Targeting enhancer switching overcomes non-genetic drug resistance in acute myeloid leukaemia.

Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Functional interdependence of BRD4 and DOT1L in MLL leukemia.

Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis

Evading the storm: BET inhibitor resistance and the leukaemia stem cell

© 2017 Dr. Chun Yew FongBromodomain and Extra Terminal protein (BET) inhibitors are first-in-class, epigenetic targeted therapies that deliver a new therapeutic paradigm by directly targeting protein-protein interactions at chromatin. Early clinical trials have shown significant promise, particularly in acute myeloid leukaemia (AML), suggesting that these compounds are likely to form an important component of future anti-cancer regimes. However, therapeutic resistance is an inevitable consequence of most cancer therapies, therefore the evaluation of resistance mechanisms is of utmost importance in order to optimise the clinical utility of this novel class of drugs. This work utilises primary murine hematopoietic stem and progenitor cells (HSPC) immortalised with MLL-AF9 to generate several clonal cell lines demonstrating robust resistance, in vitro and in vivo to the prototypical BET inhibitor, I-BET151. Resistant clones harbour cross-resistance to the chemically distinct BET inhibitor JQ1, as well as resistance to genetic knockdown of BET proteins. Moreover, resistance is stably maintained across subsequent cell generations in the absence of ongoing selective pressure. Immunophenotypic immaturity is identified in resistant clones and, through functional limiting dilution assays, resistance is definitively demonstrated to emerge from leukaemia stem cells (LSCs). This finding is further confirmed using an independently generated in vivo model of resistance and in patient derived xenograft (PDX) models of human leukaemia. The underlying mechanism of resistance is identified through examination of the transcriptome and chromatin interface utilising high throughput sequencing techniques. Consistent with the adoption of alternative transcriptional pathways, expression of key target oncogenes such as Myc remain unaltered in resistant clones despite the global loss of chromatin-bound Brd4. Alternatively, increased Wnt/β-catenin signalling in human and mouse leukaemia cells is demonstrated to account, in part, for resistance to BET inhibitors and functions to maintain expression of malignant oncogenes. Negative regulation of this pathway restores sensitivity to I-BET151 in vitro and in vivo and highlights a potential rational combination therapy strategy to circumvent or prevent the development of BET inhibitor resistance. The emergence of BET inhibitor resistance from a LSC population prompted further exploration of rational combination therapies with sound mechanistic basis. LSD1 inhibitors have demonstrated pre-clinical promise in the treatment of AML underpinned by the induction of differentiation of leukaemia cells and the loss of LSC capacity. Using the derived model BET inhibitor resistance, LSD1 inhibitors are demonstrated to function synergistically with BET inhibitors and restore sensitivity to BET inhibitor resistant clones. Induction of differentiation is demonstrated in immunophenotypic and transcriptome assays and suggest a further potential combination therapy approach to enhance the clinical utility of both drugs. Collectively, these findings give insight into the basic biology of AML, demonstrate the role of epigenetically mediated intratumoural heterogeneity and transcriptional plasticity in the evasion of targeted therapies and provide a base from which further investigation of the LSC can occur to identify vulnerabilities which may be exploited for therapeutic gain

Reuse potential of spent RO membrane for NF and UF process

With the increasing demand on reverse osmosis (RO) membranes for water purification worldwide, the number of disposed membrane elements is expected to increase accordingly. Thus, recycling and reuse of end-of-life RO membranes should be a global environmental action. In this work, we aim to reuse the spent RO membrane for nanofiltration (NF) and ultrafiltration (UF) process by subjecting the spent membrane to solvent and oxidizing solution treatment, respectively. Our results showed that solvent-treated RO membrane could perform as good as commercial NF membrane by achieving similar separation efficiencies, but with reduced water permeability due to membrane surface fouling. By degrading the polyamide layer of RO membrane, the transformed membrane could achieve high water permeability (85.6 L/m2.h.bar) and excellent rejection against macromolecules (at least 87.4%), suggesting its reuse potential as UF membrane. More importantly, our findings showed that in-situ transformation on the spent RO membrane using solvent and oxidizing solution could be safely conducted as the properties of the entire spiral wound element did not show significant changes upon prolonged exposure of these two solutions. Our findings are important to open up new possibilities for the discarded RO membranes for reuse in NF and UF process, prolonging the lifespan of spent membranes and promoting the sustainability of the membrane process

BET inhibitor resistance emerges from leukaemia stem cells

Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks. Early clinical trials have shown promise, especially in acute myeloid leukaemia, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL-AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/β-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies