bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamin 2 (DNM2) and PACSIN2 Regulate Megakaryocyte Demarcation Membrane System Formation and Platelet Production in Concert
Authors: Giannini Silvia , Bender Markus , Pluthero Fred G. , Christensen Hilary , Leung Richard , Lo Richard W. , Kormann Jan , Plomann Markus , Kahr Walter H.A. , Falet Hervé
Published: October 2019

Blood platelets are produced in the bone marrow by megakaryocytes (MKs) in a process that requires extensive intracellular membrane rearrangements. These include the formation of the demarcation membrane system (DMS), the surface-connected membrane extension that invaginates into the cell body and further develops to provide membranes for future platelets. The precise molecular mechanisms responsible for these unique membrane rearrangements remain poorly understood. We have recently shown that Dnm2fl/fl Pf4-Cre mice specifically lacking the large GTPase dynamin 2 (DNM2) in MKs develop severe macrothrombocytopenia due to impaired receptor-mediated endocytosis (RME) (Bender, Giannini et al. Blood. 2015;125(6):1014-1024). Specifically, Dnm2fl/fl Pf4-Cre MKs accumulate arrested endocytic clathrin-coated vesicles that obstruct DMS formation. The actin nucleating factor Arp2/3 complex and polymerized actin clustered with clathrin at sites of impaired RME in Dnm2fl/fl Pf4-Cre MKs. We hypothesized that a DNM2 partner recruits actin-regulatory proteins at sites of RME and investigated the contribution of the F-BAR protein PACSIN2, an internal component of the initiating DMS (Jurak Begonja, Pluthero et al. Blood. 2015;126(1):80-88), in DMS formation and platelet production, as PACSIN2 interacts with DNM2 and actin-regulatory proteins such as N-WASP and filamin A (FlnA). Pacsin2-/- mice developed mild thrombocytopenia with slightly enlarged and shallow platelets. The DMS appeared less well defined and platelet territories were not readily visualized in Pacsin2-/- MKs. Pacsin2-/- Dnm2fl/fl Pf4-Cre mice lacking both PACSIN2 and DNM2 in MKs were further generated to determine the contribution of PACSIN2 in clathrin and actin clustering in Dnm2fl/fl Pf4-Cre MKs. Strikingly, PACSIN2 genetic deletion significantly improved the severe thrombocytopenia of Dnm2fl/fl Pf4-Cre mice. Specifically, PACSIN2 deletion abrogated the accumulation of clathrin and actin clusters, thereby unclogging DMS formation, which appeared as elongated maze-like membrane tubules in Pacsin2-/- Dnm2fl/fl Pf4-Cre MKs. Our results show that DNM2 terminates PACSIN2-dependent actin polymerization that accompanies RME, thereby allowing membrane rearrangements required for DMS formation.

Disclosures

No relevant conflicts of interest to declare.
The Frequency of Folate Deficiency in Multiple Myeloma Patients
Authors: Wongrakpanich Supakanya , George Gemlyn , Chaiwatcharayut Wikrom , Candelario Nellowe , Mittal Varun , Pomerantz Sherry , Varadi Gabor
Published: October 2019

Background: At the time of being diagnosed with multiple myeloma, more than half of patients will be anemic; therefore, anemia is considered to be an important disease characteristic for multiple myeloma. Folate deficiency is one possible cause of anemia in multiple myeloma patients. The frequency of folate deficiency in multiple myeloma patients is largely unknown.

Objective: The objective of this study is to describe the frequency of serum folate deficiency in patients with multiple myeloma.

Methods: A retrospective chart review study was conducted in Einstein Medical Center, Philadelphia between the dates of 01/01/2004 and 09/25/2014. Of the 216 patients that were reviewed, 175 patients fulfilled the criteria for a diagnosis for multiple myeloma: 1) monoclonal plasma cell in the bone marrow >/= 10% 2) monoclonal protein present in the serum and/or urine 3) at least one of the following: serum calcium > 10.5 mg/l, Creatinine > 2 mg/100 ml, hemoglobin< 10g/100ml, or lytic bone lesions. The relationship between serum folate level and age at diagnosis, gender, race, disease staging defined by International staging system (ISS), percent of bone marrow infiltration, immunoglobulin (Ig) subtypes, number of death and loss to follow up, complete blood count parameters, and other metabolic parameters (glomerular filtration rate, calcium, albumin and beta-2 microglobulin) were analyzed using Chi-Square test or t-tests as appropriate.

