bluuebunny/crossref_metadata_2025 huggingface dataset:
A Zebrafish Model for Severe Congenital Neutropenia with Jagn1b-Deficiency
Authors: Doll Larissa , Aghaallaei Narges , Skokowa Julia , Welte Karl , Bajoghli Baubak
Published: November 2020

Severe congenital neutropenia (CN) is a bone marrow failure characterized by an impaired neutrophil development. This disease can be caused by different germline mutations, which one of them is Jagunal homolog 1 (JAGN1). CN patients are prone to develop life-threatening infections from birth on and the treatment of choice is recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, patients with JAGN1 deficiency poorly respond to this treatment. Therefore, the development of new therapeutic strategies to treat this disease is essential. Thus far, lack of an animal model was the main limitation to study the function of JAGN1 in neutropenia and perform high-throughput compound screening. In this work, we studied the role of jagn1b in zebrafish granulopoiesis and hematopoiesis. Two different approaches were used. First, we injected antisense morpholino that efficiently blocked the translation of jagn1b mRNA. As a second approach, we used the CRISPR-Cas9 technique to introduce mutations in the zebrafish jagn1b gene and analyzed the crispants. Both of these approaches were tested in wild-type embryos as well as the transgenic mpo:GFP reporter line, in which neutrophils are labeled with GFP. Our results showed that interference with Jagn1b reduced the number of neutrophils, without affecting the early myelopoiesis and monocytes/macrophages. Further analysis revealed that apoptosis was enhanced in embryos injected with the jagn1b morpholino, however, this effect was not restricted to the hematopoietic tissues. Our results also suggested that unfolded protein response (UPR) was activated because the expression levels of genes involved in this pathway such as chop, atf4a and atf4b were significantly upregulated in the jagn1b morphants, when compared with the wild-type counterparts. Overall, we have successfully established a novel in vivo model for studying the role of JAGN1 in granulopoiesis, and the possible role of Jagn1b in UPR response might be useful to find new targets for the development of therapeutic strategies for CN.

Disclosures

No relevant conflicts of interest to declare.
Excellent Clinical and Pharmacokinetic Real-World Experience after Switching to Rurioctocog ALFA Pegol
Authors: Benitez Hidalgo Olga , Lopez Jaime Francisco Jose , Martínez García María Fernanda , Juarez Gimenez Juan Carlos , Alvarez Martinez Eva , Suito Milagros , Bosch Francesc , Gironella Mesa Mercedes
Published: November 2020

Background:Prophylactic treatment with standard half-life coagulation factors (SHL) requires 3-4 weekly periodic infusions. Extended half-life (EHL) factor VIII (FVIII) provides improvements in half-life (t1/2) and area under the curve (AUC) of 1.3 and 1.25 times compared to standard half-life (SHL) products. Rurioctocog alfa pegol is an extended half-life (EHL) rFVIII (rFVIII-EHL) treatment that could be a more convenient and cost-effective therapeutic alternative than SHL, since it would reduce the number of weekly infusions (NWI).

Aims:The aim of this study is to analyze pharmacokinetic (PK) parameters and compare NWI and bleeding rates after switching from SHL to this EHL in hemophilia A (HA) patients.

Methods:In a prospective study, the number of weekly infusions (NWI), annualized bleeding rate (ABR), annualized joint bleeding rate (AJBR), and PK parameters were compared between previous treatment with FVIII-SHL vs rFVIII-EHL (Rurioctocog alfa pegol).

The assessment of the PK parameters: volume of distribution (Vd (dL/kg)), clearance (Cl (dL/h/kg)) and half-life time (t½ (h)) was performed by using the pharmacokinetic poblacional software online myPKFiT®.

Results:Nineteen patients from 2 Spanish hospitals (n=12 Vall d'Hebron Hospital, n=7 Hospital Regional Universitario de Málaga) were recruited. Median age was 30 years (range, 12-54), with five pediatric patients, and 18 had severe HA (one moderate HA).

Sixteen patients were under prophylactic treatment with Advate® and 1 with Kovaltry®. Two patients were switched from on-demand treatment to prophylaxis.

The median use of rFVIII-EHL was 15.63 month.

The PK parameters after the switch to the rFVIII-EHL in entire cohort and pediatric cohort are shown in Table 1.

The t ½ of rFVIII-EHL was positively correlated to preinfusion plasma levels of von Willebrand factor antigen (vWF:Ag) (Figure 1).

ABR and AJBR were reduced, as well as weekly infusion frequency (33.3%, IQR:0-50%) (Table 2). Of note, 57.9% of patients presented zero joint bleeding events after switchingtorFVIII-EHL.

Conclusions:In this prospective analysis rFVIII- EHL presents as a therapeutic alternative that allows reducing ABR and AJBR as well as weekly infusion frequency according to PK patient parameters.

Disclosures

Bosch: Roche:Honoraria;Celgene:Honoraria;Jansen:Honoraria;Abbvie:Honoraria;Novartis:Honoraria;Astra Zeneca:Honoraria;Takeda:Honoraria.Gironella Mesa:Bristol Myers Squibb:Honoraria, Speakers Bureau;Janssen:Consultancy, Honoraria.
Antithrombin Supplementation in Adults with Acute Lymphoblastic Leukemia (ALL) Following Asparaginase Therapy: A Meta-Analysis
Authors: Mosalem Osama , Mujer Mark , Abu Rous Fawzi , Cole Craig E.
Published: November 2020

Background:

Aspagarinase is a fundamental component for the treatment of patients with acute lymphoblastic leukemia (ALL). Venous thromboembolism (VTE) is a known complication of asparaginase therapy during treatment for ALL. This is attributed to the depletion of anticoagulants, particularly acquired antithrombin deficiency following asparaginase administration. The incidence of thrombosis following asparaginase is more prevalent in adults versus children and is estimated to be around 5-35 % and 2.4-8% respectively. Multiple studies have investigated the efficacy and safety of antithrombin supplementation. The recent THROMBOTECT study showed that prophylactic use of antithrombin (AT) or low molecular weight heparin (LMWH) is associated with significant risk reduction of thromboembolism in children and adolescents during induction chemotherapy of ALL. Our study sought to review the cohort studies comparing VTE in adults with and without AT supplementation in ALL receiving asparaginase and specifically evaluate the efficacy of this strategy.

