Chinese scientists have discovered that quadruplexed DNA can be used as a new target drug, and the beneficiary group will exceed PARP inhibitor drugs

Returning to China to start a business is Xu Hong's next destination.

"Now that I've left Canada, I'm back home to form a company to develop quadruplex DNA drugs," said a former scientist at the BC Cancer Research Center in Canada. We will continue to work in the field of quadruplexed DNA to bring good news to patients as soon as possible. ”

Figure | Xu Hong (Source: Xu Hong)

Before returning to China, she co-authored a Nature Communications paper [1] in which she summarized the results of the Phase 1 clinical trial of CX-546, a new target cancer drug, and answered a key question: can quadruplex DNA be used as a new target? The answer is yes.

Quadruplex DNA structures have been discovered for more than 50 years, and research in this area has been very active for nearly 20 years. However, there are currently no drugs on the market that target quadruplex DNA. Therefore, the results of clinical trials of CX-5461, a quadruplex DNA stabilizer, are of great interest.

Figure | Clinical trial results of CX-5461 in solid tumors show low side effects and high tolerability in humans (Source: Nature Communications)

The CX-5461's "bumpy" life

The story of the CX-5461 begins with two threads. One line is the American company Cylene, the company that invented the CX5461. CX5461 is derived from norfloxacin, a quinolone antibiotic that inhibits bacterial gyrase. It was then engineered by Abbott Biologics in the United States into a cytotoxic, human topoisomerase inhibitor, A-62176.

Based on this, Cylene further expanded its planar circular structure and engineered it into a molecule CX3543 that binds to tetraplex DNA, and conducted Phase I and Phase II clinical trials of CX3543 in 2007. Since CX3543 binds to ribosomal quadruplex DNA and inhibits RNA polymerase I, and CX3543 is enriched in the nucleolar region, this disrupts the nucleolar structure and causes cell death.

Therefore, Cylene believed that the inhibition of RNA polymerase I was a good direction to inhibit tumors, and then optimized CX3543 to CX5461 in the direction of RNA polymerase I inhibitors, and then followed this mechanism to design the first clinical trial of CX5461, which was conducted in Australia in 2013 and was mainly used to treat patients with leukemia and lymphoma because of the RNA polymerase I in these cancers Transcription levels are higher than in other cancers.

However, the efficacy rate of this clinical trial was not satisfactory. Of the 16 patients, only one patient had partial tumor suppression (PR) and one patient was stable (SD). Cylene was also closed in 2013, and the CX5461 patent was resold to Taiwan's Shenghua Company.

The CX5461 is on the BC Cancer Center in Canada, where Xu Hong's former owner is. While working there, she focused on the function of the oncogene breast cancer 2 gene-BRCA2. In order to identify tumors that can treat BRCA2 deficiency, she and her colleagues have been conducting synthetic lethal screening of BRCA2 since 2005.

Initially, a genome-wide small nucleic acid screen was performed, and later a small molecule compound screen was performed, and tumors with BRCA2 gene defects were found from them, which are very sensitive to CX5461 and CX3543. At the time, she and her colleagues did not know that the CX5461 was derived from the CX3543 or that the CX5461 had the ability to stabilize the quadruplex DNA.

At the time, the literature described CX5461 as an inhibitor of RNA polymerase I. So, they began to investigate why tumors with defective BRCA2 genes are intolerant to RNA polymerase I inhibition.

However, studies in this area did not yield the same results as the CX5461. Moreover, other RNA polymerase I inhibitors are not synthetically lethal to tumors with BRCA2 gene defects.

Since RNA polymerase I is involved in transcribing ribosomal DNA regions, there are a large number of sequences that can form quadruplex DNA. Therefore, she intends to test whether the CX5461 has the effect of stabilizing tetraplex DNA.

Later, Shankar Balasubramanian, a professor in the Department of Chemistry at the University of Cambridge in the United Kingdom, confirmed that CX5461 was a tetraplex DNA stabilizer after laboratory testing. Moreover, pyridostatin, another tetraplex DNA stabilizer invented by the Shankar lab, is also synthetically lethal to the BRCA2 gene.

So far, she and her colleagues believe that the synthetic lethality of CX5461 and BRCA2 genes is due to the stabilization of tetraplex DNA by CX5461. In 2017, the 12th year of BRCA2 gene research with her team, she and her team published a paper in Nature Communications describing the results of CX5461's selective inhibition of BRCA2 gene-deficient tumors and analyzing the mechanism of action, of which Xu Hong is the first author [2].

