bluuebunny/crossref_metadata_2025 huggingface dataset:
IFNγ-Induced Necroptosis Contributes to Hematopoietic Stem and Progenitor Cell Death and Bone Marrow Failure
Authors: O'Donnell Joanne , Roderick Justine E. , Pasparakis Manolis , Kelliher Michelle
Published: October 2019

RIPK1 has important kinase-dependent and kinase-independent scaffolding functions that prevent or activate necroptosis or apoptosis. Complete RIPK1 deficiency results in cell death and widespread inflammation yet tissue specific RIPK1 deletion can result in apoptosis, necroptosis and/or systemic inflammation, depending on the cell type. We have previously demonstrated that a hematopoietic RIPK1 deficiency results in constitutive activation of RIPK3 and MLKL and induction of necroptosis (Roderick et al, PNAS, 2014). These mice exhibit elevated serum TNFα and IFNγ levels, hematopoietic stem and progenitor cell (HSPC) loss, and ultimately succumb to bone marrow failure (BMF). When mice with a hematopoietic RIPK1 deficiency were placed on a RIPK3 deficient background, plasma pro-inflammatory cytokine and chemokine levels were reduced, HSPC numbers increased and BMF was significantly delayed. These mouse genetic data demonstrate that necroptotic death contributes to BMF in the mouse. To identify the receptor/ligands that trigger necroptosis, we generated mice with a hematopoietic RIPK1-deficiency on the Tnfr1-/-and Tnfr1-/-Tnfr2-/-genetic backgrounds. An absence of TNF signaling failed to prevent necroptosis and consequently, vav-iCre Ripk1f/f Tnfr1-/- Tnfr2-/-mice succumbed to BMF. Because type II interferons can also induce necroptosis, we generated vav-iCre Ripk1f/f Ifngr1-/- mice. These mice appear phenotypically normal demonstrating that an absence of IFNγ signaling prevents HSPC necroptosis and BMF.Collectively, these data may implicate IFNγ-mediated, RIPK3-dependent necroptosis in human BMF syndromes and raise the intriguing possibility that the progressive HSPC elimination observed in these patients reflects in part, IFNγ-induced necroptotic death.

Disclosures

No relevant conflicts of interest to declare.
Telomere Length and Somatic Mutation in Aplastic Anemia- Correlation with Response to Treatement
Authors: Park Hee Sue , Park Si Nae , Im Kyoungok , Kim Jung Ah , Hwang Sang Mee , Lee Dong Soon
Published: October 2019

Background: Telomere length (TL) of cell reflects the cellular age and short TL suggest the potential susceptibility to DNA damage. We investigated whether 1) the treatment response is associated with TL of hematopoietic cells and 2) whether somatic mutations are associated with TL in patients with aplastic anemia (AA).

Methods: We measured the mean TL and heterogeneity of bone marrow nucleated cells at the single cell level by quantitative fluorescence in situ hybridization. A total of 146 AA patients were enrolled; AA (n=146) and normal peripheral blood control (n=147). TL was expressed as telomere/centromere ratio. We analyzed the average and the distribution of TL belonging to the lowest 10th percentile of cell population. Along with measurement of TL, target sequencing for 88 hematopoiesis-related genes was performed to detect somatic mutations.

Results: We investigated the frequencies of cytogenetic aberrations and somatic mutations in 437 patients with aplastic anemia (AA) with prognostic relevance. Clonality was determined by G-banding/ fluorescent in situ hybridization (FISH) (n=280 patients), and targeted capture sequencing for 88 hematopoiesis-related genes (n=77). Of 437 patients, 20 (4.6%) showed disease progression and monosomy 7(50%) was the most predominant cytogenetic evolution at disease transformation. One third of patients (25/77, 32.4%) presented at least 1 mutation and the number of mutated genes was 18 and frequently mutated genes was NOTCH1 (3/25, 12%), NF1 (3/25, 12%), SCRIB (2/25, 8%), BCOR (2/25, 8%), DIS3 (2/25, 8%), DNMT3A (2/25, 8%), MED12 (2/25, 8%). In contrast to that 4.6% of AA patients showed disease progression, 4.0% of patients with SM showed disease progression. The telomere length of bone marrow nucleated cells was measured at single cell level by FISH (n=146) patients. Mean TL of AA (T/C ratio 6.99) was significantly shorter than normal control (T/C ratio 11.24). Patients with cytogenetic aberrations (CA) and/or somatic mutation (SM) showed significantly shorter TL compared to AA with normal karyotypes. However, the severity of AA did not correlate with TL. Patients with TL<6.88 showed significantly lower CR to IST (p=0.008) (Fig.1)

Conclusion: Patients with TL attrition showed poor response to IST. Short TL can be used not only as a biomarker for AA, but also as a predictive marker for treatment response to IST. In addition, CA and/or SM was strongly associated with adverse prognosis or treatment response, so we strongly suggest to adopt more sensitive test for the detection of minor clonal cell population in AA.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Oncogenic Splicing of the Apoptosis-Stimulating Protein of p53-2 (ASPP2) Is an Initiating Step in Leukemogenesis Facilitating Acquisition of Structural Genomic Mutations
Authors: Kampa-Schittenhelm Kerstin Maria , Mau-Holzmann Ulrike , Lopez Charles , Schittenhelm Marcus M.
Published: October 2019

ASPP2 is an independent haploinsufficient tumor suppressor which initiates induction of apoptosis after cellular damage in a TP53-dependent manner. We recently reported the identification of two C-terminally truncated splice variants of ASPP2 with high frequency in acute leukemias. Tantalizingly, these variants lack the functionally relevant TP53 binding sites, suggesting a dominant-negative, oncogenic function. The most prevalent isoform lacking exon 17 (ASPP2k) is detected in up to 60% of acute leukemias. A cell sort for the CD34+ stem cell fraction confirmed ASPP2k-expression in the leukemia-initiating clone. Immunoblotting revealed translation of ASPP2k into a genuine protein isoform.

