Biology subjects

Hafner, M.

Publications and source records attributed to Hafner, M..

7 recordsLinked to original sources

bioRxiv2018

The splicing factor U2AF1 contributes to cancer progression through a non-canonical role in translation regulation

Somatic mutations in the genes encoding components of the spliceosome occur frequently in human neoplasms, including myeloid dysplasias and leukemias and less often in solid tumors. One of the affected factors, U2AF1, is involved in splice site selection, and the most common change, S34F, alters a conserved nucleic acid binding domain, recognition of the 3-splice site, and alternative splicing of many mRNAs. However, the role that this mutation plays in oncogenesis is still unknown. Here, we have uncovered a non-canonical function of U2AF1, showing that it binds mature mRNA in the cytoplasm and negatively regulates mRNA translation. This splicing-independent role of U2AF1 is altered by the S34F mutation, and polysomal profiling indicates the mutation affects translation of hundreds of mRNA. One functional consequence is increased synthesis of the secreted chemokine interleukin 8 which contributes to metastasis, inflammation, and cancer progression in mice and humans.

cell biology↗

bioRxiv2018

Cheminformatics tools for analyzing and designing optimized small molecule libraries

Libraries of highly annotated small molecules have many uses in chemical genetics, drug discovery and drug repurposing. Many such libraries have become available, but few data-driven approaches exist to compare these libraries and design new ones. In this paper, we describe such an approach that makes use of data on binding selectivity, target coverage and induced cellular phenotypes as well as chemical structure and stage of clinical development. We implement the approach as R software and a Web-accessible tool (http://www.smallmoleculesuite.org) that uses incomplete and often confounded public data in combination with user preferences to score and create libraries. Analysis of six kinase inhibitor libraries using our approach reveals dramatic differences among them, leading us to design a new LSP-OptimalKinase library that outperforms all previous collections in terms of target coverage and compact size. We also assemble a mechanism of action library that optimally covers 1852 targets of the liganded genome. Using our tools, individual research groups and companies can quickly analyze private compound collections and public libraries can be progressively improved using the latest data.

systems biology↗

bioRxiv2018

CD95L mRNA is toxic to cells

CD95/Fas ligand binds to the death receptor CD95 to induce apoptosis in sensitive cells. We previously reported the CD95L mRNA is enriched in sequences that, when converted to si/shRNAs, kill all cancer cells by targeting critical survival genes (Putzbach et al., 2017). We now report expression of full-length CD95L mRNA, itself, is highly toxic to cells and induces a similar form of cell death. We demonstrate that small (s)RNAs derived from CD95L are loaded into the RNA induced silencing complex (RISC) RISC which is required for the toxicity and that processing of CD95L mRNA into sRNAs is independent of both Dicer and Drosha. We provide evidence that in addition to the CD95L transgene a number of endogenous protein coding genes involved in regulating protein translation, particularly under low miRNA conditions, can be processed to sRNAs and loaded into the RISC suggesting a new level of cell fate regulation involving RNAi.

cancer biology↗

bioRxiv2017

Therapeutically advantageous secondary targets of abemaciclib identified by multi-omics profiling of CDK4/6 inhibitors

FDA approval of multiple drugs differing in chemical structures but targeting the same protein raises the question whether such drugs have sufficiently similar mechanisms of action to be considered functionally equivalent. In this paper we compare three recently approved inhibitors of the cyclin-dependent kinases CDK4/6 - palbociclib, ribociclib, and abemaciclib - that are becoming important therapies for the treatment of hormone-receptor positive breast and potentially other cancers. We find that transcriptional and proteomic changes induced by the three drugs differ significantly and that abemaciclib has unique cellular activities including induction of cell death (even in pRb-deficient cells), arrest in the G2 phase of the cell cycle, and reduced drug adaptation. These activities appear to arise from inhibition of kinases other than CDK4/6 including CDK2/Cyclin A/E and CDK1/Cyclin B.\n\nSIGNIFICANCEThe target profiles of most drugs are established relatively early in their development and are not systematically revisited at the time of approval. Scattered reports suggest that palbociclib, ribociclib, and abemaciclib differ in pharmacokinetics, dosing, and adverse effects but the three drugs are generally regarded as similar. Our finding that the drugs differ substantially in mechanism of action - abemaciclib retains activities of the earlier-generation drug alvocidib - suggests the potential for different uses in the clinic: in particular, abemaciclib may show activity in patients progressing on palbociclib or ribociclib. More generally, our approach relying on data from five distinct phenotypic and biochemical assays strongly suggests that a multi-faceted approach is necessary to get a reliable picture the target spectrum of kinase inhibitors.

cancer biology↗

bioRxiv2017

DHX36 binding at G-rich sites in mRNA untranslated regions promotes translation

Translation efficiency can be affected by mRNA stability and secondary structures, including so-called G-quadruplex (G4) structures. The highly conserved and essential DEAH-box helicase DHX36/RHAU is able to resolve G4 structures on DNA and RNA in vitro, however a system-wide analysis of DHX36 targets and function is lacking. We globally mapped DHX36 occupancy in human cell lines and found that it preferentially binds to G-rich sequences in the coding sequences (CDS) and 5' and 3' untranslated regions (UTR) of more than 4,500 mRNAs. Functional analyses, including RNA sequencing, ribosome footprinting, and quantitative mass spectrometry revealed that DHX36 decreased target mRNA stability. However, target mRNA accumulation in DHX36 KO cells did not lead to a significant increase in ribosome footprints or protein output indicating that they were translationally incompetent. We hypothesize that DHX36 resolves G4 and other structures that interfere with efficient translation initiation.

bioinformatics↗

bioRxiv2017

A multi-center study on factors influencing the reproducibility of in vitro drug-response studies

Evidence that some influential biomedical results cannot be repeated has increased interest in practices that generate data meeting findable, accessible, interoperable and reproducible (FAIR) standards. Multiple papers have identified examples of irreproducibility, but practical steps for increasing reproducibility have not been widely studied. Here, seven research centers in the NIH LINCS Program Consortium investigate the reproducibility of a prototypical perturbational assay: quantifying the responsiveness of cultured cells to anti-cancer drugs. Such assays are important for drug development, studying cell biology, and patient stratification. While many experimental and computational factors have an impact on intra- and inter-center reproducibility, the factors most difficult to identify and correct are those with a strong dependency on biological context. These factors often vary in magnitude with the drug being analyzed and with growth conditions. We provide ways of identifying such context-sensitive factors, thereby advancing the conceptual and practical basis for greater experimental reproducibility.

cancer biology↗

bioRxiv2017

CD95L mRNA And CD95L Derived si- and shRNAs Kill Cancer Cells Through An RNAi Mechanism By Targeting Survival Genes

Over 80% of multiple tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that using specific toxic RNAi-active sequences present in the genome can kill cancer cells.

cancer biology↗