bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct Regulatory Networks Govern Human Hematopoietic Stem Cell Across Development
Authors: Zandi Sasan , Dick John E. , Notta Faiyaz , Takayama Naoya
Published: October 2019

Introduction: Much of our fundamental understanding of stem cell biology comes from studies of hematopoiesis where single cells produce differentiated progeny while still retaining the ability to produce daughter stem cells (self-renewal). The cardinal property of a stem cell, whether normal or malignant, is self-renewal; the key biological process that ensures the ability of the stem cell to maintain long-term clonal growth. However, our understanding of the molecular basis of self-renewal in human hematopoiesis is limited.

At the embryonic stage fetal liver is the main source of hematopoiesis; from week 6 of gestation until before birth. At this stage HSCs are in a different microenvironment but capable of self-renewing and differentiation to the full spectrum of blood lineages. While murine studies uncovered several intrinsic differences between fetal and adult HSCs, a comprehensive analysis of human HSC compartment across development is lacking.

In this study we have combined HSC purification methods and xenograft quantitative assay in conjunction with low input RNA sequencing and Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) to provide a comprehensive functional and molecular outlook of human stem cell compartment across development.

Results: We followed the dynamics of four sub-fractions of CD34+CD38- divided by CD90 and CD49f expression across human blood development: fetal liver (hFL) and adult bone marrow (hBM). Using xenograft model, we identified human long, intermediate and short term HSCs in hFL and hBM. 5 single CD90+CD49f+ hFL cells were capable of sustaining the multilineage graft for over 52 weeks up to tertiary recipient, while BM cells only last for 20 weeks in the primary recipient. The frequency of LT-HSC in the CD90+CD49f+ compartment goes from 1/8 in hFL to 1/50 in hBM. hFL CD90-CD49f+ cells showed an intermediate repopulation capacity up to 44 weeks in secondary recipient.

On average 10% of hFL long term HSC (LT-HSC) were in S/G2/M phase, in contrast only 0.4% of BM LT-HSC were in S/G2/M phase indicating that hFL HSCs are 20 times more in cycle compare to BM.

We found that 320 genes were expressed differentially between LT-HSC and multipotent progenitors (MPP) in hBM as oppose to only 32 genes found to be differentially expressed in hFL (FDR<0.1). Interestingly, we found only 2 genes in common between these two groups.

ERRBS showed an overall increase in methylation of HSC compartment in hBM compare to hFL and gradual demethylation of lineage associated genes in MPP.

Conclusion: Our data indicate that there are distinct regulatory networks that govern hFL and hBM HSC self-renewal. We found very little differences in gene expression between all hFL HCS compartments (average 20 genes) compare to hBM (average 224), indicating that by adulthood self-renewal is becoming more restricted to the LT-HSC compartment.

Disclosures

No relevant conflicts of interest to declare.
A Dynamic Intron Retention Program in the Mammalian Megakaryocyte and Erythrocyte Lineages
Authors: Edwards Christopher R , Middleton Rob , An Xiuli , Mishra Tejaswini , Mohandas Narla , Hardison Ross C. , Rasko John , Blobel Gerd A.
Published: October 2019

Intron retention (IR), the least studied form of alternative splicing, has recently been shown to have important biological roles in a variety of cell types. While it can alter a gene's protein-coding sequence, it is becoming particularly well-known for its potential to impact gene expression by destabilizing mRNAs through the nonsense-mediated decay pathway or by promoting their retention in the nucleus. A complex, dynamic, and biologically important IR program has been described in maturing mammalian granulocytes, but it is unknown whether IR occurs broadly in other hematopoietic lineages. We therefore globally assessed IR in the mammalian erythroid and megakaryocyte lineages. Intron Retention Finder, a bioinformatics tool that measures IR in RNA-seq datasets, was used to analyze IR in primary cells of the erythroid and megakaryocyte lineages as well as their common progenitor cells. Both lineages exhibit an extensive differential IR program involving hundreds of introns and genes. Complex IR patterns were seen in murine erythropoiesis from the megakaryocytic-erythroid branch point throughout the terminal maturation stages. Within the terminally differentiating proerythroblast to orthochromatic erythroblast stages, hundreds of introns saw their retention level increase as cells differentiate while a smaller set exhibited an opposing trend. Similarly complex patterns including a dramatic IR increase in orthochromatic erythroblasts were observed during human terminal erythroid differentiation, but not involving the murine orthologous introns or genes. Despite the common origin of erythroid cells and megakaryocytes and their overlapping gene expression patterns, the megakaryocytic IR program is entirely distinct from that of the erythroid lineage with regards to introns, genes, and affected gene ontologies. This suggests that the dynamic IR patterns are not simply the result of general maturational changes, but rather may arise via lineage-specific mechanisms. Importantly, we observed an inverse relationship between IR and gene expression changes, supporting the hypothesis that IR serves to regulate mRNA levels. Our findings add a new dimension to the megakaryocyte and erythroid transcription programs by expanding the mechanisms of gene control to include this understudied form of alternative splicing.

