bluuebunny/crossref_metadata_2025_split huggingface dataset:
Deficiency of Cux1, Encoded on Human Chromosome 7q, Causes Aberrant Hematopoietic Stem Cell Function and Spontaneous Myeloproliferative Disease in Mice
Authors: An Ningfei , Khan Saira , Imgruet Molly , Gurbuxani Sandeep , Burgess Michael R. , McNerney Megan E. 
Published: April 2021

Abstract
Loss of all or part of chromosome 7 [-7/del(7q)] is a common, high-risk cytogenetic abnormality identified in a variety myeloid malignancies, including myelodysplastic syndrome (MDS), acute myeloid leukemia, therapy-related myeloid neoplasms, juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML). CUX1 is a non-clustered, homeodomain-containing transcription factor encoded on the commonly deleted region of 7q22. CUX1 inactivating mutations occur across solid tumors and myeloid neoplasms, and carry a poor prognosis in myeloid malignancies independently of 7q status. Previously, we reported that CUX1 is a conserved, haploinsufficient, myeloid tumor suppressor. To determine the mechanism by which CUX1 regulates leukemogenesis, we generated an inducible, shRNA-transgenic, Cux1 -knockdown mouse model. Insufficient Cux1 levels caused early mortality in mice, due to a spontaneous myeloproliferative disorder with features of CMML/JMML and MDS. Cux1 deficient mice had a significant increase in myelomonocytic cell numbers in the peripheral blood, bone marrow, and spleen. Cux1 deficiency led to extramedullary hematopoiesis, splenomegaly, and an increase in megakaryocyte numbers and megakaryocyte dysplasia. In addition, Cux1 is required for normal red cell development, as Cux1 knockdown led to a normocytic anemia due to an erythroblast maturation arrest. To identify CUX1 transcriptional targets, we performed gene expression profiling after CUX1 knockdown in CD34+ human hematopoietic stem and progenitor cells (HSPCs). Differentially expressed genes were enriched for genes deregulated in JMML and -7/del(7q) MDS, in addition to altered expression of quiescent, proliferative, and myeloid differentiation pathways. Consistent with these gene signatures, Cux1 maintains hematopoietic stem cell quiescence and represses HSPC proliferation, self-renewal, and granulocyte/monocyte differentiation. Thus Cux1 has pleotropic roles in multiple myeloid lineages. Deletion of CUX1, in the context of -7/del(7q), may facilitate myeloid transformation through aberrant transcriptional regulation of HSPC differentiation and proliferation.

Disclosures
No relevant conflicts of interest to declare.
Cytokine Release Syndrome and Tumor Responses in a First-in-Man Trial of a Novel Affinity-Enhanced TCR-Gene Transduced T Cell Transfer Targeting NY-ESO-1 Antigen
Authors: Kageyama Shinichi , Ishihara Mikiya , Kitano Shigehisa , Miyahara Yoshihiro , Kato Hidefumi , Mishima Hideyuki , Yamamoto Noboru , Hattori Hiroyoshi , Iwase Hiroaki , Funakoshi Takeru , Kojima Takashi , Inoue Naoki , Kidokoro Tomohide , Tomura Daisuke , Okamoto Sachiko , Chono Hideto , Nukaya Ikuei , Mineno Junichi , Ikeda Hiroaki , Watanabe Takashi , Shiku Hiroshi 
Published: April 2021

