bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural and Functional Studies Of The Intrinsically Disordered Protein AF9 In MLL-AF9 Leukemia
Authors: Kuntimaddi Aravinda , Lokken Alyson , Zhang Shubin , Thorpe Jeremy , Leach Benjamin , Lumba Bhavna , Hemenway Charles S. , Adli Mazhar , Zeleznik-Le Nancy J. , Bushweller John H. 
Published: October 2019

Abstract
Mixed lineage leukemias are characterized by the creation of a chimeric fusion protein where MLL is fused in frame to over 60 different fusion partners, leading to the disruption of HOX gene regulation. AF9 is one of the most common MLL fusion partners, and MLL-AF9 leukemia is acute and aggressive with a poor overall prognosis. The mechanism by which AF9 regulates normal transcription and contributes to dysregulated transcription is poorly understood. We have shown that the C-terminal domain of AF9 binds to four different proteins, two of which (Dot1L – an H3K79 methyltransferase and AF4 – which recruits P-TEFb), are transcriptional activators, whereas the other two (CBX8 – which is a part of the PRC1 repressive complex and BCOR- BCL6 corepressor), are generally transcriptional repressors, suggesting that AF9 acts as a protein signaling hub. We have previously shown that the C-terminal domain of AF9 is an intrinsically disordered protein (IDP) meaning that it is unstructured on its own but undergoes coupled folding and binding upon interacting with its binding partners1. Our goal is to use structure-function studies to further understand AF9 protein interactions and to elucidate which of these interactions with AF9 are critical for MLL-AF9 leukemogenesis.
We have determined NMR solution structures of complexes of Dot1L and CBX8 with AF9. Both Dot1L and CBX8 complexes form mixed alpha-beta structures, similar to that of our previously solved AF4-AF9 complex structure where AF4, Dot1L, and CBX8 all have a consensus LXVXIXL sequence and form a ß-strand and an extended ß-turn. Intriguingly, Dot1L contains three separate binding motifs for AF9 interaction, including one repeat motif separated by seven amino acids. Our 15N-1H HSQC NMR experiments show that each of these three binding sites on Dot1L adopts a similar fold with AF9 yet has vastly different binding affinities with AF9. We have created several structure-guided mutations on AF9 that differentially block interactions of its binding partners. A single charge reversal mutation on AF9 that blocks all protein interactions with AF9 was introduced into MLL-AF9 in vitro colony forming assays, and was able to abrogate the serial replating capacity of MLL-AF9. Other mutations that selectively block the interaction of various proteins with AF9, show differential effects in colony forming assays and HOX gene expression suggesting the importance of certain AF9 protein interactions in MLL-AF9 leukemogenesis.
1. Leach, B. I. et al. Leukemia fusion target AF9 is an intrinsically disordered transcriptional regulator that recruits multiple partners via coupled folding and binding. Structure21, 176-183, doi:10.1016/j.str.2012.11.011 (2013).

Disclosures:
No relevant conflicts of interest to declare.
Heatshockproteins-60 and 90 Are Overexpressed In Leukemia Stem Cells and Stabilize Oncogene Signaling
Authors: Kampa-Schittenhelm Kerstin M , Grandl Tanja , Akmut Figen , Illing Barbara , Schittenhelm Marcus M 
Published: October 2019

Abstract

Introduction
Activating mutations of the KIT class III receptor tyrosine kinase (TK) are associated with the pathophysiology of acute leukemia, especially core binding factor leukemia (CBFL), and systemic mastocytosis (SM). Despite considerable antiproliferative and proapoptotic activity of several KIT TK inhibitors in vitro, clinical efficacy in AML and SM is generally moderate. We hypothesized that resistance to therapy is promoted by activation of alternative signaling pathways. Previously we reported that KIT TK inhibition results in significantly increased phosphorylation of heat shock protein (HSP) family members and that KIT is a client protein of phosphorylated (p)HSP90 putatively stabilizing KIT protein function in the presence of KIT TK-inhibitors (Kampa-Schittenhelm et al., ASH annual meeting 2010). This prompted us to further test the HSP inhibitor IPI-504 in KIT dependent CBFL, including the leukemic stem cell fraction.