Disclosures

No relevant conflicts of interest to declare.
CD34 Is a Ligand for Vascular Selectins on Human Hematopoietic Stem/Progenitor Cells
Authors: AbuSamra Dina Bashir
Published: October 2019

The cell surface glycoprotein CD34 is widely recognized as a marker of hematopoietic stem/progenitor cells (HSPCs), but its physiological role on HSPCs remains elusive. Although CD34 is well recognized for mediating homing of L-selectin+ naïve T cells to high endothelial venules of secondary lymphoid organs, the natural ligand for HSPC specific-CD34 is unknown. Studies in both humans and mice indicate that vascular selectins (E-selectin and P-selectin) are constitutively expressed on marrow endothelial cells and intravital studies revealed that HSPC migration to marrow occurs at specialized microvascular beds expressing E-selectin. Given that the identity of these glycoprotein E-selectin ligands (E-selL) on human HSPCs is limited, we used mass spectrometry (MS)-based proteomics to fully characterize all these potential ligands expressed on primary human CD34+ HSPCs and on the CD34+ KG1a cell line (human acute myelogenous leukemia that serves as a model for human HSPCs). We obtained a very rich resource for further investigation and chose to highlight CD34 as an E-selL candidate. The physiological binding of CD34 to E-selectin was addressed at both the molecular level and cellular level using novel binding assays. At the molecular level we developed a novel-binding assay to capture endogenous candidate glycoprotein ligands from whole cell lysates prepared from human HSPCs onto a surface plasmon resonance (SPR) chip and characterized its binding kinetics to recombinant E-sel. At the cellular level we used the blot rolling assay to monitor the ability of cells stably transfected with E-selectin (CHO-E) to roll on CD34 immunoprecipitated from HSPCs to demonstrate that CD34 binds to E-selectin with affinity comparable to well-described E-selectin ligands. Further biochemical analysis demonstrated that E-selectin binding was restricted to a specific glycoform that expresses sialyl Lewis X (sLex). This interaction was dependent on calcium, sialylation and O-glycans as removal of these abolished binding to E-selectin. CD34 binding to the other vascular selectin, P-selectin was dependent on O-glycan and tyrosine sulfation making CD34 the first selectin ligand since P-selectin glycoprotein ligand-1 (PSGL-1) reported to bind all three selectins. Among the stem cell specific processes such as self-renewal and differentiation that CD34 has been implicated to play a significant role in, these data are the first to define a role for human CD34 in mediating the direct migration of HSPCs to the bone marrow and suggests that a natural ligand for CD34 on human HSPCs are vascular selectins.

Disclosures

No relevant conflicts of interest to declare.
A Subcutaneously Administered Investigational RNAi Therapeutic (ALN-CC5) Targeting Complement C5 for Treatment of PNH and Complement-Mediated Diseases: Interim Phase 1 Study Results
Authors: Hill Anita , Taubel Jorg , Bush Jim , Borodovsky Anna , Kawahata Noriyuki , Mclean Helen , Powell Christine , Chaturvedi Prasoon , Warner Garvin , Garg Pushkal , Sorensen Benny
Published: October 2019

Introduction: Uncontrolled complement activation plays a pivotal role in a variety of disorders such as PNH and aHUS. ALN-CC5 is a subcutaneous (SC) investigational RNAi therapeutic targeting complement C5 (C5). In preclinical studies, ALN-CC5 has demonstrated decreased terminal complement activity. Based on the literature, preventing the generation of the terminal complex protects against intravascular hemolysis and complement-mediated tissue damage. The purpose of this study is to evaluate the safety and tolerability of ALN-CC5 in normal healthy volunteers.

Material and methods: A multi-centered, placebo controlled, double blind phase 1 clinical study in healthy volunteers is ongoing. Several cohorts of healthy volunteers in Part A, a single ascending dose study and Part B, a weekly multiple ascending dose study have been completed. Primary endpoints are safety and tolerability. Secondary endpoints are pharmacokinetics, reduction of circulating C5, reduction in hemolytic, CAP and CCP activity.

Results: In Part A, 20 healthy volunteers were randomized (1:3) to placebo or single SC dose of 50, 200, 400, 600 or 900mg of ALN-CC5 and followed for at least 70 days. In Part B, 12 healthy volunteers were randomized (1:3) to placebo or 5 weekly doses of 100, 200 or 400mg of ALN-CC5. No SAEs or study discontinuations occurred and overall ALN-CC5 was considered safe and generally well tolerated. A dose dependent and 94% mean maximum C5 knockdown was achieved following weekly administration. Updated safety and tolerability data as well as C5 knockdown, and changes in CAP, CCP and hemolytic activity from the study will be presented.

Conclusion: Collectively, these initial results suggest that the use of a novel RNAi therapeutic targeting C5 is a promising approach for inhibiting complement in PNH, aHUS and other complement-mediated diseases. The subcutaneous route of administration and infrequent dosing make this a potentially encouraging therapy.

Disclosures

Hill: Alnylam: Consultancy. Off Label Use: ALN-CC5 is an investigational RNAi therapeutic targeting complement C5.. Borodovsky:Alnylam Pharmaceuticals: Employment, Equity Ownership. Kawahata:Alnylam Pharmaceuticals: Employment, Equity Ownership. Mclean:Alnylam Pharmaceuticals: Employment, Equity Ownership. Powell:Alnylam Pharmaceuticals: Employment, Equity Ownership. Chaturvedi:Alnylam Pharmaceuticals: Employment, Equity Ownership. Warner:Alnylam Pharmaceuticals: Employment, Equity Ownership. Garg:Alnylam Pharmaceuticals: Employment, Equity Ownership. Sorensen:Alnylam Pharmaceuticals: Employment, Equity Ownership.
The Molecular Mechanism of Transcriptional Activation By MLL-AEP Fusion Proteins
Authors: Yokoyama Akihiko , Okuda Hiroshi
Published: October 2019

Chromosomal translocations generate a variety of mixed lineage leukemia (MLL) fusion genes, which cause aggressive leukemia. Although >70 different fusion partners have been identified, the majority of the cases are caused by the chimeric genes of MLL and a component of the AEP co-activator complex (hereafter referred to as AEP), which comprises of AF4 family proteins (e.g. AF4, AF5Q31), ENL family proteins (e.g. ENL, AF9), and the P-TEFb elongation factor. MLL-AEP fusion proteins constitutively activate their target genes by recruiting AEP components to their target chromatin, whereas wild-type MLL recruits AEP in a context-dependent manner. In the hematopoietic lineage, MLL fusion proteins aberrantly activate a subset of genes implicated in the hematopoietic stem cell (HSC) program, such as HOXA9 and MEIS1. Constitutive expression of these HSC program genes in hematopoietic progenitors has been shown to induce leukemia in a mouse model. It has been speculated that MLL-AEP activates transcription of those HSC program genes by aberrantly activating transcription elongation. However, it is largely unclear how AEP activates transcription.

