A TIAM1-TRIM28 complex mediates epigenetic silencing of protocadherins to promote migration of lung cancer cells.

Lung cancer is the leading cause of cancer deaths. Its high mortality is associated with high metastatic potential. Here, we show that the RAC1-selective guanine nucleotide exchange factor T cell invasion and metastasis-inducing protein 1 (TIAM1) promotes cell migration and invasion in the most common subtype of lung cancer, non-small-cell lung cancer (NSCLC), through an unexpected nuclear function. We show that TIAM1 interacts with TRIM28, a master regulator of gene expression, in the nucleus of NSCLC cells. We reveal that a TIAM1-TRIM28 complex promotes epithelial-to-mesenchymal transition, a phenotypic switch implicated in cell migration and invasion. This occurs through H3K9me3-induced silencing of protocadherins and by decreasing E-cadherin expression, thereby antagonizing cell-cell adhesion. Consistently, TIAM1 or TRIM28 depletion suppresses the migration of NSCLC cells, while migration is restored by the simultaneous depletion of protocadherins. Importantly, high nuclear TIAM1 in clinical specimens is associated with advanced-stage lung adenocarcinoma, decreased patient survival, and inversely correlates with E-cadherin expression.

Coordinated alterations in RNA splicing and epigenetic regulation drive leukaemogenesis.

Transcription and pre-mRNA splicing are key steps in the control of gene expression and mutations in genes regulating each of these processes are common in leukaemia1,2. Despite the frequent overlap of mutations affecting epigenetic regulation and splicing in leukaemia, how these processes influence one another to promote leukaemogenesis is not understood and, to our knowledge, there is no functional evidence that mutations in RNA splicing factors initiate leukaemia. Here, through analyses of transcriptomes from 982 patients with acute myeloid leukaemia, we identified frequent overlap of mutations in IDH2 and SRSF2 that together promote leukaemogenesis through coordinated effects on the epigenome and RNA splicing. Whereas mutations in either IDH2 or SRSF2 imparted distinct splicing changes, co-expression of mutant IDH2 altered the splicing effects of mutant SRSF2 and resulted in more profound splicing changes than either mutation alone. Consistent with this, co-expression of mutant IDH2 and SRSF2 resulted in lethal myelodysplasia with proliferative features in vivo and enhanced self-renewal in a manner not observed with either mutation alone. IDH2 and SRSF2 double-mutant cells exhibited aberrant splicing and reduced expression of INTS3, a member of the integrator complex3, concordant with increased stalling of RNA polymerase II (RNAPII). Aberrant INTS3 splicing contributed to leukaemogenesis in concert with mutant IDH2 and was dependent on mutant SRSF2 binding to cis elements in INTS3 mRNA and increased DNA methylation of INTS3. These data identify a pathogenic crosstalk between altered epigenetic state and splicing in a subset of leukaemias, provide functional evidence that mutations in splicing factors drive myeloid malignancy development, and identify spliceosomal changes as a mediator of IDH2-mutant leukaemogenesis.

A three-gene leukaemic stem cell signature score is robustly prognostic in chronic myelomonocytic leukaemia.

Leukaemic stem cell (LSC) gene expression has recently been linked to prognosis in patients with acute myeloid leukaemia (17-gene LSC score, LSC-17) and myelodysplastic syndromes. Although chronic myelomonocytic leukaemia (CMML) is regarded as a stem cell disorder, the clinical and biological impact of LSCs on CMML patients remains elusive. Making use of multiple independent validation cohorts, we here describe a concise three-gene expression signature (LSC-3, derived from the LSC-17 score) as an independent and robust prognostic factor for leukaemia-free and overall survival in CMML. We propose that LSC-3 could be used to supplement existing risk stratification systems, to improve prognostic performance and guide management decisions.

Encouraging outcomes of using a small-donor single graft in pediatric kidney transplantation.

