bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppression of EZH2 Accelerates MYC-Driven Lymphomagenesis By Inhibition of Apoptosis
Authors: Appelmann Iris , Scuoppo Claudio , Thapar Vishal , Ledezma Daniela , Lujambio Amaia , Lowe Scott W. , Chicas Agustin
Published: November 2019

EZH2 is the catalytic component of the polycomb repressive complex 2 (PRC2), which also contains the non-catalytic subunits suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). This complex methylates histone H3 at lysine 27 (H3K27) which, together with H3K4 methylation, generates a bivalent “code” that primes genes for either expression or silencing. EZH2 is highly expressed in stem cells and many proliferating cells, downregulated in differentiated cells and frequently altered in cancer in ways that point to a context dependent role for this gene. For instance, overexpression of EZH2 has been described in prostate and breast cancer, where this overexpression is associated with invasive growth metastatic potential and poor clinical outcome.

More recently, both gain and loss of function mutations of EZH2 were identified in human cancer. In particular, activating mutations of EZH2 affecting a tyrosine at position 641 located within the SET domain (Y641) were observed in lymphoma. In striking contrast to wild type EZH2 which catalyzes the monomethylation of H3K27 very efficiently and shows less efficient catalytic activity in the subsequent di- and trimethylation reactions, the mutation generates a neomorphic protein with enhanced catalytic activity and efficiency for di- and tri-methylation, hinting toward a “cooperation” of both wild type and mutant EZH2 to increase total H3K27me3 levels and representing a functional equivalent of EZH2 overexpression in human lymphoma.

Gain of function mutations in EZH2 are frequent in Diffuse Large B-cell Lymphoma (DLBCL; 22%) and in Follicular Lymphoma (FL; 7%) and recent data implicate EZH2 as an oncogene in DLBCL and FL lymphomas.

In contrast to DLBCL and FL, EZH2 mutations have so far never been identified in MYC-driven B-cell Non-Hodgkin Lymphoma (B-NHL), and EZH2 expression is suppressed in Burkitt’s Lymphoma (BL), a lymphoma for which a MYC translocation is pathognomonic, suggesting that in this context EZH2 could have a role different from its role in DLBCL and FL. We probed the role of EZH2 in MYC-driven lymphomagenesis by mimicking the loss of function mutations by RNAi mediated suppression of EZH2 and the gain of function mutations by over-expression of the Y641 mutant.

Our results show that suppression of EZH2, but not overexpression of the EZH2 Y641 mutant, accelerates MYC-driven lymphomagenesis by attenuating apoptosis. Our model recapitulates the transcriptional signature of a subset of B-NHLs driven by MYC overactivation and EZH2 suppression. Taken together, our data imply EZH2 as a tumor suppressor in the context of MYC activation and thus raise a warning for the use of EZH2-targeted therapies in some B-NHLs subtypes.

Disclosures

No relevant conflicts of interest to declare.
Predictive Value of Interim 18f-FDG PET-CT Scans on Diffuse Large B-Cell Lymphoma Treated with R-CHOP: A Prospective Study of Chinese Southwest Oncology Group (CSWOG)
Authors: Huang He , Lin Jia Tian , Guo Chengcheng , Hong Huangming , Prem Raj Shrestha , Li Xueying , Zhang Mengping , Li Shanshan , Fang Xiaojie , Wang Zhao , Xia Zhongjun , Lin Tongyu
Published: November 2019

Purpose

18F-FDG PET-CT has been widely used for pre-treatment staging and post-treatment response assessment in diffuse large B cell lymphoma (DLBCL), but the predictive value of interim PET-CT remained controversial and most of studies were retrospective.

Patients and Methods

Newly-diagnosed DLBCL patients treated with R-CHOP regimen were included in our prospective study to evaluate the predictive value of interim PET-CT. All patients were evaluated with PET-CT scans before treatment and after every 2 cycles of R-CHOP. PET-CT positivity or negativity was related to survival using Kaplan-Meier analysis.

Results

From Feb 2008 to Jan 2013, 149 patients were included. After 2 cycles of R-CHOP, the PET-CT evaluation showed CR in 82 patients. Among the remaining 67 non-CR patients, 31 achieved CR after 4 cycles. At the end of treatment, PET-CT evaluation showed CR in 121, PR in 21, SD in 3 and PD in 4 patients. With a median follow-up of 20.6 months (range 1.5-60.5 months), patients with negative PET-2 (PET-CT scan after 2 cycles) had a superior 2-year PFS than those with positive PET-2 (86.6% vs. 67.0%, p=0.019) and a tendency of superior 2-year OS without statistical differences (91.9% vs. 85.2%, p=0.330). The 2-year PFS and OS for negative PET-4 (PET-CT scan after 4 cycles) compared with positive PET-4 group were 84.8% vs. 51.9% (p=0.001) and 93.1% vs. 73.0% (p=0.027) respectively. PET-CT scans were interpreted using the International Harmonization Project (IHP) criteria above. The second analysis applying the Five-Point Scale (5PS) criteria showed that 2-year PFS for score 1-2 (uptake＜mediastinum), score 3 (mediastinum＜uptake≤liver) and score 4-5 (uptake＞liver) group were 86.6%, 75.8% and 71.0% according to PET-2, and 84.9%, 50.0% and 52.7% according to PET-4. PFS of score 3 group was not significant different from the other two groups in PET-2 (both P＞0.05), but significantly inferior to score 1-2 group (p=0.046) and similar to score 4-5 group (p=0.767) in PET-4. In the multivariate analysis, only PET-4 (95%CI, 1.07-5.44) and IPI score (95%CI, 1.53-10.06) remained independent predictive factors for PFS.

Conclusion

PET-CT after 4 cycles of R-CHOP in patients with DLBCL is highly predictive of PFS and should be considered in clinical practice.