Methods:

We performed a systematic search using PubMed, Google Scholar, EMBASE, SCOPUS and ClinicalTrials.gov without language restriction up until July 20th 2020. A random effects model was utilized to calculate risk ratio (RR) and mean difference (MD) with 95% confidence interval (CI).

Results:

Eight studies fulfilled our inclusion criteria (table 1) and were retrospective cohort studies. In adult patients, with ALL treated with asparaginase, the incidence of VTE was significantly lower in those who received AT [RR=0.46 (95 % CI= 0.31-0.70; p= 0.0002) (figure 1.1). The threshold for AT supplementation was 60-70% in most of the studies, with the exception of two studies where the threshold was 50%.

Conclusion:

In our meta-analysis of the current available data,Antithrombin supplementation in adults with ALL receiving asparaginase was associated with a significant decrease in the risk of venous thromboembolism compared to those who didn't receive thromboprophylaxis. Our data suggest the benefit of antithrombin supplementation in adults with ALL treated with asparaginase. We suggest that this strategy be implemented in a large prospective clinical trial to further confirm the benefit of this intervention.

Disclosures

No relevant conflicts of interest to declare.
Assay Interference By Emicizumab in Aptt and Chromogenic Based FVIII:C and Inhibitor Assays
Authors: Poirier Brian F , Robinson Mary , Tiefenbacher Stefan , McEwan Erin , Cogswell Caroline , Gode Roxy , Burger Emily , Allison Tyler , Littlefield Jennifer , Pikas Amanda , Schmoker Andrea
Published: November 2020

Background:Emicizumab, a humanized bispecific antibody, has been approved for the prophylactic treatment of hemophilia A in patients with and without inhibitors. Both activated partial thromboplastin time (APTT) based FVIII:C and inhibitor assays, as well as select chromogenic FVIII:C assays have been reported to be affected in the presence of emicizumab.

Aims:To assess the effect of emicizumab spiked at 0, 25, 50, and 100 ug/mL into congenital FVIII deficient plasma, containing FVIII inhibitor at 0, 1 and 5 BU, in seven commonly used APTT and chromogenic based FVIII:C assays. Samples were also tested in an APTT and chromogenic based FVIII Nijmegen inhibitor assay.

Methods:Four IVD approved, APTT based FVIII:C assays and three chromogenic FVIII:C assays were evaluated (Table 1). In addition, FVIII inhibitor levels were measured by Nijmegen Bethesda assay using either APTT (Actin®FSL, Siemens Healthcare) or chromogenic based (FVIII Chromogenic Assay, Siemens Healthcare) FVIII:C assays.

Results:Reagent and dose-dependent increases in FVIII:C were observed for all APTT reagents tested as well as for the BIOPHEN FVIII:C chromogenic assay. The presence of low or high titer inhibitors did not impact APTT based FVIII:C measured. Neither of the two chromogenic FVIII:C assays, using bovine based FX and FIXa (i.e. FVIII Chromogenic Assay and Coatest®SP4) demonstrated measurable FVIII levels at any of the emicizumab concentrations tested. FVIII Nijmegen Bethesda inhibitor results using APTT based FVIII:C, when tested in the presence of emicizumab, consistently produced false negative results (Table 1) whereas results determined using the FVIII Chromogenic Assay measured inhibitor titers at expected concentrations.

Conclusion:Confirming previously published results, emicizumab interferes in the APTT based FVIII:C and Nijmegen Bethesda inhibitor assays. Even at low emicizumab concentrations, false negative inhibitor titers are measured in APTT based inhibitor assays. Using a bovine based chromogenic FVIII:C assay is imperative when measuring both FVIII:C and FVIII inhibitors in samples containing emicizumab.

Disclosures

Tiefenbacher: Laboratory Corporation of America Holdings:Current Employment, Current equity holder in private company;Novo Nordisk:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees;Shire:Honoraria;Octapharma:Honoraria;Siemens Healthcare:Membership on an entity's Board of Directors or advisory committees;BioMarin:Membership on an entity's Board of Directors or advisory committees.
Platelet Decrease and Efficacy of Platelet-Rich Plasma Return for Peripheral Blood Stem Cell Apheresis
Authors: Shima Takahiro , Sakoda Teppei , Henzan Tomoko , Kunisaki Yuya , Maeda Takahiro , Akashi Koichi , Miyamoto Toshihiro
Published: November 2020

Peripheral blood stem cell (PBSC) transplantation is a key treatment option for hematological diseases and widely performed in clinical practice. Platelet loss is the major complication of PBSC apheresis, and platelet-rich plasma (PRP) return is recommended in case of severe platelet decrease following apheresis; however, little is known about the frequency and severity of platelet loss nor the efficacy of PRP return post-apheresis. To address these questions, we assessed changes in platelet counts following PBSC-related apheresis in 270 allogeneic (allo)- and 105 autologous (auto)-PBSC settings. We also evaluated efficacy of PRP transfusion on platelet recovery post-apheresis.

Platelet counts reduced up to 70% post-apheresis in both allo- and auto-PBSC settings, while severe platelet count decrease (< 50 x 109/L) was only observed in auto-PBSC patients (Figure 1).

