bluuebunny/crossref_metadata_2025 huggingface dataset:
Is Initial CD3+ T Cell Dose in Donor Lymphocyte Infusion for Relapsed Hematological Malignancies a Prognostic Determinant?
Authors: Atilla Erden , Ataca Pinar , Toprak Selami Kocak , Yuksel Meltem Kurt , Topcuoglu Pervin , Arslan Onder , Ozcan Muhit , Demirer Taner , Ilhan Osman , Beksac Meral , Akan Hamdi , Gurman Gunhan , Civriz Bozdag Sinem
Published: October 2019

Introduction: Patients after relapsing allogeneic hematopoietic stem cell transplantation (AHSCT) have a treatment option of donor lymphocyte infusion (DLI). DLI is a form of adoptive immunotherapy which induces graft versus leukemia effect. Severe forms of graft versus host disease (GVHD) and marrow aplasia are known complications. In this study, we aimed to detect the effect of the infused CD3+ T cell dose on response, GVHD and overall survival (OS) of hematological malignancies after AHSCT.

Methods: We retrospectively evaluated 78 DLI procedures in 57 patients with different hematological malignancies for relapse after AHSCT from June 2000 through June 2015. Initial DLI CD3+ cell dose/kg recipient body weight was ≤ 1x10^7 (n=20; Group 1), >1.0 to ≤ 5 x10^7 (n=29; Group 2), 5-10 x10^7 (n=29; Group 3). Chi-square test was used in comparison between groups. P<.05 was considered statistically significant.

Results: The median age during DLI was 33,8 ± 13,2 (range 16-67 years) in 57 patients (38M/19F). The median time interval from HCT to DLI was 8.08 (2-69) months. Ten patients (18%) had bone marrow while 47 patients (82%) had peripheral blood as stem cell source. All patients received transplants from HLA-matched related siblings. There was no difference found between the three groups according to age, diagnosis, stem cell source and conditioning regimen. Achieving complete remission after DLI was detected in thirty two (56%) patients. Disease status after DLI, acute and chronic GVHD rates were not statistically different between the groups. Overall survival which was not statistically significant according to initial DLI cell dose at 2 years were 25%, 38% and 24%, respectively.

Conclusion: An initial DLI CD3+ cell dose of 10 x10e7 or higher has been shown improve overall survival but increase GVHD rates with no effect on risk of relapse in previous reports. Response rate, GVHD and survival outcomes were found to be similar in comparison of three different DLI dose groups in our study.

Disclosures

No relevant conflicts of interest to declare.
Related or Unrelated Donor Results in Similar Outcomes after Hsct; Single Center Experience in Colombia
Authors: Abello Virginia , Rosales Carmen , Rosales Manuel , Figueroa Javier , Cordoba Iris , Borda Molina Andres Armando , Pedraza Enrique , Esguerra Herman , Villamizar Liceth
Published: October 2019

INTRODUCTION

ASTC is the standard treatment in multiple hematologic diseases; unfortunately most patients do not have a compatible family donor, URD ASCT is an alternative to those patients. There is evidence from developed countries that outcomes may be similar regardless the donor, but there is not enough information from Latin America regarding survival after URD ASCT. We compared survival at 1 and 3 years from patients transplanted using related or unrelated donors in a single center in Colombia.

MATERIALS AND METHODS

Paired analysis was performed comparing one URD ASCT with one or two RD ASCT with similar characteristic in terms of age, diagnosis (AML, ALL, aplasia, CML, MDS), EBMT risk, year of transplant and conditioning regimen. Chi-square or Fisher exact test was used to compare the groups (p<0.05). The survival curves of Kaplan-Meier and log-rank test (p<0.05) were used to estimate the probabilities of one (OS1) and three (OS3) overall survival in different groups according to EMBT score risk.

RESULTS

28 URD ASCT were performed between 2011 and 2014, 24 of those (18 matched and 6 missmatched URD ASCT) were paired with 41 RD ASCT. Of those 62% were males, mean age was 33.4 years, 70.8% patients were in early stages, almost 60% had more than 12 months interval between diagnosis and transplant. Most common diagnoses were acute lymphoid leukemia and acute myeloid leukemia. 80% of donors where found through NMDP. No significant differences in sex, age, disease status (early, intermediate, late), time interval from diagnosis to transplantation, and patient-donor sex combination, ranked according to EBMT risk score, were find between the tow groups.

Median follow-up was 322 days (range 165-693), for the whole group. There were no significant differences in the OS1 or OS3 between URD o RD ASCT, in different EBMT score groups. Low risk: OS1: URD 86.6% vs RD 75.8%, p = 0.46; OS3: 86.6% vs 68.9%, p = 0.28. High risk OS1 75% vs 63.6%, p=0.92; OS3 75% vs 63.6%, p=0.92.

CONCLUSIONS

In this cohort of patients, the type of donor had no influence in the outcome measured in terms of OS. This is the first experience with URD ASCT in Colombia; longer follow up of this cohort is needed to confirm this results, but our findings suggest this transplantation modality is a real alternative to patients in our country in need of a transplant without an HLA identical related donor.

Disclosures

No relevant conflicts of interest to declare.
Influence of Pretransplantation Serum Ferritin on Children Beta-Thalassemia Major Undergoing Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Ruan Yongsheng , Wu Xuedong , Feng Xiaoqin , He Yuelin , Li Chunfu
Published: October 2019

Objectives: To evaluate the influence of pretransplantation serum ferritin on children β-thalassemia major (β-TM) undergoing allogeneic hematopoietic stem cell transplantation.