(Clinical Trail Registration Number: CTR-TRC-11001687)

Disclosures

No relevant conflicts of interest to declare.
Identification of Cell-Type-Specific Mutations in Angioimmunoblastic T-Cell Lymphoma
Authors: Nguyen Tran Bich , Sakata-Yanagimoto Mamiko , Asabe Yukitsugu , Yoshida Kenichi , Muto Hideharu , Nakamoto-Matsubara Rie , Izutsu Koji , Nakamura Naoya , Takeuchi Kengo , Ogawa Seishi , Chiba Shigeru
Published: November 2019

[Backgrounds] Angioimmunoblastic T-cell lymphoma (AITL) is a distinct subtype of peripheral T-cell lymphoma (PTCL), characterized by generalized lymphadenopathy and autoimmune-like manifestations. Regarding genetic lesions of AITL, frequent mutations in TET2, IDH2, DNMT3A and RHOA have been identified. In some PTCL cases, TET2 and DNMT3A mutations were identified in cell populations beyond the CD4+ T-lymphocytes, in which the tumor cells are contained, suggesting that TET2 and DNMT3A mutations occurred earlier than the commitment to CD4+ T lymphocytes.

[Objective] We performed this study to identify the cell-type-specific mutations and further explore mutational profiles in AITL and AITL-related cancer.

[Methods] The dataset of targeted sequencing was analyzed for 76 genes in 79 PTCL samples. Mutational origin was analyzed by cell sorting and laser microdissection.

[Results] Targeted sequencing identified 168 mutations in 33 genes. Recurrent mutations, in addition to the already known frequent mutations in RHOA/TET2/IDH2/DNMT3A, were found in ODZ1 [4/79 (5%)], Notch1, NAV2, and MTERFD3 [3/79 (4%) for each], MLL2, TET3, FAT2, and LAMA2 [2/79 (3%) for each]. TET2/DMNT3A mutations showed statistically higher allelic burden than the newly identified mutations, suggesting precedence of TET2/DNMT3A mutations.

Cell sorting and laser microdissection, followed by amplicon sequencing, revealed that TET2/DNMT3A mutations were identified in both tumor cell-enriched and –depleted populations while RHOA and IDH2 mutations were confined to tumor cell-enriched populations. Most of the newly identified mutations were similarly classified into the above-mentioned two types. It is noteworthy that we found some mutations only in T-cell lymphoma cell-depleted CD20-positive population but not in the tumor-cell-enriched PD-1-positive population.

[Conclusion and discussion] Differentiation stages that mutational events arise are likely to be multiple in AITL and AITL-related lymphoma. Moreover, in AITL, Epstein-Bar virus-infected B cells often grow in an oligoclonal manner, sometimes resulting in monoclonal proliferation with fully malignant features. Detection of B-cell specific mutations might suggest premalignant status of B cells in these cases.

Disclosures

No relevant conflicts of interest to declare.
Clinical Prognostic Models in Diffuse Large B-Cell Lymphoma Patients Are Still Essential in the Rituximab Era
Authors: Lage Luis Alberto de Padua Covas , Costa Renata Oliveira , Hallack Neto Abrahão Elias , Siqueira Sheila , Santucci Rodrigo , de Paula Henrique Moura , Pereira Juliana
Published: November 2019

Introduction: To evaluate a new enhanced IPI proposed by the National Comprehensive Cancer Network (NCCN-IPI) in DLBCL patients, we compared the international prognostic index (IPI), R-IPI and NCCN-IPI in DLBCL patients treated with rituximab, cyclophosphamide, hidroxydaunorubicin, vincristine and prednisone (R-CHOP).

Methods: From June 2008 to November 2011 we retrospectively evaluated 146 DLBCL patients treated with R-CHOP-21 referred for cancer treatment in a single university institution in Brazil. Patient's clinical data were assessed to calculate the IPI, R-IPI and NCCN-IPI.

Results: Patient's median age was 58.9 years (range 16 – 86); 85 (57.8%) were female. According to IPI, risk categories were low (n=41, 28.1%), low-intermediate (n=43, 29.5%), high-intermediate (n=37, 25.3%) and high (n=25, 17.1%). Using R-IPI, risk categories were very good (n=19, 13%), good (n=65, 44.5%) and poor (n=62, 42.5%). According to NCCN-IPI, risk categories were low (n=12, 8.2%), low-intermediate (n=52, 35.6%), high-intermediate (n=62, 42.5%) and high (n=20, 13.7%). At 30 months (median follow up 17.7 months - range 0.6-58.2 months) the overall survival (OS) was 75.5%. The progression-free survival (PFS) at a median follow-up of 16.3 months (range 0.6-52.4) was 68.3% for all patients. Using IPI, the OS at 30 months did not differ between low and low-intermediate risk patients (96.8% vs. 82.2%; p=0.136); however, it was higher than the OS of high-intermediate risk (n=37; 96.8% vs 74.1% p=0.11) and high-risk (n=25; 96.8% vs 41% p < 0.001) patients (Figure 1). The NCCN-IPI demonstrated significant differences in OS (p < 0,001) and PFS (p<0.001) among low-intermediate, high-intermediate, and high risk groups, with the high-risk group exhibiting worse OS (32.1% in 30 months) (Figure 2). According to IPI, the OS in high-risk patients was 41%.

Figure 1: OS and PFS according to International Prognostic Index (IPI) Figure 1:. OS and PFS according to International Prognostic Index (IPI)

Figure 2: OS and PFS according to NCCN-IPI Figure 2:. OS and PFS according to NCCN-IPI

Figure 3 Figure 3.

Conclusion: In our study the NCCN-IPI, but not the IPI or R-IPI was able to discriminate a high-risk group of DLBCL patients treated with R-CHOP with worse OS.