Abstract
Adoptive cell transfers of receptor gene-engineered T cells include chimeric antigen receptor-gene transduced T (CAR-T) cell therapy and TCR-gene transduced T (TCR-T) cell therapy. In CD19-CAR-T cell therapy, high incidence of cytokine release syndrome (CRS) is associated with in vivo CAR-T cell proliferation and its clinical efficacy. In human TCR-T cell therapies, there have not been well known about CRS and its association with in vivo T cell kinetics or tumor responses.
We have been developing a novel-type affinity-enhanced NY-ESO-1-specific TCR, and an original retrovirus vector that encodes siRNA to silence endogenous TCR creation. The NY-ESO-1 TCR is mutated for high affinity with replacements of G50A and A51E in CDR2 region, which is restricted with HLA-A*02:01 and A*02:06. We extensively examined potential cross-reactivities to different antigen-peptides in preclinical studies, and the high-affinity NY-ESO-1 TCR did not recognize analogous peptides. The new generation retroviral TCR-vector provides enhanced expression of transduced tumor-specific TCRs and an inhibition effect of formations of self-reactive TCRs.
This is a first-in-man clinical trial of the novel NY-ESO-1-specfic TCR-T cell transfer to evaluate the safety, in vivo cell kinetics and clinical responses. It is designed as a cell-dose escalation from 5 x108 to 5 x109 cells. NY-ESO-1-expressing refractory cancer patients were enrolled, with 3+3 cohort design. Cyclophosphamide with/without fludarabine were administered prior to the TCR-T cell transfer as pre-conditioning.
Six patients were treated with the NY-ESO-1 TCR-T cell transfer, and evaluated for the safety and in vivo cell kinetics. The TCR-T cells appeared in peripheral blood with a dose-dependent manner, associated with in vivo proliferation in an early phase. In three patients given 5x108 cells, no toxicities were seen. Two patients receiving 5x109 cells developed early-phase CRS (G2), with elevations of serum IL-6 and IFN-gamma. They were managed the treatment of anti-IL-6 receptor monoclonal antibody, tocilizumab. In a patient who developed CRS, an event of lung injury (G3) occurred, which was associated with marked infiltration of the NY-ESO-1 TCR-T cells. It was successfully treated with steroid.
Two synovial sarcoma patients exhibited tumor responses of PRs. In one patient, progression-free survival lasted more than 8 months.
In summary, the affinity-enhanced NY-ESO-1 TCR-T cell transfer exhibited CRSs in association with in vivo cell proliferation and sequential tumor responses.

Disclosures
No relevant conflicts of interest to declare.
Bortezomib, Lenalidomide and Low-Dose Dexamethasone (VRD) Versus Lenalidomide and Low-Dose Dexamethasone (Ld) for Newly-Diagnosed Multiple Myeloma- a Randomized Phase III Study
Authors: Mookerjee Anjali , Gupta Ritu , Jasrotia Shivali , Sahoo Ranjit , Kumar Rakesh , Thulkar Sanjay , Sharma Om Dutt , Kumar Lalit 
Published: April 2021

Abstract
Background: In this prospective study, we compared VRD with Ld as induction therapy for newly-diagnosed Multiple myeloma patients. The primary objective of this study is to compare the progression-free survival in the 2 arms.
Methods: Between September 2014 and Oct 2016, 144 patients have been recruited and randomly assigned to receive 4 cycles of either Bortezomib 1.3 mg/m2 SC on days 1, 8, 15 and 22 with Lenalidomide 15mg/day from day 1 to 14 (Arm A) or Lenalidomide 25 mg/day from day 1 to 21 (Arm B). Patients in both arms received oral dexamethasone 40 mg on days 1,8,15 and 22. Both treatment regimens were 28-day cycles. All patients received 75 mg aspirin daily, acyclovir prophylaxis and monthly zoledronic acid. Response assessment was done at the end of the 4th cycle using the International Myeloma Working Group (IMWG) uniform response criteria. The study was approved by the Institute Ethics Committee (Ref IEC/NP-264/01-08-2014, RP-7/2014).
Results: These are the results from an analysis of 143 patients (arm A-74, arm B-69). Baseline characteristics of patients were similar in both arms with respect to age, gender, ISS and DS stage, immunoglobulin subtype and serum LDH. Patients' median age is 56 years (range 31-70) in arm A and 52 years (range 28-69) in arm B. Gender M/F: Arm A 54/20 and 43/26 in arm B. ISS stage III 51 (68.9%) arm A vs 44 (63.8%) arm B. Serum LDH raised to &gt;250 u/L was observed in 25 (44.6%) vs 31 (52.5%) in arms A and B, p=0.4. Revised staging including ISS and serum LDH at baseline: stage III 47 (81%) and 37 (65%) in arms A and B respectively. 14 (18.9%) and 19 (27.5%) of patients had light chain myeloma in arms A and B respectively. Overall response rates (sCR+CR+VGPR+PR) is 78.4% vs 73.9% in arms A and B respectively, p=0.6; sCR + CR 21 (28.4%) and 21 (30.4%) respectively, p=0.86. Median follow-up 17.1 months (range 1 to 33). Median overall survival (OS) is 30.2 months (95% CI 28.2 to 32.2) and 28.6 months (95%CI 26 to 31.3) in arms A and B respectively, p=0.3. Median progression-free survival (PFS) was 27.8 months (95%CI 25.4 to 30.2) and 28 months (95%CI 24.6 to 31.4) respectively, p=0.3. Estimated one-year OS is 88% vs 85% in arms A and B, and PFS 83% vs 72%, respectively. Grade 3 anemia occurred in one patient in arm B, and grade 3 deep vein thromboses in one patient in arm A. One patient in arm A developed grade 4 myelosuppression leading to therapy change at the end of the first cycle.
Conclusion: In this analysis - response rates and median progression-free survival are similar in both arms.

