bluuebunny/crossref_metadata_2025 huggingface dataset:
MSI2 Is Required for Maintaining the Activated Myelodysplastic Syndrome Stem Cell
Authors: Ho Tzu-Chieh , Taggart James , Amin Elianna , Xu Haiming , Barlowe Trevor , Perez Alexander R , Tivnan Patrick , Okabe Rachel , Lengner Christopher , Verma Amit , Roboz Gail J , Guzman Monica L. , Leslie Christina S , Nimer Stephen D. , Kharas Michael G.
Published: October 2019

+ The first four authors contributed equally to this project.

Myelodysplastic syndromes (MDS) are a group of blood cell disorders, characterized by ineffective hematopoiesis and severe cytopenias, which often transform to acute leukemia. MDS is also considered to be a clonal stem cell disease driven by alterations that are both genetic and epigenetic. However, it remains unclear how stem cell function is dysregulated and what factors drive these alterations in MDS HSCs. MSI2 is an important RNA-binding protein in normal HSC maintenance and can promote aggressive myeloid leukemia. Our preliminary data indicate that MSI2 expression is increased in high-risk MDS compared to low-risk MDS and correlates with poor survival. In order to model the role of MSI2 in MDS, we utilized the NUP98-HOXD13 transgenic (NHD13) model, which recapitulates many salient features of MDS including, leukopenia, severe anemia, erythroid dysplasia and leukemic transformation. Despite the lethal MDS or AML disease found in primary NHD13 animals, bone marrow cells transplanted into congenic mice generate a non-lethal MDS that rarely transform. Depletion of Msi2 utilizing a conditional knockout (NHD13-Msi2f/f -MX1-Cre) reversed the MDS phenotype and after one month the diseased HSPCs were eliminated. Conversely, we found that tetracycline inducible MSI2 overexpression in the context of the NHD13 transgene (NHD13/MSI2 mice) resulted in a worse MDS disease and a fully penetrant and lethal transformation to an AML, which was further accelerated during serial transplantation. AML arising in NHD13/MSI2 mice remained dependent on sustained MSI2 overexpression as mice removed from doxycycline demonstrated improved survival. Most interestingly, MSI2 overexpression expanded and maintained a more activated (G1) MDS hematopoietic stem and progenitor compartment (HSPC) in NHD13 cells. Gene expression profiling of the LSKs (Lineagelo, c-Kit+, Sca1+) before disease progression identified 891 significant genes, of which 137 genes were up-regulated (log2 fold change > 0) and 754 genes were down-regulated (log2 fold change <= 0). Furthermore, Gene Set Enrichment Analysis (GSEA) demonstrated a more progenitor like gene expression signature, enrichment in an NRAS activated signature, and a reduced quiescent phenotype. Unsupervised hierarchical clustering of the NHD13/MSI2 LSK gene signature in MDS patients resulted in four distinct clusters. Clusters segregated MSI2 high expressing MDS patients and this "MSI2 high cluster" predicted poor survival. In summary, our findings suggest that MSI2 plays a critical functional role in the maintenance of the hematopoietic stem and progenitor compartment in MDS and highlights it as a novel therapeutic target in this disease.

Disclosures

No relevant conflicts of interest to declare.
JQ1, a Selective Bromodomain Inhibitor, Augment the Immunogenicity of Mantle Cell Lymphoma By Influencing the Expression of PD-L1
Authors: Wang Hongwei , Cheng Fengdong , Cheng Jie , Villagra Alejandro , Tao Jianguo , Bradner James E. , Sotomayor Eduardo M.
Published: October 2019

Mantle cell lymphoma (MCL) is a rare and aggressive form of non-Hodgkin's B-cell lymphoma and represents 4% to 8% of NHL. New therapeutic strategies are under development to improve the clinical outcome of patients with this disease. Recent studies have highlighted the importance of BRD/acetyl-lysine binding in orchestrating molecular interactions in chromatin biology and regulating gene transcription. The Bromodomain (BRD) is a diverse family of evolutionary conserved protein-interaction modules, which recognizes acetylated lysine residues such as those on the N-terminal tails of histones.

We have recently determined the functional consequences of inhibiting Bromodomains in MCL. First, we evaluated the effects of JQ1, a selective small-molecule bromodomain inhibitor upon MCL's immunogenicity and antigen-specific CD4+ T-cell responses. In vitro treatment of MCL cells with increasing concentrations of JQ1 resulted in a decreased expression of the tolerogenic molecule PD-L1 as compared to control cells. Second, we evaluated the capabilities of JQ1-treated MCL cells to present cognate antigen to naïve or anergic antigen-specific CD4+ T-cells. We found that treatment of MCL cells with JQ1 enhances their antigen-presenting capabilities leading to effective priming of naïve CD4+ T-cells and restoring the responsiveness of tolerant T-cells as determined by their increased production of IL-2 and IFN-gamma in response to cognate antigen. More importantly, in vivo studies clearly demonstrated a strong anti-tumor effect in JQ1-treated MCL tumor bearing mice. Mechanistically, we found a decreased STAT3 phosphorylation and c-Myc expression in JQ1-treated MCL cells, which in turn might be responsible for the diminished expression of PDL1 and augmentation of MCL's immunogenicity.

Taken together, we have found that MCL cells treated with the Bromodomain specific inhibitor JQ1 are more inflammatory, display lower expression of the immunosuppressive molecule PDL1 and, are capable of restoring the responsiveness of tolerant T-cells. More importantly, JQ1 treatment in vivo inhibits tumor growth in MCL tumor bearing animals. Our studies therefore have unveiled a previously unknown immunological effect of BRD inhibitors and have identified a novel immunotherapeutic approach in MCL.