BACKGROUND: The use of small pediatric kidneys as single grafts for transplantation is controversial, due to the potential risk for graft thrombosis and insufficient nephron mass. METHODS: Aiming to test the benefits of transplanting these kidneys, 375 children who underwent kidney transplantation in a single center were evaluated: 49 (13.1%) received a single graft from a small pediatric donor (≤ 15 kg, SPD group), 244 (65.1%) from a bigger pediatric donor (> 15 kg, BPD group), and 82 (21.9%) from adult living donors (group ALD). RESULTS: Groups had similar baseline main characteristics. After 5 years of follow-up, children from the SPD group were comparable to children from BPD and ALD in patient survival (94%, 96%, and 98%, respectively, p = 0.423); graft survival (89%, 88%, and 93%, respectively, p = 0.426); the frequency of acute rejection (p = 0.998); the incidence of post-transplant lymphoproliferative disease (p = 0.671); the odds ratio for severely increased proteinuria (p = 0.357); the rates of vascular thrombosis (p = 0.846); and the necessity for post-transplant surgical intervention prior to discharge (p = 0.905). The longitudinal evolution of eGFR was not uniform among groups. The three groups presented a decrease in eGFR, but the slope of the curve was steeper in ALD children. At 5 years, the eGFR of the ALD group was 10 ml/min/1.73m2 inferior to the others. At that time, the eGFR from the SPD group was statistically similar to the BPD group (p = 0.952). CONCLUSION: In a specialized transplant center, the use of a single small pediatric donor kidney for transplantation is as successful as bigger pediatric or adult living donors, after 5 years of follow-up. A higher resolution version of the Graphical abstract is available as Supplementary information.

Pharmacological interventions for preventing venous thromboembolism in people undergoing bariatric surgery.