Disclosures
No relevant conflicts of interest to declare.
Bok Promotes Erythropoiesis in a Mouse Model of Myelodysplastic Syndrome
Authors: Kang Seong-Ho , Perales Oscar , Michuad Michael , Katz Samuel G. 
Published: April 2021

Abstract
BCL-2 Ovarian Killer (BOK) is a pro-apoptotic member of the BCL-2 family of proteins best characterized for its putative ability to induce apoptosis in response to Endoplasmic Reticulum (ER) stress, when stabilized from ER-associated degradation (ERAD). Although ER stress appropriately activates the unfolded protein response (UPR) in BOK-disrupted cells, as measured by PERK and eIF2-alpha phosphorylation, downstream effector signaling, including ATF4 and CHOP, is defective. A functional role for BOK as a tumor suppressor is suggested by its genetic location in one of the 20 most frequent, focally deleted chromosomal regions across all human cancers. To evaluate the consequences of BOK loss in the pathogenesis of myelodysplasia (MDS) and Acute Myeloid Leukemia (AML), we used the Nup98-HoxD13 (NHD13) transgenic mouse model of MDS/AML. In this model, both overexpression of anti-apoptotic BCL-2 and deletion of pro-apoptotic PUMA rescue cytopenias, but surprisingly delay progression to AML. In contrast, AML developed in 36.7% of NHD13 mice lacking BOK between the age of 8 and 13 months with a similar overall survival to the NHD13 mice. However, the loss of BOK exacerbated the anemia of the NHD13 mice, which raised a potential connection between BOK and the regulation of erythropoiesis in cells experiencing stress from the NHD13 translocation. NHD13 mice deficient for BOK exhibited significantly lower hemoglobin (Hb), lower mean cell hemoglobin concentration (MCHC) and higher mean cell volume (MCV) than NHD13 mice, whereas other lineages were unaffected. Mouse colony forming unit assays revealed there is a decreased amount of erythroid progenitor stem cells (BFU-E) in the bone marrow of NHD13-transgenic/BOK-deficient mice, which hinted at a diminished ability to produce RBCs in the absence of BOK. Isolation of various stages of erythroid progenitors in the bone marrow by CD44/TER119 FACS sorting revealed that both NHD13 and NHD13-transgenic/BOK-deficient mice have an increase in proerythroblasts relative to more mature red blood cells. Preliminary RT-QPCR analysis shows decreased expression of UPR components in the RBC progenitors of both BOK-deficient and NHD13-transgenic/BOK-deficient mice. Interestingly, CHOP is not only a component of the UPR, but also an erythropoietin target gene necessary for erythroid differentiation. These results suggest that in addition to its pro-apoptotic function, BOK may have other regulatory roles within the cell, and specifically a role in regulating erythropoiesis when certain RBC progenitors experience ER stress.

Disclosures
Katz: Gene-in-Cell: Equity Ownership.
The Flow Cytometric Osmotic Fragility Test Is an Effective Screening Method for Dehydrated Hereditary Stomatocytosis
Authors: Utsugisawa Taiju , Iwasaki Takuya , Aoki Takako , Okamoto Yoshio , Kawakami Takahiro , Ogura Hiromi , Kanno Hitoshi 
Published: April 2021

Abstract
Introduction: Dehydrated hereditary stomatocytosis (DHSt) or hereditary xerocytosis (HX) is a form of congenital hemolytic anemia characterized by red blood cell (RBC) dehydration. Heterozygous mutations in PIEZO1, a mechanically-activated ion channel, cause DHSt. Recently, KCNN4, which encodes the Gardos channel, has been found to be the second pathogenic gene for DHSt. DHSt is characterized by an alteration in the RBC morphology in target cells, stomatocytes, and/or echinocytes, and RBC deformability assessments by ektacytometry as well as RBC ion flux measurements are currently the standard laboratory tests for DHSt, but their use in laboratories is limited. The flow cytometric osmotic fragility (FCM-OF) test is a useful diagnostic test for hereditary spherocytosis (HS) and also for hereditary elliptocytosis (HE). In this study, we showed that the FCM-OF test could also successfully diagnose DHSt.
Subjects: A total of 46 cases of RBC membrane disorders were examined, and tentative diagnoses were made based on the RBC morphology, acid glycerol lysis time, and eosin 5'-maleimide binding tests, resulting in HS (n=31), HE (n=6), and DHSt (n=9).
Methods: The number of RBCs in isotonic and hypotonic buffers were measured by flow cytometry. The degree of osmotic fragility was expressed as the "percentage residual RBCs (%RRC)". We confirmed the DHSt diagnosis by the massively paralleled sequencing using our custom panels targeting 68 hemolytic anemia-related genes with the next-generation sequencer.
Results: Both HS and HE patients showed a decrease in %RRC; HS (18.0±8.9%, p&lt;0.001) and HE (41.8±15.7%, p&lt;0.001) compared to normal control (66.7±1.5%). DHSt patients showed a significant increase (112.6±34.5%, p&lt;0.001) in FCM-OF. Additionally, next-generation sequencing revealed consistent causative gene mutations for DHSt; PIEZO1 (p.R2488Q and p.E2496ELE) or KCNN4 (p.P204R, p.A279T and p.R352H).
Discussion: We examined 77 patients with congenital hemolytic anemia recently, and 59 cases were confirmed by diagnostic tests (76.6%). The results were as follows: 48 cases of RBC membrane abnormality (62.3%), 6 cases of RBC enzymopathy (7.8%), and 5 cases of hemoglobinopathy (6.5%). Of the cases of RBC membrane disorders, 31 cases of HS, 9 cases of DHSt, and 8 cases of HE were identified. These observations suggest that DHSt is the second-most common RBC membranopathy in Japan, and that the FCM-OF test and targeted sequencing efficiently discriminate DHSt from other RBC membrane disorders.

Disclosures
No relevant conflicts of interest to declare.
Restriction of HIV-1 Infection in Sickle Cell Disease Trait
Authors: Kumari Namita , Khan Javed , Ammosova Tatiana , Ahmad Asrar , Diaz Sharmin , Rana Sohail , Nekhai Sergei 
Published: April 2021

