In a new study published in Nature, Sivaraj Sivaramakrishnan, a professor in the College of Biological Sciences, along with graduate student Fred Sadler and co-authors Michael Ritt and Yatharth Sharma, uncovered the role of the third intracellular loop in the GPCR’s signaling mechanism, suggesting the possibility of a more targeted approach to drug discovery and a paradigm shift for new therapeutics.

“Typical GPCR drugs act as on or off switches for cellular signaling outcomes,” said Sivaramakrishnan. “Drugs that leverage the loop effectively can act as signaling dimmer switches to more precisely control drug responses.”

The authors developed new biochemical and biophysical tools, combined with computational measurements by collaborators Ning Ma and Nagarajan Vaidehi at the City of Hope Cancer Center. They tracked how the third intracellular loop changes in shape, or conformation, through the receptor signaling process. In a breakthrough for the field, their data show that the loop acts as a kind of gate to ensure that receptors activate the correct type of G protein signaling at the right intensity.

“A key advantage of this loop is that it is highly unique, even among closely related receptors, making it an outstanding drug target,” said Sadler. “Developing drugs through this newly discovered mechanism would allow for far more targeted therapeutics.”

Funding was provided by the National Institutes of Health and the University of Minnesota Graduate School.

Writing in the March 16, 2023 issue of Science, a team of scientists, led by senior study author Don Cleveland, PhD, Distinguished Professor of Medicine, Neurosciences and Cellular and Molecular Medicine at University of California San Diego School of Medicine, with colleagues and elsewhere, demonstrate that stathmin-2 loss can be rescued using designer DNA drugs that restore normal processing of protein-encoding RNA.

“With mouse models we engineered to misprocess their stathmin-2 encoding RNAs, like in these human diseases, we show that administration of one of these designer DNA drugs into the fluid that surrounds the brain and spinal cord restores normal stathmin-2 levels throughout the nervous system,” Cleveland said.

Cleveland is broadly credited with developing the concept of designer DNA drugs, which act to either turn on or turn off genes associated with many degenerative diseases of the aging human nervous system, including ALS, AD, Huntington’s disease and cancer.

Several designer DNA drugs are currently in clinical trials for multiple diseases. One such drug has been approved to treat a childhood neurodegenerative disease called spinal muscular atrophy.

The new study builds upon ongoing research by Cleveland and others regarding the role and loss of TDP-43, a protein associated with ALS, AD and other neurodegenerative disorders. In ALS, TDP-43 loss impacts the motor neurons that innervate and trigger contraction of skeletal muscles, causing them to degenerate, eventually resulting in paralysis.

“In almost all of instances of ALS, there is aggregation of TDP-43, a protein that functions in maturation of the RNA intermediates that encode many proteins. Reduced TDP-43 activity causes misassembly of the RNA-encoding stathmin-2, a protein required for maintenance of the connection of motor neurons to muscle,” said Cleveland.

“Without stathmin-2, motor neurons disconnect from muscle, driving paralysis that is characteristic of ALS. What we have now found is that we can mimic TDP-43 function with a designer DNA drug, thereby restoring correct stathmin-2 RNA and protein level in the mammalian nervous system.”

Specifically, the researchers edited genes in mice to contain human STMN2 gene sequences and then injected antisense oligonucleotides — small bits of DNA or RNA that can bind to specific RNA molecules, blocking their ability to make a protein or changing how their final RNAs are assembled — into cerebral spinal fluid. The injections corrected STMN2 pre-mRNA misprocessing and restored stathmin-2 protein expression fully independent of TDP-43 function.

“Our findings lay the foundation for a clinical trial to delay paralysis in ALS by maintaining stathmin-2 protein levels in patients using our designer DNA drug,” Cleveland said.