Disclosures

No relevant conflicts of interest to declare.
Brentuximab Vedotin Followed By ABVD in Patients with Previously Untreated Hodgkin Lymphoma. a Pilot Phase II Study
Authors: Zinzani Pier Luigi , Luminari Stefano , Merli Francesco , Pesce Emanuela Anna , Chauvie Stephane , Pellegrini Cinzia , Marcheselli Luigi , Capodanno Isabella , Ilariucci Fiorella , Salati Massimiliano , Argnani Lisa , Federico Massimo
Published: November 2019

active and well tolerated single agent in the treatment of heavily pretreated Hodgkin lymphoma patients. In this pilot phase II study patients with previously untreated HL underwent sequential regimen consisting in 2 cycles of BV before doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD) +/- radiotherapy (RT). The primary endpoint of the study was the response to BV assessed by positron emission tomography (PET) after the 2 cycles (PET2), defined as reduction of Deauville score or, in case of no change in Deauville score, as any reduction in standard uptake value (SUV) intensity compared to basal SUV.

Between April and October 2013, 12 patients with a median age at diagnosis of 36 years (range, 19-70), 11 stage I-IIA and 1 stage IIIA, were enrolled. BV was administered as scheduled and at the full dose of 1.8 mg/kg in all patients. After the 2 cycles of BV, the overall response rate was 91%, comprising of ten (83%) complete responses and one (8%) partial metabolic response. The non responding patient had stage III and showed a new lesion at PET2. After ABVD +/- RT, the overall response rate was 100% with 11 complete responses and 1 partial response (converting from progression disease). At a median follow up of 8 months, all 11 patients are still in complete response, while the remaining one relapsed.

During BV therapy, the only grade 3 adverse events were transient and asymptomatic increase in liver transaminases (n=3, 25%) and gamma glutamyl transpeptidase (n=2, 17%). During ABVD +/- RT grade III-IV neutropenia occurred in 9 (75%) patients. All toxicities were transient and resolved with growth factor support.

Two cycles of BV as first line-treatment in limited stage HL induced an outstanding complete response rate with limited toxicity and reducing the need of chemotherapy. Waiting for a longer follow up to assess the duration of response, our data (sequential administration of immunotherapy and chemotherapy) should be considered the starting point for further studies in first line therapy for limited stage HL patients.

Disclosures

No relevant conflicts of interest to declare.
Germ-Line and Somatic Mutations in Familial Myeloproliferative Neoplasms (MPNs). a Pilot Study
Authors: Nand Sucha , Diaz Manuel O. , Marinier David E. , Walshauser Mark A. , Rosenfeld Amy B , Michaelis Laura C , Stiff Patrick J , Gai Xiaowu
Published: November 2019

Background: Familial MPNs are uncommon disorders that, like sporadic cases, are characterized by clonal hematopoiesis and presence of somatic mutations, e.g. JAK2, CALR, MPL and occasionally TET2. There is little information, however, about germ-line mutations in these families that may explain the low penetrance hereditary predisposition.

Methods: We studied five families with MPNs, each with at least 2 affected members. After obtaining an informed consent, clinical data was obtained from the patients’ electronic medical records. Blood and buccal samples were collected from patients and unaffected relatives. Exome sequencing was performed on the blood DNA samples using Agilent SureSelect Human All Exon V5+UTRs exome capture kit followed by massively parallel sequencing with Illumina HiSeq 2000. Sanger sequencing was then done on both the blood and buccal swab DNA samples to validate selected gene variants and to differentiate the nature of those variants (germ line or somatic).

Results: The 5 families participating in this study had the following diagnoses: 1. Mother: polycythemia vera (PV); son: essential thrombocythemia (ET), 2. Mother: primary myelofibrosis (MF); daughter: unclassifiable MPN (UMPN), 3. Father: PV; son: PV, 4. Sister: MF; sister: MF, 5. Two aunts: MF; niece; UMPN. Six patients were positive for JAK2, V617F mutation. Blood and buccal samples were collected from 5 patients and 4 relatives. In all 5 families, the pro-band was younger at the time of diagnosis than his/her affected relatives. The clinical course of the MPNs appeared to be similar to the sporadic form.

Exome sequencing revealed TET2 mutations in 2 probands. In addition, novel non-synonymous mutations in several candidate genes, KMT2D, KMT2C, NBEAL1, NBEAL2, AHNAK2, RNF213, were identified in the blood samples from the patients but not their unaffected relatives. These include two novel KMT2D mutations in two unrelated families. These 2 mutations were also found in the matching buccal swab samples, indicating that they are germ line mutations.

Discussion: KMT2D and KMT2C mutations have been previously identified as somatic mutations in lymphoid malignancies, including non-Hodgkin’s lymphomas (Morin 2011), and as germ line compound heterozygote mutations in infant MLL and ALL (Valentine 2014). About 32% of diffuse large cell lymphoma and 89% of follicular lymphoma have somatic mutations of KMT2D. NBEAL2 germ line mutations are associated with familial gray platelet syndrome, where some patients have myelofibrosis (Gunay-Aygun 2011). To our knowledge, this is the first report describing germ line mutations in familial MPNs. The possible role of these mutations in predisposition to MPN will be discussed. Studies on additional families with MPNs are planned.

