Cre matters: Neuroblastoma oncogene activation controls what type of tumor forms

Neuroblastoma is the most common type of cancer in newborns and infants. Many genes are linked to the disease, hindering development of effective targeted treatments. MYCN is amplified in 50% of high-risk neuroblastoma cases, but some tumors lack this connection to MYCN and instead often rely on a related but distinct gene: c-MYC.

“Cells with MYCN amplification do not express c-MYC in most cases. They’re in the same gene family, largely overlapping in function, but not quite identical,” said Jun Yang, MD, PhD, St. Jude Department of Surgery. “A small fraction of high-risk neuroblastoma patients, however, do not have MYCN amplification in their cancer, but do highly express c-MYC.” This discrepancy highlighted for Yang a gap in the available neuroblastoma models, all of which depended on MYCN, prompting his laboratory to set out to create a c-MYC–based model for research. 

The findings, published recently in Cancer Research, took Yang’s lab on a journey of discovery they did not anticipate.

Neuroblastoma originates from neuroendocrine cells, which produce neurotransmitters such as dopamine and tyrosine. To trigger the expression of c-MYC in neuroendocrine cells, the researchers used an enzyme called Cre recombinase (an industry standard for the direct activation of genes in model organisms) tailored for tyrosine-producing cells.

However, the model did not behave as expected. “We observed tumor growth but found that it developed in the pancreas,” said Yang. “Neuroblastoma rarely occurs in the pancreas.” Histology and single-nuclear RNA sequencing results confirmed that the models were, in fact, developing an extremely rare type of pancreatic neuroendocrine tumor called somatostatinoma, which occurs in approximately 1 in 40 million individuals annually. 

The unexpected finding led the researchers to question the tools they used to make the model, in particular, the Cre recombinase. Cre recombinase is a bacterial enzyme turned molecular biology scissors which recognizes particular DNA sequences called LoxP sites, cuts out the genetic material flanked by these sites, and then recombines them. With correct “floxing”, the recombinase can be directed to remove oncogene supressors, such as that for c-MYC. Different types of Cre recombinase, such as tyrosine hydroxylase Cre and dopamine b-hydroxylase Cre, are designed to trigger oncogene activation in specific tissues, often at specific developmental stages.

“For this model, we used tyrosine hydroxylase Cre, which is classically used to generate MYCN tumors,” explained Yang. “So, we tested other types of Cre lineages and, eventually, landed on an improved dopamine b-hydroxylase Cre. Finally, we got neuroblastoma tumor growth.”

The model using dopamine b-hydroxylase Cre successfully reflected features of neuroblastoma, including sensitivity to drugs used to treat the disease. “We tested approved treatments for neuroblastoma, including difluoromethylornithine and anti-GD2 immunotherapy, to see whether the model would behave as expected,” explained first author Jie Fang, PhD, Department of Surgery. “We saw the strong therapeutic effect in the presence of both therapies that we anticipated.”

Because the research led the investigators down this surprising avenue, the findings have multiple implications. Creating a model that allows researchers to study c-MYC–driven neuroblastoma (and somatostatinoma) is vital for developing new therapeutics. Additionally, the impact of how different Cre recombinases can be used to make different tumors explains why these models have struggled in the past. This highlights an important consideration for other labs using this system. 

“c-MYC–driven tumor mouse models have been anticipated for 30 years, but nobody could generate them. Our experience explains why it’s been so difficult,” Yang said. “It’s a strong indication that the Cre used matters so much and that, maybe for others, the idea has been right, but the tool they were using was not.”