. As expected, compensation between the STAG members was observed at a number of sites within transcriptionally active topologically associated domains, with an increase in STAG1 binding peaks (~17,000 to >25,000 sites) within the mutant model, despite no changes in average global enrichment. This data was integrated with RNA-seq of the isogeneic parental and mutant cells to determine the transcriptional impact of changes in cohesin complex binding on gene expression within active and repressive domains.
Assessment of the active and repressive super-enhancer associated marks H3K27ac and H3K27me3 showed increased global levels of both marks without increase in the polycomb repressive complex 2 member, EZH2 total protein levels. These findings indicate that increased H3K27ac and H3K27me3 marks, in this model, are due to altered domain boundaries, associated with local activation of both H3K9ac and H3K9me3, leading to aberrant gene expression and consequently a neomorphic state within these mutant cells.
With recent further stratification of de-novo AML, secondary AML and high-risk MDS emerging, this study on the aberrant and non-canonical nature of the cohesin complex provides avenues for an attractive therapeutic target and personalised treatment approach for patients within the chromatin subgroup.

Disclosures
No relevant conflicts of interest to declare.
Lung Vaso-Occlusion in Sickle Cell Disease Mediated By Arteriolar Neutrophil-Platelet Micro-Emboli
Authors: Bennewitz Margaret F. , Vats Ravi , Tutuncuoglu Egemen , Gladwin Mark T. , Sundd Prithu 
Published: October 2019

Abstract
Background: Vaso-occlusive crisis (VOC) is the primary reason for emergency medical care by sickle cell disease (SCD) patients. SCD patients hospitalized with VOC often develop acute chest syndrome (ACS), a form of acute lung injury, suggesting a role for pulmonary vaso-occlusion in the onset of ACS. However, the cellular, molecular and biophysical mechanism of pulmonary vaso-occlusion is unknown.
Methods: SCD transgenic or non-sickle control mice were intravenously (IV) challenged with 2 to 3 ng of bacterial lipopolysaccharide (LPS). Fluorescent anti-mouse Ly-6G and CD49b mAbs were administered IV for in vivo staining of circulating neutrophils and platelets, respectively. Multiphoton excitation enabled quantitative fluorescence intravital lung microscopy (qFILM) was used to determine the molecular mechanism of pulmonary vaso-occlusion in live mice. Function-blocking anti-mouse P-selectin mAb (Fab fragments) was administered IV to assess the role of platelet P-selectin in promoting pulmonary vaso-occlusion.
Results: A nanogram dose of IV LPS selectively triggered pulmonary vaso-occlusion in SCD but not control mice. Remarkably, pulmonary vaso-occlusion involved occlusion of the pre-capillary pulmonary arteriole bottle-necks (junction of an arteriole and capillaries) by large neutrophil-platelet embolic aggregates. IV administration of Fab fragments of function blocking anti-P-selectin mAb led to the resolution of pulmonary vaso-occlusion, which was primarily mediated by the attenuation of large neutrophil-platelet aggregates into smaller aggregates that are not stopped by the arteriolar bottle-necks.
Conclusion: These results establish the relevance of neutrophil-platelet aggregation in pulmonary arterioles in promoting pulmonary vaso-occlusion in SCD and also highlight the therapeutic potential of inhibiting platelet P-selectin to prevent ACS in SCD patients hospitalized with VOC.
Acknowledgments: This study was supported by 1R01HL128297-01 (P.S.), AHA 11SDG7340005 (P.S.), VMI startup funds (P.S.). M.F.B. was supported by NIH-NHLBI training grant T32HL110849 and NIH-NHLBI F32 NRSA 1F32HL131216-01.