Disclosures

No relevant conflicts of interest to declare.
Protooncogene MYB Activates the Transcription of ROR1 in B Cell Malignancies
Authors: Watanabe Ken , Iida Megumi , Suwa Shihoko , Kato Miyu , Miura Osamu , Fukuda Tetsuya
Published: November 2019

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a highly conserved gene that is associated with cell survival. ROR1 is uniquely expressed in some B cell malignancies and epithelial cancer cells although it is barely detected in normal adult tissues. The precise mechanisms of this ectopic expression have remained unknown. We checked ROR1 expression by FACS analysis and by quantitative RT-PCR. ROR1 was highly expressed in the samples from CLL,MCL,SMZL,and BL. Expression levels of ROR1 mRNA and cell surface ROR1 protein showed clear positive correlation (R2=0.55) suggesting that ROR1 expression is regulated mainly transcriptionally. Then, we cloned the 2.5-kb fragment upstream of ROR1 transcriptional start site and performed promoter assay using ROR1 positive and negative cell lines. The promoter activity was observed not only in ROR1 positive lines but also in negative lines, which suggests that this region may not be the only regulatory element. Thus, we hypothesized that conserved regions in intron 1, which is about 230 kb long, may play important roles in regulation of transcription. BLAST search identified 19 regions conserved between human and rat intron 1 of ROR1. Two regions, designated as CRS3 and CRS7, further showed homology with chicken ROR1 gene. Among them, CRS3 region had enhancer activity selectively in ROR1 positive B cell lines. A consensus nucleotide sequence for the binding of protooncogene MYB was found within CRS3 region. Actually, the protein binding to CR3S sequence was verified by EMSA assay using nuclear extract from MYB positive cells. Immunoblot analysis showed that ROR1 positive B cell lines had high expression of MYB. In addition, the expression levels of MYB protein and ROR1 mRNA were positively correlated in 7 CLL samples analyzed (R2=0.49). We introduced shRNA of MYB into ROR1 positive EW36 cell line. MYB shRNA could decrease CRS3 luciferase reporter activity. As a result, MYB shRNA decreased ROR1 expression as well as MYB expression. Collectively, MYB induced ROR1 expression and might contribute to tumorgenesis of B cell malignancies.

Disclosures

No relevant conflicts of interest to declare.
Hypermethylation of TAp73 Suppresses ABL1-Involved DNA Damage Response in Multiple Myeloma
Authors: Hao Mu , Qin Yu , Zang Meirong , Xu Yan , An Gang , Li Changhong , Yang Ye , Gu Zhimin , Zhan Fenghuang , Qiu Lugui
Published: November 2019

Background: More recently, multiple myeloma (MM) cells evade apoptosis despite pervasive DNA damage was demonstrated. However, the relevance of ongoing DNA damage and the mechanisms by which apoptosis is suppressed remain to be fully elucidated. p53 deletion and mutations do not appear to be a pivotal event in the evolution from pre-malignancy toward malignancy in MM. The protooncogene ABL1 was an alternative pathway to p53 down stream of ATM/ATR, which is commonly translocated in Chronic Myelogenous Leukemia (CML). ABL1 forms a complex with the tumor suppressor gene TP73, which belongs to the p53 family. P73 is expressed as multiple isoforms due to the usage of two different promoters, P1 promoter of TAp73 inducing apoptosis and P2 promoter of Δ Np73 promoting survival. In this study, we explored the role of ABL1/p73 axis in MM cells evading ongoing DNA damage induced apoptosis.

Materials and methods: Real-time-PCR was used to detect the ABL1 and miR-203a expression in MM primary samples and MM cell lines. Immunofluency staining was performed to detect the ɣ-H2A.X level in MM cells. Flow cytometry was performed to detect the apoptosis in MM cells. Bisulfite Pyrosequencing and Methylation Specific-PCR were used to detect the p73 promoter methylation.

Results: Our results revealed that ABL1 level was up-regulated both in primary MM samples and MM cell lines (-1.25±0.28 vs. 0.06±0.24, p=0.02). MiR-203 which suppresses ABL1 expression was down-regulated (0.01±0.01 vs. 0.97±0.08, p=0.01). MM cell lines and primary cells showed high ɣ-H2A.X staining. Immunofluency staining showed that ABL1 relocalized in the nucleus of MM cells after treated with doxorubicin. The apoptosis of MM cells was significantly up-regulated (7.8±2.1)% vs. (25.4±4.5)%, p<0.05. Doxorubicin treatment combined with ABL1 inhibitor (STI571) suppressed the apoptosis significantly, (25.4±4.5)% vs. (12.2±3.4)%, p=0.03. Bisulfite Pyrosequencing and MS-PCR of 42 newly diagnosed MM patient sample revealed that the P1 promoter of p73 was hypermethylated compared with normal plasma cells (86% ±7% vs. 58%±4%, p=0.032). RT-qPCR and western blotting showed that Δ Np73 levels were significantly higher than TAp73 (148.6±19.3 vs. 6.8±2.4, p=0.021) in plasma cells of those patients and MM cell lines.

Conclusion: Hypermethylation of p73 promoter suppresses TAp73 expression. The deregulated of ABL1-p73 pathway in MM cells resulted in marked reduction of apoptosis induced by ongoing DNA damage.

Disclosures

No relevant conflicts of interest to declare.
PML-RARα Repressed Microrna 126 Mediates Differentiation in Acute Promyelocytic Leukemia By Targeting the Protooncogene C-Myb
Authors: Schödel Cindy , Hartmann Jens-Uwe , Gerloff Dennis , Braeuer-Hartmann Daniela , Katzerke Christiane , Wurm Alexander Arthur , Müller-Tidow Carsten , Tenen Daniel G. , Niederwieser Dietger , Behre Gerhard
Published: November 2019

