bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting Iron/Heme in Infectious Disease
Authors: Soares Miguel
Published: October 2019

Inflammation and immunity can be associated with varying degrees of hemolysis and as such with heme release from cell-free hemoglobin (Hb), i.e. free heme. Accumulation of free heme in plasma can lead, eventually, to tissue iron (Fe) overload, oxidative stress, and tissue damage. Presumably, these deleterious effects contribute critically to the pathogenesis of systemic infections, as illustrated for severe sepsis (1) as well as for severe forms of malaria (2). Free heme sensitizes parenchyma cells to undergo programmed cell death in response to pro-inflammatory agonists (1). This cytotoxic effect is driven by the intracellular accumulation of free radicals, which sustain the activation of the c-Jun N-terminal Kinase (JNK) signaling transduction pathway. While heme catabolism by heme oxygenase1 (HO-1) prevents heme driven programmed cell death, this cytoprotective effect requires the co-expression of ferritin H chain (FTH), which controls the pro-oxidant labile Fe released from the protoporphyrin IX ring of heme (3). The antioxidant effect of FTH restrains JNK activation, while JNK activation inhibits FTH expression, a crosstalk that controls metabolic adaptation to cellular Fe overload during systemic infections (4).

1. Larsen, R. et al., Sci Transl Med 2 (51), 51ra71 (2010).

2. Pamplona, A. et al., Nat Med 13 (6), 703 (2007); Ferreira, A. et al., J Mol Med 86 (10), 1097 (2008); Ferreira, A. et al., Cell 145 (April 29), 398 (2011).

3. Gozzelino, R. et al., Cell Host Microbe 12 (5), 693 (2012)

4.Medzhitov, R, Schneider, DS, and Soares. Science 335, 936 (2012).

Disclosures:

No relevant conflicts of interest to declare.
Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia
Authors: Thompson Craig B.
Published: October 2019

An essential feature of acute myeloid leukemia (AML) is the profound block to differentiation exhibited by leukemic cells. This AML-associated arrest in the normal process of cellular differentiation is not observed in more chronic forms of leukemia. Chronic forms of leukemia and lymphoma are marked by the increased propensity to enter the cell cycle as the malignant cells move to more mature states of differentiation and the increased resistance to apoptosis these cells exhibit. Considerable recent evidence has suggested that profound epigenetic remodeling occurs as either a result or a cause of cellular differentiation. Altered DNA methylation has now been established as a hallmark of acute leukemia and yet very little is known concerning the mechanisms through which this occurs. In 2010, in collaboration with others, we found that neomorphic mutations of the citrate metabolism genes IDH1 and IDH2 induce DNA hypermethylation and impair differentiation in hematopoietic cells. IDH mutations create a block to DNA and histone demethylation as a result of the production of 2-hydroxyglutarate (2HG). 2HG acts as a competitive inhibitor of α-ketoglutarate-dependent enzymes. The epigenetic effects of 2HG are caused in part though inhibition of TET2, a DNA demethylase enzyme also mutated in leukemia. IDH 1/2- and TET2-mutant primary AML cells displayed a similar defect in epigenetic programming consisting of global hypermethylation and a gene-specific methylation signature. This work identifies IDH1/2- and TET2-mutant leukemias as a biologically distinct disease subtype, and links cancer metabolism with epigenetic control of gene expression. The implications of this work and the identification of additional metabolic genes involved in epigenetic deregulation in leukemia will be discussed.

Disclosures:

Thompson: NIH: Research Funding; Keystone Symposia: Consultancy; NCI: Research Funding; Damon Runyon : Research Funding; Ludwig: Membership on an entity’s Board of Directors or advisory committees; HHMI: Membership on an entity’s Board of Directors or advisory committees; Merck: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Agios: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; MSKCC: Employment; Pfizer: Patents & Royalties; BMS: Patents & Royalties; Repligen: Patents & Royalties.
Lessons from the Mouse Model
Authors: Rommens Johanna M. , Tourlakis Marina E. , Liu Holly , Gandhi Rikesh , Durie Peter R.
Published: October 2019

Shwachman-Diamond syndrome (SDS) is a member of the growing list of inherited diseases that result from mutations in genes that encode components of the protein synthesis machinery. Collectively referred to as ribosomopathies, these diseases have selective organ involvement and serious hematological issues with increased susceptibility to malignancy. SDS is caused by mutations in the highly conserved SBDS gene. Recent studies have implicated SBDS in facilitating ribosomal subunit joining for translation initiation. We study mouse models with disease-associated alleles to determine the consequences of loss of Sbds and gain insight into ribosomopathy phenotypes. Mice that are homozygous for a disease-associated allele (SbdsR126T/R126T) die at birth. Growth issues and developmental delay are evident in mid-to-late-stage embryos, and analysis of ribosome profiles indicate disturbed protein synthesis in all tissues examined. SDS-related pathologies in the fetal liver, bone, and brain are also observed. For example, flow cytometry analyses and culturing of fetal liver cells show growth impairment of myeloid lineage progenitors. Ossification is delayed in the developing phalanges. Poor proliferation is evident in early neurons and death due to apoptosis occurs in differentiating neurons, as early as E14.5. As with the brain, the fetal spleen also shows disproportionate growth restriction. These responses reflect varying underlying molecular pathways that hinge on p53 activation. Breeding to Trp53 (p53) deficient mice attenuates the SDS phenotypes but does not rescue the overall growth impairment or the perinatal death. Loss of Sbds leads to a range of organ responses, with engagement of stress pathways at different times and stages. As the severity of the organ pathology does not correlate directly with alteration of 80S or polysome component peak levels in ribosome profiles, we conclude that the organ phenotype reflects limitations in gene expression and timing of expression, or results from disturbed regulation that is specific to cell type and respective protein synthesis demand.

