bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Novel Therapeutic Targets in Atm-Deficient Lymphomas Using a Whole Genome CRISPR/CAS-9 Screen
Authors: Stankovic Tatjana , Davies Nicholas , Tee Louise J , Beggs Andrew D , Taylor Malcolm 
Published: November 2019

ATM is a principal DNA damage response protein that synchronises a complex network of cellular responses to double stranded DNA breaks. ATM gene is recurrently mutated in a wide range of lymphoid malignancies, including B-cell chronic lymphocytic leukemia (CLL), T-prolymphocytic leukaemia (T-PLL), mantle cell lymphoma (MCL) and diffuse B cell lymphoma (DLBCL). ATM pathway is utilized by many DNA damaging agents and consequently inactivation of this pathway can lead to chemoresistance. Furthermore, in the absence of ATM tumour cells exhibit genomic instability that can lead to clonal selection and evolution even under current targeted treatments. Consequently there is clear need to understand dependency pathways in ATM-deficient tumours and apply tailored targeted therapies that will specifically eliminate those tumour cells.
We have previously presented a novel murine model of ATM-deficiency that spontaneously generate lymphoid tumours, mostly DLBCL. These tumours have been successfully propagated both in recipient mice and in vitro, where several cell lines have been generated.
Genome editing methods, such as CRISPR/CAS-9, permit the targeted disruption of specific genes. Protocols for genome wide screens have been developed based on this technology which can be used to identify genes that are essential for cellular survival. As such, these screens can be used to identify dependency pathways for tumours with specific genetic lesions.
Using lentiviral transduction we established two cell lines that stably expressed CAS-9. We then performed a genome wide CRISPR screen using the GeCKO library to identify novel therapeutic targets in these Atm-deficient tumours. This library consists of 130,209 unique single guide RNA (sgRNAs), targetting 20,611 genes including 1176 miRNAs.
A comparative analysis was performed of sgRNA drop-out following 15 cellular doublings. This revealed a number of pathways including those already known to be synthetically lethal with ATM deficiency, such as ATR and PARP. Pathway analysis of the top genes from this drop-out analysis identified oxidative phosphorylation, the spliceosome, ribosome biogenesis, N-glycan biosynthesis, pyrimidine metabolism and purine metabolism as the most significantly affected pathways. Furthermore, the drop-out screen revealed a number of miRNAs, including MiR-3470a, Mir-3971, MiR-669f and MiR-719.
These data provide a unique molecular assessment of the dependency of ATM-deficient lymphomas and provide a number of novel putative therapeutic targets for treating such tumours.

Disclosures
No relevant conflicts of interest to declare.
UAF1 DNA Binding Activity Is Critical for RAD51-Mediated Homologous DNA Pairing
Authors: Liang Fengshan , Miller Adam S , Tang Carolilne , Sung Patrick , Kupfer Gary M. 
Published: November 2019

Background: In the Fanconi anemia (FA) DNA repair pathway, DNA damage induces the mono-ubiquitination of the FANCI-FANCD2 (ID2) heterodimer by the FA core complex through its inherent E3 ligase activity. The timely deubiquitination of ID2 by USP1-UAF1 deubiquitinase complex is also critically important for the FA DNA repair. UAF1 has a DNA binding activity, which is required for FANCD2 deubiquitination. UAF1 also enhances RAD51-mediated homologous DNA pairing in a manner that is dependent on complex formation with RAD51AP1. UAF1 deficient cells are impaired for DNA repair by homologous recombination (HR).The biochemical and cellular functions of UAF1 DNA binding activity in HR remain elusive.
Methods:UAF1 wild type and DNA binding mutant proteins were purified and used to define its biochemical properties in HR. In vitroD-loop formation and synaptic complex assembly assay were performed to discover the DNA binding of UAF1 in RAD51 recombinase enhancement. U2OS-DR-GFP cell lines with impaired UAF1 or RAD51AP1DNA binding were generated to examine HR efficiency and DNA damage resistance.
Results:UAF1 preferentially binds an HR-intermediate-like DNA substrate (D-loop, Fig.1). The DNA binding deficient mutant of UAF1 is unable to stimulate RAD51AP1 promotion of RAD51-mediated D-loop (Fig. 2) and the ability to recruit homologous DNA to form the presynaptic complex formation in HR (Fig. 3). In cells, the UAF1 DNA-binding mutant is compromised for the ability to repair DNA damage and to implement HR (Fig. 4). Such activity correlates with the ability to confer resistance to DNA cross linking agents such as mitomycin C (Fig. 4). The DNA binding of UAF1 and RAD51AP1 have a coordinated role in HR-directed DNA damage repair (Fig. 5).
Conclusions: UAF1 DNA binding activity is indispensable for its function in enhancing RAD51-mediated homologous DNA pairing within the context of the UAF1-RAD51AP1 complex. UAF1 DNA binding deficiency causes DNA damage sensitivity and impairs HR efficiency in cells.
Translational Applicability:Our findings reveal a critical role of UAF1 DNA binding in DNA repair and genome maintenance. The identification of UAF1's role in repair will enable targeted efforts to improve molecular approaches for FA therapy.