BACKGROUND: Venous thromboembolism (VTE), which comprises deep vein thrombosis (DVT) and pulmonary embolism (PE), is the leading cause of preventable death in hospitalised people and the third most common cause of mortality in surgical patients. People undergoing bariatric surgery have the additional risk factor of being overweight. Although VTE prophylaxis in surgical patients is well established, the best way to prevent VTE in those undergoing bariatric surgery is less clear. OBJECTIVES: To evaluate the benefits and harms of pharmacological interventions (alone or in combination) on venous thromboembolism and other health outcomes in people undergoing bariatric surgery compared to the same pharmacological intervention administered at a different dose or frequency, the same pharmacological intervention or started at a different time point, another pharmacological intervention, no intervention or placebo. SEARCH METHODS: We used standard, extensive Cochrane search methods. The latest search date was 1 November 2021. SELECTION CRITERIA: We included randomised controlled trials (RCTs) and quasi-RCTs in males and females of any age undergoing bariatric surgery comparing pharmacological interventions for VTE (alone or in combination) with the same pharmacological intervention administered at a different dose or frequency, the same pharmacological intervention started at a different time point, a different pharmacological intervention, no treatment or placebo. DATA COLLECTION AND ANALYSIS: We used standard Cochrane methods. Our primary outcomes were 1. VTE and 2. major bleeding. Our secondary outcomes were 1. all-cause mortality, 2. VTE-related mortality, 3. PE, 4. DVT, 5. adverse effects and 6. quality of life. We used GRADE to assess certainty of evidence for each outcome. MAIN RESULTS: We included seven RCTs with 1045 participants. Data for meta-analysis were available from all participants. Four RCTs (597 participants) compared higher-dose heparin to standard-dose heparin: one of these studies (139 participants) used unfractionated heparin (UFH) and the other three (458 participants) used low-molecular-weight heparin (LMWH). One study compared heparin versus pentasaccharide (198 participants), and one study compared starting heparin before versus after bariatric surgery (100 participants). One study (150 participants) compared combined mechanical and pharmacological (enoxaparin) prophylaxis versus mechanical prophylaxis alone. The duration of the interventions ranged from seven to 15 days, and follow-up ranged from 10 to 180 days. Higher-dose heparin versus standard-dose heparin Compared to standard-dose heparin, higher-dose heparin may result in little or no difference in the risk of VTE (RR 0.55, 95% CI 0.05 to 5.99; 4 studies, 597 participants) or major bleeding (RR 1.19, 95% CI 0.48 to 2.96; I2 = 8%; 4 studies, 597 participants; low-certainty) in people undergoing bariatric surgery. The evidence on all-cause mortality, VTE-related mortality, PE, DVT and adverse events (thrombocytopenia) is uncertain (effect not estimable or very low-certainty evidence). Heparin versus pentasaccharide Heparin compared to a pentasaccharide after bariatric surgery may result in little or no difference in the risk of VTE (RR 0.83, 95% CI 0.19 to 3.61; 1 study, 175 participants) or DVT (RR 0.83, 95% CI 0.19 to 3.61; 1 study, 175 participants). The evidence on major bleeding, PE and mortality is uncertain (effect not estimable or very low-certainty evidence). Heparin started before versus after the surgical procedure Starting prophylaxis with heparin 12 hours before surgery versus after surgery may result in little or no difference in the risk of VTE (RR 0.11, 95% CI 0.01 to 2.01; 1 study, 100 participants) or DVT (RR 0.11, 95% CI 0.01 to 2.01; 1 study, 100 participants). The evidence on major bleeding, all-cause mortality and VTE-related mortality is uncertain (effect not estimable or very low-certainty evidence). We were unable to assess the effect of this intervention on PE or adverse effects, as the study did not measure these outcomes. Combined mechanical and pharmacological prophylaxis versus mechanical prophylaxis alone Combining mechanical and pharmacological prophylaxis (started 12 hours before surgery) may reduce VTE events in people undergoing bariatric surgery compared to mechanical prophylaxis alone (RR 0.05, 95% CI 0.00 to 0.89; number needed to treat for an additional beneficial outcome (NNTB) = 9; 1 study, 150 participants; low-certainty). We were unable to assess the effect of this intervention on major bleeding or morality (effect not estimable), or on PE or adverse events (not measured). No studies measured quality of life. AUTHORS' CONCLUSIONS: Higher-dose heparin may make little or no difference to venous thromboembolism or major bleeding in people undergoing bariatric surgery when compared to standard-dose heparin. Heparin may make little or no difference to venous thromboembolism in people undergoing bariatric surgery when compared to pentasaccharide. There are inadequate data to draw conclusions about the effects of heparin compared to pentasaccharide on major bleeding. Starting prophylaxis with heparin 12 hours before bariatric surgery may make little or no difference to venous thromboembolism in people undergoing bariatric surgery when compared to starting heparin after bariatric surgery. There are inadequate data to draw conclusions about the effects of heparin started before versus after surgery on major bleeding. Combining mechanical and pharmacological prophylaxis (started 12 hours before surgery) may reduce VTE events in people undergoing bariatric surgery when compared to mechanical prophylaxis alone. No data are available relating to major bleeding. The certainty of the evidence is limited by small sample sizes, few or no events, and risk of bias concerns. Future trials must be sufficiently large to enable analysis of relevant clinical outcomes, and should standardise the time of treatment and follow-up. They should also address the effect of direct oral anticoagulants and antiplatelets, preferably grouping them according to the type of intervention.

Blast cells surviving acute myeloid leukemia induction therapy are in cycle with a signature of FOXM1 activity.