Disclosures:

No relevant conflicts of interest to declare.
Linking Defective Ribosome Maturation to Shwachman-Diamond Syndrome
Authors: Warren Alan John
Published: October 2019

Ribosomes are RNA-protein machines that translate the genetic information encoded by the mRNA template in all living cells. Recent high-resolution structures of the ribosome have revolutionized our understanding of protein translation. However, the mechanisms of ribosome assembly and the surveillance mechanisms that monitor this process and couple it to growth are poorly understood. Causative mutations and deletions of genes involved in ribosome biogenesis define an emerging group of disorders known as the ribosomopathies. Recent work from my laboratory strongly supports the hypothesis that Shwachman-Diamond syndrome (SDS) is a ribosomopathy caused by defective maturation of the large ribosomal subunit. Elucidation of the specific function of the SBDS protein that is deficient in SDS is revealing unexpected new insights that extend our understanding of the mechanisms underlying the late cytoplasmic steps of ribosome assembly and the quality control surveillance pathways that monitor 60S maturation. Genetic dissection of this pathway may inform novel therapeutic strategies for SDS.

1. Wong C.C., Traynor D., Basse N., Kay R.R., Warren A.J. Defective ribosome assembly in Shwachman-Diamond syndrome. Plenary Paper, Blood. 2011 Oct 20;118(16):4305-12.

2. Finch A.J., Hilcenko C., Basse N., Drynan L.F., Goyenechea B., Menne T.F., González Fernández Á., Simpson P., D’Santos C.S., Arends M.J., Donadieu J., Bellanné-Chantelot C., Costanzo M., Boone C., McKenzie A.N., Freund S.M., Warren A.J. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes and Development (2011) 25: 917-929.

3. Menne T.M., Goyenechea B., Sánchez-Puig N., Wong C.C., Tonkin L.M., Ancliff P., Brost R.L., Costanzo M., Boone C. and Warren A.J. The Shwachman-Bodian-Diamond syndrome protein mediates translational activation of ribosomes in yeast. Nature Genetics (2007) 39: 486-95.

Disclosures:

No relevant conflicts of interest to declare.
Ontogeny of Hematopoiesis
Authors: Dzierzak Elaine
Published: October 2019

The current challenge in hematopoietic transplantation and regeneration therapies is acquiring and/or producing a reliable and plentiful source of hematopoietic stem cells (HSCs). Given that HSCs from bone marrow, peripheral, or umbilical cord blood undergo only limited/no expansion ex vivo, there is a high interest in understanding how the adult cohort of multipotent self-renewing HSCs are generated and expanded during embryonic development. The development of HSCs in vertebrate embryos begins in the major vasculature. HSCs are generated in a short window of developmental time starting at embryonic day E10.5 until E12 in the mouse embryo, and from gestational weeks four to six in the human embryo. The first HSCs, which are as potent as bone marrow HSCs in transplantation procedures, are generated in the aorta-gonad-mesonephros (AGM) region. HSCs are found in the major vasculature – aorta, vitelline artery, and umbilical artery – subsequent to the appearance of hematopoietic cell clusters closely associated with the lumenal walls of these vessels. The relationship of HSCs to these clusters and the identification of the precursors to HSCs have been recently established through genetic, phenotypic, and real-time imaging studies. Remarkably, HSCs and hematopoietic progenitors arise directly from a subset of endothelial cells (hemogenic endothelial cells) in a natural transdifferentiation event. They are made through a process called endothelial to hematopoietic cell transition (EHT). EHT and HSC generation is in part regulated through ventral-derived developmental signals and a group of pivotal (core) transcription factors, including Runx1 and Gata2. Conditional knockout strategies show that these transcription factors are required for the generation of vascular hematopoietic clusters and HSCs, suggesting a role in hematopoietic fate induction and/or cell expansion. Interestingly, whereas both Runx1 and Gata2 are required for HSC generation, only Gata2 remains essential in HSCs after their production. We are profiling hemogenic endothelial and HSCs by RNA sequencing so as to understand the complete genetic program that leads to generation of HSCs. These results will be discussed in the context of developmental signaling pathways (BMP4, Hedgehog, etc.) that appear to impact HSC generation and expansion, and the localized dynamic expression and function of Gata2 and Runx1 in vascular endothelial and hematopoietic cluster cells.

Disclosures:

No relevant conflicts of interest to declare.
Role of Bcl-2 Family in Lymphoid Malignancies
Authors: Strasser Andreas , Kelly G. , Glaser S. , Grabow S. , Delbridge A. , Adams J.M. , Herold M.
Published: October 2019