Almost one tenth of acute myeloid leukemias (AML) are related to t(15;17) translocation. This aberration leads to the PML-RARα fusion protein, which causes a transcriptional block of differentiation and could be resolved by all-trans-retinoic acid (ATRA). MicroRNAs (miRs) are small noncoding RNAs which are ascribed to be important regulators of cell proliferation, development, differentiation and apoptosis. MiR-126 is regarded as tumor suppressor in diverse tissues, but relatively little is known about its functional role in normal and malignant hematopoiesis. In this report, we reveal that oncogenic PML-RARα fusion protein is a repressor of miR-126 expression. Thus, we could demonstrate a significant downregulation of miR-126 expression in presence of the PML-RARα fusion protein in APL versus other AML patient samples and in a PML-RARα Knock in mouse model. We further show that ATRA induced differentiation of APL cell line NB4 comes along with an increase of miR-126 expression. The transient overexpression of miR-126 enhances ATRA stimulated differentiation of myeloid cells, whereas the opposite effect was noticed when endogenous expression of miR-126 was knocked down. Most interestingly, we observed a significant upregulation of miR-126 expression in APL blasts during ATRA treatment of APL patients. C-Myb proto-oncogene was found to be a putative target of miR-126 by analysing the 3’UTR. We could show a reduced c-Myb protein level after enforced expression of miR-126. Further, we reveal that miR-126 leads to a posttransciptional downregulation of c-Myb expression by directly targeting its 3`UTR. Our data suggest miR-126 as an important regulator of differentiation in acute promyelocytic leukemia, targeting the proto-oncogene c-Myb and recommend microRNAs to be potential therapeutic agents.

Disclosures

No relevant conflicts of interest to declare.
A Novel Suppressive Long Noncoding RNA within the IGF1R Gene Locus Is Imprinted in Acute Myelocytic Leukemia
Authors: Sun Jingnan , Hu Jifan , Li Wei , SUN Yunpeng
Published: November 2019

Background：In acute myeloid leukemia (AML) cells, the insulin-like growth factor type I receptor (IGF1R) is one of the most abundantly phosphorylated receptor tyrosine kinases, promoting cell growth and therapeutic resistance through the PI3K/Akt signaling pathway. However, little is known regarding the molecular mechanisms underlying IGF1R gene dysregulation in AML. Long non-coding RNAs have been implicated in a variety of important biological processes by epigenetic mechanisms.

Materials and methods：AML cell lines used in this study, KG-1, KG-1a were purchased from ATCC. The full length of IRAIN lncRNA was characterized by Marathon cDNA Amplification Kit. For qPCR, cDNA samples were amplified using CFX96TM real-time system (BIO-RAD) by SYBR PrimeScript™ RT-PCR Kit (TaKaRa). Both reverse transcription-associated trap (RAT) and chromatin conformation capture (3C) assays were used to examine the chromosome architecture of IGF1R and DNA-RNA interaction. The protocol of AMLpatients bone marrow and peripheral blood cell samples was approved by the Human Medical Ethical Review Committee from Jilin University First Hospital and informed consent was obtained from each AML patient and normal donor.

Results：

1. we discovered a novel intragenic 5366bp long noncoding RNA (lncRNA) within the IGF1R locus, named IRAIN, which is transcribed in an antisense direction from an intronic promoter.

2. we demonstrated IRAIN was expressed exclusively from the paternal allele, with the maternal counterpart being silenced in normal donor and AML patients.

3. we confirmed that IRAIN interacts with IGF1R chromatin DNA and is involved in the formation of an intrachromosomal enhancer/promoter loop. Knockdown of IRAIN lncRNA with shRNA abolishes this intrachromosomal interaction.

4. We demonstrated IRAIN was downregulated both in AML cell lines and high risk AML patients. Furthermore，the expression of IRAIN was dramatical higher in APL patients achieved molecular remission.

Conclusion：Our results identify IRAIN as a new imprinted suppressive lncRNA involved in long range IGF1R DNA interactions in AML. As a putative tumor suppressor and a novel treatment target， further studies are needed to delineate the specific role of this newly identified lncRNA in the uregulation of the IGF pathway in AML.

Disclosures

No relevant conflicts of interest to declare.
Differential Response of Fanconi Anemia Hematopoietic Stem and Progenitor Cells to Oxidative and Oncogenic Stresses
Authors: Du Wei , Amarachintha Surya , Ozlem Erden , Pang Qishen
Published: November 2019

Members of the Fanconi anemia (FA) protein family are involved in DNA damage response. A common damage to DNA in vivo is oxidative stress, and compelling evidence suggests that FA cells are in an in vivo pro-oxidant state. In response to oncogenic activation, normal cells induce genetically encoding programs that prevent deregulated proliferation and thus protect multicellular organisms from cancer progression. How FA cells respond to oxidative DNA damage and oncogenic stress is largely unknown. By employing an in vivo stress-response model expressing the Gadd45b-luciferase transgene, we show here that hematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene Fanca or Fancc differentially responded to oxidative and oncogenic stresses. Compared to wild-type controls, Fanca-/- or Fancc-/- HSPCs exhibited a persistent response to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. In contrast, using two inducible models of oncogenic activation (LSL-K-rasG12D and MycER), we identify a short-lived response of FA HSPCs to oncogenic insults both in vitro and in vivo. Mechanistic studies revealed that loss of Fanca or Fancc impaired oncogenic stress-induced senescence (OIS), and genetic correction of Fanca or Fancc deficiency restored OIS in HSPCs. Furthermore, FA deficiency compromised K-rasG12D-induced arginine methylation of p53 mediated by the protein arginine methyltransferase 5 (PRMT5). Finally, forced expression of PRMT5 in HSPCs from LSL-K-rasG12D/CreER-Fanca-/- mice prolonged oncogenic response and delayed leukemia development in recipient mice. Taken together, our study demonstrates differential responses of HSPCs to oxidative and oncogenic stresses and identifies the FA pathway as an integral part of this versatile cellular mechanism.

Disclosures

No relevant conflicts of interest to declare.
Activity of ABT-199 and Acquired Resistance in Follicular Lymphoma Cells
Authors: Bodo Juraj , Zhao Xiaoxian , Smith Mitchell R. , Hsi Eric D.
Published: November 2019

ABT-199, a platelet-sparing pro-apoptotic BH3 mimetic shows promise in the treatment of B-cell malignancy such as chronic lymphocytic leukemia (CLL). Given the central role of BCL2 expression in follicular lymphoma (FL), we studied the effects of ABT-199 on proliferation, induction of apoptosis, and signaling pathways in the t(14;18)+ FL cell lines WSU-FSCCL and FC-TxFL2 and in primary FL cells. We also developed an ABT-199-resistant FC-TxFL2 cell line by continuous treatment with ABT-199.