Disclosures

No relevant conflicts of interest to declare.
RAB14 and RAB5 Gtpases Regulate Human Erythropoiesis, Potentially Via Opposing Roles in Endosomal Recycling
Authors: Kim MinJung , Kingsbury Tami J. , Cheng Wen-Chih , Tan Yee Sun , Taylor Brittany M. , Koshatwar Sampada , Civin Curt I.
Published: October 2019

Previously, we reported that the erythroid-expressed miRs, miR-144 and miR-451, target the RAB14 GTPase during human erythropoiesis in the human TF1 erythropoietic model cell line and in primary CD34+ hematopoietic stem-progenitor cells. In response to erythropoietin, endogenous RAB14 levels decreased during erythropoiesis, and RAB14 knockdown increased the numbers of erythroid (CD34- CD71hi CD235ahi) cells, increased b-hemoglobin expression, and decreased ETO2 expression (Kim, BJH, 2015). Taken together, our findings revealed that RAB14 functions as a physiologic inhibitor of human erythropoiesis. RAB GTPases comprise a >60 member subfamily of the large RAS oncogene family, and multiple RAB GTPases play important roles in vesicle trafficking, signal transduction and receptor recycling. RAB14 is a component of the intermediate compartment of the endosomal recycling pathway, between the RAB4/RAB5-dependent early endosome and the RAB11-dependent recycling endosome (Linford, Dev. Cell, 2012). The RAB GTPases are responsible for directing cargo proteins to the recycling vs. degradation pathway during endosome maturation. To test the role of another endosomal RAB GTPases in human erythropoiesis, we examined the consequences of knocking down RAB5 protein levels. In contrast to our findings with RAB14, knockdown of RAB5C decreased the numbers of erythroid cells generated during erythropoiesis, indicating that RAB5C enhances physiologic erythropoiesis. Consistent with this observation, mRNA and protein levels for all three RAB5 isoforms (i.e. RAB5A, B, C) increased during erythropoiesis. The opposing erythropoietic effects of RAB14 and RAB5 GTPases may be due to their contrary roles in endosomal recycling, in that internalization of cell surface receptors is dependent on RAB5 GTPase, whereas RAB14 GTPase is involved in recycling of receptors back to the cell surface.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Utility of Lung Ultrasound for Evaluating Acute Chest Syndrome in Pediatric Patients with Sickle Cell Disease
Authors: Cohen Stephanie G. , Malik Zayir M , Hagbom Robert , Alazraki Adina L , Zmitrovich April G. , Adisa Olufolake , Morris Claudia R.
Published: October 2019

Background: Acute chest syndrome (ACS) is a common complication in patients with sickle cell disease (SCD) and is a leading cause of morbidity and mortality. Physical assessment alone is not sensitive for diagnosing ACS and therefore Chest x-ray (CXR) is recommended because of the difficulty of diagnosing ACS on clinical grounds alone. Children with SCD are repeatedly exposed to diagnostic radiation for the evaluation of ACS. Focused chest ultrasound (US) has been used to evaluate for lung consolidation. If lung US can identify patients with ACS this application could potentially limit radiation exposure in patients with SCD at risk for ACS. We evaluated the utility of physician performed US as compared to CXR to identify patients with SCD who have ACS.

Methods: This is a prospective observational study that took place from November 2014-July 2015 in 2 urban pediatric emergency departments (EDs). The study population consisted of a convenience sample of patients with SCD from birth to 18 years of age at risk for ACS and who received a CXR for suspected ACS. Medical students and clinicians with training in lung sonography consented patients and performed a focused study to evaluate for lung consolidation. A blinded expert in point-of-care US reviewed for quality assurance and agreement. Sensitivity, specificity, and likelihood ratios were calculated for test performance characteristics of ultrasound using CXR as a reference standard. Inter-observer agreement (κ) between enrolling sonologists and reviewer was also calculated.

Results: 85 patients were enrolled for a total of 98 cases. Median age was 7 years (IQR 2-13 years) and 53% of patients were male. The prevalence of ACS by CXR was 14%. Lung US was able to detect consolidation with a sensitivity of 86% (95% CI, 56-97%), specificity of 95% (95% CI, 87%-98%), positive likelihood ratio (LR) 18 (95% CI, 7-48) and negative LR 0.2 (95% CI, 0.04-0.5). The agreement between enrolling novice sonologists' interpretation and blinded reviewer's interpretation was very good with a Cohen κ of 0.86 (95% CI, 0.7-1).

Conclusions: Focused lung US was able to identify ACS with high specificity. There was very good agreement between novice and expert sonologist interpretation. Lung US may decrease the need for CXR in patients at risk for ACS. Further studies are needed to see how this test performs within current clinical practice guidelines.