Results: Information on folate status was available for 78 consecutive multiple myeloma patients. Of them, 67 patients (85.9%) had a normal serum folate level, and 11 patients (14.1%) had laboratory-defined folate deficiency (serum folate level < 3.5 ng/mL). The mean hemoglobin level in the patients who had low folate level was 9.39 g/dL compared with 10.08 in the patients who had normal folate level (P=0.580). There were no statistical differences between the normal and the folate deficiency group in terms of laboratory parameters, baseline characteristics, staging, treatment modalities, and overall survival. However, the patients who had low folate levels were younger than the patients who had normal folate levels (61.55 years versus 69.88 years, P = 0.028).

Conclusion: Folate deficiency is considered to be a contributing cause of anemia in multiple myeloma patients. The findings from our study may emphasize the need for serum folate measurement and folate replacement therapy in multiple myeloma patients.

Disclosures

No relevant conflicts of interest to declare.
The Frequency of Genetic Anomalies According to Clonal Plasma Cells' Phenotype in Patients with Newly Diagnosed (ND) Multiple Myeloma (MM)
Authors: Garifullin Andrei , Martynkevich Irina , Voloshin Sergei , Kuvshinov Alexei , Martynenko Ludmila , Kleina Elizaveta , Abdulkadyrov Kudrat
Published: October 2019

Background. Genetic anomalies (GA) are primary link of pathogenesis in MM. GA lead to formation of clonal plasma cells, which has different phenotype.

Aim. To estimate the incidence of GA and their correlation with clonal plasma cells' phenotype in patients with ND MM. Methods. We analysed 22 patients with ND MM (median age 57 years, range 38-80; male/female - 1:1.75). Cytogenetic analysis was performed on bone marrow samples using standard GTG-method. Metaphase FISH analysis was performed according to the manufacturer's protocol using DNA probes: LSI 13(RB1)13q14, IGH/CCND1, IGH/FGFR3, LSI TP53 (17q13.1). 8-color immunophenotypic by flow cytometry using antibody to CD45, CD38, CD138, CD56, CD19, CD20, CD27 and CD117 antigenes.

Results. Translocation t(11;14) was detected in 3/14 (21.4%) patients, del(13q) - 2/14 (14.3%), t(11;14) - 3/14 (21.4%), hypodyploidy - 1/20 (5%), del(17р) - 0% patients. Clonal plasma cells' phenotype CD38+CD138+CD45- was detected in 100%. Expression CD56+ was revealed in 11/22 (50%) patients, CD19+ in 9/22 (40.9%), CD117+ in 5/22 (22.7%), CD20+ in 1/22 (4.5%), CD27+ in 1/22 (4.5%). The frequency of GA didn't depend on clonal plasma cells' phenotype and was 27.3%(3/11) in CD56+ phenotype, 23.8%(5/21) - CD20-, 23.8%(5/21) - CD27-, 23.5%(4/17) - CD117-, 23%(3/13) - CD19-, 22.2%(2/9) - CD19+, 20%(1/5) - CD117+, 18.2%(2/11) - CD56-, 0%(0/1) - CD20+, 0%(0/1) - in CD27+ phenotype. Patients of standard risk group according to mSMART 2.0 with GA had CD19-negative plasma cells' phenotype vs. CD19-positive phenotype in patients of intermediate and high-risk groups (p<0.05). 3-years overall survival in standard risk group with CD19- phenotype was 92,3%, CD19+ - 77,7% (p>0.05).

Conclusion . Identification of GA, which has adverse forecast, correlates with CD19+ plasma cells phenotype. The combined definition of plasma cells phenotype and GA can improve the system of risk stratification in MM.

Disclosures

No relevant conflicts of interest to declare.
Cytometric Response in Multiple Myeloma Cells to Low and Standard Doses of Bortezomib
Authors: Beffermann Nicole , Ocqueteau Mauricio , Ramirez Pablo , Galleguillos Mauricio , Sarmiento Mauricio
Published: October 2019

Bortezomib is a very useful drug in the treatment of multiple myeloma (MM) patients. Used in combination with other antineoplastic drugs it has a well documented impact in progression free survival (PFS) and overall survival (OS) of any age patients, elegible or not for hematopoietic cell transplant. Standard dose (1,3 mg/m2) is used in almost all patients and low dose (0,7-0,8mg/m2) is reserved to patients with kidney disease and neuropathy.