The growth of tested cell lines and primary FL cells was significantly inhibited with nanomolar concentrations of ABT-199. FC-TxFL2 cells (IC50 = 7 nM) were more sensitive to ABT-199 treatment than WSU-FSCCL cells (IC50 = 110 nM). The increased sensitivity was associated with higher levels of BIM pro-apoptotic protein. ABT-199 induced rapid phosphorylation of JNK kinase and substantial decrease of mitochondrial potential in FC-TxFL2 cells. Inhibition of JNK activation further augmented the apoptosis induced with ABT-199. All other tested anti-apoptotic (such as BCL-2, BCL-xL, MCL-1) and pro-apoptotic proteins (such as Bax, Puma, Bak, Bad, Noxa, Bid, Bok) were similarly expressed in both cell lines and did not change upon ABT-199 treatment. Surprisingly, cleavage of Bid was observed, which points also to activation of extrinsic apoptotic pathway. In cells with acquired resistance after continuous ABT-199 exposure, we identified increased levels of MCL-1, and elevated phosphorylation of BCL-2 on T56 and of AKT on S473, but also evidence for increased autophagy, as assessed by LC3BI/II expression, compared to parental cells. These cells remained resistant several weeks after omitting ABT-199 from culture medium.

In a FL murine xenograft model, ABT-199 showed anti-tumor activity. Treatment significantly decreased tumors volumes compared to untreated mice. Subsequently isolated xenograft FC-TxFL2 cells previously exposed to ABT-199 showed increased resistance to ABT-199. However, unlike in vitro exposed cells, the resistance quickly diminished after cultivation without ABT-199. Next, the ability to overcome ABT-199 resistance with epigenetic modifiers such as decitabine and vorinostat was tested. A strong synergistic effect in potentiation of apoptosis with these therapeutics was observed.

Despite the theoretic rationale for BH3 mimetic activity in FL, activity in patients with FL has not been as dramatic as has been observed for CLL. Relatively easily acquired resistance observed in our model may explain this failure. Therefore, combinations that can prevent resistance acquisition should be intensively studied and moved quickly to clinical trials.

Disclosures

Smith: Abbvie: Research Funding.
Reactive Oxygen Species Produced By Myeloid Cells Block CD20-Antibody Dependent NK Cell-Mediated Cytotoxicity Against Chronic Lymphocytic Leukemia Cells
Authors: Werlenius Olle , Aurelius Johan , Simpanen Maria , Riise Rebecca E , Akhiani Ali A , Martner Anna , Andersson Per-Ola , Hellstrand Kristoffer , Thorén Fredrik B
Published: November 2019

Introduction:

The addition of the anti-CD20 antibodies rituximab or ofatumumab to chemotherapy has improved the outcome for patients with chronic lymphocytic leukemia (CLL), but the efficacy of anti-CD20 monoclonal antibodies (mAbs) as single agents is limited. Immunosuppressive reactive oxygen species (ROS), produced by myeloid cells, including monocytes, may be a limiting factor for the efficacy of ADCC-dependent therapy. With the aim of reducing a potentially inhibitory role of ROS on mAb-dependent NK cell cytotoxicity against CLL cells, we performed in vitro cytotoxicity experiments in the presence of monocytes and the ROS formation inhibitor histamine dihydrochloride (HDC).

Methods:

After informed consent, blood samples were collected from Binet stage A CLL patients seen at Sahlgrenska University Hospital, Gothenburg, Sweden. NK cells, monocytes and CLL cells were isolated from PBMCs by immunomagnetic negative selection or flow cytometry. ROS production by monocytes was assessed by isoluminol-enhanced chemiluminescense upon stimulation with the chemotactic peptide fMLF in the presence or absence of CLL cells, rituximab (Roche), ofatumumab (GSK), HDC or the NADPH oxidase inhibitor DPI. NK cell death was assessed by flow cytometry after over-night culture of monocytes and lymphocytes. Autologous ADCC assays were performed with NK cells, monocytes and CFSE-labeled CLL cells in the presence or absence of rituximab, ofatumumab, HDC, catalase and interleukin-2 (IL-2). CLL cell death was determined by flow cytometry after live/dead staining. The study was approved by the local ethical review board of Gothenburg, and conducted in accordance with the declaration of Helsinki.

Results:

Rituximab and ofatumumab both triggered extracellular ROS production by monocytes. ROS production was blocked by the NADPH oxidase inhibitor DPI. Furthermore, monocytes induced ROS-dependent apoptosis in adjacent NK cells. In the cytotoxicity experiments, rituximab-mediated NK cell dependent cytotoxicity against CLL cells was significantly inhibited by the presence of autologous monocytes. This suppression, as well as the induction of NK cell apoptosis, were dependent on ROS, since both HDC and catalase significantly increased cytotoxicity and rescued NK cells from monocyte-induced apoptosis. Expectedly, IL-2 augmented NK cell-mediated ADCC.

Conclusion:

The results imply that CLL patients harbor immunosuppressive myeloid cells that reduce CD20-antibody dependent NK cell-mediated cytotoxicity against leukemic cells by producing ROS. We propose that treatment with HDC, preferably in conjunction with NK cell stimulatory cytokines, may improve anti-CD20-based immunotherapy in CLL.