Generation of isoform-specific antibodies allows to discriminate ASPP2k from the wildtype isoforms. Interestingly, ASPP2k is the sole isoform expressed in some native leukemia samples. Other samples reveal a heterozygous expression pattern suggesting a dominant-negative function of the C-terminally truncated ASPP2k isoform.

Forced expression of ASPP2k using a HisMax plasmid vector in murine IL3-dependent pro-B Ba/F3 cells induced a more aggressive phenotype with mitotic failure and perturbed cellular proliferation compared to the empty vector controls. Of note, IL3 was successfully weaned in the ASPP2k cells - indicating that autoactivating genomic alterations must have occurred upholding cellular integrity and viability. Fractionated gamma irradiation accelerated this process. In contrast, the empty vector strains did not survive IL3 weaning. Chromosomal analyses of IL3-independent Ba/F3 ASPP2kcells revealed structural alterations including monosomies and additional marker chromosomes.

Furthermore, we demonstrate that ASPP2k is stress-inducible. Changing culture conditions (e.g. temperature) of cell lines or ex vivo native AML samples resulted in a dramatic increase of the ASPP2k isoform. Consecutive treatment of cells with daunorubicin lead to relative resistance of pre-stressed cells compared to their counterparts cultured under standard conditions. Vice versa, specific knock-down of ASPP2k resulted in a significant increase in induction of apoptosis upon chemotherapy compared to empty vector controls.

Our data demonstrate that ASPP2k plays a distinctive role as an anti-apoptotic regulator of the TP53 checkpoint rendering cells to a more aggressive phenotype as evidenced by proliferation and apoptosis rates - and facilitates acquisition of genomic mutations, a first initiating step in leukemogenesis.

Disclosures

No relevant conflicts of interest to declare.
Loss of Function Cohesin Complex Gene Mutations Create Neomorphic Cell States Advantageous to Oncogenesis
Authors: Smith James S , Savage Kienan I , Thompson Alexander , Mills Ken I
Published: October 2019

Multiple large scale sequencing projects have identified a significant number of mutations in Acute Myeloid Leukaemia (AML) and high-risk Myelodysplastic syndromes (MDS) patients. Emerging from this are those affecting members of the cohesin complex, predominantly STAG2, and it has been highlighted that the poorer overall survival of this chromatin structure group is second only to patients carrying mutations affecting TP53. The Cohesin complex is an evolutionary conserved, multimeric protein complex consisting of SMC1, SMC3, RAD21 and STAG1/ STAG2. The complex plays a pivotal role during mitosis, ensuring sister chromatid cohesion however, substantial data exists highlighting roles in DNA damage repair, homologous recombination and most significantly, contribution to long-range interactions between cis-regulatory elements of the genome.

Here, we have used the CRISPR/Cas9 genome editing system to create an isogenic model featuring a premature stop codon within STAG2 at a region which presents as highly mutated in patient data sets. This stop codon generates a loss of function phenotype due to the haplo-insufficient X-linked nature of the STAG2 gene. Following model validation, the impact on genome wide distribution of the cohesin complex was assessed using ChIP-Seq for STAG1, STAG2, SMC3 & the transcriptional insulator CTCF.

A modest increase in overall CTCF binding from ~67,000 to ~72,000 high confidence peaks was observed, despite a ~7-fold decrease in enrichment, indicating decreased global insulator activity as a result of the inactivating STAG2 mutation. As expected, compensation between the STAG members was observed at a number of sites within transcriptionally active topologically associated domains, with an increase in STAG1 binding peaks (~17,000 to >25,000 sites) within the mutant model, despite no changes in average global enrichment. This data was integrated with RNA-seq of the isogeneic parental and mutant cells to determine the transcriptional impact of changes in cohesin complex binding on gene expression within active and repressive domains.

Assessment of the active and repressive super-enhancer associated marks H3K27ac and H3K27me3 showed increased global levels of both marks without increase in the polycomb repressive complex 2 member, EZH2 total protein levels. These findings indicate that increased H3K27ac and H3K27me3 marks, in this model, are due to altered domain boundaries, associated with local activation of both H3K9ac and H3K9me3, leading to aberrant gene expression and consequently a neomorphic state within these mutant cells.

With recent further stratification of de-novo AML, secondary AML and high-risk MDS emerging, this study on the aberrant and non-canonical nature of the cohesin complex provides avenues for an attractive therapeutic target and personalised treatment approach for patients within the chromatin subgroup.

Disclosures

No relevant conflicts of interest to declare.
Lung Vaso-Occlusion in Sickle Cell Disease Mediated By Arteriolar Neutrophil-Platelet Micro-Emboli
Authors: Bennewitz Margaret F. , Vats Ravi , Tutuncuoglu Egemen , Gladwin Mark T. , Sundd Prithu
Published: October 2019

Background: Vaso-occlusive crisis (VOC) is the primary reason for emergency medical care by sickle cell disease (SCD) patients. SCD patients hospitalized with VOC often develop acute chest syndrome (ACS), a form of acute lung injury, suggesting a role for pulmonary vaso-occlusion in the onset of ACS. However, the cellular, molecular and biophysical mechanism of pulmonary vaso-occlusion is unknown.

Methods: SCD transgenic or non-sickle control mice were intravenously (IV) challenged with 2 to 3 ng of bacterial lipopolysaccharide (LPS). Fluorescent anti-mouse Ly-6G and CD49b mAbs were administered IV for in vivo staining of circulating neutrophils and platelets, respectively. Multiphoton excitation enabled quantitative fluorescence intravital lung microscopy (qFILM) was used to determine the molecular mechanism of pulmonary vaso-occlusion in live mice. Function-blocking anti-mouse P-selectin mAb (Fab fragments) was administered IV to assess the role of platelet P-selectin in promoting pulmonary vaso-occlusion.