BACKGROUND: Disease relapse remains common following treatment of acute myeloid leukemia (AML) and is due to chemoresistance of leukemia cells with disease repopulating potential. To date, attempts to define the characteristics of in vivo resistant blasts have focused on comparisons between leukemic cells at presentation and relapse. However, further treatment responses are often seen following relapse, suggesting that most blasts remain chemosensitive. We sought to characterise in vivo chemoresistant blasts by studying the transcriptional and genetic features of blasts from before and shortly after induction chemotherapy using paired samples from six patients with primary refractory AML. METHODS: Leukemic blasts were isolated by fluorescence-activated cell sorting. Fluorescence in situ hybridization (FISH), targeted genetic sequencing and detailed immunophenotypic analysis were used to confirm that sorted cells were leukemic. Sorted blasts were subjected to RNA sequencing. Lentiviral vectors expressing short hairpin RNAs were used to assess the effect of FOXM1 knockdown on colony forming capacity, proliferative capacity and apoptosis in cell lines, primary AML cells and CD34+ cells from healthy donors. RESULTS: Molecular genetic analysis revealed early clonal selection occurring after induction chemotherapy. Immunophenotypic characterisation found leukemia-associated immunophenotypes in all cases that persisted following treatment. Despite the genetic heterogeneity of the leukemias studied, transcriptional analysis found concerted changes in gene expression in resistant blasts. Remarkably, the gene expression signature suggested that post-chemotherapy blasts were more proliferative than those at presentation. Resistant blasts also appeared less differentiated and expressed leukemia stem cell (LSC) maintenance genes. However, the proportion of immunophenotypically defined LSCs appeared to decrease following treatment, with implications for the targeting of these cells on the basis of cell surface antigen expression. The refractory gene signature was highly enriched with targets of the transcription factor FOXM1. shRNA knockdown experiments demonstrated that the viability of primary AML cells, but not normal CD34+ cells, depended on FOXM1 expression. CONCLUSIONS: We found that chemorefractory blasts from leukemias with varied genetic backgrounds expressed a common transcriptional program. In contrast to the notion that LSC quiescence confers resistance to chemotherapy we find that refractory blasts are both actively proliferating and enriched with LSC maintenance genes. Using primary patient material from a relevant clinical context we also provide further support for the role of FOXM1 in chemotherapy resistance, proliferation and stem cell function in AML.

Towards Providing Effective Data-Driven Responses to Predict the Covid-19 in São Paulo and Brazil.

São Paulo is the most populous state in Brazil, home to around 22% of the country's population. The total number of Covid-19-infected people in São Paulo has reached more than 1 million, while its total death toll stands at 25% of all the country's fatalities. Joining the Brazilian academia efforts in the fight against Covid-19, in this paper we describe a unified framework for monitoring and forecasting the Covid-19 progress in the state of São Paulo. More specifically, a freely available, online platform to collect and exploit Covid-19 time-series data is presented, supporting decision-makers while still allowing the general public to interact with data from different regions of the state. Moreover, a novel forecasting data-driven method has also been proposed, by combining the so-called Susceptible-Infectious-Recovered-Deceased model with machine learning strategies to better fit the mathematical model's coefficients for predicting Infections, Recoveries, Deaths, and Viral Reproduction Numbers. We show that the obtained predictor is capable of dealing with badly conditioned data samples while still delivering accurate 10-day predictions. Our integrated computational system can be used for guiding government actions mainly in two basic aspects: real-time data assessment and dynamic predictions of Covid-19 curves for different regions of the state. We extend our analysis and investigation to inspect the virus spreading in Brazil in its regions. Finally, experiments involving the Covid-19 advance in other countries are also given.

Climatic dynamics and topography control genetic variation in Atlantic Forest montane birds.

Montane organisms responded to Quaternary climate change by tracking suitable habitat along elevational gradients. However, it is unclear whether these past climatic dynamics generated predictable patterns of genetic diversity in co-occurring montane taxa. To test if the genetic variation is associated with historical changes in the elevational distribution of montane habitats, we integrated paleoclimatic data and a model selection approach for testing the demographic history of five co-distributed bird species occurring in the southern Atlantic Forest sky islands. We found that changes in historical population sizes and current genetic diversity are attributable to habitat dynamics among time periods and the current elevational distribution of populations. Taxa with populations restricted to the more climatically dynamic southern mountain block (SMB) had, on average, a six-fold demographic expansion, whereas the populations from the northern mountain block (NMB) remained constant. In the current configuration of the southern Atlantic Forest montane habitats, populations in the SMB have more widespread elevational distributions, occur at lower elevations, and harbor higher levels of genetic diversity than NMB populations. Despite the apparent coupling of demographic and climatic oscillations, our data rejected simultaneous population structuring due to historical habitat fragmentation. Demographic modeling indicated that the species had different modes of differentiation, and varied in the timing of divergence and the degree of gene flow across mountain blocks. Our results suggest that the heterogeneous distribution of genetic variation in birds of the Atlantic Forest sky islands is associated with the interplay between topography and climate of distinct mountains, leading to predictable patterns of genetic diversity.

Targeted nanopore sequencing for the identification of ABCB1 promoter translocations in cancer.

BACKGROUND: Resistance to chemotherapy is the most common cause of treatment failure in acute myeloid leukemia (AML) and the drug efflux pump ABCB1 is a critical mediator. Recent studies have identified promoter translocations as common drivers of high ABCB1 expression in recurrent, chemotherapy-treated high-grade serous ovarian cancer (HGSC) and breast cancer. These fusions place ABCB1 under the control of a strong promoter while leaving its open reading frame intact. The mechanisms controlling high ABCB1 expression in AML are largely unknown. We therefore established an experimental system and analysis pipeline to determine whether promoter translocations account for high ABCB1 expression in cases of relapsed human AML. METHODS: The human AML cell line THP-1 was used to create a model of chemotherapy resistance in which ABCB1 expression was driven by a promoter fusion. The THP-1 model was used to establish a targeted nanopore long-read sequencing approach that was then applied to cases of ABCB1high HGSC and AML. H3K27Ac ChIP sequencing was used to assess the activity of native promoters in cases of ABCB1high AML. RESULTS: Prolonged in vitro daunorubicin exposure induced activating ABCB1 promoter translocations in human THP-1 AML cells, similar to those recently described in recurrent high-grade serous ovarian and breast cancers. Targeted nanopore sequencing proved an efficient method for identifying ABCB1 structural variants in THP-1 AML cells and HGSC; the promoter translocations identified in HGSC were both previously described and novel. In contrast, activating ABCB1 promoter translocations were not identified in ABCB1high AML; instead H3K27Ac ChIP sequencing demonstrated active native promoters in all cases studied. CONCLUSIONS: Despite frequent high level expression of ABCB1 in relapsed primary AML we found no evidence of ABCB1 translocations and instead confirmed high-level activity of native ABCB1 promoters, consistent with endogenous regulation.

Phenotypic and Genetic Structure Support Gene Flow Generating Gene Tree Discordances in an Amazonian Floodplain Endemic Species.

Before populations become independent evolutionary lineages, the effects of micro evolutionary processes tend to generate complex scenarios of diversification that may affect phylogenetic reconstruction. Not accounting for gene flow in species tree estimates can directly impact topology, effective population sizes and branch lengths, and the resulting estimation errors are still poorly understood in wild populations. In this study, we used an integrative approach, including sequence capture of ultra-conserved elements (UCEs), mtDNA Sanger sequencing and morphological data to investigate species limits and phylogenetic relationships in face of gene flow in an Amazonian endemic species (Myrmoborus lugubris: Aves). We used commonly implemented species tree and model-based approaches to understand the potential effects of gene flow in phylogenetic reconstructions. The genetic structure observed was congruent with the four recognized subspecies of M. lugubris. Morphological and UCEs data supported the presence of a wide hybrid zone between M. l. femininus from the Madeira river and M. l. lugubris from the Middle and lower Amazon river, which were recovered as sister taxa by species tree methods. When fitting gene flow into simulated demographic models with different topologies, the best-fit model indicated these two taxa as non-sister lineages, a finding that is in agreement with the results of mitochondrial and morphological analyses. Our results demonstrated that failing to account for gene flow when estimating phylogenies at shallow divergence levels can generate topological uncertainty, which can nevertheless be statistically well supported, and that model testing approaches using simulated data can be useful tools to test alternative phylogenetic hypotheses.