Impaired apoptosis is considered one of the prerequisites for the development of most, if not all, cancers, but the mechanisms that guarantee the sustained survival of most cancer cells remain unknown. Members of the Bcl-2 family are key regulators of apoptosis and include proteins essential for cell survival and those required to initiate cell death. Studies with transgenic mice have shown that over-expression of Bcl-2 or related pro-survival family proteins, such as Bcl-xL or Mcl-1, can promote tumorigenesis, particularly in conjunction with mutations that deregulate cell cycle control, such as deregulated c-myc expression. It is, however, not known whether expression of pro-survival Bcl-2 family members under endogenous control is required to maintain the survival of cells undergoing neoplastic transformation. Using Eµ-myc transgenic mice, a well-characterized model of human Burkitt’s lymphoma, we investigated the role of endogenous Bcl-2 in lymphoma development. Bcl-2 was found to be dispensable for the development of Eµ-myc pre-B/B lymphoma. In contrast, loss of Bcl-xL and even, more remarkable, loss of a single allele of Mcl1 greatly impaired lymphoma development. Experiments with inducible knockout mice demonstrated that Mcl-1 but not Bcl-xL is essential for the sustained survival and expansion of Myc-driven malignant pore-B/B lymphoma. Remarkably, even loss of one Mcl1allele greatly impaired lymphoma growth. These findings were translated into using lymphoid malignancies by using inducible expression of selective antagonists of distinct pro-survival Bcl-2 family members. Such studies showed that Mcl-1 is also critical for the sustained survival and expansion of Burkitt Lymphoma, a Myc-driven malignancy. These observations indicate that (even relatively weak) targeting of Mcl-1 may be an attractive strategy for the treatment of Myc-driven hematological malignancies.

Disclosures:

Strasser: Genentech Inc: Consultancy.
Control of the Stem Cell Gene Expression Program
Authors: Young Richard A.
Published: October 2019

The gene expression programs that establish and maintain specific cell states in humans are controlled by thousands of transcription factors, cofactors, and chromatin regulators. Our understanding of the DNA elements and regulators that control gene expression programs has advanced considerably in the last year. I will describe new insights into the roles that these elements and regulators play in global gene control during normal development and in various diseases. These insights provide opportunities for the development of novel diagnostics and therapeutics.

Disclosures:

Young: Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Enzon Pharmaceuticals: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.
Epigenetics of Hematopoiesis, Stem Cell Reprogramming, and Cancer
Authors: Feinberg Andrew P.
Published: October 2019

Since the discovery of altered DNA methylation in cancer in 1982, most studies of cancer epigenetics have focused on epimutations which could serve as surrogates of mutation. In the past decade, we and our collaborators have been leading efforts to develop whole-genome approaches to epigenetic analysis of human disease, that include novel approaches to array-based analysis and whole-genome bisulfite sequencing. This has led to the first whole-genome bisulfite sequencing methylation map of the cancer genome. Surprising results have been the discovery of CpG island shores and large hypomethylated blocks corresponding to nuclear lamina/heterochromatin regions, and accounting for the vast majority of epigenetic alterations in cancer. These results point to the possibility that at least solid tumors represent epigenetically a single process with a common molecular characteristic; namely increased epigenetic plasticity that allows selection of the tumor cells at the expense of the host. This view can explain most of the hallmarks of cancer, as well as offering a strategy for powerful new approaches to risk detection and treatment. This whole-genome epigenetic approach has also revealed that the classical lineage structure of hematopoiesis is very well reflected in DNA methylation, but that there are very surprising dynamic changes in this process, with waves of both increasing and decreasing methylation changes. This work has also revealed a molecular basis for phenotypic memory in induced pluripotent stem cells, as well as a strong connection between reprogramming differentially methylated regions, or R-DMRs, and tissue-specific differentially methylated regions, or T-DMRs.

Disclosures:

No relevant conflicts of interest to declare.
Insights from Familial Leukemia and Myelodysplastic Syndromes
Authors: Horwitz Marshall
Published: October 2019

Most malignant hematopoietic disorders arise spontaneously. Nevertheless, hereditary factors substantively contribute to risk for myelodysplastic syndrome (MDS) and leukemia, and their molecular genetic identification can help unravel pathogenesis. In some instances, germline mutations causing Mendelian forms of MDS and leukemia are similar to those occurring somatically in sporadic cases, indicating their potential to function as drivers of malignancy. In other circumstances, genes involved in hereditary predisposition to MDS and leukemia are infrequently implicated in acquired cases, suggesting they may contribute to parallel pathways of malignant transformation. Hereditary mutations in genes encoding transcription factors directing hematopoietic differentiation, including RUNX1, CEBPA, and GATA2 in MDS and AML, and PAX5 in acute lymphoblastic leukemia, indicate that disturbed hematopoietic stem cell dynamics may initiate leukemogenesis. On the other hand, for at least some leukemia-predisposing syndromes – including congenital neutropenia when caused by mutations in the gene, ELANE, encoding neutrophil elastase – terminal effectors of hematopoietic differentiation can lead to malignancy, implying that contributions to malignant transformation may go beyond the stem cell. The molecular genetic basis of hereditary leukemic disorders and how they mesh with current concepts of disease mechanism and other insights ensuing from cancer genome sequencing will be discussed.

Disclosures:

Horwitz: United States Government: Consultancy, United States ex rel. Horwitz v. Amgen Inc., Civil Action No. C07-0248 (W.D. Wash.) Other.
Adoptive Transfer of T-Cell-Receptor Engineered Human T Cells Specifically Reduces Viral Titers in HLA-Transgenic NSG Mice Infected with a Humanized Cytomegalovirus
Authors: Thomas Simone , Lemmermann Niels , Klobuch Sebastian , Podlech Jürgen , Theobald Matthias , Reddehase Matthias J , Herr Wolfgang
Published: October 2019

Reactivation of latent human cytomegalovirus (HCMV) infection is a frequent complication in patients after allogeneic hematopoietic stem cell transplantation (HSCT). Preclinical research in murine models as well as clinical phase I/II trials have shown that the adoptive transfer of virus-specific CD8+ T cells is a therapeutic option for preventing HCMV disease. However, the feasibility of HCMV-specific immunotherapy is currently limited in clinical routine due to technical restrictions. It has also limitations, if the donor is HCMV-seronegative or carries only low numbers of HCMV-specific memory T cells. In this situation, grafting non-reactive T cells by virus-antigen specific T-cell receptors (TCR) may be an alternative means to transfer HCMV-specific T-cell function into HSCT recipients. Nevertheless, improvement of clinical protocols is needed before HCMV-specific cell therapy can be implemented in general clinical practice.

