Promoter-centred chromatin interactions associated with EVI1 expression in EVI1+3q- myeloid leukaemia cells.

EVI1 expression is associated with poor prognosis in myeloid leukaemia, which can result from Chr.3q alterations that juxtapose enhancers to induce EVI1 expression via long-range chromatin interactions. More often, however, EVI1 expression occurs unrelated to 3q alterations, and it remained unclear if, in these cases, EVI1 expression is similarly caused by aberrant enhancer activation. Here, we report that, in EVI1+3q- myeloid leukaemia cells, the EVI1 promoter interacts via long-range chromatin interactions with promoters of distally located, active genes, rather than with enhancer elements. Unlike in 3q+ cells, EVI1 expression and long-range interactions appear to not depend on CTCF/cohesin, though EVI1+3q- cells utilise an EVI1 promoter-proximal site to enhance its expression that is also involved in CTCF-mediated looping in 3q+ cells. Long-range interactions in 3q- cells connect EVI1 to promoters of multiple genes, whose transcription correlates with EVI1 in EVI1+3q- cell lines, suggesting a shared mechanism of transcriptional regulation. In line with this, CRISPR interference-induced silencing of two of these sites minimally, but consistently reduced EVI1 expression. Together, we provide novel evidence of features associated with EVI1 expression in 3q- leukaemia and consolidate the view that EVI1 in 3q- leukaemia is largely promoter-driven, potentially involving long-distance promoter clustering.

FISH-negative BCR::ABL1-positive e19a2 chronic myeloid leukaemia: the most cryptic of insertions.

BACKGROUND: Chronic myeloid leukaemia (CML) is one of the most well characterised human malignancies. Most patients have a cytogenetically visible translocation between chromosomes 9 and 22 which generates the pathognomonic BCR::ABL1 fusion gene. The derivative chromosome 22 ('Philadelphia' or Ph chromosome) usually harbours the fusion gene encoding a constitutively active ABL1 kinase domain. A small subset of patients have no visible translocation. Historically, these 'Philadelphia chromosome negative' patients caused diagnostic confusion between CML and other myeloproliferative neoplasms; it is now well established that the BCR::ABL1 fusion gene can be generated via submicroscopic intrachromosomal insertion of ABL1 sequence into BCR, or, more rarely, of BCR into ABL1. The fusion genes arising from cryptic insertions are not detectable via G-banded chromosome analysis [karyotype] but can nevertheless always be detected using fluorescence in situ hybridisation (FISH) and/or qualitative reverse transcriptase PCR. CASE PRESENTATION: A 43-year-old female presented with suspected CML in 2007; however, contemporaneous gold standard laboratory investigations, G-banded chromosome analysis and FISH, were both negative. The reverse transcriptase quantitative PCR (RT-qPCR) assay available at the time, which was capable of detecting the common BCR::ABL1 transcripts (e13a2/e14a2), was also negative. Upon review in 2009, the newly recommended reverse transcriptase multiplex PCR (capable of detecting all BCR::ABL1 transcripts including the atypical ones) subsequently detected an e19a2 fusion. The patient then responded to tyrosine kinase inhibitor therapy. In contrast, FISH studies of both samples with three commercially available probes remained consistently negative. Retrospective whole genome sequencing, undertaken as part of the 100,000 Genomes Project, has now revealed that the patient's BCR::ABL1 fusion gene arose via a uniquely small insertion of 122 kb ABL1 sequences into BCR. CONCLUSIONS: We present a patient with suspected chronic myeloid leukaemia whose genetic investigations were originally negative at the time of diagnosis despite the use of contemporaneous gold standard methods. This is the first report of a FISH-negative, BCR::ABL1 positive CML which demonstrates that, even after sixty years of research into one of the most well understood human malignancies, whole genome sequencing can yield novel diagnostic findings in CML.

Chromatin-based, in cis and in trans regulatory rewiring underpins distinct oncogenic transcriptomes in multiple myeloma.

Multiple myeloma is a genetically heterogeneous cancer of the bone marrow plasma cells (PC). Distinct myeloma transcriptome profiles are primarily driven by myeloma initiating events (MIE) and converge into a mutually exclusive overexpression of the CCND1 and CCND2 oncogenes. Here, with reference to their normal counterparts, we find that myeloma PC enhanced chromatin accessibility combined with paired transcriptome profiling can classify MIE-defined genetic subgroups. Across and within different MM genetic subgroups, we ascribe regulation of genes and pathways critical for myeloma biology to unique or shared, developmentally activated or de novo formed candidate enhancers. Such enhancers co-opt recruitment of existing transcription factors, which although not transcriptionally deregulated per se, organise aberrant gene regulatory networks that help identify myeloma cell dependencies with prognostic impact. Finally, we identify and validate the critical super-enhancer that regulates ectopic expression of CCND2 in a subset of patients with MM and in chronic lymphocytic leukemia.

Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control.

