bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Hematopoietic Stem Cells by Interferons and by DNA Methylation: Implications for Bone Marrow Failure
Authors: Goodell Margaret A.
Published: October 2019

Bone marrow failure (BMF), the inability to regenerate the differentiated cells of the blood, has a number of genetic and environmental etiologies, such as mutation of telomere-associated protein genes and immune-related aplastic anemia. Recently, mutations in DNA methyltransferase 3A (DNMT3A) have been found to be associated with approximately 15% of cases of primary myelofibrosis (MF), which can be a cause of BMF. The role of DNMT3A more broadly in hematopoiesis, and specifically in BMF, is currently poorly understood. DNMT3A is one of two de novo DNA methylation enzymes important in developmental fate choice. We showed that Dnmt3a is critical for normal murine hematopoiesis, as hematopoietic stem cells (HSCs) from Dnmt3a knockout (KO) mice displayed greatly diminished differentiation potential while their self-renewal ability was markedly increased1, in effect, leading to failure of blood regeneration or BMF. Combined with loss of Dnmt3b, HSCs exhibited a profound differentiation block, mediated in part by an increase of stabilized b-catenin. While we did not initially observe bone marrow pathology or malignancy development in mice transplanted with Dnmt3a KO HSCs, when we aged a large cohort of mice, all mice succumbed to hematologic disease within about 400 days. Roughly one-third of mice developed frank leukemia (acute lymphocytic leukemia or acute myeloid leukemia), one-third developed MDS, and the remainder developed primary myelofibrosis or chronic myelomonocytic leukemia. The pathological characteristics of the mice broadly mirror those of patients, suggesting the Dnmt3a KO mice can serve as a model for human DNMT3A-mutation associated disease. Strikingly, bone marrow of mice with different disease types exhibit distinct DNA methylation features. These will findings and the implications for disease development will be discussed. We are currently investigating the factors that drive different outcomes in the mice, including stressors such as exposure to interferons. We have hypothesized that HSC proliferation accelerates the Dnnmt3a-associated disease phenotypes. We have previously shown that interferons directly impinge on HSCs in the context of infections. Interferons activate HSCs to divide, generating differentiated progeny and cycling HSCs. Repeated interferon stimulation may permanently impair HSC function and bias stem cell output. When combined with loss of Dnmt3a, interferons may promote BMF. We will discuss broadly how external factors such as aging and infection may collaborate with specific genetic determinants to affect long-term hematopoiesis and malignancy development.

Reference: Challen GA, Sun D, Jeong M, et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 2012; 44: 23-31

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Evolution and Immunogenetics of Natural Killer Cell Receptors in Health and Disease
Authors: Parham Peter
Published: October 2019

Natural killer (NK) cells are phenotypically diverse lymphocytes that contribute to innate immunity, adaptive immunity and placental reproduction. Unlike B and T cells, NK cells do not use rearranging genes to make diverse antigen receptors that are clonally expressed. Instead, NK cells express diverse combinations of a variety of receptors that are encoded by conventional non-rearranging genes. Several of these receptors are specific for conserved and variable determinants of major histocompatibility complex (MHC) class I molecules. In humans, the killer-cell immunoglobulin-like receptors (KIR) are a diverse and polymorphic family of NK-cell receptors that recognize determinants of human leukocyte antigen (HLA)-A, B and C, the polymorphic human MHC class I molecules. HLA-A, B and C are the most polymorphic of human genes, and they correlate with susceptibility to a wide range of diseases and clinical outcomes, including allogeneic hematopoietic cell transplantation (HCT). During NK-cell development, interactions between epitopes of HLA class I and KIR educate the NK cells to recognize the normal expression of these epitopes on healthy cells, and to respond to unhealthy cells in which that expression is perturbed. In the context of HCT, certain types of HLA class I mismatch enable donor-derived NK cells to make an alloreactive and beneficial graft-versus-leukemia response. Although it is likely that all placental mammals have NK cells, only a small minority of these species has a diverse KIR family like that in humans. These comprise the simian primates: New World monkeys, Old World monkeys and the great apes. Under pressure from diverse and rapidly evolving pathogens, both the MHC class I and KIR gene families have been driven to evolve rapidly. Consequently, much of their character is species-specific. This is especially true for the human KIR gene family, which is qualitatively different from that of our closest relatives, the chimpanzees. Whereas chimpanzee KIR haplotype diversity represents variations on a theme of genes encoding robust MHC class I receptors, humans have an even balance between group A KIR haplotypes encoding robust HLA class I receptors and group B KIR haplotypes encoding receptors that, to varying degree, have been subject to natural selection for reduced functional recognition of HLA class I. A balance of A and B is present in all human populations and thus appears essential for the long-term survival and competitiveness of human communities. Whereas the A KIR haplotypes correlate with successful defense against viral infection, maternal B KIR haplotypes correlate with reproductive success and donor B KIR haplotypes improve the outcome of allogeneic HCT as therapy for acute myeloid leukemia.

Disclosures

No relevant conflicts of interest to declare.
Neutrophil Extracellular Traps and von Willebrand Factor in Thrombosis
Authors: Wagner Denisa D.
Published: October 2019

For many years, my lab has been studying the interplay between inflammation and thrombosis. These processes occur together, stimulate each other and share cellular and molecular components. The latest example of a common functional component is neutrophil extracellular traps (NETs). NETs are chromatin released together with toxic granular components from highly stimulated neutrophils. Originally found to trap/sequester invading pathogens, they were soon also seen to be part of sterile inflammatory and thrombotic processes. They interact with von Willebrand factor (VWF), which is also involved in platelet and leukocyte recruitment and is crucial for venous thrombus development after inferior vena cava stenosis. It is possible that NETs and VWF work together and cleavage of either by DNase 1 or ADAMTS13 is beneficial in ischemia/reperfusion injury. This will be discussed together with the role of NETs and the enzyme that generates them (PAD4) in animal models of deep vein thrombosis, myocardial infarction and in physiological wound healing. Interestingly, we observed that various cancers in mice prime neutrophils for NETosis. This causes cancer-associated thrombosis, and the production of NETs may affect tumor biology.