Disclosures
No relevant conflicts of interest to declare.
Amlexanox As a Possible Breakthrough Drug for MLL/AF4-Positive Acute Lymphoblastic Leukemia
Authors: Tamai Hayato , Yamaguchi Hiroki , Miyake Koichi , Takatori Miyuki , Kitano Tomoaki , Yui Syunsuke , Fukunaga Keiko , Nakayama Kazutaka , Inokuchi Koiti 
Published: October 2019

Abstract
Background: MLL/AF4-positive acute lymphoblastic leukemia (ALL) is associated with poor prognosis even after allogeneic hematopoietic stem cell transplantation. Previously, we reported that this ALL shows resistance to TNF-α, which is the factor involved in the graft versus leukemia (GVL) effect or tumor immunity, by upregulation of S100A6 expression followed by interference with the p53-caspase pathway. Amlexanox, an anti-allergic drug, was reported to inhibit the translocation pathway of endogenous S100A6 in endothelial cells.
Aims: This study was performed to examine the effects of Amlexanox on MLL/AF4-positive ALL.
Methods: In vitro analysis, cell growth of MLL/AF4-positive ALL cell lines ( SEM and RS4;11) were analyzed with TNF-α (10 ng/mL) and Amlexanox (0, 10, and 100 µg/mL).The effect of Amlexanox on S100A6 and p53-caspase pathways were examined by Western blotting (WB) analysis. In vivo analysis MLL/AF4-positive transgenic mice model, which show CD45R/B220+leukemia by 12 months of age we established and human peripheral blood mononuclear cell (Hu-PBMC) NOD/SCID mice transplanted with SEM-Luc were examined to compare mice fed diet containing Amlexanox (0.02%) with mice fed control diet.
Results: There were no significant differences between the growth of SEM or RS4;11 cells in the absence or presence of 10 µg/mL of Amlexanox in vitro under 10 ng/mL of TNF-α. However, both cells showed significant growth inhibition by 10 ng/mL of TNF-α in the presence of 100 µg/mL of Amlexanox (P = 0.0085 for SEM, P = 0.0196 for RS4;11) WB analysis showed that S100A6 was activated in the presence of 10 ng/mL TNF-α, and activated S100A6 was decreased and both acetyl-p53/p53 ratio and cleaved caspase 3/caspase 3 ratio were increased in cells treated with 100 µg/mL of Amlexanox under 10 ng/mL of TNF-α in the MLL/AF4-positive human ALL cell lines. In vivo, MLL/AF4-positive transgenic mice fed a diet containing Amlexanox (0.02%) developed significantly less volume of CD45R/B220+ leukemia at the age of 1 year in comparison with mice fed control diet (P<0.001 for BM and .P<0.001 for spleen). Hu-PBMC NOD/SCID mice transplanted with SEM-Luc in the Amlexanox group showed significantly longer survival than those in the control group (P < 0.014).
Conclusions: Amlexanox may be a breakthrough drug for MLL/AF4-positive ALL because it inhibits the resistance of MLL/AF4-positive ALL to TNF-α by downregulating S100A6 expression followed by upregulating the p53-caspase pathway.Specifically, allogeneic hematopoietic stem cell transplantation is expected to show beneficial effects in combination with Amlexanox.

Disclosures
No relevant conflicts of interest to declare.
Molecular Signature of Dormancy in CD34+CD38- Acute Myeloid Leukaemia Cells
Authors: Al-Asadi Mazin Gh , Castellanos Marcos , May Sean T , Russell Nigel H , Seedhouse Claire H , Pallis Monica 
Published: October 2019

