Discovery of a hidden tumor suppressor in the genome of the virus that causes Merkel cell carcinoma

Most often we think about cancer as developing from cells that acquire cancer-driving mutations, but there’s a subset of cancers driven by another culprit: viruses. To date, “seven human viruses have been identified that cause cancer,” explains Dr. Nicholas Salisbury, a postdoctoral fellow in Dr. Denise Galloway’s lab. Epstein-Barr virus (EBV) was the first of these to be discovered, whereas human papillomavirus (HPV), perhaps the most well-known of the cancer-causing viruses, was discovered a decade later, in the 1970s. The most recent virus to join this family, Merkel cell polyomavirus (MCV), was only discovered in 2008 and causes about 80% of Merkel cell carcinoma (MCC) cases. MCC is a rare, aggressive skin cancer that, unlike other skin cancers, is predominantly driven by viral infection rather than UV rays. After Merkel cell polyomavirus infects its host cell, it highjacks the cellular machinery to replicate its own genome and causes no evident disease. In a small subset of cases, Merkel cell viral DNA becomes integrated into the host genome and pushes the cell towards becoming cancerous. Dr. Salisbury shares that most people will “get infected with Merkel cell polyomavirus at some point during childhood through skin-to-skin contact, but only about 1 in 100,000 will develop cancer.” Unlike HPV, there is no vaccine available for Merkel cell polyomavirus. And as the newest cancer-causing virus on the block, we still have so much to learn about Merkel cell polyomavirus and what leads it to cause cancer so that more effective treatment options can be developed.

Merkel cell carcinoma is caused by expression of two viral genes: small and large tumor antigens, often referred to as small T and large T. However, it wasn’t until about 5 years after Merkel cell polyomavirus was first discovered that the Galloway lab found another gene encoded within Large T in an alternate reading frame, which they named the Alternate Large T Open reading frame, or ALTO for short. Comparing the genomes of different polyomaviruses, the Galloway lab found that the ALTO sequence was fairly similar to a gene in the mouse polyomavirus (Middle T) that can cause tumors in mice. Dr. Salisbury reasoned that “since Middle T and ALTO are evolutionarily related, we initially believed that ALTO might have the same function,” predicting ALTO was also an oncogene. In a recent PNAS article led by Salisbury, he set off to test this hypothesis. Ultimately in this study, Dr. Salisbury found that his initial hypothesis was incorrect—but what he uncovered could lead to the development of a new and much needed therapy for Merkel cell carcinoma.

This research started off as “a basic biology question: what is the function of this gene?” shares Dr. Salisbury. As he explains, “this virus has two distinct biologies. First, it can infect and replicate in human cells, but without causing symptoms or overt clinical disease. Second, it integrates into the human genome and no longer replicates but causes cancer.” From previous work knocking out ALTO in Merkel cell polyomavirus and infecting cells with this mutant virus, the Galloway lab knew it wasn’t essential for viral replication. Given its similarity to oncogenic mouse Middle T, it seemed likely that ALTO might play an essential role in causing cancer. For Dr. Salisbury, the first hint that this story might not be so straightforward was when he started to look at ALTO protein expression in MCC cells using an antibody the lab developed. “When the virus infected cells in culture, ALTO could be detected as little speckles throughout the cell. However, when looking at patient-derived MCC cells, ALTO could never be detected,” Dr. Salisbury notes. While he admits some people questioned why he wanted to study an oncovirus protein that didn’t appear to be expressed in the cancer, Dr. Salisbury continued his work anyway. “To me, the fact that the ALTO DNA sequence was present in these cancer cells, but they were not producing the protein, suggested something interesting was going on. There was likely a reason why ALTO expression had been shut down.” To get at why these cancer cells didn’t express ALTO, but did express small T and large T, Dr. Salisbury transfected various MCC cell lines with an ALTO expression construct to force expression of this mysterious viral gene. Restoring ALTO expression in MCC cells caused growth arrest, revealing that ALTO is in fact a tumor suppressor, not an oncogene.

Next, the authors used RNA-sequencing to try to understand how ALTO induced growth arrest in MCC cell lines and uncovered that it activated the NF-κB pathway, an important mediator of inflammatory and anti-viral responses. But why was a viral protein activating a pathway that would alert the immune system to a viral infection? Feeling as though he’d hit a roadblock, Dr. Salisbury did what many of the best scientists do: he put the project in the freezer and pursued other research that had a clearer direction. About two years later, Dr. Salisbury returned to the ALTO project with a fresh perspective and determined to get to the bottom of this mystery. He had found a paper that showed that activating the NF-κB pathway in skin cells (keratinocytes) activates not only immune signaling but also induces growth arrest. It was now starting to make sense why the cancer cells would need ALTO to be turned off to survive.

To get at the mechanism of how ALTO activates the NF-κB pathway, Dr. Salisbury identified protein binding partners of ALTO using proximity-labeling coupled with mass spectrometry. He found several binding partners that interact with Epstein-Barr virus protein LMP1, which also activates the NF-κB pathway and inhibits viral replication. With this link, the story began to come together. Dr. Salisbury found that ALTO’s interactions with several LMP1 binding partners were important for NF-κB pathway activation and that NF-κB signaling appeared to downregulate expression of Merkel cell polyomavirus early genes, small T and large T, which promote viral replication or cancer depending on the context. Altogether, this work uncovered that ALTO must be silenced in order for MCC to develop. ALTO acts as a tumor suppressor by activating the NF-κB pathway, downregulating the viral oncogenes and causing growth arrest in cancer cells.

“While this started as a basic science question, we now have new science that we are trying to translate into a new therapy,” Salisbury shares. For localized MCC, surgery and radiation are often successful at treating this cancer. However, for advanced MCC patients, immunotherapies are often used. Unfortunately, about half of patients do not respond to immunotherapy. For this subset of non-responsive patients with minimal immune infiltration, an ALTO-based therapy could be beneficial, as the Galloway research team has shown that ALTO can prevent cancer cell growth and may help boost an immune response against the cancer in combination with immunotherapy. Dr. Salisbury is looking forward to continuing to study ALTO from the basic biology perspective and understand how ALTO gets turned off in cancers. Ultimately, he looks forward to translating these findings into an ALTO-based therapy for this rare cancer type with limited treatment options.

This work was supported by the National Institutes of Health and the Brave Like Gabe Foundation.

Fred Hutch/UW/Seattle Children’s Cancer Consortium member Dr. Denise Galloway contributed to this work.

Salisbury NJH, Amonkar S, Landazuri Vinueza J, Carter JJ, Roman A, Galloway DA. Polyomavirus ALTOs, but not MTs, downregulate viral early gene expression by activating the NF-κB pathway. Proc Natl Acad Sci U S A. 2024.