Disclosures

No relevant conflicts of interest to declare.
Shared Mechanisms of RNA Biology
Authors: Dreyfuss Gideon
Published: October 2019

Our recent observation that most primary RNA polymerase II (polII) nascent transcripts (pre-mRNAs and long non-coding RNAs, lncRNAs) in animal cells fail to produce full gene-length transcripts when U1 snRNP (U1) is inhibited, revealed a new dimension in gene expression regulation. This essential U1 protective activity, which allows transcription to go farther (telescripting), suppresses premature cleavage and polyadenylation from cryptic signals (PASs) in introns. It is separate from U1’s splicing function; both depend on U1’s base pairing through its 5’ sequence, but 5’ modified U1 variants that cannot function in splicing are still effective in telescripting. Our studies have further shown that partial U1 inhibition with an antisense oligonucleotide to its 5’-end dose-dependently shortens mRNAs. Widespread mRNA shortening of 3’ untranslated regions (3’UTRs) occurs in cancer, proliferating cells, and activated immune cells and neurons. 3’UTRs have many mRNA regulatory elements, including miRNA targets that generally repress translation, and their removal contributes to oncogenicity. We have investigated the potential role of U1 telescripting in these phenomena. We found that moderate U1 decrease shortened 3’UTRs of hundreds of genes in HeLa cancer cells while its over-expression lengthened 3’UTRs of >2,000 genes that are already shortened in these cells. Concomitantly, U1 increase dose-dependently attenuated protein synthesis, cell migration and invasiveness, while its decrease drastically enhanced these cancer cell characteristics. U1-dependent 3’UTR length changes recapitulated cancer-causing miRNA target deregulation in numerous oncogenes. The strong correlation between U1’s bi-directional effects on nascent transcripts’ length and cell phenotype suggests that mRNA shortening in cancer can be explained by a deficit in U1 telescripting. Our findings establish telescripting’s major role in gene regulation. Notably, the ability of U1 modulation alone to profoundly modify cancer aggressiveness suggests telescripting as a potential target for moderating tumorigenesis.

Disclosures

No relevant conflicts of interest to declare.
Stem Cell Rejuvenation
Authors: Wagers Amy J.
Published: October 2019

Effective functioning of the body’s tissues and organs depends upon innate regenerative processes that maintain proper cell numbers during homeostasis and replace damaged cells after injury. In many tissues, regenerative potential is determined by the presence and functionality of dedicated populations of stem and progenitor cells, which respond to exogenous cues to produce replacement cells when needed. Understanding how these unspecialized precursors are maintained and regulated is essential for understanding the fundamental biology of tissues. In addition, this knowledge has practical implications, as stem cell regenerative potential can be exploited therapeutically by transplantation to replenish the stem cell pool or by pharmacological manipulation to boost the repair activity of cells already present in the tissue. Ongoing work in my laboratory focuses on defining how changes in stem cell activity impact tissue regeneration throughout life, and identifying physiological and pathological signals that modulate regenerative function in an age-dependent manner. Our recent data using parabiosis and transplantation models suggests that the circulatory system serves as a major source of such signals. In particular, exposure of aged tissues, including skeletal muscle, cardiac muscle and neural cells, to a “youthful” systemic environment appears to reverse many indicators of age-related pathology and restores robust regeneration following injury. While prior studies have identified a handful of systemic “aging” factors, discovery of the humoral “rejuvenating” factors that act on tissue stem cells to restore regenerative function has been relatively more elusive. In recent work, we identified that the circulating hormone Growth Differentiation Factor 11 (GDF11) is a rejuvenating factor for skeletal muscle and other tissues. Supplementation of systemic GDF11 levels, which normally decline with age in mice and humans, was sufficient to restore stem cell function in the skeletal muscle and other tissues, and improved physical function in aged mice. Taken together, these data reveal critical mechanisms in the regulation of aging by blood-borne factors and identify a promising therapeutic target candidate for the reversal of age-related tissue and stem cell dysfunction.

Disclosures

No relevant conflicts of interest to declare.
Macrophages and Infection Control
Authors: Ramakrishnan Lalita
Published: October 2019

We have developed a zebrafish model for the study of infection focusing on mycobacteria, which are intracellular pathogens of macrophages, inducing their aggregation into hallmark structures called granulomas. Exploiting the optical transparency and genetic tractability of the developing zebrafish, we can visualize macrophage-pathogen interactions in great detail in live animals and study the consequences of genetic perturbations. We have identified pathways through which macrophages restrict infection and also those that alter macrophage migration, microbicidal capacity, susceptibility to apoptosis and necrosis so as to render them as tools for bacterial expansion. The complex interactions we have uncovered provide new insights into macrophage biology that are relevant not only for tuberculosis and other infectious diseases but also for the pathogenesis of noninfectious inflammatory diseases in which macrophages are increasingly recognized to play an integral role.