We next analyzed the relationship between severe platelet (< 50 x 109/L) after apheresis and several clinical factors by using univariate and multivariate analysis for auto-PBSC patients. As shown in Table 1, in univariate analysis, severe platelet counts following auto-PBSC apheresis was found more frequently in patients with lower platelet count, lower percentage of CD34+ cells in PB at pre-apheresis, repeated round of apheresis, and smaller number of collected CD34+ cells. On the other hand, in multivariate analysis, the white blood cell (WBC) counts pre-apheresis was the only significant risk factor of severe platelet count following apheresis (p = 0.038).

We finally analyzed the transitions of platelet counts in the setting of apheresis. The median platelet counts at pre-apheresis, post-apheresis, and post-PRP return were 187.0 x 109/L, 132.0 x 109/L, and 154.0 x 109/L for allo-PBSC apheresis, and 147.0 x 109/L, 111.0 x 109/L, and 127.0 x 109/L for auto-PBSC apheresis (p < 0.0001 for all, allo-PBSC donors and auto-PBSC patients, respectively) (Figure 2), indicating that PRP return post-apheresis facilitated a rapid platelet recovery in both allo- and auto-settings.

Collectively, our data suggest that WBC counts pre-apheresis is a useful predictor for severe platelet decrease following auto-PBSC apheresis and that PRP return is an effective mean to facilitate platelet recovery post-apheresis.

Disclosures

No relevant conflicts of interest to declare.
Ctdna Monitoring in Predicting Relapse of Primary Mediastinal B-Cell Lymphoma
Authors: Pang Diwen , Jiang Xinmiao , Huang Ling , Teng Yan , Liu Sichu , Chen Feili , Wei Xiaojuan , Guo Hanguo , Li Wenyu
Published: November 2020

Background: Primary mediastinal B-cell lymphoma (PMBCL) is a rare subtype of aggressive B-cell lymphoma. The majority of relapses occur within the first few months resulting in a dismal prognosis, thus we need to identify the risk of early relapse. Circulating tumor DNA (ctDNA)-based response assessment may enable response adapted therapy and early prognostication. In this study, we aim to clarify the role of ctDNA-monitoring in early prediction of relapse.

Methods: We retrospectively analyzed the characteristics of 38 patients with PMBCL. The genetic sequencing and ctDNA monitoring were conducted by target next-generation sequencing (NGS).

Results: The median age was 32 years old and 26 (68.4%) were female. Most cases (31/38, 81.6%) were diagnosed with stage I or II disease and fourteen (36.8%) cases present with extranodal involvement. The 5-year PFS and OS for PMBCL patients were 65.7 and 72.1%, respectively. High IPI score were associated with poor survival by univariate analysis. The complete response (CR) rate and overall response rate of R-CHOP was similar to R-EPOCH (69.2% vs. 70.0%, P =1.000 and 84.6% vs. 95.0%, P =0.547, respectively), however the 5-year PFS and OS rate for R-EPOCH seems longer than those for the R-CHOP (PFS 82.9% vs. 53.8%, P=0.0569; OS 92.9% vs. 60.6%, P=0.0567). Radiotherapy was less delivered in the patients who received R-EPOCH than R-CHOP (5.0% vs.53.8%, P=0.003) and had no impact on prognosis. We performed genetic sequencing on twenty-three cases. STAT6(15/23, 65.2%), SOCS1(13/23, 56.5%), TNFAIP3(13/23, 56.5%) were the most common affected genes in both response and refractory/relapsed patients. Cell cycle, FoxO signaling pathway and TNF signaling pathway were more frequently involved in refractory patients. ctDNA monitoring was conducted in 16 cases and fifteen cases show undetectable ctDNA after a median of 2 (range from 1 to 5) cycles and one refractory case presented with durable positive ctDNA. Among 4 relapse cases, 3 had prior detectable ctDNA half a month earlier before imaging manifestations of relapse. Regarding prediction of relapse, positive predictive value of ctDNA is 100%.

Conclusions: Intensive induction chemotherapy can improve prognosis of PMBCL and ctDNA precisely predicted relapse. As most cases relapse within a few months, ctDNA-monitoring is crucial to improve prognosis.

Disclosures

No relevant conflicts of interest to declare.
Re-Evaluating the Prognosis and Transformation Risk of Chronic Eosinophilic Leukemia
Authors: O'Leary Daniel , Goyal Amrita
Published: November 2020

Chronic eosinophilic leukemia, not otherwise specified, (CEL-NOS) is a rare myeloproliferative neoplasm defined by an autonomous clonal expansion of eosinophil precursors. CEL-NOS has long been considered an aggressive disease with a poor prognosis and high risk of transformation to blast phase. This impression was reinforced by a case series of 10 patients with CEL-NOS in which median overall survival was 22 months and in which one half of patients transformed to acute leukemia at a median of 20 months from time of diagnosis (Helbig et al, American Journal of Hematology, 2012). However, this has not been confirmed in any population-based or large cohort studies.

We sought to evaluate whether this poor prognosis is consistent across a larger patient database. We identified 487 patients with CEL-NOS in the Surveillance Epidemiology and End Result-18 database. Patients were predominantly male (60.5%) with a median age of 57 at time of diagnosis. Overall survival at 2 years from diagnosis for the cohort was 92.7% with a disease-specific survival of 97.2%. At 5 years overall survival remained relatively high (88.7%) as did disease-specific survival (95.6%). Median follow-up was 56.5 months (range 0-191 months). Median survival for patients who died of CEL was 5 months (range 0-46 months). This raises the possibility of a dichotomous patient population wherein there is one group of patients with a good prognosis, and a second group with aggressive disease and significantly poorer prognosis. Of note, the majority of the patients who died from CEL-NOS (21/23) died within two years of diagnosis.