Methods
Protein expression levels of (p)HSPs in leukemic blasts of high-risk and CBFL patients were studied by flow cytometry focusing on the CD34+/CD38- leukemia stem/progenitor cell fraction. Cellular proliferation and induction of apoptosis in leukemia cells treated with the HSP90 inhibitor IPI-504 was determined by XTT- and annexin V-based assays.

Results
(p)HSP90/60 levels were preferentially upregulated in CBFL associating with KIT dysregulation. Consequently, HSP90 inhibition with IPI-504 potently degraded KIT expression causing a direct antiproliferative and antiapoptotic effect in CBFL in in vitro and ex vivo models. Efficacy of IPI-504 was potentiated when combined with TK inhibitors. Importantly, high expression of (p)HSP90 and HSP60 was particularly observed in the CD34+/CD38- putative leukemia stem cell fraction arguing for a function as protection mechanism to conserve (leukemic) progenitor cell function upon cell stress such as antileukemic treatment.

Conclusion
HSPs are upregulated in CBFL, and IPI-504 induces antiproliferative and proapoptotic effects in primary leukemia samples. Importantly, in particular the putative malignant progenitor cell pool in KIT-associated acute leukemia expresses high levels of (p)HSP90, identifying HSP90 inhibition as an attractive novel strategy to overcome the therapy-refractory behavior of malignant stem/progenitor cells. Our results provide a rationale for the evaluation of HSP90 inhibitors such as IPI-504 in CBFL.

Disclosures:
No relevant conflicts of interest to declare.
A HoxD11 Homeobox Gene Mutation Associated With Congenital Absent Radius But Without The Thrombocytopenia Of The TAR Syndrome Or a HoxA11 Mutation
Authors: Fleischman Roger A. 
Published: October 2019

HoxA11 and HoxD11 are homeobox genes critical for normal development of the forearm and thus are potential candidate genes for involvement in the pathogenesis of the thrombocytopenia/absent radius (TAR) syndrome. However, we previously reported an absence of coding sequence mutations in either HoxA11 or HoxD11 in a series of 10 unrelated TAR syndrome patients (Fleischman RA et al., Br J Haematol., 116:367-75, 2002). Despite this negative finding, interest in the potential role of homeobox genes in the TAR syndrome has been supported by a report of a HoxA11 mutation occurring in two kindreds with amegakaryocytic thrombocytopenia and radio-ulnar synostosis, a less pronounced more proximal pattern of radial malformation (Thompson AA and Nguyen LT. Nat Genet., 26:397-8, 2000). Unlike HoxA11, however, no mutations in the human HoxD11 gene have been described thus far that would help elucidate the potential role of this paralogous gene in megakaryopoiesis or the TAR syndrome.
We now describe a novel mutation in human HoxD11 that results in a polyalanine sequence expansion, (GCG)6→ (GCG)8, and report that this mutation is associated with a unilateral absent radius in the affected propositus. A familial syndrome is suggested in this kindred, moreover, by the prior observation of a bilateral absent radius in a deceased maternal aunt. This mutation was not present in more than 100 unrelated normal subjects or 8 other unrelated individuals with sporadic absence of the radius. Two other living maternal relatives also carried the mutation but did not exhibit any radial defects, a finding consistent with autosomal dominance with incomplete penetrance, an inheritance pattern reported for short polyalanine expansion mutations in the related homeobox gene HoxD13 which cause synpolydactyly. In contrast to the reported HoxA11 mutation, however, neither the propositus nor the mutation carriers of this HoxD11 mutation exhibited thrombocytopenia or any other cytopenias or congenital defect. The results suggest that at least one class of mutation in human HoxD11 may be sufficient to cause an absent radius syndrome but unlike the reported HoxA11 mutation, does not adversely affect megakaryopoiesis. The findings further suggest that additional studies of the TAR syndrome may be necessary to exclude as yet undetected non-coding mutations in promoter or enhancer sequences that alter the expression of HoxA11, HoxD11 or other homeobox genes critical for radial development and/or megakaryopoiesis.
This work was supported by a VA Merit Award.

Disclosures:
No relevant conflicts of interest to declare.
X-Reactivation Impacts Human iPSC Differentiation Potential Towards Blood
Authors: Moraghebi Roksana , Kirkeby Agnete , Larsson Marcus , Herbst Andreas , Parmar Malin , Soneji Shamit , Woods Niels-Bjarne 
Published: October 2019

Abstract
To determine novel key regulators that direct ES/iPS cell differentiation to hematopoietic lineages, we compared the gene expression profiles of multiple iPS cell lines with defferential blood forming capacity. We generated multiple iPS cell lines from amniotic fluid derived mesenchymal stromal cells (AF-iPS) which differentiated towards hematopoietic lineages using our standardized and highly reproducible differentiation protocol. Of the 9 AF-iPS cell lines derived from an individual female patient, the average efficiency of CD45+ hematopoietic cells was 14.2 +/- 9% (range 1.6 to 26.3%).
To elucidate the possible reasons for this diversity in efficiency, we grouped the AF-iPS cell lines on the basis of lowest and highest blood differentiation capacity and compared their gene expression profiles by microarray. We found very few changes above 1.5-fold, but interestingly, among the 11 genes that were over-expressed in the AF-iPSC lines with poor blood differentiation efficiency 10 were located on X chromosome, and the remaining one reported to be involved in Notch signalling.
A combination of cumulative sum analysis and the location of differentially expressed genes on the X chromosome identified putative regions of reactivation at multiple, but distinct locations. The possibility of X-reactivation in these female lines was reinforced further where lower levels of XIST were seen in AF-iPSC lines shown to have low blood forming potential, however only half of the iPS cell lines with high blood differentiation capacity showed normal XIST expression when compared to the amniotic fluid mesenchymal starting cell material.
To determine whether the block in differentiation was tissue specific we tested the differentiation capacity of the AF-iPSC lines towards neuronal lineages. Intriguingly we found neural cell differentiation was not hampered within all lines with poor blood potential suggesting that the over-expression of genes as a consequence of X-reactivation can impart a specific negative effect on differentiation towards the blood lineages from pluripotency stage, while not having an effect on neuronal cell development.
To further define the source of this block, we have begun working knocking down the overexpressed genes on X chromosome in lines with poor blood differentiation potential to determine whether the efficiency can be increased (or fully rescued) with one, or a combination of these 11 candidate genes. These results have implications for the identification and selection of female iPS lines suitable for therapeutic purposes.

Disclosures:
No relevant conflicts of interest to declare.
In Vitro Generation Of Osteoclasts From Interleukin-3 (IL-3)-Dependent Mouse Hematopoietic Cells
Authors: Wang Shunqing , Hong Huixian 
Published: October 2019

Abstract

Objective
This work aims to develop a new strategy to generate murine osteoclasts in vitro using IL-3-dependent cells prepared by 6-day IL-3 treatment of murine bone marrow cells

Methods
1. Here, we describe an alternative method for in vitro generation of osteoclasts, which involves the use of interleukin (IL)-3-dependent murine bone marrow cells. Bone marrow cells, isolated from 6- to 8-week old C57BL/6 were cultured in α-MEM containing 10% FBS in tissue culture dishes overnight to remove stromal cells. Then, non-adherent bone marrow cells were harvested and continued in α-MEM containing 10% FBS without (control) or with IL-3 (1 ng/ml) for 6 days. While no cells survived in the control culture after the 6-day culturing, the IL-3-treated culture gave rise to a significant number of surviving cells. These IL-3-depedent cells were capable of differentiating to osteoclasts in response to M-CSF and RANKL stimulation. Moreover, these IL-3-dependent cells can be further expanded by plating them in non-treated plastic dishes followed with M-CSF treatment; they continued to survive and proliferate in non-treated plastic dishes in the presence of M-CSF for up to 4 days. After 4-day M-CSF treatment, these cells can be lifted by EDTA, and they were still able to differentiate into osteoclasts upon subsequent stimulation of M-CSF and RANKL.
2. We performed the in vitro bone resorption assay, Semiquantitative Reverse Transcription (RT)-PCR, Western Analysis, Infection of Murine Bone Marrow Cells (BMCs) to test whether the osteoclasts generated from IL-3-dependent murine bone marrow cells are different from the osteoclasts generated from traditional method.

Results
1. IL-3 can maintain the survival of murine bone marrow cells for up to 6 days and these cells still keep their capacity to generate osteoclasts. The capacity of IL-3-dependent cells to form osteoclasts decreases with time of IL-3 treatment and IL-3 dependent cells can be further expanded by M-CSF without significant loss of the osteoclastogenic potential.
2. IL-3-dependent cells can form functional osteoclasts. RANKL induces the expression of osteoclast genes in IL-3-dependent cells. RANKL activates some of RANK signaling pathways in IL-3-dependent cells. Importantly, we found that IL-3 dependent murine bone marrow cells can be infected by retrovirus encoding GFP.

Conclusions
1) We have developed a new strategy to generate murine osteoclasts in vitro using IL-3-dependent cells prepared by 6-day IL-3 treatment of murine bone marrow cells.
2) IL-3-dependent cells can be infected by retrovirus, permitting further experimental manipulations to express or knock down genes in IL-3-dependent cells for studying the molecular mechanism controlling differentiation and proliferation of osteoclast precursors or delineating molecular events in early osteoclastogenesis.

Disclosures:
No relevant conflicts of interest to declare.
Methylene Blue Selectively Inhibits B Cell Development and E2A Activity
Authors: Soans Eroica , Choi John K 
Published: October 2019

Abstract

Introduction
E2A, a basic helix loop helix transcription factor, is essential for B lymphocyte proliferation, survival and differentiation. Mutations in E2A are associated with B cell leukemia and lymphomas. Our preliminary data suggests that E2A activity is altered by HSP70 and since HSP70 ATPase activity is inhibited by methylene blue (MB), we hypothesized that MB will alter E2A activity and thus alter B cell development.

Methods
We studied the effects of MB on normal and leukemic B cells compared to its effect on myeloid cells. The cell lines used were REH, NALM-6 and BAF3-p185 cells compared to myeloid cell lines, CCRF and CMK for the proliferation assay. To study MB effect on normal B and myeloid progenitors, methylcellulose colony formation assays with murine bone marrow cells (strain 129S1/SVImJ, 8-12 weeks old mice) were performed. We also treated mice with MB in their drinking water (1000 μM) up to 16 weeks to study the in-vivo effect on B cells as compared to granulocyte, macrophages, T cells and NK cells in the peripheral blood by flow cytometry analysis. To understand the mechanisms by which MB alters B cell proliferation, we examined the effects of MB on cell cycle progression, apoptosis and E2A activity in B cells. For cell progression NALM-6 cells were treated with 4 μM MB for 0, 6, 12 and 24 hrs and assayed using BRDU staining by flow cytometry analysis. Further, apoptosis was measured using Annexin V staining by flow cytometry analysis in NALM-6 cells up to 48hrs post treatment with MB. Lastly, E2A activity was measured using an E2A-specific luciferase reporter assay in 293T cells.

Results
MB decreased the proliferation of precursor B cell lines, REH, NALM-6 and BAF3-p185 cells compared to myeloid cell lines, CCRF and CMK. Similar effects of MB were also seen with normal B and myeloid progenitors as assayed by methylcellulose colony formation assays with murine bone marrow cells. Similarly, there is a significant decrease in peripheral B cells as compared to granulocytes, macrophages, T cells and NK cells when the mice were treated with 1000 μM of methylene blue in their drinking water as compared to those that remained untreated. MB caused apoptosis and decreased the S phase fraction of treated NALM-6 cells. In addition, we found that MB inhibited E2A activity using an E2A-specific luciferase reporter assay.

Conclusion
Our findings suggest that MB is a specific inhibitor of B cell proliferation, survival and development. This effect may be mediated by the inhibition of E2A activity by methylene blue.

Disclosures:
No relevant conflicts of interest to declare.
Genetic Modifiers Of Beta-Thalassaemia Associated With Management Without Blood Transfusion: Ten Years Experience
Authors: Ansari Saqib Hussain , Shamsi Tahir S , Khan Mohammed Tahir , Munzir Saima , Erum Sajida , Perveen Kousar , Parveen Sadia , Farzana Tasneem , Ashraf Mushtaq 
Published: October 2019

Abstract

Background
Long survival with improve quality life in treatment of thalassaemia patients can be procure by blood transfusion and sedulous chelation but, it is cumbrous. Therapeutic manoeuvers designed to stabilize or increase hemoglobin without transfusion and to reduce the blood transfusion. Efforts have been undertaken to find an alternative approach to transfusion in β-thalassaemia.

Methods
On the basis of our previous studies evaluating the safety & efficacy of Hydroxyurea (HU) in beta thalassemia patients which is an oral Hb-F augmentation agent, at 10-15 mg/kg/day was used on 473 patients for Mean duration of 3 years under the guidelines of Helsinki’s declaration. Response was measured by using transfusion requirement prior to 6 months period of enrollment in the study as control. Patients were finally divided into Complete Responders (CR), partial responders (PR) and non-responders(NR).Patients’ genetic profile was investigated. Chi square test was applied to check association between variables, p-value ≤0.05 considered significant.

Results
Complete responders were 176, PR were 194 while 103 were NR. Most common genetic mutations observed were IVS1-5 (46%) either homozygous or heterozygous, Fr 8-9 (14.3%), IVS1-1 (11%), Cd 30 (5.5%), Fr 41-42 (5.5%), Del-619 (5%) &Cap+1 (1.9%). Astounding results were found when association of these mutations with response of HU was observed. Response was found to be closely related with gene mutations Cd-30 (61.5 % Responders), IVS1-1 (58 % Responders), Cap+1 (44.4 % Responders), IVS1-5 (33.6 % Responders). Homozygous Xmn-I polymorphism was observed in 21% & heterozygous in 24% of the patients, while 55 % had no Xmn-I polymorphism. Xmn-I polymorphism was found to be significantly associated with Response 67 % (p-value 0.00).

Conclusion
Hydroxyurea showed promising results in this long term follow up of large cohort of patients. We suggest screening all newly diagnosed thalassaemic children for Genetic characteristic in order to offer Hb-F Augmenting agent as an alternative to blood transfusion.

Disclosures:
No relevant conflicts of interest to declare.
Distinct Chemokine Receptor Profile In Chronic Lymphocytic Leukaemia and Richter Transformed Diffuse Large B Cell Lymphomas Compared To Germinal Center B Cells and De Novo Diffuse Large B Cell Lymphomas
Authors: Wenzl Kerstin , Troppan Katharina , Deutsch Alexander JA , Linkesch Werner , Neumeister Peter , Beham-Schmid Christine 
Published: October 2019

Abstract
Chemokine receptors are G-protein-coupled cell surface receptors, which dissociate upon activation by their ligands and cause downstream signaling. Recently, several studies have revealed the crucial contribution of chemokine receptors and their ligands in normal B-cell differentiation and development of hematopoietic malignancies. The Richter syndrome (RS) represents the clinico-pathologic transformation of chronic lymphocytic leukaemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). Due to the lack of knowledge on the chemokine receptors in RS, we aimed to investigate their expression profile in Richter syndrome patients. Therefore, we investigated the mRNA expression levels of 18 known chemokine receptors (CCR1-CCR9, CXCR1-CXCR7, XCR1, CX3CR1) by using semi-quantitative real time PCR on seven samples of paired (CLL and transformed DLBCL) RS samples, and six samples of de novo DLBCL, additionally four CLL samples –all of them transformed into DLBCL-, and eight transformed DLBCL samples –all of them originated from CLL- were included. Four samples of peripheral B cells and four samples of germinal center B cells (GCB) served as non-neoplastic controls.
11 out of 18 chemokine receptors were differentially expressed in CLL (RL) and transformed DLBCL originated from CLL (RH) compared to GCBs. RL exhibited an at least 15-fold higher expression of CCR2, CCR4, CCR7, CXCR3, and XCR1, as well as a de novo expression of CCR3, CCR8, CXCR1, CXCR2, and CX3CR1 compared to GCB. Additionally to these chemokine receptors, CCR1 also showed significant higher expression levels comparing GCB and RH samples. Only one chemokine receptor was found to be differentially expressed in our seven paired RS samples: CCR6 showed a trend of up-regulation in CLL components. Interestingly, the transformed DLBCL originated from CLL were characterized by a significant up-regulation of six chemokine receptors (CCR3, CCR7, CXCR1, CXCR3, CXCR4, and CX3CR1) and a down-regulation of CCR5 compared to DLBCL.
Our data indicate that the chemokine receptor expression profile of CLL and transformed DLBCL, originated from CLL samples, differs substantially from those of non neoplastic germinal center B cells and de novo DLBCL, suggesting other cellular origin and an impact on more aggressive clinical course of Richter syndrome patients. Hence, these multiple deregulated CC and CXC receptor might serve as useful prognostic tool to separate high malignant Richter syndrome from de novo DLBCL.

Disclosures:
No relevant conflicts of interest to declare.