Disclosures

No relevant conflicts of interest to declare.
The Role of Receptor Interacting Protein Kinase in Myelopoiesis in Health and Disease
Authors: Silke John , Ma Chunyan , Brumatti Gabriela , Lalaoui Najoua , Ekert Paul G
Published: October 2019

Ripk1 -/-mice die at birth from systemic inflammation and this was not transferable in hematopoietic reconstitution experiments. One caveat to interpreting this result is our observation that Ripk1-/- progenitors failed to engraft lethally irradiated hosts properly. Competitive and serial transplantation experiments suggest that Ripk1-/- hematopoietic stem and progenitor cells (HSPC) have a cell-intrinsic defect. Blocking TNF allowed Ripk1-/- progenitors to reconstitute effectively supporting the idea that the cell-intrinsic defect is due to hyper-sensitivity to TNF induced death1. TNF can induce both caspase-8 dependent apoptosis and RIPK3/MLKL dependent necroptosis. These results prompted us to determine whether RIPK1 and TNF induced cell death had a role in leukemogenesis. We therefore generated mouse models of AML leukemia by retroviral infection of fusion MLL-ENL constructs into E14 fetal liver cells from Ripk1-/-, Ripk3-/-, Mlkl-/-, Casp8-/-Ripk3-/-, Casp8-/-Mlkl-/- and Ripk3-/-Mlkl-/- mice and transplanting them into sub-lethally irradiated wild type hosts2. Except for the Ripk1-/- MLL-ENL there was no difference in the development of leukemia from these genotypes compared to MLL-ENL wild type. Surprisingly however, given the defective reconstitution potential of Ripk1-/- HSPC, leukemia induced lethality occurred more than two times faster in Ripk1-/- MLL-ENL (20 days) compared to wild type MLL-ENL (50 days). No differences in blood profile, histology, myeloid markers or cell cycling were observed. Even more surprisingly, when we retransplanted these leukemias, Ripk1-/- MLL-ENL had a prolonged disease onset (over 80 days) compared to wild type (around 25 days). I will describe experiments to discover the mechanism behind these intriguing findings.

1. Rickard JA, O'Donnell JA, Evans JM, Lalaoui N, Poh AR, et al. RIPK1 Regulates RIPK3-MLKL-Driven Systemic Inflammation and Emergency Hematopoiesis. Cell. 2014;157:1175-1188.

2. Zuber J, Radtke I, Pardee TS, Zhao Z, Rappaport AR, et al. Mouse models of human AML accurately predict chemotherapy response. Genes Dev. 2009;23:877-889.

Disclosures

No relevant conflicts of interest to declare.
Regulation of Platelet Procoagulant Activity
Authors: Heemskerk Johan
Published: October 2019

Platelets play prominent and unique roles in the kinetics of the coagulation process. Modulated by the flow conditions, they control coagulation in various ways: (1) by integrin activation and ensuing clot retraction; (2) via exposure of the procoagulant phospholipid phosphatidylserine (PS), which causes binding of a specific set of coagulation factors at the platelet surface with as a result tenase activation and thrombin generation; (3) by initiating fibrin fiber formation at the platelet surface, a process specifically catalyzed by coated platelets. Recent evidence points to fibrin as an active structural network, which provides binding sides for plasma factors and platelets and, thereby, provides an important feedback mechanism for the stimulation of platelet activation. This overview will focus on novel insights into the signaling and activation processes in platelets that regulate these distinct procoagulant functions. One key mechanism is the activation and secondary closure of integrin alphaIIb-beta3. Another key mechanism concerns the calcium-dependent exposure of PS, which is accompanied by swelling of the platelets. Together, these processes control not only platelet adhesion, but also the accessibility for coagulation factors. Major distortions in these procoagulant functions are seen in platelets from a patient with the Scott syndrome, a rare bleeding disorder associated with mutations in the calcium-dependent ion channel protein anoctamin-6 (gene ANO6, previously TMEM16A). These platelets are deficient in calcium-induced PS exposure, swelling, integrin inactivation and in the formation of thrombin and fibrin at the platelet surface. These dysfunctions are precisely phenocopied in mice lacking anoctamin-6 expression. Advanced platelet proteomics analysis indicated that the deficiency in anoctamin-6 was accompanied by decreased calpain-dependent cleavage of a whole spectrum of intracellular proteins, and an increased phosphorylation state of many signaling and cytoskeletal proteins. However, calcium signaling in the Scott platelets was unchanged. In contrast, reports on patients with a gain in platelet procoagulant function, the Stormorken syndrome, indicate a link with genetic mutation in the calcium signaling protein STIM1. The possible consequences of these novel insights for our understanding of haemorrhagic and thrombotic deseases will be discussed.

Disclosures

No relevant conflicts of interest to declare.
Iron Requirement in Hypoxic Conditions
Authors: Gassmann Max
Published: October 2019

While highlanders in the Andes and Himalayas are well adapted to live at high altitude, adequate acclimatization time to slowly adjust to hypoxic conditions is among the most important aspects for ascending mountaineers. Accordingly, most reports emphasize on mechanisms that cope with reduced oxygenation. Notably, during a journey to high altitude coverage of the elevated iron demand is equally critical. Thus, it's crucial to focus on the crosstalk between oxygen and iron homeostasis. I will discuss the role of iron in the oxygen sensing process and erythropoietin (Epo) synthesis as well as in gene expression control mediated by the hypoxia-inducible factor-2 (HIF-2). The blood hormone Epo that is abundantly expressed by the kidney under hypoxic conditions stimulates erythropoiesis in the bone marrow, a process that requires high quantities of iron. To ensure that sufficient iron is provided, hypoxia-induced soluble factors - such as the novel Epo-controlled erythroferrone that is expressed in erythroid precursor cells - reach the liver where they reduce expression of the iron hormone hepcidin. In turn, suppression of hepcidin allows both, elevated iron release from storage organs and enhanced absorption of dietary iron by enterocytes. As recently observed in sojourners at high altitude, however, iron uptake may be hampered by reduced appetite and gastrointestinal bleeding. Overall, adequate systemic iron availability is an important prerequisite to adjust not only to high-altitude induced hypoxia but to treat disease-related hypoxic conditions.

