Evolution of Molecular Targeted Cancer Therapy: Mechanisms of Drug Resistance and Novel Opportunities Identified by CRISPR-Cas9 Screening

Abstract

Molecular targeted therapy has revolutionized the landscape of cancer treatment due to better therapeutic responses and less systemic toxicity. However, therapeutic resistance is a major challenge in clinical settings that hinders continuous clinical benefits for cancer patients. In this regard, unraveling the mechanisms of drug resistance may identify new druggable genetic alterations for molecularly targeted therapies, thus contributing to improved therapeutic efficacies. The recent rapid development of novel methodologies including CRISPR-Cas9 screening technology and patient-derived models provides powerful tools to dissect the underlying mechanisms of resistance to targeted cancer therapies. In this review, we updated therapeutic targets undergoing preclinical and clinical evaluation for various cancer types. More importantly, we provided comprehensive elaboration of high throughput CRISPR-Cas9 screening in deciphering potential mechanisms of unresponsiveness to molecularly targeted therapies, which will shed light on the discovery of novel opportunities for designing next-generation anti-cancer drugs.

Keywords: CRISPR-Cas9 screening; drug resistance; molecular targeted therapy; patient-derived organoid; patient-derived xenograft (PDX).

Figure 1

The overview of major types of molecular targeted therapies to inhibit tumour angiogenesis, overgrowth and immune evasion. The potential targets are indicated in red. VEGF, Vascular endothelial growth factor; EGF, Epidermal growth factor; TRAIL, TNF-related apoptosis-inducing ligand; DRs, Death receptors; RTKs, Receptor tyrosine kinases; PD-1, Programmed cell death protein 1; PD-L1, Programmed death-ligand 1; CTLA4, Cytotoxic T-Lymphocyte Associated Protein 4; TAAs, Tumor-associated antigens; TSAs, Tumor-specific antigens.

Figure 2

A schematic diagram illustrates the workflow of high-throughput CRISPR-Cas9 screening for novel regulators of drug sensitivity. CRISPR-Cas9 sgRNA libraries are packed into lentiviral vectors and transfected into Cas9- or dCas9-expressing cancer cells. In the case of loss-of-function screening, CRISPR-Cas9-mediated genome editing leads to gene knockout (or transcriptional inhibition) in individual cells, which are subsequently selection by various drugs. The residual drug-resistant cells are collected. The abundance of cells with different sgRNAs is determined in the drug-treated and control pool. Cells with sgRNAs targeting genes that cause drug resistance upon knockout (or transcriptional inhibition) will be enriched while those resulting in enhanced sensitivity to the drug will be depleted in the final pool. For gain-of-function screening, activation of gene expression by dCas9-mediated recruitment of transcriptional activation domains to transcriptional start site. Other procedures are similar to loss-of-function screening. The unique sgRNA sequence in the genome serves as a genetic barcode for high-throughput phenotyping by next-generation sequencing. Essential genes for drug sensitivity are identified for further validation. dCas9, nuclease-dead Cas9. sgRNA, single guide RNA.