bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oncogenic Role for the Lck/ZAP70/PLCG2 Signaling Pathway in Pre-B-ALL Pathogenesis
Authors: Duque-Afonso Jesus , Wei Michael C. , Lin Chiou-Hong , Feng Jue , Buechele Corina , Wong Stephen Hon-Kit , Bassik Michael C , Cleary Michael L. 
Published: October 2019

Abstract
Although the treatment and prognosis of patients with pediatric acute lymphoblastic leukemia (ALL) have improved during the last decades, there is still a clinical need for more effective/selective and less toxic therapies.
To address this, we have interrogated various signaling pathways in human ALL cells and mouse strains that express E2A-PBX1, which is present in 5-7% of pediatric ALL. Phospho-flow analysis revealed basal hyper-phosphorylation levels of PLCγ2 in mouse E2A-PBX1 leukemias, consistent with hyper-activation of upstream signaling pathways. Efficient shRNA-mediated depletion of PLCγ2 reduced colony formation of mouse E2A-PBX1+ leukemias in vitro and increased disease-free survival after secondary bone marrow transplantation in vivo. Furthermore, PLCγ2-depleted human ALL cell lines including E2A-PBX1+ cells, showed reduced proliferation. These data suggest a pathogenic role of hyperactivated PLCγ2 in pre-B-ALL.
Bioinformatics analysis of E2A-PBX1 target genes in human ALLs revealed an enrichment of B- and T-cell activation pathways, which include the SRC-family kinase LCK and the cytoplasmic kinase ZAP70, upstream of PLCγ2. Comparative analyses of global transcriptional profiles in human primary and mouse leukemias and preleukemias induced by the E2A-PBX1 oncogene identified the signaling kinase ZAP70 as one of the earliest and most consistently up-regulated genes in E2A-PBX1 leukemias. Using a candidate gene approach, we identified LCK with increased expression levels in E2A-PBX1 leukemia cells compared to normal B-cell progenitors.
Mouse and human E2A-PBX1 leukemia cells were dependent on the E2A-PBX1 target genes ZAP70 and LCK for proliferation and survival as confirmed by shRNA knock-down experiments. Hence, efficient depletion of these genes resulted in a decrease of phosphorylated PLCγ2, suggesting therapeutic targets in E2A-PBX1 leukemias. Combined suppression of ZAP70 and LCK using double-shRNA experiments showed an additive effect on inhibition of cell proliferation and decrease of phosphorylated PLCγ2. These results provide a rationale for combination therapy to block this hyper-activated signaling pathway at different levels.
Several small molecule inhibitors were evaluated for their effects on PLCγ2 upstream pathways in E2A-PBX1 leukemia cells. SRC-family kinase inhibitors including dasatinib were most effective in reducing phosphorylation of PLCγ2 and inhibiting cell proliferation. Furthermore, dasatinib showed promising preclinical efficacy in vitro in colony forming assays and in vivo after secondary bone marrow transplantation of leukemias.
In summary, our studies demonstrate that the proliferation and survival of E2A-PBX1 leukemias are dependent on PLCγ2 and upstream signaling pathways, which are suitable for pharmacological inhibition.

Disclosures
No relevant conflicts of interest to declare.
Type-2 Phosphatidylserine (PS)-Positive Erythrocytes and Their Association with Markers of Hemolysis and Hemostatic Activation in Children with Sickle Cell Disease (SCD)
Authors: Setty Yamaja B. , Gayennebetal Suhita , Key Nigel S. , Stuart Marie 
Published: October 2019