However, bortezomib doses used in phase 1 and 2 initial studies were described between 0,7 and 1,3 mg/m2 and were equally effective.

The aim of this study was to evaluate the cytometric response of MM naive patients to two different bortezomib doses.

We retrospectively analyzed the flow cytometry of fourty eight patients with naive MM treated with VCD scheme (Bortezomib-Cyclophosphamide-Dexametasone), without kidney failure nor neuropathy, of whom 21 received low doses of bortezomib (0.8mg/m2) and 27 standard doses (1.3 mg/m2). Flow cytometry was analyzed at diagnosis and at the end of treatment according to the expression of several clusters of differentiation (CD), establishing the presence of plasmoblastic myeloma clones (>95% of plasma cells with immunophenotype CD19 (-) CD56(+) CD45(-) CD38(+) and restriction of intra cytoplasmatic kappa and or lambda light chains) and normal mature plasma cells (CD19(+) CD56(-) CD45 (+) CD38 (++) with a policlonal expression of kappa/lambda light chains). Cytometric complete response was defined as normalization of the immunophenotype of plasma cells and absence of pathological cells.

We found no statistical differences between the 2 groups in flow cytometric response (p>0.1), as shown in figure 1.

This retrospective analysis suggests that lower doses of bortezomib could have similar effects in disease control, at least in cytometric response, to standard doses. Further studies should be made to evaluate clinical response and overall survival in patients treated with low doses compared to standard doses of bortezomib. In our country high costs of new generation antineoplastic drugs makes it necessary to find less expensive and equally effective schemes in order to make these well known beneficial treatments available to a greater number of patients.

Disclosures

No relevant conflicts of interest to declare.
Direct Comparison of Wharton Jelly and Bone Marrow Derived Mesenchymal Stromal Cells to Enhance Engraftment of Cord Blood CD34+ Transplants
Authors: van der Garde Mark , Van Pel Melissa , Millan Rivero Jose , de Graaf-Dijkstra Alice , Slot Manon , Kleinveld Yoshiko , Watt Suzanne , Roelofs Helene , Zwaginga Jaap Jan
Published: October 2019

Co-transplantation of CD34+ hematopoietic stem and progenitor cells (HSPC) and mesenchymal stromal cells (MSC) enhances HSPC engraftment. For these applications, MSC are mostly obtained from bone marrow. However, MSC can also be sourced from Wharton's jelly (WJ) of the human umbilical cord, which as a 'waste product', is cheaper to acquire and without significant burden to the donor. Here, we evaluated the ability of WJ MSC to enhance HSPC engraftment. First, we compared cultured human WJ MSC with human bone marrow-derived MSC (BM MSC) for in vitro marker expression, immunomodulatory capacity and differentiation into three mesenchymal lineages. Although we confirmed that WJ MSC have a more restricted differentiation capacity, both WJ MSC and BM MSC expressed similar levels of surface markers and exhibited similar immune inhibitory capacities. Co-transplantation of either WJ MSC or BM MSC with CB CD34+ cells into NOD-SCID mice showed faster recovery of human platelets and CD45+ cells in the peripheral blood and a 3-fold higher engraftment in the BM, blood and spleen six weeks after transplantation when compared to transplantation of CD34+ cells alone. Upon co-incubation, both MSC sources increased the expression of adhesion molecules on CD34+ cells, although SDF-1-induced migration of CD34+ cells remained unaltered. Interestingly, there was an increase in CFU-GEMM when CB CD34+ cells were cultured on monolayers of WJ MSC in the presence of exogenous thrombopoietin, and an increase in BFU-E when BM MSC replaced WJ MSC in such cultures. Our results suggest that WJ MSC is likely to be a practical alternative for BM MSC to enhance CB CD34+ cell engraftment.

Disclosures

No relevant conflicts of interest to declare.
Effect of RNAi-Mediated Knockdown of MCM7 on Human Leukemia Cells and Its Mechanism
Authors: Tian Liang , Chen Baoan , Liu Juan , Cheng Jian , Xia Guohua
Published: October 2019

Background: MCM7 is one of the subunits of the MCM2-7 complex that are essential for DNA replication licensing and control of cell cycle progression. It also participates in mRNA transcription and DNA damage regulation. It is found that MCM7 was highly expressed in many cancer cells including Leukemia. Thus, lentivirus-mediated siRNA targeting MCM7 was used to suppress its endogenous expression in K562 cells and develop a novel therapeutic strategy for leukemia.

