The NCOR-HDAC3 co-repressive complex modulates the leukemogenic potential of the transcription factor ERG.

The ERG (ETS-related gene) transcription factor is linked to various types of cancer, including leukemia. However, the specific ERG domains and co-factors contributing to leukemogenesis are poorly understood. Drug targeting a transcription factor such as ERG is challenging. Our study reveals the critical role of a conserved amino acid, proline, at position 199, located at the 3' end of the PNT (pointed) domain, in ERG's ability to induce leukemia. P199 is necessary for ERG to promote self-renewal, prevent myeloid differentiation in hematopoietic progenitor cells, and initiate leukemia in mouse models. Here we show that P199 facilitates ERG's interaction with the NCoR-HDAC3 co-repressor complex. Inhibiting HDAC3 reduces the growth of ERG-dependent leukemic and prostate cancer cells, indicating that the interaction between ERG and the NCoR-HDAC3 co-repressor complex is crucial for its oncogenic activity. Thus, targeting this interaction may offer a potential therapeutic intervention.

The accuracy of a diagnosis of pneumonia in the emergency department.

BACKGROUND: Achieving a high degree of diagnostic accuracy of infections in the emergency department (ED) is crucial since a delay in diagnosis can lead to increased mortality, whereas overdiagnosis can lead to antibiotic overprescription. Limited data are available as to ED diagnostic accuracy of infections. The aim of this study was to demonstrate the degree of discordance of an ED diagnosis of pneumonia in relation to an internal medicine ward's discharge diagnosis of hospitalized adults. METHODS: We reviewed the records of all adults hospitalized in internal wards from November 2017-January 2018 diagnosed with an acute infection by an ED physician. The primary outcome was the discordance degree of an ED pneumonia diagnosis compared to the internal ward discharge diagnosis. The influence comorbidities and clinical characteristics on the diagnostic discordance were also evaluated. RESULTS: The study included 875 adults; 434 were admitted with an ED diagnosis of a specific infection. Pneumonia was the most frequent ED diagnosis (n = 195, 45%), of them, 56 (29%) were discordant diagnosis in the internal ward. CONCLUSION: Interpretation of chest X-rays with the assistance of a radiologist might help in reducing overdiagnosis and minimize antibiotic overprescription, thus improving the ED diagnostic accuracy of pneumonia.

NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis.

Cancer-Associated Fibroblasts (CAFs) were shown to orchestrate tumour-promoting inflammation in multiple malignancies, including breast cancer. However, the molecular pathways that govern the inflammatory role of CAFs are poorly characterised. In this study we found that fibroblasts sense damage-associated molecular patterns (DAMPs), and in response activate the NLRP3 inflammasome pathway, resulting in instigation of pro-inflammatory signalling and secretion of IL-1ß. This upregulation was evident in CAFs in mouse and in human breast carcinomas. Moreover, CAF-derived inflammasome signalling facilitated tumour growth and metastasis, which was attenuated when NLRP3 or IL-1ß were specifically ablated. Functionally, CAF-derived inflammasome promoted tumour progression and metastasis by modulating the tumour microenvironment towards an immune suppressive milieu and by upregulating the expression of adhesion molecules on endothelial cells. Our findings elucidate a mechanism by which CAFs promote breast cancer progression and metastasis, by linking the physiological tissue damage response of fibroblasts with tumour-promoting inflammation.

Inflammatory Activation of Astrocytes Facilitates Melanoma Brain Tropism via the CXCL10-CXCR3 Signaling Axis.

Melanoma is the deadliest skin cancer due to its high rate of metastasis, frequently to the brain. Brain metastases are incurable; therefore, understanding melanoma brain metastasis is of great clinical importance. We used a mouse model of spontaneous melanoma brain metastasis to study the interactions of melanomas with the brain microenvironment. We find that CXCL10 is upregulated in metastasis-associated astrocytes in mice and humans and is functionally important for the chemoattraction of melanoma cells. Moreover, CXCR3, the receptor for CXCL10, is upregulated in brain-tropic melanoma cells. Targeting melanoma expression of CXCR3 by nanoparticle-mediated siRNA delivery or by shRNA transduction inhibits melanoma cell migration and attenuates brain metastasis in vivo. These findings suggest that the instigation of pro-inflammatory signaling in astrocytes is hijacked by brain-metastasizing tumor cells to promote their metastatic capacity and that the CXCL10-CXCR3 axis may be a potential therapeutic target for the prevention of melanoma brain metastasis.