Methods: A retrospective analysis of 266 HLA-matched children with β-TM from January 2009 and November 2014 was performed. Transplantation conditioning regimen of these children was the NF-08-TM protocol. Median follow-up time was 28 months (3~62months). We observed the relationship between pretransplantation serum ferritin level and transplantation complications which included infection, graft versus host disease(GVHD),veno-occlusive disease(VOD) and death.

Results: Transplantation-related death occurred in 18 of 266 patients (6.8%). Five-year overall survival (OS) was found to be 92.8%. Among various complications, only infection was significantly associated with the high serum ferritin level (t=-2.673, P=0.008), especially serum ferritin above 3449.5μg/L(P=0.000). Meanwhile infection was the most common complication and severe infection would be main cause of deaths.

Conclusions: NF-08-TM conditioning regimen was the optimization for HLA-matched β-TM patients. High pretransplantation serum ferritin level would bring high infection occurrence.

Disclosures

No relevant conflicts of interest to declare.
The Thrombotic Microangiopathy Registry of North America
Authors: Metjian Ara , Tanhehco Yvette , Pham Huy Phu , Aqui Nicole A. , Bhoj Vijay , Onwuemene Oluwatoyosi , Marques Marisa , Arepally Gowthami M
Published: October 2019

The thrombotic microangiopathies (TMAs) are rare, life-threatening thrombotic disorders of diverse etiologies. Systematic studies of TMA have been difficult to perform due to their rare occurrence, disease heterogeneity and, lack of an organized research network in the United States. Whereas a multi-institutional approach has been used in Europe and Canada, TMA research in the US has been largely single-center based. To overcome the limitations of single institutional registries and to expand the number of TMA patients available for study, four academic centers convened during the 2013 ASFA annual meeting to establish the TMA Registry of North America (TRNA) with the following goals: (1) design and develop a registry to define "best practices" for the diagnosis, treatment, and management of TMA; (2) develop a platform for conducting observational and interventional clinical trials; (3) and, establish a national bio-repository of samples from patients with TMA to facilitate future studies. This abstract reports the first multi-institutional network in the United States designed to study TMA. Members met through bimonthly tele-conferences to develop a clinical registry using REDCap (Research Electronic Data Capture), a HIPAA-compliant, internet-based software program for data entry. To facilitate a cohesive and streamlined review of IRB applications, the TRNA utilized IRBshare, a portal for rapid approval of multi-site investigations. IRB consent included participation in a bio-repository arm for collecting blood and apheresate. Following approval through the Duke IRB in June 2014, study documents were uploaded to the IRBshare website. Since August 2015, 16 study participants with TMA have been consented, 13 of which have had their clinical information entered into the database. Preliminary data shows our population is 73% African-American and female. Average laboratory values at presentation included hemoglobin of 9.7 g/dL, platelets 53 x10^9/L, and LDH 1,150 u/L. ADAMTS13 testing was performed in 77% (10/13), of which 60% (6/10) measured at <10%. Current efforts include expansion of network sites to expand the registry and to develop clinical protocols for future studies. With its clinical and IRB infrastructure in place, the TRNA, which is the first U.S. research network designed to study TMA, is poised to perform cooperative observational and interventional trials in the near future.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Secondary Malignancies in Mantle Cell Lymphoma
Authors: Talakokkla Sandhya , Mahatara Renuka , Welch Christine A , Nagarajan Arun
Published: October 2019

Background: Mantle cell lymphoma is a B cell lymphoma (CD5 +) which represents 5-10 % of Non-Hodgkin's lymphoma. Most patients have advanced disease at presentation and thus carry a poor prognosis. This type of uncommon malignant lymphoma has a distinct and recurring cytogenetic abnormality involving t(11, 14) q(13, 32). Although extra nodal involvement is common, not many cases of MCL having concomitant other malignancies are reported. We hypothesized that patients with MCL have chronic immunosuppression, comparable to chronic lymphocytic leukemia patients, and therefore are at risk for developing secondary malignancies similar to CLL patients. The aim of our study is to report the retrospective analysis of patients diagnosed with MCL and the associated secondary malignancies before or after the diagnosis of MCL.

Patients and methods: The records of 41 patients who presented with MCL to "The David Lee Cancer Center" at CAMC, WVU with MCL from 2000 - 2015 were retrospectively reviewed. The number of other malignancies presenting before or after the diagnosis of MCL were analyzed.

Results: The population with MCL was represented by 41, all Caucasian patients, which were 75.6% male (n = 31) and had an average age of 68.6 ± 11.3 years. Mean follow up for all patients was 23.9 ± 32.43 months. A total of 14.6% (n=6), 83.3% males, experienced a second primary. Within the second primaries 50.0% were GI cancers which included pancreatic cancer and cholangiocarcinoma while 16.7% were prostate cancer, diffuse large B-cell lymphoma or adenocarcinoma of lungs. The time to second primaries varied with 50% of the cases being diagnosed simultaneously with MCL, while 33.3% were diagnosed prior to MCL at an average time of 34.5 months, and 16.7% were diagnosed post MCL diagnosis at an average time of 18 months.

Conclusion:

Patients with MCL are typically CD5 positive, CD10 negative, and CD23 negative which make it different from other lymphomas like small cell lymphoma, B-cell CLL that are CD 23 positive. Rare cases of MCL may be CD5 negative or CD23 Positive. We hypothesized that MCL patients have an increased risk for developing secondary cancers due to their disease biology and from underlying chronic immunosuppression. Patients with MCL have twice the risk of developing secondary malignancies and an increased frequency of certain types of cancers such as cholangiocarcinoma & pancreatic cancer.