Disclosures
No relevant conflicts of interest to declare.
Osteoclast-Induced Immunosuppression Occurs through Dysregulation of Immune Checkpoint Axes in Multiple Myeloma
Authors: Mukaihara Kenta , D'Agostino Mattia , Panaroni Cristina , Fulzele Keertik , Mori Tomoaki , Siu Ka Tat , Chattopadhyay Shrikanta , Raje Noopur 
Published: November 2019

Osteolytic bone disease has a significant impact on both quality of life and overall survival of patients with multiple myeloma (MM). Given that bone and immune cells share the same microenvironment and interact with each other via cytokines, signaling molecules and regulatory proteins in the bone marrow (Tsukasaki M et al., Nat Rev Immunol 2019), we sought to elucidate the effect of osteoclasts (OCs) on the immune microenvironment. Specifically, T cells significantly suppress differentiation of OCs through signaling crosstalk between RANKL and IFN-γ (Takayanagi H et al., Nature 2000). However, little is known regarding the effect of OCs on the immune system.
Here we investigated the effect of OCs on immune cells, especially on T cells. Dysregulation of several checkpoint molecules has been shown in MM (Kwon M et al., J Immunol 2017). We, therefore, assessed the expression of various immune checkpoint receptors, such as PD-1, TIGIT, OX40 and CD137 on T cells in co-culture with or without autologous OCs by multi-color flow cytometry. OC co-culture significantly increased co-inhibitory checkpoint (PD-1 and TIGIT) through direct contact, while decreasing co-stimulatory checkpoint (OX40 and CD137) in CD3+ T cells. Expression of the checkpoint ligand, PD-L1 was significantly increased on MM cells in the presence of both T cells and OCs compared to the presence of T cells alone. Conversely, OCs in the absence of T cells did not induce significant increase of PD-L1 on MM cells. According to previous literature, PD-L1 expression on MM cells is induced in part by IFN-γ (Liu J et al., Blood 2007). Interestingly, our data demonstrated that OCs activated IFN-γ producing T cells in co-culture conditions. Furthermore, we observed that T cell and OC-mediated upregulation of PD-L1 on MM cells was partially reversed by using IFN-γ neutralizing antibody. This implies that OCs indirectly induce PD-L1 upregulation on MM cells by enhancing IFN-γ secretion from T cells. In addition, we found that naïve CD4+ T cells have a higher propensity to differentiate into Th17 lineage in the presence of Th17 differentiation cytokines when co-cultured with OCs. Moreover, we observed an increased expansion of Th17 cells in co-culture with OCs. Those Th17 cells also showed a similar pattern of dysregulation of immune checkpoint axes.
Our study demonstrates that OCs positively regulate co-inhibitory checkpoint molecules and negatively regulate co-stimulatory molecules on T cells. These findings indicate that OCs play an important role in inhibiting T cell-mediated antitumor immunity. Targeting OCs may help restore impaired immune surveillance in MM in addition to their critical role in preventing lytic bone lesions in MM. These data may support the role of antiresorptives in immune surveillance with indirect anti-tumor effects.