Only three of the patients studied developed acute leukemia, two with acute myeloid leukemia and one acute lymphoblastic leukemia; all three died of their second malignancy within 28-60 months. An additional 11 patients developed lymphoma, 2 Hodgkin lymphoma and 9 non-Hodgkin lymphoma (NHL), including peripheral T-cell lymphoma (3/9), diffuse large B-cell lymphoma (3/9), angioimmunoblastic T-cell lymphoma (2/9), and Burkitt's lymphoma (1/9).

This data suggests that the current impression of CEL-NOS as a rare and aggressive disease with a poor prognosis may be more nuanced than previously appreciated. The limitations of SEER data are significant and include concerns regarding both accuracy of diagnosis and collection bias. However, the incongruence between current disease perception and the large-scale data available suggest that further study is both warranted and necessary. Next steps would include the creation of a prospective national registry of patients with CEL-NOS. This would assist both in giving appropriate disease-counseling for patients and in better assessing which patients might benefit from treatment with chemotherapy.

Disclosures

No relevant conflicts of interest to declare.
Interferon Induction By HbS, SERINC5 Upregulation and Reduction of HIV-1 Nef and AP2 Contribute to HIV-1 Restriction in Sickle Cell Disease
Authors: Kumari Namita , Jerebtsova Marina , Wang Songping , Diaz Sharmin , Nekhai Sergei
Published: November 2020

Concerted action of numerous positively acting cellular factors is essential for Human immunodeficiency virus type 1 (HIV-1) replication but in turn is challenged by anti-viral restriction factors. Previously we showed that ex vivo one round HIV-1 replication and replication of fully competent T-tropic HIV-1(IIIB) is significantly reduced in peripheral blood mononuclear cells (PBMCs) obtained from patients with Sickle Cell Disease (SCD). Further, we identified and confirmed CDKN1A (p21) and CH25H as host restriction factors expressed in SCD PBMCs that may contribute to the HIV-1 inhibition, in addition to the previously reported SAMHD1 and IKBα. Since CH25H is an interferon stimulated gene (ISG), we analyzed IRFs and interferon expression in SCD PBMCs. Higher levels of IRF7 and IFNβ mRNA were observed in SCD PBMCs compared to controls. We probed further to ascertain if hemin or sickle Hb was responsible for interferon response. We found upregulation of IFNβ in THP-1 - derived macrophages treated with lysates of HbSS RBCs or purified HbS as compared to untreated or HbA treated controls. HbSS RBCs lysates and purified HbS inhibited HIV-1 gag mRNA expression in monocyte-derived macrophages infected with HIV-1(Ba-L). Recent clinical study showed increased levels of CD4 in HIV-1 infected SCD patients in Africa. Thus we analyzed CD4 levels in HIV-1 IIIB infected SCD PBMCs, and found them to be higher compared to controls. Levels of HIV-1 nef mRNA, that controls CD4 expression was lower in HIV-1 IIIB infected SCD PBMCs. As Nef counteracts SERINC3/5 restriction factor, we analyzed its expression as well as the expression of AP2 clathrin adaptor that is required for Nef mediated internalization of CD4. AP2 expression was lower and SERINC5 expression was higher in SCD PBMCs.

CONCLUSIONS: SCD PBMCs could resist HIV-1 infection because of the increased IFNβ production by macrophages exposed to HbSS or sickle cell RBCs. SCD PBMC have increased levels of SERNIC5 and lower levels of HIV-1 Nef and host AP2 expression that, culumlatively, can increased CD4 levels and lead to the overall improved immunological health of SCD patients.

ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 1SC1HL150685, 5U54MD007597, 1UM1AI26617 and P30AI087714). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Disclosures

No relevant conflicts of interest to declare.
Response Adapted Therapy in ETV6/RUNX1-Positive Childhood Acute Lymphoblastic Leukemia Treated per Modified St Jude Total XV Study
Authors: Abu Shanap Mayada , Sultan Iyad , Abu-Ghosh Amal , Hashem Hasan , Tbakhi Abdelghani , Rihani Rawad
Published: November 2020

Introduction: ETV6-RUNX1 is the most common genetic aberration in childhood acute lymphoblastic leukemia (ALL), occurring in approximately 25% of cases with a precursor-B phenotype. The presence of ETV6-RUNX1 has been associated with a relatively low rate of relapse in multiple studies. Relapses tend to occur late and have a better salvage rate than other ALL subtypes. Moreover, conflicting data in literature regarding the prognostic significance of ETV6-RUNX1 after accounting for age, initial count and treatment intensity. We sought to study the clinical features, therapy response in newly diagnosed ETV6-RUNX1-positive treated at King Hussein Cancer Center.

Methods: Patients were treated per modified St Jude Total (XV) study, risk stratification was further refined by including minimal residual disease (MRD)measurements on day 15 and day 46 of remission induction therapy. Patients with the ETV6- RUNX1 fusion or hyperdiploidy without CNS or testicular disease and a satisfactory early MRD decline (<1% on day 15 and <0.01% on day 46) were classified as being low-risk for relapse and were treated on lower -risk arm regardless of age and leukocyte count.

