CRISPR Makes Resistant Lung Cancer Cells Vulnerable to Chemotherapy

Know thy enemy: NRF2

Kelly Banas knows NRF2 like the back of her hand, and for good reason. In addition to the present study, it was the topic of her doctoral dissertation. It was this in-depth knowledge of NRF2 that allowed the team to design an effective CRISPR knockout strategy with a level of precision not normally seen in knockout studies.

NRF2 is a master regulator of hundreds of other genes that are involved in many cytoprotective and metabolic pathways. Under normal physiological conditions, NRF2 protects cells from oxidative stress, toxic insult, and chemotherapy, keeping the cells in homeostasis.

»Cancer cells hijack the NRF2 pathway and dysregulate that function. When NRF2 becomes overly expressed and accumulates within the cell, the cell is able to fight off these toxic insults. That’s really where the cancer becomes chemoresistant. Later-stage patients have much higher levels of NRF2,« Banas explains.

NRF2’s key role in the progression of cancer made it an obvious target for CRISPR-based disruption. The team’s goal was to provide better outcomes for the late-stage NSCLC patients for whom treatment options are limited, by knocking out NRF2 to make the cancer cells vulnerable to chemotherapy. To test their strategy, they used two relevant cell lines: human lung adenocarcinoma A549 cells and human lung squamous cell carcinoma NCI-H1703 cells, delivering CRISPR reagents as a ribonucleoprotein (RNP) complex.

»Chemotherapy is traditionally used for stage 3 NSCLC and people become resistant to it early on. You want to knock NRF2 out [in cancer cells] because it’s a stress-response protein. The cell goes into survival mode and shuts itself off. If you destroy NRF2, it can’t avoid the toxic stress of chemo. Chemotherapy is traditionally used for stage 3 NSCLC and people get resistant to it early on - it’s really about the cell’s ability to withstand stress,« Kmiec comments.

An interesting aspect of the team’s research is that in a couple of the tumour types they investigated, NRF2 possesses a unique PAM site that does not exist in the genomes of healthy cells. Using this to her advantage, Kelly Banas was able to demonstrate 99% cleavage activity in the tumour genome and 0% in the normal genome, i.e., of healthy control cells. This unique PAM exists in a small but significant percentage of tumours, including squamous cell carcinoma, providing an exciting opportunity to use the tumour’s DNA against itself while eliminating possible off-target editing.

Not all knockouts are created equal

As Banas and her co-authors describe in their article, gene knockouts are anything but simple. In fact, even with purposeful design, gene knockout can result in a myriad of outcomes, each of which can have a significant impact on the response to treatment.

»A transcription factor like NRF2 needs to enter the nucleus. We could [delete] a binding domain, but the protein could still get into the nucleus and wreak havoc. That’s why we went after the nuclear entry signal. I think that’s something that can be missing in the field that could be causing inconsistent results. People think if there is a PAM site somewhere, that’s where they should knock the gene out, but they need to think about functional domains, « Kmiec says.

A key discovery of the study is that CRISPR-based disruption of NRF2 can result in exon skipping. A dual sgRNA approach to target exon 4 – which codes for the nuclear entry signal – resulted in deletion of a 103-base pair fragment, including exonic splicing enhancer sequences.

»We started out targeting NRF2 to knock it out at the genetic level. What we saw was frameshifting indels in the genome, but this wasn’t enough to completely knock it out. At that point, we looked at the mRNA and protein levels to get a better understanding of what was going on. Then we saw these exon skipping events happening,« Banas describes.

Although the mechanism behind CRISPR-induced exon skipping has not been fully elucidated, previous papers have described similar exon skipping events as a result of CRISPR edits.

»Depending on where you’re targeting, you can lose essential splicing sites that are necessary to transcribe, regulate and modify the mRNA downstream,« Banas adds.

Exon skipping became the focus of the study because of its effect on chemosensitivity of the cells. Banas discovered that depending on which exons are skipped and which functional domains of NRF2 remain, the response to chemotherapy differs. Even a single nucleotide difference in the genome of NRF2-knockout cells can mean the difference between exon skipping occurring or not, based on the impact on exonic splicing enhancer sequences. Of course, characterising the huge range of outcomes of CRISPR edits was a major challenge, as Kmiec explains:

»This was an inordinate amount of work. We were looking at over 400 clones that Dr. Banas had to isolate individually, on 96 well plates, and then grow them all. But she told us in a few lab meetings that something weird was happening and the protein was being truncated, and because we’re committed to the truth, we said, ‘Go find it’. Dr. Banas had the courage of her convictions and her training to pursue this and figure it out,« Kmiec says.

Exon skipping may regulate NSCLC response to chemotherapy

After performing the CRISPR edits and examining the range of outcomes at the genomic, transcriptomic, and protein levels, Banas and the team used a cell viability and proliferation assay to assess the response of various clonal populations to treatment with the drug cisplatin. Cisplatin is a common chemotherapy agent and is part of the standard regimen for NSCLC.

As they expected, the responses of the different clonal populations of NRF2-disrupted cells varied considerably, but exon skipping was not a major factor in the outcome that would impact clinical application. In particular, cells in which exon 4 of NRF2 was skipped displayed heightened sensitivity to cisplatin, because this exon encodes the nuclear entry signal that allows NRF2 to function.