Disclosures
No relevant conflicts of interest to declare.
Using Lean Six Sigma to Develop a Patient Centered Medical Home for Adults with Sickle Cell Disease
Authors: Johnson Shirley , Hartigan Sarah , Holt Emily , Sop Daniel , McHenry Chantal , Lipato Thokozeni , Elliott Taylor , Ferlis Mica , Mcmanus Caitlin , Smith Wally R 
Published: November 2019

Background: Adults with sickle cell disease (SCD) suffer poor quality, disparities, higher utilization and mortality, and unmet medical need. Thus, in 2017 Virginia Commonwealth University Health (VCU) used pilot results and axioms of the Patient-Centered Medical Home (PCMH) and Lean Six Sigma quality improvement to build an Adult SCD Medical Home.
Methods and Results: VCU funded the program in October 2017. We implemented it over the next fifteen months. We first used a Six Sigma QI cycle called DMAIC (Define, Measure, Analyze, Improve and Control). Define: we defined three improvement targets in year one: inpatient, emergency department (ED), and ambulatory care. Behavioral health, program evaluation, ambulatory clinical policies, and pediatric to adult transition were added in year two. We identified ourselves or recruited hospital partners as champions/leaders for each care target area. Each formed a multidisciplinary implementation team. The inpatient care team actually preceded Medical Home funding. Where feasible, each team conducted PDSA cycles to test and improve best practices or metrics. Measure: we ranked 567 adult SCD patients based on their CY 2017 30-day readmissions, length of stay, 3-day ED returns, ED discharges, inpatient days and discharges, outpatient visits, ED use, and charges. Each team also developed metrics. Analyze: The top 50 highest utilizers by charges were targeted for intervention. Improve: We aimed for six 12-month intervention patient results: 1) reduce readmissions by 15%; 2) reduce average length of stay by 1.5 days; 3) reduce charges by 15%; 4) improve compliance with SCD inpatient guidelines; 5) improve quality, safety, and financial reporting; 6) improve patient experience. Leaders also used Six Sigma tools weekly to oversee teams, identify resources, plan, and hire staff: for example, we ranked the urgency of steps using "Now, Later, Latest" process charts, and documented progress using "Quick Wins" communication logs. Control: Processes were continuously amended. Stakeholders met and supported us monthly to quarterly. We distributed an annual report. Metric results are presented in a separate abstract.
Conclusion: Lean Six Sigma QI principles were effective for developing and implementing an adult SCD medical home. We believe the above processes could be replicated elsewhere.
Figure

Disclosures
Lipato: Novartis: Honoraria. Smith:Novartis: Consultancy, Honoraria.
Regulation of Erythroblast Protein Expression By an RNA Splicing Mechanism, Decoy Exon-Mediated Intron Retention
Authors: Parra Marilyn , Zhang Weiguo , Vu Jonathan , DeWitt Mark , Conboy John G 
Published: November 2019

The erythroid transcriptome is extensively remodeled during terminal erythropoiesis by dynamic changes in RNA splicing of cassette exons and retained introns. Mechanistic studies of these RNA processing networks will provide new insight into pathways that impact structure and function of the erythroid proteome during erythroblast differentiation. We previously showed that up-regulation of EPB41 exon 16 splicing imparts new functionality to the encoded protein, enhancing protein-protein interactions that mechanically strengthen the red cell membrane. More recently, RNA-seq analysis revealed that numerous erythroid transcripts exhibit up-regulation of intron retention (IR) events, some of which are controlled by a decoy exon-mediated mechanism that can reduce the output of translated mRNA so as to limit protein expression. Here we demonstrate that modulation of decoy-mediated IR quantitatively affects protein expression in primary human erythroid cells. We first studied the OGT gene (O-GlcNAc transferase), a key regulator of O-GlcNAC homeostasis. OGT expression responds to pharmacological inhibitors by regulating intron 4 retention, by a mechanism requiring a intronic splicing silencer (1) that functions as a decoy exon (2) to nonproductively engage annotated splice sites at the ends of the intron, thereby blocking excision and enforcing its retention. In erythroid CD34+ progenitors at day 7 of culture, we found that treatment with an OGT inhibitor (OSMI-1) reduced OGT IR and increased spliced OGT RNA and OGT protein. Conversely, an inhibitor of the antagonistic OGA enzyme (thiamet-G) induced greater OGT IR and reduced OGT protein expression. These results are similar to what was reported previously in established cell lines (1), and suggest that modulation of IR can vary mRNA and protein expression in primary cells &gt;5-fold. To further explore the model, we independently blocked OGT IR in erythroid cultures by electroporation of an antisense morpholino directed against the OGT decoy exon 5' splice site. RT-qPCR and western blot analysis confirmed substantial reduction in IR, coupled with an increase in spliced RNA and elevated OGT protein expression, compared to control cells or cells treated with an irrelevant morpholino. The OGT-specific MO also substantially blocked IR induced by thiamet-G. These results show that pharmacological- or antisense-mediated alteration in IR can significantly change protein expression in primary erythroblast cultures. We propose that the abundance of IR transcripts in late erythropoiesis represents a widespread modulation of protein output by post-transcriptional pathways operating at the level of intron retention. Park SK, et al. (2017) Cell Rep. 20: 1088-99.Parra M et al. (2018) RNA 24: 1255-65.