Here we describe a novel preclinical mouse model that will help to evaluate new HCMV-immunotherapy options. Due to the strict species specificity of CMV, we constructed a recombinant murine CMV (mCMV) coding for the HLA-A2 restricted pp65495-503 (NLV) peptide epitope of HCMV. This peptide is expressed with flanking amino acids within the mCMV-IE2-protein in the context of experimental infection with mCMV-IE2/NLV. The NLV peptide was found to be presented by infected murine fibroblasts from NOD/SCID/IL-2Rγc-/--HLA-A2-transgenic (NSG-A2) mice as shown by T cell specific recognition in ELISPOT and cytotoxicity assays. Interestingly, this presentation proved to be susceptible to the inhibitory function of mCMV immune evasion proteins. NSG-A2 mice were then used to evaluate antiviral control by HCMV-NLV-specific TCR (TCRNLV)-engineered human T cells. After infection with mCMV-IE2-NLV, the NSG-A2 mice showed high viral titers in liver, spleen, lungs, and salivary glands as well as a dramatic virus-induced morbidity and mortality. In contrast, after intravenous injection of TCRNLV-transduced human CD4+/CD8+ T cells, viral infection was reduced in all tested organs, and mice showed improved survival. These findings suggest that the NLV peptide is processed and presented by stromal and parenchymal cells of the NSG-A2 mice and promise validity of the model for preclinical evaluation of adoptive transfer protocols, for instance cell therapy with TCR-engineered T cells alone or in combination with therapeutic vaccination.

Disclosures

No relevant conflicts of interest to declare.
Consolidative Autologous Stem Cell Transplant for Double Hit Lymphoma
Authors: Chen Andy I , Okada Craig , Gay Nate , Reiss Phil , Spurgeon Stephen , Fan Guang , Braziel Rita M. , Maziarz Richard T.
Published: October 2019

Double hit lymphoma (DHL), defined as B cell lymphoma with a c-MYC rearrangement plus a IGH/BCL2 rearrangement, confers a poor prognosis. Outcomes with R-CHOP are dismal with reported 2 year EFS <30% in multiple series. To improve upon these results, Burkitt type regimens such as R-HyperCVAD and dose adjusted (DA) R-EPOCH have been attempted, but 2 year EFS remains < 50% in early reports. In addition, the SWOG-9704 study found a benefit for consolidative autologous stem cell transplant (autoSCT) in high risk DLBCL, likely including some DHL, although this reported benefit has not been confirmed in three other randomized European studies.

Since 2010 the treatment algorithm at Oregon Health & Science University for fit patients with DHL has been DA-R-EPOCH followed by consolidative autoSCT in responding patients. Here we report the first 16 patients treated with consolidative autoSCT. At diagnosis, the median age was 58, and the median IPI was 3. All patients had rearrangements of c-MYC and IGH/BCL2. One of the c-MYC rearrangements was a classical t(8;14); the translocation partners in the other cases were unknown. Three patients also had a ‘triple hit’ with an additional BCL6 rearrangement. Fourteen patients received DA-R-EPOCH with intrathecal prophylaxis for induction. One received R-CHOP at another institution, and one received R-HyperCVAD. 15 of 16 patients were in CR after induction. Stem cell mobilization was performed with G-CSF +/- plerixafor, and BEAM was utilized as the high dose conditioning regimen.

With a median follow-up of 18 months, the estimated 2 year PFS is 91 %, and the 2 year OS is 91 % in the 16 patients who underwent consolidative autoSCT. There were no deaths from non-relapse mortality, and the only relapse was 6 months post-transplant. All other patients undergoing autoSCT remain alive and in remission.

An additional 15 patients also underwent induction with DA-R-EPOCH but did not proceed to autoSCT. Four patients who had intended to undergo autoSCT were refractory to induction. Of the remaining 11 patients, 9 did not pursue autoSCT because of age and/or comorbidities, and 1 each declined out of personal preference or insurance denial. Four of these 11 have relapsed or died.

Our results suggest that consolidative autoSCT, especially after DA-R-EPOCH induction, is an effective treatment strategy for DHL in fit patients and should be explored in prospective studies.

Disclosures

No relevant conflicts of interest to declare.
Gemcitabine in Combination with Oxaliplatin (GEMOX) As a Salvage Regimen in Patients with Relapsed/Refractory Hodgkin's Lymphoma
Authors: Ozdemir Evren , Aslan Alma , Turker Alev , Barista Ibrahim , Kars Ayse
Published: October 2019