Transcriptomic rationale for synthetic lethality-targeting ERCC1 and CDKN1A in chronic myelomonocytic leukaemia.

Despite the absence of mutations in the DNA repair machinery in myeloid malignancies, the advent of high-throughput sequencing and discovery of splicing and epigenetics defects in chronic myelomonocytic leukaemia (CMML) prompted us to revisit a pathogenic role for genes involved in DNA damage response. We screened for misregulated DNA repair genes by enhanced RNA-sequencing on bone marrow from a discovery cohort of 27 CMML patients and 9 controls. We validated 4 differentially expressed candidates in CMML CD34+ bone marrow selected cells and in an independent cohort of 74 CMML patients, mutationally contextualized by targeted sequencing, and assessed their transcriptional behavior in 70 myelodysplastic syndrome, 66 acute myeloid leukaemia and 25 chronic myeloid leukaemia cases. We found BAP1 and PARP1 down-regulation to be specific to CMML compared with other related disorders. Chromatin-regulator mutated cases showed decreased BAP1 dosage. We validated a significant over-expression of the double strand break-fidelity genes CDKN1A and ERCC1, independent of promoter methylation and associated with chemorefractoriness. In addition, patients bearing mutations in the splicing component SRSF2 displayed numerous aberrant splicing events in DNA repair genes, with a quantitative predominance in the single strand break pathway. Our results highlight potential targets in this disease, which currently has few therapeutic options.

Recent chapters of Neotropical history overlooked in phylogeography: Shallow divergence explains phenotype and genotype uncoupling in Antilophia manakins.

Establishing links between phenotypic and genotypic variation is a central goal of evolutionary biology, as they might provide important insights into evolutionary processes shaping genetic and species diversity in nature. One of the more intriguing possibilities is when no genetic divergence is found to be associated with conspicuous phenotypic divergence. In that case, speciation theory predicts that phenotypic divergence may still occur in the presence of significant gene flow-thereby resulting in little genomic divergence-when genetic loci underpinning phenotypes are under strong divergent selection. However, a finding of phenotypic distinctiveness with weak or no population genetic structure may simply result from low statistical power to detect shallow genetic divergences when small data sets are used. Here, we used a subgenomic data set of 2,386 ultraconserved elements to explore genomewide divergence between two species of Antilophia manakins, which are phenotypically distinct yet evidently lack strong genetic differentiation according to previous studies based on a limited number of loci. Our results revealed clear population structure that matches the two phenotypes, supporting the idea that smaller data sets lacked the power to detect this recent divergence event (likely <100 k ya). Indeed, we found little or no introgression between the species, as well as evidence of genomewide divergence. One implication of our study is that the Araripe plateau may be a hot spot of cryptic-diverging forest Cerrado populations. Besides their use in biogeography, subgenomic data sets may help redefine local conservation programmes by revealing cryptic population structure that may be key to population management.

Striking pseudogenization in avian phylogenetics: Numts are large and common in falcons.

Nuclear copies of mitochondrial genes (numts) are a well-known feature of eukaryotic genomes and a concern in systematics, as they can mislead phylogenetic inferences when inadvertently used. Studies on avian numts initially based on the chicken genome suggest that numts may be uncommon and relatively short among birds. Here we ask how common numts are in falcons, based on recently sequenced genomes of the Saker falcon (Falco cherrug) and Peregrine falcon (F. peregrinus). We identified numts by BLASTN searches and then extracted CYTB, ND2 and COI sequences from them, which were then used for phylogeny inference along with several sequences from other species in Falconiformes. Our results indicate that avian numts may be much more frequent and longer than previously thought. Phylogenetic inferences revealed multiple independent nuclear insertions throughout the history of the Falconiformes, including cases of sequences available in public databases and wrongly identified as authentic mtDNA. New sequencing technologies and ongoing efforts for whole genome sequencing will provide exciting opportunities for avian numt research in the near future.