Abstract
Chemotherapy drugs tend to spare cells in a state of dormancy (G0 phase of the cell cycle). Relapse of acute myeloid leukaemia (AML) is likely in part due to dormant cells evading remission-induction chemotherapy. Dormant AML cells have been identified in the bone marrow endosteal region which is characterised by an excess of TGFβ1 and a shortage of nutrients. We developed and characterized an in-vitro model of AML cell dormancy by exploiting these features.
Following preliminary investigation of several cell lines, the CD34+CD38- line TF1a was selected for in depth investigation. TF1a cells showed 72% inhibition of proliferation (p<0.001), with features of dormancy, in response to 72 hours TGFβ1+mTOR inhibitor treatment (mTOR pathway inhibition mimics major effects of nutrient scarcity).This treatment caused loss of Ki-67, as well as upregulating ALDH and CD34 and causing nuclear translocation of FOXO3a. In contrast to conventional serum-withdrawal assays for dormancy, the treatment had no impact on cell viability, assessed by Annexin V assay. Nor did the treatment lead to cell differentiation, assessed by CD11b staining and morphology.
Using whole human genome expression microarray and by intersecting differentially regulated genes in dormancy-induced TF1a cells in comparison to their proliferating (untreated) counterparts, we identified 240 genes which are significantly up-regulated >2 fold including genes involved in stemness, chemoresistance and tumour suppressor genes in addition to genes involved in canonical cell cycle regulation. There was striking upregulation of genes involved in adhesion and migration: raised expression levels of SPP1 (the gene coding for osteopontin), ITGB3, ITGB4, ITGA3 and CD44 in dormant cells were confirmed by real-time PCR. The most upregulated gene was SPP1/osteopontin (16 fold). Immunocytochemistry of biopsy material from AML patients confirmed high levels of osteopontin in the cytoplasm of blasts near the paratrabecular bone marrow. Osteopontin and other genes identified in this model, including well-characterised genes (e.g. CD44, CD47, CD123, ABBC3 and CDKN2B) as well as little-known ones (e.g. PTPRU, ITGB3 and BTG2), are potential therapeutic targets in dormant AML cells.

Disclosures
No relevant conflicts of interest to declare.
Mapping the Functional Cofactors of Oncogenic EZH2 in Natural Killer/ T Cell Lymphoma (NKTL)
Authors: Li Boheng , Yan Junli , Chung Tae-Hoon , Lee Pei Tsung , Chng Wee Joo 
Published: October 2019

Abstract
EZH2, the catalytic subunit of the PRC2 complex, has been shown to overexpress in different types of cancers. Oncogenic role of EZH2 originally was thought to exhibit through its Histone H3 K27 methyltransferase activity. In recent years, several studies, including ours, have highlighted new oncogenic roles of EZH2, either PRC2-independent, or as co-activator for other transcription factors in some context of cancers. Specifically in natural killer/ T cell lymphoma (NKTL), we have already uncovered that oncogenic role of EZH2 is independent of its methyltransferase function, and JAK3 switches EZH2 from a transcriptional repressor to an activator through direct phosphorylation thus conferring the lymphoma cells with growth advantage. What's more, we showed that JAK3, EZH2 and RNA POL II are in the same complex, whereas JAK3 does not stay together with PRC2 complex. In this study, in order to map all the interacting partners of PRC2 complex as well as EZH2-POL II complex, we used NKTL cell lines for SILAC Mass Spectrometry to identify interactome of EZH2, RNA POL II and PRC2 component SUZ12, respectively. Then we overlapped EZH2 and SUZ12 interactome for canonical factors assisting PRC2-mediated gene repression, as well as EZH2 and POL II interactome for non-canonical factors which might play a role in EZH2-regulated gene activation. Some of those non-canonical factors have been shown to display extraordinary high expression in EBV-positive lymphocytes among other cancerous and normal tissues (GTEx RNA-seq expression data), or in NKTL patient sample than normal NK cells (our own GEP data). Intriguingly, GO analysis of these three interactomes indicates that the major function of EZH2 in NKTL is realized mainly through the EZH2-POL II complex, rather than the PRC2 complex. These data reinforced the oncogenic role of EZH2-POL II complex in NKTL, and suggested new players in EZH2-mediated oncogenesis.

Disclosures
No relevant conflicts of interest to declare.
Rituximab Maintenance after First Line Immunochemotherapy Improves Overall Survival in Patients with Follicular Lymphoma
Authors: Vilmar Adam , Arboe Bente , Josefsson Par , Poulsen Christian , Christensen Jacob Haaber , Jørgensen Judit , Jensen Paw , Starklint Jørn , Pedersen Per Troellund , Pedersen Michael , Brown Peter de Nully 
Published: October 2019