Results: A nanogram dose of IV LPS selectively triggered pulmonary vaso-occlusion in SCD but not control mice. Remarkably, pulmonary vaso-occlusion involved occlusion of the pre-capillary pulmonary arteriole bottle-necks (junction of an arteriole and capillaries) by large neutrophil-platelet embolic aggregates. IV administration of Fab fragments of function blocking anti-P-selectin mAb led to the resolution of pulmonary vaso-occlusion, which was primarily mediated by the attenuation of large neutrophil-platelet aggregates into smaller aggregates that are not stopped by the arteriolar bottle-necks.

Conclusion: These results establish the relevance of neutrophil-platelet aggregation in pulmonary arterioles in promoting pulmonary vaso-occlusion in SCD and also highlight the therapeutic potential of inhibiting platelet P-selectin to prevent ACS in SCD patients hospitalized with VOC.

Acknowledgments: This study was supported by 1R01HL128297-01 (P.S.), AHA 11SDG7340005 (P.S.), VMI startup funds (P.S.). M.F.B. was supported by NIH-NHLBI training grant T32HL110849 and NIH-NHLBI F32 NRSA 1F32HL131216-01.

Disclosures

No relevant conflicts of interest to declare.
Amlexanox As a Possible Breakthrough Drug for MLL/AF4-Positive Acute Lymphoblastic Leukemia
Authors: Tamai Hayato , Yamaguchi Hiroki , Miyake Koichi , Takatori Miyuki , Kitano Tomoaki , Yui Syunsuke , Fukunaga Keiko , Nakayama Kazutaka , Inokuchi Koiti
Published: October 2019

Background: MLL/AF4-positive acute lymphoblastic leukemia (ALL) is associated with poor prognosis even after allogeneic hematopoietic stem cell transplantation. Previously, we reported that this ALL shows resistance to TNF-α, which is the factor involved in the graft versus leukemia (GVL) effect or tumor immunity, by upregulation of S100A6 expression followed by interference with the p53-caspase pathway. Amlexanox, an anti-allergic drug, was reported to inhibit the translocation pathway of endogenous S100A6 in endothelial cells.

Aims: This study was performed to examine the effects of Amlexanox on MLL/AF4-positive ALL.

Methods: In vitro analysis, cell growth of MLL/AF4-positive ALL cell lines ( SEM and RS4;11) were analyzed with TNF-α (10 ng/mL) and Amlexanox (0, 10, and 100 µg/mL).The effect of Amlexanox on S100A6 and p53-caspase pathways were examined by Western blotting (WB) analysis. In vivo analysis MLL/AF4-positive transgenic mice model, which show CD45R/B220+leukemia by 12 months of age we established and human peripheral blood mononuclear cell (Hu-PBMC) NOD/SCID mice transplanted with SEM-Luc were examined to compare mice fed diet containing Amlexanox (0.02%) with mice fed control diet.

Results: There were no significant differences between the growth of SEM or RS4;11 cells in the absence or presence of 10 µg/mL of Amlexanox in vitro under 10 ng/mL of TNF-α. However, both cells showed significant growth inhibition by 10 ng/mL of TNF-α in the presence of 100 µg/mL of Amlexanox (P = 0.0085 for SEM, P = 0.0196 for RS4;11) WB analysis showed that S100A6 was activated in the presence of 10 ng/mL TNF-α, and activated S100A6 was decreased and both acetyl-p53/p53 ratio and cleaved caspase 3/caspase 3 ratio were increased in cells treated with 100 µg/mL of Amlexanox under 10 ng/mL of TNF-α in the MLL/AF4-positive human ALL cell lines. In vivo, MLL/AF4-positive transgenic mice fed a diet containing Amlexanox (0.02%) developed significantly less volume of CD45R/B220+ leukemia at the age of 1 year in comparison with mice fed control diet (P<0.001 for BM and .P<0.001 for spleen). Hu-PBMC NOD/SCID mice transplanted with SEM-Luc in the Amlexanox group showed significantly longer survival than those in the control group (P < 0.014).

Conclusions: Amlexanox may be a breakthrough drug for MLL/AF4-positive ALL because it inhibits the resistance of MLL/AF4-positive ALL to TNF-α by downregulating S100A6 expression followed by upregulating the p53-caspase pathway.Specifically, allogeneic hematopoietic stem cell transplantation is expected to show beneficial effects in combination with Amlexanox.

Disclosures

No relevant conflicts of interest to declare.
Molecular Signature of Dormancy in CD34+CD38- Acute Myeloid Leukaemia Cells
Authors: Al-Asadi Mazin Gh , Castellanos Marcos , May Sean T , Russell Nigel H , Seedhouse Claire H , Pallis Monica
Published: October 2019

Chemotherapy drugs tend to spare cells in a state of dormancy (G0 phase of the cell cycle). Relapse of acute myeloid leukaemia (AML) is likely in part due to dormant cells evading remission-induction chemotherapy. Dormant AML cells have been identified in the bone marrow endosteal region which is characterised by an excess of TGFβ1 and a shortage of nutrients. We developed and characterized an in-vitro model of AML cell dormancy by exploiting these features.

Following preliminary investigation of several cell lines, the CD34+CD38- line TF1a was selected for in depth investigation. TF1a cells showed 72% inhibition of proliferation (p<0.001), with features of dormancy, in response to 72 hours TGFβ1+mTOR inhibitor treatment (mTOR pathway inhibition mimics major effects of nutrient scarcity).This treatment caused loss of Ki-67, as well as upregulating ALDH and CD34 and causing nuclear translocation of FOXO3a. In contrast to conventional serum-withdrawal assays for dormancy, the treatment had no impact on cell viability, assessed by Annexin V assay. Nor did the treatment lead to cell differentiation, assessed by CD11b staining and morphology.

