The Armed Forces Institute of Pathology

Blood smear from an adult female with myelodysplastic syndrome.

September 13, 2023

Weill Cornell Researchers Develop New Technique to Investigate Impact of Abnormal RNA Splicing on Disease

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Researchers in the Landau lab, led by Dr. Dan Landau, hematology and medical oncology, recently developed a new technique to investigate how mutations in RNA splicing can lead to clonal hematopoiesis and myelodysplastic syndrome — a topic with greater implications regarding the pathophysiology of cancer.  

Clonal hematopoiesis is a symptomless condition with mutated blood cells that acts as a precursor to the rarer type of blood cancer, myelodysplastic syndrome, where patients have low levels of functional blood cells. 

RNA splicing refers to the cutting up and reassembling of genetic transcripts in cells. It is a naturally occurring process required to prepare genetic transcripts for use by cells and can lead to the formation of distinct proteins with different functions. 

Prior work with bulk sequencing, an approach that enables researchers to study transcriptomes across cell populations, revealed that many cancers possess mutations in RNA splice factors, proteins that regulate the splicing process. They found that the most prevalent mutation in MDS is in SF3B1, an essential splice factor. The mutation causes abnormal splicing and ultimately produces nonviable protein products. However, bulk sequencing has two major limitations. 

“In clonal hematopoiesis and MDS, the bone marrow has both normal cells and mutated cells. With bulk sequencing, it’s difficult to know whether the change is in normal or mutated cells,” Landau said. 

The other challenge is that there isn’t just one cell type but instead a whole range of cell types, and because bulk sequencing averages across these cell types, researchers can have difficulty telling which cells the aberrant splicing is coming from. 

To address these limitations, the Landau lab developed genotyping of transcriptomes, a single-cell method, in 2019. GoT allowed them to simultaneously examine a cell’s genetic sequence and determine the presence of the SF3B1 mutation. 

In the current study, the researchers expanded GoT to profile gene mutations and gene expression patterns in cells and to map splicing changes in mutant cells compared to the normal cells. The researchers also integrated a separate method for detecting protein surface markers on individual cells. Their new technique, called GoT-Splice, enables greater precision in characterizing abnormal splicing events and how they arise in cell development processes.   

“We wanted to capture the variation across cell types and compare mutant and normal cells to see differences at the splicing and RNA regulatory levels,” said Dr. Mariela Cortés-López, a postdoctoral researcher in the Landau lab postdoc. “We wanted to see what could explain the phenotypes we observed in these diseases, how the mutations are connected to physical effects on patients.” 

One of the mutations identified in MDS was in a protein called BAX, which is a critical regulator of programmed cancer cell death. With the splicing abnormality, BAX resisted cell death instead of causing it, suggesting on a clinical level that patients with a mutation for BAX will be less responsive to specific drug treatments. 

The researchers performed the same experiments with clonal hematopoiesis in which a mutated blood cell clones itself to create a population of identical cells. They found that even in early stage clonal hematopoiesis, splicing mutations in MDS — including in BAX — are already present, even though the condition is benign. The trajectory for this line of work is to better understand the cloning process and characterize the effects of the mutations in the absence of visible disease. 

The Landau lab will continue to explore the applications of GoT-Splice to examine splice factor mutations in cancer and understand its effects in patients.

“As a physician-scientist, I have the patients in my mind,” Landau said. “It’s important for me to think about the translational implications of our research and how to get the knowledge back into the clinic. We want to make fundamental discoveries about how the body works, and we also want them to have an impact on our patients.”

Kaitlin Chung in a Sun Science Staff and can be reached at [email protected]