bluuebunny/crossref_metadata_2025_split huggingface dataset:
BTK Inhibitors Induce ABC-DLBCL Cell Apoptosis By Inhibiting CYLD Phosphorylation
Authors: Xu Xin 
Published: November 2019

BTK inhibitors induce ABC-DLBCL cell apoptosis by inhibiting CYLD phosphorylation
Xin Xu1, Zhigang Zhu1, Feng Liu1 , Weijie Zhong1,Huabao Xiong2, Tiefeng Feng3, and Qingshan Li3
1Geriatrics Department, GuangZhou First People's Hospital, GuangDong, 510180, People's Republic of China.
2Precision Immunology Institute, Mount Sinai School of Medicine, New York, NY 10029, U.S.A.
3Hemotology Department, GuangZhou First People's Hospital, GuangDong, 510180, People's Republic of China.
Activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL) is a common aggressive diffuse large B cell lymphoma (DLBCL). Even though CD20 antibody (Rituximab) combination therapy improved the total prognosis of DLBCL, there are still many relapsed and refractory ABC-DLBCL cases. The new treatment options are urgently needed.
Pathogenesis of ABC-DLBCL is different from germinal center B cell-like diffuse large B cell lymphoma (GCB-DLBCL), which is related to activation of B cell antigen receptor(BCR) signaling. The Bruton tyrosine kinase(BTK) plays a central role in BCR signaling. Inhibition of BTK function will affect BCR signaling activity, which is involved in maintaining the malignant phenotype in B-cell lymphoma. Now in clinic, BTK inhibitors like Ibrutinib or Acalabrutinib are used extensively in chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma(MCL) as first line therapy or second line therapy. But in DLBCL its role is unclear now.Some DLBCL clinic trials got the better PFS and OS in BTK inhibitor combination therapy group(combined with Rituximab or RCHOP) but some got the negative results. Whether BTK inhibitors can definitely improve ABC-DLBCL survival and the mechanism how BTK inhibitors inducing cell death are needed further investigations.
CYLD (cylindromatosis) is a tumor suppressor, its activity is inhibited by phosphorylation, which is related to apoptosis regulation pathway NF-kB in many kinds of tumors.In this study, we found that CYLD to be constitutively phosphorylated in ABC-DLBCL cell line models (HBL-1 and OCI-Ly10) as well as primary samples from patients with non-GCB DLBCL. We subsequently examined the effect of BTK inhibitors Ibrutinib (PCI-32765) and Acalabrutinib (ACP-196) on CYLD phosphorylation.
We found that BTK inhibitors Ibrutinib and Acalabrutinib both induced ABC-DLBCL apoptosis by down-regulating CYLD phosphorylation in vivo and in vitro, even in the Rituximab resistant ABC-DLBCL cell line.Combination of BTK inhibitors and Rituximab would enhance this effect. Knockdown of CYLD in ABC-DLBCL cells reduced the level of cell death induced by BTK inhibitors, which showed that this cell death induced by BTK inhibitors was CYLD dependent.
Through this study, we conclude that BTK inhibitors induce CYLD inactivation by regulating CYLD phosphorylation is clinical beneficial in ABC-DLBCL therapy, especially the relapsed or refractory ABC-DLBCL cases after Rituximab therapy.

Disclosures
No relevant conflicts of interest to declare.
Endocytosis of the Thrombopoietin Receptor Mpl Regulates Both Megakaryocyte and Erythroid Maturation in Mice
Authors: Eaton Nathan , Lee-Sundlov Melissa M. , Dlugi Theresa A. , Wieser Jon , Ramsey Haley E. , Sola-Visner Martha , Hoffmeister Karin M. , Falet Hervé 
Published: November 2019