Disclosures

No relevant conflicts of interest to declare.
Therapeutic Efficacy of Anfibatide in a Murine Model of Thrombotic Thrombocytopenic Purpura
Authors: Zheng Liang , Mao Yingying , Li Mandy , Dai Xiangrong , Li Benjamin , Zheng X. Long
Published: October 2019

Thrombotic thrombocytopenic purpura (TTP) is a rare but potentially fatal blood disorder, resulting from severe deficiency of plasma ADAMTS13 activity. Plasma infusion or exchange is the mainstay of treatment. However, complications associated with the administration of blood products and with the placement of a central venous line remain to be a major concern. To address the unmet clinical need, we test hypothesis that anfibatide, a novel snake venom-derived glycoprotein (GP) Ib antagonist, may be efficacious in treating TTP. Here, we show that purified anfibatide from snake venom inhibited murine platelet agglutination induced by botrocetin in the presence of recombinant murine VWF in a concentration-dependent manner. At 6-9 micro grams per ml (final concentrations), anfibatide nearly completely abolished botrocetin-induced murine platelet agglutination (Fig. 1A). As expected, anfibatide at the same concentrations showed little or no inhibitory effect on the protease-activating receptor 4 (PAR4) agonist (AYPGKF)-induced murine platelet aggregation. In vivo, an intravenously infused radiolabeled anfibatide exhibited a half-life of 5-7 hours depending on the initial loading dose. This information provided pharmacokinetic basis for therapeutic strategy for TTP in the murine model. An infusion of purified anfibatide at the dose of 60-90 ng per gram of body weight twice daily significantly reduced the rate of thrombocytopenia in Adamts13-/- mice triggered by intravenous infusion of a bacterial toxin, i.e. shigatoxin-2 (Stx2) (250 pg per gram body weight). As shown, 5/5 (100%) Adamts13-/- mice after being challenged with Stx2 and but treated with normal saline (control) exhibited severe thrombocytopenia (defined by 50% reduction of platelet counts from the baseline). Similarly, treatment of Adamts13-/- mice with anfibatide at the dose of 30 ng per gram body weight twice daily had little or no effect in preventing thrombocytopenia. However, treatment of the same mice with anfibatide at doses of 60-90 ng per gram body weight twice daily resulted in significantly reduced rate of severe thrombocytopenia to 11%-37.5% (Fig. 1B). Our ongoing effort is to determine the efficacy of anfibatide in treatment of acquired TTP associated with inhibitors. We conclude that anfibatide when given at the optimal doses and interval can efficiently prevent Stx2-induced TTP syndrome in the murine model. These findings support a rationale for further development of anfibatide as a novel therapeutic for TTP in humans.

Disclosures

Li: 3Lee's Pharmaceutical Holdings Limited, Shatin, Hong Kong: Employment. Dai:4Zhaoke Pharmaceutical Co. Limited, Hefei, Anhui, China: Employment. Li:3Lee's Pharmaceutical Holdings Limited, Shatin, Hong Kong, China: Employment.
Exosome-Transferred APOC3-Ncrna Complex Mediates Iron Regulation in Beta-Thalassemia Major
Authors: Peng Ching-Tien
Published: October 2019

Background: Exosomes are small membrane vesicles (50-90 mm in diameter) containing bioactive proteins and genetic materials that may be transferred to accept cells, resulting in potent biological effects in the circulatory system. Exosome-transferred APOC3-ncRNA may be a crucial function in iron regulation. However, the role in β-thalassemia major is remains unclear.

Aim: The aim of this study was to investigate how Exosome-transferred APOC3-ncRNA adapts to iron regulation in β-thalassemia major.

Design: Using Proteomics, RNA-sequencing and lncRNA Q-PCR array, we demonstrated expression of exosomes-transferred APOC3-ncRNAs in plasma of β-thalassemia major patients (n = 40). Identification of exosome by Dynamic light scattering (DLS), Flow cytometry and western blots. Lentiviral infection, ncRNAs transfection and in vitro studies revealed that knockdown or overexpression obviously increased or ameliorated exosomes-induced iron regulation. Bioinformatics analysis, luciferase assay and in vitro studies revealed that ncRNAs functioned as a repressing mediator and formed a feedback loop with ncRNAs and target gene to mediate iron regulation.

Results: We observed that ncRNA is differential expressed in the exosomes of β-thalassemia major. We analyzed the effects of ex vivo-derived exosomes for iron regulation by monocytes/hepatocytes model. In vitro model showed that exosomes were internalized by THP-1/HuH-7 cells, and regulates the IRP1/FPN/HAMP. In turn, Exosome-transferred APOC3-ncRNA mediates inflammatory cytokine expression and iron regulation via p38/pAKT/TRAF6/NF-kB pathway. Knockdown of APOC3-ncRNA reversed IRP1/FPN/HAMP expression.

Conclusions: This is the first example of extracellular APOC3/ncRNA regulating gene expression via circulatory exosomes-transferred to accept cells. Increased Exosome-transferred APOC3-ncRNA expression is one of the key factors that might have contributed to abnormal iron regulation in β-thalassemia major patients.

Disclosures

No relevant conflicts of interest to declare.
Single Cell RNA seq for Analysis of Cell Fate Decisions
Authors: Grimes H. Leighton , Harinder Singh , Olsson Andre , Salomonis Nathan , Aronow Bruce J. , Chaudhry Viren
Published: October 2019

Single-cell RNA-Seq has the potential to become a dominant approach in probing diverse and complex developmental compartments. Its unbiased and comprehensive nature could enable developmental ordering of cellular and regulatory gene hierarchies without prior knowledge. To test general utility we performed single-cell RNA-seq of murine hematopoietic progenitors focusing on the myeloid developmental hierarchy. Using novel unsupervised clustering analysis, ICDS, we correctly ordered known hierarchical states as well as revealed rare intermediates. Regulatory state analysis suggested that the transcription factors Gfi1 and Irf8 function antagonistically to control homeostatic neutrophil and macrophage production, respectively. This prediction was validated by complementary genetic and genomic experiments in granulocyte-macrophage progenitors. Using knock-in reporters for Gfi1 and Irf8 and clonogenic analyses coupled with single-cell RNA-seq we distinguished regulatory states of bi-potential progenitors from their lineage specifying or committed progeny. Thus single-cell RNA-Seq is a powerful developmental tool to characterize hierarchical and rare cellular states along with the regulators that control their dynamics.

Disclosures

No relevant conflicts of interest to declare.
PML-Rara Drives Acute Promyelocytic Leukemia Genesis By Enhanceosome Depletion Leading to 3D Chromatin Reorganization
Authors: Wang Ping , Tang Zhonghui , Zhang Hui , Ruan Yijun
Published: October 2019

Dynamic changes in chromatin looping structure are identified to reflect interactions of enhancers to target promoters. Such changes modulate hematopoietic lineage differentiation and are known to be mediated by some critical transcription factor, such as GATA1 and MYC. How chromatin topology changes in cancer such as leukemia is poorly understood on a global genomic view. Moreover, how oncogenic transcription factors, such as PML-RARA, AML1-ETO and others, manipulate gene regulation by globally altering the topology of chromatin, which contribute to leukemogenesis in acute myeloid leukemia (AML) remains to be described. Here we identified PML-RARA involved chromosomal interactions spanning hundreds of kilobases between promoters and distal regulatory elements. The RNAPII mediated chromatin connectivity between transcriptionally active genes and its distal regulatory elements were lost in the reorganized chromatin structure caused by PML-RARA, while the chromatin topological associated domain mediated by CTCF remained stable. PML-RARA mediated chromatin loops eliminated the occupancy of myeloid specific transcription factors (such as PU.1, IRF1 and CEBPB et al.) as well as coordinately general transcriptional factors (such as P300 and RNAPII) at the distal regulatory elements and respective promoters. Finally we show that interacting loci designated as super enhancers become depleted by PML-RARA looping to suppress myeloid differentiation regulomes. These distinct results based on 3D genome architecture uncover novel modes of how PML-RARA disrupts the regulome of myeloid differentiation to contribute to leukemogenesis. This study provides new insights and accessible tools to delineate the aberrant regulome originated from the chaotic gene regulatory interactions leading to cancer genesis.

Disclosures

No relevant conflicts of interest to declare.
Inhibition of GSK-3 Signalling Enhances Sensitivity of Non-Promyelocitic Acute Myeloid Leukemia (AML) Cell to Chemotherapy
Authors: Takam Kamga Paul , Dal Collo Giada , Cassaro Adriana , Adamo Annalisa , Gatti Alessandro , Carusone Roberta , Di Trapani Mariano , Midolo Martina , Resci Federica , Bonifacio Massimiliano , Krampera Mauro
Published: October 2019

Background: GSK-3 is a serine-threonine kinase involved in metabolic regulation as well as in the control of many pathways associated to cancer development, including Notch Wnt/β-catenin, Hedgehog, and AKT. It has been demonstrated that association of GSK-3 inhibitors with All-trans-retinoic acid (ATRA) significantly improves ATRA-mediated differentiation and cell death of acute promyelocytic (APL) leukaemia cells. However, little is currently known about the contribution of GSK-3 role to non-promyelocytic AML cell response to treatment with chemotherapeutic agents.

Aims: In this study, we aim to validate GSK-3 signalling as potent successful therapeutic target in non-promyelocytic AML. For this purpose we tested different GSK-3 for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatments.

Methods: GSK-3 expression was analyzed by Western blot or flow cytometry inAML cell lines (HL-60, THP1, U937) or primary non-promyelocyticAML blast cells (30 samples). AML cellscultured alone or in presence ofhuman bone marrow mesenchymal stromal cells (hBM-MSCs) were treated with GSK-3 inhibitors, including LiCL, AR-A014418, SB 216763, in association or not with Cytarabine (Ara-C) or Idarubicin. Cell proliferation and cell death were measured by CFSE dilution and TOPRO-3/Annexin-V staining, respectively.

Results: Flow cytometry and Western blot analysis in AML samples revealed high expression levels of all GSK-3forms, including total GSK-3α, (Ser21) GSK-3α, total GSK-3β, and (Ser 21) GSK-3β; theseforms were all down-modulated when AML cells were cultured in presence of hBM-MSCs, thus suggesting that GSK-3 plays an important role in transducting micro-environmental signals in AML cells interacting with bone marrow stroma. The treatment of AML cells with increasing concentrations of each GSK-3 inhibitors decreased AML cell viability in a dose-dependent manner; interestingly, hBM-MSCs or peripheral blood mononuclear cells were less sensitive to GSK-3inhibitors. The addition of each inhibitor increased dramatically the AML cell apoptotic rate induced by the addition of Ara-C or Idarubicin in vitro. Notably, LiCl and AR-A014418 were capable of abrogating hBM-MSC-mediated AML cell resistance to apoptosis induced by Ara-C or Idarubicin.

Conclusion: Overall our data clearly demonstrated that inhibition of GSK-3 reduced proliferation and chemoresistance of non promyelocytic AML cells. Thus GSK-3 inhibition represents a therapeutic strategy not only for APL but also for other AML subtypes.

Disclosures

Bonifacio: Ariad Pharmaceuticals: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Amgen: Consultancy.
Expression of MPL in Leukemia Stem Cells and Its Role in Stemness Maintainance
Authors: Li Huan , Rao Qing , Yu Pei , Chen Shuying , Li Zheng , Xing Haiyan , Wang Min , Wang Jianxiang
Published: October 2019

Osteoblast cells play an important role in bone marrow niche. The interaction between osteoblast and leukemia cells promotes leukemia development, which is mediated by some cytokines including TPO. It has become evident that TPO-MPL signaling is essential for the quiescence and self-renewal of hematopoietic stem cells, however, its expression pattern and the role in leukemia stem cells have not been reported. This study was aimed to determine the expression of MPL in acute myeloid leukemia (AML) and investigate the role of MPL in the leukemia stem cells' quiescence, drug resistance and self renewal.

The expression levels of CD34, CD38 and MPL were detected by flow cytometry in bone marrow cells from 57 newly diagnoses AML patients. The correlation between MPL and CD34, CD38 expression in AML patients were analyzed. The results showed that expression of MPL in AML patients was higher significantly than that in 13 normal donors (P<0.05). Expression of MPL in CD34 positive AML patients was obviously higher than that in CD34 negative AML patients (P<0.01). MPL was higher expressed in CD34+ cells than that in CD34- cells significantly (P<0.0001). We also detected the expression of MPL in different populations of leukemia cells in AML1-ETO9a mouse leukemia model established in our lab. We found that the ratios of MPL positive cells in Lin-c-kit+ and Lin-c-kit+sca-1+ populations were significantly higher than that in total leukemia cells. In addition, in chemotherapy treated AML1-ETO9a mice, the proportion of Lin-c-kit+MPL+ leukemia cells were increased 23.5 folds than that in untreated leukemia mice, which indicates that MPL+Lin-c-kit+ LSCs population could be enriched by chemotherapy. Furthermore, MPL+ and MPL- cells in Lin-c-kit+ leukemia population were sorted by flow cytometry and the colony formation and quiescence state were determined. The results showed that MPL+Lin-c-kit+ cells produced significantly more colonies in the second round of colony formation (p<0.05) than MPL-Lin-c-kit+ cells. The G0 phase accumulation of MPL+Lin-c-kit+ cells was significantly higher than that of MPL-Lin-c-kit+ cells (p<0.01). Above results indicate that MPL+ leukemia cells display more clonogenic potential and maintain quiescence.

These data demonstrate that as a receptor of TPO, MPL is highly expressed in leukemia stem cells and MPL positive leukemia stem cells could be enriched by chemotherapy. MPL positive leukemia stem cells exhibit more clonogenic potential, quiescence and drug resistance. It suggests TPO-MPL mediated interaction of osteoblast and leukemia cells take a role in the stemness of leukemia stem cells.

Disclosures

No relevant conflicts of interest to declare.
RUNX1 Is a Candidate Transcriptional Effector in Juvenile Myelomonocytic Leukemia
Authors: Dorantes Acosta Elisa , Huang Hui , Garcia Sara P. , Stieglitz Elliot , Loh Mignon L. , Yuan G C , Cantor Alan B.
Published: October 2019

Juvenile Myelomonocytic Leukemia (JMML) is an aggressive pediatric myeloproliferative neoplasm. Mutations leading to constitutive activation of the non-receptor tyrosine phosphatase Shp2 (PTPN11) and/or RAS signaling are driver events. Yet, the key downstream transcriptional effectors of this aberrant signaling have not been defined. We previously found that Shp2 dephosphorylates the hematopoietic transcription factor RUNX1, leading to its activation. Overexpression of a mutant RUNX1 molecule that mimics constitutive RUNX1 tyrosine dephosphorylation in murine hematopoietic stem/progenitor cells led to massive expansion of myelomonocytic cells and growth under low cytokine conditions. Likewise, RUNX1 is known to be activated by extracellular signal-related kinase (ERK) mediated phosphorylation, which is downstream of activated RAS. To further investigate the role of RUNX1 in JMML, we analyzed gene expression datasets derived from CD34+ cells from JMML patients versus healthy controls. Thirty-four genes were down regulated and 13 were up regulated in the JMML versus control CD34+ cells using a cut-off of a log2-fold change > 0 and false-discovery rate (FDR) adjusted p-value ≤ 0.1. H3K27Ac and K3K4Me1 peaks located between -50 kb 5' of the transcriptional start site (TSS) to +50 kb 3' of the transcriptional end site (TES) of the differentially expressed genes were used to identify putative enhancer elements. DNA-binding factor motif enrichment analysis was then performed at these regions. This revealed a significant enrichment for RUNX DNA consensus binding sequences in the putative enhancer elements (p-value 8.05e-03). There was also significant enrichment for ETS family transcription factors, including Spi1 (PU.1) (p-values 1.94e-06 to 1.21e-03) and for Serum Response Factor (SRF) (p-value 1.11e-04). ETS factors frequently physically and functionally cooperate with RUNX transcription factors. Analysis of the proximal promoters showed only enrichment for ETS factors (p-value 2.42e-04 to 4.02e-03) and BATF::JUN (4.64e-03). Examination of ChIP-seq datasets for RUNX1 and PU.1 in CD34+ cells from healthy controls demonstrated that many of these putative sites are occupied by RUNX1, and some with PU.1. Overall, this data supports a model in which RUNX1 (and likely ETS factors) are important transcriptional effectors of aberrant cell signaling in JMML.