Abstract
Introduction
Patients with follicular lymphomas (FL) often follow an indolent disease course initiated with the wait-and-watch approach. Chemotherapy, including rituximab (R), is inevitably required in most patients though. R-maintenance is indicated for patients achieving a partial or complete remission based results from randomized clinical trials and confirmed by meta-analyses demonstrating a prolonged progression free survival. A benefit in overall survival (OS), however, has yet to be established in the first line setting.
Patients and methods
We completed a retrospective analysis in patients extracted from the Danish National Lymphoma Register. Eligible patient data included histopathological confirmation of FL diagnosed in the period 2000-2014 who received R-Chemotherapy as first line treatment and surviving a minimum 1 year after treatment initiation. A Cox regression model was performed, adjusted for age, gender, stage, response rates and treatment period (pre-maintenance era (year 2000-2010) vs. maintenance era (year 2011-2014)) with hazard ratio (HR) of OS as the primary endpoint. All patients were followed until June 2016.
Results
1037 patients were extracted from the register. 481 patients received R-maintenance (RM-group) and 556 patients did not. The median age was 62 (26-90), 526 (51%) were male, LDH was elevated in 325 (31%) and 436 (42%) had a high FLIPI index. The median follow-up time was 6.3 years. The five year OS for all patients was 86%. A significant change in the chemotherapy regimens was observed between the two groups, since Bendamustine was more frequently used in the maintenance group. A significant lower HR was observed in the RM group with a HR of 0.63 (95% CI 0.44 - 0.88, P = 0.008). Furthermore, gender proved of significance, with a HR for men of 1.78 (95% CI 1.32-2.40, P < 0.001).
Conclusions
Rituximab maintenance treatment following first line immunochemotherapy in patients with follicular lymphoma improved survival in a large population based cohort of patients and consolidates this strategy as part of the standard of care in this patient group.

Disclosures
No relevant conflicts of interest to declare.
BCR/ABL Can Promote CD19+ Cell Growth but Not Render Them Long-Term Stemness
Authors: Li Donghe , Zhao Xuemei , Jiao Bo , Liu Ping , Ren Ruibao 
Published: October 2019

Abstract
Cancer stem cells are a subpopulation of malignant cells that have the capacity of both self-renewal and reconstitution of the cancer. Eradication of cancer stem cells is crucial for curing the malignant disease. Previous studies in hematopoietic malignancies showed that leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) chronic phase are originated from a hematopoietic stem cell (HSC), while LSCs in acute myeloid leukemia (AML) can either be derived from HSCs or be transformed from myeloid progenitors. But in acute B-lymphoblastic leukemia (B-ALL), the origin of leukemia stem cells is not clear. In this study, we tested whether BCR/ABL could transform B-lineage committed CD19+ cells to LSCs. We found that transducing BCR/ABL in CD19+ cells can promote their colony formation in vitro and induce B-ALL like disease in vivo. However, only BCR/ABL transduced whole bone marrow cells, but not CD19+ cells, can be transplanted multiple times in recipient mice, and the frequency of long-term LSCs from the latter ranges from 1/135 to 1/629. These studies suggest that LSCs in BCR/ABL+ B-ALL may originate from CD19- hematopoietic stem/progenitor cells and that CD19 chimeric antigen receptor (CAR) modified T cell therapy may not be effective in eradicating LSCs in BCR/ABL+ B-ALL.

Disclosures
No relevant conflicts of interest to declare.
PML-Rara/Nrf2-Regulated Mir-125b Targets CEBPA and Influences Acute Promyelocytic Leukemia Cell Proliferation
Authors: Chengwu Zeng , Zhang Yikai , Qin Tianyu , Yang Lijian , Chen Shaohua , Li Yangqiu 
Published: October 2019