There is growing evidence that measurement of SARS-CoV-2 viral copy number can inform clinical and public health management of SARS-CoV-2 carriers and COVID-19 patients. Here we show that quantification of SARS-CoV-2 is feasible in a clinical setting, using a duplex RT-qPCR assay which targets both the E gene (Charité assay) and a human RNA transcript, RNase P (CDC assay) as an internal sample sufficiency control. Samples in which RNase P is not amplified indicate that sample degradation has occurred, PCR inhibitors are present, RNA extraction has failed or swabbing technique was insufficient. This important internal control reveals that 2.4 % of nasopharyngeal swabs (15/618 samples) are inadequate for SARS-CoV-2 testing which, if not identified, could result in false negative results. We show that our assay is linear across at least 7 logs and is highly reproducible, enabling the conversion of Cq values to viral copy numbers using a standard curve. Furthermore, the SARS-CoV-2 copy number was independent of the RNase P copy number indicating that the per-swab viral copy number is not dependent on sampling- further allowing comparisons between samples. The ability to quantify SARS-CoV-2 viral copy number will provide an important opportunity for viral burden-guided public health and clinical decision making.

Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions.

Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that MIR300 has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. MIR300 is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates MIR300 in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating TUG1 long noncoding RNA that uncouples and limits MIR300 function to cytostasis. Genetic and pharmacologic MIR300 modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis in vitro and in patient-derived xenografts; hence, the importance of MIR300 and PP2A activity for CML development and therapy.

Treatment, prevention and public health management of impetigo, scabies, crusted scabies and fungal skin infections in endemic populations: a systematic review.

We conducted a systematic review of the treatment, prevention and public health control of skin infections including impetigo, scabies, crusted scabies and tinea in resource-limited settings where skin infections are endemic. The aim is to inform strategies, guidelines and research to improve skin health in populations that are inequitably affected by infections of the skin and the downstream consequences of these. The systematic review is reported according to the PRISMA statement. From 1759 titles identified, 81 full text studies were reviewed and key findings outlined for impetigo, scabies, crusted scabies and tinea. Improvements in primary care and public health management of skin infections will have broad and lasting impacts on overall quality of life including reductions in morbidity and mortality from sepsis, skeletal infections, kidney and heart disease.

Nous avons effectué une analyse systématique du traitement, de la prévention et du contrôle de santé publique des infections cutanées comprenant l'impétigo, la gale, la gale en croûte et la teigne, dans des cadres à ressources limitées où les infections cutanées sont endémiques. Le but étant d'informer les stratégies, les directives et la recherche pour améliorer la santé de la peau dans les populations qui sont touchées de manière inéquitable par les infections cutanées et leurs conséquences plus tard. La revue systématique est rapportée selon la déclaration PRISMA. Sur 1759 titres recensés, 81 études en texte intégral ont été passées en revue et les principaux résultats rapportés concernant l'impétigo, la gale, la gale en croûte et la teigne. Les améliorations apportées dans la prise en charge des infections de la peau dans les soins de santé primaires et les soins de santé publique auront des répercussions vastes et durables sur la qualité de vie en général, notamment une réduction de la morbidité et de la mortalité dues au sepsis, aux infections du squelette, aux maladies du rein et du cœur.

Lineage-Specific Genes Are Prominent DNA Damage Hotspots during Leukemic Transformation of B Cell Precursors.

In human leukemia, lineage-specific genes represent predominant targets of deletion, with lymphoid-specific genes frequently affected in lymphoid leukemia and myeloid-specific genes in myeloid leukemia. To investigate the basis of lineage-specific alterations, we analyzed global DNA damage in primary B cell precursors expressing leukemia-inducing oncogenes by ChIP-seq. We identified more than 1,000 sensitive regions, of which B lineage-specific genes constitute the most prominent targets. Identified hotspots at B lineage genes relate to DNA-DSBs, affect genes that harbor genomic lesions in human leukemia, and associate with ectopic deletion in successfully transformed cells. Furthermore, we show that most identified regions overlap with gene bodies of highly expressed genes and that induction of a myeloid lineage phenotype in transformed B cell precursors promotes de novo DNA damage at myeloid loci. Hence, we demonstrate that lineage-specific transcription predisposes lineage-specific genes in transformed B cell precursors to DNA damage, which is likely to promote the frequent alteration of lineage-specific genes in human leukemia.

Protocol for the systematic review of the prevention, treatment and public health management of impetigo, scabies and fungal skin infections in resource-limited settings.