Disclosures

Loh: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.
NPM1 Mutation Contributes to Hematological Dysfunction By Disrupting H3K79 Methylation
Authors: Xu Jie , Zhang Wu , Yan Xiaojing , Zhao Chen , Zhu Jiang , Chen Zhu , Chen Sai-Juan , Hu Jiong
Published: October 2019

NPM1 is one of the most frequent acquired mutated genes in acute myeloid leukemia (AML). Previous studies have shown that NPM1 mutation (NPMc+) established the distinctive gene expression signatures, which were associated with mixed lineage leukemia (MLL)-target genes, like MEIS1 and HOXA cluster. In AML carrying MLL fusion-oncoproteins, DOT1L-mediated histone 3 lysine 79 (H3K79) methylation is implicated in the regulation of MLL-target genes. Compared with MLL abnormalities, NPM1 variants preserve the similar transcriptional characteristics. However, whether NPM1 mutation could affect the histone modification of H3K79 methylation is unknown. In this study, we showed that NPM1 mutation dysregulated the homeostasis of hematopoietic stem and progenitor cells and resulted in ageing-related myeloproliferation in NPMc+ transgenic mouse model. Interestingly, through scanning the chromatin modification related gene profiling, di- and tri- methylated H3K79 were significantly elevated in bone marrow (BM) Lin-Sca-1+c-Kit+ cells (LSKs) of NPMc+ mice comparing to wild type (WT). Meanwhile, in the leukemia cell lines and AML primary BM samples, we confirmed that NPM1 mutated cells expressed the higher level of H3K79 methylation. In vitro assays also indicated that the decrease or increase of methylated H3K79 could be regulated respectively by knockdown or overexpression of NPM1 mutant but not WT. Importantly, with DOT1L inhibitor treatment, reduced di- and tri- methylated H3K79 was observed in OCI-AML3 (NPMc+) strains but not OCI-AML2 (NPM1 WT) cells. In contrast with OCI-AML2, DOT1L inhibitor significantly promoted the cell apoptosis and restrained the cell cycle of OCI-AML3. Moreover, by the means of murine BM colony formation assay, DOT1L inhibitor obviously weakened myeloid cell proliferation in NPMc+ mice, while colony number in WT group did not change. Also, leukemia development was repressed in OCI-AML3-xenografted NOD/SCID mice with the treatment of DOT1L inhibitor. Taken together, NPM1 mutation contributes to hematological dysfunction by disrupting H3K79 methylation, which could be largely attenuated by DOT1L inhibitor.

Disclosures

No relevant conflicts of interest to declare.
DNA Epigenome Editing Using Crispr-Cas Suntag-Directed DNMT3A
Authors: Huang Yung-Hsin , Jianzhong Su , Lei Yong , Gundry Michael C , Zhang Xiaotian , Jeong Mira , Li Wei , Goodell Margaret A
Published: October 2019

DNA methylation, an epigenetic modification, has widespread effects on gene expression during development. However, our ability to assign specific function to regions of DNA methylation is limited by the poor correlation between global patterns of DNA methylation and gene expression. To overcome this barrier, we utilized nuclease-deactivated Cas9 protein fused to repetitive peptide epitopes (SunTag) recruiting multiple copies of antibody-fused de novo DNA methyltranferase 3A (DNMT3A) (CRISPR-Cas SunTag-directed DNMT3A) to amplify local DNMT3A concentration and to methylate genomic sites of interest. Here, we demonstrated that CRISPR-Cas SunTag-directed DNMT3A not only dramatically increased CpG methylation but also, to our surprise, CpH (H =A or C or T) methylation at the HOXA5 lociin human embryonic kidney 293T cells (HEK293T). Furthermore, using a single sgRNA, CRISPR-Cas SunTag-directed DNMT3A was capable of methylating 4.5 kb genomic regions, surpassing previous targeted methylation tools whose activity is limited to 200bp. Using reduced representation bisulfite sequencing (RRBS) and RNA-seq, we concluded that CRISPR-Cas SunTag-directed DNMT3A methylated regions of interest without affecting global DNA methylome and transcriptome. This effective and precise tool enables site-specific manipulation of DNA methylation and may be used to address the relationship beteween DNA methylation and gene expression.

Disclosures

No relevant conflicts of interest to declare.
GFI1b As a Novel Oncosuppressor in AML
Authors: Thivakaran Aniththa , Botezatu Lacramioara , Hoenes Judith Maria , Al-Matary Yahya Saleh , Olberding Nadine , Schütte Judith , Köster Renata , Opalka Bertram , Dührsen Ulrich , Khandanpour Cyrus
Published: October 2019

The proper differentiation of hematopoietic stem cells (HSCs) is regulated by a concert of different so called transcription factors (TFs). A disturbed function of these TFs can be the basis of malignant diseases such as acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Growth Factor Independence 1b (Gfi1b) is a repressing transcription factor, with a key role in maintaining the quiescence of HSCs and the proper emergence and maturation of erythrocytes and platelets. Here we show that low expression of GFI1B in blast cells is associated with an inferior prognosis of AML and MDS patients. Using three different mice models of human AML (Nup98-HoxD13, MLL-AF9 and expression of a mutated K-Ras), we could show that reduced expression of Gfi1b accelerates AML development in mice and the latency is even more shortened when Gfi1b is conditionally deleted. Using a limiting dilution assay of transplantation of different number of Gfi1b-wildtype and Gfi1b-deficient cells, we could show that loss of Gfi1b significantly enhanced stemness of leukemic cells. Since Gfii1b is involved in epigenetic regulation of gene expression, we analyzed effect of loss of Gfi1b on an epigenetic level by analyzing the whole genome using Chip-Seq. We found that loss of Gfi1b leads to genome wide increased level of H3K9 acetylation of genes and hence expression of these genes involved in leukemia development. On a molecular level, we found that loss of Gfi1b not only increases the levels of reactive oxygen species (ROS), but also induces gene expression changes of key AML-pathways such as the p38/ AKT pathways. These results demonstrate that Gfi1b functions as an oncosuppressor in MDS/AML development.