Disclosures

No relevant conflicts of interest to declare.
Myeloid Dysfunction in Sickle Cell Disease
Authors: Romano Alessandra , Parrinello Nunziatina Laura , Conticello Concetta , Rosso Rossellina , Caterina Perrotta , Colletta Grazia , Ximenes Benedetta , La Cava Piera , Meli Carmela RITA , Di Raimondo Francesco
Published: November 2019

Background

In sickle cell disease (SCD), profound anaemia and severe hemosiderosis cause functional and physiological abnormalities in various organ systems, including immune system. Infectious complications are common, constitute the second most common cause of mortality and a main cause of morbidity. During the haemolytic crisis, large amount of arginase (s-Arg-1) are released, potentially acting as immunosuppressor molecule.

Despite its clinical impact, only a few is known about myeloid dysregulation in SCD.

Aim Detecting immunological impairment at the steady state evaluating myeloid and lymphoid cells in peripheral blood of SCD patients.

Material and Methods

Between May and June 2014,peripheral blood obtained from 30 consecutive SCD patients at the steady state plus 30 healthy subjects was studied for evaluation of myeloid subpopulations and lymphoid paresis.

Myeloiddys function was evaluated as percentage and absolute count of circulating myeloid suppressor cells (MDSC) in peripheral blood assessed by flow cytometry as follows: im-MDSC (CD34+/CD11b+/CD13+/CD14-/ HLA-DR-/CD45+), neutrophilic-like N-MDSC (CD11b+/CD13+/CD15+/CD14-/HLA-DR-/Lin-) and monocytic-likemo-MDSC (CD14+/HLA-DRlow/-), phagocytic activityof granulocytes using a commercially available kit (Phagotest R), amount of Arg-1 expressed in mature granulocytes by RT-PCR and circulating s-Arg-1 using a commercially available ELISA kit (Biovendor).

Results

The capability of phagocytosis of granulocytes wassignificantly reduced compared to healthy subjects (p=0.001). G-MDSC subset was not increased, while mo-MDSC subpopulation was increased in SCD (p=0.001) but not in thalasso-SCD.

s-ARG-1 was increased in both SCD and thalasso-SCD (respectively 203±3 ng/mL and 248±6ng/mL, p=0.003) and positively correlated with the amount of HbS (r=0.7, p=0.002). Arg-1 expression in granulocytes was increased (20 times higher than healthy controls, p=0.002)

Conclusion

SCD and thalasso-SCD caucasian patients exhibiti mmunosuppressive myeloid markers including reduced phagocytosis, increased amount of mo-MDSC, Arg-1 expression in granulocytes and circulating s-Arg-1. Further analysis are ongoing to detect if the same myeloid impairment occurs during vase occlusive crisis and in a different genetic background, like in Afro-Americans.

Disclosures

No relevant conflicts of interest to declare.
Clinical and Laboratory Evaluation of Patients with Familial Hemophagocytic Lymphohistiocytosis in a Single Center; Striking the Importance of Central Nervous System and Hepatic Involvement
Authors: Beken Burcin , Aytac Selin , Kuskonmaz Barýs , Balta Gunay , Cetin Mualla , Gurgey Aytemiz , Uckan Cetinkaya Duygu , Gumruk Fatma
Published: November 2019

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive, life-threatening condition characterized by deficient immune response. The major underlying defect in FHL is impaired Natural Killer (NK) cell and T lymphocyte degranulation and/or cytotoxicity, and the only curative treatment is hematopoetic stem cell transplantation (HSCT).

In this retrospective study, 57 patients who were diagnosed with FHL at Hacettepe University Ýhsan Dogramacý Children’s Hospital Pediatric Hematology Department, between November 1994 and December 2012 were evaluated for their clinical and laboratory findings, factors affecting the response to treatment, prognosis and survival.

Median age of these patient’s was 18 months (0.16-226 months). Mutation analysis were performed in 37 patients and of these, 11 had UNC13D, 10 had PRF1 and three had STX11 gene mutation. Fourty four persent of the patients had central nervous system (CNS) involvment on admission and spinal cord involvement was also seen on five patients. The median age of the patients who had spinal cord involvement was 11 years (7.5-14 years) and all of these have neurological symptoms, positive cerebrospinal fluid (CSF) and brain MRI findings related to FHL. One of the patients had spinal cord involvement on admission, the other four patients developed spinal cord involvement at follow up and all of these four patients were died despite HSCT.

Remission was achieved in 24 patients with the treatment protocols, HLH-1994 and HLH-2004, in a median time of 76 days (min-max:15-705 days). When factors affecting the remission time were evaluated, remission time was prolonged 3.1 times in patients with ferritin level ≥1500 mg/dL. The five year overall survival was 25% and HSCT was performed in 18 patients (32%) and five year survival rate was 44% in the transplant group.

When patients were grouped according to their ages [Group 1 (0-24 months) and Group 2 (>24 months)]; there were 33 patients below two years and 24 patients above two years. Patients in Group 1 had higher ferritin (p=0.001) and AST (p=0.043) levels whereas lower fibrinogen (p=0.023) levels on admission, survival rate was also lower in these patients (p=0.017). When we analyze the patients in Group 1, we found that patients with hepatic involment had poor prognosis (p=0.002).

In conclusion, FHL is a disease with high mortality rates and the only curative treatment is HSCT. When FHL is suspected, genetic studies should be done and not only CNS but also spinal cord imaging should be performed as well. Treatment must be started immediately in patients who has poor prognostic factors including, age below two years, ferritin level above 1500 mg/dL, and spinal cord, bone marrow or hepatic involvement.

Disclosures

No relevant conflicts of interest to declare.
Unusual Presentation for Unusual Infection: Disseminated Mycobacterium Avium-Intracellulare complex (MAC) in Patient with Sickle Cell Anemia Mimicking Blood Transfusion Reaction
Authors: Edrees Nayf , Howard Thomas H.
Published: November 2019

Nontuberculous mycobacteria (NTM) are ubiquitous free living soil and water– borne organisms that cause numerous clinical syndromes including lymphadenitis, skin, soft tissue and pulmonary infections, however disseminated infection is almost exclusively in patient with severe immunocompromise (i.e:HIV, Hematological malignancy, and bone marrow transplant).