An ERG Enhancer-Based Reporter Identifies Leukemia Cells with Elevated Leukemogenic Potential Driven by ERG-USP9X Feed-Forward Regulation.

Acute leukemia is a rapidly progressing blood cancer with low survival rates. Unfavorable prognosis is attributed to insufficiently characterized subpopulations of leukemia stem cells (LSC) that drive chemoresistance and leukemia relapse. Here we utilized a genetic reporter that assesses stemness to enrich and functionally characterize LSCs. We observed heterogeneous activity of the ERG+85 enhancer-based fluorescent reporter in human leukemias. Cells with high reporter activity (tagBFPHigh) exhibited elevated expression of stemness and chemoresistance genes and demonstrated increased clonogenicity and resistance to chemo- and radiotherapy as compared with their tagBFPNeg counterparts. The tagBFPHigh fraction was capable of regenerating the original cellular heterogeneity and demonstrated increased invasive ability. Moreover, the tagBFPHigh fraction was enriched for leukemia-initiating cells in a xenograft assay. We identified the ubiquitin hydrolase USP9X as a novel ERG transcriptional target that sustains ERG+85-positive cells by controlling ERG ubiquitination. Therapeutic targeting of USP9X led to preferential inhibition of the ERG-dependent leukemias. Collectively, these results characterize human leukemia cell functional heterogeneity and suggest that targeting ERG via USP9X inhibition may be a potential treatment strategy in patients with leukemia. SIGNIFICANCE: This study couples a novel experimental tool with state-of-the-art approaches to delineate molecular mechanisms underlying stem cell-related characteristics in leukemia cells.

A novel method for detecting the cellular stemness state in normal and leukemic human hematopoietic cells can predict disease outcome and drug sensitivity.

Acute leukemia is an aggressive blood malignancy with low survival rates. A high expression of stem-like programs in leukemias predicts poor prognosis and is assumed to act in an aberrant fashion in the phenotypically heterogeneous leukemia stem cell (LSC) population. A lack of suitable genome engineering tools that can isolate LSCs based on their stemness precludes their comprehensive examination and full characterization. We hypothesized that tagging endogenous stemness-regulatory regions could generate a genome reporter for the putative leukemia stemness-state. Our analysis revealed that the ERG + 85 enhancer region can serve as a marker for stemness-state and a fluorescent lentiviral reporter was developed that can accurately recapitulate the endogenous activity. Using our novel reporter, we revealed cellular heterogeneity in several leukemia cell lines and patient-derived samples. Alterations in reporter activity were associated with transcriptomic and functional changes that were closely related to the hematopoietic stem cell (HSC) identity. Notably, the differentiation potential was skewed towards the erythro-megakaryocytic lineage. Moreover, an ERG + 85High fraction of AML cells could regenerate the original cellular heterogeneity and was enriched for LSCs. RNA-seq analysis coupled with in silico drug-screen analysis identified 4HPR as an effective inhibitor of ERG + 85High leukemia growth. We propose that further utilization of our novel molecular tool will identify crucial determinants of LSCs, thus providing a rationale for their therapeutic targeting.

Attenuated DNA damage responses and increased apoptosis characterize human hematopoietic stem cells exposed to irradiation.