Method: Lentivirus-mediated siRNA targeting MCM7 were designed and infected K562 cells. Down-regulation of MCM7 mRNA and protein expression in K562 cells were determined by Real-time PCR and Western blot analysis. The proliferation of the transfected K562 cells was assessed by CCK8 assay. Cell cycle progression and apoptosis were calculated by flow cytometry. The related protein levels of the transfected K562 cells were detected by Western blot. The stably transfected K562 cells were injected into nude mice. The transplanted tumors were measured and weighed, then Western blot and Immunocytochemistry were used to evaluated the expression of the proteins for mechanism.

Results: The mRNA and protein expression of MCM7 in the stably transfected K562 cells decreased with the percentage of 92.3±1.88 and 91.18%±0.93 respectively. Compared with control and negative control groups, the proliferative rate of K562 cells was significantly reduced after LV-MCM7-RNAi infection. The cell cycle progression of K562 cells was blocked, with a massive increase in the percentage of cells in the G0/G1 phase and decrease in S phase. The apoptosis rate of the transfected K562 cells was significantly higher. PLK1 and pPLK1 protein were down-regulated in response to MCM7 inhibition; P53 and bax protein were up-regulated simultaneously. In vivo, the tumor size and weight dramatically decreased in the MCM7-RNAi group than in negative control group. PLK1, pPLK1 proteins in transplanted tumors were down- regulated and P53,bax were up-regulated correspondingly.

Conclusions: Lentivirus-mediated MCM7 shRNA has effectively down regulated the expression of MCM7 gene on mRNA and protein levels. Moreover, the silence of MCM7 has led to the significant decrease in proliferation, blocking the cell cycle progression of K562 cells in vitro. Simultaneity, the apoptosis of the leukemia cell was enhanced. Both of the functions above cause the inhibition of the tumor growth in nude mice. Overall, these results suggest that the proliferation and cell cycle of K562 cells could be regulated by silencing MCM7 gene which is a promising gene therapeutic method to treat gastric cancer.

Disclosures

No relevant conflicts of interest to declare.
Mortality Associated with Venous Thromboembolism in Hospitalized Pediatric Patients: A Single Tertiary Care Institute Experience
Authors: Chadha Raminder , Goel Ruchika , Takemoto Clifford M.
Published: October 2019

Background: Hospital-associated venous thromboembolism (HA-VTE) is a major cause of morbidity and mortality in adults; there is a paucity of data about mortality risk in children.

Objective: To determine clinical factors associated with mortality in children with HA-VTE.

Methods: We conducted retrospective chart review of patients <=21 years with HA-VTE admitted to the Johns Hopkins Hospital from 1992-2013. ICD-9 codes were used to identify cases with deep venous thrombosis and pulmonary embolism; diagnosis was verified by reviewing hospital records and radiologic imaging. HA-VTE was defined as 1) VTE diagnosed ≥48 hours after hospital admission or 2) VTE diagnosed within 90 days of hospital discharge. Univariate and conditional multivariable logistic regression analyses with backward elimination were used to estimate the risk factors for mortality and adjusted Odds Ratios (adjOR) calculated.

Results: Of 330 HA-VTE cases, there were 55 deaths (16.7%); 18 deaths (5.5%) were attributable to VTE. Median follow up was 2.9 years. Within 30 days of VTE diagnosis, all-cause mortality was 4.2% and VTE-attributable mortality was 1.8%. Acute systemic infection (adjOR 11.3; 95%CI 4.7-26.8) and malignancy (adjOR 3.9; 95%CI 1.6-9.5) were associated with increased adjusted odds of all-cause mortality (Table 1). For deaths specifically attributable to VTE, acute systemic infection (adjOR 18.6; 95%CI 4.9-71.4) was associated with increased adjusted odds of mortality.

Conclusion: The majority of VTE-attributable deaths occurred over 30 days from VTE diagnosis. Acute systemic infection was a significant predictor of VTE-related mortality in HA-VTE. Further studies are warranted to identify additional risks for VTE-related mortality to develop prevention strategies.