Disclosures

Dührsen: Roche: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding. Khandanpour:Max-Eder: Research Funding; Hospital of Essen university: Research Funding.
Ribosomal Protein L22 Loss Predisposes Stem Cells to Transformation By Altering Metabolism
Authors: Harris Bryan , Fahl Shawn , Perrigoue Jaqueline , Sykes Stephen M. , Verma Amit , Wiest David L
Published: October 2019

While mutations in ribosomal proteins (RP) have long been associated with increased cancer risk, the molecular basis for this association remains unclear. The prevailing view is that mutations in RP increase cancer risk either by impairing the biogenesis or function of the ribosome. Here, we report that loss of the RP, Rpl22, is frequently observed in human myelodysplastic syndrome (MDS) and is associated with reduced survival in acute myelogenous leukemia (AML). The link between Rpl22 loss and aggressiveness of myeloid disease is causal, since it is replicated in mice lacking Rpl22. Importantly, the development of an MDS-like syndrome in Rpl22-deficient mice and the accelerated development of myeloid leukemia are not caused by reduced protein synthesis in hematopoietic stem cells (HSC). Instead, whole transcriptome analysis of HSC reveals that Rpl22 loss alters the expression of genes controlling cell metabolism and results in an increase in fatty acid oxidation. Moreover, the increased fatty acid oxidation is required for the increased self-renewal and reduced differentiation displayed by Rpl22-deficient HSC, as well as the survival of Rpl22-deficient myeloid leukemia cells. Together, these findings indicate that RP are capable of suppressing transformation potential in ways that do not involve effects on global protein synthesis. Indeed, Rpl22 suppresses transformation by suppressing lipid oxidation, suggesting that MDS and AML patients with reduced Rpl22 expression might benefit from pharmacologic interventions targeting lipid oxidation.

Disclosures

No relevant conflicts of interest to declare.
Identifying Mechanisms of Glucocorticoid Resistance in Relapsed Pediatric T-ALL
Authors: Roderick Justine E. , Gallagher Kayleigh M , Tang Katherine M , Kugler-Umana Olivia , Zhu Lihua J , Green Michael R , Kelliher Michelle
Published: October 2019

While great strides have been made in the improvement of outcome for newly diagnosed pediatric acute lymphoblastic leukemia (ALL) patients, prognosis for relapsed leukemia patients remains poor. The synthetic glucocorticoid (GC) dexamethasone is part of the standard treatment for pediatric ALL and patient response to glucocorticoid treatment has proved to be a reliable prognostic indicator. Identifying the biological pathways responsible for glucocorticoid resistance may reveal novel therapeutic targets to prevent and treat relapsed ALL. Although genomic analyses of relapsed patients and matched diagnosis-relapse patient pairs have begun to define the genomic landscape of relapsed disease, discerning "driver from passenger" genetic lesions remains challenging. To identify glucocorticoid resistance genes in an unbiased, high-throughput manner, we conducted a genome wide, survival based, shRNA screen in dexamethasone sensitive murine T-ALL cells. Our preliminary data identify several hundred genes capable of mediating GC resistance, including several known GC resistance genes Nr3c1, Rcan1, Btg1 and Mllt10, thereby validating our experimental approach. Candidate genes identified in the screen including EP300 (p300), GATA3 and IKZF1 are known leukemia suppressors in pediatric ALL and the EP300 paralog CREBBP and IKAROS have been linked to GC resistance, indicating that suppressor genes involved in human leukemia and GC resistance are identified in our mouse screen. Consistently, we found the expression of several screen hits significantly decreased and/or mutated in relapse patient samples. Novel dexamethasone resistance genes identified in the screen interfere with GC-induced transcription (Stat3, Ikzf1), promote pluripotency (Esrrb, Sox2) or stimulate cAMP signaling (Adcy3, Gnas, Creb1). Silencing of these genes in multiple mouse T-ALL cell lines has no detectable effects on leukemic growth/survival in vitro, but confers resistance to dexamethasone treatment in vitro and in vivo. Moreover, we show that silencing of some candidate dexamethasone resistance genes accelerates leukemogenesis in vivo, demonstrating that leukemia suppressor genes were identified. Effect(s) of silencing or inhibiting these novel dexamethasone resistance genes/pathways in human T-ALL cell lines, primary patient samples and xenografts will be discussed. We predict that targeting these dexamethasone resistance pathways may re-sensitize relapse pediatric T-ALL cells to dexamethasone and/or contribute to more effective patient stratification to prevent relapse and induction failure.