Abstract
We recently showed that in Sickle Cell Disease (SCD), activation of anti-viral restriction factor SAMHD1 and NF-κB inhibitor, IkBα prevented ex vivo HIV-1 infection. SAMHD1 activation was due to its reduced phosphorylation by CDK2, whose activity was inhibited by reduced intracellular iron levels in SCD peripheral blood mononuclear cells (PBMC). We also found that a hazard ratio associated with HIV infection among 11,412 SCD trait subjects was 1.0% comparing to &gt;2% HIV-1 infection among African-Americans in USA. We compared 9 HbAS HIV-1 infected individuals with 107 HbAA or HbAC HIV-1 infected individuals who were enrolled at Howard University clinic. While hemoglobin levels in these two groups were not significantly different, HIV-1 viral load was significantly lower in HbAS group as well the number of HIV-1 associated complications (Hospitalization) were reduced. In the present study, we analyzed ex vivo HIV-1 infection of SCD trait PBMC and determined the expression of iron and hypoxia-regulated host factors including HO-1, NFκB, IKK, IKBα that are implicated in HIV-1 replication. We also analyzed CDK2 activity, SAMHD1 phosphorylation and RNR2 expression in SCD trait PBMCs. One round HIV-1 infection was significantly reduced in in SCD trait PBMC. Expression of HO-1 and IKBα was upregulated in SCD trait whereas IKK and NF-κB expression was downregulated. While CDK2 activity and SAMHD1 phosphorylation were not changed, RNR2 expression was reduced. Expression levels of HIV-1 env and gag mRNA were significantly lower in HIV-1 infected SCD trait subjects. In the same patients, HO-1 and IKBα mRNA levels were upregulated comparing to HIV-1+ HbAA or HbAC subjects. Our findings suggest that HIV-1 infection might be deregulation in SCD trait and that iron metabolism and hypoxia might play a role in this deregulation.
This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005, and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH.

Disclosures
No relevant conflicts of interest to declare.
Impairment in the Mesenchymal Bone-Marrow Niche of Beta-Thalassemia Patients and Association with Prolonged Iron Exposure
Authors: Crippa Stefania , Rossella Valeria , Silvestri Laura , Aprile Annamaria , Scaramuzza Samantha , Rivis Silvia , Avanzini maria Antonietta , Zecca Marco , Marktel Sarah , Ciceri Fabio , Aiuti Alessandro , Ferrari Giuliana , Bernardo Maria Ester 
Published: April 2021

Abstract
Bone marrow (BM) contains a population of mesenchymal stromal cells (MSC) that, together with endothelial cells and osteoblasts, provide a specific microenvironment to support hematopoietic stem cell homeostasis. Beta-thalassemia (BT) is an hereditary blood disorder characterized by reduced or absent synthesis of hemoglobin beta-chains. Data on the mesenchymal compartment in BT patients are scarce. We isolated and characterized MSCs from BT and healthy donor (HD) BM samples. BT-MSCs showed a reduced clonogenic capacity, delay in colony formation, lower numbers of CFU-Fs and longer population doubling time. Similarly, we observed an altered differentiation capacity into adipocytes and osteoblasts. Both HD- and BT-MSCs express the canonical mesenchymal markers. On the contrary, the expression of CD146 and CD271 was extremely reduced in BT-MSCs, indicating a pauperization of the most primitive stem cell pool. We analyzed the expression of genes involved in the crosstalk between MSCs and HSCs and found a reduced expression of Cxcl12, SCF and Angp1 in BT-MSCs. Several genes relevant for MSC functionality were also downregulated in BT-MSCs. We demonstrated that MSCs are able to uptake and store iron. ROS levels were increased in BT-MSCs possibly due to altered anti-oxidant response and iron overload associated with blood transfusions. Importantly, higher level of ROS and reduced expression of primitive markers were observed in HD-MSCs cultured in presence of iron, underlying the importance of iron level for normal MSC function. In conclusion, we showed an impairment in the mesenchymal niche of BT-BM possibly associated with prolonged iron exposure.

Disclosures
Marktel: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Ciceri: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding.
Pathological Role of Halted Proteasome Machinery in Sickle Cell Disease
Authors: Song Anren , Angelo d'Alessandro , Sun Kaiqi , Liu Hong , Peng Zhangzhe , Kellems Rodney , Juneja Harinder S. , Xia Yang 
Published: April 2021