Using whole human genome expression microarray and by intersecting differentially regulated genes in dormancy-induced TF1a cells in comparison to their proliferating (untreated) counterparts, we identified 240 genes which are significantly up-regulated >2 fold including genes involved in stemness, chemoresistance and tumour suppressor genes in addition to genes involved in canonical cell cycle regulation. There was striking upregulation of genes involved in adhesion and migration: raised expression levels of SPP1 (the gene coding for osteopontin), ITGB3, ITGB4, ITGA3 and CD44 in dormant cells were confirmed by real-time PCR. The most upregulated gene was SPP1/osteopontin (16 fold). Immunocytochemistry of biopsy material from AML patients confirmed high levels of osteopontin in the cytoplasm of blasts near the paratrabecular bone marrow. Osteopontin and other genes identified in this model, including well-characterised genes (e.g. CD44, CD47, CD123, ABBC3 and CDKN2B) as well as little-known ones (e.g. PTPRU, ITGB3 and BTG2), are potential therapeutic targets in dormant AML cells.

Disclosures

No relevant conflicts of interest to declare.
Mapping the Functional Cofactors of Oncogenic EZH2 in Natural Killer/ T Cell Lymphoma (NKTL)
Authors: Li Boheng , Yan Junli , Chung Tae-Hoon , Lee Pei Tsung , Chng Wee Joo
Published: October 2019

EZH2, the catalytic subunit of the PRC2 complex, has been shown to overexpress in different types of cancers. Oncogenic role of EZH2 originally was thought to exhibit through its Histone H3 K27 methyltransferase activity. In recent years, several studies, including ours, have highlighted new oncogenic roles of EZH2, either PRC2-independent, or as co-activator for other transcription factors in some context of cancers. Specifically in natural killer/ T cell lymphoma (NKTL), we have already uncovered that oncogenic role of EZH2 is independent of its methyltransferase function, and JAK3 switches EZH2 from a transcriptional repressor to an activator through direct phosphorylation thus conferring the lymphoma cells with growth advantage. What's more, we showed that JAK3, EZH2 and RNA POL II are in the same complex, whereas JAK3 does not stay together with PRC2 complex. In this study, in order to map all the interacting partners of PRC2 complex as well as EZH2-POL II complex, we used NKTL cell lines for SILAC Mass Spectrometry to identify interactome of EZH2, RNA POL II and PRC2 component SUZ12, respectively. Then we overlapped EZH2 and SUZ12 interactome for canonical factors assisting PRC2-mediated gene repression, as well as EZH2 and POL II interactome for non-canonical factors which might play a role in EZH2-regulated gene activation. Some of those non-canonical factors have been shown to display extraordinary high expression in EBV-positive lymphocytes among other cancerous and normal tissues (GTEx RNA-seq expression data), or in NKTL patient sample than normal NK cells (our own GEP data). Intriguingly, GO analysis of these three interactomes indicates that the major function of EZH2 in NKTL is realized mainly through the EZH2-POL II complex, rather than the PRC2 complex. These data reinforced the oncogenic role of EZH2-POL II complex in NKTL, and suggested new players in EZH2-mediated oncogenesis.

Disclosures

No relevant conflicts of interest to declare.
Rituximab Maintenance after First Line Immunochemotherapy Improves Overall Survival in Patients with Follicular Lymphoma
Authors: Vilmar Adam , Arboe Bente , Josefsson Par , Poulsen Christian , Christensen Jacob Haaber , Jørgensen Judit , Jensen Paw , Starklint Jørn , Pedersen Per Troellund , Pedersen Michael , Brown Peter de Nully
Published: October 2019

Introduction

Patients with follicular lymphomas (FL) often follow an indolent disease course initiated with the wait-and-watch approach. Chemotherapy, including rituximab (R), is inevitably required in most patients though. R-maintenance is indicated for patients achieving a partial or complete remission based results from randomized clinical trials and confirmed by meta-analyses demonstrating a prolonged progression free survival. A benefit in overall survival (OS), however, has yet to be established in the first line setting.

Patients and methods

We completed a retrospective analysis in patients extracted from the Danish National Lymphoma Register. Eligible patient data included histopathological confirmation of FL diagnosed in the period 2000-2014 who received R-Chemotherapy as first line treatment and surviving a minimum 1 year after treatment initiation. A Cox regression model was performed, adjusted for age, gender, stage, response rates and treatment period (pre-maintenance era (year 2000-2010) vs. maintenance era (year 2011-2014)) with hazard ratio (HR) of OS as the primary endpoint. All patients were followed until June 2016.

Results

1037 patients were extracted from the register. 481 patients received R-maintenance (RM-group) and 556 patients did not. The median age was 62 (26-90), 526 (51%) were male, LDH was elevated in 325 (31%) and 436 (42%) had a high FLIPI index. The median follow-up time was 6.3 years. The five year OS for all patients was 86%. A significant change in the chemotherapy regimens was observed between the two groups, since Bendamustine was more frequently used in the maintenance group. A significant lower HR was observed in the RM group with a HR of 0.63 (95% CI 0.44 - 0.88, P = 0.008). Furthermore, gender proved of significance, with a HR for men of 1.78 (95% CI 1.32-2.40, P < 0.001).

Conclusions

Rituximab maintenance treatment following first line immunochemotherapy in patients with follicular lymphoma improved survival in a large population based cohort of patients and consolidates this strategy as part of the standard of care in this patient group.

Disclosures

No relevant conflicts of interest to declare.
BCR/ABL Can Promote CD19+ Cell Growth but Not Render Them Long-Term Stemness
Authors: Li Donghe , Zhao Xuemei , Jiao Bo , Liu Ping , Ren Ruibao
Published: October 2019

Cancer stem cells are a subpopulation of malignant cells that have the capacity of both self-renewal and reconstitution of the cancer. Eradication of cancer stem cells is crucial for curing the malignant disease. Previous studies in hematopoietic malignancies showed that leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) chronic phase are originated from a hematopoietic stem cell (HSC), while LSCs in acute myeloid leukemia (AML) can either be derived from HSCs or be transformed from myeloid progenitors. But in acute B-lymphoblastic leukemia (B-ALL), the origin of leukemia stem cells is not clear. In this study, we tested whether BCR/ABL could transform B-lineage committed CD19+ cells to LSCs. We found that transducing BCR/ABL in CD19+ cells can promote their colony formation in vitro and induce B-ALL like disease in vivo. However, only BCR/ABL transduced whole bone marrow cells, but not CD19+ cells, can be transplanted multiple times in recipient mice, and the frequency of long-term LSCs from the latter ranges from 1/135 to 1/629. These studies suggest that LSCs in BCR/ABL+ B-ALL may originate from CD19- hematopoietic stem/progenitor cells and that CD19 chimeric antigen receptor (CAR) modified T cell therapy may not be effective in eradicating LSCs in BCR/ABL+ B-ALL.

Disclosures

No relevant conflicts of interest to declare.
PML-Rara/Nrf2-Regulated Mir-125b Targets CEBPA and Influences Acute Promyelocytic Leukemia Cell Proliferation
Authors: Chengwu Zeng , Zhang Yikai , Qin Tianyu , Yang Lijian , Chen Shaohua , Li Yangqiu
Published: October 2019

Acute promyelocytic leukemia (APL) is characterized by the presence of PML-RARA fusion protein. The PML-RARA oncoprotein is known to be the initiating factor for APL development. We have previously found that miR-125b is highly expressed in APL and upregulated by PML-RARA. However, the molecular mechanisms by which PML-RARA controls miR-125b expression had been uncharacterized. In the present study, we further confirmed that expression of PML-RARA resulted in a significant increase in primary miR-125b expression, and we found that the nuclear transcription factor E2-related factor 2 (Nrf2) targeting genes (HO1) was also upregulated (Figure 1A). Consistently, HO1 and miR-125b are transcriptionally upregulated by proteasome inhibitor (MG132) which has been showed to activate Nrf2 (Figure 1B). Knockdown of Nrf2 can inhibit the effect of PML-RARA expression and MG132 (Figure 1C). Thus, these results indicated that Nrf2 may mediate PML-RARA-induced miR-125b expression. Furthermore, we demonstrated the importance of miR-125b in APL cell proliferation (Figure 1D), we showed that promoting leukemic cell grow is associated with AKT pathway and MAPK signaling (Figure 1E). Subsequent bioinformatic and RNA-seq data analysis (Figure 1F and G) identified CEBPA as a putative target of miR-125b, and we further experimentally verified using luciferase reporter constructs (Figure H). In conclusion, we identified the mechanisms responsible for the high expression and the function of miR-125b in APL, suggesting that the expression of miR-125b could be a primary oncogenic event in APL leukemogenesis.

This study was supported by grants from the National Natural Science Foundation of China (No.81400102), the China Postdoctoral Science Foundation (No.2015M570751), and the Medical Scientific Research Foundation of Guangdong Province, China (A2015420).

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
RSK Inhibition Suppresses AML Proliferation through Activation of DNA Damage Pathways and S Phase Arrest
Authors: Dutta Ritika , Castellanos Maria , Tiu Bruce , Chae Hee-Don , Davis Kara L. , Sakamoto Kathleen M.
Published: October 2019

The 90 kDa Ribosomal S6 Kinase (RSK), downstream of the ERK signaling pathway, has recently been implicated in a wide variety of cancers, ranging from lung cancer to medulloblastoma, as a driver of cancer cell proliferation and survival. However, its role in Acute Myeloid Leukemia (AML) remains unknown. Thus, the goal of this study was to characterize RSK-dependent signaling pathways in AML, with the overall hypothesis that disruption of this pathway represents a potential strategy for the treatment of AML. The RSK family consists of four gene isoforms, RSK1-4 (RPS6KA1 (RSK1), RPS6KA2 (RSK3), RPS6KA3 (RSK2), RPS6KA4 (RSK4). Knockdown (KD) of RSK1 by shRNA in HL-60 and KG-1 cell lines resulted in reduced AML cell growth in vitro. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were injected with 2x106 HL-60 or KG-1 RSK1KD cells and vector control transduced cells in order to investigate the effects of RSK1 KD on AML cell growth and survival in vivo. Mice injected with RSK1 KD cells exhibited prolonged survival by 17 and 21 days respectively for HL-60 and KG-1 cell induced disease (p=0.0023 and 0.0018 respectively). These data indicate that RSK1 knockdown inhibits leukemia progression, and RSK1 is required for maximal proliferation of AML cells in vivo. Pharmacological inhibition of total RSK (RSK1-4) by the small molecule inhibitor BI-D1870 reduced AML cell growth and induced cell death in both AML cell lines and patient samples after treatment for 48 hours. The IC50 for growth inhibition was 1.8 uM for MOLM-13, 1.6 uM for MV-4-11, and 1.9 uM for HL-60 cells. In methylcellulose colony assays, normal hematopoietic stem and progenitor cell proliferation was not affected by RSK inhibition up to a concentration of 15 uM, establishing an approximately 10-fold therapeutic index. To elucidate the mechanism by which RSK inhibition suppresses AML proliferation, we performed cell cycle analysis with HL-60 cells. RSK inhibition by BI-D1870 resulted in delayed S-phase progression and accumulation of cells in late S-phase with increased pH2AX, cPARP, and CDK2/Cyclin A expression, as measured by flow cytometry. These data indicate that inhibition of RSK leads to activation of DNA damage pathways and arrest in S-phase, resulting in apoptosis. Inhibition of CDK activity rescued S-phase arrest, demonstrating that activation and dysregulation of CDK are crucial mediators of RSK inhibitor-induced S-phase arrest. In summary, this is the first study to demonstrate that RSK plays an important role in maintaining AML cell survival and proliferation and to position RSK as a promising target for treatment of AML.