The thrombopoietin receptor Mpl plays a critical role in thrombopoiesis, as it is the primary signaling constituent in megakaryocyte (MK) differentiation from hematopoietic stem and progenitor cells (HSPCs) and maintains circulating blood thrombopoietin levels via clathrin-mediated endocytosis. Thus, thrombopoiesis is tightly controlled by Mpl cell-surface expression on HSPCs, MKs, and platelets. Previous work by our group has shown that Dnm2fl/flPF4-Cre (Dnm2Plt-/-) mice, lacking the large endocytic GTPase dynamin 2 (DNM2) specifically within MKs and platelets, develop a variety of clinical phenotypes that closely resemble myelofibrosis, such as bone marrow fibrosis, marked HSPC expansion, MK hyperplasia, extramedullary hematopoiesis, and severe splenomegaly (Bender, Giannini, et al. Blood. 2015;125(6):1014-1024). Dnm2Plt-/- mice also displayed elevated plasma TPO levels and constitutive JAK2 activation in platelets, due to defective Mpl endocytosis. Here, the role of Mpl endocytosis in the maintenance of normal hematopoiesis was assessed by generating Dnm2Plt-/- mice in the Mpl-/- background. At 3 weeks of age, Mpl-/- Dnm2Plt-/- mice displayed significantly reduced HSPCs, a near complete depletion of bone marrow MKs, similar to Mpl-/- mice, indicating that Mpl is the primary receptor contributing to the aberrant hyperproliferative phenotype of Dnm2Plt-/- mice. However, Mpl-/- Dnm2Plt-/- mice also showed severe anemia, defects in erythroblast maturation, grossly elevated plasma erythropoietin (EPO) levels, and splenomegaly, resulting in early fatality by 3 to 4 weeks of age, suggesting that Mpl contributes to normal erythropoiesis in young mice. Mpl-/- and Mpl+/- Dnm2Plt-/- mice displayed reduced erythroblast development at 3 weeks of age, which returned to normal with adulthood. Taken together, the data shows that DNM2-dependent Mpl endocytosis is required for steady-state hematopoiesis and provides novel insights into a developmentally-controlled role for Mpl in normal erythropoiesis.

Disclosures
Sola-Visner: Sysmex America, Inc.: Other: Laboratory equipment on loan, Research Funding.
Adiponectin Inhibits the Differentiation and Maturation of Osteoclasts Via mTOR Pathway in Multiple Myeloma
Authors: Fu Rong , Zhaoyun Liu , Liu Hui , Li Yanqi , Wang Yangyang , Xing Rui , Mi Fu , Shao Zonghong , Song Jia , Xiang Chenhuan 
Published: November 2019

Background: To investigate the correlation of adipose cytokines (visfatin, leptin, and adiponectin) with multiple myeloma bone disease (MBD) and explore the effects and mechanism of adiponectin on the differentiation and maturation of osteoclasts in multiple myeloma (MM).
Methods: The levels of visfatin, leptin and adiponectin were measured .Their relationships between the index of myeloma tumor load and bone disease were analyzed. RT-PCR was used to detect the expressions of RANKL, OSCAR, TRAP and Cathepsin K genes. Flow cytometry (FCM) was used to detect the expressions of AdipoR1 and phosphorylation of mTOR pathway related proteins mTOR and 4EBP1.
Results :There were no significant linear correlation between leptin, visfatin and the indexes of myeloma tumor load and bone disease. Serum adiponectin levels were significantly higher in newly diagnosed multiple myeloma patients and negatively linear correlated with β2-microglobulin.There is a positive linear correlation relationship between the adiponectin and osteocalcin. The number of maturation osteoclasts in adiponectin group was less than the control group. Adiponectin also inhabits the mRNA level of osteoclast related factor- RANKL, OSCAR, TRAP and Cathepsin K. Flow cytometry showed that adiponectin increased the expression of AdipoR1 on the surface of osteoclast precursor((26.21±4.27)% vs (29.86±6.23)%, p<0.05) and reduced the expression of p-mTOR ((7.89±1.00)% vs (5.91±1.26)%, p<0.05) and p-4EBP1 ((26.78±5.00)% vs (22.49±4.24)%, p<0.05).
Conclusion: Adiponectin inhibits the differentiation and maturation of osteoclasts via increasing the expression of AdipoR1 and reducing the phosphorylation levels of mTOR and 4EBP1 proteins in MM patients.

Disclosures
No relevant conflicts of interest to declare.
The Lost or Absence of MRD<0.1% at Any Time after 2 Cycles of Consolidation Chemotherapy in CBFB-MYH11-Positive Acute Myeloid Leukemia Indicates Poor Prognosis
Authors: Duan Wenbing , Jia Jinsong , Wang Jing , Liu Xiaohong , Gong Lizhong , Jiang Qian , Zhao Ting , Wang Yu , Zhang Xiaohui , Xu Lanping , Liu Yanrong , Qin Yazhen , Huang Xiaojun , Jiang Hao 
Published: November 2019

Objective: To investigate the long-term prognostic factors of CBFB-MYH11-positive acute myeloid leukemia in low、 intermediate risk patients.
Methods:58 CBFB-MYH11-positive patients were analyzed retrospectively in 1525 acute myeloid leukemia patients who were diagnosed in Peking University Institute of Hematology from October 2013 to August 2018, and the clinical feature、induction remission rate and survival were compared.
Results: 58 (8%) patients with CBFB-MYH11-positive were found in 1525 newly diagnosed actue myeloid leukemia, 41 males and 17 females were included. The median age was 38(17-66) years , and median follow-up time was 29.8(4.8-74.4)months, 25(43.1%) patients received allogenic hematopoietic stem cell transplantation, in which there were 10 patients with c-kit mutation. According to the NCCN guideline, there were 40 patients in low risk group and 18 patients in intermediate risk group (16 patients with c-kit mutation). The rate of complete remission (CR) in the first induction chemotherapy was 98.3%(57/58), the cumulative remission rate of two cycles was 100%, the relapse rate was 25.9%(15/58), and the mortality rate was 19.0% (11 ≤ 58). After 1 and 2 cycles of consolidation, the level of CBFB-MYH11/ABL decreased to less than 0.18% and 0.094% respectively, which were related to the low recurrence rate. Among the 33 patients who did not undergo allogenic hematopoietic stem cell transplantation after two courses of consolidation, the recurrence rate of patients with CBFB-MYH11/ABL level > 0.1% (n =21) was significantly higher than that of patients with < 0.1% (n = 12) (61.9% VS 0%, P =0. 001), and the relapse-free survival(RFS) (3-year RFS rate 37.6% VS 100%, P =0.005 ) 、event-free survival (EFS)(3-year EFS rate 33. 3% VS 100%, P = 0.004) were significantly decreased. The RFS and EFS were lower than those of patients received allogenic hematopoietic stem cell transplantation (n=25) (3-year RFS rate 31.4% VS 84.6%, P = 0.000; 3-year EFS rate 31.4% VS 76.0%, P =0.004).
Conclusions: To the CBFB-MYH11-positive acute myeloid leukemia patients, if the level of CBFB-MYH11/ABL gene presented more than 0.1% at any time after 2 cycles of consolidation, poor prognosis was related to, and allogenic hematopoietic stem cell transplantation could improve the survival.

Disclosures
No relevant conflicts of interest to declare.
Serum Citrulline As a Biomarker for Blood Stream Infections in Pediatric Hematopoietic Stem Cell Transplant Patients
Authors: Singla Shyamli , Wong Justin , Jiang Shuang , Hussain Sarah , Coughlin Laura , Zhan Xiaowei , Koh Andrew Y. 
Published: November 2019