Disclosures

No relevant conflicts of interest to declare.
Pharmacomechanical Catheter Directed Thrombolysis for Pregnancy Related Proximal Lower Extremity Deep Venous Thrombosis: Effectiveness in the Prevention of Post Thrombotic Syndrome
Authors: Spectre Galia , Bloom Allan , Roth Batia , Varon David , Kalish Yosef , Da'as Nael , Tzitershpiler Tal
Published: October 2019

Pharmacomechanical catheter-directed thrombolysis (PCDT) reduces the incidence of post thrombotic syndrome (PTS) after proximal deep vein thrombosis (DVT); however its efficacy in PTS prevention for pregnancy related DVT is unknown. Most of the patients with DVT in pregnancy present with femoral or iliofemoral involvement and the prevalence after of PTS after pregnancy related DVT in the lower extermity is 42%. The purpose of this study is to compare PTS outcome in women with pregnancy related DVT with and without PCDT.

Methods: Women with pregnancy related proximal (femoral/iliofemoral) DVT who underwent PCDT and anticoagulation or anticoagulation alone, were evaluated for PTS using the Villalta scale and VEINES-QOL/Sym questionnaire.

Results: Eleven women with iliofemoral DVT underwent PCDT and anticoagulation, two during the first trimester and nine postpartum. Eighteen women (13 with iliofemoral DVT) were treated with anticoagulation only. There was no difference in age, number of pregnancies, trimester, duration of anticoagulation or thrombophilia between the groups. The time between DVT diagnosis and study inclusion was longer in the control group - median 50.5 (range 16-120) months , compared to study group: median 27 (range 11-64) months. None of the women in the study group developed PTS. Six of 18 patients in the control group developed PTS (33.3%) P=0.03, four of whom developed severe PTS (Villalta scale ≥15). One patient in each group developed recurrent DVT, and one patient in the study group developed a calf hematoma. A reduced frequency of lower extremity symptoms was observed in the study group by VEINES-Sym questionnaire P=0.01, but there was no difference in VEINES-QOL questionnaire (p=0.11)

Conclusion: This study suggests a reduction in PTS in women with pregnancy related proximal DVT who were treated with PCDT and anticoagulation compared to women who were treated with anticoagulation alone

Disclosures

No relevant conflicts of interest to declare.
TLR Activation By MSC Derived Extracellular Vesicles Drives Myeloid Commitment
Authors: Yoon Young Me , Goloviznina Natalya A , Butler John , Verghese Santhosh Chakkaramakkil , Kurre Peter
Published: October 2019

Hematopoietic stem and progenitor cells (HSPC) are responsive to paracrine signaling in the bone marrow microenvironment. We recently showed that leukemia derived extracellular vesicles (EVs) directly regulate HSPC in the leukemic niche and confer a net loss of progenitor function in part through direct action of a highly abundant miRNA on the hematopoietic transcription factor c-Myb. These observations motivated us to explore the potential regulation of HSPC by EVs present during steady state hematopoiesis in the unperturbed bone marrow. Mesenchymal stromal cells (MSC) comprise a diverse set of cells secreting supportive factors central to the maintenance of HSPC. We systematically characterized EVs released by murine MSCs as a candidate population and demonstrated that EVs themselves carry cytokines, predominantly interleukin (IL) -1, -2, -4,-6 and -17A, as well as G-CSF and Interferon g. As a functional corollary, we found that MSC EVs led to preferential granulocyte and monocyte differentiation in co-culture with C-kit enriched HSPCs, when compared to vesicle free condition (termed S100). These observations were further supported by the expansion of KSL (c-kit+,Sca-1+,lin-) and MPP2-4 (CD48+, CD150+/-) progenitor populations. To determine the in vivo relevance of these findings we performed a series of competitive transplantation experiments, that demonstrated that MSC-EV exposed HSPCs outperformed HSPCs cultured with EV depleted media, with striking advantage in early, but a significant deficit in later (week 16) myeloid recovery, compared to the S100 condition. These observations were broadly consistent with recent evidence for HSPC regulation by toll like receptors (TLRs) and we confirmed that the observed deficit in progenitor phenotypes was partially reversed in MSC-EV co-cultures with HSPCs from animals with genetic disruption of TLR4 or MyD88. EV-TLR engagement further induced a NF-kB response with downstream transcriptional activation of canonical transcriptional targets (STAT1, IRF1, -9) and significantly upregulated the release of IL-3, -6, IFNg, and G-CSF in exposed WT HSPC, compared with S100. Finally, we performed deconvolution microscopy studies of co-cultured HSPC and demonstrated the Pearson's spatial correlation between fluorescently tagged MSC-EV and TLR4 as well as the early and late endosomal marker proteins, Rab5 and Rab7, respectively. We conclude that MSC-derived EV trafficking of TLR contributes to the homeostatic regulation of HSPCs in the bone marrow microenvironment as a mechanism of tonic regulation of hematopoiesis.

Disclosures

No relevant conflicts of interest to declare.
SF3B1 Interactions with Chromatin Are Dynamic and Regulated in a Cell Cycle-Dependent Manner
Authors: Murthy Tushar , Bluemn Theresa , Pillai Manoj M. , Minella Alex C
Published: October 2019

Splicing factor 3B1 (SF3B1) is a member of the U2 snRNP complex that is a key regulator of RNA splicing. RNA splicing begins with the recognition of splice sites (ss) at the 5' and 3' ends of introns and ends with the removal of introns and joining of exons flanking them. SF3B1 plays an important role in this process by facilitating the recognition of the 3'ss. SF3B1 is frequently mutated in numerous cancers as well as the myelodysplastic syndromes (MDS). Mutations within the HEAT domain of the protein potentially contribute to disease pathogenesis. In addition to influencing splicing by binding to pre-mRNA, SF3B1 has been shown to affect splicing of exons by associating with them directly on chromatin via histone/nucleosome interactions. However, it is not understood if or how SF3B1 association with chromatin is regulated. Given that N-terminal serine and threonine residues on SF3B1 are known substrates of cyclin E-Cdk2, which phosphorylates histone subunits and other chromatin associated proteins, we hypothesized that CDK2 activity regulates SF3B1-nucleosome interactions. Although SF3B1 is phosphorylated during splicing catalysis, the function of this phosphorylation has remained unknown. We have now discovered, using nucleosome preparations and histone subunit co-immunoprecipitation assays in synchronized cells, that endogenous SF3B1 interacts with nucleosomes in a highly cell-cycle dependent manner, while total cellular abundance of SF3B1 remains invariant during cell cycle progression. In human and mouse cells, including hematopoietic cell lines, SF3B1 is excluded from chromatin during both G0 (quiescence) and G2/M phases of cell cycle. Notably, SF3B1 is enriched within chromatin maximally during S-phase. Unexpectedly, we found that the inhibition of Cdc2 (Cdk1) during G2/M enforces the SF3B1-chromatin interaction, pointing to a direct role for Cdc2 in restraining this interaction during mitosis. Further, SF3B1 loading onto chromatin during early cell cycle progression from G0 to S-phase is inhibited by Cdk2 inhibition. Thus, Cdk2 and Cdc2 appear to have antagonistic roles in controlling SF3B1-chromatin interactions during the cell cycle. Our findings suggest that Cdk activity may regulate the recruitment of the spliceosome machinery in order to coordinate splicing of particular transcripts with cell cycle progression. Current studies are focusing on how disease-associated mutations in the HEAT domain of SF3B1 affect the dynamics of its cell cycle-dependent interaction with nucleosomes and corresponding alterations to splicing outcomes.

Disclosures

No relevant conflicts of interest to declare.
TAFII250 Is Critical in AML1-ETO Mediated Leukemogenesis
Authors: Xu Ye , Man Na , Martinez Concepcion , Martinez Camilo , Beckedorf Felipe , Cheng Guoyan , Greenblatt Sarah Merin , Wang Lan , Shiekhattar Ramin , Nimer Stephen D.
Published: October 2019

Background: Rearrangement between chromosome 8 and 21 is the most frequently observed chromosomal translocation. In AML patients, it leads to the formation of a novel fusion protein AML1-ETO. AML1-ETO functions as a transcriptional regulator to suppress myeloid differentiation and to promote self-renewal of hematopoiesis stem cells, which are of importance in leukemia development. However, AML1-ETO has no enzymatic function, thus targeting AML1-ETO directly is technically difficult. TAFII250, a largest subunit of the transcription factor IID complex (TFIID) serves to bring other components of basal transcription machinery to the preinitiation complex during the transcription initiation. And TAFII250 also plays a crucial role in the expression of genes involved in cell cycle and apoptosis. Yet despite the importance of these processes, there is no direct evidence implicating TAFII250 in cancer development. We have reported that acetylation of AML1-ETO on lysine 43 is critical for AML1-ETO mediated leukemogenesis and used a peptide pulldown strategy to identify proteins that preferring interact with acetylated AML1-ETO and identified TAFII250. Here, we are going to report the functions of TAFII250 in AML1-ETO induced leukemogenesis.

Methods: We used Kasumi-1 and SKNO-1 cell lines derived from t(8; 21)AML patients, and a mouse AML1-ETO exon 9a (AE9a) expressing cell line developed from bone marrow cells of mice injected with bone marrow cells infected with AE9a to perform in vitro and in vivo studies.

Results: We demonstrate that TAFII250 associates with AML1-ETO in AML cells through its bromodomain recognizing acetylated lysine 43 on AML1-ETO. The knockdown of TAFII250 completely abolished the proliferation of AML1-ETO expressing cells and has little influence on cell growth of non AML1-ETO expressing cells K562 and CD34+ cells. In addition, when we examined cleaved caspase 3 and PARP by western and measured annexin-V through flow cytometry, we found that the deficiency of TAFII250 induces apoptosis in AML1-ETO expressing cells. Further, the co-immunoprecipitation assay revealed that the deficiency of TAFII250 blocks the binding of AML1-ETO to CBFb (core binding factor beta subunit) and the recruitment of AML1-ETO at the promoter regions of a subset of its target genes. Consequently, loss of TAFII250 interferes with the expression of these genes. The depletion of TAFII250 also has negative effect on the self-renewal of leukemic cells, promoting their differentiation. Most importantly, the loss of TAFII250 severely impairs leukemia development in AE9a expressing cells.

Conclusions: Together, these results reveal an essential role of TAFII250 in AML1-ETO mediated leukemogenesis and imply that TAFII250 might be a therapeutic target for AML1-ETO expressing AMLs.