Using an extensive structure/function analysis, we revealed that a serine-rich domain of the AF4 family proteins, termed pSER, is an essential functional component of MLL-AEP-dependent gene activation and leukemic transformation. Through biochemical purification, we have identified Selectivity Factor 1 (SL1) as a novel factor associated with the pSER domain. SL1 comprises TBP and four TBP-associated factors (TAF1A, TAF1B, TAF1C, TAF1D), and is known as a core component of the pre-initiation complex (PIC) of RNA polymerase I (RNAP1). In the presence of UBF, SL1 forms a PIC on the promoters of ribosomal RNA genes, to drive RNAP1-dependent transcription. However, its role in RNAP2-dependent transcription was unknown. The initiation of RNAP2-dependent transcription in eukaryotes occurs through the loading of TBP to the promoter, via a direct association with the TATA element or through as-yet-unidentified mechanisms. Our results demonstrate that AEP facilitates the initiation of RNAP2-dependent transcription via the loading of TBP onto the TATA element, through SL1 activity. MLL-AEP fusion proteins utilize this TBP-loading function to activate transcription initiation in leukemic transformation. The wild-type AEP complex activates gene expression in the same manner in the physiological conditions. Taken together, our results unveil a novel role of SL1 as a TBP-loading factor in RNAP2-dependent gene activation, and a previously unknown transcription initiation mechanism involving AEP, which is more important than its transcription elongation activities for leukemic transformation. These findings greatly advance our understanding of the molecular mechanism of MLL fusion-dependent leukemic transformation, which was previously interpreted simply as mis-regulated transcription elongation.

Disclosures

Yokoyama: Dainipon Sumitomo Pharma Co., Ltd.: Research Funding.
CDKN2 Gene Deletion Impair Prognosis in Adult Ph+ Acute Lymphoblastic Leukemia Even in Tyrosine Kinase Inhibitors Era
Authors: Xu Na , Li Yuling , Zhou Xuan , Zhu Hongqian , Li Lin , Lu Qisi , Cao Rui , Li Huan , Liu Qifa , Xiaoli Liu
Published: October 2019

Background: Philadelphia chromosome-positive (BCR-ABL1+) ALLs benefit from the addition of a specific tyrosine kinase inhibitors (TKIs) to chemotherapy, However, treatment failures and relapses are common and new markers are needed to identify patients with poor prognosis in prospective trials. The 9p21 locus, encoding important tumor suppressors (CDKN2A/B), is a major target of inactivation in the pathogenesis of many human tumors, but only few report investigated this deletion effect on clinical prognosis in Ph+ ALL.

Purpose: Many studies found that deletion of CDKN2 was associated with poor prognosis in ALL, and CDKN2 deletion also as a frequent cytogenetic aberration in Ph+ ALL patients. Here we study about the prognostic significance of the CDKN2 in Ph+ ALL in TKIs era.

Patients and Methods: To explore the frequency and size of alterations affecting this locus in adult BCR-ABL1-positive ALL and to investigate their prognostic value, 135 patients (98 denovo and 37 relapsed cases) were analyzed by Paired diagnosis-relapse samples were interphase fluorescence in situ hybridization(I-FISH).

Results: The prevalence of CDKN2 deletions in all study population was 27.4% (37/135). There is no difference between patients with CDKN2 deletion and wild-type patients in sex, age, white blood cells(WBC) count, BM blast percentage, and induction complete remission(CR) rate. Compared with patients with wild-type CDKN2, the patients with CDKN2 deletion had higher rates of hepatosplenomegaly, CD20 expression (p<0.05). Deletions of CDKN2 were significantly associated with poor outcomes including decreased overall survival (OS) (P<0.001), lower disease free-survival (DFS) (P<0.001), and increased cumulative incidence of relapse (P=0.003). In case of 37 patients with CDKN2 deletion, 20 patients treated with chemotherapy and Dasatinib followed by allogeneic hematopoietic stem cell transplantation (Allo-HSCT), and another 13 patients treated with chemotherapy and Imatinb followed by Allo-HSCT,there were no difference associated with OS(P=0.813) and DFS(p=0.513) between the above groups.

Conclusions: Deletion of CDKN2 by I-FISH is a frequent event in Ph-positive ALL, and frequently acquired during leukemia progression and are a poor prognostic marker of long-term outcomes even though treated by adding dasatinib. We also found CDKN2 deletion patients had higher CD20 expression, this group patients may be benefit from rituximab.

Disclosures

No relevant conflicts of interest to declare.
Increased Use of Allogeneic Transplant in CR2 Improves the Outcome for Children with Acute Myeloid Leukaemia
Authors: Vedi Aditi , Mitchell Richard , Oswald Cecelia , Marshall Glenn M , Trahair Toby , Ziegler David S.
Published: October 2019

Improvements in Outcome for Paediatric de novo Acute Myeloid Leukaemia Aditi Vedi1,2, Richard Mitchell1, Cecelia Oswald1, Glenn Marshall1,2, Toby Trahair1, David S Ziegler1,2 1Kids Cancer Centre, Sydney ChildrenÕs Hospital, Randwick, NSW, Australia, 2 School of Women and Children's Health, University of New South Wales, Randwick, NSW, Australia ABSTRACT The treatment for paediatric acute myeloid leukaemia (AML) has not changed significantly over the past 3 decades, yet outcomes have improved with cure rates increasing from 30% to over 50% of all newly diagnosed children over this period. This improvement in survival has been attributed to both treatment intensification and improved supportive care over the decades, although the precise impact of each remains unknown. Our group has retrospectively analysed a unique cohort of patients with de novo AML diagnosed in childhood (n=276), all treated with the same chemotherapy protocol over a 25-year period from 1986-2012. The contemporary cohort (2000-12), compared to historical cohorts (1986-99) had significantly improved overall survival (OS, 75% vs. 50%, p = 0.01), lower disease related mortality (38% vs. 19%, p = 0.02) and were significantly more likely to receive allogeneic transplant after relapse (SCT, 73% vs. 12%, p <0.0001). Allogeneic transplant post relapse was associated with a significantly improved survival across the entire cohort (OS 50% for allogeneic SCT vs. 12% for autologous or none, p<0.0001). There was no significant difference between the contemporary and historical cohorts in treatment related mortality (13% vs. 7%, p = 0.42) or relapse rates after induction (50% in older cohort vs. 40% in recent era, p=0.25), suggesting consistency of induction treatment efficacy and toxicity across the two periods. This data suggests improved survival in paediatric AML in the modern era has predominantly resulted from increased use of allogeneic SCT after relapse rather than from improved supportive care and is independent of chemotherapy intensification.

Disclosures

No relevant conflicts of interest to declare.
Drug Response Profiling to Identify Selective Pharmacological Activity in Drug Resistant ALL
Authors: Frismantas Viktoras , Dobay Maria Pamela , Rinaldi Anna , Joachim Kunz , Marovca Blerim , Horvath Peter , Delorenzi Mauro , Cario Gunnar , Schrappe Martin , Stanulla Martin , Kulozik Andreas E. , Muckenthaler Martina U. , Stackelberg Arend , Eckert Cornelia , Radimerski Thomas , Bornhauser Beat , Bourquin Jean-Pierre
Published: October 2019

With the rapid development of new therapeutic agents, better strategies are needed to select patients with high risk or relapsed acute lymphoblastic leukemia (ALL) for experimental therapy. We developed a large-scale drug response profiling platform that is based on an established ALL co-culture system on bone marrow stromal cells, with the advantage of standardized conditions in serum-free medium. Comparing the profiles of 61 ALL samples that were enriched for relapsed and refractory ALL, we identified informative patterns of drug sensitivity and resistance comparing between clinical and cytogenetic ALL subtypes. We detected strong and selective activity for therapeutic agents that could be used in the clinic. These include a BCP-ALL with particular sensitivity to SMAC mimetics and a T-cell ALL subsets with very high sensitivity to dasatinib or topotecan. Here we focus on identification of ALLs subtypes that are critically dependent on BCL2, based on their response patterns to the BCL2-specific BH3-mimetic venetoclax (ABT-199). A subset of BCP-ALL, including MLL-AF4 and TCF3-HLF positive ALL cases were highly sensitive to venetoclax. As reported by others recently, we also detected a smaller subgroup of T-ALL that were highly sensitive to BCL2 inhibition and confirm that these are not restricted to early thymic precursor ALL. Sensitivity to venetoclax in vitro correlated with anti-leukemic activity in ALL xenografts in vivo. Moreover combination testing on our platform indicated synergistic activity of venetoclax with conventional anti-leukemic agents such as dexamethasone and vincristine, and with the new class BRD4 inhibitors. In vivo, combination of venetoclax with dexamethasone and vincristine completely prevented disease progression of very aggressive TCF3-HLF ALL cases compared to a transient delay in disease progression that we observed in the control arms with single agent venetoclax or dexamethasone + vincristine treatment. Based on our results, we propose that drug activity profiling should be evaluated prospectively in clinical phase I and II settings as a tool to develop more personalized approaches for patients with resistant disease that are at urgent medical needs.

Disclosures

No relevant conflicts of interest to declare.
TCRab Deficient CAR T-Cells Targeting CD123: An Allogeneic Approach of Adoptive Immunotherapy for the Treatment of Acute Myeloid Leukemia (AML)
Authors: Galetto Roman , Lebuhotel Céline , Gouble Agnès , Mencia-Trinchant Nuria , Nicole Cruz M , Roboz Gail J. , Guzman Monica L. , Smith Julianne
Published: October 2019

The remissions achieved using autologous T-cells expressing chimeric antigen receptors (CARs) in patients with advanced B cell leukemia and lymphomas have encouraged the use of CAR technology to treat different types of cancers by targeting distinct tumor-specific antigens. Since the current autologous approach utilizes CAR T-cells manufactured on a "per patient" basis, we propose an alternative approach based on the use of a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic "off-the-shelf" CAR T-cell-based frozen products.

In the present work we have adapted this allogeneic platform to the production of T-cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed on tumor cells from the majority of patients with Acute Myeloid Leukemia (AML).

Multiple antigen recognition domains were screened in the context of different CAR architectures to identify candidates displaying activity against cells expressing variable levels of the CD123 antigen. The three lead candidates were tested in an orthotopic human AML cell line xenograft mouse model. From the three candidates that displayed comparable activity in vitro, we found two candidates capable of eradicating tumor cells in vivo with high efficiency.

Subsequently, Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology was used to inactivate the TCRα constant (TRAC) gene, eliminating the potential for engineered T-cells to mediate Graft versus Host Disease (GvHD). Editing of the TRAC gene can be achieved at high frequencies, and allows efficient amplification of TCR-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. In addition, we show that TCR-deficient T-cells display equivalent in vitro and in vivo activity to non-edited T-cells expressing the same CAR.

