All cancers are powered by mutation. A single change in DNA — a swapped letter in the genetic code — can disable a cellular safeguard or trigger unchecked growth. For decades, the search for the origins of these mutations focused on environmental factors such as ultraviolet radiation and tobacco smoke, but in more than half of cancer types, a significant source of mutation appears to come from within: a family of enzymes known as APOBEC3. Rather than initiating tumors outright, these enzymes are thought to accelerate the genetic changes that help cancers evolve and resist treatment.
“They’re part of the cell’s innate immune system and normally target viral DNA,” explains Christopher Belica, a graduate student in the Department of Biochemistry, Molecular Biology and Biophysics. “The problem is that when these enzymes are overexpressed or misregulated, they can start targeting the human genome instead of viral genomes.”
Studying APOBEC3 enzymes, however, is not straightforward. Scientists can readily detect the distinctive genetic fingerprints these enzymes leave in cancer genomes, but the proteins themselves are small and difficult to observe at high resolution. That makes it challenging to understand their structure, which is essential for determining how they bind DNA and, ultimately, how to block their activity. Working with Professor Hideki Aihara and collaborators in Professor Reuben Harris’ lab at the University of Texas Health Science Center at San Antonio, Belica has focused on overcoming this challenge. His research centers on developing more effective tools to track and control APOBEC3B, a member of the APOBEC3 family strongly linked to cancer.
Before he could get to the problem of visualizing how APOBEC3B works, Belica first needed a better way to measure its activity. The work culminated in a 2024 publication describing a new biochemical tool, the RADD assay (short for Real-time APOBEC3-mediated DNA Deamination), which enables researchers to monitor the enzyme’s activity in real time. Traditional methods required multiple steps and hours of processing. Belica’s technique streamlined the process into a single reaction that produces an immediate fluorescent signal when the enzyme alters a DNA base. “The primary idea was to find a more information-rich way of doing things,” Belica explains, “but it ultimately saves a lot of time as well.”
Now, backed by an NIH fellowship, Belica is investigating how APOBEC3B can be shut down. He has collaborated with Dr. Ryan Abdella, who manages the University of Minnesota’s cryoEM facility, to determine the enzyme’s three-dimensional structure using cryo-electron microscopy. Even with this cutting-edge technology, APOBEC3B is a small target, making it difficult to distinguish clearly amid the sea of microscopic particles. To overcome this, Belica identified nanobodies (tiny proteins originally derived from alpacas) that bind tightly to APOBEC3B. By pairing these nanobodies with larger support proteins, he effectively increases the size of the overall APOBEC3B complex, making it large enough to isolate and visualize in detail.
The nanobodies serve another purpose as well. By linking two nanobodies, Belica created a paired structure that appears to interfere with the enzyme’s ability to bind to DNA. “We call them nanopinchers because they’re kind of like a crab claw,” Belica explains of the ongoing research. “And we’ve found that they show inhibition on par with some of the stronger viral inhibitors of APOBEC3B.”
The implications are significant. If APOBEC3B helps tumors evolve by accelerating mutation, then slowing it down could limit a cancer’s ability to adapt and resist treatment. Translating those findings into clinical therapies will require years of continued research and collaboration. Yet by building tools to observe and block the enzyme, Belica’s work lays the foundation for a new strategy that could one day make cancer treatments more precise and more durable. –Jonathan Damery