bluuebunny/crossref_metadata_2025_split huggingface dataset:
Therapeutic Targeting of the Histone Ubiquitination-Methylation Axis in T Cell Leukemia
Authors: Ntziachristos Panagiotis , Arcipowski Kelly , Martinez Carlos Alberto , Zhu Yixing , Volk Andrew G. , Kumar Suresh , Thomas Paul M , Van Vlierberghe Pieter , Hijiya Nobuko , Marshall Stacy , Wang Jack , Wu Jian , Kelleher Neil L , Crispino John 
Published: October 2019

Abstract
Acute lymphoblastic leukemia (ALL) is a highly aggressive blood cancer affecting children and adults. Certain high-risk disease subsets have poor outcomes and often debilitating therapy-related toxicities stemming from direct inhibition of the oncogenes. We hypothesize that the process of oncogenic transformation is driven by aberrant activity of oncogene-associated chromatin modifying partners. These changes create a chromatin environment unique to the malignant state and, therefore, disruption of critical oncogenic chromatin signatures would not likely affect healthy tissues. We have generated strong evidence for the intertwined roles between the NOTCH1 oncogenic pathway and deubiquitinase enzymes in T cell leukemia, members of the ubiquitin-specific proteases (USP) family in particular. Members of the USP family physically interact with NOTCH1 and the lysine 27 on histone H3 (H3K27) demethylase JMJD3 and this methylation-ubiquitination biology-related axis coordinates regulation of transcriptional initiation and elongation, vital for the survival of leukemia cells. Interestingly transcription of USP genes is positively controlled by NOTCH1 creating a feedback loop in leukemia.
We have further characterized this oncogenic axis using a combination of small molecule inhibitors and genetic engineering of USPs in ALL cell lines, primary patient samples and primagraft models of disease. We are able to show that a) USP activity is important for certain oncogenic pathways (such as NOTCH1) in leukemia; b) Oncogenes and USP enzymes co-bind certain areas in the leukemia genome; c) Ubiquitination of histone H2B acts in a combinatorial fashion with H3K27me, is a major epigenetic change affected by the USP activity in leukemia and controls d) transcriptional elongation. Finally we demonstrate that f) chemical inhibition or down-regulation of USPs affect leukemia growth in vitro and in vivo. Ongoing and future studies include manipulation of USP levels in mouse models of leukemia as well as combinatorial use of USP inhibitors with chemotherapeutic regiments in vitro using matched diagnosis-relapsed primary samples and in xenograft studies.
Information gained from these studies will lend rationale towards the use of small molecule inhibitors against USP proteins in clinical trials for the treatment of aggressive and relapsed ALL.

Disclosures
Kumar: Progenra Inc.: Employment. Wang:Progenra Inc.: Employment. Wu:Progenra Inc.: Employment.
The miRNA-193 Family Is a Potent Tumor-Suppressor and a Biomarker for Poor Prognosis in Acute Myeloid Leukemia
Authors: Jammal Razan , Krowiorz Kathrin , Haetscher Nadine , Emmrich Stephan , Rouhi Arefeh , Heuser Michael , Bothur Sabrina , Bullinger Lars , Döhner Konstanze , Lai Courteney , Maetzig Tobias , Obulkasim Askar , Martins Vera , Mulaw Medhanie A. , Stary Jan , Baruchel Andre , De Haas Valerie , Reinhardt Dirk , Döhner Hartmut , Ruschmann Jens , Zwaan Michel , van den Heuvel-Eibrink Marry , Heckl Dirk , Fornerod Maarten , Humphries R. Keith , Rieger Michael A , Kuchenbauer Florian , Klusmann Jan-Henning 
Published: October 2019