Results: Seventy patients (n=70) with ETV6-RUNX1-positive treated at our institution between May 2006 to October 2017. The median age at diagnosis, 5.8 years (range, 1.5-10.8). ETV6-RUNX1 was associated with favorable age between 1- and 6-years in 55 patients (79%), male gender in 41 patients (59%), initial leukocyte count <10 in 39 patients (56%), CNS status 1 in 100% of patients. The majority, n= 58 (83%) of patients had NCI standard risk (SR) and n=12 (17%) had NCI high risk (HR). Sixty-six patients (94%) had MRD <1% at day 15 and all patients achieved remission with MRD<0.01% at day 46 of remission induction. All patients were treated as LR ALL per modified St Jude total XV protocol. At median follow up of (36 months; range, 26 to 154), in the subgroups of ETV6-RUNX1-positive patients classified as NCI standard risk (SR)and NCI high risk (HR) the 5-year EFS estimates were 94.1% ± 3.2 and 82%±1(P=.13), 5-year OS estimates were 100% and 92% ± 8 (p=0.31) respectively.

Conclusion: MRD treatment schema in ETV6/RUNX1 -positive patients have excellent outcomes if they achieved favorable response at day 15 and end of remission induction regardless of the age and Initial leukocyte count. Treatment reduction is feasible and declared safe for children with NCI-HR who are predicted to have a low risk of relapse on the basis of rapid clearance of MRD post remission induction therapy.

Disclosures

No relevant conflicts of interest to declare.
An Essential Role for the RNA Editor-Exonuclease Axis in Terminal Erythroid Differentiation
Authors: Han Areum , Yermalovich Alena V. , Lundin Vanessa , Pearson Daniel S. , Hachimi Mariam , Sousa Patricia , Hilton Brianna , Morse Michael , Atwater John , Zhang Yosra , Hunter Pheobe , Morris Vivian , Osborne Jihan K. , Frame Jenna M. , Jing Ran , Huang Yu Chung , Kubaczka Caroline , North Trista E. , Schlaeger Thorsten , Daley George Q.
Published: November 2020

Erythropoiesis is an intricate process by which lineage-committed erythroid progenitors become mature red blood cells. Reticulocytes are terminal-staged, immature red blood cells with residual RNA after enucleation. In the absence of pathology, reticulocytes are efficiently processed into mature red blood cells and typically represent a small percentage of cells in human peripheral blood. In contrast, when differentiated in vitro from pluripotent stem cells or CD34+ progenitor cells, red cells tend to arrest at the reticulocyte stage. Recent studies have highlighted that uridylation by Terminal Uridylyl Transferases (TUTases) occurs on a broad spectrum of RNA classes in mammalian cells. Oligo-uridylated RNA is recognized by exoribonucleases and targeted for decay. We posited that the machinery behind RNA degradation that accompanies terminal erythropoiesis might involve RNA tail editors coupled to exonuclease activity. Utilizing constitutional murine knockout models, we observed that blood from the TUTase Zcchc6 RNA editor knockout embryos exhibited reticulocytosis and a terminal maturation defect, as documented by FACS, histology, and hematological profiling. Murine strains deficient in the downstream exonuclease Dis3l2 phenocopied the RNA decay defect of the Zcchc6 KO. Conditional knockout murine models of the TUTase-Dis3l2 axis driven by the red cell specific Erythropoietin Receptor-Cre exhibited comparable phenotypes, suggesting a cell intrinsic and niche-independent role for the TUTase-Dis3l2 axis in promoting red blood cell maturation. We are modulating the expression of this axis by various methods to optimize modeling of hemoglobinopathies such as sickle cell anemia.

Disclosures

No relevant conflicts of interest to declare.
Exploring the Role of a Novel Tumor Suppressor - OSR2 in T-ALL
Authors: Sinha Arpan A. , Andrade Pilar I. , Malone-Perez Megan , Ahmed Syed T , Frazer J. Kimble
Published: November 2020

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, representing >25% of all cancers in children 0-14 years. Despite major advancements in pediatric ALL treatment, it remains the second most lethal childhood cancer, accounting for ~25% of deaths. The two types of ALL are precursor-B, or B-ALL, and precursor-T, or T-ALL, which have distinct molecular landscapes. Of these types, T-ALL comprises about 15% and 25% of pediatric and adult cases, respectively, and is historically considered more aggressive and treatment-resistant, with an inferior prognosis. In the precision medicine era, it is imperative to identify genetic alterations and aberrant gene expression patterns, to better understand tumor biology and improve treatment outcomes by identifying new therapeutic targets.

Our study investigates a novel transcription factor, odd-skipped related transcription factor 2 (OSR2), which we hypothesize is a putative T-ALL tumor suppressor. We are using a zebrafish T-ALL model expressing transgenic human MYC (hMYC) regulated by a lymphoblast-specific promoter, rag2. Prior work in zebrafish and human T-ALL found low OSR2 levels in ~95% of T-ALL. Based on this, we then used RNA-seq to analyze 10 hMYC zebrafish T-ALL, confirming low-to-absent osr2 in all 10 T-ALL relative to wild-type (WT) T cells. We further confirmed decreased osr2 expression by qRT-PCR of additional T-ALL and WT thymocytes. We hypothesized that if OSR2 suppresses T-ALL, impaired zebrafish Osr2 function might increase T-ALL incidence and shorten latency. To test this, we bred osr2-mutant fish to rag2:hMYC transgenic animals to create three genotypes: heterozygous osr2-mutant (osr2het) fish, heterozygous hMYC (hMYChet) fish, and compound-heterozygote (osr2het;hMYChet) fish. We screened these genotypes for T-ALL incidence by serial fluorescence microscopy, with T-ALL subsequently confirmed by fluorescence-based flow cytometry. By 7 months of age, we found 9/18 (50%) of double-heterozygous fish developed T-ALL, compared to 0/7 hMYChet fish (p = 0.026); osr2het fish also did not develop T-ALL. Together, our findings suggest osr2 allelic loss accelerates MYC-driven T-ALL, supporting our hypothesis that osr2 is a T-ALL tumor suppressor.

