Researchers develop a new machete technique to cut the cancer genome and study copy number alterations

Researchers develop a new machete technique to cut the cancer genome and study copy number alterations

MACHETE is a new CRISPR-based technique developed by researchers at the Sloan Kettering Institute (SKI) to study large-scale genetic deletions efficiently in laboratory models.

People already call it the Paper Machete.

Still, lead authors Francisco “Pancho” Barriga and Kaloyan Tsanov of the Sloan Kettering Institute don’t want the name of their new research technique to overshadow their findings, which shed new light on a genetic change that contributes to about 15 percent of all cancers, and could help identify patients who are likely to respond to immunotherapies.

MACHETE is what the duo call the CRISPR-based method they developed to study copy number alterations, or CNAs, which are large-scale genetic changes that frequently occur in cancer.

The acronym MACHETE stands for Molecular Alteration of Chromosomes with Tandem Engineering Elements. It’s a new way of cutting out specific significant sections of the genetic code to reflect the changes that arise in cancer and other human diseases.

This means that, for the first time, there is a simple and efficient way to study CNA deletions in laboratory models, such as the pancreatic cancer and melanoma mouse models used in their study, which was published in nature cancer on November 7, 2022.

“At first, we didn’t even want to put MACHETE in the title of our paper, to better highlight the fascinating biology we found,” says Dr. Barriga, a postdoctoral fellow at the Institute, which is the experimental research arm of the Cancer Center. Memorial Sloan Kettering (MSK).

Still, that didn’t stop a fellow lab paste a photo of Danny Trejo’s famous character, Machete, on his desk after first submitting the work. Nor did another scientist tweet a preprint copy of the article on the actor’s Twitter handle. (The gesture was not reciprocated, unfortunately.)

Beyond Cancerous Single Gene Mutations

To understand the ways in which the study broke new ground regarding one of the most common copy number alterations in human cancers, and what it might mean for patients in the future, it is necessary to appreciate the underlying biology.

Many people think of mutations in cancer as small “typos” in the genetic code that affect the activity of a single gene, either turning it on or off. And for decades, researchers have also focused primarily on these little mistakes that cause many types of cancer.

However, copy number alterations can affect dozens of genes simultaneously and duplicate or delete large sections of individual chromosomes.

A typical tumor carries an average of 24 different CNAs that affect up to 30% of its genome, the researchers note.

“Point mutations are relatively easier to study than CNAs,” says Dr. Tsanov, who, like Dr. Barriga, is a postdoc in the lab of researcher Scott Lowe, lead author of the study and chair of the Program. of Cancer Biology and Genetics of the Institute. “But CNAs are just as important, they are much more complex.”

Tumors with a higher level of CNA, also known as CNA load, are linked to recurrence and worse outcomes in breast, prostate, endometrial, clear cell renal, thyroid and colorectal cancer, previous MSK research found.

But again, the size and variety of the changes, affecting thousands, or even millions, of DNA base pairs rather than just the alteration of a single letter in the DNA sequence, has made them very difficult. to recreate in laboratory models for detailed study.

A potential new approach to studying large-scale deletions occurred to Dr. Barriga while riding home from the lab on the Roosevelt Island tram.

“I was wondering, ‘How can we select cells with the expected deletions even if they are very rare?’ ” he says. “I had an idea and put together the initial concept of what the overall strategy would be that night. When we tried it, it just worked. I may never have something that smooth for the rest of my career.”

Thinking of the right combination of words to make the MACHETE acronym work took more time, he jokes. co-worker

But the duo knew they were right when the genetic changes made by MACHETE in a mouse model of pancreatic ductal adenocarcinoma resulted in the same results as a similar natural mutation in a different mouse model that Dr. Tsanov was studying for. a separate project.

“It was really a close collaboration from there,” says Dr. Tsanov. The research team also included more than a dozen other scientists from MSK, the Cancer Research Institute of Ontario, New York University Grossman School of Medicine and the Princess Margaret Cancer Center in Toronto.

Research may help identify which patients will benefit from immunotherapy

After inserting the genetically “mashed” cells into the pancreas of laboratory mice, the mice developed cancer. The genetic alterations removed a portion of chromosome 9, and with it a gene known as CDKN2A — a well-established tumor suppressor gene. As expected, this shut down the cells’ innate ability to prevent tumor cell growth.

This large portion also deleted the genetic code for a group of interferons, proteins that cause immune cells to fight invaders, such as cancer cells, whose importance the scientists wanted to prove.

One of the most prevalent CNAs in people affects this chromosomal region, 9p21.3, and about half of patients develop tumors that also lack these interferons.

“We have learned of CDKN2A mutations for a long time, and it was already remarkable how well they worked,” says Dr. Lowe, who is also a Howard Hughes Medical Institute investigator. “This study says there is much more, with important therapeutic implications.”

The additional loss of interferons creates a one-two punch that makes tumors invisible to immune system defenders and helps cancer spread, the researchers found.

“These interferons have been difficult to study because they are encoded by a group of 16 genes,” adds Dr. Lowe. “The use of MACHETE revealed an important way in which developing cancer cells evade recognition by the immune system and can also lead to resistance to immunotherapies designed to reactivate the immune system to attack cancer.”

Co-author Dana Pe’er, a computational biologist at the Sloan Kettering Institute, was instrumental in helping the team understand how disruption of interferon genes affected immune cells and helped cancer evade the immune system, says Dr. Lowe .

In addition to pancreatic cancer, the findings held true for a mouse model of melanoma.

Research suggests, then, that patients whose interferon region is still intact may be better candidates for immunotherapy than those who have lost it. Immunotherapies can work wonders, but one of the big challenges has been identifying which patient cancers will respond to them and which won’t.

Even state-of-the-art genomic tests, such as MSK-IMPACT®, however, they do not normally collect information on this group of interferon genes. Further study could show whether adding it to sequencing tests could help identify patients who are most likely to benefit from immunotherapy, Dr. Lowe says.

Meanwhile, CNA deletions have been linked to a variety of human genetic disorders called chromosomal deletion syndromes. “So MACHETE provides a new framework for investigating large deletion events beyond cancer,” says Dr. Barriga, pointing to a half-dozen other research groups. they are already using the technique developed by the Sloan Kettering Institute.

This work was supported by the David M. Rubenstein Center for Pancreatic Cancer Research; the Marie-Josée and Henry R. Kravis Center for Molecular Oncology; NIH/NCI Cancer Center Support Grant (P30 CA008748); the Howard Hughes Medical Institute; the National Institutes of Health (grant P01CA13106); cycle for survival®; a GMTEC Postdoctoral Fellowship; a GMTEC Postdoctoral Fellowship funded by Shulamit Katzman; an MSK Translational Research Oncology Training Fellowship (5T32CA160001-08); a Young Investigator Award from the Edward P. Evans Foundation; the Jane Coffin Childs Memorial Fund for Medical Research; the William C. and Joyce C. O’Neil Charitable Trust; the MSKS Single Cell Sequencing Initiative; the La Caixa Junior Leader Postdoctoral Grant (LCF/BQ/PI20/11760006); and Agilent’s thought leader program.

Dr. Lowe is a scientific consultant with shares in Blueprint Medicines, Constellation Pharmaceuticals, Faeth Therapeutics, Mirimus, ORIC Pharmaceuticals, and PMV Pharmaceuticals. The other authors of the study declare that there are no conflicts of interest.

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