Scientists in the U.S. have shown how CRISPR-Cas9 gene editing can be used to restore the effectiveness of first-line chemotherapies against lung cancer, by knocking down a tumor gene that acts as a master regulator of genes involved in the development of resistance.

Eric B. Kmiec, Ph.D., and colleagues at the Gene Editing Institute of the Helen F. Graham Cancer Center & Research Institute at Christiana Care Health System, used CRISPR-Cas9 technology to disable the NRF2 gene in laboratory-grown lung cancer cells. Tests showed that the NRF2-knockout cells were less able to proliferate in culture, and were more sensitive to chemotherapeutic agents including cisplatin and carboplatin. The NRF2-deficient cancer cells also grew more slowly than unmodified cancer cells when transplanted into mice. Tumor growth stopped completely for 16 days in recipient animals treated using different forms of chemotherapy, and there was “a dramatic decrease in tumor volume,” the researchers write in their published paper in Molecular Therapy Oncolytics.

“Our goal is to see if CRISPR can be used with chemotherapy to provide a safe, affordable way to give patients who are not responding to treatment at least a fighting chance against this very challenging cancer,” said Dr. Kmiec, who is research lead and the director of the Gene Editing Institute. “We believe that finding ways to use CRISPR to improve existing treatments will lead to some of the first benefits for patients while we tackle the vital ethical issues around the use of CRISPR for edits that can be passed on through DNA. This is an exciting step in the journey of exploring the health benefits of gene editing.” Dr. Kmiec and co-authors Pawel Bailk Ph.D., Yichen Wang Ph.D., and Kelly Banas Ph.D., reported on their work in a paper titled, “Functional Gene Knockout of NRF2 Increases Chemosensitivity of Human Lung Cancer A549 Cells In Vitro and in a Xenograft Mouse Model.”

Lung cancer is the leading cause of cancer mortality in the U.S., accounting for more than 1 in 4 cancer deaths, and killing more people than breast, prostate, and colon cancer combined, the authors wrote. Chemotherapy remains a key treatment option for lung cancer, but in most cases tumors will become resistant to chemotherapeutic agents. Studies have linked the development of drug resistance with upregulation of different genes that are involved in shipping drugs out of cells, or that act to direct gene transcription.

One of these genes, nuclear factor erythroid 2-related factor (NRF2), is considered to represent a master regulator of another 100–200 genes involved in how cells respond to oxidative and/or electrophilic stress. Its targets include genes that control efflux pumps, while NRF2 is also known to regulate the expression of genes involved in degrading proteins and detoxification.

Prior research has built a case for NRF2 as a potential target for improving cancer response to existing treatments. Chemotherapy has been shown to activate the transcriptional activity of NRF2 target genes, “often triggering a cytoprotective response,” the authors write. Environmental stress and adverse growth conditions can also trigger upregulation of NRF2. “The upregulation of NRF2 expression leads to an enhanced resistance of cancer cells to chemotherapeutic drugs, which by their very action induce an unfavorable environment for cell proliferation.”

Prior research has, in addition, shown that NRF2 inhibitors can boost the effectiveness of chemotherapy against cancer in vitro and in xenografts, while “NRF2 is also known to be the central contributor in the resistance to cisplatin in bladder cancer,” the team added.

With this in mind, the scientists devised a strategy that would combine CRSPR-directed gene editing to disable the NRF2 gene, with traditional chemotherapy. “The overall strategy is to design and utilize a CRISPR/Cas9 gene-editing tool to disable the NRF2 gene in lung cancer cells, rendering it incapable of producing a functional protein,” they stated.

To this end they used CRISPR technology to functionally disable both copies of the NRF2 gene in A549 lung cancer cells. A549 cells are normally resistant to chemotherapy, but the team reasoned that cells with the NRF2 gene knocked down should be more sensitive to chemotherapeutic drugs, as the genes responsible for pumping the drugs out of the cancer cells would remain inactivated. Their in vitro tests confirmed that while wild-type A549 cells were resistant to monotherapy using either cisplatin or carboplatin, and were also resistant to all but the highest doses of cisplatin and vinorelbine combination therapy, the NRF2-knockout A549 cells were sensitive to both single and combined treatments. “In the genetically engineered knockout cell lines, we clearly observed an increase in chemosensitivity in a dose-dependent fashion,” the team wrote.

The team then transplanted either wild-type or NRF2-knockout A549 cells into mice, and again evaluated the effects of chemotherapy on the resulting tumors. They found that while the tumors formed by wild-type A549 cells were resistant to chemotherapy, the NRF2 knockout xenografts grew more slowly, even without the addition of chemotherapy. When cisplatin therapy was administered for 16 days the NRF2-knockout tumors stopped growing over the course of treatment. “Proliferation of the implanted cells was arrested,” the authors wrote. Similar results were observed in CRISPR-modified A549 cell-derived xenograft tumors treated using cisplatin and vinorelbine combination therapy. Carboplatin also reduced proliferation and growth trend in the NRF2-knockout tumors. “These results suggest that the combination of gene editing in chemotherapy produces an enhanced chemosensitivity in A549 cells, both in cell culture and in a xenograft mouse model,” the researchers stated.

NRF2 is a well recognized as a potential target for boosting the effectiveness of anticancer drugs, the authors note. “To our knowledge, however, we are the first to generate NRF2-deficient A549 cells bearing either heterozygous or homozygous knock-outs … Our results provide some support for the notion that the combination of gene-editing activity and chemotherapy acts synergistically to reduce tumor cell growth.”

The authors acknowledge that appropriate delivery systems for the CRISPR constructs will be needed before the approach could be considered for clinical applications. “… it is imperative that the development of CRISPR-directed gene editing of multipurpose human genes for combinatorial therapy of lung cancer be coupled with the evolution of more effective delivery methods of these genetic tools to lung tissues,” they wrote.

And while the potential applications of CRISPR to not just disable genes, but to replace sections of DNA, are evident, Dr. Kmiec’s team is, for now, applying CRISPR-based technologies at their most “native form,” which is to cut out sections of genetic code. Their application of CRISPR to improve drug response represents what Dr. Kmiec calls the “low-hanging fruit” of CRISPR patient applications. “We think it’s best to start with CRISPR therapies that involve relatively conservative uses of this powerful tool,” he commented. “This approach can also hopefully help contain costs and provide a level of safety and reliability that is reassuring for patients and increases the chance that insurance companies will provide coverage.”

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