Disclosures

No relevant conflicts of interest to declare.
Incidence of Invasive Fungal Infection (IFI) in ACUTE Myeloid Leukemia (AML) on Primary Prophylaxis with Voriconazole in Tertiriary Care Center in Pakistan
Authors: Riaz Shazia , Saeed Maria , Imran Ahmed
Published: November 2020

OBJECTIVE:

The aim of this study was to establish the incidence of IFI in AML patients receiving voriconazole prophylaxis in developing country.

METHOD:

This prospective study was conducted at The Children's Hospital, Lahore from July 2019 to December 2019 under Prospective AML registry approved by Institutional Review Board. All, consecutive patients of AML, either newly diagnosed or previously diagnosed cases with persistent fever and prolonged neutropenia, were included. Moreover, all patients of AML receiving chemotherapy also received voriconazole prophylaxis. Data of 64 patients was computed in accordance with age, gender, FAB classification, chemotherapy regimen and anti-fungal therapy. EORTC/MSG criteria was used to determine and classify IFI as proven, probable, or possible after performing CT chest and PNS, x-ray PNS, and abdominal ultrasound. CT scan was independently reviewed by a trained radiologist. Data was analyzed by SPSS version 22.

RESULTS:

Total 64 patients of AML were included in the study. The mean age of presentation was 6 years, range 1.5years to 16 years with 45 % males. Out of 64 patients, fungal infection was suspected in 50 patients (78%) due to persistent fever and prolonged neutropenia. In suspected 50 patients, probable and possible fungal infections were found in 22(34.37%) Patients. Eighteen 18/22 (81.81 %) were probable and 4/22 (18.18 %) were possible fungal infection. Majority of the cases 13/22 (59%) patients developed fungal infection during or after course of first induction therapy.

CONCLUSION:

Patients with high-risk hematologic malignancies especially AML are more at risk to develop IFI. In our study despite primary prophylaxis with voriconazole, incidence was higher than that reported in the literature from developed countries. Our data suggests that there is need to further improve supportive care measures and implement IFI preventive measures to decrease the incidence of IFI and improve survival.

Disclosures

No relevant conflicts of interest to declare.
Aberrant Cell Cycle Programming Confers Rapid Lethality in the EuSOX11+ CCND1 MCL Mouse Model
Authors: Edwards Donna R , Kuo Pei-yu , Lagana Alessandro , Park Seongjee , Nedumaran Pavithra , Leshchenko Violetta , Christie Stephanie , Jatiani Shashidhar , Teruya-Feldstein Julie , Parekh Samir
Published: November 2020

Mantle Cell Lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation. This hallmark oncogenic event transposes CCND1 (11q13) under the control of the immunoglobin heavy chain (IGH) locus (14q32) resulting in constitutive cyclin D1 expression. Although Cyclin D1 (CCND1) overexpression is a key hallmark of MCL, CCND1 overexpressing murine models do not effectively recapitulate the MCL phenotype. Our published data demonstrated that SOX11 binds and regulates components in multiple oncogenic pathways in MCL (Kuo et. al., Oncogene 2015). Furthermore, we recently demonstrated that SOX11 promotes BCR signaling to drive MCL-like pathogenesis utilizing a SOX11 overexpressing (Eu-SOX11) murine model (Kuo et. al., Blood 2018). Given that the majority of classic human MCL co-express CCND1 and SOX11, we hypothesize that these oncogenes may cooperate to drive the pathogenesis of classical MCL.

To study in vivo cooperation between CCND1 and SOX11, we crossed Eu-SOX11 mice with Eu-CCND1 mice to generate Eu-SOX11:CCND1 double transgenic (DT) mice. We have previously reported an significant increase in the fraction of CD5+CD19+CD23- MCL cells in the peripheral blood, spleens, lymph nodes and bone marrow of Eu-SOX11 mice and this fraction was further enhanced in DT mice (Fig.1A). We have now conducted a 2-year survival analysis on all 4 genotypes and found significantly reduced survival in DT as compared to Eu-SOX11 mice (Fig.1B). Median survival in DT mice is 16.5 months as compared to 19.7 months in Eu-SOX11 mice.

Taken together, our results demonstrate a B-cell specific in vivo cooperation between SOX11 and CCND1 towards promoting a lethal MCL phenotype. RNA sequencing of splenocytes from DT versus SOX11-Tg mice showed significant enrichment of E2F1 target genes (p<.02) as a top oncogenic pathway (Fig.1C). Our previous results with CDK4/6 inhibition demonstrate a profound reduction in S phase in MCL cells (Divakar et. al., Leukemia 2016).

We have developed small molecule SOX11 inhibitors using a SOX11-DNA homology model we built using the crystal structure of the SOX4-DNA binding domain as a template. SOX11 small molecular inhibitors in combination with CDK4 inhibitors will be used to further dissect the mechanism of cooperation between SOX11 and CCND1 and develop a therapeutic strategy for MCL that is mechanistically distinct from BTK or Bcl-2 inhibition.

