Nature Cancer has unveiled a new CRISPR-based technology that can cut the cancer genome and study changes in copy number

Research focus

• MACHETE is a new CRISPR-based technique that can efficiently study large-scale gene deletions
  in laboratory models.
  

• Using MACHETE, the researchers found that a prominent deletion would normally eliminate a group of interferon genes, resulting in poorer prognosis in mouse models of pancreatic cancer and melanoma
  .
  

• Intact tumor interferons may be biomarkers of immunotherapy response
  .
  

This technique is called the Machete Paper
.

Still, 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
.
Their findings reveal a genetic change that causes about 15 percent of cancers, which may help identify patients
who respond to immunotherapy.

MACHETE is a CRISPR-based method they developed to study copy number alterations (CNAs), large-scale genetic changes
that often occur in cancer.

An acronym
for MACHETE's Molecular Alteration of Chromosomes with Engineered Tandem Elements.
This is a new method that can cut out important target parts of the genetic code that reflect changes
that occur in cancer and other human diseases.

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

"In the beginning, we didn't even want to put MACHETE in our paper to better highlight the important biological properties we found," said
Dr.
Barriga of the institute.

Still, that didn't stop a lab buddy from sticking a photo of Danny Trejo's famous character: Machete to his
desk after the first presentation of his work.
Another scientist also tweeted a preprint of the paper to the actor's Twitter account
.
(Unfortunately, this move did not receive a response)
。

Beyond single-gene cancer mutations

To understand this study's new breakthrough in the most common copy number alterations in human cancers, and what it might mean for future patients, we need to understand basic biology
.

Many people think of a mutation in cancer as a small "typo" in the genetic code that affects the activity of a single gene — either activating it or shutting it
down.
For decades, researchers have also focused on these tiny mistakes
that lead to a variety of cancers.

However, changes in copy number can affect dozens of genes at once, replicating or deleting most of
a single chromosome.

The researchers noted that a typical tumor carries an average of 24 different CNAs, affecting up to 30 percent
of its genome.

"Point mutations are relatively easier to study than CNAs," Dr.
Tsanov said, "but CNAs are just as important — they're just much
more complex.
" ”

Previous studies at MSK have found that tumors with higher levels of CNA (also known as CNA burden) are associated with
recurrence and progression of breast, prostate, endometrial, clear cell carcinoma of the kidney, thyroid, and colorectal cancers.

But the magnitude and variety of these changes affect thousands, if not millions, of DNA base pairs, not just a single letter in the DNA sequence, making them difficult to regenerate in laboratory models for in-depth study
.

On his way home from the lab on the Roosevelt Island Trolley, Dr.
Barriga thought of a potential new way
to study large-scale gene deletions.

"I wondered: 'How can we select cells with expected deletions, even if they are very rare?' I had an idea and sketched out a preliminary concept
of the overall strategy for the night.
We tried it and it worked great
.
I may never have such a smooth event in the rest of my career
.
”

HE JOKED THAT IT TOOK LONGER TO COME UP WITH THE RIGHT COMBINATION OF WORDS TO MAKE THE ACRONYM MACHETE WORK
.

But when MACHETE was used to genetically alter a mouse model with pancreatic ductal adenocarcinoma and the results were identical to those produced by a similar mutation that occurred naturally in another mouse model that Dr.
Tsanov studied for another project, the two knew they had found
something.

"Since then, it's really been a close collaboration," Dr.
Tsanov said
.
The research team also included more than a dozen other scientists
from MSK, the Ontario Cancer Institute, New York University Grossman School of Medicine and the Princess Margaret Cancer Center in Toronto.

Research may help determine which patients will benefit from immunotherapy

After inserting genetically "macheted" cells into the pancakes of lab mice, the mice developed cancer
.
The genetic alteration removes a segment of chromosome 9, along with a gene called CDKN2A, a recognized tumor suppressor gene
.
As expected, this shuts down the cell's innate ability to
block tumor cells from appearing.

The slice also removes the genetic code from a set of interferons — a protein that triggers immune cells to fight off invaders, such as cancer cells — and the scientists wanted to test the importance of
interferon.

One of the most common CNAs in humans affects this chromosomal region - 9p21.
3, and about half of patients develop tumors in which these interferons are also missing
.

"We have known about CDKN2A mutations for a long time, and the way they work is already remarkable, and this study shows that there is much more to it with important therapeutic implications
.
"

The researchers found that the extra loss of interferon formed a combination punch that made the tumor invisible to defenders in the immune system and helped the cancer spread
.

"Studying these interferons is difficult because they are encoded by clusters of 16 genes, and using MACHETE reveals the main ways in which developing cancer cells avoid being recognized by the immune system, which may also lead to resistance to immunotherapies designed to reactivate the immune system to attack cancer
.
"

Dr.
Lowe said computational biologist Dana Pe'er was instrumental
in helping the research team understand how disruption of the interferon gene affects immune cells and helps cancers evade the immune system.

In addition to pancreatic cancer, the findings also apply to mouse models
of melanoma.

Studies have shown that patients with intact interferon regions may be better candidates for immunotherapy
than those with missing interferon regions.
Immunotherapy can work wonders, but one of the biggest challenges is determining which patients' cancers work and which don't
.

However, even the most advanced genomic tests, such as MSK-IMPACT®, typically do not collect information
about this interferon genome.
Dr.
Lowe noted that further research could show whether adding it to sequencing tests could help identify patients
who are more likely to benefit from immunotherapy.

Meanwhile, CNA deletion has been linked
to a variety of human genetic disorders known as chromosomal deletion syndrome.
MACHETE thus provides a new framework for studying large gene deletion events outside of cancer," said
Dr.
Barriga.
He noted that six other research groups are already using technology
developed at the Sloan Kettering Institute.