Abstract
Deregulated microRNA (miRNA) expression has been implicated in the pathogenesis of acute myeloid leukemia (AML). We previously showed that miR-193b is a STAT5-regulated miRNA that controls hematopoietic stem and progenitor cell (HSPC) expansion by modulating cytokine receptor signaling. Here we demonstrate that the miR-193 family members miR-193a and 193b are potent tumor suppressors in AML. Both miRNAs were downregulated in several cytogenetically-defined subgroups of pediatric and adult AML (n=202), whereas low miR-193b expression was an independent indicator for poor prognosis and survival. Accordingly, ectopic retroviral Hoxa9-Meis1 expression in HSPCs from miR-193b-/- mice resulted in a more aggressive disease with significantly shortened latency and survival as compared to miR-193bWT/WT HSPCs. Inversely, ectopic miR-193 expression in leukemic cells belonging to various AML subgroups decreased leukemic growth in vitro and prolonged survival of mice suffering from Hoxa9-Meis1-induced leukemia through a G1/S phase block. These effects were mediated by targeting c-KIT, KRAS and SOS2 - key factors of the KIT-RAS-RAF-MEK-ERK signaling cascade - as well as the downstream cell cycle regulator CCND1. Knockdown of each of these genes partially recapitulated the anti-proliferative effect of ectopic lentiviral miR-193 expression. As the tumor suppressive function is independent of patient age or AML cytogenetic background, these observations suggest an opportunistic role for miR-193 in future AML therapies. With the notion that a single miRNA can control aberrant MAPK signaling at multiple levels, restoring miR-193 expression in AML cells with constitutive activation of this cascade would assure high antileukemic efficacy, while avoiding the fast development of resistance mechanisms.

Disclosures
Heuser: Bayer Pharma AG: Research Funding; Novartis: Consultancy, Research Funding; BerGenBio: Research Funding; Tetralogic: Research Funding; Karyopharm Therapeutics Inc: Research Funding; Celgene: Honoraria; Pfizer: Research Funding. Mulaw:NuGEN: Honoraria. Baruchel:Jazz: Consultancy; Servier: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Baxalta: Research Funding.
Microrna-494 Activation Suppresses Bone Marrow Stromal Cell-Mediated Drug Resistance in Acute Myeloid Leukemia Cells
Authors: Tian Chen , Zheng Guoguang , You M. James , Zhang Yizhuo 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) is not sensitive to chemotherapy partially because of the protection of AML cells by mesenchymal stromal cells (MSCs). Our previous studies found that MSCs protected AML cells from apoptosis through the c-Myc-dependent pathway. However, the mechanism by which MSCs regulate c-Myc in AML cells is still unknown. To elucidate the mechanism, we performed microRNA array analysis of AML cell lines and validated by TaqMan realtime PCR. The results showed that the expression of microRNA-494 (miR-494) in AML cells after coculture with MSCs was down-regulated. Reporter gene analysis confirmed miR-494 as one of the regulators of c-Myc. In the coculture system, activation of miR-494 in AML cells suppressed proliferation and induced apoptosis of AML cells in vitro. After addition of mitoxantrone to the coculture system, the proliferation of AML cells with miR-494 activation was suppressed more than that of control cells. After subcutaneous injection of AML cell lines in combination with MSC, tumor growth was suppressed in mice injected with miR-494-overexpressing AML cells. The rate of tumor formation was even lower after mitoxantrone treatment in the miR-494 overexpressing group. Moreover, miR-494 activation resulted in a decrease of leukemic cell counts in peripheral blood and bone marrow and prolonged survival in mice injected with miR-494-overexpressing AML cellls and MSCs compared to the control mice. Our results indicate that miR-494 suppresses drug resistance in AML cells by down-regulating c-Myc through interaction with MSCs and that miR-494 therefore is a potential therapeutic target.

Disclosures
No relevant conflicts of interest to declare.
Altered Expression of Immune Checkpoint Molecules Including Programmed Cell Death-1 (PD-1) and Its Ligands PD-L1/PD-L2 in Waldenstrom's Macroglobulinemia
Authors: Jalali Shahrzad , Price-Troska Tammy , Novak Anne J. , Ansell Stephen M 
Published: October 2019