Signaling through retinoic acid receptors in cardiac development: Doing the right things at the right times.

Retinoic acid (RA) is a terpenoid that is synthesized from vitamin A/retinol (ROL) and binds to the nuclear receptors retinoic acid receptor (RAR)/retinoid X receptor (RXR) to control multiple developmental processes in vertebrates. The available clinical and experimental data provide uncontested evidence for the pleiotropic roles of RA signaling in development of multiple embryonic structures and organs such eyes, central nervous system, gonads, lungs and heart. The development of any of these above-mentioned embryonic organ systems can be effectively utilized to showcase the many strategies utilized by RA signaling. However, it is very likely that the strategies employed to transfer RA signals during cardiac development comprise the majority of the relevant and sophisticated ways through which retinoid signals can be conveyed in a complex biological system. Here, we provide the reader with arguments indicating that RA signaling is exquisitely regulated according to specific phases of cardiac development and that RA signaling itself is one of the major regulators of the timing of cardiac morphogenesis and differentiation. We will focus on the role of signaling by RA receptors (RARs) in early phases of heart development. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Vaginolysin drives epithelial ultrastructural responses to Gardnerella vaginalis.

Gardnerella vaginalis, the bacterial species most frequently isolated from women with bacterial vaginosis (BV), produces a cholesterol-dependent cytolysin (CDC), vaginolysin (VLY). At sublytic concentrations, CDCs may initiate complex signaling cascades crucial to target cell survival. Using live-cell imaging, we observed the rapid formation of large membrane blebs in human vaginal and cervical epithelial cells (VK2 and HeLa cells) exposed to recombinant VLY toxin and to cell-free supernatants from growing liquid cultures of G. vaginalis. Binding of VLY to its human-specific receptor (hCD59) is required for bleb formation, as antibody inhibition of either toxin or hCD59 abrogates this response, and transfection of nonhuman cells (CHO-K1) with hCD59 renders them susceptible to toxin-induced membrane blebbing. Disruption of the pore formation process (by exposure to pore-deficient toxoids or pretreatment of cells with methyl-ß-cyclodextrin) or osmotic protection of target cells inhibits VLY-induced membrane blebbing. These results indicate that the formation of functional pores drives the observed ultrastructural rearrangements. Rapid bleb formation may represent a conserved response of epithelial cells to sublytic quantities of pore-forming toxins, and VLY-induced epithelial cell membrane blebbing in the vaginal mucosa may play a role in the pathogenesis of BV.

Multilocus tests of Pleistocene refugia and ancient divergence in a pair of Atlantic Forest antbirds (Myrmeciza).

The Atlantic Forest (AF) harbours one of the most diverse vertebrate faunas of the world, including 199 endemic species of birds. Understanding the evolutionary processes behind such diversity has become the focus of many recent, primarily single locus, phylogeographic studies. These studies suggest that isolation in forest refugia may have been a major mechanism promoting diversification, although there is also support for a role of riverine and geotectonic barriers, two sets of hypotheses that can best be tested with multilocus data. Here we combined multilocus data (one mtDNA marker and eight anonymous nuclear loci) from two species of parapatric antbirds, Myrmeciza loricata and M. squamosa, and Approximate Bayesian Computation to determine whether isolation in refugia explains current patterns of genetic variation and their status as independent evolutionary units. Patterns of population structure, differences in intraspecific levels of divergence and coalescent estimates of historical demography fit the predictions of a recently proposed model of refuge isolation in which climatic stability in the northern AF sustains higher diversity and demographic stability than in the southern AF. However, a pre-Pleistocene divergence associated with their abutting range limits in a region of past tectonic activity also suggests a role for rivers or geotectonic barriers. Little or no gene flow between these species suggests the development of reproductive barriers or competitive exclusion. Our results suggests that limited marker sampling in recent AF studies may compromise estimates of divergence times and historical demography, and we discuss the effects of such sampling on this and other studies.