Disclosures

Teruya-Feldstein: Edge Anthem: Consultancy. Parekh:Karyopharm: Research Funding; Celgene: Research Funding; Foundation Medicine: Consultancy.
RNF5 Defines Acute Myeloid Leukemia Growth and Susceptibility to Histone Deacetylase Inhibitors
Authors: Khateb Ali , Deshpande Anagha , Feng Yongmei , Lazar Ikrame , Lee Joo Sang , Fabre Bertrand , Li Yan , Finlay Darren , Fujita Yu , Zhang Tongwu , Pass Ian , Livneh Ido , Burian Carol , Mason James R. , Almog Ronit , Horesh Nurit , Ofran Yishai , Brown Kevin , Vuori Kristiina , Jackson Michael , Ruppin Eytan , Deshpande Aniruddha J. , Ronai Ze'ev A.
Published: November 2020

Acute myeloid leukemia (AML) remains an incurable blood cancer largely due to rapid emergence of resistance to conventional treatments. Thus, new therapeutic modalities are greatly needed to halt AML development. Here, using genetic and xenograft mouse models, we reveal that inhibition of the ubiquitin ligase RNF5 in human AML cell lines and in MLL-AF9-driven AML severely decreased the leukemogenic potential of those cells and prolonged survival of model leukemic mice. These findings suggest the possibility that targeting a single gene, namely RNF5, could effectively inhibit different AML subtypes. We initially focused on RNF5 as its expression is upregulated in AML patient cohorts as well as in AML-derived cell lines compared with normal hematopoietic cells. Furthermore, high RNF5 expression in AML patient specimens correlated with poor prognosis, relapse and short overall patient survival. By contrast, specimens from AML patients who responded to therapy exhibited low RNF5 levels. In vitro, RNF5 loss impaired the clonogenic potential of MLL-AF9-transduced bone marrow cells and markedly attenuated growth and survival of AML but not CML or T-ALL cell lines, in which RNF5 is also highly expressed. High-throughput screen and bioinformatics analysis identified RNF5 and ER-associated degradation (ERAD) components, as augmenting AML cell sensitivity to histone deacetylase (HDAC) inhibition. Indeed, inhibition of RNF5 sensitized AML cells to HDAC inhibitors. Correspondingly, a favorable prognosis was observed in AML patients exhibiting low expression of RNF5 and HDAC. Collectivity, our studies identify a potential new therapeutic modality based on targeting RNF5 to inhibit AML and suggest that RNF5 expression could serve as a prognostic marker and means to stratify patients for treatment with HDAC inhibitors.

Disclosures

Ofran: AbbVie: Membership on an entity's Board of Directors or advisory committees. Vuori:Bionano Genomics: Membership on an entity's Board of Directors or advisory committees.
Biological interpretation of deep neural network for phenotype prediction based on gene expression
Authors: Hanczar Blaise , Zehraoui Farida , Issa Tina , Arles Mathieu
Published: November 2020

Background

The use of predictive gene signatures to assist clinical decision is becoming more and more important. Deep learning has a huge potential in the prediction of phenotype from gene expression profiles. However, neural networks are viewed as black boxes, where accurate predictions are provided without any explanation. The requirements for these models to become interpretable are increasing, especially in the medical field.

Results

We focus on explaining the predictions of a deep neural network model built from gene expression data. The most important neurons and genes influencing the predictions are identified and linked to biological knowledge. Our experiments on cancer prediction show that: (1) deep learning approach outperforms classical machine learning methods on large training sets; (2) our approach produces interpretations more coherent with biology than the state-of-the-art based approaches; (3) we can provide a comprehensive explanation of the predictions for biologists and physicians.

Conclusion

We propose an original approach for biological interpretation of deep learning models for phenotype prediction from gene expression data. Since the model can find relationships between the phenotype and gene expression, we may assume that there is a link between the identified genes and the phenotype. The interpretation can, therefore, lead to new biological hypotheses to be investigated by biologists.
Hidden paths to endless forms most wonderful: Complexity of bacterial motility shapes diversification of latent phenotypes
Authors: Rendueles Olaya , Velicer Gregory J.
Published: November 2020

Background

Evolution in one selective environment often latently generates phenotypic change that is manifested only later in different environments, but the complexity of behavior important to fitness in the original environment might influence the character of such latent-phenotype evolution. UsingMyxococcus xanthus,a bacterium possessing two motility systems differing in effectiveness on hard vs. soft surfaces, we test (i) whether and how evolution while swarming on one surface—the selective surface—latently alters motility on the alternative surface type and (ii) whether patterns of such latent-phenotype evolution depend on the complexity of ancestral motility, specific ancestral motility genotypes and/or the selective surface of evolution. We analysze an experiment in which populations established from three ancestral genotypes—one with both motility systems intact and two others with one system debilitated—evolved while swarming across either hard or soft agar in six evolutionary treatments. We then compare motility-phenotype patterns across selective vs. alternative surface types.

Results

Latent motility evolution was pervasive but varied in character as a function of the presence of one or two functional motility systems and, for some individual-treatment comparisons, the specific ancestral genotype and/or selective surface. Swarming rates on alternative vs. selective surfaces were positively correlated generally among populations with one functional motility system but not among those with two. This suggests that opportunities for pleiotropy and epistasis generated by increased genetic complexity underlying behavior can alter the character of latent-phenotype evolution. No tradeoff between motility performance across surface types was detected in the dual-system treatments, even after adaptation on a surface on which one motility system dominates strongly over the other in driving movement, but latent-phenotype evolution was instead idiosyncratic in these treatments. We further find that the magnitude of stochastic diversification at alternative-surface swarming among replicate populations greatly exceeded diversification of selective-surface swarming within some treatments and varied across treatments.

Conclusion

Collectively, our results suggest that increases in the genetic and mechanistic complexity of behavior can increase the complexity of latent-phenotype evolution outcomes and illustrate that diversification manifested during evolution in one environment can be augmented greatly by diversification of latent phenotypes manifested later.
BrrICE1.1 is associated with putrescine synthesis through regulation of the arginine decarboxylase gene in freezing tolerance of turnip (Brassica rapa var. rapa)
Authors: Yin Xin , Yang Yunqiang , Lv Yanqiu , Li Yan , Yang Danni , Yue Yanling , Yang Yongping
Published: November 2020

Background

In the agricultural areas of Qinghai-Tibet Plateau, temperature varies widely from day to night during the growing season, which makes the extreme temperature become one of the limiting factors of crop yield. Turnip (Brassica rapa var. rapa) is a traditional crop of Tibet grown in the Tibet Plateau, but its molecular and metabolic mechanisms of freezing tolerance are unclear.