Disclosures

No relevant conflicts of interest to declare.
MYC-Dependent PI3K and MCL-1 Feedbacks Attenuate BET Inhibitors Activity in Diffuse Large B-Cell Lymphoma
Authors: Derenzini Enrico , Mondello Patrizia , Liu Yuxuan , Scallion Mary , Asgari Zahra , Philip John , Hilden Patrick , Djaballah Hakim , Ouerfelli Ouathek , de Stanchina Elisa , Seshan Venkatraman , Hendrickson Ronald , Zelenetz Andrew D. , Younes Anas
Published: October 2019

MYC overexpression is a poor prognostic predictor in Diffuse Large B-Cell Lymphoma (DLBCL). MYC-targeting with bromodomain and extraterminal protein family (BET) inhibitors is a promising strategy for the treatment of MYC-driven cancers, including lymphomas. However, preclinical and emerging data from early clinical trials demonstrated a modest antiproliferative activity in vitro and in vivo. We hypothesized that BET inhibition may induce feedback survival mechanisms preventing or attenuating cell death that could be exploited for designing future, more effective, combination strategies. In a high-throughput combinatorial drug screening experiment, we found that phosphatidylinositol 3-kinase (PI3K) pathway inhibitors enhanced the antiproliferative effects of BET inhibitors (JQ1, I-BET 151, CPI-203) with a strong class effect. JQ1 upregulated the mRNA expression of several upstream components of the PI3K pathway, including PIK3CA, PIK3R1, PDK1 in a large panel of DLBCL and Burkitt lymphoma cell lines. These effects translated in increased pathway activation as demonstrated by increased levels of the phosphorylated forms of downstream targets GSK3α/β, TSC2, P70S6K, and by increased concentrations of chemokines known to be regulated by PI3K in cell culture supernatants (CCL3 and CCL4). This effect was reversed by submicromolar doses of the PI3K inhibitor BKM-120. MYC silencing recapitulated the effects of BET inhibitors on PI3K pathway gene expression, activation and chemokine secretion. These data indicate that BET inhibition induces PI3K activation by a MYC-dependent feedback. We also observed transcriptional upregulation of the antiapoptotic gene Myeloid Leukemia 1 (MCL-1) following BET inhibition or MYC depletion, suggesting a second MYC-dependent mechanism. RNAi-mediated MCL-1 silencing or co-treatment with a small molecule MCL-1 inhibitor (UMI-77) enhanced the effects of BET inhibitors in DLBCL cell lines by inducing apoptosis. Using SILAC-based quantitative mass spectrometry, we found that BET inhibitors at submicromolar doses downregulated several E2 ubiquitin conjugating enzymes including UBE2C. RNAi mediated UBE2C knockdown induced MCL-1 upregulation in DLBCL cells. The enhanced in vitro effect of combining BETi and PI3Ki was reproduced in TMD8 mouse xenografts. To our knowledge, this is the first study demonstrating MYC-dependent regulation of the PI3K pathway, MCL-1 and the ubiquitin system upon BET inhibition. Our study revealed previously unknown mechanisms of action of BET inhibitors uncovering novel MYC-dependent survival feedback loops, and providing a framework for future combination strategies.

Disclosures

Zelenetz: Gilead Sciences: Research Funding.
Survival Outcomes in Grade 3 Follicular Lymphoma Prior to and after the Introduction of Rituximab - an Analysis Based on the Surveillance, Epidemiology, and End Result Registry Database
Authors: Giri Upama , Martin Michael G.
Published: October 2019

Background

Follicular lymphoma (FL) is the most common form of indolent lymphoma, however Grade 3 FL (FL3) has more aggressive behavior and its response to therapy is controversial. Several studies have reported improved clinical outcomes in patients with FL3 treated with rituximab (R), approved in early 2006, as a first line therapy for diffuse large B-cell lymphoma. The aim of this population based study is to compare the overall survival and disease specific survival in patients diagnosed with FL3 prior to and after 2006.

Materials and methods

We used the Surveillance, Epidemiology, and End Results 18 database to identify cases diagnosed with FL3 between 1983 and 2013, limiting the selection to patients aged more than 15 years with single primary malignancy. The data was split into two cohorts - Cohort 1 comprised of cases diagnosed between 1983 and 2005, with a study cutoff of December 2005, and Cohort 2 comprised of cases diagnosed between 2006 and 2013. The Overall Survival (OS) and Disease Specific Survival (DSS) for the two cohorts were calculated using the Kaplan-Meier method and the results were compared using the Log Rank test. The Cox proportional hazards regression model was used to identify factors other than time of diagnosis, which had a significant impact on survival times.

Results

Cohort 1 included 3,959 cases with death occurring in 1,319 cases during the study period (1,013 deaths due to disease specific factors) and Cohort 2 included 3,050 cases with death occurring in 509 cases (374 deaths due to disease specific factors) (table 1). The 5-year OS and DSS survivals for Cohort 1 were 59.3% and 67.7%, and for Cohort 2 were 78.3% and 84% respectively (figure 1). The differences in both OS and DSS survival rates were confirmed to be significant using the Log Rank test (p<<0.005 in both instances).

Cox regression analysis was performed using age at diagnosis, sex, race, marital status, primary site (nodal/extra nodal) and Ann Arbor stage as covariates, after removing cases with missing values. The test results showed that age at diagnosis, sex, Ann Arbor stage (p<<0.005 for all three) and primary site (p<0.05) had a significant impact on OS. However, DSS was not significantly influenced by primary site. Males had an increased risk of mortality, as did an increase in age and advanced Ann Arbor stage (table 2).

Conclusion

A significant increase in OS and DSS was observed in patients diagnosed with FL3 after the introduction of rituximab. Survival outcomes were also influenced by age at diagnosis, sex and Ann Arbor stage.

Figure Figure.

Disclosures

No relevant conflicts of interest to declare.