Abstract
Introduction: Type-2 phosphatidylserine (PS)-positive red cells are a subpopulation of erythrocytes that are highly positive for PS, contain low levels of fetal hemoglobin, are specific for sickle cell disease (SCD) and have been identified in the dense red cell fraction. Studies have implicated PS-positive red cells in enhancing anemia due to phagocytosis and hemolysis. Shielding of red cell PS by diannexin, a synthetic homodimer of human annexin-V, has been demonstrated to provide protection against hemolysis and prevent activation of prothrombinase.
Methods: Using flow cytometry, we measured the levels of type-1 (red cells with low PS positivity) and type-2 PS-positive red cells in 50 children with SCD (31 with HbSS and 19 with HbSC), and assessed their association with various markers of hemolysis and hemostatic activation. Markers of hemolysis evaluated included plasma lactate dehydrogenase (LDH), reticulocyte count, and hemoglobin. Whole blood tissue factor (WBTF), pro-thrombin fragment F1+2, and D-dimer were evaluated as markers of hemostatic activation.
Results: We demonstrate that the levels of type-2 PS-positive red cells are significantly increased in HbSS patients (1.37 ± 0.97%, p<0.01) compared to children with HbSC disease (0.32 ± 0.21%) and age- and race-matched controls (0.15 ± 0.15%, n=19). WBTF and D-dimer showed significant associations with both type-1 and -2 red cells with no significant differences in the strength of their association. However, significantly greater correlations were noted between type-2 PS red cells and hemolytic markers compared to those noted with type-1 (Steiger's Z=3.05 to 4.59, p<0.01). In addition our in vitro studies demonstrate increased osmotic fragility of these red cells.
Table 1. Association of PS-positive RBCs with markers of hemolysis and hemostatic activation Biomarker Type-1 PS-positive RBCs Type-2 PS-positive RBCs Markers of Hemolysis LDH r = 0.44, p<0.002 r = 0.63, p<0.00001 % Reticulocyte r = 0.43, p=0.002 r = 0.66, p<0.00001 Hemoglobin r =-0.35, p=0.014 r =-0.63, p<0.00001 Markers of Hemostatic Activation WBTF r = 0.41, p=0.008 r = 0.56, p<0.0002 F1+2 r = 0.26, p=0.07 r = 0.31, p<0.03 D-dimer r = 0.46, p<0.001 r = 0.56, p<0.0005
Conclusions: Type-2 PS-positive red cells are elevated in SCD and the number of these cells correlates significantly with both markers of hemolysis and hemostasis. These findings provide a patho-physiologic link between the intravascular hemolytic milieu of SCD and the hemostatic perturbations previously noted in this disease.

Disclosures
No relevant conflicts of interest to declare.
Mathematical Models of Cancer Evolution and Cure
Authors: Shain Kenneth H. 
Published: October 2019

You cannot cure what you do not understand. So how can mathematical modeling address this pressing issue? The advances in therapeutic success in multiple myeloma over the last decades have hinged on an an army of researchers identifying a critical genetic, epigenetic and biochemical signaling factors within of MM cells as well as the tumor microenvironment (TME). Unfortunately, despite these large scale efforts we do not yet offer our patients curative intent therapy. The inability to provide curative therapy, especially in the setting of HRMM, is characterized by evolving resistance to lines of sequential therapy as a result of alternating clonal dynamics following the failure of initial therapy to eradicate minimal residual disease (MRD). Recent results underline the importance of tumor heterogeneity, in the form of pre-existing genotypically (and phenotypically) distinct sub-populations that translate to drug-resistant phenotypes leading to treatment failure. This phenomenon of “clonal tides”, has been well characterized using contemporary molecular techniques demonstrating that clonal evolution progresses by different evolutionary patterns across patients. Thus, resistance to therapy is a consequence of Darwinian dynamics- influenced by tumor heterogeneity, genomic instability, the TME (ecosystem), and selective pressures induced by therapy. Such evolutionary principles can be analyzed and exploited by mathematical models to personalize therapeutic options for patients with MM. Currently available clinical decision support tools and physician acumen are not able to account for the shear amount of information available. Mathematical models, however, provide a critical mechanism(s) to account of the large number of aspects to help predict and manage MM- accounting for what we do not know. Models can be designed with the specific intent of characterizing intra-tumoral heterogeneity, changing ecosystems, and clinical parameters over time to create patient-specific clinical predictions much like hurricane prediction models. This can only be achieved by creating mathematical models parameterized by longitudinal data of a number of parameters. The novel application of mathematical models based on Darwinian dynamics can be imputed with data to 1) predict progression events (risk of progression to from smoldering to active MM), 2) relapse, and 3) predictions of clinical response of MM patients for the optimizing therapeutics for cure or optimal control of MM; thus, providing invaluable clinical decision support tools.