Failure to precisely repair DNA damage in self-renewing Hematopoietic Stem and early Progenitor Cells (HSPCs) can disrupt normal hematopoiesis and promote leukemogenesis. Although HSPCs are widely considered a target of ionizing radiation (IR)-induced hematopoietic injury, definitive data regarding cell death, DNA repair, and genomic stability in these rare quiescent cells are scarce. We found that irradiated HSPCs, but not lineage-committed progenitors (CPs), undergo rapid ATM-dependent apoptosis, which is suppressed upon interaction with bone-marrow stroma cells. Using DNA repair reporters to quantify mutagenic Non-Homologous End Joining (NHEJ) processes, we found that HSPCs exhibit reduced NHEJ activities in comparison with CPs. HSPC-stroma interactions did not affect the NHEJ capacity of HSPCs, emphasizing its cell autonomous regulation. We noted diminished expression of multiple double strand break (DSB) repair transcripts along with more persistent 53BP1 foci in irradiated HSPCs in comparison with CPs, which can account for low NHEJ activity and its distinct control in HSPCs. Finally, we documented clonal chromosomal aberrations in 10% of IR-surviving HSPCs. Taken together, our results revealed potential mechanisms contributing to the inherent susceptibility of human HSPC to the cytotoxic and mutagenic effects of DNA damage.

Impact of late pregnancy haemoglobin A1c at 29-30 weeks' gestation on adverse pregnancy outcomes among women with pre-existing diabetes: a retrospective analysis.

This study was to assess the relationship between late pregnancy haemoglobin A1C (HbA1C) at 29-30 weeks of gestation and adverse pregnancy outcomes (APOs) in 272 pregnant women with pre-existing diabetes. HbA1C ≥6.1% was associated with significantly increased risk of preterm delivery, Caesarean section, large for gestational age (LGA), neonatal respiratory distress, neonatal hypoglycaemia, and composite adverse neonatal outcome (p < .05). The risk of pre-eclampsia increased significantly at the lower HbA1C cut-off of > 5.6% (p = .039). Reduction of HbA1C cut-off from 6.1% to 5.6% improved the sensitivity but reduced the specificity for prediction of APOs. Overall, the receiver operating characteristic (ROC) curves demonstrated the moderate predictive value of late pregnancy HbA1C for APOs. In conclusion, elevated late pregnancy HbA1C levels at 29-30 gestational weeks had a negative impact on APOs in pregnant women with pre-existing diabetes. However, HbA1C cut-off levels of neither ≥6.1% nor >5.6% were ideal for predicting APOs. Impact statement What is already known on this subject: Poorly controlled diabetes is associated with adverse pregnancy outcomes (APOs). Periconceptual haemoglobin A1C (HbA1C) correlates well with the risk of foetal anomaly but is not predictive of APOs at time of delivery. New evidence suggested that late pregnancy HbA1C is predictive of APOs but the definitions of a late pregnancy gestational week and target HbA1C cutpoint remain in doubt. What the results of this study add: This study investigated the relationship between late pregnancy HbA1C levels at 29-30 weeks of gestation and the APOs among pregnant women with pre-existing diabetes. Late pregnancy HbA1C ≥ 6.1% correlated with the risk of APOs but the increased risk of pre-eclampsia only became significant at the lower cut-off of >5.6%. Reducing HbA1C cut-off from 6.1% to 5.6% improved the sensitivity but reduced the specificity for prediction of APOs. Overall, late pregnancy HbA1C had a moderate predictive value for APOs. What the implications are of these findings for clinical practice and/or further research: HbA1C cut-off levels of neither ≥6.1% nor >5.6% were ideal in predicting APOs among pregnant women with pre-existing diabetes. As HbA1C levels tend to drop in pregnancy, caution should be taken when interpreting HbA1C in pregnancy. More multi-centred studies are required to explore the respective glycaemic target for each APO and to determine the ideal timing for late pregnancy HbA1C measurement.

DNA-damage response in hematopoietic stem cells: an evolutionary trade-off between blood regeneration and leukemia suppression.