We have performed an initial evaluation of the expression of CD123 in AML patients and found an average cell surface expression of CD123 was of 67% in leukemic blasts (95% CI 48-82), 71% in CD34+CD38+ cells (95% CI 56-86), and 64% in CD34+CD38- (95% CI 41-87). Importantly, we have found that CD123 surface expression persists in CD34+CD38-CD90- cells after therapy in at least 20% of patients in remission (n=25), thus emphasizing the relevance of the target. Currently, the sensitivity of primary AML cells to CAR T-cells is being tested.

Finally, we will also present our large scale manufacturing process of allogeneic CD123 specific T-cells from healthy donors, showing the feasibility for this off-the-shelf T-cell product that could be available for administration to a large number of AML patients.

Disclosures

Galetto: Cellectis SA: Employment. Lebuhotel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Smith:Cellectis: Employment, Patents & Royalties.
UCH-L1 Cooperates with BCL6 and Identifies Patients with Aggressive Germinal Center Diffuse Large B-Cell Lymphoma
Authors: Bedekovics Tibor , Hussain Sajjad , Feldman Andrew L , Galardy Paul J.
Published: October 2019

Gene expression profiling has identified two major subclasses of diffuse large B-cell lymphoma. Cases resembling germinal center B-cells (GCB-DLBCL) generally occur in younger patients, have a distinct molecular pathophysiology, and have improved outcomes compared with those similar to activated post-germinal center cells (ABC-DLBCL). We previously found that the ubiquitin hydrolase UCH-L1 is frequently overexpressed in mature B-cell malignancies and is a potent oncogene in mice. The cause for its overexpression in lymphoma, and whether it impacts the outcome of patients with DLBCL is unknown. Here we show that UCH-L1 reflects germinal center lineage in lymphoma and is an oncogenic biomarker of aggressive GCB-DLBCL. We find that UCH-L1 is specifically induced in germinal center B-cells in mice and humans, and that its expression correlates highly with the GCB subtype in DLBCL. Despite the typically good outcomes of GCB-DLBCL, increased UCHL1 identifies a subgroup with early relapses independent of MYC expression, suggesting biologic diversity in this subset of disease. We also find that UCH-L1 synergizes with BCL6 in a mouse model of germinal center B-cell lymphoma, but not with the development of multiple myeloma derived from post-germinal center cells. Consistent with this, forced Uchl1 overexpression had a substantial impact on gene expression in germinal center B-cells including pathways of cell cycle progression, cell death and proliferation, and DNA replication. These data demonstrate a novel role for UCH-L1 outside of the nervous system and suggest its potential use as a biomarker and therapeutic target in DLBCL.

Disclosures

Galardy: Mission Therapeutics: Research Funding.
Intermediate Dose Methotrexate Improves Overall Survival and Progression-Free Survival of Patients with Diffuse Large B Cell Lymphoma Treated with the R-CHOP or CHOP Regimen
Authors: Dann Eldad J , Heffes Vered , Mashiach Tatiana , Benyamini Noam , Avivi Irit , Horowitz Netanel A
Published: October 2019

Introduction: Patients (pts) with diffuse large B cell lymphoma (DLBCL) and high International Prognostic Index (IPI) or extra-nodal localization are at a higher risk for relapse with central nervous system (CNS) involvement. The current policy at the Rambam Health Care Campus (Haifa, Israel) is to give DLBCL pts 6 cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) together with 4 doses of intrathecal (IT) methotrexate (MTX), which may be followed by 2 additional cycles of an intermediate dose (3 g/m2) of intravenous MTX (ID-MTX). The present study aimed to compare the outcome of DLBCL pts with risk factors for CNS relapse who did or did not receive prophylaxis with ID-MTX.

Methods: We retrospectively evaluated a cohort of DLBCL pts treated at Rambam between the years 1991 and 2012. Overall survival (OS), progression-free survival (PFS) and CNS involvement at relapse were estimated in 203 of 355 pts [96 females, 107 males; median age 59 (range18-90) years]. The study included all pts with the primary diagnosis of DLBCL and risk factors for CNS relapse, i.e., Waldeyer ring (5%), breast (2%), testicular (3%), orbital (1%) involvement, bone marrow involvement (25%), stage IV disease (63%), LDH > normal (68%), ≥1 extra-nodal site (26%), IPI ≥3 (41%). Ten percent of pts had stage I-IE disease, 15% - stage II-IIE, 12% - stage III, 63% - stage IV, 38% had B symptoms. Pts with CNS involvement at diagnosis were excluded from the study.

Results: One hundred and fifty patients (74%) were treated with R-CHOP. In this group, 40% of pts with IPI=0/1, 29% with IPI=2 and 47% with IPI ≥3 received ID-MTX prophylaxis. Sixty four pts received ID-MTX (32%), 14 pts (7%) received IT MTX only, 125 pts had no prophylaxis (62%). At a median follow-up of 92 months, 5% of pts had CNS relapse with no difference in incidence between the study groups. Pts with IPI≥3 treated with ID-MTX had a reduced overall relapse rate and significantly better PFS and OS (table 1).

Conclusion: The addition of 2 cycles of ID-MTX to the R-CHOP regimen improved both PFS and OS of DLBCL pts with IPI≥3. A randomized controlled study is warranted to provide stronger evidence.