Co-authors include: Michael W. Baughn, Jone López-Erauskin, Melinda S. Beccari, Roy Maimon, Sonia Vazquez-Sanchez, Jonathan W. Artates and Eitan Acks, all at Ludwig Institute for Cancer Research-UC San Diego and UC San Diego; Ze’ev Melamed, Ludwig Institute for Cancer Research-UC San Diego, UC San Diego, and The Hebrew University of Jerusalem; Karen Ling, Paayman Jafar-nejad, Frank Rigo and C. Frank Bennett, all at Ionis Pharmaceuticals; Aamir Zuberi, Maximilliano Presa, Elena Gonzalo-Gil and Cathleen Lutz, all at The Jackson Laboratory; Som Chaturvedi, Mariana Bravo-Hernández, Vanessa Taupin and Stephen Moore, all at UC San Diego; L. Sandra Ndayambaje and Ana R. Agra de Almeida Quadros, Harvard Medical School; Clotilde Lagier-Tourenne, Harvard University and Broad Institute of Harvard University and Massachusetts Institute of Technology.

Yet no one is certain why this disease is suddenly affecting so many young people. In a new paper published in Science, Dana-Farber Cancer Institute researchers outline the complexities of the disease and the research needed to map out a path toward understanding it.

“The rising incidence of young-onset colorectal cancer is extremely concerning, and it is urgent that the scientific community comes together to better understand the underlying causes and biology,” said co-author Kimmie Ng, MD, MPH, associate chief of gastrointestinal oncology and director of the Young-Onset Colorectal Cancer Center at Dana-Farber. The Center provides expert care for patients and conducts the multidisciplinary research required to understand colorectal cancer in young adults and develop new ways to prevent, detect and treat it.

Young-onset Colorectal Cancer: a unique challenge

Young-onset colorectal cancer (CRC), also called early-onset CRC, differs from later-onset CRC in several ways, according to the authors. Young-onset disease is often more aggressive, presents on the left side of the colon rather than the right, and often presents with rectal bleeding and abdominal pain.

At a molecular level, however, studies have shown conflicting results that suggest both similarities and differences in the genetic mutations that drive the diseases. This is likely due to the complexity of the disease, according to the authors, and future research should account for this variability.

More study is also needed to determine if CRC risk factors for young people are similar to those for older adults. Obesity and environmental exposures, for instance, have been associated with young-onset disease, but other factors could also play a role, such as increased antibiotic use or the frequency of Cesarean sections, both of which could influence the microbiome. To begin to understand the risk factors, the authors suggest that investigations should include a combination of genetics, environmental exposures, diet and lifestyle measures, as well as immune system interactions and the microbiome composition.

One clear difference is that young-onset CRC is typically discovered after the disease has advanced. This is due in part to the fact that screening for colorectal cancer starts at age 45 in the U.S., so the disease often goes undetected in younger people.

“It’s important not to dismiss the idea that a young person could have colorectal cancer even though the disease is still more common in older adults,” said co-author Marios Giannakis, MD, PhD, a gastrointestinal oncologist at Dana-Farber.

Responding with multidisciplinary research involving diverse populations

To account for the complexity of young-onset CRC, Ng and Giannakis said that research should be multidisciplinary and include many areas of investigation simultaneously. For instance, genome-wide association studies, which aim to find risk genes for the disease, should also include data about environmental exposures that could also increase risk.

These types of studies could point to new ways to identify young people who are at high risk of young-onset disease and should be screened for CRC. “Risk stratification is going to be very important as we think about screening for young-onset disease,” said Giannakis.

Clinical studies should also include the collection of blood, tissue, and stool samples from patients over time to shed light on the role of immune cells, environmental exposures and the microbiome in disease onset, progression, and treatment response. Ng and Giannakis encourage global collaborations aimed at facilitating the collection of these specimens, such as the Count Me In Colorectal Cancer Project, which directly partners with patients in the U.S. and Canada and makes all data available for research.

Ng and Giannakis also call for more effort in ensuring diverse populations are included in studies of young-onset CRC. Studies show underrepresented minorities are disproportionally burdened by young-onset CRC and non-Hispanic Black patients have a higher mortality rate when compared to non-Hispanic whites.

“Although each of these steps require commitment and perseverance,” said the authors, “it is the growing numbers of young patients bravely battling this disease that will be the compass that keeps us on the path towards better understanding, preventing, and treating young-onset colorectal cancer.”