Disclosures
No relevant conflicts of interest to declare.
Prediction of Malingant Plasma Cell Biology Related Survival in AL-Amyloidosis
Authors: Beck Susanne , Emde Martina , Moreaux Jerome , Seckinger Anja , Hose Dirk 
Published: November 2019

Background. Survival in AL-amyloidosis is thought to be primarily determined by signs and symptoms caused by deposition of amyloid light chains, most prominently in the heart. In contrast, molecular characteristics of the underlying malignant plasma cell disease have been described, but are mostly considered less important.
Aim of our study is to predict malignant plasma cell biology related survival in AL-amyloidosis by establishing the first gene expression based risk stratification (HDAL) and assessing its independence from clinical serum parameter assessing risk.
Methods. CD138+-purified plasma cell samples of 919 patients with malignant plasma cell diseases, i.e. 195 AL-amyloidosis and 724 symptomatic myeloma patients were investigated by gene expression profiling, 124 AL-amyloidosis patients by RNA-sequencing. Gene expression profiling data of AL-amyloidosis patients were spitted in a training (TG, n=99) and a validation group (VG, n=96). A two-step model according to Rème et al. was applied on the training group, including a running log rank test for gene selection and a multi-cut-off running log-rank algorithm for optimal cut-off-selection, leading to a selection of 15 genes associated with good and 44 genes with adverse prognosis. The resulting HDAL-score was validated on the independent VG and our myeloma patient cohort using survival estimates for censored data. Differences between curves were assessed using the Log-rank test. The continuous RNA-Seq HDAL-score (R-HDAL) was subsequently derived to validate the survival association of the selected genes. HDAL was tested for independence with serum parameter assessing clinical staging systems by multivariate Cox regression.
Results. Categorical split of the HDAL-score delineates three significantly predictive groups of 48%, 29% and 23% of AL-amyloidosis patients with a median survival of 105, 53 and 3 months in the training, and 72, 33 and 6 months in the validation group, respectively. In symptomatic myeloma patients, HDAL significantly stratifies two groups with a median survival of 128 and 78 months. Thus, HDAL is a malignant plasma biology related derived risk stratification. HDAL and R-HDAL are significantly predictive for survival as continuous parameters. Categorial and continuous HDAL are individually independent predictive from clinical staging systems, i.e. Mayo 2004, 2012 and Euro score, and the assessed serum parameters, i.e. NT-ProBNP, cTNT and dFLC.
In conclusion, malignant plasma cell biology related and amyloid deposition mediated survivals in AL-amyloidosis are independent. Prognosis driven by the first component can significantly be assessed by transcriptome profiling (HDAL or R-HDAL).
We thank U. Hegenbart and S. Schönland for clinical collaboration in this work.

Disclosures
Moreaux: Diag2Tec: Other: Co-founder of Diag2Tec company.
Efficacy of Fecal Microbiota Transplant in Hematological Cancers Patients with Recurrent Clostridioides Difficile Infection: A Systemic Review and Meta-Analysis
Authors: Tariq Raseen , Furqan Fateeha , Jamshed Saad , Khanna Sahil 
Published: November 2019