Table 1. All patients Pts No. PFS % P *HR 95% CI P OS % #HR 95% CI ID-MTX 65 60 1 88 1 IT MTX 15 56 0.09 1.92 0.9-4.1 0.008 43 3.27 1.4-7.8 No prophylaxis 123 42 0.04 1.58 1.0-2.49 0.002 51 2.49 1.4-4.4 R-CHOP Data IPI≥3 no prophylaxis 35 20 1 26 1 IPI≥3 +ID-MTX 31 58 0.004 0.38 0.29-0.85 0.001 67 0.29 0.14-0.6 No.: number; *HR: hazard ratio for progression; #HR for mortality; CI: confidential interval

Disclosures

No relevant conflicts of interest to declare.
Chemotherapy Alone Is Effective to Treat Localized and Disseminated Ocular Adnexa Lymphomas
Authors: Ma Wei-Li
Published: October 2019

Background:

The treatment strategies for ocular adnexa lymphomas (OALs), including conjunctiva, orbital, and lacrimal gland lymphomas, remain controversial. In this study, we assessed the efficacies and outcomes of different modality treatment, including chemotherapy, radiotherapy, and surgery for OALs.

Methods and Materials:

Between Jan. 1990 to Dec. 2013, patients receiving first-line chemotherapy (oral alkylating agents, rituximab-based treatment, or CHOP [cyclophosphamide, doxorubicin, vincristine, and steroid]-like regimens), radiotherapy (30 to 50 Gy, in 1.8- to 2.0- Gy per daily fractions), and surgery for newly diagnosed OAL were included in this study. The clinicopathologic features, including patients' characteristics, primary lymphoma locations, pathology subtypes, treatment modality, event-free survival (EFS) following first-line therapy, and OS (OS) were analyzed.

Results:

There were 56 men and 37 women with median age of 58 years were included. Of them, 79 patients (85%) were diagnosed with low-grade mucosa-associated lymphoid tissue (MALT) lymphoma, 5 (5%) with high-grade transformed MALT lymphoma, and 9 (10%) with diffuse large B cell lymphoma. Orbit was the most common involved location (45 patients, 48%), followed by conjunctiva (35 patients, 38%), and lacrimal gland (13 patients, 14%). Of 79 patients with stage I-IIE1 disease, 22 received chemotherapy, 34 with radiotherapy, and 23 patients with other modality treatments (18 with surgery, 4 with combined radiotherapy and chemotherapy, and 1 with intra-lesional injection of rituximab). The 5-year EFS for chemotherapy, radiotherapy, and other modality treatment was 89.2%, 89.7%, and 83.1%, respectively, whereas the 5-year OS for chemotherapy, radiotherapy, and other modality treatment was 100%, 90.4%, and 87.5%, respectively. Of 14 patients with stage IIE2 to IV disease, the 5-year EFS for chemotherapy alone (n=9) and combined radiotherapy and chemotherapy (n=5) were 68.6%, 80%, respectively, whereas the 5-year OS for both group was 80.0%. Among 42 patients receiving radiotherapy, 7 (16.7%) patients developed cataract, 4 (9.5%) patients with keratitis, and 2 (4.8%) patients with maculopathy. In multivariate analysis, high-grade transformation (P=0.049) was the significant factor for shorter EFS of 1st -line treatment. Old age (> 60 years) (p = 0.014), advanced stage (stages III and IV) (p = 0.03), and high-grade transformation (p = 0.018) were the prognostic factors of poor OS.

Conclusions:

In addition to radiotherapy, our data indicate that chemotherapy as first-line therapy provides good disease control and less radiotherapy-associated side effects for patients with stage I to IIE1 OALs. Old age, advanced stage, and high-grade histology subtype remain poor prognostic factors for OALs, and new treatment strategies for these patients are warranted.

Disclosures

No relevant conflicts of interest to declare.
JQ1, a Potent c-MYC Inhibitor Overcomes Rituximab-Chemotherapy Resistance in Lymphoma Pre-Clinical Models
Authors: Gu Juan J , Zhang Qunling , Mavis Cory , Czuczman Myron S. , Hernandez-Ilizaliturri Francisco J.
Published: October 2019

Background: The poor clinical outcomes of patients with aggressive B-cell lymphoma in the post-rituximab era, stress the need to identify and/or optimize novel targeted agents. Several retrospective and prospective clinical studies had demonstrated that C-myc expression correlates with a poor clinical outcome in patients with newly diagnosed or relapsed/refractory diffuse large B-cell lymphoma (DLBCL). To this end, we evaluated the therapeutic effects of targeting C-myc using JQ1, a novel bromodomain inhibitor in rituximab-sensitive or -resistant models.

Methods: A panel of rituximab-sensitive (RSCL) or rituximab-resistant (RSCL) cell lines was exposed to JQ1 (0-100 µM) for 24-72 hrs. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 values were calculated using the GraphPad Prism6 software. Subsequently lymphoma cells were exposed to JQ1 or vehicle and various chemotherapy agents such as doxorubicin (0.5, 1, 2µM), dexamethasone (1µM), Ibrutinib (1µM), bortezomib (10-20nM) or carfilzomib (10nM) for 48 hours. Coefficient of synergy was calculated using the CalcuSyn software.

Results: In vitro exposure of RRCL and to a lesser degree RSCL to JQ1 resulted in a dose- and time-dependent cell death. Strong synergistic activity was observed when JQ1 was combined with doxorubicin, dexamethasone bortezomib or carfilzomib in vitro. Cell cycle analysis demonstrated that in vitro of RSCL or RRCL to JQ1 resulted in G1 cell cycle arrest.