After seeing the paper, Dr. Laurence Hurley, one of the founders of Cylene Corporation, was very excited. Dr. Lawrence Hurley, the inventor of CX3543, believes that previous clinical trials of CX3543 and CX5461 have been ineffective due to misconceptions about the drug mechanism of CX3543 and CX5461.

At that time, the drug mechanism she introduced in her paper brought back the original intention of the molecular design of CX3543 and CX5461, that is, CX3543 and CX5461 are tetraplex DNA stabilizers. At this point, the CX5461 finally overlaps in the U.S. and Canada, and the mechanism and application of the CX5461 are unanimous.

In 2016, Hong's team conducted a clinical trial of CX5461 in solid tumors in Canada. Although CX5461 was in Phase 1 clinical trials in Australia, it was directed at patients with hematologic malignancies and lymphomas. The clinical trial for patients with solid tumors started with a phase I clinical trial with dose escalation and was completed at the end of 2019.

Not long ago, in June 2022, she and her colleagues published a summary paper of the clinical trial at Nature Communications, which is the result presented at the beginning of this article.

More than 50 years after its discovery, the discovery of a drug that shows promising clinical applications for quadruplex DNA structures is a great encouragement to scientists in the field and will attract more people to work in the field.

It is important to know that the target of small molecule and large molecule targeted drugs has always been protein. If the special secondary structure of DNA could also be targeted, the concept of drug targets would be revolutionized.

In addition to quadruplex DNA, there are many specific non-traditional structures on DNA, as well as quadruplexes on RNA, as well as other special secondary and tertiary structures. In the future, these nucleic acid higher-order structures are also expected to be elucidated in detail and become drug targets, just like the three-dimensional structures of proteins.

Figure | Efficacy data from the CX-5461 clinical trial are encouraging (Source: Nature Communications).

Quadruplex DNA drugs will benefit a wider range of patients than PARP inhibitors

Xu Hong said that the focus of clinical trials is nothing more than safety and efficacy. On both counts, the CX-5461 delivers encouraging results.

First of all, in terms of safety, the dose of CX-5461 can be tolerated by the human body is very high, reaching 475mg/㎡. There are no tertiary side effects greater than 10% other than photosensitivity. The appearance of photosensitivity is due to the patient's failure to do a good job of sun protection. If sun protection is in place, photosensitivity side effects can be avoided altogether. This is a significant advantage over chemotherapy drugs, which often have a tertiary side effect rate of more than 50%.

In targeted drugs, such as DNA repair enzyme PARP (poly ADP-ribose polymerase) inhibitors, there is a probability of 20%-40% of the side effects above the third level. Therefore, from a safety perspective, the low toxicity of CX-5461 is attractive to cancer patients.

According to reports, the main design consideration of the phase I clinical trial is safety, so the drug dose of patients is not optimal, and the patients recruited are not all patients who are sensitive to quadruplex DNA drugs.

Of the 40 patients with solid tumors, a total of 4 patients had a partial tumor response (PR) and 11 patients had stable disease (SD). Among them, 4 patients achieved 6 months of stable tumor non-growth, i.e., stable SD.

In 40 patients, the disease control rate was 28%. If only tumors with BRCA2 gene mutations were considered, PR plus SD was 82% of the 11 patients whose tumors were measurable. Of the 7 patients with BRCA1 gene mutations, 4 were stable (57%).

Tumors with BRCA1 mutations appear less sensitive than tumors with BRCA2 mutations, which may reflect different characteristics of the BRCA1 gene from the BRCA2 gene. However, the sample size is not large enough to be conclusive.

Another important point is that the results of the Phase 1 trial have enabled a proof of concept of the quadruplex DNA drug mechanism in humans. CX-5461 is a stabilizer of quadruplex DNA, which stabilizes the replication and transcription of DNA.

The homologous recombination repair pathway involving BRCA1, BRCA2, PALB2, and RAD51 can mediate the crossover of the quadruplex structure in the DNA template, thereby bypassing the quadruplex DNA and allowing DNA replication to continue.

Figure | BRCA2, a revert mutation in the PALB2 gene mediates resistance to CX-5461 (source: Nature Communications).

On the other hand, stable quadruplex DNA has the potential to form DNA breaks, which require homologous recombination repair pathways, as well as other DNA damage repair pathways. This is why patients with BRCA2 and PALB2-deficient tumors in clinical trials are very sensitive to CX-5461.

In a clinical trial, a patient with breast cancer metastases with a mutation in the PALB2 reading frame had tumors shrink and achieve PR after treatment with CX-5461, but the course of the disease recurred. Sequencing of samples of metastatic hepatocellular carcinoma cells showed that the resistant tumor produced additional mutations on PALB2, thus restoring the reading frame of PALB2.

Another patient with a misaligned reading frame mutation in the BRCA2 gene initially responded well to CX5461 and was stable. Later, the reading frame is restored due to reversion mutations produced in the BRCA2 gene, which mediates the development of drug resistance. The discovery of reverting mutations in progressive tumors suggests that BRCA2 and PALB2 deficiencies are indeed key genes mediating CX-5461 sensitivity.

Recently, the related paper was titled "Results of the phase I CCTG IND.231 trial of CX-5461 in patients with advanced solid tumors enriched for DNA-repair." deficiencies, published in Nature Communications.

Figure | Related paper (source: Nature Communications).

Hong Xu serves as a co-author, and Lesley Seymour, director of the New Drug Research Program of the Canadian Cancer Trials Group, serves as the corresponding author.

It should be noted that for BRCA1, BRCA2 and homologous recombination-deficient tumors, the efficacy of currently commercially available PARP inhibitor drugs is quite good. It has been suggested that, based on data from Phase 1 clinical trials, CX5461 appears to be less effective than PARP inhibitors.

However, in clinical trials of PARP inhibitors, most patients who are sensitive to platinum drugs are selected. However, the CX5461 clinical trial did not screen patients for prior chemotherapy history. Also, CX5461 was not compared head-to-head with a PARP inhibitor in this clinical trial, so it was not possible to compare the benefits and disadvantages of the two drugs.

Furthermore, because CX5461 is mechanistically distinct from the DNA repair enzyme PARP inhibitor, the population of susceptible patients is necessarily different. It has been reported that some patients with tumors that are not sensitive to PARP inhibitors respond well to CX5461.

Therefore, from a mechanistic point of view, quadruplex DNA drugs will benefit a wider range of patients than PARP inhibitor drugs, including not only homologous recombination repair defects (HRD), but also other DNA damage repair defects, such as non-homologous sticky end joining (NHEJ) and tumors with quadruplex DNA unwinding defects.

Figure | CX-5461 is stable in humans with a long half-life (Source: Nature Communications).

Ten years of sharpening a sword, fortunately with the help of experts, successfully transformed the results

"I've been working on the CX5461 for 15 years, starting in 2007. For a long time, research was in a difficult situation, and colleagues did not see any hope and left one after another. I hadn't published a single paper in 6 years, and I moved on to other topics halfway through.

However, I never gave up until the task I had set was completed, but fortunately, everyone was very happy with the final result. And as a researcher of basic theories, I feel very fortunate to see the results being used in clinical practice. ”

In addition, she experienced a long period of silent work while studying the genetic mechanisms of CX5461 and BRCA2, but when everyone saw the good efficacy of CX5461, the participation of many heavyweight experts also promoted the in-depth understanding and clinical application of the drug.

She continued, "We worked with the Shankar Balasubramanian team at the University of Cambridge, one of the most authoritative laboratories in the field of quadruplex DNA, to test and confirm CX5461 as a quadruplex DNA stabilizer by Shankar Laboratories;

Dr. Sam Aparicio, Director of the Breast Cancer Research Unit at BC Cancer Centre in Canada, was my leader at the time, and he was instrumental in the launch of the CX5461 clinical trial in Canada; Dr. Lawrence Hurley, one of the founders of the quadruplex DNA field and inventor of the CX3543, collaborated with us to provide experience with previous clinical trials of the CX3453 and CX5461. ”

As mentioned earlier, once the successful clinical application of quadruplex DNA drugs is of great significance to the field of quadruplex DNA and even the broader field of nucleic acid research, it will also bring a revolutionary impetus to the concept of drug targets in the pharmaceutical industry.

Xu Hong's next career is to promote the druggability development and clinical application of quadruplex DNA drugs. She said: "Through years of work, I have a better understanding of the advantages and disadvantages of this type of drug. Compared with other cancer drugs on the market, quadruplex DNA drugs have characteristics that other drugs do not have, and will occupy an important position in the drug market in the future to address the clinical needs of patients. ”

Resources:

1.Hilton, J., Gelmon, K., Bedard, P.L. et al. Results of the phase I CCTG IND.231 trial of CX-5461 in patients with advanced solid tumors enriched for DNA-repair deficiencies. Nat Commun 13, 3607 (2022). https://doi.org/10.1038/s41467-022-31199-2 

2.Xu, H., Di Antonio, M., McKinney, S. et al. CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours. Nat Commun 8, 14432 (2017). https://doi.org/10.1038/ncomms14432