Disclosures

No relevant conflicts of interest to declare.
Tyrosine 842 Residue in the Activation Loop of FLT3-ITD Is Indespensible for Oncogenic Transformation
Authors: Kazi Julhash U. , Rönnstrand Lars
Published: October 2019

The receptor tyrosine kinase FLT3 is mutated in around 30% of acute myeloid leukemia patients. The so-called internal tandem duplication (ITD) mutation in the juxtamembrane domain is the most common type of mutation in FLT3. Other oncogenic mutations include point mutations in the kinase domain. One of the hallmark of receptor activation is phosphorylation on several tyrosine residues in the receptor. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. The tyrosine residue Y842, which is located in the activation loop of FLT3, and is well conserved in all receptor tyrosine kinases. It has been demonstrated that tyrosine phosphorylation of the activation loop is essential for the activity of some but not all receptor tyrosine kinases. In this report we show that phosphorylation on Y842 residue is dispensable for receptor activity and stability. However, Y842 plays critical roles in oncogenic FLT3-ITD mediated transformation. We have shown that a Y-to-F mutation (Y842F) results in reduced cell viability and proliferation. This mutation also led to dramatic decrease in in vitro colony formation in semi-solid medium. Furthermore, cells carrying the Y842F mutant of FLT3-ITD displayed significant delay in tumor formation in nude mice. Gene expression analysis, using microarrays, showed that mutation of Y842 causes suppression of anti-apoptotic genes. Finally, we showed that phosphorylated Y842 is a binding site for SHP2 and mutation of this site results in impaired activation of signaling through the RAS/ERK pathway. Collectively, our data suggest that Y842 in FLT3 plays an important role in FLT3-mediated RAS/ERK signaling and cellular transformation.

Disclosures

No relevant conflicts of interest to declare.
Clinical Features and Outcomes of Isolated Myeloid Sarcoma in the United States: Analysis Using a National Dataset
Authors: Goyal Gaurav , Bartley Adam C , Al-Kali Aref , Hogan William J , Litzow Mark , Go Ronald S.
Published: October 2019

Introduction

Isolated myeloid sarcoma is a rare form (<1%) of acute myeloid leukemia presenting as extramedullary tumor. Contemporary clinical data are mostly limited to institutional case series. Using the National Cancer Database, the largest public cancer registry covering >70% of all newly diagnosed cancers in the United States, we determined the patterns of anatomical presentation and clinical outcome of myeloid sarcoma.

Methods

We identified patients with a histologically confirmed diagnosis of isolated myeloid sarcoma from 2004-2013 using International Classification of Diseases for Oncology version 3 (ICD-O-3) code: 9930. To allow at least 1 year of follow-up, only patients diagnosed from 2004-2012 were included in the survival analysis using Kaplan-Meier estimates.

Results

A total of 746 patients were included in the study. The median age of patients was 59 years (range, 41 to 73) and 56% were males. The anatomical distribution and median overall survival of patients are depicted in the Table. The top 3 most common sites of presentation were connective/soft tissues (31.3%), skin/breast (12.3%), and digestive system (10.3%). Compared to other races, Blacks were more likely to have presentation in bones/joints (11.8% vs 6.3% in others), lymph nodes/spleen (22.1% vs. 9%), and less likely in skin/breast (4.4% vs. 13.8%). Asians were more likely to present with cardiopulmonary/mediastinal disease as compared to other races (13.6% vs. 4.2%). According to outcomes, we can categorize the patients into 3 groups: good (median overall survival >30 months: reproductive and digestive systems), intermediate (median overall survival 15-30 months: head/neck and kidney/bladder/retroperitoneum/adrenal), and poor (median overall survival <15 months: nervous system, connective/soft tissue, and bones/joints). There was no significant difference in overall survival between males and females (P =0.06). Among the races, Blacks had the worst overall survival (P =0.02; Figure).

Conclusions

This is the largest registry-based study on isolated myeloid sarcoma in the United States. Isolated myeloid sarcoma has a diverse anatomic clinical presentation and the overall survival varied significantly according to sites of presentation and racial subgroups. The results of our study may aid the prognostication of patients for treatment decision making and in the understanding of the biological differences by anatomic sites of presentation.

Table Anatomical distribution and median overall survival of isolated myeloid sarcoma Table. Anatomical distribution and median overall survival of isolated myeloid sarcoma Figure Overall survival by sex and race in isolated myeloid sarcoma Figure. Overall survival by sex and race in isolated myeloid sarcoma

Disclosures

Al-Kali: Onconova Therapeutics, Inc.: Research Funding; Celgene: Research Funding.
Genome-Wide Genotype-Based Risk Model for Survival in Core Binding Factor Acute Myeloid Leukemia Patients
Authors: Park Silvia , Hangseok Choi , Kim Hee-Je , Ahn Jae-Sook , Kim Hyeoung Joon , Kim Sung-Hyun , Mun Yeung-Chul , Jung Chul Won , Kim Dong Hwan
Published: October 2019

Introduction: The present study attempted to build a single nucleotide polymorphism (SNP)-based risk model for predicting overall survival (OS) and event free survival (EFS) in patients with core binding factor acute myeloid leukemia (CBF-AML).

Methods: Adopting genome-wide SNP array using Affymetrix SNP array 6.0, we analyzed 868,157 SNPs with respect to OS and EFS in 104 patients with CBF-AML. Significant SNPs were identified from single SNP analysis. The risk model was constructed with incorporation of six SNPs and three clinical factors (age, c-kit exon 17 mutation, and LDH) for OS, and six SNPs and three clinical factors (age, WBC, and LDH) for EFS. The model was further defined into low and high risk groups based on risk scores.

Results: The median age was 39 years, and the subgroup of t(8;21) and inv(16) or t(16;16) was assessed in 68 (65.4%) and 36 patients (34.6%). Finally 6 SNPs per each OS (rs4353685, rs4908185, rs7709207, rs12034, rs1554844, and rs17241868) and EFS (rs13385610, rs11210617, rs11169282, rs7709207, rs4438401 and rs16894846) were incorporated into the risk model. OS was significantly different in favor of the low risk group (80.4%) compared to the high risk group (22.0% at 3 years; p= 8.75 x 10-13; HR 8.67). For EFS, there was also a significant difference between the low (75.0%) versus high risk group (17.1% at 3 years; p=5.95 x 10-13; HR 7.67).