Conclusions: In summary, our data suggests that targeting C-myc expression using JQ1 results in anti-tumor activity against RSCL and RRCL. In addition, JQ1 exhibited synergistic activity when combined with chemotherapy agents (doxorubicin or dexamethasone) or targeted agents (bortezomib or carfilzomib). On going studies are aimed to study the mechanisms by which c-myc inhibition results in cell death in RSCL and RRCL. JQ1 is a distinct targeted agent undergoing clinical evaluation in patients with relapsed/refractory lymphomas. Molecular studies dissecting the cellular pathways affected by JQ1 are important in order to further advance the clinical development of c-myc inhibitors in lymphoid malignancies. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund)

Disclosures

Czuczman: Boehringer-Ingelheim: Other: Advisory Board; Immunogen: Other: Advisory board; MorphoSys: Consultancy; Celgene: Employment.
In Vitro and In Vivo Characterization of CD19/CD3 Tandab AFM11 and CD19/CD16A Tandab AFM12 Targeting NHL
Authors: Zhao Xing , Rajasekaran Narendiran , Reusch Uwe , Weichel Michael , Ellwanger Kristina , Marschner Jens-Peter , Treder Martin , Kohrt Holbrook
Published: October 2019

Introduction: CD19 is expressed by B cells from early development through differentiation into plasma cells, and represents a validated target for the development of therapeutic antibodies to treat B cell malignancies such as Non Hodgkin Lymphoma (NHL) and acute lymphoblastic leukemia (ALL). Different CD19-targeting T-cell engagers are investigated in clinical studies for the treatment of NHL or ALL, including Affimed's AFM11, a bispecific CD19/CD3 TandAb antibody, which is currently investigated in a phase 1 dose escalation study. Indeed, Affimed's bispecific tetravalent platform comprises not only T-cell engaging TandAbs with two binding sites for CD3, but also NK-cell recruiting TandAbs with two binding sites for CD16A. In the present study, Affimed's AFM11, was characterized and compared in in vitro and in vivo studies with the CD19/CD16A TandAb AFM12.

Methods: Analogous to the CD19/CD3 TandAb AFM11, a bispecific tetravalent TandAb AFM12 was constructed with two binding sites for CD19 and two sites for CD16A. Both TandAbs were characterized side by side for their biophysical properties, binding affinities to CD19+ tumor target cells and to their respective effector cells by flow cytometry. Kinetics and dose-response characteristics were evaluated in in vitro cytotoxicity assays. Potency and efficacy of both TandAbs were compared on different CD19+ tumor target cell lines using primary human effector cells. To compare the efficacy of AFM11 and AFM12 a patient-derived tumor xenograft model was developed.

Results: AFM12 mediated efficacious target cell lysis with a very fast on-set in vitro. Lysis induced by AFM11 was less efficacious (lower specific lysis than AFM12) but reproducibly more potent (lower EC50 value). In addition to the potency and efficacy of AFM11 and AFM12, different aspects of safety, such as effector cell activation in the presence and absence of target cells were investigated and will be described.

Conclusions:

Affimed's CD19/CD3 and CD19/CD16A TandAbs with identical anti-CD19 tumor-targeting domains but different effector cell-recruiting domains represent interesting molecules to study T-cell- or NK-cell-based immunotherapeutic approaches. The comparison of AFM11 and AFM12 demonstrated that AFM12-mediated lysis was fast and efficacious, whereas AFM11 showed a higher potency. In summary, the NK-cell recruiting TandAb AFM12 represents an alternative to T-cell recruiting molecules, as it may offer a different side effect profile, comparable to that of AFM13, the first NK-cell TandAb clinically investigated.

Disclosures

No relevant conflicts of interest to declare.
Preclinical Analysis of Linsitinib for the Treatment of Polycythemia Vera
Authors: Basu Titiksha , Gruender Albert , Dierks Christine , Pahl Heike L
Published: October 2019

Even after the discovery of JAK2V617F in majority of Polycythemia Vera (PV) patients and the generation of JAK2 inhibitors, the treatment of PV remains to be improved. An increased understanding of aberrant signaling in PV cells may yield novel drug targets for this patient population. The formation of erythroid colonies in the absence of exogenous erythropoietin (Epo), so called Epo-independent erythroid colonies (EECs), is a pathognomonic hallmark of PV. Over 20 years ago, Axelrad and colleagues proposed that EEC formation is not due to Epo-independence. Rather, they postulated that PV cells are hypersensitive to Insulin Growth Factor-1 (IGF-1). Subsequently, increased IGF-1 receptor (IGF-1R) phosphorylation was demonstrated in PV MNCs. In addition, serum concentrations of IGF-1 binding protein (IGF-1BP) are significantly elevated in PV patients. It has been shown that IGF-1 cooperates with JAK2V617F to increase JAK/STAT signaling, elevating constitutive phosphorylation of STAT factors and thereby augmenting aberrant proliferation. We therefore hypothesized that inhibition of IGF-1 signaling would ameliorate the PV phenotype, even in the presence of JAK2V617F.

The dual IGF-1R and insulin receptor (IR) kinase inhibitor linsitinib (formerly called OSI-906) was used to investigate the contribution of the IGF-1 pathway to PV pathophysiology. In methylcellulose colony assays, linsitinib significantly reduced the EEC formation from PV MNCs. In contrast, in the presence of EPO, colony formation from either PV or healthy control MNCs was not affected. These data suggest that linsitinib specifically inhibits growth of the PV clone. Linsitinib likewise significantly reduced the cytokine independent proliferation of Ba/F3 cells expressing JAK2V617F but did not affect proliferation of Ba/F3 cells expressing either JAKWT or JAK2V617F in the presence of cytokine (mIL3). These data again argue that IGF-1 signalling is required for aberrant, cytokine independent growth. In addition to reducing proliferation, Linsitinib also induced apoptosis in IL3-independent JAK2V617F transduced Ba/F3 cells. Using the JAK2V617F bone marrow transplant (BMT) model, we investigated the effect of linsitinib on the PV phenotype in vivo. Linsitinib treatment significantly reduced the elevated RBC count, hematocrit and hemoglobin in this PV mouse model. In addition, the white blood cell count was decreased.

The data presented here provide a pre-clinical rationale for expanding the investigation of linsitinib as well as similar IGF-1 pathway inhibitors for the treatment of PV. The effect of combining JAK2 and IGF-1 inhibition may prove especially promising in this patient population and is currently being investigated.

Disclosures

No relevant conflicts of interest to declare.
A Novel Erythroid Regulator MLF1IP Promotes Proliferation in Mice By Regulating Cell Cycle
Authors: Zhang Tianjiao , Ma Xiaotang , Li Bing , Huang Gang , Gale Robert Peter , Zhang Yue , Xu Zefeng , Qin Tiejun , Prchal Josef T. , Xiao Zhijian
Published: October 2019

Purpose: MLF1IP is a novel constitutive kinetochore component essential in physiological progress of cell cycle. Mouse homologue appears to be lineage-specifically expressed, but little is known about how it functions in hematopoiesis and whether it is involved in hematopoietic disorders. The goal of the study was to confirm biological function of MLF1IP in human K562 erythroleukemic cells, analyzed hematological abnormalities in MLF1IP transgenic mice and potential mechanisms underlying these abnormalities, and evaluated MLF1IP expression in erythroid colonies from PV and MDS.

Methods: MLF1IP was down-regulated by lentivirus in K562 cell line. The proliferation of K562 cells was analyzed by cell colony formation assay. VP16 was used to induce apoptosis of K562 cells. The entire coding region of human MLF1IP was cloned into the HS21/45-vav plasmid to generate transgenic mice, which were crossed with wild-type C57BL/6J mice, negative siblings of transgenic mice were used as controls. Genotypes of the transgenic mice were identified by PCR of tail genomic DNA with primers specific for transgenic MLF1IP sequence. Relative quantitative real-time PCR was used to detect mRNA level in BM cells. Western blot were carried out to check the expression of cell cycle associated proteins. Flow cytometry analyses were used to assess cell cycles and proportions of different stages of erythroblast populations. Absolute quantitative real-time PCR were used to examine MLF1IP expression in colonies from healthy donors and subjects.

Results: MLF1IP knock-down K562 cells are arrested in G2/M and have reduced colony-forming activity. MLF1IP knockdown had no detectable effect on apoptosis in K562 cells, while significantly enhanced etoposide-induced apoptosis was seen in MLF1IP-knockdown K562 cells at 48 h after treatment compared with that in same treated control cells. MLF1IP transgenic mice show increased hemoglobin, with normal levels of platelets and neutrophils. Erythroid progenitors and early-stage erythroblasts in bone marrow are markedly increased. These mice have increased cyclin D2, reduced p27 and p21 and increased cycling, suggesting that enhanced MLF1IP expression leads to erythroid expansion via accelerating cell cycle progression in bone marrow cells. Interestingly, MLF1IP expression in colony forming unit-erythropoiesis (CFU-E) is increased in polycythemia vera (PV) and decreased in low-risk myelodysplastic syndrome (MDS), consistent with erythropoiesis characteristics of these disorders.

Conclusion: Our data demonstrate that MLF1IP expression promotes erythroid proliferation via regulating cell cycle status of erythroid cells, implicating abnormal MLF1IP levels in erythropoiesis disorders. Correction of MLF1IP expression could be a novel therapy target in diverse dysfunctional erythropoiesis conditions.

Disclosures

No relevant conflicts of interest to declare.
Regulation of Transcription Factors Foxp1, E2A and Egr-1 By microRNA-191 Constrains B Lymphocyte Development and Suppresses Development of Diffuse Large B Cell Lymphoma
Authors: Lyszkiewicz Marcin , Blume Jonas , Zietara Natalia , Witzlau Katrin , Puchalka Jacek , Saran Namita , Duber Sandra , Bishnudeo Roy , Weiss Siegfried , Krueger Andreas
Published: October 2019

Development of B-cells in the bone marrow is a tightly controlled, multi-step process. Whereas its major transcriptional regulators are well characterized, less is known about its post-transcriptional regulation. It has been demonstrated that B cell development critically depends on microRNAs. Yet, the role of individual members still remains to be uncovered.

Relatively subtle regulatory effects of miRNAs are well-visible in the pathways which require tight control. Disruption of such a pathway, where the protein function (i.e. transcription factor) highly depends on the gene dosage, is major cause of malignant transformation.

We identified transcription factors Foxp1, E2A and Egr1 as direct targets of microRNA miR-191. Reduced expression of these transcription factors through ectopic expression of miR-191 in B-cell progenitors resulted in numerous secondary effects, such as downregulation of Rag1, Rag2, Dntt and Il7r alpha genes. In consequence, we observed impaired immunoglobulin heavy chain rearrangement and limited responsiveness to IL-7 of B cell progenitors resulting in partial developmental arrest and predisposition of immature B-cells to death during negative selection. Finally, we showed that co-overexpression of E2A or FoxP1 and miR-191 restored normal B cell development, supporting the hypothesis that both transcription factors are functionally relevant targets of miR-191.

Having established a regulatory cascade comprising miR-191 at the top of hierarchy and the transcription factors Foxp1, E2A and Egr1 as its direct targets we sought to identify a pathophysiological scenario in which this cascade might be explored therapeutically. Given that the most aggressive forms of human diffuse large B cell lymphoma (DLBCL) are FOXP1 dependent, we assessed whether miR-191 was able to limit growth of DLBCL. Of note, in vivo ectopic expression of miR-191 prevented the growth of DLBCL. Consistent with murine data, expression of the three target genes FOXP1, EGR1 and TCF3 (encoding E2A) was decreased upon miR-191 overexpression. Interestingly, treatment with the histone deacetylase inhibitor vorinostat revealed decreased resistance to cell death of DLBCL lines overexpressing miR-191 in vitro.

Thus, our data describe a new regulatory pathway, where miR-191 by fine-tuning key B-lineage transcription factors acts as modulator of B-cell development as well as tumor suppressor in B-lineage malignancy.

Disclosures

No relevant conflicts of interest to declare.