Self-renewing and multipotent hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis. Their enormous regenerative potential coupled with lifetime persistence in the body, in contrast with the Progenitors, demand tight control of HSCs genome stability. Indeed, failure to accurately repair DNA damage in HSCs is associated with bone marrow failure and accelerated leukemogenesis. Recent observations exposed remarkable differences in several DNA-damage response (DDR) aspects between HSCs and Progenitors, especially in their DNA-repair capacities and susceptibility to apoptosis. Human HSCs in comparison with Progenitors exhibit delayed DNA double-strand break rejoining, persistent DDR signaling activation, higher sensitivity to the cytotoxic effects of ionizing radiation and attenuated expression of DNA-repair genes. Importantly, the distinct DDR of HSCs was also documented in mouse models. Nevertheless, physiological significance and the molecular basis of the HSCs-specific DDR features are only partially understood. Taking radiation-induced DDR as a paradigm, this review will focus on the current advances in understanding the role of cell-intrinsic DDR regulators and the cellular microenvironment in balancing stemness with genome stability. Pre-leukemia HSCs and clonal hematopoiesis evolvement will be discussed as an evolutionary compromise between the need for lifelong blood regeneration and DDR. Uniquely for this review, we outline the differences in HSCs-related DDR as highlighted by various experimental systems and attempt to provide their critical analysis.

SMYD2 lysine methyltransferase regulates leukemia cell growth and regeneration after genotoxic stress.

The molecular determinants governing escape of Acute Myeloid Leukemia (AML) cells from DNA damaging therapy remain poorly defined and account for therapy failures. To isolate genes responsible for leukemia cells regeneration following multiple challenges with irradiation we performed a genome-wide shRNA screen. Some of the isolated hits are known players in the DNA damage response (e.g. p53, CHK2), whereas other, e.g. SMYD2 lysine methyltransferase (KMT), remains uncharacterized in the AML context. Here we report that SMYD2 knockdown confers relative resistance to human AML cells against multiple classes of DNA damaging agents. Induction of the transient quiescence state upon SMYD2 downregulation correlated with the resistance. We revealed that diminished SMYD2 expression resulted in the upregulation of the related methyltransferase SET7/9, suggesting compensatory relationships. Indeed, pharmacological targeting of SET7/9 with (R)-PFI2 inhibitor preferentially inhibited the growth of cells expressing low levels of SMYD2.Finally, decreased expression of SMYD2 in AML patients correlated with the reduced sensitivity to therapy and lower probability to achieve complete remission. We propose that the interplay between SMYD2 and SET7/9 levels shifts leukemia cells from growth to quiescence state that is associated with the higher resistance to DNA damaging agents and rationalize SET7/9 pharmacological targeting in AML.

Quercetin alters the DNA damage response in human hematopoietic stem and progenitor cells via TopoII- and PI3K-dependent mechanisms synergizing in leukemogenic rearrangements.

Quercetin (Que) is an abundant flavonoid in the human diet and high-concentration food supplement with reported pro- and anti-carcinogenic activities. Topoisomerase II (TopoII) inhibition and subsequent DNA damage induction by Que was implicated in the mixed lineage leukemia gene (MLL) rearrangements that can induce infant and adult leukemias. This notion raised concerns regarding possible genotoxicities of Que in hematopoietic stem and progenitor cells (HSPCs). However, molecular targets mediating Que effects on DNA repair relevant to MLL translocations have not been defined. In this study we describe novel and potentially genotoxic Que activities in suppressing non-homologous end joining and homologous recombination pathways downstream of MLL cleavage. Using pharmacological dissection of DNA-PK, ATM and PI3K signalling we defined PI3K inhibition by Que with a concomitant decrease in the abundance of key DNA repair genes to be responsible for DNA repair inhibition. Evidence for the downstream TopoII-independent mutagenic potential of Que was obtained by documenting further increased frequencies of MLL rearrangements in human HSPCs concomitantly treated with Etoposide and Que versus single treatments. Importantly, by engaging a tissue engineered placental barrier, we have established the extent of Que transplacental transfer and hence provided the evidence for Que reaching fetal HSPCs. Thus, Que exhibits genotoxic effects in human HSPCs via different mechanisms when applied continuously and at high concentrations. In light of the demonstrated Que transfer to the fetal compartment our findings are key to understanding the mechanisms underlying infant leukemia and provide molecular markers for the development of safety values.

Factors associated with placenta praevia in primigravidas and its pregnancy outcome.

