BRD4-mediated repression of p53 is a target for combination therapy in AML.

Acute myeloid leukemia (AML) is a typically lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report enhanced toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETi's ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML.

BET Inhibition Suppresses S100A8 and S100A9 Expression in Acute Myeloid Leukemia Cells and Synergises with Daunorubicin in Causing Cell Death.

S100A8 and S100A9 are both members of the S100 family and have been shown to play roles in myeloid differentiation, autophagy, apoptosis, and chemotherapy resistance. In this study we demonstrate that the BET-bromodomain inhibitor JQ1 causes rapid suppression of S100A8 and S100A9 mRNA and protein in a reversible manner. In addition, we show that JQ1 synergises with daunorubicin in causing AML cell death. Daunorubicin alone causes a dose- and time-dependent increase in S100A8 and S100A9 protein levels in AML cell lines which is overcome by cotreatment with JQ1. This suggests that JQ1 synergises with daunorubicin in causing apoptosis via suppression of S100A8 and S100A9 levels.

DIS3 isoforms vary in their endoribonuclease activity and are differentially expressed within haematological cancers.

DIS3 (defective in sister chromatid joining) is the catalytic subunit of the exosome, a protein complex involved in the 3'-5' degradation of RNAs. DIS3 is a highly conserved exoribonuclease, also known as Rrp44. Global sequencing studies have identified DIS3 as being mutated in a range of cancers, with a considerable incidence in multiple myeloma. In this work, we have identified two protein-coding isoforms of DIS3. Both isoforms are functionally relevant and result from alternative splicing. They differ from each other in the size of their N-terminal PIN (PilT N-terminal) domain, which has been shown to have endoribonuclease activity and tether DIS3 to the exosome. Isoform 1 encodes a full-length PIN domain, whereas the PIN domain of isoform 2 is shorter and is missing a segment with conserved amino acids. We have carried out biochemical activity assays on both isoforms of full-length DIS3 and the isolated PIN domains. We find that isoform 2, despite missing part of the PIN domain, has greater endonuclease activity compared with isoform 1. Examination of the available structural information allows us to provide a hypothesis to explain this altered behaviour. Our results also show that multiple myeloma patient cells and all cancer cell lines tested have higher levels of isoform 1 compared with isoform 2, whereas acute myeloid leukaemia and chronic myelomonocytic leukaemia patient cells and samples from healthy donors have similar levels of isoforms 1 and 2. Taken together, our data indicate that significant changes in the ratios of the two isoforms could be symptomatic of haematological cancers.

Epigenetic Guardian: A Review of the DNA Methyltransferase DNMT3A in Acute Myeloid Leukaemia and Clonal Haematopoiesis.

Acute myeloid leukaemia (AML) is a haematological malignancy characterized by clonal stem cell proliferation and aberrant block in differentiation. Dysfunction of epigenetic modifiers contributes significantly to the pathogenesis of AML. One frequently mutated gene involved in epigenetic modification is DNMT3A (DNA methyltransferase-3-alpha), a DNA methyltransferase that alters gene expression by de novo methylation of cytosine bases at CpG dinucleotides. Approximately 22% of AML and 36% of cytogenetically normal AML cases carry DNMT3A mutations and around 60% of these mutations affect the R882 codon. These mutations have been associated with poor prognosis and adverse survival outcomes for AML patients. Advances in whole-exome sequencing techniques have recently identified a large number of DNMT3A mutations present in clonal cells in normal elderly individuals with no features of haematological malignancy. Categorically distinct from other preleukaemic conditions, this disorder has been termed clonal haematopoiesis of indeterminate potential (CHIP). Further insight into the mutational landscape of CHIP may illustrate the consequence of particular mutations found in DNMT3A and identify specific "founder" mutations responsible for clonal expansion that may contribute to leukaemogenesis. This review will focus on current research and understanding of DNMT3A mutations in both AML and CHIP.

Haemolytic anaemia: don't leave it out in the cold.

This clinical case highlights the diagnostic odyssey of an adolescent girl presenting to A&E with non-specific headaches and chest pain. The case will describe the steps in decision making from admission to follow-up.

Low Oxygen Affinity Variant Haemoglobin in an elderly woman presenting with low oxygen saturation.

An asymptomatic 81-year-old woman was referred by her general practitioner regarding a pulse-oximetry oxygen saturation (SpO2) of 74%. An arterial blood gas analysis (ABG) on air showed PaO2 12.9 kPa, oxygen saturation 80%, with normal pH, PaCO2, methaemoglobin and carboxyhaemoglobin levels. After a normal chest x-ray, tinzaparin was administered empirically for possible occult pulmonary embolus. This diagnosis was subsequently excluded with an unremarkable computed tomography pulmonary angiogram (CTPA). She was further investigated as an out patient. DNA globin-gene analysis identified a variant haemoglobin revealed to be haemoglobin Saint Mande (HbSM). Following reassurance regarding the benign nature of her condition, she has remained well.

The 3' to 5' Exoribonuclease DIS3: From Structure and Mechanisms to Biological Functions and Role in Human Disease.

