Pathogen and human NDPK-proteins promote AML cell survival via monocyte NLRP3-inflammasome activation.

A history of infection has been linked with increased risk of acute myeloid leukaemia (AML) and related myelodysplastic syndromes (MDS). Furthermore, AML and MDS patients suffer frequent infections because of disease-related impaired immunity. However, the role of infections in the development and progression of AML and MDS remains poorly understood. We and others previously demonstrated that the human nucleoside diphosphate kinase (NDPK) NM23-H1 protein promotes AML blast cell survival by inducing secretion of IL-1ß from accessory cells. NDPKs are an evolutionary highly conserved protein family and pathogenic bacteria secrete NDPKs that regulate virulence and host-pathogen interactions. Here, we demonstrate the presence of IgM antibodies against a broad range of pathogen NDPKs and more selective IgG antibody activity against pathogen NDPKs in the blood of AML patients and normal donors, demonstrating that in vivo exposure to NDPKs likely occurs. We also show that pathogen derived NDPK-proteins faithfully mimic the catalytically independent pro-survival activity of NM23-H1 against primary AML cells. Flow cytometry identified that pathogen and human NDPKs selectively bind to monocytes in peripheral blood. We therefore used vitamin D3 differentiated monocytes from wild type and genetically modified THP1 cells as a model to demonstrate that NDPK-mediated IL-1ß secretion by monocytes is NLRP3-inflammasome and caspase 1 dependent, but independent of TLR4 signaling. Monocyte stimulation by NDPKs also resulted in activation of NF-κB and IRF pathways but did not include the formation of pyroptosomes or result in pyroptotic cell death which are pivotal features of canonical NLRP3 inflammasome activation. In the context of the growing importance of the NLRP3 inflammasome and IL-1ß in AML and MDS, our findings now implicate pathogen NDPKs in the pathogenesis of these diseases.

Valproic acid disables the Nrf2 anti-oxidant response in acute myeloid leukaemia cells enhancing reactive oxygen species-mediated killing.

BACKGROUND: We previously demonstrated the in vitro killing of AML cells by the combination of the lipid-lowering agent bezafibrate (BEZ) and the contraceptive hormone medroxyprogesterone acetate (MPA). A phase II trial demonstrated in vivo safety and efficacy of BEZ and MPA (BaP) in elderly, relapsed/refractory AML and high-risk myelodysplastic syndrome (MDS) patients. However, we observed dose-limiting toxicities in a second trial that attempted to improve outcomes via escalation of BaP doses. Thus we sought to identify a third repurposed drug that potentiates activity of low dose BaP (BaP 0.1 mM). METHODS AND RESULTS: We demonstrate that addition of a commonly used anti-epileptic, valproic acid (VAL) to low dose BaP (BaP 0.1 mM)(VBaP) enhanced killing of AML cell lines/primary AML cells to levels similar to high dose BaP (BaP 0.5 mM). Similarly, addition of VAL to BaP 0.1 mM enhanced reactive oxygen species (ROS), lipid peroxidation and inhibition of de novo fatty acid synthesis. Overexpression of Nrf2 in K562 and KG1a completely inhibited ROS production and rescued cells from VAL/BaP 0.1 mM/VBaP killing. CONCLUSIONS: Given the good safety data of low-dose BaP in elderly/relapsed/refractory AML patients, and that VAL alone is well-tolerated, we propose VBaP as a novel therapeutic combination for AML.

Biochemical and Molecular Analysis of the Hb Lepore Boston Washington in a Syrian Homozygous Child.

Hemoglobin (Hb) Lepore is composed of two normal α chains and two 뫧 fusion globins that arise from unequal crossover events between the δ- and ß-globin genes. The Hb Lepore is widespread all over the world and in many ethnic groups. It includes some of the few clinically significant Hb variants that are associated with a ß-thalassemia phenotype. Here, we describe the first occurrence of Hb Lepore Boston Washington in a Syrian individual. The patient, a 10-year-old child, shows severe anemia with a Hb level of 6.85 g/dL and typical thalassemic red cell indices. The diagnostic procedure implies hematological, biochemical, and molecular analysis, including multiplex ligation-dependent probe amplification (MLPA) assay, GAP-PCR, and DNA sequencing. This latter allowed us to define the correct structure of the hybrid δß-globin gene. The knowledge of the spectrum of mutations associated with different geographical areas is the prerequisite to set up large-scale screening programs and be able to offer genetic counseling to couples at risk.

The New -474(C→T) Substitution Discovered in the HBG2 Promoter of a Sardinian δß-Thalassemia Carrier.

During a screening for hemoglobinopathies, we found a carrier of the Sardinian δß-thalassemia condition. The proband's hematology and hemoglobin (Hb) profile agreed with those of the other carriers previously identified during our diagnostic program except for the fetal Hb (HbF) composition, which consisted of both α2Aγ2 and α2Gγ2 instead of nearly 100% α2Aγ2. In order to explain the unusual γ-chain ratio, sequencing of the Gγ promoter was carried out and revealed two nucleotide substitutions in cis: C→T at position -474 and A→G at position -309 from the Cap site. The latter had previously been observed in subjects with raised HbF levels, although it has not yet been evaluated at functional level. We used the luciferase assay to determine whether the two mutations modify the transcriptional activity of the Gγ promoter. Results indicated that the observed in vivo Gγ-globin production cannot be translated into increased in vitro promoter function, suggesting that the assessed mutations cannot be considered as functional single nucleotide polymorphisms per se; instead, a more complex regulatory mechanism might be involved.

Hb F-Avellino [(G)γ41(C7)Phe → Leu; HBG2: c.124 T > C]: A New Hemoglobin Variant Observed In A Healthy Newborn.

Here we describe Hb F-Avellino [(G)γ41(C7)Phe → Leu; HBG2: c.124 T > C], a new hemoglobin (Hb) variant observed in a healthy newborn. The proband's hemolysate was found to be mildly unstable by the isopropanol test. The occurrence of the variant was assessed by both chromatographic and electrophoretic methods. DNA sequencing analysis of the (G)γ gene showed a T to C transition at codon 41 (TTC > CTC) corresponding to the Phe → Leu substitution. Normal functional properties have been hypothesized.

The First Mitogenome of the Cyprus Mouflon (Ovis gmelini ophion): New Insights into the Phylogeny of the Genus Ovis.

Sheep are thought to have been one of the first livestock to be domesticated in the Near East, thus playing an important role in human history. The current whole mitochondrial genome phylogeny for the genus Ovis is based on: the five main domestic haplogroups occurring among sheep (O. aries), along with molecular data from two wild European mouflons, three urials, and one argali. With the aim to shed some further light on the phylogenetic relationship within this genus, the first complete mitochondrial genome sequence of a Cypriot mouflon (O. gmelini ophion) is here reported. Phylogenetic analyses were performed using a dataset of whole Ovis mitogenomes as well as D-loop sequences. The concatenated sequence of 28 mitochondrial genes of one Cypriot mouflon, and the D-loop sequence of three Cypriot mouflons were compared to sequences obtained from samples representatives of the five domestic sheep haplogroups along with samples of the extant wild and feral sheep. The sample included also individuals from the Mediterranean islands of Sardinia and Corsica hosting remnants of the first wave of domestication that likely went then back to feral life. The divergence time between branches in the phylogenetic tree has been calculated using seven different calibration points by means of Bayesian and Maximum Likelihood inferences. Results suggest that urial (O. vignei) and argali (O. ammon) diverged from domestic sheep about 0.89 and 1.11 million years ago (MYA), respectively; and dates the earliest radiation of domestic sheep common ancestor at around 0.3 MYA. Additionally, our data suggest that the rise of the modern sheep haplogroups happened in the span of time between six and 32 thousand years ago (KYA). A close phylogenetic relationship between the Cypriot and the Anatolian mouflon carrying the X haplotype was detected. The genetic distance between this group and the other ovine haplogroups supports the hypothesis that it may be a new haplogroup never described before. Furthermore, the updated phylogenetic tree presented in this study determines a finer classification of ovine species and may help to classify more accurately new mitogenomes within the established haplogroups so far identified.

A Novel Heme Pocket Hemoglobin Variant Associated with Normal Hematology: Hb Zara or α91(FG3)Leu→Ile (α2) (HBA2: c.274C > A).

We report a new hemoglobin (Hb) variant on the HBA2 gene, Hb Zara [α91(FG3)Leu→Ile (α2); HBA2: c.274C > A], which was found in a Caucasian man from Croatia. It was observed by routine cation exchange chromatography as an abnormal 21.8% fraction overlapping Hb A2, and associated with normal hematology. It was slightly unstable by the standard isopropanol precipitation test. DNA analysis revealed the CTT > ATT mutation at codon 91 on an α2 gene of a normal α-globin gene arrangement. This new variant represents the sixth described mutation at codon α91 and fourth on the α2 locus. As a result of the slight instability due to the significant role of the α91 residue in the α1ß2 contact, the level of the Hb Zara variant was lower than levels observed for several stable variants codified by the α2 locus.

A new unstable variant of the fetal hemoglobin HBG2 gene: Hb F-Turritana [(G) γ64(E8)Gly→Asp, HBG2:c.194G>A] found in cis to the Hb F-Sardinia gene [(A) γ(E19)Ile→Thr, HBG1:c.227T>C].

A new variant of the fetal hemoglobin (Hb) was observed in a newborn baby subjected to phototherapy due to jaundice, by means of electrophoretic and chromatographic techniques. The variant Hb resulted unstable by the isopropanol stability test. After HBG2 gene sequencing, the G to A transversion at codon 64, position eight of the E helix, was found, which corresponds to the Asp for Gly amino acid substitution. The new variant was called Hb F-Turritana [(G) γ64(E8)Gly→Asp, HBG2:c.194G>A]. Incoming aspartic acid residue, bulky and negatively charged, may be responsible for alteration of the heme pocket steric configuration and for instability. The new abnormal HBG2 gene was found to be associated in cis with the mutated HBG1 gene, which characterizes the Hb F-Sardinia [(A) γ (E19)Ile→Thr, HBG1:c.227T>C] variant.