Disclosures

No relevant conflicts of interest to declare.
Comparing the Desmopressin (DDAVP) Challenge Test to DDAVP Clinical Response in Pediatric Patients with Von Willebrand's Disease: A Single Center Experience
Authors: Nadella Divya , Al-Ghafry Maha , Imran Hamayun , Siddiqui Abdul H.
Published: October 2019

Introduction

Von Willebrand's disease (VWD) is the most common inherited bleeding disorder. It is characterized by both quantitative and qualitative defects of the von Willebrand Factor (VWF) and generally manifests as menorrhagia, epistaxis and easy bruising. Initial treatment involves the use of desmopressin (DDAVP) that induces the release of VWF from the endothelium. Patients undergo a DDAVP challenge test that involves intravenous DDAVP (IV-DDAVP) infusion with multiple blood draws to determine an increase in the VWD panel. Due to the DDAVP challenge test being a costly and lengthy process, we sought to evaluate the prognostic utility of the challenge test in patients who responded favorably.

Methods

A retrospective cohort study was conducted in pediatric patients aged 18 years or younger with a laboratory diagnosis of VWD who underwent the DDAVP challenge test between 2005 and 2015 at our institution. VWD panel results pre and post DDAVP challenge test were obtained from clinical records. A complete response was defined as a 2-fold increase at 1-3 hours or at least 1.5-fold increase at 4-6 hours post-infusion. Patients' clinical records were reviewed for initial presentation and response to DDAVP. SPSS version 23.0.0 was used to analyze demographic and laboratory data, with frequencies and paired t-tests performed for comparison where appropriate.

Results

Seventy two pediatric patients were identified with a diagnosis of VWD: 34 (47%) were VW type 1, 51 (72%) were females and 50 (69%) were Caucasian. Challenge results were available for 48 patients (66%): Post-DDAVP levels were significantly higher than baseline (p = <0.001) in the VWD panel for either times (1-3 or 4-6 hours). Thirty-seven patients (51%) had both challenge results and clinical symptoms reported with 78% (29/37) considered responders and 22% (8/37) considered non-responders to IV-DDAVP. 86% (25/29) of responders were initially placed on intranasal DDAVP (IN-DDAVP) alone while the others received combined therapy with addition of a plasma derived product (PDP). All non-responders initially received DDAVP therapy with some improvement in their clinical symptoms. Eleven (38% of responders) and four (50% of non-responders) were switched to a PDP, mostly for better symptom control. Six responders (16%) had side-effects with DDAVP.

Conclusion

A high proportion of our patients with VWD responded to the DDAVP challenge test, but subsequently required therapy with a PDP; and half of non-responders were able to use DDAVP. In our pediatric patient population, an initial IV-DDAVP challenge test did not identify those that would respond clinically to IN-DDAVP therapy long-term. Thus, more studies addressing the utility of the challenge test in VWD would be needed before drawing further conclusions on its prognostic value.

Disclosures

No relevant conflicts of interest to declare.
Amino Acid Transporter X Is Required for Hematopoietic Stem Cell Maintenance through Regulating Specific Amino Acids Level
Authors: Li Zhenrui , Takubo Keiyo , Qian Pengxu , Suda Toshio , Li Linheng
Published: October 2019

Hematopoietic stem cells (HSCs) maintenance is required to preserve stem cell pool and compensate the dynamic loss of blood cells. Previous studies of HSCs maintenance mainly focus on the quiescent versus active state of HSCs and accumulated evidence indicates that metabolism plays a critical role in coordinating divergent stem cell states. While recent reports largely emphasized the role of catabolic glycolysis on long-term (LT) HSC maintenance, we found that free amino acids are enriched in primitive stem cell by ~1.5 fold. Given that amino acid metabolism in HSCs is largely unknown, we first cultured bone marrow (BM) cells with individual amino acid deprived medium to study the function of individual amino acids on HSCs in vitro. Surprisingly, we found that specific amino acids, including valine, methionine and threonine (VMT), are essential for maintaining primitive HSCs, as removing them (VMT) individually from media dramatically reduced primitive HSC number by over 95%. Thus, we hypothesize that specific amino acids are critical for preserving the stem cell pool and maintaining their function. To test it, we transplanted equal number of cells cultured with complete or individual VMT deprived media into lethally irradiated recipient mice and found VMT deprivation in vitro impaired stem cell repopulation ability. We also identified the amino acid transporter X (AATX) that is specifically expressed in HSCs and maintain VMT levels within the cell. Furthermore, inhibition of AATX reduced LT-HSC (LSK CD34- Flk2-) number in vivo. BM transplantation indicated that AATX inhibition impaired stem cell long-term reconstitution ability by over 2 fold. Our studies uncovered a role of amino acid metabolism in HSC maintenance and discovered the underlying molecular mechanism related to the amino acid transport. This finding may impact clinical treatment of blood disorders including leukemia.

Disclosures

No relevant conflicts of interest to declare.
Prmt5 Negatively Regulates Erythropoiesis By Multiple Mechanisms, Including Controlling DNA Methyltransferase 3A Protein Levels
Authors: Hamard Pierre-Jacques , Luciani Luisa , Liu Fan , Cheng Guoyan , Hatlen Megan A. , Guryanova Olga A , Zhao Xinyang , Levine Ross L. , Nimer Stephen D.
Published: October 2019

The shift in sites of hematopoiesis during embryonic development leads to primitive hematopoiesis within the fetal liver at E12.5, which generates primarily erythrocytes. Definitive hematopoiesis, which occurs within the bone marrow, follows at birth. During the erythroid differentiation of fetal hematopoiesis, progenitor cell maturation is accompanied by global DNA demethylation, a process necessary for fetal liver erythrocyte formation and accompanied by diminishing expression of the de novo DNA methyltransferases, Dnmt3a and Dnmt3b.

