In cancer, the context 'makes' the mutation

In cancer, it’s tempting to divide genes into two camps: those that suppress cancer and those that drive it. But cancer is more complicated. In a recent study, investigators at Fred Hutchinson Cancer Research Center showed how the same gene can restrain tumor growth or promote it, depending on its accompanying mutations.

The findings highlight the idea that "context really determines [gene] function,” said Dr. Bob Eisenman, an expert in the genetics of cancer, who co-led the project with Hutch lung cancer expert Dr. David MacPherson. “The context [in which a genetic change] is occurring is clearly a major determinant of its outcome,” Eisenman said. The study was published May 28 in the journal Cancer Cell.

The team examined a gene called Max, a mandatory partner of a protein in cells called Myc, which, when elevated, can cause unrestrained tumor growth. Yet, counterintuitively, some tumors carry defective copies of the Max gene. Using strains of lab mice created to model human small-cell lung cancer, the researchers found that while Max co-pilots Myc-driven cancers, it throws a brake on tumors caused by other factors. Removing it from these other cancer cells enabled them to rewire their metabolism to enhance growth.

“Max is clearly a potent tumor suppressor in certain contexts,” MacPherson said. “That occurs through a mechanism that's independent of Myc and likely mediated by repression of key target genes.”

A Max-imal paradox

Eisenman is an expert in the cellular and genetic changes that turn healthy cells into cancer cells. In particular, he focuses on the role that Myc can play in this process. Myc is a protein that can turn on genes involved in cell growth and proliferation. In many tumors, genetic and cellular changes that raise Myc levels help cells grow unfettered, making Myc one of the best-known tumor-driving genes.

Thirty years ago, Eisenman’s lab first described the gene for Max, the protein partner that makes Myc’s functions possible.  

Because Myc must work with Max, researchers assumed it was just as much of a cancer driver as Myc. Yet counterintuitively, some tumors carry defective copies of Max, whether inherited or acquired later in life.

“The idea that loss of Max would somehow be involved in the genesis of yet other cancers didn't seem to make any sense,” Eisenman said. “It was very paradoxical.”

Defective Max is primarily associated with neuroendocrine tumors, which arise from cells that release hormones in response to signals from nerves. One example is small-cell lung cancer, or SCLC, a deadly disease that currently lacks targeted treatments. This is MacPherson’s area of expertise. He seeks to better understand how small-cell lung cancer develops in the hopes of discovering weaknesses that new treatments could target.

The genetics of small-cell lung cancer are varied. In some patients, increased Myc drives their cancer. But tumors in a subset of patients have a defective Max gene. Could normal Max also suppress tumor development? If so, how? And how could turning it off possibly promote tumor development?  Eisenman and MacPherson teamed up to find out.

Context is key

Postdoctoral fellows Drs. Arnaud Augert and Haritha Mathsyaraja spearheaded the efforts to untangle Max’s role in small-cell lung cancer development. They created mouse models of small-cell lung cancer using several known SCLC-linked mutations, with or without accompanying Max mutations. At the same time, Augert used CRISPR, the precise gene-editing tool, to screen SCLC mutations to reveal those most likely to be driving tumor formation. Max emerged as a prime suspect.

Using their mice, the team found that, yes, Max is a cancer driver — and a cancer suppressor. It just depends on context.

They compared two mouse strains that grow small-cell lung tumors driven by mutations common in human SCLC. In one model, the scientists accelerated tumor formation by increasing Myc levels. Removing Max from tumors with amped-up Myc slowed tumor formation, extending tumor-free survival by about 22 days. (Further investigation showed that Myc-dependent tumors may require the presence of Max: Despite the researchers’ genetic manipulations, Max was never completely lost from tumors that relied on Myc to grow.)

Conversely, when the team removed Max from tumors with normal levels of Myc, this accelerated their growth. It took only five months for tumors to appear in these mice, compared to about 10 months for mice whose tumors still had a working Max gene.

In previous work in a model of lymphoma, Mathsyaraja had showed that Myc absolutely needs Max to drive tumor formation.

The current findings underscore that “context is critical,” Eisenman said.

Max loss rewires cell metabolism

To understand how Max can sometimes restrain tumor growth, the scientists looked to see which genes were turned on at unusually high levels in SCLC tumors missing Max. Mathsyaraja and Augert quickly spotted a theme: metabolism.

In particular, the SCLC cells missing Max had amplified the molecular reactions that form serine, a building block of proteins. The cells also ramped up a serine-based mechanism for constructing nucleotides, the building blocks of DNA.

“So if you can imagine what a cancer cell wants to do, it wants to make more proteins and more nucleotides [in order to grow],” said Hutch colleague Dr. Lucas Sullivan, who collaborated with the team to study the metabolic perturbations seen in the SCLC cells lacking Max. “This signature is a phenomenon that's been observed across many, many cancers.”

This signature gave the cells missing Max an unexpected survival “superpower,” said Eisenman: the ability to thrive without serine. Usually, cells import a lot of this building block from their environment. But the team found that because SCLC cells without Max can build their own serine, they can grow even when it’s missing. In contrast, cells with normal Max can’t manufacture enough serine to grow without importing it; under these conditions, they die.

Conversely, losing Max made the cells much more sensitive to a drug that disrupts nucleotide production, further suggesting that this pathway contributes to their enhanced growth.

Exploring Max’s molecular network

Though the researchers have solved the conundrum of how Max can be defective in cancer, they’ve revealed several more. For one, they were surprised that cells missing Max could even elevate these metabolic pathways. That's because Myc, which needs Max to function, is known to regulate them.

But Myc is the center of a complex web of molecular interactions, and Max also interacts with many other molecules involved in turning genes on and off. It collaborates with some, like Myc, and blocks the activity of others.

“My guess would be, therein lies the answer, that [loss of Max] is really disrupting the function of a more complex network,” Eisenman said. The team is currently working to untangle this web and clarify the molecular mechanisms that underpin Max’s role as a tumor suppressor.

As a first step toward potential therapeutics, they also want to understand whether the metabolic and molecular changes they see in SCLC cells missing Max are common to other neuroendocrine tumors. Why is it that Max appears to act as a tumor suppressor in neuroendocrine tumors but not in others?

“Probably in most cancer types, it's really going to be deleterious for the cells to lose Max,” MacPherson said. It’s likely, he said, that Max suppresses tumor formation for only a subset of tumors: “One of the unknowns is, what's the common link amongst that subset?”

To explore commonalities, the researchers are currently developing models of other neuroendocrine tumor types with and without Max. MacPherson suspects that a shared molecular circuitry in neuroendocrine cells may make it possible for Max to take on a tumor-suppressive role it can’t assume in other cell types.

The researchers are also actively seeking to determine if losing Max creates unique and possibly druggable vulnerabilities in neuroendocrine tumor cells.

While Max’s shifting roles is making it difficult to pin down, Sullivan is hopeful.

“This happens a lot when studying metabolism,” he said. “There's a lot of metabolic flexibility and cancer cells can behave differently in different situations. … While there are no absolute rules that we can universally take advantage of, these kinds of tissue-specific interactions actually give us a case for optimism. They imply that tumors arising in different tissues interact differently with their genetic network. It suggests that there's the possibility of some specific vulnerability for Max-null [without Max] cancers.”

The National Cancer Institute funded this work.