Disclosures:
Shain: Celgene: Consultancy , Speakers Bureau ; Amgen/Onyx: Consultancy , Speakers Bureau ; Takeda: Consultancy , Speakers Bureau ; Signal Genetics: Consultancy , Research Funding.
Exploring the Function of Sestrin/Gator As Novel Regulators of Hematopoiesis
Authors: Kurrle Nina , Schnütgen Frank , Heidler Juliana , Poser Ina , Wempe Frank , Yepes Diego , Wittig Ilka , Oellerich Thomas , Rieger Michael A , von Melchner Harald , Serve Hubert 
Published: October 2019

Abstract
The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that responds to multiple environmental cues such as reactive oxygen species (ROS) and thereby regulates many fundamental biological processes including cell growth and autophagy. mTOR is found in two distinct multiprotein complexes, mTORC1 and mTORC2, of which mTORC1 has been established to play an important role in the regulation of hematopoiesis. For example, mTORC1 inhibition, combined with activation of canonical Wnt-signaling, was shown to increase long term repopulating (LT)-HSC self-renewal, whereas its activation depletes LT-HSCs.
The activity of mTORC1 is tightly controlled by multiple layers of upstream regulators, including the recently discovered GTPase activating protein (GAP) activity towards Rags (GATOR) complex, which is an AMP-Kinase/Tuberous sclerosis complex (TSC)-independent mTORC1 inhibitor induced by amino acid deprivation. GATOR consists of two subcomplexes, GATOR1 and GATOR2, whereby GATOR2 inhibits GATOR1. Inactivation of GATOR2 prevents mTORC1 activation by amino acids, whereas inactivation of GATOR1 constitutively activates mTORC1.
Sestrins (Sesn1, Sesn2 and Sesn3) are a family stress-inducible, redox-sensitive proteins that are involved in cellular- or organism-level adaptation to diverse metabolic challenges. They have been identified as direct interactors of GATOR2 and shown to inhibit mTORC1 by preventing GATOR2 from inhibiting GATOR1 in presence of amino acids. In quantitative affinity purification-mass spectrometry (AP-MS) and coimmunoprecipitation experiments with HeLa- and mouse embryonic stem cells harboring in situ GFP-tagged Sesn2, WDR59 and NPRL3 alleles, we could confirm the Sesn2/GATOR interaction under nearly physiological conditions.
To analyze the function of Sestrin/GATOR during hematopoiesis in more detail, we isolated Lin- Sca+ hematopoietic cells from the bone marrow of Sesn2-/- mice and performed serial replating experiments and competitive hematopoietic repopulation experiments in lethally irradiated mice and observed that Sesn2-/- progenitor cells proliferate significantly faster than their wild type counterparts albeit only during the initial engraftment phase. At later stages, the wild type cells took over, exceeding the Sesn2-/- cells by 3-4 fold in peripheral blood, bone marrow and spleen three months after transplantation. This suggests that the increased proliferative potential of Sesn2-/- progenitor cells leads to a depletion of the LT-HSC pool, strikingly resembling the phenotype of activated mTORC1.

Disclosures
No relevant conflicts of interest to declare.
Lysine Methyltransferase Kmt5a Restricts Myeloid-Biased Output of Lymphoid-Primed Multipotent Progenitors
Authors: Borikar Sneha , Philip Vivek , Kuffler Lauren , Trowbridge Jennifer J. 
Published: October 2019

Abstract
Distinct, lineage-biased subsets of multipotent progenitor cells (MPP) dynamically respond to the demands of the hematopoietic system to replenish mature hematopoietic cells as needed. It currently remains unclear as to whether distinct epigenetic mechanisms regulate lineage-specific expansion and differentiation from MPPs. Focusing on lymphoid-primed multipotent progenitor cells (LMPP/MPP4), we performed a lentiviral shRNA screen of 15 epigenetic factors, selected based on differential expression between myeloid-restricted and lymphoid-restricted progenitors. Following a 48 hour infection with lentiviral shRNA constructs or a non-targeting control, the lineage potential of lymphoid-primed multipotent progenitors was interrogated by myeloid and lymphoid colony forming unit (CFU) assays. From this screen, knockdown of the lysine methyltransferase Kmt5a most dramatically altered lineage output from lymphoid-primed multipotent progenitors through an expansion of myeloid lineage colonies without altering lymphoid colony production. To confirm target specificity, two independent shRNA hairpins targeting distinct locations of the Kmt5a transcript demonstrated that knockdown of Kmt5a (97.1% and 99.5% versus non-targeting control shRNA) increased macrophage colony production by 1.94 and 1.95 fold, respectively (P < 0.01 and P < 0.05, n = 3). Preliminary single cell culture experiments support that the enhanced myeloid lineage output from lymphoid-primed multipotent progenitors occurs at the single-cell level through increased cloning efficiency of myeloid-biased cells. Our results suggest that Kmt5a functions to restrict myeloid lineage output from lymphoid-primed multipotent progenitors. Mechanistically, KMT5A is responsible for monomethylation of histone H4K20 and the methylation of non-histone proteins (ex. p53K376). Our ongoing work aims to distinguish between these histone and non-histone targets to determine the precise mechanisms restricting myeloid lineage output from lymphoid-primed multipotent progenitors. This work has direct implications for a better understanding of the molecular drivers of transient myeloid lineage reprogramming of lymphoid-primed multipotent progenitors during hematopoietic regeneration, age associated myeloid lineage skewing of hematopoiesis, and myeloid malignancies.