Abstract
Although proteasome machinery is a conserved cellular component to maintain their normal function, its function in erythrocyte under stress conditions is largely unknown, especially in sickle cell disease (SCD). To determine whether proteasome machinery is altered in SCD erythrocyte, we conducted western blot to detect total ubiquitinated proteins on the erythrocyte membrane in both mice and humans with or without SCD. We found that ubiquitinated proteins were significantly accumulated in SCD mice and humans compared to WT mice and normal controls, indicating that proteasome machinery is halted in SCD. Next, to determine which specific proteins are ubiquitinated and accumulated in SCD, we conducted robust and nonbiased proteomic profiling by immunoprecipitation ubiquitinated proteins followed by proteomics analysis. We found significant accumulation of several categories of ubiquitinated proteins on the erythrocyte membrane in SCD, including cytoskeleton proteins (Spectrin, Actin, Ankryin), glycolytic enzymes (GAPDH, 2,3-BPG mutase, Pyruvate Kinase, G6PD), transporters (Band3, large neutral AA transporter, calcium transporter, ENT1), hemoglobin, components of proteasome machinery [E2, E3 ligases, and valosin-containing protein (p97)]. Finally, to determine the effect of halted proteasome machinery in SCD functionally, we conducted in vitro hypoxia induced red blood cell (RBC) sickling assay. We found that inhibition of RBC proteasome machinery by targeting p97 using CB-5083 or targeting proteasome using MG132 increases SCD RBC sickling. Overall, our findings reveal a novel role of halted proteasome machinery in the pathophysiology of SCD and open up new therapies for the disease.

Disclosures
No relevant conflicts of interest to declare.
Hairy Platelet-Derived Extracellular Vesicles Promote Lung Vaso-Occlusion in Sickle Cell Disease
Authors: Sundd Prithu , Jimenez Maritza Ann , Bennewitz Margaret F. , Brzoska Tomasz , Tutuncuoglu Egemen , Kato Gregory J. , Gladwin Mark T 
Published: April 2021

Abstract
Background: Acute chest syndrome (ACS) is a type of acute lung injury and the leading cause of mortality in Sickle Cell Disease (SCD). Current treatments for ACS are primarily supportive, and there is a critical need for rescue therapies. ACS is often a sequela of acute systemic vaso-occlusive crisis and preceded by thrombocytopenia. However, the role of platelets in the pathogenesis of ACS remains largely unknown.
Methods: We used our validated model of vaso-occlusive crisis in transgenic, humanized SCD mice, which is triggered by intravenous challenge with nanogram levels of the TLR4 ligand, lipopolysaccharide (LPS). Platelet-neutrophil aggregates and blood flow in the lung microcirculation was visualized in real time in vivo, using multi-photon-excitation microscopy of intact lung in live SCD mice. SCD or control human blood was perfused through microfluidic channels in vitro and neutrophil-platelet aggregation was visualized using fluorescence microscopy. Platelet derived extracellular vesicles were characterized using nanoparticle tracking and biochemical approaches.
Results: We have made a novel finding that the arrest of blood flow and injury in the lung is secondary to blockade of pulmonary arterioles by platelet-neutrophil aggregates. Using in vitro microfluidic studies, we confirmed that platelet-neutrophil aggregation is higher in LPS-treated SCD patient blood compared with healthy controls, and this correlates with increased numbers of platelet-derived extracellular vesicles (EVs) that express IL-1β. Our studies also reveal that platelet-neutrophil aggregation in pulmonary arterioles of SCD mice is associated with an increase in peripheral blood levels of platelet-derived EVs containing IL-1β. Remarkably, inhibition of TLR4 or TLR4/NLRP3-inflammasome activated caspase-1, or inhibition of IL-1β signaling, attenuated release of platelet EVs and platelet-neutrophil aggregation in the lung arterioles of SCD mice in vivo and SCD human blood in vitro .
Conclusions: TLR4 and NLRP3-inflammasome-mediated caspase-1 activation in platelets during vaso-occlusive crisis leads to release of IL-1β-containing EVs into the circulation. These circulating platelet EVs promote platelet-neutrophil aggregation in pulmonary arterioles, which results in arrest of blood flow in the lung, leading to ACS. Therapeutic inhibition of TLR4/NLRP3-caspase-1 signaling in platelets or IL-1β signaling is a potential therapy for ACS in SCD patients.
Acknowledgments: This study was supported by 1R01HL128297-01 (P.S.) and VMI startup funds (P.S.).

Disclosures
No relevant conflicts of interest to declare.