Abstract
Waldenstrom's Macroglobulinemia (WM) is an indolent B-cell malignancy that is characterized by bone marrow infiltration of lymphoplasmacytic cells and excessive synthesis of IgM. Despite improved treatment strategies and increased understanding of the underlying molecular mechanisms contributing to malignant disease progression, WM remains incurable. While whole exome sequencing analysis has revealed the genomic events associated with malignant B cell transformation in WM, the bone marrow (BM) microenvironment is also emerging as a major player in the pathogenesis of WM. Previously, our lab has reported that an array of cytokines, including IL-21 and IL-6 are increased in the WM BM and such increases are associated with high proliferation rates and IgM secretion by WM cells. In this study, we aimed to identify how immune checkpoint molecules in the BM microenvironment, specifically programmed cell death-1 (PD-1) and its ligands PD-L1 and PD-L2, contribute to malignant cell expansion in WM.
Using Real-Time PCR analysis, we show that the expression of PD-1, PD-L1 and PD-L2 are all significantly elevated in the cells of WM BM as compared to their normal BM counterparts. Interestingly, PD-L1 expression is also upregulated in the malignant WM cells. These findings were further confirmed by flow cytometry analysis. Subsequent in vitro analysis showed that PD-L1 and PD-L2 are cleaved from the WM cells and these soluble ligands (sPD-L1 and sPD-L2) are able to bind to the cells in the BM microenvironment, including T-cells. This results in reduced activation and proliferation of T-cells, and is accompanied by a reduction in cyclin A and p-ERK levels. In validation of the in vitro data, we also found sPD-L1 and sPD-L2 in the bone marrow plasma obtained from WM patients. While sPD-L2 was found to be present in both WM and normal BM plasma (though in a reduced level in WM), sPD-L1 was exclusive to the WM samples, implying that the function of these molecules are differentially regulated in WM. Furthermore, overexpression of PD-L1 in a WM cell line, MWCL.1, resulted in an increase in IgM secretion. Interestingly, treatment with IL-21 increased the expression of PD-L1 in WM cells, indicating that elevated IL-21 in WM BM could contribute to increased expression of PD-L1 by malignant WM cells.
In summary, our data support a role for PD-1 ligands as immune regulatory molecules in WM. Further studies are ongoing to explore the molecular mechanisms by which PD-1/PD-L1/PD-L2 signaling affects the pathophysiology of WM cells.

Disclosures
No relevant conflicts of interest to declare.
Enhancer-Promoter Looping Deciphers Dosage of the Haploinsufficient Transcription Factor, CUX1
Authors: Arthur Robert K , An Ningfei , Kahn Saira , McNerney Megan E. 
Published: October 2019

Abstract
One third of tumor suppressor genes encode haploinsufficient transcriptional regulators, including transcription factors and chromatin remodelers. This presents a major barrier in oncology, as tumor suppressor genes and transcription factors are inherently difficult to target therapeutically. It remains unknown how a 50% reduction of a transcriptional regulator translates at the cis-regulatory level into a malignant transcriptional program. It is imperative to address this question, in order to predict and target aberrant downstream pathways. CUX1 encodes a quintessential haploinsufficient transcription factor that is recurrently mutated or deleted, monoallelically, in high-risk myeloid neoplasms and across solid tumor types. We hypothesized that the transcriptional response to changes in CUX1 level is encoded within the cis-regulatory architecture of dosage-sensitive CUX1 target genes. In this study, we determined that CUX1 primarily binds distal enhancers, in a tissue-specific manner. CUX1 binding sites are significantly enriched for co-binding with transcriptional activators and cohesin components. Haploinsufficiency of CUX1 in K562 myeloid leukemia cells led to altered expression of mitotic and apoptotic genes with concomitant increased cellular proliferation. Surprisingly, ChIP-seq of CUX1 in the haploinsufficient state revealed a unimodal decrease in CUX1 occupancy genome-wide, with no relevance to differential gene expression. Thus we used a machine-learning algorithm to identify characteristics of CUX1 binding sites at dosage-sensitive target genes and revealed a relationship with distance to the transcription start site, chromatin accessibility, and enhancer activity. Finally, we demonstrate that CUX1 binding sites at dosage-sensitive genes loop to the promoter, and those genes with an intermediate number of Hi-C loops are most responsive to changes in CUX1 abundance. Overall, these data point to a novel mechanism of transcription factor dose-responsiveness mediated by enhancer-promoter looping.

Disclosures
No relevant conflicts of interest to declare.
Heterotrimeric G-Proteins Are Indispensable for FLT3-ITD autophosphorylation and Oncogenic Function
Authors: Rehage Maike , Wingert Susanne , Haetscher Nadine , Bothur Sabrina , Serve Hubert , Grez Manuel , Schwaeble Joachim , Rieger Michael A 
Published: October 2019