Table 1. Odds Ratio Estimates of All-cause Mortality in Hospital Associated Venous Thromboembolism in Children in a Tertiary Care Hospital. Putative Risk Factor Odds Ratio 95% WaldConfidence Limits *Systemic Infection 11.3 4.7 26.8 Cancer 3.9 1.6 9.5 Pulmonary Embolism 0.7 0.3 2.0 Age (each year increase) 0.9 0.9 1.1 Male gender versus female 0.7 0.3 1.5 Central Venous Catheter 1.4 0.6 3.2 Hospital readmission within 90 days 1.5 0.6 3.7 *Meningitis, abscess, pneumonia, osteomyelitis, bacteremia, fungemia, and pyelonephritis

Disclosures

Takemoto: Novo Nordisk: Research Funding; Mast Therapeutics: Speakers Bureau.
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

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

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

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

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

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

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

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.
Runx1 Is Strictly Required for Cbfb-MYH11 Induced Leukemia Development
Authors: Zhen Tao , Kwon Erika Mijin , Hyde R. Katherine , Zhao Ling , Lopez Guadalupe , Alemu Lemlem , Liu Pu Paul
Published: October 2019

Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia (AML M4Eo), which generates a CBFB-MYH11 fusion gene. The prevailing hypothesis for the mechanism of leukemia development by CBFbeta-SMMHC, the fusion protein encoded by CBFB-MYH11, is that CBFbeta-SMMHC is a dominant negative repressor of RUNX1, a transcription factor that physically interacts with CBFbeta and CBFbeta-SMMHC. If this hypothesis is correct, reducing RUNX1 activity should facilitate leukemogenesis by CBFB-MYH11. In fact, loss-of-function mutations in RUNX1 are common in human AML, but not in inv(16) AML. However, we previously demonstrated that CBFB-MYH11 has RUNX1-repression independent functions (Hyde et al., Blood 115:1433, 2010). Moreover, we recently showed that a dominant negative allele of Runx1, Runx1-lz, delayed leukemogenesis by CBFB-MYH11 in a mouse model (Hyde et al., Leukemia 29:1771, 2015). These findings challenge the RUNX1-repression model for CBFbeta-SMMHC mediated leukemogenesis. However, our previous findings are not conclusive since the Runx1+/lz mice used in the previous study have one wild-type Runx1 allele, and still retain some Runx1 function.

To definitively address this question, we crossed Cre-based conditional Runx1 knockout mice (Runx1f/f) with Cre-based conditional Cbfb-MYH11 knockin mice (Cbfb+/56M) and Mx1-Cre mice to generate Runx1f/f, Mx1-Cre, Cbfb+/56Mmice, which express CBFbeta-SMMHC but not Runx1 after pIpC (poly I:C) treatment to induce Cre expression. Runx1f/f, Mx1-Cre, Cbfb+/56Mmice had more severe platelet deficiencies and higher numbers of Lin-/Sca1-/C-kit+ progenitors and Lin-/Sca1+/C-kit+ hematopoietic stem cells in the bone marrow when comapred with Runx1f/f, Mx1-Cre mice. Unexpectedly Runx1f/f, Mx1-Cre, Cbfb+/56Mmice also developed severe macrocytic anemia within two weeks after pIpC induction, which was lethal in about 1/3 of the mice. However, none of the Runx1f/f, Mx1-Cre, Cbfb+/56M mice developed leukemia up to one year after pIpC treatment. In contrast, all Mx1-Cre, Cbfb+/56M mice developed leukemia with an average survival of 4 months, as reported previously. These results suggest that Runx1 is strictly required for Cbfb-MYH11 induced leukemogenesis.

To further study the mechanism of leukemogenesis, we performed RNA-Seq on C-kit+ bone marrow cells isolated from mice two weeks after pIpC treatment, to explore the global gene expression changes caused by Runx1 knockout on Cbfb-MYH11 expressing mice. Our preliminary data analysis showed that 1688 genes were differential expressed (Padj ≤0.05, FC ≥ 2) between Runx1f/f, Mx1-Cre, Cbfb+/56M and Mx1-Cre, Cbfb+/56M mice. Interestingly, many of these genes (48%) are Runx1 target genes. The above results suggest that mis-regulating the expression of Runx1 target genes contributes to leukemogenesis by CBFbeta-SMMHC.