Introduction:
Fecal microbiota transplant (FMT) has been shown to be an effective treatment for recurrent Clostridioides difficile infection (CDI). Immunosuppression is one of the risk factors for CDI. However, most of the trials evaluating efficacy of FMT have excluded immunocompromised patients. Patients with hematological cancers represent a major class of immunocompromised patients. Hence, we performed a meta-analysis to evaluate the efficacy of FMT in patients with hematological cancer for the treatment of recurrent CDI.
Methods:
A systematic search of Medline, Embase, and Web of Science was performed from January 2000 up to December 2018. Articles included for meta-analysis were case series and case reports that assessed efficacy of FMT in hematological cancer patients were included. Study quality was assessed using the Newcastle-Ottawa scale. The main outcomes were pooled proportions of patients achieving cure after first FMT. The cure rate was defined as resolution of diarrhea without any recurrence.
Results:
10 studies (5 case series and 5 case reports) were included in the analysis, making up 29 patients who underwent 38 total FMT procedures. The follow up period ranged from 1- 14 months. Of the included patients, 17 had leukemia, 10 had lymphoma and 2 patients had multiple myeloma and myelodysplastic syndrome each. 7 of these patients had undergone hematopoietic stem cell transplant.
The pooled cure rate was 82% [confidence interval (CI) 67-94%] after 1 FMT with no heterogeneity (I2=0%). Minor publication bias was seen on visual inspection of funnel plot.
FMT was generally well tolerated by these patients. No serious side effects were reported in any study. One of the studies reported death due to cardiac arrest 5 days after FMT which was thought to be unrelated to the procedure. Mild side effects including mild abdominal pain, transient diarrhea, fecal urgency, constipation and nausea were reported in 5 patients.
Conclusion:
Based on limited observational data, FMT seems to be an effective and safe modality for management of recurrent CDI in patients with hematological cancers. However, further prospective clinical trials are needed to establish its safety as a first line therapy for recurrent CDI in this population.
Figure

Disclosures
Jamshed: Takeda Pharmaceutical: Honoraria. Khanna:Rebiotix, Inc: Research Funding; Probio Tech, LLC: Consultancy; Facile Therapeutics: Consultancy; Shire, Plc: Consultancy.
In-Hospital Complications and Outcomes of Autologous Stem Cell Transplantation in Multiple Myeloma Patients Older Than 65-Years Old in the United States: National Inpatient Sample Analysis, 2011-2015
Authors: Bakalov Veli , Tang Amy , Shaikh Hira G. , Shah Shrunjal , Chahine Zena , Kaplan Robert B. , Lister John , Sadashiv Santhosh 
Published: November 2019

Background. Use of Autologous Stem Cell Transplantation (ASCT) continues to increase in elderly patients with multiple myeloma. The main goal of our study was to describe in-hospital complications and outcomes after ASCT in patients younger and older than 65-years old utilizing the Nationwide Inpatient Sample (NIS) database.
Methods. Cohort selection. The NIS database for the years 2011 to 2015 was queried for the analysis. We used International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) in order to identify patients with MM as a primary diagnosis for the hospitalization, and ASCT as a primary diagnosis of the hospitalization. In order to determine comorbidities in selected population we used Clinical Classifications Software (CCS) in conjunction with ICD-9-CM codes. Statistical Analysis.Complex weights were used throughout all calculations, enabling appropriate national projections. Chi-squared and independent t-tests were used for univariate analysis where appropriate. In our study p-value <0.05 was considered statistically significant. Data were analyzed using SAS v9.4 (SAS Institute, Cary, NC).
Results.A total of 3,664 patients with MM receiving ASCT were identified in our cohort. Males represented 57.1% of the population, majority of the patients were white, with private insurance, located at the urban teaching hospitals (Table 1). Main insurance in patients older than 65 years old was Medicare. Main comorbidities during hospitalization were anemia, hypertension, electrolyte abnormalities, acute kidney injury, cardiac dysrhythmias, sepsis and pneumonia (Table 2). Overall in-hospital mortality was low, 0.7%, and was significantly higher in patients older than 65 years old (1.3% vs 0.4%, p-value 0.002), however when adjusted for the baseline characteristics with multivariate logistic regression analysis in-hospital mortality was no longer statistically significant (Table 3).
Conclusions. We found that patients older than 65 years with MM receiving ASCT are more susceptible to in-hospital complications. However we found that in-hospital outcomes are not statistically significant when adjusted to baseline characteristics, suggesting that the procedure is safe even in the elderly population. Findings of our study can be used to design future randomized controlled trials.

Disclosures
No relevant conflicts of interest to declare.