Abstract
Heterotrimeric G-proteins transmit signals of G-protein coupled receptors and regulate many basic cellular functions. However, their role in normal and malignant hematopoietic stem cells remains obscure. Activating mutations in the heterotrimeric G-protein Gaq were found in various cancers and its expression is enhanced in diffuse large B-cell lymphoma and T-ALL. Our previous data suggested the involvement of heterotrimeric G-proteins in Flt3-ITD-mediated leukemic transformation. FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3-ITD) is a frequent oncoprotein in acute myeloid leukemia causing constitutive active STAT5 signaling. Here, we investigated a novel role of Gaq in Flt3-ITD-induced leukemic transformation.
We could show that Gaq is indispensable for aberrant FLT3-ITD activation and oncogenic function as Gaq activity is necessary to maintain the autophosphorylation of FLT3-ITD. Gaq abrogation resulted in diminished cell proliferation and colony formation as well as delayed leukemogenesis in vivo of Flt3-ITD leukemic cells. Importantly, the growth inhibition could be rescued by addition of IL3 and did not occur in the presence of FLT3 ligand-activated FLT3 wildtype receptor, demonstrating the specificity of Gaq requirement for FLT3-ITD oncogenic signaling. Interestingly, co-immunoprecipitations revealed a direct physical interaction between FLT3-ITD and Gaq which did not require phosphorylation of the receptor tyrosine kinase. Hence, FLT3-ITD hyperphosphorylation seems to be rather a consequence of the interaction than a prerequisite. Flt3-ITD-induced transformation of murine hematopoietic stem/progenitor cells (HSPCs) strictly depended on the presence of Gaq, and the ablation of Gaq/11 in transplanted Flt3-ITD-transduced HSPCs from conditional Gaq/11 double knock-out mice delayed leukemic burden. These findings of an unexpected, yet critical, role of Gaq place the molecule as an important target for antileukemic strategies.

Disclosures
No relevant conflicts of interest to declare.
Bacteremia, Not Coagulation Proteases Contribute to In Vivo Complement Activation in Sepsis
Authors: Keshari Ravi Shankar , Silasi-Mansat Robert , Lupu Cristina , Taylor Fletcher B. , Lupu Florea 
Published: October 2019

Abstract
Bacterial sepsis induces strong activation of coagulation, complement and fibrinolytic systems that contribute to disseminated intravascular coagulation, organ damage and death. While the contact of the blood with pathogens or pathogen-associated molecular patterns (PAMPs) can trigger the activation of both systems, a bidirectional complement-coagulation crosstalk is believed to occur. Although the role of complement activation products as positive regulators of coagulation is documented, direct activation of the complement proteins by thrombin or other hemostatic proteases was alluded but not demonstrated in vivo. Here we aimed to: (i) determine if in vivo generation of thrombin and other hemostatic proteases can activate the complement proteins and (ii) discriminate between the direct effect of the pathogen/PAMPs vs. hemostatic proteases on complement activation in a clinically relevant model of sepsis. We have compared the time-course of complement activation markers (C3b, C5a and C5b-9 terminal complex) in plasma of baboons exposed to 1010 cfu/kg (LD100) E. coli vs. intravenous infusion of factor Xa/PC:PS, a potent procoagulant stimulus. In baboons challenged with LD100 E. coli, complement activation markers C3b, C5a and C5b-9 reached maximum levels after 2 hrs (see figure). Complement activation coincided with the peak of bacteremia and LPS, but not with markers of thrombin generation (TAT and fibrinogen consumption; see figure) or fibrinolysis (FDP, D-dimers), which reached peak levels after 6 hours. Differently, infusion of FXa/PC:PS (36.6 pmol/L FXa and 56.3 nmol/L PC/PS per kg body weight) induced a rapid burst of thrombin and almost full consumption of fibrinogen during the first 10 min post-infusion, with no increase of complement activation markers. Based on these data we conclude that in vivo activation of the coagulation cascade does not support complement activation as was postulated by previous in vitro studies. Therefore, we conclude that pathogens and PAMPs are the main activators of the complement during sepsis while direct activation by hemostatic proteases is minor or absent.
Figure Figure.

Disclosures
No relevant conflicts of interest to declare.
Therapeutic Blockade of Macrophage Colony Stimulating Factor (CSF-1) Delays AML Progression in Mice In Vivo
Authors: Goh Sal Lee , Levesque Jean-Pierre , Petitt Allison R , Barbier Valarie , Winkler Ingrid G 
Published: October 2019