Disclosures

No relevant conflicts of interest to declare.
Microrna (miR)-17-92 Contributes to Therapy Resistance in Burkitt Lymphoma Cells
Authors: Miles Rodney R. , Rodic Vladimir , Barth Matthew J. , Cairo Mitchell S. , Hermiston Michelle L.
Published: October 2019

Background

Burkitt lymphoma (BL) is curable, but patients with relapse or refractory disease have very poor outcomes. The underlying biological differences leading to relapse/refractory disease are unknown. We previously identified a potential association between microRNA (miR)-17-92 and relapse in patient samples. MiRs encoded by this gene have been implicated in therapy resistance, so we are seeking relationships between miRNA expression and therapy resistance in BL cells.

Methods

The miR-17-92 locus was targeted for deletion in Raji BL cells using custom CRISPR-Cas9 lentiviral vectors. Single cell-derived clones were established and locus deletion was determined by genomic DNA PCR. Inducible miR-17-92 expression in Raji cells was established by transduction of a cumate switch inducible lentivector system containing the miR-17-92 gene along with a CymR repressor expression vector (Raji/CymR/miR-17-92). MiR expression was assessed by TaqMan assay, and protein expression and caspase-3 cleavage were assessed by western blotting. Cells were treated with 4HC, the active metabolite of cyclophosphamide, or doxorubicin, and viability was assessed by AlamarBlue assay.

Results

Genomic DNA PCR verified hemizygous deletion of the miR-17-92 gene in several Raji BL clones; no homozygous deleted clones were identified. In two hemizygous clones R5 and R24, miR-17 expression was decreased to 0.24-fold and 0.37-fold, respectively, relative to the parental Raji line. The miR-17-92 target proteins PTEN and Bim were increased in the R5 and R24 clones by western blotting. Compared to Raji, R5 and R24 showed increased basal caspase-3 cleavage and much greater induction of caspase-3 cleavage with cytotoxic chemotherapy (10 µM 4HC). In addition, 0.5 and 1 µM doxorubicin showed a dose dependent induction of cleaved caspase in R5 and R24 cells that was much greater than in parental Raji cells. The IC50 for doxorubicin in R5 and R24 miR-17-92 hemizygous cells was markedly decreased (0.00025 and 0.00032 µM, respectively) compared to parental Raji cells (0.064 µM). Finally, miR-17 expression was inducible by treating cumate repressor vector transduced Raji/CymR/miR-17-92 cells with 30 µg/mL cumate (+2.8-fold at 3 days, +7.5-fold at 5 days).

Conclusion

We established miR-17-92 hemizygous-deleted cell lines that show increased PTEN and pro-apoptotic Bim, increased basal caspase-3 cleavage, and increased chemosensitivity. We have established Raji BL cell lines with inducible miR-17-92 expression to extend these experiments by determining the effect of increased miR-17-92 on chemosensitivity. Our findings show that high miR-17-92 expression in BL cells can contribute to therapy resistance, which may be important for understanding refractory BL in patients and could lead to more effective targeted therapies.

Disclosures

Cairo: Celgene: Research Funding.
Limited Role of Workup Including PET-CT, Esophagogastroduodenoscopy and Bone Marrow Biopsy in Staging of Ocular Extranodal Marginal Zone Lymphoma of Mucosa-Associated Lymphoid Tissue (MALT) Type
Authors: Chae Heejung , Suh Cheolwon , Yoon Dok Hyun , Hong Jung Yong , Park Chan-Sik , Huh Jooryung , Sa Ho-Seok , Kim Kyoung Won
Published: October 2019

Background

Lymphoma of the ocular adnexa accounts for approximately 1 to 2% of non-Hodgkin lymphomas and the most common subtype is extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) type (80-90%). Ocular MALT lymphoma is mostly localized disease with indolent clinical course. The workup for non-gastric MALToma is usually done similar to the workup for other non-Hodgkin lymphoma. However, as there are paucity of data in the role of PET, esophagogastroduodenoscopy and bone marrow biopsy,weaimed toinvestigate these issues in patients with ocular MALT lymphoma.

Patients and Method

One hundred thirty patients diagnosed as ocular MALT lymphoma between Jan 2001 and Dec 2015 at a single tertiary hospital were included in this retrospective analysis. We retrospectively reviewed clinical characteristics, diagnosis, natural history of disease, treatment and outcomes.

Result

The median age of all patients was 48 years (range, 20-70 years). Fifty-nine (45%) were male patients. The majority of patients (n=87, 67%) presented with stage I disease, followed by bilateral involvement, defined as stage I+I (n=21, 16%), stage IV (n=16, 12%), and stage II (n=6, 5%). Nodal involvement was present in 8% (n=10), and 12% (n=16) of patents accompanied extra-nodal involvement other than orbits at initial diagnosis. Positron emission tomography (PET) had low sensitivity in detecting primary orbit lesions (n=44/120, 37%). Moreover, only in 3% of cases (n=4), PET imaging after CT scans on neck, chest, abdomen and pelvis could provide additional clinically meaningful information that led to upstaging. Accompanying gastric MALToma was found in 2% (n=2/82) of patients by EGD. Those two patients all survived until last follow-up. Bone marrow (BM) involvement was found in 6% (n=7/120). None of them had clinically significant cytopenia. With a median follow-up period of 74 months, 5-year overall survival (OS) rate was 99% in all patients. There was no significant difference in 5-year OS rates according to BM involvement (100% with and 99% without BM involvement).

Conclusion

PET-CT, EGD and BM biopsy has limited role in staging workup of primary ocular MALT lymphoma. Those workups should be performedonly in selected cases.