Results

Here, based on the changes in transcriptional and metabolic levels of Tibetan turnip under freezing treatment, the expression of the arginine decarboxylase gene BrrADC2.2 exhibited an accumulative pattern in accordance with putrescine content. Moreover, we demonstrated that BrrICE1.1 (Inducer of CBF Expression 1) could directly bind to the BrrADC2.2 promoter, activating BrrADC2.2 to promote the accumulation of putrescine, which was verified by RNAi and overexpression analyses for both BrrADC2.2 and BrrICE1.1 using transgenic hair root. The function of putrescine in turnip was further analyzed by exogenous application putrescine and its inhibitor DL-α-(Difluoromethyl) arginine (DFMA) under freezing tolerance. In addition, the BrrICE1.1 was found to be involved in the ICE1-CBF pathway to increase the freezing stress of turnip.

Conclusions

BrrICE1.1 could bind the promoter of BrrADC2.2 or CBFs to participate in freezing tolerance of turnip by transcriptomics and targeted metabolomics analyses. This study revealed the regulatory network of the freezing tolerance process in turnip and increased our understanding of the plateau crops response to extreme environments in Tibet.
Chitosan regulates metabolic balance, polyamine accumulation, and Na+ transport contributing to salt tolerance in creeping bentgrass
Authors: Geng Wan , Li Zhou , Hassan Muhammad Jawad , Peng Yan
Published: November 2020

Background

Chitosan (CTS), a natural polysaccharide, exhibits multiple functions of stress adaptation regulation in plants. However, effects and mechanism of CTS on alleviating salt stress damage are still not fully understood. Objectives of this study were to investigate the function of CTS on improving salt tolerance associated with metabolic balance, polyamine (PAs) accumulation, and Na+ transport in creeping bentgrass (Agrostis stolonifera).

Results

CTS pretreatment significantly alleviated declines in relative water content, photosynthesis, photochemical efficiency, and water use efficiency in leaves under salt stress. Exogenous CTS increased endogenous PAs accumulation, antioxidant enzyme (SOD, POD, and CAT) activities, and sucrose accumulation and metabolism through the activation of sucrose synthase and pyruvate kinase activities, and inhibition of invertase activity. The CTS also improved total amino acids, glutamic acid, and γ-aminobutyric acid (GABA) accumulation. In addition, CTS-pretreated plants exhibited significantly higher Na+ content in roots and lower Na+ accumulation in leaves then untreated plants in response to salt stress. However, CTS had no significant effects on K+/Na+ ratio. Importantly, CTS enhanced salt overly sensitive (SOS) pathways and also up-regulated the expression of AsHKT1 and genes (AsNHX4, AsNHX5, and AsNHX6) encoding Na+/H+ exchangers under salt stress.

Conclusions

The application of CTS increased antioxidant enzyme activities, thereby reducing oxidative damage to roots and leaves. CTS-induced increases in sucrose and GABA accumulation and metabolism played important roles in osmotic adjustment and energy metabolism during salt stress. The CTS also enhanced SOS pathway associated with Na+ excretion from cytosol into rhizosphere, increased AsHKT1 expression inhibiting Na+ transport to the photosynthetic tissues, and also up-regulated the expression of AsNHX4, AsNHX5, and AsNHX6 promoting the capacity of Na+ compartmentalization in roots and leaves under salt stress. In addition, CTS-induced PAs accumulation could be an important regulatory mechanism contributing to enhanced salt tolerance. These findings reveal new functions of CTS on regulating Na+ transport, enhancing sugars and amino acids metabolism for osmotic adjustment and energy supply, and increasing PAs accumulation when creeping bentgrass responds to salt stress.
BRD4 and PIN1 gene polymorphisms are associated with high pulse pressure risk in a southeastern Chinese population
Authors: Qiu Jin-jia , Yang Rui-zhi , Tang Yi-jie , Lin Ying-yi , Xu Hao-jie , Zhang Na , Liang Min , Cai Hong-da , Zeng Kai , Wu Xiao-dan
Published: November 2020

Background

BRD4 and PIN1 have been described to be involved in inflammation and vascular endothelial cell dysfunction, which in turn may increase pulse pressure.

Hypothesis

Genetic mutations within the BRD4 and PIN1 genes could affect the risk of high pulse pressure.

Methods

A total of four single nucleotide polymorphisms (SNPs) (BRD4: rs4808278; PIN1: rs2233678, rs2287838, and rs2233682) were genotyped in a cohort of 666 hypertensive patients and 232 normotensive controls with Chinese Han origin. Generalized multifactor dimensionality reduction (GMDR) was used to screen the best interaction combination among the four SNPs within the BRD4 and PIN1 genes and diabetes. Logistic regression analysis was performed to calculate the odds ratio (ORs) (95% confidence interval (CI)) for the association between the four SNPs.

Results

Adjusted for age, weight, waist circumference, drinking, smoking, hypertension, and diabetes, high pulse pressure risk was significantly higher for carriers with the rs4808278-TT genotype in BRD4 than those with wild genotypes (OR: 0.400, 95% CI: 0.217–0.737, P* < 0.05). However, we did not find any significant association of rs2233678, rs2287838, and rs2233682 in PIN1 with high pulse pressure susceptibility after covariate adjustment. GMDR analysis indicated a significant three-locus model (P = 0.0107) involving rs4808278, rs2233678, and diabetes, the cross-validation consistency of the three-locus models was 9/10, and the testing accuracy was 57.47%.

Conclusions

Genetic mutations within BRD4 (rs4808278) could affect the susceptibility to high pulse pressure in a southeastern Chinese population.