Differentiation block in acute myeloid leukemia regulated by intronic sequences of FTO.

Iroquois transcription factor gene IRX3 is highly expressed in 20-30% of acute myeloid leukemia (AML) and contributes to the pathognomonic differentiation block. Intron 8 FTO sequences ∼220kB downstream of IRX3 exhibit histone acetylation, DNA methylation, and contacts with the IRX3 promoter, which correlate with IRX3 expression. Deletion of these intronic elements confirms a role in positively regulating IRX3. RNAseq revealed long non-coding (lnc) transcripts arising from this locus. FTO-lncAML knockdown (KD) induced differentiation of AML cells, loss of clonogenic activity, and reduced FTO intron 8:IRX3 promoter contacts. While both FTO-lncAML KD and IRX3 KD induced differentiation, FTO-lncAML but not IRX3 KD led to HOXA downregulation suggesting transcript activity in trans. FTO-lncAMLhigh AML samples expressed higher levels of HOXA and lower levels of differentiation genes. Thus, a regulatory module in FTO intron 8 consisting of clustered enhancer elements and a long non-coding RNA is active in human AML, impeding myeloid differentiation.

Identification of curaxin as a potential new therapeutic for JAK2 V617F mutant patients.

Myelofibrosis is a myeloproliferative neoplasm (MPN) which typically results in reduced length and quality of life due to systemic symptoms and blood count changes arising from fibrotic changes in the bone marrow. While the JAK2 inhibitor ruxolitinib provides some clinical benefit, there remains a substantial unmet need for novel targeted therapies to better modify the disease process or eradicate the cells at the heart of myelofibrosis pathology. Repurposing drugs bypasses many of the hurdles present in drug development, such as toxicity and pharmacodynamic profiling. To this end we undertook a re-analysis of our pre-existing proteomic data sets to identify perturbed biochemical pathways and their associated drugs/inhibitors to potentially target the cells driving myelofibrosis. This approach identified CBL0137 as a candidate for targeting Jak2 mutation-driven malignancies. CBL0137 is a drug derived from curaxin targeting the Facilitates Chromatin Transcription (FACT) complex. It is reported to trap the FACT complex on chromatin thereby activating p53 and inhibiting NF-kB activity. We therefore assessed the activity of CBL0137 in primary patient samples and murine models of Jak2-mutated MPN and found it preferentially targets CD34+ stem and progenitor cells from myelofibrosis patients by comparison with healthy control cells. Further we investigate its mechanism of action in primary haemopoietic progenitor cells and demonstrate its ability to reduce splenomegaly and reticulocyte number in a transgenic murine model of myeloproliferative neoplasms.

Therapeutic targeting of EP300/CBP by bromodomain inhibition in hematologic malignancies.

CCS1477 (inobrodib) is a potent, selective EP300/CBP bromodomain inhibitor which induces cell-cycle arrest and differentiation in hematologic malignancy model systems. In myeloid leukemia cells, it promotes rapid eviction of EP300/CBP from an enhancer subset marked by strong MYB occupancy and high H3K27 acetylation, with downregulation of the subordinate oncogenic network and redistribution to sites close to differentiation genes. In myeloma cells, CCS1477 induces eviction of EP300/CBP from FGFR3, the target of the common (4; 14) translocation, with redistribution away from IRF4-occupied sites to TCF3/E2A-occupied sites. In a subset of patients with relapsed or refractory disease, CCS1477 monotherapy induces differentiation responses in AML and objective responses in heavily pre-treated multiple myeloma. In vivo preclinical combination studies reveal synergistic responses to treatment with standard-of-care agents. Thus, CCS1477 exhibits encouraging preclinical and early-phase clinical activity by disrupting recruitment of EP300/CBP to enhancer networks occupied by critical transcription factors.