Abstract
Acute promyelocytic leukemia (APL) is characterized by the presence of PML-RARA fusion protein. The PML-RARA oncoprotein is known to be the initiating factor for APL development. We have previously found that miR-125b is highly expressed in APL and upregulated by PML-RARA. However, the molecular mechanisms by which PML-RARA controls miR-125b expression had been uncharacterized. In the present study, we further confirmed that expression of PML-RARA resulted in a significant increase in primary miR-125b expression, and we found that the nuclear transcription factor E2-related factor 2 (Nrf2) targeting genes (HO1) was also upregulated (Figure 1A). Consistently, HO1 and miR-125b are transcriptionally upregulated by proteasome inhibitor (MG132) which has been showed to activate Nrf2 (Figure 1B). Knockdown of Nrf2 can inhibit the effect of PML-RARA expression and MG132 (Figure 1C). Thus, these results indicated that Nrf2 may mediate PML-RARA-induced miR-125b expression. Furthermore, we demonstrated the importance of miR-125b in APL cell proliferation (Figure 1D), we showed that promoting leukemic cell grow is associated with AKT pathway and MAPK signaling (Figure 1E). Subsequent bioinformatic and RNA-seq data analysis (Figure 1F and G) identified CEBPA as a putative target of miR-125b, and we further experimentally verified using luciferase reporter constructs (Figure H). In conclusion, we identified the mechanisms responsible for the high expression and the function of miR-125b in APL, suggesting that the expression of miR-125b could be a primary oncogenic event in APL leukemogenesis.
This study was supported by grants from the National Natural Science Foundation of China (No.81400102), the China Postdoctoral Science Foundation (No.2015M570751), and the Medical Scientific Research Foundation of Guangdong Province, China (A2015420).
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.
RSK Inhibition Suppresses AML Proliferation through Activation of DNA Damage Pathways and S Phase Arrest
Authors: Dutta Ritika , Castellanos Maria , Tiu Bruce , Chae Hee-Don , Davis Kara L. , Sakamoto Kathleen M. 
Published: October 2019

Abstract
The 90 kDa Ribosomal S6 Kinase (RSK), downstream of the ERK signaling pathway, has recently been implicated in a wide variety of cancers, ranging from lung cancer to medulloblastoma, as a driver of cancer cell proliferation and survival. However, its role in Acute Myeloid Leukemia (AML) remains unknown. Thus, the goal of this study was to characterize RSK-dependent signaling pathways in AML, with the overall hypothesis that disruption of this pathway represents a potential strategy for the treatment of AML. The RSK family consists of four gene isoforms, RSK1-4 (RPS6KA1 (RSK1), RPS6KA2 (RSK3), RPS6KA3 (RSK2), RPS6KA4 (RSK4). Knockdown (KD) of RSK1 by shRNA in HL-60 and KG-1 cell lines resulted in reduced AML cell growth in vitro. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were injected with 2x106 HL-60 or KG-1 RSK1KD cells and vector control transduced cells in order to investigate the effects of RSK1 KD on AML cell growth and survival in vivo. Mice injected with RSK1 KD cells exhibited prolonged survival by 17 and 21 days respectively for HL-60 and KG-1 cell induced disease (p=0.0023 and 0.0018 respectively). These data indicate that RSK1 knockdown inhibits leukemia progression, and RSK1 is required for maximal proliferation of AML cells in vivo. Pharmacological inhibition of total RSK (RSK1-4) by the small molecule inhibitor BI-D1870 reduced AML cell growth and induced cell death in both AML cell lines and patient samples after treatment for 48 hours. The IC50 for growth inhibition was 1.8 uM for MOLM-13, 1.6 uM for MV-4-11, and 1.9 uM for HL-60 cells. In methylcellulose colony assays, normal hematopoietic stem and progenitor cell proliferation was not affected by RSK inhibition up to a concentration of 15 uM, establishing an approximately 10-fold therapeutic index. To elucidate the mechanism by which RSK inhibition suppresses AML proliferation, we performed cell cycle analysis with HL-60 cells. RSK inhibition by BI-D1870 resulted in delayed S-phase progression and accumulation of cells in late S-phase with increased pH2AX, cPARP, and CDK2/Cyclin A expression, as measured by flow cytometry. These data indicate that inhibition of RSK leads to activation of DNA damage pathways and arrest in S-phase, resulting in apoptosis. Inhibition of CDK activity rescued S-phase arrest, demonstrating that activation and dysregulation of CDK are crucial mediators of RSK inhibitor-induced S-phase arrest. In summary, this is the first study to demonstrate that RSK plays an important role in maintaining AML cell survival and proliferation and to position RSK as a promising target for treatment of AML.

Disclosures
No relevant conflicts of interest to declare.