DIS3 is a conserved exoribonuclease and catalytic subunit of the exosome, a protein complex involved in the 3' to 5' degradation and processing of both nuclear and cytoplasmic RNA species. Recently, aberrant expression of DIS3 has been found to be implicated in a range of different cancers. Perhaps most striking is the finding that DIS3 is recurrently mutated in 11% of multiple myeloma patients. Much work has been done to elucidate the structural and biochemical characteristics of DIS3, including the mechanistic details of its role as an effector of RNA decay pathways. Nevertheless, we do not understand how DIS3 mutations can lead to cancer. There are a number of studies that pertain to the function of DIS3 at the organismal level. Mutant phenotypes in S. pombe, S. cerevisiae and Drosophila suggest DIS3 homologues have a common role in cell-cycle progression and microtubule assembly. DIS3 has also recently been implicated in antibody diversification of mouse B-cells. This article aims to review current knowledge of the structure, mechanisms and functions of DIS3 as well as highlighting the genetic patterns observed within myeloma patients, in order to yield insight into the putative role of DIS3 mutations in oncogenesis.

The RAG Model: A New Paradigm for Genetic Risk Stratification in Multiple Myeloma.

Molecular studies have shown that multiple myeloma is a highly genetically heterogonous disease which may manifest itself as any number of diverse subtypes each with variable clinicopathological features and outcomes. Given this genetic heterogeneity, a universal approach to treatment of myeloma is unlikely to be successful for all patients and instead we should strive for the goal of personalised therapy using rationally informed targeted strategies. Current DNA sequencing technologies allow for whole genome and exome analysis of patient myeloma samples that yield vast amounts of genetic data and provide a mutational overview of the disease. However, the clinical utility of this information currently lags far behind the sequencing technology which is increasingly being incorporated into clinical practice. This paper attempts to address this shortcoming by proposing a novel genetically based "traffic-light" risk stratification system for myeloma, termed the RAG (Red, Amber, Green) model, which represents a simplified concept of how complex genetic data may be compressed into an aggregate risk score. The model aims to incorporate all known clinically important trisomies, translocations, and mutations in myeloma and utilise these to produce a score between 1.0 and 3.0 that can be incorporated into diagnostic, prognostic, and treatment algorithms for the patient.

The genetic architecture of multiple myeloma.

Multiple myeloma is a malignant proliferation of monoclonal plasma cells leading to clinical features that include hypercalcaemia, renal dysfunction, anaemia, and bone disease (frequently referred to by the acronym CRAB) which represent evidence of end organ failure. Recent evidence has revealed myeloma to be a highly heterogeneous disease composed of multiple molecularly-defined subtypes each with varying clinicopathological features and disease outcomes. The major division within myeloma is between hyperdiploid and nonhyperdiploid subtypes. In this division, hyperdiploid myeloma is characterised by trisomies of certain odd numbered chromosomes, namely, 3, 5, 7, 9, 11, 15, 19, and 21 whereas nonhyperdiploid myeloma is characterised by translocations of the immunoglobulin heavy chain alleles at chromosome 14q32 with various partner chromosomes, the most important of which being 4, 6, 11, 16, and 20. Hyperdiploid and nonhyperdiploid changes appear to represent early or even initiating mutagenic events that are subsequently followed by secondary aberrations including copy number abnormalities, additional translocations, mutations, and epigenetic modifications which lead to plasma cell immortalisation and disease progression. The following review provides a comprehensive coverage of the genetic and epigenetic events contributing to the initiation and progression of multiple myeloma and where possible these abnormalities have been linked to disease prognosis.

A dynamic multi-compartmental model of DNA methylation with demonstrable predictive value in hematological malignancies.

Recent advances have highlighted the central role of DNA methylation in leukemogenesis and have led to clinical trials of epigenetic therapy, notably hypomethylating agents, in myelodysplasia and acute myeloid leukemia. However, despite these advances, our understanding of the dynamic regulation of the methylome remains poor. We have attempted to address this shortcoming by producing a dynamic, six-compartmental model of DNA methylation levels based on the activity of the Dnmt methyltransferase proteins. In addition, the model incorporates the recently discovered Tet family proteins which enzymatically convert methylcytosine to hydroxymethylcytosine. A set of first order, partial differential equations comprise the model and were solved via numerical integration. The model is able to predict the relative abundances of unmethylated, hemimethylated, fully methylated, and hydroxymethylated CpG dyads in the DNA of cells with fully functional Dnmt and Tet proteins. In addition, the model accurately predicts the experimentally measured changes in these abundances with disruption of Dnmt function. Furthermore, the model reveals the mechanism whereby CpG islands are maintained in a hypomethylated state via local modulation of Dnmt and Tet activities without any requirement for active demethylation. We conclude that this model provides an accurate depiction of the major epigenetic processes involving modification of DNA.

The polo-like kinase inhibitor BI 2536 exhibits potent activity against malignant plasma cells and represents a novel therapy in multiple myeloma.

OBJECTIVE: Polo-like kinase 1 (Plk1) is a regulator of the cell cycle that has been implicated in the pathology of many cancers. We have investigated whether this kinase plays a role in multiple myeloma (MM) using the Plk1 inhibitor BI 2536. MATERIALS AND METHODS: We have used six MM cell lines and six patient-derived samples to determine the effects of the Plk1 inhibitor, BI 2536, on cell viability, apoptosis, and cytokinesis. We have also examined the effect of the microenvironment on these parameters and the effects of BI 2536 in combination with other antimyeloma agents. RESULTS: We show that MM cell lines and patient samples express PLK1 and that cell death by apoptosis occurs when Plk1 is inhibited. Cells treated with BI 2536 accumulate in the G(2)/M phase of the cell cycle causing endoduplication. The effects of BI 2536 are not abrogated when cells are cultured on extracellular matrix components, in the presence of interleukin-6, or with bone marrow stromal cells. CONCLUSIONS: Plk1 inhibition leads to cell death in MM cell lines and patient myeloma samples. Our data suggest that inhibition of Plk1 may have potential use as a therapeutic strategy in multiple myeloma.