Disclosures

No relevant conflicts of interest to declare.
Peptidomimetic Blockade of MYB in Acute Myeloid Leukemia
Authors: Ramaswamy Kavitha , Forbes Lauren , Brown Fiona C , Koche Richard , Gindin Tatyana , Minuesa Gerard , Kharas Michael G. , Krivtsov Andrei , Armstrong Scott A. , Still Eric , de Stanchina Elisa , Isenhart Randi , Guo Guangwu , Knoechel Birgit , Kentsis Alex
Published: October 2019

Aberrant gene expression is a hallmark of acute leukemias. However, therapeutic strategies for its blockade are generally lacking, in large part due to the pharmacologic challenges of drugging transcription factors. MYB-driven gene trans-activation with CREB-binding protein (CBP) is required for the initiation and maintenance of a variety of acute lymphoblastic and myeloid leukemias, including refractory MLL-rearranged leukemias. Using structure-guided molecular design, we developed a prototypical peptidomimetic inhibitor MYBMIM that interferes with the assembly of the molecular MYB:CBP complex at μM concentrations and rapidly accumulates in the nuclei of AML cells. We found that treatment of AML cells with MYBMIM, but not with its inactive near-isosteric analogue TG3, led to the displacement and dissociation of MYB:CBP complex in cells, causing rapid downregulation of MYB-dependent gene expression including MYC and BCL2 oncogenes. This was associated with obliteration of H3K27Ac-driven oncogenic enhancers induced by CBP and enriched for MYB binding sites (Figure 1A; p=1e-118). Both human MLL-rearranged and non-rearranged AML cells, but not normal CD34+ umbilical cord blood progenitor cells, underwent sustained mitochondrial apoptosis in response to MYBMIM treatment, an effect that could be partially blocked by ectopic expression of BCL2. We observed that MYBMIM treatment (50 mg/kg/day) impeded leukemia progression and extended survival of immunodeficient mice engrafted with primary patient-derived MLL-rearranged leukemia cells (Figure 1B; p=3.8e-3). These findings emphasize the exquisite dependence of human AML on MYB:CBP transcriptional dysregulation, and establish a pharmacologic approach for its therapeutic blockade.

Figure MYBMIM blocks oncogenic gene expression (A) and impedes leukemogenesis in vivo(B). Figure. MYBMIM blocks oncogenic gene expression (A) and impedes leukemogenesis in vivo(B).

Disclosures

Armstrong: Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy.
Identification of TRA-1-60-Positive Cells As a Potent Refractory Population in Follicular Lymphomas
Authors: Takata Katsuyoshi , Iioka Hidekazu , Miyata-Takata Tomoko , Miki Yukari , Yoshino Tadashi , Maeda Yoshinobu , Saito Ken , Steidl Christian , Kondo Eisaku
Published: October 2019

Despite receiving Rituximab-combined chemotherapy, approximately half of follicular lymphoma (FL) patients suffer tumor recurrence, a major clinical issue. Retrieval of lymphoma stem-like cells from these poorly understood, intractable lymphomas would thus greatly ameliorate the cancer therapeutics. Here we demonstrate that the TRA-1-60-expressing cells are a unique population in FLs, converge to the conventional stem cell marker Oct3/4 and ALDH1-positive population, and strongly resist current agents against B-cell lymphomas.

TRA-1-60 expression was detected in minor populations of resting B-lymphocytes inside germinal centers in reactive lymphoid tissue, whereas scattered TRA-1-60-expressing cells were detected in the marginal area of neoplastic follicles of FLs, close to small blood vessels. Translocation t(14;18) was thus detected in DNAs from the TRA-positive cells in all five patients' samples, which evinced the identical pattern of gene fusion as that of TRA-negative cells in the same tissue in 4 of 5 fresh frozen FL samples. Retrospective histological comparison between the recurrent and cognate primary tissues revealed that the number of TRA-1-60-positive lymphoma cells from R-CHOP treated FL subjects had increased four-fold relative to primary tissue, a finding corroborated by in vitro assay on Rituximab treated 2 cell lines, wherein TRA-positive cell numbers increased over ten to thirty-fold compared to the untreated sample. Moreover, TRA-1-60-positive cell population was markedly resistant to current therapeutic agents for B-cell lymphomas. Concordantly, small numbers of TRA-1-60-positive cells implanted subcutaneously into NOD/SCID mice evinced potent tumor-initiating capacity in vivo, where tumors were twelve-fold larger in volume (p=0.0021<0.005) and thirteen-fold heavier in weight (p=0.0015<0.005) compared to those xenografted from the TRA-negative cells.

In conclusion, while further investigation for molecular insights will be necessary, it is now clear that TRA-1-60 should be a prominent focus among cellular markers in follicular lymphoma research, by which we ultimately hope to explain clinical intractability against combination-Rituximab and other therapies, and to better understand the stemness among a range of lymphoma cell types.

Disclosures

Maeda: Mundipharma KK: Research Funding.