This study's objective was to evaluate the efficacy and safety of gemcitabine in combination with oxaliplatin (GEMOX) as a salvage regimen in patients with relapsed or refractory Hodgkin's lymphoma. Twenty-five patients were enrolled. All patients had received ≥ 2 prior chemotherapy regimens, had an ECOG performance status ≤ 2 and had adequate organ function. Patients received intravenous gemcitabine (1000 mg/m2) and oxaliplatin (100 mg/m2) on days 1 and 15, every 4 weeks. The median age was 29 years (range, 18-64) and 16 (68%) were male. Twenty-one (84%) had primary refractory disease (n=13) or relapsed within 12 months after initial therapy (n=8). All had previous platinum-based salvage chemotherapy (ICE, 23; DHAP, 2). Ten patients (40%) had relapsed/refractory disease following autologous stem cell transplantation (SCT). None had previous brentuximab vedotin treatment. Twenty-one (84%) patients were refractory or progressive on the last treatment. Median number of previous lines of chemotherapy was 2 (range, 2-4). Median number of GEMOX cycles administered to the patients was 3 (range, 2-6). Treatment response was evaluated with PET-CT before and 2-3 cycles after treatment, and those patients who demonstrated a response continued to receive a maximum of 6 courses of GEMOX or bridged to SCT. Of 25 patients, 2 (8%) had complete response, 9 (36%) had partial response and the remaining patients had refractory/progressive disease with an overall response rate of 44%. Seven of the 10 patients who had relapsed/refractory disease after autologous SCT achieved a response (CR, 2; PR, 5). The median time to progression for responding patients was 3 months (range, 1-40 months). One patient is disease free for 40 months. Three patients were successfully bridged to SCT (autologous, 2; allogeneic, 1). Main toxicity was hematological. Grade ≥ 3 hematologic toxicity occurred in 10 patients: thrombocytopenia (36%), neutropenia (16%) and anemia (8%). Among these, 7 had previous autologous SCT. One patient had grade 4 neutropenia and thrombocytopenia. Treatment cycle postponed in 6 patients without dose reduction because of hematological toxicity. Seven patients (28%) needed G-CSF support. One patient developed febrile neutropenia. No treatment-related deaths occurred. GEMOX was shown to be an effective salvage regimen in patients with relapsed/refractory Hodgkin's lymphoma, producing an overall response rate of 44%. It is an active regimen in patients who had relapsed/refractory disease after autologous SCT. Although, the median PFS time was short, some patients can be bridged to SCT and some can get long-term PFS. Hematological toxicity was common, especially in patients with previous autologous SCT.

Disclosures

Off Label Use: Gemcitabine in Hodgkin's Lymphoma Oxaliplatin in Hodgkin's Lymphoma.
Targeting Myddosome Self-Assembly in Waldenstrom's Macroglobulinemia
Authors: Liu Xia , Hunter Zachary , Xu Lian , Chen Jie , Chen Jiaji , Tsakmaklis Nicholas , Patterson Christopher J , Castillo Jorge J , Buhrlage Sara , Gray Nathanael , Treon Steven P , Yang Guang
Published: October 2019

Background: MYD88 mutations are present in over 95% of patients with Waldenstrom's Macroglobulinemia (WM), and promote Myddosome self-assembly that triggers NFκB-dependent survival through BTK and IRAK1/IRAK4 (Blood 122(7):1222-32). While current therapeutic strategies are aimed at blocking these downstream kinases, peptidomimetics that interfere with Myddosome self-assembly may offer a more targeted approach for blocking aberrant MYD88 signaling.

Methods: We expressed by lentiviral transduction mini-peptides of MYD88 Toll/Interleukin-1 Receptor (TIR) or Death Domain (DD) sequences in mutated MYD88 WM and wild-type MYD88 control cells (Figure 1). We used phospho-flow analysis to evaluate for changes in pBTK, pIRAK1/IRAK4, and pNFKB, and determined cell growth and survival by Alamar Blue Assay, Annexin V, and cleaved Caspase 3 staining.

Results: Transduction of TIR or DD mini-peptides in mutated MYD88 WM cells but not wild-type MYD88 control cells reduced NFKB activation and tumor cell growth, and prompted Annexin V and/or cleaved Caspase 3 staining. TIR interfering mini-peptides impacted BTK but not IRAK1/IRAK4 activation, whereas DD interfering mini-peptides showed an opposite effect.

Conclusions: The findings demonstrate differences in BTK versus IRAK1/IRAK4 directed NFKB signaling in response to Myddosome self-assembly in MYD88 mutated WM cells. The feasibility of directly targeting MYD88 homodimerization to block aberrant MYD88 signaling was also recognized, and suitable peptide sequences for the development of peptidomimetics that interfere with Myddosome self-assembly and signaling were identified. The findings provide a framework for direct targeting of the Myddosome in MYD88 mutated WM disease.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Mirna Regulation of Blood Group AB0 Genes
Authors: Kronstein-Wiedemann Romy , Nowakowska Paulina , Milanov Peter , Seifried Erhard , Tonn Torsten
Published: October 2019

In clinical transfusion and transplantation medicine, AB0 is the most important blood group system. A and B antigens are synthesized by glycosyltransferases A and B encoded by co-dominantly inherited A and B genes. The 0 phenotype derives from a single nucleotide deletion; the resulting stop codon in the exon 7 of glycosyltransferase A leads to a lack of glycosylation activity to A and/or B. The DNA structure of the three major alleles of the human blood group AB0 system was first described in 1990. Besides the 4 major AB0 blood groups (A, B, AB, 0), weak expression variants commonly occur. Beside introns and exons, two regions were found to be critical for transcriptional activity of AB0 genes: a promotor sequence upstream of the translation start site and a CBF/NF-Y-binding enhancer element. However, the molecular basis for the weak expression levels of AB0 blood group receptors is not fully understood. Here we asked whether microRNA´s may play a role in the regulation of blood group A and B antigens. MiRNA's are small, non-protein encoding RNA sequences that bind to the 3' untranslated region (UTR) of target mRNAs resulting in translation repression or mRNA degradation. By distinct complementary approaches, including gene array analysis and overexpression of glycosyltransferase specific micro-RNAs (miRNAs) in primary hematopoietic stem cells, we identify here that miR-331-3p and miR-1908-5p directly target glycosyltransferase A and B. Consistently, overexpression of miR-331-3p and -1908-5p in hematopoietic stem cells (HSCs) leads to a 30-50% reduction of blood group A antigens per cell in differentiated RBCs. Downregulation of endogenous miR-1908-5p but not miR-331-3p leads to increased amount of blood group A antigen per red blood cell compared to control cells. This discrepancy between the effects of downregulation of miR-1908-5p and of miR-331-3p is likely explained by the constitutive expression levels of both miRNAs during erythropoiesis. In particular, miR-1908-5p is expressed throughout erythroid differentiation, while miR-331 displays low expression, thus, further downregulation of miR-331-3p could not further affect blood group A antigen levels. Furthermore miR-331-3p and -1908-5p were enhanced in red blood cells of Aweak variants. Sequencing of the 3'UTR of 6 Aweak variants revealed the presence of more miRNA binding sites for miR-1908-5p compared to normal controls.

These data identify microRNAs as regulators of blood group glycosyltransferase expression, thereby extending our understanding of blood group regulation in carriers of weak blood group variants. Regulating blood groups by microRNAs could pave the way for the development of universal blood.

Disclosures

No relevant conflicts of interest to declare.
Mechanism Governing Heme Synthesis Reveals a GATA Factor-Heme Circuit That Controls Differentiation
Authors: Bresnick Emery H , Tanimura Nobuyuki , Miller Eli , Igarashi Kazuhiko , Yang David T , Burstyn Judith , Dewey Colin
Published: October 2019

Metal ion-containing macromolecules have fundamental roles in essentially all biological processes throughout the evolutionary tree. For example, iron-containing heme is a cofactor in enzyme catalysis and electron transfer and an essential hemoglobin constituent. To meet the intense demand for hemoglobin assembly in red blood cells, the cell type-specific factor GATA-1 activates transcription of Alas2, encoding the rate-limiting enzyme in heme biosynthesis, 5-aminolevulinic acid synthase-2 (ALAS-2). Using genetic editing to unravel mechanisms governingheme biosynthesis, we discovered a GATA factor- and heme-dependent circuit that establishes the erythroid cell transcriptome. CRISPR/Cas9-mediated ablation of two Alas2 intronic cis-elements strongly reduced GATA-1-induced Alas2 transcription and heme biosynthesis. Surprisingly, the cis-element ablation impaired GATA-1 regulation of other vital constituents of the erythroid cell transcriptome, including components mediating autophagy and nutrient transport. Bypassing ALAS-2 function in Alas2 cis-element-mutant cells by providing its catalytic product 5-aminolevulinic acid rescued heme biosynthesis and a sector of the GATA-1-dependent genetic network. A component of the gene cohort deregulated in heme deficiency could not be reactivated by rescuing heme biosynthesis, and we are testing whether irreversible changes in their chromatin states upon heme deficiency confer stable repression. Heme amplified GATA-1 function by downregulating the heme-sensing transcriptional repressor Bach1 and via a Bach1-insensitive mechanism. Based on this dual mechanism, we developed a paradigm in which a fundamentally critical process in hematology, heme biosynthesis, is intricately linked to the actions of a quintessential regulator of a cell type-specific transcriptional program that controls cellular differentiation. As known mechanistic insights into GATA factor mechanisms do not predict whether a GATA-1 target gene will be heme- or Bach1-sensitive, these studies revealed a new mode of GATA factor function that controls erythroid cell development and function. We will discuss how this mechanism is being dissected in biologically important murine and human systems.

Disclosures

No relevant conflicts of interest to declare.
Congenital Neutrophilia Arising from a T618I Germline Mutation in the CSF3R Gene
Authors: McMahon Daniel P , Lance Amanda , Price Andrea E. , Gerber Jonathan M. , Baxter Sarah A , Druhan Lawrence J , Steuerwald Nury M , Avalos Belinda R
Published: October 2019

Recently, somatic mutations in the granulocyte colony-stimulating factor receptor (CSF3R) have been identified in the majority of patients with chronic neutrophilic leukemia (CNL). The T618I mutation is the most common of these mutations, and it has been suggested that this specific mutation be added to the current WHO criteria for the diagnosis of CNL. In vitro studies of cells transfected with the T618I mutation have demonstrated that the mutant receptor confers a hyperproliferative phenotype.

We report here a case of congenital neutrophlia with an associated germline T618I CSF3R mutation. The patient, currently a 10 year old female, has had an elevated but stable leukocytosis (WBC 24,000 - 35,000) since birth, comprised of approximately 80% neutrophils. She has been healthy, with no increase in frequency of infections. Bone marrow studies performed at 5 months and 10 years of age have shown the marrow to be hypercellular, with normal karyotype/FISH (including BCR/ABL negative) and no dysplastic features. Additionally, no mutations have been detected in JAK2, MPL, CALR, PDGFA/B, and FGFR1.

DNA was extracted from whole blood and buccal cells collected from the patient, and using next generation sequencing a T618I mutation in CSF3R was identified in both her buccal and blood cells, confirmed by Sanger sequencing. The T618I mutation was present at an approximately 50% allele frequency in both tissues. Using a combination of next generation and Sanger sequencing, the patient's DNA was also analyzed for the presence of mutations in ASXL1, SETBP1, and TET2; as these have been reported in combination with the T618I mutation in many cases of CNL. However, no such additional mutations were detected in our patient.

Congenital neutrophilia arising from another activating mutation in CSF3R has previously been reported in a family from France (Plo et al., J Exp Med 2009). The mutation identified in that family was reported to be at amino acid position 617 (T617N) (numbering from the methionine 1, which includes the leader sequence, this correlates to a T640N mutation). Similar to the T618I mutation in our patient, none of the 12 affected family members (ages 8-80 years) progressed to an acute leukemia. Collectively, these observations suggest that activating CSF3R mutations, including the T618I mutation, may result in chronic neutrophilic leukemia but that subsequent disease progression may be due to other, cooperating mutations.

Disclosures

No relevant conflicts of interest to declare.
Role of Prostate Apoptosis Response-4 Tumor Suppressor in the Survival and Growth of Chronic Lymphocytic Leukemia
Authors: Nooti Sunil K , McKenna Mary K , Frissora Frank W , Gachuki Beth W , Alhakeem Sara S , Greene Joseph T , Muthusamy Natarajan , Rangnekar Vivek M , Bondada Subbarao
Published: October 2019

Chronic Lymphocytic Leukemia (CLL) is characterized by accumulation of clonally expanded population of CD5+ CD19+ B lymphocytes in peripheral blood and secondary lymphoid organs, with a majority of circulating cells in a non-dividing resting stage. However CLL is no longer considered a static disease that results from simple accumulation of long-lived lymphocytes, but rather, is a dynamic process with a birth rate of about 0.1-2% of the entire CLL clone per day. The Eµ-Tcl1 mouse serves as an excellent model for the development of CLL as they develop a CLL like disease by 9-13 months of age, due to overexpression of the oncogene, T cell Leukemia 1 (Tcl1), in B cells through the Ig VH promoter and Eµ enhancer (Bichi et al. PNAS. 2002). In an adoptive transfer model, intravenous or intraperitoneal injection of primary CD5+ CD19+ CLL cells from the Eµ-Tcl1 CLL mouse into recipient syngeneic mice leads to development of a CLL like disease within 3-5 weeks of transfer. We have characterized the growth of CLL cells in these mice by periodic submandibular bleeding, ultrasonography of spleen and flow cytometry. We find that Eµ-Tcl1 CLL cells express more Prostate apoptosis response-4 (Par-4), a pro-apoptotic tumor suppressor protein, than normal B-1 or B-2 cells in mice. Par-4 is silenced by promoter methylation in more than 30% of all cancers. Par-4 has been shown to be secreted and to induce apoptosis selectively in various types of cancer cells but not in normal cells. Although we find that Eµ-Tcl1 CLL cells constitutively secrete Par-4, they are resistant to Par-4 mediated apoptosis. We show that CLL cells have constitutively active B-cell receptor signaling and that inhibition of BCR signaling with FDA approved drugs (i.e. Dasatinib, Ibrutinib, and Fostamatinib) causes a decrease in Par-4 protein and mRNA levels and increases apoptosis. Interestingly, systemic Par-4 appears to inhibit CLL growth in vivo, since adoptively transferred Eµ-Tcl1 CLL cells grew better in Par-4 null mice, despite excellent Par-4 expression and secretion by the transferred cells. Thus there were three times more CLL cells in the bone marrow and twice as many in the spleens of Par-4 null mice compared to their wild type counterparts. We conclude that even though Par-4 is pro-apoptotic in the CLL microenvironment, intracellular Par-4 is either rendered inactive or is potentially pro-survival in Eµ-Tcl1 CLL cells. (Supported in part by NIH grants)

Disclosures

No relevant conflicts of interest to declare.
The Effect of Proteasome Subunit Beta Type 1 P11A Polymorphism on the Survival of Multiple Myeloma Patients Treated with First Line Bortezomib Based Chemotherapy
Authors: Varga Gergely , Mikala Gabor , Szombath Gergely , Balassa Katalin , Kiss Katalin Piroska , Tordai Attila , Andrikovics Hajnalka , Masszi Tamas
Published: October 2019

Background: Proteasome inhibitors have a fundamental role in the treatment of multiple myeloma however there are still patients who fail to achieve a good response and have a dismal prognosis. Markers predicting the outcome of individual patients are needed to identify those who would benefit more from a different approach. Proteasome subunit beta type 1 (PSMB1) rs12717 single nucleotide polymorphism (C/G substitution resulting in a P11A change) was reported recently to be associated with greater clinical benefit in relapsed follicular lymphoma patients treated with bortezomib containing combination (Coiffier et al Clin Cancer Res 2013). To the best of our knowledge there is only one report with limited number of myeloma patients showing no association between PSMB1 P11A genotype and survival (Lichter et al Blood 2012).

Aims and Methods: We analyzed the association of PSMB1 P11A polymorphism and treatment outcome of 220 consecutive myeloma patients having had first line chemotherapy in our unit. PSMB1 P11A polymorphism was tested using LightCycler melting analysis. Statistical analyses were performed using the SPSS (version 20.0) software package.

Results: The distribution of presentation prognostic markers such as age, ISS and FISH were even among the three genotype groups. 149 patients had bortezomib-based chemotherapy, mainly VTD (n=76) and MPV (n=47); 63% of these patients had ASCT consolidation. 71 patients had non-bortezomib-containing induction with either thalidomide, lenalidomide or other protocols including MP and VAD; only 28% of these patients had ASCT. In a recessive model the median PFS was 727 (635-818) days in carriers of the wild type C allele either in homozygous of heterozygous form (C/C&C/G, n=188), and only 491 (366-615) days in the homozygous variant G allele carriers (G/G, n=32, p=0.01). In multivariate analysis, C allele carriers had favorable PFS compared to G/G genotype patients with a hazard ratio of 1.402 (1.093-1.799), p=0.008 besides age, ISS, FISH, bortezomib treatment and ASCT. When we analyzed separately the bortezomib-treated and non-bortezomib-treated patients the benefit in PFS was only present in the bortezomib group [Fig 1, PFS 747 (656-837) days in C/C&C/G and 448 (279-616) days in G/G patients, p=0.002], and not in patients treated without bortezomib (p=0.559). We performed statistical interaction testing which showed significant interaction between bortezomib exposure and PSMB1 P11A (p=0.035).

Conclusion: PSMB1 P11A is a novel predictive marker in multiple myeloma which could help to identify patients having a suboptimal response to bortezomib who might benefit from alternative management.

Disclosures

No relevant conflicts of interest to declare.
Upregulation of Microparticles, Tissue Factor, Adhesion Molecules, Nitric Oxide and Adiponectin in End Stage Renal Disease
Authors: Fareed Jawed , Bansal Vinod , Syed Daneyal , Hoppensteadt Debra , Guler Nil
Published: October 2019

Introduction: End stage renal disease (ESRD) represents the final stage of chronic kidney disease characterized by kidney failure (GFR <15 mL/min/1.73 m2). To understand the pathophysiology of hemostatic dysregulation in ESRD, we measured the circulating levels of microparticles (MP), tissue factor (TF), adhesion molecules, such as p-selectin (P-Sel), soluble ICAM (s-ICAM), nitric oxide (NO) and adiponectin (AD).

Methods: Citrated blood plasma samples were collected from 119 ESRD patients undergoing maintenance hemodialysis to profile various thrombotic inflammatory biomarkers. 100 normal plasma samples were collected from healthy individuals and were used as controls. MP levels were measured using an annexin binding method (Hyphen Biomedical, Paris, France). NO were measured using a kit from R&D systems (Minneapolis, Minnesota) and ELISA based methods for TF, P-Sel, s-ICAM and adiponectin were obtained from R&D systems. Chromogenic substrate methods were used to measure the heparin levels and thrombin generation.

Results: MP levels were elevated in the ESRD group (28.1nm + 6.1nm vs. the control 8.9nm +1.3nm). Tissue factor levels were found to be increased in the ESRD group (20.4±6.1pg/ml) vs the control (11.9±2.8pg/ml). The nitric oxide level was markedly higher in the ESRD group (32±17uM) vs the controls (7±3uM). The p-selectin levels were also elevated in the ESRD group (46±20ng/ml) vs the control (31±3ng/ml). The soluble ICAM levels were higher in the ESRD group (250±112ng/ml) vs the control (180±19ng/ml). Interestingly, the adiponectin levels were also increased in the ESRD group (19.2±9.3ug/ml) vs the control (11.2±4.1ug/ml). Measured variations in thrombin generation parameters were noted. Detectable levels of heparin (.05 - 0.20 U/ml) were present in the ESRD group.

Summary/Conclusions: These studies suggest that MP, TF, NO, p-selectin and s-ICAM levels are increased in the ESRD patient. It is of interest to note that despite that a significant number of ESRD patients were diabetic; the AD levels were increased in this group. These results also suggest that while ESRD represents a pro-inflammatory/hypercoagulable state, the repeated administration of heparin and other drugs may contribute to the regulation of the hemostatic process and inflammatory balance in these patients.

Disclosures

No relevant conflicts of interest to declare.
Enhanced NF-Kb Non-Canonical Signaling Impairs Hematopoietic Stem Cell Self-Renewal
Authors: Xiu Yan , Zhao Chen
Published: October 2019

We previously demonstrated that the NF-B non-canonical signaling way positively and intrinsically regulates hematopoietic stem/progenitor cell (HSPC) self-renewal and maintains stromal/osteoblastic niches (Stem Cells 2012 30:709-18). These results lead us to think that persistent activation of NF-B non-canonical signaling would have favorable effects on the HSPC pool size and self-renewal capacity. NF-B-inducing kinase (NIK) plays a critical role in non-canonical NF-B signaling by directly phosphorylating IKK. It is constitutively degraded by TRAF3 in unstimulated cells to prevent unwanted NF-B activation. To investigate the enhanced NF-kB non-canonical signaling specifically in hematopoietic cells, we crossed Vav-Cre mice with a mouse strain in which a mutated form of NIK lacking the TRAF3-binding domain is expressed under the control of the ROSA26 promoter after Cre-mediated deletion of the LoxP-flanked STOP cassette (NIKΔT3Cre mice). In contrast to what we expected in these preliminary studies, the NIKΔT3Cre mice rapidly developed anemia, pancytopenia, with a reduced HSPC pool and marrow cellularity and postnatal lethality, mimicking many of the findings in humans with bone marrow failure syndrome, and different from recently published mice with deficiency in A20, which also activates NF-B signaling. Furthermore, the NIK activated HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Further analysis showed that the mutant cells are proliferate faster and predispose to apoptosis than wild type cells. These observations suggest that finely controlled NF-B activity is crucial for HSC maintenance. Currently, we are focusing on the analysis of the underlying molecular mechanisms.

Disclosures

No relevant conflicts of interest to declare.