Conclusion: A genome-wide SNP based risk model can stratify CBF-AML patients according to their OS and EFS in 104 patients.

Figure 1 Overall survival and event free survival by risk model composed of SNPs and clinical risk factors Figure 1. Overall survival and event free survival by risk model composed of SNPs and clinical risk factors Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
The Macrophage-Specific CD163 Is an Endocytic Receptor for Von Willebrand Factor (VWF) Cleaving Protease ADAMTS13
Authors: Verbij Fabian , Sorvillo Nicoletta , Kaijen Paul , Hrdinova Johana , Fijnheer Rob , ten Brinke Anja , Meijer Alexander B. , van Alphen Floris P. , van der Berg Timo K. , Graversen Jonas H. , Moestrup Soren K. , Voorberg Jan
Published: October 2019

Von Willebrand Factor (VWF) cleaving protease ADAMTS13 is responsible for proteolysis of ultra large VWF multimers in the circulation. ADAMTS13 is synthesized by hepatic stellate cells in the liver. Also endothelial cells have been suggested to contribute to the synthesis of ADAMTS13. In patients with acquired thrombotic thrombocytopenic purpura auto-reactive antibodies are formed primarily against the spacer domain of ADAMTS13. Previously we have shown that monocyte-derived dendritic cells were able to endocytose ADAMTS13 via the macrophage mannose receptor and subsequently process it into peptides and present it on MHC class II. However, it is currently unclear which receptor contributes to the clearance of ADAMTS13 from the circulation. The half-life of ADAMTS13 was measured following plasma infusion in patients with congenital TTP and was found to vary between 2.1 and 3.3 days.

Internalization of ADAMTS13 by tissue-resident macrophages may contribute to its clearance from the circulation. Here we investigated endocytic mechanisms contributing to the uptake of ADAMTS13 by macrophages. Human monocyte-derived macrophages (MDMs) were used to monitor the uptake of fluorescently labelled recombinant ADAMTS13 by flow cytometry. Internalization of ADAMTS13 was blocked upon addition of the cell-permeable dynamin-inhibitor dynasore. Partial blockage of ADAMTS13 internalization was observed employing mannan; uptake however was not affected by a blocking antibody directed towards the macrophage mannose receptor CD206. A pull-down with ADAMTS13 and subsequent mass spectrometric analysis identified the hemoglobin scavenger receptor CD163 as a candidate receptor for ADAMTS13. CD163 is primarily expressed by the monocyte-macrophage lineage and is highly expressed on type-2 macrophages present in the bone marrow, the red pulp of the spleen and in the liver on Kupffer cells. Blocking experiments with a monoclonal anti-CD163 antibody EDHu-1 resulted in a decreased ADAMTS13 internalization by macrophages. Pronounced inhibition of ADAMTS13 uptake by EDHu-1 was observed in macrophages in which the expression of CD163 was boosted upon incubation with IL-10. In agreement with these findings CD163-expressing CHO cells but not CHO CD163-/- cells were capable of rapidly internalizing ADAMTS13. Surface plasmon resonance revealed high affinity binding of ADAMTS13 to soluble CD163 containing scavenger receptor cysteine-rich (SRCR) domain 1-9. Our results position CD163 as a novel binding partner for ADAMTS13 and suggest that binding of ADAMTS13 to CD163 promotes its internalization by macrophages.

Disclosures

No relevant conflicts of interest to declare.
Cyclin D2 Overexpression Recapitulates Mantle Cell Lymphoma in Mice
Authors: Pieters Tim , Goossens Steven , T'Sas Sara , Berx Geert , Haigh Jody J. , Vlierberghe Pieter Van
Published: October 2019

Mantle cell lymphoma (MCL) is a highly aggressive subtype of B-cell lymphoma that is characterized by a poor response to current treatment regimens. Most MCLs carry a prototypical translocation, t(11;14), which juxtaposes the CCND1 gene towards the immunoglobulin heavy chain (IGH) locus, resulting in cyclin D1 overexpression. Strikingly, MCL has not been recapitulated in transgenic mouse models of Ccnd1 overexpression alone. Notably, a subset of MCL patients are cyclin D1 negative but instead overexpress cyclin D2 (encoded by CCND2)as a consequence of recurrent genomic rearrangements involving the CCND2 locus.

Here, we developed a novel conditional ROSA26-driven Ccnd2 overexpression mouse model and showed that hematopoietic-specific Ccnd2 activation is sufficient to drive MCL formation in mice. Starting from 36 weeks, these mice develop huge B-cell lymphomas and these tumor cells have the typical MCL morphology, are Sox11 positive and disseminate into other organs, all typical features of MCL. In addition, preliminary shallow sequencing analysis revealed a somatic Crlf2 deletion as a cooperative genetic event in one of the murine Ccnd2-driven MCL tumors.

In this study, we want to further validate and characterize this novel Ccnd2-driven mouse model for MCL and test putative synergisms between Ccnd2 overexpression and other recurrent cooperating genetic lesions that occur in human MCL, such as loss of p53 or SOX11 activation. Noteworthy, the MCL cells from this mouse model also contain a luciferase reporter, allowing accurate in vivo tracing of tumor cells in xenograft experiments. These xenograft experiments can be used as preclinical models, in which bioluminescence is used to asses the tumor burden and to monitor tumor regression upon drug treatment. In conclusion, we have developed a novel mouse model in which Ccnd2 overexpression faithfully recapitulates MCL and this model will allow us to understand the molecular mechanisms that drive MCL and identify and test novel drugs to treat this aggressive and until now incurable disease.

