Scientists Use CRISPR-Based Technique to Examine Cancer Genome

MACHETE, or Molecular Alteration of Chromosomes with Engineered Tandem Elements, is a new CRISPR-based technique developed by Sloan Kettering Institute (SKI) researchers Francisco “Pancho” Barriga and Kaloyan Tsanov. Their work, recently published in Nature Cancer, presents a method designed to study copy number alterations (CNAs), which are large-scale genetic changes that can happen in cancer, in mouse models.

Copy number alterations are not mere changes in a single gene’s genetic code. These mutations can affect dozens of genes simultaneously, deleting or duplicating large chunks of individual chromosomes. The team notes that, on average, tumors carry about 24 different CNAs that impact up to 30% of their genome. “Point mutations are relatively easier to study than CNAs,” says Dr. Tsanov who, like co-author Dr. Barriga, is a postdoc in the lab of investigator Scott Lowe, the study’s senior author and Chair of the Institute’s Cancer Biology & Genetics Program. “But CNAs are just as important — they’re just a lot more complex.”

Tumors that experience “CNA burden,” or a higher level of CNAS, are more likely to come back or worsen in multiple types of cancer. With such broad scale alterations in genetic code, it has been challenging for researchers to find a method to study them effectively.

“I wondered: ‘How can we select cells with the intended deletions even if they are very rare?’” Dr. Barringa recalls. “I got an idea and drew up the initial concept for what would be the general strategy that evening. When we tried it, it just worked. I may never have something come together that smoothly for the rest of my career.”

Dr. Barringa decided to investigate how MACHETE could induce genetic changes in mouse models of pancreatic ductal adenocarcinoma and, ultimately, found similar results to a mutation that colleague Dr. Tsanov was studying for a separate project. “It was really a close collaboration from there on in,” Dr. Tsanov says. The research team also included more than a dozen other scientists from MSK, the Ontario Institute for Cancer Research, New York University Grossman School of Medicine, and the Princess Margaret Cancer Centre in Toronto.

The team began by deleting a segment of chromosome 9 possessing a gene known as CDKN2a, a well-established tumor suppressor. “We have known about CDKN2A mutations for a long time, and it was already remarkable how they worked,” says Dr. Lowe. “This study says there is so much more to it, with important therapeutic implications.”

This broad deletion also removed code for a cluster of interferons, or proteins that assist the immune system in fighting off cancerous cells. After inserting these “macheted” cells into the mouse models’ pancreas, they developed cancer. With so much of the immune system’s support removed, the tumors appeared “invisible” to defenders and allowed cancer to spread more quickly. These findings reigned true for both pancreatic and melanoma mouse models.

“It’s been hard to study these interferons because they’re encoded by a cluster of 16 genes,” Dr. Lowe adds. “Using MACHETE revealed a major way in which developing cancer cells avoid being recognized by the immune system, and which can also lead to resistance to immunotherapies aimed at reactivating the immune system to attack the cancer.”

These findings suggest that patients with an in-tact interferon region may be better candidates for immunotherapy than those without it. Immunotherapies are incredibly helpful for treating cancer, but that’s not the case for all patients. By investigating which candidates would be a better fit, medical teams can spend more time providing apt treatment to the individuals needing it most.