In our current study of hematopoietic cell specific Prmt5 knockout mice, we have identified Prmt5 as a master regulator of erythropoiesis; the cell-specific deletion of Prmt5 in fetal liver cells is embryonic lethal as Prmt5-null embryos have severe anemia and increased expression of Dnmt3a and Dnmt3b proteins. RNA-seq and pathway analysis studies revealed profound defects in several critical pathways that regulate normal hematopoiesis, including the tumor suppressor p53 pathway. Methyl-seq studies are currently being conducted to determine the effects of the enforced expression of key DNA methyltransferases on global DNA methylation and gene expression in erythroid progenitors and how it leads to a block in erythrocyte maturation. To decipher the extent of Dnmt3a or p53's involvement in the observed phenotypes, we have generated double knockout mouse models that are being analyzed. Mechanistically, p53 has been shown to be directly methylated by Prmt5, a modification that affects its tumor suppressor activity. Here we have found that Dnmt3a is also a substrate of Prmt5 and the effects of the di methylation of Dnmt3a on its function are currently under investigation. Thus, we have uncovered a potential functional interaction between DNA methylation and protein arginine methylation triggered by Prmt5 that regulates primitive erythropoiesis.

Disclosures

Levine: Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.
MLL Is Essential for NUP98-HOXA9-Induced Leukemia
Authors: Shima Yutaka , Yumoto Minori , Kitabayashi Issay
Published: October 2019

NUP98, a component of the nuclear pore complex (NPC), plays an important role in molecular trafficking between the cytoplasm and the nucleus. NUP98 has two Phe-Gly (FG) repeat domains, which are characteristic of NPC proteins. The NUP98 gene is rearranged and fused to several partner genes, such as HOXA9 and DDX10, in acute myeloid leukemia and myelodysplastic syndromes. Leukemia with NUP98 rearrangement is associated with poor prognosis. However, the molecular mechanism of leukemogenesis induced by NUP98-fusions remains unclear.

To investigate the role of NUP98 of NUP98 fusion proteins in leukemogenesis, we generated a set of deletion mutants of NUP98-HOXA9 lacking each FG repeat domain. These mutants were transduced into normal murine progenitor cells and their activity in the immortalization of murine cells was examined by colony-formation assays. Deletion of the second FG domain resulted in a dramatic delay in leukemia induction when cells were transplanted into mice, suggesting that the second FG repeat domain is crucial for immortalization in vitro and leukemogenesis in vivo. qPCR and ChIP analyses showed that the FG repeat domain is necessary for Hoxa gene activation and recruitment to the Hoxa gene locus. To identify interacting proteins of the FG repeat domain of NUP98-HOXA9, protein complexes containing full-length and mutant NUP98-HOXA9 were purified. Mass spectrometry analysis showed that full-length NUP98-HOXA9, but not the mutant lacking the second FG repeat, interacted with MLL. ChIP analysis also showed co-localization of NUP98-HOXA9 and MLL at the Hoxa gene locus in NUP98-HOXA9-expressing cells. Furthermore, analysis of Mll null mice showed that MLL is essential for the recruitment of NUP98-HOXA9 to the Hoxa gene locus and for NUP98-HOXA9-induced immortalization and leukemogenesis. These results suggest that NUP98 fusion proteins interact with MLL to activate HOXA genes during leukemogenesis.

Disclosures

Kitabayashi: Daiichi Sankyo Co., Ltd.: Research Funding.
Clinical Significance of Co-Existence of PHF6 and NOTCH1 mutations in Adult T-Cell Acute Lymphoblastic Leukemia
Authors: Ge Zheng , Li Min , Xiao Lichan , Zhang Run , Li Jianyong
Published: October 2019

Objective: T-cell acute lymphoblastic leukemia (T-ALL) is caused by collaboration of multiple genetic abnormalities in the transformation of T-cell progenitors. PHF6 is founded as a new key tumor suppressor and mutated in T-ALL. The clinical significance of PHF6 mutations has not been fully determined in adult T-ALL. This study aimed to screen the PHF6 mutations in adult T-ALL and explore the associations of PHF6 mutations with other genetic lesions, as well as their clinical relevance in adult T-ALL patients.

Methods: We amplified the exons of PHF6, NOTCH1, FBXW7, PTEN and JAK1 following by DNA sequencing to identify the genomic mutations and examined the PHF6 mRNA level by qPCR in adult T-ALL patients. We also analyzed the correlations of PHF6 and NOTCH1 mutations with clinical features using a χ2 test and survival curve using the Kaplan-Meier method.

Results: The 27.1% (16/59) PHF6 mutations including 10 novel mutations were detected in Chinese adult T-ALL. Six of 16 (37.5%) were frame-shift mutations, which could result in the deletion of the protein. We also observed PHF6 expression was significantly lower in T-ALL patients with PHF6 mutations compared with wide type cases (0.00423 vs. 0.06464, P=0.035) , indicating PHF6 mutations could be loss of function. Moreover, PHF6 mutation was significantly associated with NOTCH1 mutation(P=0.035). We further analyzed the domains involving co-existence mutations of NOTCH1 with PHF6. The most commonly mutated domains in NOTCH1 co-existed with PHF6 were HD-N only 6/12 (50.0%), followed by HD-C only 2/12(16.7%), PEST only 2/12(16.7%), HD-C+PEST 1/12(8.3%) and HD-N+HD-C 1/12(8.3%), indicating that HD domain (especially HD-N) of NOTCH1 may contribute to the synergistic effect on oncogenesis of the two genes. Furthermore, the patients with co-existence of PHF6 and NOTCH1 mutations had lower hemoglobin and higher incidence of splenomegaly or lymphadenopathy compared to that without co-existence of the mutations (95.0 vs 122.0, P=0.007; 81.8% vs 38.3%, P=0.009; 90.9% vs 44.7%, P=0.006). Importantly, the patients with co-existence of mutations in PHF6 and NOTCH1 (PHF6mutNOTCH1mut) had significant shorter event-free survival (EFS) compared with that without co-existence (non-PHF6mutNOTCH1mut)(2.0 months vs. 12.0 months, P=0.027).

Conclusion: PHF6 is inactivated in T-ALL due to its low expression and mutations. PHF6 mutation is co-existed with NOTCH1 mutations, and the patients with PHF6mutNOTCH1mut had a poor prognosis. Our results indicated synergistic effect of PHF6 and NOTCH1 mutations on leukemogenesis and PHF6mutNOTCH1mut may be potential prognostic marker in adult T-ALL.

Disclosures

No relevant conflicts of interest to declare.
Predictors of Early Death and Survival Among Children, Adolescents and Young Adults with Acute Myeloid Leukemia in California, 1988-2011: A Population-Based Study
Authors: Abrahão Renata , Keogh Ruth H , Lichtensztajn Daphne Y , Marcos-Gragera Rafael , Medeiros Bruno C. , Coleman MIchel P , Ribeiro Raul C. , Keegan Theresa HM
Published: October 2019

Background A better understanding of factors associated with early death and survival may guide future health policy aimed at improving outcomes among children, adolescents and young adults with acute myeloid leukemia (AML).

Methods We examined trends in early death and survival among 3,935 patients aged 0-39 years diagnosed with de novo AML in California during 1988-2011. We estimated overall survival and applied logistic and Cox regression to evaluate the association between sociodemographic and selected clinical factors with early death and survival.

Results There was a trend towards decline in early death from 9.7% in 1988-1995 to 7.1% in 2004-2011 (P=0.062). Survival improved substantially over time, but 5-year survival was still only 50% (95% CI 47%-53%) even in the most recent calendar period. Overall, the main factors associated with poor outcomes were older age at diagnosis, treatment at hospitals not affiliated with National Cancer Institute (NCI)-designated cancer centers, and black race/ethnicity. For patients diagnosed during 1996-2011, survival was lower for patients without health insurance and those who did not receive a hematopoietic stem cell transplant (HSCT). The association between survival and sociodemographic and clinical factors were stronger among patients aged 30-39 years.

Conclusions Mortality after AML remained strikingly high and increased with age. To improve outcomes, strategies may include wider insurance coverage, treatment at hospitals affiliated with NCI-designated cancer centers and access to HSCT for high-risk patients. Collaborative clinical trials aimed to evaluate the efficacy and toxicity of novel agents for each subtype of disease are warranted.

Disclosures

No relevant conflicts of interest to declare.
Alvocidib Potentiates the Activity of Azacytidine in an MCL-1-Dependent Fashion
Authors: Kim Wontak , Soh Katherine K. , Bearss Jeremiah J. , Lee Ye Sol , Peterson Peter , Whatcott Clifford J. , Siddiqui-Jain Adam , Weitman Steven , Bogenberger James M , Tibes Raoul , Bearss David J. , Warner Steven L.
Published: October 2019

Despite significant efforts, the clinical mechanism of action of hypomethylating agents such as 5-azacytidine (5-aza) is still poorly understood. 5-aza is currently indicated for the treatment of patients with myelodysplastic syndrome (MDS). While 5-aza has achieved good single-agent activity in acute myeloid leukemia (AML), complete response rates remain low when used as a single agent. In a recent report aimed at identifying rational therapeutic combinations with 5-aza, Bogenberger and colleagues identified multiple BCL-2 family member/BH3-containing therapeutic targets, which synergize with 5-aza when inhibited genetically or pharmacologically. The CDK9 inhibitor, alvocidib, has achieved significant improvement in complete response rates of newly diagnosed AML patients when administered before cytarabine and mitoxantrone (FLAM regimen) in a randomized multi-center Phase 2 trial when compared to 7+3 standard of care treatment. Recent reports suggest that the transcriptional repression of key anti-apoptotic proteins (eg., MCL-1) mediated by alvocidib's CDK9 inhibition, drive the pro-apoptotic activity of alvocidib in the FLAM regimen. We, therefore, hypothesized that alvocidib and 5-aza would synergize therapeutically in the treatment of AML by means of transcriptional repression of MCL-1 and sensitization to 5-aza. In this report, we demonstrate that treatment of AML cell lines with alvocidib inhibits both mRNA and protein expression of MCL-1 in a time and concentration-dependent fashion. Pre-treatment of cells with alvocidib, to repress MCL-1 expression prior to 5-aza treatment, reduced the 5-aza cell viability EC50 more than 2.5-fold, from 1.8 µM to 0.6 µM in MV4-11 cells. The alvocidib/5-aza combination also resulted in synergistic increases in caspase activity relative to either single agent within the combination, at multiple dose levels. Therefore, following reports suggesting inhibition of BCL-2 family members including MCL-1, sensitizes cells to 5-aza, our data suggest that the alvocidib/5-aza combination may constitute a viable therapeutic regimen. We also conclude that a CDK9 inhibitor/5-aza combination may be an effective clinical approach for the treatment of AML.

Disclosures

Kim: Tolero Pharmaceuticals: Employment. Soh:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Lee:Tolero Pharmaceuticals: Employment. Peterson:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Weitman:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment.
The Highly Potent Bromodomain (BRD) Inhibitor FV-281 Displays Preclinical Efficacy in Acute Myeloid Leukemia (AML)
Authors: Wang Zezhou , Dove Peter , Shamas-Din Aisha , Hurren Rose , Wang Xiaoming , MacLean Neil , Gronda Marcela , Bo Feng , Li Janessa , Skorupski Clarissa , Hadri Wissam , O'Neill David , Aman Ahmed , Al-awar Rima , Minden Mark D. , Slassi Malik , Schimmer Aaron D.
Published: October 2019

Recent studies have demonstrated the therapeutic potential of targeting BRD2, BRD3 and BRD4 in hematological malignancies including AML. Here, we report a novel orally bioavailable BRD inhibitor, FV-281, that displays preclinical efficacy in AML in vitro and in vivo.

In a binding screen assay of 32 bromodomains (BromoScanTM), FV-281 selectively bound BRD2, BRD3 and BRD4 with Kd values of 2.7 to 7.3 nM and was 2-5 fold more potent than the leading bromodomain inhibitor, OTX015. In AML cell lines (OCI-AML2, Tex, HL-60 and MV4-11), FV-281 reduced cell growth and viability after 72 hour incubation with IC50 values < 50 nM and was 6-fold more potent than OTX015. FV-281 was not cytotoxic to normal hematopoietic cells (n=4) with all IC50 values > 10 µM. In contrast, FV-281 induced cell death as measured by Annexin V/PI staining in 3 of 4 primary AML samples with IC50values < 850 nM.

Within 4 hours of incubation with FV-281, c-Myc mRNA and protein level in OCI-AML2 cells were reduced by >95% and >80%, respectively. Following FV-281 washout, c-Myc protein expression returned to baseline within 20 hours with no loss of cell viability observed. Thus, the FV-281 is a reversible bromodomain inhibitor and sustained target inhibition is required to induce cell death. Likewise, after 6 and 48 hours of incubation with OCI-AML2 cells, FV-281 reduced Bcl-2 mRNA and protein level by >60% and >90%, respectively.

To assess in vivo efficacy and toxicity, MV4-11 AML cells were engrafted subcutaneously in NOD-SCID mice (n=10). Once tumors were palpable, mice were treated with FV-281 orally (30mg/kg/d x 2 weeks) or vehicle control. FV-281 suppressed tumor growth without evidence of overt toxicity. In addition, primary AML cells were engrafted into the femurs of NOD-SCID mice (n=10). Three weeks after implantation, mice were treated with FV-281 orally (30mg/kg, 5 of 7 days, x 4 weeks) or vehicle control. FV-281 decreased AML engraftment in the injected and non-injected femur without evidence of overt toxicity (% human CD45+/CD19-/CD33+ in non-injected femur treated vs control: 20% vs 60%, p<0.0001).

Thus, FV-281 is a novel oral reversible bromodomain inhibitor with significant in vivo activity in murine models of AML. These data support evaluation of this agent in upcoming phase I clinical trials.

Disclosures

Wang: Fluorinov Pharma Inc.: Employment. Dove:Fluorinov Pharma Inc.: Employment. Hadri:Fluorinov Pharma Inc.: Employment. O'Neill:Fluorinov Pharma Inc.: Employment. Slassi:Fluorinov Pharma Inc.: Employment.
Extensive Chronic Graft-Versus-Host-Disease Significantly Increases the Risk of Severe and Multifocal Genital Tract HPV Disease in Long-Term Survivors of Allogeneic Stem Cell Transplantation
Authors: Shanis Dana L , Anandi Prathima , Grant Caitlin , Pophali Priyanka A , Koklanaris Eleftheria , Savani Bipin N. , Barrett Austin John , Battiwalla Minoo , Stratton Pamela
Published: October 2019

Background: High rates of cervical HPV disease in women after allogeneic stem cell transplantation (SCT) have been reported, but risk factors related to severe, multifocal, including vaginal and vulvar, HPV disease are not defined.

Objective: To determine rates and risk factors for multifocal and severe HPV disease in post-transplant women.

Methods: In a prospective long-term study after SCT, gynecologic history and assessment, cervical cytology and HPV testing were obtained with follow-up colposcopy and surgery as indicated for abnormal results. Prior HPV disease, genital graft-versus-host-disease (gGVHD), chronic GVHD (cGVHD) and immunosuppression treatment (IST) >3years were assessed for their association with extent and severity of genital HPV disease. Logistic regressions were used for multivariate analysis.

Results: Sixty five long term (>3 year) SCT survivors were studied prospectively on protocol. Patients received allogeneic transplantation from HLA-identical sibling donors with most undergoing myeloablative total body irradiation (94%) and T lymphocyte-depleted peripheral blood stem cells in 91% Of 65 women, 62 had gynecologic assessment with 8 (13%) having prior history of HPV disease; 16 (26%) had gGVHD. 20 women (32%) had acute GVHD, 46 (74%) had cGVHD; extent was limited in 23(37%) and extensive in 23(37%). 26(42%) had cGVHD requiring IST >3years. Of 21(34%) women with HPV disease after transplant, 12 required surgery and 7 had multifocal disease. Extensive chronic GVHD (but not acute GVHD) was found to significantly impact occurrence (OR=3.5, p=0.038), high-grade severity (OR=7.1, p=0.024) or multifocal HPV disease (OR=14.6, p=0.017).

Conclusion: Women who have undergone SCT have an increased risk of genital HPV disease, with highest rates in women with extensive cGVHD. Likely as a result of chronic immune dysregulation and the temporal nature of HPV, these women are at high risk of severe, multifocal disease, which if untreated may progress to genital cancer. Thus, gynecologic assessment as well as possible post-transplant HPV vaccination are critical aspects of care for women with significant GVHD post-transplant.

Support: Intramural programs of NHLBI, Clinical Center and NICHD, NCT00106925

Disclosures

Stratton: Allergan: Research Funding.
Impairment of IL-17 Immunity to Candida and IFN-g Immunity to Mycobacterium in Humans with Bi-Allelic Rorc mutations
Authors: Okada Satoshi , Markle Janet , Kobayashi Masao , Bustamante Jacinta , Casanova Jean-Laurent
Published: October 2019

Inborn errors of human IL-17 immunity underlie chronic mucocutaneous candidiasis (CMC), which is characterized by infections of the skin, nails, oral and genital mucosae with Candida albicans. Inborn errors of human IFN-γ immunity underlie Mendelian susceptibility to mycobacterial disease (MSMD), a rare congenital disorder characterized by susceptibility to infections by poorly virulent intracellular pathogens such as Mycobacterium bovis Bacille Calmette-Guérin vaccines (BCG) and non-tuberculosis Mycobacterium. Rarely, patients may be affected by both candidiasis and mycobacteriosis, including some patients with IL-12p40 and IL-12Rβ1 deficiencies that impair IFN-γ immunity and IL-17 immunity. We studied seven patients from three unrelated consanguineous kindreds with this unusual combination of infectious diseases without known genetic disorder. We combined whole exome sequencing and genome wide linkage analysis, and discovered bi-allelic loss-of-function mutations in RORC, which encodes the transcription factors RORγ and RORγT. All of the seven patients suffered from severe mycobacterial infections, and six also exhibited mild CMC. RORγT is the key transcription factor of Th17 cells, which produce IL-17 and IL-22. Therefore, as predicted by the mouse model, RORγT deficiency prevented the development of IL-17-producing T cells, which accounts for the patients' CMC. Consistent with the phenotype of Rorc-/- mice, these patients presented with mild T cell lymphopenia, small thymus, lack of palpable axillary and cervical lymph nodes, and absence of MAIT and iNKT cells.

The patients' severe infections with mycobacteria were not predicted by previous studies of Rorc-/- mice. To explain this unexpected phenotype, we focused on IFN-γ immunity. Leukocytes from RORC-/- patients showed impaired IFN-γ production in response to a mycobacterial challenge, and this defect is probably attributable to the functional impairment of CD4+ CCR6+ CXCR3+ αβ T cells, γδT cells, or both. These findings also suggested that IFN-γ treatment may be beneficial for RORC-/- patients. Moreover, this phenotype does not seem to be human-specific, as we also found that Rorc-/- mice deficient were susceptible to mycobacterial infection. We thus discovered that human RORC is essential not only for the development of IL-17-producing lymphocytes protecting the mucocutaneous barriers against Candida, but also for the activation of IFN-g-producing T cells, and for systemic protection against Mycobacterium.

Disclosures

No relevant conflicts of interest to declare.
Unstaged Diffuse Large B Cell Lymphoma in the United States: Predictors and Patient Outcomes
Authors: Shah Binay K. , Bista Amir , Sharma Sandhya
Published: October 2019

Background: Treatment and prognosis of diffuse large B cell lymphoma (DLBCL) depends on the stage of lymphoma. We conducted this study to examine unstaged DLBCL in the United States.

Methods and methodology: We used Surveillance Epidemiology and End Result (SEER) 18 registries to select patients with DLBCL diagnosed during January 2000 to December 2012. We used LRD Summary stage 2000 was used to determine stage of the disease - localized, regional, distant or unstaged. We used Logistic regression to investigate factors associated with unstaged DLBCL. We used Cox Proportional Hazard model to compare survival outcomes.

Results: Among 67765 patients, 3194 (4.71%) were unstaged. Age (60+years), "Others" and Caucasian races, single or single/divorced/widow marital status, metropolitan residence, median household income> $50,000, lymph node as the primary site and cased with other primaries before diagnosis of DLBCL were the factors associated with unstaged cases (Table 1). The 5- year relative survival rate for unstaged patients was inferior to those with localized and regional disease, and superior to those with distant disease (HRs of 0.58, 0.66 and 1.24 for localized, regional and distant respectively when compared to unstaged cases).

Conclusion: In this large population-based study, 4.71% patients with DLBCL had unstaged disease. Patients with unstaged DLBC had significantly inferior survival rates compared to patients with localized and regional stage.

Table 1. Factors associated with unstaged DLBCL cases Parameters Unadjusted OR (95% CI) P value Adjusted OR (95% CI) P value Age (60+ Vs. <60 years) 1.478 (1.363 - 1.602) <0.001 1.458 (1.335 - 1.592) <0.001 Sex (Female Vs. Male) 1.063 (0.990 - 1.141) 0.093 0.983 (0.911 - 1.059) 0.646 Race Caucasians Reference Reference African American 0.804 (0.691 - 0.935) 0.005 0.835 (0.715 - 0.974) 0.022 Others 1.109 (0.976 - 1.261) 0.112 1.257 (1.104 - 1.431) 0.001 Marital Status Married Reference Reference Single 1.026 (0.927 - 1.135) 0.662 1.208 (1.086 - 1.345) 0.001 Single/divorced/widow 1.249 (1.152 - 1.355) <0.001 1.185 (1.087 - 1.291) <0.001 Rural/Urban Rural Reference Reference Urban 0.878 (0.651 - 1.183) 0.393 0.896 (0.661 - 1.214) 0.479 Metropolitan 0.882 (0.667 - 1.165) 0.882 1.028 (0.767 - 1.379) 0.852 Median annual household income Upto 25,000 Reference Reference >25,000-50,000 01.009 (0.753 - 1.354) 0.951 0.927 (0.675 - 1.271) 0.636 >50,000 0.758 (0.563 - 1.021) 0.068 0.673 (0.486 - 0.933) 0.017 Sequence (Not first or only primary vs. first or only primary) 1.261 (1.156 - 1.377) <0.001 1.219 (1.115 - 1.334) <0.001 Site of primary Lymph nodes Reference Reference Extra-lymphatic 0.760 (0.704 - 0.821) <0.001 0.748 (0.693 - 0.808) <0.001 Unknown primary 6.295 (4.569 - 8.672) <0.001 6.727 (4.865 - 9.300) <0.001

Disclosures

No relevant conflicts of interest to declare.
ATP11C Encodes a Major Flippase in Human Erythrocyte and Its Genetic Defect Causes Congenital Non-Spherocytic Hemolytic Anemia
Authors: Arashiki Nobuto , Takakuwa Yuichi , Ogura Hiromi , Utsugisawa Taiju , Miyano Satoru , Ogawa Seishi , Kojima Seiji , Ohga Shouichi , Mohandas Narla , Kanno Hitoshi
Published: October 2019

Flippases are members of the P-IV ATPase family of proteins, and contribute to localization of phosphatidylserine (PS) in inner leaflet by its ATP-dependent active transport from outer to inner leaflet of lipid bilayer of erythrocyte membranes. It is critical that erythrocytes maintain PS in the inner monolayer to ensure their 120-day survival in circulation since externalization of PS will be recognized as an eat-me signal, resulting in phagocytosis by splenic macrophages. In the present study, we identified that ATP11C gene encodes a major flippase in human erythrocytes. A 13 years-old boy was referred to our hospital for consultation for work up of an undiagnosed congenital hemolytic anemia. Since extensive biochemical and molecular analysis failed to identify hemoglobin, erythrocyte membranes and enzyme abnormalities for the pathogenesis of hemolysis, we performed the whole exome analysis by massively parallel sequencing. We identified that the proband is hemizyogous, and the mother is heterozygous for a missense mutation of ATP11C, c.1253C>A, corresponding to a single amino acid substitution, p.Thr418Asn. Flipping activity as measured by PS internalization was decreased to 10% in the red cells of the proband compared to a normal control, clearly demonstrating that ATP11C encodes a major flippase in the human erythrocyte membranes. The PS-positive erythrocytes were not significantly increased in the whole blood but only in the most dense senescent cells, suggesting that PS exposure did not occur until very late stages of lifespan. We showed that PS exposure mediated by Ca2+-stimulated phospholipid scrambling was not different between red cells of the proband and control. Taken together, our findings imply that suppressed scrambling activity rather than flippase activity is the major contributor to maintainance of PS in inner leaflet of normal red cells during their 120-day lifespan, and that PS exposure to cell surface as an 'eat-me' signal depends primarily on scramblase activity at the end of lifespan. Importantly, our study has enables us to identify the major flippase of human erythrocyte membrane.

Disclosures

No relevant conflicts of interest to declare.
Mlkl Pores Release Neutrophil Extracellular Traps in Necroptotic Neutrophils
Authors: D'Cruz Akshay A , Bliss-Moreau Meghan , Ericcson Maria , Croker Ben A
Published: October 2019

Neutrophil extracellular traps (NETs) are networks of extracellular nuclear DNA and microbicidal proteins released from neutrophils in response to tissue damage and infection. Despite evidence of pathogenic roles for NETs in systemic lupus erythematosus, rheumatoid arthritis, diabetes, artherosclerosis and Alzheimer's disease, the major biochemical pathways controlling their formation remains poorly understood. Apoptosis does not contribute to NET formation but the role of regulated non-apoptotic cell death pathways such as necroptosis is not known. We have investigated the role of positive and negative regulators of necroptosis including receptor-interacting protein kinase-3 (RIPK3), mixed lineage kinase domain-like (MLKL), receptor-interacting protein kinase-1 (RIPK1) and Caspase-8. Using immunogold electron microscopy, flow cytometry, imaging flow cytometry and fluorescence microscopy, we demonstrate that necroptosis can drive NET formation via MLKL pore formation at the cell surface. This process is caspase-independent but reactive oxygen species-dependent. Genetically-modified mouse peripheral blood and bone marrow neutrophils were used to show that Caspase-8 and RIPK1 negatively regulate NET formation driven by RIPK3 and MLKL. Mice that lack MLKL are deficient in necroptosis and NET formation, and were sensitive to methicillin-resistant Staphylococcus aureus (MRSA). Neutrophil-specific Caspase-8-deficiency also leads to increased susceptibility to MRSA due to increased rates of necroptotic neutrophil death. Killing of MRSA by necroptotic neutrophils is sensitive to DNase, and is dependent on MLKL, suggesting that necroptosis-driven NET formation contributes to the bactericidal activity of neutrophils. Human peripheral blood neutrophils also generate NETs that are sensitive to pharmacological inhibitors of necroptosis, suggesting that targeting necroptosis in general may help combat autoimmune responses to DNA. This study provides a framework to investigate the role of extracellular DNA release and cell death in the setting of infection, autoimmunity and autoinflammatory disease.

Disclosures

No relevant conflicts of interest to declare.