AIM: To examine the factors associated with placenta praevia in primigravidas and also compare the pregnancy outcomes between primigravidas and nonprimigravidas. METHOD: A retrospective cohort study was conducted in women who underwent caesarean section for major placenta praevia in a tertiary university hospital from January 2007 till December 2013. Medical records were reviewed. RESULT: Among 243 with major placenta praevia, 56 (23.0%) were primigravidas and 187 (77.0%) were nonprimigravidas. Factors associated with placenta praevia in the primigravidas were history of assisted conception (P = 0.02) and history of endometriosis (P = 0.01). For maternal outcomes, the nonprimigravidas required earlier delivery than primigravidas (35.76 ± 2.54 weeks versus 36.52 ± 1.95 weeks, P = 0.03) and had greater blood loss (P = 0.04). A vast majority of the primigravidas had either posterior type II or type III placenta praevia. As for neonatal outcomes, the Apgar score at 1 minute was significantly lower for the nonprimigravidas (7.89 ± 1.72 versus 8.39 ± 1.288.39 ± 1.28, P = 0.02). CONCLUSION: This study highlighted that endometriosis and assisted conception were highly associated with placenta praevia in primigravida. Understanding the pregnancy outcomes of women with placenta praevia can assist clinicians in identifying patients who are at higher risk of mortality and morbidity. Identifying potential risk factors in primigravida may assist in counseling and management of such patients.

Comparison between AmniSure placental alpha microglobulin-1 rapid immunoassay and standard diagnostic methods for detection of rupture of membranes.

Objective. To determine the diagnostic accuracy of placental alpha microglobulin-1 assay and standard diagnostic methods for detecting rupture of membrane. Study Design. Prospective diagnostic study, between June 2011 to November 2011 at a tertiary centre. Initial evaluation included both the standard diagnostic methods for rupture of membranes and placental alpha microglobulin-1 immunoassay. The actual rupture of membranes was diagnosed on review of the medical records after delivery (absence of membrane or a positive pad chart). Main Outcome Measures. Placental alpha microglobulin-1 immunoassay and standard diagnostic methods for diagnosis of rupture of membrane. Results. A total of 211 patients were recruited. At initial presentation, 187 patients (88.6%) had ruptured membranes, while 24 patients (11.4%) had intact membranes. Placental alpha microglobulin-1 immunoassay confirmed rupture of membranes at initial presentation with a sensitivity of 95.7% (179 of 187), specificity of 100% (24 of 24), positive predictive value of 100% (179 of 179), and negative predictive value of 75.0% (24 of 32). By comparison, the conventional standard diagnostic methods had a sensitivity of 78.1% (146 of 187), specificity of 100% (24 of 24), positive predictive value of 100% (146 of 146), and negative predictive value of 36.9% (24 of 65) in diagnosing rupture of membrane. Conclusion. Placental alpha-microglobulin-1 immunoassay is a rapid and accurate method for confirming the diagnosis of rupture of membrane. It was superior to conventional standard diagnostic methods (pooling, nitrazine, and ferning), the nitrazine test alone or fern test alone.

Umbilical vein oxytocin in the management of retained placenta: an alternative to manual removal of placenta?

PURPOSE: Retained placenta is potentially life threatening due to possible complications associated with manual removal. Our aim was to determine whether umbilical vein injection of oxytocin in saline reduces the need for manual removal of placenta. METHODS: This was a randomised controlled trial conducted at a tertiary hospital from December 2002 to March 2004. A total of 61 women delivering singletons, who had no sign of placental separation 20 min after vaginal delivery, were randomised to receive either intra-umbilical oxytocin 100 IU diluted in 30 ml of saline or controlled cord traction only. Manual removal was done if the placenta was not expelled in another 30 min in both arms. RESULTS: There was a significant reduction in the rate of subsequent manual removal of placenta (30 vs. 67.7%, p < 0.05), incidence of uterine atony (3.3 vs. 25.8%, p < 0.05) and the need for uterotonic agents (33.3 vs. 64.5%, p < 0.05) in the oxytocin group when compared with the control group. No significant differences were found in the need for blood transfusion, uterine curettage, incidence of postpartum haemorrhage and haemoglobin level reduction. CONCLUSION: Intra-umbilical vein oxytocin injection is clinically effective for the management of a retained placenta.