Unbiased compound-protein interface mapping and prediction of chemoresistance loci through forward genetics in haploid stem cells.

Moritz Horn,Ulrich Elling,Stephan Miethe,Martin Peifer,Josef M Penninger,Virginia Kroef,Nicole Schuller,Kira Allmeroth,Martin S Denzel

doi:10.18632/oncotarget.24305

Abstract

Forward genetic screens in haploid mammalian cells have recently emerged as powerful tools for the discovery and investigation of recessive traits. Use of the haploid system provides unique genetic tractability and resolution. Upon positive selection, these screens typically employ analysis of loss-of-function (LOF) alleles and are thus limited to non-essential genes. Many relevant compounds, including anti-cancer therapeutics, however, target essential genes, precluding positive selection of LOF alleles. Here, we asked whether the use of random and saturating chemical mutagenesis might enable screens that identify essential biological targets of toxic compounds. We compare and contrast chemical mutagenesis with insertional mutagenesis.Selecting mutagenized cells with thapsigargin, an inhibitor of the essential Ca2+ pump SERCA2, insertional mutagenesis retrieved cell clones overexpressing SERCA2. With chemical mutagenesis, we identify six single amino acid substitutions in the known SERCA2-thapsigargin binding interface that confer drug resistance. In a second screen, we used the anti-cancer drug MG132/bortezomib (Velcade), which inhibits proteasome activity. Using chemical mutagenesis, we found 7 point mutations in the essential subunit Psmb5 that map to the bortezomib binding surface. Importantly, 4 of these had previously been identified in human tumors with acquired bortezomib resistance. Insertional mutagenesis did not identify Psmb5 in this screen, demonstrating the unique ability of chemical mutagenesis to identify relevant point mutations in essential genes.Thus, chemical mutagenesis in haploid embryonic stem cells can define the interaction of toxic small molecules with essential proteins at amino acid resolution, fully mapping small molecule-protein binding interfaces.

Highlights

Studying protein-protein and protein-small molecule interactions is of critical importance to understand fundamental biological processes and to advance drug development strategies
Random insertional mutagenesis shows that Atp2a2/SERCA2 overexpression suppresses thapsigargin toxicity
We asked whether ENU-based mutagenesis combined with generation sequencing can give deeper insights into structure-function relations and chemoresistance mechanisms compared to classical gene trap-based approaches

Summary

Introduction

Studying protein-protein and protein-small molecule interactions is of critical importance to understand fundamental biological processes and to advance drug development strategies. A detailed understanding of the interaction of chemotherapeutic compounds with their respective protein targets is critical (i) for on- and offtarget toxicity studies during drug development, (ii) for target deconvolution, and (iii) for the anticipation of chemoresistance mutations in treated patients. Biochemical and biophysical approaches have been successfully applied to unravel such interactions and to define binding interfaces [1]. Most of these strategies require previous knowledge of interacting partners and come with a certain bias, are highly labor intensive, and can fail to resolve the specific interaction surface. Can an unbiased genomic approach point to physical interactions and predict chemoresistance?

Objectives

Methods

Results

Conclusion