BACKGROUND: Impetigo, scabies, and fungal skin infections disproportionately affect populations in resource-limited settings. Evidence for standard treatment of skin infections predominantly stem from hospital-based studies in high-income countries. The evidence for treatment in resource-limited settings is less clear, as studies in these populations may lack randomisation and control groups for cultural, ethical or economic reasons. Likewise, a synthesis of the evidence for public health control within endemic populations is also lacking. We propose a systematic review of the evidence for the prevention, treatment and public health management of skin infections in resource-limited settings, to inform the development of guidelines for the standardised and streamlined clinical and public health management of skin infections in endemic populations. METHODS: The protocol has been designed in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols statement. All trial designs and analytical observational study designs will be eligible for inclusion. A systematic search of the peer-reviewed literature will include PubMed, Excertpa Medica and Global Health. Grey literature databases will also be systematically searched, and clinical trials registries scanned for future relevant studies. The primary outcome of interest will be the clinical cure or decrease in prevalence of impetigo, scabies, crusted scabies, tinea capitis, tinea corporis or tinea unguium. Two independent reviewers will perform eligibility assessment and data extraction using standardised electronic forms. Risk of bias assessment will be undertaken by two independent reviewers according to the Cochrane Risk of Bias tool. Data will be tabulated and narratively synthesised. We expect there will be insufficient data to conduct meta-analysis. The final body of evidence will be reported against the Grades of Recommendation, Assessment, Development and Evaluation grading system. DISCUSSION: The evidence derived from the systematic review will be used to inform the development of guidelines for the management of skin infections in resource-limited settings. The evidence derived will be intended for use by clinicians, public health practitioners and policy makers in the treatment of skin infections and the development of skin infection control programmes. The review will identify any gaps in the current evidence to provide direction for future research. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42015029453.

Abnormal villous morphology mimicking a hydatidiform mole associated with paternal trisomy of chromosomes 3,7,8 and unipaternal disomy of chromosome 11.

BACKGROUND: Pregnancies affected by non-molar chromosomal abnormality may sometimes demonstrate abnormal chorionic villous morphology that is similar to partial hydatidiform mole. Determination of the underlying aetiology may be difficult in such cases. CASE PRESENTATION: This report describes a case referred to the regional trophoblastic disease unit as a possible hydatidiform mole that demonstrated both villous dysmorphology and abnormal p57(KIP2) expression. Molecular genotyping revealed that while most chromosomes in the villous tissue were diploid and biparental, chromosomes 3, 7 and 8 were trisomic with an additional paternally derived chromosome. In contrast chromosome 11 showed uniparental disomy of paternal origin a situation more usually associated with complete hydatidiform moles. This unusual case highlights that exceptions may occur to the general rules of both histological morphology and immunoprofile, and that these can be resolved by detailed molecular genetic investigations. CONCLUSION: The findings confirm that trisomic pregnancies may demonstrate morphological villous features similar to hydatidiform mole, and that loss of p57(KIP2) expression occurs due to an absence of maternally transcribed genes on chromosome 11 and can therefore be independent of androgenetic complete hydatidiform mole.

A differential role for CD248 (Endosialin) in PDGF-mediated skeletal muscle angiogenesis.

CD248 (Endosialin) is a type 1 membrane protein involved in developmental and pathological angiogenesis through its expression on pericytes and regulation of PDGFRß signalling. Here we explore the function of CD248 in skeletal muscle angiogenesis. Two distinct forms of capillary growth (splitting and sprouting) can be induced separately by increasing microcirculatory shear stress (chronic vasodilator treatment) or by inducing functional overload (extirpation of a synergistic muscle). We show that CD248 is present on pericytes in muscle and that CD248-/- mice have a specific defect in capillary sprouting. In contrast, splitting angiogenesis is independent of CD248 expression. Endothelial cells respond to pro-sprouting angiogenic stimulus by up-regulating gene expression for HIF1α, angiopoietin 2 and its receptor TEK, PDGF-B and its receptor PDGFRß; this response did not occur following a pro-splitting angiogenic stimulus. In wildtype mice, defective sprouting angiogenesis could be mimicked by blocking PDGFRß signalling using the tyrosine kinase inhibitor Imatinib mesylate. We conclude that CD248 is required for PDGFRß-dependant capillary sprouting but not splitting angiogenesis, and identify a new role for CD248 expressed on pericytes in the early stages of physiological angiogenesis during muscle remodelling.

PP2A-activating drugs selectively eradicate TKI-resistant chronic myeloid leukemic stem cells.

The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase-independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression--but not activity--of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/ß-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase-independent and PP2A-mediated inhibition of JAK2 and ß-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1-positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/ß-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.

Combined inhibition of p97 and the proteasome causes lethal disruption of the secretory apparatus in multiple myeloma cells.

Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs) have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER)-associated degradation (ERAD) and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48) has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma.

Combining BCR-ABL1 transcript levels at 3 and 6 months in chronic myeloid leukemia: implications for early intervention strategies.

Several groups have shown that that the BCR-ABL1 transcript level measured at 3 or 6 months after starting treatment with tyrosine kinase inhibitors strongly predicts clinical outcomes for patients with chronic myeloid leukemia. In this work, we asked whether the prognostic value of the 3-month transcript level could be improved by combining the 3- and 6-month results. We classified patients treated with imatinib and patients treated with dasatinib according to their transcript levels at 3 months and 6 months. The patients who met the 3-month landmark but failed the 6-month one had outcomes identical to those of patients who met both landmarks, whereas the patients who failed the first landmark but met the second one had prognoses similar to those who failed both landmarks. In summary, early intervention strategies can be based robustly just on the transcript level at 3 months. This trial was registered at www.clinicaltrials.gov as # NCT01460693.