Ceramide-induced integrated stress response overcomes Bcl-2 inhibitor resistance in acute myeloid leukemia.

Inducing cell death by the sphingolipid ceramide is a potential anti-cancer strategy, but the underlying mechanisms remain poorly defined. Here, we show that triggering accumulation of ceramide in acute myeloid leukaemia (AML) cells by inhibition of sphingosine kinase induces an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This leads to transcription of the BH3-only protein, Noxa, and degradation of the pro-survival Mcl-1 protein on which AML cells are highly dependent on for survival. Targeting this novel ISR pathway in combination with the Bcl-2 inhibitor venetoclax synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anti-cancer effects of ceramide and pre-clinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.Alexander C. Lewis, Victoria S. Pope, Melinda N. Tea, Manjun Li, Gus O. Nwosu, Thao M. Nguyen, Craig T. Wallington-Beddoe, Paul A. B. Moretti, Dovile Anderson, Darren J. Creek, Maurizio Costabile, Saira R. Ali, Chloe A. L. Thompson-Peach, B. Kate Dredge, Andrew G. Bert, Gregory J. Goodall, Paul G. Ekert, Anna L. Brown, Richard D'Andrea, Nirmal Robinson, Melissa R. Pitman, Daniel Thomas, David M. Ross, Briony L. Gliddon, Jason A. Powell, and Stuart M. Pitso

Role of the miR-200 family in mediating EMT in response to TGF-beta

Presentation SY-10 at Symposium 3: microRNA regulation of cytokine gene expressionPhilip A. Gregory, Cameron P. Bracken, Andrew G. Bert, Emily L. Paterson, Natasha Kolesnikoff, Gelareh Farshid, Yeesim Khew-Goodall, Gregory J. Goodal

Limit analysis of spherical pressure vessels with protruding nozzles and associated defects

SIGLELD:1769.7F(CEGB-RD/B--5038N81)(microfiche). / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Insufficiently complex unique-molecular identifiers (UMIs) distort small RNA sequencing

The attachment of unique molecular identifiers (UMIs) to RNA molecules prior to PCR amplification and sequencing, makes it possible to amplify libraries to a level that is sufficient to identify rare molecules, whilst simultaneously eliminating PCR bias through the identification of duplicated reads. Accurate de-duplication is dependent upon a sufficiently complex pool of UMIs to allow unique labelling. In applications dealing with complex libraries, such as total RNA-seq, only a limited variety of UMIs are required as the variation in molecules to be sequenced is enormous. However, when sequencing a less complex library, such as small RNAs for which there is a more limited range of possible sequences, we find increased variation in UMIs are required, even beyond that provided in a commercial kit specifically designed for the preparation of small RNA libraries for sequencing. We show that a pool of UMIs randomly varying across eight nucleotides is not of sufficient depth to uniquely tag the microRNAs to be sequenced. This results in over de-duplication of reads and the marked under-estimation of expression of the more abundant microRNAs. Whilst still arguing for the utility of UMIs, this work demonstrates the importance of their considered design to avoid errors in the estimation of gene expression in libraries derived from select regions of the transcriptome or small genomes.Klay Saunders, Andrew G. Bert, B. Kate Dredge, John Toubia, Philip A. Gregory, Katherine A. Pillman, Gregory J. Goodall, Cameron P. Bracke

Extensive transcriptional responses are co-ordinated by microRNAs as revealed by Exon-Intron Split Analysis (EISA)

Epithelial-mesenchymal transition (EMT) has been a subject of intense scrutiny as it facilitates metastasis and alters drug sensitivity. Although EMT-regulatory roles for numerous miRNAs and transcription factors are known, their functions can be difficult to disentangle, in part due to the difficulty in identifying direct miRNA targets from complex datasets and in deciding how to incorporate 'indirect' miRNA effects that may, or may not, represent biologically relevant information. To better understand how miRNAs exert effects throughout the transcriptome during EMT, we employed Exon-Intron Split Analysis (EISA), a bioinformatic technique that separates transcriptional and post-transcriptional effects through the separate analysis of RNA-Seq reads mapping to exons and introns. We find that in response to the manipulation of miRNAs, a major effect on gene expression is transcriptional. We also find extensive co-ordination of transcriptional and post-transcriptional regulatory mechanisms during both EMT and mesenchymal to epithelial transition (MET) in response to TGF-β or miR-200c respectively. The prominent transcriptional influence of miRNAs was also observed in other datasets where miRNA levels were perturbed. This work cautions against a narrow approach that is limited to the analysis of direct targets, and demonstrates the utility of EISA to examine complex regulatory networks involving both transcriptional and post-transcriptional mechanisms.Katherine A. Pillman, Kaitlin G. Scheer, Emily Hackett-Jones, Klay Saunders, Andrew G Bert, John Toubia, Holly J Whitfield, Sunil Sapkota, Laura Sourdin, Hoang Pham, Thuc D. Le, Joseph Cursons, Melissa J. Davis, Philip A. Gregory, Gregory J. Goodall and Cameron P. Bracke