Disclosures

No relevant conflicts of interest to declare.
Chimeric Antigen Receptor-Modified T Cells for the Treatment of Acute Myeloid Leukemia Expressing CD33
Authors: Song Degang , Swartz Michael H. , Biesecker Steve G. , Borda Fernando , Shah Rutul R. , Emtage Peter , Wierda William G. , Cooper Laurence J.N. , Chan Tim
Published: October 2019

Relapsed acute myeloid leukemia (AML) is an aggressive disease with very poor outcomes. Redirection of T-cell specificity via chimeric antigen receptor (CAR) has shown promising anti-tumor activity in clinical trials, particularly for B cell linage malignancies. CD33 is a transmembrane protein expressed on normal and malignant myeloid-derived cells as well (as on subsets of activated T cells and NK cells). Since this protein is commonly expressed on AML cells, we sought to evaluate the efficacy of targeting AML with CD33-specific CAR-T cells. We generated a lentiviral construct to co-express CD33-specific CAR and a kill switch based on a tag derived from the epidermal growth factor receptor. The latter allows for the conditional elimination of CAR-T cells in vivo. Following transduction of primary T cells, we confirmed CAR and kill switch co-expression by flow cytometry and western blot analyses. Elimination of genetically modified T cells was demonstrated using the clinically-available antibody, cetuximab. CD33 CAR-T cells demonstrated specific cytotoxicity to CD33+ target cell lines. CD33 CAR-T cells were also activated to produce IFNg, TNF, and IL-2 cytokines in response to CD33+ target cells. Furthermore, adoptive transfer of CD33 CAR-T in immunocompromised (NSG) mice bearing established CD33+(CD19neg) AML (MOLM-13) tumor resulted in reduction of tumor burden and improvement of overall survival, compared to control mice receiving CD19 CAR-T cells or no immunotherapy (Figure). Sampling of blood demonstrated the persistence of the CD33 CAR-T cells with no detection of AML (MOLM-13) tumor cells. These pre-clinical data demonstrate the effectiveness of CD33 CAR-T cells in targeting CD33+ AML tumor cells and provide a rationale for future clinical evaluation in AML patients with unmet medical need.

Disclosures

Song: Intrexon Corporation: Employment, Equity Ownership. Swartz:Intrexon Corporation: Employment, Equity Ownership. Biesecker:Intrexon Corporation: Employment, Equity Ownership. Borda:Intrexon Corporation: Employment. Shah:Intrexon Corporation: Employment, Equity Ownership. Wierda:Genentech: Research Funding; Gilead: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Acerta: Research Funding. Cooper:MD Anderson Cancer Center: Employment; Intrexon: Equity Ownership; Sangamo BioSciences: Patents & Royalties; Targazyme,Inc.,: Equity Ownership; City of Hope: Patents & Royalties; ZIOPHARM Oncology: Employment, Equity Ownership, Patents & Royalties; Miltenyi Biotec: Honoraria; Immatics: Equity Ownership. Chan:Intrexon Corporation: Employment, Equity Ownership.
Foxp3+ regulatory T Cells Maintain Bone Marrow Microenvironment for B Cell Differentiation from Hematopoietic Stem Cells
Authors: Nishikii Hidekazu , Pierini Antonio , Yokoyama Yasuhisa , Kimura Takaharu , Kwon Hye-Sook , Alvarez Maite , Baker Jeanette , Shizuru Judith , Wu Joy , Chiba Shigeru , Negrin Robert S.
Published: October 2019

Background: Foxp3+regulatory T cells (Treg) are a subpopulation of T cells, which regulate the immune system, maintain self-tolerance and enhance immune tolerance after transplantation. It was also reported that recipient derived Treg could provide immune privilege niche to allogeneic hematopoietic stem cells (HSC) after transplantation. However, the precise role of Treg in hematopoiesis has not been fully elucidated.

Methods: We used Foxp3-DTR mice (B6, CD45.2) for in vivo depletion of Treg through diphtheria toxin (DT) injection and investigated whether Treg depletion would affect hematopoiesis derived from HSC. To investigate whether Treg depletion affects the function of the bone marrow microenvironment, we transplanted wild type bone marrow cells into lethally irradiated Foxp3-DTR mice after Treg depletion.

Results: We found 1) a significant defect on B cell progenitors including mature B cells (IgM+B220+, P<0.001), pre-B cells (IgM-B220+CD19+cKit-, P<0.001) and pro-B cells (IgM-B220+CD19+cKit+, P<0.05), 2) LT-HSC population (CD34-/lowFlit3-cKit+Sca1+Lin-) was significantly expanded (p<0.01) and entered into cell cycle, 3) the residual Foxp3-CD4+ or CD8+ T cells in the bone marrow had an activated immune phenotype and clustered at sinusoids when bone marrow cells from Treg depleted mice were analyzed. Expanded LT-HSC from Treg depleted mice had reduced long-term reconstitution capacity when we performed competitive repopulation experiments using purified LT-HSC from Foxp3-DTR mice with or without Treg depletion (100 cells/mice, CD45.2), total bone marrow cells (2x10e5/mice, B6-F1, CD45.1/CD45.2) and congenic recipient mice (lethally irradiated B6, CD45.1). B cell reconstitution was also severely abrogated following transplantation using Treg depleted mice as recipients (p<0.01). In those mice, we observed a significant reduction of IL-7 production (p<0.01). Interestingly, we found that a subpopulation of CD45-TER119-CD31- ICAM1+ perivascular stromal cells are a major source of IL-7 in the bone marrow. ICAM1+ perivascular stromal cells also secrete SCF and CXCL12, which is crucial for the maintenance of LT-HSC. In Treg depleted BM cells, a significant reduction in IL-7 secretion from ICAM1+ perivascular stromal cells was observed, suggesting that this population is the target of activated T cells after Treg depletion.

Conclusions: These data demonstrate that Treg play a key role in B cell differentiation from HSCs by maintaining the immunological homeostasis in the bone marrow microenvironment. These data provide new insights into Treg biology and function in normal and stress hematopoiesis.

