Functional-genetic approaches to understanding drug response and resistance
Introduction
Ten years after the first description of a complete cancer genome [1], extensive next-generation sequencing efforts have provided detailed maps of genetic and epigenetic aberrations in tens of thousands of human cancers [2, 3, 4]. In parallel, the advent of genome editing and high-throughput screening technologies revolutionize the identification and functional characterization of cancer drivers and dependencies. Collectively, these technological advances accelerate the nomination of potential therapeutic targets. At the same time, drug development remains a slow and extremely costly process that typically takes 10–15 years and fails for more than 95% of candidate drugs [5,6], often at late stages due to lack of efficacy or clinical safety [7]. High attrition rates are a major cause for skyrocketing drug development costs, which have been estimated to exceed $2.5 billion per newly approved drug [8]. In addition, the clinical efficacy of approved cancer therapeutics remains variable between patients and limited by the widespread development of drug resistance. While there is broad consensus that curative cancer therapies will require personalized combination therapies, mechanisms and predictive biomarkers determining sensitivity and resistance remain incompletely understood for most drugs in clinical use.
To tackle these challenges, recently developed functional-genetic tools, in concert with other emerging technologies such as single-cell and time-resolved transcriptomics, establish a versatile toolkit to rigorously study and validate new candidate targets before drug development. For established compounds, functional-genetic screens provide a transformative approach for deciphering response biomarkers, resistance mechanisms and synergistic target interactions, and thereby guide the development of personalized therapies and rational drug combinations. Here, we review promising applications and remaining limitations of recently established functional-genetic tools in drug and therapy development.
Section snippets
Genetic tools for deciphering mechanisms of drug resistance
Multiplexed RNAi or CRISPR screens based on viral delivery of complex short-hairpin (sh) or guide (g) RNA libraries have revolutionized the functional annotation of the human genome. Particularly robust are proliferation-based positive selection screens detecting the enrichment of sh/gRNAs that induce a competitive advantage in cell proliferation and/or survival (Figure 1). When performed in presence of a selective pressure (e.g. a cancer therapeutic), such screens enable the systematic
Exploring drug sensitivity and synergy using functional genetic screens
The search for genetic events that render cells (particularly) sensitive to a specific drug is of great interest and relevance, both for the identification of predictive response biomarkers and for the development of synergy-based combination therapies. Functional genetic screens enable the systematic identification of genes required for proliferation and/or survival in a given cell type, and such ‘drop-out’ screens using different platforms (RNAi, CRISPR, CRISPRi) have meanwhile been used to
Other emerging tools for probing candidate targets and drugs
Complementing advances in genetic screening, other recent developments fundamentally change the way we can investigate molecular functions of candidate targets and drugs. Chemical-genetic methods for targeted protein degradation, such as the auxin-inducible degron (AID) and the dTAG systems [59,60] can be used to rapidly eliminate degron-tagged proteins via the ubiquitin proteasome system within minutes. Such time scales are not feasible using loss-of-function tools acting at the DNA or RNA
Epilogue and outlook
The development of multiplexed functional-genetic screening methods has turned cancer cell lines into one of the most versatile systems for high-throughput genetic studies. In biomedical research, RNAi-based or CRISPR-based screens will continue to deliver candidate therapeutic targets at an unprecedented rate. The availability of orthogonal loss-of-function tools [50] also facilitates rigorous validation studies in cell culture and animal models, which are an important measure to increase the
Conflict of interest statement
M.H. declares no conflict of interest. J.Z. is a scientific advisor and shareholder of Mirimus Inc., an inventor on patent application EU17166629.0-1403 covering SLAM-seq and related methods (licensed to Lexogen GmbH) and receives research support from Boehringer Ingelheim.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We are thankful to all members of the Zuber laboratory for fruitful discussions. Research in the Zuber lab is supported by the European Research Council (ERC-StG-336860), the Austrian Science Fund (SFB grant F4710), and generous institutional funding from Boehringer Ingelheim. We apologize to authors whose work could not be cited because of space limitations.
References (68)
- et al.
Cell-of-origin patterns dominate the molecular classification of 10,000 tumors from 33 types of cancer
Cell
(2018) - et al.
Innovation in the pharmaceutical industry: new estimates of R&D costs
J Health Econ
(2016) - et al.
Ebola virus entry requires the cholesterol transporter Niemann-Pick C1
Nature
(2011) - et al.
An Integrated genome-wide CRISPRa approach to functionalize lncRNAs in drug resistance
Cell
(2018) - et al.
MED12 controls the response to multiple cancer drugs through regulation of TGF-β receptor signaling
Cell
(2012) - et al.
Transcriptional plasticity promotes primary and acquired resistance to BET inhibition
Nature
(2015) - et al.
Combined CRISPRi/a-based chemical genetic screens reveal that Rigosertib is a microtubule-destabilizing agent
Mol Cell
(2017) - et al.
RNA targeting with CRISPR-Cas13
Nature
(2017) - et al.
Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL
Cell
(2003) - et al.
Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems
Science
(2016)