Background: Invasive microbial infections remain a major cause of morbidity and mortality in hematopoietic stem cell transplant (HSCT) patients. Microbes residing in the GI tract are believed to be the source of mucosal barrier injury-associated blood stream infections (mBSI) in HSCT and cancer patients. Three host factors are critical for preventing microbial translocation from the gut: neutrophils, intact gut barrier function, and gut microbiota homeostasis. Currently there are no reliable biomarkers that can identify patients who will develop mBSIs.
Objectives/Hypothesis: Since all HSCT patients develop severe neutropenia (ANC<100), we hypothesized that HSCT patients who develop mBSI will have more impaired gut barrier function and a greater imbalance in gut microbiota populations (as evidenced by depletion of beneficial obligate anaerobe commensals) compared to counterparts who do not develop mBSI.
Methods: We designed a prospective, non-randomized, case control pilot study for HSCT patients < 20 years of age. Serum citrulline, which has been used as a surrogate to measure overall gut function, was obtained 1-2 times weekly. Bacterial genomic DNA was isolated from stool samples collected every 7 days. Amplicons (16S rRNA V4 variable region) were generated and sequenced using the Illumina MiSeq platform. Microbial taxonomic assignments were determined using QIIME software and further characterized into relative abundances of obligate (beneficial commensal) versus facultative (pathogenic) anaerobes. Clinical characteristics were recorded.
Results: Of the 78 HSCT patients enrolled on the study, 27% (21 patients) developed BSI. Of the patients with confirmed blood stream infections, 16 patients were categorized as mBSI, and ultimately 11 patients were included in our analysis. 48 patients did not develop a BSI and had evaluable citrulline levels and constituted our control group. Patients with mBSI had a more significant decline in citrulline levels during the period from the start of conditioning to the development of mBSIs (linear mixed effect model, p = 0.048, likelihood ratio test) compared to counterparts without mBSI. We found no significant association between levels of facultative (pathogenic) anaerobes and the development of mBSI in this cohort.
Conclusions: Decreased citrulline levels, which suggest impaired gut epithelial function, are associated with the development of severe, invasive bacterial infections. These data suggest that measuring citrulline may have utility as a biomarker for predicting mBSI in HSCT patients.

Disclosures
Koh: Merck: Consultancy.
Hematopoietic Cell Cluster Identifier (HCCI): A Knowledgebase Computational Tool to Facilitate the Characterization of Hematopoietic Cell Populations in Single Cell RNA-Sequencing
Authors: Kumar Gaurav , Ertel Adam , Naranjo Nicole , Shahid Shuvanon , Languino Lucia , Fortina Paolo 
Published: November 2019

Single cell transcriptional profiling is critical to interrogate cell types, states and functionality in a complex tissue or disease. Despite rapid advancements in single cell RNA sequencing (scRNA-seq) analysis, major challenge remains in identification of distinct cell types based on specific molecular signatures. Few databases are available to facilitate cell-type characterization, however, their broad all-cells encompassing structure is deemed often inept. At present, manual curation is the only option. To address this, we developed a knowledgebase computational tool, HCCI, that identifies and characterizes up to 28 different hematopoietic cell types in a scRNA-seq dataset. Utilizing the knowledgebase comprised of ~1500 marker genes obtained from data mining, HCCI first uses a voting algorithm to search and match genes from a given cluster to a single or more possible cell type. Next, it normalizes score of each cluster-cell type pair to provide a percentage denoting a cluster to a specific cell-type. To demonstrate this, we utilized a dataset of Peripheral Blood Mononuclear Cells (PBMC) available from 10X Genomics. As the cell populations are already known, this was an ideal dataset to evaluate the performance of HCCI. As expected, unsupervised clustering and UMAP algorithms identified 9 different clusters of cells conforming to the existing knowledge of this dataset. Differentially expressed genes from each cluster were input into HCCI, and the following unique clusters were identified: naïve CD4 (cluster 1), M0 macrophage (cluster 2), CD8 T-cells (cluster 3), naïve B-cell (cluster 4), cytotoxic T-cell (cluster 5), M1 macrophage (cluster 6), activated NK cell (cluster 7), monocyte (cluster 8) and plasmacytoid dendritic cell (cluster 9). Here using HCCI, an improvement in classification was observed for clusters 2,3,6,8 and 9 which were originally classified only in major blood cell types. Classification of clusters 1,4,5 and 7 remained unchanged. The classification by HCCI was observed to be much precise when compared to the manual curation performed originally. To our knowledge, HCCI is the only available tool for characterization of hematopoietic cells from scRNA-seq data.

