Mouse Model May Shed Light on APC mutations in brain metastases

A mouse model under development one day may unlock the role of adenomatous polyposis coli (APC) mutations in melanoma brain metastases. A $100,000 pilot award from the Melanoma Research Alliance (MRA), Washington, DC, will allow University of Minnesota researchers to lay groundwork for additional future funding.1

“What this means is, we can afford to do the initial development of the tools, model, and resources so that we can apply for multimillion dollar federal research funds. Without this award, we wouldn’t be able to do that,” said lead investigator James Robinson, PhD. He is assistant professor at The Hormel Institute, University of Minnesota in Austin, Minnesota.

Immune checkpoint inhibitors have revolutionized melanoma treatment, but few patients experience complete responses. Patients with mutations in the APC and beta-catenin genes suffer from a higher incidence of brain metastases than do patients without these mutations, which can alter cell growth, identity, movement, and death. However, Robinson said, it is unclear whether these mutations drive metastatic development, particularly to the brain, which remains a common point of checkpoint-inhibitor failure.

If one could identify patients with APC mutations sooner, he said, such patients may benefit from more aggressive treatment or mutation-specific treatment. Because APC mutations reside in the relatively well-understood Wnt signaling pathway, Robinson added, many drugs already in clinical trials or under investigation for other diseases could merit melanoma trials, depending on which mutations investigators choose to target.

The team has developed a unique mouse model for use in this research.2 In transgenic mouse models, tumors arise over time from multiple cells. Conversely, Robinson and colleagues use a chicken virus to produce melanoma from melanocytes. “We can put in different genetic alterations, such as these mutations we’re studying, or the typical mutations found in melanoma—for instance, BRAFV600E or deletion of PTEN—and study the whole melanoma from the point of initiation.” Once investigators have preliminary data from this project, they plan to screen melanoma samples nationally for the presence of these mutations and to ascertain whether and why these mutations are causing metastases.

“In human samples,” Robinson said, “you can look to see what actually happens in the patients with mutations. But it’s hard to do a direct, controlled experiment because there are obviously thousands of different changes that occur in tumors, and melanomas are the single most mutated human tumor. So it’s sometimes hard to see the woods for the trees.”

The mouse model allows researchers to ask direct questions such as such as how changing one base pair in a single gene impacts a melanoma tumor. “We’re going to look at all the immune components of the brain metastases in the primary tumors using state-of-the-art genomic techniques that allow you to interrogate different populations of tumor cells and normal cells within the tumor.” For example, antibody assays will allow investigators to differentiate the CD45 immune-cell component from the melanoma-cell component of the tumors.

Multiplexed ion beam imaging (MIBI) technology will enable investigators to screen up to 40 antigens simultaneously, using 40 antibodies at once in a paraffin-embedded tissue slice to explore whether immune changes or changes intrinsic to APC-mutated cancer cells themselves are driving metastases. “Are the cancer cells growing? Are they more motile, more invasive? Or do they survive in circulation to become metastatic cancer cells, as opposed to primary tumor cells? None of this is known.”

Working from a single paraffin block which may contain pieces of tumors sampled decades ago, the MIBI process will allow researchers to place a section of tumor on a slide, then use PCR DNA sequencing and, if necessary, whole genomic sequencing to determine what mutations and cell populations (such as CD4 and CD8 T cells and macrophages) are present. “The point of the pilot grant is to produce the model and pilot these technologies in a small number of samples so we can persuade the National Institutes of Health (NIH) to award sufficient funds to be able to do it on a national basis.”

With any study of this kind, Robinson said, key elements include securing samples from as many centers as possible and having the funding and ability to perform the actual screening, along with the mouse experiments. His team plans to begin applying for federal funding in October.

“The way that pilot grants work,” he said, “you usually expect not to get NIH funding the first time around.” Therefore, Robinson hopes that a February 2023 submission finds success. “If not, we have the other year of MRA funding, so we can continue to take this forward and apply in 2024 too.”

Disclosures:

Robinson reports no relevant financial interests.

References:

1. Melanoma Research Alliance. Melanoma Research Alliance announces $13 million in grants to advance melanoma prevention, detection & treatment.https://www.curemelanoma.org/assets/Uploads/MRA-Grant-Awards-2022.pdf. May 19, 2022. Accessed June 16, 2022.

2. Grigore F, Yang H, Hanson ND, VanBrocklin MW, Sarver AL, Robinson JP. BRAF inhibition in melanoma is associated with the dysregulation of histone methylation and histone methyltransferases. Neoplasia. 2020;22(9):376-389. doi:10.1016/j.neo.2020.06.006