Disclosures
No relevant conflicts of interest to declare.
Coordinated Repression of Cyclin-Dependent Kinase Inhibitors By PML/RARa in Acute Promyelocytic Leukemia
Authors: Tan Yun , Jin Wen , Wang Xiaoling , Wang Kankan 
Published: October 2019

Abstract
Inappropriate cell proliferation during oncogenesis is often attributed to dysregulation of components of the cell cycle machinery, which is strictly controlled by the activity of cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinases inhibitors (CKI). In our previous study, we have reported that the oncogenic fusion protein PML/RARa directly targets and represses the expression of CDKN2D, one of the CKI member, in acute promyelocytic leukemia (APL). Since different members of CKIs can mediate separate pathways to negatively regulate CDKs so as to control the cell cycle progression, thus implicating that multiple components of CKIs might be affected during the malignant transformation. However, the transcription regulation of the overall machinery of CKIs in APL remains unclear. Here, we report that PML/RARa coordinately represses multiple members of CKIs by directly binding to their regulatory regions. To identify potential CKIs that were dys-regulated in APL cells, we first studied the chromatin status of all the CKIs in APL by chromatin immunoprecipitation combined with next generation sequencing (ChIP-seq) using antibodies against histone modification markers (H3K4me3, H3K4me1 and H3K27me3). We found that the regulatory regions of CDKN2C, CDKN2D and CDKN1A were accessible in APL, suggesting that these CKIs were dynamically regulated in APL. We then performed ChIP-seq using antibody against PML/RARa and found that these three CKIs were occupied with PML/RARa binding, indicating that PML/RARa coordinately regulated CKIs in APL. To further study the role of PML/RARa in transcription regulation of CKIs, we used luciferase reporter assays to detect the effect of PML/RARα on CKIs transcription regulation and found that PML/RARα directly inhibited the promoter activity of CDKN2C and CDKN2D. Furthermore, we demonstrated that ATRA treatment increased the expression of CDKN2C, CDKN2D and CDKN1A through releasing the PML/RARα binding on chromatin in NB4 cells. Ectopic expression of these three CKIs could induce both cell cycle arrest and differentiation. Finally, the finding on the transcriptional regulation of CDKN1A, CDKN2C and CDKN2D in NB4 cells was validated in primary APL patient samples. Collectively, this study emphasizes that PML/RARa represses the CKI machinery and leads to cell cycle progression and differentiation block of APL. The coordinated regulation of multi-factors involved in the same pathway may shed new lights on the study of pathogenesis and target therapy of APL.

Disclosures
No relevant conflicts of interest to declare.
Cdkn3 Knockout Mice Develop Hematopoietic Malignancies
Authors: Sun Zejin , Cerabona Donna , He Ying , Nalepa Grzegorz 
Published: October 2019

Abstract
Cyclin dependent kinase inhibitor 3 (CDKN3) is a dual-specificity cell cycle regulatory phosphatase. In interphase, CDKN3 prevents premature G1/S transition by dephosphorylating interphase cyclin-dependent kinases (CDKs) to prevent premature inactivation of the RB pathway. During cell division, CDKN3 dephosphorylates the key mitotic kinase CDK1 at threonine-161 to extinguish CDK1 activity at the exit from mitosis. CDKN3 knockdown in cultured cells impairs the spindle assembly checkpoint (SAC), accelerates cell cycle progression and causes chromosomal instability, suggesting that it may function as a tumor suppressor. However, since CDKN3 has been reported as overexpressed in some malignancies and mutated or silenced in others, it is unclear whether it functions as an oncogene or a tumor suppressor.
To understand the in vivo role of CDKN3 in carcinogenesis, we generated the first Cdkn3 conditional knockout mouse model. We found that Cdkn3-/- mice were viable, non-dysmorphic and born at expected Mendelian ratios, indicating that this gene is dispensable for normal embryonic development. In agreement with the postulated role of this phosphatase in cell cycle progression and regulation of CDKs, we found that Cdkn3-/- cells had increased CDK1, CDK2 and CDK4 activity; increased inhibitory phosphorylation of Rb; increased DNA replication and proliferation; and impaired SAC. Increased CDK activity and accelerated cell cycle progression caused genomic instability reflected by increased frequency of in vivo micronucleation during hematopoiesis as well as higher frequency of aneuploidy and multinucleation and accumulation of supernumerary centrosomes in Cdkn3-/- cells cultured ex vivo. Cdkn3-/- mice had increased myeloid colony-forming units in progenitor assays. Long-term observation of Cdkn3-/- mice revealed an increased risk of death from a variety of hematopoietic (leukemia and lymphoma) and non-hematopoietic (lung, prostate and ovarian) malignancies.
Our findings establish Cdkn3 as an in vivo tumor suppressor in bone marrow and a variety of other tissues. In the long term, Cdkn3-/- mice will serve as a tool to dissect the function of this phosphatase in cell cycle control in more detail, and may prove useful in preclinical studies of chemotherapy of CDK-hyperactive, genomically unstable leukemia and lymphoma.

Disclosures
No relevant conflicts of interest to declare.
A Phase II Trial of Bendamustine, Carboplatin and Dexamethasone (BCD) for Refractory or Relapsed Peripheral T-Cell Lymphoma (BENCART): A Consortium for Improving Survival of Lymphoma (CISL) Trial
Authors: Park Byeong-Bae , Kim Won Seog , Suh Cheolwon , Yoon Dok Hyun , Won Jong-Ho , Yang Deok-Hwan , Do Young-Rok , Lee Won Sik , Kim Jeong-A , Kim Min Kyoung , Jo Jae-Cheol , Koh Youngil 
Published: October 2019

Abstract
Background
There is no standard salvage chemotherapy for relapsed or refractory peripheral T-cell lymphomas (PTCLs). In BETLY trial, which is sole prospective trial for bendamustine in refractory or relapsed T-cell lymphoma, bendamustine showed an encouraging high response rate across the two major PTCL subtypes, independent of age and prior treatment, with acceptable toxicity in refractory or relapsed PTCLs. We planned this trial to investigate the efficacy and toxicity of bendamustine, carboplatin and dexamethasone (BCD) for relapsed or refractory PTCLs, which would be expected to show more promising clinical outcomes than that of bendamustine single therapy.
Patients and Methods
Patients with relapsed or refractory PTCLs with more than one previous regimen regardless stem cell transplantation are eligible and totally 30 patients are needed. BCD treatment, which consist of bendamustine 80mg/m2 intravenously (i.v.) on Days 1 and 2, carboplatin AUC 5.0 i.v. on Day 1, and dexamethasone 40 mg orally on Days 1-4, was planned up to six treatment cycles without progressive disease and Peg-GCSF was supported at every cycle. Autologous stem cell transplantation (ASCT) after 3 cycles of BCD could be proceeded for eligible patients.
Results
Twenty-eight eligible patients were evaluated for toxicity and response. The diagnoses of participants included 15 cases of PTCL-NOS (54%), five cases of angioimmunoblastic T-cell lymphoma (18%) and four cases of ALK-negative anaplastic large cell lymphoma (14%) among others. The median age of the patients was 59 years (range, 23-75 years). After treatments with BCD, which delivered a median of three BCD cycles, there were 8 patients with complete responses (CR; 30%) and seven with partial responses (PR; 25%). The overall response rate (RR) was 55%. Five patients preceded to ASCT and three patients finally achieved CR. The median progression free survival was 4.8 months with a median follow-up duration of 4.5 months. In a total of 85 cycles of BCD, grade 3 or 4 neutropenia, thrombocytopenia and anemia occurred in 17.6%, 38.8% and 16.5% of cycles, respectively. Only one patients experienced febrile neutropenia.
Conclusions
BCD is an effective salvage regimen for relapsed or refractory PTCLs and can be administered with acceptable toxicity.

Disclosures
No relevant conflicts of interest to declare.