Disclosures

No relevant conflicts of interest to declare.
AFM13 Is the Most Advanced Bispecific NK-Cell Engaging Antibody in Clinical Development Substantially Enhancing NK-Cell Effector Function and Proliferation
Authors: Pahl Jens , Reusch Uwe , Gantke Thorsten , Kerber Anne , Koch Joachim , Treder Martin , Cerwenka Adelheid
Published: October 2019

Introduction:

AFM13 is an NK-cell engaging CD30/CD16A bispecific tetravalent TandAb antibody currently in phase 2 clinical development in Hodgkin lymphoma (HL) and other CD30+ malignancies. It engages NK-cells through CD16A with high affinity and specificity and confers significantly stronger NK-cell activation compared to other therapeutic antibodies. We have previously shown synergistic efficacy when NK-cell activation by AFM13 is combined with check-point modulation such as anti-PD-1 treatment, which is known to unleash T cell and NK-cell activity. The goal of this study was to identify further candidates for combination treatments and biomarkers that potentially indicate NK-cell responses to AFM13 treatment.

Methods:

AFM13-mediated NK-cell cytotoxicity and IFN-γ production after 4-hour interaction with HL cell lines was measured by 51Cr release assays and flow cytometry, respectively. Expression of NK-cell receptors, NK-cell proliferation (CFSE dilution) and expansion (absolute cell counts) was analyzed by flow cytometry.

Results:

The interaction of NK-cells with AFM13-coated tumor cells up-regulated the expression of NK-cell receptors such as CD25, CD69, CD137/4-1BB as well as molecules that may serve as NK-cell check-points when compared with the unrelated NK-cell binding TandAb AFM12 that does not bind to target cells. Importantly, CD16A engagement by AFM13 enhanced the proliferation and expansion potential of NK-cells when subsequently incubated with IL-15 or with particularly low doses of IL-2. NK-cell cytotoxicity and IFN-γ production was substantially increased towards CD30+ tumor cells in the presence of AFM13. Even target cells resistant to naïve and IL-2/IL-15-activated NK-cells were susceptible to AFM13-induced NK-cell cytotoxicity. AFM13 concentrations of as low as 10-2 µg/mL resulted in maximal activity while AFM13 was significantly more potent than native anti-CD30 IgG1 antibody. NK-cell activation by IL-2 or IL-15 had a synergistic effect on AFM13-mediated cytotoxicity.

Conclusion:

AFM13 specifically enhances the cytotoxic, proliferative and cytokine-producing potential of NK-cells. Our data indicate that the distinctive modulation of NK-cell receptors can be utilized to monitor NK-cell responses during AFM13 therapy and provides candidates for therapeutic combination strategies. Moreover, the combination with low doses of IL-2 or with IL-15 may expand the quantity of tumor-reactive NK-cells after AFM13 treatment and promote NK-cell functionality in the tumor microenvironment in cancer patients.

Disclosures

Reusch: Affimed: Employment, Patents & Royalties: Patents. Gantke:Affimed GmbH: Employment. Kerber:Affimed: Employment. Koch:Affimed: Employment. Treder:Affimed: Employment. Cerwenka:Affimed: Research Funding.
Arginase 1 Is a Marker of Myeloid-Mediated Immunosuppression with Prognostic Meaning in Classic Hodgkin Lymphoma
Authors: Romano Alessandra , Parrinello Nunziatina L , Tibullo Daniele , Vetro Calogero , Giallongo Cesarina , La Cava Piera , Chiarenza Annalisa , Villari Loredana , Tripodo Claudio , Motta Giovanna , Caruso Laura , Gallamini Andrea , Pileri Stefano A , Di Raimondo Francesco
Published: October 2019

Purpose : Neutrophilia is hallmark of classic Hodgkin Lymphoma (cHL), but its precise characterization remains elusive. We aimed at investigating the immunosuppressive role of high-density neutrophils in HL.

Experimental design : First, N-HL function was evaluated in vitro, showing increased arginase (Arg-1) expression and activity compared to healthy subjects. Second, we measured serum level of Arg-1 (s-Arg-1) by ELISA in two independent, training (N=40) and validation (N=78) sets.

Results : s-Arg-1 was higher in patients with advanced stage (p=0.045), B-symptoms (p=0.0048) and a positive FDG-PET scan after two cycles of chemotherapy (PET-2, p=0.012). Baseline levels of s-Arg-1 >200 ng/mL resulted in 92% sensitivity and 56% specificity to predict a positive PET-2.

Patients showing s-Arg-1 levels > 200 ng/mL had a shorter progression free survival (PFS). In multivariate analysis, PET-2 and s-Arg-1 at diagnosis were the only statistically significant prognostic variables related to PFS (respectively p=0.0004 and p=0.012).

Moving from PET-2 status and s-Arg-1 level we constructed a prognostic score to predict long-term treatment outcome: low s-Arg-1 and negative PET-2 scan (score 0, N=63), with a 3-Y PFS of 89.5%; either positive PET-2 or high s-Arg-1 (score 1, N=46) with 3-Y PFS of 67.6%, and both positive markers (score 2, N=9) with a 3-Y PFS of 37% (p=0.0004).

Conclusions : We conclude that N-HL are immunosuppressive through increased Arg-1 expression, a potential novel potential biomarker for HL prognosis.

Disclosures

Vetro: MLL Munich Leukemia Laboratory: Employment. Chiarenza:Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy.