Target Identification and Drug Discovery with CRISPR

A primary driver of cancer initiation is mutations in the genetic code and resulting gene products, leading to the dysregulation of oncogenic pathways that promote cell growth and survival. For drug developers, this provides an opportunity to use the molecular features of a tumor to guide therapy, leading to better clinical outcomes.

The drug discovery process begins with target identification—identifying those molecules, including genes, protein, or RNA, that when inhibited or activated elicit a therapeutic effect. Targets can be identified from data mining, genetic association, studying mRNA expression profiles, or performing in vitro cell-based mechanistic studies to look at pathway and phenotypic analysis. A good target should be:

• safe—does not lead to toxic side effects when drugged
• efficacious—results in an effective therapeutic outcome
• druggable—accessible to a drug molecule resulting in measured biological effect

But drug discovery is a long, complex, and expensive process, taking up to 15 years and costing in excess of $1 billion to bring a drug to clinical use. The 90% attrition rate in cancer drug development is largely due to a lack of target efficacy, which has resulted in reduced development of cancer drugs, and current projections estimate a negative rate of return on drug development by 2020.¹

Unbiased target identification with CRISPR

Functional genomics aims to link genotype with biological phenotype, and recent developments in RNAi, and lately CRISPR-Cas9, has given researchers the ability to screen whole genomes to investigate the functional consequences of gene expression. This opens up an alternative avenue for target identification—CRISPR-Cas9 screening with genome-wide sgRNA libraries allows gain of function and loss of function studies to be performed with high specificity by most laboratories. Delivery of the sgRNA library by lentiviral vector results in the stable integration of the transgene, effectively providing each cell with a barcode allowing screening to be performed in a pooled format. Following the screen, the sgRNA that have been enriched or depleted can be easily identified from the pool by NGS, enabling both positive and negative screening. This robust and unbiased means of identifying targets in tumors promises to improve success and accelerate development of new drugs.

At the recent American Association for Cancer Research meeting in Atlanta, René Bernards from Netherlands Cancer Institute spoke about the different ways CRISPR can be used in translational cancer research, and its impact on target identification and drug discovery.

CRISPR-Cas9 and synthetic lethality

Synthetic lethality is the phenomenon where disruption of two independent genes separately will not be lethal to a cell or organism, but in combination results in death. In cancer cells, the oncogenic mutations that drive the cancer will, in some cases, be synthetically lethal in combination with the disruption of a second gene. A well-known example is the selective sensitivity of BRCA-1 mutant tumors to PARP enzyme inhibition, showing that synthetic lethality can be leveraged as a therapeutic target in the clinic. The main advantage of targeting synthetic lethal genes for cancer is that they are less likely to result in toxic side effects, as wild-type cells should not be susceptible to the therapy.

CRISPR-Cas9 can be used to identify those genes that confer synthetic lethality with the use of isogenic cell pairs, which are identical apart from a single mutation. By performing a CRISPR-Cas9 screen in these cell pairs, those sgRNA that are depleted in the mutant cell line compared to wild type would indicate genes that are synthetically lethal in the cancer cell line, and so a potential target for therapeutic agents.

Key Takeaways

• Identifying target molecules associated with disease states is the first step in the drug discovery process
• Drug discovery is hampered by a lack of targets with clinical efficacy
• CRISPR-Cas9 screening provides a robust and unbiased means of elucidating the functional consequences of gene expression with high specificity
• Genome-wide sgRNA CRISPR libraries can be used to perform loss of function or gain of function screens to identify targets for drug discovery
• CRISPR-Cas9 screens can be utilized to determine the molecular basis of cellular processes such as synthetic lethality, drug interactions, senescence, and cancer-specific signaling pathways, to identify targets to leverage for clinical therapeutics

However, a caveat to keep in mind is that the mutation introduced to create the isogenic cell-line pair must be selected carefully, as it may not be a driver of the oncogenic process and its loss not specifically associated with the synthetic lethal interaction. To control for this, scientists can instead select cell lines with existing oncogenic mutations and correct them to wild type, or screen a cell-line panel containing multiple different mutant and wild-type cell lines. Performing a CRISPR-Cas9 screen in this way would allow identification of common hits across all the cell lines—those genes whose knockout in the mutant cell line confers synthetic lethality, suggestive of highly effective targets. Recent work by Behan et al., involved CRISPR-Cas9 screening 324 cell lines from 30 cancer types across 19 different tissues to identify those genes required for cell fitness.² The authors then developed a data-driven framework to systematically prioritize targets based on their effects in a cancer-specific context, genomic biomarkers, potential toxicity, and availability and suitability of pharmaceutical intervention. From this work, the authors identified Werner-syndrome ATP-dependent helicase as a potential synthetic lethal target.

CRISPR-Cas9 for potent drug combinations

As well as genotype-dependent screening, CRISPR-Cas9 can be used to explore gene modulation that enhances the efficacy of existing therapies. For example, screens that look for the loss of sgRNAs in the presence of a drug treatment will find those gene knockouts that sensitize to a particular treatment—for example, Szlachta et al., used a CRISPR knockout screen to identify a number of genes whose knockout sensitizes pancreatic cancer cells to MEK inhibiters.³ By contrast, screens that identify sgRNAs that are enriched in the presence of a drug provide insights into potential resistance mechanisms. An earlier paper from Hou et al., identified genes that function in resistance to FLT3 inhibitors—drugs that are currently in clinical trial for acute myeloid leukemia.⁴

Altered cellular states for potential cancer therapy

In addition to targets that elicit cell death when drugged, scientists are now also looking to exploit cell senescence clinically. Senescence is a program triggered by stresses that prevent abnormal cells from further proliferation resulting in their entering irreversible growth arrest. Senescent cells have altered metabolic pathways and different gene-expression pathways—and are differentially sensitive to pharmacological treatments.

In work from René Bernards lab, Wang et al., used a CRISPR library targeting 446 enzymes involved in chromatin remodeling and modulation of epigenetic marks, to identify SMARCB1 as a gene whose knockout induces senescence.⁵ Subsequently ABT263, a specific inhibitor of anti-apoptotic proteins was found to massively induce apoptosis in melanoma cells made senescent through SMARCB1 depletion but had no effect on the wild-type parental cells.

Building confidence with orthologous approaches

Screening using CRISPR-Cas9 mediated disruption is an unbiased and robust means of interrogating entire genomes for target identification. The combination of efficient gene ablation, and rapid analysis by NGS has enabled scientists to identify a number of putative drug targets.

These screens do, however, have some limitations. One potential issue is that complete gene knockout may not effectively mimic the effect of a drug as René Bernards warns in his 2019 AACR talk—“Not a single drug exists that will fully 100% inhibit a gene so suppression of gene activity is more likely to be accomplished with a given drug than complete inhibition of a gene product. Therefore, shRNA is more likely to reflect what you could accomplish with a drug compared to a complete knockout.”

But the off-target activity of shRNA complicates analysis—so perhaps the best of both worlds for target identification is CRISPRi, where a catalytically inactive Cas9 is targeted to genes to suppress their transcription. For the highest level of confidence in targets, scientists will rightly seek to combine multiple orthologous approaches—shRNA, CRISPR-Cas9 knockout, CRISPRi, genetrap, and others, prior to moving on to the time-consuming process of validation.

References

1. Hay, M., Thomas, D. W., Craighead, J. L., Economides, C. & Rosenthal, J. Clinical development success rates for investigational drugs. Nature Biotechnology 32, 40–51 (2014)

2. Behan, F. M. et al. Prioritization of cancer therapeutic targets using CRISPR–Cas9 screens. Nature 568, 511–516 (2019)

3. Szlachta, K. et al. CRISPR knockout screening identifies combinatorial drug targets in pancreatic cancer and models cellular drug response. Nature Communications 9, (2018)

4. Hou, P. et al. A Genome-Wide CRISPR Screen Identifies Genes Critical for Resistance to FLT3 Inhibitor AC220. Cancer Research 77, 4402–4413 (2017)

5. Wang, L. et al. High-Throughput Functional Genetic and Compound Screens Identify Targets for Senescence Induction in Cancer Cell Reports 21, 773–783 (2017)