Mycobacterium avium complex (MAC) is hard to diagnose as it is considered slow grower NTM.

We describe a case of disseminated Mycobacterium avium-intracellulare complex infection in teenager with sickle hemoglobinopathy with unique presentation mimicking pRBCs transfusion reaction.

Patient presented on three different occasions with tachycardia, hypotension and fever within 2-24 hours following pRBCs pheresis, all three episodes were investigated and were negative for transfusion reactions.

Patient had central venous catheter (CVC), frequent admissions for vaso-occlusive painful episode, on hydrocortisone for adrenal insufficiency and off Hydroxyurea for two months.

Diagnosis of mycobacterium avium complex bacteremia was confirmed by two positive blood cultures, whole body CT scan showed liver nodules, spleen nodules and lung nodules. Pulmonary dissemination was confirmed by Biopsy and culture, Lymphocyte markers showed severe lymphopenia with absolute CD4 count of 64.

Patient underwent treatment with three month of four antibiotics followed by 9 months of three antibiotics with removal of the central line, follow up scan showed decrease size and numbers of nodules, patient started tolerating pheresis within one month of the antibiotics initiation.

NTM infection should be added to the list of pathogens in sickle cell patients with CVCs and fever and should be considered in frequent pRBC transfusion like reaction with negative workup.

Routine blood culture can identify rapid growing NTM but specific mycobacterial blood culture is required in case of other NTM species (slow grower). As dissemination almost always occurs in those with impaired cellular immunity, HIV testing and lymphocyte markers should be performed

Removal of involved CVCs is essential for the treatment as well as appropriate antimicrobial medications.

Disclosures

No relevant conflicts of interest to declare.
A Novel Notch/NRF2 Mechanism Mediates Reductive Stress in Acute Myeloblastic Leukemia (AML): A Therapeutic Strategy to Overcome Chemoresistance
Authors: Kannan Sankaranarayanan , Zweidler-McKay Patrick A.
Published: November 2019

Background: Chronic oxidative stress (COS) is the consequence of prolonged elevation of reactive oxygen species (ROS). In the context of AML, COS leads to sustained induction of antioxidant pathways to compensate for COS. The presence of abundant antioxidants may contribute to chemoresistance in AML. NRF2 is a master regulator of the antioxidant response, is induced by oxidative stress and functions as a cyto-protective mechanism in acute oxidative stress. In AML, sustained NRF2-activation can be viewed as a pathological maladaptation as it leads to elevated reductive metabolite formation, termed reductive stress (RS). In this study we describe a novel link between NRF2-induced reductive stress and the Notch pathway in AML.

Approach: Redox balance was measured in AML patient samples in the setting of Notch activation (via DLL1 ligand) and/or NRF2 inhibition (via Brusatol).

Results: AML samples demonstrate increased reductive metabolites (i.e. reductive stress) compared to controls and Notch activation decreases aberrant reductive metabolite levels to control baseline (GSH/GSSG ratio, panel A). Mechanistically, Notch signaling induced increased ROS formation, decreased NRF2 protein levels, and decreased expression of multiple NRF2-responsive antioxidants (data not shown). Importantly, Notch activation also enhanced the efficacy of an NRF2 inhibitor Brusatol (from *P≤0.05 to **P≤0.01) in combination with ROS-producing chemotherapeutic doxorubicin, eradicating >98% of AML cells within 48 hours (** P≤0.01; panel B).

Conclusions: We describe the aberrant accumulation of reductive metabolites in human AML as a potential cytoprotective maladaptation, i.e. reductive stress. Furthermore, we demonstrate a novel mechanism whereby Notch activation regulates the antioxidant response via suppression of the master regulator NRF2. These data establish the pivotal role of Notch/NRF2 in the regulation of antioxidant responses in AML, which have potential clinical significance for ROS-inducing chemotherapies.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Impact of Chemotherapy Delay on Overall Survival for AML with I DH1/2 Mutations: A Study in Adult Chinese Patients
Authors: Wang Jing Han , Guo Qi , Ma Zhi Xin , Ma Qiu Ling , Yu Meng Xia , Yin Xiu Feng , Lu Sha Sha , Xie Qiong Hong , Jiang Yue Hong , Shen Dan , Ma li Ya , Shi Hui , Yu Wen Juan , Lou Ye Jiang , Li Ying , Yang Min , Xu Gai Xiang , Mao Li Ping , Li Jian Hu , Wang Fan Ping , Wang Dong Mei , Wei Ju Ying , Tong Hong Yan , Huang Jian , Jin Jie
Published: November 2019

The effect of time from diagnosis to treatment (TDT) on overall survival of patients with acute myeloid leukemia (AML) remain obscure. Furthermore, whether chemotherapy delay impacts overall survival (OS) of patients with a special molecular subtype has not been investigated. Here, we enrolled 364 cases of AML to assess the effect of TDT on OS by fractional polynomial regression in the context of clinical parameters and genes of FLT3 ITD, NPM1, CEBPA, DNMT3a, and IDH1/2 mutations. Results of the current study show IDH1/2 mutations are associated with older age, M0 morphology, an intermediate cytogenetic risk group, and NPM1 mutations. TDT associates with OS for AML patients in a nonlinear pattern with a J shape. Moreover, adverse effect of delayed treatment on OS was observed in patients with IDH1/2 mutations, but not in those with IDH1/2 wild type. Therefore, initiating chemotherapy as soon as possible after diagnosis might be a potential strategy to improve OS in AML patients with IDH1/2 mutations.

Disclosures

No relevant conflicts of interest to declare.