Disclosures

Negrin: Stanford University: Patents & Royalties.
Hepatitis in Association with Prolonged 6-Mercaptopurine Therapy
Authors: McILVANIE SAMUEL K. , MacCARTHY JOHN D.
Published: October 2019

Four patients are presented who developed toxic hepatitis while being treated for leukemia with 6-mercaptopurine and steroids. The onset was rapid and suggested an acute hepatitis with intrahepatic blockage. Pathologic examination of the liver in two patients who expired with hepatitis is presented and the pathology described. In the third autopsied case the hepatitis had resolved while receiving massive steroid therapy, even though the leukemia progressed rapidly when 6-mercaptopurine was withdrawn. Early recognition of the toxic hepatitis becomes especially important whero 6-mercaptopurine is the first drug to be used in the management of the patient with acute leukemia.
Abnormal Human Hemoglobins. I. Biosynthesis in Vitro of Fe59-Labeled Human Hemoglobin A and Hemoglobin S
Authors: KRAUS LORRAINE M. , MORRISON DEMPSIE B. , KRAUS ALFRED P.
Published: October 2019

The synthesis of Fe59 labeled-hemoglobin during incubation of bone marrow from normal and sickle cell anemia (crisis and non-crisis) patients has been investigated. The in vitro synthesis of hemoglobin S in the steady state (noncrisis) sickle cell anemia individual was found to proceed at a much faster rate than the synthesis of hemoglobin A from normal individuals. In the crisis of sickle cell anemia, the uptake of Fe59 and synthesis of hemogobin S was approximately at the same level as synthesis of hemoglobin A from the normal individual. The results of this study indicate that one of the mechanisms involved in sickle cell anemia crisis is a decrease in synthesis of hemoglobin S as compared to the synthesis of hemoglobin S during the steady state of noncrisis.
The Collection and Storage of Viable Human Fetal Hematopoietic Tissue for Intravenous Use
Authors: FERREBEE JOSEPH W. , LOCHTE HARRY L. , SWANBERG HERBERT , THOMAS E. DONNALL
Published: October 2019

Livers and spleens from human fetuses in the 12 to 20 weeks’ gestation period were preserved in a sterile condition in glycerol and serum at -70 C. for periods of several weeks. Studies of DNA synthesis rates with C14-formate, and radioautographs after incubation with tritiated thymidine, revealed that hematopoietic elements concentrated in these tissues at this stage of gestation maintained their viability during these periods of storage. Intravenous infusion of cellular suspensions prepared from stored frozen tissue, or from fresh tissue in this same age group, produced no observed untoward clinical event in the three patients studied.

Some of the arguments favoring the use of fetal hematopoietic tissue, in contrast to adult marrow, in the attempt to restore marrow function after radiation injury are briefly reviewed.
Correlation of Radioactive Hemin Formation with Morphologic Alterations in Cultures of Human Bone Marrow
Authors: POWSNER EDWARD R. , BERMAN LAWRENCE
Published: October 2019

A method for measurement of radioactive hemin formation has been applied to the in vitro system for culture of human erythroblasts previously described. The results of these measurements have been shown to correlate with a morphologic parameter of maturation, the increase of orthochromic cells, in the same in vitro cell culture system.

The significance of these findings as well as the differences in the times of formation of heme and the increase of orthochromic cells has been discussed.

It is proposed that both the formation of radioactive hemin and the increase of orthochromic cells may be useful in the measurement of different aspects of the effects of various plasmas on the maturation of normal and abnormal human erythroblasts.
The Relationship of Tumor Necrosis to Red Blood Cell Changes in the Hamster
Authors: SHERMAN JOSEPH D.
Published: October 2019

1. Sterile, cell-free saline extracts of the necrotic portion of methylcholanthrene-induced hamster sarcoma injected into non-tumor bearing hamsters produced a statistically significant decrease in hemoglobin, hematocrit and red blood cell levels.

2. These changes in hemoglobin, hematocrit and red blood cells are comparable to those observed during sarcoma growth.

3. Sterile, cell-free, extracts of viable sarcoma injected into hamsters failed to produce alterations in the hemoglobin, hematocrit or red blood cell values.

4. The necrosis produced during sarcoma growth in the hamster cheek pouch is concluded to be responsible for alterations in the red blood cell counts, hemoglobin and hematocrit values.
Studies on Erythropoiesis. XII. The Effect of Transfusion-Induced Polycythemia in the Mother on the Fetus
Authors: JACOBSON LEON O. , MARKS EDNA K. , GASTON EVELYN O.
Published: October 2019

Erythropoiesis is reduced to zero in the mouse by repeated homologous red cell transfusion.

Mating of transfusion-induced polycythemic mice was allowed, and observations were made on maternal and fetal erythropoiesis. It was observed that erythropoiesis in the pregnant, polycythemic mouse was suppressed throughout pregnancy, this observation suggesting that erythropoietin production was concomitantly suppressed.

Erythropoiesis in the fetuses of polycythemic mothers was initiated by 10 days and maintained in an active state throughout fetal life. Hematologic values of these fetuses at birth were higher than those of the newborn normal pregnant mice of the same strain.

It is possible that the fetus initiates and maintains erythropoietin independently of erythropoietin from the maternal circulation. The higher hematologic values of the newborn of polycythemic mothers are probably the result of the greater availability of iron from the breakdown of transfused cells, since it was found that comparable hematologic values were achieved in the newborn when iron-dextran (Imferon) was injected into the maternal tissues during pregnancy.
Long-Term Mixed Cultures of Human Hemic Cells, with Granulocytic, Lymphocytic, Plasmocytic and Erythrocytic Series Represented
Authors: BROOKE JOHN H. , OSGOOD EDWIN E.
Published: October 2019

New culture strains containing humman hemic cells of monocytic, lymphocytic, granulocytic, plasmocytic and erythrocytic series are described with suggestions as to maintenance of the desired cell series in culture.

The addition of cells from lymphocytic, plasmocytic and erythrocytic series to a previously well established culture has made possible long-term culture of these cell types, since this procedure aids in maintaining the necessary high gradient factor for their growth and multiplication.

Although the life span of these cells has been considerably shortened in culture and immature forms predominate, the cells show recognizable characteristics of the cell series of origin. A high population density must be maintained in cultivation of the mixed cultures or the desired cell type may be lost.