Disclosures
No relevant conflicts of interest to declare.
Cell-Intrinsic Inflammation Drives Progression from Myelodysplastic Syndromes to Leukemia
Authors: Barreyro Laura , Sampson Avery , Hueneman Kathleen , Choi Kwangmin , Christie Susanne , Wang Dehua , Huang Gang , Starczynowski Daniel T. 
Published: November 2019

Myelodysplastic Syndromes (MDS) often progress to Acute Myeloid leukemia (AML) typically following acquisition of mutations in RUNX1, NRAS/KRAS or AXSL1. Inflammatory processes associated with autoimmune diseases or natural aging are thought to contribute to the development of MDS and progression to AML. Moreover, dysregulation of innate immune signaling is broadly observed in hematopoietic stem and progenitor cells (HSPC) from MDS patients. However, how dysregulation of innate immune signaling and/or inflammatory signals might lead to malignant transformation and overt AML has not been fully elucidated. We have developed a mouse model of cell-intrinsic inflammation-driven progression from MDS to AML that will allow us to determine the mechanisms of oncogenic transformation. MicroRNA miR-146a is a 5q gene that is frequently deleted in MDS and AML and its deletion promotes dysregulation of innate immune signaling and a systemic inflammatory state. Deletion of miR-146a results in myeloproliferation and marrow failure in mice associated with attrition of HSPC, but alone is insufficient to induce overt AML. Using a genetically engineered approach, we show that deficiency of miR-146a (miR-146a-/-) combined with a C-terminal truncation of the RUNX1 (S291fsX300) protein causes anemia, thrombocytopenia and ineffective erythropoiesis mimicking human MDS in younger mice, but then the mice progresses to a fatal AML. The inflammatory environment induced by miR-146a deletion was exacerbated in mice with miR-146a-/-expressing RUNX1-S291fsX300 (RUNX1-mut) and correlated with the increased incidence and aggressiveness of hematologic malignancies. In particular, the levels of IFNγ were increased in plasma in mice with miR-146a-/-expressing RUNX1-mut in comparison to mice reconstituted with miR-146a-/-or RUNX1-mut-expressing BM cells. As previously reported, miR-146a-/-cells exhausted during serial transplantation, however RUNX1-mut rescued the hematopoietic repopulation deficiency of miR-146a-/-HSPC, suggesting that the RUNX1 mutation promotes malignant transformation to AML under inflammatory conditions. To establish whether cell-intrinsic immune signaling is required for RUNX1-mut;miR-146a-/- leukemic cells, we evaluated an inhibitor (NSC697923) that targets TRAF6, a key target of miR-146a in MDS/AML, via inhibition of its co-factor UBE2N. Treatment with NSC697923 or a RUNX1 inhibitor (Ro 5-3335) abolished colony formation by RUNX1-mut;miR-146a-/- leukemic cells, indicating that cell-intrinsic innate immune signaling is not only required for initiating AML, but is also critical for sustaining the leukemic phenotype. These findings describe the first evidence that dysregulation of innate immune signaling and/or inflammatory signals contribute to the progression and maintenance of AML.

Disclosures
Starczynowski: Kurome Therapeutics: Consultancy.
Rezafungin Prevention of Pneumocystis Pneumonia and Pneumocystis reactivation Using Different Doses and Durations of Prophylaxis in a Mouse Model
Authors: Cushion Melanie T , Ashbaugh Alan , Ong Voon 
Published: November 2019

Rezafungin (RZF) is a novel echinocandin in development for prevention of invasive fungal disease caused by Candida, Aspergillus, and Pneumocystis species in patients undergoing blood and marrow transplantation. RZF has a favorable safety and tolerability profile, a low risk of drug-drug interactions and, with its stability, pharmacokinetics that allow for once-weekly dosing with broad distribution to the lung and other fungal target organs.
We previously demonstrated RZF efficacy in preventing Pneumocystis infection in an immunosuppressed mouse model. The present study addressed whether Pneumocystis infection in a similar immunosuppressed mouse model could re-activate after 2 to 8 wks of prophylactic therapy using different dosing regimens of RZF.
C3H/HeN mice were infected with P. murina intranasally at 2 x 106/50 µl after immunosuppression with dexamethasone. Mice were administered vehicle as a negative control (C/S), trimethoprim/sulfamethoxazole (T/S) as a positive control, caspofungin (CASPO) as a comparator echinocandin or RZF intraperitoneally at time of challenge. Study drug administration was stopped at 2, 4, 6, and 8 weeks, at which time mice were immunosuppressed for an additional 6 weeks to allow any residual P. murina to re-activate. Mice were then euthanized and lungs were processed for analysis of nuclei and asci.
All RZF dose regimens at all timepoints significantly reduced both nuclei and asci burdens versus the C/S group (Figure 1). After 4 weeks of RZF prophylaxis (plus 6 wks additional immunosuppression [week 10 timepoint]; Figure 1), both RZF 20 mg/kg groups (3x/wk and 1x/wk) had prevented P. murina organisms from activating an infection. After 6 and 8 weeks of RZF prophylaxis (week 12 and 14 timepoints; Figure 1), no re-activation of infection was present in any of the study groups. After 2 and 4 weeks of prophylaxis (week 8 and 10 timepoints), there was a significant reduction of nuclei and asci counts in all groups of RZF versus CASPO. After 2, 4, and 6 weeks of RZF prophylaxis, there was a significant reduction of nuclei and asci counts between all groups of RZF versus T/S 1x/week. There was a significant benefit in survival between the RZF group at 20 mg/kg/3x/week versus CASPO at week 14 (Figure 2).
In this study, prophylaxis with RZF for as little as 4 weeks prevented P. murina organisms from developing infection after cessation of therapy and showed more efficacy than CASPO. These results in the mouse model of Pneumocystis pneumonia provide evidence that RZF can prevent Pneumocystis reactivation and that such regimens hold promise for prophylaxis against Pneumocystis in at-risk patients undergoing blood and marrow transplantation.

Disclosures
Cushion: Cidara Therapeutics: Consultancy, Research Funding.
Maintenance Sorafenib Is Superior to Prophylactic DLI in Improving the Prognosis of FLT3-ITD-Positive AML after Allogeneic HSCT
Authors: Shi Jimin , Cao Liqin , Luo Yi , Zhao Yanmin , Tan Yamin , Yu Jian , Lai Xiaoyu , Zhu Yuanyuan , Hu Yongxian , He Jingsong , Sun Jie , Zheng Weiyan , Wei Guoqing , Huang He 
Published: November 2019

Background: Compared with traditional chemotherapy, allogeneic hematopoietic stem cell transplantation (allo-HSCT) can improve the prognosis of patients with FLT3-ITD positivity. Relapse after transplantation is still an important factor affecting survival. Prophylactic donor lymphocyte infusion (DLI) and maintenance sorafenib are two methods for preventing relapse after allo-HSCT in FLT3-ITD-positive acute myeloid leukemia (AML) patients, and their roles in these patients after allo-HSCT need to be further studied. Methods: From January 2014 to December 2018, 69 FLT3-ITD-positive patients who received allo-HSCT at our center were included in this retrospective study. All patients were divided into four groups according to the different treatments received: group A (23 patients who received maintenance sorafenib), group B (12 patients who received prophylactic DLI), group C (33 patients who received neither prophylactic DLI nor maintenance sorafenib), and group D (1 patient who received a combination of prophylactic DLI with maintenance sorafenib), because there was only one patient in group D, it was not included in the analysis. Results: For all patients, the 2-year estimated overall survival (OS) and leukemia-free survival (LFS) were 79.9% and 78.3%, respectively, and the 2-year cumulative incidence of relapse (CIR) was 17.2%. The 2-year estimated OS, LFS and CIR in group A, group B and group C were 95.7%vs75%vs66.8%, 95.7%vs74.1%vs70.2% and 4.3%vs6.7%vs26.1%, respectively. Group A was the best one in OS, LFS and CIR. Between the group A and group C, there were significant difference in OS(P=0.03) and LFS(P=0.04), but not in CIR(P=0.06).The OS in group A was significantly higher than group B(P=0.047), but the difference was not statistically in LFS and CIR (P=0.14 and P=0.21). Conclusion: Maintenance sorafenib after allo-HSCT was associated with improved outcomes for FLT3-ITD positive AML patients and was superior to prophylactic DLI in promoting FLT3-ITD-positive patients obtain a better survival after allo-HSCT.
Figure

Disclosures
No relevant conflicts of interest to declare.