Abstract
Macrophage colony-stimulating factor (M-CSF or CSF-1) plays a role in regulating innate immune responses promoting macrophage growth and differentiation. We hypothesized CSF-1 may also play a role in growth and progression of Acute Myeloid Leukaemia (AML). The aim of this study is to investigate the role of CSF-1 in survival and chemo-resistance of leukaemia stem and progenitor cells (LSPC). We further hypothesized that blocking CSF-1R signalling in LSPC may dampen leukaemia survival in vitro and delay leukaemia progression in vivo.
AML was induced in mice by injecting murine Haematopoietic Stem and Progenitor Cells (HSPC) transduced with either MLL-AF9 or AML1- ETO fusion oncogenes for the development of either monomyelocytic or granulocytic leukaemia respectively. We found CSF-1R, the main receptor for CSF-1, was expressed on these acute myeloid leukaemia cells, thus it is possible that CSF-1 provide supportive microenvironment for leukemic growth. To identify whether CSF-1 in the bone marrow (BM) niche is essential for growth of malignant LSPC, we harvested normal or- leukaemic blasts from BM for in vitro studies using Long-Term Culture-Initiating-Cell (LT-CIC) assays. In these assays AML LSPC or normal HSPC cells were co-cultured with mesenchymal stromal cells (MSC) from either wildtype mice (MSC that produce CSF-1) or MSC from OP/OP mice (unable to produce functional CSF-1). We found normal (wild-type) HSPC were able to proliferate, survive and produce LT-CIC in the absence of niche-provided CSF-1, however AML blasts could not, unless rescued by addition of recombinant CSF-1 (100 ng/mL) in vitro. Together these data suggest CSF-1 signalling may be critical for AML LSPC but not normal HSPC.
Next we investigated in mice whether therapeutic CSF-1 blockade could similarly dampen AML survival or progression in vivo. Cohorts of mice were injected with luciferase-expressing monomyelocytic (MLL-AF9) BM leukaemic blasts, then 7 days later administered the small molecule CSF-1 antagonist (GW2580, 160mg/kg daily for 10 days) or vehicle control. Leukaemia progression was tracked by biweekly bioluminescence and testbleeds for appearance of GFP+ leukaemia blasts in blood. We found therapeutic blockade of CSF-1 significantly reduced tumour burden in these mice by both bioluminescence and testbleed analysis. Mice were also monitored for duration of survival. As anticipated by the observed reduction in leukaemia burden, therapeutic CSF-1 blockade also significantly extended the duration of overall mouse survival (P<0.005, n= 8 mice/ group).
Together these studies suggest therapeutic CSF-1 blockade may show promise as an adjunct therapy to help reduce tumour burden and improve success of AML leukaemia therapies.

Disclosures
Winkler: GlycoMimetics: Research Funding.
Improved Sensitivity in Detection of FMS-like Tyrosine Kinase Internal Tandem Duplication of a Method Using Next-Generation Sequencing Data
Authors: Shin Junghoon , Kim Daeyoon , Hong Yoojin , Koh Youngil , Yun Hongseok , Shin Dong-Yeop , Yoon Sung-Soo , Kim Inho , Lee Junglim , Kim Byung-Su 
Published: October 2019

Abstract
We developed new ITD detection algorithm (ITDetect) based on whole exome sequencing (WES) data for FMS-like tyrosine kinase internal tandem duplication (FLT3-ITD). ITDetect is based on BWA and is specified for ITD detection including FLT3-ITD. We validated and compared result of ITDetect with other ITD detecting algorithms using nested polymerase chain reaction (PCR) method. Nested PCR uses two types of primer specified for FLT3-ITD detection.
In 81 acute myeloid leukemia patients with WES data, FLT3-ITD was positive in 11 patients (13%) when called with ITDetect, all of whom were validated with nested PCR. Meanwhile FLT3-ITD was positive only in 7/81 patients by conventional PCR. The concordance rate of ITDetect and nested PCR was 95% (77/81). ITDetect showed better ITD detection performance when compared with previously reported ITD callers. In large AML cohort (n=213), patients who were positive for FLT3-ITD with nested-PCR but not with conventional PCR had shorter survival outcomes than patients who were negative for FLT3-ITD with nested PCR, suggesting clinical significance of sensitive FLT3-ITD detection.
In conclusion, we developed more sensitive detection methods for FLT3-ITD based on WES data that is clinically meaningful. Utilization of more sensitive detection method than conventional PCR in clinic should be considered.
Figure FLT3-ITD detection procedure. Figure. FLT3-ITD detection procedure. Figure Performance of various NGS ITD detectors. Figure. Performance of various NGS ITD detectors. Figure Survival comparison between patients positive for FLT3-ITD by nested PCR method but negative by conventional PCR method and those negative by both methods. Figure. Survival comparison between patients positive for FLT3-ITD by nested PCR method but negative by conventional PCR method and those negative by both methods.

Disclosures
No relevant conflicts of interest to declare.